Immune effector cells derived from genetically engineered induced pluripotent stem cells using membrane-bound IL12 and uses thereof

Abstract

Genetically engineered induced pluripotent stem cells (iPSCs) expressing a chimeric antigen receptor (CAR) and membrane-bound IL-12, and their derivative cells, as well as methods for producing and using the same are provided. Compositions, polypeptides, vectors, and manufacturing methods are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 350,172, filed on June 8, 2022, which is incorporated herein by reference in its entirety.

[0002] This application provides immune effector cells derived from genetically modified induced pluripotent stem cells (iPSCs) that express membrane - bound IL - 12, and their derivative cells. Also provided is the use of iPSCs or their derivative cells that express chimeric antigen receptors for allogenic cell therapy. Also provided are related vectors, polynucleotides, and pharmaceutical compositions.

[0003] Reference to Electronically Submitted Sequence Listing This application contains a sequence listing submitted electronically via EFS - Web, which is an ASCII - formatted sequence listing with the file name "Sequence Listing_ST26.xml", created on June 6, 2023, and a size of 210 kb. The sequence listing submitted via EFS - Web is part of the specification and is incorporated herein by reference in its entirety.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference.

Background Art

[0005] Chimeric antigen receptors (CARs) significantly enhance the antitumor activity of immune effector cells. A CAR is an engineered receptor typically containing an extracellular targeting domain linked to a linker peptide, a transmembrane (TM) domain, and one or more intracellular signaling domains. Traditionally, the extracellular domain consists of an antigen-binding fragment (such as single-chain Fv, scFv) of an antibody specific for a given tumor-associated antigen (TAA) or cell surface target. While the extracellular domain confers tumor specificity to the CAR, the intracellular signaling domain activates T cells genetically engineered to express the CAR upon TAA / target engagement. The engineered immune effector cells are reinfused into cancer patients, where they specifically engage and kill cells expressing the CAR's TAA target (Maus et al., Blood. 2014 Apr 24;123(17):2625-35, Curran and Brentjens, J Clin Oncol. 2015 May 20;33(15):1703-6).

[0006] Autologous patient-specific CAR-T therapy has emerged as a powerful and potentially curative therapy for cancer, particularly CD19-positive hematological malignancies. However, autologous T cells must be generated on a custom-made basis, which remains a significant limiting factor for large-scale clinical applications due to production costs and the risk of production failure. The development and broader application of CAR-T technology are also limited by several other major drawbacks, including, for example, a) inefficient antitumor responses in solid tumors, b) limited penetration and susceptibility of adoptively transferred CAR T cells to the immunosuppressive tumor microenvironment (TME), c) poor persistence of CAR-T cells in vivo, d) severe adverse events in patients, including cytokine release syndrome (CRS) and graft-versus-host disease (GVHD) mediated by CAR-T, and e) the time required for manufacturing.

[0007] Cytokines such as interleukin-2 (IL-2), IL-12, and IL-15 have been investigated to improve the antitumor activity of adoptive T cell therapy (ACT). Considering that IL-12 is a potent mediator of activated immune cells and can greatly enhance the activity of immune cells against tumor cells, it is particularly attractive for such purposes. IL-12 is a heterodimeric protein composed of the p35 (IL-12A) and p40 (IL-12B) subunits and was first characterized as a potent activator of natural killer (NK) cells. Since then, IL-12 has also been shown to promote the differentiation of CD4 T cells into interferon-γ (IFN-γ)-producing type 1 helper cells (TH1), increase CD8 T cell cytotoxicity, upregulate antigen presentation, and reprogram myeloid-derived suppressor cells (MDSC) into a phenotype that instructs T cells. When NK cells are exposed to IL-12, a cytokine produced by dendritic cells and macrophages, the activity of NK cells that kill sensitive targets such as cancer cells increases 20- to 100-fold. Thus, IL-12 is often used in NK or T cell therapy.

[0008] However, systemic exposure to IL12 can have negative effects throughout the body, and its therapeutic use is limited by these systemic effects. The clinical utility of IL-12 is limited by severe toxicity upon systemic administration. To safely utilize IL-12 for cancer treatment, several groups have investigated the ability to selectively stimulate antitumor immune responses in the tumor microenvironment. This includes efforts to genetically engineer tumor-specific T cells to selectively drive IL-12 expression upon encountering tumor antigens. See L. Zhang, et al. “Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment.” Mol. Ther.19, 751-759 (2011). This significantly improved the effectiveness of T cell therapy in a mouse tumor model. Clinical evaluation of tumor-infiltrating lymphocytes (TILs) genetically engineered to produce IL-12 in this manner resulted in objective clinical responses at cell doses that were 1 / 10 to 1 / 100 of those required for past TIL therapies, including in patients who had previously failed standard TIL therapy. L. Zhang, et al, “Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma.” Clin. Cancer Res.21, 2278-2288 (2015). However, despite this promising efficacy, insufficient control of overall IL-12 expression in patients led to severe IFN-γ-related toxicities, and further development of this approach was halted. To safely utilize IL-12 for cancer immunotherapy, several researchers have taken the approach of tethering IL-12 to the surface of tumor-specific T cells directly prior to adoptive transfer by binding the cytokine to an antibody that binds to a cell surface receptor, thereby controlling the cytokine dose level and activity profile of IL-12.See, for example, Jones et al., Sci. Adv. 8, eabi8075 (2022). Alternatively, the IL-12 protein can be fused to a transmembrane domain (TM), such as the EGFR transmembrane domain, or a signaling domain. International Publication Nos. WO 2018 / 068008 and WO 2020 / 160350 disclose T cells engineered to express membrane-bound IL-12, and methods of treating cancer using such cells. However, the T cells disclosed therein are only suitable for autologous therapy.

[0009] Another approach investigated for controlling cytokine expression is the concept of slow-release of cytokines, which allows release upon protease cleavage by attaching the cytokine to the outer side of the cell membrane via a cleavable linker. See, for example, A. Gonzalez et al, Senti Bio Abstract #584 AACR Annual Meeting 2022.

[0010] Accordingly, there is an unmet need for therapeutically sufficient and functional allogeneic antigen-specific immune cells having membrane-bound IL-12 for effective use in immunotherapy. SUMMARY OF THE INVENTION

[0011] In one general aspect, genetically engineered induced pluripotent stem cells (iPSCs) or their derivative cells are provided. The cells comprise (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (ii) a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide that is at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide that is at least 90% similar thereto, and a membrane-bound interleukin 12 (IL-12) encoding a second exogenous polynucleotide comprising a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides, and (iii) a deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes.

[0012] In certain embodiments, the polynucleotide encoding membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for activation-inducible release of IL-12 through protease ADAM17. ADAM17 is expressed by activated lymphocytes and is directly involved in the release of other immune mediators such as TNFa that are similarly presented in a membrane-tethered form. When this membrane-tethered IL-12 is expressed on engineered iNK or T cells, it remains cell-associated. Upon cell activation and increased ADAM17 expression, the protease cleaves the membrane stalk and releases IL-12 into the extracellular space. This type of regulation ensures that the activity of IL-12 is confined to the space around the tumor, where the engineered immune cells associate with their targets on the tumor cells that trigger their activation.

[0013] In certain embodiments, the iPSC cells or their derivative cells further comprise a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

[0014] In certain embodiments, one or more of the exogenous polynucleotides are integrated into one or more loci on the chromosome of the cell, preferably, one or more loci are those of one or more genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided that at least one of the exogenous polynucleotides is integrated into a locus of a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, the integration resulting in the expression of the deleted or reduced gene, more preferably, one or more of the exogenous polynucleotides are integrated into the loci of CIITA, AAVS1, and B2M genes, the integration resulting in the deleted or reduced expression of one or more of CIITA and B2M genes. In some embodiments, one or more of the exogenous polynucleotides are integrated into the loci of CIITA, CLYBL, and B2M genes.

[0015] In certain embodiments, the iPSC is reprogrammed from total peripheral blood mononuclear cells (PBMCs).

[0016] In certain embodiments, the iPSC is derived from reprogrammed T cells.

[0017] In certain embodiments, the CAR comprises (i) a signal peptide, (ii) an extracellular domain comprising a binding domain that specifically binds to an antigen, (iii) a hinge region, (iv) a transmembrane domain, (v) an intracellular signaling domain, and (vi) a co-stimulatory domain such as a co-stimulatory domain comprising a CD28 signaling domain.

[0018] In certain embodiments, the signal peptide is the GMCSFR signal peptide.

[0019] In certain embodiments, the extracellular domain comprises a VHH domain.

[0020] In certain embodiments, the hinge region comprises the CD28 hinge region.

[0021] In certain embodiments, the transmembrane domain comprises the CD28 transmembrane domain.

[0022] In certain embodiments, the intracellular signaling domain comprises the CD3ζ intracellular domain.

[0023] In certain embodiments, the co-stimulatory domain comprises the CD28 signaling domain.

[0024] In certain embodiments, the CAR is (i) a signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1; (ii) a hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22; (iii) a transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24; (iv) an intracellular signaling domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6; (v) a co-stimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 and comprising.

[0025] In certain embodiments, the CAR comprises: (i) a signal peptide comprising the amino acid sequence of SEQ ID NO: 1; (ii) an extracellular domain comprising a scFV or VHH domain; (iii) a hinge region comprising the amino acid sequence of SEQ ID NO: 22; (iv) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24; (v) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6; and (vi) a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 20.

[0026] In certain embodiments, HLA-E has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 66. Preferably, HLA-E has the amino acid sequence of SEQ ID NO: 66.

[0027] In certain embodiments, HLA-G has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 69. Preferably, HLA-G has the amino acid sequence of SEQ ID NO: 69.

[0028] In certain embodiments, the second exogenous polynucleotide comprises a first polypeptide comprising a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IL-12 alpha subunit p35 or SEQ ID NO: 102, a second polypeptide comprising a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IL-12 beta subunit p40 or SEQ ID NO: 103, and a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 100, and encodes a membrane-bound interleukin 12 (IL-12). In certain embodiments, the second exogenous polynucleotide sequence encoding membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101. Preferably, the second exogenous polynucleotide is integrated at the locus of a gene selected from the group consisting of the AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, TAPI, TAP2, Tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, preferably the AAVS1 or CLYBL gene locus.

[0029] In certain embodiments, IL-12 is fused to a transmembrane domain such as the EGFR transmembrane domain. In certain aspects, the IL-12 / TM subunit is further fused to a signaling domain (SD). For example, the signaling domain is the CD3ζ, CD28, and / or 4-1BB signaling domain. In certain aspects, the signaling domain comprises the CD3ζ and 4-1BB signaling domains. In some aspects, the signaling domain is 4-1BB.

[0030] In certain embodiments, a first exogenous polynucleotide is integrated into the locus of the AAVS1 gene, (i) a second exogenous polypeptide is integrated into the locus of the CIITA gene, and (ii) a third exogenous polypeptide is integrated into the locus of the B2M gene, wherein the integration of the exogenous polynucleotide results in the deletion or reduction of the expression of CIITA and B2M, and preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0031] In certain embodiments, a first exogenous polynucleotide is integrated into the locus of the CIITA gene, (i) a second exogenous polypeptide is integrated into the locus of the AAVS1 gene, and (ii) a third exogenous polypeptide is integrated into the locus of the B2M gene, wherein the integration of the exogenous polynucleotide results in the deletion or reduction of the expression of CIITA and B2M, and preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0032] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the B2M gene, (i) the second exogenous polypeptide is integrated at the locus of the AAVS1 gene, (ii) the third exogenous polypeptide is integrated at the locus of the CIITA gene, wherein integration of the exogenous polynucleotide results in deletion or reduction of CIITA and B2M expression, preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0033] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the CIITA gene, (i) the second exogenous polypeptide is integrated at the locus of the CLYBL gene, (ii) the third exogenous polypeptide is integrated at the locus of the B2M gene, wherein integration of the exogenous polynucleotide results in deletion or reduction of CIITA and B2M expression, preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0034] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the B2M gene, (i) the second exogenous polypeptide is integrated at the locus of the CLYBL gene, (ii) the third exogenous polypeptide is integrated at the locus of the CIITA gene, wherein integration of the exogenous polynucleotide results in deletion or reduction of CIITA and B2M expression, preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0035] In certain embodiments, the derived cells are natural killer (NK) cells or T cells.

[0036] Optionally, the genetically engineered iPSC or its derivative cells further comprise a third exogenous polynucleotide encoding HLA-E having the amino acid sequence of SEQ ID NO: 66 or HLA-G having the amino acid sequence of SEQ ID NO: 69. Preferably, the third exogenous polynucleotide is integrated at the locus of a gene selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, TAPI, TAP2, Tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, the TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, preferably the AAVS1 or CLYBL gene locus.

[0037] In certain embodiments, the second exogenous polynucleotide comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 97 or 99. In certain embodiments, the third exogenous polynucleotide comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 67 or 70.

[0038] In certain embodiments, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0039] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the CIITA gene, the second exogenous polynucleotide is integrated at the locus of the AAVS1 gene, and the third exogenous polynucleotide is integrated at the locus of the B2M gene, wherein the integration of the exogenous polynucleotide results in the deletion or reduction of the expression of the CIITA and B2M genes. Preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0040] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the CIITA gene, the second exogenous polynucleotide is integrated at the locus of the CLYBL gene, and the third exogenous polynucleotide is integrated at the locus of the B2M gene, wherein the integration of the exogenous polynucleotide results in the deletion or reduction of the expression of the CIITA and B2M genes. Preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0041] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the B2M gene, the second exogenous polynucleotide is integrated at the locus of the AAVS1 gene, and the third exogenous polynucleotide is integrated at the locus of the CIITA gene, wherein the integration of the exogenous polynucleotide results in the deletion or reduction of the expression of the CIITA and B2M genes. Preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0042] In certain embodiments, the first exogenous polynucleotide is integrated into the locus of the B2M gene, the second exogenous polynucleotide is integrated into the locus of the CLYBL gene, and the third exogenous polynucleotide is integrated into the locus of the CIITA gene, wherein the integration of the exogenous polynucleotide results in the deletion or reduction of the expression of the CIITA and B2M genes. Preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0043] In certain embodiments, the cells also optionally contain an exogenous polynucleotide encoding a safety switch. Since cell therapies such as CAR-T therapy have a long or infinite half-life and thus the toxicity can be progressive, the cells are engineered to contain a safety switch to eliminate the infused cells in case of adverse events. Thus, the CAR cells are engineered to contain a gene for an artificial cell death polypeptide (a "suicide gene"), which is a molecule encoded by a gene that enables the selective destruction of CAR cells, enables the selective disappearance of genetically modified cells, and prevents collateral damage to neighboring cells and / or tissues. The artificial cell death polypeptide can mediate the induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some cases, the artificial cell death polypeptide is activated by an exogenous molecule, such as an antibody, antiviral drug, or radioisotope conjugate drug, that initiates apoptosis and / or cell death of the therapeutic cells when activated. In one example, the artificial cell death polypeptide contains a viral enzyme recognized by an antiviral drug. In certain embodiments, the viral enzyme is herpes simplex virus thymidine kinase (HSV-TK) (Bonini et al, Science. 1997 Jun 13;276(5319):1719-24). In another example, the safety switch includes an inactivated cell surface receptor that contains an epitope specific for a monoclonal antibody, preferably a truncated epidermal growth factor (tEGFR) variant.In certain embodiments, the inactivated cell surface protein is selected from the group of monoclonal antibody-specific epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab.

[0044] In certain embodiments, the inactivated cell surface protein is a truncated epidermal growth factor (tEGFR) variant. In certain embodiments, the tEGFR variant has, or consists of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71. Preferably, the tEGFR variant has, or consists of, the amino acid sequence of SEQ ID NO: 71.

[0045] In certain embodiments, the inactivated cell surface receptor preferably comprises an epitope specific for a monoclonal antibody operably linked to a cytokine such as IL-15 by a self-protease peptide sequence. Examples of self-protease peptides include, but are not limited to, peptide sequences selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), The peptide sequence selected from the group consisting of zosea asignavirus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flashery virus 2A (BmIFV2A), and combinations thereof. In one embodiment, the self-protease peptide is the self-protease peptide of porcine teschovirus-1 2A (P2A). In certain embodiments, the self-protease peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 73, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO: 73.

[0046] In certain embodiments, the cell may also optionally contain a fifth exogenous polynucleotide encoding a cytokine such as IL-15 or a membrane-bound IL-15 fusion protein.

[0047] As used herein, "interleukin-15" or "IL-15" refers to a cytokine that regulates the activation and proliferation of T and NK cells, or a functional portion thereof. The "functional portion" (the "bioactive portion") of a cytokine refers to a portion of the cytokine that retains one or more functions of the full-length or mature cytokine. Such functions of IL-15 include promotion of NK cell survival, regulation of the activation and proliferation of NK cells and T cells, and support of NK cell development from hematopoietic stem cells. As will be understood by those skilled in the art, the sequences of various IL-15 molecules are known in the art. In certain embodiments, IL-15 is wild-type IL-15. In certain embodiments, IL-15 is human IL-15. In certain embodiments, IL-15 is membrane-bound IL-15. In certain embodiments, IL-15 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 72, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO: 72.

[0048] In certain embodiments, the inactivated cell surface receptor comprises a truncated epidermal growth factor (tEGFR) variant operably linked to interleukin-15 (IL-15) by a self-protease peptide sequence. In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 74, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO: 74. In certain embodiments, the tEGFR variant consists of the amino acid sequence of SEQ ID NO: 71, the self-protease peptide has the amino acid sequence of SEQ ID NO: 73, and IL-15 comprises the amino acid sequence of SEQ ID NO: 72.

[0049] In certain embodiments, the iPSC or derivative has a deletion or reduced expression of one or more of the B2M and / or CIITA genes.

[0050] In certain embodiments, the derived cells are natural killer (NK) cells or T cells.

[0051] Also, (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (ii) a first polypeptide comprising an interleukin-12 (IL-12) alpha subunit p35 or a polypeptide that is at least 90% similar thereto, a second polypeptide comprising an IL-12 beta subunit p40 or a polypeptide that is at least 90% similar thereto, and a membrane-bound IL-12 comprising a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides, or a second exogenous polynucleotide encoding a membrane-bound IL-12 fused to an ADAM17 protease cleavage site peptide, (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G), (iv) optionally, a fourth exogenous polynucleotide encoding a safety switch, (v) optionally, a fifth exogenous polynucleotide encoding a cytokine and are induced pluripotent stem cells (iPSCs), natural killer (NK) cells, or T cells, (vi)(a) The first, second, and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA, and B2M genes, whereby the expression of CIITA and B2M is deleted or reduced; (b) The first, second, and third exogenous polynucleotides are integrated into the loci of the CLYBL, CIITA, and B2M genes, whereby the expression of CIITA and B2M is deleted or reduced; (c) The first, second, and third exogenous polynucleotides are integrated into the loci of the CIITA, AAVS1, and B2M genes, whereby the expression of CIITA and B2M is deleted or reduced; (d) The first, second, and third exogenous polynucleotides are integrated into the loci of the CIITA, CLYBL, and B2M genes, whereby the expression of CIITA and B2M is deleted or reduced; (e) The first, second, and third exogenous polynucleotides are integrated into the loci of the B2M, AAVS1, and CIITA genes, whereby the expression of CIITA and B2M is deleted or reduced; or (f) The first, second, and third exogenous polynucleotides are integrated into the loci of the B2M, CLYBL, and CIITA genes, whereby the expression of CIITA and B2M is deleted or reduced. Also provided are induced pluripotent stem cells (iPSCs), natural killer (NK) cells, or T cells.

[0052] In certain embodiments, the present disclosure provides iPSCs, natural killer (NK) cells, or T cells comprising a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the iPSCs, natural killer (NK) cells, or T cells comprise a second exogenous polynucleotide encoding a membrane-bound interleukin-12 (IL-12) having the amino acid sequence of SEQ ID NO: 96; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 98; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 108; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 110; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 112; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 114; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 116; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 118; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 120; a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 122; or a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 124.In certain embodiments, the iPSC, natural killer (NK) cell, or T cell optionally comprises a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66 and / or an exogenous polynucleotide encoding human leukocyte antigen G (HLA-G) having the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the iPSC, natural killer (NK) cell, or T cell optionally comprises a fourth exogenous polynucleotide encoding an IL-15 protein according to SEQ ID NO: 72. In certain embodiments, one or more of the exogenous polynucleotides comprised by the iPSC, natural killer (NK) cell, or T cell are integrated at the loci of the CIITA and B2M genes, whereby expression of CIITA and / or B2M is deleted or reduced.

[0053] Also provided are iPSCs, natural killer (NK) cells, or T cells comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising an IL-12 alpha subunit p35 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 102, a second polypeptide comprising an IL-12 beta subunit p40 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 103, and a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides having the amino acid sequence of SEQ ID NO: 100, a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12); (iii) a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; and (iv) a fourth exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and an interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 72, wherein the first, second, and third exogenous polynucleotides are integrated at the loci of (a) the AAVS1, CIITA, and B2M genes, respectively; (b) the AAVS1, CIITA, and B2M genes, respectively; (c) the CLYBL, CIITA, and B2M genes, respectively; (d) the CIITA, AAVSI, and B2M genes, respectively; (e) the CIITA, CLYBL, and B2M genes, respectively; (f) the B2M, AAVS1, and CIITA genes, respectively; or (g) the B2M, CLYBL, and CIITA genes, respectively, whereby the expression of CIITA and B2M is deleted or reduced, and a fourth exogenous polynucleotide.

[0054] In certain embodiments, (i) the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, (ii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70, and the first, second, and third exogenous polynucleotides are integrated at the loci of (a) the AAVS1, CIITA, and B2M genes, respectively, (b) the AAVS1, CIITA, and B2M genes, respectively, (c) the CLYBL, CIITA, and B2M genes, respectively, (d) the CIITA, AAVSI, and B2M genes, respectively, (e) the CIITA, CLYBL, and B2M genes, respectively, (f) the B2M, AAVS1, and CIITA genes, respectively, or (g) the B2M, CLYBL, and CIITA genes, respectively.

[0055] Also provided is a composition comprising the cells of the present application.

[0056] In certain embodiments, the composition of the present application further comprises or can be used in combination with one or more other therapeutic agents. Examples of such other therapeutic agents include, but are not limited to, peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), siRNA, oligonucleotides, mononuclear blood cells, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

[0057] Also provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof the cells of the present application or the composition of the present application.

[0058] In certain embodiments, the cancer is non-Hodgkin lymphoma (NHL).

[0059] Also provided is a method of producing the derivative cells of the present application, the method comprising differentiating the iPSCs of the present application under conditions of cell differentiation to thereby obtain the derivative cells.

[0060] Also provided is a method of obtaining a genetically engineered iPSC of the present application, the method comprising introducing a first, a second, and optionally a third exogenous polynucleotide into iPSC cells, thereby obtaining a genetically engineered iPSC. The genetically engineered iPSC of the present application can be obtained using any genetic engineering method. Preferably, the genetic engineering includes targeted editing, more preferably, the targeted editing includes deletion, insertion, or in / del, and the targeted editing is carried out by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional modification of these methods.

[0061] Also provided is a method of differentiating induced pluripotent stem cell (iPSC) cells into NK cells by subjecting the cells to a differentiation protocol that includes adding recombinant human IL-12 during the last 24 hours of culture. Preferably, the recombinant IL-12 is IL12 p70.

[0062] Also provided are CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs) that include (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (ii) a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide that is at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide that is at least 90% similar thereto, and a membrane-bound interleukin 12 (IL-12) encoding a second exogenous polynucleotide that includes a transmembrane domain fused to the ends of the first and / or second IL-12 subunit polypeptides, and (iii) a deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes.

[0063] Other embodiments of the present application include genetically engineered iPSCs or their derivative cells for use in treating cancer in a subject in need thereof.

[0064] In some embodiments, the engineered iPSC-derived cells of the present invention have improved anti-tumor immunity, increased persistence, increased resistance to immune cells, or increased immune tolerance, or the genetically engineered iPSCs may have increased resistance to T and / or NK cells. Specifically, the IL-12 transgene of the present invention, when transfected into iPSCs and differentiated into NK cells according to the present invention, exhibits increased anti-tumor immunity, increased persistence, decreased exhaustion, and increased continuous killing when compared to NK cells derived from iPSC cells that do not use the IL-12 transgene of the present invention. The genetically engineered iPSCs of the present invention have the potential to differentiate into non-pluripotent cells including hematopoietic cells having the same functional targeted genome editing. In some embodiments, the genetically engineered iPSCs of the present invention have the potential to differentiate into mesoderm cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, or B cells.

[0065] The foregoing summary, and the following detailed description of the preferred embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present application is not limited to the exact embodiments shown in the drawings.

Brief Description of the Drawings

[0066]

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DETAILED DESCRIPTION OF THE INVENTION

[0067] Throughout the background and the specification, various publications, papers, and patents are cited or described, and each of these references is hereby incorporated by reference in its entirety into this specification. The discussion of documents, acts, materials, devices, articles, etc. contained in this specification is for the purpose of providing context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0068] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. If not, the specific terms used in this specification have the meaning described in the specification.

[0069] It should be noted that the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents unless the context clearly indicates otherwise.

[0070] Unless otherwise indicated, any numerical values such as concentrations or concentration ranges described in this specification should be understood to be modified by the term "about" in all cases. Thus, the numerical values typically include ±10% of the recited values. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). When used in this specification, the use of a numerical range clearly includes all individual numerical values within that range, including all possible sub-ranges, integers, and fractional values within such a range, unless the context clearly indicates otherwise.

[0071] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to all elements in the series. One of ordinary skill in the art will be able to understand and confirm numerous equivalents to the specific embodiments of the applications described herein without performing more than routine experimentation. Such equivalents are intended to be encompassed by this application.

[0072] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", or "containing", or any other variation thereof, are to be understood as implying the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements and can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated otherwise, "or" as used herein refers to an inclusive or and not an exclusive or. For example, condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0073] As used herein, the conjunction "and / or" between a plurality of recited elements is understood to include both individual and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options fall within the scope of this meaning and are thus understood to satisfy the requirements of the term "and / or" as used herein. Simultaneous applicability of multiple of the options also falls within the scope of this meaning and is thus understood to satisfy the requirements of the term "and / or".

[0074] As used throughout the specification and claims, the term "consists of" as used herein, or variations such as "consist of" or "consisting of", indicates that it includes any recited integer or group of integers, but cannot add additional integers or groups of integers to the specified method, structure, or composition.

[0075] As used throughout the specification and claims, the term "consists essentially of" as used herein, or variations such as "consist essentially of" or "consisting essentially of", indicates the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not substantially change the basic or novel characteristics of the specified method, structure, or composition. See M.P.E.P. § 2111.03.

[0076] As used herein, "subject" means any animal, preferably a mammal, most preferably a human. The term "mammal" as used herein includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0077] Also, when referring to the dimensions or characteristics of the components of the preferred invention, the terms "about", "approximately", "generally", "substantially", and similar terms as used herein are intended to indicate that the recited dimensions / characteristics are not strict boundaries or parameters and do not exclude minor variations therefrom that would be understood by one of ordinary skill in the art as being functionally the same or similar. At a minimum, such references, including numerical parameters, will include variations that would not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0078] In the context of two or more nucleic acid or polypeptide sequences (e.g., CAR polypeptides and the CAR polynucleotides encoding them), the term "identical" or percent "identity" refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or by visual inspection, are the same or have the same amino acid residues or nucleotides at the specified percentage.

[0079] For sequence comparison, typically one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, coordinates of subsequences are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0080] Optimal alignment of arrays for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0081] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as possible while increasing the cumulative alignment score.

[0082] The cumulative score is calculated for nucleotide sequences using parameters M (reward score for matching residue pairs, always >0) and N (penalty score for mismatched residues, always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of word hits in each direction stops when: the cumulative alignment score decreases by amount X from its maximum achieved value, the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) has, by default, a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and uses comparison of both strands. For amino acid sequences, the BLASTP program has, by default, a word length (W) of 3, an expectation (E) of 10, and uses the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0083] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see also, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum total probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0084] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide when, for example, the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0085] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, protein, or cell) is substantially separated from, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues. Thus, "isolated" nucleic acids, peptides, proteins, and cells include nucleic acids, peptides, proteins, and cells purified by standard purification methods and the purification methods described herein. An "isolated" nucleic acid, peptide, protein, and cell can still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell and is part of the composition. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0086] The term "polynucleotide" as used herein is also synonymously referred to as "nucleic acid molecule", "nucleotide", or "nucleic acid", and refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that can be single-stranded, or more typically double-stranded or a mixture of single-stranded and double-stranded regions, including DNA and RNA. Further, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone is modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses typically the chemical, enzymatic, or metabolic modified forms of polynucleotides found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also often includes relatively short nucleic acid chains often referred to as oligonucleotides.

[0087] "Construct" refers to a polymer or molecular complex containing a polynucleotide that is delivered to host cells either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, which can be replicated and / or expressed in the target cell. The term "vector" as used herein includes the construct to be delivered. A vector can be a linear or circular molecule. A vector can be integrative or non-integrative. 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, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0088] "Integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted at a preselected site or "integration site" within the chromosomal or mitochondrial DNA of the cell. As used herein, the term "integration" further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of the endogenous sequence or nucleotides at the integration site. If a deletion is present at the insertion site, "integration" can further include the replacement of the deleted endogenous sequence or nucleotides with one or more inserted nucleotides.

[0089] As used herein, the term "exogenous" is intended to mean that the referenced molecule or the referenced activity has been introduced into the host cell or is non-native to the host cell. A molecule can be introduced, for example, by introduction of the coding nucleic acid into the host genetic material such as by integration into the host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, this term when used in reference to the expression of a coding nucleic acid refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to the referenced molecule or activity that is present in the host cell in its native form. Similarly, when used in reference to the expression of a coding nucleic acid, this term refers to the expression of a coding nucleic acid that is naturally contained within the cell and not exogenously introduced.

[0090] As used herein, a "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed in vivo into RNA and in some instances translated into a polypeptide. Genes or polynucleotides of interest include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode an miRNA, shRNA, native polypeptide (i.e., a polypeptide found in nature), or a fragment thereof, a variant polypeptide (i.e., a mutant of a native polypeptide having less than 100% sequence identity to the native polypeptide), or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0091] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that one function is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA when it can affect the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation.

[0092] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term encompasses the transcription of a gene into RNA. This term also encompasses the translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. An expressed CAR can be within the cytoplasm of a host cell, within an extracellular environment such as the growth medium of a cell culture, or can be immobilized on the cell membrane.

[0093] As used herein, the terms "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by one of ordinary skill in the art. The conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. This term also encompasses amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, for example, conjugation with a labeling component. Also included within the definition are polypeptides containing, for example, one or more analogs of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0094] The peptide sequences described in this specification are described according to the normal convention where the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Amino acid isomers are known, but the L-form of the amino acid is represented unless otherwise clearly indicated.

[0095] As used herein, the term "engineered immune cell" refers to an immune cell, also called an immune effector cell, that has been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the cell's total genetic material.

[0096] I. Induced pluripotent stem cells (iPSCs) and immune effector cells iPSCs have unlimited self-renewal capacity. The use of iPSCs enables the generation, by cell engineering, of a controlled cell bank of modified cells that can be expanded and differentiated into the desired immune effector cells, supplying large quantities of allogeneic therapeutic products.

[0097] Genetically engineered iPSCs and their derivative cells are provided herein. The selected genomic modifications provided herein enhance the therapeutic properties of the derivative cells. The derivative cells are functionally improved and are suitable for allogeneic, off-the-shelf cell therapy according to the combination of selective modalities introduced into the cells at the level of iPSCs through genomic manipulation. This approach can help to reduce side effects mediated by CRS / GVHD and prevent long-term autoimmunity while providing excellent efficacy.

[0098] As used herein, the term "differentiation" refers to the process by which unspecialized ( "uncommitted") or less specialized cells acquire the characteristics of specialized cells. Examples of specialized cells include blood cells or muscle cells. Differentiated or induced-differentiated cells have taken a more specialized ( "committed") position within the cell lineage. When applied to the process of differentiation, the term "committed" refers to a cell that, in the differentiation pathway, under normal circumstances, continues to differentiate into a specific cell type or a subset of cell types and, under normal circumstances, cannot differentiate into a different cell type or revert to a less differentiated cell type to the point where it can no longer do so. As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or the cell mass or embryo proper. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, namely, the ectoderm, mesoderm, and endoderm. Pluripotency is a continuous connection of developmental potential within the range of more primitive and more pluripotent cells that can give rise to a complete organism (such as embryonic stem cells) from incomplete or partially pluripotent cells (such as epiblast stem cells or EpiSCs) that cannot give rise to a complete organism.

[0099] As used herein, the term "reprogramming" or "dedifferentiation" refers to a method of increasing the potential of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an un-reprogrammed state. In other words, a reprogrammed cell is in a less differentiated state than the same cell in an un-reprogrammed state.

[0100] As used herein, the term "induced pluripotent stem cell" or "iPSC" means a cell that has been induced, changed, or reprogrammed from a differentiated adult, neonatal, or fetal cell such that the stem cell can differentiate into cells of all three germ layers or cortical layers: mesoderm, endoderm, and ectoderm. The generated iPSCs do not refer to cells as found in nature.

[0101] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" or "HPC" refer to cells that are committed to the hematopoietic system but are capable of further hematopoietic differentiation. Examples of hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hemocytoblasts), myeloid precursors, megakaryocyte precursors, erythroid precursors, and lymphoid precursors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), as well as lymphoid lineages (T cells, B cells, NK cells). As used herein, "CD34+ hematopoietic progenitor cells" refers to HPCs that express CD34 on their surface.

[0102] As used herein, the term "immune cell" or "immune effector cell" refers to a cell involved in an immune response. Examples of immune responses include, for example, promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.

[0103] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a type of white blood cell that matures in the thymus and has various roles in the immune system. T cells may have roles including, for example, the identification of specific foreign antigens in the body and the activation and inactivation of other immune cells. T cells can be any T cells, such as cultured T cells, for example primary T cells, or T cells from a cultured T cell line, such as Jurkat, SupTl, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell, at any stage of development, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as 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 (γδ T cells), etc. Further types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0104] "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. These are characterized by their secretion profiles after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" was originally identified as a differentiation antigen on T lymphocytes but is also found on other cells including monocytes / macrophages and is a 55kD glycoprotein. The CD4 antigen is a member of the immunoglobulin supergene family and has been implicated as an associative recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, these define the helper / inducer subset.

[0105] "CD8+ T cells" refers 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 on thymocytes as well as on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is an associative recognition element in major histocompatibility complex class I-restricted interactions.

[0106] As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of the T cell receptor (TCR chain). NK cells can also refer to genetically engineered NK cells such as NK cells modified to express a chimeric antigen receptor (CAR). NK cells can also be differentiated from stem cells or progenitor cells.

[0107] As used herein, the term "gene knock-in" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties in a source cell or iPSC, and can be retained in iPSCs derived from the source cell and / or hematopoietic cells derived from iPSCs. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and iPSCs derived from the source cell can further differentiate into a specific cell type including any hematopoietic cell. iPSCs derived from the source cell, and differentiated cells derived therefrom, may be collectively referred to as "derived" or "descendant" cells depending on the context. For example, throughout this application, derived effector cells, or derived NK or "iNK" cells, or derived T or "iT" cells are cells differentiated from iPSCs as compared to their primary counterparts obtained from natural / native sources such as peripheral blood, cord blood, or other donor tissues. As used herein, gene knock-in conferring preferential therapeutic properties is incorporated into iPSCs either through reprogramming of selected source cells specific to the donor, disease, or treatment response, or by introducing a genetically modified modality using genome editing into the iPSCs.

[0108] The parental cell line of induced pluripotent stem cells (iPSCs) can be generated from peripheral blood mononuclear cells (PBMCs) or T cells using any known method for introducing reprogramming factors into non-pluripotent cells, such as the episomal plasmid-based processes previously described in U.S. Patent Nos. 8,546,140, 9,644,184, 9,328,332, and 8,765,470, the entire disclosures of which are incorporated herein by reference. The reprogramming factors can be in the form of polynucleotides and are thus introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, 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, the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs, and genome editing is introduced by creating one or more targeted integrations and / or in / dels at one or more selected sites. In another embodiment, as described in U.S. Patent Nos. 9,206,394 and 1,078,7642, the entire disclosures of which are incorporated herein by reference, the iPSCs are obtained from human T cells having antigen specificity and a rearranged TCR gene (hereinafter also referred to as "T-iPS" cells).

[0109] According to certain embodiments, the present application relates to induced pluripotent stem cells (iPSCs) or derivatives thereof comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide that is at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide that is at least 90% similar thereto, and a membrane-bound interleukin-12 (IL-12) encoding a second exogenous polynucleotide comprising a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides; and (iii) a deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes.

[0110] II. Expression of Chimeric Antigen Receptor (CAR) According to embodiments of the present application, iPSCs or derivatives thereof comprise a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR that targets a tumor antigen. In one embodiment, the CAR targets the CD19 antigen.

[0111] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds an antigen or target, a transmembrane domain, and an intracellular signaling domain. Association of the extracellular domain of the CAR with a target antigen on the surface of a target cell results in clustering of the CAR and delivery of an activation stimulus to the CAR-containing cell. The CAR can redirect the specificity of immune effector cells and initiate the proliferation, cytokine production, phagocytosis, and / or production of molecules that mediate the cell death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner.

[0112] As used herein, the term "signal peptide" refers to the leader sequence at the amino terminus (N-terminus) of the nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequently to cell surface expression, either cotranslationally or post-translationally.

[0113] As used herein, the terms "extracellular antigen-binding domain", "extracellular domain", or "extracellular ligand-binding domain" refer to the portion of the CAR that is located external to the cell membrane and is capable of binding to an antigen, target, or ligand.

[0114] As used herein, the term "hinge region" or "hinge domain" refers to the portion of the CAR that connects two adjacent domains of the CAR protein, namely the extracellular domain and the transmembrane domain of the CAR protein.

[0115] As used herein, the term "transmembrane domain" refers to the portion of the CAR that extends across the cell membrane and anchors the CAR to the cell membrane.

[0116] As used herein, the terms "intracellular signaling domain", "cytoplasmic signaling domain", or "intracellular signaling domain" refer to the portion of the CAR that is located internal to the cell membrane and is capable of transmitting effector signals.

[0117] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., NK cell or T cell) that provides a primary cytoplasmic signaling sequence that regulates primary activation of a receptor in a stimulatory manner with respect to at least some aspects of an immune cell signaling pathway. Stimulatory molecules include two distinct classes of cytoplasmic signaling sequences, namely those that initiate antigen-dependent primary activation (referred to as "primary signaling domains") and those that act in an antigen-independent manner to provide a secondary co-stimulatory signal (referred to as "co-stimulatory signaling domains").

[0118] In certain embodiments, the extracellular domain comprises an antigen-binding domain and / or an antigen-binding fragment. The antigen-binding fragment can be, for example, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen. The antigen-binding fragments of the present application, among other things, when administered alone or in combination with other anti-cancer therapies, exhibit high-affinity binding to tumor antigens, high specificity for tumor antigens, complement-dependent cytotoxicity (CDC) against cells expressing tumor antigens, antibody-dependent phagocytosis (ADPC), and / or the ability to stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), as well as the ability to inhibit tumor growth in subjects and animal models in which it is needed, and possess one or more desirable functional properties.

[0119] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules that are monoclonal or polyclonal, human, humanized, chimeric, and hybrid antibodies, as well as antibody fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) according to the amino acid sequence of their heavy chain constant domains. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, the antibodies of the present application can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3, or IgG4. The antibody light chains of vertebrate species can be assigned to one of two clearly different types, namely kappa and lambda, based on the amino acid sequence of their constant domains. Thus, the antibodies of the present application can contain a kappa or lambda light chain constant domain. According to certain embodiments, the antibodies of the present application include heavy chain and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, an antibody contains an antigen-binding region composed of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domain is also alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domain is also alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0120] As used herein, the term "isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a particular tumor antigen substantially does not contain antibodies that do not bind to that tumor antigen). Further, an isolated antibody substantially does not contain other cellular materials and / or chemicals.

[0121] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the present application can be prepared by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals such as transgenic mice or rats having a genome containing a human heavy chain transgene and a light chain transgene.

[0122] As used herein, the term "antigen-binding fragment" refers to an antibody fragment such as, for example, diabody, Fab, Fab’, F(ab’)2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single-domain antibody (sdAb), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, minibody, nanobody, domain antibody, bivalent domain antibody, light chain variable domain (VL), variable domain of a camel antibody (V H H), or any other antibody fragment that binds an antigen but does not contain the complete antibody structure. An antigen-binding fragment can bind the same antigen to which the parent antibody or parent antibody fragment binds.

[0123] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).

[0124] As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region or comprises only the heavy chain variable region.

[0125] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, produced using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0126] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified such that its antigen-binding properties are retained while its antigenicity in the human body is reduced and its sequence homology to that of a human antibody is increased.

[0127] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of an antibody from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0128] As used herein, the term "multispecific antibody" refers to an antibody that includes a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence among the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence among the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody includes a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0129] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to two or fewer epitopes or two or fewer antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises half or a fragment of an antibody having binding specificity for a first epitope and half or a fragment of an antibody having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises an scFv or a fragment thereof having binding specificity for a first epitope, and an scFv or a fragment thereof having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a V H H and a V H H having binding specificity for a second epitope.

[0130] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to a tumor antigen" refers to a dissociation constant (K -7 d) of 1×10 -8 M or less, preferably 1×10 -9 M or less, more preferably 5×10 -9 M or less, 1×10 -10 M or less, 5×10 -10Refers to an antigen-binding domain or antigen-binding fragment that binds to a tumor antigen with a KD of less than M. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka), expressed as molar concentration (M). The KD value of an antibody can be determined using methods in the art in view of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, for example, a Biacore® system, or by using biolayer interferometry technology, for example, an Octet RED96 system.

[0131] The smaller the KD value of the antigen-binding domain or antigen-binding fragment, the higher the affinity of the antigen-binding domain or antigen-binding fragment for the target antigen.

[0132] In various embodiments, antibodies or antibody fragments suitable for use in the CARs of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab’) fragments, disulfide-bonded Fv (sdFv), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals (“SMIP™”), intracellular antibodies, minibodies, single-domain antibody variable domains, nanobodies, VHH, diabodies, tandem diabodies (TandAb®), anti-idiotype (anti-Id) antibodies (including, e.g., anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the foregoing. Antibodies and / or antibody fragments can be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, variable domains of camel antibodies and humanized versions, variable domains of shark antibodies and humanized versions, and variable domains of camelized antibodies.

[0133] In some embodiments, the antigen-binding fragment is a Fab fragment, Fab’ fragment, F(ab’)2 fragment, scFv fragment, Fv fragment, dsFv diabody, VHH, VNAR, single-domain antibody (sdAb) or nanobody, dAb fragment, Fd’ fragment, Fd fragment, heavy-chain variable region, isolated complementarity-determining region (CDR), diabody, triabody, or decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0134] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or a nanobody. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0135] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0136] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH.

[0137] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises an scFv.

[0138] In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.

[0139] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise an scFv.

[0140] Alternative scaffolds for immunoglobulin domains that exhibit similar functional features such as high affinity and specific binding to a target biomolecule can also be used in the CARs of the present disclosure. Such scaffolds have been shown to provide molecules with improved characteristics such as higher stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that can be used in the CARs of the present disclosure include tenascin type III domains derived from engineered tenascin (e.g., Centyrin™), scaffolds derived from engineered gamma-B crystallin or scaffolds derived from engineered ubiquitin (e.g., Affilins), the tenth fibronectin type III (10Fn3) domain derived from engineered fibronectin (e.g., monobodies, Adnectins™, or AdNexins™), engineered ankyrin repeat motif-containing polypeptides (e.g., DARPins™), the A domain derived from engineered low density lipoprotein receptor (LDLR-A) (e.g., Avimers™), lipocalins (e.g., anticalins), Kunitz domains derived from engineered protease inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2), the Z domain derived from engineered protein A (Affibodies™), polypeptides derived from Sac7d (e.g., Nanoffitins® or affitins), the SH2 domain derived from engineered Fyn (e.g., Fynomers®), CTLD3 (e.g., tetranectin), thioredoxin (e.g., peptide aptamers), KALBITOR®, beta-sandwiches (e.g., iMab), miniproteins, C-type lectin-like domain scaffolds, engineered antibody mimics, and any genetically engineered counterparts of the foregoing that retain their binding functionality (each of which is incorporated by reference in its entirety, Worn A, Pluckthun A, J Mol Biol 305: 989-1010 (2001), Xu L et al., Chem Biol 9: 933-42 (2002), Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004), Binz H et al., Nat Biolechnol 23: 1257-68 (2005), Hey T et al., Trends Biotechnol 23:514-522 (2005), Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005), Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006), Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007), Skerra, Current Opin. in Biotech., 2007 18: 295-304, Byla P et al., J Biol Chem 285: 12096 (2010), Zoller F et al., Molecules 16: 2467-85 (2011)).

[0141] In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0142] In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a leader sequence. The leader sequence can be located at the N-terminus of the extracellular tag-binding domain. The leader sequence can optionally be cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of leader sequences known to those of skill in the art can be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence can be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from the human immunoglobulin heavy chain (HC).

[0143] In some embodiments, the leader sequence is derived from GMCSFR. In one embodiment, the GMCSFR leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 1.

[0144] In some embodiments, the first polypeptide of the CAR of the present disclosure is a transmembrane domain fused in-frame between an extracellular tag-binding domain and a cytoplasmic domain.

[0145] The transmembrane domain can be derived from a protein that contributes to the extracellular tag-binding domain, a protein that contributes to a signaling or co-signaling domain, or a completely different protein. In some examples, the transmembrane domain can be selected or modified by amino acid substitution, deletion, or insertion to minimize interaction with other members of the CAR complex. In some examples, the transmembrane domain can be selected or modified by amino acid substitution, deletion, or insertion to avoid binding of a protein that naturally associates with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and / or an optimal distance between domains connected to the transmembrane domain.

[0146] The transmembrane domain may be derived from either natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains particularly useful in the present disclosure may be derived from the α, β, or ζ chains of the T cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154 (i.e., including at least its transmembrane region). Alternatively, the transmembrane domain may be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan, and / or valine can be found at each end of the synthetic transmembrane domain.

[0147] In some embodiments, it may be desirable to utilize a transmembrane domain of the ζ, η, or FcεR1γ chain containing a cysteine residue capable of disulfide bonding so that the resulting chimeric protein can form a disulfide-linked dimer with itself or with an unmodified version of the ζ, η, or FcεR1γ chain or a related protein. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such a domain to the transmembrane domain of the same or a different surface membrane protein in order to minimize interaction with other members of the receptor complex. In other cases, it may be desirable to use the transmembrane domains of ζ, η, or FcεR1γ and -β, MB1 (Igα.), B29, or CD3-γ, ζ, or η in order to maintain physical association with other members of the receptor complex.

[0148] In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 23. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 24.

[0149] In some embodiments, the first polypeptide of the CAR of the present disclosure includes a spacer region between the extracellular tag-binding domain and the transmembrane domain, and the tag-binding domain, linker, and transmembrane domain are in-frame with each other.

[0150] As used herein, the term "spacer region" generally refers to any oligopeptide or polypeptide that functions to link a tag-binding domain to a transmembrane domain. The spacer region can be used to provide greater flexibility and accessibility to the tag-binding domain. The spacer region can contain up to 300 amino acids, preferably 10 - 100 amino acids, and most preferably 25 - 50 amino acids. The spacer region can be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the spacer region can be a synthetic sequence corresponding to a naturally occurring spacer region sequence, or a completely synthetic spacer region sequence. Non-limiting examples of spacer regions that can be used in accordance with the present disclosure include a portion of the human CD8α chain, the partial extracellular domain of CD28, the FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, the Ig hinge, or a functional fragment thereof. In some embodiments, additional linker amino acids are added to the spacer region to ensure that the antigen-binding domain is at an optimal distance from the transmembrane domain. In some embodiments, if the spacer is derived from an Ig, the spacer can be mutated to prevent Fc receptor binding.

[0151] In some embodiments, the spacer region contains a hinge domain. The hinge domain can be derived from CD8α, CD28, or immunoglobulin (IgG). For example, the IgG hinge can be from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0152] In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or a functional fragment thereof. In certain embodiments, the IgG hinge is from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the hinge domain comprises CH1, CH2, CH3, and / or the hinge region of an immunoglobulin. In certain embodiments, the hinge domain comprises the core hinge region of an immunoglobulin. The term "core hinge" can be used interchangeably with the term "short hinge" (also known as "SH"). Non-limiting examples of suitable hinge domains are the core immunoglobulin hinge regions comprising EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgG1, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP)3 (SEQ ID NO: 59) from IgG3, and ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes). In certain embodiments, the hinge domain is a fragment of an immunoglobulin hinge.

[0153] In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 21. In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 22.

[0154] In some embodiments, the transmembrane domain and / or the hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD28.

[0155] In certain aspects, the first polypeptide of the CAR of the present disclosure comprises a cytoplasmic domain comprising at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more co-stimulatory signaling domains.

[0156] The cytoplasmic domain is responsible for activating at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR is located. The term "effector function" refers to the specialized functions of a cell. The effector functions of T cells can be, for example, cytolytic activity or helper activity including cytokine secretion. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform specialized functions. Usually, the entire signaling domain is present, while in many cases it is not necessary to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain as long as it transmits the effector function signal. Thus, the term, intracellular signaling domain, means any truncated portion of the signaling domain that is sufficient to transmit the effector function signal.

[0157] Non-limiting examples of signaling domains that can be used in the CARs of the present disclosure include, for example, signaling domains derived from DAP10, DAP12, the Fc epsilon receptor I gamma chain (FCER1G), FcRβ, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

[0158] In some embodiments, the cytoplasmic domain comprises a CD3ζ signaling domain. In one embodiment, the CD3ζ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 6, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 6.

[0159] In some embodiments, the cytoplasmic domain further comprises one or more co-stimulatory signaling domains. In some embodiments, the one or more co-stimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.

[0160] In one embodiment, the co-stimulatory signaling domain is derived from 41BB. In one embodiment, the 41BB co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 8.

[0161] In one embodiment, the co-stimulatory signaling domain is derived from IL2Rb. In one embodiment, the IL2Rb co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 9.

[0162] In one embodiment, the co-stimulatory signaling domain is derived from CD40. In one embodiment, the CD40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 10.

[0163] In one embodiment, the co-stimulatory signaling domain is derived from OX40. In one embodiment, the OX40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 11.

[0164] In one embodiment, the co-stimulatory signaling domain is derived from CD80. In one embodiment, the CD80 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 12.

[0165] In one embodiment, the co-stimulatory signaling domain is derived from CD86. In one embodiment, the CD86 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 13.

[0166] In one embodiment, the co-stimulatory signaling domain is derived from CD27. In one embodiment, the CD27 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 14.

[0167] In one embodiment, the co-stimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 15.

[0168] In one embodiment, the co-stimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 16.

[0169] In one embodiment, the co-stimulatory signaling domain is derived from DAP10. In one embodiment, the DAP10 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 17.

[0170] In one embodiment, the co-stimulatory signaling domain is derived from DAP12. In one embodiment, the DAP12 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 18.

[0171] In one embodiment, the co-stimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 19.

[0172] In some embodiments, the CAR of the present disclosure comprises one co-stimulatory signaling domain. In some embodiments, the CAR of the present disclosure comprises two or more co-stimulatory signaling domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six, or more co-stimulatory signaling domains.

[0173] In some embodiments, the signaling domain and the costimulatory signaling domain can be arranged in any order. In some embodiments, the signaling domain is upstream of the costimulatory signaling domain. In some embodiments, the signaling domain is downstream of the costimulatory signaling domain. In cases where two or more costimulatory domains are included, the order of the costimulatory signaling domains can be exchanged.

[0174] Non-limiting exemplary CAR regions and sequences are provided in Table 1.

[0175] [Table 1-1]

[0176] [Table 1-2]

[0177] [Table 1-3]

[0178] [Table 1-4]

[0179] In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen. The antigen-binding domain of the second polypeptide can bind to multiple antigens or multiple epitopes in one antigen. For example, the antigen-binding domain of the second polypeptide can bind to 2, 3, 4, 5, 6, 7, 8, or more antigens. As another example, the antigen-binding domain of the second polypeptide can bind to 2, 3, 4, 5, 6, 7, 8, or more epitopes in the same antigen.

[0180] The options for the antigen-binding domain can depend on the type and number of antigens that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize an antigen that acts as a cell surface marker on a target cell associated with a particular medical condition. In certain embodiments, the CARs of the present disclosure can be genetically modified to target a desired tumor antigen by engineering a desired antigen-binding domain that specifically binds to an antigen (such as one on a tumor cell). Non-limiting examples of cell surface markers that can serve as targets for the antigen-binding domain in the CARs of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0181] In some embodiments, the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen.

[0182] In some embodiments, the antigen-binding domain binds to at least one tumor antigen. In some embodiments, the antigen-binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors.

[0183] In some embodiments, the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.

[0184] In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies. Non-limiting examples of tumor antigens associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2. Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, nectin-4, and B7H4. Non-limiting examples of tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigens associated with liver cancer include claudin 18.2, GPC-3, EpCAM, cMET, and AFP. Non-limiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70. Non-limiting examples of tumor antigens associated with bladder cancer include nectin-4 and SLITRK6. Non-limiting examples of tumor antigens associated with renal cell carcinoma include CD70, FOLR1, SLITICR6, and nectin-4.

[0185] Additional examples of antigens that can be targeted by an antigen-binding domain include, but are not limited to, alpha-fetoprotein, A3, A33 antibody-specific antigen, Ba733, BrE3-antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-I), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, PAM-4 antibody-specific antigen, placental growth factor, p53, prostate acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomsen-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A-antigen, angiogenesis marker, oncogene marker, or oncogene product.

[0186] In one embodiment, the antigen targeted by the antigen-binding domain is CD19. In one embodiment, the antigen-binding domain comprises an anti-CD19 scFv. In one embodiment, the anti-CD19 scFv comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 2. In one embodiment, the anti-CD19 scFv comprises a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 4, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 4. In one embodiment, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 7, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 7.

[0187] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens can be cell receptors and cells that produce antibodies directed against "self". In some embodiments, the antigen is associated with an autoimmune disease or disorder such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes mellitus, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Crohn's disease, or ulcerative colitis.

[0188] In some embodiments, the autoimmune antigens that can be targeted by the CARs disclosed herein include, but are not limited to, platelet antigens, myelin protein antigens, Sm antigens in snRNPs, islet cell antigens, rheumatoid factors, and anti-citrullinated proteins, citrullinated proteins and peptides, such as CCP-1, CCP-2 (cyclic citrullinated peptide), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage, such as collagen II, IX, and XI, circulating serum proteins, such as RF (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components, such as RA33 / hnRNP A2, Sm, eukaryotic translation elongation factor 1 alpha 1, stress proteins, such as HSP-65, -70, -90, BiP, inflammatory / immunological factors, such as B7-H1, IL-1 alpha, and IL-8, enzymes, such as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors, such as lipocortin 1, neutrophil nuclear proteins, such as lactoferrin and 25-35 kD nuclear proteins, granular proteins, such as bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factors, histones, ribosomal P protein, cardiolipin, vimentin, nucleic acids, such as dsDNA, ssDNA, and RNA, ribonucleoproteins and proteins, such as Sm antigens (including but not limited to SmD' and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro (SSA), and La (SSB) antigens.

[0189] In various embodiments, the scFv fragments used in the CARs of the present disclosure can include a linker between the VH and VL domains. The linker can be a peptide linker and can include any naturally occurring amino acids. Exemplary amino acids that can be included within the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and Thr. The linker should have a sufficient length to link the VH and VL in a manner that forms the correct conformation with respect to each other to retain a desired activity such as binding to an antigen. The linker can be about 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 35 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15 to 20 amino acids in length. Exemplary linkers that can be used are Gly-rich linkers, Gly- and Ser-containing linkers, Gly- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0190] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 3. In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker comprises the amino acid sequence set forth in SEQ ID NO: 25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 25.

[0191] Other linker sequences may include a portion of an immunoglobulin hinge region, CL, or CH1 derived from any immunoglobulin heavy or light chain isotype. Exemplary linkers that may be used include any of SEQ ID NOs: 26-56 in Table 1. Further linkers are described, for example, in WO 2019 / 060695, which is incorporated herein by reference in its entirety.

[0192] III. Safety Switch / Artificial Cell Death Polypeptide According to embodiments of the present application, iPSC cells or their derivative cells optionally contain an exogenous polynucleotide encoding a safety switch, which may contain an artificial cell death polypeptide. Cell therapies such as CAR-T therapy have a long or infinite half-life and thus the toxicity can be progressive. Therefore, the cells are engineered to include a safety switch to eliminate the infused cells in case of adverse events. Thus, the CAR cells are engineered to contain a gene for a safety switch (i.e., a "suicide gene"), which is a molecule encoded by a gene that enables selective destruction of the CAR cells and selective disappearance of the genetically modified cells, preventing collateral damage to neighboring cells and / or tissues. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some cases, the safety switch is activated by an exogenous molecule, such as an antibody, an antiviral drug, or a radioisotope conjugate drug, that initiates apoptosis and / or cell death of the therapeutic cells when activated. In one example, the artificial cell death polypeptide contains a viral enzyme recognized by an antiviral drug. In certain embodiments, the viral enzyme is herpes simplex virus thymidine kinase (HSV-TK) (Bonini et al, Science. 1997 Jun 13;276(5319):1719-24). In another example, the safety switch includes an inactivated cell surface receptor containing an epitope specific for a monoclonal antibody, preferably a truncated epidermal growth factor (tEGFR) variant.

[0193] As used herein, the term "artificial cell death polypeptide" refers to an engineered protein designed to prevent potential toxicity or other harmful effects in cell therapy. Artificial cell death polypeptides can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some cases, artificial cell death polypeptides are activated by exogenous molecules, such as antibodies, that initiate apoptosis and / or cell death of therapeutic cells when activated.

[0194] In certain embodiments, the artificial cell death polypeptide comprises an inactivated cell surface receptor that contains an epitope specifically recognized by an antibody, particularly a monoclonal antibody, also referred to herein as an epitope specific for the monoclonal antibody. When expressed by iPSCs or their derivative cells, the inactivated cell surface receptor has an inactive or significantly impaired signaling, yet can still be specifically recognized by the antibody. Specific binding between the antibody and the inactivated cell surface receptor enables elimination of iPSCs or their derivative cells by ADCC and / or ADCP mechanisms, as well as direct killing using antibody-drug conjugates with toxins or radionuclides.

[0195] In certain embodiments, the inactivated cell surface receptor comprises an epitope selected from epitopes specifically recognized by antibodies including, but not limited to, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab.

[0196] The epidermal growth factor receptor, also known as EGFR, ErbB1, and HER1, is a cell surface receptor for members of the epidermal growth factor family of extracellular ligands. As used herein, "truncated EGFR", "tEGFR", "short EGFR", or "sEGFR" refers to an inactive EGFR variant lacking the EGF binding domain and intracellular signaling domain of EGFR. Exemplary tEGFR variants contain residues 322 - 333 of domain 2, all of domains 3 and 4, and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope. Expression of the tEGFR variant on the cell surface enables cell elimination by an antibody that specifically binds to tEGFR, such as cetuximab (Erbitux®), if desired. Due to the absence of the EGF binding domain and intracellular signaling domain, tEGFR is inactive when expressed by iPSCs or their derivative cells.

[0197] Exemplary inactivating cell surface receptors of the present application include tEGFR variants. In certain embodiments, expression of an inactivating cell surface receptor in engineered immune cells expressing a chimeric antigen receptor (CAR) induces apoptosis of the engineered immune cells when the cells are contacted with an anti - EGFR antibody. Methods of using inactivating cell surface receptors are described in International Publication No. WO 2019 / 070856, International Publication No. WO 2019 / 023396, and International Publication No. WO 2018 / 058002, the disclosures of which are incorporated herein by reference. For example, an anti - EGFR antibody can be administered to a subject previously receiving the engineered immune cells of the present disclosure containing a heterologous polynucleotide encoding an inactivating cell surface receptor including a tEGFR variant, in an amount effective to remove the previously administered engineered immune cells in the subject.

[0198] In certain embodiments, the anti - EGFR antibody is cetuximab, matuzumab, necitumumab, or panitumumab, and preferably, the anti - EGFR antibody is cetuximab.

[0199] In certain embodiments, the tEGFR variant comprises, or consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO:71, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO:71.

[0200] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the CD79b epitope comprises, or consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO:78, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO:78.

[0201] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the CD20 epitope comprises, or consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO:80, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO:80.

[0202] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of the Her2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the epitope specific for the monoclonal antibody comprises, or consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO:82, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO:82.

[0203] In certain embodiments, the inactivated cell surface protein is a truncated epidermal growth factor (tEGFR) variant. In certain embodiments, the tEGFR variant has, or consists of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71. Preferably, the tEGFR variant has, or consists of, the amino acid sequence of SEQ ID NO: 71.

[0204] IV. Cytokine Expression In some embodiments, the iPSC cells or their derivative cells optionally contain an exogenous polynucleotide encoding a cytokine such as interleukin-15 or interleukin-2.

[0205] As used herein, "interleukin-15" or "IL-15" refers to a cytokine that regulates the activation and proliferation of T and NK cells, or a functional portion thereof. A "functional portion" (a "bioactive portion") of a cytokine refers to a portion of a cytokine that retains one or more functions of the full-length or mature cytokine. Such functions of IL-15 include promoting NK cell survival, regulating the activation and proliferation of NK and T cells, and assisting in NK cell development from hematopoietic stem cells. As will be understood by those skilled in the art, the sequences of various IL-15 molecules are known in the art. In certain embodiments, IL-15 is wild-type IL-15. In certain embodiments, IL-15 is human IL-15. In certain embodiments, IL-15 comprises an amino acid sequence that is at least 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72.

[0206] In some embodiments, IL-15 is in a membrane-bound form, and all or a functional portion of the IL-15 protein is fused to all or a portion of a transmembrane protein that tethered the expressed IL-15 as a cell membrane-bound polypeptide (mbIL15), for example, the construct described in U.S. Patent No. 9,629,877 B2, which is incorporated herein by reference in its entirety during the filing of this application.

[0207] As used herein, "interleukin-2" refers to a cytokine that regulates the activation and proliferation of T and NK cells, or a functional portion thereof. In certain embodiments, IL-2 is wild-type IL-2. In certain embodiments, IL-2 is human IL-2. In certain embodiments, IL-2 comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76, preferably the amino acid sequence of SEQ ID NO: 76.

[0208] In certain embodiments, the cytokine may be linked to a safety switch such that the cytokine is preferably included in an inactivated cell surface receptor that contains an epitope specific for a monoclonal antibody and is operably linked to the cytokine by a self - protease peptide sequence. Examples of self - protease peptides include, but are not limited to, peptide sequences selected from the group consisting of porcine teschovirus - 1 2A (P2A), foot - and - mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), Bombyx mori infectious flacherie virus 2A (BmIFV2A), and combinations thereof. In one embodiment, the self - protease peptide is the self - protease peptide of porcine teschovirus - 1 2A (P2A). In certain embodiments, the self - protease peptide comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73.

[0209] In certain embodiments, the inactivated cell surface receptor comprises a truncated epidermal growth factor (tEGFR) variant that is operably linked to interleukin - 15 (IL - 15) or IL - 2 by a self - protease peptide sequence. In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74, preferably the amino acid sequence of SEQ ID NO: 74.

[0210] In some embodiments, the inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77, preferably the amino acid sequence of SEQ ID NO: 77.

[0211] In some embodiments, the inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 21.

[0212] In certain embodiments, the inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 23.

[0213] In certain embodiments, the inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, transmembrane region, and cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises multiple epitopes specifically recognized by an antibody, the epitopes can have the same or different amino acid sequences, and the epitopes can be linked together via a peptide linker such as a flexible peptide linker having the sequence of (GGGGS)n [where n is an integer from 1 to 8] (SEQ ID NO: 25). In some embodiments, the inactivated cell surface receptor further comprises a cytokine such as IL-15 or IL-2. In certain embodiments, the cytokine is in the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked, directly or indirectly, via an autocatalytic protease peptide sequence such as those described herein, to an epitope specifically recognized by an antibody. In some embodiments, the cytokine is indirectly linked to the epitope by connecting to the transmembrane region via an autocatalytic protease peptide sequence.

[0214] Non-limiting exemplary inactivated cell surface receptor regions and sequences are provided in Table 2.

[0215]

Table 2-1

[0216]

Table 2-2

[0217] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79.

[0218] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 81, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO: 81.

[0219] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 83, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO: 83.

[0220] III. HLA Expression In certain embodiments, the iPSCs or their derivative cells of the present application can be further modified by introducing a third exogenous polynucleotide encoding one or more proteins associated with immune evasion, such as non-classical HLA class I proteins (such as HLA-E and HLA-G). Specifically, disruption of the B2M gene eliminates surface expression of all MHC class I molecules, rendering the cells vulnerable to lysis by NK cells through the "loss of self" response. Expression of exogenous HLA-E may confer resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol. 2017 Aug; 35(8): 765-772).

[0221] In certain embodiments, the iPSC or its derivative cells comprise a third exogenous polypeptide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G). In certain embodiments, HLA-E comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65. In certain embodiments, HLA-G comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68, preferably comprising SEQ ID NO: 68.

[0222] In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-E via a linker. In certain embodiments, the third exogenous polypeptide comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66.

[0223] In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-G via a linker. In certain embodiments, the third exogenous polypeptide comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 69.

[0224] IV. Membrane-bound IL-12 Interleukin-12 (IL-12) is a heterodimeric molecule composed of an alpha chain (p35 subunit) and a beta chain (p40 subunit) that are covalently linked by disulfide bridges to form a biologically active 70 kDa dimer. Biologically, IL-12 is an inflammatory cytokine produced by various cells of the immune system, including phagocytes, B cells, and activated dendritic cells, in response to infection (Colombo and Trinchieri (2002), Cytokine & Growth Factor Reviews, 13: 155-168 and Hamza et al., “Interleukin-12 a Key Immunoregulatory Cytokine in Infection Applications” Int. J. Mol. Sci. 11;789-806 (2010)). IL-12 plays an essential role in mediating the interaction between the natural and acquired arms of the immune system, acting on T cells and natural killer (NK) cells to enhance the proliferation and activation of cytotoxic lymphocytes and the production of other inflammatory cytokines, particularly interferon-gamma (IFN-gamma).

[0225] IL-12 has been tested in human clinical trials as an immunotherapeutic agent for the treatment of a variety of cancers, including kidney, colon, and ovarian cancers, melanoma, and T-cell lymphoma (Atkins et al. (1997), Clin. Cancer Res., 3: 409-17, Gollob et al. (2000), Clin. Cancer Res., 6: 1678-92, Hurteau et al. (2001), Gynecol. Oncol., 82: 7-10, and Youssoufian, et al. (2013) Surgical Oncology Clinics of North America, 22(4):885-901), as well as as an adjuvant for cancer vaccines (Lee et al. (2001), J. Clin. Oncol. 19: 3836-47). However, IL-12 is toxic when administered systemically as a recombinant protein. Trinchieri, Adv. Immunol. 1998; 70:83-243. Since IL-12 is a heterodimeric molecule composed of an alpha chain (p35 subunit) and a beta chain (p40 subunit), co-expression of the two subunits is required for the production of a biologically active heterodimer. Expression of recombinant IL-12 has been achieved using a bicistronic vector containing the p40 and p35 subunits, which can be separated by an IRES (internal ribosome entry site) sequence to allow for independent expression of both subunits from a single vector. However, the use of the IRES sequence may impair protein expression. Mizuguchi et al. Mol Thera (2000); 1: 376-382. Furthermore, unequal expression of the p40 and p35 subunits may result in the formation of homodimeric proteins (e.g., p40-p40), which may have an inhibitory effect on IL-12 signaling. Gillessen et al. Eur. J. Immunol. 25(1):200-6 (1995).

[0226] As an alternative to the bicistronic expression of the IL-12 subunits, functional single-chain IL-12 fusion proteins have been generated by linking the p40 and p35 subunits using a disulfide, (Gly4Ser)3, or Gly6Ser linker. Lieschke et al., (1997), Nature Biotechnology 15, 35-40, Lode et al (1998), PNAS 95, 2475-2480. (These forms of the IL-12 conformation of p40-linker-p35 or p35-linker-p40 can be referred to herein as "conventional single-chain IL-12 (scIL-12).

[0227] Membrane-anchored IL-12 protein sequences that can be used in various embodiments include the amino acid sequence of wild-type IL-12, as well as analogs and derivatives thereof. For example, the IL12 polypeptide may be modified (e.g., genetically, synthetically, or recombinantly) to increase its sensitivity to proteases and reduce the biological half-life of the IL12 complex compared to the corresponding IL12 lacking protease sensitivity. The protease-sensitive form of IL12 is described in International Publication No. WO 2017 / 062953, the contents of which are incorporated herein by reference in their entirety.

[0228] According to the present invention, the p35 and p40 subunits of the IL-12 protein, preferably those in the conformation of conventional single-chain IL-12, are tethered to the cell membrane by fusing scIL-12 to a transmembrane domain (TM) such as the EGFR transmembrane domain. In certain embodiments, the ρ35 / TM subunit is further fused to a signaling domain (SD). For example, the signaling domain is a CD3ζ, CD28, and / or 4-1BB signaling domain. In certain embodiments, the signaling domain comprises a CD3ζ and a 4-1BB signaling domain. In some embodiments, the signaling domain is 4-1BB.

[0229] As used herein, the term "transmembrane domain (TM)" broadly refers to an amino acid sequence about 15 residues in length that spans the plasma membrane. More preferably, the transmembrane domain comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domains of the present disclosure may be derived from either natural or synthetic sources. The transmembrane domain can be derived from any protein that is naturally membrane-bound or transmembrane. In some embodiments, the transmembrane domain can be derived from EGFR.

[0230] Alternatively, the transmembrane domains of the present disclosure may be synthetic. In some aspects, the synthetic sequences may predominantly contain hydrophobic residues such as leucine and valine.

[0231] The amino acid sequence of p70 IL-12 fused to EGFR transmembrane is as follows.

[0232]

Chemical formula

[0233] The corresponding nucleotide sequence of p70 IL-12 fused to EGFR transmembrane is as follows.

[0234]

Chemical formula

[0235]

Chemical formula

[0236] As an alternative construct according to the present invention, the membrane-bound IL-12 p70 protein may incorporate a protease cleavage site for activation-induced release through protease ADAM17. ADAM17 is expressed by activated lymphocytes and is directly involved in the release of other immune mediators such as TNFa, which is also presented in a membrane-tethered form. When this membrane-tethered IL12 is expressed on engineered iNK cells, it remains cell-associated. Upon cell activation and increased ADAM17 expression, the protease cleaves the membrane stalk, releasing IL12 into the extracellular space. This type of regulation ensures that the activity of IL12 is confined to the space around the tumor, where the engineered immune cells associate with their targets on the tumor cells that trigger their activation.

[0237] An exemplary amino acid sequence of the membrane IL-12 p70 ADAM17 protease cleavage site fusion protein is as follows.

[0238]

Chemical formula

[0239] The corresponding nucleotide sequence of the membrane IL-12 p70 ADAM17 protease cleavage site fusion protein is as follows.

[0240]

Chemical formula

[0241]

Chemical formula

[0242] TNFα and TGFα are two substrates that can be cleaved by ADAM17. In certain embodiments, the membrane-bound IL-12 p70 protein can include TNFα or TGFα, or a portion of one or both of them, for activation-induced release through the protease ADAM17. An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α short scaffold protease cleavage site fusion protein is as follows.

[0243]

Chemical formula

[0244] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α short scaffold protease cleavage site fusion protein is as follows.

[0245]

Chemical formula

[0246]

Chemical formula

[0247] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site fusion protein is as follows.

[0248]

Chemical formula

[0249] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site fusion protein is as follows.

[0250]

Chemical formula

[0251] [Chemical formula]

[0252] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold / TACEtide protease cleavage site fusion protein is as follows.

[0253] [Chemical formula]

[0254] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α / TACEtide scaffold protease cleavage site fusion protein is as follows.

[0255] [Chemical formula]

[0256] [Chemical formula]

[0257] [Chemical formula]

[0258] An exemplary amino acid sequence of membrane IL-12 p70 having a TNF-α scaffold protease cleavage site fusion protein is as follows.

[0259] [Chemical formula]

[0260] The corresponding nucleotide sequence of membrane IL-12 p70 having a TNF-α scaffold protease cleavage site fusion protein is as follows.

[0261] [Chemistry]

[0262] [Chemistry]

[0263] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site and an EGFR transmembrane domain fusion protein is as follows.

[0264] [Chemistry]

[0265] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site and an EGFR transmembrane domain fusion protein is as follows.

[0266] [Chemistry]

[0267] [Chemistry]

[0268] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold tandem protease cleavage site fusion protein is as follows.

[0269] [Chemistry]

[0270] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α scaffold tandem protease cleavage site fusion protein is as follows.

[0271] [Chemistry]

[0272]

Chem.

[0273] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site (variant 1) fusion protein is as follows.

[0274]

Chem.

[0275] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site (variant 1) fusion protein is as follows.

[0276]

Chem.

[0277]

Chem.

[0278]

Chem.

[0279] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site (variant 2) fusion protein is as follows.

[0280]

Chem.

[0281] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site (variant 2) fusion protein is as follows.

[0282]

Chem.

[0283]

Chem.

[0284] An exemplary amino acid sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site (variant 3) fusion protein is as follows.

[0285]

Chem.

[0286] The corresponding nucleotide sequence of membrane IL-12 p70 having a TGF-α scaffold protease cleavage site (variant 3) fusion protein is as follows.

[0287]

Chem.

[0288]

Chem.

[0289] In certain embodiments, the exogenous polynucleotide encoding the mbIL-12 protein or the membrane IL-12 p70 ADAM17 protease cleavage site fusion protein is integrated into one or more loci on the chromosome of the cell, preferably, one or more loci are those of one or more genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, more preferably, one or more of the exogenous polynucleotides are integrated into the loci of the AAVS1 or CLYBL gene.

[0290] V. Other Optional Genome Editing In one embodiment of the cells described above, genome editing at one or more selected sites may include the insertion of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of genome-engineered iPSCs or their derivative cells. In some embodiments, the genome-engineered iPSCs generated using the above method include one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, B cell CD20, ErbB2, or CD79b, and when the genome-engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safe harbor loci including AAVSl, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR, or RUNX1. Other exogenous polynucleotides encoding proteins may include PET reporters, constitutive cytokines, inhibitory checkpoint inhibitor proteins such as PD1, PD-L1, and CTLA4, and those encoding proteins that target the CD47 / signal regulatory protein alpha (SIRPα) axis. In some other embodiments, the genome-engineered iPSCs generated using the methods provided herein include in / dels in one or more endogenous genes related to targeting modalities, receptors, signaling molecules, transcription factors, drug target candidates, regulation and modulation of the immune response, or proteins that suppress the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derivative cells.

[0291] VI. Promoter In some embodiments, the exogenous polynucleotide for insertion is operably linked to one or more exogenous promoters, including (1) CMV, EF1a, PGK, CAG, UBC, or other constitutive, inducible, temporally specific, tissue-specific, or cell type-specific promoters, or (2) one or more endogenous promoters contained within a selected site, including AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors.

[0292] VII. Targeted genome editing at selected loci in iPSCs According to embodiments of the present application, one or more of the exogenous polynucleotides are integrated into one or more loci on the chromosomes of the iPSCs.

[0293] Genome editing, or genomic editing, or gene editing are used interchangeably herein and are a type of genetic manipulation in which DNA is inserted, deleted, and / or substituted in the genome of a target cell. Targeted genome editing (which is interchangeable with "targeted genomic editing" or "targeted gene editing") enables insertion, deletion, and / or substitution at a pre-selected site in the genome. If the endogenous sequence is deleted or disrupted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down due to the deletion or disruption of the sequence. Thus, targeted editing can also be used to precisely disrupt the expression of an endogenous gene. The term "targeted integration" is used herein as well and refers to a process that includes the insertion of one or more exogenous sequences at a pre-selected site in the genome, with or without deletion of the endogenous sequence at the insertion site.

[0294] Targeted editing can be achieved either through nuclease-independent methods or through nuclease-dependent methods. In nuclease-independent targeted editing methods, homologous recombination is guided through the enzymatic machinery of the host cell by homologous sequences flanking the exogenous polynucleotide to be inserted.

[0295] Alternatively, targeted editing can be achieved at a higher frequency through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. 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 (in / dels) of a few endogenous nucleotides. In comparison, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homologous arms, the exogenous genetic material can be introduced into the genome by homologous recombination during homologous recombination repair (HDR), resulting in "targeted integration".

[0296] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) systems. Additionally, the DICE (double integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration.

[0297] ZFN is a targeted nuclease comprising a nuclease fused to a zinc finger DNA binding domain. The term "zinc finger DNA binding domain" or "ZFBD" means a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within the zinc finger binding domain, the structure of which is stabilized through coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not occur in nature, and its design / composition results primarily from the application of rational criteria, such as substitution rules and computerized algorithms, for processing information in a database that preserves information on existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261. See also International Publication Nos. 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536, and 03 / 016496. A "selected" zinc finger domain is a domain that is not found in nature, and its generation results primarily from empirical processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the entire disclosures of which are incorporated herein by reference. The most recognized example of ZFNs in the art is the fusion of the Fokl nuclease with a zinc finger DNA binding domain.

[0298] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. "Transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" means the polypeptide domain of a TAL effector protein that mediates 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, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA binding domain depends on the effector-variable incomplete 34-amino acid repeats, which contain polymorphisms at selected repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of a Fokl nuclease and a TAL effector DNA binding domain.

[0299] Another example of a targeted nuclease found to be useful in the subject methods is a targeted Spoll nuclease, which is a polypeptide comprising a Spoll polypeptide having nuclease activity fused to a DNA binding domain, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc., that is specific for a DNA sequence of interest. See, e.g., U.S. Patent Application Publication No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0300] Further examples of targeted nucleases suitable for the present application include, but are not limited to, Bxbl, phiC3 l, R4, PhiBTl, and Wp / SPBc / TP90l-l, whether used individually or in combination.

[0301] Other non-limiting examples of engineered nucleases include naturally occurring and recombinant nucleases, CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr, restriction endonucleases, meganucleases, homing endonucleases, and the like. As an example, CRISPR / Cas9 requires two main components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex that is recruited to a target DNA sequence that includes the PAM and a seed region near the PAM. The crRNA and tracrRNA combine to form a chimeric guide RNA (gRNA) that can direct Cas9 to target the selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction. As another example, CRISPR / Cpf1 includes two main components: (1) the Cpf1 endonuclease and (2) the crRNA. When co-expressed, the two components form a ribonucleoprotein (RNP) complex that is recruited to a target DNA sequence that includes the PAM and a seed region near the PAM. The crRNA combines to form a chimeric guide RNA (gRNA) that can direct Cpf1 to target the selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction.

[0302] MAD7 is an engineered Cas12a variant that is derived from the bacterium Eubacterium rectale, has a preference for 5'-TTTN-3' and 5'-CTTN-3' PAM sites, and does not require tracrRNA. See, for example, PCT Publication No. WO 2018 / 236548, the disclosure of which is incorporated herein by reference.

[0303] DICE-mediated insertion provides for unidirectional integration of exogenous DNA that is tightly restricted to the small attB and attP recognition sites of each of a pair of recombinases, such as phiC31 and Bxbl. Since these target att sites do not occur naturally in the mammalian genome, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0304] One aspect of the present application 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 method of targeted integration comprises introducing the construct into a cell to enable site-specific homologous recombination by the enzymatic machinery of the cell host. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site to enable ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site to enable TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a gRNA comprising a Cpf1 expression cassette and a guide sequence specific to a desired integration site to enable Cpf1-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a gRNA comprising a Cas9 expression cassette and a guide sequence specific to a desired integration site to enable Cas9-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing into a desired integration site in the cell a construct comprising one or more att sites of a pair of DICE recombinases, introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing an expression cassette for DICE recombinase to enable DICE-mediated targeted integration.

[0305] Genomic safe harbors that can be adapted to the predictable expression of newly integrated DNA without theoretical harmful effects on the host cell or organism are examples of sites for targeted integration, although not limited thereto, and are intragenic or extragenic regions of the human genome. In certain embodiments, the genomic safe harbor for targeted integration is one or more loci of a gene selected from the group consisting of the AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR, and RUNX1 genes.

[0306] In other embodiments, the site of targeted integration is selected for deletion or reduced expression of the endogenous gene at the insertion site. As used herein, the term "deletion" with respect to gene expression refers to any genetic modification that abolishes gene expression. Examples of "deletion" of gene expression include, for example, removal or deletion of the DNA sequence of the gene that abolishes gene expression, insertion of an exogenous polynucleotide sequence at the locus of the gene, and one or more substitutions within the gene.

[0307] Genes for target deletion include, but are not limited to, genes for major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must match for histocompatibility in an allogeneic recipient in order to avoid the problem of allogeneic rejection. "MHC deficiency", including MHC-class I deficiency, or MHC-class II deficiency, or both, refers to cells that lack, no longer maintain, or have a reduced level of surface expression of the complete MHC complex containing the MHC class I protein heterodimer and / or the MHC class II heterodimer, where the attenuated or reduced level is lower than the level naturally detectable by other cells or by synthetic methods. MHC-class I deficiency can be achieved by a functional deletion of any region of the MHC-class I locus (chromosome 6p2l) or, but not limited to, by deleting or reducing the expression level of one or more MHC-class I-related genes including the beta-2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 gene, and the tapasin gene. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC-class I heterodimers. B2M null cells are MHC-I deficient. MHC-class II deficiency can be achieved by, but not limited to, a functional deletion or reduction of MHC-II-related genes including RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through activation of the transcription factor RFX5 required for the expression of class II proteins. CIITA null cells are MHC-II deficient. In certain embodiments, one or more of the exogenous polynucleotides are integrated into one or more loci of a gene selected from the group consisting of the B2M, TAP1, TAP2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby deleting or reducing the expression of the gene upon integration.

[0308] In certain embodiments, the exogenous polynucleotide is integrated into one or more loci on the chromosome of the cell, preferably, one or more loci are loci of genes selected from the group consisting of the AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRa or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided that at least one of the one or more loci is a locus of a gene selected from the group consisting of MHC genes, such as genes selected from the group consisting of the B2M, TAP1, TAP2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes. Preferably, one or more exogenous polynucleotides are integrated into loci of MHC class I-related genes, such as the beta-2 microglobulin (B2M) gene, the TAP 1 gene, the TAP 2 gene, or the Tapasin gene, and loci of MHC-II-related genes, such as the RFXANK, CIITA, RFX5, RFXAP, or CIITA genes, and optionally further into loci of safe harbor genes selected from the group consisting of the AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR, and RUNX1 genes. More preferably, one or more of the exogenous polynucleotides are integrated into loci of the CIITA, AAVS1, and B2M genes, or loci of the CIITA, CLYBL, and B2M genes.

[0309] In certain embodiments, (i) the first exogenous polynucleotide (CAR) is integrated at the locus of the CIITA or B2M gene, (ii) the second exogenous polypeptide (mbIL-12) is integrated at the locus of the AAVS1 or CLYBL gene, and (iii) the third exogenous polypeptide (i.e., the HLA-E and / or HLA-G transgene) is integrated at the locus of the B2M or CIITA gene, wherein integration of the exogenous polynucleotide results in deletion or reduction of expression of the CIITA and B2M genes.

[0310] In certain embodiments, (i) the first exogenous polynucleotide encodes a CAR, (ii) the second exogenous polynucleotide comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 97 or 99, and (iii) the third exogenous polynucleotide comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 67 or 70.

[0311] In certain embodiments, (i) the first exogenous polynucleotide encodes a CAR, (ii) the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and (iii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0312] VIII. Derived cells In another aspect, the present invention relates to cells and derivative cells derived from the differentiation of iPSCs. As described above, the genome editing introduced into iPSC cells is retained in the derivative cells. In certain embodiments of derivative cells obtained from iPSC differentiation, the derivative cells include, but are not limited to, hematopoietic cells such as HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, B cells, antigen-presenting cells (APCs), monocytes, and macrophages. In certain embodiments, the derivative cells are immune effector cells such as NK cells or T cells.

[0313] In certain embodiments, the present application provides a natural killer (NK) cell or T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide that is at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide that is at least 90% similar thereto, and a membrane-bound interleukin 12 (IL-12) encoding a second exogenous polynucleotide comprising a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides; and (iii) a deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes. In certain embodiments, a natural killer (NK) cell or T cell comprising a polynucleotide encoding membrane-bound IL-12 is fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide for activation-induced release of IL-12 through the protease ADAM17. ADAM17 is expressed by activated lymphocytes and is directly involved in the release of other immune mediators such as TNFa, which is also presented in a membrane-tethered form. When this membrane-tethered IL-12 is expressed on engineered iNK or T cells, it remains cell-associated. Upon cell activation and increased ADAM17 expression, the protease cleaves the membrane stalk, releasing IL-12 into the extracellular space. This type of regulation ensures that the activity of IL-12 is confined to the space around the tumor, where the engineered immune cells associate with their targets on the tumor cells that trigger their activation.

[0314] In certain embodiments, the NK cell or T cell further comprises a third exogenous polynucleotide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G).

[0315] Also provided are NK cells or T cells comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a membrane-bound interleukin 12 (IL-12) encoding a second exogenous polynucleotide comprising a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides; and (iii) a deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes.

[0316] In another embodiment, also provided is an NK or T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising an IL-12 alpha subunit p35 having at least 90% sequence identity to SEQ ID NO: 102, a second polypeptide comprising an IL-12 beta subunit p40 having at least 90% sequence identity to SEQ ID NO: 103, and a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides having the amino acid sequence of SEQ ID NO: 100, a second exogenous polynucleotide encoding membrane-bound interleukin-12 (IL-12), or a second exogenous polynucleotide encoding membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for activation-inducible release of IL-12 through protease ADAM17, encoding a protein having the amino acid sequence of SEQ ID NO: 98; (iii) a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66 or 69; and (iv) a fourth exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin-15 (IL-15) having the amino acid sequence of SEQ ID NO: 72, wherein the first, second, and third exogenous polynucleotides are integrated at the loci of (a) the AAVS1, CIITA, and B2M genes, respectively, (b) the AAVS1, CIITA, and B2M genes, respectively, (c) the CLYBL, CIITA, and B2M genes, respectively, (d) the CIITA, AAVSI, and B2M genes, respectively, (e) the CIITA, CLYBL, and B2M genes, respectively, (f) the B2M, AAVS1, and CIITA genes, respectively, or (g) the B2M, CLYBL, and CIITA genes, respectively, whereby the expression of CIITA and B2M is deleted or reduced, and the fourth exogenous polynucleotide.

[0317] In certain embodiments, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 97 or 99, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67 or 70.

[0318] Also provided are CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs) comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising the IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising the IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12) comprising a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides; and (iii) a deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes.

[0319] In certain embodiments, the CD34+ HPCs further comprise a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

[0320] In certain embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to the CD19 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain such as a co-stimulatory domain comprising the CD28 signaling domain.

[0321] Also provided is a method of producing derivative cells. The method comprises differentiating iPSCs under conditions of cell differentiation to thereby obtain derivative cells.

[0322] The iPSCs of the present application can be differentiated by any method known in the art. Exemplary methods are described in U.S. Patent No. 8,846,395, U.S. Patent No. 8,945,922, U.S. Patent No. 8,318,491, International Publication No. 2010 / 099539, International Publication No. 2012 / 109208, International Publication No. 2017 / 070333, International Publication No. 2017 / 179720, International Publication No. 2016 / 010148, International Publication No. 2018 / 048828, and International Publication No. 2019 / 157597, each of which is incorporated herein by reference in its entirety. The differentiation protocol can use feeder cells or be feeder-free. As used herein, "feeder cells" or "feeder" refers to a type of cell that, when co-cultured with a second type of cell, provides stimuli, growth factors, and nutrients for the support of the second cell type, thereby providing an environment in which the second type of cell can grow, expand, or differentiate.

[0323] In another embodiment of the invention, the iPSC-derived cells of the invention are NK cells prepared by a method of differentiating iPSC cells into NK cells by subjecting the cells to a differentiation protocol that includes adding recombinant human IL-12 p70 during the last 24 hours of culture. By including IL-12 in the differentiation protocol, cells primed with IL-12 exhibit more rapid cell killing compared to those differentiated in the absence of IL-12 (Figure 8A). Furthermore, cells differentiated using the IL-12 condition exhibit improved cancer cell growth inhibition (Figure 8B).

[0324] IX. Polynucleotides, Vectors, and Host Cells (1) Nucleic acid encoding a CAR In another general aspect, the invention relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful in the invention according to the embodiments of the present application. Those skilled in the art will understand that the coding sequence of the CAR can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the CAR of the present application can be modified without changing the amino acid sequence of the protein.

[0325] In certain embodiments, the isolated nucleic acid encodes CAR-targeted CD19. In certain embodiments, the isolated nucleic acid encoding the CAR comprises a polynucleotide sequence that is at least 90% identical, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 62, preferably the polynucleotide sequence of SEQ ID NO: 62.

[0326] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding a CAR useful in the invention according to the embodiments of the present application. Any vector known to those skilled in the art in view of the present disclosure, such as a plasmid, cosmid, phage vector, or viral vector, can be used. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element for establishing the conventional functions of an expression vector, e.g., a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressive promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used in the present invention for the production of CARs in cells. Recombinant expression vectors according to the embodiments of the present application can be generated using conventional cloning techniques or artificial gene synthesis.

[0327] In certain embodiments, the present application provides a vector for targeted integration of a CAR useful for the invention according to the embodiments of the present application. In certain embodiments, the vector comprises, in 5' to 3' order, an exogenous polynucleotide having (a) a promoter, (b) a polynucleotide sequence encoding a CAR according to the embodiments of the present application, and (c) a terminator / polyadenylation signal.

[0328] In certain embodiments, the promoter is the CAG promoter. In certain embodiments, the CAG promoter comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 63, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical. Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta-actin.

[0329] In certain embodiments, the terminator / polyadenylation signal is the SV40 signal. In certain embodiments, the SV40 signal comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 64, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical. Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0330] In certain embodiments, the polynucleotide sequence encoding the CAR comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 62, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical.

[0331] In some embodiments, the vector further comprises a left homology arm and a right homology arm adjacent to the exogenous polynucleotide. As used herein, "left homology arm" and "right homology arm" refer to a pair of nucleic acid sequences that are adjacent to the exogenous polynucleotide and facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. The sequences of the left and right homology arms can be designed based on the target integration site. In some embodiments, the left or right homology arm is homologous to the sequence on the left or right side of the integration site.

[0332] In certain embodiments, a membrane-bound IL12 transgene (e.g., according to SEQ ID NO: 97 or 99) is integrated into the AAVS1 locus, and the left homology arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 104, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, and the right homology arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 105, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical. In certain embodiments, a membrane-bound IL12 transgene (e.g., according to SEQ ID NO: 97 or 99) is integrated into the CLYBL locus, and the left homology arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 106, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, and the right homology arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 107, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical.

[0333]

Table 3-1

[0334]

Table 3-2

[0335]

Table 3-3

[0336]

Table 3-4

[0337] In certain embodiments, the vector comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 97 or 99, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, preferably the polynucleotide sequence of SEQ ID NO: 97 or 99.

[0338] (2) Nucleic acid encoding mbIL-12 In another general aspect, the present invention relates to an isolated nucleic acid encoding an mbIL-12 protein useful in the inventions according to the embodiments of the present application. Those skilled in the art will understand that the coding sequence of an inactivated cell surface receptor can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding mbIL-12 of the present application can be modified without changing the amino acid sequence of the protein.

[0339] In certain embodiments, the isolated nucleic acid encodes a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for the activation-induced release of IL-12 through protease ADAM17.

[0340] In certain embodiments, mbIL-12 consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96, 98, 102, or 103.

[0341] In certain embodiments, the isolated nucleic acid encodes a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for the inducible release of IL-12 through protease ADAM17, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98.

[0342] (3) Nucleic acid encoding a safety switch In certain embodiments, the isolated nucleic acid encodes a safety switch such as an inactivated cell surface receptor having a cleaved epidermal growth factor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab, or panitumumab, preferably cetuximab.

[0343] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin.

[0344] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, such as an epitope specifically recognized by rituximab.

[0345] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of the Her2 receptor, such as an epitope specifically recognized by trastuzumab.

[0346] In certain embodiments, the self-protease peptide sequence is porcine teschovirus-1 2A (P2A).

[0347] In certain embodiments, the cleaved epidermal growth factor (tEGFR) variant consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0348] In certain embodiments, the epitope specific for the monoclonal antibody that is specifically recognized by polatuzumab vedotin consists of an amino acid sequence that is at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78.

[0349] In certain embodiments, the epitope specific for the monoclonal antibody that is specifically recognized by rituximab consists of an amino acid sequence that is at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80.

[0350] In certain embodiments, the epitope specific for the monoclonal antibody that is specifically recognized by trastuzumab consists of an amino acid sequence that is at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82.

[0351] In certain embodiments, IL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72.

[0352] In certain embodiments, the self-protease peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73.

[0353] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74.

[0354] In certain embodiments, the isolated nucleic acid encoding the inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90% identical, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, to SEQ ID NO: 75, preferably the polynucleotide sequence of SEQ ID NO: 75.

[0355] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79.

[0356] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for the invention according to embodiments of the present application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used in the present invention for the production of inactivated cell surface receptors in cells. Recombinant expression vectors according to embodiments of the present application can be generated using conventional cloning techniques or artificial gene synthesis.

[0357] In certain aspects, the present application provides a vector for targeted integration of an inactivated cell surface receptor useful for the invention according to embodiments of the present application. In certain embodiments, the vector comprises, in 5' to 3' order, (a) a promoter, (b) a polynucleotide sequence encoding an inactivated cell surface receptor such as an inactivated cell surface receptor comprising a truncated epidermal growth factor (tEGFR) variant and interleukin 15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by a self-cleaving protease peptide sequence such as porcine teschovirus-1 2A (P2A), and (c) a terminator / polyadenylation signal.

[0358] In certain embodiments, the promoter is the CAG promoter. In certain embodiments, the CAG promoter comprises a polynucleotide sequence that is at least 90% identical, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta-actin.

[0359] In certain embodiments, the terminator / polyadenylation signal is the SV40 signal. In certain embodiments, the SV40 signal comprises a polynucleotide sequence that is at least 90% identical, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0360] In certain embodiments, the polynucleotide sequence encoding the inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90% identical, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, to SEQ ID NO: 75.

[0361] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm adjacent to the exogenous polynucleotide.

[0362] In certain embodiments, the left homologous arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 84, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical. In certain embodiments, the right homologous arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 85, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical.

[0363] In certain embodiments, the vector comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 86, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, preferably the polynucleotide sequence of SEQ ID NO: 86.

[0364] (4) Nucleic acid encoding the HLA construct In another general aspect, the invention relates to an isolated nucleic acid encoding an HLA construct useful in the invention according to the embodiments of the present application. Those skilled in the art will understand that the coding sequence of the HLA construct can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the HLA construct of the present application can be modified without changing the amino acid sequence of the protein.

[0365] In certain embodiments, the isolated nucleic acid encodes an HLA construct that includes a signal peptide, such as an HLA-G signal peptide, operably linked to an HLA coding sequence, such as the coding sequence of mature B2M and / or mature HLA-E. In some embodiments, the HLA coding sequence encodes HLA-G and B2M operably linked by a 4×GGGGS linker and / or B2M and HLA-E operably linked by a 3×GGGGS linker. In certain embodiments, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence that is at least 90% identical, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence that is at least 90% identical, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0366] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an HLA construct useful for the invention according to the embodiments of the present application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used in the present invention for the production of HLA constructs in cells. Recombinant expression vectors according to the embodiments of the present application can be generated using conventional cloning techniques or artificial gene synthesis.

[0367] In certain embodiments, the present application provides vectors for targeted integration of HLA constructs useful for the invention according to embodiments of the present application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in 5' to 3' order, (a) a promoter, (b) a polynucleotide sequence encoding an HLA construct, and (c) a terminator / polyadenylation signal.

[0368] In certain embodiments, the promoter is the CAG promoter. In certain embodiments, the CAG promoter comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 63, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical. Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta-actin.

[0369]

[0370] In certain embodiments, the polynucleotide sequence encoding the HLA construct comprises a signal peptide such as the HLA-G signal peptide, mature B2M, and mature HLA-E, wherein HLA-G and B2M are operably linked by a 4×GGGGS linker (SEQ ID NO: 31), and the B2M transgene and HLA-E are operably linked by a 3×GGGGS linker (SEQ ID NO: 25). In certain embodiments, the HLA construct comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 67, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLA construct comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 70, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0371] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm adjacent to the exogenous polynucleotide.

[0372] In certain embodiments, the left homologous arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 87, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical. In certain embodiments, the right homologous arm comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 88, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical.

[0373] In certain embodiments, the vector comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 89, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, preferably the polynucleotide sequence of SEQ ID NO: 89.

[0374] (5) Host cell In another general aspect, the present application provides a host cell comprising an isolated nucleic acid encoding a vector of the present application and / or a construct of the present application. Any host cell known to those skilled in the art in view of the present disclosure can be used for the recombinant expression of the exogenous polynucleotide of the present application. According to certain embodiments, a recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.

[0375] Examples of host cells include, for example, recombinant cells containing a vector of the present application or an isolated nucleic acid useful for the production of the vector or construct of interest, or engineered iPSCs or their derivative cells containing one or more isolated nucleic acids of the present application integrated into one or more chromosomal loci. The host cells of the isolated nucleic acids of the present application can also be immune effector cells such as T cells or NK cells containing one or more isolated nucleic acids of the present application. Immune effector cells can be obtained by differentiation of the engineered iPSCs of the present application. Any suitable method in the art can be used for differentiation in view of the present disclosure. Immune effector cells can also be obtained by transfecting immune effector cells with one or more isolated nucleic acids of the present application.

[0376] X. Compositions In another general aspect, the present application provides a composition comprising an isolated polynucleotide of the present application, a host cell, and / or an iPSC of the present application or its derivative cells.

[0377] In certain embodiments, the composition further comprises one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, dsRNA (double-stranded RNA), siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising a polynucleotide of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

[0378] In certain embodiments, the composition is a pharmaceutical composition comprising an isolated polynucleotide of the present application, a host cell, and / or an iPSC or a derivative cell thereof of the present application, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising an isolated polynucleotide of the present application, an isolated polypeptide of the present application, a host cell of the present application, and / or an iPSC or a derivative cell thereof of the present application, together with a pharmaceutically acceptable carrier. Polynucleotides, polypeptides, host cells, and / or iPSCs or derivative cells thereof of the present application, and compositions comprising them are also useful for the manufacture of pharmaceuticals for the therapeutic applications referred to herein.

[0379] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient, or diluent depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the compositions described herein or the biological activity of the compositions described herein. According to certain embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in polynucleotides, polypeptides, host cells, and / or iPSCs or derivative cells thereof can be used.

[0380] The combination of a pharmaceutically active ingredient and a pharmaceutically acceptable carrier is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g. 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include buffering agents, diluents, solvents, isotonicity regulators, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in the formulation of the pharmaceutical compositions of the present application.

[0381] XI. Method of Use In another general aspect, the present application provides a method of treating a disease or condition in a subject in need thereof. The method includes administering to the subject in need thereof a therapeutically effective amount of the cells of the present application and / or the compositions of the present application. In certain embodiments, the disease or condition is cancer. The cancer can be, for example, a solid or liquid cancer. The cancer can be selected from the group consisting of, for example, lung cancer, gastric cancer, colon cancer, liver cancer, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, endometrial cancer, prostate cancer, thyroid cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), Hodgkin lymphoma / disease (HD), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myelogenous leukemia (AML), and other liquid tumors. In a preferred embodiment, the cancer is non-Hodgkin lymphoma (NHL).

[0382] According to embodiments of the present application, the composition includes a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and / or an iPSC or a derivative cell thereof. As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or medical response in a subject. A therapeutically effective amount can be determined in an empirical and routine manner in relation to the described purpose.

[0383] As used herein in connection with the cells of the present application and / or the pharmaceutical compositions of the present application, a therapeutically effective amount means an amount of the cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0384] According to certain embodiments, a therapeutically effective amount refers to an amount of treatment sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of the disease, disorder, or condition being treated, or symptoms associated therewith, (ii) reducing the duration of the disease, disorder, or condition being treated, or symptoms associated therewith, (iii) preventing the progression of the disease, disorder, or condition being treated, or symptoms associated therewith, (iv) causing regression of the disease, disorder, or condition being treated, or symptoms associated therewith, (v) preventing the occurrence or onset of the disease, disorder, or condition being treated, or symptoms associated therewith, (vi) preventing recurrence of the disease, disorder, or condition being treated, or symptoms associated therewith, (vii) reducing hospitalization of a subject having the disease, disorder, or condition being treated, or symptoms associated therewith, (viii) reducing the length of hospitalization of a subject having the disease, disorder, or condition being treated, or symptoms associated therewith, (ix) increasing survival of a subject having the disease, disorder, or condition being treated, or symptoms associated therewith, (xi) inhibiting or reducing the disease, disorder, or condition being treated, or symptoms associated therewith in a subject, and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another treatment.

[0385] The therapeutically effective amount or dosage may vary depending on various factors such as the disease, disorder, or condition being treated, the means of administration, the target site, the physiological state of the subject (e.g., including age, weight, health), whether the subject is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The treatment dosage is optimally titrated to optimize safety and efficacy.

[0386] According to certain embodiments, the compositions described herein are formulated to be appropriate for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be appropriate for intravenous, subcutaneous, or intramuscular administration.

[0387] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in any convenient manner known to those skilled in the art. For example, the cells of the present application can be administered to a subject by aerosol inhalation, injection, ingestion, infusion, implantation, and / or transplantation. The compositions containing the cells of the present application can be administered by transarterial, subcutaneous, intradermal, intratumoral, intra-articular, intramedullary, intramuscular, intrapleurally, intravenous (i.v.) injection, or intraperitoneally. In certain embodiments, the cells of the present application can be administered with or without using lymphocyte depletion of the subject.

[0388] The pharmaceutical compositions containing the cells of the present application can be provided in a sterile liquid preparation, typically an isotonic aqueous solution containing a cell suspension, or optionally as an emulsion, dispersion, etc., which are typically buffered to a selected pH. The composition can contain a carrier suitable for the integrity and viability of the cells and for the administration of the cell composition, such as water, saline, phosphate buffered saline, etc.

[0389] Sterile injectable solutions can be prepared by incorporating the cells of the present application into a suitable amount of a suitable solvent, optionally together with various other ingredients as required. Such compositions can contain pharmaceutically acceptable carriers, diluents, or excipients such as sterile water, saline, glucose, dextrose, etc., which are suitable for use with the cell composition and for administration to a subject such as a human. Suitable buffering agents for providing the cell composition are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the present application.

[0390] The cells of the present application and / or the pharmaceutical composition of the present application can be administered in any physiologically acceptable vehicle. The cell population containing the cells of the present application can include a purified population of cells. Those skilled in the art can easily determine the cells in the cell population using various well-known methods. The purity range in the cell population containing the genetically modified cells of the present application can be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art. For example, a decrease in purity may require an increase in dosage.

[0391] The cells of the present application are generally administered as a dosage based on the number of cells per kilogram of body weight (cells / kg) of the subject to whom the cells and / or the pharmaceutical composition containing the cells are administered. Generally, the cell dosage is about 10 4 ~ about 10 10 cells / 1 kg of body weight, for example, about 10 5 ~ about 10 9 , about 10 5 ~ about 10 8 , about 10 5 ~ about 10 7 , or about 10 5 ~ about 10 6 in the range. Generally, in the case of systemic administration, a higher dosage is used than for local administration of the immune cells of the present application to the region of the tumor and / or cancer. Exemplary dosage ranges include, but are not limited to, 1×10 4 ~ 1×10 8 , 2×10 4 ~ 1×10 8 , 3×10 4 ~ 1×10 8 , 4×10 4 ~ 1×10 8 , 5×10 4 ~ 6×10 8 , 7×10 4 ~ 1×10 8 , 8×10 4 ~ 1×10 8、9×10 4 ~1×10 8 、1×10 5 ~1×10 8 、1×10 5 ~9×10 7 、1×10 5 ~8×10 7 、1×10 5 ~7×10 7 、1×10 5 ~6×10 7 、1×10 5 ~5×10 7 、1×10 5 ~4×10 7 、1×10 5 ~4×10 7 、1×10 5 ~3×10 7 、1×10 5 ~2×10 7 、1×10 5 ~1×10 7 、1×10 5 ~9×10 6 、1×10 5 ~8×10 6 、1×10 5 ~7×10 6 、1×10 5 ~6×10 6 、1×10 5 ~5×10 6 、1×10 5 ~4×10 6 、1×10 5 ~4×10 6 、1×10 5 ~3×10 6 、1×10 5 ~2×10 6 、1×10 5 ~1×10 6 、2×10 5 ~9×10 7 、2×10 5 ~8×10 7 、2×10 5 ~7×10 7 、2×10 5 ~6×10 7 、2×10 5 ~5×10 7 、2×10 5 ~4×107 , 2×10 5 ~4×10 7 , 2×10 5 ~3×10 7 , 2×10 5 ~2×10 7 , 2×10 5 ~1×10 7 , 2×10 5 ~9×10 6 , 2×10 5 ~8×10 6 , 2×10 5 ~7×10 6 , 2×10 5 ~6×10 6 , 2×10 5 ~5×10 6 , 2×10 5 ~4×10 6 , 2×10 5 ~4×10 6 , 2×10 5 ~3×10 6 , 2×10 5 ~2×10 6 , 2×10 5 ~1×10 6 , 3×10 5 ~3×10 6 Examples include 2×10 to 4×10, 2×10 to 3×10, 2×10 to 2×10, 2×10 to 1×10, 2×10 to 9×10, 2×10 to 8×10, 2×10 to 7×10, 2×10 to 6×10, 2×10 to 5×10, 2×10 to 4×10, 2×10 to 4×10, 2×10 to 3×10, 2×10 to 2×10, 2×10 to 1×10, 3×10 to 3×10 cells / kg, etc. Furthermore, the dosage can be adjusted to account for whether a single dose or multiple doses are being administered. The exact determination of what constitutes an effective dose may depend on the individual factors of each subject.

[0392] As used herein, the terms "treat", "treating", and "treatment" are all intended to refer to the amelioration or improvement of at least one measurable physical parameter related to cancer, which may or may not be distinguishable in a subject. The terms "treat", "treating", and "treatment" can also refer to causing a regression of, preventing the progression of, or at least slowing the progression of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to the alleviation, prevention of the occurrence or onset, or reduction in the duration of one or more symptoms associated with a disease, disorder, or condition such as a tumor or more preferably cancer. In certain embodiments, "treat", "treating", and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to increasing the survival of a subject having a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to eliminating a disease, disorder, or condition in a subject.

[0393] The cells of the present application and / or the pharmaceutical composition of the present application can be administered in combination with one or more additional therapeutic agents. In certain embodiments, the one or more therapeutic agents are selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), siRNAs, oligonucleotides, mononuclear blood cells, vectors containing one or more polynucleotides of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

[0394] Embodiments The present application provides the following non-limiting embodiments.

Example

[0395] [Example 1] Generation of the mbIL-12 cell line Generation of iPSCs The parental cell line of induced pluripotent stem cells (iPSCs) was generated from peripheral blood mononuclear cells (PBMCs) using a process based on episomal plasmids previously described in U.S. Patent Nos. 8,546,140, 9,644,184, 9,328,332, and 8,765,470, the complete disclosures of which are incorporated herein by reference.

[0396] Generation of Vectors (Plasmids) A gene fragment (gBlock) encoding the gene of interest along with a promoter, a terminator, and homologous arms was designed and chemically synthesized by IDT, Inc. The gBlock gene fragment was assembled into the pUC19 plasmid according to the manufacturer's protocol using the In-Fusion® Cloning HD Plus Kit (Takara Bio, Shiga, Japan). The reaction product, i.e., the expression construct, from In-Fusion Cloning was transformed into Stbl3 bacterial cells (Thermo Fisher, Waltham, Massachusetts) for amplification according to the manufacturer's protocol. The vector (plasmid) from the amplified expression construct was purified from the bacterial cell culture according to the manufacturer's protocol using the HiSpeed Plasmid Maxi Prep Kit (Qiagen, Hilden, Germany). Research-grade sequencing was performed on the purified plasmid DNA and evaluated by restriction digestion to confirm the transgene sequence. The concentration of the purified plasmid DNA was measured by absorbance. Furthermore, the ratios of absorbance at A260 / A280 nm and A260 / A230 nm were measured to evaluate the residual RNA and protein levels, respectively.

[0397] AAVS1-Targeted Plasmid Gene block DNA fragments from IDT for each membrane IL12 variant were cloned into the p1392 vector for site-specific insertion of the transgene into the AAVS1 locus, enabling strong expression using the CAG promoter and the SV40 terminator. The AAVS1-targeting plasmid contains the CAG promoter (SEQ ID NO: 63), the SV40 terminator / polyadenylation (SEQ ID NO: 64), and mbIL-12 (SEQ ID NO: 97) or the membrane-bound IL-12 p70 ADAM17 protease cleavage site fusion protein (SEQ ID NO: 99).

[0398] Establishment of mbIL-12 iPSC cell lines The p1392 AAVS1 homologous recombination repair (HDR) vector specifically targets intron 1 of the AAV1 locus (PPP1R12C gene) for site-specific integration of the transgene into the cell, enabling geneticin antibiotic selection of the correctly engineered cells. To facilitate site-specific integration of the transgene, an HDR vector containing two versions of membrane IL12 was electroporated into iPSC005 cells together with the Cpf1 / AAVS1 gRNA ribonucleoprotein complex.

[0399] After 4 days of culture, the cells were incubated with 500 μg / ml of geneticin to kill and remove the cells that had not successfully incorporated the transgene into the AAVS1 locus. The surviving cells showing correct insertion of the transgene were expanded and analyzed for membrane IL12 expression by flow cytometry. The results are shown in FIGS. 2A - C.

[0400] Subsequently, for functional characterization, the cells were differentiated into HPCs and then into iNK cells.

[0401] Surface detection of IL12 at the HPC stage on day 9 is shown in FIG. 3.

[0402] PCR confirmation of p1514 transgene insertion into iPSC PCR primers were designed to amplify the tmIL12 sequence in the genomic DNA of iPSC1294 cells based on the genomic map of Figure 4A.

[0403] Primer 1514 forward and 1514 R will amplify a band of approximately 1700 bp, while primer 1514 forward and 1514 R2 will amplify a band of approximately 600 bp. The results in Figure 4B confirm the presence of the transgene in iPSCs.

[0404] Furthermore, junction PCR was performed to confirm the insertion of the transgene at the correct locus (see, for example, the genomic map in Figure 5A).

[0405] The results are shown in Figure 5B.

[0406] Surface detection of IL12 at the iNK stage on day 14 is shown in Figure 6A.

[0407] Surface detection of IL12 at the iNK stage on day 21 is shown in Figure 6B.

[0408] The expression of the transgene decreased during differentiation into iNK on day 21.

[0409] To confirm the expression in cells engineered with membrane-tethered IL12 (p1514, Figure 1B) cleavable by ADAM17, iPSCs were incubated with various concentrations of the ADAM17 inhibitor TAPI-1 and then analyzed by flow cytometry for IL12 detection on the surface of the engineered iPSCs.

[0410] The effect of 50 uM TAPI-1 on tmIL12 expression is shown in Figure 7. Lower concentrations had no effect on cell health or tmIL12 detection.

[0411] [Example 2] Cytokines enhanced the cytotoxicity assay Interleukin-12 is a cytokine that stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells. To determine whether IL-12 had an effect on the target cell cytotoxicity of CAR / IL-15 iNK cells, iNK cells were differentiated in a standard protocol (without IL-12) or with the inclusion of 10 ng / ml of recombinant human IL-12 p70 (PeproTech, Rocky Hill, NJ) during the last 24 hours of culture. iNK cells were used in the Incucyte killing assay to determine the efficacy of killing the Raji CD19+ B cell leukemia cell line (ATCC, Manassas, VA). Cells primed with IL-12 showed more rapid killing of Raji cells compared to those differentiated in the absence of IL-12 (Figure 8A).

[0412] iNK cells primed with IL-12 were further tested for their effect on in vivo tumorigenesis. The Burkitt lymphoma cell line Raji labeled with luciferase was transplanted intravenously (iv) into female NSG™ mice on day 0 of the study. On days 1, 4, and 7 of the study, mice were injected intravenously with 1 × 10 7 unprimed or IL12-primed CAG-CAR-IL15 iNK cells. During the duration of the study, starting on day 1, mice were supplemented intraperitoneally with recombinant human IL-2 (100,000 IU, PeproTech #200-02) three times a week. The untreated group served as a control. Prior to imaging using an IVIS SpectrumCT (Perkin Elmer), mice were injected with luciferin (VivoGlo™, Promega). The reaction between the luciferin substrate and the firefly luciferase enzyme produced by Raji tumor cells generates light that is measured as a bioluminescent signal. Data are presented as mean whole body bioluminescence average luminance ± SD. At the end of the 20-day study, 50% and 62% tumor growth inhibition was observed in the unprimed and IL-12-primed iNK treatments, respectively ( *p < 0.05, ** p < 0.01) (Figure 8B).

Claims

**Claim 1** An induced pluripotent stem cell (iPSC) or a derivative cell thereof, comprising an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12) comprising: (i) a first polypeptide comprising a polypeptide that is at least 90% similar to the IL-12 alpha subunit p35, (ii) a second polypeptide comprising a polypeptide that is at least 90% similar to the IL-12 beta subunit p40, and (iii) a transmembrane domain fused to the ends of the first and / or second IL-12 subunit polypeptides. **Claim 2** The iPSC or derivative cell thereof according to claim 1, wherein the polynucleotide encoding the membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for activation-induced release of the IL-12 through protease ADAM17. **Claim 3** An induced pluripotent stem cell (iPSC) or a derivative cell thereof, comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12) comprising: a first polypeptide comprising a polypeptide that is at least 90% similar to the IL-12 alpha subunit p35, a second polypeptide comprising a polypeptide that is at least 90% similar to the IL-12 beta subunit p40, and a transmembrane domain fused to the ends of the first and / or second IL-12 subunit polypeptides; and (iii) deletion or reduced expression of one or more of the B2M, TAP1, TAP2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes. **Claim 4** A membrane-bound interleukin-12 (IL-12) comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide that is at least 90% similar thereto, and a second polypeptide comprising an IL-12 beta subunit p40 or a polypeptide that is at least 90% similar thereto, which is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for inducible release of the IL-12 through protease ADAM17; and (iii) one or more deletions or reduced expressions of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CII TA, RFX5, and RFXAP genes, preferably deletions or reduced expressions of the B2M and CII TA genes, of the iPSC or a derivative cell thereof according to claim 1.

5. The iPSC or a derivative cell thereof according to claim 1 or 2, further comprising a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

6. The iPSC or a derivative cell thereof according to claim 1 or 2, further comprising an exogenous polynucleotide encoding a safety switch.

7. The iPSC or a derivative cell thereof according to claim 7, wherein the safety switch comprises an exogenous polynucleotide encoding an inactivated cell surface receptor comprising an epitope specific for a monoclonal antibody.

8. The iPSC or a derivative cell thereof according to claim 7, wherein the safety switch comprises an exogenous polynucleotide encoding an inactivated cell surface receptor comprising an epitope specific for a monoclonal antibody and interleukin-15 (IL-15), and the inactivated cell surface receptor and IL-15 are operably linked by a self-protease peptide sequence.

9. The iPSC or a derivative cell thereof according to any one of claims 1 to 7, wherein the transmembrane domain (TM) is an EGFR transmembrane domain.

10. The iPSC or a derivative cell thereof according to claim 9, wherein the transmembrane domain (TM) is further fused to a signal transduction domain (SD).

11. The iPSC or a derivative cell thereof according to claim 10, wherein the signal transduction domain is a CD3ζ, CD28, and / or 4-1BB signal transduction domain.

12. (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a first polypeptide comprising a polypeptide that is at least 90% similar to interleukin-12 alpha subunit p35 or IL-12 alpha subunit p35, a second polypeptide comprising a polypeptide that is at least 90% similar to interleukin-12 beta subunit p40 or IL-12 beta subunit p40, and a transmembrane domain fused to the termini of the first and / or second IL-12 subunit polypeptides, a membrane-bound interleukin-12 (IL-12), or a second exogenous polynucleotide encoding a membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide; (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G); (iv) optionally, a fourth exogenous polynucleotide encoding a safety switch; (v) optionally, a fifth exogenous polynucleotide encoding a cytokine and comprising an induced pluripotent stem cell (iPSC), a natural killer (NK) cell, or a T cell, wherein one or more of the exogenous polynucleotides are integrated into the loci of the CIITA and B2M genes, whereby the expression of CIITA and B2M is deleted or reduced, an induced pluripotent stem cell (iPSC), a natural killer (NK) cell, or a T cell.

13. (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) i. membrane-bound interleukin-12 (IL-12) having the amino acid sequence of SEQ ID NO: 96; ii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 98; iii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 108; iv. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 110, v. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 112, vi. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 114, vii. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 116, viii. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 118, ix. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 120, x. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 122, or xi. Membrane-bound IL-12 fused with a polynucleotide encoding an ADAM17 protease cleavage site peptide having the amino acid sequence of SEQ ID NO: 124 and a second exogenous polynucleotide encoding the same, (iii) Optionally, a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66 and / or an exogenous polynucleotide encoding human leukocyte antigen G (HLA-G) having the amino acid sequence of SEQ ID NO: 69, (iv) Optionally, a fourth exogenous polynucleotide encoding the IL-15 protein according to SEQ ID NO: 72 and an iPSC, natural killer (NK) cell, or T cell comprising wherein one or more of the exogenous polynucleotides are integrated into the loci of the CIITA and B2M genes, whereby the expression of CIITA and / or B2M is deleted or reduced, an iPSC, natural killer (NK) cell, or T cell.

14. The safety switch includes an exogenous polynucleotide encoding an inactivated cell surface receptor comprising an epitope specific for a monoclonal antibody and interleukin 15 (IL-15), wherein the inactivated cell surface receptor and IL-15 are operably linked by a self-protease peptide sequence. The iPSC, natural killer (NK) cell, or T cell according to any one of claims 12 to 13.

15. One or more of the exogenous polynucleotides are integrated into one or more loci on the chromosome of the cell selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAP1, TAP2, Tapasin, NLRRC5, RFXANK, CII TA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, provided that at least one of the exogenous polynucleotides is integrated into the locus of a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CII TA, RFX5, and RFXAP genes, thereby resulting in deletion or reduced expression of the gene. The iPSC or derivative cell according to claim 1 or 2.

16. (i) One or more of the exogenous polynucleotides are integrated into the loci of the CII TA, AAVS1, and B2M genes. (ii) One or more of the exogenous polynucleotides are integrated into the loci of the CII TA, CLYBL, and B2M genes. The iPSC or derivative cell according to claim 1 or 2.

17. The CAR includes (i) a signal peptide, (ii) an extracellular domain including a binding domain that specifically binds to the antigen, (iii) a hinge region, (iv) a transmembrane domain, (v) an intracellular signaling domain, (vi) a co-stimulatory domain such as a co-stimulatory domain including a CD28 signaling domain The iPSC or derivative cell according to any one of claims 1 to 15.

18. The iPSC or derivative cell according to claim 17, wherein the signal peptide is a GMCSFR signal peptide.

19. The iPSC or derivative cell according to claim 17, wherein the extracellular domain comprises a VHH domain.

20. The iPSC or derivative cell according to claim 17, wherein the hinge region comprises a CD28 hinge region.

21. The iPSC or derivative cell according to claim 17, wherein the transmembrane domain comprises a CD28 transmembrane domain.

22. The iPSC or derivative cell according to claim 17, wherein the intracellular signaling domain comprises a CD3ζ intracellular domain.

23. The iPSC or derivative cell according to claim 17, wherein the costimulatory domain comprises a CD28 signaling domain.

24. The CAR is (i) the signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, (ii) the hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, (iii) the transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, (iv) the intracellular signaling domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6, and (v) the costimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 and the iPSC or derivative cell according to claim 17.

25. The CAR is (i) the signal peptide comprising the amino acid sequence of SEQ ID NO: 1, (ii) an extracellular domain comprising an scFv or VHH domain; (iii) the hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, (iv) the transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24, (v) the intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6, (vi) the co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 20 An iPSC or a derivative cell according to claim 17, comprising

26. The inactivated cell surface protein is selected from the group of monoclonal antibody-specific epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab. An iPSC or a derivative cell according to claim 14.

27. The inactivated cell surface protein is a cleaved epidermal growth factor (tEGFR) mutant. An iPSC or a derivative cell according to claim 26.

28. The self-protease peptide sequence is porcine teschovirus-1 2A (P2A). An iPSC or a derivative cell according to claim 26.

29. The tEGFR mutant consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

71. An iPSC or a derivative cell according to claim 26.

30. The cytokine comprises an IL-15 protein comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

72. An iPSC or a derivative cell according to any one of claims 1 to 15.

31. The self-protease peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

73. An iPSC or a derivative cell according to claim 28.

32. The iPSC or derivative cell according to claim 5, wherein the HLA-E comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 66, or the HLA-G comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

69.

33. The derivative cell according to any one of claims 1 to 32, which is a natural killer (NK) cell or a T cell.

34. A composition comprising the cell according to any one of claims 1 to 33.

35. The composition according to claim 34, further comprising or used in combination with one or more therapeutic agents selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), siRNAs, oligonucleotides, mononuclear blood cells, vectors containing one or more target polynucleotides, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

36. A method for treating cancer in a subject in need thereof, comprising administering to the subject in need thereof the cell according to any one of claims 1 to 33 or the composition according to any one of claims 34 and 35.

37. The method according to claim 36, wherein the cancer is non-Hodgkin lymphoma (NHL).

38. The method according to claim 36, wherein the cancer is selected from lung cancer, gastric cancer, colon cancer, liver cancer, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, endometrial cancer, prostate cancer, thyroid cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin lymphoma (NHL), Hodgkin lymphoma / disease (HD), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myelogenous leukemia (AML), and other liquid tumors.

39. A method for producing the derivative cell according to claim 33, comprising differentiating the iPSC cell according to claim 1 or 2 under conditions of cell differentiation to thereby obtain the derivative cell.

40. The method according to claim 39, wherein the iPSC is obtained by genome engineering of the iPSC, and the genome engineering includes targeted editing. **Claim 41** The method according to claim 39, wherein the iPSC is edited by targeted editing including deletions, insertions, or in / dels performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods. **Claim 42** CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs), comprising a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and an exogenous polynucleotide encoding membrane-bound interleukin 12 (IL-12) comprising a transmembrane domain fused to the ends of the first and / or second IL-12 subunit polypeptides.

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