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

Abstract

Genetically engineered induced pluripotent stem cells (iPSCs) and derived cells that express chimeric antigen receptor (CAR) and membrane-bound IL-12 are provided, as well as methods of making and using the same. Compositions, polypeptides, vectors, and methods of preparation are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 350,172, filed on June 8, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present application provides immune effector cells derived from induced pluripotent stem cells (iPSC) and their derivatives derived from genetically modified to express membrane-bound IL-12. The present application also provides the use of iPSC or its derivative cells to express chimeric antigen receptors for allogeneic cell therapy. The present application also provides related vectors, polynucleotides and pharmaceutical compositions.

[0004] References to electronically submitted sequence listings

[0005] This application contains a sequence listing, which is electronically submitted via EFS-Web in an ASCII formatted sequence listing, with the file name "Sequence Listing_ST26.xml", creation date June 6, 2023, and a size of 210 kb. The sequence listing submitted via EFS-Web is part of this specification, and is incorporated herein by reference in its entirety.

[0006] Incorporation by reference

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Background Art

[0008] Chimeric antigen receptor (CAR) significantly enhances the anti-tumor activity of immune effector cells. CAR is an engineered receptor, generally comprising an extracellular targeting domain, a transmembrane (TM) domain, and one or more intracellular signaling domains connected to a linker peptide. Traditionally, the extracellular domain is composed of an antigen binding fragment (such as a single-chain Fv, scFv) of an antibody specific for a specific tumor-associated antigen (TAA) or a cell surface target. The extracellular domain gives CAR tumor specificity, and when contacted with TAA / targets (engagement), the intracellular signaling domain activates T cells that have been genetically engineered to express CAR. Engineered immune effector cells are re-injected into cancer patients, where they specifically contact and kill cells expressing CAR's TAA targets (Maus et al., Blood. 2014Apr 24; 123 (17): 2625-35; Curran and Brentjens, JClin Oncol. 2015May 20; 33 (15): 1703-6).

[0009] Autologous, patient-specific CAR-T therapy has emerged as a powerful, potentially curative cancer therapy, particularly for CD19-positive hematological malignancies. However, autologous T cells must be generated on a custom basis, which remains a significant limiting factor for large-scale clinical application due to production costs and the risk of production failure. The development of CAR-T technology and its broader application are also limited by several other key shortcomings, including, for example, a) low efficiency of anti-tumor 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 CAR-T-mediated cytokine release syndrome (CRS) and graft-versus-host disease (GVHD), and e) the time required for manufacturing.

[0010] Cytokines such as interleukin-2 (IL-2), IL-12, and IL-15 have been studied for improving the antitumor activity of adoptive T cell therapy (ACT). Given that IL-12 is a potent mediator of activated immune cells and can greatly enhance the activity of immune cells against tumor cells, IL-12 is particularly attractive for this purpose. IL-12 is a heterodimeric protein composed of p35 (IL-12A) and p40 (IL-12B) subunits, and was initially 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 type 1 helper cells (TH1) that produce interferon-γ (IFN-γ), increase the cytotoxicity of CD8 T cells, upregulate antigen presentation, and reprogram MDSCs into a T cell-supporting phenotype. When NK cells are exposed to IL-12, a cytokine produced by dendritic cells and macrophages, the activity of NK cells to kill sensitive targets such as cancer cells increases 20-100 times. Therefore, IL-12 is often used in NK or T cell therapy.

[0011] However, systemic exposure to IL12 may have negative effects on the whole body, and its therapeutic use is limited by these systemic effects. The clinical application of IL-12 is limited by severe toxicity when administered systemically. In order to safely utilize IL-12 for cancer treatment, some research groups have studied the ability to selectively stimulate anti-tumor immune responses in the tumor microenvironment. This includes genetically engineering tumor-specific T cells to selectively drive the expression of IL-12 when 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 improves the efficacy of T cell therapy in mouse tumor models. Clinical evaluation of tumor-infiltrating lymphocytes (TILs) genetically engineered to produce IL-12 in this way produced objective clinical responses at 10-100 times lower cell doses than those required for previous TIL therapies (including patients who had previously failed standard TIL therapies). 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 the encouraging efficacy, insufficient control of IL-12 expression in patients resulted in severe IFN-γ-related toxicity, and further development of this approach was halted. In order to safely utilize IL-12 for cancer immunotherapy, some researchers have adopted a method of controlling the cytokine dose level and activity spectrum of IL-12 by directly tying the cytokine to the surface of tumor-specific T cells before adoptive transfer, which is achieved by tying IL-12 to antibodies that bind to cell surface receptors. See, for example, Jones et al., Sci. Adv. 8, eabi 8075 (2022). Alternatively, the IL-12 protein can be fused to a transmembrane domain (TM) such as an EGFR transmembrane domain or a signaling domain. WO2018 / 068008 and WO2020 / 160350 disclose T cells engineered to express membrane-bound IL-12 and methods of treating cancer with such cells. However, the T cells disclosed therein are only suitable for autologous therapy.

[0012] Another concept that has been studied for controlling cytokine expression is to attach the cytokine to the outside of the cell membrane via a cleavable linker, thereby allowing release upon protease cleavage to control the release of the cytokine. See, for example, A. Gonzalez et al, Senti Bio Abstract #584 AACR Annual Meeting 2022.

[0013] Therefore, there is an unmet need for therapeutically sufficient and functional alloantigen-specific immune cells with membrane-bound IL-12 for effective use in immunotherapy. Summary of the invention

[0014] In a general aspect, a genetically engineered induced pluripotent stem cell (iPSC) or a derived cell thereof is provided. The cell comprises: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, and the second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, wherein the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of B2M and CIITA genes.

[0015] In certain embodiments, the polynucleotides encoding membrane-bound IL-12 are fused to the polynucleotides encoding ADAM17 protease cleavage site peptides for inducing activation and releasing IL-12 by protease ADAM17. ADAM17 is expressed by activated lymphocytes and directly participates in the release of other immune mediators (such as TTNFa), which are similarly present in a membrane-anchored form. When this membrane-bound IL-12 is expressed on engineered iNK or T cells, it remains in contact with the cell. When cell activation and ADAM17 expression increase, protease cuts the membrane stem (membrane stalk) and releases IL-12 to the extracellular space. This type of regulation ensures that the activity of IL-12 is confined to the space around the tumor, where engineered immune cells contact their targets on tumor cells, thereby triggering its activation.

[0016] In certain embodiments, the iPSC cell or a cell derived therefrom further comprises a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or a human leukocyte antigen G (HLA-G).

[0017] In certain embodiments, one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are one or more loci selected from the following one or more genes: AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, 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 gene, provided that at least one of the exogenous polynucleotides is integrated at a locus selected from the following genes: B2M, TAP 1, 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 gene, provided that at least one of the exogenous polynucleotides is integrated at a locus selected from the following genes: 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, and the integration leads to the deletion or reduced expression of the genes, more preferably, one or more of the exogenous polynucleotides are integrated at the loci of CIITA, AAVS1 and B2M genes, and the integration leads to the deletion or reduced expression of one or more of CIITA and B2M genes. In some embodiments, one or more of the exogenous polynucleotides are integrated at the loci of CIITA, CLYBL and B2M genes.

[0018] In certain embodiments, iPSCs are reprogrammed from whole peripheral blood mononuclear cells (PBMCs).

[0019] In certain embodiments, iPSCs are derived from reprogrammed T-cells.

[0020] 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,

[0021] (v) an intracellular signaling domain; and (vi) a costimulatory domain, such as a costimulatory domain comprising a CD28 signaling domain.

[0022] In certain embodiments, the signal peptide is a GMCSFR signal peptide.

[0023] In certain embodiments, the extracellular domain comprises a VHH domain.

[0024] In certain embodiments, the hinge region comprises a CD28 hinge region.

[0025] In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0026] In certain embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular domain.

[0027] In certain embodiments, the costimulatory domain comprises a CD28 signaling domain.

[0028] In certain embodiments, the CAR comprises:

[0029] (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;

[0030] (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;

[0031] (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;

[0032] (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; and

[0033] (v) a 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.

[0034] 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 costimulatory domain comprising the amino acid sequence of SEQ ID NO: 20.

[0035] In certain embodiments, HLA-E has an amino acid sequence that has 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.

[0036] In certain embodiments, HLA-G has an amino acid sequence that has 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.

[0037] In certain embodiments, (i) a second exogenous polynucleotide comprises a polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide, and a transmembrane domain, wherein the first polypeptide comprises IL-12 alpha subunit p35 or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 102, and the second polypeptide comprises IL-12 beta subunit p40 or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 103, wherein the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide, and the transmembrane domain is identical to SEQ ID NO: 104. NO: 100 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, the second exogenous polynucleotide encoding membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide, and the ADAM17 protease cleavage site peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 101. Preferably, the second exogenous polynucleotide is integrated at a locus selected from the following genes: AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, 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 gene, preferably at the locus of AAVS1 or CLYBL gene.

[0038] In certain embodiments, IL-12 is fused to a transmembrane domain, such as an 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 a CD3 ζ, CD28, and / or 4-1 BB signaling domain. In particular aspects, the signaling domain comprises a CD3 ζ and a 4-1 BB signaling domain. In some aspects, the signaling domain is 4-1 BB.

[0039] In certain embodiments, the first exogenous polynucleotide is integrated at the locus of the AAVS1 gene; (i) the second exogenous polypeptide is integrated at the locus of the AAVS1 gene; and (ii) the third exogenous polypeptide is integrated at the locus of the B2M gene; wherein the integration of the exogenous polynucleotides causes the loss or reduced expression of CIITA and B2M, 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; (i) the second exogenous polypeptide is integrated at the locus of the AAVS1 gene; and (ii) the third exogenous polypeptide is integrated at the locus of the B2M gene; wherein the integration of the exogenous polynucleotides causes the loss or reduced expression of CIITA and B2M, 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; (i) the second exogenous polypeptide is integrated at the locus of the AAVS1 gene; and (ii) the third exogenous polypeptide is integrated at the locus of the CIITA gene; wherein the integration of the exogenous polynucleotides causes the absence or reduced expression of CIITA and B2M, 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 at the locus of the CIITA gene; (i) the second exogenous polypeptide is integrated at the locus of the CLYBL gene; and (ii) the third exogenous polypeptide is integrated at the locus of the B2M gene; wherein the integration of the exogenous polynucleotides causes the loss or reduced expression of CIITA and B2M, 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 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; and (ii) the third exogenous polypeptide is integrated at the locus of the CIITA gene; wherein the integration of the exogenous polynucleotides causes the absence or reduced expression of CIITA and B2M, 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.

[0044] In certain embodiments, the derivative cell is a Natural Killer (NK) cell or a T cell.

[0045] Optionally, the genetically engineered iPSC or a cell derived therefrom further comprises 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 a locus of a gene selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, 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 at a locus of the AAVS1 or CLYBL gene.

[0046] 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.

[0047] 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.

[0048] 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 polynucleotides causes the CIITA and B2M genes to be deleted or have reduced 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.

[0049] 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 polynucleotides causes the CIITA and B2M genes to be deleted or their expression to be reduced, 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.

[0050] 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 polynucleotides causes the CIITA and B2M genes to be deleted or have reduced 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.

[0051] 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 CLYBL gene; and the third exogenous polynucleotide is integrated at the locus of the CIITA gene; wherein the integration of the exogenous polynucleotides causes the CIITA and B2M genes to be deleted or the expression of the genes is reduced, 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.

[0052] In certain embodiments, the cell also optionally includes an exogenous polynucleotide encoding a safety switch. Since cell therapy (such as CAR-T therapy) has a long or uncertain half-life, toxicity may be progressive. Cells have been engineered to include a safety switch to eliminate injected (infused) cells when adverse events occur. Therefore, CAR cells have been engineered to include genes ("suicide genes") of artificial cell death polypeptides, which are a kind of genetically encoded molecules that allow selective destruction of CAR cells, thereby allowing selective elimination of genetically modified cells to prevent collateral damage to neighboring cells and / or tissues. Artificial cell death polypeptides can mediate the induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcription 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, antiviral drugs or radioisotope conjugated drugs), and when activated, they trigger apoptosis and / or cell death of therapeutic cells. In one example, artificial cell death polypeptides include viral enzymes recognized by antiviral drugs. 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 comprises an inactivated cell surface receptor comprising a monoclonal antibody specific epitope, preferably a truncated epidermal growth factor (tEGFR) variant.In certain embodiments, the inactivated cell surface protein is selected from a monoclonal antibody specific epitope selected from ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol), daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab epitopes specifically recognized by the anti-cancer drugs vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab.

[0053] In certain embodiments, the inactivated cell surface protein is a truncated epidermal growth factor (tEGFR) variant. In certain embodiments, the tEGFR variant has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71 or is composed of an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Preferably, the tEGFR variant has the amino acid sequence of SEQ ID NO: 71 or is composed of the amino acid sequence of SEQ ID NO: 71.

[0054] In certain embodiments, the inactivated cell surface receptor comprises a monoclonal antibody specific epitope, which is operably connected to a cytokine (such as IL-15), preferably connected by an autoprotease peptide sequence. Examples of autoprotease peptides include, but are not limited to, peptide sequences selected from type 1 porcine teschovirus 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), type A equine rhinitis virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), Flacherie virus 2A (BmIFV2A) and combinations thereof. In one embodiment, the autoprotease peptide is an autoprotease peptide of type 1 porcine teschovirus 2A (P2A). In certain embodiments, the autoprotease 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:71, preferably the amino acid sequence of SEQ ID NO:73.

[0055] In certain embodiments, the cells may also optionally contain a fifth exogenous polynucleotide encoding a cytokine, such as IL-15 or a membrane-bound IL-15 fusion protein.

[0056] As used herein, "interleukin-15" or "IL-15" refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. A "functional portion" ("biologically active portion") of a cytokine refers to a cytokine portion that retains one or more functions of a full-length or mature cytokine. Such functions of IL-15 include promoting NK cell survival, regulating NK cell and T cell activation and proliferation, and supporting the development of NK cells from hematopoietic stem cells. Those skilled in the art will appreciate that 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%, 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.

[0057] In certain embodiments, the inactivated cell surface receptor comprises a truncated epidermal growth factor (tEGFR) variant, which is operably linked to interleukin-15 (IL-15) via an autoprotease peptide sequence. In a specific embodiment, 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 SEQ ID NO: 74 amino acid sequence. In certain embodiments, the tEGFR variant consists of the amino acid sequence of SEQ ID NO: 71, the autoprotease peptide has the amino acid sequence of SEQ ID NO: 73, and IL-15 comprises the amino acid sequence of SEQ ID NO: 72.

[0058] In certain embodiments, the iPSC or derivative has a deletion or reduced expression of one or more of the B2M and / or CIITA genes.

[0059] In certain embodiments, the derivative cell is a Natural Killer (NK) cell or a T cell.

[0060] Also provided is an induced pluripotent stem cell (iPSC), natural killer (NK) cell or T cell, comprising:

[0061] (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR);

[0062] (ii) a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain,

[0063] The first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto,

[0064] The second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto,

[0065] The transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide;

[0066] Alternatively, the protein encodes membrane-bound interleukin 12 (IL-12) fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide.

[0067] (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G);

[0068] (iv) an optional fourth exogenous polynucleotide encoding a safety switch; and

[0069] (v) an optional fifth exogenous polynucleotide encoding a cytokine;

[0070] (vi) among which

[0071] (a) the first, second and third exogenous polynucleotides are integrated into the loci of AAVS1, CIITA and B2M genes, thereby deleting or reducing the expression of CIITA and B2M;

[0072] (b) the first, second and third exogenous polynucleotides are integrated into the loci of CLYBL, CIITA and B2M genes, thereby deleting or reducing the expression of CIITA and B2M;

[0073] (c) the first, second and third exogenous polynucleotides are integrated into the loci of CIITA, AAVS1 and B2M genes, thereby causing the deletion or reduced expression of CIITA and B2M;

[0074] (d) the first, second and third exogenous polynucleotides are integrated into the loci of CIITA, CLYBL and B2M genes, thereby deleting or reducing the expression of CIITA and B2M;

[0075] (e) the first, second and third exogenous polynucleotides are integrated into the loci of B2M, AAVS1 and CIITA genes, thereby causing the deletion or reduced expression of CIITA and B2M; or

[0076] (f) The first, second and third exogenous polynucleotides are integrated into the loci of B2M, CLYBL and CIITA genes, thereby deleting or reducing the expression of CIITA and B2M.

[0077] In certain aspects, the present disclosure provides an iPSC, a natural killer (NK) cell, or a T cell, comprising: a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the iPSC, a natural killer (NK) cell, or a T cell comprises a second polynucleotide encoding: i. a membrane-bound interleukin 12 (IL-12) having an amino acid sequence of SEQ ID NO: 96;

[0078] ii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 98;

[0079] iii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 108;

[0080] iv. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 110;

[0081] v. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 112;

[0082] vi. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 114;

[0083] vii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 116;

[0084] viii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 118;

[0085] ix. Membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 120;

[0086] x. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 122; or

[0087] xi. Membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 124. In certain embodiments, iPSCs, natural killer (NK) cells, or T cells optionally include a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having an amino acid sequence of SEQ ID NO: 66, and / or an exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G) having an amino acid sequence of SEQ ID NO: 69. In certain embodiments, iPSCs, natural killer (NK) cells, or T cells optionally include a fourth exogenous polynucleotide encoding an IL-15 protein of SEQ ID NO: 72. In certain embodiments, one or more of the exogenous polynucleotides contained in iPSCs, natural killer (NK) cells, or T cells are integrated into the loci of the CIITA and B2M genes, thereby deleting or reducing expression of CIITA and / or B2M.

[0088] Also provided is an iPSC, a natural killer (NK) cell or a T cell, comprising:

[0089] (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR);

[0090] (ii) a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain,

[0091] The first polypeptide comprises IL-12 alpha subunit p35, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 102, the second polypeptide comprises IL-12 beta subunit p40, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 103, and the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide, and the transmembrane domain has the amino acid sequence of SEQ ID NO: 100;

[0092] (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having an amino acid sequence of SEQ ID NO: 66;

[0093] (iv) a fourth exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autologous protease 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;

[0094] Wherein, the first, second and third exogenous polynucleotides are integrated at the loci of the following genes:

[0095] (a) AAVS1, CIITA and B2M genes, (b) AAVS1, CIITA and B2M genes, (c) CLYBL, CIITA and B2M genes, (d) CIITA, AAVSI and B2M genes, (e) CIITA, CLYBL and B2M genes, (f) B2M, AAVS1 and CIITA genes, or (g) B2M, CLYBL and CIITA genes, thereby eliminating or reducing the expression of CIITA and B2M.

[0096] In certain embodiments, (i) the second exogenous polynucleotide comprises a polynucleotide sequence of SEQ ID NO: 97 or 99; and (ii) the third exogenous polynucleotide comprises a polynucleotide sequence of SEQ ID NO: 67 or 70; and the first, second and third exogenous polynucleotides are integrated at the loci of the following genes: (a) AAVS1, CIITA and B2M genes, respectively, (b) AAVS1, CIITA and B2M genes, respectively, (c) CLYBL, CIITA and B2M genes, respectively, (d) CIITA, AAVSI and B2M genes, respectively, (e) CIITA, CLYBL and B2M genes, respectively, (f) B2M, AAVS1 and CIITA genes, respectively, or (g) B2M, CLYBL and CIITA genes, respectively.

[0097] A composition is also provided, which comprises the cell of the present application.

[0098] In certain embodiments, the composition of the present application may further include one or more other therapeutic agents, or 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, dsRNAs (double-stranded RNAs), siRNAs, oligonucleotides, mononuclear blood cells, carriers comprising one or more polynucleic acids of interest, antibodies, chemotherapeutics or radioactive moieties or immunomodulatory drugs (IMiD).

[0099] Also provided is a method for treating cancer in a subject in need thereof, the method comprising administering the cell or composition of the present application to the subject in need thereof.

[0100] In certain embodiments, the cancer is non-Hodgkin lymphoma (NHL).

[0101] A method for preparing the derivative cells of the present application is also provided, the method comprising differentiating the iPSCs of the present application under cell differentiation conditions, thereby obtaining the derivative cells.

[0102] A method for obtaining a genetically engineered iPSC of the present application is further provided, the method comprising introducing a first exogenous polynucleotide, a second exogenous polynucleotide and an optionally present third exogenous polynucleotide into an iPSC cell, thereby obtaining a genetically engineered iPSC. Any genetic engineering method can be used to obtain the genetically engineered iPSC of the present application. Preferably, genetic engineering includes targeted editing, more preferably, targeted editing includes deletion, insertion or insertion / deletion (in / del), and wherein targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination or any other functional changes of these methods.

[0103] Also provided is a method of differentiating induced pluripotent stem cell (iPSC) cells into NK cells, the method being to subject the cells to a differentiation protocol comprising adding recombinant human IL-12 in the last 24 hours of culture. Preferably, the recombinant IL-12 is IL12p70.

[0104] Also provided is a CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC), 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, and the second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, and the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes.

[0105] Other embodiments of the present application include a genetically engineered iPSC or a cell derived therefrom for use in treating cancer in a subject in need thereof.

[0106] In some embodiments, the engineered iPSC-derived cells of the present invention have improved anti-tumor immunity, improved persistence, improved resistance to immune cells or improved immune resistance; or, the genome-engineered iPSC may have improved resistance to T cells and / or NK cells. In particular, once transfected into iPSC and differentiated into NK cells according to the present invention, the IL-12 transgene of the present invention exhibits improved anti-tumor immunity, improved persistence, reduced exhaustion and improved continuous killing compared to NK cells derived from iPSC cells without the IL-12 transgene of the present invention. The genome-engineered iPSC of the present invention has the potential to differentiate into non-pluripotent cells containing hematopoietic lineage cells with the same functional targeted genome editing. In some embodiments, the genome-engineered iPSC of the present invention has the potential to differentiate into mesoderm cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem / progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells or B cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings.

[0108] Figure 1A-1B Shown are plasmid vector maps containing the mbIL12 transgene and p70 (A) without the ADAM17 cleavage site (p1513), and (B) with the ADAM17 cleavage site (p1514) for insertion into the AAVS1 locus.

[0109] Figures 2A-2C Flow cytometry results of membrane-bound IL12 expression in iPSCs are shown. iPSCs were transduced with (A) p1513 plasmids, (B) p1514 plasmids, or (C) were not transduced. After 4 days of culture, cells were incubated with 500ug / ml of Geneticin to screen out cells that did not successfully integrate the transgene into the AAVS1 locus. Surviving cells were amplified (indicating that the transgene was correctly inserted) and the expression of membrane IL12 was analyzed by flow cytometry.

[0110] Figure 3 Shown are the results of IL12 expression in cells transduced with p1513 (left) or p1514 (right) after 9 days in culture (HPC stage).

[0111] Figures 4A-4BShown are (A) the genomic map on which the PCR primers designed for amplifying the transmembrane IL12 sequence in iPS cell genomic DNA were based, and (B) the gel electrophoresis results of amplifying iPSC genomic DNA using the 1514 forward primer and one of the 1514R or 1514R2 primers, confirming the presence of the transgene in the iPS cells.

[0112] Figure 5A-5B Shown are (A) a genomic map of the primer sites used to perform ligation PCR, where one primer is specific for the transgene sequence and the other primer is specific for the genomic sequence outside the homology arm, and (B) gel electrophoresis results of the PCR products of the ligation PCR reaction to confirm the insertion of the transgene at the correct locus.

[0113] Figure 6A-6B Shown are the results of IL12 expression in cells transduced with p1513 (left) or p1514 (right) after (A) 14 days and (B) 21 days in culture (iNK stage).

[0114] Figure 7 Flow cytometry results showing the effect of TAPI-1 on IL12 surface expression in control cells (left) or cells incubated with 50 uM of the ADAM17 inhibitor TAPI-1 (right).

[0115] Figures 8A-8B The cytotoxicity of iNK cells expressing CAG-CAR-IL15 in the presence or absence of human recombinant IL12 is shown. Fig. 8A Shown is a graph demonstrating the time-dependent death of Raji cells when cultured with CAG-CAR-IL15 iNK cells with or without IL12. Figure 8B Shown are graphs demonstrating tumor growth measured as mean whole body luminescence mean radiance of mice infused with IL12-primed and unprimed CAG-CAR-IL15 iNK cells. DETAILED DESCRIPTION

[0116] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, regulations, materials, devices, articles and the like that have been included in this specification is only for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these materials form part of the prior art with respect to any invention disclosed or claimed.

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Otherwise, certain terms used herein have the meaning as shown in the specification.

[0118] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0119] Unless otherwise indicated, any numerical value, such as the concentration or concentration range described herein, should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include ± 10% of the value. 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). As used herein, unless the context clearly indicates otherwise, the use of numerical ranges clearly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within such ranges.

[0120] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of the application described herein using no more than routine experimentation. This application is intended to cover such equivalents.

[0121] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, will be understood to mean including the stated integer or group of integers but not excluding any other integer or group of integers, and are intended to be non-exclusive or open. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. In addition, unless expressly stated to the contrary, "or" refers to an inclusive or and not an exclusive or. For example, a condition A or B is satisfied by any of: 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).

[0122] As used herein, the connection term "and / or" between multiple reference elements is understood to cover both individual and combination 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 are understood to fall within the meaning, thus satisfying the requirements of the term "and / or" as used herein. The concurrent applicability of more than one option is also understood to fall within the meaning, thus satisfying the requirements of the term "and / or".

[0123] As used herein, when used throughout the specification and claims, the term "consists of" or variations such as "consist of" or "consisting of" means that any listed integer or group of integers is included, but no additional integers or groups of integers can be added to the specified method, structure or composition.

[0124] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" when used throughout the specification and claims means including any recited integer or group of integers, and optionally including any recited integer or group of integers that does not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0125] As used herein, "subject" means any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses 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.

[0126] It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to dimensions or features of components of the preferred invention indicate that the described dimensions / features are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations that will 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.).

[0127] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., CAR polypeptides and CAR polynucleotides encoding them) refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0128] For sequence comparison, usually a sequence is used as a reference sequence, and the test sequence is compared with it. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0129] Optimal alignment of sequences for comparison can be performed, 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 search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0130] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are 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 analysis 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 match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits are used as seeds for starting searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0131] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction stops when: the cumulative alignment score decreases by the amount X from its maximum achieved; the cumulative score becomes 0 or less due to the accumulation of one or more negative scoring 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) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0132] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, 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 smallest total probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest 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.

[0133] As described below, another 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. Thus, for example, when the two peptides differ only in conservative substitutions, the polypeptide is generally substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0134] As used herein, the term "isolated" refers to a biological component (such as a nucleic acid, peptide, protein or cell) that has been substantially separated, produced or purified from other biological components of the organism in which the component naturally occurs, i.e., from 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 purification methods described herein. "Isolated" nucleic acids, peptides, proteins and cells can be part of a composition, and if the composition is not part of the native environment of the nucleic acid, peptide, protein or cell, it is still isolated. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0135] As used herein, the term "polynucleotide" is 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 without limitation 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 containing DNA and RNA, which can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "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 with a backbone modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and uncommon bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells."Polynucleotide" also includes relatively short nucleic acid chains, commonly referred to as oligonucleotides.

[0136] "Construct" refers to a macromolecule or molecular complex comprising a polynucleotide to be delivered in vitro or in vivo to a host cell. 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, where it can replicate and / or express. The term "vector" as used herein includes a construct to be delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. 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, etc.

[0137] "Integration" refers to the stable insertion of one or more nucleotides of the construct into the cell genome, that is, covalently linked to the nucleic acid sequence in the cell chromosome DNA. "Targeted integration" refers to the insertion of the nucleotides of the construct into the chromosome or mitochondrial DNA of the cell at a pre-selected site or "integration site". As used herein, the term "integration" further refers to the process of inserting one or more exogenous sequences or nucleotides of the construct in the case where the integration site has or does not have a missing endogenous sequence or nucleotide. In the case where the insertion site has a missing, "integration" may further include replacing the missing endogenous sequence or nucleotide with one or more inserted nucleotides.

[0138] As used herein, the term "exogenous" means that the molecule or activity referred to is introduced into the host cell, or is not native to the host cell. For example, the molecule can be introduced by introducing the encoding nucleic acid into the host genetic material, such as by integrating into the host chromosome or as a non-chromosomal genetic material such as a plasmid. Therefore, when used with respect to the expression of encoding nucleic acids, the term refers to the introduction of an expressible form of the encoding nucleic acid into the cell. The term "endogenous" refers to the molecule or activity referred to that exists in its native form in the host cell. Similarly, when used with respect to the expression of encoding nucleic acids, the term refers to the expression of encoding nucleic acids naturally contained in the cell that are not introduced exogenously.

[0139] As used herein, a "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that is transcribed into RNA under the control of appropriate regulatory sequences and translated into a polypeptide in vivo in certain circumstances. The gene or polynucleotide of interest may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode a miRNA, shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a natural polypeptide mutant having less than 100% sequence identity with a natural polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

[0140] "Operably linked" refers to the association of a nucleic acid sequence with a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA when the promoter is able to 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 the sense or antisense direction.

[0141] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses the translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed CAR can be in the cytoplasm of a host cell, enter an extracellular environment such as a growth medium for a cell culture, or be anchored to a cell membrane.

[0142] As used herein, the term "peptide", "polypeptide" or "protein" may refer to a molecule composed of amino acids and may be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art.

[0143] The peptide sequences described herein are written according to the usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, unless otherwise expressly indicated, the L-form of the amino acid is represented.

[0144] As used herein, the term "engineered immune cell" refers to an immune cell that has been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell, also known as an immune effector cell.

[0145] I. Induced Pluripotent Stem Cells (IPSCs) and Immune Effector Cells

[0146] iPSCs have unlimited self-renewal capacity. Using iPSCs, cells can be engineered to generate a controlled pool of modified cells that can be expanded and differentiated into desired immune effector cells, providing large quantities of homogeneous allogeneic therapeutic products.

[0147] Genetically engineered iPSC and its derived cells are provided herein. The selected genome modification provided herein enhances the therapeutic properties of derived cells. After the combination of selective modalities is introduced into cells at the iPSC level by genome engineering, the derived cells are functionally improved and suitable for allogeneic off-the-shelf cell therapy. This method can help reduce CRS / GVHD-mediated side effects and prevent long-term autoimmunity while providing good efficacy.

[0148] As used herein, the term "differentiation" is the process by which unspecialized ("uncommitted") or less specialized cells acquire specialized cell characteristics. Specialized cells include, for example, blood cells or muscle cells. Differentiation or differentiation-induced cells are cells with a more specialized ("committed") position within a cell lineage. When applied to a differentiation process, the term "committed" refers to a cell that has proceeded to a certain point in a differentiation pathway, and under normal circumstances, it will continue to differentiate into a specific cell type or a subset of a cell type, and under normal circumstances, may not differentiate into different cell types or revert to less differentiated cell types. As used herein, the term "pluripotent" refers to the ability of a cell to correctly form all lineages of a body or somatic cell or embryo. For example, an embryonic stem cell is a pluripotent stem cell that can form cells from each of the three germ layers of the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incomplete or partially pluripotent cells that cannot give rise to a complete organism (e.g., epiblast stem cells or EpiSCs) to more primitive, more potent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0149] As used herein, the term "reprogramming" or "dedifferentiation" refers to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can be differentiated into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state compared to the same cell in a non-reprogrammed state.

[0150] As used herein, the term "induced pluripotent stem cells" or iPSCs means that stem cells are generated from differentiated adult, neonatal or fetal cells that have been induced or altered or reprogrammed to be able to differentiate into tissues of all three germ layers of mesoderm, endoderm and ectoderm or the dermis. The iPSCs generated do not refer to cells found in nature.

[0151] The term "hematopoietic stem and progenitor cell", "hematopoietic stem cell", "hematopoietic progenitor cell" or "hematopoietic precursor cell" or "HPC" refers to a cell that is differentiated into the hematopoietic lineage but is capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem / cells (HSCs) are multipotent stem cells that can give rise to all blood cell types, including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). As used herein, "CD34+ hematopoietic progenitor cells" refers to HPCs that express CD34 on their surface.

[0152] As used herein, the term "immune cell" or "immune effector cell" refers to a cell that participates in an immune response. An immune response includes, for example, promoting an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.

[0153] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a leukocyte that completes maturation in the thymus and has various effects in the immune system. The effects that T cells can have include, for example, identifying specific foreign antigens in the body and activating and inactivating other immune cells. T cells can be any T cells, such as cultured T cells, for example, primary T cells, or T cells from cultured T cell lines, for example, Jurkat, SupT1, etc., or obtained from mammalian T cells. T cells can be CD3+ cells. T cells can be any type of T cells, and can be T cells at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, immature (naive) T cells, regulatory T cells, gamma delta T cells (gd T cells), etc. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express T cell receptors (TCR) or chimeric antigen receptors (CAR). T cells can also differentiate from stem cells or progenitor cells.

[0154] "CD4+T cells" refer to a subset of T cells that express CD4 on their surface and are associated with a cell-mediated immune response. They are characterized by a secretory profile after stimulation, which may include the secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein that was originally defined as a differentiation antigen on T-lymphocytes, but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is considered to be an associative recognition element in the MHC (major histocompatibility complex) class II restricted immune response. On T-lymphocytes, they define an auxiliary / inducing subset.

[0155] "CD8+ T cells" refers to a subset of T cells that express CD8, MHC class I restricted, on their surface and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T-lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the cognate recognition element in the major histocompatibility complex class I restricted interaction.

[0156] 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 T cell receptors (TCR chains). NK cells may also refer to genetically engineered NK cells, such as NK cells modified to express chimeric antigen receptors (CARs). NK cells may also differentiate from stem cells or progenitor cells.

[0157] As used herein, the term "genetic imprint" refers to the genetic or epigenetic information that contributes to preferential therapeutic attributes in source cells or iPSCs, and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, "source cells" are non-pluripotent cells that can be used to produce iPSCs by reprogramming, and source cell-derived iPSCs can be further differentiated into specific cell types, including any hematopoietic lineage cells. Source cell-derived iPSCs and cells differentiated therefrom are sometimes collectively referred to as "derived" or "derived" cells according to context. For example, when used throughout the application, compared to their primary counterparts obtained from natural / natural sources such as peripheral blood, cord blood or other donor tissues, derived effector cells, or derived NK or "iNK" cells or derived T or "iT" cells are cells differentiated from iPSCs. As used herein, genetic imprints conferring preferential therapeutic properties are incorporated into iPSCs by reprogramming selected source cells specific for donor, disease or therapeutic response, or by introducing patterns of genetic modifications into iPSCs using genome editing.

[0158] Any known method for introducing reprogramming factors into non-pluripotent cells can be used to generate induced pluripotent stem cell (iPSC) parental cell lines from peripheral blood mononuclear cells (PBMC) or T-cells, such as previously described in U.S. Patent Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470 based on episomal plasmid processes, the complete disclosures of which are incorporated herein by reference. Reprogramming factors can be in the form of polynucleotides, and are therefore introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes (episomes) and microcircles. In a specific embodiment, 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, iPSC is a cloned iPSC or obtained from an iPSC pool, and genome editing is introduced by performing one or more targeted integrations and / or insertions / deletions (in / del) at one or more selected sites. In another embodiment, iPSCs are obtained from human T cells with antigen specificity and recombinant TCR genes (hereinafter also referred to as "T-iPS" cells), as described in US Pat. Nos. 9,206,394 and 1,0787,642, which are incorporated herein by reference.

[0159] According to a particular aspect, the present application relates to an induced pluripotent stem cell (iPSC) cell or a cell derived therefrom, 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, and the second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, and the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes.

[0160] II. Chimeric Antigen Receptor (CAR) Expression

[0161] According to an embodiment of the present application, iPSC cells or cells derived therefrom include a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR targeting a tumor antigen. In one embodiment, CAR targets the CD19 antigen.

[0162] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane domain, and an intracellular signaling domain that specifically bind to an antigen or target. The extracellular domain of CAR contacts the target antigen on the surface of the target cell, resulting in the aggregation of CAR and delivering activation stimulation to cells containing CAR. CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis, and / or the production of molecules that can mediate cell death of target antigen-expressing cells in a manner that is independent of major histocompatibility (MHC).

[0163] As used herein, the term "signal peptide" refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein that co-translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and directs subsequent surface expression.

[0164] As used herein, the term "extracellular antigen binding domain," "extracellular domain," or "extracellular ligand binding domain" refers to the portion of the CAR that is located outside the cell membrane and is capable of binding to an antigen, target, or ligand.

[0165] As used herein, the term "hinge region" or "hinge domain" refers to the portion of the CAR that connects the two adjacent domains of the CAR protein, namely the extracellular domain and the transmembrane domain of the CAR protein.

[0166] 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.

[0167] As used herein, the term "intracellular signaling domain," "cytoplasmic signaling domain," or "intracellular signaling domain" refers to the portion of a CAR that is located within the cell membrane and is capable of transducing an effector signal.

[0168] 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 for at least some aspects of the immune cell signaling pathway, regulating the primary activation of the receptor in a stimulatory manner. Stimulatory molecules include two different types of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and those that provide secondary costimulatory signals in an antigen-independent manner (referred to as "costimulatory signaling domains").

[0169] In certain embodiments, the extracellular domain comprises an antigen binding domain and / or an antigen binding fragment. For example, an antigen binding fragment can be an antibody or its antigen binding fragment that specifically binds to a tumor antigen. The antigen binding fragment of the present application has one or more desired functional properties, including but not limited to high affinity binding to tumor antigens, high specificity to tumor antigens, stimulating complement dependent cytotoxicity (CDC) for cells expressing tumor antigens, antibody dependent phagocytosis (ADPC) and / or antibody dependent cell-mediated cytotoxicity (ADCC) ability, and when administered alone or in combination with other anticancer therapies, the ability to inhibit tumor growth in subjects and animal models in need.

[0170] As used herein, the term "antibody" is used in a broad sense, including immunoglobulins or antibody molecules, including monoclonal or polyclonal human, humanized, composite and chimeric antibodies and antibody fragments. In general, antibodies are proteins or peptide chains that exhibit binding specificity to specific antigens. Antibody structure is well known. According to the heavy chain constant domain amino acid sequence, immunoglobulins can be divided into five major categories (i.e., IgA, IgD, IgE, IgG and IgM). IgA and IgG are further divided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 subclasses (sub-classified). Therefore, the antibody of the present application can be any one of the five major categories or corresponding subclasses. Preferably, the antibody of the present application is IgG1, IgG2, IgG3 or IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of vertebrate species can be divided into one of two significantly different types, i.e., κ and λ. Therefore, the antibody of the present application can contain κ or λ light chain constant domains. According to a specific embodiment, the antibody of the present application includes a heavy chain and / or light chain constant region from a rat or human antibody. In addition to the heavy and light constant domains, the antibody contains an antigen binding region consisting of a light chain variable region and a heavy chain variable region, each variable region containing three domains (i.e., complementary determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domain is alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domain is alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0171] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to a particular tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, the isolated antibody is substantially free of other cellular material and / or chemicals.

[0172] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody comprising the population is identical, except for possible naturally occurring mutations that may be present in small amounts. The monoclonal antibody of the present application can be prepared by hybridoma methods, phage display technology, single lymphocyte gene cloning technology or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas, which include B cells obtained from transgenic non-human animals such as transgenic mice or rats, which have a genome comprising a human heavy chain transgene and a light chain transgene.

[0173] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a single-domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single-domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain of a camel antibody (V H H), or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. Antigen-binding fragments are capable of binding to the same antigen to which the parent antibody or parent antibody fragment binds.

[0174] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide (eg, a linker peptide) of about 15 to about 20 amino acids.

[0175] As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region, or comprises only a heavy chain variable region.

[0176] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. This definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0177] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology to a human antibody, thereby retaining the antigen-binding properties of the antibody but reducing its antigenicity in humans.

[0178] 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 the light and heavy chains usually correspond to the variable regions of antibodies derived from one mammal (e.g., mouse, rat, rabbit, etc.), with desired specificity, affinity and capacity, while the constant region corresponds to the sequence of antibodies derived from another mammal (e.g., human) to avoid causing an immune response in that species.

[0179] As used herein, the term "multi-specific antibody" refers to an antibody comprising a variety of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the variety of immunoglobulin variable domain sequences has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence in the variety of immunoglobulin variable domain sequences has binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope do not overlap or substantially do not overlap. In one embodiment, the first epitope and the second epitope are on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In one embodiment, the multi-specific antibody comprises the third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multi-specific antibody is a bispecific antibody molecule, a tri-specific antibody molecule or a tetra-specific antibody molecule.

[0180] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. The bispecific antibody is characterized in that the first immunoglobulin variable domain sequence has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence has binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope are on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a half antibody or a fragment thereof having binding specificity to the first epitope, and a half antibody or a fragment thereof having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a scFv or fragment thereof having binding specificity for a first epitope and a scFv or fragment thereof having binding specificity for a second epitope. H H, and V having binding specificity for a second epitope H H.

[0181] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to a tumor antigen" refers to a -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller, or 1×10 -10 The term "KD" refers to the dissociation constant, which is obtained by the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). In view of the present disclosure, the KD value of an antibody can be determined using methods in the art. For example, it can be determined by using surface plasmon resonance, such as by using a biosensor system, for example, The KD of the antigen-binding domain or antigen-binding fragment is determined by using a biofilm interferometry system, such as the Octet RED96 system.

[0182] The smaller the KD value of an antigen-binding domain or antigen-binding fragment, the higher the affinity with which the antigen-binding domain or antigen-binding fragment binds to the target antigen.

[0183] In various embodiments, antibodies or antibody fragments suitable for use in the CAR 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-linked Fv (sdFv), masked antibodies (e.g., ), Small Modular ImmunoPharmaceutical ("SMIPsTM"), intracellular antibodies, mini antibodies, single domain antibody variable domains, nano antibodies, VHH, diabodies, tandem diabodies Anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of antigen-specific TCRs) and epitope binding fragments of any of the above. Antibodies and / or antibody fragments can be derived from mouse antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains. In some embodiments, the antigen binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv fragment, a Fv fragment, a dsFv double antibody, a VHH, a VNAR, a single domain antibody (sdAb) or a nanobody, a dAb fragment, a Fd' fragment, a Fd fragment, a heavy chain variable region, an isolated complementary determining region (CDR), a double antibody, a three-antibody or a ten-antibody. In some embodiments, the antigen binding fragment is a scFv fragment. In some embodiments, the antigen binding fragment is a VHH.

[0184] In some embodiments, at least one of the extracellular tag binding domain, antigen binding domain, or tag comprises a single domain antibody or nanobody. In some embodiments, at least one of the extracellular tag binding domain, antigen binding domain, or tag comprises a VHH.

[0185] In some embodiments, the extracellular tag binding domain and the tag each comprise a VHH.

[0186] In some embodiments, the extracellular tag binding domain, the tag, and the antigen binding domain each comprise a VHH.

[0187] In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises a scFv.

[0188] In some embodiments, the extracellular tag binding domain and the tag each comprise a scFv.

[0189] In some embodiments, the extracellular tag binding domain, the tag, and the antigen binding domain each comprise a scFv.

[0190] Alternative scaffolds of immunoglobulin domains that exhibit similar functional characteristics, such as high affinity and specific binding to target biomolecules, can also be used in the CARs of the present disclosure. Such scaffolds have been shown to produce molecules with improved characteristics, such as greater stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that can be used in the CARs of the present disclosure include engineered, tenascin-derived tenascin type III domains (e.g., Centyrin TM ); engineered, γ-B crystallin-derived scaffolds or engineered, ubiquitin-derived scaffolds (e.g., Affilins); engineered, fibronectin-derived tenth fibronectin type III (10Fn3) domains (e.g., monoclonal antibodies, AdNectins TM or AdNexins TM ); engineered peptides containing ankyrin repeat motifs (e.g., DARPins TM ); engineered, low-density lipoprotein receptor-derived A domain (LDLR-A) (e.g., Avimers TM ); Lipocalins (e.g., anticalins); Engineered, protease inhibitor-derived Kunitz domains (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); Engineered, protein A-derived Z domains (Affibodies TM ); Sac7d-derived polypeptides (e.g., or affitins); engineered, Fyn-derived SH2 domains (e.g., ); CTLD3 (eg, Tetranectin); Thioredoxin (eg, peptide aptamer); β-sandwiches (e.g., iMab); small proteins; C-type lectin-like domain scaffolds; engineered antibody mimics; and any genetically manipulated counterparts of the above that retain their binding functionality ( A,Pluckthun A,J Mol Biol 305:989-1010(2001); 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), each of which is incorporated herein by reference in its entirety).

[0191] In some embodiments, the replacement scaffold is Affilin or Centyrin.

[0192] In some embodiments, the first polypeptide of CAR of the present disclosure includes a leader sequence. The leader sequence may be located at the N-terminus of the extracellular tag binding domain. During cell processing and CAR localization to the cell membrane, the leader sequence may be optionally cut from the extracellular tag binding domain. Any of the various leader sequences known to those skilled in the art may be used as a leader sequence. Non-limiting examples of peptides from which leader sequences may be derived include granulocyte-macrophage colony stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α or any other protein 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 human immunoglobulin heavy chain (HC).

[0193] In some embodiments, the leader sequence is derived from GMCSFR. In one embodiment, the GMCSFR leader sequence comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, wherein the variant has 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 with SEQ ID NO: 1.

[0194] In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a transmembrane domain fused in frame between an extracellular tag binding domain and a cytoplasmic domain.

[0195] The transmembrane domain can be derived from a protein that contributes an extracellular tag binding domain, a protein that contributes a signaling or common signaling domain, or a completely different protein. In some cases, the transmembrane domain can be selected or modified by amino acid substitution, deletion or insertion to minimize interactions with other members of the CAR complex. In some cases, the transmembrane domain can be selected or modified by amino acid substitution, deletion or insertion to avoid protein binding naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow flexibility and / or optimal distance between domains connected to the transmembrane domain.

[0196] The membrane-spanning domain can be derived from a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound protein or membrane-spanning protein. The non-limiting examples of the membrane-spanning domain used in the present disclosure can be derived from (i.e., at least comprising the following membrane-spanning region): α, β or ζ chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137 or CD154 of a T cell receptor (TCR). Alternatively, the membrane-spanning domain can be synthetic, in which case it mainly comprises hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan and / or valine can be found at each end of a synthetic membrane-spanning domain.

[0197] In some embodiments, it is desirable to utilize a transmembrane domain of a ζ, η or FcεR1γ chain that contains a cysteine ​​residue capable of disulfide bonding, such that the resulting chimeric protein is capable of forming a disulfide-linked dimer with itself or with an unmodified version of ζ, η or FcεR1γ or a related protein. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In other cases, it is desirable to employ a transmembrane domain of ζ, η or FcεR1γ and -β, MB1 (Igα.), B29 or CD3-γ, ζ or η in order to maintain physical contact with other members of the receptor complex.

[0198] In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof, which has 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 with SEQ ID NO: 23. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 24 or a variant thereof, which has 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 with SEQ ID NO: 24.

[0199] In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a spacer between the extracellular tag binding domain and the transmembrane domain, wherein the tag binding domain, the linker, and the transmembrane domain are in frame with each other.

[0200] As used herein, the term "spacer" generally refers to any oligopeptide or polypeptide capable of connecting a tag binding domain to a transmembrane domain. The spacer can be used to provide more flexibility and accessibility for the tag binding domain. The spacer can contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The spacer 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 can be a synthetic sequence corresponding to a naturally occurring spacer sequence, or can be a fully synthetic spacer sequence. Non-limiting examples of spacers that can be used according to the present disclosure include a portion of a human CD8 alpha chain, a portion of the extracellular domain of CD28, a FcγRllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or a functional fragment thereof. In some embodiments, additional connecting amino acids are added to the spacer to ensure that the antigen binding domain is the optimal distance from the transmembrane domain. In some embodiments, when the spacer is derived from Ig, the spacer can be mutated to prevent Fc receptor binding.

[0201] In some embodiments, the spacer comprises a hinge domain. The hinge domain can be derived from CD8α, CD28 or an immunoglobulin (IgG). For example, an IgG hinge can be from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE or a chimera thereof.

[0202] 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 its chimera. In certain embodiments, the hinge region comprises CH1, CH2, CH3 and / or hinge region of an immunoglobulin. In certain embodiments, the hinge region comprises the core hinge region of an immunoglobulin. The term "core hinge" can be used interchangeably with the term "short hinge" (also referred to as "SH"). Non-limiting examples of suitable hinge domains are core immunoglobulin hinge regions, including 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 region is a fragment of an immunoglobulin hinge.

[0203] In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the hinge domain of CD8 comprises the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof, which has 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 with SEQ ID NO: 21. In one embodiment, the hinge domain of CD28 comprises the amino acid sequence shown in SEQ ID NO: 22 or a variant thereof, which has 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 with SEQ ID NO: 22.

[0204] In some embodiments, the transmembrane domain and / or hinge domain are derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and hinge domain are derived from CD28.

[0205] 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 further comprises one or more costimulatory signaling domains.

[0206] The cytoplasmic domain is responsible for the activation of at least one normal effector function of the host cell (e.g., T cell) in which the CAR is located. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell may be a cell lytic activity or an auxiliary activity including the secretion of a cytokine. Therefore, the term "signaling domain" refers to a protein portion that transduces effector function signals and guides cells to perform specialized functions. Although the entire signaling domain is usually present, in many cases the entire chain does not have to be used. In terms of using a truncated portion of an intracellular signaling domain, as long as it transduces an effector function signal, this truncated portion can be used to replace a complete chain. Therefore, the term intracellular signaling domain is intended to include any truncated portion of a signaling domain sufficient to transduce an effector function signal.

[0207] 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, Fcε receptor 1 γ chain (FCER1G), FcRβ, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

[0208] In some embodiments, the cytoplasmic domain comprises a CD3 zeta signaling domain. In one embodiment, the CD3 zeta 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.

[0209] In some embodiments, the cytoplasmic domain further comprises one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory 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.

[0210] In one embodiment, the costimulatory signaling domain is derived from 41BB. In one embodiment, the 41BB costimulatory 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.

[0211] In one embodiment, the costimulatory signaling domain is derived from IL2Rb. In one embodiment, the IL2Rb costimulatory 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.

[0212] In one embodiment, the costimulatory signaling domain is derived from CD40. In one embodiment, the CD40 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof, wherein the variant has 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.

[0213] In one embodiment, the costimulatory signaling domain is derived from OX40. In one embodiment, the OX40 costimulatory 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.

[0214] In one embodiment, the costimulatory signaling domain is derived from CD80. In one embodiment, the CD80 costimulatory 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.

[0215] In one embodiment, the costimulatory signaling domain is derived from CD86. In one embodiment, the CD86 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof, wherein the variant has 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.

[0216] In one embodiment, the costimulatory signaling domain is derived from CD27. In one embodiment, the CD27 costimulatory 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.

[0217] In one embodiment, the costimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS costimulatory 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.

[0218] In one embodiment, the costimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof, wherein the variant has 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.

[0219] In one embodiment, the costimulatory signaling domain is derived from DAP10. In one embodiment, the DAP10 costimulatory 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.

[0220] In one embodiment, the costimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP 12 costimulatory 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.

[0221] In one embodiment, the costimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof, wherein the variant has 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.

[0222] In some embodiments, the CAR of the present disclosure comprises a 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 2, 3, 4, 5, 6 or more co-stimulatory signaling domains.

[0223] In some embodiments, the signaling domain and the costimulatory signaling domain can be placed 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 the case of including two or more costimulatory domains, the order of the costimulatory signaling domains can be swapped.

[0224] Non-limiting exemplary CAR regions and sequences are provided in Table 1.

[0225] Table 1.

[0226]

[0227]

[0228]

[0229]

[0230] 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 more than one antigen or more than one epitope in an 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.

[0231] The selection of antigen binding domains can depend on the type and quantity of antigens defining the target cell surface.For example, antigen binding domains can be selected to identify antigens as cell surface markers on target cells associated with a specific disease state.In certain embodiments, by engineering specific binding to an antigen (e.g., an antigen on a tumor cell) desired antigen binding domain, the CAR of the present disclosure can be genetically modified to target a tumor antigen of interest.Non-limiting examples of cell surface markers that can be used as targets of antigen binding domains in the CAR of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0232] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen.

[0233] 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.

[0234] 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, two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, two or more autoimmune antigens are associated with different autoimmune diseases.

[0235] In some embodiments, tumor antigens are 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 tight junction proteins 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.

[0236] Other examples of antigens that can be targeted by the antigen binding domain include, but are not limited to, alpha-fetoprotein, A3, antigens specific for A33 antibodies, Ba 733, 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 villus 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 inhibitory factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigens specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A-antigen, angiogenesis markers, carcinogenic markers or oncogene carcinogenic products.

[0237] 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), the heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, the variant 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 with SEQ ID NO: 2. In one embodiment, the anti-CD19 scFv comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant has 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 of SEQ ID NO: 7 or a variant thereof, wherein the variant has 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.

[0238] In some embodiments, the antigen is associated with an autoimmune disease or condition. Such antigens can be derived from cell receptors and cells that produce "self" directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or condition, such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome ( 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-Barre syndrome, Crohn's disease, or ulcerative colitis.

[0239] In some embodiments, the autoimmune antigens that the CAR disclosed herein can target include, but are not limited to, platelet antigens, myelin protein antigens, Sm antigens in snRNP, islet cell antigens, rheumatoid factors, and anti-citrullinated proteins. Citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclic citrullinated peptides), fibrinogen, fibrin, vimentin, filamentous protein (fillaggrin), collagen I and II peptides, α-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeleton 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 1α1, stress proteins such as HSP-65, -70, -90, BiP, inflammatory / immune factors such as B7-H1, IL-1α and IL-8, enzymes such as calpastatin, α-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35kD nuclear protein, granule proteins such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factor, histones, ribosomal P protein, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA and RNA, ribonuclear granules and proteins such as Sm antigens (including but not limited to SmD's and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro (SSA) and La (SSB) antigens.

[0240] In various embodiments, the scFv fragment used in the CAR of the present disclosure may include a joint between the VH and VL domains. The joint may be a peptide joint and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the joint are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The length of the joint should be sufficient to connect VH and VL so that they form a correct conformation relative to each other, thereby maintaining the desired activity, such as binding to an antigen. The joint may be about 5-50 amino acids long. In some embodiments, the joint is about 10-40 amino acids long. In some embodiments, the joint is about 10-35 amino acids long. In some embodiments, the joint is about 10-30 amino acids long. In some embodiments, the joint is about 10-25 amino acids long. In some embodiments, the joint is about 10-20 amino acids long. In some embodiments, the joint is about 15-20 amino acids long. Exemplary linkers that can be used are Gly-rich linkers, linkers containing Gly and Ser, linkers containing Gly and Ala, linkers containing Ala and Ser, and other flexible linkers.

[0241] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof, and the variant has 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 with 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 shown in SEQ ID NO: 25 or a variant thereof, and the variant has 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 with SEQ ID NO: 25.

[0242] Other linker sequences can include portions of immunoglobulin hinge regions, CL or CH1 derived from any immunoglobulin heavy or light chain isotype. Exemplary linkers that can be used include any of SEQ ID NOs: 26-56 in Table 1. Additional linkers are described in, for example, International Patent Publication No. WO2019 / 060695, which is incorporated herein by reference in its entirety.

[0243] III. Safety switch / artificial cell death peptide

[0244] According to the embodiment of the present application, iPSC cells or their derivative cells optionally include an exogenous polynucleotide encoding a safety switch, and the safety switch may include an artificial cell death polypeptide. Since cell therapy (such as CAR-T therapy) has a long or uncertain half-life, toxicity may be progressive. Cells have been engineered to include a safety switch so that injected cells are eliminated when an adverse event occurs. Therefore, CAR cells have been engineered to include a gene (i.e., a "suicide gene") including a safety switch, which is a genetically encoded molecule that allows selective destruction of CAR cells, thereby allowing selective elimination of genetically modified cells to prevent collateral damage to neighboring cells and / or tissues. The safety switch can mediate the induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcription and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some cases, the safety switch is activated by an exogenous molecule (e.g., an antibody, an antiviral drug, or a radioisotope conjugated drug), and when activated, it triggers apoptosis and / or cell death of the treated cells. In one example, the artificial cell death polypeptide includes 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 comprises an inactivated cell surface receptor comprising a monoclonal antibody specific epitope, preferably a truncated epidermal growth factor (tEGFR) variant.

[0245] As used herein, the term "artificial cell death polypeptide" refers to an engineered protein intended to prevent potential toxicity or other adverse effects of 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, the artificial cell death polypeptide is activated by an exogenous molecule, such as an antibody, and when activated, it triggers apoptosis and / or cell death of the treated cells.

[0246] In certain embodiments, the artificial cell death polypeptide comprises an inactivated cell surface receptor, and the inactivated cell surface receptor comprises an epitope specifically recognized by an antibody (particularly a monoclonal antibody), also referred to herein as a monoclonal antibody specific epitope. When expressed by iPSC or its derivative cells, the inactivated cell surface receptor is inactive or significantly impaired in signal transduction, but can still be specifically recognized by the antibody. The specific binding of the antibody to the inactivated cell surface receptor can eliminate iPSC or its derivative cells through ADCC and / or ADCP mechanisms, as well as direct killing with antibody drug conjugates with toxins or radionuclides.

[0247] In certain embodiments, the inactivated cell surface receptor comprises an epitope selected from the group consisting of epitopes specifically recognized by antibodies including, but not limited to, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, vebutuximab, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, vepotuzumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab.

[0248] 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 the EGFR intracellular signaling domain. Exemplary tEGFR variants contain residues 322-333 of domain 2, all of domains 3 and 4, and a transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope. Expression of tEGFR variants on the cell surface enables, as needed, the use of antibodies that specifically bind to tEGFR, such as cetuximab Due to the absence of the EGF binding domain and the intracellular signaling domain, tEGFR is inactive when expressed by iPSCs or their derivatives.

[0249] The exemplary inactivated cell surface receptors of the present application include tEGFR variants. In certain embodiments, when the cell is contacted with an anti-EGFR antibody, the expression of the inactivated cell surface receptor in the engineered immune cells expressing the chimeric antigen receptor (CAR) induces the cell suicide of the engineered immune cells. The method of using inactivated cell surface receptors is described in WO2019 / 070856, WO2019 / 023396, and WO2018 / 058002, and its disclosure is incorporated herein by reference. For example, an effective amount of anti-EGFR antibodies that can eliminate the engineered immune cells previously administered in the subject can be administered to a subject who has previously received the engineered immune cells of the present disclosure, and the engineered immune cells include heterologous polynucleotides encoding inactivated cell surface receptors comprising tEGFR variants.

[0250] In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necituzumab, or panitumumab, preferably the anti-EGFR antibody is cetuximab.

[0251] In certain embodiments, the tEGFR variant comprises or consists of 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:71, preferably the amino acid sequence of SEQ ID NO:71.

[0252] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by vepotuzumab. In certain embodiments, the CD79b epitope 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: 78 or is composed of 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: 78, preferably an amino acid sequence of SEQ ID NO: 78.

[0253] 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%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80, preferably an amino acid sequence of SEQ ID NO: 80.

[0254] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of a Her 2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody specific epitope 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: 82 or is composed of 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: 82, preferably an amino acid sequence of SEQ ID NO: 82.

[0255] In certain embodiments, the inactivated cell surface receptor is a truncated epidermal growth factor receptor (tEGFR) variant. In certain embodiments, the tEGFR variant has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71 or is composed of an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Preferably, the tEGFR variant has an amino acid sequence of SEQ ID NO: 71 or is composed of an amino acid sequence of SEQ ID NO: 71.

[0256] IV. Cytokine Expression

[0257] In some embodiments, the iPSC cells or cells derived therefrom optionally comprise an exogenous polynucleotide encoding a cytokine, such as interleukin-15 or interleukin-2.

[0258] As used herein, "interleukin-15" or "IL-15" refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. A "functional portion" ("biologically active portion") of a cytokine refers to a cytokine portion that retains one or more functions of a full-length or mature cytokine. Such functions of IL-15 include promoting NK cell survival, regulating NK cell and T cell activation and proliferation, and supporting the development of NK cells from hematopoietic stem cells. Those skilled in the art will appreciate that 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%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72, preferably an amino acid sequence of SEQ ID NO: 72.

[0259] In some embodiments, IL-15 is in a membrane-bound form, wherein all or a functional portion of the IL-15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL-15 as a cell membrane-bound polypeptide ( mbIL15 )", such as the construct described in U.S. Pat. No. 9,629,877 B2, which is incorporated herein by reference.

[0260] As used herein, "interleukin-2" refers to a cytokine that regulates T and NK cell activation and proliferation, 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.

[0261] In certain embodiments, the cytokine can be connected to a safety switch so that it comprises an inactivated cell surface receptor operably connected to the cytokine (preferably connected via an autoprotease peptide sequence), the inactivated cell surface receptor comprising a monoclonal antibody specific epitope. Examples of autoprotease peptides include, but are not limited to, peptide sequences selected from porcine teschovirus type 1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus type A (ERAV) 2A (E2A), lycopodiella virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), malarial disease virus 2A (BmIFV2A) and combinations thereof. In one embodiment, the autoprotease peptide is an autoprotease peptide of porcine teschovirus type 1 2A (P2A). In certain embodiments, the autoprotease 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.

[0262] In certain embodiments, the inactivated cell surface receptor comprises a truncated epidermal growth factor (tEGFR) variant, which is operably linked to interleukin-15 (IL-15) or IL-2 via an autologous protease peptide sequence. In a specific embodiment, 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 an amino acid sequence of SEQ ID NO: 74.

[0263] 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.

[0264] 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 shown in SEQ ID NO: 21 or a variant thereof, wherein the variant has 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 with SEQ ID NO: 21.

[0265] 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 shown in SEQ ID NO: 23 or a variant thereof, wherein the variant has 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 with SEQ ID NO: 23.

[0266] In certain embodiments, the cell surface receptor of inactivation includes one or more epitopes specifically recognized by antibodies in its extracellular domain, transmembrane region and cytoplasmic domain.In some embodiments, the cell surface receptor of inactivation further includes the hinge region between epitope and transmembrane region.In some embodiments, the cell surface receptor of inactivation includes more than one epitope specifically recognized by antibodies, and the epitope can have the same or different amino acid sequence, and the epitope can be connected together via a peptide linker, such as a flexible peptide linker with (GGGGS)n sequence, wherein n is an integer of 1-8 (SEQ ID NO:25).In some embodiments, the cell surface receptor of inactivation further includes a cytokine, such as IL-15 or IL-2.In certain embodiments, the cytokine is in the cytoplasmic domain of the cell surface receptor of inactivation.Preferably, the cytokine is directly or indirectly (via an autoprotease peptide sequence (such as those described herein)) operably connected to the epitope specifically recognized by antibodies.In some embodiments, the cytokine is indirectly connected to the epitope via an autoprotease peptide sequence by being connected to the transmembrane region.

[0267] Non-limiting exemplary inactivated cell surface receptor regions and sequences are provided in Table 2.

[0268] Table 2.

[0269]

[0270]

[0271] In a specific embodiment, 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:79, preferably the amino acid sequence of SEQ ID NO:79.

[0272] In a specific embodiment, 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:81, preferably the amino acid sequence of SEQ ID NO:81.

[0273] In a specific embodiment, 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:83, preferably the amino acid sequence of SEQ ID NO:83.

[0274] III. HLA expression

[0275] In certain embodiments, the iPSC or its derivative cells of the present application can be further modified by introducing a third exogenous polynucleotide, wherein the third exogenous polynucleotide encodes one or more proteins associated with immune escape, such as non-classical HLA I class proteins (e.g., HLA-E and HLA-G). In particular, the destruction of the B2M gene eliminates the surface expression of all MHC class I molecules, making the cells susceptible to NK cell lysis by "missing self" reaction. Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol. 2017Aug; 35 (8): 765–772).

[0276] In certain embodiments, the iPSC or a cell derived therefrom comprises a third exogenous polynucleotide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G). In a specific embodiment, 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 a specific embodiment, 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 SEQ ID NO: 68.

[0277] In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein, wherein the mature B2M protein is fused to HLA-E via a linker. In a specific embodiment, 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.

[0278] 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 a specific embodiment, 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.

[0279] IV. Membrane-bound IL-12

[0280] Interleukin-12 (IL-12) is a heterodimeric molecule consisting of an α chain (p35 subunit) and a β chain (p40 subunit), covalently linked by a disulfide bridge to form a biologically active 70 kDa dimer. Biologically, IL-12 is an inflammatory cytokine produced by various cells of the immune system in response to infection, including phagocytes, B cells, and activated dendritic cells (Colombo and Trinchieri (2002), Cytokine & Growth Factor Reviews, 13: 155-168 and Hamza et al. "Interleukin-12a Key Immunoregulatory Cytokine in Infection Applications" Int. J. Mol. Sci. 11; 789-806 (2010). IL-12 plays a crucial role in mediating the interaction between the innate and adaptive arms of the immune system, acting on T cells and natural killer (NK) cells, enhancing the proliferation and activity of cytotoxic lymphocytes and the production of other inflammatory cytokines, especially interferon-γ (IFN-γ).

[0281] IL-12 has been used as an immunotherapeutic agent in human clinical trials for the treatment of various cancers (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), including renal, colon and ovarian cancers, melanoma and T-cell lymphoma) and as adjuvants for cancer vaccines Lee et al. (2001), J. Clin. Oncol. 19:3836-47). However, when administered systemically as a recombinant protein, IL-12 is toxic. Trinchieri, Adv. Immunol. 1998; 70:83-243. Since IL-12 is a heterodimeric molecule composed of an α chain (p35 subunit) and a β chain (p40 subunit), simultaneous expression of the two subunits is necessary to produce biologically active heterodimers. Expression of recombinant IL-12 is achieved using a bicistronic vector containing p40 and p35 subunits, which can be separated by an IRES (internal ribosome entry site) sequence to allow independent expression of the two subunits from a single vector. However, the use of an IRES sequence may impair protein expression. Mizuguchi et al. Mol Thera (2000); 1:376-382. Furthermore, unequal expression of the p40 and p35 subunits can lead to the formation of homodimeric proteins (e.g., p40-p40), which can have an inhibitory effect on IL-12 signaling. Gillessen et al. Eur. J. Immunol. 25(1):200-6(1995).

[0282] As an alternative to bicistronic expression of IL-12 subunits, functional single-chain IL-12 fusion proteins were generated by linking the p40 and p35 subunits with a disulfide bond, (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 p40-linker-p35 or p35-linker-p40 IL-12 configurations may be referred to herein as "conventional single-chain IL-12 (scIL-12)".

[0283] 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, compared to the corresponding IL12 lacking protease sensitivity, the IL12 polypeptide can be modified (e.g., by genetic engineering, synthetic engineering, or recombinant engineering) to increase sensitivity to proteases, thereby shortening the biologically active half-life of the IL12 complex. Protease-sensitive forms of IL12 are described in International Patent Publication No. WO2017062953; the contents of which are incorporated by reference in their entirety.

[0284] According to the present invention, the p35 and p40 subunits of the IL-12 protein (preferably in the traditional single-chain IL-12 configuration) are bound to the cell membrane by fusing scIL-12 to a transmembrane domain (TM) such as an EGFR transmembrane domain. In certain aspects, the p35 / TM subunits are further fused to a signaling domain (SD). For example, the signaling domain is a CD3 ζ, CD28, and / or 4-1BB signaling domain. In specific aspects, the signaling domain comprises a CD3 ζ and a 4-1BB signaling domain. In some aspects, the signaling domain is 4-1BB.

[0285] As used herein, the term "transmembrane domain (TM)" refers generally to an amino acid sequence of 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 domain of the present disclosure may be derived from a natural source or from a synthetic source. The transmembrane domain may be derived from any natural membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain may be derived from EGFR.

[0286] Alternatively, the transmembrane domains of the present disclosure may be synthetic. In some aspects, the synthetic sequence may comprise primarily hydrophobic residues, such as leucine and valine.

[0287] The amino acid sequence of p70 IL-12 fused to the EGFR transmembrane is as follows:

[0288] (SEQ ID NO:96)

[0289] The corresponding nucleotide sequence of p70 IL-12 fused to the EGFR transmembrane is as follows:

[0290]

[0291] As an alternative construct of the present invention, membrane-bound IL-12p70 can integrate a protease cleavage site for inducing release by protease ADAM17 activation. ADAM17 is expressed by activated lymphocytes and directly participates in the release of other immune mediators (such as TNFa), which similarly exist in a membrane-anchored form. When this membrane-bound IL12 is expressed on engineered iNK cells, it maintains contact with the cells. When cell activation and ADAM17 expression increase, protease cuts the membrane stem and releases IL12 into the extracellular space. This type of regulation ensures that the activity of IL12 is confined to the space around the tumor, where engineered immune cells contact their targets on tumor cells, thereby triggering its activation.

[0292] An exemplary amino acid sequence of a membrane IL-12p70 ADAM17 protease cleavage site fusion protein is as follows:

[0293] (SEQ ID NO:98).

[0294] The corresponding nucleotide sequence of the membrane IL-12p70 ADAM17 protease cleavage site fusion protein is as follows:

[0295]

[0296] TNFα and TGFα are two cleavable substrates of ADAM17. In certain embodiments, the membrane-bound IL-12p70 protein may comprise TNFα or TGFα, or a portion of one or both, for induction of release by protease ADAM17 activation. An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TGF-α short scaffold protease cleavage site is as follows:

[0297] (SEQ ID NO:108).

[0298] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α short scaffold protease cleavage site fusion protein is as follows:

[0299]

[0300] An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TGF-α scaffold protease cleavage site is as follows:

[0301] (SEQ ID NO:110).

[0302] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α scaffold protease cleavage site fusion protein is as follows:

[0303]

[0304] An exemplary amino acid sequence of a membrane IL-12p70 and TGF-α scaffold / TACEtide protease cleavage site fusion protein is as follows:

[0305] (SEQ ID NO:112).

[0306] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α scaffold / TACEtide protease cleavage site fusion protein is as follows:

[0307]

[0308] An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TNF-α scaffold protease cleavage site is as follows:

[0309] (SEQ ID NO:114).

[0310] The corresponding nucleotide sequence of the membrane IL-12p70 and TNF-α scaffold protease cleavage site fusion protein is as follows:

[0311]

[0312] An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TGF-α scaffold protease cleavage site and an EGFR transmembrane domain is as follows:

[0313] (SEQ ID NO:116).

[0314] The corresponding nucleotide sequence of the membrane IL-12p70 with the TGF-α scaffold protease cleavage site and the EGFR transmembrane domain fusion protein is as follows:

[0315]

[0316] An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TGF-α scaffold and tandem protease cleavage site is as follows:

[0317] (SEQ ID NO:118).

[0318] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α scaffold tandem protease cleavage site fusion protein is as follows:

[0319]

[0320] An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TGF-α scaffold protease cleavage site (variant 1) is as follows:

[0321] (SEQ ID NO:120).

[0322] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α scaffold protease cleavage site (variant 1) fusion protein is as follows:

[0323]

[0324] An exemplary amino acid sequence of a membrane IL-12 p70 fusion protein with a TGF-α scaffold protease cleavage site (variant 2) is as follows:

[0325] (SEQ ID NO:122).

[0326] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α scaffold protease cleavage site (variant 2) fusion protein is as follows:

[0327]

[0328] An exemplary amino acid sequence of a membrane IL-12p70 fusion protein with a TGF-α scaffold protease cleavage site (variant 3) is as follows:

[0329] (SEQ ID NO:124).

[0330] The corresponding nucleotide sequence of the membrane IL-12p70 and TGF-α scaffold protease cleavage site (variant 3) fusion protein is as follows:

[0331]

[0332] In certain embodiments, the exogenous polynucleotide encoding mbIL-12 protein or membrane IL-12p70 ADAM17 protease cleavage site fusion protein is integrated into one or more loci on the cell chromosome, preferably one or more loci are one or more loci selected from the following one or more genes: AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, 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 gene, more preferably, one or more of the exogenous polynucleotides are integrated into the locus of the AAVS1 or CLYBL gene.

[0333] V. Other optional genome editing

[0334] In one embodiment of the above-mentioned cell, genome editing at one or more selected sites may include inserting one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting moieties, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the implantation, transportation, homing, vitality, self-renewal, persistence and / or survival of genome-engineered iPSC or its derivative cells. In some embodiments, the genome-engineered iPSC produced using the above-mentioned method comprises one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b, wherein when the genome-engineered iPSC comprises two or more suicide genes, the suicide genes are integrated in different safe harbor loci, and the safe harbor loci comprise AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1. Other exogenous polynucleotides encoding proteins can include encoding PET reporters, homeostatic cytokines, inhibitory checkpoint inhibitors such as PD1, PD-L1 and CTLA4, and proteins targeting the CD47 / signal regulatory protein α (SIRPα) axis. In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein contain insertions / deletions (in / del) in one or more endogenous genes associated with proteins that regulate and modulate or inhibit the implantation, transport, homing, vigor, self-renewal, persistence and / or survival of iPSCs or their derivatives.

[0335] VI. Promoter

[0336] In some embodiments, the exogenous polynucleotide for insertion is operably linked to (1) one or more exogenous promoters, including CMV, EF1a, PGK, CAG, UBC, or other constitutive, inducible, temporal, tissue or cell type specific promoters; (2) one or more endogenous promoters contained in a selected site, including AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet the criteria of a genomic safe harbor.

[0337] VII. Targeted genome editing of selected loci in iPSCs

[0338] According to an embodiment of the present application, one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of the iPSC.

[0339] Genome editing, or genome editing, or genetic editing, are used interchangeably herein and are a kind of genetic engineering, in which DNA is inserted, deleted and / or replaced in the genome of the targeted cell. Targeted genome editing (interchangeable with "targeted genome editing" or "targeted genetic editing") enables insertion, deletion and / or replacement at preselected sites in the genome. In the targeted editing process, when the endogenous sequence is deleted or destroyed at the insertion site, the endogenous gene containing the affected sequence can be knocked out or knocked down due to sequence deletion or destruction. Therefore, targeted editing can also be used for accurately destroying endogenous gene expression. Similar use is made in this article of the term "targeted integration", which refers to the process of inserting one or more exogenous sequences at the preselected sites in the genome when the insertion site is missing or not missing the endogenous sequence.

[0340] Targeted editing can be achieved by nuclease-independent methods or by nuclease-dependent methods. In nuclease-independent targeted editing methods, homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide to be inserted and is carried out by the enzymatic machinery of the host cell.

[0341] Alternatively, a higher frequency of targeted editing can be achieved by specifically introducing double-strand breaks (DSBs) through specific rare-cutting endonucleases. This nuclease-dependent targeted editing exploits 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 / del) of a small number of endogenous nucleotides. In contrast, when a donor vector containing exogenous genetic material flanked by a pair of homologous arms is present, the exogenous genetic material can be introduced into the genome through homologous recombination during homology-directed repair (HDR), resulting in "targeted integration."

[0342] 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. In addition, the DICE (double integrase cassette exchange) system using phiC31 and Bxbl integrases is also a promising tool for targeted integration.

[0343] ZFN is a targeted nuclease comprising a nuclease fused to a zinc finger DNA binding domain. "Zinc finger DNA binding domain" or "ZFBD" refers to a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. Zinc fingers are domains of about 30 amino acids within a zinc finger binding domain, and their structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. "Designed" zinc finger domains are domains that do not exist in nature, and their design / composition is primarily derived from reasonable criteria, for example, using substitution rules and computer algorithms for processing information in databases storing existing ZFP design and binding data information. See, e.g., U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. "Selected" zinc finger domains are domains not found in nature and are generated primarily from empirical processes such as phage display, interaction traps, or hybrid selection. ZFNs are described in more detail in U.S. Pat. Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is a fusion of the Fok1 nuclease with a zinc finger DNA binding domain.

[0344] 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" refers to the polypeptide domain of the TAL effector protein, which is responsible for the binding of the TAL effector protein to DNA. Plant pathogens of the genus Xanthomonas secrete TAL effector proteins during infection. These proteins enter the plant cell nucleus, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription on these sequences via their transactivation domains. The specificity of the TAL effector DNA binding domain depends on the variable number of effectors of imperfect 34 amino acid repeats, which contain polymorphisms at selected repeat positions called repeat variable diresidues (RVDs). TALEN is described in more detail in U.S. Patent Application No. 2011 / 0145940, which is incorporated herein by reference. The most recognized TALEN example in the art is a fusion polypeptide of the Fokl nuclease and the TAL effector DNA binding domain.

[0345] Another example of a targeted nuclease used in the subject methods is a targeted Spoll nuclease, which comprises a Spoll polypeptide having nuclease activity fused to a DNA binding domain specific for a DNA sequence of interest, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. See, e.g., U.S. Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0346] Other examples of targeted nucleases suitable for use in the present application include, but are not limited to, Bxbl, phiC3 1, R4, PhiBT1, and Wp / SPBc / TP901-1, whether used alone or in combination.

[0347] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR-associated nucleases from the families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, etc. As an example, CRISPR / Cas9 requires two main components: (1) Cas9 endonuclease and (2) crRNA-tracrRNA complex. When co-expressed, the two components form a complex that is recruited to a target DNA sequence comprising a PAM and a seed region near the PAM. The crRNA and tracrRNA can combine to form a chimeric guide RNA (gRNA) that guides Cas9 to a target selected sequence. The two components can then be delivered to mammalian cells via transfection or transduction. As another example, CRISPR / Cpf1 comprises two main components: (1) CPf1 endonuclease and (2) crRNA. When co-expressed, the two components form a ribonucleoprotein (RNP) complex that is recruited to a target DNA sequence containing a PAM and a seed region near the PAM. The crRNA can combine to form a chimeric guide RNA (gRNA) that guides Cpf1 to the target selected sequence. The two components can then be delivered to mammalian cells via transfection or transduction.

[0348] MAD7 is an engineered Cas12a variant derived from the bacterium Eubacterium rectale, which has a preference for 5′-TTTN-3′ and 5′-CTTN-3′ PAM sites and does not require tracrRNA. See, e.g., PCT Publication No. 2018 / 236548, the disclosure of which is incorporated herein by reference.

[0349] DICE-mediated insertion uses a pair of recombinases, e.g., phiC31 and Bxbl, to provide unidirectional integration of foreign DNA, tightly confined to each enzyme's own small attB and attP recognition sites. Because these target att sites are not naturally present in mammalian genomes, they must first be introduced into the genome at the desired integration site. See, e.g., U.S. Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0350] 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 homology arms specific to the desired integration site, and the targeted integration method comprises introducing the construct into the cell so that site-specific homologous recombination can be performed by the cell host enzymatic mechanism. In another embodiment, the method for targeted integration in the cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to achieve ZFN-mediated insertion. In another embodiment, the method for targeted integration in the cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to achieve TALEN-mediated insertion. In another embodiment, the method for targeted integration in the cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, introducing a Cpf1 expression cassette and a gRNA comprising a guide sequence specific to the desired integration site into the cell to achieve Cpf1-mediated insertion. In another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, introducing a Cas9 expression cassette and a gRNA comprising a guide sequence specific to the desired integration site into the cell to achieve Cas9-mediated insertion. In another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more att sites for a pair of DICE recombinase into the desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for the DICE recombinase to achieve DICE-mediated targeted integration.

[0351] The site of targeted integration includes, but is not limited to, a genomic safe harbor, which is an intragenic or extragenic region of the human genome that can theoretically accommodate predictable expression of newly integrated DNA without adversely affecting the host cell or organism. In certain embodiments, the genomic safe harbor for targeted integration is a locus of one or more genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR, and RUNX1 genes.

[0352] 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 "deletions" of gene expression include, for example, removal or deletion of the DNA sequence of a gene, insertion of an exogenous polynucleotide sequence at the locus of a gene, and one or more substitutions within a gene that abolish the expression of the gene.

[0353] Genes targeted for 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 be matched for tissue compatibility in allogeneic receptors to avoid allogeneic rejection issues. "MHC deficiency", including MHC class I deficiency, or MHC class II deficiency, or both, refers to cells that lack the surface expression level of a complete MHC complex containing MHC class I protein heterodimers and / or MHC class II heterodimers, or no longer maintain their surface expression level, or reduce their surface expression level, thereby weakening or reducing the level below the level that can be naturally detected by other cells or synthetic methods. MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p2l), or by deletion or reduction of the expression level of one or more MHC class I-related genes, including but not limited to the beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene, and Tapasin gene. For example, the B2M gene encodes a common subunit that is essential for the cell surface expression of all MHC class I heterodimers. B2M naked cells are MHC-I defects. MHC class II defects can be achieved by functional deletion or reduction of MHC-II related genes, including but not limited to RFXANK, CIITA, RFX5 and RFXAP. CIITA is a transcriptional coactivator that plays a role by activating the transcription factor RFX5 required for class II protein expression. CIITA naked cells are MHC-II defects. In certain embodiments, one or more of the exogenous polynucleotides are integrated in the locus of one or more genes of the gene selected from B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP, thereby making gene deletion or reducing expression by integration.

[0354] In certain embodiments, the exogenous polynucleotide is integrated at one or more loci on the cell chromosome, preferably the one or more loci are one or more loci selected from the following genes: AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, 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 gene, with the condition that at least one of the one or more loci is a locus of an MHC gene, such as a gene selected from B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. Preferably, one or more exogenous polynucleotides are integrated into the locus of MHC class I related genes, such as beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene; and at the locus of MHC-II related genes, such as RFXANK, CIITA, RFX5, RFXAP or CIITA gene; and optionally further at the locus of a safe harbor gene selected from AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes. More preferably, one or more of the exogenous polynucleotides are integrated into the locus of CIITA, AAVS1 and B2M genes, or at the locus of CIITA, CLYBL and B2M genes.

[0355] 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., HLA-E and or HLA-G transgene) is integrated at the locus of the B2M or CIITA gene; wherein the integration of the exogenous polynucleotides deletes or reduces the expression of the CIITA and B2M genes.

[0356] 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.

[0357] In certain embodiments, (i) the first exogenous polynucleotide encodes a CAR; (ii) the second exogenous polynucleotide comprises a polynucleotide sequence of SEQ ID NO: 97 or 99; and (iii) the third exogenous polynucleotide comprises a polynucleotide sequence of SEQ ID NO: 67 or 70.

[0358] VIII. Derived cells

[0359] On the other hand, the present invention relates to cells derived from iPSC differentiation, i.e., derived cells. As described above, the genome editing introduced into iPSC cells is retained in derived cells. In certain embodiments of derived cells obtained from iPSC differentiation, derived cells are hematopoietic cells, including but not limited to HSC (hematopoietic stem / progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, B cells, antigen presenting cells (APC), monocytes and macrophages. In certain embodiments, derived cells are immune effector cells, such as NK cells or T cells.

[0360] 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 second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, and the second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, and the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes. In certain embodiments, natural killer (NK) cells or T cells include polynucleotides encoding membrane-bound IL-12, which are fused to polynucleotides encoding ADAM17 protease cleavage site peptides for inducing the release of IL-12 by protease ADAM17 activation. ADAM17 is expressed by activated lymphocytes and directly participates in the release of other immune mediators (such as TNFa), which similarly exist in a membrane-anchored form. When this membrane-bound IL-12 is expressed on engineered iNK or T cells, it remains in contact with the cell. When cell activation and ADAM17 expression increase, protease cuts the membrane stem 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 engineered immune cells contact their targets on tumor cells, thereby triggering its activation.

[0361] 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).

[0362] The present application also provides a NK cell or T cell, which comprises: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, and the second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, and the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes.

[0363] In another embodiment, a NK or T cell is provided, 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises an IL-12α subunit p35 having at least 90% sequence identity to SEQ ID NO: 102, and the second polypeptide comprises an IL-12β subunit p40 having at least 90% sequence identity to SEQ ID NO: 103, wherein the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide, and the transmembrane domain has the amino acid sequence of SEQ ID NO: 100, or the second polynucleotide encoding membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide, for inducing the release of IL-12 by activation of the protease ADAM17, wherein the second exogenous polynucleotide encodes a peptide having SEQ ID NO:98; (iii) a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having an amino acid sequence of SEQ ID NO:66 or 69; (iv) a fourth exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having an amino acid sequence of SEQ ID NO:71, an autologous protease peptide having an amino acid sequence of SEQ ID NO:73, and a fourth exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having an amino acid sequence of SEQ ID NO:74. NO:72 amino acid sequence of interleukin 15 (IL-15); wherein the first, second and third exogenous polynucleotides are integrated at the loci of the following genes: (a) AAVS1, CIITA and B2M genes, respectively, (b) AAVS1, CIITA and B2M genes, respectively, (c) CLYBL, CIITA and B2M genes, respectively, (d) CIITA, AAVSI and B2M genes, respectively, (e) CIITA, CLYBL and B2M genes, respectively, (f) B2M, AAVS1 and CIITA genes, respectively, or (g) B2M, CLYBL and CIITA genes, respectively, thereby causing the absence or reduced expression of CIITA and B2M.

[0364] 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.

[0365] Also provided is a CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC), 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, wherein the first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, and the second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, and the transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes.

[0366] 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).

[0367] In certain embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to a CD19 antigen; (iii) a hinge region, (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain, such as a costimulatory domain comprising a CD28 signaling domain.

[0368] A method for preparing derivative cells is also provided, wherein the method comprises differentiating iPSCs under cell differentiation conditions, thereby obtaining derivative cells.

[0369] The iPSC of the present application can be differentiated by any method known in the art. Exemplary methods are described in US8846395, US8945922, US8318491, WO2010 / 099539, WO2012 / 109208, WO2017 / 070333, WO2017 / 179720, WO2016 / 010148, WO2018 / 048828 and WO2019 / 157597, each of which is incorporated herein by reference as a whole. Differentiation protocols may use feeder cells or may not contain feeders. As used herein, "feeder cells" or "feeders" are terms describing a type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, expand or differentiate, because feeder cells provide stimulation, growth factors and nutrients to support the second cell type.

[0370] In another embodiment of the present invention, the iPSC-derived cells of the present invention are NK cells, which are prepared by a method of differentiating iPSC cells into NK cells, wherein the method is by subjecting the cells to a differentiation protocol, the differentiation protocol comprising adding recombinant human IL-12p70 in the last 24 hours of culture. By including IL-12 in the differentiation protocol, cells primed with IL-12 exhibit faster cell killing ( Fig. 8A ). In addition, cells differentiated using IL-12 conditioning showed improved cancer cell growth inhibition ( Figure 8B ).

[0371] IX. Polynucleotides, Vectors and Host Cells

[0372] (1) Nucleic acid encoding CAR

[0373] In another general aspect, the present invention relates to a separated nucleic acid, which encodes a chimeric antigen receptor (CAR) that can be used for the present invention according to the embodiments of the present application. It will be appreciated by those skilled in the art that the coding sequence of CAR can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, it will be appreciated by those skilled in the art that the nucleic acid sequence encoding the CAR of the present application can be changed without changing the amino acid sequence of the protein.

[0374] In certain embodiments, the isolated nucleic acid encodes a CAR targeting CD 19. In a specific embodiment, the isolated nucleic acid encoding the CAR comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 62, preferably a polynucleotide sequence of SEQ ID NO: 62.

[0375] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding a CAR that can be used for the present invention according to the embodiments of the present application. Any vector known to those skilled in the art according to the present disclosure, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector may include any element to establish the conventional functions of the expression vector, for example, a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter may 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 herein to produce CAR in cells. Conventional cloning techniques or artificial gene synthesis can be used to produce recombinant expression vectors according to the embodiments of the present application.

[0376] In a specific aspect, the present application provides a vector for targeted integration of the CAR of the present invention according to an embodiment of the present application. In certain embodiments, the vector comprises an exogenous polynucleotide having (a) a promoter in the order of 5' to 3'; (b) a polynucleotide sequence encoding a CAR according to an embodiment of the present application; and (c) a terminator / polyadenylation signal.

[0377] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 63. Other promoters may also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin.

[0378] 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%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 64. Other terminator sequences may also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0379] In certain embodiments, the polynucleotide sequence encoding CAR comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:62.

[0380] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide. As used herein, "left homology arm" and "right homology arm" refer to a pair of nucleic acid sequences, which flank the exogenous polynucleotide and promote the integration of the exogenous polynucleotide into the specified chromosomal locus. The sequence of the left and right arm homology arms can be designed based on the integration site of interest. In some embodiments, the left homology arm or the right homology arm is homologous to the left or right sequence of the integration site.

[0381] In certain embodiments, a membrane-bound IL12 transgene (e.g., according to SEQ ID NO:97 or 99) is integrated at the AAVS1 locus and (i) the left homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:104, and the right homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:105. In certain embodiments, a membrane-bound IL12 transgene (e.g., according to SEQ ID NO: 97 or 99) is integrated at the CLYBL locus and (i) the left homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 106, and the right homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 107.

[0382] Table 3.

[0383]

[0384]

[0385]

[0386]

[0387] In a specific embodiment, the vector comprises a polynucleotide sequence that is at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 97 or 99, preferably a polynucleotide sequence of SEQ ID NO: 97 or 99.

[0388] (2) Nucleic acid encoding mb IL-12

[0389] In another general aspect, the present invention relates to an isolated nucleic acid encoding the mbIL-12 protein used in the present invention according to the embodiments of the present application. Those skilled in the art will appreciate that the coding sequence of the inactivated cell surface receptor can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, those skilled in the art will appreciate that the nucleic acid sequence encoding the mbIL-12 of the present application can be changed without changing the amino acid sequence of the protein.

[0390] In certain embodiments, the isolated nucleic acid encodes membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for inducing release of IL-12 by protease ADAM17 activation.

[0391] 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.

[0392] In certain embodiments, the isolated nucleic acid encodes membrane-bound IL-12, which is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for inducing the release of IL-12 by activation of the protease ADAM17, and which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:98.

[0393] (3) Nucleic acid encoding safety switch

[0394] In certain embodiments, the isolated nucleic acid encodes a safety switch, such as an inactivated cell surface receptor with a truncated epidermal growth factor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, nexituzumab or panitumumab, preferably cetuximab.

[0395] 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 vepotuzumab.

[0396] 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.

[0397] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of a Her 2 receptor, such as an epitope specifically recognized by trastuzumab.

[0398] In certain embodiments, the autologous protease peptide sequence is porcine teschovirus type 1 2A (P2A).

[0399] In certain embodiments, the truncated epidermal growth factor (tEGFR) variant consists of an amino acid sequence that has 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.

[0400] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by Vipotuzin consists of 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:78.

[0401] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by rituximab consists of 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:80.

[0402] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of 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:82.

[0403] In certain embodiments, IL-15 comprises an amino acid sequence that has 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.

[0404] In certain embodiments, the autoprotease peptide has an amino acid sequence that has 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.

[0405] In certain embodiments, the polynucleotide 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.

[0406] In a specific embodiment, the isolated nucleic acid encoding an inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90%, 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.

[0407] In certain embodiments, the polynucleotide 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.

[0408] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding a cell surface receptor that can be used for inactivation of the present invention according to the embodiments of the application. Any vector known to those skilled in the art according to the disclosure, such as a plasmid, a cosmid, a phage vector or a 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 to establish the conventional functions of the expression vector, for example, a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker and an 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 herein to produce inactivated cell surface receptors in cells. Conventional cloning techniques or artificial gene synthesis can be used to produce recombinant expression vectors according to the embodiments of the application.

[0409] In a specific aspect, the present application provides a vector for targeted integration of an inactivated cell surface receptor of the present invention according to an embodiment of the present application. In certain embodiments, the vector comprises an exogenous polynucleotide having (a) a promoter in the order of 5' to 3'; (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 connected by a self-protease peptide sequence, such as porcine Teschovirus 2A (P2A) type 1, and (c) a terminator / polyadenylation signal.

[0410] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 63. Other promoters may also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin.

[0411] 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%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 64. Other terminator sequences may also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0412] In certain embodiments, the polynucleotide sequence encoding the inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:75.

[0413] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.

[0414] In certain embodiments, the left homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 84. In certain embodiments, the right homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 85.

[0415] In a specific embodiment, the vector comprises a polynucleotide sequence that is at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:86, preferably the polynucleotide sequence of SEQ ID NO:86.

[0416] (4) Nucleic acid encoding HLA construct

[0417] In another general aspect, the present invention relates to an isolated nucleic acid encoding an HLA construct useful for the present invention according to embodiments of the present application. One skilled in the art will appreciate that the coding sequence of the HLA construct can be altered (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, one skilled in the art will appreciate that the nucleic acid sequence encoding the HLA construct of the present application can be altered without changing the amino acid sequence of the protein.

[0418] In certain embodiments, the isolated nucleic acid encodes an HLA construct comprising a signal peptide, such as an HLA-G signal peptide, operably linked to an HLA coding sequence, such as a coding sequence for mature B2M and / or mature HLA-E. In some embodiments, the HLA coding sequence encodes HLA-G and B2M, which are operably linked via a 4X GGGGS linker, and / or B2M and HLA-E, which are operably linked via a 3X GGGGS linker. In a specific embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 67, preferably a 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%, such as 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.

[0419] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an HLA construct that can be used for the present invention according to the embodiments of the present application. Any vector known to those skilled in the art according to the present disclosure, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector may include any element to establish the conventional functions of the expression vector, for example, a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker and an origin of replication. The promoter may 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 herein to produce HLA constructs in cells. Conventional cloning techniques or artificial gene synthesis can be used to produce recombinant expression vectors according to the embodiments of the present application.

[0420] In a specific aspect, the present application provides a vector that can be used for targeted integration of the HLA construct of the present invention according to the embodiments of the present application. In certain embodiments, the vector comprises an exogenous polynucleotide having (a) a promoter in the order of 5' to 3'; (b) a polynucleotide sequence encoding the HLA construct; and (c) a terminator / polyadenylation signal.

[0421] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 63. Other promoters may also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin.

[0422] 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%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 64. Other terminator sequences may also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0423] In certain embodiments, the polynucleotide sequence encoding the HLA construct comprises a signal peptide, such as an HLA-G signal peptide, mature B2M and mature HLA-E, wherein HLA-G and B2M are operably linked by a 4X GGGGS linker (SEQ ID NO: 31), and the B2M transgene and HLA-E are operably linked by a 3X GGGGS linker (SEQ ID NO: 25). In a specific embodiment, the HLA construct comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 67, preferably a polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLA construct comprises a polynucleotide sequence that is at least 90%, such as 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.

[0424] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.

[0425] In certain embodiments, the left homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 87. In certain embodiments, the right homology arm comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 88.

[0426] In a specific embodiment, the vector comprises a polynucleotide sequence that is at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:89, preferably the polynucleotide sequence of SEQ ID NO:89.

[0427] (5) Host cells

[0428] In another general aspect, the application provides a host cell comprising the vector of the application and / or the isolated nucleic acid encoding the construct of the application. Any host cell known to those skilled in the art in view of the disclosure can be used for the recombinant expression of the exogenous polynucleotides of the application. According to a specific embodiment, the recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein it is stably integrated into the host cell genome, thereby effectively expressing the recombinant nucleic acid.

[0429] Examples of host cells include, for example, recombinant cells containing the vector or isolated nucleic acid of the present application, which can be used to produce a vector or construct of interest; or engineered iPSC or its derivative cells containing one or more isolated nucleic acids of the present application, preferably integrated in one or more chromosomal loci. The host cell of the isolated nucleic acid of the present application may also be an immune effector cell, such as a T cell or NK cell, comprising one or more isolated nucleic acids of the present application. Immune effector cells can be obtained by differentiation of the engineered iPSC of the present application. In view of the present disclosure, any suitable method in the art can be used for differentiation. Immune effector cells can also be obtained by transfecting immune effector cells with one or more isolated nucleic acids of the present application.

[0430] X. Composition

[0431] In another general aspect, the present application provides a composition comprising the isolated polynucleotide, host cell and / or iPSC or derivative cell thereof of the present application.

[0432] In certain embodiments, the composition further comprises one or more therapeutic agents selected from peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), siRNAs, oligonucleotides, mononuclear blood cells, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

[0433] In certain embodiments, the composition is a pharmaceutical composition, which includes the polynucleotides of the separation of the application, host cells and / or iPSC or its derivative cells of the application and a pharmaceutically acceptable carrier. The term "pharmaceutical composition" as used herein refers to a product comprising the polynucleotides of the separation of the application, the polypeptides of the separation of the application, the host cells of the application and / or iPSC or its derivative cells of the application and a pharmaceutically acceptable carrier. The polynucleotides, polypeptides, host cells and / or iPSC or its derivative cells of the application and compositions comprising them can also be used to prepare drugs for therapeutic applications mentioned herein.

[0434] 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 substance known in the art for pharmaceutical preparations. It will be understood that the characteristics of the carrier, excipient or diluent depend on the route of administration for a specific application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic substance that does not interfere with the effectiveness of the compositions described herein or the biological activity of the compositions described herein. According to specific embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for polynucleotides, polypeptides, host cells and / or iPSCs or cells derived therefrom can be used.

[0435] The preparation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present application.

[0436] XI. How to use

[0437] In another general aspect, the application provides a method for treating a disease or condition of a subject in need. The method includes administering a therapeutically effective amount of the cell of the application and / or the composition of the application to a subject in need. In certain embodiments, the disease or condition is cancer. For example, cancer can be a solid cancer or a liquid cancer. For example, cancer can be 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, and non-Hodgkin lymphoma (NHL), Hodgkin lymphoma / disease (HD), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors. In a preferred embodiment, cancer is non-Hodgkin lymphoma (NHL).

[0438] According to the embodiments of the present application, the composition comprises a therapeutically effective amount of isolated polynucleotides, isolated polypeptides, host cells and / or iPSCs or their derivative cells. As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that induces a desired biological or medical response in a subject. The therapeutically effective amount can be determined empirically and conventionally according to the purpose.

[0439] As used herein, with respect to the cells and / or pharmaceutical compositions of the present application, a therapeutically effective amount refers to the amount of the cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0440] According to specific embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition being treated, or symptoms associated therewith; (ii) reduce the duration of the disease, disorder or condition being treated, or symptoms associated therewith; (iii) prevent the progression of the disease, disorder or condition being treated, or symptoms associated therewith; (iv) cause regression of the disease, disorder or condition being treated, or symptoms associated therewith; (v) prevent the development or onset of the disease, disorder or condition being treated, or symptoms associated therewith; (vi) prevent recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospital stay of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increase survival of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder or condition being treated, or symptoms associated therewith, in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect of another therapy.

[0441] The therapeutically effective amount or dosage can vary depending on various factors, such as the disease, disorder or condition to be treated, the mode of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0442] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to the subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous or intramuscular administration.

[0443] The cell of the present application and / or the pharmaceutical composition of the present application can be administered in any convenient manner known to those skilled in the art. For example, the cell of the present application can be administered to a subject by aerosol inhalation, injection, uptake, infusion (transfusion), implantation and / or transplantation. The composition comprising the cell of the present application can be administered via artery, subcutaneous, intradermal, intratumor, intranodal, intramedullary, intramuscular, intrapleural, by intravenous (iv) injection or intraperitoneal administration. In certain embodiments, the cell of the present application can be administered with or without lymphocyte removal (lymphodepletion) of the subject.

[0444] Pharmaceutical compositions comprising the cells of the present application can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally emulsions, dispersions, etc., typically buffered to a selected pH. The composition can include a carrier, e.g., water, saline, phosphate-buffered saline, etc., suitable for the integrity and viability of the cells, and suitable for administration of the cell composition.

[0445] Sterile injectable solutions can be prepared as needed by incorporating the cells of the present application into an appropriate amount of suitable solvents containing various other ingredients. Such compositions can include pharmaceutically acceptable carriers, diluents or excipients such as sterile water, normal saline, glucose, dextrose, etc., suitable for use with cell compositions, and suitable for administration to subjects such as people. Suitable buffers for providing cell compositions are well known in the art. Any vehicle, diluent or additive used is compatible with the integrity and vigor of the cells maintaining the present application.

[0446] The cell of the present application and / or the pharmaceutical composition of the present application can be administered in any physiologically acceptable vehicle. The cell group comprising the cell of the present application can include a purified cell group. Those skilled in the art can easily determine the cell in the cell group using various well-known methods. The purity range of the cell group comprising the genetically modified cell of the present application can be from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95% or from about 95% to about 100%. Those skilled in the art can easily adjust dosage, for example, purity reduction may require dosage increase.

[0447] The cells of the present application are generally administered at a dose based on cells / kilogram (cell / kg) of subject body weight, and the cells and / or pharmaceutical compositions comprising the cells are administered to the subject. Generally, the cell dose is about 10 4 to about 10 10 cells / kg body weight, for example, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 or about 10 5 to about 10 6 , depending on the route and site of administration. Generally speaking, in the case of systemic administration, the dose used is higher than regional administration, where the immune cells of the present application are administered in the area of ​​the tumor and / or cancer. Exemplary dosage ranges include, but are not limited to, 1 x 10 4 -1x 10 8、2x10 4 -1x 10 8 、3x 10 4 -1x 10 8 、4x 10 4 -1x 10 8 、5x 10 4 -6x 10 8 、7x 10 4 -1x 10 8 、8x 10 4 -1x 10 8 、9x 10 4 -1x 10 8 、1x 10 5 -1x 10 8 、1x 10 5 -9x 10 7 、1x 10 5 -8x 10 7 、1x 10 5 -7x 10 7 、1x 10 5 -6x10 7 、1x 10 5 -5x 10 7 、1x 10 5 -4x 10 7 、1x 10 5 -4x 10 7 、1x 10 5 -3x 10 7 、1x 10 5 -2x 10 7 、1x 10 5 -1x 10 7 、1x 10 5 -9x 10 6 、1x 10 5 -8x 10 6 、1x 10 5 -7x 10 6 、1x 10 5 -6x 10 6 、1x 10 5 -5x 10 6 、1x10 5 -4x 10 6 、1x 10 5 -4x 10 6 、1x 10 5 -3x 10 6 、1x 105 -2x 10 6 , 1x 10 5 -1x 10 6 , 2x 10 5 -9x 10 7 , 2x 10 5 -8x 10 7 , 2x 10 5 -7x 10 7 , 2x 10 5 -6x 10 7 , 2x 10 5 -5x 10 7 , 2x 10 5 -4x 10 7 , 2x 10 5 -4x10 7 , 2x 10 5 -3x 10 7 , 2x 10 5 -2x 10 7 , 2x 10 5 -1x 10 7 , 2x 10 5 -9x 10 6 , 2x 10 5 -8x 10 6 , 2x 10 5 -7x 10 6 , 2x 10 5 -6x 10 6 , 2x 10 5 -5x 10 6 , 2x 10 5 -4x 10 6 , 2x 10 5 -4x 10 6 , 2x 10 5 -3x 10 6 , 2x10 5 -2x 10 6 , 2x 10 5 -1x 10 6 , 3x 10 5 -3x 10 6 Cells / kg, etc. In addition, the dose can be adjusted to take into account whether a single dose is administered or whether multiple doses are administered. What is considered an effective dose can be accurately determined based on individual factors for each subject.

[0448] As used herein, the terms "treat, treating, treatment" all mean to improve or reverse at least one measurable physical parameter associated with cancer, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms "treat, treating, treatment" may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a specific embodiment, "treat, treating, treatment" refers to alleviating, preventing the development or onset of one or more symptoms associated with a disease, disorder, or condition (such as a tumor or more preferably cancer), or reducing the duration. In a specific embodiment, "treat, treating, treatment" refers to preventing the recurrence of a disease, disorder, or condition. In a specific embodiment, "treat, treating, treatment" refers to increasing the survival of a subject suffering from a disease, disorder, or condition. In a specific embodiment, "treat, treating, treatment" refers to eliminating a disease, disorder, or condition in a subject.

[0449] The cell of the present application and / or the pharmaceutical composition of the present application can be co-administered with one or more additional therapeutic agents. In certain embodiments, one or more therapeutic agents are selected from peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), siRNAs, oligonucleotides, mononuclear blood cells, carriers comprising one or more polynucleic acids of interest, antibodies, chemotherapeutics or radioactive moieties or immunomodulatory drugs (IMiD).

[0450] Implementation

[0451] This application provides the following non-limiting embodiments.

[0452] Example

[0453] Example 1. mbIL-12 cell line development

[0454] iPSC development

[0455] Induced pluripotent stem cell (iPSC) parental cell lines were generated from peripheral blood mononuclear cells (PBMCs) using an episomal plasmid-based process as 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.

[0456] Vector (plasmid) production

[0457] Gene fragments (gBlocks) encoding the transgene of interest, as well as promoters, terminators, and homology arms were designed and synthesized by chemical synthesis at IDT. The gBlock gene fragments were assembled into pUC19 plasmids using Cloning HD Plus kit (Takara Bio; Shiga, Japan) according to the manufacturer's protocol. The reaction products from In-Fusion Cloning, i.e., expression constructs, were transformed into Stbl3 bacterial cells (Thermo Fisher; Waltham, Massachusetts) for amplification according to the manufacturer's protocol. The vectors (plasmids) from the amplified expression constructs were purified from bacterial cell cultures using HiSpeed ​​Plasmid Maxi Prep kit (Qiagen; Hilden, Germany) according to the manufacturer's protocol. The purified plasmid DNA was sequenced at the research level and evaluated by restriction digestion to confirm the transgenic sequence. The concentration of the purified plasmid DNA was measured by absorbance. In addition, the absorbance ratios of A260 / A280 nm and A260 / A230 nm were measured to evaluate residual RNA and protein levels, respectively.

[0458] AAVS1 targeting plasmid

[0459] DNA fragments from IDT gene-blocks for each membrane IL12 variant were cloned into the p1392 vector for site-specific insertion of the transgene into the AAVS1 locus, allowing strong expression using the CAG promoter and SV40 terminator. The AAVS1 targeting plasmid contained the CAG promoter (SEQ ID NO: 63), SV40 terminator / polyadenylation (SEQ ID NO: 64) and either mbIL-12 (SEQ ID NO: 97) or a membrane-bound IL-12p70 ADAM17 protease cleavage site fusion protein (SEQ ID NO: 99).

[0460] Establishment of mbIL-12 iPSC cell line

[0461] The p1392 AAVS1 homology-directed repair (HDR) vector specifically targets intron 1 of the AAV1 locus (PPP1R12C gene) for site-specific integration of the transgene within the cell and allows for Geneticin antibiotic selection of the forward engineered cells. The HDR vector containing two membrane IL12 forms was electroporated into iPSC005 cells together with the Cpf1 / AAVS1 gRNA ribonucleoprotein complex to promote site-specific integration of the transgene.

[0462] After 4 days of culture, the cells were incubated with 500 ug / ml of Geneticin to kill cells that failed to successfully integrate the transgene into the AAVS1 locus. Surviving cells (indicating that the transgene was correctly inserted) were expanded and analyzed by flow cytometry for membrane IL12 expression. Figure 2A -C.

[0463] Subsequently, the cells were differentiated into HPCs and then into iNK cells for functional characterization.

[0464] Figure 3 IL12 surface detection at day 9 of the HPC stage is shown.

[0465] PCR confirmation of p1514 transgene insertion in iPSCs:

[0466] based on Figure 4A Based on the genome map in the literature, PCR primers were designed to amplify the tmIL12 sequence in the genomic DNA of iPSC1294 cells.

[0467] Primers 1514 Forward and 1514R will amplify a band of ∼1700 bp, while primers 1514 Forward and 1514R2 will amplify a band of ∼600 bp. Figure 4B The results confirmed the presence of transgenes in iPSCs.

[0468] In addition, a junction PCR was performed to confirm that the transgene was inserted into the correct locus (see, e.g. Figure 5A genome map in ).

[0469] The results are as follows Figure 5B shown.

[0470] Fig. 6A IL12 surface detection at day 14 of the iNK stage is shown.

[0471] Figure 6B IL12 surface detection at day 21 of the iNK stage is shown.

[0472] During differentiation to day 21 iNK cells, the expression of the transgene decreased.

[0473] To verify that ADAM17 can cleave membrane-tethered IL12 (p1514; Figure 1B ) in engineered cells, iPSCs were incubated with different concentrations of the ADAM17 inhibitor TAPI-1, and then the detection of IL12 on the surface of engineered iPSCs was analyzed by flow cytometry.

[0474] Figure 7Shown is the effect of 50uM TAPI-1 on tmIL12 expression. Lower concentrations had no effect on cell health or tmIL12 detection.

[0475] Example 2. Cytokine-enhanced cytotoxicity assay

[0476] Interleukin-12 is a cytokine that stimulates T cells and natural killer (NK) cells to produce interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To determine whether IL-12 has an effect on the target cytotoxicity of CAR / IL-15iNK cells, iNK cells were differentiated according to the standard protocol (without IL-12) or with the addition of 10 ng / ml recombinant human IL-12p70 (PeproTech; Rocky Hill, NJ) during the last 24 hours of culture. iNK cells were used in the Incucyte killing assay to determine the killing efficacy against the Raji CD19+ B cell leukemia cell line (ATCC; Manassas, VA). Cells primed with IL-12 exhibited more rapid killing of Raji cells (P < 0.05) compared to those differentiated in the absence of IL-12. Fig. 8A ).

[0477] The effect of IL-12-primed iNK cells on in vivo tumorigenesis was further tested. On day 0 of the study, luciferase-labeled Burkitt's lymphoma cell line Raji was intravenously (iv) implanted into female NSGs. TM On days 1, 4, and 7 of the study, mice were intravenously infused with 1x10 7 The mice were supplemented with recombinant human IL-2 (100,000 IU, PeproTech #200-02) intraperitoneally three times a week starting from day 1 during the study. The untreated group served as a control. The mice were injected with luciferin (VivoGlo) before imaging using IVIS SpectrumCT (Perkin Elmer). TM , Promega). The reaction of luciferin substrate with firefly luciferase produced by Raji tumor cells produces light measured as a bioluminescent signal. Data are expressed as mean whole body bioluminescence mean radiance ± SD. At the end of the study on day 20, 50% and 62% tumor growth inhibition was observed for unprimed and IL-12 primed iNK treatment, respectively (*p<0.05, **p<0.01) ( Figure 8B ).

Claims

1. An induced pluripotent stem cell (iPSC) or a cell derived therefrom, comprising: (i) an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, The first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, The second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, The transmembrane domain is fused to the terminus of the first and / or second IL-12 subunit polypeptide. 2 . The iPSC or a cell derived therefrom according to claim 1 , wherein a polynucleotide encoding membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for inducing release of the IL-12 by activation of the protease ADAM17.

3. The iPSC or a cell derived therefrom according to claim 1, 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, The first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, The second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, The transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes.

4. The iPSC or a cell derived therefrom according to claim 1, 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide and a second polypeptide, The first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, The second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, The polynucleotide is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide, for inducing the release of the IL-12 by activation of the protease ADAM17; and (iii) deletion or reduced expression of one or more of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes. 5 . The iPSC or a cell derived therefrom according to claim 1 , further comprising a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G). The iPSC or a cell derived therefrom according to claim 1 or 2, further comprising an exogenous polynucleotide encoding a safety switch.

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

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

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

10. The iPSC or a cell derived therefrom according to claim 9, wherein the transmembrane domain TM It is further fused to the signaling domain (SD).

11. The iPSC or cell derived therefrom of claim 10, wherein the signaling domain is a CD3ζ, CD28 and / or 4-1BB signaling domain.

12. An induced pluripotent stem cell (iPSC), natural killer (NK) cell or T cell, 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), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, The first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, The second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, The transmembrane domain is fused to the end of the first and / or second IL-12 subunit polypeptide, or 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) an optional fourth exogenous polynucleotide encoding a safety switch; and (v) an optional fifth exogenous polynucleotide encoding a cytokine; One or more of the exogenous polynucleotides are integrated into the gene loci of CIITA and B2M genes, thereby causing the deletion or reduced expression of CIITA and B2M.

13. An iPSC, a natural killer (NK) cell or a T cell, comprising (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding: i. membrane-bound interleukin 12 (IL-12) having an 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 an 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 an amino acid sequence of SEQ ID NO: 108; iv. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 110; v. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 112; vi. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 114; vii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 116; viii. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 118; ix. Membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 120; x. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 122; or xi. membrane-bound IL-12 fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide having an amino acid sequence of SEQ ID NO: 124; (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; and (iv) optionally, a fourth exogenous polynucleotide encoding the IL-15 protein of SEQ ID NO: 72; One or more of the exogenous polynucleotides are integrated into the gene loci of CIITA and B2M genes, thereby causing the deletion or reduced expression of CIITA and / or B2M.

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

15. The iPSC or derived cell of claim 1 or 2, wherein one or more of the exogenous polynucleotides are integrated at one or more loci on a chromosome of the cell, the loci being selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCRa or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, with the proviso that at least one of the exogenous polynucleotides is integrated at a locus selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby resulting in the deletion or reduced expression of the genes.

16. The iPSC or derived cell of claim 1 or 2, wherein (i) one or more of the exogenous polynucleotides are integrated into the loci of CIITA, AAVS1 and B2M genes, (ii) one or more of the exogenous polynucleotides are integrated at the loci of CIITA, CLYBL and B2M genes.

17. The iPSC or derived cell of any one of claims 1-15, wherein the CAR comprises: (i) a signal peptide, (ii) an extracellular domain comprising a binding domain that specifically binds to an antigen, (iii) hinge region, (iv) transmembrane domain, (v) an intracellular signaling domain, and (vi) a costimulatory domain, such as a costimulatory domain comprising a CD28 signaling domain.

18. The iPSC or derived cell of claim 17, wherein the signal peptide is a GMCSFR signal peptide.

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

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

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

22. The iPSC or derivative cell of claim 17, wherein the intracellular signaling domain comprises a CD3 zeta intracellular domain.

23. The iPSC or derivative cell of claim 17, wherein the co-stimulatory domain comprises a CD28 signaling domain.

24. The iPSC or derivative cell of claim 17, wherein the CAR comprises: (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; and (v) a 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.

25. The iPSC or derivative cell of claim 17, wherein 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 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) 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 costimulatory domain comprising the amino acid sequence of SEQ ID NO:

20.

26. The iPSC or derived cell of claim 14, wherein the inactivated cell surface protein is selected from a monoclonal antibody specific epitope selected from an epitope specifically recognized by ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, velizumab, 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.

27. The iPSC or derived cell of claim 26, wherein the inactivated cell surface protein is a truncated epidermal growth factor (tEGFR) variant.

28. The iPSC or derived cell of claim 26, wherein the autologous protease peptide sequence is porcine Teschovirus type 1 2A (P2A).

29. The iPSC or derivative cell of claim 26, wherein the 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 SEQ ID NO:

71.

30. The iPSC or derivative cell of any one of claims 1-15, wherein 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.

31. The iPSC or derivative cell of claim 28, wherein the autoprotease 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.

32. The iPSC or derivative cell of 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 of any one of claims 1-32, wherein the derivative cell is a Natural Killer (NK) cell or a T cell.

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

35. The composition of claim 34, further comprising or used in combination with one or more therapeutic agents selected from peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), siRNAs, oligonucleotides, mononuclear blood cells, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

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

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

38. The method of 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, and non-Hodgkin lymphoma (NHL), Hodgkin lymphoma / disease (HD), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

39. A method for preparing the derivative cell of claim 33, comprising differentiating the iPSC cell of claim 1 or 2 under cell differentiation conditions, thereby obtaining the derivative cell.

40. The method of claim 39, wherein the iPSC is obtained by genome engineering iPSC, wherein the genome engineering comprises targeted editing.

41. The method of claim 39, wherein the iPSC is edited by targeted editing comprising deletion, insertion, or insertion / deletion by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

42. A CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC), comprising: An exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12), wherein the membrane-bound interleukin 12 (IL-12) comprises a first polypeptide, a second polypeptide and a transmembrane domain, The first polypeptide comprises IL-12α subunit p35 or a polypeptide at least 90% similar thereto, The second polypeptide comprises IL-12β subunit p40 or a polypeptide at least 90% similar thereto, The transmembrane domain is fused to the terminus of the first and / or second IL-12 subunit polypeptide.

Images