Genetically engineered cells and uses thereof

Abstract

Genetically engineered induced pluripotent stem cells (iPSCs) and their derivatives that express chimeric antigen receptors (CARs) and methods of using same are provided. Compositions, polypeptides, vectors, and methods of production are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 120,799, filed December 3, 2020, U.S. Provisional Patent Application No. 63 / 120,948, filed December 3, 2020, and U.S. Provisional Patent Application No. 63 / 120,980, filed December 3, 2020, each of which is incorporated by reference herein in its entirety.

[0002] Technical Field This application provides genetically engineered induced pluripotent stem cells (iPSCs) and their derivatives. Use of iPSCs or their derivatives to express chimeric antigen receptors for allogeneic cell therapy is also provided. Related vectors, polynucleotides, and pharmaceutical compositions are also provided.

[0003] Reference to an electronically submitted sequence listing This application contains a Sequence Listing that has been submitted electronically via EFS-Web as a Sequence Listing in ASCII format having a size of 113 kb with filename "CNTY-001-WO-01_SequenceListing_ST25" and a creation date of November 1, 2021. The Sequence Listing submitted via EFS-Web is a part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Chimeric antigen receptors (CARs) significantly enhance the antitumor activity of immune effector cells. CARs are engineered receptors that typically contain an extracellular targeting domain linked to a linker peptide, a transmembrane (TM) domain, and one or more intracellular signaling domains. Traditionally, the extracellular domain consists of an antigen-binding fragment of an antibody (e.g., single-chain Fv, scFv) specific for a given tumor-associated antigen (TAA) or cell surface target. The extracellular domain confers tumor specificity to the CAR, while the intracellular signaling domain activates T cells engineered to express the CAR upon TAA / target engagement. The engineered immune effector cells are reinfused into the cancer patient, where they specifically associate with and kill cells expressing the CAR's TAA target (Maus et al., Blood. 2014 Apr 24;123(17):2625-35; Curran and Brentjens, J Clin Oncol. 2015 May 20;33(15):1703-6).

[0005] Autologous, patient-specific CAR-T therapy has emerged as a powerful and potentially curative treatment for cancer, particularly for CD19-positive hematologic malignancies. However, autologous T cells must be generated on a custom-made 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 important drawbacks, including a) inefficient antitumor responses in solid tumors, b) limited penetration and sensitivity 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.

[0006] Thus, there is an unmet need for therapeutically sufficient and functional antigen-specific immune cells for effective use in immunotherapy. Summary of the Invention

[0007] In one general aspect, a genetically engineered induced pluripotent stem cell (iPSC) or a derivative thereof is provided, comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope, preferably a truncated epidermal growth factor receptor (tEGFR) variant, and interleukin-15 (IL-15), wherein the inactivated cell surface receptor and IL-15 are operably linked by an autoprotease peptide, such as the autoprotease peptide of the porcine teschovirus-1 2A (P2A) peptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably deletion or reduced expression of the B2M and CIITA genes.

[0008] Also provided is an iPSC cell, or a derivative thereof, comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) that targets the CD19 antigen; (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide, such as the autoprotease peptide of porcine teschovirus-1 2A (P2A) peptide; and (iii) a deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, preferably a deletion or reduced expression of the B2M and CIITA genes.

[0009] In certain embodiments, the iPSC cell or a derivative thereof further comprises a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) or human leukocyte antigen G (HLA-G).

[0010] In certain embodiments, one or more of the exogenous polynucleotides are integrated into one or more loci on a cellular chromosome, preferably the one or more loci are selected from the group consisting of AAVS1, 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 proviso that at least one of the exogenous polynucleotides is integrated into the locus of a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, wherein the integration results in deletion or reduced expression of the gene; more preferably, one or more of the exogenous polynucleotides is integrated into the locus of the CIITA, AAVS1, and B2M genes, wherein the integration results in deletion or reduced expression of one or more of the CIITA and B2M genes.

[0011] In certain embodiments, iPSCs are reprogrammed from whole peripheral blood mononuclear cells (PBMCs).

[0012] In certain embodiments, the iPSCs are derived from reprogrammed T cells.

[0013] In certain embodiments, the CAR comprises: (i) a signal peptide, such as a signal peptide that includes or is a GMCSFR signal peptide; (ii) an extracellular domain that includes a binding domain that specifically binds to a CD19 antigen; (iii) a hinge region, such as a hinge region that includes a CD28 hinge region; (iv) a transmembrane domain, such as a transmembrane domain that includes a CD28 transmembrane domain; (v) an intracellular signaling domain, such as an intracellular signaling domain that includes a CD3ζ intracellular domain; and (vi) a costimulatory domain, such as a costimulatory domain that includes a CD28 signaling domain.

[0014] In certain embodiments, the extracellular domain comprises an scFv derived from an antibody that specifically binds to the CD19 antigen.

[0015] In certain embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to an antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain, such as a costimulatory domain comprising a CD28 signaling domain.

[0016] In certain embodiments, the signal peptide comprises or is a GMCSFR signal peptide.

[0017] In certain embodiments, the extracellular domain comprises a VHH domain.

[0018] In certain embodiments, the hinge region comprises a CD28 hinge region.

[0019] In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0020] In certain embodiments, the intracellular signaling domain comprises a CD3ζ intracellular domain.

[0021] In certain embodiments, the costimulatory domain comprises a CD28 signaling domain.

[0022] In certain embodiments, the CAR is (i) a signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; (ii) an extracellular 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: 7; (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 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; (v) 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 (vi) 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. Includes.

[0023] In certain embodiments, the CAR is (i) a signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; (ii) a hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22; (iii) a transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24; (iv) an intracellular signaling domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; 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. Includes.

[0024] 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 an 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.

[0025] In certain embodiments, the CAR comprises: (i) a signal peptide comprising the amino acid sequence of SEQ ID NO: 1; (ii) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 7; (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.

[0026] In certain embodiments, the inactivated cell surface protein is selected from the group consisting of ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. and selected from the group of monoclonal antibody-specific epitopes selected from epitopes specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab.

[0027] In certain embodiments, the inactivated cell surface protein is a truncated epidermal growth factor (tEGFR) variant.

[0028] In certain embodiments, the tEGFR variant has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71. Preferably, the tEGFR variant has or consists of the amino acid sequence of SEQ ID NO: 71.

[0029] In certain embodiments, the IL-15 has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72. Preferably, the IL-15 comprises the amino acid sequence of SEQ ID NO: 72.

[0030] In certain embodiments, the autoprotease peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73. Preferably, the autoprotease peptide has the amino acid sequence of SEQ ID NO: 73.

[0031] In certain embodiments, the iPSCs or derivatives have a deletion or reduced expression of one or more of the B2M and / or CIITA genes.

[0032] 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 the IL-15 comprises the amino acid sequence of SEQ ID NO:72.

[0033] In certain embodiments, the 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, the HLA-E has the amino acid sequence of SEQ ID NO: 66.

[0034] In certain embodiments, the 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, the HLA-G has the amino acid sequence of SEQ ID NO: 69.

[0035] In certain embodiments, the genetically engineered iPSCs or derivatives thereof are (1) a first exogenous polynucleotide encoding a CAR having (i) a signal peptide comprising the amino acid sequence of SEQ ID NO: 1; (ii) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 7; (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; and (2) A second exogenous polynucleotide encoding a tEGFR variant consisting of the amino acid sequence of SEQ ID NO: 71 and IL-15 comprising the amino acid sequence of SEQ ID NO: 72, wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide comprising the amino acid sequence of SEQ ID NO: 73. Including, Here, the first and second exogenous polynucleotides are integrated into the locus of two genes selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably the B2M and CIITA genes, and this integration results in deletion or reduced expression of the two genes.

[0036] In certain embodiments, (i) 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: 75; and (ii) 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.

[0037] In certain embodiments, a first exogenous polynucleotide is integrated into the locus of the AAVS1 gene; (i) a second exogenous polypeptide is integrated into the locus of the CIITA gene; and (ii) a third exogenous polypeptide is integrated into the locus of the B2M gene; wherein integration of the exogenous polynucleotides deletes or reduces the expression of CIITA and B2M, and preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75 and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0038] In certain embodiments, the derivative cells are natural killer (NK) cells or T cells.

[0039] Optionally, the genetically engineered iPSCs or derivative cells thereof further comprise a third exogenous polynucleotide encoding HLA-E having the amino acid sequence of SEQ ID NO: 66 or HLA-G having the amino acid sequence of SEQ ID NO: 69. Preferably, the third exogenous polynucleotide is integrated into the locus of a gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H1 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 genes, preferably the AAVS1 gene.

[0040] In certain embodiments, the first 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: 62. 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: 75. 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.

[0041] In certain embodiments, the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0042] In certain embodiments, a first exogenous polynucleotide is integrated into the locus of the AAVS1 gene; a second exogenous polynucleotide is integrated into the locus of the CIITA gene; and a third exogenous polynucleotide is integrated into the locus of the B2M gene; wherein integration of the exogenous polynucleotides deletes or reduces expression of the CIITA and B2M genes, and preferably, the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0043] In certain embodiments, the derivative cells are natural killer (NK) cells or T cells.

[0044] (i) a first exogenous polynucleotide encoding a CAR 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: 61; (ii) a second exogenous polynucleotide encoding a tEGFR variant comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71, an autoprotease 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: 73, and IL-15 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, wherein the tEGFR is operably linked to the IL-15 via the autoprotease peptide; and (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) 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: 66, or HLA-G 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: 69. Also provided are induced pluripotent stem cells (iPSC) cells or iPSC-derived natural killer (NK) cells or T cells (i.e., iNK or iT) comprising: wherein the first, second and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA and B2M genes, thereby deleting or reducing the expression of the CIITA and B2M genes.

[0045] In certain embodiments, the iPSCs, NK cells, or T cells of the present application are (i) a first exogenous polynucleotide encoding a CAR having the amino acid sequence of SEQ ID NO: 61; (ii) a second exogenous polynucleotide encoding a tEGFR variant having or consisting of the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and an IL-15 having the amino acid sequence of SEQ ID NO: 72; and (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66 Including, wherein the first, second and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA and B2M genes, respectively, thereby deleting or reducing the expression of the CIITA and B2M genes.

[0046] In certain embodiments, the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0047] (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin-15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) optionally, a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66. Also provided are iPSCs, natural killer (NK) cells, or T cells comprising: wherein the first, second and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA and B2M genes, thereby deleting or reducing the expression of CIITA and B2M.

[0048] In certain embodiments, (i) the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and (ii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67, and the first, second, and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA, and B2M genes, respectively.

[0049] Compositions comprising the cells of the present application are also provided.

[0050] In certain embodiments, the compositions of the present application may further comprise or be used in combination with one or more other therapeutic agents, including, but not limited to, peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), siRNAs, oligonucleotides, mononuclear blood cells, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

[0051] Also provided is a method of treating cancer in a subject in need thereof, comprising administering to a subject in need thereof a cell of the present application or a composition of the present application.

[0052] In certain embodiments, the cancer is non-Hodgkin's lymphoma (NHL).

[0053] Also provided is a method for producing a derivative cell of the present application, the method comprising differentiating an iPSC of the present application under conditions for cell differentiation, thereby obtaining a derivative cell.

[0054] The present application also provides a method for obtaining genetically engineered iPSCs, comprising introducing a first, second, and optionally a third exogenous polynucleotide into iPSC cells, thereby obtaining genetically engineered iPSCs. The genetically engineered iPSCs of the present application can be obtained using any genetic engineering method. Preferably, the genetic engineering includes targeted editing, and more preferably, the targeted editing includes deletion, insertion, or in / del, where the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0055] Also provided is a method for differentiating induced pluripotent stem cells (iPSCs) into NK cells by subjecting the cells to a differentiation protocol that includes the addition of recombinant human IL-12 for the final 24 hours of culture. Preferably, the recombinant IL-12 comprises or is IL12p70.

[0056] Also provided are: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope and interleukin-15 (IL-15), wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide; and (iii) CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs) comprising a deletion or reduced expression of one or more of the following genes: B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP.

[0057] Other embodiments of the present application include genetically engineered iPSCs or derivatives thereof for use in treating cancer in a subject in need thereof.

[0058] In some embodiments, engineered iPSCs or derived cells of the present invention have improved persistence, increased immune cell resistance, or increased immune resistance; or genomically engineered iPSCs may have increased resistance to T and / or NK cells. In particular, the IL-15 transgene of the present invention, when transfected into iPSCs according to the present invention and differentiated into NK cells, exhibits increased persistence, reduced attrition, and increased serial killing when compared to NK cells derived from iPSCs without the IL-15 transgene of the present invention. Genomically engineered iPSCs of the present invention have the potential to differentiate into non-pluripotent cells, including hematopoietic cells, with the same functional targeted genome editing. In some embodiments, genomically engineered iPSCs of the present invention have the potential to differentiate into mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0059] In one general aspect, a polynucleotide encoding an artificial cell death polypeptide is provided. In certain embodiments, the polynucleotide encodes an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope and interleukin-15 (IL-15), wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide.

[0060] In certain embodiments, the inactivated cell surface receptor is selected from the group consisting of ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. and selected from the group of monoclonal antibody-specific epitopes selected from epitopes specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab.

[0061] In certain embodiments, the inactivated cell surface receptor is a truncated epidermal growth factor receptor (tEGFR) variant.

[0062] In certain embodiments, the autoprotease peptide comprises or is the porcine teschovirus-1 2A (P2A) peptide.

[0063] In certain embodiments, 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, preferably the amino acid sequence of SEQ ID NO: 71.

[0064] In certain embodiments, the IL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72.

[0065] In certain embodiments, 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, preferably the amino acid sequence of SEQ ID NO: 73.

[0066] In certain embodiments, the polynucleotide consists of operably linked polynucleotides encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin-15 (IL-15) having the amino acid sequence of SEQ ID NO: 72.

[0067] Also provided is a polynucleotide encoding an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody selected from the group consisting of cetuximab, matuzumab, necitumumab, panitumumab, polatuzumab vedotin, rituximab, and trastuzumab, and IL-15, wherein the epitope and cytokine are operably linked by a P2A sequence.

[0068] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 79, 81, and 83.

[0069] Proteins encoded by the polynucleotides of the present application are also provided.

[0070] Also provided are induced pluripotent stem cells (iPSCs) or derivatives thereof comprising a polynucleotide of the present application.

[0071] Also provided are vectors comprising the polynucleotides of the present application.

[0072] In certain embodiments, the vector comprises: (i) promoters; (ii) terminator and / or polyadenylation signal sequences; (iii) the left-hand homologous sequence; and (iv) Right homologous sequence Further includes:

[0073] In certain embodiments, the left-hand homologous sequence comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:84.

[0074] In certain embodiments, the right homologous sequence comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:85.

[0075] In certain embodiments, the vector comprises a polynucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:86.

[0076] 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 accompanying drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Brief explanation of the drawings]

[0077] [Figure 1-1]Schematic diagrams showing vectors (plasmids) according to embodiments of the present application. Figure 1A shows a CIITA-targeting transgene plasmid with a CMV early enhancer / chicken β-actin (CAG) promoter, an SV40 terminator / polyadenylation signal, and a tEGFR-IL15 coding sequence. Figure 1B shows an AAVS1-targeting transgene plasmid with a CAG promoter, an SV40 terminator / polyadenylation signal, and an anti-CD19 scFv chimeric antigen receptor (CAR) coding sequence. Figure 1C shows a B2M-targeting transgene plasmid with a CAG promoter, an SV40 terminator / polyadenylation signal, and a peptide-B2M-HLA-E coding sequence. [Figure 1-2] (As mentioned above.) [Figure 2] Graph demonstrating CAR-iNK cell-mediated target cell cytotoxicity over time in Reh cells and CD19 knockout (CD19KO) Reh cells. [Figure 3-1] Graphs showing the functionality of iNK cells expressing CAR-IL15 compared to iNK cells expressing CAR alone. Figure 3A shows a graph showing the concentration of IL-15 (pg / ml / 1x1e6 cells / 24 hours) released from CAR iNK cells and CAR / IL15 iNK cells. Figure 3B shows a graph showing the percentage of iNK cells in the blood and lungs of mice injected with CAR iNK cells or CAR-IL15 iNK cells after 20 days. Figure 3C shows a graph showing the percentage of iNK cells in the lungs of mice injected with CAR iNK cells or CAR-IL15 iNK cells with and without recombinant IL-15. [Figure 3-2] (As mentioned above.) [Figure 4-1]Figure 4A shows graphs depicting the proliferation and serial killing of CAG-CAR-IL-15 iNK cells. Figure 4A shows graphs depicting the serial killing of CD19+ Reh cells over time by CAG-CAR / IL15-iNK cells. Figure 4B shows graphs depicting increased proliferation of CAG-CAR / IL-15 iNK cells compared to CAG-CAR iNK cells. Figure 4C shows graphs depicting increased target serial killing of CD19+ Raji cells over time by CAG-CAR / IL-15 iNK cells compared to CAG-CAR iNK cells. [Figure 4-2] (As mentioned above.) [Figure 5] Figure 5A shows graphs depicting the cytotoxicity of CAG-CAR-IL15-expressing iNK cells in the presence and absence of human recombinant IL12. Figure 5A shows graphs depicting the killing of Raji cells over time when cultured with CAG-CAR-IL15 iNK cells in the presence and absence of IL12. Figure 5B shows graphs depicting tumor growth, measured as mean whole-body luminescence average brightness, in mice injected with IL12-primed and unprimed CAG-CAR-IL15 iNK cells. [Figure 6] Figure 6 shows graphs showing cetuximab-induced cell elimination in CAG-CAR-expressing iNK cells and CAG-CAR-IL15-tEGFR-expressing iNK cells. Figure 6A shows graphs showing the percentage of Annexin-V staining in CAG-CAR-expressing cells. Figure 6B shows graphs showing the percentage of Annexin-V staining in CAG-CAR-IL15-tEGFR-expressing cells. [Figure 7] Graphs showing an Incucyte-based assay measuring the reduction of Nuclight Red K562 cells over time at effector-to-target ratios of (A) 20:1, (B) 10:1, and (C) 1:1. Normalized target cell counts as a percentage of target cell-only counts for four iNK1248-iPSC611 and three PB-NK averages. Each data point is the average of three replicates, and error bars represent the standard error of the mean. [Figure 8]1 is a graph showing a flow-based NK purity check of PB-NK and iNK1248-iPSC611 isolated from three PBMC donors. [Figure 9] Graph showing an Incucyte-based assay measuring the reduction of Nuclight Red target cells over time at four effector-to-target ratios. Normalized target cell counts as a percentage of target cell-only counts in Reh and Reh-CD19KO cells cocultured with iNK1248-iPSC611 at effector-to-target ratios of (A) 10:1, (B) 5:1, (C) 1:1, and (D) 1:5. Each data point is the average of three replicates, and error bars represent the standard error of the mean. [Figure 10] Graph showing an Incucyte-based assay measuring the reduction of Nuclight Red target cells over time at four effector-to-target ratios. Normalized target cell counts as a percentage of target cell-only counts in NALM6 and NALM6-CD19KO cells cocultured with iNK1248-iPSC611 at effector-to-target ratios of (A) 10:1, (B) 5:1, (C) 1:1, and (D) 1:5. Each data point is the average of three replicates, and error bars represent the standard error of the mean. [Figure 11] Figure 1 shows the cumulative fold expansion of iNK1248-iPSC611 and WT iNK1487-iPSC005 during a 21-day retention assay without exogenous IL2 support. Cells were cultured in basal NKCM at 37°C and 5% CO2 for 14 days. Every 3-4 days, cells from all conditions were harvested, counted in ViCell Blue, resuspended at 0.5 x e6 / mL in the appropriate medium, and then replated. Cumulative fold change was calculated after 21 days. [Figure 12]This graph shows the cumulative fold expansion of iNK1248-iPSC611 and WT iNK1487-iPSC005 during a 21-day survival assay. Cells were cultured in NKCM containing one of six IL2 concentrations: (A) 10 nM, (B) 3 nM, (C) 1 nM, (D) 0.3 nM, (E) 0.1 nM, or (F) 0 nM, at 37°C and 5% CO2 for 21 days. Every 3–4 days, cells from all conditions were harvested, counted using ViCell Blue, resuspended in the appropriate medium at 0.5 × e6 / mL, and then replated. Cumulative fold change was calculated after 21 days. [Figure 13] Graph showing the gating strategy for the ADCC assay. Cells were gated for lymphocytes, followed by exclusion of doublets, followed by gating for CellTrace Violet (CTV)+ iNK, and finally gating for LIVE / DEAD™ Near-IR+ to determine the % of therapeutic iNK targets killed. FSC-A = forward scatter area, SSC-A = side scatter area, FSC-H = forward scatter height, CTV = CellTrace Violet, NIR = Near-IR. [Figure 14] 1 is a graph showing EGFR staining on therapeutic iNK cells. EGFR PE levels on EGFR-stained therapeutic iNK (black histogram) compared to unstained therapeutic iNK (gray histogram) or unedited WT iNK (dashed line). [Figure 15] Figure 1 shows a graph depicting cetuximab-mediated ADCC of therapeutic iNK cells. Specific cytolysis of therapeutic iNK cells mediated by cetuximab (closed triangles) compared to human IgG1 isotype control (open triangles). IL-2-activated PBMCs were co-cultured with therapeutic iNK at an E:T ratio of 25:1 for 16 hours to determine specific iNK cell killing. Each data point represents the mean of triplicate wells, with error bars ± standard deviation. [Figure 16] 1 is a graph showing the sensitivity of select WT iNK cells to complement cytotoxicity mediated by anti-HLA-ABC Ab. [Figure 17]Figure 1 shows the gating strategy for the allo-evation CTL cytotoxicity and activation assay. Cells were gated on quantitation beads and lymphocytes. Doublets within lymphocytes were excluded, followed by gating on LIVE / DEAD™ Near-IR negativity, followed by CTV to identify iNK cells and TCRαβ to identify T cells. Within T cells, CD4 negative, CD8 positive cells were followed by CD25 to identify activated CD8+ T cells. Key assay parameters, quantitation beads, viable iNK cells, and activated CD8+ T cells are displayed. FSC-A = forward scatter area, SSC-A = side scatter area, FSC-H = forward scatter height, FSC-W = forward scatter width, LD = LIVE / DEAD™ Near-IR, CTV = CellTrace Violet. [Figure 18-1] 18A-B are graphs showing CTL-mediated lysis of iNK cells. Assessment of specific iNK lysis by FACS. Figure 18A shows gating for iNK and T cells. Figure 18B shows specific lysis of iNK cells co-cultured with CTL at a CTL:iNK ratio of 5:1. Each symbol represents one donor; open bars are parental wild-type iNK cells, and shaded bars are edited β2MKO iNK cells. [Figure 18-2] (As mentioned above.) [Figure 19] 19A and 19B are graphs showing activation of iNK-specific CTLs in cocultures. Figure 19A shows histogram plots of CD25 expression on CD8+ T cells. The dashed line indicates T cells cultured alone, the solid open histogram indicates T cells cocultured with parental wild-type iNK cells, and the shaded histogram indicates T cells cocultured with edited β2MKO iNK cells. Figure 19B shows the frequency of activated T cells in cocultures with parental iNK cells (open bars), β2MKO iNK cells (shaded bars), or targetless T cells alone (hatched bars). Each symbol represents one donor. [Figure 20]Graph showing the gating strategy for the allophore cytotoxicity assay. Cells were gated on lymphocytes, followed by exclusion of doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally gating on LIVE / DEAD™ Near-IR+ to determine the % of iNK targets killed. FSC-A = forward scatter area, SSC-A = side scatter area, FSC-H = forward scatter height, CTV = CellTrace Violet, NIR = Near-IR. [Figure 21] Graph showing HLA-E staining on therapeutic iNK cells. HLA-E = open histogram, mouse IgG1 isotype control = gray filled histogram. [Figure 22] 1 is a graph showing NKG2A staining of PBMCs. PBMC samples were gated on viable lymphocytes (data not shown) and then gated on CD3-CD56+ cells ("NK cells"). The frequency of NKG2A-expressing NK cells was then determined based on FMO. [Figure 23] Figure 1 shows the cell death of therapeutic iNK cells (gray bars) compared to WT (black bars) and iNK lacking β2M (white bars). Freshly thawed PBMCs were co-cultured with therapeutic iNK at an E:T ratio of 25:1 in the presence of 10 ng / mL IL-15 for 72 hours, and cell death of edited iNK compared to WT was determined. Each data point is the average of triplicate wells. [Figure 24] Graph showing the mean percent weight change for untreated mice (●) or mice treated intravenously with 10×10 (▽) and 15×10 (◆) (cryostored) iPSC611. Means for treatment groups with ≥50% are plotted. Arrows indicate dosing days. [Figure 25] Graph showing mean whole-body average brightness for untreated mice (●) and mice treated intravenously with 10×106 (▽) and 15×106 (◆) (cryostored) iPSC611 cells once a week for three doses. Groups are plotted through day 21, the final imaging time point at which untreated controls remained and at which %TGI was calculated. Arrows indicate the day of dosing. [Figure 26] Figure 1 shows the survival rate of NALM6-bearing mice treated with iPSC611. Mice were left untreated or treated intravenously with 10x10 and 15x10 cryopreserved iPSC611 cells once a week for three consecutive days. Mice were humanely euthanized when they became moribund or showed signs of excessive tumor burden, as a surrogate indicator of survival. [Figure 27] Graph showing the retention of iPSC611 in the lungs and blood of NALM6-bearing mice. Mice were left untreated or received a single intravenous injection of 15×106 cryopreserved iPSC611. One week after injection, lungs and blood were collected for FACS analysis. The number of iNKs per 100,000 lymphocytes for individual mice is plotted (circles), and the group mean is represented by a bar. [Figure 28] 10 is a graph showing the average percent weight change of mice treated intravenously with 15×10 6 iPSC611 and given IP administration of PBS (●) or 40 mg / kg cetuximab (□). [Figure 29] This graph shows the presence of iPSC611 in the lungs and blood of NSG mice. Mice were left untreated (untreated) or received a single intravenous dose of 15 x 10 iPSC611 on day 1. On days 2 and 3, mice were treated IP with 20 mL / kg PBS (●) or 40 mg / kg cetuximab (□). All mice received rhIL-2 on days 1 and 3. On day 5, lungs and blood were collected and processed for FACS analysis and detection of iPSC611. There was a significant 96% reduction in iNK cells in the lungs of cetuximab-treated mice (p=0.0002) and a 95% reduction in iNK cells in the blood (p=0.0321). Data are expressed as the number of iNK cells per 100,000 lymphocytes per mouse and plotted with the mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0078] Detailed Description Various publications, articles, and patents are cited or described in the background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, acts, materials, devices, articles and the like which is included within the specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0079] 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. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0080] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0081] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, described herein are understood to be modified in all instances by the term "about." Thus, numerical values ​​typically include ±10% of the recited 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, the use of numerical ranges explicitly includes all individual numerical values ​​within such ranges and fractions of those values, including all possible subranges, integers within the range, unless the context clearly dictates otherwise.

[0082] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize that they will be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the application.

[0083] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variations thereof, are understood to refer to the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be inclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive "or," not an exclusive "or." For example, condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0084] As used herein, the conjunction "and / or" between listed elements is understood to encompass both individual options and combinations of options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first option without the second option. The second option refers to the applicability of the second option without the first option. The third option refers to the applicability of the first option and the second option together. Any one of these options falls within this meaning and is therefore understood to meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within this meaning and is therefore understood to meet the requirements of the term "and / or."

[0085] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates the inclusion of any listed integer or group of integers, but does not allow for additional integers or groups of integers to be added to the specified method, structure, or composition.

[0086] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that does not materially alter the basic or novel nature of the specified method, structure, or composition. See MPEP § 2111.03.

[0087] As used herein, "subject" means any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein 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, and others, more preferably humans.

[0088] It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to a size or characteristic of a preferred inventive component do not delimit the described size / characteristic, but rather exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters include variations that do not vary to the least significant digit using mathematical and industrial principles recognized in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0089] The terms "identical" or "percent identical" in the context of two or more nucleic acid or polypeptide sequences (e.g., CAR polypeptides and CAR polynucleotides that encode them) refer to two or more sequences or subsequences that are the same when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or as determined by visual inspection, or two or more sequences or subsequences that have a specified percentage of amino acid residues or nucleotides that are the same.

[0090] For sequence comparison, typically one sequence acts as reference sequence, and it is compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, if necessary, the coordinate of subsequence is designated, and the parameter of sequence algorithm program is designated.Then, sequence comparison algorithm calculates the sequence identity of test sequence and reference sequence based on designated program parameter.

[0091] Optimal sequence alignment 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 similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (see Ausubel).

[0092] Examples of suitable algorithms for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms 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 published through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned with words of the same length in database sequences, match or meet a certain positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0093] Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted when the cumulative alignment score falls below its maximum achieved value by an amount X; the accumulation of one or more negatively-scoring residue alignments causes the cumulative score to fall below zero; 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program for amino acid sequences uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0094] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0095] Another indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, when two peptides differ only by conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions.

[0096] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, protein, or cell) is substantially separated from, produced separately from, or purified to remove other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues, of the organism in which it naturally occurs. "Isolated" nucleic acids, peptides, proteins, and cells therefore include nucleic acids, peptides, proteins, and cells purified by standard purification methods and those described herein. "Isolated" nucleic acids, peptides, proteins, and cells may be part of a composition, but are still isolated if this composition is not part of the nucleic acid, peptide, protein, or cell's natural environment. The term also encompasses chemically synthesized nucleic acids as well as nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell.

[0097] As used herein, the term "polynucleotide," which is synonymously referred to as "nucleic acid molecule," "nucleotide," "nucleic acid," or "polynucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions 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 backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms of viral and cellular DNA and RNA characteristic of these. "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0098] A "construct" refers to a polymer or molecular complex containing a polynucleotide to be delivered to a host cell either in vitro or in vivo. A "vector," as used herein, refers to any nucleic acid construct capable of directing the delivery or transport of foreign genetic material to a target cell, where the foreign genetic material can be replicated and / or expressed. The term "vector," as used herein, includes the construct to be delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and others.

[0099] By "integration" is meant that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal DNA of the cell. By "targeted integration" is meant that the nucleotides of the construct are inserted into a preselected site or "integration site" of the chromosome or mitochondrial DNA of the cell. The term "integration" as used herein further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of endogenous sequences or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted endogenous sequences or nucleotides with one or more inserted nucleotides.

[0100] As used herein, the term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into or foreign to the host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host genetic material, for example, by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid in an expressible form into a cell. The term "endogenous" refers to a referenced molecule or activity that is present in its native form in a host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is naturally contained within a cell and that has not been exogenously introduced.

[0101] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of an appropriate regulatory sequence, is transcribed into RNA in vivo and optionally translated into a polypeptide. A gene of interest or a polynucleotide may include, but is not limited to, a prokaryotic sequence, a cDNA derived from eukaryotic mRNA, a genomic DNA sequence derived from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may encode an miRNA, an shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a variant of a natural polypeptide that has less than 100% sequence identity with the natural polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0102] "Operably linked" refers to the linkage of nucleic acid sequences into a single nucleic acid fragment, such that the function of one is affected by the other.For example, if a promoter can affect the expression of a coding sequence or functional RNA, the promoter is operably linked to the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter).The coding sequence can be operably linked to the regulatory sequence in sense or antisense direction.

[0103] The term "expression" as used herein refers to the biosynthesis of gene products. This term encompasses the transcription of genes into RNA. This term also encompasses the translation of RNA into one or more polypeptides, and further encompasses all natural post-transcriptional and post-translational modifications. Expressed CAR can be in the cytoplasm of host cells, in an extracellular environment such as the growth medium of cell culture, or anchored in the cell membrane.

[0104] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to an amino acid polymer of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, either naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art, are also included in this definition.

[0105] 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, it is the L-amino acids that are represented unless otherwise explicitly indicated.

[0106] As used herein, the term "engineered immune cells" refers to immune cells, also called immune effector cells, that have been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0107] As used herein, "porcine teschovirus-1 2A peptide" or "P2A peptide" or "P2A" refers to a picornavirus "self-cleaving peptide." The average length of a P2A peptide is 18-22 amino acids. The P2A peptide was first identified in foot-and-mouth disease virus (FMDV), a member of the picornavirus family (Ryan et al., J Gen Virol, 1991, 72(Pt 11):2727-2732). It has been reported that the ribosome skips synthesis of the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, leading to cleavage between the 2A peptide and the peptide immediately downstream (see, e.g., Donnelly et al., J Gen Virol, 2001, 82:1013-1025). Exemplary P2A peptides useful in the present application comprise an amino acid sequence that is at least 90%, e.g., 90%, 91%, 92%, 93%, 04%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73. In some embodiments, a P2A peptide useful in the present application comprises the amino acid sequence of SEQ ID NO: 73.

[0108] Induced pluripotent stem cells (IPSCs) and immune effector cells iPSCs have unlimited self-renewal capacity. The use of iPSCs enables cell manipulation to produce controlled cell banks of modified cells that can be expanded and differentiated into desired immune effector cells, providing large quantities of homogeneous allogeneic therapeutic products.

[0109] Genetically engineered iPSCs and their derived cells are provided herein. Selected genomic modifications provided herein enhance the therapeutic properties of the derived cells. The derived cells are functionally improved and suitable for allogeneic off-the-shelf cell therapy after a combination of selective modalities is introduced into the cells by genomic engineering at the iPSC level. This approach can help reduce side effects mediated by CRS / GVHD and prevent long-term autoimmunity while providing excellent efficacy.

[0110] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell. Specialized cells include, for example, blood cells or muscle cells. A differentiated or differentiation-induced cell is one that occupies a more specialized ("committed") position within a cell lineage. The term "committed," when applied to the differentiation process, refers to a cell that has progressed to a point in the differentiation pathway where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell to properly form all lineages of the body or soma or embryo. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0111] As used herein, the term " reprogramming " or " dedifferentiation " refers to the method of increasing the capacity of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell capacity has greater developmental plasticity (i.e., can differentiate into more cell types) compared with the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0112] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells produced from differentiated adult, neonatal, or fetal cells that have been induced, changed, or reprogrammed into cells that can differentiate into tissues of all three germ layers or layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to the cells as they are found in nature.

[0113] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" or "HPCs" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, pluripotent hematopoietic stem cells (hemocyte blasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that 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" refer to HPCs that express CD34 on their surface.

[0114] 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, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.

[0115] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and has various roles in the immune system. T cells may have roles including, for example, identifying specific foreign antigens in the body and activating and deactivating other immune cells. T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell, 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 (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (gd T cells), and others, and can be at any developmental stage. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tern cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0116] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by their secretory profile after stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein 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 implicated as a binding recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, they define helper / inducer subsets.

[0117] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the binding recognition element in major histocompatibility complex class I-restricted interactions.

[0118] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of a T cell receptor (TCR chain). NK cells may also refer to genetically engineered NK cells, such as NK cells modified to express a chimeric antigen receptor (CAR). NK cells may also differentiate from stem or progenitor cells.

[0119] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to preferential therapeutic traits in source cells or iPSCs and may be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs by reprogramming, which can further differentiate 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, depending on the context. For example, as used throughout this application, derived effector cells, or derived NK or "iNK" cells, or derived T or "iT" cells, are cells differentiated from iPSCs, compared to their primary counterparts obtained from natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, genetic imprints that confer preferential therapeutic traits are incorporated into iPSCs either by reprogramming selected source cells that are donor, disease, or treatment response specific, or by using genome editing to introduce genetically modified modalities into iPSCs.

[0120] Induced pluripotent stem cell (iPSC) parent cell lines can be generated from peripheral blood mononuclear cells (PBMCs) or T cells using any known method for introducing reprogramming factors into non-pluripotent cells, such as the episomal plasmid-based process previously described in U.S. Patent Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference. 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, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs, and the genome editing is introduced by creating one or more targeted integrations and / or in / dels at one or more selected sites. In another embodiment, the iPSCs are obtained from human T cells with antigen specificity and rearranged TCR genes (also referred to herein as "T-iPS" cells), as described in U.S. Patent Nos. 9,206,394 and 10,787,642, which are hereby incorporated by reference.

[0121] According to certain aspects, the present application relates to an induced pluripotent stem cell (iPSC) cell, or a derivative thereof, comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide, such as the porcine teschovirus-1 2A (P2A) peptide; and (iii) a deletion or reduced expression of the B2M and CIITA genes.

[0122] I. Chimeric Antigen Receptor (CAR) Expression According to embodiments of the present application, the iPSC cells or their derivatives comprise a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR that targets a tumor antigen. In one embodiment, the CAR targets the CD19 antigen.

[0123] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular signaling domain. Association of the extracellular domain of the CAR with the target antigen on the target cell surface results in CAR clustering and delivers an activating stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger the production of molecules that can mediate proliferation, cytokine production, phagocytosis, and / or cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0124] 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- or post-translationally directs the nascent CAR protein to the endoplasmic reticulum and subsequent surface expression.

[0125] As used herein, the terms "extracellular antigen-binding domain," "extracellular domain," or "extracellular ligand-binding domain" refer to the portion of a CAR that is located on the outside of the cell membrane and that is capable of binding to an antigen, target, or ligand.

[0126] As used herein, the term "hinge region" or "hinge domain" refers to the portion of a CAR that connects two adjacent domains of the CAR protein, namely the extracellular domain and the transmembrane domain of the CAR protein.

[0127] As used herein, the term "transmembrane domain" refers to the portion of a CAR that extends through and anchors the CAR to the cell membrane.

[0128] As used herein, the terms "intracellular signaling domain," "cytoplasmic signaling domain," or "intracellular signaling domain" refer to the portion of a CAR that is located inside the cell membrane and is capable of transducing an effector signal.

[0129] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., an NK cell or a T cell) that provides a primary cytoplasmic signaling sequence that stimulatorily regulates primary activation of receptors for at least some aspect of an immune cell signaling pathway. Stimulatory molecules contain two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and sequences that act antigen-independently to provide secondary activation of costimulatory signals (referred to as "costimulatory signaling domains").

[0130] In certain embodiments, the extracellular domain comprises an antigen-binding domain and / or antigen-binding fragment. The antigen-binding fragment may be, for example, an antibody or antigen-binding fragment thereof 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 the tumor antigen, high specificity for the tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing the tumor antigen, and the ability to inhibit tumor growth in subjects and animal models in need thereof when administered alone or in combination with other anti-cancer therapies.

[0131] As used herein, the term "antibody" is used broadly and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal, human, humanized, composite, and chimeric antibodies and antibody fragments. Generally, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. The structure of antibodies is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, antibodies of the present application can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, kappa and lambda, based on the amino acid sequence of their constant domains. Thus, the antibodies of the present application may contain kappa or lambda light chain constant domains. In certain embodiments, the antibodies of the present application comprise the heavy and / or light chain constant regions of a rat or human antibody. In addition to the heavy and light chain constant domains, the antibodies contain an antigen-binding region made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0132] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0133] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in small amounts. The monoclonal antibodies of the present application can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse or rat, whose genome includes human heavy chain and light chain transgenes.

[0134] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single-domain antibody (sdAb), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibody, minibody, nanobody, domain antibody, bivalent domain antibody, light chain variable domain (VL), variable domain of a camelid antibody (VL), H H), or any other antibody fragment that binds to an antigen but does not contain the entire antibody structure. An antigen-binding fragment is capable of binding to the same antigen as that bound by the parent antibody or parent antibody fragment.

[0135] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).

[0136] As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region, or that comprises only a heavy chain variable region.

[0137] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, and made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0138] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase its sequence homology with the sequence of a human antibody, thereby retaining the antigen-binding properties of the antibody but reducing its antigenicity in the human body.

[0139] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of antibodies derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0140] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In embodiments, the first and second epitopes overlap or substantially overlap. In embodiments, the first and second epitopes do not overlap or substantially do not overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In embodiments, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0141] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or no more than two antigens. Bispecific antibodies are characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In embodiments, the first and second epitopes overlap or substantially overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, a bispecific antibody comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In embodiments, a bispecific antibody comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In embodiments, a bispecific antibody comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope. In embodiments, a bispecific antibody comprises a VFv or fragment thereof having binding specificity for a first epitope. H H, and V with binding specificity for a second epitope H Contains H.

[0142] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to a tumor antigen" is one that binds to a tumor antigen in an amount of 1×10 -7 M or less, preferably 1 × 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10"KD" refers to an antigen-binding domain or antigen-binding fragment that binds with a KD of M or less. The term "KD" refers to the dissociation constant, obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as a molar concentration (M). KD values ​​for antibodies can be determined using methods in the art in light of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, e.g., by using a biosensor system, e.g., a Biacore® system, or by using biolayer interferometry technology, e.g., an Octet RED96 system.

[0143] 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.

[0144] In various embodiments, antibodies or antibody fragments suitable for use in the CARs of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), masked antibodies (e.g., Probody®), small modular immunopharmaceuticals ("SMIP™"), intrabodies, minibodies, single-domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against 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, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0145] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity-determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0146] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or nanobody, hi some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0147] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0148] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises an scFv.

[0149] In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.

[0150] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise an scFv.

[0151] Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high affinity and specific binding of target biomolecules, can also be used in the CARs of the present disclosure. Such scaffolds have been shown to result in 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™); engineered gamma-B crystallin-derived scaffolds or engineered ubiquitin-derived scaffolds (e.g., Affilin™); engineered fibronectin-derived 10th fibronectin type III (10Fn3) domains (e.g., monobodies, AdNectin™, or AdNexin™); engineered ankyrin repeat motif-containing polypeptides (e.g., DARPin™); engineered low-density lipoprotein receptor-derived A domain (LDLR-A) (e.g., Avime™); lipocalins (e.g., anticalins); engineered proteases. Kunitz domains from enzyme inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); engineered Z domains from Protein A (Affibody™); Sac7d-derived polypeptides (e.g., Nanoffitin® or affitin); engineered SH2 domains from Fyn (e.g., Fynomer®); CTLD3 (e.g., tetranectin); thioredoxin (e.g., peptide aptamers); KALBITOR®; β-sandwiches (e.g., iMabs); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimetics; and any engineered counterparts of the above that retain their binding functionality (Woern A, Pluckthun A, J Mol Biol 305: 989-1010 (2001);Xu L et al., Chem Biol 9: 933-42 (2002);Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004);Binz H et al., Nat Biolechnol 23: 1257-68 (2005);Hey T et al., Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Skerra, Current Opin. in Biotech., 2007 18: 295-304; Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85 (2011), each of which is incorporated by reference in its entirety.

[0152] In some embodiments, the alternative scaffold is affilin or centirin.

[0153] In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a leader sequence. The leader sequence may be located at the N-terminus of the extracellular tag-binding domain. The leader sequence may optionally be cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of leader sequences known to those skilled in the art may be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from a human immunoglobulin heavy chain (HC).

[0154] In some embodiments, the leader sequence is derived from GMCSFR. In one embodiment, the GMCSFR leader sequence comprises the amino acid sequence set forth in SEQ ID NO:1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1.

[0155] In some embodiments, the first polypeptide of a CAR of the present disclosure comprises a transmembrane domain fused in-frame between an extracellular tag-binding domain and a cytoplasmic domain.

[0156] The transmembrane domain can be derived from the protein that contributes to the extracellular tag binding domain, the protein that contributes to the signal transduction or co-signal transduction domain, or from a completely different protein.In some cases, the transmembrane domain can be selected or modified by amino acid substitution, deletion, or insertion to minimize the interaction 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 the binding of the protein that is naturally associated with the transmembrane domain.In certain embodiments, the transmembrane domain comprises additional amino acids to allow flexibility and / or optimal distance between the domains that are connected to the transmembrane domain.

[0157] The transmembrane domain can be derived from either natural or synthetic origin. If the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains particularly useful in the present disclosure can be derived from (i.e., at least include the transmembrane region of) the α, β, or ζ chain of the T cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it contains primarily hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan, and / or valine can be found at each end of a synthetic transmembrane domain.

[0158] In some embodiments, it may be desirable to utilize the transmembrane domain of the zeta, eta, or FcεR1γ chain, which contains cysteine ​​residues capable of disulfide bonding, allowing the resulting chimeric protein to form disulfide-linked dimers with itself or with unmodified versions of the zeta, eta, or FcεR1γ chain of related proteins. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains with transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex. In other cases, it may be desirable to employ the transmembrane domains of zeta, eta, or FcεR1γ and -β, MB1 (Igα), B29, or CD3-γ, zeta, or eta to maintain physical association with other members of the receptor complex.

[0159] In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:23. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:24, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:24.

[0160] In some embodiments, the first polypeptide of a CAR of the present disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, wherein the tag-binding domain, linker, and transmembrane domain are in frame with each other.

[0161] The term "spacer region," as used herein, generally refers to any oligopeptide or polypeptide that functions to link a tag-binding domain to a transmembrane domain. Spacer regions can be used to provide greater flexibility and accessibility to the tag-binding domain. Spacer regions can contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. Spacer regions can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region. Alternatively, spacer regions can be synthetic sequences that correspond to naturally occurring spacer region sequences or can be entirely synthetic spacer region sequences. Non-limiting examples of spacer regions that can be used according to the present disclosure include portions of the human CD8 α chain, partial extracellular domains of CD28, FcyRlla receptors, IgG, IgM, IgA, IgD, IgE, Ig hinges, or functional fragments thereof. In some embodiments, additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is at an optimal distance from the transmembrane domain, hi some embodiments, if the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.

[0162] In some embodiments, the spacer region comprises a hinge domain. The hinge domain can be derived from CD8α, CD28, or immunoglobulin (IgG). For example, the IgG hinge can be from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0163] 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 chimeras thereof. In certain embodiments, the hinge domain comprises the CH1, CH2, CH3 and / or hinge region of an immunoglobulin. In certain embodiments, the hinge domain comprises the core hinge region of an immunoglobulin. The term "core hinge" can be used interchangeably with the term "short hinge" (also known as "SH"). Non-limiting examples of suitable hinge domains are 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 domain is a fragment of an immunoglobulin hinge.

[0164] In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO:21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:21. In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO:22, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:22.

[0165] In some embodiments, the transmembrane domain and / or hinge domain are derived from CD8 or CD28. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD28.

[0166] In certain aspects, the first polypeptide of the CAR of the present disclosure comprises a cytoplasmic domain that includes at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more costimulatory signaling domains.

[0167] The cytoplasmic domain is responsible for activating at least one of the normal effector functions of a host cell (e.g., a T cell) carrying the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytotoxic activity or helper activity, including cytokine secretion. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Although the entire signaling domain is usually present, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the signaling domain sufficient to transmit the effector function signal.

[0168] 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, Fc epsilon receptor I gamma chain (FCER1G), FcRβ, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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 having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 10.

[0174] 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.

[0175] 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.

[0176] 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 having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 13.

[0177] 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.

[0178] 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.

[0179] 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 having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 16.

[0180] 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.

[0181] In one embodiment, the costimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP12 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.

[0182] 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 having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 19.

[0183] In some embodiments, the CAR of the present disclosure comprises one costimulatory signaling domain.In some embodiments, the CAR of the present disclosure comprises two or more costimulatory signaling domains.In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.

[0184] In some embodiments, the signal transduction domain and the costimulatory signal transduction domain can be arranged in any order. In some embodiments, the signal transduction domain is upstream of the costimulatory signal transduction domain. In some embodiments, the signal transduction domain is downstream of the costimulatory signal transduction domain. When two or more costimulatory domains are included, the order of the costimulatory signal transduction domains can be switched.

[0185] Non-limiting exemplary CAR regions and sequences are provided in Table 1.

[0186] [Table 1-1]

[0187] [Table 1-2]

[0188] [Table 1-3]

[0189] 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. In 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.

[0190] The selection of antigen binding domains can depend on the type and number of antigens that define the surface of target cells. For example, antigen binding domains can be selected to recognize antigens that act as cell surface markers on target cells associated with a particular disease state. In certain embodiments, the CARs of the present disclosure can be genetically modified to target tumor antigens of interest by engineering a desired antigen binding domain that specifically binds to the antigen (e.g., on tumor cells). Non-limiting examples of cell surface markers that can serve as targets for the antigen binding domains in the CARs of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0191] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen.

[0192] 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.

[0193] In some embodiments, the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.

[0194] In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies. Non-limiting examples of tumor antigens associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2. Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, nectin-4, and B7H4. Non-limiting examples of tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigens associated with liver cancer include claudin 18.2, GPC-3, EpCAM, cMET, and AFP. Non-limiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70. Non-limiting examples of tumor antigens associated with bladder cancer include nectin-4 and SLITRK6.

[0195] Further examples of antigens that can be targeted by the antigen-binding domain include alpha-fetoprotein, A3, antigen specific for the A33 antibody, 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, Flt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits nit, 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, antigen specific for PAM-4 antibody, placenta 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 marker, oncogene marker or oncogene product.

[0196] 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) comprising the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2. In one embodiment, the anti-CD19 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:4, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4. In one embodiment, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO:7, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:7.

[0197] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cells that produce cell receptors and "self"-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Crohn's disease, or ulcerative colitis.

[0198] In some embodiments, autoimmune antigens that can be targeted by the CARs disclosed herein include platelet antigens, myelin protein antigens, Sm antigens in snRNPs, islet cell antigens, rheumatoid factors, and anti-citrullinated proteins, citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclic citrullinated peptides), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), articular cartilage components such as collagen II, IX, and XI, circulating serum proteins such as RF (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33 / hnRNP A2, Sm, eukaryotic translation elongation factor 1 alpha 1, stress proteins such as HSP-65, -70, -90, BiP, inflammatory / immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin, and 25-35 kD nuclear protein , particulate proteins such as bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factors, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonucleopartides and proteins such as Sm antigens (including, but not limited to, SmD antigen and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro(SSA), and La(SSB) antigens.

[0199] In various embodiments, the scFv fragment used in the CAR of the present disclosure may include a linker between the VH domain and the VL domain. The linker may be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be long enough to connect the VH and VL so that they form the correct conformation relative to each other and thereby retain the desired activity, such as antigen binding. The linker may be about 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 35 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15 to 20 amino acids in length. Exemplary linkers that can be used are Gly-rich linkers, Gly- and Ser-containing linkers, Gly- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0200] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO:3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3. In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker comprises the amino acid sequence set forth in SEQ ID NO:25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:25.

[0201] Other linker sequences can include portions of an immunoglobulin hinge region, CL, or CH1, 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, for example, in WO 2019 / 060695, which is incorporated herein by reference in its entirety.

[0202] II. Artificial Cell Death Polypeptides According to an embodiment of the present application, the iPSC cell or a derivative thereof comprises a second exogenous polynucleotide encoding an artificial cell death polypeptide.

[0203] As used herein, the term "artificial cell death polypeptide" refers to an engineered protein designed to prevent potential toxic or otherwise adverse effects of a cell therapy. Artificial cell death polypeptides can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. Optionally, the artificial cell death polypeptide is activated by an exogenous molecule, e.g., an antibody, which, upon activation, induces apoptosis and / or cell death of therapeutic cells.

[0204] In certain embodiments, the artificial cell death polypeptide comprises an inactivated cell surface receptor that includes an epitope specifically recognized by an antibody, particularly a monoclonal antibody, also referred to herein as a monoclonal antibody-specific epitope. When expressed by iPSCs or their derivatives, the inactivated cell surface receptor is inactive or significantly defective in signal transduction, yet can still be specifically recognized by the antibody. Specific binding of the antibody to the inactivated cell surface receptor allows for the elimination of iPSCs or their derivatives not only by ADCC and / or ADCP mechanisms, but also by direct killing using antibody-drug conjugates with toxins or radionuclides.

[0205] In certain embodiments, the inactivated cell surface receptor is selected from the group consisting of ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. The antibody comprises an epitope selected from those specifically recognized by antibodies including, but not limited to, vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab.

[0206] The epidermal growth factor receptor, also known as EGFR, ErbB1, and HER1, is a cell surface receptor for members of the epidermal growth factor family of extracellular ligands. As used herein, "truncated EGFR," "tEGFR," "short EGFR," or "sEGFR" refers to an inactive EGFR variant that lacks the EGF-binding and intracellular signaling domains of EGFR. An exemplary tEGFR variant contains domains 322-333 of domain 2, all of domains 3 and 4, and the transmembrane domain of the native EGFR sequence, which contains the cetuximab-binding epitope. Expression of the tEGFR variant on the cell surface allows for cell ablation, if desired, with an antibody that specifically binds to tEGFR, such as cetuximab (Erbitux®). Due to the absence of the EGF-binding and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or their derived cells.

[0207] Exemplary inactivated cell surface receptors of the present application include tEGFR variants. In certain embodiments, expression of an inactivated cell surface receptor in engineered immune cells expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cells when the engineered immune cells are contacted with an anti-EGFR antibody. Methods using inactivated cell surface receptors are described in International Publication Nos. 2019 / 070856, 2019 / 023396, and 2018 / 058002, the disclosures of which are incorporated herein by reference. For example, a subject who has previously received engineered immune cells of the present disclosure comprising a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an amount of an anti-EGFR antibody effective to eliminate the previously administered engineered immune cells in the subject.

[0208] In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab.

[0209] 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%, 82%, 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.

[0210] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the CD79b epitope comprises or consists of an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78, preferably the amino acid sequence of SEQ ID NO: 78.

[0211] 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 at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80, preferably the amino acid sequence of SEQ ID NO: 80.

[0212] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of the Her2 receptor or ErbB, e.g., an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody-specific epitope comprises or consists of an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82, preferably the amino acid sequence of SEQ ID NO: 82.

[0213] In some embodiments, the inactivated cell surface receptor further comprises a cytokine such as interleukin-15 or interleukin-2.

[0214] As used herein, "interleukin-15" or "IL-15" refers to a cytokine, or a functional portion thereof, that regulates the activation and proliferation of T and NK cells. A "functional portion" ("biologically active portion") of a cytokine refers to a portion of a cytokine that retains one or more functions of the full-length or mature cytokine. Such functions for IL-15 include promoting the survival of NK cells, regulating the activation and proliferation of NK cells and T cells, as well as supporting NK cell development from hematopoietic stem cells. As will be recognized by those skilled in the art, the sequences of various IL-15 molecules are known in the art. In certain embodiments, the IL-15 is wild-type IL-15. In certain embodiments, the IL-15 is human IL-15. In certain embodiments, the IL-15 comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0215] As used herein, "interleukin-2" refers to a cytokine, or a functional portion thereof, that regulates the activation and proliferation of T and NK cells. In certain embodiments, the IL-2 is wild-type IL-2. In certain embodiments, the IL-2 is human IL-2. In certain embodiments, the IL-2 comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0216] In certain embodiments, the inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably by an autoprotease peptide. Examples of autoprotease peptides include, but are not limited to, peptide sequences selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), and combinations thereof. In one embodiment, the autoprotease peptide comprises or is the autoprotease peptide of porcine teschovirus-1 2A (P2A) peptide. In certain embodiments, the autoprotease peptide comprises an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0217] In certain embodiments, the inactivated cell surface receptor comprises a truncated epidermal growth factor (tEGFR) variant operably linked to interleukin-15 (IL-15) or IL-2 by an autoprotease peptide. In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:74, preferably the amino acid sequence of SEQ ID NO:74.

[0218] In some embodiments, the inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0219] In some embodiments, the inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO:21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:21.

[0220] In certain embodiments, the inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:23.

[0221] In certain embodiments, the inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, transmembrane region, and cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises more than one epitope specifically recognized by an antibody, and the epitopes may have the same or different amino acid sequences, and the epitopes may be linked to each other via a peptide linker, such as a flexible peptide linker having the sequence (GGGGS)n, where n is an integer between 1 and 8 (SEQ ID NO: 25). In some embodiments, the inactivated cell surface receptor further comprises a cytokine, such as IL-15 or IL-2. In certain embodiments, the cytokine is located in the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked to the epitope specifically recognized by the antibody directly or indirectly via an autoprotease peptide, such as those described herein. In some embodiments, the cytokine is indirectly linked to the epitope by being linked to the transmembrane domain via an autoprotease peptide.

[0222] Non-limiting exemplary regions and sequences of inactivated cell surface receptors are provided in Table 2.

[0223] [Table 2-1]

[0224] [Table 2-2]

[0225] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0226] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0227] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 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.

[0228] III. HLA Expression In certain embodiments, the iPSCs or their derivatives of the present application can be further modified by introducing a third exogenous polynucleotide encoding one or more proteins involved in immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). In particular, disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving the cells vulnerable to lysis by NK cells through a "loss of self" response. Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol. 2017 Aug;35(8):765-772).

[0229] In certain embodiments, the iPSCs or derived cells thereof comprise a third exogenous polypeptide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G). In certain embodiments, the HLA-E comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the HLA-G comprises an amino acid sequence at least 90%, e.g., 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.

[0230] In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-E via a linker. In certain embodiments, the third exogenous polypeptide comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:66.

[0231] In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-G via a linker. In certain embodiments, the third exogenous polypeptide comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:69.

[0232] IV. Other Optional Genome Editing In one embodiment of the above cells, the genome editing at one or more selected sites may include insertion of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genomically engineered iPSCs or their derivative cells.

[0233] In some embodiments, the exogenous polynucleotide for insertion is operably linked to (1) one or more exogenous promoters including CMV, EFla, PGK, CAG, UBC, or other constitutive, inducible, temporal, tissue, or cell type specific promoters; or (2) one or more endogenous promoters contained within selected sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of a genome safe harbor. In some embodiments, the genomically engineered iPSCs generated using the above methods contain one or more different exogenous polynucleotides encoding proteins, including caspase, thymidine kinase, cytosine deaminase, B cell CD20, ErbB2, or CD79b; where, when the genomically engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safeguard loci, including AAVSI, CCR5, ROSA26, collagen, HTRP, H11, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. Other exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitor proteins, such as PD1, PD-L1, and CTLA4, as well as proteins targeting the CD47 / signal-regulatory protein alpha (SIRPα) axis. In some other embodiments, the genomically engineered iPSCs generated using the methods provided herein contain in / dels in one or more endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response regulation and modulation, or proteins that inhibit engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells.

[0234] V. Targeted Genome Editing at Selected Loci in iPSCs According to embodiments of the present application, one or more of the exogenous polynucleotides are integrated into one or more loci on the chromosomes of the iPSCs.

[0235] Genome editing, or genomic editing, or gene editing, as used interchangeably herein, is a type of genetic manipulation in which DNA is inserted, deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (interchangeably with "targeted genomic editing" or "targeted gene editing") allows for insertion, deletion, and / or replacement at a preselected site in the genome. If an endogenous sequence is deleted or disrupted at the insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or knocked down due to the sequence deletion or disruption. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. Similarly, the term "targeted integration" is also used herein to refer to a process involving the insertion of one or more exogenous sequences into a preselected site in the genome, with or without deletion of the endogenous sequence at the insertion site.

[0236] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach, in which homologous recombination is guided via the enzymatic machinery of the host cell by homologous sequences flanking the exogenous polynucleotide to be inserted.

[0237] Alternatively, targeted editing can be achieved more frequently by specifically introducing double-strand breaks (DSBs) using specific low-frequency-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, NHEJ often leads to random insertion or deletion (in / del) of a small number of endogenous nucleotides. In comparison, in the presence of a donor vector containing exogenous genetic material flanked by pairs of homologous arms, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) via homologous recombination, resulting in "targeted integration."

[0238] 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), and RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) systems. Moreover, the DICE (double integrase cassette exchange) system, which utilizes phiC31 and Bxbl integrases, is also a promising tool for targeted integration.

[0239] ZFN is a targeted nuclease comprising a nuclease fused with a zinc finger DNA binding domain. By "zinc finger DNA binding domain" or "ZFBD" is meant a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within the zinc finger binding domain, whose 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. A "designed" zinc finger domain is a domain that does not exist in nature, whose design / composition is primarily the result of rational criteria, such as the application of substitution rules and computer algorithms for processing information within database storage information of existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; also see International Publication Nos. WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. A "selected" zinc finger domain is a domain that is not found in nature, and its production is primarily due to empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of Fokl nuclease with the zinc finger DNA binding domain.

[0240] TALENs are targeted nucleases containing a nuclease fused to a TAL effector DNA-binding domain. By "transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" is meant the polypeptide domain of a TAL effector protein responsible for binding to DNA. TAL effector proteins are secreted by the plant pathogen Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA-binding domain, and activate gene transcription at these sequences via their transactivation domain. The specificity of the TAL effector DNA-binding domain depends on an effector-variable number of imperfect 34 amino acid repeats, which contain polymorphisms at select repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of a Fokl nuclease with a TAL effector DNA binding domain.

[0241] Another example of a targeted nuclease for use in the subject methods is a targeted Spoll nuclease, a polypeptide comprising a Spoll polypeptide with nuclease activity fused to a DNA-binding domain, e.g., a zinc finger DNA-binding domain, a TAL effector DNA-binding domain, etc., which has specificity for a DNA sequence of interest. See, e.g., U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0242] Further examples of target nucleases suitable for the present application include, but are not limited to, Bxbl, phiC3 l, R4, PhiBTl, and Wp / SPBc / TP90l-l, whether used individually or in combination.

[0243] Other non-limiting examples of targeting nucleases include natural and recombinant nucleases; CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and others. For example, CRISPR / Cas9 requires two main components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When coexpressed, these two components form a complex that is recruited to target DNA sequences containing a PAM and a seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to the target sequence of choice. These two components can then be delivered into mammalian cells via transfection or transduction. As another example, CRISPR / Cpf1 contains two main components: (1) the CPf1 endonuclease and (2) the crRNA. When coexpressed, these two components form a ribonucleoprotein (RNP) complex that is recruited to target DNA sequences containing a PAM and a seeding region near the PAM. The crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpf1 to a target sequence of choice. These two components can then be delivered into mammalian cells via transfection or transduction.

[0244] MAD7 is an engineered Cas12a variant originating from the bacterium Eubacterium rectale that prefers 5'-TTTN-3' and 5'-CTTN-3' PAM sites and does not require tracrRNA (see, e.g., PCT Publication No. 2018 / 236548, incorporated herein by reference).

[0245] DICE-mediated insertion uses a pair of recombinases, such as phiC31 and Bxbl, to provide unidirectional integration of exogenous DNA, but this unidirectional integration is strictly limited to each enzyme's own small attB and attP recognition sites. Because these target att sites do not naturally occur in mammalian genomes, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, incorporated herein by reference.

[0246] One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arms specific to the desired integration site, and the method of targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by the cellular host enzyme machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a gRNA comprising a Cpf1 expression cassette and a guide sequence specific to the desired integration site into the cell to enable Cpf1-mediated insertion. In another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a Cas9 expression cassette and a gRNA comprising a guide sequence specific to the desired integration site into the cell to enable Cas9-mediated insertion. In yet another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more att sites of a pair of DICE recombinase into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable DICE-mediated targeted integration.

[0247] The site for targeted integration includes, but is not limited to, genomic safety region.Genomic safety region is the intragenic or extragenic region of human genome that can accommodate the predictable expression of newly integrated DNA, theoretically without causing any adverse effects to host cell or organism.In certain embodiments, the genomic safety region for targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR and RUNX1 genes.

[0248] In other embodiments, the site for targeted integration is selected to delete or reduce the expression of endogenous gene at insertion site.As used herein, the term " deletion " in relation to gene expression refers to any genetic modification that negates gene expression.Examples of the " deletion " of gene expression include, for example, the removal or deletion of the DNA sequence of gene, the insertion of exogenous polynucleotide sequence at gene locus, and one or more substitutions within gene, which negates gene expression.

[0249] Genes for targeted 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 histocompatible-matched in the allogeneic recipient to avoid allogeneic rejection problems. "MHC deficiency," including MHC class I deficiency or MHC class II deficiency, or both, refers to cells that lack, no longer maintain, or have reduced levels of surface expression of complete MHC complexes, including MHC class I protein heterodimers and / or MHC class II heterodimers, such that the reduced or reduced levels are lower than those naturally detectable 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 6p21) or by deletion or reduced expression levels of one or more MHC class I-related genes, including, but not limited to, the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin gene. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers. B2M-null cells are MHC-I-deficient. MHC class II deficiency can be achieved by 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 functions via activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-null cells are MHC-II-deficient. In certain embodiments, one or more exogenous polynucleotides are integrated into one or more loci of a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby deleting or reducing the expression of the integrated gene.

[0250] In certain embodiments, the exogenous polynucleotide is integrated into one or more loci of a chromosome of the cell, preferably the one or more loci of a gene selected from the group consisting of AAVS1, 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 genes, with the proviso that at least one of the one or more loci is an MHC gene, e.g., a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes. Preferably, one or more exogenous polynucleotides are integrated into the locus of an MHC class-I-related gene, such as the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, or tapasin gene; and the locus of an MHC-II-related gene, such as the RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally, the locus of a safety region gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, and RUNX1 genes. More preferably, one or more exogenous polynucleotides are integrated into the locus of the CIITA, AAVS1, and B2M genes.

[0251] In certain embodiments, (i) a first exogenous polynucleotide is integrated into the locus of the AAVS1 gene; (ii) a second exogenous polypeptide is integrated into the locus of the CIITA gene; and (iii) a third exogenous polypeptide is integrated into the locus of the B2M gene; wherein integration of the exogenous polynucleotides deletes or reduces expression of the CIITA and B2M genes.

[0252] In certain embodiments, (i) the first 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: 62; (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: 75; 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.

[0253] In certain embodiments, (i) the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; (ii) the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and (iii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0254] derived cells In another aspect, the present invention relates to cells derived from iPSC differentiation, i.e., derivative cells. As described above, the genome editing introduced into iPSC cells is retained in the derivative cells. In certain embodiments of the derivative cells obtained from iPSC differentiation, the derivative cells are hematopoietic cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, B cells, antigen-presenting cells (APCs), monocytes, and macrophages. In certain embodiments, the derivative cells are immune effector cells, such as NK cells or T cells.

[0255] 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 truncated epidermal growth factor (tEGFR) variant and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide, such as the autoprotease peptide of porcine teschovirus-1 2A (P2A) peptide; and (iii) a deleted or reduced expression of an MHC class I-related gene and an MHC class II-related gene, e.g., an MHC class-I-related gene selected from the group consisting of the B2M gene, the TAP1 gene, the TAP2 gene, and the tapasin gene, and an MHC-II-related gene selected from the group consisting of the RFXANK gene, the CIITA gene, the RFX5 gene, the RFXAP gene, and the CIITA gene, preferably the B2M gene and the CIITA gene.

[0256] 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).

[0257] Also provided is an NK cell or T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of SEQ ID NO: 61; (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin-15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66, wherein the first, second and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA and B2M genes, respectively, thereby deleting or reducing the expression of CIITA and B2M.

[0258] In certain embodiments, the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0259] Also provided are CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs), comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope and interleukin-15 (IL-15), wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide; and (iii) a deletion or reduced expression of one or more of the following genes: B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP.

[0260] 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).

[0261] In certain embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to the CD19 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain, e.g., a costimulatory domain comprising a CD28 signaling domain.

[0262] Also provided is a method for producing derivative cells, which comprises differentiating iPSCs under conditions for cell differentiation, thereby obtaining derivative cells.

[0263] The iPSCs of the present application can be differentiated by any method known in the art.Exemplary methods are described in U.S. Patent No. 8846395, U.S. Patent No. 8945922, U.S. Patent No. 8318491, International Publication No. WO2010 / 099539, WO2012 / 109208, WO2017 / 070333, WO2017 / 179720, WO2016 / 010148, WO2018 / 048828 and WO2019 / 157597, each of which is incorporated herein by reference in its entirety.Differentiation protocols can use feeder cells or not include feeder cells. As used herein, "feeder cells" or "feeder" is a term describing one cell type that is co-cultured with a second cell type to provide an environment in which the second cell type can grow, expand, or differentiate, as feeder cells provide stimuli, growth factors, and nutrients for the support of a second cell type.

[0264] In another embodiment of the present invention, the iPSC-derived cells of the present invention are NK cells prepared by a method of differentiating iPSC cells into NK cells by subjecting the cells to a differentiation protocol that includes the addition of recombinant human IL-12p70 for the final 24 hours of culture. By including IL-12 in the differentiation protocol, IL-12-primed cells exhibit rapid cell killing compared to cells differentiated in the absence of IL-12 (Figure 5A). Furthermore, cells differentiated using IL-12 conditions exhibit improved inhibition of cancer cell growth (Figure 5B).

[0265] Polynucleotides, Vectors, and Host Cells (1) a nucleic acid encoding a CAR In another general aspect, the present invention relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful in the present invention according to an embodiment of the present application. Those skilled in the art will recognize that the coding sequence of the CAR can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, those skilled in the art will understand that the nucleic acid sequence encoding the CAR of the present application can be altered without changing the amino acid sequence of the protein.

[0266] In certain embodiments, the isolated nucleic acid encodes a CAR that targets CD 19. In certain embodiments, the isolated nucleic acid encoding the CAR comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 62, preferably the polynucleotide sequence of SEQ ID NO: 62.

[0267] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding a CAR useful in the invention according to embodiments of the present application. In view of the present disclosure, any vector known to those skilled in the art can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element for establishing the conventional function of an expression vector, such as 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 a CAR in a cell. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present application.

[0268] In certain aspects, the present application provides vectors for targeted integration of CARs useful in the inventions according to embodiments of the present application. In certain embodiments, the vector comprises, from 5' to 3', an exogenous polynucleotide having: (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to embodiments of the present application; and (c) a terminator / polyadenylation signal.

[0269] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence with SEQ ID NO: 63.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0270] In certain embodiments, terminator / polyadenylation signal is SV40 signal.In certain embodiments, SV40 signal comprises at least 90% identical polynucleotide sequence with SEQ ID NO: 64, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence.Other terminator sequences can also be used, examples of which include but are not limited to BGH, hGH and PGK.

[0271] In certain embodiments, the polynucleotide sequence encoding the CAR comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:62.

[0272] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm flanking the exogenous polynucleotide. As used herein, "left homologous arm" and "right homologous arm" refer to a pair of nucleic acid sequences flanking the exogenous polynucleotide, which nucleic acid sequences facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. The sequences of the left and right homologous arms can be designed based on the intended integration site. In some embodiments, the left or right homologous arm is homologous to the left or right sequence of the integration site.

[0273] In certain embodiments, the left homologous arm comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 90. In certain embodiments, the right homologous arm comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 91.

[0274] In certain embodiments, the vector comprises a polynucleotide sequence that is at least 85%, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:92, preferably the polynucleotide sequence of SEQ ID NO:92.

[0275] (2) a nucleic acid encoding an inactivated cell surface receptor In another general aspect, the present invention relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful in the invention according to the embodiments of the present application. Those skilled in the art will recognize that the coding sequence of an inactivated cell surface receptor can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will recognize that the nucleic acid sequence encoding the inactivated cell surface receptor of the present application can be altered without changing the amino acid sequence of the protein.

[0276] In certain embodiments, the isolated nucleic acid encodes any of the inactivated cell surface receptors described herein, e.g., those comprising a monoclonal antibody-specific epitope, and a cytokine, e.g., IL-15 or IL-2, wherein the monoclonal antibody-specific epitope and the cytokine are operably linked by an autoprotease peptide.

[0277] In some embodiments, the isolated nucleic acid is an antibody, e.g., ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab Encodes an inactivated cell surface receptor containing an epitope specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab.

[0278] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epidermal growth factor receptor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab, or panitumumab, preferably cetuximab.

[0279] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, e.g., epitopes that are specifically recognized by polatuzumab vedotin.

[0280] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, eg, epitopes specifically recognized by rituximab.

[0281] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of the Her2 receptor, eg, epitopes specifically recognized by trastuzumab.

[0282] In certain embodiments, the autoprotease peptide comprises or is the porcine teschovirus-1 2A (P2A) peptide.

[0283] In certain embodiments, the truncated epidermal growth factor (tEGFR) variant consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0284] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by polatuzumab vedotin consists of an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:78.

[0285] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by rituximab consists of an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:80.

[0286] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82.

[0287] In certain embodiments, the IL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:72.

[0288] In certain embodiments, the autoprotease peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:73.

[0289] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74.

[0290] In certain embodiments, the isolated nucleic acid encoding the inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90%, e.g., 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.

[0291] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:79.

[0292] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful in the invention according to embodiments of the present application. Any vector known to those of skill in the art in light of the present disclosure can be used, for example, a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as 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 into 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 generate recombinant expression vectors according to embodiments of the present application.

[0293] In certain aspects, the present application provides vectors for targeted integration of inactivated cell surface receptors, useful for the inventions according to embodiments of the present application. In certain embodiments, the vector comprises, from 5' to 3', an exogenous polynucleotide having: (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, for example, an inactivated cell surface receptor comprising a truncated epidermal growth factor receptor (tEGFR) variant, and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide, for example, the porcine teschovirus-1 2A (P2A) peptide; and (c) a terminator / polyadenylation signal.

[0294] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence with SEQ ID NO: 63.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0295] In certain embodiments, terminator / polyadenylation signal is SV40 signal.In certain embodiments, SV40 signal comprises at least 90% identical polynucleotide sequence with SEQ ID NO: 64, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence.Other terminator sequences can also be used, examples of which include but are not limited to BGH, hGH and PGK.

[0296] In certain embodiments, the polynucleotide sequence encoding the inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:75.

[0297] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.

[0298] In certain embodiments, the left homologous arm comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 84. In certain embodiments, the right homologous arm comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 85.

[0299] In certain embodiments, the vector comprises a polynucleotide sequence that is at least 85%, e.g., 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.

[0300] (3) a nucleic acid encoding an HLA construct In another general aspect, the present invention relates to an isolated nucleic acid encoding an HLA construct useful in the invention according to the embodiments of the present application. Those skilled in the art will recognize that the coding sequence of an HLA construct can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will recognize 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.

[0301] In certain embodiments, the isolated nucleic acid encodes an HLA construct comprising a signal peptide, e.g., an HLA-G signal peptide, operably linked to an HLA coding sequence, e.g., the coding sequence for mature B2M and / or mature HLA-E. In some embodiments, the HLA coding sequence encodes HLA-G and B2M operably linked by a 4xGGGGS linker, and / or B2M and HLA-E operably linked by a 3xGGGGS linker. In certain embodiments, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO:67, preferably the polynucleotide sequence of SEQ ID NO:67. In another embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:70, preferably the polynucleotide sequence of SEQ ID NO:70.

[0302] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an HLA construct useful in the invention according to embodiments of the present application. Any vector known to those skilled in the art in light of the present disclosure can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids into 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 generate recombinant expression vectors according to embodiments of the present application.

[0303] In certain aspects, the present application provides vectors for targeted integration of HLA constructs useful in the inventions according to embodiments of the present application. In certain embodiments, the vector comprises, from 5' to 3', an exogenous polynucleotide having: (a) a promoter; (b) a polynucleotide sequence encoding the HLA construct; and (c) a terminator / polyadenylation signal.

[0304] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence with SEQ ID NO: 63.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0305] In certain embodiments, terminator / polyadenylation signal is SV40 signal.In certain embodiments, SV40 signal comprises at least 90% identical polynucleotide sequence with SEQ ID NO: 64, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence.Other terminator sequences can also be used, examples of which include but are not limited to BGH, hGH and PGK.

[0306] In certain embodiments, the polynucleotide sequence encoding the HLA construct comprises a signal peptide, such as the HLA-G signal peptide, mature B2M, and mature HLA-E, wherein HLA-G and B2M are operably linked by a 4xGGGGS linker (SEQ ID NO: 31), and the B2M transgene and HLA-E are operably linked by a 3xGGGGS linker (SEQ ID NO: 25). In certain embodiments, the HLA construct comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the 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.

[0307] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.

[0308] In certain embodiments, the left homologous arm comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 87. In certain embodiments, the right homologous arm comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 88.

[0309] In certain embodiments, the vector comprises a polynucleotide sequence that is at least 85%, e.g., 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.

[0310] (4) Host cells In another general aspect, the present application provides a host cell comprising an isolated nucleic acid encoding a vector of the present application and / or a construct of the present application. Any host cell known to those of skill in the art in light of the present disclosure can be used for recombinant expression of the exogenous polynucleotide of the present application. According to certain embodiments, the recombinant expression vector is transformed into the host cell by conventional methods, such as chemical transfection, heat shock, or electroporation, where the vector is stably integrated into the host cell genome, thereby efficiently expressing the recombinant nucleic acid.

[0311] Examples of host cells include, for example, recombinant cells containing a vector or isolated nucleic acid of the present application, useful for producing a vector or construct of interest; or engineered iPSCs or their derivatives containing one or more isolated nucleic acids of the present application, preferably integrated into one or more chromosomal loci. Host cells for the isolated nucleic acids of the present application can also be immune effector cells, such as T cells or NK cells, containing one or more isolated nucleic acids of the present application. Immune effector cells can be obtained by differentiation of the engineered iPSCs of the present application. Any suitable method in the art can be used for differentiation in light of the present disclosure. Immune effector cells can also be obtained by transfecting one or more isolated nucleic acids of the present application into immune effector cells.

[0312] composition In another general aspect, the application provides compositions comprising an isolated polynucleotide of the application, a host cell of the application and / or an iPSC or a derived cell thereof.

[0313] In certain embodiments, the composition further comprises one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (IMiD).

[0314] In certain embodiments, the composition is a pharmaceutical composition comprising the isolated polynucleotide of the present application, the host cell of the present application and / or the iPSC or derived cell thereof, and a pharmaceutically acceptable carrier. The term "pharmaceutical composition," as used herein, refers to a product comprising the isolated polynucleotide of the present application, the isolated polypeptide of the present application, the host cell of the present application, and / or the iPSC or derived cell thereof of the present application together with a pharmaceutically acceptable carrier. The polynucleotides, polypeptides, host cells, and / or the iPSC or derived cell thereof of the present application and compositions comprising them are also useful for the manufacture of medicaments for the therapeutic applications mentioned herein.

[0315] As used herein, the term "carrier" refers to any excipient, diluent, bulking agent, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It is understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the compositions described herein. In certain embodiments, any pharmaceutically acceptable carrier suitable for use with polynucleotides, polypeptides, host cells, and / or iPSCs or their derivative cells can be used in light of the present disclosure.

[0316] The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, from 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 adjusters, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present application.

[0317] How to use In another general aspect, the present application provides a method of treating a disease or condition in a subject in need thereof. The method includes administering a therapeutically effective amount of a cell of the present application and / or a composition of the present application to a subject in need thereof. In certain embodiments, the disease or condition is cancer. The cancer can be, for example, a solid cancer or a liquid cancer. The cancer can be selected from the group consisting of lung cancer, gastric cancer, colon cancer, liver cancer, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, endometrial cancer, prostate cancer, thyroid cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma / disease (HD), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myelogenous leukemia (AML), and other liquid tumors. In a preferred embodiment, the cancer is non-Hodgkin's lymphoma (NHL).

[0318] According to an embodiment of the present application, the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and / or an iPSC or its derivative. 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. A therapeutically effective amount can be empirically and routinely determined for a given purpose.

[0319] As used herein in reference to the cells of the present application and / or pharmaceutical compositions of the present application, a therapeutically effective amount means the amount of cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0320] According to certain embodiments, a therapeutically effective amount refers to the amount of therapy sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition being treated, or the symptoms associated therewith; (ii) reducing the duration of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iv) reversing the disease, disorder, or condition being treated, or the symptoms associated therewith; (v) preventing the occurrence or onset of the disease, disorder, or condition being treated, or the symptoms associated therewith. (vi) prevent the recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increase the survival of a subject having 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 efficacy of another therapy.

[0321] The therapeutically effective amount or dosage can vary depending on a variety of factors, such as the disease, disorder, or condition to be treated, the means 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 medications being administered, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages are optimally designed to optimize safety and efficacy.

[0322] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0323] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered by any convenient method known to those of skill in the art. For example, the cells of the present application can be administered to a subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation, and / or transplantation. Compositions comprising the cells of the present application can be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intrapleurally, by intravenous (iv) injection, or intraperitoneally. In certain embodiments, the cells of the present application can be administered with or without lymphodepletion of the subject.

[0324] Pharmaceutical compositions comprising the cells of the present application can be provided in a sterile liquid formulation, typically buffered to a selected pH, typically an isotonic aqueous solution with a cell suspension, or optionally as an emulsion, dispersion, or the like. The composition can include a carrier suitable for maintaining cell integrity and viability and for administration of the cell composition, e.g., water, saline, phosphate-buffered saline, etc.

[0325] Sterile injectable solutions can be prepared by incorporating the cells of the present application, optionally with various other ingredients, in an appropriate amount of an appropriate solvent. Such compositions may contain pharmaceutically acceptable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, etc., suitable for use in cell compositions and administration to subjects, such as humans. Buffers suitable for providing cell compositions are well known in the art. Any vehicle, diluent, or additive used is compatible with maintaining the integrity and viability of the cells of the present application.

[0326] The cells and / or pharmaceutical compositions of the present application can be administered in any physiologically acceptable medium. Cell populations containing the cells of the present application may include purified populations of cells. Those skilled in the art can easily determine the purity of cells in a cell population using various well-known methods. The purity range of a cell population containing genetically modified cells of the present application may be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. Dosages can be easily adjusted by those skilled in the art; for example, a decrease in purity may require an increased dosage.

[0327] The cells of the present application are generally administered as a dose based on cells per kilogram (cells / kg) of body weight of the subject to whom the cells and / or pharmaceutical compositions comprising the cells are administered. Generally, cell doses range from about 10 to 150 mg / kg, depending on the mode and location of administration. 4 ~about 10 10 Pieces / kg body weight, e.g., about 10 5 ~about 10 9 , about 10 5 ~about 10 8 , about 10 5 ~about 10 7 , or about 10 5 ~about 10 6 Generally, higher doses are used for systemic administration than for local administration, where the immune cells of the present application are administered to the area of ​​the tumor and / or cancer. Exemplary dose ranges include 1 x 10 4 ~1×10 8 , 2 × 10 4 ~1×10 8 , 3×10 4 ~1×10 8 , 4×10 4 ~1×10 8 , 5×10 4 ~6×10 8 , 7×10 4 ~1×10 8 , 8×10 4 ~1×10 8 , 9×10 4 ~1×10 8、1×10 5 ~1×10 8 、1×10 5 ~9×10 7 、1×10 5 ~8×10 7 、1×10 5 ~7×10 7 、1×10 5 ~6×10 7 、1×10 5 ~5×10 7 、1×10 5 ~4×10 7 、1×10 5 ~4×10 7 、1×10 5 ~3×10 7 、1×10 5 ~2×10 7 、1×10 5 ~1×10 7 、1×10 5 ~9×10 6 、1×10 5 ~8×10 6 、1×10 5 ~7×10 6 、1×10 5 ~6×10 6 、1×10 5 ~5×10 6 、1×10 5 ~4×10 6 、1×10 5 ~4×10 6 、1×10 5 ~3×10 6 、1×10 5 ~2×10 6 、1×10 5 ~1×10 6 、2×10 5 ~9×10 7 、2×10 5 ~8×10 7 、2×10 5 ~7×10 7 、2×10 5 ~6×10 7 、2×10 5 ~5×10 7 、2×10 5 ~4×10 7 、2×10 5 ~4×107 , 2 × 10 5 ~3×10 7 , 2 × 10 5 ~2×10 7 , 2 × 10 5 ~1×10 7 , 2 × 10 5 ~9×10 6 , 2 × 10 5 ~8×10 6 , 2 × 10 5 ~7×10 6 , 2 × 10 5 ~6×10 6 , 2 × 10 5 ~5×10 6 , 2 × 10 5 ~4×10 6 , 2 × 10 5 ~4×10 6 , 2 × 10 5 ~3×10 6 , 2 × 10 5 ~2×10 6 , 2 × 10 5 ~1×10 6 , 3×10 5 ~3×10 6 These dosages include, but are not limited to, doses per kg, etc. Moreover, dosages can be adjusted to account for whether a single dose or multiple doses are administered. The precise determination of what is considered an effective dose can be based on factors individual to each subject.

[0328] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to the restoration or reversal of at least one measurable physical parameter associated with cancer that may, but is not necessarily, be discernible in the subject. The terms "treat," "treating," and "treatment" may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the alleviation, prevention of the onset or onset, or reduction in the duration of one or more symptoms associated with a disease, disorder, or condition, e.g., a tumor or, more preferably, cancer. In certain embodiments, "treat," "treating," and "treatment" refer to the prevention of recurrence of a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to an increase in survival time of a subject with a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0329] The cells and / or pharmaceutical compositions of the present application can be administered in combination with one or more additional therapeutic agents, hi certain embodiments, the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). [Example]

[0330] Abbreviation

[0331] [Table 3]

[0332] [Example 1] Cell line generation iPSC generation Induced pluripotent stem cell (iPSC) parent cell lines were generated from peripheral blood mononuclear cells (PBMCs) using an episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference.

[0333] Vector (plasmid) production Gene fragments (gBlocks) encoding the transgene of interest along with promoters, terminators, and homology arms were designed and chemically synthesized at IDT, Inc. The gBlock gene fragments were assembled into pUC19 plasmids using the In-Fusion® Cloning HD Plus Kit (Takara Bio; Shiga, Japan) according to the manufacturer's protocol. The reaction product from In-Fusion Cloning, i.e., the expression construct, was transformed into Stbl3 bacterial cells (Thermo Fisher; Waltham, MA) according to the manufacturer's protocol for amplification. The vector (plasmid) from the amplified expression construct was purified from the bacterial cell culture using the HiSpeed ​​Plasmid Maxi Prep Kit (Qiagen; Hilden, Germany) according to the manufacturer's protocol. The purified plasmid DNA was subjected to research-grade sequencing and assessed by restriction digestion to confirm the sequence of the transgene. The concentration of the purified plasmid DNA was measured by absorbance. Moreover, the absorbance ratios at A260 / A280nm and A260 / A230nm were measured to assess residual RNA and protein levels, respectively.

[0334] CIITA targeting plasmid The CIITA targeting plasmid contains a CAG promoter (SEQ ID NO: 63), an SV40 terminator / polyadenylation region (SEQ ID NO: 64), and the tEGFR-IL15 coding sequence. The tEGFR-IL15 transgene encodes tEGFR-IL15 (SEQ ID NO: 71), which contains residues 322-333 of domain 2, all of domains 3 and 4, and the transmembrane domain of the native EGFR sequence. The tEGFR-IL15 transgene is followed by an in-frame P2A peptide sequence (SEQ ID NO: 73), followed by the full-length IL-15 sequence (SEQ ID NO: 72). A schematic diagram of the CIITA targeting transgene plasmid is shown in Figure 1A.

[0335] AAVS1 targeting plasmid The AAVS1 targeting plasmid contains a CAG promoter (SEQ ID NO: 63), an SV40 terminator / polyadenylation region (SEQ ID NO: 64), and an anti-CD19 scFv chimeric antigen receptor (CAR) sequence (SEQ ID NO: 62). The encoded CAR contains the FMC63 scFv followed by the GMCSFR signal peptide linked to residues 114-220 of CD28 and residues 52-163 of CD3 zeta isoform 3. A schematic diagram of the AAVS1 targeting transgene plasmid is shown in Figure 1B.

[0336] B2M targeting plasmid The B2M targeting plasmid contains a CAG promoter (SEQ ID NO: 63), an SV40 terminator / polyadenylation (SEQ ID NO: 64), and a peptide-B2M-HLA-E coding sequence (SEQ ID NO: 67). The protein encoded by the B2M transgene (SEQ ID NO: 66) contains a signal peptide from HLA-G, followed by the 9-amino acid peptide VMAPRTLIL linked with a 4xGGGGS linker, the mature B2M sequence linked with a 3xGGGGS linker, and then the mature HLA-E sequence (SEQ ID NO: 65). A schematic diagram of the B2M targeting transgene plasmid is shown in Figure 1C.

[0337] Insertion of the transgene into B2M (exon 2) and CIITA (exon 1) disrupts the coding sequence and prevents translation of the full-length sequence. Loss of B2M expression prevents intrinsic MHC class I assembly and disrupts expression. Loss of CIITA prevents transcription of the HLA II gene and prevents MHC class II expression. Insertion of the transgene into intron 1 of the AAVS1 locus does not result in any alteration of the coding sequence. Homology arm sequences were designed to be located 5' and 3' to the Cpf1 genomic nuclease cleavage site and contain 500 to 1200 bp of target locus-specific sequence.

[0338] Establishment of CAR-engineered iPSC cell lines The establishment of cell lines consisted of transfection, electroporation, expansion of CAR-engineered iPSCs, cell sorting, and cell cloning steps. Three consecutive rounds of transfection and electroporation were performed using the relevant purified plasmid DNA and recombinant Cpf1 ultra / guide RNA ribonucleoprotein (RNP) complexes specific for a single target locus (either B2M, CIITA, or AAVS1). Using the Benchling™ software design tool, guide RNAs (gRNAs) with all off-target site scores below 2 (0-100) were selected (Table 3). The majority of potential off-target sites were intergenic.

[0339] [Table 4]

[0340] Briefly, a vial of iPSC cells from a parental cell line was thawed and placed in Complete Essential 8™ medium (Thermo Fisher) with H1152 Rho kinase inhibitor, pelleted, and resuspended in Complete Essential 8 medium. The cell suspension was then transferred to a well of a vitronectin-coated 6-well plate containing Complete Essential 8 medium with H1152 and incubated at 37°C, 5% CO2, and low O2. Cells from one well were expanded into a T-75 flask. When the flask reached 60-70% confluence, it was expanded and placed into another T-75 flask. When this flask reached 60-70% confluence, the cells were used for transfection. H1152 was added to the T-75 flask containing the iPSC cells, and the cells were incubated at 37°C, 5% CO2, and low O2.

[0341] Following incubation, cells were washed with DPBS, dissociated from the flask, and resuspended in Complete Essential 8 medium. Cells were counted and plated at the appropriate cell density for transfection onto vitronectin-coated T-75 flasks containing Complete Essential 8 medium with H1152. Lipofectamine stem cell reagent (Thermo Fisher) and purified plasmid DNA were prepared in Opti-MEM (Thermo Fisher) and incubated. The transfection mix containing purified plasmid DNA was added to the cells, which were then incubated at 37°C, 5% CO2, and low O2.

[0342] After transfection, cells were washed with DPBS, dissociated from the flask, and resuspended in Complete Essential 8 medium. Cells were then washed with Opti-MEM, counted, washed with additional Opti-MEM, and resuspended in Opti-MEM at a cell density appropriate for electroporation. Ribonucleoprotein (RNP) complexes were generated by combining Alt-R® CRISPR-Cpf1 crRNA with Alt-R® Cpf1 Ultranuclease (IDT; Coralville, IA). The RNP complex and Cpf1 electroporation enhancer were added to the transfected cells and electroporated. The electroporated cells were then added to wells of a prewarmed, vitronectin-coated 24-well plate containing Complete Essential 8 medium and NU7026 and incubated at 37°C, 5% CO2, and low O2.

[0343] Cells were cultured in Complete Essential 8 medium on vitronectin-coated plates for a minimum of 10 days to allow for homologous recombination repair. After the cells on the 24-well plates reached 60-70% confluence, they were dissociated, expanded, and placed into a single well of a 6-well plate. After reaching confluence, a single well of the 6-well plate was expanded and placed into a T-75 flask. Cells were maintained in culture for a minimum of 10 days, after which the cultures were analyzed by flow cytometry for the presence of the inserted transgene and / or the absence of the deleted endogenous gene. Cells were then subjected to flow cytometric sorting to isolate the modified population.

[0344] After each round of transfection and electroporation, expanded engineered cells were sorted for stable integrants by fluorescence-activated cell sorting (FACS) using transgene-specific antibodies. Sorting after each round of engineering includes markers from previous rounds, and multiple rounds of sorting may be required to sufficiently enrich the population for all respective markers. Sorting was performed on a MacsQuant Tyto cell sorter (Miltenyi Biotech; Bergisch Gladbach, Germany) using fluorescently labeled antibodies against fusion proteins of human HLA-E, human EGFR, and human CD19-Fc.

[0345] After completing all three manipulation steps and the required rounds of selection, single-cell clones were isolated by limiting dilution cloning. Cells were washed once with DPBS and dissociated from the plates. They were resuspended in Complete Essential 8 medium, filtered through a 70 μm cell strainer, counted, and diluted to a final density of 1,000 cells / mL in Complete Essential 8 medium. A 200 μL aliquot of cells was then transferred to 9 mL of StemFit® (Amsbio; Abington, United Kingdom) with 1 mL of Clone® supplement (StemCell Technologies; Vancouver, Canada), plated at 100 μL / well, and allowed to rest for 24 hours. After resting, the medium was changed every 48 hours until colonies reached approximately 2 mm in diameter, at which point wells with single colonies were identified by visual inspection and split into duplicate vitronectin-coated plates containing Complete Essential 8 medium. The medium was changed daily until the cells reached greater than 50% confluency. One plate was used to expand single cell lines in 6-well plates for use in the process, and the other was used for characterization.

[0346] Hematopoietic progenitor cell (HPC) differentiation iPSC cells thawed from a cryopreserved cell bank were grown on vitronectin-coated plates in E8 medium supplemented with H1152 Rho kinase inhibitor. iPSC cells were passaged twice by dissociation using TrypLE™ (Thermo Fisher Scientific) and replated onto vitronectin plates with E8 medium plus H1152. After dissociation with TrypLE and two passages, iPSC cells were treated with TrypLE once more and resuspended at an optimal concentration in HDM-I medium supplemented with H1152. HDM medium contains IMDM medium, Ham's F12 medium, vitamin A-free CTS B27 supplement, non-essential amino acids, magnesium 2-ascorbate phosphate, monothioglycerol, and heparin. HDM-I medium contains HDM plus CHIR99021 GSK3 inhibitor, FGF2, and VEGF. The resuspended cells were then seeded into appropriate containers depending on the scale. The next day (D1), 80% of the medium was replaced with fresh HDM-I medium. On days 2, 3, and 4, 80% of the medium was removed and replaced with HDM-II medium (HDM medium + BMP4, FGF2, and VEGF). On day 5, HPCs may begin to bud from the cell aggregates and appear in the culture. HPCs were harvested 2 days after they began to appear, but were harvested on day 8 or later. Starting on day 5, 80% of the medium was removed daily, and any HPCs present in the removed medium were collected by centrifugation. The cells were resuspended in HDM-III (HDM + BMP4, SCF, TPO, FLT3L, and IL3) and added back to the culture. HPCs were then harvested on days 8 or 9 (depending on the day of their initial appearance).

[0347] Differentiation and activation of natural killer (NK) cells and T cells HPCs were differentiated to generate NK or T cells. Cells were thawed, washed, and seeded into RetroNectin / DLL4-coated G-Rex bioreactors. Notch signaling, specific cytokines, and growth factors were used for differentiation into lymphoid lineages and subsequent maturation and activation of NK or T cells. During harvest, the cultures were concentrated, washed, formulated using defined cryopreservation media, and filled into AT vials using an M1 filling station. Vials were visually inspected, cryopreserved in a controlled-rate freezer, and stored in the vapor phase of an LN2 freezer.

[0348] NK and T cells can also be differentiated using feeder cells. Briefly, K562 myeloid leukemia cells engineered to express class I molecules, CD64, 4-1BBL, and transmembrane domains are cultured with HCP for a sufficient time to promote the differentiation of NK or T cells.

[0349] [Example 2] CD19-targeted cytotoxicity assay To demonstrate CD19-specific target cell killing, cytotoxicity was measured using the IncuCyte® assay (Essen Bioscience Inc.; Ann Arbor, MI). A CD19-knockout Reh B-cell leukemia cell line was established. For use in the Incucyte assay, cells were also transduced with NucLightRed using lentivirus from Essen Biosciences (Sartorious). Parental and CD19-knockout Reh B-cell leukemia cells were then cocultured with iNK cells expressing the FMC63 CD28z CAR (anti-CD19) at a 1:1 effector-to-target cell ratio. Target cell death was measured for 72 hours. CAR iNK cells effectively killed CD19-positive target cells (Figure 2).

[0350] [Example 3] CAR / IL-15 iNK assay To test the ability of iNK cells engineered to express an IL-15 transgene (CAR / IL-15 iNK) to release IL-15, CAR iNK or CAR / IL-15 iNK cells were cultured alone in medium or cocultured with K562 myeloid leukemia cells (ATCC) at a 1:1 effector-to-target ratio. After 24, 28, 72, or 96 hours of incubation, supernatants were collected and assayed for IL-15 concentrations using an MSD immunoassay (catalog number K151URK-4) according to the manufacturer's protocol (Meso Scale Diagnostic; Rockville, MD). In both medium alone and with K562 targets, iNK cells engineered to express the IL-15 transgene demonstrated superior IL-15 release into the medium (Figure 3A).

[0351] To examine the in vivo persistence of CAR / IL-15 iNK cells, CAR iNK or CAR / IL-15 iNK cells (10 E 6 cells) were injected intravenously into immune-deficient NSG™ mice (The Jackson Laboratory; Bar Harbor, ME) on day 0. At day 20 post-injection, human CD45 + CD56 + Blood and lungs were analyzed for the presence of infused iNK cells using fluorescence-activated cell sorting (FACS). They were detectable after 20 days only if the infused cells carried the human IL-15 transgene (Figure 3B).

[0352] To further examine the effect of the IL-15 transgene on the persistence of CAG-CAR / IL-15 cells in vivo, 1 × 10 7Mice were intravenously injected with CAG-CAR or CAG-CAR / IL-15 cells. Half of the mice were supplemented with exogenous recombinant human IL-15 (1 μg / mouse, intraperitoneally, daily) for the duration of the study. On study day 8, lungs were harvested and processed for flow cytometry analysis. Samples were stained with Fixable LiveDead NearIR (Thermo Fisher), anti-huCD45, and anti-huCD56. During analysis, iNK cells were defined as CD56 / CD45 double-positive cells and recorded as a percentage of the live cell population (Figure 3C).

[0353] To test the ability of CAR / IL-15 iNK cells to cause killing over multiple rounds of target challenge, we set up serial killing assays using bulk cultures for repeated stimulations, in parallel with Incucyte assays for quantification of cytolytic activity at each round. CAR / IL-15 iNK cells were cultured with irradiated Reh cells (2 Gy) at an effector-to-target (E:T) ratio of 1:1 for 3–4 days. Results showed that CAR / IL-15-iNK cells caused serial killing for seven rounds before exhaustion (Figure 4A). At the end of bulk culture, Reh target cells were undetectable. CAR / IL-15 iNK cells were counted using ViCell Blue to track expansion and enable the setup of subsequent bulk cultures and Incucyte assays. Using cells recovered from previous bulk cultures as the effector population, Incucyte-based killing assays were set up in parallel with each bulk culture at multiple E:T ratios of 5:1, 1:1, and 1:5.

[0354] Next, we compared CAR / IL-15-iNK cells with CAR-iNK cells that do not express IL-15. CAR / IL-15-iNK cells showed superior proliferation compared to CAR-iNK cells (Figure 4B). CAR / IL-15-iNK cells also showed superior sequential killing of Raji cells compared to CAR-iNK cells (Figure 4C).

[0355] [Example 4] Cytokine-enhanced cytotoxicity assay Interleukin-12 is a cytokine that stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells. To determine whether IL-12 had an effect on target cytotoxicity of CAR / IL-15 iNK cells, iNK cells were differentiated using standard protocols (without IL-12) or with the inclusion of 10 ng / ml recombinant human IL-12p70 (PeproTech; Rocky Hill, NJ) for the final 24 hours of culture. The iNK cells were used in an Incucyte killing assay to determine their efficacy in killing the Raji CD19+ B-cell leukemia cell line (ATCC; Manassas, VA). IL-12-primed cells demonstrated rapid killing of Raji cells compared to cells differentiated in the absence of IL-12 (Figure 5A).

[0356] IL-12-primed iNK cells were further examined for their effect on tumor formation in vivo. Luciferase-labeled Burkitt's lymphoma cell line Raji was implanted intravenously (iv) into female NSG™ mice on study day 0. Mice were injected with 1 x 10 7 Unprimed or IL12-primed CAG-CAR-IL15 iNK cells were intravenously infused on days 1, 4, and 7 of the study. Starting on day 1, mice were supplemented with intraperitoneal recombinant human IL-2 (100,000 IU, PeproTech #200-02) three times a week for the duration of the study. An untreated group served as a control. Mice were injected with luciferin (VivoGlo™, Promega) and then imaged using an IVIS SpectrumCT (Perkin Elmer). The reaction of the luciferin substrate with the firefly luciferase enzyme produced by the Raji tumor cells produces light, which is measured as the bioluminescence signal. Data are expressed as the mean whole-body bioluminescence mean brightness ± SD. At the end of the study on day 20, 50% and 62% tumor growth inhibition was observed with unprimed and IL-12 primed iNK treatment, respectively (*p<0.05, **p<0.01) (Figure 5B).

[0357] [Example 5] Elimination assay CAR / IL15 iNK cells were engineered to express tEGFR as a depletion feature, intended to act as a target for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by dosing with cetuximab, an EGFR inhibitor. iNK cells with or without EGFR expressed from the transgene were cocultured with human PBMCs. Increasing doses of cetuximab were added to promote ADCC, and the cells were incubated for 3 hours. Control cells were treated with human IgG1. The results are shown in Figures 6A-6B. Only EGFR-expressing iNK cells exhibited dose-dependent cell death (Annexin V staining) in the ADCC assay. This data demonstrates that cetuximab can be used to efficiently deplete CAR / IL15 / tEGFR iNK cells.

[0358] [Example 6] MHC class I and class II deletions The plasmid constructs described herein are designed to target the integration of exogenous polypeptides useful in the invention of this application and simultaneously delete or reduce the expression of MHC class I and class II genes. Genome manipulation of IPSCs using B2M and CIITA targeting plasmids is performed as described above. After differentiation into NK cells, confirmation of MHC class I and II expression is confirmed using flow cytometry using antibodies specific for HLA I (alpha chain) and HLA II (alpha or beta chain).

[0359] [Example 7] Nonclassical HLA expression The iNK cells of the present application are engineered to further express non-classical HLA proteins, HLA-E or HLA-G, which are confirmed at all stages by flow cytometry using antibodies specific for HLA-E or HLA-G.

[0360] [Example 8] iNK-mediated lysis of K562 cells To demonstrate the ability of iNK cells to induce basic NK cell functionality, we evaluated iNK clone iNK1248-iPSC611 and primary peripheral blood NK (PB-NK) cells from three PBMC donors for their ability to kill K562 cells using the Incucyte live imaging platform. The Incucyte platform allows real-time quantification of fluorescently labeled target cells, whose depletion serves as a measure of target lysis.

[0361] Generation and expansion of K562 cell line Chronic myeloid leukemia (CML) cell line K562 was obtained from ATCC. K562 cells were transduced with NucLight Red lentivirus according to the standard Sartorius protocol. Transduced cells were selected and cultured in IMDM medium containing 1 μg / mL puromycin. Cells were cultured at a cell density of 1e5-1e6 cells / mL, splitting every 2-3 days.

[0362] Isolation of NK PBMCs from three donors were thawed at 37°C and centrifuged at 300g for 3 minutes. The supernatant was aspirated, and cells were resuspended in RPMI + 10% FBS 10ng / mL Il-15 and allowed to rest overnight. NK cells were isolated from resting PBMCs using CD56 MicroBeads, human (Miltenyi, part 130-050-401) according to the manufacturer's recommended protocol.

[0363] NK purity check CAR-iNK clones and PB-NK donors were plated at 100,000 cells / well in 96-well U-bottom plates (BD falcon 353077). All wash steps were performed by centrifuging at 300 x G for 3 minutes and flicking the supernatant into a sink. Cells were washed twice in PBS and stained with 100 μl of LIVE / DEAD™ Fixable Near-IR Viability Stain (Thermo Fisher) diluted 1:1000 in PBS for 15 minutes at room temperature (RT). Cells were washed twice in BD FACS stain buffer BSA (BD). TrustainFcX, a human Fc receptor blocker, was diluted 1:100 in BD FACS stain, and 50 μl of the dilution was added to each well and incubated at 4°C for 30 minutes. Cells were washed twice in BD FACS stain buffer BSA (BD). A staining cocktail was made by diluting mAbs against CD16, CD4, CD19, CD45, CD3, CD56, and CD14 1:100 in BD FACS staining buffer. Cells were stained with 50 μl of the staining cocktail and incubated at 4°C for 30 minutes, protected from light. Cells were washed twice using BD FACS staining buffer and fixed in 100 μl of BD stabilizing fixative. All samples were run at the same voltage using a BD Symphony A3 Lite. Flow cytometry data were analyzed using FlowJo 10.7.2.

[0364] [Table 5]

[0365] Setting up the Incucyte assay 4 x 10 for 20:1 E:T wells 5 / well, 2 x 10 per 10:1 E:T well 5 / well, and 2 x 10 for 1:1 E:T wells 4CAR-iNK and PB-NK effector cell clones were added to wells of a 96-well flat-bottom plate (BD catalog no. 353072) in 100 μL of NKCM medium at a final effector count of 1 / well. NKCM assay medium was made with 500 mL of IMDM and 500 mL of Ham's F-12 nutrient mix as the base medium. The base medium was supplemented with 2% CTS B-27 supplement, XenoFree, vitamin A-free, 1% MEM non-essential amino acid solution, 250 μM Mg-2-phosphate ascorbate, 100 μM mono-thioglycerol, 1% GlutaMax, and 2 mM nicotinamide.

[0366] Next, 2 x 10 4 K562-NLR cells were added to each well in 100 μL of NKCM media. Triplicate wells were used for each CAR-iNK effector cell assay. The assay plate was allowed to rest at room temperature for 15 minutes to allow the cells to settle. The plate was placed in an Incucyte S3 instrument in a 37°C incubator with 5% CO2. The instrument scan type was set to whole-well read with image phase and red channel with a red capture time of 400 ms. The instrument scan frequency was set to read every 3 hours for 72 hours. Whole-well analysis parameters were selected for the Incucyte assay. The RCU threshold was set to 2.0, the radius was set to 100 μm, and edge splitting was on.

[0367] analysis NLR counts / well data for all wells and all time points were exported from the Incucyte 2020A software and pasted into Microsoft Excel. To calculate values ​​for normalized target counts as a percentage of the mean NLR counts in target cell-only wells, each triplicate value was divided by the mean of the target cell-only wells appropriate for each target cell line (N=3), and this value was multiplied by 100. Data were graphed using GraphPad Prism software (version 8). Triplicate normalized NLR target count values ​​were graphed on the Y-axis with each time point on the X-axis.

[0368] result iNK1248-iPSC611 and PB-NK cells demonstrated the ability to kill K562 cells at effector-to-target ratios of 20:1, 10:1, and 1:1. As shown in Figure 7A-C, the Incucyte-based assay measured the reduction of Nuclight Red K562 cells over time at effector-to-target ratios of (A) 20:1, (B) 10:1, and (C) 1:1. Normalized target cell counts as a percentage of target cell counts represent the average of four iNK1248-iPSC611 and three PB-NK cells.

[0369] In terms of purity, PB-NK cells were 87%–96% CD45+ / CD56+ after isolation. iNK1248-iPSC611 were 98.8% CD45+ / CD56+. PB-NK cells were 20.15%–0.195% CD3+ and 19.1%–0.075% CD3+ / CD56+. iNK1248-iPSC611 were 0.08% CD3+ and 0.048% CD3+CD56+ (Figure 8).

[0370] [Example 9] In Vitro Depletion of CD19+ Cells Using CAR-iNK Cells The CAR-iNK clone, iNK1248-iPSC611, was engineered to express an anti-CD19 chimeric antigen receptor (CAR) and target CD19+ cancers. Using the Incucyte live cell imaging platform, we demonstrated the cytolytic activity of iNK1248-iPSC611 at multiple effector-to-target (E:T) ratios by real-time quantification of fluorescently labeled target cells, whose depletion serves as a measure of the efficacy of effector-target cell killing. CAR-mediated lysis of CD19+ target cells was demonstrated using an isogenic pair of Reh and NALM6 cell lines, either naively expressing CD19 or CD19 knockout (KO).

[0371] NucLight Red transduction of target cell lines Reh and NALM6 cells were obtained from ATCC. Cell lines were transduced with Incucyte NucLightRed lentiviral reagent (EF1α; promoter, puromycin selection) according to the manufacturer's protocol at an MOI of 3 in medium containing 8 μg / mL polybrene. NLR-transduced cells were selected, and cell lines were cultured in RPMI with 10% FBS and 1 μg / mL puromycin.

[0372] Generation of Reh-CD19KO and NALM6-CD19KO target lines Reh-CD19KO and NALM6-CD19KO were generated using the Lonza CRISPR-Cas-9 system on parental Reh and NALM6 cells (previously transduced with NucLight Red) using the manufacturer's protocol for the Amaxa 4-D Nucleofector. The target sequence for the custom CD19KO crRNA used was GCTGTGCTGCAGTGCCTCAA. CD19+ cells were depleted using the Human CD19 Positive Selection Kit II according to the manufacturer's protocol, and CD19 expression was assayed by flow cytometry. Cell lines were cultured in RPMI-10% FBS, 1 μg / mL puromycin.

[0373] [Table 6]

[0374] Setting up the Incucyte CAR-iNK killing assay 2×10 5 (10:1), 1×10 5 (5:1), 2×10 4 (1:1), or 4 × 10 3 (1:5) of each CAR-iNK effector cell clone was added to wells of a 96-well flat-bottom plate (BD Cat. No. 353072) in 100 μL of NKCM medium, followed by 2 × 10 4Reh-NLR, Reh-CD19KO-NLR, NALM6-NLR, or NALM6-CD19KO-NLR cells were added in 100 μL of NKCM medium. Triplicate wells were used for each CAR-iNK effector cell assay. The assay plate was allowed to rest at room temperature for 15 minutes to allow cells to settle. The plate was placed in an Incucyte S3 instrument in a 37°C incubator with 5% CO2. The instrument scan type was set to whole-well read with image phase and a red channel capture time of 400 ms. The instrument scan frequency was set to read every 2 hours for 72 hours. Whole-well analysis parameters were selected for the Incucyte assay. The RCU threshold was set to 2.0, the radius was set to 100 μm, and edge splitting was turned on.

[0375] analysis NLR counts / well data for all wells and all time points were exported from the Incucyte 2020A software and pasted into Microsoft Excel. To calculate values ​​for normalized target counts as a percentage of the mean NLR counts in target cell-only wells, each triplicate value was divided by the mean of the target cell-only wells appropriate for each target cell line (N=3), and this value was multiplied by 100. Data were graphed using GraphPad Prism software (version 8). Triplicate normalized NLR target count values ​​were graphed on the Y-axis with each time point on the X-axis.

[0376] result Antigen-specific lysis of both Reh and NALM6 cells by iNK1248-iPSC611 cells was demonstrated across a range of effector-to-target ratios, with CD19+ cells killed faster and more completely than matched CD19KO lines at each E:T ratio tested.

[0377] The four E:T ratios showed a range of cytolytic activity against Reh cells (Figure 9). Lower cytolytic activity was observed in Reh CD19KO cells compared to parental Reh cells. Figure 9 shows the results of an Incucyte-based assay measuring the reduction of Nuclight Red target cells over time at four effector-to-target ratios. Normalized target cell counts as a percentage of target cell-only counts in Reh and Reh-CD19KO cells cocultured with iNK1248-iPSC611 at E:T ratios of (A) 10:1, (B) 5:1, (C) 1:1, and (D) 1:5. Figure 10 shows the results of an Incucyte-based assay measuring the reduction of Nuclight Red target cells over time at four effector-to-target ratios. Normalized target cell counts as a percentage of target cell-only counts in NALM6 and NALM6-CD19KO cocultured with iNK1248-iPSC611 at E:T ratios of (A) 10:1, (B) 5:1, (C) 1:1, and (D) 1:5.

[0378] [Example 10] In Vitro Persistence of iNK Cells The single-cell iNK clone iNK1248-iPSC611 was engineered to secrete the NK homeostatic cytokine IL-15. In a 21-day persistence assay, the in vitro persistence of iNK1248-iPSC611 was compared to bulk unengineered "wild-type" (WT) iNK iNK1487-iPSC005 cells and the NK cell leukemia line KHYG-1 in the presence of varying levels of IL-2 (10 nM to 0 nM). Cells were harvested every 3–4 days, counted with ViCell Blue, and replated in fresh medium. The viable cell counts collected from ViCell Blue were used to calculate cumulative fold expansion.

[0379] 21-day residual assay 1.5×10 6iNK1248-iPSC611, WT iNK1487-iPSC005, or KHYG-1 immortalized NK cells were added at 0.5 × e6 cells / mL to individual wells of a 24-well plate in a total of 3 mL of NKCM containing six different concentrations of IL2. 6 KHYG-1 immortalized NK cells were also added to separate wells of a 24-well plate at 0.5× e6 cells / mL in a total of 3 mL of RPMI + 10% HI FBS + 1× Pen Strep containing six different concentrations of IL2. Both plates were transferred to an incubator set at 37°C with 5% CO2.

[0380] After 72 or 96 hours, cells were harvested and transferred to 15 mL conical tubes. The cells were centrifuged at 300 g for 10 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 3 mL of basal RPMI assay medium. 200 microliters of cells were removed and counted using ViCell Blue.

[0381] After counting, cells were centrifuged again at 300g for 10 minutes. The supernatant was aspirated, and cells were resuspended at 0.5 x e6 cells / mL in NKCM assay medium or RPMI assay medium containing the corresponding concentration of IL2. Cells at 0.5 x e6 cells / mL were replated at 3 mL / well. If cells were resuspended at 0.5 x e6 cells / mL in a volume less than 3 mL, the entire volume was plated. If the resuspension volume was less than 200 uL, the cell line was not replated. At the end of day 14, the assay was terminated and cells were discarded.

[0382] analysis Cell counts and cell viability were obtained using ViCell Blu. The population used to calculate fold change was viable cells / mL. Data were graphed using GraphPad Prism software (version 8.4.3).

[0383] result The single-cell clone iNK1248-IPSC611 persisted in vitro longer than WT iNK1487-iPSC005 in the absence of exogenous IL-2 (Figure 11). Cells were cultured in basal NKCM at 37°C with 5% CO2 for 14 days. Every 3–4 days, cells from all conditions were harvested, counted using ViCell Blue, resuspended at 0.5 × e6 / mL in the appropriate medium, and then replated. After 21 days, cumulative fold changes were calculated. The single-cell clone iNK1248-IPSC611 persisted in vitro longer than WT iNK1487-iPSC005 in the absence of exogenous IL-2, indicating that the IL-15 transgene was functional and exhibited its intended mode of action, i.e., high persistence. IL-15 released by iNK1248-IPSC611 is sufficient to support homeostatic survival of cells but is insufficient to trigger mitogenic expansion.

[0384] Exogenous IL-2 support increased the survival of both iNK1248-iPSC611 and WT iNK1487-iPSC005 (Figures 12A-F). Cells were cultured in NKCM containing one of six IL-2 concentrations: 10 nM (Figure 12A), 3 nM (Figure 12B), 1 nM (Figure 12C), 0.3 nM (Figure 12D), 0.1 nM (Figure 12E), or 0 nM (Figure 12F) at 5% CO2 and 37°C for 21 days. Every 3–4 days, cells from all conditions were harvested, counted using ViCell Blue, resuspended in the appropriate medium at 0.5 × e6 cells / mL, and then replated. After 21 days, cumulative fold changes were calculated. Supporting iNK1248-iPSC611 with exogenous IL-2 increased the persistence of both iNK1248-iPSC611 and WT iNK1487-iPSC005, indicating that additional homeostatic cytokines are required to enable limited mitogen-expanded proliferation of iNK1248-iPSC611. To determine whether the combination of engineered IL-15 and exogenous IL-2 induces uncontrolled proliferation of therapeutic iNK cells, we cultured the cells for 2 weeks in the presence of IL-2 and compared iNK1248-iPSC611 with the IL-2-dependent NK leukemia line KHYG-1. KHYG-1, but not iNK1248-iPSC611, demonstrated logarithmic growth over the 2-week culture period.

[0385] [Example 11] In Vitro Depletion of Therapeutic iNK Cells by Cetuximab Antibody-dependent cellular cytotoxicity (ADCC) is a cellular immune defense mechanism in which target cells coated with antibodies that recognize cell surface antigens are lysed by effector cells bearing Fc receptors. ADCC can be mediated by a variety of immune cells, including natural killer (NK) cells, neutrophils, macrophages, and eosinophils, through the recognition of bound immunoglobulins via their Fc receptors, particularly CD16 (FcγRIII).

[0386] Cetuximab is a chimeric mouse-human antibody that targets the extracellular domain of the epidermal growth factor receptor (EGFR). It has been demonstrated to mediate ADCC against EGFR-expressing tumor cell lines via its human IgG1 Fc region (Kurai, 2007).

[0387] The following experiment was performed to assess whether iPSC-derived NK (iNK) development candidates (e.g., therapeutic iNK) express EGFR and are susceptible to ADCC mediated by cetuximab compared to an isotype control antibody when cultured with interleukin (IL)-2-activated peripheral blood mononuclear cells (PBMCs).

[0388] Isolation and culture of primary effector cells Peripheral mononuclear blood cells (PBMCs) were collected from buffy coats (Bloodworks Northwest) of consenting healthy adult donors by centrifugation over a Ficoll-Hypaque density gradient. Prior to use in experiments, cells were cultured at 1 × 10 in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 55 mM b-mercaptoethanol (Life Technologies) in the presence of 10 ng / mL IL-2 (Peprotech). 6 The cells were cultured overnight at 100 cells / mL.

[0389] ADCC assay iNK cells were labeled with 2.5 mM CTV (Life Technologies) and plated at 2.5 × 10 as targets in triplicate in 96-well flat-bottom plates (Corning). 4 Cells were plated at 1000 / well. Cetuximab (Selleckchem) or human IgG1 isotype control (Invivogen) was pre-incubated with therapeutic iNK targets at concentrations ranging from 10 pg / mL to 10 mg / mL for 30 min before effector cell addition. IL-2-activated effector PBMCs were added in triplicate wells / condition at an effector:target (E:T) ratio of 25:1, and cultures were maintained at 5% CO2, 37% C. oThe cells were incubated in an incubator for 16 hours. Dead cells were identified by flow cytometry using LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher) according to the manufacturer's protocol. Samples were acquired on a Symphony A3 (BD ​​Biosciences) and analyzed with FlowJo version 10.7.1 software.

[0390] Flow cytometry For determination of antibody bound per cell (ABC), 2 x 10 5 Therapeutic iNK cells were labeled with EGFR-PE (Novus Biologicals) for 15 minutes at room temperature in the dark, washed with cell staining buffer (BioLegend), and fixed with fixation buffer (BioLegend) for 10 minutes at room temperature in the dark. A single tube of BD Quantibrite beads (BD Biosciences) was rehydrated in 500 mL of PBS according to the manufacturer's protocol. Labeled therapeutic iNK cells and the BD Quantibrite PE tube were acquired on a Symphony A3 (BD ​​Biosciences) using the same voltage and settings, and all samples were analyzed with FlowJo version 10.7.1 software. Using known PE-to-antibody ratios, the number of PE molecules per cell could be converted to antibody counts per cell. Quantibrite beads were gated by FSC-A versus SSC-A. PE fluorescence was then visualized as a histogram, and gates were set for each of four distinct peaks. The geometric mean fluorescence was exported for each PE peak and used to calculate ABC.

[0391] For ADCC assay analysis, cells were transferred to 96-well round-bottom plates (Falcon), washed in 1x PBS, pH 7.2 (Life Technologies), and resuspended in PBS containing LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher) according to the manufacturer's protocol. Prior to antibody addition, nonspecific binding to Fc receptors (FcRs) was blocked using Human TruStain FcX Fc Receptor Blocking Solution (BioLegend). Cells were incubated with antibodies against CD56 and CD16 for 20 minutes at room temperature and washed three times with cell staining buffer (BioLegend) before fixation with fixation buffer (BioLegend). Samples were collected using a Symphony A3 (BD ​​Biosciences), and all FCS files were analyzed using FlowJo version 10.7.1 software.

[0392] Lymphocytes were gated based on forward scatter area (FSC-A) and side scatter area (SSC-A). Singlets were excluded based on a forward scatter area (FSC-A) versus forward scatter height (FSC-H) gate. CTV + Therapeutic iNK targets or CTV - Gating on effector cells followed by CTVs positively labeled for LIVE / DEAD Fixable Near-IR + Gating was performed on therapeutic iNK cells. The % of therapeutic iNK targets killed was determined by gating on lymphocytes from cells, followed by excluding doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally gating on LIVE / DEAD™ Near-IR+, as shown in Figure 13. FSC-A = forward scatter area, SSC-A = side scatter area, FSC-H = forward scatter height, CTV = CellTrace Violet, NIR = Near-IR.

[0393] analysis To calculate the number of antibodies bound per cell (ABC), a linear regression of Log10 PE molecules / bead was plotted against Log10 geometric mean-PE using the following formula: y = mx + c, where y equals Log10 fluorescence and x equals Log10 PE molecules / bead. Using the above formula, the number of antibodies bound per cell for each sample was determined by interpolating the ABC value based on the geometric mean fluorescence value for each sample.

[0394] LIVE / DEAD NIR + CTV + The specific cell lysis rate for the ADCC assay was calculated as (Kim, 2007) using the following formula, where targets are considered dead iNK and spontaneous iNK cell death is determined by iNK cells cultured without the addition of effector cells (E:T of 0:1):

[0395] result Using geometric mean fluorescence intensity values, an ABC value of 7,341 was calculated for therapeutic iNK by Quantibrite bead technology (Figure 14 and Table 6). Figure 14 shows EGFR PE levels on EGFR-stained therapeutic iNK (black histogram) compared with unstained therapeutic iNK (gray histogram) or unedited WT iNK (dashed line). EGFR expression on therapeutic iNK cells was observed by flow cytometry with an ABC value of 7,341. This level of EGFR was sufficient to observe cetuximab-mediated ADCC activity with an EC50 of 2.0 ng / mL in cocultures of iNK with IL-2-activated PBMCs.

[0396] [Table 7]

[0397] The addition of cetuximab to co-cultures of IL-2-activated PBMCs and iNK cells induced ADCC of therapeutic iNK targets with an EC of 2.0 ng / mL. 50IL-2-activated PBMCs were co-cultured with therapeutic iNK at an E:T ratio of 25:1 for 16 hours to determine the specific cell killing rate of iNK. Each data point represents the mean of triplicate wells, with error bars ± standard deviation. Although some background killing was observed at the highest antibody concentrations, addition of the human IgG1 isotype control did not mediate ADCC of the therapeutic iNK targets.

[0398] [Example 12] Antibody and complement escape using B2M knockout Allogeneic cell therapy products derived from induced pluripotent stem cells (iPSCs) have the potential to be used as off-the-shelf treatments for many diseases, but they can generate a severe immune response by the host due to incompatibility in human leukocyte antigen (HLA) genes. In addition to the immune response mediated by CD8 T cells against HLA class I molecules, some patients may have pre-existing antibodies (Abs) against these polymorphic proteins (1, 2). If Abs against HLA class I molecules are indeed present, there is the potential for complement-mediated cytotoxicity (CDC) of effector cells. A strategy to eliminate Ab binding to HLA class I molecules is by deleting beta-2 microglobulin (b2M), which encodes a subunit common to HLA class I proteins and is required for cell surface expression.

[0399] The CDC assay is a simple method for measuring how well Abs induce cell killing in the presence of complement proteins (3). Plasma, as well as serum, contains the full range of complement proteins known as the complement cascade. However, these molecules are unstable, and therefore, collected serum samples must be promptly frozen before use in a CDC assay. Alternatively, rabbit complement can be used as a reagent in the assay in place of human complement. Using a common pan-HLA-ABC Ab to model potential HLA class I titers from patients (4), iNK cells were tested to demonstrate the sensitivity of wild-type (WT) HLA class I-expressing iNK cells and the protection from CDC of B2M knockout (KO) clone 611 iNK cells.

[0400] Complement-mediated cytotoxicity assay iNK cells, WT 005 and clone 611, were diluted to 4x10e6 cells / mL in RPMI-1640 basal medium. iNK cells were seeded at 200K cells / well in polypropylene, U-well, 96-well plates (50uL / well). Samples were seeded in triplicate. Abs were diluted to 40ug / mL in RPMI-1640 and dispensed at 50uL / well (final 10ug / mL). Baby rabbit complement (BRC) was thawed immediately before use, then diluted 1:5 in RPMI-1640 and dispensed at 100uL / well (final 10% BRC). The final volume in each well was 200uL. For both iNK cell types, there were four conditions: A, no addition (RPMI-1640 only); B, isotype Ab + BRC; C, anti-HLA-ABC Ab + BRC; and D, anti-CD52 Ab + BRC. Cells were then incubated at 37°C, 5% CO for 1 hour.

[0401] After the incubation period, plates were centrifuged at 1200 RPM for 1 minute, decanted, and the RPMI-1640 along with the BRC was removed and replaced with 200 uL / well of RPMI-1640 + 10% heat-inactivated FBS. Cells were then counted using trypan blue to score both live and dead cells.

[0402] analysis Cell viability was graphed and statistically analyzed using GraphPad Prism software. Student's T-test was used to assess statistical significance of differences in viability. Differences between samples were considered significant when the probability value (p) was ≤ 0.05.

[0403] result Upon thawing and centrifugation, cells were resuspended in 1 mL of Easysep buffer and counted for viability using trypan blue. Prior to use in the CDC assay, cells were found to have high viability: WT005: 24.6 x 10e6 / mL, 95% viability; Clone 611 iNK cells: 22.6 x 10e6 / mL, 93% viability.

[0404] Removal of B2M from iNK cells protects them from complement-mediated cytotoxicity in the presence of Abs against HLA-ABC molecules and complement. As shown in Figure 16, both freshly thawed WT005 and clone 611 iNK cells were found to maintain high viability when cultured alone or with isotype Abs plus BRC in RPMI-1640 for 1 hour. In contrast, only WT005 iNK cells were killed in the presence of HLA-ABC Abs plus BRC, whereas clone 611 iNK cells were ineffective. To demonstrate that clone 611 iNK cells were still susceptible to complement-mediated killing, we included Abs against CD52. Addition of anti-CD52 Abs plus BRC resulted in the killing of both iNK cells.

[0405] [Example 13] Comparison of CTL activation and iNK cell lysis between β2M-deficient iPSC-derived NK cells and β2M-expressing wild-type iNK cells Allogeneic cell therapy products derived from induced pluripotent stem cells (iPSCs) have the potential to be used as off-the-shelf treatments for many diseases, but they can generate a severe immune response by the host due to incompatibility in human leukocyte antigen (HLA) genes (Lanza, et al. Nat Rev Immunol. 2019 Dec;19(12):723-733). In particular, direct lysis of mismatched class I HLA-bearing cells occurs via activation of host CD8+ T cells that interact with class I HLA molecules (Felix, et al. Nat Rev Immunol. 2007 Dec;7(12):942-53). Activation of host CD8 T cells is disrupted by the deletion of beta-2 microglobulin (β2M), which encodes a subunit common to all class I HLA genes and is required for their surface expression (Krangel, et al. Cell. 1979 Dec;18(4):979-91; and Zijlstra, et al. Nature. 1989 Nov 23;342(6248):435-8).

[0406] Here, we culture iPSC-derived NK (iNK) cells gene-edited to be β2M-deficient (KO) with CD8+ cytotoxic lymphocytes (CTL) derived from peripheral blood mononuclear cells (PBMC) from multiple donors to determine whether they induce CTL activation and lysis of iNK cells compared with wild-type iNK cells expressing β2M.

[0407] Generation of effector cytotoxic lymphocytes (CTLs) Cryopreserved peripheral mononuclear blood cells (PBMCs) isolated from consented healthy adult donors were purchased (StemCell Technologies) and stored in liquid nitrogen until use. CTLs specifically reactive with the parent iPSC line were generated. Briefly, 5 × 10 7T cells were isolated from PBMCs using a human T cell isolation kit (StemCell Technologies) according to the manufacturer's instructions and primed three times by co-culturing with parental iPSC-derived iNK cells in medium containing IL2. Following another round of T cell isolation, T cells were expanded with Immunocult anti-CD2 / CD3 / CD28 stimulation reagent (StemCell Technologies) in medium containing IL2, IL7, and IL15. The expanded cells were cryopreserved at 107 cells / ml in CS-10 (StemCell Technologies) buffer.

[0408] Allo-escape cytotoxicity and CTL activation assays iNK cells were labeled with 5 μM CTV (Life Technologies) according to the manufacturer's instructions and plated at 5 × 10 cells in 96-well U-bottom plates (Falcon) as targets. 4 Cells / well were plated in duplicate. Cryopreserved CTLs were thawed and added at a 5:1 effector:target (E:T) ratio in triplicate wells / condition, and cultures were incubated for 48 hours in a 5% CO2, 37% CO2 incubator.

[0409] Flow cytometry Cells were washed in 1x PBS pH 7.2 (Life Technologies) and resuspended in PBS containing LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher) according to the manufacturer's protocol. Prior to antibody addition, nonspecific binding to Fc receptors (FcRs) was blocked using Human TruStain FcX Fc Receptor Blocking Solution (BioLegend). Cells were incubated with antibodies against TCRab, CD4, CD8, and CD25 for 20 minutes at room temperature, washed three times with cell staining buffer (BioLegend), and then fixed with fixation buffer (BioLegend). Samples were collected using a Symphony A3 (BD ​​Biosciences), and all FCS files were analyzed using FlowJo version 10.7.1 software.

[0410] Lymphocytes and quantification beads were gated based on forward scatter height (FSC-H) and side scatter area (SSC-A). Singlets were excluded based on a forward scatter area (FSC-A) vs. forward scatter height (FSC-H) gate. Viable cells were gated as LIVE / DEAD NIR stain-negative. T cells (TCRαβ positive, CTV negative) and iNK cells (CTV positive and TCRαβ negative) were gated based on CTV and TCRαβ. Within the T cell gate, CD8 positive and CD4 negative cells were selected. Within the CD8+ T cell population, CD25 expression was assessed, and the CD25 positive gate was determined to capture minimal positive background events among T cells cultured alone without target (Figure 17). Quantification beads and lymphocytes were gated from the cells as shown in Figure 17. Within lymphocytes, doublet exclusion was followed by gating on LIVE / DEAD™ Near-IR negative, followed by gating on CTV to identify iNK cells and TCRαβ to identify T cells. Within T cells, CD4 negative, CD8 positive cells were gated followed by gating on CD25 to identify activated CD8+ T cells. Key assay parameters are shown: quantitation beads, live iNK cells, and activated CD8+ T cells. FSC-A = forward scatter area, SSC-A = side scatter area, FSC-H = forward scatter height, FSC-W = forward scatter width, LD = LIVE / DEAD™ Near-IR, CTV = CellTrace Violet.

[0411] analysis The number of viable iNK cells for each well was normalized by dividing the acquired CTV+ gate event count by the event count from the quantification bead gate. The average of duplicate wells for each donor condition was used for calculations. Specific lysis of iNK cells by CTLs was determined by the following formula:

[0412] Here, iNK cis the normalized CTV+ event count in a given iNK:CTL co-culture condition; iNK a is the normalized CTV+ event count in the corresponding control iNK-only condition. To determine the significance of the assay results, p-values ​​were determined by Student's unpaired t-test of the assay values, n=3 individual donors.

[0413] result Specific killing of parental iNK cells was observed at 86-98%, corresponding to 64-84% activation of CTLs when co-cultured with parental iNK cells. Specific killing of 0.5-21% was observed among edited β2MKO iNKs, corresponding to 1-3% activation of CTLs co-cultured with β2MKO iNK cells. Both iNK killing and CTL activation were significantly reduced when β2MKO iNK cells were used as targets.

[0414] iNK cells were incubated alone or with CTLs at a 5:1 CTL:iNK ratio for 48 hours, and viable iNK cells were then measured by flow cytometry (Figure 18A). Parental iNK cells showed 86-98% specific lysis, whereas β2MKO iNK cells showed 0.5-21% specific lysis (Figure 18B).

[0415] CTLs were incubated alone or with iNK at a 5:1 CTL:iNK ratio for 48 hours, and then CD8+ T cell activation by CD25 expression was measured by flow cytometry (Fig. 19A). 64-84% of CTLs were activated by parental wild-type iNK, whereas 1-3% were activated by β2MKO iNK, and 0.5-5% were activated in the absence of target cells (Fig. 19B).

[0416] [Example 14] β2M - / - / HLA-E + PBMC-mediated killing of iNK cells Allogeneic cell therapy products derived from induced pluripotent stem cells (iPSCs) have the potential to be used as off-the-shelf treatments for many diseases, but they can generate a severe immune response by the host due to incompatibility in human leukocyte antigen (HLA) genes. A strategy to eliminate host CD8 T cell activation is by deleting beta-2 microglobulin (β2M), which encodes a subunit common to class I major histocompatibility complex (MHC) and is required for MHC class I surface expression (Krangel, et al. Cell. 1979 Dec;18(4):979-91; and Zijlstra, et al. Nature. 1989 Nov 23;342(6248):435-8).

[0417] However, a limitation of this approach is that although the rejection of engineered iPSC cell products by CD8 can be overcome, these MHC class I-negative cells can be lysed by natural killer (NK) cells due to a "missing self" (Bix, et al. Nature 349, 329-331 (1991); Liao, et al. Science 253, 199-202 (1991)).

[0418] One approach to limit lysis by host NK cells is overexpression of HLA-E on the surface of iPSC-derived cell products (Gornalusse, et al. Nat Biotechnol. 2017 Aug;35(8):765-772; Hoerster, et al. Front Immunol. 2021 Jan 29;11:586-168). HLA-E presents peptides derived from the signal sequences of other HLA class I molecules and is a minimally polymorphic ligand that binds to the inhibitory NK receptor complex CD94 / NKG2A (Braud, et al. Nature 349, 329-331 (1991); Miller, et al. J Immunol. 2003 Aug 1;171(3):1369-75).

[0419] Here, β2M - / -iPSC-derived NKs (iNKs) that have been edited to be β2M but express HLA-E (e.g., therapeutic iNKs) will be cultured with peripheral blood mononuclear cells (PBMCs) to determine whether these cells are less sensitive to killing by PBMCs compared to iNKs that lack β2M and do not express HLA-E.

[0420] Isolation and culture of primary effector cells Peripheral mononuclear blood cells (PBMCs) were collected from buffy coats (Bloodworks Northwest) of consenting healthy adult donors by centrifugation over a Ficoll-Hypaque density gradient and cryopreserved in a Cryostor CS10.

[0421] Allogeneic escape cytotoxicity assay iNK cells were labeled with 2.5 μM CTV (Life Technologies) according to the manufacturer's instructions, and 2.5 × 10 cells were used as targets. 4 Cells were plated in triplicate in 96-well flat-bottom plates (Corning) at 1000 / well. Cryopreserved PBMCs were thawed and added to triplicate wells / conditions at a 25:1 effector:target (E:T) ratio, and cultures were incubated for 72 hours in a 5% CO2, 37% CO2 incubator. Dead cells were identified by flow cytometry using LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher) according to the manufacturer's protocol. Samples were acquired on a Symphony A3 (BD ​​Biosciences) and analyzed with FlowJo version 10.7.1 software.

[0422] Flow cytometry For determination of antibody bound per cell (ABC), 1 x 10 5iNK cells were labeled with mouse IgG1 PE isotype control (BioLegend) or HLA-E PE (BioLegend) for 15 minutes at room temperature in the dark, washed with cell staining buffer (BioLegend), and fixed with fixation buffer (BioLegend) for 10 minutes at room temperature in the dark. A single tube of BD Quantibrite beads (BD Biosciences) was reconstituted in 500 mL of PBS according to the manufacturer's protocol. Labeled iNK cells and the BD Quantibrite PE tube were acquired on a Symphony A3 (BD ​​Biosciences) using the same voltage and settings, and all samples were analyzed with FlowJo version 10.7.1 software. By using a known ratio of PE to antibody, the number of PE molecules per cell could be converted to the number of antibody molecules per cell. Quantibrite beads were gated by FSC-A vs. SSC-A. PE fluorescence was then visualized as a histogram, and gates were set for each of four distinct peaks. The geometric mean fluorescence for each PE peak was exported and used for the calculation of ABC.

[0423] For NK cell phenotyping, cells were transferred to 96-well round-bottom plates (Falcon), washed in 1x PBS pH 7.2 (Life Technologies), and resuspended in PBS containing LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher) according to the manufacturer's protocol. Prior to antibody addition, nonspecific binding to Fc receptors (FcRs) was blocked using Human TruStain FcX Fc Receptor Blocking Solution (BioLegend). Cells were incubated with antibodies against CD3, CD56, and CD16 for 20 minutes at room temperature and washed three times with cell staining buffer (BioLegend) before fixation with fixation buffer (BioLegend). Samples were collected using a Symphony A3 (BD ​​Biosciences), and all FCS files were analyzed using FlowJo version 10.7.1 software.

[0424] For analysis of the allophore cytotoxicity assay, cells were transferred to a 96-well round-bottom plate (Falcon), washed in 1x PBS, pH 7.2 (Life Technologies), and resuspended in PBS containing LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher) according to the manufacturer's protocol. Prior to antibody addition, nonspecific binding to Fc receptors (FcRs) was blocked using human TruStain FcX Fc receptor blocking solution (BioLegend). Cells were incubated with antibodies against CD56 and CD16 for 20 min at RT and washed three times with cell staining buffer (BioLegend) before fixation with fixation buffer (BioLegend). Samples were collected using a Symphony A3 (BD ​​Biosciences), and all FCS files were analyzed using FlowJo version 10.7.1 software.

[0425] Lymphocytes were gated based on forward scatter area (FSC-A) and side scatter area (SSC-A). Singlets were excluded based on a forward scatter area (FSC-A) vs. forward scatter height (FSC-H) gate. CTV + iNK target or CTV - Gating on effector cells followed by CTVs positively labeled for LIVE / DEAD Fixable Near-IR + Gating was performed on iNK cells (Figure 20). Cells were gated on lymphocytes, followed by exclusion of doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally gating on LIVE / DEAD™ Near-IR+ to determine the % of iNK targets killed. FSC-A = forward scatter area, SSC-A = side scatter area, FSC-H = forward scatter height, CTV = CellTrace Violet, NIR = Near-IR.

[0426] analysis To calculate the number of antibodies bound per cell (ABC), a linear regression of Log10 PE molecules per bead was plotted against Log10 geometric mean-PE using the following formula: y = mx + c, where y equals Log10 fluorescence and x equals Log10 PE molecules per bead. The number of antibodies bound per cell for each sample was determined by using the above formula and interpolating the ABC value based on the geometric mean fluorescence value for each sample after subtracting the isotype control background value.

[0427] LIVE / DEAD NIR for each iNK group + CTV + Cell death for the allogeneic escape assay was calculated by determining the mean rate relative to targets (killed iNK) and dividing by the mean rate relative to LIVE / DEAD NIR+CTV+ WT iNK targets. Results are expressed as "cell death relative to WT iNK."

[0428] result For iNK cells edited from line 004, HLA-E expression was measured by flow cytometry with an ABC value of 3.625. HLA-E expression on therapeutic iNK cells was sufficient to observe reduced cell death when cultured with PBMCs compared to HLA-E-negative β2M KO iNK.

[0429] The ABC value for therapeutic iNK cells expressing HLA-E was calculated to be 3,625 by Quantibrite using the geometric mean fluorescence intensity value (Figure 21; HLA-E = open histogram, mouse IgG1 isotype control = gray-filled histogram).

[0430] HLA-E binds to the heterodimer CD94 / NKG2A, an inhibitory receptor expressed on NK cells. CD94 can also pair with NKG2C to form an activating receptor, which was not evaluated here. The frequency of NKG2A-expressing NK cells in the PBMC environment was measured for two donors. Cryopreserved PBMCs were thawed and cells expressing NK cell markers (CD3 - CD56 + CD16 + / - ) and assessed the frequency of NKG2A-expressing NK cells. In donor 1, 63.7% of NK cells expressed NKG2A, whereas in donor 2, 44.1% of NKG2A expressed NKG2A. + NK cells were included (Figure 22). PBMC samples were gated on viable lymphocytes and then gated on CD3-CD56+ cells ("NK cells"). The frequency of NKG2A-expressing NK cells was then determined based on FMO.

[0431] Donor-mismatched PBMCs and edited iNK cells were incubated at an E:T ratio of 25:1 for 72 hours, and iNK cell viability was measured by flow cytometry. iNK cells lacking surface HLA (b2M KO, open bars) showed approximately a 2.25- and 1.5-fold increase in cell death (black bars) compared to WT cells upon PBMC coculture with donors 1 and 2, respectively. Therapeutic iNK cells expressing HLA-E reduced cell death to the level of WT iNK cells (gray bars) (Figure 23 and Table 7). Freshly thawed PBMCs were cocultured with therapeutic iNK cells at an E:T ratio of 25:1 in the presence of 10 ng / mL IL-15 for 72 hours, and cell death of edited iNK cells compared to WT cells was determined as described in the Methods. Each data point is the average of triplicate wells.

[0432] [Table 8]

[0433] [Example 15] In Vivo Evaluation of the Antitumor Efficacy of iNK Cells The objective of this study was to evaluate the in vivo antitumor efficacy of cryopreserved iPSC611CD19iNK cells. The second objective of this study was to evaluate the 7-day persistence of cryopreserved iPSC611CD19iNK cells after a single dose.

[0434] animal For this study, female NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl We used 100% SzJ mice (Jackson Labs, Bar Harbor, Maine, USA). At the start of the study, mice were 7-9 weeks old and had an average starting weight of 23 grams. Animals were allowed to acclimate for 1 week before any experimental procedures were performed.

[0435] Autoclaved water and irradiated food (Laboratory Autoclavable Rodent Diet 5010, Lab Diet) were provided ad libitum, and the animals were maintained on a 12-hour light-dark cycle. Cages, bedding, and water bottles were autoclaved before use and changed every two weeks. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

[0436] tumor NALM6-Fluc-Puro (ALL) tumor cells (Imanis Life Sciences, CL151) were maintained in RPMI 1640 medium with 10 mM HEPES, 2.5 μg / mL puromycin, and 10% (v / v) HI FBS. Each mouse received 1 × 10 cells in a total volume of 0.2 mL of serum-free RPMI 1640 medium. 5 NALM6-Fluc-Puro cells were administered.

[0437] Efficacy Study Design and Treatment The day of tumor cell implantation was designated as study day 0. NALM6-Fluc-Puro tumor cells were implanted intravenously, and bioluminescence signals (range 64,120–141,400 p / s / cm) were measured. 2 / sr;Average=92,649±19,925p / s / cm 2Mice were randomized into treatment groups of N=10 by RT-PCR (RT-PCR / sr).

[0438] On days 1, 8, and 15 after i.v. implantation of NALM6-Fluc-Puro tumor cells, 10 × 10 6 or 15 x 10 6 Cryopreserved iPSC611 therapeutic iNK cells were thawed and resuspended in a volume of 0.2 mL of Ringer's lactate / 5% human serum albumin and injected intravenously (Groups 2 and 3). Group 1 served as an untreated control (Table 8, Efficacy Study Design).

[0439] [Table 9]

[0440] All mice received intraperitoneal recombinant human IL-2 (PeproTech® 200-02) at a dose of 100,000 international units (IU) in 0.2 mL per mouse on days 1, 2, 4, 7, 8, 10, 12, 15, 17, 19, 21, 23, 25, and 28. Briefly, lyophilized rhIL-2 (1 mg) was centrifuged at 2000 g for 1 minute, resuspended and solubilized in 1 mL of 100 mM acetic acid, and then mixed with 4 mL of 0.1% BSA in PBS. 1 mL aliquots were frozen at −80° C. until use, at which point the aliquots were thawed at ambient temperature and mixed with 3 mL of PBS for a final concentration of 500,000 IU / mL.

[0441] Tumor burden was assessed by bioluminescence imaging using an IVIS Lumina S5 (Perkin Elmer®). Briefly, mice were injected i.p. with 150 mg / kg D-luciferin (VivoGlo™ luciferin, Promega™), anesthetized with 2.5–3.5% vaporized isoflurane in oxygen, and imaged ventrally and dorsally 20 min after luciferin injection. Total whole-body bioluminescence was calculated by adding the mean brightness of the ventral and dorsal images.

[0442] Animal weight and bioluminescence were monitored twice weekly. Animals were monitored daily for clinical signs. Individual animals were removed from the study and humanely euthanized if they were moribund or had lost ≥ 20% of their original body weight on three consecutive measurements.

[0443] In some cases, supportive nutrition and hydration were provided to ensure the well-being of the mice throughout the study. All mice were provided with HydroGel® ad libitum on treatment days (days 1, 8, and 15).

[0444] Residue Testing An additional cohort of satellite animals was designated for tissue sampling to assess single-dose retention of iPSC611 cells. On day 0, 10 female NSG mice were implanted intravenously with NALM6-Fluc-Puro cells as described above. On day 1, mice received 15 x 10 6 Group 1 received a single intravenous injection of cryopreserved iPSC611 cells (Group 3). Group 1 remained the untreated control (Table 9, residual study design). All animals received recombinant human IL-2, formulated as described above, on days 1, 3, 5, and 7.

[0445] [Table 10]

[0446] On day 8, all tested mice and one naive age-matched mouse were humanely euthanized and samples were collected. Whole blood was collected by cardiac puncture into lithium heparin-coated tubes (BD 365965). Lungs were flushed in situ with PBS via the right ventricle, trimmed, and placed in 2.4 mL of 1x Buffer S (Miltenyi Biotech GmbH, 130-095-927) on ice until processing. Cervical lymph nodes were collected and placed in 2.4 mL of 1x Buffer S on ice until processing.

[0447] Blood was processed by transferring to a 96-well, 2 mL deep-well plate containing 1.5 mL of PBS. The plate was centrifuged at 300 g for 5 minutes, and the supernatant was decanted. The cell pellet was resuspended in 750 μL of ACK lysis solution and incubated at room temperature for 5 minutes, at which point 750 μL of PBS was added to each well. The plate was centrifuged at 300 g for 5 minutes, and the supernatant was decanted. ACK lysis was repeated twice as described above. After ACK lysis was complete, the resulting cell pellet was resuspended in 150 μL of PBS and transferred to a 96-well, U-bottom plate for FACS staining and analysis.

[0448] [Table 11]

[0449] Tissues were processed using a Miltenyi Biotech GmbH lung dissociation kit. Briefly, 1x Buffer S was prepared by mixing 1 mL of 20x Buffer S with 19 mL of sterile water. Enzyme D was reconstituted with 3 mL of 1x Buffer S, gently inverting every minute until dissolved. Enzyme A was reconstituted with 1 mL of 1x Buffer S, gently inverting every minute until dissolved. Tissues were individually collected in 1x Buffer S in gentleMACS C tubes. Immediately before processing, 100 μL of Enzyme D and 15 μL of Enzyme A were added to each tube. Tubes were placed in a gentleMACS Dissociator using program "m_lung_01." The tubes were then placed on a MACSmix Tube Rotator for 30 minutes at 37°C, followed by further mechanical dissociation using the gentleMACS Dissociator using program "m_lung_02." The sample was then filtered through a MACS SmartStrainer (70 μm) placed on a 50 mL tube and washed with 10 mL of PBS. The suspension was centrifuged at 300 × g for 10 min, the supernatant was aspirated, and the cell pellet was collected at 10 × 10 6 The cells were resuspended in PBS at 1000 cells / mL.

[0450] Cell suspensions from blood, lung, and cervical lymph nodes were plated at approximately 1e6 cells / well in 96-well U-bottom plates (BD falcon 353077). All washing steps were performed by centrifugation at 300 x G for 3 minutes, and the supernatant was shaken off into a sink. Cells were washed twice in PBS and stained with 50 μl of LIVE / DEAD™ Fixable Near-IR viability dye (Thermo Fisher) diluted 1:1000 in PBS for 15 minutes at room temperature (RT). 50 μl of Fc receptor blocker (Innovex NB309) was added to each well and incubated for 20 minutes at 4°C. Cells were washed twice in BD FACS staining buffer BSA (BD). A staining cocktail was made by diluting mAbs against CD45 and CD56 1:20 in BD FACS staining buffer. Cells were stained with 50 μl of the staining cocktail and incubated for 30 minutes at 4°C, protected from light. Cells were washed twice using BD FACS staining buffer and fixed in 100 μl of BD stabilizing fixative. All samples were run at the same voltage using a BD Symphony A3 Lite, and all events were collected. Flow cytometry data were analyzed using FlowJo 10.7.2.

[0451] iNK cells were defined as viable CD45+ and CD56+ singlets and expressed as the number of iNK cells per 100K viable lymphocytes. The lower limit of detection (LLOD) was defined as the maximum + 1 standard deviation (SD) of the control group that did not receive iNK treatment. Samples above the LLOD were plotted in GraphPad Prism.

[0452] analysis Graph body weight as a percentage change in mean group weight using the formula: where "W" represents the mean weight of a treatment group on a particular day and "W0" represents the mean weight of the same treatment group at the start of treatment.

[0453] Tumor growth inhibition (TGI) is defined as the difference between the whole-body mean luminance of the treatment and control groups, calculated as %TGI = (1 - T / C) 100, where T is the mean luminance of the treatment group and C is the mean luminance of the control group.

[0454] For survival assessment, results are plotted as percentage survival versus days after tumor implantation. Adverse clinical signs indicative of excessive tumor burden (such as ruffled / shaggy coat, hunched posture, lethargy, or hind limb weakness) are used as a surrogate endpoint for death. Kaplan Meier survival analysis is used to determine median survival.

[0455] Life extension rate (ILS) is %ILS=S T / S C Calculate as follows: [wherein, S T is the median survival time in the treatment group, and S C is the median survival in days in the control group.] Animals that did not reach the surrogate endpoint due to adverse clinical signs or death unrelated to treatment or tumor burden were censored for survival assessment.

[0456] Tumor bioluminescence data, body weight, survival time, and residuals were graphically displayed and statistically analyzed using GraphPad Prism software (version 9.0.1). Statistical significance for tumor bioluminescence was assessed using a conventional two-way analysis of variance (ANOVA) and Tukey's multiple comparison with a 95% confidence interval. Differences between groups were considered significant when the probability value (p) was ≤ 0.05. Statistical significance for survival probability was assessed using the Mantel-Cox test with the Gehan-Breslow-Wilcoxon test. Statistical significance for residuals was assessed using a conventional two-way analysis of variance (ANOVA) and Tukey's multiple comparison with a 95% confidence interval. Differences between groups were considered significant when the probability value was ≤ 0.05.

[0457] result Cryogenically stored iPSC611 cells were well tolerated as determined by body weight and clinical findings. iPSC611 demonstrated significant antitumor efficacy at both dose levels. A significant survival benefit was observed in mice treated with iPSC611 cells. Cryogenically stored iPSC611 had limited in vivo persistence 1 week after injection.

[0458] Group mean body weight changes of NALM6-Fluc-Puro tumor-bearing mice treated with iPSC611 cells or tumor-only controls are graphically shown in Figure 24 (untreated mice (●), or 10 × 10 6 Pieces (▽) and 15 x 10 6 Mean percent weight change in mice treated intravenously with iPSC611 (cryopreserved) cells (◆). Means present in ≥50% of treatment groups are plotted. Arrows indicate the day of dosing. No significant weight loss (>10% loss from the start of treatment) was observed in any of the groups receiving iPSC611 cells or in tumor-only controls.

[0459] Statistically significant antitumor activity was observed in 10 × 10 6 pieces and 15 x 10 6 10×10 iPSC611 cryopreserved cells (Table 11). Tumor growth is shown in Figure 25 (untreated mice (●) and 10×10 6 Pieces (▽) and 15 x 10 6 Mean whole-body average brightness (◆) of mice treated intravenously with iPSC611 (cryopreserved) cells once a week for three sessions. Groups are plotted through the final imaging time point, day 21, at which point the untreated control group remained and %TGI was calculated. Arrows indicate the day of dosing.

[0460] [Table 12]

[0461] The percent survival (%ILS) was calculated for all treatment groups. 6 pieces and 15 x 10 6A higher survival rate was observed for the group receiving cryopreserved iPSC611 cells than for the tumor-only control (Table 12, Figure 26).

[0462] [Table 13]

[0463] We evaluated the persistence of fresh and cryopreserved iPSC611 in blood and tissues 1 week after iNK injection into NALM6 tumor-bearing mice. Low recovery of viable cells was observed for cervical lymph node samples; therefore, these were not analyzed.

[0464] FACS analysis of lungs and blood showed limited retention of cryopreserved iPSC611 (Figure 27). Mice were left untreated or treated with 15x10 6 Each mouse received a single intravenous dose of iPSC611 cryopreserved cells. One week after injection, lungs and blood were collected for FACS analysis. The number of iNK cells per 100,000 lymphocytes was plotted for each individual mouse (o), with the average per group represented by a bar. One week after injection, iNK cells were detected in the lungs and blood of two of the five mice injected with iPSC611.

[0465] [Example 16] In Vivo Assessment of iNK Cell Depletion The purpose of this study was to evaluate the in vivo depletion of cryopreserved iPSC611 CD19iNK cells using Erbitux (cetuximab).

[0466] animal For this study, female NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl We used 10- to 12-week-old (SzJ) mice (Jackson Labs, Bar Harbor, Maine, USA) at the start of the study. At the start of the study, the mice were 10–12 weeks old and had an average starting weight of 24.3 grams. The animals were allowed to acclimate for 1 week before any experimental procedures were performed.

[0467] Autoclaved water and irradiated food (Laboratory Autoclavable Rodent Diet 5010, Lab Diet) were provided ad libitum, and the animals were maintained on a 12-hour light-dark cycle. Cages, bedding, and water bottles were autoclaved before use and changed every two weeks. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

[0468] Study design and treatment Mice were randomized into groups of N=5 by body weight (range 23.1-25.7 grams; mean=24.3±0.85 grams) (Table 13, Study Design).

[0469] The day of implantation of iPSC611 cells was designated as study day 1. On day 1, 15 x 10 cells were thawed and resuspended in a volume of 0.2 mL of lactated Ringer's / 5% human serum albumin. 6 Cryopreserved iPSC611 cells were intravenously injected into mice (Groups 2 and 3). Group 1 was left as an untreated control.

[0470] All mice in groups 1, 2, and 3 received intraperitoneal administration of recombinant human IL-2 (PeproTech® 200-02) at a dose of 100,000 international units (IU) / mouse in 0.2 mL on days 1 and 3. Briefly, lyophilized rhIL-2 (1 mg) was centrifuged at 2000 g for 1 minute, resuspended and solubilized in 1 mL of 100 mM acetic acid, and then mixed with 4 mL of 0.1% BSA in PBS. 1 mL aliquots were frozen at -80°C until use, at which point the aliquots were thawed at ambient temperature and mixed with 3 mL of PBS for a final concentration of 500,000 IU / mL.

[0471] Mice received intraperitoneal antibody therapy on days 2 and 3. Group 2 was dosed IP with 20 mL / kg PBS. Group 3 was dosed IP with 40 mg / kg cetuximab in a volume of 20 mL / kg.

[0472] Animal weights were recorded daily. Animals were monitored daily for clinical signs.

[0473] [Table 14]

[0474] Sampling On day 5, all mice were humanely euthanized and samples were collected. Blood was collected by cardiac puncture into lithium heparin-coated tubes (BD Microtainer 365965). Lungs were flushed in situ with PBS via the right ventricle, trimmed, and placed in PBS + 2% FBS on ice until processing.

[0475] Blood was processed through two rounds of ACK lysis according to the following protocol: Blood was transferred to a 2 ml deep-well plate, and the tubes were rinsed with 1 ml of PBS. The deep-well plates were centrifuged at 300 x g for 3 minutes. The supernatant was removed, and 1 ml of ACK was added to each well. The plate was incubated for 2 minutes, and then 1 ml of PBS was added to stop the osmotic lysis. The plate was centrifuged at 300 x g for 3 minutes, and the supernatant was removed. ACK lysis was repeated one or two more times as needed. Samples were resuspended in 200 μL of BD FACS stain buffer and transferred to a 96-well U-bottom plate for staining.

[0476] Lungs were processed to a single-cell suspension using mechanical dissociation and mild enzymatic digestion. Briefly, lung tissue was transferred to a dish without medium and minced (<1 mm in size) using a razor blade or scalpel until a uniform paste was formed. The minced tissue was transferred to 2 mL of digestion medium containing 10% collagenase / hyaluronidase, 15% DNase I solution (1 mg / mL), and 75% RPMI 1640 medium and incubated at 37°C for 20 minutes on a shaking platform. The tissue was passed through a 70 μm nylon mesh strainer on a 50 mL conical tube using the rubber end of a syringe plunger to obtain a cell suspension. The suspension was then filtered through a new 70 μm nylon mesh strainer on a 50 mL conical tube and rinsed with 10 mL of RPMI. The cell suspension was transferred to a 15 mL conical tube and centrifuged at 500 × G for 10 minutes at room temperature with the brake on low. The supernatant was removed and discarded. Cells were resuspended in 10 mL of PBS and counted to 10 x 10 6 The cells were adjusted to cells / mL and subjected to a single ACK lysis step before plating, staining, and FACS analysis.

[0477] [Table 15]

[0478] Cell suspensions from lungs were plated at approximately 1e6 cells / well in 96-well U-bottom plates (BD falcon 353077). All washing steps were performed by centrifugation at 300×G for 3 minutes and shaking the supernatant into a sink. Cells were washed twice in PBS and stained with 50 μl of LIVE / DEAD™ Fixable Near-IR viability dye (Thermo Fisher) diluted 1:1000 in PBS for 15 minutes at room temperature (RT). 50 μl of Fc receptor blocker (Innovex NB309) was added to each well and incubated for 20 minutes at 4°C. Cells were washed twice in BD FACS staining buffer BSA (BD). A staining cocktail was made by diluting mAbs against CD45 and CD56 1:20 in BD FACS staining buffer. Cells were stained with 50 μl of the staining cocktail and incubated for 30 minutes at 4°C, protected from light. Cells were washed twice using BD FACS staining buffer and fixed in 100 μl of BD stabilizing fixative. All samples were run at the same voltage using a BD Symphony A3 Lite, and all events were collected. Flow cytometry data were analyzed using FlowJo 10.7.2.

[0479] iNK cells were defined as viable singlets that were CD45+ and CD56+ and expressed as the number of iNK cells per 100K viable lymphocytes. The lower limit of detection (LLOD) was defined as the maximum + 1 standard deviation (SD) of the control group that did not receive iNK treatment. Samples above the LLOD were plotted in GraphPad Prism.

[0480] analysis Graph body weight as a percentage change in mean group weight using the formula: where "W" represents the mean weight of a treatment group on a particular day and "W0" represents the mean weight of the same treatment group at the start of treatment.

[0481] Body weights and residues were graphed and statistically analyzed using GraphPad Prism software (version 9.0.1). Statistical significance of removal was assessed using an unpaired one-tailed t-test with Welch's correction with a 95% confidence interval. Differences between groups were considered significant when the probability value was ≦0.05.

[0482] result iPSC611 cells were significantly reduced in the lungs and blood of mice treated with cetuximab. The group mean weight changes of mice receiving IP administration of PBS (●) or 40 mg / kg cetuximab (□) are shown graphically in Figure 28 (15 × 10 6 (Average weight change in mice intravenously treated with iPSC611). No significant weight loss (>10% loss from the start of treatment) was observed in any of the groups receiving iPSC611 cells or antibody.

[0483] Four days after injection of iPSC611 into NSG mice, the presence of iNK cells in the blood and lungs was assessed. FACS analysis of the lungs showed a significant 96% reduction in iNK counts in the lungs of mice treated with cetuximab compared to PBS-treated mice (p=0.0002). As shown in Figure 29, FACS analysis of the blood showed a significant 95% reduction in iNK counts in the blood of mice treated with cetuximab compared to PBS-treated mice (p=0.0321).

[0484] It will be appreciated by those skilled in the art that modifications may be made to the above-described embodiments without departing from the broad inventive concept thereof. It is therefore understood that the invention is not limited to the particular embodiments disclosed, and it is intended to cover modifications within the spirit and scope of the invention as defined herein.

Claims

1. (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope and interleukin 15 (IL-15), wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide; and (iii) deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes; An induced pluripotent stem cell (iPSC) or a derived cell thereof comprising:

2. The method further comprises a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G), Optionally, 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; Optionally, 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.

2. The iPSC or derived cell of claim 1.

3. One or more of the exogenous polynucleotides are (i) integrated into one or more loci on a chromosome of the cell, wherein the one or more loci are selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, and TCR a 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 said exogenous polynucleotides is integrated into the locus of a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby resulting in the deletion or reduced expression of said gene; or (ii) integrated into the CIITA, AAVS1 and B2M gene loci; 3. An iPSC or derived cell according to claim 1 or 2.

4. The CAR, (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to an antigen; (iii) hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain Including, Optionally, (a) the signal peptide comprises a GMCSFR signal peptide; or (b) the extracellular domain comprises a VHH domain that specifically binds to the antigen; or (c) the hinge region comprises a CD28 hinge region; or (d) the transmembrane domain comprises a CD28 transmembrane domain; or (e) the intracellular signaling domain comprises a CD3ζ intracellular domain; or (f) the costimulatory domain comprises a CD28 signaling domain; Optionally, The CAR is (A) (i) the signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1; (ii) the hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22; (iii) the transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24; (iv) the intracellular signaling domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6; and (v) the costimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

20. or (B) (i) the signal peptide comprising the amino acid sequence of SEQ ID NO: 1; (ii) the extracellular domain comprising an scFv or VHH domain that specifically binds to the antigen; (iv) the transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24; (v) the intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6; and (vi) the costimulatory domain comprising the amino acid sequence of SEQ ID NO:

20. Including, 4. The iPSC or derived cell of any one of claims 1 to 3. (i) the inactivated cell surface protein is Ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab and / or selected from the group of monoclonal antibody specific epitopes selected from the epitopes specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab. a truncated epidermal growth factor (tEGFR) variant, optionally 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; and / or (ii) the autoprotease peptide comprises a porcine teschovirus-1 2A (P2A) peptide, and optionally 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; and / or (iii) the IL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72; Optionally, 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:

75.

5. The iPSC or derived cell of any one of claims 1 to 4. (i) the first exogenous polynucleotide is integrated into the locus of the AAVS1 gene; (ii) the second exogenous polypeptide is integrated into the locus of the CIITA gene; and (iii) the third exogenous polypeptide is integrated into the locus of the B2M gene; The integration of the exogenous polynucleotides deletes or reduces the expression of CIITA and B2M, and preferably, the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO:

67.

6. An iPSC or derived cell according to any one of claims 1 to 5.

7. An iPSC or derived cell described in any one of claims 1 to 6, wherein the derived cell is a CD34+ hematopoietic progenitor cell (HPC), a natural killer (NK) cell or a T cell.

8. The method of claim 7, wherein the first exogenous polynucleotide encoding the CAR targets a CD19 antigen, and optionally: a) the extracellular domain comprises an scFv derived from an antibody that specifically binds to the CD19 antigen; b) the CAR is (i) the signal peptide comprising the amino acid sequence of SEQ ID NO: 1; (ii) the extracellular domain comprising the amino acid sequence of SEQ ID NO: 7; (iii) the hinge region comprising the amino acid sequence of SEQ ID NO: 22; (iv) the transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24; (v) the intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6; and (vi) the costimulatory domain comprising the amino acid sequence of SEQ ID NO:

20. Including, c) the inactivated cell surface is a truncated epidermal growth factor receptor (tEGFR); and / or d) the first 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:62; 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:75; and 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; 8. An iPSC or derived cell according to any one of claims 1 to 7.

9. (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of SEQ ID NO: 61; (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin-15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) optionally, a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO:

66. Including, The derivative cells are natural killer (NK) cells or T cells.

9. An iPSC or derived cell according to any one of claims 1 to 8.

10. An iPSC or derived cell according to any one of claims 1 to 9, further comprising one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

11. An iPSC or derived cell according to any one of claims 1 to 9, or a composition according to claim 10, for use in a method for treating cancer in a subject in need thereof.

12. A method for producing a derivative cell derived from the induced pluripotent stem cells (iPSCs) of any one of claims 1 to 9, comprising differentiating the iPSCs under conditions for cell differentiation, thereby obtaining the derivative cell; Optionally, the iPSCs are obtained by genomic engineering of unmodified iPSCs, wherein the genomic engineering comprises targeted editing, and optionally the targeted editing comprises deletions, insertions, or in / dels performed by CRISPR, ZFNs, TALENs, homing nucleases, homologous recombination, or any other functional variation of these methods. The manufacturing method.

13. A method for differentiating induced pluripotent stem cells (iPSCs) described in any one of claims 1 to 9 into NK cells, the method comprising subjecting the iPSCs to a differentiation protocol, including culturing the iPSCs in a medium containing recombinant human IL-12p70 for the final 24 hours of culture under the differentiation protocol.

14. A polynucleotide encoding an inactivated cell surface receptor containing a monoclonal antibody-specific epitope and interleukin 15 (IL-15), comprising: the monoclonal antibody-specific epitope and the IL-15 are operably linked by an autoprotease peptide; Optionally, (i) The inactivated cell surface receptor is selected from the group consisting of ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. comprising an epitope specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab; and / or (ii) the inactivated cell surface receptor comprises a truncated epidermal growth factor receptor (tEGFR) variant, optionally 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, or wherein the tEGFR variant consists of the amino acid sequence of SEQ ID NO: 71; and / or (iii) the autoprotease peptide comprises a porcine teschovirus-1 2A (P2A) peptide, and optionally 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 or consisting of the amino acid sequence of SEQ ID NO: 73; and / or (iii) the IL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72, or the IL-15 consists of the amino acid sequence of SEQ ID NO: 72; The polynucleotide.

15. A protein encoded by the polynucleotide described in claim 14.

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