Genetically engineered cells bearing anti-CD19 / anti-CD22 chimeric antigen receptors and uses thereof

Abstract

Provided are genetically engineered induced pluripotent stem cells (iPSCs) and derived cells thereof that express a chimeric antigen receptor (CAR), and methods of using the same. Compositions, polypeptides, vectors, and methods of manufacture 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 / 294,618, filed December 29, 2021, and U.S. Provisional Patent Application No. 63 / 350,156, filed June 8, 2022, each of which is incorporated by reference in its entirety herein.

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

[0003] Reference to Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing with the file name "SequenceListing_ST26.xml" and a creation date of December 23, 2022, having a size of 381 kb. The Sequence Listing submitted via EFS-Web is a part of the specification and is incorporated herein by reference in its entirety.

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

[0005] Chimeric antigen receptors (CARs) have shown remarkable activity in the treatment of acute lymphoblastic leukemia by enhancing the antitumor activity of immune effector cells. Autologous, patient-specific CAR-T therapy has emerged as a potent and potentially curative therapy for cancer, especially CD19- and CD22-related malignancies. In particular, CAR-T cells with dual antigen targeting (e.g., using two CARs by co-administration or co-transduction into immune cells) have shown potential to overcome antigen downregulation in ALL, a common cause of treatment failure. 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 has also been limited due to several other important drawbacks, including, for example, a) inefficient anti-tumor 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 some aspects, the disclosure provides a method for treating a tumor comprising administering to a subject a therapeutic agent comprising: one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting a CD22 antigen; and optionally, (i) a CD19 antigen binding domain encoded by the one or more exogenous polynucleotides; (ii) a deletion or reduced expression of one or more of the following genes: B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, RFXAP; (iii) an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G); (iv) Provided is an induced pluripotent stem cell (iPSC) or a derived cell thereof comprising at least one of the following exogenous polynucleotides: an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or an NKG2D protein; (v) a deletion or reduced expression of one or more of the NKG2A or CD70 genes; (vi) an exogenous polynucleotide encoding a cytokine; (vii) an exogenous polynucleotide encoding a safety switch; and (viii) an exogenous polynucleotide encoding a PSMA cell tracer. In some embodiments, a CD19 antigen binding domain, where (i) the CAR is a bispecific CAR comprising a CD19 antigen binding domain, or (ii) the one or more exogenous polynucleotides encode an additional CAR comprising a CD19 antigen binding domain. In some embodiments, the CAR comprises an anti-CD22 VHH domain, and / or the CD19 antigen binding domain comprises an anti-CD19 VHH domain. In some embodiments, the cytokine comprises an IL-15 protein. In some embodiments, the IL-15 protein comprises an inactivated cell surface receptor comprising a monoclonal antibody specific epitope and interleukin 15 (IL-15), and the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide. In some embodiments, the IL-15 protein comprises a fusion polypeptide comprising IL-15 and IL-15 receptor alpha (IL-15Rα).In some 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. In some embodiments, the iPSC or derived cell comprises a deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. In some embodiments, the iPSC or derived cell comprises an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G). In some embodiments, the CD16 is a CD16 variant protein. In some embodiments, the CD16 variant protein is a high affinity CD16 variant. In some embodiments, the CD16 variant protein is a non-cleavable CD16 variant. In some embodiments, the CD16 variant protein comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A. In some embodiments, the CD16 variant protein comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 181 and 182. In some embodiments, the iPSC or derived cell comprises an exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by an autoprotease peptide. In some embodiments, the NKG2D protein is a wild-type NKG2D protein. In some embodiments, the NKG2D protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 184. In some embodiments, the autoprotease peptide is selected from the group consisting of a porcine teschovirus-1 2A (P2A) peptide, a foot and mouth disease virus 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a flacherie virus 2A (BmIFV2A) peptide.In some embodiments, the autoprotease peptide is a P2A peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 186. In some embodiments, the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 186. In some embodiments, one or more of the exogenous polynucleotides are integrated into one or more loci on a chromosome of the cell selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, 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, 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 AAVS1, B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, thereby resulting in deletion or reduced expression of the gene. In some embodiments, one or more of the exogenous polynucleotides are integrated into the locus of the AAVS1 and B2M genes. In some embodiments, the iPSCs or derived cells comprise a deletion or reduced expression of one or more of the B2M or CIITA genes. In some embodiments, the iPSCs or derived cells comprise a deletion or reduced expression of the B2M and CIITA genes. In some embodiments, the iPSCs are reprogrammed from whole peripheral blood mononuclear cells (PBMCs). In some embodiments, the iPSCs are derived from reprogrammed T cells. In some embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising an antigen-binding domain that targets the CD22 antigen; (iii) a hinge region; (iv) a transmembrane domain, (v) an intracellular signaling domain; and (vi) a costimulatory domain. In some embodiments, the extracellular domain comprises a VHH single domain antibody that specifically binds to the CD22 antigen.In some embodiments, the extracellular domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 96-98, 152, and 155. In some embodiments, the extracellular domain comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 99-101, 153, and 156. In some embodiments, the additional CAR comprises (i) a signal peptide; (ii) an additional 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. In some embodiments, the further extracellular domain comprises an scFv derived from an antibody that specifically binds to the CD19 antigen. In some embodiments, the further extracellular domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with one or more of SEQ ID NOs: 2, 4, and 7, or is encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with one or more of SEQ ID NOs: 145 and 147. In some embodiments, the signal peptide comprises a GMCSFR signal peptide. In some embodiments, the hinge region for each of the CAR and the further CAR is independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region. In some embodiments, the transmembrane domain for each of the CAR and the additional CAR is independently selected from the group consisting of a CD28 transmembrane domain and a Cd8 transmembrane domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular domain.In some embodiments, the costimulatory domain for each of the CAR and the additional CAR is independently selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, a DAP10 signaling domain, an IL18R1 signaling domain, and an IL18RAP signaling domain. In some embodiments, the additional CAR comprises: (i) a signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 103, or 144; (ii) 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: 2, 4, or 7; (iii) an additional extracellular domain comprising an amino acid sequence or encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 145 or 147; (iv) an additional extracellular domain comprising an amino acid sequence comprising 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, or an intracellular signaling domain encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 149; 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.In some embodiments, the additional CAR comprises (i) a signal peptide comprising the amino acid sequence of SEQ ID NO: 1, 103, or 144; (ii) an additional extracellular domain comprising the amino acid sequence of SEQ ID NO: 2, 4, and 7, or encoded by the polynucleotide sequence of SEQ ID NO: 145 and 147; (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, or encoded by the polynucleotide sequence of SEQ ID NO: 149; and (vi) a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the CAR comprises (i) a signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 103, or 144. (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 one of SEQ ID NOs: 96-98, 152, and 155; (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: 21 or 102; (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: 23 or 24; (v) 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: 6, 19 8, or 199, or an intracellular signaling domain encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 149; 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: 8, 17, 198, or 199. In some embodiments, the CAR comprises: (i) a signal peptide comprising the amino acid sequence of SEQ ID NO: 1, 103, or 144; (ii) an extracellular domain comprising the amino acid sequence of one of SEQ ID NOs: 96-98, 152, and 155; (iii) a hinge region comprising the amino acid sequence of SEQ ID NO: 21 or 102; (iv) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 23 or 24; (v) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6, 198, or 199, or an intracellular signaling domain encoded by the polynucleotide sequence of SEQ ID NO: 149; and (vi) a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 8, 17, 198, or 199. In some embodiments, the iPSC or derived cell comprises an exogenous polynucleotide encoding a safety switch.In some embodiments, the safety switch comprises an exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody specific epitope. In some 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, polatuzumab The monoclonal antibody specific epitope is selected from the group of epitopes specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab. In some embodiments, the inactivated cell surface protein is a truncated epidermal growth factor (tEGFR) variant. In some 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 with SEQ ID NO: 71. In some embodiments, the safety switch comprises (i) an intracellular domain having herpes simplex virus thymidine kinase (HSV-TK) or (ii) an inducible caspase 9 (iCasp9). In some embodiments, the iPSC or derived cell comprises an exogenous polynucleotide encoding a PSMA cell tracer, the PSMA cell tracer comprising an extracellular domain comprising a PSMA extracellular domain or a fragment thereof. In some embodiments, the iPSC or derived cell comprises a combined artificial cell death / reporter system polypeptide comprising an intracellular domain having herpes simplex virus thymidine kinase (HSV-TK) and a linker, a transmembrane region, and an extracellular domain comprising a PSMA extracellular domain or a fragment thereof.In some embodiments, (i) the HSV-TK 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: 187 or 188, or (ii) the iCasp9 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: 200 or 201. In some embodiments, the combined artificial cell death / reporter system polypeptide comprises HSV-TK fused to a truncated variant PSMA polypeptide via a linker. In some embodiments, the truncated variant PSMA polypeptide 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: 189. In some embodiments, the linker comprises an autoprotease peptide sequence selected from the group consisting of a P2A peptide sequence, a T2A peptide sequence, an E2A peptide sequence, and an F2A peptide sequence. In some embodiments, the artificial cell death / reporter system polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 190. In some embodiments, the artificial cell death / reporter system polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 191-193. In some embodiments, the artificial cell death / reporter system polypeptide comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 194-196.In some embodiments, 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 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.In some embodiments, (i) one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting a CD22 and / or CD19 antigen are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178. (ii) the exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 67 and 70; (iii) the NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or NKG (iv) the exogenous polynucleotide encoding the 2D protein comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 179, 183, and 185; (v) the exogenous polynucleotide encoding the cytokine 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: 197; v) the exogenous polynucleotide encoding the safety switch comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 194-196; and / or (vi) the exogenous polynucleotide encoding the PSMA cell tracer 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: 189.In some embodiments, (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting CD22 and / or CD19 antigens comprise one or more sequences selected from the group consisting of SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178; (ii) the exogenous polynucleotides encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprise one or more sequences selected from the group consisting of SEQ ID NOs: 67 or 70. (iii) the exogenous polynucleotide encoding the NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or the NKG2D protein comprises the polynucleotide sequence of SEQ ID NO: 179, 183, or 185; (iv) the exogenous polynucleotide encoding the cytokine comprises the polynucleotide sequence of SEQ ID NO: 197; and / or (v) the exogenous polynucleotide encoding the safety switch comprises a polynucleotide sequence having one of SEQ ID NOs: 194-196. In some embodiments, the exogenous polynucleotide is integrated into a locus independently selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD33 locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.In some embodiments, (i) one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising one or more antigen binding domains targeting CD22 and / or CD19 antigens are integrated at the locus of the AAVS1 gene; (ii) an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) is integrated at the locus of the B2M gene; (iii) an exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or NKG2D is integrated at the locus of the CD70 gene; (iv) an exogenous polynucleotide encoding a cytokine is integrated at the locus of the NKG2A gene; (v) there is a deletion or reduced expression of the CIITA gene; and (. vi) Optionally, a safety switch or PSMA is integrated into the CIITA locus. In some embodiments, the CAR is a bispecific CAR comprising a CD22 / CD19 loop. In some embodiments, the bispecific CAR comprises one or more amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 61, 96-98, 104-111, 120-131, 152, 155, 157, 159, 161, 163, 165-167, 171, and 173-175. In some embodiments, the bispecific CAR comprises one or more polynucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178. In some embodiments, the derivative cell is a natural killer (NK) cell or a T cell. In some embodiments, the derivative cell is a natural killer (NK) cell. In some embodiments, the derivative cell is a T cell. In some embodiments, the T cell is a gamma delta T cell. In some embodiments, the T cell is a gamma delta Vγ9 / Vδ1 T cell.

[0008] In some aspects, the present disclosure provides compositions comprising any of the iPSCs or derived cells of the present disclosure. In some embodiments, the compositions further comprise or are used in combination with one or more therapeutic agents selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), siRNAs, oligonucleotides, mononuclear blood cells, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs).

[0009] In some aspects, the disclosure provides an induced pluripotent stem cell (iPSC) or a derived cell thereof comprising one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) targeting CD22 and CD19 antigens; and at least one of: (i) a deletion or reduced expression of one or more of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, RFXAP genes; (ii) an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G); (iii) an exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or an NKG2D protein; (iv) a deletion or reduced expression of one or more of NKG2A or CD70 genes; (v) an exogenous polynucleotide encoding a cytokine; (vi) an exogenous polynucleotide encoding a safety switch; and (vi) an exogenous polynucleotide encoding a PSMA cell tracer. In some embodiments, the CAR is a bispecific CAR comprising a CD22 / CD19 loop. In some embodiments, the CAR comprises an anti-CD22 VHH domain. In some embodiments, each of the one or more exogenous polynucleotides comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences independently selected from the group consisting of SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178.

[0010] In some aspects, the disclosure provides a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of at least one exogenous polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that targets a CD22 antigen; and, optionally, (i) a CD19 antigen-binding domain encoded by the one or more exogenous polynucleotides; (ii) a deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes; (iii) an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G); (iv) a NK cell. Provided are CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs) comprising at least one of the following exogenous polynucleotides: (v) deletion or reduced expression of one or more of NKG2A or CD70 genes; (vi) exogenous polynucleotides encoding cytokines; (vii) exogenous polynucleotides encoding safety switches; and (viii) exogenous polynucleotides encoding PSMA cell tracers. In some embodiments, the CD34+ HPCs comprise a CD19 antigen binding domain, and (i) the CAR is a bispecific CAR comprising a CD19 antigen binding domain, or (ii) the one or more exogenous polynucleotides encode an additional CAR comprising a CD19 antigen binding domain. In some embodiments, the CAR comprises an anti-CD22 VHH domain, and / or the CD19 antigen binding domain comprises an anti-CD19 VHH domain. In some embodiments, the cytokine comprises an IL-15 protein. In some embodiments, the IL-15 protein comprises an inactivated cell surface receptor comprising a monoclonal antibody specific epitope and interleukin 15 (IL-15), and the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide. In some embodiments, the IL-15 protein comprises a fusion polypeptide comprising IL-15 and IL-15 receptor alpha (IL-15Rα).In some 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. In some embodiments, a deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. In some embodiments, an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G). In some embodiments, one or more of the exogenous polynucleotides are integrated into one or more loci on a chromosome of the cell independently selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, 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, 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 AAVS1, B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, thereby resulting in deletion or reduced expression of the gene. In some embodiments, one or more of the exogenous polynucleotides are integrated into the locus of CIITA, AAVS1 and B2M genes. In some embodiments, the CD34+ HPC comprises one or more deletions or reduced expression of B2M or CIITA genes. In some embodiments, the CAR comprises (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to CD22 antigen and, optionally, a binding domain that specifically binds to CD19 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain. In some embodiments, the extracellular domain comprises a VHH single domain antibody that specifically binds to CD22 antigen.In some embodiments, the extracellular domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 96-98, 152, and 155. In some embodiments, the extracellular domain comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 99-101, 153, and 156. In some embodiments, the bispecific CAR comprises one or more amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 61, 96-98, 104-111, 120-131, 152, 155, 157, 159, 161, 163, 165-167, 171, and 173-175. In some embodiments, the bispecific CAR comprises an amino acid sequence encoded by one or more polynucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178. In some embodiments, the additional CAR comprises (i) a signal peptide; (ii) an additional 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. In some embodiments, the additional extracellular domain comprises an scFv derived from an antibody that specifically binds to the CD19 antigen.In some embodiments, the additional extracellular domain (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 2, 4, and 7, or (ii) is encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 145 and 147.

[0011] In some aspects, the disclosure provides chimeric antigen receptor (CAR) polypeptides comprising an extracellular domain that specifically binds CD22 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178.

[0012] In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering to a subject in need thereof any of the derivative cells of the present disclosure, or any of the compositions of the present disclosure. In some embodiments, the cancer is selected from the group consisting of leukemias, such as AML, CML, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL), lymphomas, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and follicular lymphoma, and solid cancers, such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastric cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and cerebral cancer, as well as cancer of unknown primary (CUP). In some embodiments, the cancer is a B-cell malignancy, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma, follicular lymphoma. In some embodiments, the subject has minimal residual disease (MRD) after initial cancer treatment. In some embodiments, the subject does not have minimal residual disease (MRD) after one or more cancer treatments or repeated dosing.

[0013] In some aspects, the present disclosure provides a method for producing any of the derivative cells of the present disclosure, comprising differentiating iPSC cells under conditions for cell differentiation, thereby obtaining derivative cells.In some embodiments, the iPSC is obtained by genomic engineering of unmodified iPSC, and genomic engineering comprises targeted editing.In some embodiments, the targeted editing comprises deletion, insertion, or in / del carried out by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0014] In some aspects, the present disclosure provides a method of differentiating induced pluripotent stem cells (iPSCs) into NK cells, comprising subjecting the iPSCs to a differentiation protocol, including culturing the cells in a medium comprising recombinant human IL-12 for the final 24 hours of culture under the differentiation protocol. In some embodiments, the recombinant IL-12 comprises IL12p70.

[0015] The above summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Brief description of the drawings]

[0016] [Figure 1-1] Graph showing the results of VHH-Fc fusion proteins examined by flow cytometry for CD22 cell binding in Raji cells. A) PROT739 and PROT740 from the VHH phage library show binding to Raji cells with an EC50 value of approximately 2 nM. No binding was observed to Raji CD22 knockout cells. B) PROT265 and PROT810 (an affinity matured variant of PROT265) bind to Raji cells, with PROT810 showing improved binding compared to PROT265. [Figure 1-2] (As stated above.) [Diagram 2] Schematic diagram of VHH-Fc fusion proteins examined for binding to full-length CD22, CD22Δ2-3, and CD22ΔV-1. PROT265 (D04) bound to all proteins tested, suggesting that it binds to IgC domains 4-6. PROT739 (A01) bound to full-length CD22 and had weak binding to CD22Δ2-3 protein without binding to CD22ΔV-1 protein, suggesting that it binds to the IgC1-2 domain. PROT740 (E04) bound to full-length CD22 and CD22Δ2-3 protein, but not to CD22ΔV-1 protein, suggesting that it binds to the IgV-IgC1 domain. [Diagram 3] Graph showing VHH-CAR constructs transduced into Jurkat cells. Cells were tested for activation by co-culturing with cells expressing the CD22 target protein at an effector:target (E:T) ratio of approximately 4:1. Cells were stained for CD3 and CD69 expression using BV421 anti-human CD69 antibody (Biolegend) and PE-anti-CD3 clone OKT3 (Biolegend). A) Jurkat CAR-T cells (CD3+) harboring clones A01 (P952) and E04 (P953) show activation of CD69 expression after 48 hours of co-incubation with various CD22+ or CD22- cell lines. B) Jurkat CAR-T cells harboring clone D04 (D04.P262) show activation of CD69 expression after 48 hours of co-incubation with various CD22+ or CD22- cell lines. [Figure 4] Figure 1: Cytotoxicity of VHH-CAR expressing cells against Raji cells. Primary T cells were transduced with VHH-CAR lentivirus. Cells were expanded for 14 days and then co-cultured with CD22+ Raji cells at E:T ratios of 2:1 and 5:1 for 48 h. Raji cell killing was assessed by flow cytometry. A) P403 (D04), P1278 (D04_AM_D11) and P456 (positive control scFv-CAR) were transduced into T cells and assessed for cytotoxicity against Raji cells. The P403 construct (CD22_D04) showed Raji cell killing modestly above background (untransduced cells; UTD). The affinity-improved variant P1278 (CD22_D04_AM_D11) had significantly better cytotoxicity, up to 80% at an E:T ratio of 5:1. B) The P952 (CD22_CNTY_VHH1_A01) and P953 (CD22_CNTY_VHH1_E04) constructs demonstrated cytotoxicity modestly above background. [Diagram 5]Graph showing cytotoxicity of tandem VHH-CAR expressing cells against (A) Raji cells and (B) K-562 cells. VHH-CAR lentivirus was transduced into primary T cells. Cells were expanded for 14 days and then co-cultured with CD22+ Raji cells at E:T ratios of 1:2, 1:1 and 2:1 for 48 h. Target cell killing was assessed by flow cytometry. Single VHH (D04_AM_D11) CAR (P1729) demonstrated minimal killing of Raji cells. Tandem VHH of E04 - D04_AM_D11 (P1738) showed slightly improved Raji cell killing, up to 20%, at an E:T ratio of 2:1. However, for D04_AM_D11 - E04(P1730) placed in the other orientation, cytotoxicity improved to 40% at 2:1, similar to the positive control scFv. D04_AM_D11 - A01 CARs (P1732 and P1733) in either orientation showed robust cytotoxicity up to 90% at an E:T ratio of 2:1. [Figure 6-1] FIG. 1 shows various bispecific CAR constructs (targeting CD19 and CD22 antigens) used in various embodiments of the present disclosure. CAR ectodomains were engineered using selected CD22 binders in combination with FMC63. Selected CARs were expressed and tested for cytotoxic activity in T cells along with monospecific CAR-T cells. [Figure 6-2] (As stated above.) [Figure 6-3] (As stated above.) [Figure 6-4] (As stated above.) [Figure 6-5] (As stated above.) [Figure 6-6] (As stated above.) [Figure 6-7] (As stated above.) [Figure 6-8] (As stated above.) [Figure 6-9] (As stated above.) [Figure 7](A) Graph showing CD19-dependent killing of Cho-CD19 cells and Raji-CD22 knockout cells using therapeutic cells expressing mono- and bispecific CAR constructs of the present disclosure at an E:T ratio of 1.25:1. All of the bispecific CAR-T cells tested exhibited CD19-targeted cytotoxic activity comparable to FMC63 CARs (e.g., P1209). The bispecific CARs showed CD22-directed cytotoxicity comparable to monospecific tandem CARs (e.g., P1631, P1633, P1702 and P1734). (B) CD22-dependent killing of Cho-CD22 cells, Raji-CD19 knockout cells, and Nalm6-CD19 knockout cells using therapeutic cells expressing mono- and bispecific CAR constructs of the present disclosure at an E:T ratio of 1.25:1. The bispecific CAR T cells showed CD22-dependent activity comparable to CD22 monospecific CARs. P2015 and p2016 had reduced activity against CHO-CD22 and the highest activity against Raji-CD19 knockout cells and Nalm6-CD19 knockout cells. [Figure 8] Graph showing CD19 / CD22 double positive killing of Reh, Raji, Jeko, and Nalm6 cells using therapeutic cells expressing mono- and bispecific CAR constructs of the present disclosure at an E:T ratio of 1.25:1. [Figure 9] Graph showing percent expression of various mono- and bispecific CAR constructs of the present disclosure in T cells. [Figure 10-1] FIG. 1 shows (A) various mono-, bi-, and tri-specific CAR constructs of the present disclosure, and (B) percent expression of various CAR constructs in T cells. [Figure 10-2] (As stated above.) [Figure 11] FIG. 1 shows (A) various bi- and tri-specific CAR constructs of the present disclosure, and (B) target-dependent cytotoxicity of therapeutic cells expressing various CAR constructs against Cho cells. [Figure 12-1]Graph showing the results of cytotoxicity analysis of therapeutic cells expressing various mono-, bi-, and tri-specific CAR constructs of the disclosure at E:T ratios of 0 to 10:1 in (A) Cho cells (CD22- / CD19-), (B) Cho-CD22 cells (CD22+ / CD19-), (C) Cho-CD19 cells (CD22- / CD19+), (D) Raji cells (CD22+ / CD19+), (E) Raji-CD22KO cells (CD22- / CD19+), (F) Raji-CD19KO cells (CD22+ / CD19-), and (G) Jeko cells (CD22+ / CD19+). [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 12-4] (As stated above.) [Figure 13] Graph showing CD19, CD22, and CD19 / CD22 dependent killing of Reh, Jeko, Cho-CD22, Cho-CD19, Cho, Raji-CD22 KO, Raji-CD19 KO, Raji, Nalm6-CD19 KO, and Nalm6 cells using therapeutic cells expressing various mono- and bispecific CARs of the disclosure. [Figure 14-1] Graph showing the results of cumulative cytotoxicity analysis of therapeutic cells expressing various mono- and bispecific CAR constructs of the disclosure at E:T ratios of 0 to 5:1 in (A) Cho cells (CD22- / CD19-), (B) Cho-CD22 cells (CD22+ / CD19-), (C) Cho-CD19 cells (CD22- / CD19+), (D) Raji cells (CD22+ / CD19+), (E) Raji-CD19KO cells (CD22+ / CD19-), (F) Raji-CD22KO cells (CD22- / CD19+), (G) Nalm6 cells (CD22+ / CD19+), (H) Nalm6-CD19KO cells (CD22+ / CD19-), (I) Reh cells (CD22+ / CD19+), and (J) Jeko cells (CD22+ / CD19+). [Figure 14-2] (As stated above.) [Figure 14-3] (As stated above.) [Figure 14-4] (As stated above.) [Figure 14-5] (As stated above.) [Figure 15] Graphs showing the results of cytotoxicity analysis of therapeutic cells expressing various mono- and bispecific CAR constructs of the present disclosure at E:T ratios of 0 to 5:1 in (A) Cho cells (CD22- / CD19-), (B) Cho-CD22 cells (CD22+ / CD19-), and (C) Cho-CD19 cells (CD22- / CD19+). [Figure 16] Graph showing target-dependent cytotoxicity of therapeutic cells expressing mono- and bispecific CAR constructs against Cho, Cho-CD19, and Cho-CD22 cells. [Figure 17] Graph showing the results of cytotoxicity analysis of therapeutic cells expressing various mono- and bispecific CAR constructs of the present disclosure at E:T ratios of 0 to 5:1 in (A) Nalm6 cells (CD22+ / CD19+) and (B) Nalm6-CD19KO cells (CD22+ / CD19-). [Figure 18] Graph showing target-dependent cytotoxicity of therapeutic cells expressing mono- and bispecific CAR constructs against Nalm6 cells, and Nalm6-CD19 cells. [Figure 19] Graphs showing the results of cytotoxicity analysis of therapeutic cells expressing various mono- and bispecific CAR constructs of the present disclosure at E:T ratios of 0 to 5:1 in (A) Raji cells (CD22+ / CD19+), (B) Raji-CD22KO cells (CD22- / CD19+), and (C) Raji-CD19KO cells (CD22+ / CD19-). [Figure 20] Graph showing target-dependent cytotoxicity of therapeutic cells expressing mono- and bispecific CAR constructs against Raji, Raji-CD19KO, and Raji-CD22 KO cells. [Figure 21] Illustrative of an exemplary cell of the disclosure expressing an anti-CD22 / CD19 loop CAR on the cell surface. [Figure 22]1 is a graph showing that CAR expression in cells of the present disclosure engineered to express a bispecific CAR that recognizes both CD19 and CD22. First, iPSCs were genetically modified to express a transgene for a CD19 / CD22 CAR or a control CD19 CAR (or cells were not engineered for a CAR-less control). The iPSCs were differentiated into T cells (iT cells) and CAR expression was assessed by staining with either an anti-idiotypic antibody that binds to the CD19 recognition domain of the CAR (left) or an anti-VHH antibody that binds to the CD22 recognition domain of the CAR (right). Flow cytometry was used to assess cell staining. CAR-less iT cells were not stained with either antibody. CD19 / CD22 CAR-iT cells were stained with antibodies for both the CD19 and CD22 recognition domains. CD19 CAR-iT cells were stained with antibodies for the CD19 recognition domain but not for the CD22 recognition domain. These results demonstrate that CD19 / CD22 bispecific CAR-iT cells express a CAR molecule that has the potential to bind to either the CD19 or CD22 antigen on target cells. [Diagram 23]Graph showing antigen-specific cytolysis of tumor cells by CD19 / CD22 bispecific CAR-iT cells. CAR-iT cells with bispecific CARs eliminate tumor cells through recognition of either CD19 or CD22. Either CD19 CAR-iT cells or CD19 / CD22 CAR-iT cells were co-cultured with tumor cells at a 1:1 effector-to-target ratio for 24 hours. Cytolysis (%) was calculated based on the number of tumor cells present after 24 hours of co-culture with iT cells compared to the number of tumor cells present when cultured alone. Data were collected using an IncuCyte live cell imaging platform and red fluorescently labeled tumor cells. (A) When both CD19 and CD22 were present on the surface of tumor cells (Daudi B-cell lymphoma), both CD19 CAR-iT cells and CD19 / CD22 CAR-iT cells mediated tumor killing. (B) When tumor cells expressed CD22 but not CD19 (Daudi CD19 knockout cells), only bispecific CD19 / CD22 CAR-iT cells mediated tumor killing. (C) When tumor cells expressed CD19 but not CD22 (Daudi CD22 knockout cells), both CD19 and CD19 / CD22 CAR-iT cells mediated tumor killing. Thus, CD19 / CD22 bispecific CAR-iT cells can target B cell malignancies that have lost CD19 expression by targeting CD22. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 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. Discussions of documents, acts, materials, devices, articles and the like which are included within the specification are for the purpose of providing a context for the invention. Such discussions are not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Otherwise, certain terms used herein have the meanings set forth herein.

[0019] 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.

[0020] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges described herein, are to be understood in all cases to be modified 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 expressly 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 indicates otherwise.

[0021] 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.

[0022] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variations thereof, are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integers 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 to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and 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).

[0023] As used herein, the conjunction "and / or" between listed elements is understood to include 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 element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any one of these options is understood to fall within this meaning and thus 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 thus meet the requirements of the term "and / or".

[0024] 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 whole or group of wholes listed, but does not allow for additional wholes or groups of wholes to be added to the specified method, structure, or composition.

[0025] 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.

[0026] As used herein, "subject" refers to any animal, preferably a mammal, 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.

[0027] 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 are not precise boundaries or parameters of the described size / characteristic, and do not 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 from the least significant digit using mathematical and industrial principles recognized in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0028] 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 encoding them), refer to two or more sequences or subsequences that are the same when compared and aligned for maximum correspondence using one of the sequence comparison algorithms below or by visual inspection, or two or more sequences or subsequences that have a particular percentage of amino acid residues or nucleotides that are the same.

[0029] For sequence comparison, typically one sequence acts as a reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, if necessary, coordinates of partial sequence are designated, and parameters of sequence algorithm program are designated.Then, sequence comparison algorithm calculates the sequence identity of test sequence compared with reference sequence based on designated program parameters.

[0030] 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 Wisconsingenetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally as described in Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0031] Examples of algorithms suitable 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 match or meet a certain positive threshold score T when aligned with words of the same length in database sequences. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0032] Cumulative scores are calculated using the parameters M (reward score for a matching residue pair; 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 the word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X; when the cumulative score falls below zero due to the accumulation of one or more negatively scoring residue alignments; or when 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 wordlength (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 wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0033] In addition to calculating 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 minimum 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 when the minimum 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.

[0034] 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 substitution, 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 to each other under stringent conditions.

[0035] 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, such as 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 thus 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 the composition is not part of the natural environment of the nucleic acid, peptide, protein, or cell. The term also encompasses chemically synthesized nucleic acids, as well as nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell.

[0036] As used herein, the term "polynucleotide", which is referred to synonymously as "nucleic acid molecule", "nucleotide" or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that contain RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains 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 "polynucleotide." "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0037] "Construct" refers to a macromolecule or molecular complex that contains a polynucleotide to be delivered to a host cell, either in vitro or in vivo. "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 may be replicated and / or expressed. The term "vector" as used herein includes the construct to be delivered. Vectors can be linear or circular molecules. Vectors can be integrative or non-integrative. 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.

[0038] By "integration" is meant that one or more nucleotides of the construct are stably inserted into the cell genome, e.g., 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 the deletion of endogenous sequences or nucleotides at the integration site. When 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.

[0039] As used herein, the term "exogenous" is intended to mean that the referenced molecule or the referenced activity is introduced into or is 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 relation to expression of an encoding nucleic acid, the term refers to the introduction of an encoding nucleic acid in an expressible form into a cell. The term "endogenous" refers to the referenced molecule or activity that is present in its native form in the host cell. Similarly, when used in relation to expression of an encoding nucleic acid, the term refers to the expression of an encoding nucleic acid that is naturally contained within the cell and that has not been exogenously introduced.

[0040] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that is transcribed into RNA in vivo and optionally translated into a polypeptide when placed under the control of appropriate regulatory sequences. A gene or polynucleotide of interest 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 code for miRNA, shRNA, a natural polypeptide (e.g., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (e.g., a mutant 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, and so on.

[0041] "Operably linked" refers to the binding of nucleic acid sequences into a single nucleic acid fragment such that the function of one is affected by the other.For example, a promoter is operably linked to a coding sequence or functional RNA when it can affect the expression of the coding sequence or functional RNA (e.g., 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 orientation.

[0042] The term "expression" as used herein refers to the biosynthesis of a gene product. This term encompasses the transcription of a gene into RNA. This term also encompasses the translation of RNA into one or more polypeptides, and further encompasses all natural post-transcriptional and post-translational modifications. The expressed CAR may be in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or anchored in a cell membrane.

[0043] As used herein, the term "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 amino acid polymers of any length. The polymers can be linear or branched, can include modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art, are also included in this definition.

[0044] 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 expressly indicated otherwise.

[0045] As used herein, the term "engineered immune cells" refers to immune cells, also referred to as 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.

[0046] Induced pluripotent stem cells (IPSCs) and immune effector cells iPSCs have unlimited self-renewal capacity. The use of iPSCs enables cell engineering to produce controlled cell banks of modified cells that can be expanded and differentiated into desired immune effector cells, providing large quantities of homogenous allogeneic therapeutic products.

[0047] Provided herein are genetically engineered iPSCs and their derived cells. The 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 level of iPSCs. This approach can help reduce the side effects mediated by CRS / GVHD and prevent long-term autoimmunity while providing excellent efficacy.

[0048] As used herein, the term "differentiation" is 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 particular 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 the lineages of a body or soma or embryo. For example, an embryonic stem cell is 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), which are unable to give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells), which are able to give rise to a complete organism.

[0049] 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 (e.g., can differentiate into more cell types) compared to 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.

[0050] 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 or changed or reprogrammed into cells capable of differentiating into tissues of all three germ layers or layers: mesoderm, endoderm, and ectoderm. The produced iPSCs do not refer to the cells as they are found in nature.

[0051] 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 capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hemocytoblasts), myeloid progenitor cells, megakaryocyte 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 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.

[0052] 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.

[0053] 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 cultured T cell lines, 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., Thl 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 stage of development. Further types of helper T cells include cells such as Th3 (Treg), Thl7, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0054] "CD4+ T cells" refers 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 secretion 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.

[0055] "CD8+ T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and on 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.

[0056] 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 T cell receptors (TCR chains). NK cells can also refer to genetically engineered NK cells, such as NK cells modified to express chimeric antigen receptors (CARs). NK cells can also differentiate from stem or progenitor cells.

[0057] 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 can 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 be further differentiated into specific cell types, including any hematopoietic lineage cells. Source cell-derived iPSCs, and cells differentiated therefrom, are sometimes collectively referred to as "derived" or "derived" cells, depending on the context. For example, derived effector cells, or derived NK or "iNK" cells, or derived T or "iT" cells, as used throughout this application, 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 therapeutic response specific, or by introducing genetically modified modalities into iPSCs using genome editing.

[0058] Induced pluripotent stem cell (iPSC) parent cell lines may 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. Patents 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference. The reprogramming factors may be in the form of polynucleotides and are thus introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by lentiviral vectors. In some embodiments, one or more polynucleotides are introduced by episomal vectors. In various other embodiments, one or more polynucleotides are introduced by Sendai virus vectors. In some embodiments, the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs, and genome editing is introduced by creating one or more targeted integrations and / or in / dels at one or more selected sites. In another embodiment, the iPSCs are obtained from human T cells with antigen specificity and rearranged TCR genes (hereinafter also referred to as "T-iPS" cells), as described in U.S. Patent Nos. 9,206,394 and 10,787,642, which are hereby incorporated by reference in this application.

[0059] According to certain aspects, the present application relates to an induced pluripotent stem cell (iPSC) or a derived cell thereof comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a deleted or reduced expression of B2M and CIITA genes; and, optionally, (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and interleukin 15 (IL-15), wherein IL-15RA and IL-15 are operably linked.

[0060] I. Chimeric Antigen Receptor (CAR) Expression According to an embodiment of the present application, the iPSC cell or derived cell comprises one or more first exogenous polynucleotides encoding a first and a second chimeric antigen receptor (CAR), e.g., a CAR targeting one or more tumor antigens. In one embodiment, the CAR targets the CD19 antigen and the second CAR targets the CD22 antigen. In another embodiment, the CAR targets the CD19 antigen and the second CAR targets the CD22 antigen, and the targeting region (e.g., extracellular domain) of one or both of the CARs comprises an antibody fragment (e.g., VHH domain). In other embodiments, the CAR can be a bispecific CAR (e.g., CD19 / CD22 CAR) that targets two or more antigens. In one example, the therapeutic cell of the present disclosure can express a bispecific CAR that targets CD19 and CD22. The targeting domain (e.g., antigen binding region) of the CAR can comprise a VHH and / or scFv domain. In some embodiments, the CAR can be a bispecific CAR in a loop configuration.

[0061] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide that includes at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular signaling domain. The association of the extracellular domain of the CAR with the target antigen on the target cell surface results in the clustering of the CAR, delivering an activation stimulus to the CAR-containing cell. The CAR triggers the production of molecules that can redirect the specificity of immune effector cells and mediate proliferation, cytokine production, phagocytosis, and / or cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0062] As used herein, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein that co-translationally or post-translationally directs the nascent CAR protein to the endoplasmic reticulum and subsequent surface expression.

[0063] As used herein, the term "extracellular antigen-binding domain," "extracellular domain," or "extracellular ligand-binding domain" refers to the part of a CAR that is located on the outside of the cell membrane and is capable of binding to an antigen, target, or ligand.

[0064] As used herein, the term "hinge region" or "hinge domain" refers to a portion of a CAR that connects two adjacent domains of a CAR protein, e.g., the extracellular and transmembrane domains of the CAR protein.

[0065] 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.

[0066] 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 transmitting an effector signal.

[0067] 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 a receptor 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 a costimulatory signal (referred to as "co-stimulatory signaling domains").

[0068] In certain embodiments, the extracellular domain comprises an antigen-binding domain and / or an antigen-binding fragment. The antigen-binding fragment can be, for example, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen. The antigen-binding 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.

[0069] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including human, humanized, composite and chimeric antibodies and antibody fragments, that are monoclonal or polyclonal. In general, 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 (e.g., IgA, IgD, IgE, IgG and IgM) depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Thus, the antibodies of the present application can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3 or IgG4. The antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences 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 contain 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 (e.g., 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.

[0070] 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.

[0071] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies constituting 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, the 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 a human heavy chain transgene and a light chain transgene.

[0072] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain of a camelid antibody ... H H), or any other antibody fragment that binds to an antigen but does not contain the complete 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.

[0073] 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).

[0074] As used herein, the term "single domain antibody" refers to conventional single domain antibodies in the art that comprise a heavy chain variable region and a heavy chain constant region, or that comprise only a heavy chain variable region.

[0075] 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 that comprise at least one human heavy and / or light chain polypeptide.

[0076] 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.

[0077] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule 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.

[0078] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, where 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 an embodiment, the first and second epitopes are on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or substantially do not overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In an embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0079] 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. A bispecific antibody is 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 an embodiment, the first and second epitopes are on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence 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, which has binding specificity for a second epitope. H H. In embodiments, the term X / Y loop (where "X" and "Y" are antigens, e.g., CD19 and CD22) refers to the extracellular region of one scFv (either CD19 or CD22) nested between the VL and VH of the other scFv. In some embodiments, X and Y can be the same antigen. In some embodiments, X and Y can be different antigens. In some embodiments, X and Y are tumor antigens.

[0080] 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 1×10 -10 KD refers to an antigen-binding domain or antigen-binding fragment that binds with a KD of less than or equal to M. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (e.g., Kd / Ka) and is expressed as a molar concentration (M). The KD value for an antibody 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, for example by using a biosensor system, such as a Biacore® system, or by using biolayer interferometry technology, such as an Octet RED96 system.

[0081] 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 a target antigen.

[0082] 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 may 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.

[0083] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a 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 a scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0084] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single domain antibody or a nanobody. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0085] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0086] 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.

[0087] In some embodiments, the extracellular tag binding domain and the tag each comprise an scFv.

[0088] In some embodiments, the extracellular tag binding domain, the tag, and the antigen binding domain each comprise an scFv.

[0089] Alternative scaffolds of immunoglobulin domains that exhibit similar functional characteristics, such as high affinity and specific binding of target biomolecules, may 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 may 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., Avimer™); 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-sandwich (e.g., iMab); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimetics; and engineered counterparts of any of the above that retain their binding functionality (WoernA, 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).

[0090] In some embodiments, the alternative scaffold is affilin or centirin.

[0091] 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 a 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 human immunoglobulin heavy chain (HC).

[0092] 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.

[0093] In some embodiments, the first polypeptide of a CAR of the disclosure comprises a transmembrane domain fused in-frame between an extracellular tag-binding domain and a cytoplasmic domain.

[0094] 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 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 binding of the protein that is naturally associated with the transmembrane domain.In certain embodiments, the transmembrane domain contains additional amino acids to allow flexibility and / or optimal distance between the domains associated with the transmembrane domain.

[0095] The transmembrane domain may be derived from either natural or synthetic origin. If the origin is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains that are particularly useful in the present disclosure may be derived from (e.g., at least including the transmembrane regions of) the alpha or beta chain of the T cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8alpha, CD9, CD16, CD22, CD28, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises mainly hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan and / or valine may be found at each end of a synthetic transmembrane domain.

[0096] In some embodiments, it is desirable to utilize a transmembrane domain of the zeta, eta or FcεR1 gamma chain, which contains a cysteine ​​residue capable of disulfide bonding, so that the resulting chimeric protein can form a disulfide-linked dimer with itself or with an unmodified version of the zeta, eta or FcεR1 gamma chain of a related protein. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domains binding with transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex. In other cases, it is desirable to employ a transmembrane domain of zeta, eta or FcεR1 gamma and -beta, MB1 (Igα.), B29 or CD3-gamma, zeta, or eta to retain physical association with other members of the receptor complex.

[0097] 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.

[0098] In some embodiments, the first polypeptide of a CAR of the disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, where the tag-binding domain, linker, and transmembrane domain are in frame with each other.

[0099] 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, the spacer region can be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or can be a completely synthetic spacer region sequence. Non-limiting examples of spacer regions that can be used according to the present disclosure include a portion of the human CD8 alpha chain, a partial extracellular domain of CD28, the FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, Ig hinge, 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. In some embodiments, if the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.

[0100] In some embodiments, the spacer region comprises a hinge domain. The hinge domain can be derived from CD8, CD8α, CD28, or immunoglobulin (IgG). For example, the IgG hinge can be from IgG1, IgG2, IgG3, IgG4, IgG4 CH3, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimera thereof.

[0101] 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, IgG4 CH3, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera 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 include EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgG1, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 59) from IgG3, ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated by reference in its entirety for all purposes), and

[0102] [ka]

[0103] 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. In certain embodiments, the hinge domain is a fragment of an immunoglobulin hinge.

[0104] 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.

[0105] 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.

[0106] In certain aspects, the first polypeptide of the CAR of the present disclosure comprises a cytoplasmic domain that comprises at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more costimulatory signaling domains.

[0107] The cytoplasmic domain is responsible for activating at least one of the normal effector functions of the host cell (e.g., 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 secretion of cytokines. 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 not necessary in many cases 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.

[0108] 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), FcR beta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD5, CD22, CD226, CD66d, CD79A, CD79B, IL18R1, and IL18RAP. In some embodiments, the signaling domain of the CAR can comprise a TIR domain sequence from IL18R1 or IL18RAP.

[0109] In some embodiments, the cytoplasmic domain comprises a CD3 zeta signaling domain. In one embodiment, the CD3 zeta signaling domain comprises an amino acid sequence as 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.

[0110] 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, IL18R1, IL18RAP, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM. In some embodiments, the costimulatory signaling domain of the CAR can comprise a TIR domain sequence from IL18R1 or IL18RAP.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] In one embodiment, the costimulatory signaling domain is derived from DAP12. 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.

[0122] In one embodiment, the costimulatory signaling domain is derived from IL18R1 or IL18RAP. In one embodiment, the IL18R1 or IL18RAP costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 198 or 199, 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: 198 or 199.

[0123] 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.

[0124] 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.

[0125] 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 domain can be switched.

[0126] Non-limiting exemplary CAR regions and sequences are provided in Table 1, including the amino acid and nucleic acid sequences of the various CAR constructs shown in Figures 6, 10A, and 11A.

[0127] [Table 1-1]

[0128] [Table 1-2]

[0129] [Table 1-3]

[0130] [Table 1-4]

[0131] [Table 1-5]

[0132] [Table 1-6]

[0133] [Table 1-7]

[0134] [Table 1-8]

[0135] [Table 1-9]

[0136]

Table 1-10

[0137]

Table 1-11

[0138]

Table 1-12

[0139]

Table 1-13

[0140]

Table 1-14

[0141]

Table 1-15

[0142]

Table 1-16

[0143]

Table 1-17

[0144]

Table 1-18

[0145]

Table 1-19

[0146]

Table 1-20

[0147]

Table 1-21

[0148]

Table 1-22

[0149]

Table 1-23

[0150]

Table 1-24

[0151]

Table 1-25

[0152]

Table 1-26

[0153]

Table 1-27

[0154]

Table 1-28

[0155]

Table 1-29

[0156]

Table 1-30

[0157]

Table 1-31

[0158]

Table 1-32

[0159]

Table 1-33

[0160]

Table 1-34

[0161]

Table 1-35

[0162]

Table 1-36

[0163]

Table 1-37

[0164]

Table 1-38

[0165]

Table 1-39

[0166]

Table 1-40

[0167]

Table 1-41

[0168]

Table 1-42

[0169]

Table 1-43

[0170]

Table 1-44

[0171]

Table 1-45

[0172]

Table 1-46

[0173]

Table 1-47

[0174]

Table 1-48

[0175]

Table 1-49

[0176]

Table 1-50

[0177]

Table 1-51

[0178]

Table 1-52

[0179]

Table 1-53

[0180]

Table 1-54

[0181]

Table 1-55

[0182]

Table 1-56

[0183]

Table 1-57

[0184]

Table 1-58

[0185]

Table 1-59

[0186]

Table 1-60

[0187] [Table 1-61]

[0188] [Table 1-62]

[0189] [Table 1-63]

[0190] [Table 1-64]

[0191] [Table 1-65]

[0192] [Table 1-66]

[0193] [Table 1-67]

[0194] In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen.The antigen-binding domain of the second polypeptide can bind to more than one antigen or more than one epitope in an antigen.For example, the antigen-binding domain of the second polypeptide can bind to 2, 3, 4, 5, 6, 7, 8 or more antigens.As another example, the antigen-binding domain of the second polypeptide can bind to 2, 3, 4, 5, 6, 7, 8 or more epitopes in the same antigen.

[0195] The selection of antigen binding domains may depend on the type and number of antigens that define the surface of target cells. For example, antigen binding domains may be selected to recognize antigens that act as cell surface markers on target cells that are 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 through 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 may act as targets for antigen binding domains in the CARs of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0196] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] Further examples of antigens that may 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, Fit-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.

[0201] 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 with 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 with 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.

[0202] 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-Barre syndrome, Crohn's disease or ulcerative colitis.

[0203] 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, 116 longatio, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factors, keratin, Sa (cytoskeletal protein vimentin), articular cartilage components such as collagens II, IX, and XI, circulating serum proteins such as RF (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33 / hnRNP A2, Sm, eukaryotic 117 longatio, ... 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, granular protein Proteins such as bactericidal permeability enhancing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factors, histones, ribosomal P protein, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, 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.

[0204] In various embodiments, the scFv fragments used in the CAR of the present disclosure may include a linker between the VH and VL domains. 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 of sufficient length to link the VH and VL such that the VH and VL form the correct conformation relative to each other, thereby retaining the desired activity, such as binding to an antigen. The linker may be about 5-50 amino acids in length. In some embodiments, the linker is about 10-40 amino acids in length. In some embodiments, the linker is about 10-35 amino acids in length. In some embodiments, the linker is about 10-30 amino acids in length. In some embodiments, the linker is about 10-25 amino acids in length. In some embodiments, the linker is about 10-20 amino acids in length. In some embodiments, the linker is about 15-20 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.

[0205] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99% sequence identity with SEQ ID NO: 3. In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker comprises the amino acid sequence shown in SEQ ID NO: 25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99% sequence identity with SEQ ID NO: 25.

[0206] 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.

[0207] II. Artificial Cell Death Polypeptides According to an embodiment of the present application, the iPSC cell or a derived cell thereof comprises an exogenous polynucleotide encoding an artificial cell death polypeptide.

[0208] 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 may mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation and / or antibody-mediated depletion. In some cases, 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 the therapeutic cells.

[0209] In certain embodiments, the artificial cell death polypeptide comprises an inactivated cell surface receptor that comprises an epitope that is specifically recognized by an antibody, particularly a monoclonal antibody, also referred to herein as a monoclonal antibody specific epitope. When expressed by iPSCs or derived cells thereof, the inactivated cell surface receptor is inactive or significantly deficient in signal transduction, yet can be specifically recognized by the antibody. Specific binding of the antibody to the inactivated cell surface receptor allows for the elimination of iPSCs or derived cells thereof not only by ADCC and / or ADCP mechanisms, but also by direct killing using antibody drug conjugates with toxins or radionuclides.

[0210] 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, 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.

[0211] 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 domain 2 322-333, 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®). tEGFR is inactive when expressed by iPSCs or derived cells, due to the absence of the EGF-binding and intracellular signaling domains.

[0212] Exemplary inactivated cell surface receptors of the present application include tEGFR variants. In certain embodiments, expression of the inactivated cell surface receptor in engineered immune cells expressing chimeric antigen receptors (CARs) induces cell suicide of the cells when the engineered immune cells are contacted with anti-EGFR antibodies. Methods using inactivated cell surface receptors are described in WO 2019 / 070856, WO 2019 / 023396, and WO 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 effective amount of an anti-EGFR antibody to remove the previously administered engineered immune cells in the subject.

[0213] In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably, the anti-EGFR antibody is cetuximab.

[0214] 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.

[0215] 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.

[0216] 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%, such as 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.

[0217] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of the Her2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody specific epitope comprises or consists of an amino acid sequence at least 90%, such as 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.

[0218] In some embodiments, the inactivated cell surface receptor further comprises a cytokine, such as interleukin-15 or interleukin-2.

[0219] As used herein, "interleukin-15" or "IL-15" refers to a cytokine, or a functional portion thereof, that regulates activation and proliferation of T and NK cells. A "functional portion" ("biologically active portion") of a cytokine refers to a portion of the cytokine that retains one or more functions of the full-length or mature cytokine. Such functions for IL-15 include promoting survival of NK cells, regulating 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 one of skill in the art, the sequences of a variety of 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.

[0220] 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 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:76, preferably the amino acid sequence of SEQ ID NO:76.

[0221] In certain embodiments, the inactivated cell surface receptor comprises a monoclonal antibody specific epitope operably linked to a cytokine, preferably by an autoprotease peptide sequence. 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), Flacheria Disease Virus 2A (BmIFV2A), and combinations thereof. In one embodiment, the autoprotease peptide is the autoprotease peptide of Porcine Teschovirus-1 2A (P2A). In certain embodiments, the autoprotease peptide comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 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.

[0222] 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 sequence. In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 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.

[0223] In some embodiments, the inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 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.

[0224] 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 with SEQ ID NO:21.

[0225] In certain embodiments, the inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO:23.

[0226] 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, 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 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 sequence, such as those described herein. In some embodiments, the cytokine is indirectly linked to the epitope by association with the transmembrane domain via an autoprotease peptide sequence.

[0227] In some embodiments, the artificial cell death polypeptide can include an inducible caspase 9 sequence (iCasp9). Caspase 9 homodimerizes and becomes activated. The homodimer undergoes a conformational change in which one proteolytic domain of the dimer pair becomes active. Physiologically, this occurs through binding of the CARD domain of caspase 9 to APAF-1. In iCasp9, the APAF-1 domain is replaced with a modified FKBP12 that is mutated to selectively bind a chemical inducer of dimerization (CID). The presence of the CID results in homodimerization and activation. iCasp9 is based on a modified human caspase 9 fused to human FK506 binding protein (FKBP) (Straathof et al (2005) Blood 105:4247-4254). It allows conditional dimerization in the presence of a small molecule CID known as AP1903. AP1903 is an experimental drug and is considered biologically inactive since it does not interact with wild-type FKBP12. However, clinical experience with this agent is limited to a very small number of patients (Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683; and luliucci, JD et al. (2001) J. Clin. Pharmacol. 41, 870-879). AP1903 is also a relatively large polar molecule and is unlikely to cross the blood-brain barrier.

[0228] Non-limiting exemplary artificial cell death polypeptide / inactivating cell surface receptor regions and sequences are provided in Table 2.

[0229] [Table 2-1]

[0230] [Table 2-2]

[0231] [Table 2-3]

[0232] In certain embodiments, the inactivated cell surface receptor comprises 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:79, preferably the amino acid sequence of SEQ ID NO:79.

[0233] In certain embodiments, the inactivated cell surface receptor comprises 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:81, preferably the amino acid sequence of SEQ ID NO:81.

[0234] In certain embodiments, the inactivated cell surface receptor comprises 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:83, preferably the amino acid sequence of SEQ ID NO:83.

[0235] III. HLA Expression In certain embodiments, the iPSCs or derived cells of the present application can be further modified by introducing an 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 abolishes 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).

[0236] In certain embodiments, the iPSCs or derived cells thereof comprise an 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%, such as 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%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68, preferably SEQ ID NO: 68.

[0237] 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%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66.

[0238] In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused via a linker to HLA-G. 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.

[0239] IV. Other Genome Editing Options In one embodiment of the above cells, 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, pharma- ceutical 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 derived cells thereof.

[0240] 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 at selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, HII, 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 if the genomically engineered iPSCs contain more than one suicide gene, the suicide genes are integrated into different safe zone loci including AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, Hll, 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.

[0241] 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 a chromosome of the iPSC.

[0242] 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 (interchangeable 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 that involves inserting one or more exogenous sequences into a preselected site in the genome with or without deletion of the endogenous sequence at the insertion site.

[0243] 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.

[0244] Alternatively, targeted editing can be achieved at higher frequencies by specific introduction of double-strand breaks (DSBs) by 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 a pair of homologous arms, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration".

[0245] 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 (Dual Integrase Cassette Exchange) system, which utilizes phiC31 and Bxbl integrase, is also a promising tool for targeted integration.

[0246] ZFN is a targeted nuclease that comprises a nuclease fused with a zinc finger DNA binding domain. By "zinc finger DNA binding domain" or "ZFBD" is meant a polypeptide domain that sequence-specifically binds to DNA via one or more zinc fingers. A zinc finger is a domain of about 30 amino acids in a zinc finger binding domain whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2 zinc finger, C3H zinc finger, and C4 zinc finger. 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 processing algorithms for processing information in 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; see also 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, whose production is mainly 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 with the zinc finger DNA binding domain of Fokl nuclease.

[0247] TALENs are targeted nucleases that contain 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 that is responsible for binding the TAL effector protein to DNA. TAL effector proteins are secreted during infection by the plant pathogen Xanthomonas genus. 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 selected repeat positions called repeat variable-diresidues (RVDs). TALENs are described in more detail in US Patent Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of TALENs in the art is the fusion polypeptide of Fokl nuclease with the TAL effector DNA binding domain.

[0248] Another example of a targeted nuclease for use in the subject methods is a targeted Spoll nuclease, a polypeptide that includes a Spol I 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., and that 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.

[0249] 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.

[0250] 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. As an example, CRISPR / Cas9 requires two main components: (1) Cas9 endonuclease and (2) crRNA-tracrRNA complex. When co-expressed, these two components form a complex that is recruited to target DNA sequences that contain 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 a target selection sequence. These two components can then be delivered to mammalian cells via transfection or transduction. As another example, CRISPR / Cpf1 contains two main components: (1) CPf1 endonuclease and (2) crRNA. When co-expressed, these two components form a ribonucleoprotein (RNP) complex that is recruited to a target DNA sequence containing a PAM and a seeding region near the PAM. The crRNAs can be combined to form a chimeric guide RNA (gRNA) to guide Cpf1 to a target selection sequence. These two components can then be delivered into mammalian cells via transfection or transduction.

[0251] 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.

[0252] DICE-mediated insertion uses a pair of recombinases, e.g., phiC31 and Bxbl, to provide unidirectional integration of exogenous DNA, but this unidirectional integration is strictly limited to the small attB and attP recognition sites of each enzyme itself. 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, e.g., U.S. Patent Application Publication No. 2015 / 0140665, which is incorporated herein by reference.

[0253] One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by a cell host enzyme mechanism. 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 a desired integration site into a 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 a desired integration site into a cell to allow TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, introducing into the cell a gRNA comprising a Cpf1 expression cassette and a guide sequence specific to a desired integration site to allow Cpf1 mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, introducing into the cell a gRNA comprising a Cas9 expression cassette and a guide sequence specific to a desired integration site to allow Cas9 mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more att sites of a pair of DICE recombinase into a desired integration site, introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell an expression cassette for DICE recombinase to allow DICE mediated targeted integration.

[0254] The site for targeted integration includes, but is not limited to, genomic safety region.Genomic safety region is an intragenic or extragenic region of the human genome that can accommodate the predictable expression of newly integrated DNA, theoretically without adverse effects on 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, Hll, GAPDH, TCR and RUNX1 genes.

[0255] In other embodiments, the site for targeted integration is selected to delete or reduce the expression of endogenous genes at the insertion site.As used herein, the term "deletion" with respect to gene expression refers to any genetic modification that negates the expression of genes.Examples of the "deletion" of gene expression include, for example, the removal or deletion of the DNA sequence of a gene, the insertion of an exogenous polynucleotide sequence at the locus of a gene, and one or more substitutions within a gene, which negates the expression of a gene.

[0256] Genes for targeted deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be histocompatibly 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 or 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 6p2l), or 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, the TAP1 gene, the TAP2 gene, and the tapasin gene. For example, the B2M gene encodes a common subunit that is essential for cell surface expression of all MHC class I heterodimers. B2M null cells are MHC-I deficient. MHC class II deficient 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 of the 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 gene with the integration.

[0257] 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 MHC class-I related genes, such as beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene or tapasin gene; and MHC-II related genes, such as RFXANK, CIITA, RFX5, RFXAP or CIITA gene; and optionally further into the locus of a safety region gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 gene. More preferably, one or more of the exogenous polynucleotides are integrated into the locus of CIITA, AAVS1 and B2M gene.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] In some aspects, the present disclosure describes engineered iPSCs and cells derived therefrom that exogenously express recombinant CD16 and recombinant NKG2D.In some aspects, such cells also express one or more CARs (e.g., one or more CARs that comprise CD22 antigen binding domain and / or CD19 antigen binding domain).For example, in some embodiments, such cells can express a single CAR that comprises one or more antigen binding domains that bind to CD22 and / or CD19.In another example, such cells can express two or more CARs, each CAR comprises an antigen binding domain that binds to a tumor antigen, and the tumor antigen is independently selected from the group consisting of CD22 and CD19.

[0262] In addition to the method of exogenously expressing or overexpressing CD16 and NKG2D proteins and transgenes in cells, such cells and their therapeutic uses are described herein. Surface receptor CD16 (FcγRIIIA) affects human natural killer (NK) cells during maturation. NK cells bind to the Fc portion of IgG via CD16 and perform antibody-dependent cellular cytotoxicity, which is essential for the effectiveness of some anti-tumor monoclonal antibody therapies. NKG2D is a stimulatory / activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system, including NK cells and a subset of T cells. NKG2D is crucial in various aspects of innate and adaptive immune function. In some embodiments, CD16 and NKG2D are expressed from a single polynucleotide construct, which is advantageous for reducing the number of gene edits of cells.

[0263] In certain aspects, iPSCs or derived cells thereof are provided, comprising exogenous or isolated polynucleotide constructs encoding CD16 protein and NKG2D protein. In some embodiments, iPSCs or derived cells thereof expressing recombinant CD16 protein and recombinant NKG2D protein are described herein. In some embodiments, the recombinant protein is encoded by exogenous or isolated polynucleotide constructs. In some embodiments, the polynucleotide constructs encoding CD16 protein and NKG2D protein also comprise polynucleotide sequences encoding autoprotease peptides or autocleaving peptides. In some embodiments, the exogenous polynucleotide constructs encoding CD16 protein, NKG2D protein and autocleaving peptides are introduced into iPSCs or derived cells thereof. The exogenous or isolated polynucleotide constructs can be introduced into the locus of iPSCs or derived cells thereof.

[0264] In some embodiments, the iPSCs or derived cells expressing recombinant CD16 protein and recombinant NKG2D protein also express chimeric antigen receptor (CAR). In some embodiments, the cells expressing recombinant CD16 protein and recombinant NKG2D protein also express recombinant HLA-E, HLA-G, or both. In some embodiments, the iPSCs or derived cells expressing recombinant CD16 protein and recombinant NKG2D protein also express CAR and recombinant HLA-E, HLA-G, or both. In many embodiments, the cells expressing recombinant CD16 protein, recombinant NKG2D protein, and CAR also express recombinant IL-15 protein. In many embodiments, the cells express recombinant CD16 protein, recombinant NKG2D protein, CAR, recombinant IL-15 protein, and recombinant HLA-E, HLA-G, or both.

[0265] In many embodiments, the cells expressing recombinant CD16 protein, recombinant NKG2D protein, and CAR also express a recombinant fusion protein comprising IL-15 and IL-15Ra. In many embodiments, the cells express recombinant CD16 protein, recombinant NKG2D protein, CAR, recombinant fusion protein comprising IL-15 and IL-15Ra, and either or both of recombinant HLA-E and HLA-G. In some embodiments, the cells expressing recombinant CD16 protein and recombinant NKG2D protein also express recombinant IL-15 protein. In some embodiments, the cells expressing recombinant CD16 protein and recombinant NKG2D protein also express recombinant fusion protein comprising IL-15 and IL-15Ra. In some embodiments, the cells expressing recombinant CD16 protein, recombinant NKG2D protein, and recombinant IL-15 protein also express a CAR. In some embodiments, the cells expressing recombinant CD16 protein, recombinant NKG2D protein, and recombinant fusion protein comprising IL-15 and IL-15Ra also express a CAR.

[0266] In one aspect, an exogenous or isolated polynucleotide construct is provided that encodes CD16 protein and NKG2D protein. In some embodiments of the exogenous polynucleotide construct, the polynucleotide sequence encoding CD16 protein and the polynucleotide sequence encoding NKG2D protein are operably linked by a polynucleotide sequence encoding an autoprotease peptide or a self-cleaving peptide. In some embodiments, the polynucleotide construct comprises, from the 5' end to the 3' end, a polynucleotide sequence encoding CD16 protein, a polynucleotide sequence encoding an autoprotease peptide or a self-cleaving peptide, and a polynucleotide sequence encoding NKG2D protein. In some embodiments, the polynucleotide construct comprises, from the 5' end to the 3' end, a polynucleotide sequence encoding NKG2D protein, a polynucleotide sequence encoding an autoprotease peptide or a self-cleaving peptide, and a polynucleotide sequence encoding CD16 protein. In some embodiments, the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 179. In some embodiments, the exogenous polynucleotide construct encodes the amino acid sequence of SEQ ID NO: 180.

[0267] In some embodiments, the CD16 protein (also referred to as "low affinity immunoglobulin gamma Fc region receptor III-A" or "Fc gamma receptor IIIa") is a wild-type CD16 protein. In some embodiments, the human wild-type CD16 protein has an amino acid sequence as set forth in NCBI Ref. Seq. No. NP_000560.7 or UniProt No. P08637. In some cases, the coding sequence of human wild-type CD16 is set forth in NCBI Ref. No. NM_000569.8.

[0268] In some embodiments, the CD16 protein is a CD16 variant protein. In some cases, the CD16 variant protein has an amino acid sequence with wild-type CD16, such as wild-type CD16 of SEQ ID NO: 181, of at least 90%, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some cases, the CD16 variant is a high-affinity CD16 variant. In other cases, the CD16 variant is a non-cleavable CD16 variant. In some cases, the CD16 variant is a high-affinity and non-cleavable CD16 variant.

[0269] In some embodiments, the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an F158V substitution and one or more substitutions selected from F176V, S197P, D205A, S219A, T220A, and any combination thereof. In one embodiment, the CD16 variant has an F176V substitution and one or more substitutions selected from F158V, S197P, D205A, S219A, T220A, and any combination thereof. In many embodiments, the CD16 variant has an S197P substitution and one or more substitutions selected from F158V, F176V, D205A, S219A, T220A, and any combination thereof. In various embodiments, the CD16 variant has a D205A substitution and one or more substitutions selected from F158V, F176V, S197P, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a S219A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a T220A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the variant CD16 protein has the sequence of SEQ ID NO: 182. In some embodiments, the nucleic acid sequence encoding the variant CD16 protein has the sequence of SEQ ID NO: 183. In some embodiments, the wild-type CD16 protein has the sequence of SEQ ID NO: 181.

[0270] In some embodiments, the NKG2D protein (also referred to as NKG2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily K1 member 1 or KLRK1) is a wild-type NKG2D protein. In some embodiments, the human wild-type NKG2D protein has an amino acid sequence as shown in NCBI Ref. Seq. Nos. NP_001186734.1 or NP_031386.2 or UniProt No. P26718. In some cases, the coding sequence of human wild-type NKG2D is as shown in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3. In some embodiments, the NKG2D protein is a NKG2D variant protein. In some cases, the NKG2D variant protein has an amino acid sequence that has at least 90%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wild-type NKG2D, e.g., the wild-type NKG2D of SEQ ID NO: 184. In some embodiments, the NKG2D protein has the amino acid sequence of SEQ ID NO: 184. In some embodiments, the nucleic acid sequence encoding the NKG2D protein has the sequence of SEQ ID NO: 185.

[0271] As discussed above, constructs are provided herein that include an autoprotease peptide sequence that includes a 2A peptide that can induce ribosome skipping during translation of a polypeptide. The 2A peptide functions to "cleave" an mRNA transcript by causing the ribosome to skip synthesis of a peptide bond at the C-terminus between a glycine (G) and a proline (P) residue, thereby resulting in a separation between the end of the 2A sequence and the next peptide downstream. 2A peptides include, but are not limited to, Porcine Teschovirus-1 2A (P2A) peptide, Foot and Mouth Disease Virus (FMDV) 2A (F2A) peptide, Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, Thosea asigna virus 2A (T2A) peptide, Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A) peptide, and Flacheria Disease Virus 2A (BmIFV2A) peptide.

[0272] Exemplary P2a peptides can include an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 186. In some embodiments, the P2A peptide has the amino acid sequence of SEQ ID NO: 186.

[0273] derived cells In another aspect, the present invention relates to cells derived from the differentiation of iPSC, derived cells.As mentioned above, the genome editing introduced into iPSC cells is retained in the derived cells.In certain embodiments of derived cells obtained from iPSC differentiation, the derived cells are hematopoietic cells, including but not limited to HSC (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, antigen-presenting cells (APCs), monocytes and macrophages.In certain embodiments, the derived cells are immune effector cells, such as NK cells or T cells.

[0274] In certain embodiments, the application provides a natural killer (NK) cell or T cell comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a deleted or reduced expression of an MHC class I- and MHC class II-associated gene, e.g., an MHC class-I-associated gene selected from the group consisting of the B2M gene, the TAP1 gene, the TAP2 gene, and the tapasin gene, and an MHC-II-associated 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; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and interleukin 15 (IL-15), wherein the IL-15RA and IL-15 are operably linked.

[0275] In certain embodiments, the NK cell or T cell further comprises an exogenous polynucleotide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G).

[0276] Also provided is a NK cell or T cell comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of SEQ ID NO: 61; (ii) an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 202, wherein the IL-15RA and IL-15 are operably linked, wherein the exogenous polynucleotide is integrated into the locus of the AAVS1, B2M, and CIITA genes, respectively, thereby deleting or reducing expression of CIITA and B2M.

[0277] 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.

[0278] Also provided are: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) an 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 sequence; and (iii) a CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC), comprising a deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes.

[0279] In certain embodiments, the CD34+ HPCs further comprise an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

[0280] In certain embodiments, the CAR comprises: (i) a signal peptide comprising a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to a CD19 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain comprising a costimulatory domain, e.g., a CD28 signaling domain.

[0281] Methods for producing derivative cells are also provided, the methods comprising differentiating iPSCs under conditions for cell differentiation, thereby obtaining derivative cells.

[0282] 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. WO 2010 / 099539, WO 2012 / 109208, WO 2017 / 070333, WO 2017 / 179720, WO 2016 / 010148, WO 2018 / 048828 and WO 2019 / 157597, each of which is incorporated herein by reference in its entirety.Differentiation protocols may use feeder cells or may not include feeders. 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, since feeder cells provide stimuli, growth factors, and nutrients for the support of a second cell type.

[0283] 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, cells primed with IL-12 exhibit rapid cell killing compared to cells differentiated in the absence of IL-12. Furthermore, cells differentiated using IL-12 conditions exhibit improved cancer cell proliferation inhibition.

[0284] 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.It is recognized by those skilled in the art that the coding sequence of the CAR can be changed (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein.Therefore, it is understood by those skilled in the art that the nucleic acid sequence encoding the CAR of the present application can be changed without changing the amino acid sequence of the protein.

[0285] 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.

[0286] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding a CAR useful for the invention according to the embodiments of the present application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a 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 for the production of CAR in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate the recombinant expression vector according to the embodiments of the present application.

[0287] In certain aspects, the present application provides a vector for targeted integration of a CAR useful in the invention according to an embodiment 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 an embodiment of the present application; and (c) a terminator / polyadenylation signal.

[0288] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90% identical polynucleotide sequence with SEQ ID NO:63, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0289] 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.

[0290] 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.

[0291] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm adjacent to the exogenous polynucleotide. As used herein, "left homologous arm" and "right homologous arm" refer to a pair of nucleic acid sequences adjacent to the exogenous polynucleotide, which facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. The sequence of the left and right arm homologous arms can be designed based on the intended integration site. In some embodiments, the left or right arm homologous arm is homologous to the left or right sequence of the integration site.

[0292] In certain embodiments, the left homology 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 homology 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.

[0293] 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.

[0294] (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 for the invention according to the embodiment of the present application. It is recognized by those skilled in the art that the coding sequence of the inactivated cell surface receptor can be changed (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, it is understood by those skilled in the art that the nucleic acid sequence encoding the inactivated cell surface receptor of the present application can be changed without changing the amino acid sequence of the protein.

[0295] In certain embodiments, the isolated nucleic acid encodes any of the inactivated cell surface receptors described herein, e.g., those that comprise 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 sequence.

[0296] 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 and encoding an inactivated cell surface receptor that contains an epitope specifically recognized by pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab.

[0297] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epidermal growth factor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab or panitumumab, preferably cetuximab.

[0298] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, e.g., an epitope that is specifically recognized by polatuzumab vedotin.

[0299] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, eg, an epitope that is specifically recognized by rituximab.

[0300] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of the Her2 receptor, eg, an epitope specifically recognized by trastuzumab.

[0301] In certain embodiments, the autoprotease peptide sequence is porcine teschovirus-1 2A (P2A).

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] In certain embodiments, the isolated nucleic acid encoding an 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.

[0310] 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.

[0311] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for the invention according to the embodiments of the present application. Any vector known to the skilled artisan in view of the present disclosure can be used, for example, a plasmid, a cosmid, a phage vector or a 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, for example, a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein for the production of inactivated cell surface receptors in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate the recombinant expression vector according to the embodiments of the present application.

[0312] In a particular aspect, the present application provides a vector for targeted integration of an inactivated cell surface receptor, which is useful for the invention according to the embodiments of the present application. In a particular embodiment, 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 (tEGFR) variant, and interleukin 15 (IL-15), wherein the tEGFR variant and the IL-15 are operably linked by an autoprotease peptide sequence, for example, porcine teschovirus-1 2A (P2A), and (c) a terminator / polyadenylation signal.

[0313] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90% identical polynucleotide sequence with SEQ ID NO:63, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0314] 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.

[0315] 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.

[0316] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm flanking the exogenous polynucleotide.

[0317] In certain embodiments, the left homology 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: 84. In certain embodiments, the right homology 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:85.

[0318] 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.

[0319] (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 embodiment of the present application.It is recognized by those skilled in the art that the coding sequence of the HLA construct can be changed (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein.Therefore, it is understood by those skilled in the art that the nucleic acid sequence encoding the HLA construct of the present application can be changed without changing the amino acid sequence of the protein.

[0320] 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., a 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 4×GGGGS linker, and / or B2M and HLA-E operably linked by a 3×GGGGS linker. In certain embodiments, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence 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%, 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.

[0321] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an HLA construct useful for the invention according to the embodiments of the present application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, cosmid, phage vector or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element for establishing the conventional 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 for the production of HLA constructs in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to the embodiments of the present application.

[0322] In certain aspects, the present application provides a vector for targeted integration of HLA constructs useful in the invention 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 HLA construct; and (c) a terminator / polyadenylation signal.

[0323] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90% identical polynucleotide sequence with SEQ ID NO:63, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0324] 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.

[0325] 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, where 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.

[0326] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm flanking the exogenous polynucleotide.

[0327] In certain embodiments, the left homology 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 homology 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.

[0328] 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.

[0329] (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 skilled in the art in view of the present disclosure can be used for recombinant expression of an 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 effectively expressing the recombinant nucleic acid.

[0330] Examples of host cells include recombinant cells containing vectors or isolated nucleic acids of the present application, useful for producing vectors or constructs of interest, for example; or engineered iPSCs or derived cells thereof containing one or more isolated nucleic acids of the present application, preferably integrated into one or more chromosomal loci. The host cells of the isolated nucleic acids of the present application can also be immune effector cells, such as T cells or NK cells, containing one or more isolated nucleic acids of the present application. The 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. The immune effector cells can also be obtained by transfecting one or more isolated nucleic acids of the present application into immune effector cells.

[0331] composition In another general aspect, the application provides a composition comprising an isolated polynucleotide of the application, a host cell of the application, and / or an iPSC or a derived cell thereof.

[0332] 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 a radioactive moiety, or an immunomodulatory drug (iMiD).

[0333] 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 pharma- ceutically acceptable carrier. The term "pharmaceutical composition" as used herein means 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, together with a pharma- ceutically acceptable carrier. The polynucleotide, polypeptide, host cell, and / or iPSC or derived cell thereof of the present application and the composition comprising them are also useful for the manufacture of medicaments for the therapeutic applications mentioned herein.

[0334] As used herein, the term "carrier" refers to any excipient, diluent, bulking agent, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal 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 "pharmaceutical acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the compositions described herein or the biological activity of the compositions described herein. In accordance with certain embodiments, any pharmaceutical acceptable carrier suitable for use in polynucleotides, polypeptides, host cells, and / or iPSCs or derived cells thereof in light of the present disclosure may be used.

[0335] Formulation of pharma- ceutical active ingredients with pharma- ceutical 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 adjusting agents, preservatives, stabilizers, and chelating agents. One or more pharma- ceutical acceptable carriers may be used in the formulation of the pharmaceutical compositions of the present application.

[0336] How to use Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, especially histological examination. The definition of cancer cells, as used herein, includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that usually appear as solid tumors, a "clinically detectable" tumor is one that can be detected based on tumor mass, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during physical examination; and / or one that can be detected due to the expression of one or more cancer-specific antigens in samples that can be obtained from patients.

[0337] Cancerous conditions may be characterized by the abnormal proliferation of malignant cancer cells, and may include leukemias, such as AML, CML, ALL, and CLL, lymphomas, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, and solid cancers, such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and cerebral cancer, as well as cancer of unknown primary site (CUP).

[0338] Cancer cells in an individual may be immunologically distinct from normal somatic cells in the individual (e.g., cancerous tumors may be immunogenic). For example, cancer cells may be capable of inducing a systemic immune response in an individual against one or more antigens expressed by the cancer cells. The tumor antigen that induces the immune response may be specific to the cancer cells or may be shared by one or more normal cells in the individual.

[0339] An individual's cancer cells suitable for the treatments described herein may express the antigen and / or may be of the correct HLA type to bind to the antigen receptor expressed by the T-cells.

[0340] The individual suitable for the above-mentioned treatment can be a mammal.In a preferred embodiment, the individual is a human.In another preferred embodiment, non-human mammals can be used, particularly mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., mice, primates, pigs, dogs, or rabbits).

[0341] In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment. In some embodiments, the individual may not have minimal residual disease after one or more cancer treatments or repeat medications.

[0342] An individual with cancer may show at least one identifiable sign, symptom, or laboratory finding that is sufficient to diagnose cancer according to clinical standards known in the art.Examples of such clinical standards can be found in pharmaceutical textbooks such as Harrison's Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001.In some cases, diagnosing cancer in an individual may include identifying a specific cell type (e.g., cancer cell) in a sample of body fluid or tissue obtained from the individual.

[0343] Antitumor effect is a biological effect that can be manifested by a reduction in the rate of tumor growth, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or recovery from various physiological symptoms associated with a cancerous condition. "Antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies described herein, and also the T cells obtainable by the method of the present invention, in preventing the development of initial tumors.

[0344] Treatment can be any treatment and / or therapy, whether human or animal (e.g., in veterinary applications), that achieves some desired therapeutic effect, including, for example, inhibiting or slowing the progression of a condition, including a reduction in the rate of progression, a halt in the rate of progression, recovery from a condition, curing or ameliorating (partially or totally) a condition, preventing, slowing, alleviating or halting one or more symptoms and / or signs of a condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0345] Treatment can also be preventative (e.g., prophylactic). For example, an individual susceptible to or at risk of developing or recurring cancer can be treated as described herein. Such treatment can prevent or delay the development or recurrence of cancer in an individual.

[0346] In particular, treatment may include inhibition of cancer growth, including complete cancer remission, and / or inhibition of cancer metastasis. Cancer growth generally refers to any one of a number of indices that indicate changes in cancer to more advanced forms. Thus, indices for measuring inhibition of cancer growth include reduction in cancer cell survival, reduction in tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), slowing of tumor growth, destruction of tumor vasculature, improved performance in delayed-type hypersensitivity skin test, increase in activity of T cells, and reduction in levels of tumor-specific antigens. Administration of modified T cells as described herein may improve an individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the tendency of cancer growth in the individual.

[0347] 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 the cells of the present application and / or the compositions 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).

[0348] According to an embodiment of the present application, the composition comprises a therapeutically effective amount of isolated polynucleotide, isolated polypeptide, host cell, and / or iPSC or its derived cell.As used herein, the term "therapeutically effective amount" refers to the amount of active ingredient or component that induces a desired biological or medical response in a subject.Therapeutically effective amount can be empirically and routinely determined for a given purpose.

[0349] 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 an amount of the cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0350] According to certain embodiments, a therapeutically effective amount refers to an amount of a 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 onset 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 time 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.

[0351] 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 administered, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages are optimally designed to optimize safety and efficacy.

[0352] 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.

[0353] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in any convenient manner known to those skilled in the art. For example, the cells of the present application can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and / or transplantation. The composition comprising the cells of the present application can be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, 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.

[0354] 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 cell integrity and viability and administration of the cell composition, e.g., water, saline, phosphate buffered saline, etc.

[0355] Sterile injectables can be prepared by incorporating the cells of the present application in an appropriate amount of a suitable solvent, optionally with various other ingredients. Such compositions can include pharma- ceutically acceptable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, etc., suitable for use in cell compositions and for administration to subjects, such as humans. Buffers suitable for providing cell compositions are well known in the art. Any medium, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the present application.

[0356] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in any physiologically acceptable medium. The cell population comprising the cells of the present application can include a purified population of cells. One of skill in the art can readily determine the cells in the cell population using a variety of well-known methods. The range of purity in the cell population comprising the genetically modified cells of the present application can be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dosage can be readily adjusted by one of skill in the art, for example, a decrease in purity may require an increase in dosage.

[0357] 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 which the cells and / or pharmaceutical compositions containing the cells are administered. Generally, cell doses range from about 10 to about 20, 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 1×10 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 The effective dose may be determined by, but is not limited to, any of the following: per kg, etc. Moreover, the dose may be adjusted to account for whether a single dose or multiple doses are administered. The exact determination of what is considered an effective dose may be based on factors individual to each subject.

[0358] 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 related to cancer that may, but is not necessarily, be identifiable 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, such as a tumor or, more preferably, a 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 the survival time of a subject having 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.

[0359] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in combination with one or more additional therapeutic agents. In certain embodiments, the one or more therapeutic agents are selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, 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). EXAMPLES

[0360] Abbreviation

[0361] [Table 3]

[0362] [Example 1] Development of cells expressing CD22-specific VHH CAR CARs were constructed with VHH binding domains, either single or multiple, on the extracellular side of the protein construct. VHH antibody binding fragments were isolated through phage display of a VHH library and selection on soluble CD22 protein. VHH proteins were tested for protein affinity and specific binding to CD22+ cells as VHH-IgG1 Fc fusions. Epitope mapping was performed using CD22 protein variants in which unique domains were deleted from the full-length protein by binding competition. Two clones demonstrated binding to the N-terminal domains of CD22, IgV and IgC1-2, and another clone demonstrated binding to the C-terminal IgC domains 4-6. A clone binding to the C-terminal IgC domain was affinity matured by scanning mutagenesis of CDR3. This improved affinity from 30 nM to 5 nM.

[0363] VHH fragments were cloned into lentiviral vectors in frame with a CAR consisting of a hinge domain (IgG4 Fc or CD8 hinge), a transmembrane domain (CD28tm), a costimulatory domain (41BB, CD28, DAP10) and a primary signaling domain from CD3z. As monomeric VHH-CARs, low levels of T cell-mediated cytotoxicity were observed. When placed in a tandem format, VHH1-VHH1 or VHH1-VHH2, higher levels of cytotoxicity were observed, suggesting that improved affinity / avidity for CD22 on cells is essential for full target cell engagement.

[0364] Anti-CD22 VHH antibodies were isolated from a VHH phage display library through panning against the CD22 full-length ECD. Three clones of demonstrated interest were labeled D04, A01 and E04 (Table 3). The VHH sequences were fused to a human IgG1 Fc domain in a mammalian expression construct, expressed in Expi293 cells as VHH-Fc fusion proteins and purified with ProteinA (MabSelect SuRe) resin. The VHH-Fc proteins were eluted with 0.1 M sodium acetate (pH=3.5) and neutralized with 2.5 M sodium acetate (pH=7.5).

[0365] VHH-Fc fusion proteins were assessed for affinity to monomeric CD22 (Acro Biosystems) using a ForteBio Octet Red BLI instrument. VHH-Fc proteins were captured on an anti-human IgG Fc sensor at 50 nM. The sensor chip was quenched with 100 nM human IgG Fc protein, then CD22 antigen was added at 250 nM to assess binding kinetics. Association was examined for 600 s, dissociation was examined for 600 s, and Kon, Kdis, and KD were calculated. The A01 clone (PROT739) has an affinity of 96 nM, the E04 (PROT740) clone has an affinity of 23 nM, and the D04 clone (PROT265) has an affinity of 32 nM (Table 4).

[0366] [Table 4]

[0367] [Table 5]

[0368] VHH-Fc proteins were tested for binding to CD22 expressed on the cell surface. VHH-Fc proteins were serially diluted and added to Raji cells and incubated at 4C for 30 min. Cells were washed once in BD staining buffer, BSA (BD Pharmingen) and then PE-labeled anti-human IgG Fc (Jackson Immnoresearch) was added to detect VHH-Fc cell binding and incubated at 4C for 30 min. Cells were washed once and then resuspended in staining buffer containing 0.1% pluronic acid and run through a flow cytometer to detect fluorescent labeling of VHH-Fc cells. Binding curves are shown in Figure 1, which shows that all three clones bind to CD22-positive Raji cells.

[0369] To determine which domain the VHH-Fc protein is bound to, two deletion variant proteins were constructed. One variant, CD22Δ2-3, deletes IgC domains 2&3, and the other variant, CD22ΔV-1, deletes the N-terminal IgV and IgC1 domains. Binding of VHH-Fc proteins to full-length CD22 was compared to binding to these variants using a ForteBio Octet. VHH-Fc proteins were captured on an anti-human IgG Fc biosensor chip in kinetic buffer (ForteBio). The chip was quenched with human IgG Fc fragment (Jackson Immunoresearch), then CD22 protein was allowed to associate with VHH-Fc for 10 min. The biosensor chip was then transferred to kinetic buffer for dissociation of CD22 protein. PROT265(D04) bound to all proteins tested, suggesting that it binds to IgC domains 4-6. PROT739 (A01) bound to full-length CD22 and had weak binding to CD22Δ2-3 protein but not to CD22ΔV-1 protein, suggesting that it binds to the IgC1-2 domain.PROT740 (E04) bound to full-length CD22 and CD22Δ2-3 protein but not to CD22ΔV-1 protein, suggesting that it binds to the IgV-IgC1 domain (Figure 2).

[0370] The D04 clone was affinity matured by CDR scanning mutagenesis and phage display selection for higher affinity clones. Variants were isolated and expressed as VHH-Fc fusions for affinity evaluation. Several clones showed improvement, with the best clone, D04-AM-D11 (PROT810), showing a 5-fold improvement to 7 nM (Table 4). This clone also showed improved binding to CD22+ Raji cells (Figure 1B).

[0371] VHH sequences were cloned into lentiviral CAR constructs with a human IgG4 (CH3) hinge, CD28 transmembrane domain, 41BB costimulatory domain and CD3z signaling domain (Table 5). Lentivirus was produced for the various constructs and transduced into Jurkat cells to examine antigen-dependent activation of this T cell line. VHH-CAR-transduced cells were cultured for one week and then co-cultured with other tumor cell lines with or without CD22 surface expression. Jurkat cells were then stained for expression of the activation marker CD69 and examined by flow cytometry. Activated CAR-Jurkat cells were compared to untransduced control (UTD) cells that lacked the CAR. Figure 3a shows that cells with A01 (P952) and E04 (P953) VHH-CARs were activated when exposed to CD22+ cells, Daudi and Raji, but not Raji CD22 knockout or K-562 cells. The D04 VHH-CAR (D04.P262) was tested in a separate experiment (Figure 3b) and also showed activation upon exposure to Daudi and Raji cells.

[0372] [Table 6]

[0373] [Example 2] Cytotoxicity assay VHH-CARs were tested in primary T cells for killing of CD22+ tumor target cells. Primary donor T cells were transduced with lentivirus for each of the described CARs and expanded for 14 days. CAR expression was assessed by flow cytometry. CD22+ Raji cells were labeled with CellTrace Violet (ThermoFisher) and co-cultured with CAR-T cells at various E:T ratios for 48 hours. Raji cell viability was then assessed by flow cytometry (Figure 4). The P403 construct (CD22_D04) showed Raji cell killing modestly above background (UTD). The affinity-improved variant P1278 (CD22_D04_AM_D11) had significantly better cytotoxicity of up to 80% at an E:T ratio of 5:1. The P952 (CD22_CNTY_VHH1_A01) and P953 (CD22_CNTY_VHH1_E04) constructs demonstrated cytotoxicity modestly above background.

[0374] To improve the cytotoxicity profile, VHHs were assembled in tandem on the CAR protein. The A01 and E04 clones bound to an N-terminal epitope, and the D04 clone bound to a C-terminal epitope, so a combination of these VHHs was made so that the VHH-CAR could bind two different epitopes. The VHHs were assembled in tandem, separated by a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 3), and paired with different CAR domains: 1) CD8 hinge / CD28 transmembrane / 41BB / CD3z and 2) CD8 hinge / transmembrane / DAP10 / CD3z (Table 3). Lentiviruses were prepared from these constructs and transduced into primary T cells. Cytotoxicity was evaluated as described. The results showed improved cell killing of Raji cells using tandem biparatopic VHH-CARs, with the "CD22_D04_AM_D11-linker-CD22_A01" format having the most robust cell killing (Figure 5).

[0375] [Example 3] Cytotoxicity assays using therapeutic cells expressing mono- and bispecific CARs against various target cells Bispecific CAR ectodomains were engineered using selected CD22 binders in combination with FMC63 (Figure 6). Selected CARs (e.g., identified by P####) were expressed and tested for cytotoxic activity in T cells along with monospecific CAR-T cells.

[0376] Selected bispecific CARs were tested for killing of CD19+, CD22+ or CD19+ / CD22+ tumor target cells in primary T cells (Table 6).

[0377] [Table 7]

[0378] Lentivirus for each of the described CARs was transduced into primary donor T cells and expanded for 14 days. CAR expression was assessed by flow cytometry. Target-expressing cells were co-cultured with CAR-T cells at various E:T ratios for 48 hours. Cell viability was assessed by flow cytometry (Figures 9 & 10B). CD19, CD22, and CD19 / CD22-dependent cytotoxicity was assessed at an E:T ratio of 1.25:1 (Figures 7 and 8). Data represent cumulative cytotoxicity (e.g., percent target killing) for the cell lines tested. All of the bispecific CAR-T cells tested exhibited CD19-targeted cytotoxic activity comparable to FMC63 CAR P1209 (Figure 7A). The bispecific CARs exhibited CD22-dependent cytotoxicity comparable to monospecific tandem CARs P1631, P1633, P1702, and P1734 (Figure 7B). This study demonstrated the successful engineering of several bispecific CD19-CD22 CAR-T cells.

[0379] It will be appreciated by those skilled in the art that modifications may be made without departing from the broad inventive concept of the above-described embodiments. It is therefore understood that the invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the invention as defined herein.

Claims

1. An induced pluripotent stem cell (iPSC) or a CD34+ hematopoietic progenitor cell (HPC) derived therefrom, comprising an exogenous polynucleotide encoding a bispecific chimeric antigen receptor (CAR) comprising a first antigen-binding domain targeting the CD22 antigen and a second antigen-binding domain targeting the CD19 antigen.

2. a) the CAR comprises an anti-CD22 VHH domain; and / or b) the CAR comprises an anti-CD19 VHH domain; or c) the CAR comprises a single-chain variable fragment (scFv) derived from an antibody that specifically binds to the CD19 antigen; The iPSC or CD34+ HPC of claim 1.

3. The iPSC or CD34+ HPC described in claim 1, wherein the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs), optionally T cells.

4. The CAR, a) a signal peptide; b) an extracellular domain comprising said first and second antigen-binding domains; c) hinge region; d) a transmembrane domain; e) an intracellular signaling domain; and f) comprises a costimulatory domain; The iPSC or CD34+ HPC of claim 1.

5. An iPSC or CD34+ HPC as described in claim 4, wherein the extracellular domain comprises a VHH single domain antibody that specifically binds to the CD22 antigen.

6. The extracellular domain (i) 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 one of SEQ ID NOs: 96-98, 152, and 155; and / or (ii) Encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the base sequence of one of SEQ ID NOs: 99-101, 153, and 156; The iPSC or CD34+ HPC of claim 4.

7. An iPSC or CD34+ HPC as described in claim 4, wherein the extracellular domain comprises an scFv derived from an antibody that specifically binds to the CD19 antigen.

8. The extracellular domain (i) 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 one or more of SEQ ID NOs: 2, 4, and 7; and / or (ii) encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more base sequences of SEQ ID NOs: 145 and 147; The iPSC or CD34+ HPC of claim 4.

9. An iPSC or CD34+ HPC as described in claim 1, wherein the bispecific CAR comprises a CD22 / CD19 loop.

10. The bispecific CAR, (i) comprises one or more amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 96-98, 104-111, 120-131, 152, 155, 157, 159, 161, 163, 165-167, 171, and 173-175; and / or (ii) encoded by one or more polynucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 62, 99-101, 112-119, 132-143, 153, 156, 158, 160, 162, 164, 168-170, 172, and 176-178; The iPSC or CD34+ HPC of claim 1.

11. A derivative cell of the iPSC or CD34+ HPC of claim 1, the derivative cell a) Natural Killer (NK) cells; b) T cells; c) gamma delta T cells; or d) Gamma delta Vγ9 / Vδ1 T cells That is, The derivative cells.

12. A composition comprising the iPSCs or CD34+ HPCs described in claim 1.

13. Use of a derivative cell according to claim 11, or a composition comprising a derivative cell according to claim 11, in the manufacture of a medicament for treating cancer, optionally wherein the cancer is a B-cell malignancy, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma, follicular lymphoma.

14. A method for producing a derivative cell as described in claim 11, the method comprising differentiating an iPSC or CD34+ HPC as described in any one of claims 1 to 10 under conditions for cell differentiation, thereby obtaining the derivative cell.

15. A bispecific chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain comprising a first antigen-binding domain, wherein the first antigen-binding domain targets a CD22 antigen and a second antigen-binding domain targets a CD19 antigen, and wherein the polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-131, 152, 155, 157, 159, 161, 163, 165-167, 171, and 173-175.

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