Genetically engineered cells bearing anti-nectin-4 chimeric antigen receptors and uses thereof

Abstract

Genetically engineered induced pluripotent stem cells (iPSCs) and their derivatives that express anti-Nectin-4 chimeric antigen receptors (CARs) and, optionally, inhibitory CARs, as well as methods for their use, are provided. Compositions, polypeptides, vectors, and methods of production are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,989, filed November 10, 2022, which is incorporated herein by reference in its entirety.

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

[0003] Reference to an electronically submitted sequence listing This application contains a Sequence Listing that has been submitted electronically via EFS-Web as a Sequence Listing in XML format with the file name "SequenceListing_ST26" and a creation date of November 9, 2023, having a size of 536 kilobytes. The Sequence Listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Chimeric antigen receptors (CARs) have shown remarkable activity in cancer therapy by enhancing the antitumor activity of immune effector cells. CAR-T cells are engineered to target antigens expressed on cancer cells. However, some of these antigens may also be expressed at low levels in normal, healthy tissues. Therefore, CAR-T cell attack of these healthy tissues can lead to on-target and off-tumor toxicity. The most well-known example is CD19-targeting CAR-T cells for treating B-cell malignancies. Because CD19 is also expressed on normal B cells, patients may experience adverse B-cell effects (e.g., aplasia or hypogammaglobulinemia) from treatment. Other tumor antigens, such as ERBB2 (HER2) and EGFR, are expressed to some extent on epithelial cells in the lung, liver, and skin, leading to toxicity in these tissues. Careful antigen selection and engineering of the CAR construct are required to maximize specificity.

[0005] To address these challenges, embodiments of the present disclosure are designed to increase the depth and durability of responses by targeting Nectin-4, a tumor-associated marker for many tumors, including lung, breast, colon, bladder, kidney, head and neck, esophageal, and ovarian cancers. The present invention also provides cells that have been engineered to express an additional antigen-binding molecule that targets a healthy cell antigen (e.g., DSG1) to reduce off-target binding / improve tumor-targeting specificity, either by engineering the cells to express an additional inhibitory CAR in combination with an anti-Nectin-4 CAR or by engineering the cells to express a dual-targeting CAR that targets Nectin-4 and a healthy cell antigen. Summary of the Invention

[0006] In one general aspect, the disclosure provides a method for producing a chimeric antigen receptor (CAR) comprising one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that targets the Nectin-4 antigen; and (i) a deletion or reduced expression of one or more of the following genes: B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, RFXAP; (ii) an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G); (iii) a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII), cluster of differentiation (CCR), or NK cell receptor (NKCR). The present invention provides an induced pluripotent stem cell (iPSC) or a derived cell thereof, comprising at least one of the following: (i) an exogenous polynucleotide encoding CD16, and / or NKG2D protein; (iv) a deletion or reduced expression of one or more of the NKG2A or CD70, CD38, and CD33 genes; (v) an exogenous polynucleotide encoding a cytokine; (vi) an exogenous polynucleotide encoding a safety switch; (vii) an exogenous polynucleotide encoding a PSMA cell tracer; and (viii) an exogenous polynucleotide encoding a membrane-bound IL-12 polypeptide. In certain embodiments, the CAR may be a dual-targeting CAR comprising an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the cell may comprise one or more exogenous polynucleotides encoding an additional CAR comprising an antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the CAR may comprise an anti-Nectin-4 VHH domain. In certain embodiments, the cytokine may comprise IL-15. In certain embodiments, the iPSCs or derived cells thereof may further comprise an inactivated cell surface receptor that may comprise a monoclonal antibody-specific epitope, and the inactivated cell surface receptor and IL-15 may be operably linked by an autoprotease peptide.In certain embodiments, IL-15 may comprise an IL-15 and IL-15 receptor alpha (IL-15Rα) fusion polypeptide. In certain embodiments, IL-15 may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72. In certain embodiments, iPSCs or derived cells thereof may comprise a deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes. In certain embodiments, iPSCs or derived cells thereof may comprise an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G). In certain embodiments, CD16 may be a CD16 variant protein. In certain embodiments, the CD16 variant protein may be a high-affinity CD16 variant. In certain embodiments, the CD16 variant protein may be a non-cleavable CD16 variant. In certain embodiments, the CD16 variant protein may comprise wild-type CD16 having one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, and T220A. In certain embodiments, the CD16 variant protein may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 187 and 188. In certain embodiments, iPSCs or their derived cells may comprise exogenous polynucleotides encoding CD16 and NKG2D proteins, and the CD16 and NKG2D proteins may be operably linked by an autoprotease peptide. In certain embodiments, the NKG2D protein may be wild-type NKG2D protein. In certain embodiments, the NKG2D protein may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:190.In certain embodiments, the autoprotease peptide may be 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 certain embodiments, the autoprotease peptide may be a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 192. In certain embodiments, the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein may comprise a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 193. In certain embodiments, one or more of the exogenous polynucleotides may be integrated into one or more loci on a chromosome of a cell selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD33, CD38, CD70, TRAC, 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 B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby resulting in deletion or reduced expression of the gene. In certain embodiments, one or more of the exogenous polynucleotides can be integrated into the locus of AAVS1 and B2M gene.In certain embodiments, iPSC or its derived cell can comprise one or more deletions or reduced expression of B2M or CIITA gene.In certain embodiments, iPSC or its derived cell can comprise one or more deletions or reduced expression of B2M and CIITA gene.In certain embodiments, iPSCs can be reprogrammed from whole peripheral blood mononuclear cells (PBMCs). In certain embodiments, iPSCs can be derived from reprogrammed T cells. In certain embodiments, a CAR can comprise (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to the Nectin-4 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain. In certain embodiments, the extracellular domain can comprise a VHH single-domain antibody that specifically binds to the Nectin-4 antigen. In certain embodiments, the extracellular domain can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 105-130. In certain embodiments, the extracellular domain can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 131-156. In certain embodiments, the CAR can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 171-184. In certain embodiments, the additional CAR can comprise: (i) a signal peptide; (ii) an additional extracellular domain comprising a binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain. In certain embodiments, the additional extracellular domain may comprise a VHH or scFv that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the signal peptide may comprise a GMCSFR signal peptide or a MARS signal peptide.In certain embodiments, the hinge region for each of the CAR and the additional CAR can be independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region. In certain embodiments, the transmembrane domain for each of the CAR and the additional CAR can be independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain. In certain embodiments, the intracellular signaling domain can include a CD3ζ intracellular domain. In certain embodiments, the costimulatory domain for each of the CAR and the additional CAR can be independently selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain.In certain embodiments, in a CAR, (i) the signal peptide can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 97, or 98; and (ii) the extracellular domain can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 105-130. or the extracellular domain may be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131-156; (iii) the hinge region may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 21 or 96. (iv) the transmembrane domain may comprise amino acids 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) the intracellular signaling domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6. and (vi) the costimulatory domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or 17. In certain embodiments, in a CAR, (i) the signal peptide may comprise amino acids having a sequence of SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain may comprise amino acids having a sequence of one of SEQ ID NOs: 105-130; (iii) the hinge region may comprise amino acids having a sequence of SEQ ID NO: 21 or 96; (v) the transmembrane domain may comprise amino acids having the sequence of SEQ ID NO: 23 or 24; (v) the intracellular signaling domain may comprise amino acids having the sequence of SEQ ID NO: 6, or the intracellular signaling domain may be encoded by a polynucleotide having the sequence of SEQ ID NO: 101; and (vi) the costimulatory domain may comprise amino acids having the sequence of SEQ ID NO: 8 or 17. In certain embodiments, the iPSC or derivative cell may comprise an exogenous polynucleotide encoding a safety switch. In certain embodiments, the safety switch may comprise an exogenous polynucleotide encoding an inactivated cell surface receptor that may comprise a monoclonal antibody-specific epitope. In certain embodiments, the inactivated cell surface receptor is selected from the group consisting of ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. The monoclonal antibody-specific epitope may be selected from the group consisting of epitopes specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab. In certain embodiments, the inactivated cell surface receptor may be a truncated epidermal growth factor receptor (tEGFR) variant. In certain embodiments, the tEGFR variant consists of amino acids with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 71. In certain embodiments, the safety switch may comprise an intracellular domain containing herpes simplex virus thymidine kinase (HSV-TK). In certain embodiments, the iPSC or derived cells may comprise an exogenous polynucleotide encoding a PSMA cell tracer, and the PSMA cell tracer may comprise an extracellular domain comprising the PSMA extracellular domain or a fragment thereof.In certain embodiments, iPSCs, or their derivative cells, may comprise a combined artificial cell death / reporter system polypeptide comprising an intracellular domain comprising herpes simplex virus thymidine kinase (HSV-TK) and a linker, a transmembrane region, and an extracellular domain comprising the PSMA extracellular domain or a fragment thereof. In certain embodiments, the HSV-TK may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 229 or 230. In certain embodiments, the combined artificial cell death / reporter system polypeptide may comprise HSV-TK fused to a truncated variant PSMA polypeptide via a linker. In certain embodiments, the truncated variant PSMA polypeptide may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 231. In certain embodiments, the linker may comprise 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 certain embodiments, the artificial cell death / reporter system polypeptide may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 232. In certain embodiments, the artificial cell death / reporter system polypeptide can comprise amino acids 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: 233-235. In certain embodiments, the artificial cell death / reporter system polypeptide can be encoded by a polynucleotide 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: 236-238.In certain embodiments, HLA-E may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, and / or HLA-G may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69.In certain embodiments, (i) one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting the Nectin-4 antigen may comprise nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more selected from the group consisting of SEQ ID NOs: 171-184; (ii) 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 may comprise nucleotides 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; (iv) 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 may comprise nucleotides 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: 185, 189, and 19 (iv) the exogenous polynucleotide encoding the cytokine may comprise nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 239; (v) the exogenous polynucleotide encoding the safety switch may comprise nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 239; and / or (vi) the exogenous polynucleotide encodes a PSMA cell tracer, and the PSMA cell tracer may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO:236-238.In certain embodiments, (i) one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting the Nectin-4 antigen may comprise nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 171-184; (ii) an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) may comprise nucleotides having the sequence of SEQ ID NO: 67 or 70; (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 may comprise nucleotides having the sequence of SEQ ID NO: 185, 189, or 191; (iv) an exogenous polynucleotide encoding a cytokine may comprise nucleotides having the sequence of SEQ ID NO: 239; and / or (v) an exogenous polynucleotide encoding a safety switch may comprise nucleotides having the sequence of one of SEQ ID NOs: 236-238. In certain embodiments, the exogenous polynucleotide may be 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 certain embodiments, (i) one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting the Nectin-4 antigen may be integrated into 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) may be integrated into the locus of the B2M gene; in certain embodiments, (iii) an exogenous polynucleotide encoding the NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or NKG2D may be integrated into the locus of the CD70 gene; (iv) an exogenous polynucleotide encoding a cytokine may be integrated into the locus of the NKG2A gene; (v) an exogenous polynucleotide encoding a safety switch may be integrated into the locus of the CLYBL gene; and (vi) there may be a deletion or reduced expression of the CIITA gene. In certain embodiments, the one or more exogenous polynucleotides further encode one or more inhibitory CARs (iCARs) comprising at least one antigen-binding domain that targets an antigen independently selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC)1, DSC3, desmoglein (DSG)1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), and V-set and immunoglobulin domain containing 8 (VSIG8).In certain embodiments, an iCAR may comprise: (i) a signal peptide; (ii) an extracellular domain comprising an antigen-binding domain that specifically binds to at least one antigen selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC)1, DSC3, desmoglein (DSG)1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8); (iii) a hinge region; (iv) one or more transmembrane domains; (v) an intracellular signaling domain; and / or (vi) a costimulatory domain. In certain embodiments, the extracellular domain of iCAR can comprise amino acids 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: 354-363. In certain embodiments, the extracellular domain of iCAR can comprise amino acids 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: 364-373. In certain embodiments, the signal peptide of iCAR may comprise a CD8 signal peptide, a GMCSFR signal peptide, a MARS signal peptide, or an IgK signal peptide, or a variant thereof. In certain embodiments, the signal peptide of iCAR may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 97 and 292. In certain embodiments, the signal peptide of iCAR may be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 98, 327, and 378. In certain embodiments, the hinge region of the iCAR may be selected from the group consisting of a CD28 hinge region, a CD45 hinge region, a G4S-CD45 hinge region, a CD8 hinge region, and a CXC3R GPCR hinge region. In certain embodiments, the hinge region of the iCAR may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 21, 22, 288, 289, 319, and 321. In certain embodiments, the hinge region of an iCAR can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 315-318, 320, and 322. In certain embodiments, one or more transmembrane domains of an iCAR can be independently selected from the group consisting of a CD28 transmembrane domain, a CD8 transmembrane domain, a PD1 transmembrane domain, a SynNotch transmembrane domain, and a CXC3R GPCR. In certain embodiments, a transmembrane domain of an iCAR can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 23, 24, 290, 291, 323, and 325. In certain embodiments, the transmembrane domain of iCAR isIt may be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 324, 326, and 374-377. In certain embodiments, the intracellular signaling domain of an iCAR may comprise one or more of a PD1 intracellular domain, a LIRB1 intracellular domain, a TIGIT intracellular domain, a CTLA4 intracellular domain, a CSK*(YSSV) intracellular domain, a KIR2DL1 intracellular domain, a DR1 intracellular domain, a Casp8wt intracellular domain, a tCasp8 intracellular domain, a tCasp8 dimer intracellular domain, a tBid15 intracellular domain, a Casp9wt intracellular domain, a tCasp9 intracellular domain, a tCasp9 dimer intracellular domain, a SHP1 intracellular domain, a (G4S)2-SHP1 intracellular domain, a CSK intracellular domain, a (G4S)2-CSK intracellular domain, an ADAM17 cleavage site, a CD28 intracellular domain, a CD3ζ intracellular domain, a G4S3 linker, an ADAM17 protease domain, and a (G4S)3-ADAM17 protease domain. In certain embodiments, the intracellular signaling domain of iCAR may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, and 267-287. In certain embodiments, the intracellular signaling domain of iCAR may be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 266, and 293-314. In certain embodiments, the costimulatory domain of iCAR may be selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain. In certain embodiments, in an iCAR, (i) the signal peptide can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 97 or 292, or the signal peptide can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 98, 327,or 378; (ii) the extracellular domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 354-363, or the extracellular domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 364-373. (iii) the hinge region may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21, 22, 288, 289, 319, or 321, or the hinge region may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 315-318, 320, or 322. (iv) each of the one or more transmembrane domains comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence independently selected from the group consisting of SEQ ID NOs: 23, 24, 290, 291, 323, and 325, or each of the one or more transmembrane domains comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence independently selected from the group consisting of SEQ ID NOs: 324, 326, and 374-377. (v) the intracellular signaling domain may comprise amino acids with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 6, 8, and 267-287, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 266, and 293-314.In certain embodiments, in an iCAR, (i) the signal peptide can comprise amino acids having the sequence of SEQ ID NO: 97 or 292, or the signal peptide can be encoded by the polynucleotide sequence of SEQ ID NO: 98, 327, or 378; (ii) the extracellular domain can comprise amino acids having the sequence of SEQ ID NO: 354-363, or the extracellular domain can be encoded by the polynucleotide sequence of SEQ ID NO: 364-373; (iii) the hinge region can comprise amino acids having the sequence of SEQ ID NO: 21, 22, 288, 289, 319, or 321, or the hinge region can be encoded by the polynucleotide sequence of SEQ ID NO: 315-318, 320, or 322. (iv) each of the one or more transmembrane domains may comprise amino acids having a sequence independently selected from the group consisting of SEQ ID NOs: 23, 24, 290, 291, 323, and 325, or each of the one or more transmembrane domains may be encoded by a polynucleotide having a sequence independently selected from the group consisting of SEQ ID NOs: 324, 326, and 374-377; and (v) the intracellular signaling domain may comprise amino acids having a sequence independently selected from one or more of SEQ ID NOs: 6, 8, and 267-287, or the intracellular signaling domain may be encoded by a polynucleotide of one of SEQ ID NOs: 266, and 293-314. In certain embodiments, the derivative cell may be a natural killer (NK) cell or a T cell. In certain embodiments, the derivative cell may be a T cell. In certain embodiments, the T cell may be a gamma delta T cell. In certain embodiments, the T cell may be a gamma delta Vγ9 / Vδ1 T cell.

[0007] In some aspects, the present disclosure provides compositions comprising the derivative cells of the present disclosure. In some embodiments, the compositions may further comprise, or be used in combination with, one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (IMiD).

[0008] In some aspects, the present disclosure provides CD34+ hematopoietic progenitor cells (HPCs) derived from the induced pluripotent stem cells (iPSCs) of the present disclosure. In certain embodiments, the CAR may be a dual-targeting CAR comprising an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the one or more exogenous polynucleotides encode an additional CAR comprising an antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the CD34+ HPCs may further comprise an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G). In certain embodiments, one or more of the exogenous polynucleotides may be integrated into one or more loci on a chromosome of the cell independently selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCRa or b constant region, NKG2A, NKG2D, CD33, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, with the proviso that at least one of the exogenous polynucleotides may be integrated into the locus of a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby resulting in deletion or reduced expression of the gene. In certain embodiments, one or more of the exogenous polynucleotides may be integrated into the locus of the AAVS1 and B2M genes. In certain embodiments, the CD34+ HPCs may have a deletion or reduced expression of one or more of the B2M or CIITA genes.In certain embodiments, the CAR may comprise (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to the Nectin-4 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain. In certain embodiments, the extracellular domain may comprise a VHH single-domain antibody that specifically binds to the Nectin-4 antigen. In certain embodiments, the extracellular domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 105-130. In certain embodiments, the CD34+ HPCs may comprise an additional CAR comprising: (i) a signal peptide; (ii) an additional extracellular domain comprising a binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain, for example, a costimulatory domain comprising a CD28 signaling domain. In certain embodiments, the additional extracellular domain may comprise a VHH that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the CD34+ HPCs may comprise an additional exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, and the CD16 protein and the NKG2D protein may be operably linked by an autoprotease peptide. In certain embodiments, the CD16 protein may be a CD16 variant protein. In certain embodiments, the CD16 variant may be a high-affinity CD16 variant. In certain embodiments, the CD16 variant may be a non-cleavable CD16 variant. In certain embodiments, the CD16 variant may comprise one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.

[0009] In some aspects, the present disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain comprising an antigen-binding domain that specifically binds to Nectin-4. In certain embodiments, the CAR may be a dual-targeting CAR, and the extracellular domain may comprise an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In certain embodiments, the CAR may comprise (i) a signal peptide; (ii) an extracellular domain comprising an antigen-binding domain that specifically binds to the Nectin-4 antigen; (iii) a hinge region; (iv) one or more transmembrane domains; (v) an intracellular signaling domain; and / or (vi) a costimulatory domain. In certain embodiments, the extracellular domain may comprise a VHH single-domain antibody that specifically binds to the Nectin-4 antigen. In certain embodiments, the extracellular domain can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 105-130. In certain embodiments, the extracellular domain can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 131-156. In certain embodiments, the CAR can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 171-184. In certain embodiments, the signal peptide may comprise a GMCSFR signal peptide or a MARS signal peptide. In certain embodiments, the hinge region for each of the CAR and the additional CAR may be independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region. In certain embodiments, the transmembrane domain for each of the CAR and the additional CAR may be independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain.In certain embodiments, the intracellular signaling domain may comprise a CD3ζ intracellular domain. In certain embodiments, the costimulatory domains for each of the CAR and additional CAR may be independently selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain. In certain embodiments, in the CAR, (i) the signal peptide may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 97, or 98; (ii) the extracellular domain may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 105-130. or can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131-156; (iii) the hinge region can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 21 or 96. (iv) the transmembrane domain may comprise amino acids 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) the intracellular signaling domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6. and (vi) the costimulatory domain may comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or 17.In certain embodiments, in a CAR, (i) the signal peptide may comprise amino acids having the sequence of SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain may comprise amino acids having the sequence of one of SEQ ID NOs: 105-130; (iii) the hinge region may comprise amino acids having the sequence of SEQ ID NO: 21 or 96; (iv) the transmembrane domain may comprise amino acids having the sequence of SEQ ID NO: 23 or 24; (vi) the intracellular signaling domain may comprise amino acids having the sequence of SEQ ID NO: 6, or the intracellular signaling domain may be encoded by the polynucleotide of SEQ ID NO: 101; and (vii) the costimulatory domain may comprise amino acids having the sequence of SEQ ID NO: 8 or 17.

[0010] In some aspects, the present disclosure provides inhibitory chimeric antigen receptor (iCAR) polypeptides comprising an extracellular domain that includes an antigen-binding domain that specifically binds to at least one antigen selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC)1, DSC3, desmoglein (DSG)1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8). In certain embodiments, an iCAR may comprise: (i) a signal peptide; (ii) an extracellular domain comprising an antigen-binding domain that specifically binds to at least one antigen selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC)1, DSC3, desmoglein (DSG)1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8); (iii) a hinge region; (iv) one or more transmembrane domains; (v) an intracellular signaling domain; and / or (vi) a costimulatory domain. In certain embodiments, the antigen-binding domain specifically binds to at least one antigen selected from DSC1, DSC3, DSG1, and DSG3. In certain embodiments, the antigen-binding domain specifically binds to DSG1. In certain embodiments, the antigen-binding domain specifically binds to at least one antigen selected from (i) DSG1 and (ii) DSC1, DSC3, and DSG3. In certain embodiments, the extracellular domain of an iCAR may comprise amino acids 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: 354-363.In certain embodiments, the extracellular domain of iCAR can be encoded by a polynucleotide 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: 364-373. In certain embodiments, the signal peptide of iCAR can comprise a CD8 signal peptide, a GMCSFR signal peptide, a MARS signal peptide, or an IgK signal peptide, or a variant thereof. In certain embodiments, the signal peptide of iCAR can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 97 and 292. In certain embodiments, the signal peptide of iCAR can be encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 98, 327, and 378.In certain embodiments, in an iCAR, (i) the signal peptide can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 97 or 292; (ii) the extracellular domain can comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 354-363, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 364-373. and (iii) the iCAR may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 242-265, and 352, or the iCAR may comprise a sequence of amino acids encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 328-351, and 353. In certain embodiments, in an iCAR, (i) the signal peptide may comprise amino acids having the sequence of SEQ ID NO: 97, or 292; (ii) the extracellular domain may comprise amino acids having the sequence of one of SEQ ID NOs: 354-363; and / or (iii) the iCAR may comprise amino acids having the sequence of one of SEQ ID NOs: 242-265, and 352.

[0011] In some aspects, the present disclosure provides an induced pluripotent stem cell (iPSC) or a derivative thereof of the present disclosure, which may further comprise an iCAR of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising the derivative of the present disclosure.

[0012] In some aspects, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering a derivative cell of the present disclosure or a composition of the present disclosure to the subject in need thereof. In certain embodiments, the cancer may be selected from the group consisting of leukemias (e.g., AML, CML, ALL, and CLL), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma), and solid cancers (e.g., 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 (CUP). In certain embodiments, the cancer may be selected from the group consisting of bladder cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and esophageal cancer. In certain embodiments, the subject has minimal residual disease (MRD) after an initial cancer treatment. In certain embodiments, the subject does not have minimal residual disease (MRD) after one or more cancer treatments or repeated medications. In certain embodiments, the method involves administering to the subject one or more of the following: 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 method may further comprise administering a therapeutic agent selected from the group consisting of vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab. In certain embodiments, the method may further comprise administering a therapeutic agent to the subject, and the therapeutic agent may be avelumab. In certain embodiments, the cells and the therapeutic agent can be administered simultaneously. In certain embodiments, the cells and the therapeutic agent can be administered sequentially.

[0013] In some aspects, the present disclosure provides a method for producing a derivative cell of the present disclosure, comprising differentiating the iPSC of the present disclosure under conditions for cell differentiation, thereby obtaining a derivative cell.In certain embodiments, the iPSC can be obtained by genetically manipulating unmodified iPSC, and the genetic manipulation can include targeted editing of the genome of the iPSC.In certain embodiments, the targeted editing can include deletion, insertion, or in / del, performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0014] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Brief explanation of the drawings]

[0015] [Figure 1-1] 1A-1C: (A) Diagram of an exemplary cell of the present disclosure expressing an anti-Nectin-4 CAR on the cell surface. (B) Diagram of an exemplary cell of the present disclosure targeting tumor cells. (i) When an anti-Nectin-4 CAR is used to bind to the Nectin-4 antigen on the tumor cell surface, and (ii) when surface-expressed CD16 is used to bind to an antibody that modulates the tumor microenvironment, such as a tumor antigen antibody (e.g., cetuximab, trastuzumab, avelumab, and / or others) or an antibody against a checkpoint inhibitor, including PD-L1 and CTLA4 (avelumab, ipilimumab, and / or others). (C) Table of exemplary gene edits performed on cells of the present disclosure and the associated rationale for performing the gene edits. [Figure 1-2] (As mentioned above.) [Figure 1-3] (As mentioned above.) [Figure 2]2A-2C: (A) Full-length nectin4, (B) IgC1 / 2 nectin4 domains, and (C) IgC2 nectin4 domains used for VHH and scFv binding molecule discovery. [Figure 3-1] 3A-3C: (A) (Left) human IgG, the source of certain scFv binding molecules of the present disclosure, and (Right) llama IgG, the source of certain VHH binding molecules of the present disclosure; (B) schematic of VHH library construction, in which a V2.0 phage library containing approximately 2 x 10 unique sequences was constructed; and (C) phage panning against Nectin-4 protein. Three rounds of phage panning were performed against the VHH phage library using plate-bound Nectin-4-HIS protein (AcroBiosystems #NE4-H52H3), and individual colonies were screened by ELISA using periplasmic extracts (PPE). [Figure 3-2] (As mentioned above.) [Figure 3-3] (As mentioned above.) [Figure 4]

[0023] Figure 1 is a flowchart detailing the VHH CAR selection process. VHH binding molecules were selected using biophysical analysis, cell binding (fluorescence-activated cell sorting; FACS) assays, Nurkat tonic / activation, in vitro cytotoxicity assays, and / or Retrogenix / in vivo screening. [Figure 5-1]Figures 5A-5H show nectin4 cell surface expression in (A) HeLa cells, (B) K562 cells, (C) CHO-K1 cells, (D) HEPG2 cells, (E) T-47D cells, (F) OVCAR3 cells, (G) OE19 cells, (H) A431 cells, and (G) CHO-Nectin4 cells. Flow cytometry was used to detect nectin4 protein expression on the cell surface of a panel of normal and tumor cell lines using a PE anti-nectin4 detection antibody (R&D Systems, catalog number FAB2659P, clone: ​​337516, msIgG2b; 1 μg / ml). Panel (H) is a summary table of the target cell lines, their description, the percentage of nectin4-positive cells, and the nectin4 mean fluorescence intensity (MFI ratio). [Figure 5-2] (As mentioned above.) [Figure 5-3] (As mentioned above.) [Figure 5-4] (As mentioned above.) [Figure 5-5] (As mentioned above.) [Figure 6] Figure 1 shows the results of screening 14 anti-nectin-4 VHH-Fc for binding to nectin-4-positive cell lines. All 14 VHH-Fc showed specific binding to CHO-nectin-4 cells, and 12 of the 14 cell lines showed specific binding to the nectin-4-positive tumor cell line T47D. [Figure 7] Schematic of the Nurkat activation assay. The Nur77-sfGFP-PEST KI Jurkat reporter line (Nurkat cells) was engineered by lentiviral transfection to drive GFP expression from the Nur77 promoter with a panel of VHH CARs directed against nectin-4. Nurkat cells were co-cultured with target cells lacking nectin-4 or with varying nectin-4 density on the cell surface. Flow cytometry was used to quantitate the GFP signal resulting from Nurkat cell activation by CARs. [Figure 8-1]8A-8D: (A) Schematic of an exemplary anti-Nectin-4 VHH CAR of the present disclosure; and (B) results of a tonic signaling assay as a function of CAR, showing that higher levels of CAR on the cell surface can lead to artificially higher tonic signaling; (C) results of a Jurkat_Nur77 reporter assay for activation by Nectin-4 negative or positive cell lines, and (D) results of a Jurkat_Nur77 reporter assay for tonic signaling by Nectin-4 negative or positive cell lines. [Figure 8-2] (As mentioned above.) [Figure 9] Figure 1 shows the results of VHH-CAR T cell-mediated target-specific killing of Nectin-4 positive K562, HeLa, T47D, OVCAR3, OE19, A431 cell lines in vitro. [Figure 10] FIG. 1 shows the results of T cell activation by 1:1 co-culture of cells of the present disclosure expressing an anti-Nectin-4 CAR with various target cells, as measured by the percentage of cells that are CD25 positive (top row) and IL-2 expression (bottom row). [Figure 11-1] Figures 11A-11B: (A) A table showing the binding affinity of various anti-nectin-4 binding molecules of the present disclosure to human and mouse nectin-4 and target epitopes, and (B) a diagram showing the binding of various anti-nectin-4 binding molecules to nectin-4. [Figure 11-2] (As mentioned above.) [Figure 12-1]Figures 12A-12B: (A) Results of a Nectin-4 binding specificity study measuring VHH_Fc protein binding to A431, A549, Capan-2, HEPG2, Jurkat, MOLM-13, NALM-6, OE19, OVCAR3, and U-2 OS cell lines. To aid in lead VHH characterization and selection, a cell-based specificity FACS screen was established. VHH-Fc cell-binding dose-response curves (DRCs) were generated for a diverse panel of human cell lines derived from various tissue / organ types. VHH-Fc showing nonspecific binding to target-negative lines can be flagged as potential off-target binding, while VHH-Fc showing minimal nonspecific binding to target-negative lines can be prioritized as lead candidates. (B) Table of target cell lines used in the binding specificity screen. [Figure 12-2] (As mentioned above.) [Figure 13-1] Figures 13A-13B: (A) Nectin-4 antigen density was assessed in various solid tumor and control cell lines, as well as primary human keratinocytes (PHKs), using Quantibrite PE (Beckton Dickinson). Quantibrite beads were coated with four calculated PE levels (low, medium-low, medium-high, and high). These calculated PE / bead values ​​and fluorescence intensity in flow cytometry were used to generate standard curves to estimate antigen density on various target cell lines. (B) Anti-nectin-4 phycoerythrin (PE) / cell (normalized to isotype) for various target cell lines, which were separated into groups of cells that were nectin-4 negative or had low, medium, or high levels of nectin-4. [Figure 13-2] (As mentioned above.) [Figure 14-1]Figures 14A-14H: Cytotoxicity of Nectin4 CAR across various effector-to-target cell ratios for (A) TUCCSUP cells, (B) HELA cells, (C) HT1197 cells, (D) T24 cells, (E) HT1376 cells, (F) OVCAR cells, (G) OE19 cells, and (H) T47D cells. The T24 tumor cell line expresses similar levels of Nectin4 compared to primary human keratinocytes. This line has been used as a surrogate for Nectin4 expression in human keratinocytes to aid in the selection of binding molecules that kill tumors without significant skin toxicity. [Figure 14-2] (As mentioned above.) [Figure 15-1] Figures 15A-15D: (A) Cumulative cytotoxicity as a percentage of target cells killed, (B) cumulative interferon gamma (IFN) secretion, and (C) cumulative IL2 secretion of effector cells expressing various anti-Nectin-4 VHH CARs. [Figure 15-2] (As mentioned above.) [Figure 15-3] (As mentioned above.) [Figure 16-1] Figures 16A-16C: (A) Cumulative cytotoxicity as a percentage of target cells killed, (B) cumulative interferon gamma (IFN) secretion, and (C) cumulative IL2 secretion of effector cells expressing various anti-Nectin-4 VHH CARs with either 41BB or CD28 costimulatory domains. [Figure 16-2] (As mentioned above.) [Figure 16-3] (As mentioned above.) [Figure 17-1] Figures 17A-17C: Efficacy screening results of primary T cells expressing anti-Nectin-4 VHH CAR in the OVCAR-3 xenograft tumor model, including (A) tumor burden, (B) mouse weight change, and (C) percentage of CAR-positive cells. [Figure 17-2] (As mentioned above.) [Figure 17-3] (As mentioned above.) [Figure 18-1]Figures 18A-18B: (A) All single cell types with NECTIN4 gene expression >50 tpm (transcripts per million) across 30 tissues, and (B) all single cell types in bronchial or lung tissue with NECTIN4 gene expression >10 tpm. Data taken from the publicly available Human Protein Atlas single-cell RNA-sequencing atlas of normal tissues. [Figure 18-2] (As mentioned above.) [Figure 19-1] Figures 19A-19B: Gene expression in (A) suprabasal keratinocyte cells (B) basal keratinocyte cells of normal skin tissue compared with median tumor gene expression across patients with bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, or pancreatic cancer indications. Median patient gene expression is calculated from bulk RNA-sequencing measurements of human tumors by The Cancer Genome Atlas Program. Suprabasal and basal keratinocyte cell gene expression is calculated from single-cell RNA-sequencing atlases of normal tissues from the Human Protein Atlas. Only surfaceome genes, which encode plasma membrane proteins that are at least partially exposed to the extracellular space, are shown. DSG1 is highlighted in blue to emphasize its high gene expression in normal suprabasal and basal skin keratinocytes and its low gene expression across most of the indicated cancer indications. [Figure 19-2] (As mentioned above.) [Figure 19-3] (As mentioned above.) [Figure 19-4] (As mentioned above.) [Figure 19-5] (As mentioned above.) [Figure 19-6] (As mentioned above.) [Figure 19-7] (As mentioned above.) [Figure 19-8] (As mentioned above.) [Figure 20-1]Figures 20A-20B: (A) Geometric means of patient gene expression for bladder cancer, breast cancer, esophageal cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, or pancreatic cancer indications compared to the geometric means of suprabasal and basal keratinocyte gene expression in the skin. Gene expression is expressed as TPM. Only surfacesome genes that encode plasma membrane proteins that are at least partially exposed to the extracellular space are shown. (B) Candidate gene targets for inhibitory CARs that prevent lysis of skin keratinocytes. The tumor TPM column shows median patient gene expression calculated from bulk RNA-sequencing measurements of human tumors by The Cancer Genome Atlas Program. Patients with bladder cancer, breast cancer, esophageal cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, or pancreatic cancer indications are included. The Keratinocyte TPM column shows the average gene expression in skin suprabasal and basal keratinocyte cells according to the Human Protein Atlas single-cell RNA sequencing atlas of normal tissues. The Fold Difference column shows the value obtained by dividing the "Keratinocyte TPM" column by the "Tumor TPM" column. Genes are sorted in descending order of fold difference, with all surfacesome genes having a difference greater than 50-fold. The Expected Cell Type column annotates the single cell type of the 30 tissues with the highest expression of this gene, as measured by the Human Protein Atlas single-cell RNA sequencing dataset. All displayed genes are part of the surfacesome genes, which encode plasma membrane proteins that are at least partially exposed to the extracellular space. However, some genes may also be localized to other membranes of the cell. The Expected Subcellular Localization column annotates information about the predicted subcellular localization of each gene-encoded protein according to the Human Protein Atlas. The protein data column provides annotations from additional data sources regarding protein expression and surface presentation in skin keratinocyte cells. The "Notes" column provides an analysis of the relative detection levels of gene-encoded proteins across skin keratinocytes, in different layers of the skin, and in different cell types of the body.Shaded rows indicate genes that are most likely to be presented as proteins on the surface of skin keratinocytes according to this analysis. [Figure 20-2] (As mentioned above.) [Figure 20-3] (As mentioned above.) [Figure 20-4] (As mentioned above.) [Figure 20-5] (As mentioned above.) [Figure 20-6] (As mentioned above.) [Figure 20-7] (As mentioned above.) [Figure 21] Figure 1 shows tumor gene expression distribution across patients in The Cancer Genome Atlas for ADRB2, DSC1, DSC3, DSG1, DSG3, GDPD2, HCAR3, LY6D, NECTIN4, and VSIG8 genes. Results are shown separately for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, and pancreatic cancer indications. The number of patients included in each indication is listed below the indication (n = number of patients). Gene expression is displayed in tpm from bulk RNA sequencing. [Figure 22] Figure 1 shows tumor gene expression distribution across patients in The Cancer Genome Atlas dataset for the genes DSG1 and Nectin4. Results are shown separately for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, and pancreatic cancer indications. The number of patients included in each indication is listed below the indication name (n = number of patients). Gene expression is displayed in tpm from bulk RNA sequencing. [Figure 23] Figure 1 shows tumor gene co-expression of DSG1 and NECTIN4 for each patient in The Cancer Genome Atlas for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, and pancreatic cancer indications. The number of patients included in each indication is listed below the indication (n = number of patients). Gene expression is expressed in tpm from bulk RNA sequencing. [Figure 24] This figure shows tumor Nectin4 and DSG1 protein expression across patients in the Human Protein Atlas, measured by immunohistochemistry protein microarray and classified by pathologists as undetectable, low expression, moderate expression, or high expression. Results are plotted separately for each patient according to cancer indication. Each cancer indication includes 4–12 patients, indicated by the length of the bar for that indication. Some potential cancer indications for Nectin4-targeted therapy are indicated by arrows. Note that DSG1 was only detected in skin cancer, head and neck cancer, and one lung cancer patient. Here, lung cancer can include both small cell lung cancer and non-small cell lung cancer indications. [Figure 25-1] Figure 1 shows DSG1 and NECTIN4 gene expression in normal tissues from the Genotype-Tissue Expression project, measured in tpm by bulk RNA sequencing. Box plots show the 25th to 75th percentile of gene expression across patients, with the median marked. Box plot whiskers are 1.5 interquartile distances long, with outlier patients outside this range indicated by dots. The number of individuals in each tissue is shown (n = number of individuals). DSG1 and NECTIN4 show consistently high gene expression in both sun-exposed and non-sun-exposed skin, exceeding gene expression in all other normal tissues across individuals. [Figure 25-2] (As mentioned above.) [Figure 25-3] (As mentioned above.) [Figure 26]Figure 1 shows gene expression of DSG1, NECTIN4, and PRF1 in several lines of induced pluripotent stem cells differentiated into T cells. Gene expression was measured by bulk RNA sequencing and is expressed in units of tpm. In all T cells differentiated from induced pluripotent stem cells, DSG1 and NECTIN4 gene expression was very low (<1 tpm) relative to PRF1. Gene expression is shown at both day 28 (D28) and day 35 (D35-aAPC) of T cell differentiation cultured with irradiated artificial antigen-presenting cells. [Figure 27] Figure 1 shows Nectin4 and DSG1 gene expression in cross-tissue cell-type clusters from the Human Protein Atlas single-cell RNA-sequencing dataset for 30 normal human tissues. Only cell types with Nectin4 gene expression above 10 tpm are shown. The tissues in which each cell type was found in the dataset are annotated to the right of the plot. A vertical dashed line indicates 10 tpm. [Figure 28-1] Figures 28A-28B: (A) AQP4, DSG1, and NECTIN4 gene expression in all cell type clusters for skin (left) and lung (right) tissue samples from the Human Protein Atlas single-cell RNA sequencing dataset. The relative abundance of each cell type in each tissue is annotated as a percentage next to the cluster name. Gene expression is displayed in units of tpm. (B) Information from multiple sources indicates that DSG1 or alternative desmosomal genes, CLDN10B, and AQP4 are ideal targets for inhibitory CARs for NECTIN4 therapy. Note that each inhibitory CAR target is suitable for preventing lysis of various normal cell types derived from various tissues of the body. [Figure 28-2] (As mentioned above.) [Figure 28-3] (As mentioned above.) [Figure 28-4] (As mentioned above.) [Figure 29-1]Figure 1 shows Nectin4 expression in each normal tissue cell type in the Human Protein Atlas, as measured by immunohistochemistry protein microarray and classified by pathologists as undetected, low expressing, moderate expressing, or high expressing. The normal tissue cell types with the greatest Nectin4 protein expression detected by this method are outlined at the top of the plot. [Figure 29-2] (As mentioned above.) [Figure 29-3] (As mentioned above.) [Figure 29-4] (As mentioned above.) [Figure 30] Figure 1 shows tumor NECTIN4 protein expression across patients for all cancer indications in the Human Protein Atlas, measured by immunohistochemistry protein microarray and classified by pathologists as undetectable, low expression, moderate expression, or high expression. Results are plotted separately for each patient according to cancer indication. Each cancer indication included 4-12 patients, as indicated by the length of the bar for that indication. [Figure 31-1] Figure 1 shows DSG1 and NECTIN4 gene expression in tumor tissues from The Cancer Genome Atlas, measured in tpm by bulk RNA sequencing. Box plots show the 25th to 75th percentiles of gene expression across patients, with median values ​​marked. Box plot whiskers are 1.5 interquartile distances long, and outlier patients outside this range are indicated by dots. The number of individuals for each cancer indication is shown (n = number of individuals). [Figure 31-2] (As mentioned above.) [Figure 32-1]Figures 32A-32C: Gene co-expression of DSG1 and NECTIN4 by patient in The Cancer Genome Project. (A) Tumors from patients with lung cancer indications, including small cell and non-small cell lung cancer, (B) tumors from selected NECTIN4-expressing cancer indications, and (C) all normal solid tissue data from cancer patients in the dataset. Gene expression is displayed in tpm from bulk RNA sequencing. Note that the number of samples available for normal solid tissue data is indicated by n (n = number of samples). [Figure 32-2] (As mentioned above.) [Figure 32-3] (As mentioned above.) [Figure 32-4] (As mentioned above.) [Figure 33-1] Figures 33A-33C: Gene expression in normal tissues from the Genotype-Tissue Expression Project, measured by bulk RNA sequencing in tpm. (A) DSC3 and NECTIN4, (B) CLCA4 and NECTIN4, and (C) DSC1 and NECTIN4. Box plots show the 25th to 75th percentiles of gene expression across patients, with median values ​​marked. Box plot whiskers are 1.5 interquartile distances long, with outlier patients outside this range indicated by dots. The number of individuals in each tissue is shown (n = number of individuals). [Figure 33-2] (As mentioned above.) [Figure 33-3] (As mentioned above.) [Figure 33-4] (As mentioned above.) [Figure 33-5] (As mentioned above.) [Figure 33-6] (As mentioned above.) [Figure 33-7] (As mentioned above.) [Figure 33-8] (As mentioned above.) [Figure 33-9] (As mentioned above.) [Figure 34]Figure 1 shows NECTIN4 gene expression in single-cell clusters from 30 normal tissues within the Human Protein Atlas single-cell RNA-sequencing dataset, where combinatorial inhibitory CARs can provide protection from NECTIN4-targeted activating CARs. Only single-cell clusters with NECTIN4 > 50 tpm and DSG1 < 50 tpm are shown. [Figure 35-1] Figure 35 shows median tumor gene expression across patients compared to the weighted geometric mean of gene expression in normal tissue cell types with NECTIN4 > 50 tpm and DSG1 < 50 tpm, as displayed in Figure 35, for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, or pancreatic cancer indications. Patient median gene expression was calculated from bulk RNA-sequencing measurements of human tumors by The Cancer Genome Atlas Program. Weighted geometric means of normal tissue cell types were calculated from the Human Protein Atlas single-cell RNA-sequencing atlas of 30 normal tissues. Weights are calculated for each cell type and gene as follows: (weighted tpm) = (tpm) * (relative abundance of this lung cell type among all lung cells) * (tpm of NECTIN4) / (tpm of this gene). Only surfacesome genes encoding plasma membrane proteins at least partially exposed to the extracellular space are shown. CLDN10 is highlighted in blue to emphasize its high gene expression in normal NECTIN4-high / DSG1-low expressing cell types and its low gene expression in most of the cancer indications shown. [Figure 35-2] (As mentioned above.) [Figure 35-3] (As mentioned above.) [Figure 36]Figure 37 shows the geometric means of gene expression in patient tumors, including bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, or pancreatic cancer indications, compared to the geometric means of gene expression in normal tissue cell types with NECTIN4 > 50 tpm and DSG1 < 50 tpm, as displayed in Figure 35. Patient gene expression medians were calculated from bulk RNA-sequencing measurements of human tumors by The Cancer Genome Atlas Program. Weighted geometric means of normal lung cell types were calculated from the Human Protein Atlas single-cell RNA-sequencing atlas of normal tissues. Weights are calculated for each cell type and gene as follows: (weighted tpm) = (tpm) * (relative abundance of this lung cell type among all lung cells) * (tpm of NECTIN4) / (tpm of this gene). Only surfacesome genes encoding plasma membrane proteins at least partially exposed to the extracellular space are shown. CLDN10 is highlighted in blue to highlight its high gene expression in normal NECTIN4 high / DSG1 low cell types and low gene expression in cancer. [Figure 37] Tumor gene expression distribution across patients in The Cancer Genome Atlas dataset for the genes CLDN10 and NECTIN4. Results are shown separately for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, and pancreatic cancer indications. The number of patients included in each indication is listed below the indication (n = number of patients). Gene expression is displayed in tpm from bulk RNA sequencing. [Figure 38-1]Figure 1 shows CLDN10 and NECTIN4 gene expression in normal tissues from the Genotype-Tissue Expression project, measured by bulk RNA sequencing in tpm. Box plots show the 25th to 75th percentiles of gene expression across patients, with median values ​​marked. Box plot whiskers are 1.5 interquartile distances long, and outlier patients outside this range are indicated by dots. The number of individuals in each tissue is shown (n = number of individuals). [Figure 38-2] (As mentioned above.) [Figure 38-3] (As mentioned above.) [Figure 39-1] Figures 39A-39E: Gene co-expression of DSG1 and / or CLDN10 in NECTIN4-expressing cell types from 30 normal tissues as measured by single-cell RNA sequencing. Shown are all single-cell type clusters with (A) NECTIN4 > 100 tpm and (DSG1 or CLDN10 > 100 tpm), (B) NECTIN4 > 50 tpm and (DSG1 or CLDN10 > 50 tpm), (C) NECTIN4 > 20 tpm and (DSG1 or CLDN10 > 20 tpm), (D) NECTIN4 > 10 tpm and (DSG1 or CLDN10 > 10 tpm), and (E) NECTIN4 above the indicated tpm and DSG1 and CLDN10 below the indicated tpm. Data are taken from the publicly available Human Protein Atlas single-cell RNA sequencing atlas of normal tissues. [Figure 39-2] (As mentioned above.) [Figure 39-3] (As mentioned above.) [Figure 39-4] (As mentioned above.) [Figure 39-5] (As mentioned above.) [Figure 39-6] (As mentioned above.) [Figure 39-7] (As mentioned above.) [Figure 39-8] (As mentioned above.) [Figure 39-9](As mentioned above.) [Figure 39-10] (As mentioned above.) [Figure 39-11] (As mentioned above.) [Figure 40] Figure 1 shows gene expression of AQP4, CLDN10, DSG1, and NECTIN4 in several lines of induced pluripotent stem cells differentiated into T cells. Gene expression is expressed in tpm, as measured by bulk RNA sequencing. Gene expression of all genes is very low (<1 tpm) in all samples. Gene expression is shown for both day 28 (D28) and day 35 (D35-aAPC) of T cell differentiation cultured with irradiated artificial antigen-presenting cells. [Figure 41-1] Figure 1 shows tumor NECTIN4, DSG1, DSC1, ADRB2, DSC3, LY6D, DSG3, and CLCA4 protein expression in patient tumors in the Human Protein Atlas, measured by immunohistochemistry protein microarray and classified by pathologists as undetectable, low expression, moderate expression, or high expression. Results are plotted separately for each patient according to cancer indication. Each cancer indication included 4–12 patients, as indicated by the length of the bar for that indication. [Figure 41-2] (As mentioned above.) [Figure 42-1]Figure 18B shows median tumor gene expression across patients compared to the weighted geometric mean of gene expression in normal lung cell types (excluding bronchial cells, shown in Figure 18B) with NECTIN4 gene expression greater than 10 for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, or pancreatic cancer indications. Patient median gene expression was calculated from bulk RNA-sequencing measurements of human tumors by The Cancer Genome Atlas Program. Weighted geometric means of normal lung cell types were calculated from the Human Protein Atlas single-cell RNA-sequencing atlas of normal tissues. Weights are calculated for each cell type and gene as follows: (weighted tpm) = (tpm) * (relative abundance of this lung cell type among all lung cells) * (tpm of NECTIN4) / (tpm of this gene). Only surfacesome genes encoding plasma membrane proteins at least partially exposed to the extracellular space are shown. AQP4 is highlighted in blue to emphasize its high gene expression in normal lung cell types and its low gene expression in most of the cancer indications shown. [Figure 42-2] (As mentioned above.) [Figure 42-3] (As mentioned above.) [Figure 42-4] (As mentioned above.) [Figure 43]Figure 4 shows the geometric mean of gene expression in patient tumor indications shown in Figure 44, including bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, or pancreatic cancer indications, compared to the weighted geometric mean of gene expression in normal lung cell types (excluding bronchial cells, shown in Figure 18B) with NECTIN4 gene expression greater than 10. Patient gene median expression was calculated from bulk RNA-sequencing measurements of human tumors by The Cancer Genome Atlas Program. Weighted geometric means of normal lung cell types were calculated from the Human Protein Atlas single-cell RNA-sequencing atlas of normal tissues. Weights are calculated for each cell type and gene as follows: (weighted tpm) = (tpm) * (relative abundance of this lung cell type among all lung cells) * (tpm of NECTIN4) / (tpm of this gene). Only surfacesome genes encoding plasma membrane proteins at least partially exposed to the extracellular space are shown. AQP4 is highlighted in blue to highlight its high gene expression in normal NECTIN4 high / DSG1 low cell types and low gene expression in cancer. [Figure 44] Figure 1 shows tumor gene expression distribution across patients in The Cancer Genome Atlas dataset for the genes AQP4 and NECTIN4. Results are shown separately for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, and pancreatic cancer indications. The number of patients included in each indication is listed below the indication (n = number of patients). Gene expression is displayed in tpm from bulk RNA sequencing. [Figure 45]Figure 1 shows tumor gene expression distribution across patients in The Cancer Genome Atlas dataset for the genes AQP4, DSG1, and NECTIN4. Results are shown separately for bladder, breast, esophageal, head and neck, non-small cell lung, ovarian, and pancreatic cancer indications. The number of patients included in each indication is listed below the indication name (n = number of patients). Gene expression is displayed in tpm from bulk RNA sequencing. [Figure 46-1] Figures 46A-46B: Co-expression of (A) AQP4 and NECTIN4 and (B) DSG1 and AQP4 genes by patient in The Cancer Genome Project patient tumors. Gene expression is displayed in tpm from bulk RNA sequencing. The number of patients included in each indication is listed below the indication (n = number of patients). [Figure 46-2] (As mentioned above.) [Figure 47-1] Figure 1 shows AQP4 and NECTIN4 gene expression in normal tissues from the Genotype-Tissue Expression project, measured by bulk RNA sequencing in tpm. Box plots show the 25th to 75th percentiles of gene expression across patients, with median values ​​marked. Box plot whiskers are 1.5 interquartile distances long, with outlier patients outside this range indicated by dots. The number of individuals in each tissue is shown (n = number of individuals). Data for lung and bladder tissues are highlighted by boxes. [Figure 47-2] (As mentioned above.) [Figure 47-3] (As mentioned above.) [Figure 48-1]Gene expression of AQP4, DSG1, and NECTIN4 in all cell types in (left) skin and (right) lung. Data are taken from the publicly available Human Protein Atlas single-cell RNA-sequencing atlas of normal tissues. The percentage of all cells in the tissue that comprise each cell type is annotated, and cell types are listed in descending order of abundance from top to bottom. [Figure 48-2] (As mentioned above.) [Figure 49-1] Figures 49A-49C: Gene expression of AQP4, DSG1, and NECTIN4 in all cell types of (A) normal brain and (B) normal breast, and (C) 30 normal tissues showing NECTIN4 expression above 50 tpm. Data were obtained from the publicly available Human Protein Atlas single-cell RNA sequencing atlas of normal tissues. The percentage of all cells in the tissue that comprise each cell type is annotated, and cell types are listed in descending order of abundance from top to bottom. [Figure 49-2] (As mentioned above.) [Figure 49-3] (As mentioned above.) [Figure 49-4] (As mentioned above.) DETAILED DESCRIPTION OF THE INVENTION

[0016] Various publications, articles, and patents are cited or described in the background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, acts, materials, devices, articles and the like which is included within the specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0018] 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.

[0019] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, described herein are understood to be modified in all instances by the term "about." Thus, numerical values ​​typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all individual numerical values ​​within such ranges and fractions of those values, including all possible subranges, integers within the range, unless the context clearly dictates otherwise.

[0020] 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.

[0021] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variations thereof, are understood to refer to the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be inclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive "or," not an exclusive "or." For example, condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0022] As used herein, the conjunction "and / or" between listed elements is understood to encompass both individual options and combinations of options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first option without the second option. The second option refers to the applicability of the second option without the first option. The third option refers to the applicability of the first option and the second option together. Any one of these options falls within this meaning and is therefore understood to meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within this meaning and is therefore understood to meet the requirements of the term "and / or."

[0023] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates the inclusion of any listed integer or group of integers, but does not allow for additional integers or groups of integers to be added to the specified method, structure, or composition.

[0024] 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.

[0025] As used herein, "subject" means any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and others, more preferably humans.

[0026] It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to a size or characteristic of a preferred inventive component do not delimit the described size / characteristic, but rather exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters include variations that do not vary to the least significant digit using mathematical and industrial principles recognized in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0027] The term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that is transplanted into a cell. Generally, the CAR of the present disclosure comprises one or more extracellular domains containing an antigen-binding domain, one or more intracellular domains containing one or more costimulatory and / or signaling domains, and a scaffold comprising multiple transmembrane domains and intracellular or extracellular loops on which one or more extracellular or intracellular domains are disposed. The antigen-binding domain of the CAR targets a specific antigen. The targeting region may comprise a full-length heavy chain, a Fab fragment, an scFv, a bivalent single-chain antibody, or a diabody, each of which is specific for a target antigen (e.g., nectin-4 or DSG1). The antigen-binding domain may be derived from the same or a different species in which the CAR will be used.

[0028] The terms "binding molecule" or "specifically binds" or "specific for," with reference to the antigen-binding domain of a ligand such as an antibody, a fragment thereof, or a CAR, refer to an antigen-binding domain that recognizes and binds to a specific antigen but does not substantially recognize or bind to other molecules in a sample. An antigen-binding domain that specifically binds to an antigen from one species may also bind to that antigen from another species. This cross-species reactivity does not contradict the definition of the antigen-binding domain being specific. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (allelic variants, splice variants, isoforms, etc.). This cross-reactivity does not contradict the definition of the antigen-binding domain being specific.

[0029] The terms "engineered cells" and "genetically modified cells" can be used interchangeably herein. These terms refer to the inclusion and / or expression of a foreign gene or nucleic acid sequence that alters the genotype or phenotype of the cell or its progeny. In particular, these terms refer to cells, preferably T cells, that have been engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins that are not naturally expressed in these cells. For example, T cells have been engineered to express artificial constructs such as chimeric antigen receptors on their cell surface. For example, a sequence encoding a CAR can be delivered to cells using a retroviral or lentiviral vector.

[0030] The term "target," as used herein, refers to an antigen or epitope associated with a cell that is to be specifically recognized by an antigen-binding domain, e.g., the antigen-binding domain of an antibody or a CAR. The antigen or epitope for antibody recognition may be bound to the cell surface, or may be secreted, part of the outer cell membrane, or shed from the cell.

[0031] As used herein, the term "dual-targeting" refers to a protein (e.g., a chimeric protein) capable of binding to two different antigens. Specifically, the dual-targeting proteins of the present disclosure (e.g., a CAR with two or more tumor or cancer antigen-binding domains) are not naturally occurring and are produced by genetic engineering or other methods. In one embodiment, the primary cells, engineered iPSCs, or derived cells of the present disclosure may contain one or more exogenous polynucleotides encoding a CAR having a first antigen-binding domain that specifically binds to Nectin-4 and a second antigen-binding domain that specifically binds to DSG1. This is in contrast to other examples of the present disclosure in which the primary cells, engineered iPSCs, or derived cells contain one or more polynucleotides encoding a first CAR having a first antigen-binding domain that specifically binds to Nectin-4 and a second CAR having a second antigen-binding domain that specifically binds to DSG1.

[0032] The terms "identical" or "percent identical" in the context of two or more nucleic acid or polypeptide sequences (e.g., CAR polypeptides and CAR polynucleotides that encode them) refer to two or more sequences or subsequences that are the same when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or as determined by visual inspection, or two or more sequences or subsequences that have a specified percentage of amino acid residues or nucleotides that are the same.

[0033] For sequence comparison, typically one sequence acts as reference sequence, and it is compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, if necessary, the coordinate of subsequence is designated, and the parameter of sequence algorithm program is designated.Then, sequence comparison algorithm calculates the sequence identity of test sequence and reference sequence based on designated program parameter.

[0034] Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (see Ausubel).

[0035] Examples of suitable algorithms for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is published through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned with words of the same length in database sequences, match or meet a certain positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0036] Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted when the cumulative alignment score falls below its maximum achieved value by an amount X; the accumulation of one or more negatively-scoring residue alignments causes the cumulative score to fall below zero; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program for amino acid sequences uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0037] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0038] Another indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, when two peptides differ only by conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions.

[0039] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, protein, or cell) is substantially separated from, produced separately from, or purified to remove other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues, of the organism in which it naturally occurs. "Isolated" nucleic acids, peptides, proteins, and cells therefore include nucleic acids, peptides, proteins, and cells purified by standard purification methods and those described herein. "Isolated" nucleic acids, peptides, proteins, and cells may be part of a composition, but are still isolated if this composition is not part of the nucleic acid, peptide, protein, or cell's natural environment. The term also encompasses chemically synthesized nucleic acids as well as nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell.

[0040] As used herein, the term "polynucleotide," which is synonymous with "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- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms of viral and cellular DNA and RNA characteristic of these. "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0041] A "construct" refers to a polymer or molecular complex containing a polynucleotide to be delivered to a host cell either in vitro or in vivo. A "vector," as used herein, refers to any nucleic acid construct capable of directing the delivery or transport of foreign genetic material to a target cell, where the foreign genetic material can be replicated and / or expressed. The term "vector," as used herein, includes the construct to be delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and others.

[0042] By "integration" is meant that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal DNA of the cell. By "targeted integration" is meant that the nucleotides of the construct are inserted into a preselected site or "integration site" of the chromosome or mitochondrial DNA of the cell. The term "integration" as used herein further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of endogenous sequences or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted endogenous sequences or nucleotides with one or more inserted nucleotides.

[0043] As used herein, the term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into or foreign to the host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host genetic material, for example, by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid in an expressible form into a cell. The term "endogenous" refers to a referenced molecule or activity that is present in its native form in a host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is naturally contained within a cell and that has not been exogenously introduced.

[0044] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of an appropriate regulatory sequence, is transcribed into RNA in vivo and optionally translated into a polypeptide. A gene of interest or a polynucleotide may include, but is not limited to, a prokaryotic sequence, a cDNA derived from eukaryotic mRNA, a genomic DNA sequence derived from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may encode an miRNA, an shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a variant of a natural polypeptide that has less than 100% sequence identity with the natural polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0045] "Operably linked" refers to the linkage of nucleic acid sequences into a single nucleic acid fragment, such that the function of one is affected by the other.For example, if a promoter can affect the expression of a coding sequence or functional RNA, the promoter is operably linked to the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter).The coding sequence can be operably linked to the regulatory sequence in sense or antisense direction.

[0046] The term "expression" as used herein refers to the biosynthesis of gene products. This term encompasses the transcription of genes into RNA. This term also encompasses the translation of RNA into one or more polypeptides, and further encompasses all natural post-transcriptional and post-translational modifications. Expressed CAR can be in the cytoplasm of host cells, in an extracellular environment such as the growth medium of cell culture, or anchored in the cell membrane.

[0047] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to an amino acid polymer of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, either naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art, are also included in this definition.

[0048] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, it is the L-amino acids that are represented unless otherwise explicitly indicated.

[0049] As used herein, the term "engineered immune cells" refers to immune cells, also called immune effector cells, that have been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0050] Induced pluripotent stem cells (IPSCs) and immune effector cells iPSCs have unlimited self-renewal capacity. The use of iPSCs enables cell manipulation to produce controlled cell banks of modified cells that can be expanded and differentiated into desired immune effector cells, providing large quantities of homogeneous allogeneic therapeutic products.

[0051] Genetically engineered iPSCs and their derived cells are provided herein. Selected genomic modifications provided herein enhance the therapeutic properties of the derived cells. The derived cells are functionally improved and suitable for allogeneic off-the-shelf cell therapy after a combination of selective modalities is introduced into the cells by genomic engineering at the iPSC level. This approach can help reduce side effects mediated by CRS / GVHD and prevent long-term autoimmunity while providing excellent efficacy.

[0052] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell. Specialized cells include, for example, blood cells or muscle cells. A differentiated or differentiation-induced cell is one that occupies a more specialized ("committed") position within a cell lineage. The term "committed," when applied to the differentiation process, refers to a cell that has progressed to a point in the differentiation pathway where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell to properly form all lineages of the body or soma or embryo. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0053] As used herein, the term " reprogramming " or " dedifferentiation " refers to the method of increasing the capacity of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell capacity has greater developmental plasticity (i.e., can differentiate into more cell types) compared with the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0054] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells produced from differentiated adult, neonatal, or fetal cells that have been induced, changed, or reprogrammed into cells that can differentiate into tissues of all three germ layers or layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to the cells as they are found in nature.

[0055] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" or "HPCs" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, pluripotent hematopoietic stem cells (hemocyte blasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). As used herein, "CD34+ hematopoietic progenitor cells" refer to HPCs that express CD34 on their surface.

[0056] 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.

[0057] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and has various roles in the immune system. T cells may have roles including, for example, identifying specific foreign antigens in the body and activating and deactivating other immune cells. T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (gd T cells), and others, and can be at any developmental stage. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tern cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0058] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by their secretory profile after stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is implicated as a binding recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, they define helper / inducer subsets.

[0059] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the binding recognition element in major histocompatibility complex class I-restricted interactions.

[0060] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of a T cell receptor (TCR chain). NK cells may also refer to genetically engineered NK cells, such as NK cells modified to express a chimeric antigen receptor (CAR). NK cells may also differentiate from stem or progenitor cells.

[0061] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to preferential therapeutic traits in source cells or iPSCs and may be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs by reprogramming, which can further differentiate into specific cell types, including any hematopoietic lineage cells. Source cell-derived iPSCs and cells differentiated therefrom are sometimes collectively referred to as "derived" or "derived" cells, depending on the context. For example, as used throughout this application, derived effector cells, or derived NK or "iNK" cells, or derived T or "iT" cells, are cells differentiated from iPSCs, compared to their primary counterparts obtained from natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, genetic imprints that confer preferential therapeutic traits are incorporated into iPSCs either by reprogramming selected source cells that are donor, disease, or treatment response specific, or by using genome editing to introduce genetically modified modalities into iPSCs.

[0062] Induced pluripotent stem cell (iPSC) parent cell lines can be generated from peripheral blood mononuclear cells (PBMCs) or T cells using any known method for introducing reprogramming factors into non-pluripotent cells, such as the episomal plasmid-based process previously described in U.S. Patent Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference. Reprogramming factors can be in the form of polynucleotides and are therefore introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs, and the genome editing is introduced by creating one or more targeted integrations and / or in / dels at one or more selected sites. In another embodiment, the iPSCs are obtained from human T cells with antigen specificity and rearranged TCR genes (also referred to herein as "T-iPS" cells), as described in U.S. Patent Nos. 9,206,394 and 10,787,642, which are hereby incorporated by reference.

[0063] According to certain aspects, the present application relates to an induced pluripotent stem cell (iPSC) cell or a derivative thereof, comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) an exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide sequence, such as porcine teschovirus-1 2A (P2A); and (iii) a deletion or reduced expression of the B2M and CIITA genes.

[0064] I. Chimeric Antigen Receptor (CAR) Expression According to an embodiment of the present application, iPSC cells or derived cells thereof comprise one or more exogenous polynucleotides encoding chimeric antigen receptors (CARs), wherein the CARs target the nectin-4 antigen. 2+ The nectin family is a cell adhesion molecule (CAM) involved in cell-cell-independent interactions. There are four nectins in the nectin family. Nectins 1-3 are abundant in normal adult tissues Nectin-4 is primarily expressed during fetal development, and its expression declines in adult tissues (low levels are expressed in skin, bladder, placenta, oral mucosa, and tonsils).

[0065] Nectin interacts with other cell surface molecules, including cadherins, integrins, and growth factor receptors. These interactions help modulate cell adhesion, migration, and proliferation. Nectin-4 dimers bind to nectin-1 or nectin-4 on neighboring cells. Nectin-4 also binds to TIGIT on immune cells, and this interaction leads to the inhibition of NK cells.

[0066] Therefore, Nectin-4 is a suitable target for the CAR of the present invention because it is frequently expressed in bladder cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and esophageal cancer. The highest expression levels of Nectin-4 are found in bladder cancer, breast cancer, lung cancer, and pancreatic cancer. Clinical validation of Nectin-4 as a tumor target has been demonstrated by the approval of enfortumab vedotin for the treatment of urothelial cancer.

[0067] Thus, in one embodiment, the CAR targets the Nectin-4 antigen and the targeting region (e.g., the extracellular domain) of the CAR comprises an antibody fragment (e.g., a VHH domain). In other embodiments, the iPSCs or derived cells thereof comprise one or more first exogenous polynucleotides encoding a single CAR that targets the Nectin-4 antigen.

[0068] In some embodiments, the iPSCs or their derivative cells comprise one or more first exogenous polynucleotides encoding a CAR (e.g., targeting Nectin 4) and an additional CAR that targets another antigen. In some embodiments, the antigen targeted by the additional CAR is selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. In other embodiments, the iPSCs or their derivative cells comprise one or more first exogenous polynucleotides encoding a dual-targeting CAR that targets the Nectin 4 antigen and another antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6. Each of the binding domains of the CAR, the additional CAR, or the dual-targeting CAR can be independently selected from, for example, an scFv and a VHH.

[0069] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular signaling domain. Association of the extracellular domain of the CAR with the target antigen on the target cell surface results in CAR clustering and delivers an activating stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger the production of molecules that can mediate proliferation, cytokine production, phagocytosis, and / or cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0070] As used herein, the term "signal peptide" refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein that co- or post-translationally directs the nascent CAR protein to the endoplasmic reticulum and subsequent surface expression.

[0071] As used herein, the terms "extracellular antigen-binding domain," "extracellular domain," or "extracellular ligand-binding domain" refer to the portion of a CAR that is located on the outside of the cell membrane and that is capable of binding to an antigen, target, or ligand.

[0072] As used herein, the term "hinge region" or "hinge domain" refers to the portion of a CAR that connects two adjacent domains of the CAR protein, namely the extracellular and transmembrane domains of the CAR protein.

[0073] 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.

[0074] As used herein, the terms "intracellular signaling domain," "cytoplasmic signaling domain," or "intracellular signaling domain" refer to the portion of a CAR that is located inside the cell membrane and is capable of transducing an effector signal.

[0075] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., an NK cell or a T cell) that provides a primary cytoplasmic signaling sequence that stimulatorily regulates primary activation of receptors for at least some aspect of an immune cell signaling pathway. Stimulatory molecules contain two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and sequences that act antigen-independently to provide secondary activation of costimulatory signals (referred to as "costimulatory signaling domains").

[0076] In certain embodiments, the extracellular domain comprises an antigen-binding domain and / or antigen-binding fragment. The antigen-binding fragment may be, for example, an antibody or antigen-binding fragment thereof that specifically binds to a tumor antigen. The antigen-binding fragment of the present application has one or more desired functional properties, including, but not limited to, high-affinity binding to the tumor antigen, high specificity for the tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing the tumor antigen, and the ability to inhibit tumor growth in subjects and animal models in need thereof when administered alone or in combination with other anti-cancer therapies.

[0077] As used herein, the term "antibody" is used broadly and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal, human, humanized, composite, and chimeric antibodies and antibody fragments. Generally, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. The structure of antibodies is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, antibodies of the present application can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, kappa and lambda, based on the amino acid sequence of their constant domains. Thus, the antibodies of the present application may contain kappa or lambda light chain constant domains. In certain embodiments, the antibodies of the present application comprise the heavy and / or light chain constant regions of a rat or human antibody. In addition to the heavy and light chain constant domains, the antibodies contain an antigen-binding region made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0078] 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.

[0079] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in small amounts. The monoclonal antibodies of the present application can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse or rat, whose genome includes human heavy chain and light chain transgenes.

[0080] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single-domain antibody (sdAb), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibody, minibody, nanobody, domain antibody, bivalent domain antibody, light chain variable domain (VL), variable domain of a camelid antibody (VL), H H), or any other antibody fragment that binds to an antigen but does not contain the entire antibody structure. An antigen-binding fragment is capable of binding to the same antigen as that bound by the parent antibody or parent antibody fragment.

[0081] 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).

[0082] As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region, or that comprises only a heavy chain variable region.

[0083] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, and made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0084] 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.

[0085] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of antibodies derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0086] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In embodiments, the first and second epitopes overlap or substantially overlap. In embodiments, the first and second epitopes do not overlap or substantially do not overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In embodiments, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0087] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or no more than two antigens. Bispecific antibodies are characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In embodiments, the first and second epitopes overlap or substantially overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, a bispecific antibody comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In embodiments, a bispecific antibody comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In embodiments, a bispecific antibody comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope. In embodiments, a bispecific antibody comprises a VFv or fragment thereof having binding specificity for a first epitope. H H and V with binding specificity for a second epitope H and H. In embodiments, the term X / Y loop (where "X" and "Y" are antigens such as nectin-4, and antigens selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6) refers to the extracellular region of one scFv nested between the VL and VH of another 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.

[0088] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to a tumor antigen" is one that binds to a tumor antigen in an amount of 1×10 -7 M or less, preferably 1 × 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 "KD" refers to an antigen-binding domain or antigen-binding fragment that binds with a KD of M or less. The term "KD" refers to the dissociation constant, obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as a molar concentration (M). KD values ​​for antibodies can be determined using methods in the art in light of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, e.g., by using a biosensor system, e.g., a Biacore® system, or by using biolayer interferometry technology, e.g., an Octet RED96 system.

[0089] The smaller the KD value of an antigen-binding domain or antigen-binding fragment, the higher the affinity with which the antigen-binding domain or antigen-binding fragment binds to the target antigen.

[0090] In various embodiments, antibodies or antibody fragments suitable for use in the CARs of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), masked antibodies (e.g., Probody®), small modular immunopharmaceuticals ("SMIP™"), intrabodies, minibodies, single-domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the above. Antibodies and / or antibody fragments can be derived from mouse antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0091] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity-determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0092] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or nanobody, hi some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0093] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0094] 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.

[0095] In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.

[0096] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise an scFv.

[0097] Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high affinity and specific binding of target biomolecules, can also be used in the CARs of the present disclosure. Such scaffolds have been shown to result in molecules with improved characteristics, such as greater stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that can be used in the CARs of the present disclosure include engineered tenascin-derived tenascin type III domains (e.g., Centyrin™); engineered gamma-B crystallin-derived scaffolds or engineered ubiquitin-derived scaffolds (e.g., Affilin™); engineered fibronectin-derived 10th fibronectin type III (10Fn3) domains (e.g., monobodies, AdNectin™, or AdNexin™); engineered ankyrin repeat motif-containing polypeptides (e.g., DARPin™); engineered low-density lipoprotein receptor-derived A domain (LDLR-A) (e.g., Avime™); lipocalins (e.g., anticalins); engineered proteases. Kunitz domains from enzyme inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); engineered Z domains from Protein A (Affibody™); Sac7d-derived polypeptides (e.g., Nanoffitin® or affitin); engineered SH2 domains from Fyn (e.g., Fynomer®); CTLD3 (e.g., tetranectin); thioredoxin (e.g., peptide aptamers); KALBITOR®; β-sandwiches (e.g., iMabs); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimetics; and any engineered counterparts of the above that retain their binding functionality (Woern A, Pluckthun A, J Mol Biol 305: 989-1010 (2001);Xu L et al., Chem Biol 9: 933-42 (2002);Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004);Binz H et al., Nat Biolechnol 23: 1257-68 (2005);Hey T et al., Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Skerra, Current Opin. in Biotech., 2007 18: 295-304; Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85 (2011), each of which is incorporated by reference in its entirety.

[0098] In some embodiments, the alternative scaffold is affilin or centirin.

[0099] In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a leader sequence. The leader sequence may be located at the N-terminus of the extracellular tag-binding domain. The leader sequence may optionally be cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of leader sequences known to those skilled in the art may be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from a human immunoglobulin heavy chain (HC).

[0100] 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.

[0101] In some embodiments, the first polypeptide of a CAR of the present disclosure comprises a transmembrane domain fused in-frame between an extracellular tag-binding domain and a cytoplasmic domain.

[0102] The transmembrane domain can be derived from the protein that contributes to the extracellular tag binding domain, the protein that contributes to the signal transduction or co-signal transduction domain, or from a completely different protein.In some cases, the transmembrane domain can be selected or modified by amino acid substitution, deletion, or insertion to minimize the interaction with other members of the CAR complex.In some cases, the transmembrane domain can be selected or modified by amino acid substitution, deletion, or insertion to avoid the binding of the protein that is naturally associated with the transmembrane domain.In certain embodiments, the transmembrane domain comprises additional amino acids to allow flexibility and / or optimal distance between the domains that are connected to the transmembrane domain.

[0103] The transmembrane domain can be derived from either natural or synthetic origin. If the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains particularly useful in the present disclosure can be derived from (i.e., at least include the transmembrane region of) the α, β, or ζ chain of the T cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it contains primarily hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan, and / or valine can be found at each end of a synthetic transmembrane domain.

[0104] In some embodiments, it may be desirable to utilize the transmembrane domain of the zeta, eta, or FcεR1γ chain, which contains cysteine ​​residues capable of disulfide bonding, allowing the resulting chimeric protein to form disulfide-linked dimers with itself or with unmodified versions of the zeta, eta, or FcεR1γ chain of related proteins. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains with transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex. In other cases, it may be desirable to employ the transmembrane domains of zeta, eta, or FcεR1γ and -β, MB1 (Igα), B29, or CD3-γ, zeta, or eta to maintain physical association with other members of the receptor complex.

[0105] 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.

[0106] In some embodiments, the first polypeptide of a CAR of the present disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, wherein the tag-binding domain, linker, and transmembrane domain are in frame with each other.

[0107] The term "spacer region," as used herein, generally refers to any oligopeptide or polypeptide that functions to link a tag-binding domain to a transmembrane domain. Spacer regions can be used to provide greater flexibility and accessibility to the tag-binding domain. Spacer regions can contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. Spacer regions can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region. Alternatively, spacer regions can be synthetic sequences corresponding to naturally occurring spacer region sequences or can be entirely synthetic spacer region sequences. Non-limiting examples of spacer regions that can be used according to the present disclosure include portions of the human CD8 α chain, partial extracellular domains of CD28, FcyRlla receptors, IgG, IgM, IgA, IgD, IgE, Ig hinges, or functional fragments thereof. In some embodiments, additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is at an optimal distance from the transmembrane domain, hi some embodiments, if the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.

[0108] 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 a chimera thereof.

[0109] 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, incorporated herein by reference in its entirety for all purposes), and ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:96), 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.

[0110] 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.

[0111] 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.

[0112] In certain aspects, the first polypeptide of the CAR of the present disclosure comprises a cytoplasmic domain that includes at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more costimulatory signaling domains.

[0113] The cytoplasmic domain is responsible for activating at least one of the normal effector functions of a host cell (e.g., a T cell) carrying the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytotoxic activity or helper activity, including cytokine secretion. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Although the entire signaling domain is usually present, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the signaling domain sufficient to transmit the effector function signal.

[0114] 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, and CD79b.

[0115] In some embodiments, the cytoplasmic domain comprises a CD3 zeta signaling domain. In one embodiment, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO:6, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6.

[0116] In some embodiments, the cytoplasmic domain further comprises one or more costimulatory signaling domains, in some embodiments, the one or more costimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] In one embodiment, the costimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP12 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 18.

[0128] 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.

[0129] 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.

[0130] In some embodiments, the signal transduction domain and the costimulatory signal transduction domain can be arranged in any order. In some embodiments, the signal transduction domain is upstream of the costimulatory signal transduction domain. In some embodiments, the signal transduction domain is downstream of the costimulatory signal transduction domain. When two or more costimulatory domains are included, the order of the costimulatory signal transduction domains can be switched.

[0131] Non-limiting exemplary CAR regions and sequences are provided in Table 1, including the amino acid and nucleic acid sequences of various CAR constructs of the present disclosure.

[0132] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 [Table 1-30] [Table 1-31] [Table 1-32] [Table 1-33] [Table 1-34]

[0133] In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen. The antigen-binding domain of the second polypeptide can bind to more than one antigen or more than one epitope in an antigen. For example, the antigen-binding domain of the second polypeptide can bind to 2, 3, 4, 5, 6, 7, 8 or more antigens. In another example, the antigen-binding domain of the second polypeptide can bind to 2, 3, 4, 5, 6, 7, 8 or more epitopes in the same antigen.

[0134] The selection of antigen binding domains can depend on the type and number of antigens that define the surface of target cells. For example, antigen binding domains can be selected to recognize antigens that act as cell surface markers on target cells associated with a particular disease state. In certain embodiments, the CARs of the present disclosure can be genetically modified to target tumor antigens of interest by engineering a desired antigen binding domain that specifically binds to the antigen (e.g., on tumor cells). Non-limiting examples of cell surface markers that can serve as targets for the antigen binding domains in the CARs of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0135] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen.

[0136] 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.

[0137] 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.

[0138] In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies. Non-limiting examples of tumor antigens associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2. Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, nectin-4, and B7H4. Non-limiting examples of tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigens associated with liver cancer include claudin 18.2, GPC-3, EpCAM, cMET, and AFP. Non-limiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70. Non-limiting examples of tumor antigens associated with bladder cancer include nectin 4 and SLITRK6. Non-limiting examples of tumor antigens associated with glioblastoma include Cd133, EGFr, CD70, and IL13Ra2. Non-limiting examples of tumor antigens associated with renal cell carcinoma include nectin 4, SLITRK6, CD70, and FOLR1. Non-limiting examples of tumor antigens associated with ovarian cancer include nectin 4, mesothelin, FSHR, and FOLR1. Non-limiting examples of tumor antigens associated with hepatocellular carcinoma include GPC3.

[0139] Further examples of antigens that can be targeted by the antigen binding domain include alpha-fetoprotein, A3, antigen specific for the A33 antibody, Ba 733, BrE3-antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CS Ap, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, Fit-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits , hypoxia inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-I), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, nectin 4, 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.

[0140] In one embodiment, the antigen targeted by the antigen-binding domain is Nectin 4. In one embodiment, the antigen-binding domain comprises an anti-Nectin 4 VHH. In other embodiments, the antigen-binding domain comprises an anti-Nectin 4 scFv. In one embodiment, the anti-Nectin 4 antigen-binding domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 105-130, 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 one of SEQ ID NOs: 105-130. In one embodiment, the anti-Nectin-4 antigen-binding domain comprises an amino acid sequence encoded by a polynucleotide sequence set forth in one of SEQ ID NOs: 131-156, 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 one of SEQ ID NOs: 131-156.

[0141] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cells that produce cell receptors and "self"-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Crohn's disease, or ulcerative colitis.

[0142] 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, fillaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), articular cartilage components such as collagen II, IX, and XI, circulating serum proteins such as RF (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33 / hnRNP. A2, Sm, eukaryotic translation elongation factor 1 alpha 1, stress proteins such as HSP-65, -70, -90, BiP, inflammatory / immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin, and 25-35 kD nuclear protein , particulate proteins such as bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factors, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonucleopartides and proteins such as Sm antigens (including, but not limited to, SmD antigen and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro(SSA), and La(SSB) antigens.

[0143] In various embodiments, a CAR of the present disclosure may comprise an scFv domain or fragment thereof, and the scFv domain or fragment thereof used in the CAR may comprise a linker between the VH domain and the VL domain. The linker may be a peptide linker and may comprise any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be long enough to connect the VH and VL so that they form the correct conformation relative to each other and thereby retain the desired activity, such as antigen binding. The linker may be approximately 5 to 50 amino acids in length. In some embodiments, the linker is approximately 10 to 40 amino acids in length. In some embodiments, the linker is approximately 10 to 35 amino acids in length. In some embodiments, the linker is approximately 10 to 30 amino acids in length. In some embodiments, the linker is approximately 10 to 25 amino acids in length. In some embodiments, the linker is approximately 10 to 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.

[0144] In one embodiment, the CAR may comprise a linker, and the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity 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 set forth in SEQ ID NO: 25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 25.

[0145] 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: 3, 25-56, 99, and 102-104 in Table 1. Additional linkers are described, for example, in WO 2019 / 060695, which is incorporated herein by reference in its entirety.

[0146] II. Inhibitory chimeric antigen receptor (iCAR) expression Inhibitory chimeric antigen receptors (iCARs) are genetically engineered receptors used in cell-based cancer therapies. They are a modification of conventional chimeric antigen receptor (CAR) technology that can be used to enhance immune cell resistance to cancer. In contrast to CARs (e.g., synthetic receptors expressed on the surface of immune cells to enhance their ability to recognize and attack cancer cells), iCARs are designed to inhibit T cell activation when the T cell encounters a target antigen. iCARs consist of several components, including an antigen-binding domain (e.g., extracellular domain), a signal peptide, a hinge region, a transmembrane domain, and an internal domain (e.g., inhibitory domain). The inhibitory domain is typically derived from immune checkpoint molecules such as PD-1 (programmed cell death protein 1) or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), which are known to suppress immune cell activity. The incorporation of the inhibitory component in iCARs allows for better control of the immune response against cancer cells by reducing the risk of off-target effects and toxicity associated with cell-based cancer therapies. Non-limiting exemplary iCAR regions and sequences, including the amino acid and nucleic acid sequences of various iCAR constructs of the present disclosure, are provided in Tables 4-6. SP = signal peptide; H = hinge; and TMD = transmembrane domain.

[0147] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 Table 2-32 Table 2-33 Table 2-34 Table 2-35 Table 2-36 Table 2-37 Table 2-38 Table 2-39 Table 2-40 Table 2-41 Table 2-42 Table 2-43 Table 2-44 Table 2-45 Table 2-46 Table 2-47 Table 2-48 Table 2-49 Table 2-50 Table 2-51 Table 2-52 Table 2-53 Table 2-54 Table 2-55 Table 2-56 Table 2-57 Table 2-58 Table 2-59 Table 2-60 Table 2-61

[0148] Table 3-1 Table 3-2

[0149] Table 4-1 Table 4-2 Table 4-3 Table 4-4

[0150] In some aspects, the present disclosure provides induced pluripotent stem cells (iPSCs) or their derivatives comprising one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting the Nectin-4 antigen. In some embodiments, the one or more exogenous polynucleotides further encode one or more inhibitory CARs (iCARs). In some embodiments, the one or more iCARs comprise at least one antigen-binding domain targeting an antigen. In some embodiments, the antigen targeted by at least one antigen-binding domain of the iCAR is independently selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC) 1, DSC3, desmoglein (DSG) 1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), and V-set and immunoglobulin domain-containing 8 (VSIG8).

[0151] In some embodiments, iCARs contain a signal peptide. A signal peptide is a short amino acid sequence included in the design of a chimeric antigen receptor (CAR) to facilitate correct processing and targeting of the engineered protein. The signal peptide guides the proper localization and presentation of the CAR on the cell surface, allowing it to recognize cancer cells and initiate an immune response. Signal peptides used in CARs are often derived from antibodies or other surface proteins that naturally contain such targeting sequences. Typically, they are attached to the N-terminus of the CAR construct, at the beginning of the protein sequence, and can be 15 to 30 amino acids long, with a central hydrophobic region flanked by positively charged residues. Any signal peptide known to those skilled in the art can be used in combination with the engineered iPSCs or their derivative cells of the present disclosure. However, various signal peptide sequences can be tested to optimize CAR expression and function. In some embodiments, the signal peptide of an iCAR comprises a CD8 signal peptide, a GMCSFR signal peptide, a MARS signal peptide, or an IgK signal peptide, or variants thereof. In some embodiments, the signal peptide of iCAR comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 97 and 292. In some embodiments, the signal peptide of iCAR is encoded by a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 98 and 327.

[0152] In some embodiments, an iCAR comprises an extracellular domain comprising at least one antigen-binding domain that specifically binds to one or more of the following: adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC)1, DSC3, desmoglein (DSG)1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8). For example, an iCAR may comprise an extracellular domain comprising a first binding domain that specifically binds to ADRB2 and a second binding domain that binds to DSG1. In another example, an iCAR may comprise an extracellular domain comprising a first binding domain that specifically binds to DSC1 and a second binding domain that binds to HCAR3. In some embodiments, the iCAR comprises an extracellular domain comprising a binding domain that specifically binds to adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC)1, DSC3, desmoglein (DSG)1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, or immunoglobulin domain-containing 8 (VSIG8). For example, the iCAR comprises an extracellular domain comprising a binding domain that specifically binds to DSG1. In another example, the iCAR comprises an extracellular domain comprising a binding domain that specifically binds to AQP4. In some embodiments, the extracellular domain of iCAR comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 354-363.In some embodiments, at least a portion of the extracellular domain of iCAR is encoded by a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 364-373.

[0153] In some embodiments, the iCAR comprises a hinge region. The hinge domain connects the recognition and functional elements of the CAR, and its structural flexibility and length allow for antigen binding ability and spatial orientation. The hinge provides structural flexibility between the target recognition domain and the cell, allowing free rotation and orientation of the antigen binding site. Generally, the hinge domain connects the antigen binding motif (usually an scFv) to the transmembrane region of the CAR and is approximately 12 to 60 amino acids in length (longer hinges provide greater flexibility). In some embodiments, the hinge region of the iCAR is selected from the group consisting of a CD28 hinge region, a CD45 hinge region, a G4S-CD45 hinge region, a CD8 hinge region, and a CXC3R GPCR hinge region. In some embodiments, the hinge region of iCAR comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 21, 22, 288, 289, and 321. In some embodiments, the hinge region of iCAR is encoded by a polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 315-318, 320, and 322.

[0154] In some embodiments, iCARs contain one or more transmembrane domains. The transmembrane domains anchor the CAR to the cell membrane and transmit signals from antigen binding to cell activation through connections with cytoplasmic signaling sequences. The transmembrane domains are often derived from CD3-zeta, CD4, CD8, or CD28 proteins, may contain a hydrophobic amino acid sequence that spans the lipid bilayer, may be approximately 20-30 amino acids long, and may be integrated into the membrane to form an alpha-helical structure with charged residues at both ends that anchor the helix. In some embodiments, the transmembrane domains can enable CAR dimerization or multimerization, which can amplify signal transduction. Modifying the transmembrane properties (e.g., hydrophobicity, length, flexibility, and / or dimerization ability) can help optimize CAR expression and function. In some embodiments, the one or more transmembrane domains of the iCAR are independently selected from the group consisting of a CD28 transmembrane domain, a CD8 transmembrane domain, a PD1 transmembrane domain, a SynNotch transmembrane domain, and a CXC3R GPCR. In some embodiments, the transmembrane domain of iCAR comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 23, 24, 290, 291, 323, and 325. In some embodiments, the transmembrane domain of iCAR is encoded by a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 324, 326, and 374-377.

[0155] In some embodiments, iCARs comprise an intracellular signaling domain. The intracellular signaling domain initiates and regulates CAR-engineered cellular responses through ITAM and costimulatory interactions, which can be optimized to improve therapeutic benefit. Specifically, the intracellular signaling domain is the region of the CAR that initiates cell activation after the CAR binds to its target antigen. Generally, the intracellular signaling domain may contain an immunoreceptor tyrosine-based activation motif (ITAM) that mediates signal transduction and often includes a costimulatory domain such as CD3-zeta and / or CD28 or 4-1BB. When the CAR binds to its antigen, the ITAM tyrosine is phosphorylated, initiating a cell activation cascade via enzymes such as ZAP70, leading to transcription factor activation. The inclusion of a costimulatory signaling domain (e.g., CD28, 4-1BB) together with CD3-zeta can provide a synergistic signal, enhancing cell activation, cytokine production, proliferation, and persistence. Varying the combination and order of ITAM and costimulatory domains allows for tuning of CAR signaling strength and balancing efficacy and safety. In some embodiments, the intracellular signaling domain of the iCAR comprises one or more of a PD1 intracellular domain, a LIRB1 intracellular domain, a TIGIT intracellular domain, a CTLA4 intracellular domain, a CSK*(YSSV) intracellular domain, a KIR2DL1 intracellular domain, a DR1 intracellular domain, a Casp8wt intracellular domain, a tCasp8 intracellular domain, a tCasp8 dimer intracellular domain, a tBid15 intracellular domain, a Casp9wt intracellular domain, a tCasp9 intracellular domain, a tCasp9 dimer intracellular domain, a SHP1 intracellular domain, a (G4S)2-SHP1 intracellular domain, a CSK intracellular domain, a (G4S)2-CSK intracellular domain, an ADAM17 cleavage site, a CD28 intracellular domain, a CD3ζ intracellular domain, a G4S3 linker, an ADAM17 protease domain, and a (G4S)3-ADAM17 protease domain.In some embodiments, the intracellular signaling domain of iCAR comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 6, 8, and 267-287. In some embodiments, the intracellular signaling domain of iCAR is encoded by a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 266, 293-318, 320, and 322. In some embodiments, the iCAR comprises a costimulatory domain. In some embodiments, the costimulatory domain of the iCAR is selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain.

[0156] III. Artificial Cell Death Polypeptides According to embodiments of the present application, the iPSC cell or a derived cell thereof may comprise a second exogenous polynucleotide encoding an artificial cell death polypeptide.

[0157] As used herein, the term "artificial cell death polypeptide" refers to an engineered protein designed to prevent potential toxic or otherwise adverse effects of a cell therapy. Artificial cell death polypeptides can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. Optionally, the artificial cell death polypeptide is activated by an exogenous molecule, e.g., an antibody, which, upon activation, induces apoptosis and / or cell death of therapeutic cells.

[0158] In certain embodiments, the artificial cell death polypeptide comprises an inactivated cell surface receptor that includes an epitope specifically recognized by an antibody, particularly a monoclonal antibody, also referred to herein as a monoclonal antibody-specific epitope. When expressed by iPSCs or their derivatives, the inactivated cell surface receptor is inactive or significantly defective in signal transduction, yet can still be specifically recognized by the antibody. Specific binding of the antibody to the inactivated cell surface receptor allows for the elimination of iPSCs or their derivatives not only by ADCC and / or ADCP mechanisms, but also by direct killing using antibody-drug conjugates with toxins or radionuclides.

[0159] In certain embodiments, the inactivated cell surface receptor is selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. The inactivated cell surface receptor comprises an epitope selected from epitopes specifically recognized by antibodies including, but not limited to, vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, or ustekinumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab. In certain embodiments, the inactivated cell surface receptor comprises an epitope specifically recognized by trastuzumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope specifically recognized by bevacizumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope specifically recognized by avelumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by ipilimumab.

[0160] The epidermal growth factor receptor, also known as EGFR, ErbB1, and HER1, is a cell surface receptor for members of the epidermal growth factor family of extracellular ligands. As used herein, "truncated EGFR," "tEGFR," "short EGFR," or "sEGFR" refers to an inactive EGFR variant that lacks the EGF-binding and intracellular signaling domains of EGFR. An exemplary tEGFR variant contains domains 322-333 of domain 2, all of domains 3 and 4, and the transmembrane domain of the native EGFR sequence, which contains the cetuximab-binding epitope. Expression of the tEGFR variant on the cell surface allows for cell ablation, if desired, with an antibody that specifically binds to tEGFR, such as cetuximab (Erbitux®). Due to the absence of the EGF-binding and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or their derived cells.

[0161] Exemplary inactivated cell surface receptors of the present application include tEGFR variants. In certain embodiments, expression of an inactivated cell surface receptor in engineered immune cells expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cells when the engineered immune cells are contacted with an anti-EGFR antibody. Methods using inactivated cell surface receptors are described in International Publication Nos. 2019 / 070856, 2019 / 023396, and 2018 / 058002, the disclosures of which are incorporated herein by reference. For example, a subject who has previously received engineered immune cells of the present disclosure comprising a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an amount of an anti-EGFR antibody effective to eliminate the previously administered engineered immune cells in the subject.

[0162] In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab.

[0163] In certain embodiments, the tEGFR variant comprises or consists of an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 71, preferably the amino acid sequence of SEQ ID NO: 71.

[0164] 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%, 92%, 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.

[0165] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the CD20 epitope comprises or consists of an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 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.

[0166] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of the Her2 receptor or ErbB, e.g., an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody-specific epitope comprises or consists of an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 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.

[0167] IV. Cytokine Expression In some embodiments, the iPSC cells or derivatives thereof optionally comprise an exogenous polynucleotide encoding a cytokine, such as interleukin-15 or interleukin-2.

[0168] As used herein, "interleukin-15" or "IL-15" refers to a cytokine, or a functional portion thereof, that regulates the activation and proliferation of T and NK cells. A "functional portion" ("biologically active portion") of a cytokine refers to a portion of a cytokine that retains one or more functions of the full-length or mature cytokine. Such functions for IL-15 include promoting the survival of NK cells, regulating the activation and proliferation of NK cells and T cells, as well as supporting NK cell development from hematopoietic stem cells. As will be recognized by those skilled in the art, the sequences of various IL-15 molecules are known in the art. In certain embodiments, the IL-15 is wild-type IL-15. In certain embodiments, the IL-15 is human IL-15. In certain embodiments, the IL-15 comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:72, preferably the amino acid sequence of SEQ ID NO:72.

[0169] In some embodiments, IL-15 is in a membrane-bound form in which all or a functional portion of the IL-15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL-15 as a cell membrane-bound polypeptide (mbIL15), e.g., the construct described in U.S. Pat. No. 9,629,877 B2, which is hereby incorporated by reference.

[0170] As used herein, "interleukin-2" refers to a cytokine, or a functional portion thereof, that regulates the activation and proliferation of T and NK cells. In certain embodiments, the IL-2 is wild-type IL-2. In certain embodiments, the IL-2 is human IL-2. In certain embodiments, the IL-2 comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:76, preferably the amino acid sequence of SEQ ID NO:76.

[0171] In certain embodiments, the inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably via 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), flacherie 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%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73.

[0172] 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 at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:74, preferably the amino acid sequence of SEQ ID NO:74.

[0173] In some embodiments, the inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77, preferably the amino acid sequence of SEQ ID NO: 77.

[0174] In some embodiments, the inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO:21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:21.

[0175] In certain embodiments, the inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:23.

[0176] In certain embodiments, the inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, transmembrane region, and cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises more than one epitope specifically recognized by an antibody, and the epitopes may have the same or different amino acid sequences, and the epitopes may be linked to each other via a peptide linker, such as a flexible peptide linker having the sequence (GGGGS)n, where n is an integer between 1 and 8 (SEQ ID NO: 25). In some embodiments, the inactivated cell surface receptor further comprises a cytokine, such as IL-15 or IL-2. In certain embodiments, the cytokine is located in the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked to the epitope specifically recognized by the antibody directly or indirectly via an autoprotease peptide sequence, such as those described herein. In some embodiments, the cytokine is indirectly linked to the epitope by being linked to the transmembrane domain via an autoprotease peptide sequence.

[0177] Non-limiting exemplary regions and sequences of inactivated cell surface receptors are provided in Table 2.

[0178] [Table 5-1] [Table 5-2]

[0179] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79.

[0180] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81, preferably the amino acid sequence of SEQ ID NO: 81.

[0181] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, preferably the amino acid sequence of SEQ ID NO: 83.

[0182] V. HLA Expression In one aspect, MHC1 and / or MHCII knockout and / or knockdown can be incorporated into cells for use in "allogeneic" cell therapy, in which cells are harvested from a subject, engineered to knockout or knockdown, e.g., B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP gene expression, and then returned to a different subject. The knockout or knockdown of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes described herein can (1) prevent graft-versus-host responses, (2) prevent host-versus-graft responses, and / or (3) improve cell safety and efficacy. Thus, in certain embodiments, the disclosed invention includes independently knocking out and / or knocking down one or more genes selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes in iPSC cells. In certain embodiments, the disclosed method includes independently knocking out and / or knocking down two genes selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes in iPSC cells, particularly B2M and CIITA, to achieve class I and II HLA disruption. In certain embodiments, the iPSCs or their derivatives can be further modified by introducing exogenous polynucleotides encoding one or more proteins involved in immune escape, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). In particular, disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving 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;35(8):765-772).

[0183] Incorporating MHC I and / or MHC II knockout and / or knockdown into cells for use in "allogeneic" cell therapy will enable potential cell products to avoid recognition and destruction by the host immune system. The reduced alloreactivity possible through the use of this technology will allow for repeated administration of CAR-modified cell therapies to improve therapeutic potential. Combined with the extended killing capacity of optimized immune cells derived from a single genetically engineered cell clone, the cells will have the potential for repeated dosing to maximize response and durability of efficacy. Moreover, this technology may enable dosing in patients with limited or no immune preconditioning regimens.

[0184] Thus, in certain embodiments, the iPSCs or derivatives thereof of the present application can be further modified by introducing a third exogenous polynucleotide encoding one or more proteins involved in immune escape, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).

[0185] In certain embodiments, the iPSCs or derived cells thereof comprise a third exogenous polypeptide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G). In certain embodiments, the HLA-E comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the HLA-G comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68, preferably SEQ ID NO: 68.

[0186] In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-E via a linker. In certain embodiments, the third exogenous polypeptide comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:66.

[0187] In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-G via a linker. In certain embodiments, the third exogenous polypeptide comprises an amino acid sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:69.

[0188] VI. Other Optional Genome Editing In one embodiment of the above cells, the genome editing at one or more selected sites may include insertion of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genomically engineered iPSCs or their derivative cells.

[0189] In some embodiments, the exogenous polynucleotide for insertion is operably linked to (1) one or more exogenous promoters including CMV, EFla, PGK, CAG, UBC, or other constitutive, inducible, temporal, tissue, or cell type specific promoters; or (2) one or more endogenous promoters contained within selected sites including AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of a genome safe harbor. In some embodiments, the genomically engineered iPSCs generated using the above methods contain one or more different exogenous polynucleotides encoding proteins, including caspase, thymidine kinase, cytosine deaminase, B cell CD20, ErbB2, or CD79b; where, when the genomically engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safeguard loci, including AAVSI, CCR5, ROSA26, collagen, HTRP, H11, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. Other exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitor proteins, such as PD1, PD-L1, and CTLA4, as well as proteins targeting the CD47 / signal-regulatory protein alpha (SIRPα) axis.

[0190] In one embodiment, the cells may contain an exogenous polynucleotide encoding CD16 protein and / or NKG2D protein, which may be operably linked by an autoprotease peptide as disclosed in co-pending patent application No. PCT / US23 / 68079. Thus, in some embodiments, the cells of the present invention may include genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD16 and recombinant NKG2D. The surface receptor CD16 (FcγRIIIA) influences 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 mostly expressed on cells of the cytotoxic arm of the immune system, including NK cells and subsets of T cells. NKG2D is crucial for diverse 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 a cell.

[0191] In some embodiments, the polynucleotide construct encoding the CD16 protein and the NKG2D protein also comprises a polynucleotide sequence encoding a self-protease peptide or a self-cleaving peptide. In some embodiments, the exogenous polynucleotide construct encoding the CD16 protein, the NKG2D protein, and the self-cleaving peptide is introduced into iPSCs or their derived cells. The exogenous or isolated polynucleotide construct can be introduced into the locus of iPSCs or their derived cells.

[0192] In some embodiments, the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 185. In some embodiments, the exogenous polynucleotide construct encodes the amino acid sequence of SEQ ID NO: 186.

[0193] 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 the amino acid sequence 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.

[0194] 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 the wild-type CD16 of SEQ ID NO: 187, that has 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.

[0195] 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: 188. In some embodiments, the nucleic acid sequence encoding the variant CD16 protein has the sequence of SEQ ID NO: 189. In some embodiments, the wild-type CD16 protein has the sequence of SEQ ID NO: 187.

[0196] 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 the amino acid sequence set forth 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 set forth in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3. In some embodiments, the NKG2D protein is an NKG2D variant protein. In some cases, the NKG2D variant protein has an amino acid sequence having 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 a wild-type NKG2D, e.g., the wild-type NKG2D of SEQ ID NO: 190. In some embodiments, the NKG2D protein has the amino acid sequence of SEQ ID NO: 190. In some embodiments, the nucleic acid sequence encoding the NKG2D protein has the sequence of SEQ ID NO: 191.

[0197] As discussed above, constructs are provided herein that contain an autoprotease peptide sequence containing a 2A peptide that can induce ribosome skipping during polypeptide translation. The 2A peptide functions to "cleave" mRNA transcripts by causing the ribosome to skip peptide bond synthesis at the C-terminus between glycine (G) and proline (P) residues, thereby separating the end of the 2A sequence from the next downstream peptide. 2A peptides include, but are not limited to, the porcine teschovirus-1 2A (P2A) peptide, the foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, the equine rhinitis A virus (ERAV) 2A (E2A) peptide, the Thosea asigna virus 2A (T2A) peptide, the cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and the flacherie virus 2A (BmIFV2A) peptide.

[0198] 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: 192. In some embodiments, the P2A peptide has the amino acid sequence of SEQ ID NO: 192.

[0199] Another optional genome editing technique is to insert a polynucleotide encoding membrane-bound interleukin-12 (IL-12), which comprises a first polypeptide comprising the IL-12 alpha subunit p35, a second polypeptide comprising the IL-12 beta subunit p40, and a transmembrane fused to the end of the first and / or second IL-12 subunit polypeptide, as disclosed in co-pending patent application No. PCT / US23 / 68105. In certain embodiments, the polynucleotide encoding membrane-bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for activation-induced release of IL-12 by the protease ADAM17. ADAM17 is expressed by activated lymphocytes and is directly involved in the release of other immune mediators, such as TNFα, which are also presented in membrane-anchored forms. When this membrane-anchored IL-12 is expressed on engineered iNK or T cells, it remains associated with the cells. Upon cell activation and increased ADAM17 expression, proteases cleave the membrane stalk, releasing IL-12 into the extracellular space. This type of regulation ensures that IL-12 activity is limited to the peritumoral space and that engineered immune cells engage targets on tumor cells that trigger activation. Thus, the cells of the present invention may further comprise (i) an exogenous polynucleotide encoding a membrane-bound interleukin-12 (IL-12) comprising a first polypeptide comprising IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a transmembrane domain fused to the terminus of the first and / or second IL-12 subunit polypeptide.

[0200] 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.

[0201] VII. Targeted genome editing at selected loci in iPSCs According to embodiments of the present application, one or more of the exogenous polynucleotides are integrated into one or more loci on the chromosomes of the iPSCs.

[0202] Genome editing, or genomic editing, or gene editing, as used interchangeably herein, is a type of genetic manipulation in which DNA is inserted, deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (interchangeably with "targeted genomic editing" or "targeted gene editing") allows for insertion, deletion, and / or replacement at a preselected site in the genome. If an endogenous sequence is deleted or disrupted at the insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or knocked down due to the sequence deletion or disruption. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. Similarly, the term "targeted integration" is also used herein to refer to a process involving the insertion of one or more exogenous sequences into a preselected site in the genome, with or without deletion of the endogenous sequence at the insertion site.

[0203] 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.

[0204] Alternatively, targeted editing can be achieved more frequently by specifically introducing double-strand breaks (DSBs) using specific low-frequency-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, NHEJ often leads to random insertion or deletion (in / del) of a small number of endogenous nucleotides. In comparison, in the presence of a donor vector containing exogenous genetic material flanked by pairs of homologous arms, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) via homologous recombination, resulting in "targeted integration."

[0205] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) systems. Moreover, the DICE (double integrase cassette exchange) system, which utilizes phiC31 and Bxbl integrases, is also a promising tool for targeted integration.

[0206] ZFN is a targeted nuclease comprising a nuclease fused with a zinc finger DNA binding domain. By "zinc finger DNA binding domain" or "ZFBD" is meant a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within the zinc finger binding domain, whose structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not exist in nature, whose design / composition is primarily the result of rational criteria, such as the application of substitution rules and computer algorithms for processing information within database storage information of existing ZFP designs and binding data. For example, see U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; also see International Publication Nos. WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. A "selected" zinc finger domain is a domain that is not found in nature, and its production is mainly due to empirical processes such as phage display, interaction trap or hybrid selection. ZFN is described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of Fokl nuclease with the zinc finger DNA binding domain.

[0207] TALENs are targeted nucleases containing a nuclease fused to a TAL effector DNA-binding domain. By "transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" is meant the polypeptide domain of a TAL effector protein responsible for binding to DNA. TAL effector proteins are secreted by the plant pathogen Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA-binding domain, and activate gene transcription at these sequences via their transactivation domain. The specificity of the TAL effector DNA-binding domain depends on an effector-variable number of imperfect 34 amino acid repeats, which contain polymorphisms at select repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of a Fokl nuclease with a TAL effector DNA binding domain.

[0208] Another example of a targeted nuclease for use in the subject methods is a targeted Spoll nuclease, a polypeptide comprising a Spoll polypeptide with nuclease activity fused to a DNA-binding domain, e.g., a zinc finger DNA-binding domain, a TAL effector DNA-binding domain, etc., which has specificity for a DNA sequence of interest. See, e.g., U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0209] 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.

[0210] Other non-limiting examples of targeting nucleases include natural and recombinant nucleases; CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and others. For example, CRISPR / Cas9 requires two main components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When coexpressed, these two components form a complex that is recruited to target DNA sequences containing a PAM and a seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to the target sequence of choice. These two components can then be delivered into mammalian cells via transfection or transduction. As another example, CRISPR / Cpf1 contains two main components: (1) the CPf1 endonuclease and (2) the crRNA. When coexpressed, these two components form a ribonucleoprotein (RNP) complex that is recruited to target DNA sequences containing a PAM and a seeding region near the PAM. The crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpf1 to a target sequence of choice. These two components can then be delivered into mammalian cells via transfection or transduction.

[0211] 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).

[0212] DICE-mediated insertion uses a pair of recombinases, such as phiC31 and Bxbl, to provide unidirectional integration of exogenous DNA, but this unidirectional integration is strictly limited to each enzyme's own small attB and attP recognition sites. Because these target att sites do not naturally occur in mammalian genomes, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, incorporated herein by reference.

[0213] One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arms specific to the desired integration site, and the method of targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by the cellular host enzyme machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a gRNA comprising a Cpf1 expression cassette and a guide sequence specific to the desired integration site into the cell to enable Cpf1-mediated insertion. In another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a Cas9 expression cassette and a gRNA comprising a guide sequence specific to the desired integration site into the cell to enable Cas9-mediated insertion. In yet another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more att sites of a pair of DICE recombinase into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable DICE-mediated targeted integration.

[0214] The site for targeted integration includes, but is not limited to, genomic safety region.Genomic safety region is the intragenic or extragenic region of human genome that can accommodate the predictable expression of newly integrated DNA, theoretically without causing any adverse effects to host cell or organism.In certain embodiments, the genomic safety region for targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR and RUNX1 genes.

[0215] In other embodiments, the site for targeted integration is selected to delete or reduce the expression of endogenous gene at insertion site.As used herein, the term " deletion " in relation to gene expression refers to any genetic modification that negates gene expression.Examples of the " deletion " of gene expression include, for example, the removal or deletion of the DNA sequence of gene, the insertion of exogenous polynucleotide sequence at gene locus, and one or more substitutions within gene, which negates gene expression.

[0216] Genes for targeted deletion include, but are not limited to, genes for major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be histocompatible-matched in the allogeneic recipient to avoid allogeneic rejection problems. "MHC deficiency," including MHC class I deficiency or MHC class II deficiency, or both, refers to cells that lack, no longer maintain, or have reduced levels of surface expression of complete MHC complexes, including MHC class I protein heterodimers and / or MHC class II heterodimers, such that the reduced or reduced levels are lower than those naturally detectable by other cells or synthetic methods. MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p21) or by deletion or reduced expression levels of one or more MHC class I-related genes, including, but not limited to, the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin gene. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers. B2M-null cells are MHC-I-deficient. MHC class II deficiency can be achieved by functional deletion or reduction of MHC-II-related genes, including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions via activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-null cells are MHC-II-deficient. In certain embodiments, one or more exogenous polynucleotides are integrated into one or more loci of a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby deleting or reducing the expression of the integrated gene. Other genes that can be targeted for deletion include NKG2A, CD38, CD70, and CD33.

[0217] 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, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD33, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, with the proviso that at least one of the one or more loci is an MHC gene, e.g., a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP genes. Preferably, one or more exogenous polynucleotides are integrated into the locus of an MHC class-I-related gene, such as the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, or tapasin gene; and the locus of an MHC-II-related gene, such as the RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally, the locus of a safety region gene selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, and RUNX1 genes. More preferably, one or more exogenous polynucleotides are integrated into the locus of the CIITA, AAVS1, and B2M genes.

[0218] In certain embodiments, (i) a first exogenous polynucleotide is integrated into the locus of the AAVS1 gene or the CLYBL 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.

[0219] 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 one or more sequences selected from the group consisting of SEQ ID NOs: 131-156, 171-184; (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.

[0220] In certain embodiments, (i) the first exogenous polynucleotide comprises a polynucleotide sequence of one or more sequences selected from the group consisting of SEQ ID NOs: 131-156, and 171-184; (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.

[0221] VIII. Derived cells In another aspect, the present invention relates to cells derived from iPSC differentiation, i.e., derivative cells. As described above, the genome editing introduced into iPSCs is retained in the derivative cells. In certain embodiments of the derivative cells obtained from iPSC differentiation, the derivative cells are hematopoietic cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, B cells, antigen-presenting cells (APCs), monocytes, and macrophages. In certain embodiments, the derivative cells are immune effector cells, such as NK cells or T cells.

[0222] In certain embodiments, the present application provides natural killer (NK) cells or T cells comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide sequence, such as the autoprotease peptide sequence of porcine teschovirus-1 2A (P2A); and (iii) a deleted or reduced expression of an MHC class I-related gene and an MHC class II-related gene, e.g., an MHC class-I-related gene selected from the group consisting of the B2M gene, the TAP1 gene, the TAP2 gene, and the tapasin gene, and an MHC-II-related gene selected from the group consisting of the RFXANK gene, the CIITA gene, the RFX5 gene, the RFXAP gene, and the CIITA gene, preferably the B2M gene and the CIITA gene.

[0223] In certain embodiments, the NK cell or T cell further comprises a third exogenous polynucleotide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G).

[0224] Also, an NK cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 157 to 170; (ii) a second exogenous polynucleotide encoding a truncated epidermal growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin-15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66, Also provided are NK cells or T cells in which the first, second, and third exogenous polynucleotides are integrated into the loci of the AAVS1, CIITA, and B2M genes, respectively, thereby deleting or reducing the expression of CIITA and B2M.

[0225] In certain embodiments, the first exogenous polynucleotide comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 131-156 and 171-184; 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.

[0226] Also provided are CD34+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cells (iPSCs), comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope and interleukin-15 (IL-15), wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide sequence; and (iii) a deletion or reduced expression of one or more of the following genes: B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP.

[0227] In certain embodiments, the CD34+ HPCs further comprise a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

[0228] In certain embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to Nectin-4; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain, e.g., a costimulatory domain comprising a CD28 signaling domain.

[0229] Methods for producing derivative cells are also provided, which include differentiating iPSCs under conditions for cell differentiation, thereby obtaining derivative cells.

[0230] The iPSCs of the present application can be differentiated by any method known in the art. Exemplary methods are described in U.S. Patent Nos. 8,846,395, 8,945,922, 8,318,491, WO 2010 / 099539, WO 2012 / 109208, WO 2017 / 070333, WO 2017 / 179720, WO 2016 / 01014, each of which is incorporated herein by reference in its entirety. These differentiation protocols are described in WO2019 / 157597, WO2022 / 120334, WO2022 / 133169, WO2022 / 216624, WO2022 / 216514, and WO2022 / 216524. Differentiation protocols may use feeder cells or may be feeder-free. As used herein, "feeder cells" or "feeders" are terms 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 the second cell type.

[0231] In another embodiment of the present invention, the iPSC-derived cells of the present invention are NK cells prepared by a method of differentiating iPSCs into NK cells by subjecting the iPSCs 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, the IL-12-primed cells exhibit rapid cell killing compared to cells differentiated in the absence of IL-12. Furthermore, cells differentiated using IL-12 conditions exhibit improved inhibition of cancer cell growth.

[0232] IX. Polynucleotides, Vectors, and Host Cells (1) a nucleic acid encoding a CAR In another general aspect, the present invention relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful in the present invention according to an embodiment of the present application. Those skilled in the art will recognize that the coding sequence of the CAR can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, those skilled in the art will understand that the nucleic acid sequence encoding the CAR of the present application can be altered without changing the amino acid sequence of the protein.

[0233] In certain embodiments, the isolated nucleic acid encodes a CAR that targets Nectin 4. In certain embodiments, the isolated nucleic acid encoding the CAR comprises a polynucleotide sequence that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to one or more sequences selected from SEQ ID NOs: 131-156, and 171-184.

[0234] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding a CAR useful in the invention according to embodiments of the present application. In view of the present disclosure, any vector known to those skilled in the art can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element for establishing the conventional function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce a CAR in a cell. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present application.

[0235] In certain aspects, the present application provides vectors for targeted integration of CARs useful in the inventions according to embodiments of the present application. In certain embodiments, the vector comprises, from 5' to 3', an exogenous polynucleotide having: (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to embodiments of the present application; and (c) a terminator / polyadenylation signal.

[0236] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence with SEQ ID NO: 63.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0237] 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.

[0238] 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 one or more selected from the group consisting of SEQ ID NOs: 131-156 and 171-184.

[0239] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm flanking the exogenous polynucleotide. As used herein, "left homologous arm" and "right homologous arm" refer to a pair of nucleic acid sequences flanking the exogenous polynucleotide, which nucleic acid sequences facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. The sequences of the left and right homologous arms can be designed based on the intended integration site. In some embodiments, the left or right homologous arm is homologous to the left or right sequence of the integration site.

[0240] In certain embodiments, the left homologous arm comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 84, 87, 90, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213 or 215. In certain embodiments, the right homologous arm comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 85, 88, 91, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216.

[0241] 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. Table 3 provides a list of exemplary homology arm sequences and corresponding guide sequences to facilitate integration of an exogenous polynucleotide at various loci.

[0242] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21 Table 6-22 Table 6-23 Table 6-24

[0243] (2) a nucleic acid encoding an inactivated cell surface receptor In another general aspect, the present invention relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful in the invention according to the embodiments of the present application. Those skilled in the art will recognize that the coding sequence of an inactivated cell surface receptor can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will recognize that the nucleic acid sequence encoding the inactivated cell surface receptor of the present application can be altered without changing the amino acid sequence of the protein.

[0244] In certain embodiments, the isolated nucleic acid encodes any of the inactivated cell surface receptors described herein, e.g., a monoclonal antibody-specific epitope, and / or a cytokine, e.g., IL-15 or IL-2, wherein the monoclonal antibody-specific epitope and cytokine are optionally operably linked by an autoprotease peptide sequence.

[0245] In some embodiments, the isolated nucleic acid is selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, and certolizumab. The isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody such as pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, or ustekinumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by cetuximab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by trastuzumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by bevacizumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by avelumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by ipilimumab.

[0246] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epidermal growth factor receptor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab, or panitumumab, preferably cetuximab.

[0247] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, e.g., epitopes that are specifically recognized by polatuzumab vedotin.

[0248] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, eg, epitopes specifically recognized by rituximab.

[0249] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of the Her2 receptor, eg, epitopes specifically recognized by trastuzumab.

[0250] In certain embodiments, the autoprotease peptide sequence is porcine teschovirus-1 2A (P2A).

[0251] 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.

[0252] 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:78.

[0253] 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:80.

[0254] 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] In certain embodiments, the isolated nucleic acid encoding the inactivated cell surface receptor comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:75, preferably the polynucleotide sequence of SEQ ID NO:75.

[0259] 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.

[0260] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful in the invention according to embodiments of the present application. Any vector known to those of skill in the art in light of the present disclosure can be used, for example, a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids into cells are known in the art and can be used herein to produce inactivated cell surface receptors in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present application.

[0261] In certain aspects, the present application provides vectors for targeted integration of inactivated cell surface receptors useful in the inventions according to embodiments of the present application. In certain embodiments, the vector comprises, from 5' to 3', an exogenous polynucleotide having: (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, for example, an inactivated cell surface receptor comprising a truncated epidermal growth factor receptor (tEGFR) variant, and interleukin-15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide sequence, for example, porcine teschovirus-1 2A (P2A); and (c) a terminator / polyadenylation signal.

[0262] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence with SEQ ID NO: 63.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0263] 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.

[0264] 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.

[0265] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm flanking the exogenous polynucleotide.

[0266] In certain embodiments, the left homologous arm comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 84. In certain embodiments, the right homologous arm comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 85.

[0267] 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.

[0268] (3) a nucleic acid encoding an HLA construct In another general aspect, the present invention relates to an isolated nucleic acid encoding an HLA construct useful in the invention according to the embodiments of the present application. Those skilled in the art will recognize that the coding sequence of an HLA construct can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will recognize that the nucleic acid sequence encoding the HLA construct of the present application can be altered without changing the amino acid sequence of the protein.

[0269] In certain embodiments, the isolated nucleic acid encodes an HLA construct comprising a signal peptide, e.g., an HLA-G signal peptide, operably linked to an HLA coding sequence, e.g., the coding sequence for mature B2M and / or mature HLA-E. In some embodiments, the HLA coding sequence encodes HLA-G and B2M operably linked by a 4xGGGGS linker, and / or B2M and HLA-E operably linked by a 3xGGGGS linker. In certain embodiments, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO:67, preferably the polynucleotide sequence of SEQ ID NO:67. In another embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO:70, preferably the polynucleotide sequence of SEQ ID NO:70.

[0270] In another general aspect, the present application provides a vector comprising a polynucleotide sequence encoding an HLA construct useful in the invention according to embodiments of the present application. Any vector known to those skilled in the art in light of the present disclosure can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids into cells are known in the art and can be used herein to produce HLA constructs in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present application.

[0271] In certain aspects, the present application provides vectors for targeted integration of HLA constructs useful in the inventions according to embodiments of the present application. In certain embodiments, the vector comprises, from 5' to 3', an exogenous polynucleotide having: (a) a promoter; (b) a polynucleotide sequence encoding the HLA construct; and (c) a terminator / polyadenylation signal.

[0272] In certain embodiments, the promoter is a CAG promoter.In certain embodiments, the CAG promoter comprises at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical polynucleotide sequence with SEQ ID NO: 63.Other promoters can also be used, examples of which include but are not limited to EF1a, UBC, CMV, SV40, PGK1 and human beta-actin.

[0273] 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.

[0274] In certain embodiments, the polynucleotide sequence encoding the HLA construct comprises a signal peptide, such as the HLA-G signal peptide, mature B2M, and mature HLA-E, wherein HLA-G and B2M are operably linked by a 4xGGGGS linker (SEQ ID NO: 31), and the B2M transgene and HLA-E are operably linked by a 3xGGGGS linker (SEQ ID NO: 25). In certain embodiments, the HLA construct comprises a polynucleotide sequence at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLA construct comprises a polynucleotide sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0275] In some embodiments, the vector further comprises a left homologous arm and a right homologous arm flanking the exogenous polynucleotide.

[0276] In certain embodiments, the left homologous arm comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 87. In certain embodiments, the right homologous arm comprises a polynucleotide sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 88.

[0277] 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.

[0278] (4) Host cells In another general aspect, the present application provides a host cell comprising an isolated nucleic acid encoding a vector of the present application and / or a construct of the present application. Any host cell known to those of skill in the art in light of the present disclosure can be used for recombinant expression of the exogenous polynucleotide of the present application. According to certain embodiments, the recombinant expression vector is transformed into the host cell by conventional methods, such as chemical transfection, heat shock, or electroporation, where the vector is stably integrated into the host cell genome, thereby efficiently expressing the recombinant nucleic acid.

[0279] Examples of host cells include, for example, recombinant cells containing a vector or isolated nucleic acid of the present application, useful for producing a vector or construct of interest; or engineered iPSCs or their derivatives containing one or more isolated nucleic acids of the present application, preferably integrated into one or more chromosomal loci. Host cells for the isolated nucleic acids of the present application can also be immune effector cells, such as T cells or NK cells, containing one or more isolated nucleic acids of the present application. Immune effector cells can be obtained by differentiation of the engineered iPSCs of the present application. Any suitable method in the art can be used for differentiation in light of the present disclosure. Immune effector cells can also be obtained by transfecting one or more isolated nucleic acids of the present application into immune effector cells.

[0280] composition In another general aspect, the application provides compositions comprising an isolated polynucleotide of the application, a host cell of the application and / or an iPSC or a derived cell thereof.

[0281] In certain embodiments, the composition further comprises one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (iMiD).

[0282] In certain embodiments, the composition is a pharmaceutical composition comprising the isolated polynucleotide of the present application, the host cell of the present application and / or the iPSC or derived cell thereof, and a pharmaceutically acceptable carrier. The term "pharmaceutical composition," as used herein, refers to a product comprising the isolated polynucleotide of the present application, the isolated polypeptide of the present application, the host cell of the present application, and / or the iPSC or derived cell thereof of the present application together with a pharmaceutically acceptable carrier. The polynucleotides, polypeptides, host cells, and / or the iPSC or derived cell thereof of the present application and compositions comprising them are also useful for the manufacture of medicaments for the therapeutic applications mentioned herein.

[0283] As used herein, the term "carrier" refers to any excipient, diluent, bulking agent, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It is understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the compositions described herein. In certain embodiments, any pharmaceutically acceptable carrier suitable for use with polynucleotides, polypeptides, host cells, and / or iPSCs or their derivative cells can be used in light of the present disclosure.

[0284] The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, from Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity adjusters, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present application.

[0285] 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 the type of cancer that usually appears as a solid tumor, a "clinically detectable" tumor is one that can be detected based on the 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 obtained from patients.

[0286] 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, thymus cancer, bone cancer, and cerebral cancer, as well as cancer of unknown primary cancer (CUP).

[0287] Cancer cells in an individual can be immunologically distinct from normal somatic cells in the individual (i.e., cancerous tumors can be immunogenic). For example, cancer cells can induce a systemic immune response in an individual against one or more antigens expressed by cancer cells. The tumor antigen that induces the immune response can be specific to cancer cells or can be shared by one or more normal cells in the individual.

[0288] 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.

[0289] In particular, cancer cells of individuals suitable for the treatment described herein express the antigen Nectin-4. Nectin-4 is frequently expressed in bladder cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and esophageal cancer. The highest expression levels of Nectin-4 are found in bladder cancer, breast cancer, lung cancer, and pancreatic cancer. Clinical validation of Nectin-4 as a tumor target has been demonstrated by the approval of enfortumab vedotin for the treatment of urothelial cancer.

[0290] The individual suitable for the above-mentioned treatment may be a mammal. In a preferred embodiment, the individual is a human. In another preferred embodiment, a non-human mammal, particularly a mammal that has traditionally been used as a model for demonstrating therapeutic efficacy in humans (e.g., mouse, primate, pig, dog, or rabbit) may be used.

[0291] 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.

[0292] Individuals 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 involve identifying specific cell types (such as cancer cells) in samples of body fluids or tissue obtained from the individual.

[0293] An anti-tumor 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. An "anti-tumor 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 methods of the present invention, to prevent the development of an initial tumor.

[0294] 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, reducing the rate of progression, halting the rate of progression, recovering from the condition, curing or ameliorating (partially or totally) the condition, preventing, slowing, alleviating or halting one or more symptoms and / or signs of the condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0295] Treatment can also be preventative (i.e., 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 the individual.

[0296] In particular, treatment can include inhibiting cancer growth, including complete cancer remission, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indicators that indicate changes within a cancer to a more advanced form. Thus, indicators for measuring inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), slowing tumor growth, destruction of tumor vasculature, improved performance in delayed-type hypersensitivity skin tests, an increase in T-cell activity, and a decrease in the level of tumor-specific antigens. Administration of modified T-cells as described herein can improve an individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the individual's propensity for cancer growth.

[0297] The present application provides a method of treating a disease or condition in a subject in need thereof. The method includes administering a therapeutically effective amount of a cell of the present application and / or a composition of the present application to a subject in need thereof. In certain embodiments, the disease or condition is cancer. The cancer may be, for example, a solid cancer or a liquid cancer. The cancer may 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).

[0298] According to an embodiment of the present application, the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and / or an iPSC or its derivative. As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that induces a desired biological or medical response in a subject. A therapeutically effective amount can be empirically and routinely determined for a given purpose.

[0299] As used herein in reference to the cells of the present application and / or pharmaceutical compositions of the present application, a therapeutically effective amount means the amount of cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0300] According to certain embodiments, a therapeutically effective amount refers to the amount of therapy sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition being treated, or the symptoms associated therewith; (ii) reducing the duration of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iv) reversing the disease, disorder, or condition being treated, or the symptoms associated therewith; (v) preventing the occurrence or onset of the disease, disorder, or condition being treated, or the symptoms associated therewith. (vi) prevent the recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increase the survival of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder or condition being treated, or symptoms associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic efficacy of another therapy.

[0301] The therapeutically effective amount or dosage can vary depending on a variety of factors, such as the disease, disorder, or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages are optimally designed to optimize safety and efficacy.

[0302] 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.

[0303] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered by any convenient method known to those of skill in the art. For example, the cells of the present application can be administered to a subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation, and / or transplantation. Compositions comprising the cells of the present application can be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intrapleurally, by intravenous (iv) injection, or intraperitoneally. In certain embodiments, the cells of the present application can be administered with or without lymphodepletion of the subject.

[0304] Pharmaceutical compositions comprising the cells of the present application can be provided in a sterile liquid formulation, typically buffered to a selected pH, typically an isotonic aqueous solution with a cell suspension, or optionally as an emulsion, dispersion, or the like. The composition can include a carrier suitable for maintaining cell integrity and viability and for administration of the cell composition, e.g., water, saline, phosphate-buffered saline, etc.

[0305] Sterile injectable solutions can be prepared by incorporating the cells of the present application, optionally with various other ingredients, in an appropriate amount of an appropriate solvent. Such compositions may contain pharmaceutically acceptable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, etc., suitable for use in cell compositions and administration to subjects, such as humans. Buffers suitable for providing cell compositions are well known in the art. Any vehicle, diluent, or additive used is compatible with maintaining the integrity and viability of the cells of the present application.

[0306] The cells and / or pharmaceutical compositions of the present application can be administered in any physiologically acceptable medium. Cell populations containing the cells of the present application may include purified populations of cells. Those skilled in the art can easily determine the purity of cells in a cell population using various well-known methods. The purity range of a cell population containing genetically modified cells of the present application may be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. Dosages can be easily adjusted by those skilled in the art; for example, a decrease in purity may require an increased dosage.

[0307] The cells of the present application are generally administered as a dose based on cells per kilogram (cells / kg) of body weight of the subject to whom the cells and / or pharmaceutical compositions comprising the cells are administered. Generally, cell doses range from about 10 to 150 mg / kg, depending on the mode and location of administration. 4 ~about 10 10 Pieces / kg body weight, e.g., about 10 5 ~about 10 9 , about 10 5 ~about 10 8 , about 10 5 ~about 10 7 , or about 10 5 ~about 10 6Generally, higher doses are used for systemic administration than for local administration, where the immune cells of the present application are administered to the area of ​​the tumor and / or cancer. Exemplary dose ranges include 1 x 10 4 ~1×10 8 , 2 × 10 4 ~1×10 8 , 3×10 4 ~1×10 8 , 4×10 4 ~1×10 8 , 5×10 4 ~6×10 8 , 7×10 4 ~1×10 8 , 8×10 4 ~1×10 8 , 9×10 4 ~1×10 8 , 1×10 5 ~1×10 8 , 1×10 5 ~9×10 7 , 1×10 5 ~8×10 7 , 1×10 5 ~7×10 7 , 1×10 5 ~6×10 7 , 1×10 5 ~5×10 7 , 1×10 5 ~4×10 7 , 1×10 5 ~4×10 7 , 1×10 5 ~3×10 7 , 1×10 5 ~2×10 7 , 1×10 5 ~1×10 7 , 1×10 5 ~9×10 6 , 1×10 5 ~8×10 6 , 1×10 5 ~7×10 6 , 1×10 5 ~6×10 6 , 1×10 5 ~5×10 6 , 1×10 5 ~4×10 6 , 1×10 5 ~4×106 , 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 ~4×10 7 , 2 × 10 5 ~3×10 7 , 2 × 10 5 ~2×10 7 , 2 × 10 5 ~1×10 7 , 2 × 10 5 ~9×10 6 , 2 × 10 5 ~8×10 6 , 2 × 10 5 ~7×10 6 , 2 × 10 5 ~6×10 6 , 2 × 10 5 ~5×10 6 , 2 × 10 5 ~4×10 6 , 2 × 10 5 ~4×10 6 , 2 × 10 5 ~3×10 6 , 2 × 10 5 ~2×10 6 , 2 × 10 5 ~1×10 6 , 3×10 5 ~3×10 6 These dosages include, but are not limited to, doses per kg, etc. Moreover, dosages can be adjusted to account for whether a single dose or multiple doses are administered. The precise determination of what is considered an effective dose can be based on factors individual to each subject.

[0308] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to the restoration or reversal of at least one measurable physical parameter associated with cancer that may, but is not necessarily, be discernible in the subject. The terms "treat," "treating," and "treatment" may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the alleviation, prevention of the onset or onset, or reduction in the duration of one or more symptoms associated with a disease, disorder, or condition, e.g., a tumor or, more preferably, cancer. In certain embodiments, "treat," "treating," and "treatment" refer to the prevention of recurrence of a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to an increase in survival time of a subject with a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0309] The cells and / or 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). In certain embodiments, the one or more therapeutic agents comprise antibodies. In certain embodiments, the one or more therapeutic agents are selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab. The therapeutic agents may comprise one or more antibodies independently selected from the group consisting of vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab. In certain embodiments, the therapeutic agent or agents comprise cetuximab. In certain embodiments, the therapeutic agent or agents comprise trastuzumab. In certain embodiments, the therapeutic agent or agents comprise bevacizumab. In certain embodiments, the therapeutic agent or agents comprise avelumab. In certain embodiments, the therapeutic agent or agents comprise ipilimumab. [Example]

[0310] Abbreviation

[0311] [Table 7]

[0312] [Example 1] Preparation of phagemid libraries Objective: To large-scale clone VHH libraries into phagemids and generate glycerol stocks for subsequent phage production. material: Cell: MC1061F' (Lucigen #60512-2) Buffer: - D-PBS (Dulbecco's Phosphate Buffered Saline; Life Technologies #14190) - TBST (Tris-buffered saline with 0.05% Tween-20, Sigma #79039-10PAK) - PBST (D-PBS with 0.05% Tween-20) - Tween-20 (Sigma #P-7949) - PBST-M (PBST with 3% skim milk powder) - 2 x YT (Teknova #Y0167) - LB (Carb) agar plates (Teknova #L1010) - Carbenecillin, also known as "Carb" (Novagen #69101) - Concentration = 100mg / mL - Kanamycin, also known as "Kan" (Sigma #60615) - Concentration = 35mg / mL - Tetracycline, also known as "Tet" (Sigma #T3383-25g) - Concentration = 15mg / mL - IPTG (Isopropyl β-D-1-thiogalactopyranoside; Sigma #69101) - Concentration = 1M - PEG / NaCl (Teknova #P4138) - M13K07(Antibody Design Labs #NC1591729) enzyme: - NcoI (NEB #R3193L) - XhoI(NEB #R0146L) - T4 DNA ligase (Invitrogen #15224090) kit: - PCR cleanup kit (Qiagen, #28104) - QIAquick Gel Extraction Kit (Qiagen, #28704) Oligonucleotides: - PelB_F ATGAAATACCTATTGCCTACGGCAGCC - P3_Spe_R CTCAGAACCGCCACCTTCACTAGT

[0313] procedure: The VHH V2-medium library was PCR amplified using PelB_F+P3_Spe_R oligonucleotides for 13 cycles at an annealing temperature of 60°C. The VHH library fragments were gel-isolated and digested with NcoI+XhoI restriction enzymes at 37°C for 1.5 hours. The digested DNA was then column-purified. Similarly, the V2-short library (previously amplified DNA) was digested with NcoI+XhoI restriction enzymes at 37°C for 1.5 hours. The digested DNA was then column-purified. 15 μg of P222 vector was also digested with NcoI+XhoI at 37°C for 1.5 hours and isolated by gel isolation. Ligation was performed by combining 4 μg of P222 vector with 1.376 μg of VHH V2 short or medium fragments at room temperature for 6 hours, followed by overnight at 18°C. The ligation product was column purified using the Qiaquick MinElute PCR purification kit and brought to 22 uL dH20.

[0314] Library transformation 500 μL of MC1061F' cells were added to each ligation. Approximately 26 μL was aliquoted into each of 20 1 mm gap BTX electroporation cuvettes. Each aliquot was electroporated, resuspended in 1 mL of recovery medium, and then transferred to a 125 mL shake flask. Each cuvette was then filled with 1 mL of medium and transferred to a 125 mL shake flask for a total volume of approximately 40 mL. The cells were then grown by placing the flasks at 37°C for 1 hour. 10 μL of cell culture was diluted with 90 μL of medium, and six dilutions were performed. Dilutions 4, 5, and 6 were plated on LB(Carb) agar plates and incubated overnight at 37°C. V2-short chain: 4.0×10^9 transformants V2-medium chain: 1.95 x 10^9 transformants

[0315] [Table 8]

[0316] Cell cultures were grown in 1 L of 2xYT (Carb) at 37°C and the OD600 was monitored hourly. When the OD600 reached 1, samples were harvested by centrifugation at 4800 x G and resuspended in 50 mL of 2xYT (Carb, 20% glycerol). Samples were aliquoted in 5 x 10 mL volumes into 15 mL conical tubes and stored at -80 Celsius.

[0317] [Example 2] Generation of phage from VHH phagemid libraries material: cell: - MC1061F' (Lucigen #60512-2) Buffer: - D-PBS (Dulbecco's Phosphate Buffered Saline; Life Technologies #14190) - TBST (Tris-buffered saline with 0.05% Tween-20, Sigma #79039-10PAK) - PBST (D-PBS with 0.05% Tween-20) - Tween-20 (Sigma #P-7949) - PBST-M (PBST with 3% skim milk powder) - 2 x YT (Teknova #Y0167) - LB (Carb) agar plates (Teknova #L1010) - Carbenicillin, also known as "Carb" (Novagen #69101) - Concentration=100mg / mL - Kanamycin, also known as "Kan" (Sigma #60615) - Concentration = 35mg / mL - Tetracycline, also known as "Tet" (Sigma #T3383-25g) - Concentration = 15mg / mL - IPTG (Isopropyl β-D-1-thiogalactopyranoside; Sigma #69101) - Concentration = 1M - PEG / NaCl (Teknova #P4138) - M13K07(Antibody Design Labs #PH010L)

[0318] procedure: Helper phage infection: VHH phagemid libraries were prepared as shown in Figure 3B. 10 mL of library glycerol stock was thawed for each library (short and medium chain). The glycerol stock was diluted with 990 mL of 2xYT (Carb) into two 1 L flasks. Samples were grown at 37°C with shaking at 225 rpm to an OD of 0.6. 1 mL of M13K07 was added to the culture mix and grown at 37°C for 30 minutes. 1 mL of kanamycin and 1 mL of IPTG were added, and the mixture was incubated overnight at 30°C at 225 rpm.

[0319] Phage recovery: Cells from overnight cultures were centrifuged at 4400 x g for 15 minutes at 4°C. The supernatant was transferred to a new tube and the centrifugation was repeated to remove all cells. The supernatant was transferred to a 1 L bottle and 100 mL of PEG / NaCl was added. The mixture was placed on ice for 1 hour, transferred to a 50 mL centrifuge tube, and the phage was centrifuged at 13000 x g for 10 minutes at 4°C. The supernatant was discarded and the phage resuspended in 100 mL of D-PBS. The sample was centrifuged at 8000 x g for 5 minutes at 4°C, and the supernatant was collected, aliquoted into cryovials, and frozen at -80°C.

[0320] Titer: 10 μL of the remaining phage was used to perform titration on MC1061F' cells. MC1061F' cells were grown in 45 mL of 2xYT (Tet) to an OD of 0.5. 10 μL of phage was serially diluted 1:10 on plates (12 total dilutions). 10 μL of each dilution was added to 90 μL of MC1061F' cells and incubated at 37°C for 30 minutes. Each infected well was spotted onto an LB(Carb) agar plate, and the total well volume of dilutions 8–10 was plated onto separate plates and incubated overnight at 37°C. Additionally, 8th and 9th dilutions of the MC1061-infected cells were plated onto LB(Carb) agar plates. All libraries were titered up to the 9th dilution. Plate counts were as follows: V2S dilution 9:96 colonies -> 1.9×10^12pfu / mL V2M dilution 9: 124 colonies -> 2.4 x 10^12 pfu / mL

[0321] [Example 3] Phage panning and screening against Nectin-4 protein Three rounds of phage panning with the Century VHH phage library were performed against plate-bound Nectin4-HIS protein (AcroBiosystems #NE4-H52H3), and individual colonies were screened by ELISA using periplasmic extracts (PPE).

[0322] Phage panning material: Libraries (prepared according to Examples 1 and 2): VHH-V2-short chain VHH-V2-medium chain cell: - MC1061F' (Lucigen #60512-2) Buffer: - D-PBS (Dulbecco's Phosphate Buffered Saline; Life Technologies #14190) - TBST (Tris-buffered saline with 0.05% Tween-20, Sigma #79039-10PAK) - PBST (D-PBS with 0.05% Tween-20) - Tween-20 (Sigma #P-7949) - PBST-M (PBST with 5% skim milk powder) - 2 x YT (Teknova #Y0167) - LB (Carb) agar plates (Teknova #L1010) - Carbenicillin, also known as "Carb" (Novagen #69101) - Concentration = 100mg / mL - Kanamycin, also known as "Kan" (Sigma #60615) - Concentration = 35mg / mL - Tetracycline, also known as "Tet" (Sigma #T3383-25g) - Concentration = 15mg / mL - IPTG (Isopropyl β-D-1-thiogalactopyranoside; Sigma #69101) - Concentration = 1M - PEG / NaCl (Teknova #P4138) - M13K07(NEB #N0315S) enzyme: - NheI (NEB #R0131M) - SpeI (NEB #R0133M) - T4 DNA ligase (Invitrogen #15224090) kit: - PCR cleanup kit (Qiagen, #28104) - QIAquick Gel Extraction Kit (Qiagen, #28704) - QIAprep Spin Miniprep Kit (Qiagen #27106) protein - Nectin 4 (AcroBiosystems #NE4-H52H3) - Streptavidin (VWR #VWRVE497-5MG) - Human IgG (whole molecule; Jackson ImmunoResearch #009-000-003)

[0323] method: Phage selection of Nectin-4 VHH phage: On day 0, four wells of a maxisorp plate were coated with 5 μg / mL (200 μL / well) Nectin-4 and incubated overnight at 4°C. On day 1, for the first round of panning, MC1061F' cultures were started using 0.5 mL glycerol stock in 25 mL of 2xYT + 25 μL Tet in a 250 mL flask and incubated at 37°C until an OD600 of 0.6 was reached (approximately 2.5 hours). Four Eppendorf tubes were blocked with 400 μL of PBST-M per well. Nectin-4 was removed from the wells of the maxisorp plate. The Nectin-4-coated wells were blocked with 300 μL of PBST-M.

[0324] For library blocking, 200 μL of each library was added to 200 μL of PBST-M in a blocked tube containing 10 μg / mL human IgG (whole molecule) and incubated at room temperature for 45 minutes. To allow antigen binding, the PBST-M was discarded from the maxisorp plate, and 200 μL of the blocked library was added to each Nectin-4-coated well and incubated at room temperature for 45 minutes. Washing was performed by discarding the phage mix from the maxisorp plate and washing each well six times with PBS-T and once with PBS. For phage infection and propagation, 200 μL of MC1061F' cells (OD600 = 0.7) were added to each library well of the maxisorp plate and incubated at 37°C for 30 minutes. 10 μL was removed for six serial 1:10 dilutions, and 2 μL was spotted onto LB (carb / glucose) plates to determine output titers and incubated overnight at 37°C. Five-fold dilutions were plated for single-colony sequencing. The remaining MC1061F' cells were harvested and grown in 10 mL of 2xYT (Carb) to an OD of 0.6. Each culture was infected with 10 μL of helper phage (NEB) for 30 minutes at 37°C. 10 μL of IPTG and kanamycin were added, and the cultures were grown overnight at 30°C. Four wells of a maxisorp plate were coated with 2 μg / mL (200 μL / well) of nectin 4 and incubated overnight at 4°C.

[0325] On day 2, for the second round of panning, the overnight phage culture from the first round of panning was centrifuged at 6000 x g for 10 minutes. The phage was precipitated with 0.2 volumes of PEG / NaCl on ice for 30 minutes. The phage was then centrifuged at 13000 x g for 10 minutes and resuspended in 1:10 volume of PBS (this is the round 2 input phage). Round 2 panning was completed following the same steps as described for round 1 panning.

[0326] On day 3, the steps from day 2 were repeated. After phage elution in MC1061F' cells, the cells were grown overnight at 37°C for DNA preparation and pIII excision.

[0327] On day 4, each library panning plasmid DNA was miniprepped (Qiagen Plasmid Miniprep Kit). 10 uL of miniprep DNA was digested with NheI and SpeI restriction enzymes for 1 hour at 37°C.

[0328] [Table 9]

[0329] The samples were run on a 1% agarose gel and the 5 kb vector band was gel extracted. The DNA was purified in 30 uL of EB using a Qiagen gel extraction kit. 3 uL of the isolated DNA was ligated with T4 DNA ligase for 1 hour.

[0330] [Table 10]

[0331] The ligated DNA was column purified (Qiagen PCR purification kit) with 30 μL of dH2O. MC1061F' cells were electroporated in a 1 mm electroporation cuvette with 3 μL of ligated DNA at 1.8 kV, 200 ohms, and 50 μF. Cells were rescued by adding 950 μL of SOC medium at room temperature and diluted 1:10 in triplicate with 90 μL of SOC. Dilutions 2 and 3 were plated on LB(Carb) agar plates and incubated overnight at 37°C. screening material: cell: - MC1061F' (Lucigen #60512-2) Enzymes and kits: - XhoI(NEB R0416L) - NEBuilder HiFi Assembly Master Mix (E2621L) - NEB5-alpha cells (NEB #C2987H) - Q5 2x Master Mix (NEB #M0492L) - OneShot Stable3 cells (Life Technologies #C7373-03) - Qiagen Gel Extraction Kit (Qiagen, #28706) - Qiaquick Mini Prep Kit (Qiagen #27106) - Qiagen PCR Purification Kit (Qiagen #28106) Buffer: - D-PBS (Dulbecco's Phosphate Buffered Saline; Life Technologies #14190) - TBST (Tris-buffered saline with 0.05% Tween-20, Sigma #79039-10PAK) - PBST (D-PBS with 0.05% Tween-20) - Tween-20 (Sigma #P-7949) - PBST-M (PBST with 3% skim milk powder) - PPB (Teknova 101320-468) - 2 x YT (Teknova #Y0167) - LB (Carb / 20% glucose) agar medium (Teknova #L1801) - Carbenicillin, also known as "Carb" (Novagen #69101) - Concentration = 100mg / mL - IPTG (Isopropyl β-D-1-thiogalactopyranoside; Sigma #69101) - Concentration = 1M - PEG / NaCl (Teknova #P4138) Antibodies and detection reagents - Anti-Flag:HRP (Sigma Aldrich #F1804) - Nectin 4 (AcroBiosystems #NE4-H52H3) - Ultra TMB(ThermoFisher #34028) - Stop solution (ThermoFisher #N600) Oligonucleotide sequence - P268VHH_F GCTTAGCGGAGCCAGATGCGAGGTACAACTTTTGGAGTCAGGC - P268VHH_R CGGACCGTATTTGGACTCGCTCGAGACCGTCACCTGGGT - MARS_VHH_F CTTTCAGGCGCGCTGTGAGGTACAACTTTTGGAGTCAGGC - IgG1_VHH_R GTCACAACTCTTAGGTTCGCTCGAGACCGTCACCTGGGT

[0332] procedure: Periplasmic protein preparation: Bacteria for phage panning were grown in 96-deep-well plates. Each colony was grown in 1 mL of 2xYT (Carb) at 37°C with shaking until turbid. 5 μL was spotted onto an LB (Carb) rectangular agar plate and incubated overnight at 30°C. 100 μL of 2xYT (Carb) with a final concentration of 1 μL IPTG / mL was added, and the culture was grown overnight at 30°C with shaking. Cells were harvested by centrifugation at 4,800 × g for 10 minutes. The pellet was resuspended in 100 μL of PBP, and the cells were kept on ice for 20 minutes. The cell suspension was centrifuged at 4,800 × g for 10 minutes at 4°C. All supernatant was removed, and the pellet was resuspended in 100 μL of ice-cold 5 mM MgSO4. The mixture was incubated on ice for 20 minutes with occasional shaking, and then centrifuged at 4800 xg for 10 minutes at 4°C.

[0333] ELISA: Eight Maxisorp plates were coated with 100uL / well of 1ug / mL Nectin-4 overnight at 4°C. Plates were emptied, and 200uL / well of SuperBlock was added to all plates and incubated for 1 hour at room temperature. Plates were washed once with TBST in an Aquamax plate washer. 60uL of anti-Flag:HRP (1:10,000 in PBS-T with 1:10 Superblock) was added per well to the V-bottom plate of each library screening plate. 60uL / well of PPE was added to each well of the V-bottom plate and incubated for 1 hour at room temperature. 50uL of PPE / anti-Flag mix was transferred to each ELISA plate and incubated for 1 hour at room temperature. Plates were washed three times with TBST in an Aquamax plate washer. 50uL / well of TMB Ultra solution was added and incubated for 30 minutes at room temperature until the wells turned blue. 50 uL / well of stop solution was added and absorbance was read at 450 nm on a Spectramax i3x plate reader. 54 unique clones were identified from the two libraries. A subset of 20 clones was selected from V2S and V2M and cloned into P201 (SEQ ID NOs: 111-130).

[0334] [Example 4] VHH-Fc shows specific binding to CHO-Nectin4 cells Fourteen anti-nectin-4 VHH-Fc were screened for binding to nectin-4-positive cell lines, as shown in Figure 6. All 14 VHH-Fc showed specific binding to CHO-nectin-4 cells, and 12 of the 14 cell lines showed specific binding to the nectin-4-positive tumor cell line T47D. material: - BD Staining Buffer, BSA (BD Pharmingen, Cat. No. 554657) - EDTA, 0.5M (Corning, Catalog No. 46-034-CI) Pluronic™ F-68 Nonionic Surfactant (100x) (Gibco, Catalog No. 24040032) - LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Thermo, Catalog No. L10119; 1:1000 dilution) - PE Goat Anti-hu Fc (JIR, Cat. No. 109-116-098 Stock 1mg / ml, Polyclonal; 1ug / ml) - Innovex Fc receptor blocker (Innovex Biosciences, catalog number NB309) - hu IgG1, k isotype (Biolegend, Cat. No. 403502, Clone: ​​QA16A12, Concentration 1mg / ml, 6.67uM) - Staining buffer: BD staining buffer + 2mM EDTA (add 2ml of EDTA to a 500ml bottle of BD staining buffer) - Running buffer: BD staining buffer + 1 mM EDTA + 0.1% pluronic acid (add 1 ml of EDTA and 5 ml of pluronic acid to a 500 ml bottle of BD staining buffer) procedure: 200ul of cell suspension was collected and counted in VI-cell.

[0335] [Table 11]

[0336] Fifty thousand cells were transferred to a V-bottom PP 96-well plate (to perform the staining). 150 μl of FACS buffer was added to serve as a wash solution. The plate was centrifuged at 300 × g for 3 minutes at 4-8°C. The supernatant was aspirated and the plate was briefly vortexed to disperse the cell pellet. 50 μl of Innovex Fc Blocker was added and the plate was incubated at room temperature for 15-20 minutes. The plate was centrifuged at 300 × g for 3 minutes at 4-8°C. The supernatant was aspirated and the plate was briefly vortexed to disperse the cell pellet. The cells were resuspended in the staining conditions (50 nM = 7.5 μg / ml for mAb and 50 nM = 4 μg / ml for VHH-Fc). The plate was incubated at 4°C for 30 minutes, protected from light exposure. 150 μl of staining buffer was added to the wells (to serve as a wash step). The plate was centrifuged at 300 × g for 3 minutes at 4-8°C. The supernatant was aspirated, and the cells were resuspended in 50 μl of staining solution (NearIR + PE anti-hu, IgG, Fc, 1 μg / ml; or NearIR only for PE mAb control). The plate was incubated at 4°C for 30 minutes and protected from light exposure. 150 μl of staining buffer was added to the wells (serving as a wash step), and the plate was centrifuged at 300 × g for 3 minutes at 4-8°C. The supernatant was aspirated, and 200 μl of staining buffer was added to the wells (serving as a wash step). The plate was centrifuged at 300 × g for 3 minutes at 4-8°C and vortexed to disperse the cell pellet. The cells were resuspended in 35 μl of staining buffer and analyzed using an IntelliCyt iQue instrument.

[0337] [Example 5] Jurkat_Nur77 reporter assay for tonic signaling and activation by Nectin-4 positive cell lines The functional activity of anti-nectin 4 VHH-CARs was evaluated using a Nurkat activation assay. Anti-nectin 4 VHH binding molecules were identified using a phage library as described herein, and nine VHHs were selected for CAR formatting based on robust VHH-Fc binding to CHO-nectin 4 and T47D cell lines (see, e.g., Example 4 and Figure 6). The Nur77-Jurkat reporter line (Nurkat) was transfected with CAR lentivirus, and GFP expression associated with CAR expression was assessed by flow cytometry. In this reporter line, GFP expression is associated with Nur77. Therefore, background GFP expression corresponds to tonic signaling. Transfected Nurkat cells were screened for activation after co-culture with target cells.

[0338] Cells were transfected on day 0. 0.66 × 10 cells with a viability of 98% 6 Cells were counted in Vi-cell. Cells were centrifuged at 300 × g for 5 minutes to pellet Nur77-Jurkat cells and 0.45 × 10 cells were added to R10 medium containing 3 μg / ml of polybrene (Boston BioProducts, 10 mg / ml). 6 The cells were resuspended at 1000 cells / ml and plated into a 24-well plate at a total of 200,000 cells per well. 15 μl of lentivirus was added and the plate was centrifuged at 1300 x g for 45 minutes at 32°C. 1 ml of fresh R10 medium was added to a final volume of approximately 1.5 ml and incubated at 37°C.

[0339] Activation assays were set up on day 3. 50 × 10 3 Cells were stained for GFP and CAR expression (Protein A, anti-VHH, MSLN protein), respectively. Cells were also stained with Nectin4-HIS protein (Acro Biosystems #NE4-H52H3, lot 2823a-214VF1-WR; 0.4 mg / ml stock) at a concentration of 2 μg / ml. FACS staining of CD3 and GFP was performed on activated CAR-Jurkat cells.

[0340] The results for tonic signaling, activation, and IL-2 secretion are provided in Figures 8B–8D, respectively. CAR expression was confirmed for each construct, with positive CAR expression ranging from approximately 60% to 100%. Approximately 6 / 8 Nectin-4 VHH-CARs and positive control CARs bound to recombinant Nectin-4-HIS protein, with binding proportional to VHH expression (geometric mean). Constructs P3108 and P3112 showed minimal binding to Nectin-4 protein and also low CAR expression. All Nectin-4 VHH-CARs and positive control CARs demonstrated target-specific activation against CHO-Nectin-4 and all Nectin-4+ tumor cell lines. Most Nectin-4 VHH-CARs demonstrated CAR-mediated activation comparable to the positive control scFv, enfortumab. Reduced activation was observed against OVCAR3, likely due to lower Nectin-4 expression. P3106 (NEC_M_5) exhibited high CAR expression, low tonic signaling (approximately 2%), and robust activation across all Nectin4+ lines.

[0341] [Example 6] VHH-CAR T cells demonstrate target-specific killing of Nectin4+ cell lines in vitro Primary T cells were transfected with anti-Nectin4 VHH-CAR lentiviral binding molecules, including eight VHHs and two scFv positive controls (P2025 and P2026, bidirectional enfortumab). CAR expression was confirmed by flow cytometry, and transfected CAR-Ts were screened using a FACS-based cytotoxicity assay against Nectin4+ target cell lines.

[0342] Activated T cells were transfected on day 0. Cells were harvested and counted in Vi-cell: 0.7E6c / ml @ 86% viability (6.1 ml total). Cells were pelleted by centrifugation at 300 x g for 5 minutes, and TransAct was removed. T cells were resuspended in medium to 0.6E6c / ml. 250 ul of cells / well were directly plated into a 24-well plate (150,000 T cells / well) and incubated while preparing the Transdux master mix. Transdux Max master mix was prepared in a 50 ml conical tube using TransDux™ MAX Lentiviral Transfection Reagent (SBI, Cat. No. LV860A-1) and HEPES (Gibco, Cat. No. 15630-080). 250 ul of TransduxMAX master mix was added to each well of the 24-well plate. 60 ul of lentivirus was added directly to the well. No lentivirus was added to mock-transfected (untransfected; UTD) wells. The plates were carefully sealed with parafilm and centrifuged at 32°C and 1300G for 1.5 hours. After centrifugation, the parafilm was removed, and 500 μl of T cell medium (R10 + 30 U / ml IL-2) was added to each well, and the plates were incubated. Five days after transfection, CAR expression FACS analysis was performed. 50 μl (approximately 50K) of cells per sample were stained for CAR expression using Nectin4-HIS protein (Acro Biosystems #NE4-H52H3, lot 2823a-214VF1-WR; 0.4 mg / ml stock) at a concentration of 2 μg / ml.

[0343] Cytotoxicity assays were performed 12 days after transfection. Briefly, cells were counted using Vi-Cell.

[0344] [Table 12]

[0345] 4×10 61 x 10 target cells were centrifuged at 300 x g for 5 min and stained with CTV staining solution (1:1000 in PBS) for 1 x 10 6 Cells were resuspended at 1000 / mL. Cells were incubated at 37°C for 10 minutes, with the tube inverted at the 5 minute mark to ensure even staining. CTV staining was quenched with 5 ml of R10 and the cells were centrifuged. Cells were washed with 5 ml of R10 medium, resuspended in 5 ml of R10, and recounted in Vi-cell.

[0346] [Table 13]

[0347] The target cell density was increased to 0.1 × 10 using medium (RPMI + 10% HI-FBS). 6 The concentration was adjusted to 10,000 cells / ml. Cells were plated at a density of 10,000 target cells per well. Primary transfected T cells (effector cells) were also plated, and a 200ul aliquot of cells was collected from the 6-well plate and counted using a Vi-Cell.

[0348] [Table 14]

[0349] Effector cell density was adjusted based on CAR+ expression to approximately 0.2 × 10 cells using culture medium (RPMI + 10% HI-FBS). 6 The target cell suspension was adjusted to cells / ml. 100 ul of the target cell suspension was plated into a 96-well flat bottom tissue culture plate.

[0350] Figure 9 shows the results of cytotoxicity assays. VHH-CAR T cells demonstrated target-specific killing of Nectin-4+ cell lines in vitro. CAR expression was confirmed for all constructs, ranging from approximately 50% to 90%. CAR-T cell samples showed binding to recombinant Nectin-4-HIS protein, and binding was proportional to CAR expression (VHH geometric mean). P3108 and P3112 had lower CAR expression and minimal binding to recombinant Nectin-4-HIS protein. Nectin-4 VHH-CAR and positive control scFv-CAR demonstrated CAR-mediated cytotoxic activity against Nectin-4+ tumor cell lines (T-47D, OVCAR3, OE19, and A431). No off-target cell killing was observed against the Nectin-4-negative cell lines, K562 or HeLa, demonstrating binding molecule specificity. Overall, the six anti-nectin-4 VHH CAR-Ts showed robust killing of nectin-4+ tumor cell lines, comparable to enfortumab-scFv / CAR (NEC_M_5, P3106; NEC_M_8, P3107; NEC_M_44, P3109; NEC_M_46, P3110; NEC_S_31, P3113; NEC_S_55, P3114).

[0351] [Example 7] Nectin-4 binding molecule specificity screening To aid in lead VHH characterization and selection, we established a cell-based specificity FACS screen. VHH-Fc cell-binding dose-response curves (DRCs) were generated against a diverse panel of human cell lines derived from various tissue / organ types. VHH-Fc showing nonspecific binding to target-negative lines were flagged as potential off-target binding, and we proceeded with VHH-Fc showing minimal nonspecific binding to target-negative lines.

[0352] Anti-Nectin 4 VHH-Fc cell-binding molecules were screened. Specific cell binding was previously confirmed at 5 nM and 50 nM for CHO-Nectin 4 and T47D cell lines, but no binding was confirmed for Jurkat or parental CHO cells (see, e.g., Figure 6). Briefly, FACS screening was performed by harvesting all cells and distributing 50 x 10 cells per well. 3 Blocking was performed by plating cells onto a plate of 1000 cells. Blocking was performed using InvivomAb Fc blocking agent (25 μg / ml, 20-30 minutes, room temperature). Cells were washed once and incubated in VHH-Fc for 30 minutes at 4°C. Cells were then washed three times and incubated with PE anti-VHH 1 μg / ml + NearIR (1:1000) for 30 minutes at 4°C. Cells were washed twice and imaged. The results of the FAC screening are shown in Figure 12A, and a corresponding description of the cell lines used in the specificity screening is provided in Figure 12B.

[0353] A431, Capan-2, OE19, and OVCAR3 cell lines express nectin-4. None of the samples showed substantial nonspecific binding to nectin-4-negative cell lines. Geometric mean graphs revealed that PROT1735 and PROT1749 exhibited slightly increased background binding to HEPG2, Jurkat, and U-2 OS cells. There were notable differences in the binding curves to nectin-4+ cells, resulting in a range of EC50 values ​​among the clones. Some samples showed weak binding to MOLM-13 at 500 nM, which may be due to the presence of FcR and incomplete Fc blockade.

[0354] [Example 8] Determination of Nectin-4 antigen density on target cell lines As shown in Figures 13A-13B, Nectin-4 antigen density was assessed in various solid tumor and control cell lines, as well as primary human keratinocytes (PHKs), using BD's Quantibrite PE kit. Quantibrite beads were coated with four calculated PE levels: low, medium-low, medium-high, and high. These calculated PE / bead values ​​and flow cytometric fluorescence intensities were used to generate a standard curve for estimating antigen density on cell lines. material: - McCoy's 5A Medium (Gibco Catalog No. 16600082) - RPMI1640 medium (Gibco catalog number 61870036) - EMEM medium (ATCC catalog number 30-2003) - DMEM medium (Gibco catalog number 10569010) - Lebovitz's L15 medium (Gibco catalog number 11415064) - Accutase (Gibco catalog number 00-4555-56) - TrypLE Express (Gibco Catalog No. 12605028) - DPBS (Gibco Catalog Number 14190144) - HI FBS (Gibco Catalog No. 10438026) - BD Staining Buffer, BSA (BD Pharmingen, Cat. No. 554657) - QuantiBrite PE beads (BD Biosciences Catalog No. 340495, Lot No. 82820) - Anti-human nectin 4, PE conjugate (R&D Systems Cat. No. FAB2659P) - Anti-human mesothelin, PE conjugate (R&D Systems Cat. No. FAB32652P) - Mouse IgG2a Kappa Isotype Control, PE (R&D Systems Catalog No. MAB0031) - Rat IgG2b isotype control, PE (R&D Systems Cat. No. IC006P) - Human Fc-G1 (Fc-blocked) (BioXCell Catalog No. BE0096, Lot No. 74782001)

[0355] procedure: Flow cytometry was performed to evaluate Nectin-4 expression. Briefly, cells were washed twice with 150 μL of BD staining buffer. Cells were Fc receptor blocked with 50 μL of staining buffer containing 25 μg / mL BioXCell human Fc-G1. Cells were incubated at room temperature for 25 minutes and washed twice with 150 μL of BD staining buffer. Mouse IgG2b anti-human Nectin-4 PE, mouse IgG2b isotype PE, rat IgG2a anti-human mesothelin PE, and rat IgG2a isotype PE were diluted 1:25 in BD staining buffer. Cells were resuspended in 50 μL of staining agent and incubated in the refrigerator for 30 minutes. Cells and beads were washed twice with 150 μL of BD staining buffer, resuspended in 30 μL of staining buffer, and run on an Intellicyt iQue3 flow cytometer.

[0356] PE / cell calculations were also performed. The PE / bead values ​​for the fo...

Claims

1. One or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that targets the Nectin-4 antigen; and (i) deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and 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 a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII), cluster of differentiation 16 (CD16), and / or NKG2D protein; (iv) deletion or reduced expression of one or more of the NKG2A or CD70, CD38, and CD33 genes; (v) an exogenous polynucleotide encoding a cytokine; (vi) an exogenous polynucleotide encoding a safety switch; (vii) an exogenous polynucleotide encoding a PSMA cell tracer; and (viii) an exogenous polynucleotide encoding a membrane-bound IL-12 polypeptide At least one of An induced pluripotent stem cell (iPSC) or a derived cell thereof, comprising:

2. (i) the CAR is a dual-targeting CAR comprising an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; or (ii) the cells comprise one or more exogenous polynucleotides encoding an additional CAR comprising an antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; The iPSC or its derivative cell described in claim 1.

3. The iPSC or its derivative cell according to claim 1 or 2, wherein the CAR comprises an anti-nectin-4 VHH domain.

4. The iPSC or its derivative cell according to any one of claims 1 to 3, wherein the cytokine comprises IL-15.

5. The iPSC or derived cell of claim 4, further comprising an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope, wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide.

6. The iPSC or its derivative cell of claim 4, wherein the IL-15 comprises an IL-15 and IL-15 receptor alpha (IL-15Rα) fusion polypeptide.

7. 7. The iPSC or a derived cell thereof of any one of claims 4 to 6, wherein the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

72.

8. 8. The iPSC or a derived cell thereof of any one of claims 1 to 7, comprising the deletion or reduced expression of one or more of the B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.

9. The iPSC or its derivative cell according to any one of claims 1 to 8, comprising an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

10. The iPSC or its derivative cell according to any one of claims 1 to 9, wherein the CD16 is a CD16 variant protein.

11. The iPSC or its derivative cell of claim 10, wherein the CD16 variant protein is a high-affinity CD16 variant.

12. The iPSC or its derivative cell of claim 10 or 11, wherein the CD16 variant protein is a non-cleavable CD16 variant.

13. The iPSC or its derivative cell according to any one of claims 10 to 12, wherein the CD16 variant protein comprises wild-type CD16 having one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, and T220A.

14. 14. The iPSC or a derived cell thereof of any one of claims 10 to 13, wherein the CD16 variant protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 187 and 188.

15. 15. The iPSC or its derivative cell according to any one of claims 1 to 14, comprising an exogenous polynucleotide encoding the CD16 protein and the NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by an autoprotease peptide.

16. The iPSC or its derivative cell of claim 15, wherein the NKG2D protein is a wild-type NKG2D protein.

17. 16. The iPSC or a derived cell thereof of claim 15, wherein the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

190.

18. 18. The iPSC or its derivative cell according to any one of claims 15 to 17, wherein the autoprotease peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A) peptide, Foot and Mouth Disease virus 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 Falcata Virus 2A (BmIFV2A) peptide.

19. The iPSC or its derivative cell of claim 18, wherein the autoprotease peptide is a P2A peptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

192.

20. 20. The iPSC or a derived cell thereof of any one of claims 15 to 19, wherein the exogenous polynucleotides encoding the CD16 protein and the NKG2D protein comprise polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

193.

21. One or more of the exogenous polynucleotides may be selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR 21. The iPSC or derivative thereof of any one of claims 1 to 20, wherein the exogenous polynucleotide is integrated into one or more loci on a chromosome of the cell selected from the group consisting of a chromosome containing a gene selected from the group consisting of a chromosome containing a nucleotide sequence selected from the group consisting of a nucleotide sequence ...

22. 22. The iPSC or a derived cell thereof of any one of claims 1 to 21, wherein one or more of the exogenous polynucleotides are integrated into the locus of the AAVS1 and B2M genes.

23. 23. The iPSC or a derived cell thereof of any one of claims 1 to 22, having a deletion or reduced expression of one or more of the B2M or CIITA genes.

24. 24. The iPSC or a derived cell thereof of claim 23, comprising the deletion or reduced expression of the B2M and CIITA genes.

25. The iPSC of any one of claims 1 to 24, which is reprogrammed from whole peripheral blood mononuclear cells (PBMCs).

26. The iPSC according to any one of claims 1 to 25, which is derived from a reprogrammed T cell.

27. The CAR is (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to the Nectin-4 antigen; (iii) hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain 27. The iPSC or derived cell of any one of claims 1 to 26, comprising:

28. The iPSC or its derivative cell of claim 27, wherein the extracellular domain comprises a VHH single domain antibody that specifically binds to the Nectin-4 antigen.

29. 29. The iPSC or derivative thereof of claim 27 or 28, wherein the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105-130.

30. The iPSC or a derived cell thereof of claim 27 or 28, wherein (i) the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 131-156; or (ii) the CAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 171-184.

31. the further CAR is (i) a signal peptide; (ii) an additional extracellular domain comprising a binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain The iPSC or a derived cell thereof according to any one of claims 2 to 30, comprising:

32. The iPSC or its derivative cell of claim 31, wherein the additional extracellular domain comprises a VHH or scFv that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6.

33. The iPSC or its derivative cell according to any one of claims 27 to 32, wherein the signal peptide comprises a GMCSFR signal peptide or a MARS signal peptide.

34. The iPSC or a derived cell thereof according to any one of claims 27 to 33, wherein 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.

35. The iPSC or a derived cell thereof of any one of claims 27 to 34, wherein the transmembrane domain for each of the CAR and the further CAR is independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain.

36. The iPSC or its derivative cell according to any one of claims 27 to 35, wherein the intracellular signaling domain comprises a CD3ζ intracellular domain.

37. The iPSC or a derived cell thereof of any one of claims 27 to 36, wherein the costimulatory domain for each of the CAR and the further CAR is independently selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain.

38. In the CAR, (i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 105-130, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131-156; (iii) the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21 or 96; (iv) the transmembrane domain comprises amino acids 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) the intracellular signaling domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6 or is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:101; and (vi) the costimulatory domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or 17; An iPSC or a derived cell thereof according to any one of claims 27 to 37.

39. In the CAR, (i) the signal peptide comprises an amino acid sequence having SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain comprises an amino acid sequence having one of SEQ ID NOs: 105-130; (iii) the hinge region comprises an amino acid sequence of SEQ ID NO: 21 or 96; (iv) the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 23 or 24; (v) the intracellular signaling domain comprises an amino acid having the sequence of SEQ ID NO:6, or the intracellular signaling domain is encoded by a polynucleotide having the sequence of SEQ ID NO:101; and (vi) the costimulatory domain comprises an amino acid sequence of SEQ ID NO: 8 or 17; 39. The iPSC or a derived cell thereof of claim 38.

40. 40. The iPSC or a derived cell thereof of any one of claims 1 to 39, comprising an exogenous polynucleotide encoding a safety switch.

41. 41. The iPSC or derivative thereof of claim 40, wherein the safety switch comprises an exogenous polynucleotide encoding an inactivated cell surface receptor comprising a monoclonal antibody-specific epitope.

42. The inactivated cell surface protein is selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, and polatuzumab.

42. The iPSC or derived cell thereof of claim 41, wherein the iPSC or derived cell thereof is selected from the group of monoclonal antibody-specific epitopes selected from epitopes specifically recognized by vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab.

43. 43. The iPSC or derived cell thereof of claim 42, wherein the inactivated cell surface receptor is a truncated epidermal growth factor receptor (tEGFR) variant.

44. 44. The iPSC or a derived cell thereof of claim 43, wherein the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

71.

45. 45. The iPSC or a derived cell thereof of any one of claims 40 to 44, wherein the safety switch comprises an intracellular domain having herpes simplex virus thymidine kinase (HSV-TK).

46. The iPSC or its derivative cell of any one of claims 1 to 45, comprising the exogenous polynucleotide encoding the PSMA cell tracer, wherein the PSMA cell tracer comprises an extracellular domain comprising the PSMA extracellular domain or a fragment thereof.

47. The iPSC or its derivative cell of claim 46, comprising 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 the PSMA extracellular domain or a fragment thereof.

48. 48. The iPSC or a derived cell thereof of any one of claims 45 to 47, wherein the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 229 or 230.

49. The iPSC or derived cell thereof of claim 47, wherein the combined artificial cell death / reporter system polypeptide comprises the HSV-TK fused to a truncated variant PSMA polypeptide via the linker.

50. The iPSC or its derivative cell of claim 49, wherein the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

231.

51. The iPSC or its derivative cell of any one of claims 47 to 50, wherein 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.

52. 52. The iPSC or derivative thereof of any one of claims 47 to 51, wherein 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 SEQ ID NO:

232.

53. 53. The iPSC or derivative thereof of claim 52, wherein the artificial cell death / reporter system polypeptide comprises amino acids 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: 233-235.

54. 54. The iPSC or a derived cell thereof of any one of claims 47 to 53, wherein the artificial cell death / reporter system polypeptide is encoded by a polynucleotide 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: 236-238.

55. 55. The iPSC or a derived cell thereof of any one of claims 1 to 54, wherein the HLA-E comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, and / or the HLA-G comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

69.

56. (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting the Nectin-4 antigen comprise nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more selected from the group consisting of SEQ ID NOs: 171-184; (ii) the exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises nucleotides 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 exogenous polynucleotide encoding the NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or NKG2D protein comprises nucleotides 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: 185, 189, and 191; (iv) the exogenous polynucleotide encoding a cytokine comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 239; (v) the exogenous polynucleotide encoding a safety switch comprises nucleotides 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:236-238; and / or (vi) the exogenous polynucleotide encodes a PSMA cell tracer, and the PSMA cell tracer comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 231; 56. An iPSC or a derived cell thereof according to any one of claims 1 to 55.

57. (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting the Nectin-4 antigen comprise nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 171 to 184; (ii) the exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises nucleotides having the sequence of SEQ ID NO: 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 NKG2D protein comprises nucleotides having the sequence of SEQ ID NO: 185, 189, or 191; (iv) the exogenous polynucleotide encoding the cytokine comprises a nucleotide having the sequence of SEQ ID NO: 239; and / or (v) the exogenous polynucleotide encoding the safety switch comprises nucleotides having a sequence of one of SEQ ID NOs: 236-238; 57. An iPSC or a derived cell thereof according to any one of claims 1 to 56.

58. The exogenous polynucleotide is located at the AAVS1 locus, B2M locus, CIITA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD 33 locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPB 58. The iPSC or its derivative cell of claim 56 or 57, wherein the iPSC is integrated into a locus independently selected from the group consisting of the P 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.

59. (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain targeting a Nectin-4 antigen are integrated into the locus of the AAVS1 gene; (ii) the exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) is integrated into the locus of the B2M gene; (iii) the exogenous polynucleotide encoding the NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or NKG2D is integrated into the CD70 gene locus; (iv) the exogenous polynucleotide encoding the cytokine is integrated into the locus of the NKG2A gene; (v) the exogenous polynucleotide encoding a safety switch is integrated into the locus of the CLYBL gene; and (vi) the CIITA gene is deleted or has reduced expression; 59. The iPSC or a derived cell thereof of claim 58.

60. 60. The iPSC or derived cell thereof of any one of claims 1 to 59, wherein the one or more exogenous polynucleotides further encode one or more inhibitory CARs (iCARs) comprising at least one antigen-binding domain targeting an antigen independently selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC) 1, DSC3, desmoglein (DSG) 1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), and V-set and immunoglobulin domain-containing 8 (VSIG8).

61. The iCAR is (i) a signal peptide; (ii) an extracellular domain comprising an antigen-binding domain that specifically binds to at least one antigen selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC) 1, DSC3, desmoglein (DSG) 1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8); (iii) hinge region; (iv) one or more transmembrane domains; (v) an intracellular signaling domain; and / or (vi) a costimulatory domain 61. The iPSC or a derived cell thereof of claim 60, comprising:

62. 62. The iPSC or derivative thereof of claim 61, wherein the extracellular domain of the iCAR comprises amino acids 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: 354-363.

63. 63. The iPSC or a derived cell thereof of claim 61 or 62, wherein the extracellular domain of the iCAR is encoded by a polynucleotide 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: 364-373.

64. The iPSC or its derived cell according to any one of claims 61 to 63, wherein the signal peptide of the iCAR comprises a CD8 signal peptide, a GMCSFR signal peptide, a MARS signal peptide, or an IgK signal peptide, or a variant thereof.

65. 64. The iPSC or derived cell thereof of any one of claims 61 to 63, wherein the signal peptide of the iCAR comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 97 and 292.

66. 64. The iPSC or a derived cell thereof of any one of claims 61 to 63, wherein the signal peptide of the iCAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 98, 327, and 378.

67. 67. The iPSC or a derived cell thereof of any one of claims 61 to 66, wherein the hinge region of the iCAR is selected from the group consisting of a CD28 hinge region, a CD45 hinge region, a G4S-CD45 hinge region, a CD8 hinge region, and a CXC3R GPCR hinge region.

68. 68. The iPSC or a derived cell thereof of any one of claims 61 to 67, wherein the hinge region of the iCAR comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 21, 22, 288, 289, 319, and 321.

69. 68. The iPSC or a derived cell thereof of any one of claims 61 to 67, wherein the hinge region of the iCAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 315-318, 320, and 322.

70. 70. The iPSC or derived cell thereof of any one of claims 61 to 69, wherein the one or more transmembrane domains of the iCAR are independently selected from the group consisting of a CD28 transmembrane domain, a CD8 transmembrane domain, a PD1 transmembrane domain, a SynNotch transmembrane domain, and a CXC3R GPCR.

71. 70. The iPSC or a derived cell thereof of any one of claims 61 to 69, wherein the transmembrane domain of the iCAR comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 23, 24, 290, 291, 323, and 325.

72. 70. The iPSC or a derived cell thereof of any one of claims 61 to 69, wherein the transmembrane domain of the iCAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 324, 326, and 374-377.

73. the intracellular signaling domain of the iCAR is selected from the group consisting of a PD1 intracellular domain, a LIRB1 intracellular domain, a TIGIT intracellular domain, a CTLA4 intracellular domain, a CSK*(YSSV) intracellular domain, a KIR2DL1 intracellular domain, a DR1 intracellular domain, a Casp8wt intracellular domain, a tCasp8 intracellular domain, a tCasp8 dimer intracellular domain, a tBid15 intracellular domain, a Casp9wt intracellular domain, a tCasp9 intracellular domain, and a tC 73. The iPSC or a derived cell thereof of any one of claims 61 to 72, comprising one or more of an asp9 dimer intracellular domain, a SHP1 intracellular domain, a (G4S)2-SHP1 intracellular domain, a CSK intracellular domain, a (G4S)2-CSK intracellular domain, an ADAM17 cleavage site, a CD28 intracellular domain, a CD3ζ intracellular domain, a G4S3 linker, an ADAM17 protease domain, and a (G4S)3-ADAM17 protease domain.

74. 73. The iPSC or a derived cell thereof of any one of claims 61-72, wherein the intracellular signaling domain of the iCAR comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 6, 8, and 267-287.

75. 73. The iPSC or a derived cell thereof of any one of claims 61-72, wherein the intracellular signaling domain of the iCAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 266, and 293-314.

76. 76. The iPSC or derived cell thereof of any one of claims 61 to 75, wherein the costimulatory domain of the iCAR is selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain.

77. In the iCAR, (i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 97 or 292, or the signal peptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 98, 327, or 378; (ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 354-363, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 364-373; (iii) the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:21, 22, 288, 289, 319, or 321, or the hinge region is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:315-318, 320, or 322; (iv) each of the one or more transmembrane domains comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence independently selected from the group consisting of SEQ ID NOs: 23, 24, 290, 291, 323, and 325, or each of the one or more transmembrane domains is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence independently selected from the group consisting of SEQ ID NOs: 324, 326, and 374-377; (v) the intracellular signaling domain comprises amino acids 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: 6, 8, and 267-287, or the intracellular signaling domain is encoded by a polynucleotide 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: 266, and 293-314; 77. An iPSC or a derived cell thereof according to any one of claims 61 to 76.

78. In the iCAR, (i) the signal peptide comprises amino acids having a sequence of SEQ ID NO: 97 or 292, or the signal peptide is encoded by a polynucleotide sequence of SEQ ID NO: 98, 327, or 378; (ii) the extracellular domain comprises an amino acid sequence having a sequence of SEQ ID NO: 354-363, or the extracellular domain is encoded by a polynucleotide sequence of SEQ ID NO: 364-373; (iii) the hinge region comprises amino acids having a sequence of SEQ ID NO: 21, 22, 288, 289, 319, or 321, or the hinge region is encoded by a polynucleotide sequence of SEQ ID NO: 315-318, 320, or 322; (iv) each of the one or more transmembrane domains comprises an amino acid having a sequence independently selected from the group consisting of SEQ ID NOs: 23, 24, 290, 291, 323, and 325, or each of the one or more transmembrane domains is encoded by a polynucleotide having a sequence independently selected from the group consisting of SEQ ID NOs: 324, 326, and 374-377; and (v) the intracellular signaling domain comprises amino acids having a sequence of one or more of SEQ ID NOs: 6, 8, and 267-287, or the intracellular signaling domain is encoded by a polynucleotide of one of SEQ ID NOs: 266, and 293-314; 78. The iPSC or a derived cell thereof of claim 77.

79. 79. A derivative cell according to any one of claims 1 to 78 which is a natural killer (NK) cell or a T cell.

80. 80. The derivative cell of claim 79, which is a T cell.

81. 81. The derivative cell of claim 80, wherein the T cell is a gamma delta T cell.

82. 82. The derivative cell of claim 81 , wherein the T cell is a gamma delta Vγ9 / Vδ1 T cell.

83. A composition comprising a derivative cell according to any one of claims 1 to 82.

84. 84. The composition of claim 83, further comprising or used in combination with one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, small RNA, dsRNA (double-stranded RNA), siRNA, an oligonucleotide, a mononuclear blood cell, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (IMiD).

85. 79. A CD34+ hematopoietic progenitor cell (HPC) derived from the induced pluripotent stem cell (iPSC) of any one of claims 1 to 78.

86. (i) the CAR is a dual-targeting CAR comprising an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; or (ii) the one or more exogenous polynucleotides encode an additional CAR comprising an antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; The CD34+ HPC described in claim 85.

87. 87. The CD34+ HPC of claim 85 or 86, comprising an exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

88. One or more of the exogenous polynucleotides may be selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR 88. The CD34+ HPC of any one of claims 85-87, wherein the exogenous polynucleotide is integrated into one or more loci on a chromosome of the cell independently selected from the group consisting of a a or b constant region, NKG2A, NKG2D, CD33, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT gene, wherein at least one of the exogenous polynucleotides is integrated into the locus of a gene selected from the group consisting of B2M, TAP1, TAP2, tapasin, RFXANK, CIITA, RFX5, and RFXAP gene, thereby resulting in deletion or reduced expression of the gene.

89. 89. The CD34+ HPC of claim 88, wherein one or more of the exogenous polynucleotides are integrated into the AAVS1 and B2M gene loci.

90. 90. The CD34+ HPC of any one of claims 85 to 89, having a deletion or reduced expression of one or more of the B2M or CIITA genes.

91. The CAR is (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to the Nectin-4 antigen; (iii) hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain The CD34+ HPC of any one of claims 85 to 90, comprising:

92. The CD34+ HPC of claim 91, wherein the extracellular domain comprises a VHH single domain antibody that specifically binds to the Nectin4 antigen.

93. 93. The CD34+ HPC of claim 92, wherein the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105-130.

94. (i) a signal peptide; (ii) an additional extracellular domain comprising a binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a costimulatory domain, e.g., a costimulatory domain comprising a CD28 signaling domain The CD34+ HPC of any one of claims 85 to 93, having a further CAR comprising:

95. The CD34+ HPC of claim 94, wherein the additional extracellular domain comprises a VHH that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6.

96. The CD34+ HPC of any one of claims 85 to 95, further comprising 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.

97. The CD34+ HPC of claim 96, wherein the CD16 protein is a CD16 variant protein.

98. The CD34+ HPC of claim 97, wherein the CD16 variant is a high-affinity CD16 variant.

99. The CD34+ HPC of claim 97 or 98, wherein the CD16 variant is a non-cleavable CD16 variant.

100. The CD34+ HPC of any one of claims 97 to 99, wherein 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.

101. A chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain that includes an antigen-binding domain that specifically binds to Nectin-4.

102. The CAR of claim 101, wherein the CAR is a dual-targeting CAR and the extracellular domain comprises an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, folate receptor alpha, FSHR, mesothelin, and SLITRK6.

103. (i) a signal peptide; (ii) the extracellular domain comprising the antigen-binding domain that specifically binds to the Nectin-4 antigen; (iii) hinge region; (iv) one or more transmembrane domains; (v) an intracellular signaling domain; and / or (vi) a costimulatory domain The CAR according to any one of claims 101 to 102, comprising:

104. The CAR of any one of claims 101 to 103, wherein the extracellular domain comprises a VHH single domain antibody that specifically binds to the Nectin-4 antigen.

105. The CAR of any one of claims 101 to 103, wherein the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105 to 130.

106. The CAR of any one of claims 101 to 103, wherein (i) the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 131 to 156; or (ii) the CAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 171 to 184.

107. The CAR according to any one of claims 103 to 106, wherein the signal peptide comprises a GMCSFR signal peptide or a MARS signal peptide.

108. The CAR of any one of claims 103 to 107, wherein the hinge region for each of the CARs is independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region.

109. The CAR of any one of claims 103 to 108, wherein the transmembrane domain for each of the CAR and the further CAR is independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain.

110. The CAR according to any one of claims 103 to 109, wherein the intracellular signaling domain comprises a CD3ζ intracellular domain.

111. The CAR of any one of claims 103 to 110, wherein the costimulatory domain for each of the CAR and the further CAR is independently selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain.

112. In the CAR, (i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 105-130, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131-156; (iii) the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21 or 96; (iv) the transmembrane domain comprises amino acids 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) the intracellular signaling domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6, or the intracellular signaling domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:101; and (vi) the costimulatory domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or 17; The CAR according to any one of claims 103 to 111.

113. In the CAR, (i) the signal peptide comprises an amino acid sequence having SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain comprises an amino acid sequence having one of SEQ ID NOs: 105-130; (iii) the hinge region comprises an amino acid sequence of SEQ ID NO: 21 or 96; (iv) the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 23 or 24; (vi) the intracellular signaling domain comprises amino acids having the sequence of SEQ ID NO:6, or the intracellular signaling domain is encoded by the polynucleotide of SEQ ID NO:101; and (vii) the costimulatory domain comprises an amino acid sequence of SEQ ID NO: 8 or 17; The CAR described in claim 112.

114. An inhibitory chimeric antigen receptor (iCAR) polypeptide comprising an extracellular domain comprising an antigen-binding domain that specifically binds to at least one antigen selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC) 1, DSC3, desmoglein (DSG) 1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8).

115. (i) a signal peptide; (ii) the extracellular domain comprising an antigen-binding domain that specifically binds to at least one antigen selected from the group consisting of adrenoceptor beta 2 (ADRB2), aquaporin 4 (AQP4), claudin 10 (CLDN10B), desmocollin (DSC) 1, DSC3, desmoglein (DSG) 1, DSG3, glycerophosphodiester phosphodiesterase domain-containing 2 (GDPD2), hydroxycarboxylic acid receptor 3 (HCAR3), lymphocyte antigen 6 family member D (LY6D), V-set, and immunoglobulin domain-containing 8 (VSIG8); (iii) hinge region; (iv) one or more transmembrane domains; (v) an intracellular signaling domain; and / or (vi) a costimulatory domain The iCAR of claim 114, comprising:

116. The iCAR of claim 115, wherein the antigen-binding domain specifically binds to at least one antigen selected from DSC1, DSC3, DSG1, and DSG3.

117. The iCAR of claim 115, wherein the antigen-binding domain specifically binds to DSG1.

118. The iCAR of claim 115, wherein the antigen-binding domain specifically binds to at least one antigen selected from (i) DSG1, and (ii) DSC1, DSC3, and DSG3.

119. The iCAR of any one of claims 115-118, wherein the extracellular domain of the iCAR comprises amino acids 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: 354-363.

120. The iCAR of any one of claims 115-118, wherein the extracellular domain of the iCAR is encoded by a polynucleotide 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: 364-373.

121. The iCAR of any one of claims 115 to 120, wherein the signal peptide of the iCAR comprises a CD8 signal peptide, a GMCSFR signal peptide, a MARS signal peptide, or an IgK signal peptide, or a variant thereof.

122. The iCAR of any one of claims 115 to 120, wherein the signal peptide of the iCAR comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 97 and 292.

123. The iCAR of any one of claims 115 to 120, wherein the signal peptide of the iCAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 98, 327, and 378.

124. In the iCAR, (i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 97 or 292; (ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 354-363, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 364-373; and (iii) the iCAR comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 242-265, and 352, or the iCAR comprises a sequence of amino acids encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 328-351, and 353; An iCAR described in any one of claims 115 to 123.

125. In the iCAR, (i) the signal peptide comprises an amino acid sequence having SEQ ID NO: 97, or 292; (ii) the extracellular domain comprises an amino acid sequence having one of SEQ ID NOs: 354-363; and / or (iii) the iCAR comprises an amino acid sequence having one of SEQ ID NOs: 242-265, and 352; The CAR described in claim 124.

126. An induced pluripotent stem cell (iPSC) or a derived cell thereof according to any one of claims 1 to 78, further comprising an iCAR according to any one of claims 114 to 125.

127. A pharmaceutical composition comprising the derivative cell of claim 126.

128. 127. A method of treating cancer in a subject in need thereof, comprising administering to a subject in need thereof a derivative cell of any one of claims 1 to 82 and 126 or a composition of claim 83, 84 or 127.

129. 129. The method of claim 128, wherein the cancer is selected from the group consisting of 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, thymus cancer, bone cancer, and cerebral cancer, as well as cancer of unknown primary (CUP).

130. 128. The method of claim 127, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and esophageal cancer.

131. 129. The method of any one of claims 126-128, wherein the subject has minimal residual disease (MRD) after initial cancer treatment.

132. 130. The method of any one of claims 126-129, wherein the subject has no minimal residual disease (MRD) after one or more cancer treatments or repeat medications.

133. The subject is treated with ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab 131. The method of any one of claims 126-130, further comprising administering a therapeutic agent selected from the group consisting of vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab.

134. 131. The method of any one of claims 126 to 130, further comprising administering to the subject a therapeutic agent, wherein the therapeutic agent is avelumab.

135. 133. The method of claim 131 or 132, wherein the cells and the therapeutic agent are administered simultaneously.

136. 133. The method of claim 131 or 132, wherein the cells and the therapeutic agent are administered sequentially.

137. 83. A method for producing a derivative cell according to any one of claims 79 to 82, comprising differentiating an iPSC according to any one of claims 1 to 78 under conditions for cell differentiation, thereby obtaining said derivative cell.

138. 136. The method of Claim 135, wherein the iPSCs are obtained by genetically engineering unmodified iPSCs, and wherein the genetic engineering comprises targeted editing of the genome of the iPSCs.

139. 137. The method of Claim 136, wherein the targeted editing comprises a deletion, insertion, or in / del performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

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