Gene transfer vectors and methods for engineering cells

Abstract

The present invention relates to compositions and methods for use in genome engineering of induced pluripotent stem cells (iPSCs). In particular, the described methods and compositions are useful for introducing transgenes into iPSCs, such as pluripotent hematopoietic stem and / or progenitor cells (HSC / PCs), using a CRISPR nuclease-based system (e.g., a MAD7 nuclease-based system), and preparing immune effector cells derived from iPSCs.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 171,891, filed April 7, 2021, which is incorporated by reference in its entirety. [Technical field]

[0002] The present disclosure is in the field of genome engineering, and in particular the targeted modification of the genome of a cell.

[0003] Reference to an Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing with the file name "Sequence Listing_ST25.txt" and a creation date of March 31, 2022, and a size of 119 kb. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Various methods and compositions have been described for targeted cleavage of genomic DNA. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, to induce targeted deletion of cellular DNA sequences, and to facilitate targeted recombination at a given chromosomal locus. These methods often involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks in target DNA sequences, so that repair of the cleavage by error-prone processes such as non-homologous end joining (NHEJ) or repair using a repair template (homologous recombination repair or HDR) can result in knockout of a gene or insertion of a sequence of interest (targeted integration). Cleavage can occur by the use of specific nucleases, such as artificial zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR / Cas systems with artificial crRNA / tracr RNA ("single guide RNA") to induce specific cleavage.

[0005] Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs, are a type of pluripotent stem cells artificially derived from non-pluripotent cells, typically adult somatic cells, by inserting specific genes. Induced pluripotent stem cells are considered to be identical to natural pluripotent stem cells such as embryonic stem cells in many respects, such as expression of specific stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiation potential, but their full relationship to natural pluripotent stem cells is still being evaluated. IPS cells were first generated from mouse cells in 2006 (Takahashi et al., 2006) and from human cells in 2007 (Takahashi et al., 2007; Yu et al., 2007). This is cited as an important advancement in stem cell research, as it allows researchers to obtain pluripotent stem cells that are important in research and have potential therapeutic applications without the controversial use of embryos.

[0006] Human iPSC technology is a highly promising and potentially unlimited source of therapeutically viable hematopoietic cells for the treatment of numerous hematologic and non-hematologic malignancies, including cancer. To advance the promise of human iPSCs and genome-modified human iPSC technology as an allogeneic source of hematopoietic cell therapeutics, it is essential to be able to efficiently and reproducibly generate immune effector populations, including hematopoietic stem and progenitor cells (HSCs) as well as diverse subsets of T, B, NKT, and NK lymphocytes and their progenitors with desired genetic modifications. Thus, there is a need for methods and complexes for the efficient insertion of genetic elements into human iPSCs for therapeutic use. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure describes compositions and methods for use in genome engineering of cells, such as iPSCs. Specifically, the methods and compositions described relate to compositions and methods for introducing transgenes into iPSCs, such as pluripotent hematopoietic stem and / or progenitor cells (HSC / PCs), and preparing immune effector cells derived from iPSCs. More specifically, one aspect of the disclosure relates to a MAD7 / gRNA ribonucleoprotein (or ribonucleoprotein) (RNP) complex composition for inserting a transgene, the composition comprising: (I) MAD7 nuclease; (II) a guide RNA (gRNA) specific for MAD7 nuclease, the gRNA comprising a guide sequence capable of hybridizing to a target sequence of an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell (e.g., an iPSC), the guide sequence being selected from SEQ ID NOs: 120-130, and wherein when the gRNA forms a complex with the MAD7 nuclease, the guide sequence directs sequence-specific binding of the MAD7 nuclease to the target sequence; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of a target sequence in an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter operably linked to a polynucleotide sequence (3) encoding a transgene; and (4) a transcription terminator sequence. Includes.

[0008] In another aspect, provided herein is a MAD7 / gRNA ribonucleoprotein (RNP) complex composition for transgene insertion, the composition comprising: (I) A MAD7 nuclease system, comprising: (a) a sequence encoding a guide RNA (gRNA) operably linked to a first regulatory element, the gRNA comprising a guide sequence capable of hybridizing to a target sequence of an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell (e.g., an iPSC), the guide sequence being selected from SEQ ID NOs: 120-130, and when transcribed, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; (b) a sequence encoding a MAD7 nuclease operably linked to a second regulatory element; and a MAD7 nuclease system encoded by one or more vectors comprising: (II) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of a target sequence in an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter operably linked to a polynucleotide (3) encoding a transgene; and (4) a transcription terminator sequence. Includes.

[0009] In another aspect, provided herein is a MAD7 / gRNA ribonucleoprotein (RNP)-based vector system, the vector system comprising: (I) one or more vectors comprising: (a) a sequence encoding a guide RNA (gRNA), operably linked to a first regulatory element, the gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell (e.g., an iPSC), the guide sequence being selected from SEQ ID NOs: 120-130, and wherein, upon transcription, the guide sequence directs sequence-specific binding of a MAD7 complex to the target sequence; and (b) a sequence encoding a MAD7 nuclease, operably linked to a second regulatory element; and (II) a transgene vector comprising: (1) left and right polynucleotide sequences that are homologous to the left and right arms of a target sequence in the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL locus; (2) a promoter operably linked to a polynucleotide (3) encoding a transgene; and (4) a transcription terminator sequence. Includes.

[0010] In various embodiments, the first and / or second regulatory element is a promoter. In some embodiments, the first and second regulatory elements are the same. In some embodiments, the first and second regulatory elements are different.

[0011] In some embodiments of the compositions or vector systems described herein, the transgene comprises a sequence encoding a chimeric antigen receptor (CAR), optionally wherein the CAR is specific for a tumor antigen associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy.

[0012] In some embodiments, the guide sequence is specific to the AAVS1 locus. In some embodiments, the gRNA guide sequence specific to the AAVS1 locus comprises SEQ ID NO: 120.

[0013] In some embodiments of the compositions or vector systems described herein, the transgene comprises a sequence encoding a chimeric antigen receptor (CAR), optionally wherein the CAR is specific for a tumor antigen associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy, and further wherein the guide sequence is specific for the AAVS1 locus. In some embodiments, the gRNA guide sequence specific for the AAVS1 locus comprises SEQ ID NO: 120.

[0014] In some embodiments of the compositions or vector systems described herein, the transgene comprises a sequence encoding an artificial cell death polypeptide.

[0015] In some embodiments, the guide sequence is specific to the B2M or CIITA locus. In some embodiments, the gRNA guide sequence is specific to the B2M locus and comprises SEQ ID NO: 121. In some embodiments, the gRNA guide sequence is specific to the CIITA locus and comprises SEQ ID NO: 122 or 126.

[0016] In some embodiments of the compositions or vector systems described herein, the transgene comprises a sequence encoding an artificial cell death polypeptide, and the guide sequence is specific to the B2M or CIITA locus. In some embodiments, the gRNA guide sequence is specific to the B2M locus and comprises SEQ ID NO: 121. In some embodiments, the gRNA guide sequence is specific to the CIITA locus and comprises SEQ ID NO: 122 or 126.

[0017] In some embodiments of the compositions or vector systems described herein, the transgene comprises a sequence encoding an exogenous cytokine.

[0018] In some embodiments, the guide sequence is specific to the B2M or CIITA locus. In some embodiments, the gRNA guide sequence is specific to the B2M locus and comprises SEQ ID NO: 121.

[0019] In some embodiments of the compositions or vector systems described herein, the transgene comprises a sequence encoding an exogenous cytokine and the guide sequence is specific for the B2M or CIITA locus. In some embodiments, the gRNA guide sequence is specific for the B2M locus and comprises SEQ ID NO: 121.

[0020] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the CIITA locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 122 or 126.

[0021] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the NKG2A locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 124.

[0022] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the TRAC locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 125.

[0023] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the CLYBL locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 123.

[0024] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the CD70 locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 127.

[0025] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the CD38 locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 128.

[0026] In some embodiments of the compositions or vector systems described herein, the gRNA guide sequence is specific to the CD33 locus. In one embodiment, the gRNA guide sequence comprises SEQ ID NO: 129 or 130.

[0027] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of AAVS1 comprise the nucleotide sequences of SEQ ID NOs: 60 and 61, respectively, or fragments thereof.

[0028] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of B2M comprise the nucleotide sequences of SEQ ID NOs: 63 and 64, respectively, or fragments thereof.

[0029] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CIITA comprise (i) the nucleotide sequences of SEQ ID NOs: 66 and 67, respectively, or a fragment thereof, or (ii) the nucleotide sequences of SEQ ID NOs: 106 and 107, respectively, or a fragment thereof.

[0030] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CLYBL comprise the nucleotide sequences of SEQ ID NOs: 69 and 70, respectively, or fragments thereof.

[0031] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CD70 comprise the nucleotide sequences of SEQ ID NOs: 109 and 110, respectively, or fragments thereof.

[0032] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of NKG2A comprise the nucleotide sequences of SEQ ID NOs: 72 and 73, respectively, or fragments thereof.

[0033] In some embodiments of the compositions or vector systems described herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of TRAC comprise the nucleotide sequences of SEQ ID NOs: 75 and 76, respectively, or fragments thereof.

[0034] In some embodiments of the compositions or vector systems described herein, when the RNP complex is introduced into a cell, expression of an endogenous gene comprising a target sequence complementary to the guide sequence of the gRNA molecule is reduced or eliminated in said cell.

[0035] In another aspect, provided herein is one or more retroviruses comprising the vector system described herein.

[0036] In another aspect, provided herein are iPSCs transformed with a transgene by a MAD7 / gRNA ribonucleoprotein (RNP) composition described herein.

[0037] In another aspect, provided herein are iPSCs transformed with a vector system described herein or one or more retroviruses described herein.

[0038] In some embodiments of the iPSCs described herein, the transgene comprises a sequence encoding a chimeric antigen receptor (CAR). The CAR can be specific for a tumor antigen 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. In some embodiments, the tumor antigen associated with glioblastoma is selected from HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2; the tumor antigen associated with ovarian cancer is selected from FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, Nectin-4, and B7H4; the tumor antigen associated with cervical cancer or head and neck cancer is selected from GD2, MUC1, mesothelin, HER2, and EGFR; the tumor antigen associated with liver cancer is selected from claudin 18.2, GPC-3, EpCAM, cMET, and AFP; and the tumor antigen associated with hematological malignancies is selected from CD19, CD22, CD79, BCMA, GPRC5D, SLAM. The tumor antigen associated with bladder cancer is selected from Nectin-4 and SLITRK6.

[0039] In some embodiments of the iPSCs described herein, the CAR is an antigen specific for alpha-fetoprotein, A3, A33 antibody, Ba733, BrE3 antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3 , EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA The antibody may be specific for a tumor antigen selected from -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, 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 markers, cancer gene markers, and cancer gene products.

[0040] In one embodiment of the iPSCs described herein, the tumor antigen is CD19.

[0041] In another aspect, provided herein is an engineered immune effector cell or population thereof derived from iPSC described herein.In some embodiments, the immune effector cell is T cell or NK cell.In some embodiments, the T cell is CD4+ T cell, CD8+ T cell, or a combination thereof.

[0042] In another aspect, provided herein is a pharmaceutical composition comprising immune effector cells derived from iPSCs described herein.

[0043] In another aspect, provided herein is a method for preventing or treating cancer, comprising administering to an individual in need thereof a pharma- ceutical effective amount of an immune effector cell or population described herein or a pharmaceutical composition described herein. In some embodiments, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, endometrial cancer, ovarian cancer, lymphoma, and brain cancer.

[0044] In another aspect, provided herein is a gRNA comprising a guide sequence selected from the group consisting of SEQ ID NOs: 120-130. In some embodiments, the gRNA comprises a guide sequence of SEQ ID NO: 123, 124, or 125. In one embodiment, the gRNA comprises a guide sequence of SEQ ID NO: 123. In one embodiment, the gRNA comprises a guide sequence of SEQ ID NO: 124. In one embodiment, the gRNA comprises a guide sequence of SEQ ID NO: 125. [Brief description of the drawings]

[0045] [Figure 1] The AAVS1 targeting vector map is shown. [Diagram 2] A B2M targeting vector map is shown. [Diagram 3] The CIITA targeting vector map is shown. [Figure 4] The CLYBL targeting vector map is shown. [Diagram 5] NKG2A targeting vector map is shown. [Figure 6] A TRAC targeting vector map is shown. [Figure 7A]Figure 7A shows flow cytometric analysis of cells engineered to insert a CAR transgene at the AAVS1 site. Figure 7B shows flow cytometric analysis of the bulk population of engineered cells. [Figure 7B] Figure 7B shows flow cytometric analysis of cells engineered to insert a CAR transgene at the AAVS1 site. Figure 7B shows flow cytometric analysis of cells after sorting for CAR-positive cells. [Figure 7C] Figure 7C shows flow cytometry analysis of cells engineered to insert a CAR transgene at the AAVS1 site. Figure 7C shows flow cytometry analysis of CAR-positive single cell clones. [Figure 8A] Figure 8A shows flow cytometric analysis of cells engineered to insert an HLA-E transgene at the B2M site.Figure 8B shows flow cytometric analysis of the bulk population of engineered cells. [Figure 8B] Figure 8B shows flow cytometric analysis of cells engineered to insert an HLA-E transgene at the B2M site.Figure 8B shows flow cytometric analysis of cells after sorting for HLA-E positive, B2M negative cells. [Figure 8C] Figure 8C shows flow cytometric analysis of cells engineered to insert an HLA-E transgene at the B2M site, and Figure 8D shows flow cytometric analysis of HLA-E positive, B2M negative single cell clones. [Figure 9A] Figure 9A shows flow cytometric analysis of cells engineered to insert an EGFR transgene at the CIITA site.Figure 9B shows flow cytometric analysis of the bulk population of engineered cells. [Figure 9B] Figure 9B shows flow cytometric analysis of cells engineered to insert an EGFR transgene at the CIITA site.Figure 9B shows flow cytometric analysis of cells after sorting for EGFR positive cells. [Figure 9C]Figure 9C shows flow cytometric analysis of cells engineered to insert an EGFR transgene at the CIITA site, and Figure 9D shows flow cytometric analysis of EGFR-positive single cell clones. [Figure 10A] Figure 10 shows flow cytometric analysis of cells engineered to insert a PSMA transgene at the CLYBL site. Figure 10A shows flow cytometric analysis of the bulk population of engineered cells. [Figure 10B] Figure 10B shows flow cytometric analysis of cells engineered to insert a PSMA transgene at the CLYBL site.Figure 10B shows flow cytometric analysis of cells after sorting for PSMA positive cells. [Figure 11A] Figure 11 shows flow cytometric analysis of cells engineered to insert the IL15-IL15RA transgene at the NKG2A site.Figure 11A shows flow cytometric analysis of the bulk population of engineered cells. [Figure 11B] Figure 11B shows flow cytometric analysis of cells engineered to insert the IL15-IL15RA transgene at the NKG2A site.Figure 11B shows flow cytometric analysis of cells after sorting for IL15-IL15RA positive cells. [Figure 12] The CIITA targeting vector map is shown. [Figure 13] A CD70 targeting vector map is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present application provides, inter alia, compositions and methods for use in genome engineering of cells such as iPSCs. Specifically, the methods and compositions described relate to introducing nucleic acids encoding transgenes into iPSCs such as pluripotent hematopoietic stem and / or progenitor cells (HSC / PCs) and preparing immune effector cells such as T cells, NK cells, macrophages and dendritic cells derived from iPSCs. Specifically, DNA sequences encoding gene transfer vectors for genome engineering of human cell lines and methods used are disclosed. The gene transfer vectors are designed for inserting transgenes into the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, and / or CLYBL loci of human cells (e.g., iPSCs) and include promoter sequences, terminator sequences, and homology arms specific for the loci of interest. The gene transfer vectors can be used with CRISPR nuclease-based systems, such as MAD7 nuclease-based systems. Also included are novel guide sequences for use with CRISPR nuclease-based systems for the insertion of transgenes, particularly MAD7 nuclease-based systems. In some embodiments, the MAD7 nuclease-based system comprises a non-naturally occurring or engineered MAD7 nuclease.

[0047] I. definition Various publications, articles, and patents are cited or described in the Background and throughout the specification; each of these references is incorporated herein by reference in its entirety for all intended purposes. The discussion of documents, acts, materials, devices, articles, and the like included in this specification is intended to provide a context for the embodiments of the present disclosure. 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.

[0048] 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 pertains. Other specific terms used herein have the meanings described herein.

[0049] It should 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.

[0050] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Such equivalents are intended to be encompassed by the present application.

[0051] As used herein, the terms "comprise" or "comprising," "include" or "including," "has" or "having," "contain" or "containing," or any other variation thereof, are understood to mean the inclusion of a specified integer or group of integers, but not the exclusion of other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Additionally, unless expressly stated to the contrary, "or" refers to inclusive or / and not exclusive. For example, condition A or condition B is satisfied by any 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), or both A and B are true (or exist).

[0052] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any of these options are understood to be within the meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of multiple options is also understood to be within the meaning and therefore meet the requirements of the term "and / or".

[0053] As used herein, the term "consisting of," or variations thereof, when used throughout the specification and claims, means that any recited integer or group of integers is inclusive, but that no additional integer or group of integers is added to the specified method, structure, or composition.

[0054] As used herein, the term "consisting essentially of" or variations thereof, as used throughout this specification and claims, means the inclusion of any recited integer or group of integers, and further the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel characteristics of a particular method, structure, or composition. See MPEP 2111.03.

[0055] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0056] As used herein, the terms "about," "approximately," "generally," "substantially," and similar terms, when referring to a dimension or characteristic (e.g., concentration or concentration range) of a component of the present invention, are to be understood to mean that the described dimension / characteristic is not a precise boundary or parameter, but does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. Unless otherwise stated, any numerical values, such as concentrations or concentration ranges described herein, should be understood in all cases to be modified by the term "about." At a minimum, such references involving numerical parameters will include variations that would not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.). In some embodiments, numerical values ​​typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all the possible subranges, and all individual numerical values ​​within that range (including integers and fractions of values ​​within such ranges), unless the context clearly dictates otherwise.

[0057] The term "identical" or percent "identity", in the context of two or more nucleic acids (e.g., guide RNA sequences or homology arm sequences) or polypeptide sequences (e.g., CAR polypeptides and CAR polynucleotides encoding them), refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0058] For sequence comparison, typically, one sequence serves as the reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, partial sequence coordinates are designated, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence to reference sequence based on designated program parameters.

[0059] Optimal sequence alignment for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), 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 as described in Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 This can be done by the method disclosed in the Supplement (Ausubel).

[0060] Examples of algorithms suitable for determining percent 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 publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a certain positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0061] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). 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 off its maximum achieved value by an amount X; the cumulative score falls below zero due to the accumulation of alignments of one or more negative scoring residues; 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 wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0062] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0063] 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.Therefore, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitution.Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0064] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated from, produced away from, or purified away from other biological components of the organism in which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues. Thus, "isolated" nucleic acids, peptides, proteins, and cells include nucleic acids, peptides, proteins, and cells that have been purified by standard purification methods and the purification methods described herein. "Isolated" nucleic acids, peptides, proteins, and cells may be part of a composition and still be isolated, provided that the composition is not part of the natural environment of the nucleic acid, peptide, protein, or cell. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0065] As used herein, the term "polynucleotide" is synonymously referred to as "nucleic acid molecule", "nucleotide" or "nucleic acid" and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that contain RNA or DNA, or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA that contains one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases, and unconventional bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also includes relatively short nucleic acid chains often referred to as "oligonucleotides."

[0066] A "construct" refers to a macromolecule or complex of molecules that includes a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct that can direct the delivery or introduction of foreign genetic material into a target cell, where it can be replicated and / or expressed. As used herein, the term "vector" includes the construct that is delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. The 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 the like.

[0067] "Integration" or "insertion" means that one or more sequences or nucleotides of an exogenous construct are stably inserted into a cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal or mitochondrial DNA of a cell. "Targeted integration" means that the nucleotides of a construct are inserted into the chromosomal or mitochondrial DNA of a cell at a preselected site or "integration site". The term "integration" or "insertion" as used herein further refers to a process involving the insertion of one or more sequences or nucleotides of an exogenous construct, with or without the deletion of an endogenous sequence or one or more nucleotides at the integration site. If there is a deletion at the insertion site, "integration" may further include the replacement of the deleted endogenous sequence or one or more nucleotides with one or more inserted sequences or nucleotides.

[0068] As used herein, the term "exogenous" is intended to mean that the referenced molecule or the referenced activity is introduced into the host cell or is non-native to the host cell. For example, the molecule can be introduced by introduction of the encoding nucleic acid into the host genetic material, such as 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 into the cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity that is present in the host cell in its natural form. Similarly, when used in reference to expression of an encoding nucleic acid, the term "endogenous" refers to expression of an encoding nucleic acid that is naturally contained within the cell and is not exogenously introduced.

[0069] As used herein, a "transgene", "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that is transcribed into RNA in vivo and, in some cases, translated into a polypeptide when placed under the control of appropriate regulatory sequences. A gene or polynucleotide of interest may include, but is not limited to, a prokaryotic sequence, a cDNA derived from eukaryotic mRNA, a genomic DNA sequence derived from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may code for 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 having 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 selection marker, and the like.

[0070] "Operably linked" refers to the operational linkage of nucleic acid or amino acid sequences, which are in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation.

[0071] The term "expression" as used herein refers to the biosynthesis of a gene product. This term includes the transcription of a gene into RNA. This term also includes the translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide (e.g., CAR) can be in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or anchored in the cell membrane.

[0072] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and can refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other alteration or modification, such as conjugation with a labeling component). Also included in the definition are, for example, polypeptides that contain one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0073] 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-form of the amino acid that is represented unless otherwise indicated.

[0074] II. Induced pluripotent stem cells (IPSCs) and immune effector cells iPSCs have unlimited self-renewal capacity. The use of iPSCs allows for the generation of controlled cell banks of engineered cells that can be expanded and differentiated into desired immune effector cells, providing large quantities of allogeneic therapeutic products.

[0075] Provided herein are genetically engineered iPSCs and their derivative cells. Selected genomic modifications provided herein enhance the therapeutic properties of the derived cells. The derived cells are functionally improved and suitable for allogenic off-the-shelf cell therapy, receiving a combination of selective modalities introduced into the cells at the iPSC level via genomic engineering. This approach may help reduce cytokine release syndrome CRS / GVHD mediated side effects and prevent long-term autoimmunity while providing superior efficacy.

[0076] 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. Differentiated or differentiation-induced cells are cells that have taken on a more specialized ("committed") position within a cell lineage. The term "committed" as applied to the process of differentiation refers to a cell that, under normal circumstances, will continue to differentiate into a particular cell type or subset of cell types and has progressed down a differentiation pathway to a point where, under normal circumstances, it cannot 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 form all lineages of the body or somatic cells or embryo proper. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs), which are unable to give rise to an entire organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells), which are able to give rise to an entire organism.

[0077] As used herein, the term "induced pluripotent stem cell" or iPSC means that stem cells are generated from differentiated adult, neonatal or fetal cells that are induced or changed or reprogrammed into cells that can differentiate into tissues of all three germ layers or layers (mesoderm, endoderm, and ectoderm). The generated iPSCs are not cells found in nature.

[0078] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hemocytoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells).

[0079] As used herein, the term "immune cell" or "immune effector cell" refers to a cell that participates in immune response. Immune response includes, for example, promoting immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and bone marrow-derived phagocytes.

[0080] As used herein, the term "engineered immune cell" or "engineered immune effector cell" refers to an immune cell that has 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.

[0081] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a type of white blood cell that has completed maturation in the thymus and has various roles in the immune system. T cells may have roles such as, for example, identifying specific foreign antigens in the body and activating and inactivating other immune cells. T cells can be any T cell, such as cultured T cells (e.g., primary T cells, or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.)), or T cells obtained from a mammal. T cells can be CD3+ cells. T cells may be of any type and at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (gdT cells; γδT cells), and the like. Further types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). T cells may also refer to genetically engineered T cells, such as, for example, T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0082] "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a secretory profile following stimulation that may include secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4 and IL10. "CD4" is a 55 kD glycoprotein that was first 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 involved as an associative recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, they define helper / inducer subsets.

[0083] "CD8+ T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the associative recognition element in major histocompatibility complex class I restricted interactions.

[0084] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). NK cells can also refer to genetically engineered NK cells, such as NK cells modified to express chimeric antigen receptors (CARs). NK cells can also be differentiated from stem or progenitor cells.

[0085] Induced pluripotent stem cell (iPSC) parent cell line can be made from peripheral blood mononuclear cells (PBMC) or T cells using any known method for introducing reprogramming factors into non-pluripotent cells, such as episomal plasmid-based process as previously described in U.S. Patent Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference in their entirety for all intended purposes. Reprogramming factors can be in the form of polynucleotides, and thus are introduced into non-pluripotent cells by vectors such as retrovirus, Sendai virus, adenovirus, episomes, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by lentivirus vectors. In some embodiments, one or more polynucleotides are introduced by episomal vectors. In various other embodiments, one or more polynucleotides are introduced by Sendai virus vectors. In some embodiments, the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs, and genome editing is introduced by performing one or more targeted integrations and / or in / dels at one or more selected sites. In another embodiment, the iPSCs are obtained from human T cells with antigen specificity and rearranged TCR genes (hereinafter also referred to as "T-iPS" cells), as described in U.S. Patent Nos. 9,206,394 and 10,787,642 (which are incorporated herein by reference in their entirety for all intended purposes).

[0086] Derived immune effector cells In another aspect, the present disclosure relates to cells derived from differentiation of iPSCs, derivative immune effector cells. As described above, the genome editing introduced into iPSCs is retained in the derivative immune effector cells. In certain embodiments of the derivative cells obtained from differentiation of iPSCs, the derivative cells are hematopoietic cells, including but not limited to HSCs (hematopoietic stem cells and hematopoietic progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells. In certain embodiments, the derivative cells are immune effector cells, such as NK cells or T cells.

[0087] In certain embodiments, the application provides natural killer (NK) cells or T cells derived from iPSCs carrying one or more transgene inserts prepared according to the present disclosure.

[0088] A method for producing a derivative cell is also provided, which comprises differentiating iPSCs under conditions for cell differentiation, thereby obtaining a derivative cell.

[0089] The iPSCs of the present application can be differentiated by any method known in the art.Exemplary methods are described in U.S. Patent No. 8,846,395, U.S. Patent No. 8,945,922, U.S. Patent No. 8,318,491, and International Publication No. WO2010 / 099539, WO2012 / 109208, WO2017 / 070333, WO2017 / 179720, WO2016 / 010148, WO2018 / 048828 and WO2019 / 157597, each of which is incorporated herein by reference in its entirety for all intended purposes.

[0090] III. Targeted genome editing at selected loci in iPSCs According to embodiments of the present application, one or more exogenous polynucleotides are inserted into one or more loci on one or more chromosomes of the iPSCs.

[0091] Genome editing, or gene editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a target cell. Targeted genome editing, or targeted genomic editing (interchangeable with "targeted gene editing") allows for insertion, deletion, and / or replacement at a preselected site in a genome. When 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 deletion or disruption of the sequence. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. Similarly, as used herein, the terms "targeted integration" and "targeted insertion" refer to a process involving the insertion of one or more exogenous sequences at a preselected site in a genome, with or without the deletion of the endogenous sequence at the insertion site.

[0092] Targeted editing can be achieved by either nuclease-independent or nuclease-dependent approaches. In the nuclease-independent targeted editing approach, homologous recombination is guided by the homologous sequences flanking the exogenous polynucleotide to be inserted through the host cell's enzymatic machinery.

[0093] Alternatively, targeted editing can be achieved at higher frequencies through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertion or deletion (in / del) of a small number of endogenous nucleotides. In comparison, the presence of a donor vector containing exogenous genetic material flanked by a pair of homology arms (or homology arms) allows the exogenous genetic material to be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in "targeted integration."

[0094] Targeting nucleases include natural and recombinant nucleases such as CRISPR-associated nucleases from families including Cas, Cpf, Cse, Csy, Csn, Csd, Cst, Csh, Csa, Csm, and Cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like. As an example, CRISPR / Cpf1 includes two major components: (1) Cpf1 endonuclease and (2) guide nucleic acid (which can be DNA or RNA). When co-expressed, the two components form a ribonucleoprotein (RNP) complex that is recruited to a target DNA sequence that includes a PAM and a seeding region near the PAM. The guide nucleic acid can be used to guide Cpf1 to target a selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction.

[0095] One type of alternative CRISPR nuclease family, Cpf1 (also known as Cas12a), has been used for genome editing since its first report in 2015 (Zetsche et al Cell, 163(3), 759-771). Cpf1 nuclease exhibits distinct features from Cas9 nuclease, such as staggered DSBs, T-rich PAMs, and the natural use of only one guide RNA molecule to form a complex with Cpf1 and target DNA. These features allow Cpf1 nuclease to be used in target organisms or regions within the genome of organisms where lower GC content makes the use of Cas9 less feasible.

[0096] Recently, an alternative CRISPR nuclease called MAD7 has been disclosed in U.S. Patent Nos. 9,982,279 and 10,337,028, the contents of which are incorporated herein in their entirety for all intended purposes. Inscripta has made this nuclease free for all commercial or academic research. Its use for commercial genome editing is therefore of great interest. Inscripta reports that MAD7 was developed from Eubacterium rectale and its functionality has been demonstrated in E. coli, S. cerevisiae, and human HEK293T cell lines. MAD7 shares only 31% identity with Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and also shares a T-rich PAM site (5'-YTTN-3') and a 21 nucleotide protospacer (region of the gRNA that associates the nuclease with the DNA target) length. Certain embodiments of the present disclosure are particularly suitable for use with the endonuclease MAD7. This nuclease requires only the crRNA for gene editing and allows for specific targeting of AT-rich regions of the genome. MAD7 cleaves DNA with a staggered cut compared to S. pyogenes, which has a blunt cut.

[0097] Exemplary MAD7 sequences and scaffold sequences of guide nucleic acids are provided in Table 1. In general, a "scaffold sequence" includes any sequence having sufficient sequence to promote the formation of a targetable ribonucleoprotein complex. A targetable ribonucleoprotein complex may include a nucleic acid-guided nuclease (e.g., MAD7) and a guide nucleic acid including a scaffold sequence and a guide sequence. A sufficient sequence within the scaffold sequence to promote the formation of a targetable ribonucleoprotein complex may include a degree of complementarity along the length of two sequence regions within the scaffold sequence, such as, for example, one or two sequence regions involved in the formation of a secondary structure (e.g., a pseudoknot region). The one or two sequence regions may be included in or encoded on the same polynucleotide. Alternatively, the one or two sequence regions may be included in or encoded on separate polynucleotides. In some embodiments, the scaffold sequence may include any one of SEQ ID NOs: 117-119. In some embodiments, the scaffold sequence comprises the sequence of SEQ ID NO: 117, and in some embodiments, the scaffold sequence comprises the sequence of SEQ ID NO: 118. In some embodiments, the scaffold sequence comprises the sequence of SEQ ID NO:119. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0098] Thus, one aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genomic insertion utilizing MAD7 endonuclease. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired insertion site, and the method of targeted insertion comprises introducing the construct into a cell to allow site-specific homologous recombination by a cellular host enzyme machinery. In another embodiment, the method of targeted insertion in a cell comprises introducing into a cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a CRISPR MAD7 expression cassette comprising a DNA binding domain specific to a desired insertion site. Specifically, according to the present disclosure, the method of targeted insertion in a cell comprises introducing into a cell a construct comprising one or more exogenous polynucleotides for insertion into a specific locus in iPSCs by introducing into the cell MAD7 nuclease and a gRNA comprising a guide sequence specific to a desired insertion site to allow MAD7-mediated insertion.

[0099] In general, the guide nucleic acid can form a complex with a compatible nucleic acid-guided nuclease and can hybridize with a target sequence, thereby guiding the nuclease to the target sequence. The guide nucleic acid can be DNA. The guide nucleic acid can be RNA. The guide nucleic acid can include both DNA and RNA. The guide nucleic acid can include modified nucleotides or non-naturally occurring nucleotides. When the guide nucleic acid includes RNA, the RNA guide nucleic acid can be encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, a linear construct, or an editing cassette as disclosed herein. In particular, in certain embodiments of the present disclosure, the guide sequence is for use in a MAD7 / gRNA ribonucleoprotein (RNP) complex for insertion of a transgene into a specific locus in iPSCs, the MAD7 / gRNA ribonucleoprotein (RNP) complex comprising: (I) a guide RNA (gRNA) polynucleotide sequence specific for MAD7 nuclease, the polynucleotide sequence comprising a guide sequence capable of hybridizing to a safe harbor locus in iPSCs (e.g., an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus); (II) a MAD7 enzyme protein; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to the left and right arms of a target sequence at a safe harbor locus (e.g., AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus), (2) a promoter operably linked to a polynucleotide encoding a transgene of interest (3), and (4) a transcription terminator sequence. In one embodiment, the guide sequence comprises a nucleotide sequence selected from SEQ ID NOs: 120-130.

[0100] The site for targeted insertion includes, but is not limited to, genomic safe harbor, which is an intragenic or extragenic region of the human genome that can theoretically accommodate predictable expression of newly inserted DNA without adversely affecting the host cell or organism.In certain embodiments, the genomic safe harbor for targeted insertion is one or more loci of genes selected from the group consisting of AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus.

[0101] In other embodiments, the site for targeted insertion is selected to delete or reduce the expression of endogenous genes at the insertion site.As used herein, the term "deletion" with respect to gene expression refers to any genetic modification that disables the expression of gene.Examples of the "deletion" of gene expression include, for example, the removal or deletion of the DNA sequence of a gene, the insertion of an exogenous polynucleotide sequence at the locus of a gene, and one or more substitutions within a gene, which disables the expression of the gene.

[0102] Genes for targeted deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems. "MHC-deficient", including MHC-class I-deficient or MHC-class II-deficient, or both, refers to cells that lack or no longer maintain or have reduced levels of surface expression of complete MHC complexes, including MHC class I protein heterodimers and / or MHC class II heterodimers, and the reduced or reduced levels are lower than the levels that are naturally detectable by other cells or by artificial 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 β2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 gene, and the Tapasin gene. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers. B2M null cells are MHC-I deficient. MHC class II deficiency can be achieved by 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 through activation of the transcription factor RFX5, which is required for class II protein expression. CIITA null cells are MHC-II deficient. In certain embodiments, one or more exogenous polynucleotides are inserted into one or more loci of a gene selected from the group consisting of the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby deleting or reducing expression of the gene due to the insertion.

[0103] In certain embodiments, the exogenous polynucleotide is inserted into one or more loci on a chromosome of the cell, preferably the one or more loci are the loci of a gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, CD70, CD38, CD33, or TIGIT genes, with the proviso that at least one of the one or more loci is an MHC gene (e.g., B2M, TAP 1, TAP Preferably, the one or more exogenous polynucleotides are inserted into the locus of MHC class I-related genes (such as, for example, β2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or tapasin gene); and the locus of MHC-II-related genes (such as, for example, RFXANK, CIITA, RFX5, RFXAP or CIITA gene); and optionally further into the locus of a safe harbor gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR and RUNX1 genes. More preferably, the one or more exogenous polynucleotides are inserted into the locus of CIITA, AAVS1 and B2M genes.

[0104] In certain embodiments, multiple transgenes can be inserted into the targeted site for deletion of major histocompatibility complex (MHC) class I and MHC class II proteins.For example, (a) a first exogenous polynucleotide is inserted into the locus of AAVS1 gene; (b) a second exogenous polypeptide is inserted into the locus of CIITA gene; and a third exogenous polypeptide is inserted into the locus of B2M gene; the insertion of exogenous polynucleotides causes the expression of CIITA and B2M genes to be deleted or reduced.

[0105] In certain embodiments, the guide RNA for insertion into the AAVS1 locus comprises a guide sequence of SEQ ID NO: 120 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 60 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 61 or a fragment thereof.

[0106] In certain embodiments, the guide RNA for insertion into the B2M locus comprises a guide sequence of SEQ ID NO: 121 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 63 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 64 or a fragment thereof.

[0107] In certain embodiments, a guide RNA for insertion into the CIITA locus comprises a guide sequence of SEQ ID NO: 122 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 66 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 67 or a fragment thereof. In certain embodiments, a guide RNA for insertion into the CIITA locus comprises a guide sequence of SEQ ID NO: 126 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 106 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 107 or a fragment thereof.

[0108] In certain embodiments, the guide RNA for insertion into the NKG2A locus comprises a guide sequence of SEQ ID NO: 123 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 69 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 70 or a fragment thereof.

[0109] In certain embodiments, the guide RNA for insertion into the TRAC locus comprises a guide sequence of SEQ ID NO: 124 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 72 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 73 or a fragment thereof.

[0110] In certain embodiments, the guide RNA for insertion into the CLYBL locus comprises a guide sequence of SEQ ID NO: 125 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 75 or a fragment thereof, and the right homology sequence is selected from SEQ ID NO: 76 or a fragment thereof.

[0111] In certain embodiments, the guide RNA for insertion into the CD70 locus comprises a guide sequence of SEQ ID NO: 127 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 109 or a fragment thereof, and the right homology sequence is selected from SEQ ID NO: 110 or a fragment thereof.

[0112] In certain embodiments, the guide RNA for insertion into the CD38 locus comprises the guide sequence of SEQ ID NO: 128 or a variant thereof.

[0113] In certain embodiments, the guide RNA for insertion into the CD33 locus comprises the guide sequence of SEQ ID NO: 129 or 130, or a variant thereof.

[0114] Provided in Table 2 are targeting domain sequences of gRNA molecules (both RNA and DNA sequences are provided) and corresponding homology arm sequences for use in the compositions and methods of the disclosure, e.g., in altering expression of or modifying target genes in iPSCs. [Table 2-1] [Table 2-2]

[0115] Homology Arm Whether single-stranded or double-stranded, the donor template generally contains one or more regions that are homologous to regions of DNA (e.g., the target nucleic acid) within or near (e.g., flanking or adjacent to) the target sequence to be cleaved (e.g., the cleavage site). These regions of homology are referred to herein as "homology arms" and are shown diagrammatically below: [5' homology arm]-[replacement sequence]-[3' homology arm].

[0116] The homology arms of the donor template described herein may be of any suitable length, so long as they are long enough to allow efficient cleavage of the cut site on the target nucleic acid by the DNA repair process that requires the donor template. In certain embodiments, if the amplification of the homology arms, for example by PCR, is desired, the homology arms are of such a length that the amplification can be performed. In certain embodiments, if the sequencing of the homology arms is desired, the homology arms are of such a length that the sequencing can be performed. In certain embodiments, if the quantitative evaluation of the amplicons is desired, the homology arms are of such a length that the amplification of a similar number of each amplicon is achieved, for example, by having similar G / C content, amplification temperature, etc. In certain embodiments, the homology arms are double-stranded. In certain embodiments, the homology arms are single-stranded.

[0117] In certain embodiments, the 5' homology arm is 50 to 250 nucleotides in length. In certain embodiments, the 5' homology arm is about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, or about 250 nucleotides in length.

[0118] In certain embodiments, the 3' homology arm is between 50 and 250 nucleotides in length. In certain embodiments, the 3' homology arm is about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, or about 250 nucleotides in length.

[0119] The 5' and 3' homology arms may be the same length or may be different lengths. In certain embodiments, the 5' and 3' homology arms are amplified to allow quantitative evaluation of gene editing events such as targeted insertion at the target nucleic acid. In certain embodiments, quantitative evaluation of gene editing events may rely on amplifying both the 5' and 3' junctions at the targeted insertion site by amplifying all or part of the homology arms using a single pair of PCR primers in a single amplification reaction. Thus, the lengths of the 5' and 3' homology arms may be different, but the length of each homology arm should be amplifiable (e.g., using PCR) as needed. Furthermore, if amplification of both the 5' and 3' homology arms in a single PCR reaction is desired, the length difference between the 5' and 3' homology arms should allow PCR amplification using a single pair of PCR primers.

[0120] IV. Transgene for insertion in iPSCs According to an embodiment of the present application, iPSCs are engineered by inserting one or more transgenes using the MAD7 / gRNA ribonucleoprotein (RNP) complex of the present disclosure. Many different transgenes, including genes of interest, can be inserted using the RNP complex, guide sequence and homology arms according to the present disclosure. Exemplary transgenes are discussed further below.

[0121] A. Chimeric Antigen Receptors ("CARs") At least one of the transgenes that can be inserted encodes an exogenous chimeric antigen receptor (CAR), such as a CAR that targets a tumor antigen.

[0122] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide that includes at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of the target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. The CAR redirects the specificity of immune effector cells and induces proliferation, cytokine production, phagocytosis, and / or the production of molecules that can mediate cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0123] As used herein, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of the nascent CAR protein, which co-translationally or post-translationally targets the nascent protein to the endoplasmic reticulum and subsequent surface expression.

[0124] As used herein, the term "extracellular antigen-binding domain," "extracellular domain," or "extracellular ligand-binding domain" refers to the portion of a CAR that is located on the outside of the cell membrane and can bind to an antigen, target, or ligand.

[0125] As used herein, the term "hinge region" or "hinge domain" refers to the portion of a CAR that connects the two adjacent domains of the CAR protein, i.e., the extracellular and transmembrane domains of the CAR protein.

[0126] As used herein, the term "transmembrane domain" refers to the portion of a CAR that extends across and anchors the CAR to the cell membrane.

[0127] As used herein, the term "intracellular signaling domain," or "cytoplasmic signaling domain," refers to the portion of a CAR that is located inside the cell membrane and is capable of transmitting an effector signal.

[0128] As used herein, the term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells) that provides a primary cytoplasmic signaling sequence that regulates the primary activation of receptors in a stimulatory manner for at least some aspects of immune cell signaling pathways. Stimulatory molecules include two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and those that act in an antigen-independent manner to provide secondary costimulatory signals (referred to as "co-stimulatory signaling domains").

[0129] In certain embodiments, the extracellular domain comprises an antigen-binding domain and / or an antigen-binding fragment. The antigen-binding fragment can be, for example, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen. The antigen-binding fragment of the present application has one or more desirable functional properties, including, but not limited to, high affinity binding to tumor antigen; high specificity to tumor antigen; ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cellular cytotoxicity (ADCC) against cells expressing tumor antigen; and ability to inhibit tumor growth in subjects and animal models in need thereof when administered alone or in combination with other anti-cancer therapies.

[0130] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human, humanized, composite and chimeric antibodies, and antibody fragments, whether monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a particular antigen. Antibody structure 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 subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Thus, the antibodies of the present application may be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3 or IgG4. The antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Thus, the antibodies of the present application may comprise a kappa or lambda light chain constant domain. According to certain embodiments, the antibodies of the present application comprise heavy and / or light chain constant regions derived from a rat or human antibody. In addition to the heavy and light chain constant domains, the antibodies comprise an antigen-binding region composed of a light chain variable region and a heavy chain variable region, each of which comprises 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.

[0131] 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 particular tumor antigen is substantially free of antibodies that do not bind to that tumor antigen). Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0132] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the present application can be 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 that contain B cells obtained from a transgenic non-human animal, such as a transgenic mouse or rat, whose genome contains human heavy chain transgenes and light chain transgenes.

[0133] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv ’ ), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), single domain antibodies (sdAbs), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelized single domain antibodies, minibodies, nanobodies, domain antibodies, bivalent domain antibodies, light chain variable domains (VL), variable domains of camelid antibodies (VHH), or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. Antigen-binding fragments are capable of binding to the same antigen as that bound by the parent antibody or parent antibody fragment.

[0134] 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 linked by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).

[0135] As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region, or which comprises only a heavy chain variable region.

[0136] 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, made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies that contain at least one human heavy and / or light chain polypeptide.

[0137] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to have increased sequence homology with human antibody sequences such that the antigen-binding properties of the antibody are retained but the antibody is less antigenic in humans.

[0138] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody 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 an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0139] As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or substantially do not overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In an embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0140] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In certain 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 certain 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 certain embodiments, a bispecific antibody comprises a VFv or fragment thereof having binding specificity for a first epitope. H H and V with binding specificity for a second epitope. H Contains H.

[0141] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to a tumor antigen" is defined as a domain or fragment that specifically binds to a tumor antigen, the domain or fragment being greater than 1×10 -7 M or less, preferably 1×10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×10 -10KD refers to an antigen-binding domain or antigen-binding fragment that binds to a tumor antigen with a KD equal to or less than M. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods in the art in light of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, for example by using a biosensor system, such as a Biacore® system, or by using biolayer interferometry technology, such as an Octet RED96 system.

[0142] The smaller the value of the KD 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.

[0143] 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., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), 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 an antigen-specific TCR), and epitope-binding fragments of any of the above. The antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0144] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab ’ In some embodiments, the antigen-binding fragment is a F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is a scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0145] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or a nanobody.

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

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

[0148] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH.

[0149] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises an scFv.

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

[0151] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise an scFv.

[0152] Alternative scaffolds of 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 produce molecules with improved characteristics, such as higher stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that can be used in the CARs of the present disclosure include engineered tenascin-derived tenascin type III domains (e.g., Centyrin™); engineered gamma B-crystallin-derived scaffolds or engineered ubiquitin-derived scaffolds (e.g., Affilins™); engineered fibronectin type III (10Fn3) domains from fibronectin (e.g., monobodies, AdNectins™, or AdNexins™); engineered ankyrin repeat motif-containing polypeptides (e.g., DARPins™); engineered lipocalins (e.g., anticalins); Kunitz domains from engineered protease inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); engineered Z domains from Protein A (Affibodies™); Sac7d-derived polypeptides (e.g., Nanoffitins® or affitins); engineered SH2 domains from Fyn (e.g., Fynomers®); CTLDs 3(e.g., tetranectin); thioredoxin (e.g., peptide aptamers); KALBITOR®; beta sandwich (e.g., iMabs); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimetics; as well as engineered counterparts of any 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 for all intended purposes).

[0153] In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0154] 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 be optionally cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of leader sequences known to those skilled in the art may be used as a leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from human immunoglobulin heavy chain (HC).

[0155] 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.

[0156] In some embodiments, the first polypeptide of a CAR of the disclosure comprises a transmembrane domain fused in frame between the extracellular tag-binding domain and the cytoplasmic domain.

[0157] The transmembrane domain may be derived from a protein that contributes to the extracellular tag-binding domain, a protein that contributes to the signaling domain or co-signaling domain, or may be derived from a completely different protein. In some cases, the transmembrane domain may be selected or modified by amino acid substitution, deletion, or insertion to minimize interaction with other members of the CAR complex. In some cases, the transmembrane domain may be selected or modified by amino acid substitution, deletion, or insertion to avoid binding of proteins that naturally associate with the transmembrane domain. In certain embodiments, the transmembrane domain contains additional amino acids that allow flexibility and / or optimal distance between the domains connected to the transmembrane domain.

[0158] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains that are particularly useful in the present disclosure may be derived from (i.e., may include at least the transmembrane regions of) the α, β or ζ chains of the T cell receptor (TCR), CD28, CD3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan and / or valine may be found at each end of a synthetic transmembrane domain.

[0159] In some embodiments, it is desirable to utilize transmembrane domains of the zeta, eta or FcεR1 gamma chains that contain cysteine ​​residues capable of disulfide bonding, so that the resulting chimeric protein can form disulfide-linked dimers with itself or with unmodified versions of the zeta, eta or FcεR1 gamma chains or related proteins. In some cases, the transmembrane domains are selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it is desirable to use transmembrane domains of zeta, eta or FcεR1 gamma and -β, MB1 (Igα), B29 or CD3-γ, zeta or eta to retain physical association with other members of the receptor complex.

[0160] 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.

[0161] In some embodiments, the first polypeptide of a CAR of the disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, and the tag-binding domain, linker, and transmembrane domain are in-frame with each other.

[0162] 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 more 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 naturally occurring molecules, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or from all or a portion of an antibody constant region. Alternatively, the spacer region can be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or can be a completely synthetic spacer region sequence. Non-limiting examples of spacer regions that can be used in accordance with the present disclosure include portions of the human CD8 alpha chain, partial extracellular domains of CD28, FcyRllla 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. In some embodiments, if the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.

[0163] In some embodiments, the spacer region comprises a hinge domain. The hinge domain can be derived from CD8α, CD28 or immunoglobulin (IgG). For example, the IgG hinge can be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof.

[0164] In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or a functional fragment thereof. In certain embodiments, the IgG hinge is derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof. In certain embodiments, the hinge domain comprises an immunoglobulin CH1, CH2, CH3 and / or hinge region. In certain embodiments, the hinge domain comprises an immunoglobulin core hinge region. The term "core hinge" can be used interchangeably with the term "short hinge" (also known as "SH"). Non-limiting examples of suitable hinge domains are the core immunoglobulin hinge regions, including EPKSCDKTHTCPPCP (SEQ ID NO: 55) from IgG1, ERKCCVECPPCP (SEQ ID NO: 56) from IgG2, and ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP) from IgG3. 3 (SEQ ID NO:57), and ESKYGPPCPSCP (SEQ ID NO:58) from IgG4 (see Wypych et al., JBC 2008 283(23): 16194-16205; incorporated herein by reference in its entirety for all purposes). In an embodiment, the hinge domain is a fragment of an immunoglobulin hinge.

[0165] 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.

[0166] 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. [Table 3]

[0167] In certain aspects, the first polypeptide of the CAR of the present disclosure comprises a cytoplasmic domain that comprises at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more costimulatory signaling domains.

[0168] The cytoplasmic domain is responsible for activating at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Usually, the entire signaling domain is present, but in many cases it is not necessary to use the entire chain. When 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.

[0169] Non-limiting examples of signaling domains that can be used in the CARs of the present disclosure include, for example, signaling domains from DAP10, DAP12, Fcε receptor Iγ chain (FCER1G), FcR β, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

[0170] In some embodiments, the cytoplasmic domain comprises a CD3 zeta signaling domain. In one embodiment, the CD3 zeta signaling domain comprises an amino acid sequence 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In some embodiments, the costimulatory signaling domain is derived from CD40. In some embodiments, the CD40 costimulatory signaling domain comprises an 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.

[0175] 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.

[0176] In some embodiments, the costimulatory signaling domain is derived from CD80. In some embodiments, the CD80 costimulatory signaling domain comprises an 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.

[0177] In some embodiments, the costimulatory signaling domain is derived from CD86. In some embodiments, the CD86 costimulatory signaling domain comprises an 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.

[0178] In some embodiments, the costimulatory signaling domain is derived from CD27. In some embodiments, the CD27 costimulatory signaling domain comprises an 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.

[0179] In one embodiment, the costimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS costimulatory signaling domain comprises an 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.

[0180] 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.

[0181] 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.

[0182] In one embodiment, the costimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP12 costimulatory signaling domain comprises an 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.

[0183] In one embodiment, the costimulatory signaling domain is derived from 2B4 (CD244). In certain aspects, the 2B4 (CD244) costimulatory signaling domain comprises an 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.

[0184] In some embodiments, the costimulatory signaling domain is derived from CD28. In some embodiments, the CD28 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO:20, 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:20.

[0185] In one embodiment, a CAR of the present disclosure comprises a hinge region, a transmembrane region, and a costimulatory signaling domain all derived from CD28. In one embodiment, the hinge region, the transmembrane region, and the costimulatory signaling domain derived from CD28 comprise the amino acid sequence set forth in SEQ ID NO:5, or a variant thereof having at least 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:5.

[0186] 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 some embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.

[0187] 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 more than one costimulatory domain is included, the order of the costimulatory signal transduction domains can be swapped.

[0188] Non-limiting exemplary CAR regions and sequences are provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0189] In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen. The antigen-binding domain of the second polypeptide may bind to multiple antigens or multiple epitopes in the antigen. For example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more antigens. As another example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more epitopes in the same antigen.

[0190] The selection of antigen binding domains may depend on the type and number of antigens that define the surface of target cells. For example, antigen binding domains may be selected to recognize antigens that function as cell surface markers on target cells associated with a particular disease state. In an embodiment, 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 antigen binding domains in the CARs of the present disclosure include those associated with tumor cells or autoimmune diseases.

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

[0192] In some embodiments, the antigen binding domain binds to at least one tumor antigen. In some embodiments, the antigen binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors.

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

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

[0195] Further examples of antigens that the antigen binding domain may target include alpha-fetoprotein, A3, antigen specific for the A33 antibody, Ba733, BrE3 antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138. , colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic These include, but are not limited to, gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placenta growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, angiogenesis markers, oncogene markers, or oncogene products.

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

[0197] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells that produce "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 (granulomatosis with polyangiitis), myasthenia gravis, Hashimoto's disease, hyperthyroidism (Graves' disease or Graves' disease), chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease, or ulcerative colitis.

[0198] In some embodiments, autoimmune antigens that may be targeted by the CARs disclosed herein include platelet antigens, myelin protein antigens, Sm antigens in snRNPs, pancreatic islet cell antigens, rheumatoid factors, and anti-citrullinated proteins, citrullinated proteins and peptides (e.g., CCP-1, CCP-2 (cyclic citrullinated peptides)), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factors, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage (e.g., collagen II, IX and XI), circulating serum proteins (e.g., RF (IgG, IgM), fibrinogen, plasminogen, ferritin), nuclear components (e.g., RA33 / hnRNP, A2, Sm, eukaryotic translation elongation factor 1α1), stress proteins (e.g., HSP-65, -70, -90, BiP), inflammatory / immune factors (e.g., B7-H1, IL-1α, and IL-8), enzymes (e.g., calpastatin, α-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase), receptors (e.g., lipocortin 1), neutrophil nuclear proteins (e.g., lactoferrin and 25-35 kD nuclear protein), granule proteins (e.g., bactericidal permeability increasing protein, These include, but are not limited to, ribosomal protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, pancreatic islet cell antigens, rheumatoid factors, histones, ribosomal P protein, cardiolipin, vimentin), nucleic acids (e.g., dsDNA, ssDNA, and RNA), ribonucleopartides and proteins (e.g., Sm antigens (including but not limited to SmD and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro(SSA), and La(SSB) antigens).

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

[0200] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO:3, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3. In another embodiment, the linker is (G 4 S) 3 In one embodiment, (G 4 S) 3The linker comprises an amino acid sequence set forth in SEQ ID NO:25, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:25.

[0201] Other linker sequences may include portions of the hinge region, CL or CH1 of an immunoglobulin from any immunoglobulin heavy or light chain isotype. Exemplary linkers that may be used include any of SEQ ID NOs: 26-54 in Table 4. Additional linkers are described, for example, in International Publication No. WO2019 / 060695, which is incorporated herein by reference in its entirety for all intended purposes.

[0202] B. Artificial Cell Death Polypeptides Another potential transgene for insertion according to the present disclosure is an exogenous polynucleotide encoding an artificial cell death polypeptide.

[0203] As used herein, the term "artificial cell death polypeptide" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. Artificial cell death polypeptides may mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation and / or antibody-mediated depletion. In some cases, the artificial cell death polypeptide is activated by an exogenous molecule, e.g., an antibody, and upon activation, induces apoptosis and / or cell death of therapeutic cells. In certain embodiments, the mechanism of action of the artificial cell death polypeptide is metabolic, dimerization-induced, or therapeutic monoclonal antibody-mediated.

[0204] In certain embodiments, the artificial cell death polypeptide is an inactivated cell surface receptor that contains an epitope that is specifically recognized by an antibody, particularly a monoclonal antibody, also referred to herein as a monoclonal antibody specific epitope. When expressed by iPSCs or their derived cells, the inactivated cell surface receptor is inactive or significantly impaired in signal transduction, but can still be specifically recognized by the antibody. The specific binding of the antibody to the inactivated cell surface receptor allows for the elimination of iPSCs or their derived cells by ADCC and / or ADCP mechanisms, as well as direct killing by antibody drug conjugates with toxins or radionuclides.

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

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

[0207] Exemplary inactivated cell surface receptors of the present application include tEGFR variants. In certain embodiments, expression of the inactivated cell surface receptor in engineered immune cells expressing chimeric antigen receptors (CARs) induces cell suicide of the engineered immune cells when the cells are contacted with anti-EGFR antibodies. Methods using inactivated cell surface receptors are described in WO2019 / 070856, WO2019 / 023396, WO2018 / 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 may be administered an amount of anti-EGFR antibody effective to remove the previously administered engineered immune cells in the subject.

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

[0209] In certain embodiments, the tEGFR variant comprises or consists of an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:77, and preferably comprises or consists of the amino acid sequence of SEQ ID NO:77.

[0210] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the CD79b epitope comprises or consists of an amino acid sequence 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:81, and preferably comprises or consists of the amino acid sequence of SEQ ID NO:81.

[0211] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the CD20 epitope comprises or consists of an amino acid sequence 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, and preferably comprises or consists of the amino acid sequence of SEQ ID NO:82.

[0212] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of the Her2 receptor or ErbB, such as the epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody specific epitope comprises or 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:84, and preferably comprises or consists of the amino acid sequence of SEQ ID NO:84.

[0213] 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, CD20 of B cells, ErbB2 or CD79b, and when the genomically engineered iPSCs contain more than one suicide gene, the suicide genes are integrated into different safe harbor loci such as AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL.

[0214] C. Cytokines In some embodiments, the transgene for insertion encodes a cytokine, such as interleukin-15 or interleukin-2.

[0215] As used herein, "interleukin-15" or "IL-15" refers to a cytokine, or a functional portion thereof, that regulates activation and proliferation of T and NK cells. A "functional portion" ("biologically active portion") of a cytokine refers to a portion of a cytokine that retains one or more functions of the full-length or mature cytokine. Such functions of IL-15 include promoting survival of NK cells, regulating activation and proliferation of NK cells and T cells, and supporting NK cell development from hematopoietic stem cells. As will be appreciated by one of skill 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 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, and preferably comprises the amino acid sequence of SEQ ID NO:79.

[0216] As used herein, "interleukin-2" refers to a cytokine, or a functional portion thereof, that regulates the activation and proliferation of T cells and NK cells. In certain embodiments, the IL-2 is wild-type IL-2. In certain embodiments, the IL-2 is human IL-2. In certain embodiments, the IL-2 comprises an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:85, and preferably comprises the amino acid sequence of SEQ ID NO:85.

[0217] In certain embodiments, the transgene may comprise an exogenous gene encoding an inactivated cell surface receptor comprising a monoclonal antibody specific epitope operably linked to a cytokine, preferably by an autoprotease peptide sequence. Examples of autoprotease peptides include, but are not limited to, peptide sequences selected from the group consisting of porcine teschovirus-1 2A (P2A), foot and mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), Flacherie Virus 2A (BmIFV2A), and combinations thereof. In one embodiment, the autoprotease peptide is a porcine teschovirus-1 2A (P2A) autoprotease peptide. 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:78, and preferably comprises the amino acid sequence of SEQ ID NO:78.

[0218] In certain embodiments, the inactivated cell surface receptor comprises a truncated epidermal growth factor receptor (tEGFR) variant operably linked to interleukin-15 (IL-15) or IL-2 by an autoprotease peptide sequence. In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:86, and preferably comprises the amino acid sequence of SEQ ID NO:86.

[0219] In some embodiments, the inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:80, and preferably comprises the amino acid sequence of SEQ ID NO:80.

[0220] In some embodiments, the inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. In certain embodiments, 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.

[0221] In certain embodiments, the inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, 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.

[0222] In certain embodiments, the inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody within its extracellular domain, transmembrane region, and cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises multiple epitopes specifically recognized by an antibody, the epitopes can have the same or different amino acid sequences, and the epitopes can be linked to each other via a peptide linker, such as a flexible peptide linker having a sequence of (GGGGS)n, where n is an integer between 1 and 8 (SEQ ID NOs: 87, 101, 25, 31, 32, and 102-104, respectively). 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 within the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked to an epitope specifically recognized by an antibody, either 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 linking it to a transmembrane domain via an autoprotease peptide sequence.

[0223] Non-limiting exemplary regions and sequences of inactivated cell surface receptors are provided in Table 5. [Table 5-1] [Table 5-2]

[0224] 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:88, and preferably comprises the amino acid sequence of SEQ ID NO:88.

[0225] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:89, and preferably comprises the amino acid sequence of SEQ ID NO:89.

[0226] In certain embodiments, the inactivated cell surface receptor comprises an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:90, and preferably comprises the amino acid sequence of SEQ ID NO:90.

[0227] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

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

[0229] In certain embodiments, the iPSCs or derived cells thereof comprise a 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 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: 91, and preferably comprises the amino acid sequence of SEQ ID NO: 91. In certain embodiments, the HLA-G 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: 95, and preferably comprises the amino acid sequence of SEQ ID NO: 95.

[0230] In certain embodiments, the 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 exogenous polypeptide 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:93.

[0231] In other embodiments, the 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 exogenous polypeptide 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:96.

[0232] E. Other Optional Genome Editing In other embodiments of the above cells, genome editing using the RNP complex of the present disclosure may include the insertion of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptide proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, potential drug targets, or proteins that promote the engraftment, trafficking, homing, survival, self-renewal, persistence and / or survival of the genome-engineered iPSCs or their derived cells. Other transgene insertions include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitor proteins such as PD1, PD-L1, and CTLA4, as well as proteins that target the CD47 / signal regulatory protein alpha (SIRPα) axis.

[0233] V. Regulatory elements In certain embodiments, the polynucleotide encoding MAD7 nuclease, gRNA, or exogenous polynucleotide for insertion is operably linked to at least a regulatory element.The regulatory element can mediate the expression of MAD7, gRNA, and / or transgene in host cell.The regulatory element includes, but is not limited to, promoter, enhancer, initiation site, polyadenylation (poly A) tail, IRES element, response element, and termination signal.

[0234] In some embodiments, the exogenous polynucleotide for insertion is operably linked to: (1) one or more exogenous promoters, including CMV, EFla, PGK, CAG, UBC, SV40, human beta-actin, or other constitutive, inducible, stage-specific, tissue-specific, or cell type-specific promoters; or (2) one or more endogenous promoters contained in selected sites such as AAVS1, B2M, CUTA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL, or other loci that meet the criteria of the Genomic Safe Harbor.

[0235] In some embodiments, the promoter is a CAG promoter. In some embodiments, the CAG promoter 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:98.

[0236] In some embodiments, the exogenous polynucleotide for insertion is operably placed under the control of a Kozak consensus sequence. In some embodiments, the Kozak sequence comprises a polynucleotide sequence of GCCACC or a variant thereof.

[0237] In certain embodiments, the exogenous polynucleotide for insertion is operably linked to a terminator / polyadenylation signal. In some embodiments, the terminator / polyadenylation signal is an SV40 signal. In certain embodiments, the SV40 signal comprises a polynucleotide sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%) to SEQ ID NO: 99. Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0238] VI. composition In another general aspect, the application provides a composition comprising an isolated polynucleotide of the application, a host cell of the application, and / or an iPSC or a derived cell thereof.

[0239] 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 mononuclear blood cell, a feeder cell, a feeder cell component or a substitute thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

[0240] In certain embodiments, the composition is a pharmaceutical composition comprising the isolated polynucleotide of the present application, the host cell of the present application and / or the iPSC or derived cell thereof, and a pharma- ceutically acceptable carrier. The term "pharmaceutical composition" as used herein 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, together with a pharma- ceutically acceptable carrier. The polynucleotide, polypeptide, host cell, and / or iPSC or derived cell of the present application, and compositions comprising them, are also useful in the manufacture of medicaments for therapeutic applications as referred to herein.

[0241] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the compositions described herein or the biological activity of the compositions described herein. According to certain embodiments, in view of the present disclosure, any pharmaceutical acceptable carrier suitable for use in polynucleotides, polypeptides, host cells, and / or iPSCs or derived cells thereof can be used.

[0242] Formulation of pharma- ceutically active ingredients with a pharma- ceutically acceptable carrier is known in the art (e.g., Remington: The Science and Practice of Pharmacy (e.g., 21st ed. (2005) and any later editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, isotonicity agents, preservatives, stabilizers, and chelating agents. One or more pharma- ceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present application.

[0243] VII. How to use In another general aspect, the present application provides a method of treating a disease or condition in a subject in need thereof. The method includes administering a therapeutically effective amount of a cell of the present application and / or a composition of the present application to a subject in need thereof. In an embodiment, the disease or condition is cancer. The cancer can be, for example, a solid cancer or a liquid cancer. The cancer can be, for example, selected from the group consisting of lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, 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).

[0244] 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 derived cell.As used herein, the term "therapeutically effective amount" refers to the amount of active material or ingredient that induces a desired biological or pharmaceutical response in a subject.The therapeutically effective amount can be determined empirically and in a routine manner in relation to the stated purpose.

[0245] As used herein with respect to the cells of the present application and / or pharmaceutical compositions of the present application, a therapeutically effective amount means the amount of the cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0246] According to certain embodiments, a therapeutically effective amount refers to an 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 symptoms associated therewith; (ii) shortening the duration of the disease, disorder or condition being treated or symptoms associated therewith; (iii) preventing the progression of the disease, disorder or condition being treated or symptoms associated therewith; (iv) causing regression of the disease, disorder or condition being treated or symptoms associated therewith; (v) preventing the onset or onset of the disease, disorder or condition being treated or symptoms associated therewith. (vi) prevent the recurrence of the disease, disorder or condition, or symptoms associated therewith, being treated; (vii) reduce hospitalization of a subject having the disease, disorder or condition, or symptoms associated therewith, being treated; (viii) shorten the length of hospitalization of a subject having the disease, disorder or condition, or symptoms associated therewith, being treated; (ix) increase the survival rate of a subject having the disease, disorder or condition, or symptoms associated therewith, being treated; (xi) inhibit or alleviate the disease, disorder or condition, or symptoms associated therewith, being treated in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic efficacy of another therapy.

[0247] The therapeutically effective amount or dosage may vary depending on a variety of factors, such as the disease, disorder or condition being 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 pharmaceutical agents being administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0248] 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.

[0249] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in any convenient manner known to those skilled in the art. For example, the cells of the present application can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and / or transplantation. The compositions comprising the cells of the present application can be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrathoracically, 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.

[0250] Pharmaceutical compositions comprising the cells of the present application can be provided as sterile liquid preparations, typically isotonic aqueous solutions containing cell suspensions, or optionally as emulsions, dispersions, etc., which are typically buffered to a selected pH. The compositions can include carriers suitable for cell integrity and viability and administration of the cell composition, e.g., water, saline, phosphate buffered saline, etc.

[0251] Sterile injectable solutions can be prepared by incorporating the cells of the present application in an appropriate amount of an appropriate solvent, with various other ingredients as necessary. Such compositions can include pharma- ceutically acceptable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, etc., that are suitable for use with the cell composition and for administration to subjects, such as humans. Suitable buffers for providing cell compositions are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the present application.

[0252] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in any physiologically acceptable vehicle. The cell population containing the cells of the present application can include a purified cell population. Those skilled in the art can easily determine the cells in the cell population using various well-known methods. The range of purity in the cell population containing the genetically modified cells of the present application can be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. Those skilled in the art can easily adjust the dosage, for example, a decrease in purity requires an increase in the dosage.

[0253] The cells of the present application are generally administered as a dose based on cells per kilogram (cells / kg) of body weight of the subject to which the cells and / or pharmaceutical compositions comprising the cells are administered. Generally, cell doses range from about 10 to about 20, depending on the method and location of administration. 4 ~about 10 10 Cells / kg body weight range, e.g., about 10 5 ~about 10 9 , about 10 5 ~about 10 8 , about 10 5 ~about 10 7 , or about 10 5 ~about 10 6 Generally, higher doses are used for systemic administration than for regional administration, where the immune cells of the present application are administered to the area of ​​the tumor and / or cancer. Exemplary dose ranges include 1x10 4 ~1x10 8 , 2x10 4 ~1x10 8 , 3x10 4 ~1x10 8 , 4x10 4 ~1x10 8 , 5x10 4 ~6x10 8 , 7x10 4 ~1x10 8 , 8x10 4 ~1x10 8 , 9x10 4~1x10 8 、1x10 5 ~1x10 8 、1x10 5 ~9x10 7 、1x10 5 ~8x10 7 、1x10 5 ~7x10 7 、1x10 5 ~6x10 7 、1x10 5 ~5x10 7 、1x10 5 ~4x10 7 、1x10 5 ~4x10 7 、1x10 5 ~3x10 7 、1x10 5 ~2x10 7 、1x10 5 ~1x10 7 、1x10 5 ~9x10 6 、1x10 5 ~8x10 6 、1x10 5 ~7x10 6 、1x10 5 ~6x10 6 、1x10 5 ~5x10 6 、1x10 5 ~4x10 6 、1x10 5 ~4x10 6 、1x10 5 ~3x10 6 、1x10 5 ~2x10 6 、1x10 5 ~1x10 6 、2x10 5 ~9x10 7 、2x10 5 ~8x10 7 、2x10 5 ~7x10 7 、2x10 5 ~6x10 7 、2x10 5 ~5x10 7 、2x10 5 ~4x10 7 、2x105 ~4x10 7 , 2x10 5 ~3x10 7 , 2x10 5 ~2x10 7 , 2x10 5 ~1x10 7 , 2x10 5 ~9x10 6 , 2x10 5 ~8x10 6 , 2x10 5 ~7x10 6 , 2x10 5 ~6x10 6 , 2x10 5 ~5x10 6 , 2x10 5 ~4x10 6 , 2x10 5 ~4x10 6 , 2x10 5 ~3x10 6 , 2x10 5 ~2x10 6 , 2x10 5 ~1x10 6 , 3x10 5 ~3x10 6 The dosage may include, but is not limited to, cells / kg. Furthermore, the dosage may be adjusted to account for whether a single dose or multiple doses are administered. The exact amount that is considered to be an effective dose may be determined based on factors specific to each subject.

[0254] As used herein, the terms "treat", "treating", and "treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer, which may, but is not necessarily, discernible in the subject. The terms "treat", "treating", and "treatment" can also refer to causing regression, preventing progression, or at least slowing progression of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to the alleviation, prevention of onset or onset, or shortening of the duration of one or more symptoms associated with a disease, disorder, or condition, such as a tumor or, more preferably, a cancer. In certain embodiments, "treat", "treating", and "treatment" refer to the prevention of recurrence of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to an increase in the survival rate of a subject having a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0255] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in combination with one or more additional therapeutic agents. In certain embodiments, the one or more therapeutic agents are selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or substitutes thereof, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). EXAMPLES

[0256] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, but not limit, the disclosed embodiments.

[0257] Example 1. Site-specific manipulation of iPSCs using a two-step transfection process Day 1: Lipofectamine-stem transfection of donor pDNA into iPSCs Add 100 µM stock H1152 Rho inhibitor solution to a concentration of 1 µM to a T-75 flask containing iPSCs at approximately 70% confluency. Incubate the cells at 37 °C and 5% CO. 2 Low O 2 Incubate in an incubator for at least 1 hour. During incubation, allow the vitronectin-coated T75 flasks to come to room temperature for at least 15 minutes. Aspirate the coating solution from each flask and replace with 10 mL of Essential 8 complete medium + 1 μM H1152. Store the plates at 37 °C and 5% CO until use. 2 Low O 2 Place in incubator. After incubation, aspirate medium from the T-75 flask containing iPSCs and add 7 mL of 1x DPBS down the side of the flask and gently swirl to wash. Aspirate DPBS and add 2 mL of TrypLE Select directly to the cells. Incubate cells at 37°C for 3-5 minutes, followed by addition of 10 mL of Essential 8 complete medium to the flask. Dislodge cells from the plate by pipetting, then transfer to a sterile 50 mL conical tube. Centrifuge cells at 200 x g for 5 minutes. Aspirate supernatant and resuspend cells in 10 mL of Essential 8 complete medium. Count cells using an NC-200 NucleoCounter. Seed 2E6 cells into each T-75 flask. Incubate cells at 37°C, 5% CO until required for transfection. 2 Low O 2 Incubate in an incubator. Set up the transfection mix in a sterile 15 mL centrifuge tube as listed below, scale up if necessary, according to the table below: Tube #1 Opti-MEM 1250μl Lipofectamine-stem 50μl Tube #2 Opti-MEM 1250μl 5 μg pDNA

[0258] Mix tubes 1 and 2 by adding the components of tube 2 to tube 1, then incubate at ambient temperature for 10 minutes. Add the entire mixture dropwise to the appropriate flask. Gently rock the flask and incubate at 37°C, 5% CO. 2 Low O 2 Place in the incubator.

[0259] Day 2: Feeding iPSCs Allow Essential 8 complete medium to come to ambient temperature (≥15 min). Replace spent medium from iPSC cultures with 14 mL of fresh Essential 8 complete medium per vessel and immediately after feeding is completed, cultures are incubated at 37 °C in hypoxic 5% CO. 2 Return to humidified incubator. No feeding / medium change is performed on iPSC cultures on the day of passaging as this significantly reduces colony detachment.

[0260] Day 3: Preparation of ribonucleoprotein (RNP) complexes Perform electroporation 40-48 h after transfection of iPSCs with donor pDNA. Combine the following in a sterile PCR tube and mix well (multiply volumes for the appropriate number of conditions + 1 for excess): 1.4μL 1xDPBS ·1.6μL 100μM Alt-R CRISPR-MAD7 crRNA ·2μL Alt-R MAD7 Ultra Nuclease

[0261] Centrifuge the solution briefly, incubate at ambient temperature for 10-20 min, and then store at 2-8 °C until required for electroporation.

[0262] Aspirate spent media from the T-75 flask containing cells and add 7 mL of 1x DPBS to wash. Aspirate 1x DPBS and replace with 2 mL of TrypLE. Incubate the flask at 37°C and 5% CO. 2 Low O 2 Place in incubator for 3-5 min, followed by adding 10 mL of E8 complete medium and pipetting up and down 3-4 times to dislodge cells. Transfer cells to a 50 mL conical and centrifuge at 200 x g for 5 min. During centrifugation, prepare the appropriate number of coated 6-well plates by aspirating the coating solution from each well and adding 2 mL of Essential 8 complete medium + 1 µM H1152 to each well. Aspirate the supernatant and resuspend the cells in 10 mL of cold Opti-MEM medium, followed by a further centrifugation at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells in 10 mL of cold Opti-MEM medium. Count cells with an NC-200 cell counter and record.

[0263] The cells were centrifuged at 200 × g for 5 min and resuspended in 2 × 10 ng / mL Opti-MEM pre-equilibrated to ambient temperature. 6 Resuspend at a concentration of 100 cells / mL. Set the BTX ECM-830 electroporator as follows: 150V 10ms 1 pulse

[0264] For each electroporation, add the following to a BTX electroporation cuvette with a 2 mm gap width: ·5μl RNP complex 1.4 μL Cpf1 electroporation enhancer 200μL of cells

[0265] Gently tap the cuvette to ensure all contents fall to the bottom, place it in the electroporation safety stand, close the dome and press start.

[0266] Using a sterile transfer pipette with each cuvette, drop the cells into the appropriate well of the prepared 6-well plate and then incubate at 37 °C, 5% CO. 2 Low O 2 Place in the incubator.

[0267] Example 2. Editing the AAVS1 locus CAR transgene donor plasmid is specifically engineered to insert CAR into AAVS1 site. Figure 7A shows flow cytometry analysis of bulk population of cells after engineering. Figure 7B shows flow cytometry analysis of cells after sorting for CAR positive cells. Figure 7C shows flow cytometry analysis of CAR positive single cell clone.

[0268] Example 3. Editing the B2M locus HLA-E transgene donor plasmid was specifically engineered to insert HLA-E at the B2M site. Figure 8A shows flow cytometric analysis of the bulk population of engineered cells. Figure 8B shows flow cytometric analysis of cells after sorting for HLA-E positive, B2M negative cells. Figure 8C shows flow cytometric analysis of HLA-E positive, B2M negative single cell clones.

[0269] Example 4. Editing the CIITA locus EGFR transgene donor plasmid was specifically engineered to insert EGFR into the CIITA site. Figure 9A shows flow cytometry analysis of the bulk population of cells after engineering. Figure 9B shows flow cytometry analysis of cells after sorting for EGFR cells. Figure 9C shows flow cytometry analysis of EGFR positive single cell clones.

[0270] Example 5. Editing the CYBYL locus The PSMA transgene donor plasmid was specifically engineered to insert PSMA into the CLYBL site. Figure 10A shows flow cytometry analysis of the bulk population of engineered cells. Figure 10B shows flow cytometry analysis of cells after sorting for PSMA-positive cells.

[0271] Example 6. Editing the NKG2A locus IL15-IL15RA transgene donor plasmid was specifically engineered to insert IL15-IL15RA into the NKG2A site. Figure 11A shows flow cytometric analysis of the bulk population of engineered cells. Figure 11B shows flow cytometric analysis of cells after sorting for IL-15-IL15RA positive cells.

[0272] The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be encompassed within the scope of the appended claims.

[0273] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

Claims

1. A MAD7 / gRNA ribonucleoprotein (RNP) complex composition for transgene insertion, comprising: (I) MAD7 nuclease; (II) a guide RNA (gRNA) specific for MAD7 nuclease, the gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, the guide sequence being selected from SEQ ID NOs: 120-130, and wherein when the gRNA forms a complex with the MAD7 nuclease, the guide sequence directs sequence-specific binding of the MAD7 nuclease to the target sequence; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of the target sequence of an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter operably linked to a polynucleotide sequence (3) encoding a transgene; and (4) a transcription terminator sequence; The composition comprising:

2. 2. The composition of claim 1, wherein the transgene comprises a sequence encoding a chimeric antigen receptor (CAR).

3. 2. The composition of claim 1, wherein the guide sequence is specific to the AAVS1 locus.

4. 4. The composition of claim 3, wherein the guide sequence of the gRNA comprises SEQ ID NO:

120. (i) the transgene comprises a sequence encoding an artificial cell death polypeptide, and the guide sequence is specific for a B2M or CIITA locus; or 2. The composition of claim 1, wherein (ii) the transgene comprises a sequence encoding an exogenous cytokine, and the guide sequence is specific for a B2M or CIITA locus. (i) the guide sequence of the gRNA is specific to the B2M locus and comprises SEQ ID NO: 121; or (ii) the guide sequence of the gRNA is specific to the CIITA locus and comprises SEQ ID NO: 122 or 126. (i) the guide sequence of the gRNA is specific to the NKG2A locus and comprises SEQ ID NO: 124; or (ii) the guide sequence of the gRNA is specific to the TRAC locus and comprises SEQ ID NO: 125; or (iii) the guide sequence of the gRNA is specific to the CLYBL locus and comprises SEQ ID NO: 123; or (iv) the guide sequence of the gRNA is specific to the CD70 locus and comprises SEQ ID NO: 127; (v) the guide sequence of the gRNA is specific to the CD38 locus and comprises SEQ ID NO: 128; or (vi) The composition of claim 1, wherein the guide sequence of the gRNA is specific to the CD33 locus and comprises SEQ ID NO: 129 or 130.

8. (i) the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of AAVS1 comprise the nucleotide sequences of SEQ ID NOs: 60 and 61, respectively, or fragments thereof; and / or (ii) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of B2M comprise the nucleotide sequences of SEQ ID NOs: 63 and 64, respectively, or fragments thereof; and / or (iii) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CIITA comprise (a) the nucleotide sequences of SEQ ID NOs: 66 and 67, respectively, or (b) the nucleotide sequences of SEQ ID NOs: 106 and 107, respectively, or fragments thereof; (iv) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CLYBL comprise the nucleotide sequences of SEQ ID NOs: 69 and 70, respectively, or fragments thereof; and / or (v) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CD70 comprise the nucleotide sequences of SEQ ID NOs: 109 and 110, respectively, or fragments thereof; and / or (vi) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of NKG2A comprise the nucleotide sequences of SEQ ID NOs: 72 and 73, respectively, or fragments thereof; and / or (vii) The composition of claim 1, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of TRAC comprise the nucleotide sequences of SEQ ID NOs: 75 and 76, respectively, or fragments thereof.

9. The composition of claim 1, wherein, when the RNP complex is introduced into a cell, expression of an endogenous gene in the cell that comprises a target sequence complementary to the guide sequence of the gRNA molecule is reduced or eliminated.

10. The composition of claim 1 , wherein the cells are induced pluripotent stem cells (iPSCs).

11. 2. An iPSC transformed with a transgene by the composition of claim 1.

12. The iPSC of claim 11, wherein the transgene comprises a sequence encoding a chimeric antigen receptor (CAR).

13. The iPSC of claim 12, wherein the CAR is specific for a tumor antigen associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy.

14. Tumor antigens associated with glioblastoma are selected from HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2, and tumor antigens associated with ovarian cancer are selected from FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, Nectin-4, and B7H. 4, the tumor antigen associated with cervical cancer or head and neck cancer is selected from GD2, MUC1, mesothelin, HER2 and EGFR, the tumor antigen associated with liver cancer is selected from claudin 18.2, GPC-3, EpCAM, cMET and AFP, the tumor antigen associated with hematological malignancies is selected from CD19, CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123 and CD70, and the tumor antigen associated with bladder cancer is selected from Nectin-4 and SLITRK6.

15. The tumor antigens include alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba733, BrE3 antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon specific antigen-p(C SAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, E phA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonado tropin (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, PAM-4 antibody specific antigen, placental growth factor , p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, angiogenesis markers, cancer gene markers and cancer gene products.

16. The iPSC of claim 13, wherein the tumor antigen is CD19.

17. 12. An engineered immune effector cell or population thereof derived from the iPSC of claim 11.

18. 18. The engineered immune effector cell or population thereof of claim 17, wherein the immune effector cell is a T cell or a NK cell.

19. 20. The engineered immune effector cell or population thereof of claim 18, wherein the T cells are CD4+ T cells, CD8+ T cells, or a combination thereof.

20. A MAD7 / gRNA ribonucleoprotein (RNP) complex composition for transgene insertion, comprising: (I) A MAD7 nuclease system comprising: (a) a sequence encoding a guide RNA (gRNA) operably linked to a first regulatory element, said gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, said guide sequence being selected from SEQ ID NOs: 120-130, and wherein, upon transcription, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; (b) a sequence encoding a MAD7 nuclease operably linked to a second regulatory element; and a MAD7 nuclease system encoded by one or more vectors comprising: (II) A transgene vector comprising: (1) left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter operably linked to a polynucleotide (3) encoding a transgene; and (4) a transcription terminator sequence. The composition comprising:

21. A MAD7 / gRNA ribonucleoprotein (RNP)-based vector system, comprising: (I) one or more vectors comprising: (a) a sequence encoding a guide RNA (gRNA), operably linked to a first regulatory element, the gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, the guide sequence being selected from SEQ ID NOs: 120-130, and wherein, upon transcription, the guide sequence directs sequence-specific binding of a MAD7 complex to the target sequence; and (b) a sequence encoding a MAD7 nuclease, operably linked to a second regulatory element; and (II) A transgene vector comprising: (1) left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter operably linked to a polynucleotide encoding a transgene; and (4) a transcription terminator sequence. The vector system comprising:

22. 22. The composition of claim 20 or the vector system of claim 21, wherein the cell is an induced pluripotent stem cell (iPSC).

23. 22. The composition of claim 20 or the vector system of claim 21, wherein the first and / or second regulatory element is a promoter.

24. (i) the first and second regulatory elements are the same, or 22. The composition of claim 20 or the vector system of claim 21, wherein (ii) the first and second regulatory elements are different.

25. 22. The composition of claim 20 or the vector system of claim 21, wherein the transgene comprises a sequence encoding a chimeric antigen receptor (CAR).

26. 22. The composition of claim 20, or the vector system of claim 21, wherein the guide sequence is specific to the AAVS1 locus.

27. 27. The composition or vector system of claim 26, wherein the guide sequence of the gRNA comprises SEQ ID NO:

120.

28. (i) the transgene comprises a sequence encoding an artificial cell death polypeptide, and the guide sequence is specific for a B2M or CIITA locus; or 22. The composition of claim 20 or the vector system of claim 21, wherein (ii) the transgene comprises a sequence encoding an exogenous cytokine and the guide sequence is specific for a B2M or CIITA locus.

29. (i) the guide sequence of the gRNA is specific to the B2M locus and comprises SEQ ID NO: 121; or 29. The composition or vector system of claim 28, wherein (ii) the guide sequence of the gRNA is specific to the CIITA locus and comprises SEQ ID NO: 122 or 126. (i) the guide sequence of the gRNA is specific to the NKG2A locus and comprises SEQ ID NO: 124; or (ii) the guide sequence of the gRNA is specific to the TRAC locus and comprises SEQ ID NO: 125; or 22. The composition of claim 20 or the vector system of claim 21, wherein (iii) the guide sequence of the gRNA is specific to the CLYBL locus and comprises SEQ ID NO:

123. (i) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of AAVS1 comprise the nucleotide sequences of SEQ ID NOs: 60 and 61, respectively, or fragments thereof; and / or (ii) the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of B2M comprise the nucleotide sequences of SEQ ID NOs: 63 and 64, respectively, or fragments thereof; and / or (iii) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CIITA comprise (a) the nucleotide sequences of SEQ ID NOs: 66 and 67, respectively, or (b) the nucleotide sequences of SEQ ID NOs: 106 and 107, respectively, or fragments thereof; and / or (iv) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CLYBL comprise the nucleotide sequences of SEQ ID NOs: 69 and 70, respectively, or fragments thereof; and / or (v) the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of NKG2A comprise the nucleotide sequences of SEQ ID NOs: 72 and 73, respectively, or fragments thereof; and / or (vi) The composition of claim 20 or the vector system of claim 21, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of TRAC comprise the nucleotide sequences of SEQ ID NOs: 75 and 76, respectively, or fragments thereof.

32. 22. The composition of claim 20 or the vector system of claim 21, wherein, when the RNP complex is introduced into a cell, expression of an endogenous gene in the cell that comprises a target sequence that is complementary to the guide sequence of the gRNA molecule is reduced or eliminated.

33. 22. One or more retroviruses comprising the vector system of claim 21.

34. 22. An iPSC transformed with the vector system of claim 21.

35. The iPSC of claim 34, wherein the transgene comprises a sequence encoding a chimeric antigen receptor (CAR).

36. The iPSC of claim 35, wherein the CAR is specific for a tumor antigen associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy.

37. Tumor antigens associated with glioblastoma are selected from HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2, and tumor antigens associated with ovarian cancer are selected from FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, Nectin-4, and B7H. 4, the tumor antigen associated with cervical cancer or head and neck cancer is selected from GD2, MUC1, mesothelin, HER2 and EGFR, the tumor antigen associated with liver cancer is selected from claudin 18.2, GPC-3, EpCAM, cMET and AFP, the tumor antigen associated with hematological malignancies is selected from CD19, CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123 and CD70, and the tumor antigen associated with bladder cancer is selected from Nectin-4 and SLITRK6.

38. The tumor antigens include alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba733, BrE3 antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon specific antigen-p(C SAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, E phA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonado tropin (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, PAM-4 antibody specific antigen, placental growth factor , p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, angiogenesis markers, cancer gene markers and cancer gene products.

39. The iPSC of claim 34, wherein the tumor antigen is CD19.

40. 35. An immune effector cell or population thereof derived from an iPSC according to claim 34.

41. A pharmaceutical composition comprising immune effector cells derived from iPSCs according to any one of claims 11 to 16 and 34 to 39.

42. A composition for preventing or treating cancer, comprising a pharma- ceutical effective amount of an immune effector cell or a population thereof according to any one of claims 17 to 19 and 40.

43. 43. The composition of claim 42, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, endometrial cancer, ovarian cancer, lymphoma, and brain cancer.

44. A gRNA comprising a guide sequence selected from the group consisting of SEQ ID NOs: 120-130.

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