Therapeutic allogeneic cells

Abstract

The present disclosure provides allogeneic cells that, for example, when used to treat a disease in a patient, reduce, minimize, or eliminate the risk of graft-versus-host disease (GVHD) and reduce, minimize, or eliminate the risk of CD4, CD8, and NK cell rejection. The allogeneic cells may be genetically engineered to reduce expression or activity of MHC class I and / or MHC class II molecules, such as by knocking out the RFX5 gene. Methods for preparing and using such allogeneic cells are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 314,848, filed on 28 February 2022, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. The XML copy, created on 22 February 2023, is named K-1138-WO-PCT_SL.xml and has a size of 147,560 bytes.

[0003] Field of Invention This disclosure relates to the field of cell therapy, and more specifically, to genetically modified immune cells. [Background technology]

[0004] T-cell therapy is based on human T cells that have been enriched or modified to target and kill a patient's cancer cells. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to genetically engineer T cells to express constructs that direct them to specific target cancer cells. Chimeric antigen receptors (CARs) and genetically engineered T cell receptors (TCRs) containing binding domains that can interact with specific tumor antigens enable T cells to target and kill cancer cells that express specific tumor antigens.

[0005] CD19-directed chimeric antigen receptor T cells (CAR T cells) have demonstrated potent antitumor effects in the treatment of various B-cell malignancies. However, autologous CAR T therapy presents technical, manufacturing, and commercial constraints that can limit its clinical application to its maximum potential. Allogeneic CAR T therapy is an alternative strategy that overcomes the inherent limitations of autologous therapy and provides a “ready-made” approach for clinical use.

[0006] In principle, allogeneic CAR T therapy uses T cells derived from healthy human donors, and then genetically modifies them to confer specificity to tumor antigens. Furthermore, gene editing is introduced to prevent graft-versus-host disease (GVHD) and rejection of allogeneic CAR T cells by the patient's immune system. GVHD is mainly caused by the interaction between the T cell receptor (TCR) αβ protein on donor T cells and incompatible human leukocyte antigen (HLA) molecules on recipient patient cells. Conversely, the host's endogenous CD8 + T cells can interact with and eliminate donor T cell grafts that contain incompatible major histocompatibility complex (MHC) class I molecules. [Overview of the project]

[0007] Efforts have been made to develop readily available, low-immunogenic cells, particularly immune cells such as T cells and NK cells. For example, beta-2-microglobulin (B2M), a major component of MHC class I molecules, is inactivated in allogeneic T cells. Inactivation of B2M can eliminate MHC class I, which is thought to reduce or prevent rejection by incompatible CD8 T cells in the host. However, B2M knockout CAR T cells have been observed to be susceptible to host NK cell killing, as NK cells become stimulated, killing allogeneic T cells lacking MHC class I activity.

[0008] This disclosure provides, in various embodiments, a cell engineering approach superior to B2M knockout. This approach can achieve minimization or elimination of the risk of GVHD, as well as the risk of CD4, CD8, and NK cell rejection, while maintaining equivalent or even improved therapeutic activity.

[0009] Therefore, according to one embodiment of the present disclosure, allogeneic cells genetically engineered to reduce the expression or activity of MHC class I or MHC class II are provided. In some embodiments, the expression or activity of MHC class I is reduced, but not eliminated. In some embodiments, the expression and / or activity of MHC class II may be reduced, or even eliminated. Methods for preparing such cells are also provided.

[0010] One embodiment of the present disclosure provides isolated human immune cells engineered to have 10% to 80% lower MHC class I activity or expression as compared to a corresponding unengineered immune cell. In some embodiments, the cell has at least 20% lower, or at least 40% lower MHC class II activity or expression as compared to a corresponding unengineered immune cell. In some embodiments, the cell has at least 75% lower MHC class II activity or expression as compared to a corresponding unengineered immune cell or reference cell.

[0011] In some embodiments, the corresponding immune cell or reference immune cell is an immune cell that has not been engineered to reduce expression of MHC class I molecules and / or MHC class II molecules. In some embodiments, the corresponding immune cell or reference immune cell is an immune cell that is engineered to reduce expression of MHC class I, but not to reduce expression of MHC class II.

[0012] In some embodiments, the cell is a cell derived from a T cell or an NK cell, or any other immune cell such as a monocyte or macrophage, or a stem cell such as an iPSC. In some embodiments, the cell comprises an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the CAR recognizes CD19 and / or CD20. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 26 or 27, or a sequence having at least about 90% sequence identity to SEQ ID NO: 26 or 27.

[0013] In some embodiments, the CAR recognizes CLL-1. In some embodiments, the CAR includes the amino acid sequence of any of SEQ ID NOs. 65-76 or 78-83, or a sequence having at least about 90% sequence identity to any of SEQ ID NOs. 65-76 or 78-83.

[0014] In some embodiments, at least one endogenous gene among RFX5 (regulator X5), TAP1 (antigen processing-associated transporter 1), TAP2 (antigen peptide transporter 2), or CIITA (class II, major histocompatibility complex, transactivator) is inactivated or reduced. In some embodiments, both alleles of the endogenous gene are inactivated. In some embodiments, RFX5 is inactivated intracellularly. In some embodiments, the endogenous genes of TAP1, TAP2, and CIITA are not manipulated.

[0015] In some embodiments, the endogenous gene for TRAC (T cell receptor alpha constant region) is further inactivated or reduced. In some embodiments, the endogenous gene for B2M (beta-2-microglobulin) is not manipulated, or the cells have normal B2M activity.

[0016] In some embodiments, inactivation is achieved by (a) editing of endogenous genes, (b) expression of inhibitory RNA, or (c) an inhibitor, preferably an antibody. In some embodiments, editing is performed by CRISPR / Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, Cpf1, homologous recombination, single-stranded oligodeoxynucleotide (ssODN), or base editing.

[0017] Methods for cells are also provided, but are not limited to these. In some embodiments, a CAR or TCR is introduced into cells before gene editing, such as RFX5. Methods for using cells to treat diseases are also provided. [Modes for carrying out the invention]

[0018] definition

[0019] To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of these terms and other terms are provided throughout this specification.

[0020] As used herein, unless otherwise specified or evident from the context, the term “or” is understood to be inclusive and encompasses both “or” and “and.”

[0021] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of two designated features or components, with or without the other. Accordingly, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include A and B, A or B, A (alone), and B (alone). Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0022] Unless specifically stated or evident from the context, the term “about” refers to a value or composition that falls within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and this depends to some extent on how that value or composition is measured or determined, i.e., on the limits of the measurement system. For example, “about” or “essentially from” may mean within a range of 1 or more than 1 standard deviation by the practice of the art. “About” or “essentially from” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be greater or less than the stated value by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001%. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean a value of up to one order of magnitude or up to five times the stated value. Where specific values ​​or compositions are presented in this disclosure, unless otherwise specified, the meaning of “approximately” or “essentially consisting of” should be assumed to be within an acceptable margin of error for those specific values ​​or compositions.

[0023] "Administering" refers to the physical delivery of a drug, such as the modified T cells disclosed herein, to a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by injection or infusion. The term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. The administration may also be carried out, for example, once, multiple times, and / or over a longer period of time.

[0024] The terms “activated” and “activated” refer to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In one embodiment, activation may also relate to induced cytokine production and detectable effector function. The term “activated T cell” refers, among other things, to a proliferating T cell. Signals generated solely via the TCR may be insufficient for complete T cell activation, and one or more secondary or co-stimulatory signals may also be required. Thus, T cell activation includes a primary stimulatory signal mediated by the TCR / CD3 complex and one or more secondary co-stimulatory signals. Co-stimulation may be demonstrated by proliferation and / or cytokine production by T cells that have received a primary activating signal, such as stimulation mediated by the TCR / CD3 complex.

[0025] The term "allogeneic" refers to any material that originates from one individual and is then introduced into another individual of the same species, such as allogeneic T cell transplantation.

[0026] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding molecule. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are incorporated into more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are tetramers of about 150 kD, consisting of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other in a structure commonly referred to as a "Y-shape". The heavy and light chains are linked or connected to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, resulting in the dimers being linked to each other to form a tetramer. Naturally produced antibodies, for example, undergo glycosylation on this CH2 domain.

[0027] The terms “antigen-binding molecule,” “antigen-binding moiety,” “antigen-binding fragment,” or “antibody fragment” refer to any molecule containing the antigen-binding moiety (e.g., CDR) of an antibody from which the molecule originates. Antigen-binding molecules may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on tumor cells. In some embodiments, the antigen-binding molecule binds to an antigen on cells involved in hyperproliferative diseases, or to a viral or bacterial antigen. In certain embodiments, the antigen-binding molecule is a chimeric antigen receptor (CAR) or an engineered T-cell receptor (TCR). In certain embodiments, the antigen-binding molecule is 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta-3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein consisting of a cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of ImprintedSites), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30(TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, C D72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152(C TLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV) infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (CD2 subset 1, also called CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epidermal glycoprotein-2 (EGP-2), epidermal glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (EL F2M), endothialin, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrin B2, receptor tyrosine protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML), IgA receptor Fc fragment (FCAR or CD89), fibroblast-activating protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extradomain B, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, fucosyl Fucosyl GM1, GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), GP 100, GloboH glycoceramide hexasaccharide portion (GloboH), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gplOO), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cell receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPVE7), heat shock protein 70-2 mutant (mut hsp70-2), human scattering factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin-11 receptor alpha (IL-1) IRa), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, κ light chain, KCS1, kinase insertion domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-LA, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A Member 2 (LILRA2), Regmine, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, Lymphocyte antigen 6 complex, LTBR, ​​Gene locus K9 (LY6K), Ly-6, Lymphocyte antigen 75 (LY75), Melanoma cancer testicular antigen-1 (MAD-CT-1), Melanoma cancer testicular antigen-2 (MAD-CT-2), MAGE, Melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTI, Mesothelin, MAGEA3, apoptosis-induced melanoma inhibitor (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian duct inhibitor (MIS) receptor type II, neuroblastoma-derived homolog of v-myc avian myelocytosis viral oncogene (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neuronal cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary gland differentiation antigen (NY-BR-1), NY-ESO-1, tumor fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES1) p53, p53 variants, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (Macropain) subunit, beta type, advanced glycation end product receptor (RAG) E-1), RANKL, Ras variant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous receptor 1 (RU1), renal ubiquitous receptor 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen 3 recognized by T cells (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, Sonic Hedgehog (SHH), sperm protein 17 (SPA17), time-specific embryonic antigen 4- (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), sulbibin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative leading frame protein (TARP), telomerase, TIGIT TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF-beta 2, TGF-β, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, TnThe antigen-binding molecule binds to Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms tumor protein (WT1), or the X antigen family, member 1A (XAGE1). The amino acid sequences that specifically bind to the antigen are known in the art or can be prepared using methods known in the art, examples of which include immunoglobulins, variable regions of immunoglobulins (e.g., variable fragments ("Fv") or bivalent variable fragments ("Fab")), single-chain antibodies, etc. In certain embodiments, the antigen-binding molecule is an antibody fragment that specifically binds to the antigen, including one or more complementarity-determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule includes or consists of an avimer.

[0028] The terms “variable region” and “variable domain” are used interchangeably. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the amino-terminal approximately 110–120 amino acids of the mature heavy chain and approximately 90–115 amino acids of the mature light chain, which vary significantly in sequence between antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while more highly conserved regions within the variable domain are called the framework region (FR). While we do not wish to be bound to any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be primarily responsible for the antibody's interaction with and specificity to the antigen. In certain embodiments, the variable region is the human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is the primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes rodent or mouse CDR and primate (e.g., non-human primate) framework regions (FR).

[0029] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

[0030] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

[0031] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is a compromise between the two, used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on the analysis of available composite crystal structures.

[0032] "Antigen" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in a human or animal, including compositions (such as those containing tumor-specific proteins) that are injected or absorbed into a human or animal. Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is substantially not found on the surface of other normal (desired) cells and is designed to bind to the binding domain of a TCR or CAR as intended herein. Those skilled in the art will readily understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Antigens may be endogenously expressed, i.e., expressed by genomic DNA or by recombination. Antigens may be specific to specific tissues, such as cancer cells, or they may be broadly expressed. Furthermore, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen. In one particular embodiment, the antigens include 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta-3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein consisting of a cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), and CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of ImprintedSites), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30(TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, C D72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152(C TLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV) infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (CD2 subset 1, also called CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epidermal glycoprotein-2 (EGP-2), epidermal glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (EL F2M), endothialin, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrin B2, receptor tyrosine protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML), IgA receptor Fc fragment (FCAR or CD89), fibroblast-activating protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extradomain B, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, fucosyl Fucosyl GM1, GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), GP 100, GloboH glycoceramide hexasaccharide portion (GloboH), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gplOO), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cell receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPVE7), heat shock protein 70-2 mutant (mut hsp70-2), human scattering factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin-11 receptor alpha (IL-1) IRa), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, κ light chain, KCS1, kinase insertion domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-LA, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A Member 2 (LILRA2), Regmine, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, Lymphocyte antigen 6 complex, LTBR, ​​Gene locus K9 (LY6K), Ly-6, Lymphocyte antigen 75 (LY75), Melanoma cancer testicular antigen-1 (MAD-CT-1), Melanoma cancer testicular antigen-2 (MAD-CT-2), MAGE, Melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTI, Mesothelin, MAGEA3, apoptosis-induced melanoma inhibitor (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian duct inhibitor (MIS) receptor type II, neuroblastoma-derived homolog of v-myc avian myelocytosis viral oncogene (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neuronal cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary gland differentiation antigen (NY-BR-1), NY-ESO-1, tumor fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES1) p53, p53 variants, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (Macropain) subunit, beta type, advanced glycation end product receptor (RAG) E-1), RANKL, Ras variant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous receptor 1 (RU1), renal ubiquitous receptor 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen 3 recognized by T cells (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, Sonic Hedgehog (SHH), sperm protein 17 (SPA17), time-specific embryonic antigen 4- (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), sulbibin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative leading frame protein (TARP), telomerase, TIGIT TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF-beta 2, TGF-β, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, TnThe target is any molecule conjugated by an antibody, such as a Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms oncoprotein (WT1), or any or a fragment of the X antigen family, member 1A (XAGE1). The "target" is any molecule conjugated by a binding motif, antigen-binding system, CAR, or antigen-binding agent, such as an antibody.

[0033] The term "self" refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT®) method described herein comprises collecting lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0034] A “chimeric antigen receptor” or “CAR” refers to a molecule engineered to include a binding motif and means for activating immune cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR includes a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR T cells. An “extracellular domain” (or “ECD”) refers to a portion of a polypeptide that, if the polypeptide is present on the cell membrane, is understood to be located outside the cell membrane, in the extracellular space.

[0035] As used herein, the term “extracellular ligand-binding domain” refers to a ligand, such as an oligonucleotide or polypeptide capable of binding to a cell surface molecule. For example, an extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a specific disease condition (e.g., cancer). Examples of cell surface markers that can act as ligands include those associated with viruses, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0036] A “spacer” or “hinge” may follow the binding domain of a CAR, which refers to a region that moves the antigen-binding domain away from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999, 6:412-419). The hinge region in a CAR is generally located between the transmembrane (TM) domain and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, and CD7, which may be the wild-type hinge regions of these molecules or may be modified.

[0037] The “transmembrane” region or domain is the portion of the CAR that fixes the extracellular binding portion to the plasma membrane of an immunoeffector cell and facilitates the binding of the binding domain to the target antigen. The transmembrane domain may be the CD3 zeta transmembrane domain, however, other transmembrane domains that may be used include those derived from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain is the CD137 transmembrane domain. In certain embodiments, the transmembrane domain is synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine.

[0038] The term “intracellular signaling domain” or “signaling domain” refers to a portion of a chimeric antigen receptor protein that is involved in inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the transduction of an effective CAR message that binds to a target antigen into the interior of an immunoeffector cell, resulting in the release of cytotoxic factors to CAR-bound target cells or other cellular responses induced by antigen binding to an extracellular CAR domain. The term “effector function” refers to a specific function of a cell. The effector function of a T cell may be, for example, assistance or activity including cytolytic activity or cytokine secretion. Therefore, the terms “intracellular signaling domain” or “signaling domain,” as used interchangeably herein, refer to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Usually, the entire intracellular signaling domain may be used, but in many cases, it is not necessary to use the entire domain. Insofar as a cleavage portion of an intracellular signaling domain is used, such a cleavage portion may be used in place of the entire domain, insofar as it transduces an effector function signal. The term “intracellular signaling domain” means that it includes any cleavage portion of an intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domains are also known as "signal transduction domains" and are typically derived from portions of human CD3 or FcRy chains.

[0039] It is known that signals generated solely through T cell receptors are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via T cell receptors (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). Co-stimulatory cytoplasmic signaling sequences may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0040] Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in this disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0041] As used herein, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for the efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Thus, this disclosure provides exemplary costimulatory domains derived from CD3 zeta and 4-1BB, but other costimulatory domains are intended for use with the CARs described herein. Inclusion of one or more costimulatory signaling domains may enhance the efficacy and proliferation of T cells expressing CAR receptors. Intracellular signaling and co-stimulatory signaling domains can be tandem-linked to the carboxyl terminus of the transmembrane domain in any order.

[0042] While scFv-based CARs engineered to contain CD3 or FcR gamma-derived signaling domains have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to induce signals that promote T cell survival and proliferation in the absence of concomitant co-stimulatory signals. Other CARs containing binding domains, hinges, transmembrane domains, and signaling domains derived from CD3 zeta or FcR gamma, along with one or more co-stimulatory signaling domains (e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134, and CD278), can more effectively direct antitumor activity and increase cytokine secretion, lytic activity, and the survival and proliferation of CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone et al., Molecular Therapy, 2009, 17:1453-1464; Zhong et al., Molecular Therapy, 2010, 18:413-420; Carpenito et al., PNAS, 2009, 106:3360-3365).

[0043] "Co-stimulatory signals" refer to signals that, when combined with primary signals such as TCR / CD3 ligation, bring about T cell responses such as proliferation and / or upregulation or downregulation of important molecules, though not limited to these signals.

[0044] "Co-stimulatory ligands" include molecules on antigen-presenting cells that specifically bind to homologous costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses, such as proliferation, activation, and differentiation, though this is not limited to these processes. In addition to the primary signals provided by stimulating molecules, costimulatory ligands induce signals, for example, through the binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Co-stimulatory ligands are not limited to, but include 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), and MHC class I chain-related protein B ( Examples include B, MICB, OX40 ligand, PD-L2, or programmed death (PD) L1.Examples of co-stimulatory ligands, though not limited to them, include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0045] A "costimulatory molecule" is a congenital junction partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by T cells, such as proliferation, but is not limited to this.Co-stimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD33, and CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CDl Examples include la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecules, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or their fragments, cleaved forms, or combinations.

[0046] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR or within the framework region of its antibody or its antigen-binding molecule may be replaced with amino acid residues having a similar side chain. Generally, two sequences are considered "substantially similar" if they contain conserved amino acid substitutions at corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "nonpolar" side chains. Substitutions of different amino acids of the same type may be considered conserved substitutions. An exemplary amino acid classification is summarized in Table 1 below. TIFF0007912602000001.tif137128

[0047] "T cell receptors" or "TCRs" refer to antigen-recognizing molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes—α, β, γ, and δ—can rearrange to produce a wide variety of TCR proteins.

[0048] The term "heterogeneous" means that a sequence originates from any source other than those found in nature. For example, a heterogeneous sequence included as part of a costimulatory protein is an amino acid that does not exist naturally as part of the wild-type human costimulatory protein, i.e., it does not align with the wild-type human costimulatory protein. For example, a heterogeneous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human costimulatory protein coding sequence.

[0049] The term "identity" refers to the overall relationship between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules), and / or polypeptide molecules. Methods for calculating the identity percentage between two given polypeptide sequences are known. For example, the calculation of the identity percentage between two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). Then, nucleotides or amino acids at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The identity percentage between two sequences is optionally a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison or alignment of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms such as BLAST (a basic local alignment search tool). In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are identical by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0050] T cells for immunotherapy may be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, ascites, pleural fluid, splenic tissue, and tumors. Furthermore, T cells may be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units taken from a subject using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0051] "Patient" includes any human being suffering from cancer (e.g., lymphoma or leukemia). The terms "Subject" and "Patient" are used interchangeably herein.

[0052] The terms “subject” and “patient” include human and non-human animal subjects, as well as subjects with formally diagnosed disabilities, subjects without formally recognized disabilities, subjects receiving medical attention, and subjects at risk of developing disabilities.

[0053] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or whose benefits to the recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and must not be harmful to the recipient or whose benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, that is involved in the transport or delivery of a drug from one part of the body to another (for example, from one organ to another). Each carrier present in a pharmaceutical composition must be compatible with the other components of the formulation and "acceptable" in the sense that it is not harmful to the patient, or whose benefits to the recipient outweigh any harmful effects. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as tragacanth powder, malt, gelatin, talc, cocoa butter, and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as agar, magnesium hydroxide, and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solutions, polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, suitable substances used in pharmaceutical formulations.

[0054] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant subjects or population. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, but is not limited to, forms adapted for: oral administration, e.g., liquid (aqueous or non-aqueous or suspension), tablets, e.g., buccal, sublingual, and those targeting systemic absorption, bolus, powder, granules, paste for application to the tongue; parenteral administration, e.g., sterile solution or suspension, or sustained-release formulations, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin, lungs, or oral cavity; intravaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual, ocular, transdermal, or nasal, lungs, and other mucosal surfaces.

[0055] The terms “reduce” and “decrease” are used interchangeably herein and refer to any change that becomes less than the original. “Reduce” and “decrease” are relative terms and require a comparison between before and after measurement. “Reduce” and “decrease” include complete depletion.

[0056] The term “reference” describes the standard or control on which the comparison is performed. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control which is a drug, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a reference or control from the past that is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation, where the similarity is sufficient to justify the dependence on and / or comparison with the selected reference or control.

[0057] Regulatory T cells ("Treg," "Treg cells," or "Treg(plural)") refer to a lineage of CD4+ T lymphocytes involved in regulating specific immune activities, such as responses to autoimmunity, allergy, and infection. Regulatory T cells can modulate the activity of T cell populations and can also influence certain innate immune cell types. Tregs can be identified by the expression of biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically constitute about 5–10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells) can constitute as much as 20–30% of the total CD4+ T lymphocyte population.

[0058] The “therapeutic effective dose,” “effective dose,” “effective amount,” or “therapeutic effective dosage” of a therapeutic agent, such as engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or impairment resulting from the onset of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0059] The terms “transduction” and “transduced” refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Jones et al., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentiviral vector, or any combination thereof.

[0060] "Treatment" or "treating" a subject means any type of intervention or process performed on the subject, or administration of an activator to the subject, with the aim of reversing, alleviating, improving, inhibiting, slowing or preventing the onset, progression, development, severity or relapse of symptoms, complications or conditions, or biochemical signs associated with the disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment may be the treatment of a subject that does not show signs of the related disease, disorder and / or condition, and / or a subject that shows only initial signs of the disease, disorder and / or condition. In some embodiments, such treatment may be the treatment of a subject that shows one or more definitive signs of the related disease, disorder and / or condition. In some embodiments, treatment may be the treatment of a subject that has been diagnosed with the related disease, disorder and / or condition. In some embodiments, treatment may be the treatment of a subject that is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the related disease, disorder and / or condition.

[0061] The term “vector” refers to a recipient nucleic acid molecule that contains or has been modified to incorporate a provided nucleic acid sequence. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors contain sequences that direct the expression of the inserted gene into which they are operably linked. Such vectors may be referred to herein as “expression vectors.” Standard techniques can be used for vector manipulation, for example, found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference.

[0062] A "zinc finger DNA-binding protein" (or binding domain) is a protein, or domain in a larger protein, that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequences within a binding domain whose structure is stabilized via the coordination of zinc ions. Thus, each zinc finger ZFP in a multi-finger structure contains a recognition helix region for binding to DNA within the backbone. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP. The term "zinc finger nuclease" includes one ZFN and a pair of ZFNs (the members of the pair are called "left and right" or "first and second" or "pair") that dimerize to cleave a target gene.

[0063] A “TALE DNA-binding domain” or “TALE” is a polypeptide containing one or more TALE repeat domains / units. Each repeat domain, containing repeating variable duo residues (RVDs), is involved in the binding of the TALE to its congenerally targeted DNA sequence. A single “repeat unit” (also called a “repeat”) is typically 33–35 amino acids long and exhibits at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. TALE proteins may be designed to bind to target sites using canonical or non-canonical RVDs within the repeat unit. See, for example, U.S. Patents 8,586,526 and 9,458,205. Zinc finger and TALE DNA-binding domains may be “engineered” to bind to a given nucleotide sequence, for example, by manipulating the recognition helix region of a naturally occurring zinc finger protein (modification of one or more amino acids) or by manipulating the amino acids involved in DNA binding (repeating variable duo residues or RVD region). Thus, engineered zinc finger proteins or TALE proteins are proteins that do not exist in nature. Non-limiting examples of methods for manipulating zinc finger proteins and TALEs include design and selection. Designed proteins are non-naturally occurring proteins whose design / composition arises primarily from reasonable criteria. Reasonable criteria for design include the application of substitution rules and computerized algorithms for processing information in databases that store information on existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Patents 9,458,205, 8,586,526, 6,140,081, 6,453,242, and 6,534,261, as well as International Publications 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536, and 03 / 016496. The term "TALEN" includes a single TALEN and a pair of TALENs (the members of a pair are called "left and right," "first and second," or "pair") that dimerize and cleave a target gene.

[0064] The CRISPR / Cas (Clustered regularly interspaced short palindromic repeats / CRISPR-related protein) system is considered the most powerful genome editing tool due to its unparalleled editing efficiency, convenience, and the concept of its potential applications in living organisms. Guided by guide RNA (gRNA), the Cas nuclease can generate DNA double-strand breaks (DSBs) at target genomic sites in various cells (both cell lines and living organisms). These DSBs are then repaired by endogenous DNA repair systems that can be used to perform the desired genome editing.

[0065] A base editor (BE) that integrates the CRISPR / Cas system with the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) cytosine deaminase family has recently been developed, significantly enhancing the efficiency of CRISPR / Cas9-mediated gene modification. Through fusion with Cas9 nickase (nCas9) or non-catalyzed Cas9 (dCas9), the cytosine (C) deamination activity of rat APOBEC1 (rA1) can be intentionally directed to target bases in the genome, catalyzing the substitution of C to thymine (T) at these bases.

[0066] Prime editing (PE) is a genome editing technology that can modify the genome of living organisms. Prime editing directly writes new genetic information to a target DNA site. It uses a fusion protein consisting of a catalytically impaired endonuclease (e.g., Cas9) fused to an engineered reverse transcriptase, and prime editing guide RNA (pegRNA) that can identify the target site and provide new genetic information for replacing the target DNA nucleotide. Prime editing mediates targeted insertions, deletions, and base changes without requiring double-strand breaks (DSBs) or donor DNA templates. RFX5 knockout cells

[0067] Allogeneic donor cells derived from healthy donors have the potential to provide ready-made cell products that can be applied as needed, at a much lower cost compared to those derived from the donor's own cells. With advances in gene editing technology, attempts are being made to knock out or knock out specific genes in order to develop ready-made, low-immunogenic cells that are suitable for use.

[0068] Beta-2-microglobulin (β2M or B2M) is a key component of MHC class I molecules. Deletion of B2M can eliminate MHC class I, which has been shown to reduce or prevent rejection by incompatible CD8 T cells in the host.

[0069] The inventors developed allogeneic anti-CD19 CAR T cell products in which parts of both the TCR alpha constant (TRAC) locus and B2M were deleted by zinc finger nuclease (ZFN), resulting in reduced protein expression on the cell surface. However, these edited CAR T cells were observed to be sensitive to host NK cells, as they stimulated NK cells and killed allogeneic T cells lacking MHC class I expression.

[0070] Therefore, the inventors sought an alternative gene editing approach that is superior to B2M knockout and can achieve minimization or elimination of the risk of GVHD, as well as minimization or elimination of the risk of CD4, CD8, and NK cell rejection, while maintaining equivalent or even improved therapeutic activity.

[0071] Accordingly, according to one embodiment of the present disclosure, allogeneic cells are provided that are genetically engineered to reduce the expression or activity of MHC class I or MHC class II. In some embodiments, the expression or activity of MHC class I is reduced but not eliminated. In some embodiments, the expression and / or activity of MHC class II may be reduced or even eliminated. Methods for preparing such cells are also provided.

[0072] In one embodiment, MHC class I expression or activity is reduced by at least 10%, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a reference allogeneic cell (e.g., an unmanipulated cell, e.g., a TRC knockout-only T cell). In another embodiment, MHC class I expression or activity is maintained at at least 5%, or at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a reference allogeneic cell. In one embodiment, the expression or activity of MHC class I is approximately 5%-90%, 10%-80%, 20%-80%, 20%-70%, 30%-70%, 30%-60%, 40%-60%, 10%-60%, 10%-50%, 20%-60%, 20%-50%, 20%-40%, 10%-40%, or 10%-30% compared to reference allogeneic cells.

[0073] In one embodiment, MHC class II expression or activity is reduced by at least 10%, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to reference allogeneic cells. In another embodiment, MHC class II expression or activity is maintained at at least 5%, or at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to reference allogeneic cells. In one embodiment, the expression or activity of MHC class II is approximately 5%-90%, 10%-80%, 20%-80%, 20%-70%, 30%-70%, 30%-60%, 40%-60%, 10%-60%, 10%-50%, 20%-60%, 20%-50%, 20%-40%, 10%-40%, or 10%-30% compared to reference allogeneic cells.

[0074] In one embodiment, the expression or activity of both MHC class I and II is reduced by at least 10%, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to reference allogeneic cells. In one embodiment, the expression or activity of both MHC class I and II is retained at at least 5%, or at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to reference allogeneic cells.

[0075] In some embodiments, the reference immune cells are not engineered to reduce the expression of MHC class I molecules and / or MHC class II molecules. In some embodiments, the reference immune cells are engineered to reduce the expression of MHC class I but not MHC class II (e.g., TRAC and B2M knockout). In some embodiments, the reference immune cells are engineered by introducing an exogenous construct expressing a CAR or TCR, but not to reduce the expression of MHC class I or MHC class II molecules. In some embodiments, the reference immune cells are non-transduced (NTD) cells derived from a healthy donor that are not engineered to reduce the expression of MHC class I or MHC class II molecules.

[0076] In some embodiments, the expression or activity of MHC class I is reduced or knocked down, while the expression or activity of MHC class II is knocked out or eliminated.

[0077] The term MHC class I also refers to human leukocyte antigen (HLA) class I. Examples of MHC class I molecules include, but are not limited to, B2M, individual HLA molecules (e.g., HLA-A, -B, -C, -E, -G), TAP1, TAP2, and / or genes associated with naked lymphocytosis syndrome I (BLSI).

[0078] The term MHC class II also refers to human leukocyte antigen (HLA) class II. Examples of MHC class II molecules include, but are not limited to, transcription factors (e.g., RFXANK, RFXS, RFXAP, or RFX5) or transactivators (CHTA), BLS II-related genes, and / or individual HLA molecules (e.g., HLA-DP, -DQ, -DR, -DiVI, -DO-alpha and beta chains).

[0079] We investigated several candidate genes, including single knockouts of RFX5 (regulatory factor X5), TAP1 (antigen processing-associated transporter 1), TAP2 (antigen peptide transporter 2), and CIITA (class II, major histocompatibility complex, transactivator). We found that single RFX5 knockout achieved optimal downregulation of MHC class I and elimination of MHC class II expression in engineered cells. Downregulation of MHC class I was sufficient to reduce rejection by incompatible CD8 T cells but did not promote killing by NK cells. Elimination of MHC class II expression also reduced the response by incompatible CD4 T cells. Importantly, as demonstrated in experimental examples, RFX5-knockout CAR cells exhibited superior manufacturability and CAR functionality.

[0080] Accordingly, one embodiment of the present disclosure provides a method for preparing allogeneic cells with reduced activity to induce graft-versus-host disease (GVHD) or host rejection. In some embodiments, the method involves reducing (or completely eliminating) the expression / activity of genes involved in the expression of MHC class I and / or II.

[0081] In some embodiments, the gene is selected from RFX5 (regulator X5), TAP1 (antigen processing-associated transporter 1), TAP2 (antigen peptide transporter 2), or CIITA (class II, major histocompatibility complex, transactivator). In some embodiments, the gene is RFX5, and at least one of TAP1, TAP2, and CIITA is also completely or partially inactivated or inhibited. In some embodiments, only the expression or activity of RFX5 is reduced in the cell, and no genetic alteration occurs to TAP1, TAP2, and CIITA.

[0082] In some embodiments, the endogenous B2M (beta-2-microglobulin) gene within the cell is not manipulated. That is, gene editing is not performed on the B2M locus, and no inhibitor is introduced into the cell.

[0083] In some embodiments, cells are further manipulated to reduce (or completely eliminate) the expression / activity of TRAC (T cell receptor alpha constant region).

[0084] In some embodiments, the cells are derived from a healthy donor. In some embodiments, the cells are derived from / differentiated from stem cells, such as induced pluripotent stem cells (iPSCs).

[0085] In some embodiments, the cells are T cells derived from a healthy donor. Technologies for reducing gene expression / activity in cells

[0086] Methods for reducing or eliminating gene expression or activity are known in the art. In some embodiments, such reduction or elimination includes any detectable decrease in the production of a gene (e.g., RFX5). In certain examples, detectable RFX5 in cells is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a control (the amount of RFX5 detected in the corresponding cells where RFX5 is not inhibited) (e.g., a reduction of 40% to 90%, 40% to 80%, or 50% to 95%).

[0087] In a particular embodiment, the detectable TCR in a cell is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a control (the amount of TCR detected in the corresponding cell in which the TCR is not inhibited) (e.g., reduced by 40% to 90%, 40% to 80%, or 50% to 95%).

[0088] In certain embodiments, detectable B2M in cells is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a control (the amount of B2M detected in corresponding cells where B2M is not inhibited) (e.g., reduced by 40% to 90%, 40% to 80%, or 50% to 95%).

[0089] In certain embodiments, the reduction or elimination of gene expression is achieved through direct inhibition of the gene (for example, knocking down or knocking out the RFX5 gene may reduce or eliminate the expression or activity of RFX5). In other embodiments, the reduction or elimination of gene expression is achieved through indirect inhibition of the gene (for example, knocking down or knocking out the RFX5 gene may reduce the expression or activity of MHC class I molecules).

[0090] The percentage decrease and percentage increase can be calculated by methods known in the art. As a non-limiting example, the percentage decrease or reduction in molecular expression or activity in edited cells (e.g., cells containing RFX5 KO) compared to reference or corresponding cells (e.g., cells without RFX5 KO) can be calculated by subtracting the edited cell value from the reference / corresponding cell value, dividing that amount by the reference value, and then multiplying by 100 to obtain the percentage decrease. If the percentage is negative, it may mean that there was an increase rather than a decrease.

[0091] In some embodiments, gene expression or activity can be reduced using appropriate inhibitors such as small molecule inhibitors, inhibitory RNAs (e.g., siRNA, shRNA), or antibodies. In some embodiments, this can be achieved by gene editing of the target gene in one or both alleles of the target gene.

[0092] In certain embodiments, the expression of one or more RFX5 is reduced using a DNA-binding domain, for example, coupled to a nuclease domain, which specifically binds to a target site within the RFX5 gene, mediates mutations at the target site, and thereby reduces the expression of functional RFX5. Any DNA-binding domain, including but not limited to zinc finger DNA-binding domains, TALE DNA-binding domains, DNA-binding portions (sgRNAs) of CRISPR / Cas nucleases, or DNA-binding domains derived from meganucleases, may be used in the compositions and methods disclosed herein.

[0093] In certain embodiments, the DNA-binding domain comprises a zinc finger protein. Preferably, the zinc finger protein is not naturally occurring in that it has been engineered to bind to a selected target site. The engineered zinc finger-binding domain may have novel binding specificity compared to naturally occurring zinc finger proteins. The engineering method includes, but is not limited to, rational design and various types of selection. Rational design includes, for example, using a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruplet sequence.

[0094] Typically, ZFPs contain at least three fingers. Some ZFPs contain four, five, or six fingers. ZFPs with three fingers typically recognize target sites containing nine or ten nucleotides. ZFPs with four fingers typically recognize target sites containing 12 to 14 nucleotides, while ZFPs with six fingers can recognize target sites containing 18 to 21 nucleotides. ZFPs can also be fusion proteins containing one or more regulatory domains, which may be transcriptional activation domains or transcriptional repression domains.

[0095] In some embodiments, the DNA-binding domain may be derived from a nuclease. For example, recognition sequences of homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII are known. Furthermore, the DNA-binding specificity of homing endonucleases and meganucleases can be manipulated to bind to non-natural target sites.

[0096] In some embodiments, the TALEN comprises an endonuclease (e.g., FokI) cleavage domain or a cleavage half-domain. In other embodiments, the TALE-nuclease is megaTAL. These megaTAL nucleases are fusion proteins comprising a TALE DNA-binding domain and a meganuclease cleavage domain. The meganuclease cleavage domain is monomerically active and does not require dimerization for activity.

[0097] In certain embodiments, the DNA-binding domain is part of a CRISPR / Cas nuclease system containing a single guide RNA (sgRNA) that binds to DNA. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes the RNA component of the system, and the cas (CRISPR-related) locus, which encodes the protein, constitute the gene sequence of the CRISPR / Cas nuclease system. In a microbial host, the CRISPR locus contains a combination of a CRISPR-related (Cas) gene and a non-coding RNA element that can program the specificity of CRISPR-mediated nucleic acid cleavage.

[0098] sgRNAs that may be suitable for use in the cells and methods of this disclosure can be identified using CRISPR design tools. Exemplary sgRNA sequences are shown in Table 2. TIFF0007912602000002.tif75144

[0099] Type II CRISPR is one of the most well-characterized systems, performing targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs (pre-crRNA array and tracrRNA) are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA, mediating the processing of the pre-crRNA into a mature crRNA containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs a functional domain (e.g., a nuclease such as Cas) to the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA adjacent to the protospacer-adjacent motif (PAM) further required for target recognition. Finally, Cas9 mediates the cleavage of the target DNA, resulting in a double-strand break within the protospacer. The CRISPR / Cas system operates in three steps: (i) insertion of a foreign DNA sequence into the CRISPR array to prevent future attacks in a process called "adaptation," (ii) expression of related proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference by foreign nucleic acids. Therefore, in bacterial cells, some so-called "Cas" proteins are involved in the innate function of the CRISPR / Cas system, playing a role in functions such as the insertion of foreign DNA.

[0100] Non-limiting examples of nucleases include meganucleases, TALENs, and zinc finger nucleases. Nucleases may contain heterologous DNA-binding domains and cleavage domains (e.g., zinc finger nucleases, meganucleases with heterologous cleavage domains and DNA-binding domains), or the DNA-binding domain of naturally occurring nucleases may be modified to bind to a selected target site (e.g., meganucleases engineered to bind to a site different from the congeneral-binding site). Expression of chimeric antigen receptors or T cell receptors

[0101] Manipulated cells, particularly immune cells such as T cells, NK cells, and other immune cell types, can also be genetically engineered using vectors designed to express CARs that redirect cytotoxicity to tumor cells. CARs are molecules that combine antibody-based specificity against a target antigen (e.g., a tumor antigen) with a T cell receptor-activating intracellular domain to produce chimeric proteins that exhibit specific anti-tumor cell immune activity.

[0102] The CARs contemplated herein include an extracellular domain, a transmembrane domain, and an intracellular signaling domain that bind to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain). A key feature of CARs is their ability to redirect immunoeffector cell specificity, thereby inducing the production of molecules that can mediate cell death of target antigen-expressing cells in a manner independent of proliferation, cytokine production, phagocytosis, or major histocompatibility (MHC), and utilizing the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0103] In some embodiments, the CAR includes an extracellular binding domain that specifically binds to a target antigen, including but not limited to the extracellular domain of an antibody or its antigen-binding fragment, a tether ligand, or a coreceptor.

[0104] As a non-limiting example, target antigens may include: HPV oncoproteins including HPV-16 E6 and HPV-16 E7, alpha-folate receptor, 5T4, α vβ6 integrin, BCMA, TACI, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, and the EGFR family including ErbB2(HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, glypican-3 (GPC3), HLA-Al+MAGEI, HL A-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-llRα, IL-13Rα2, Lambda , Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII, one or more hinge domains or spacer domains, transmembrane domains including but not limited to transmembrane domains from CD8α, CD4, CD45, PD-1, and CD152, one or more intracellular costimulatory signaling domains including but not limited to intracellular costimulatory signaling domains from CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS), and primary signaling domains derived from CD3ζ or FcRγ. In one embodiment described herein, the CAR binds to a tumor antigen including CLL-1, CD19, CD20, CD28, CD137 (4-1BB), glypican-3 (GPC3), PSCA, or PSMA. In a specific embodiment, the CAR binds to CD19. In a specific embodiment, the CAR binds to CD20. In a specific embodiment, the CAR includes a first scFv that binds to CD19 and a second scFv that binds to CD20. Examples of CD19-binding sequences or CD20-binding sequences are shown in Table 3. TIFF0007912602000003.tif221170TIFF0007912602000004.tif193170TIFF00079126020 00005.tif155170TIFF0007912602000006.tif253170TIFF0007912602000007.tif229170

[0105] In various embodiments, the binding motif may include a heavy chain variable domain of the Disclosure (e.g., having at least 75% sequence identity to the heavy chain variable domains shown in Table 3, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity), a light chain variable domain of the Disclosure (e.g., having at least 75% sequence identity to the light chain variable domains shown in Table 3, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity), and a linker (e.g., Whitlow et al. Protein See Eng. 1993, November, 6(8):989-95. In various embodiments, the binding motif may include a leader sequence or signal sequence, a heavy chain variable domain of the Disclosure (for example, having at least 75% sequence identity to the heavy chain variable domains shown in Table 3, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity), a light chain variable domain of the Disclosure (for example, having at least 75% sequence identity to the light chain variable domains shown in Table 3, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), and a linker.

[0106] Where amino acid or nucleotide sequences of binding motifs containing the heavy chain variable domain and the light chain variable domain of the Disclosure are provided, the linker connecting the two variable domains will be evident from the sequences in consideration of the Disclosure and knowledge of the Art. Where amino acid or nucleotide sequences of binding motifs containing the heavy chain variable domain and the light chain variable domain of the Disclosure are provided, the leader sequence will be evident in consideration of the Disclosure and knowledge of the Art. To avoid misunderstanding, the heavy chain variable domain and the light chain variable domain of the Disclosure may exist in any orientation, for example, an orientation in which the heavy chain variable domain is C-terminus of the light chain variable domain, or an orientation in which the heavy chain variable domain is N-terminus of the light chain variable domain. Exemplary anti-CD20 binding motifs are shown in Table 4. TIFF0007912602000008.tif208170TIFF0007912602000009.tif208170TIFF0007912602000010.tif221170TIFF0007912602000011.tif82170

[0107] Table 5 provides exemplary nucleotide sequences encoding the binding motif, hinge, and co-stimulatory domain. TIFF0007912602000012.tif255170TIFF0007912602000013.tif249170TIFF0007912602000014.tif249170 TIFF0007912602000015.tif245170TIFF0007912602000016.tif249170TIFF0007912602000017.tif233170 TIFF0007912602000018.tif233170TIFF0007912602000019.tif241170TIFF0007912602000020.tif237170 TIFF0007912602000021.tif233170TIFF0007912602000022.tif249170TIFF0007912602000023.tif249170

[0108] The hinges may be derived from natural or synthetic sources. In some embodiments, the antigen-binding systems of the present disclosure may include hinges from, or derived from (e.g., all or fragments of) the following: CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), C D30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile) , CD100(SEMA4D), CD103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD15 8C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD22 9(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD31 4(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF18),Inducible T cell costimulators (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, or Toll ligand receptor, or fragments or combinations thereof. In certain embodiments, the CAR does not include the CD28 hinge.

[0109] Transmembrane domains may originate from either natural or synthetic sources. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Exemplary transmembrane domains include the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, and activated NK cell receptors. Body, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (C D226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, CD83 Ligands that bind to it, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1, CD150;The transmembrane domain may be derived from IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof (e.g., may include at least a transmembrane domain). In some embodiments, the transmembrane domain may be synthetic (and may include mainly hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is included at both ends of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked to or connected to the cytoplasmic domain. In some embodiments, a short oligo or polypeptide linker (e.g., 2-10 amino acid length) may form a linkage between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.

[0110] In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, for example, Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010) and Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)).

[0111] CARs may include, for example, a co-stimulatory signaling domain to enhance signaling efficacy. See U.S. Patents 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (above), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N.Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signals generated solely through TCRs may be insufficient for complete T cell activation, and secondary or co-stimulatory signals may increase activation. Accordingly, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., innate immune cell effector functions described herein). In some embodiments, a portion of such co-stimulatory signaling domains may be used insofar as the portion transduces the effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of T cell coreceptors (or fragments thereof). Non-limiting examples of such T cell coreceptors include ligands that bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0112] In certain embodiments, the CARs contemplated herein may include linker residues added between various domains, for example, between the VH domain and the VL domain, for proper interspersed conformation of the molecule. The CARs contemplated herein may include one, two, three, four, or five or more linkers. In some embodiments, the length of the linkers is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linkers have an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more.

[0113] In some embodiments, the CARs contemplated herein include an intracellular signaling domain. The “intracellular signaling domain” refers to a portion of the CAR that is involved in transducing an effective CAR message that binds to a target antigen into the interior of an immunoeffector cell to induce effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity (including the release of cytotoxic factors to CAR-bound target cells or other cellular responses induced by antigen binding to an extracellular CAR domain). In some embodiments, the signaling domain and / or activation domain include an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, for example, Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010) and Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)).In certain embodiments, preferred signaling domains include, but are not limited to, 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that binds to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1, This includes CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76), TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavage, or combinations thereof.

[0114] The components of CAR can be replaced or "swapped" with equivalent components using routine biotechnology techniques. In various embodiments, the Disclosure provides a binding motif by any one of SEQ ID NOs: 31-40, or a binding motif by a sequence having at least 75% identity to any one of SEQ ID NOs: 31-40 (e.g., 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% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity), in combination with a hinge (e.g., adjacent fusion), and optionally further in combination with a co-stimulatory domain (e.g., adjacent fusion). A small number of non-limiting examples are provided in sequences having at least 75% identity to sequence numbers 42-64, or any one of sequence numbers 42-64 (e.g., 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% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity).

[0115] Bicistronic CARs may comprise a first CAR sequence and a second CAR sequence, and are expressed as a single polypeptide containing a cleavable linker between the first and second CARs. Examples of cleavable linkers include, but are not limited to, Furin-GSG-T2A (see, e.g., Chng et al. MAbs. March-April 2015, 7(2):403-412 (incorporated herein by reference with respect to cleavable linkers), and also see Guedan et al. Mol Ther Methods Clin Dev. March 15, 2019, 12:145-156 (incorporated herein by reference with respect to bicistronic CAR design)), 2A linkers (e.g., T2A), 2A-like linkers, or their functional equivalents and combinations thereof. In some embodiments, the linker includes a picornavirus 2A-like linker, a CHYSEL sequence of porcine scoparium virus (P2A), a virus (T2A), or combinations thereof, variants, and functional equivalents.

[0116] Exemplary anti-CD20 / anti-CD19 bisistronic CARs may have or include sequences having at least 75% identity to the nucleotide and amino acid sequences described in SEQ ID NOs. 28 and 26, respectively, or sequences having at least 75% identity to SEQ ID NOs. 28 and 26 (e.g., 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% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity).

[0117] A bispecific CAR may be a single polypeptide comprising a first binding motif and a second binding motif. Exemplary anti-CD20 / anti-CD19 bispecific CARs may have or contain sequences having at least 75% identity to the nucleotide and amino acid sequences described in SEQ ID NOs. 29 and 27, respectively, or sequences having at least 75% identity to SEQ ID NOs. 29 and 27 (e.g., 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% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity).

[0118] This disclosure provides, in particular, bispecific antibodies that bind to CD20 and a second target antigen, such as CD19. The bispecific antibody includes an antibody having a first binding motif that binds to a first target antigen and a second binding motif that binds to a second target antigen. In some embodiments, the bispecific antibody includes an anti-CD20 binding motif and an anti-CD19 binding motif of this disclosure. In some embodiments, the bispecific antibody includes an anti-CD20 binding motif comprising an anti-CD20 heavy chain variable domain and an anti-CD20 light chain variable domain of this disclosure, and an anti-CD19 binding motif comprising an anti-CD19 heavy chain variable domain and an anti-CD19 light chain variable domain.

[0119] This disclosure includes nucleic acids encoding anti-CD20 binding motifs and / or anti-CD19 binding motifs provided herein. This disclosure includes nucleic acids encoding antibodies, including but not limited to nucleic acids encoding binding motifs (e.g., anti-CD20 binding motifs and anti-CD19 binding motifs). This disclosure includes nucleic acids encoding antigen-binding systems, including but not limited to nucleic acids encoding bisistronic and bispecific chimeric antigen receptors (e.g., bisistronic and bispecific chimeric antigen receptors that bind to CD20 and CD19).

[0120] This disclosure includes vectors comprising nucleic acids of this disclosure and / or vectors encoding polypeptides of this disclosure. In various embodiments, this disclosure includes vectors comprising nucleic acids encoding anti-CD20 binding motifs and / or anti-CD19 binding motifs provided herein. In various embodiments, this disclosure includes vectors comprising nucleic acids encoding antibodies provided herein, including but not limited to nucleic acids encoding binding motif molecules (e.g., anti-CD20 binding motifs or anti-CD19 binding motifs). In various embodiments, this disclosure includes vectors comprising nucleic acids encoding one or more antigen-binding systems provided herein, including but not limited to nucleic acids encoding bisistronic or bispecific chimeric antigen receptors (e.g., bisistronic and bispecific chimeric antigen receptors that bind to CD20 and CD19).

[0121] In certain embodiments, the CARs of this disclosure bind to CLL-1. Examples of CLL-1 CAR amino acid sequences are shown in Table 6. TIFF0007912602000024.tif250170TIFF0007912602000025.tif255169TIFF0007912602000026.tif250170

[0122] In some embodiments, the CARs of the present disclosure include amino acid sequences that are at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 65-76 or 78-83.

[0123] In some embodiments, the CAR or TCR of the Disclosure may further comprise a leader sequence or peptide (also referred to herein as a “signal peptide”). In certain embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 96). In some embodiments, the leader peptide comprises the amino acid sequence of SEQ ID NO: 96.

[0124] The term "effector function" refers to a specific function of a cell. The effector function of a T cell may include, for example, assisting or activating cytolytic activity or cytokine secretion. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals, directing the cell to perform its specific function. While the entire intracellular signaling domain may be used, it is often not necessary to use the entire domain. Insofar as a cleavage portion of an intracellular signaling domain is used, such a cleavage portion may be used in place of the entire domain, as long as it transduces the effector function signal. The term "intracellular signaling domain" means including any cleavage portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0125] In some embodiments, cells can be engineered to express exogenous T cell receptors (TCRs). A library of TCRs may be screened for their selectivity for target antigens. In this way, native TCRs with high binding affinity and reactivity to target antigens can be selected, cloned, and subsequently introduced into a T cell population used for adoptive immunotherapy.

[0126] In one embodiment described herein, T cells are modified by introducing a polynucleotide encoding a subunit of a TCR that can form a TCR that confers specificity to tumor cells expressing a target antigen to the T cell. In some embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to a naturally occurring subunit, insofar as the subunit retains the ability to form a TCR that confers the ability to hom to target cells to the transfected T cell and is involved in immunologically relevant cytokine signaling. The TCR also binds with high affinity to target cells displaying relevant tumor-associated peptides and can optionally mediate the efficient killing of target cells presenting relevant peptides in vivo.

[0127] Nucleic acids encoding TCRs can be isolated from their natural context on the (naturally occurring) chromosomes of T cells and incorporated into suitable vectors as described elsewhere herein. Both the nucleic acids and the vectors containing them may be transferred to cells, which may be T cells. The modified T cells can then express one or more strands (and in some embodiments, two strands) of the transduced nucleic acid or the TCR encoded by the nucleic acid. In some embodiments, the TCR is an exogenous TCR, since it is introduced into T cells that do not normally express the transduced TCR. An embodiment of the TCR is that it has high binding affinity to tumor antigens presented by the major histocompatibility complex (MHC) or similar immunological components. In contrast to TCRs, CARs are engineered to bind to target antigens in a manner independent of MHC.

[0128] The nucleic acid-encoded proteins described herein may be expressed with additional polypeptides attached to the amino-terminus or carboxyl-terminus of the α- or β-chain of the TCR, provided that the attached additional polypeptides do not interfere with the α- or β-chain's ability to form a functional T cell receptor and with MHC-dependent antigen recognition.

[0129] Antigens recognized by TCRs as intended herein include, but are not limited to, cancer antigens, including those of hematological cancers and solid tumors and virus-induced cancers. Other exemplary antigens include HPV oncoproteins, including HPV-16 E6 and HPV-16 E7, alpha-folate receptor, 5T4, α v β6 integrin, BCMA, TACI, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Examples include, but are not limited to, glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NY-ES0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII.

[0130] In some embodiments, the polynucleotide encoding the CAR or TCR is introduced into the cells after the cells have been manipulated to reduce the expression or activity of MHC class I and / or II. In some embodiments, the polynucleotide encoding the CAR or TCR is introduced into the cells before the cells have been manipulated to reduce the expression or activity of MHC class I and / or II. In some embodiments, two rounds of the operation are performed with at least one day between them (but not on the same day or within 24 hours). treatment

[0131] The cells of this disclosure, for example, allogeneic cells, can be used to treat a variety of diseases and conditions, particularly cancer. In one embodiment, cancer may include Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and bladder cancer.

[0132] In some embodiments, the cells of this disclosure may be used to treat bone marrow diseases including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroblastic leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorders, myeloid neoplasms, myeloid sarcomas, blastic plasmacytoid dendritic cell neoplasms (BPDCN), or combinations thereof. Further diseases include inflammatory and / or autoimmune diseases such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, lupus, and celiac disease.

[0133] In some embodiments, the cells of the Disclosure may be used to treat cancers arising from B cells, such as B-cell lymphoma. In some embodiments, the cells of the Disclosure may be used to treat DLBCL arising from diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and follicular lymphoma.

[0134] Another embodiment described herein is a method of treating cancer in a subject in need of treatment, comprising administering an effective amount, for example, a therapeutically effective amount, of a composition comprising a cell of the present disclosure. The amount and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, but appropriate dosages can be determined by clinical trials. In some embodiments, the cancer is characterized by expression of an antigen targeted by a CAR or TCR molecule, such as CD19 and / or CD20.

[0135] In some embodiments, the cancer is characterized by expression of an antigen targeted by a CAR or TCR molecule, such as CLL-1.

[0136] In other embodiments, a method is provided comprising administering a therapeutically effective amount of modified T cells contemplated herein, or a composition comprising the same, alone or in combination with one or more therapeutic agents, to a patient in need thereof. In certain embodiments, the cells of the present disclosure are used for the treatment of patients at risk of developing cancer. Accordingly, the present disclosure provides a method for the treatment or prevention of cancer, comprising administering a therapeutically effective amount of the modified T cells of the present disclosure to a subject in need thereof.

[0137] CAR + / CAR-T + / TCR + The target dose of cells is about 1 × 10 6 to about 2 × 10 10 cells / kg (e.g., about 1 × 10 6 cells / kg, about 2 × 10 6 cells / kg, about 3 × 10 6 cells / kg, about 4 × 10 6 cells / kg, about 5 × 10 6 cells / kg, about 6 × 10 6 cells / kg, about 7 × 10 6 cells / kg, about 8 × 10 6 cells / kg, about 9 × 10 6 cells / kg, about 1 × 10 7 cells / kg, about 2 × 10 7 cells / kg, about 3 × 10 7 cells / kg, about 4 × 107 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, approximately 9×10 7 cells / kg, approximately 1×10 8 cells / kg, approximately 2×10 8 cells / kg, approximately 3×10 8 cells / kg, approximately 4×10 8 cells / kg, approximately 5×10 8 cells / kg, approximately 6×10 8 cells / kg, approximately 7×10 8 cells / kg, approximately 8×10 8 cells / kg, approximately 9×10 8 cells / kg, approximately 1×10 9 cells / kg, approximately 2×10 9 cells / kg, approximately 3×10 9 cells / kg, approximately 4×10 9 cells / kg, approximately 5×10 9 cells / kg, approximately 6×10 9 cells / kg, approximately 7×10 9 cells / kg, approximately 8×10 9 cells / kg, approximately 9×10 9 cells / kg, 1×10 10 Cells / kg, or approximately 2 × 10⁻⁶ 10 It will be understood that the range may be cells / kg. It will also be understood that doses above and below this range may be appropriate for specific subjects.

[0138] Those skilled in the art will recognize that multiple administrations of the compositions of the present disclosure may be required to achieve the desired treatment. For example, the compositions may be administered one, two, three, four, five, six months, one year, two years, five years, ten years, or more, over a span of one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, ten years, or more.

[0139] In one embodiment, a subject requiring it is administered an effective amount of the composition to increase the cellular immune response against cancer in the subject. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. Thus, a humoral immune response primarily mediated by helper T cells that can activate B cells that result in antibody production may also be induced. To analyze the type of immune response induced by the compositions of this disclosure, various techniques well described in the art can be used, for example, as described in Current Protocols in Immunology, edited by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, and Warren Strober (2001), John Wiley & Sons, NY, NY.

[0140] Methods for administering the cell compositions described herein include any method effective in resulting in either the reintroduction of ex vivo genetically modified immune effector cells that directly express a TCR or CAR in a subject, or the reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a TCR or CAR upon introduction into a subject. One method includes transducing peripheral blood T cells with the nucleic acid constructs of this disclosure ex vivo and returning the transduced cells to a subject.

[0141] Another embodiment described herein is a method of bringing cancer cells into contact with manipulated immune cells of the Disclosure, wherein cancer cell proliferation is inhibited or reduced.

[0142] While the foregoing disclosure is described in some detail as examples and illustrations for clarity, it will be readily apparent to those skilled in the art, in light of the teachings of this disclosure, that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and are not limiting. Those skilled in the art will readily recognize a variety of non-essential parameters that can be changed or modified to produce similar results.

[0143] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference. However, the reference of references herein should not be construed as an acknowledgment that such references are prior art to this disclosure. In the event of any difference between any definition or term provided in an incorporated reference and the terms and definitions provided herein, the terms and definitions herein shall prevail. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Section 1] Isolated human immune cells engineered to have (i) 10% to 80% lower MHC class I activity or expression compared to reference cells, (ii) at least 75% lower MHC class II activity or expression compared to reference cells, and (iii) exogenous polynucleotides encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR). [Section 2] The immune cells described in item 1, which are T cells or NK cells. [Section 3] Immune cells as described in any one of the preceding paragraphs, wherein the endogenous gene RFX5 (regulatory factor X5), TAP1 (antigen processing-related transporter 1), TAP2 (antigen peptide transporter 2), or CIITA (class II, major histocompatibility complex, transactivator) is inactivated or deficient. [Section 4] The immune cells described in item 3, wherein both alleles of the endogenous gene are inactivated or deficient. [Section 5] Immune cells as described in item 3, wherein the endogenous gene for RFX5 is inactivated or deficient. [Section 6] Immune cells as described in item 3, in which the endogenous genes TAP1, TAP2, and CIITA have not been manipulated. [Section 7] Immune cells as described in any one of items 1 to 6, wherein the endogenous gene of TRAC (T cell receptor alpha constant region) is further inactivated. [Section 8] Immune cells as described in any one of the preceding paragraphs, wherein the endogenous gene for B2M (beta-2-microglobulin) is not manipulated, or the cells have normal B2M activity. [Section 9] The immune cell according to item 1, wherein the CAR recognizes CD19 and / or CD20. [Section 10] The immune cell described in item 1, wherein the CAR recognizes CLL-1. [Section 11] The immune cell according to claim 9, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 26 or 27 or a sequence having 90% sequence identity with SEQ ID NO: 26 or 27. [Section 12] The immune cell according to claim 10, wherein the CAR contains the amino acid sequence of SEQ ID NOs. 65-76 or 78-83, or a sequence having 90% sequence identity with SEQ ID NOs. 65-76 or 78-83. [Section 13] The immune cells according to any one of claims 4 to 12, wherein inactivation is achieved by (a) editing of the endogenous gene, (b) expression of inhibitory RNA, or (c) an inhibitor, preferably an antibody. [Section 14] Immune cells as described in item 13, edited by CRISPR / Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, Cpf1, homologous recombination, single-stranded oligodeoxynucleotide (ssODN), or base editing. [Section 15] The immune cells described in item 1, wherein the reference immune cells have not been edited to reduce the expression of MHC class I or MHC class II. [Section 16] A method for preparing allogeneic cells with reduced graft-versus-host disease (GVHD) or host rejection-inducing activity, comprising manipulating cells to reduce the expression or activity of MHC class I by 10% to 80% and the expression or activity of MHC class II by at least 75% compared to reference cells. [Section 17] The method according to item 16, wherein the expression or activity of RFX5 (regulatory factor X5), TAP1 (antigen processing-related transporter 1), TAP2 (antigen peptide transporter 2), or CIITA (class II, major histocompatibility complex, transactivator) is reduced in the allogeneic cells. [Section 18] The method according to item 17, wherein both alleles of the endogenous gene are inactivated or deleted. [Section 19] The method according to item 17 or 18, for reducing the expression or activity of RFX5 in the cells. [Section 20] The method according to item 16, wherein the endogenous genes TAP1, TAP2, and CIITA are not manipulated. [Section 21] The method according to any one of claims 16 to 20, further comprising reducing the expression or activity of TRAC (T cell receptor alpha constant region) in the cells. [Section 22] The method according to any one of items 16 to 21, wherein the endogenous gene for B2M (beta-2-microglobulin) in the cells has not been manipulated. [Section 23] The method according to any one of claims 16 to 22, wherein the reduction is achieved by (a) gene editing, (b) expression of inhibitory RNA, or (c) an inhibitor, preferably an antibody. [Section 24] The method according to item 23, wherein editing is performed by CRISPR / Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, Cpf1, homologous recombination, single-stranded oligodeoxynucleotide (ssODN), or base editing. [Section 25] The method according to any one of items 16 to 24, wherein the allogeneic cells are T cells or NK cells. [Section 26] The method according to claim 16, further comprising introducing an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) into the allogeneic cells. [Section 27] The method according to paragraph 26, wherein the CAR recognizes CD19 and / or CD20. [Section 28] The method according to claim 27, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 26 or 27, or an amino acid sequence having 90% identity with SEQ ID NO: 26 or 27. [Section 29] The method according to paragraph 26, wherein the CAR recognizes CLL-1. [Section 30] The method according to claim 29, wherein the CAR comprises the amino acid sequence of SEQ ID NOs. 65-76 or 78-83, or a sequence having 90% sequence identity with SEQ ID NOs. 65-76 or 78-83. [Section 31] The method according to any one of claims 26 to 30, wherein the CAR or TCR is introduced into the cells before gene editing. [Section 32] A method for treating cancer in a patient requiring treatment, comprising administering to the patient an immune cell as described in any one of items 1 to 15. [Section 33] The method according to item 32, wherein the immune cells are not originally derived from the patient. [Section 34] The method according to paragraph 32, wherein the cancer is lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin lymphoma, or non-Hodgkin lymphoma. [Section 35] A method for bringing cancer cells into contact with immune cells described in any one of items 1 to 15, wherein the proliferation of the cancer cells is inhibited or reduced. [Examples]

[0144] Example 1. Testing of RFX5 knockout CAR T cells

[0145] In this example, we evaluated the in vitro efficacy of a research-grade zinc finger nuclease (ZFN) targeting RFX5 (regulatory factor X5) deletion, as well as its ability to protect against T cell and NK cell-mediated rejection when used in allogeneic (MHC-incompatible) CAR-T cell products.

[0146] Methods: Healthy donor T cells were transduced with CD19 / CD20 bicistronic CARs (using a lentiviral vector (LLV)), followed by electroporation with ZFNs targeting TRAC (T cell receptor alpha constant region) and RFX5. Edited and unedited CAR T cells were grown for 14 days, and the expression of CAR, TCRαβ, MHC class I, and MHC class II was examined on days 7 and 10. The majority of cells were frozen on day 10 for all functional and allogeneic reactivity assays, but a small number of cells were kept alive until day 14 to confirm cell viability, maintaining growth under the same conditions as unedited cells throughout the entire production process. CAR functionality was evaluated by co-culturing CAR-T cells with CD19 / CD20-positive tumor target cell lines (Raji WT) using cytotoxicity (24 and 48 hours), proliferation (120 hours), and cytokine secretion (24 hours) at various E:T ratios. The persistence and cytotoxicity of CAR-T cells were evaluated by continuous stimulation of CAR-T cells every 3-4 days with CD19 / CD20-positive Raji MHC knockout tumor cells at a 1:1 E:T ratio. Furthermore, the killing of edited and unedited CAR T cells by MHC-incompatible CD8 T cells and NK cells was measured.

[0147] Results: RFX5 knockout (KO) CAR-T cells possessed similar manufacturability and CAR functionality to β2M KO CAR-T cells. As shown in Tables 7A-C, RFX5 knockout (KO) allo-CAR-T cells exhibited better viability and similar proliferation rates compared to B2M KO allo-CAR-T cells. TIFF0007912602000027.tif45128TIFF0007912602000028.tif37128TIFF0007912602000029.tif52128

[0148] Furthermore, as shown in Table 8, RFX5 or B2M KO did not affect the expression of exogenous CAR molecules. TIFF0007912602000030.tif101152

[0149] Tables 9A-C show that CD19 / CD20 B2M KO and RFX5 KO CAR-T had similar activity. TIFF0007912602000031.tif34169TIFF0007912602000032.tif28170TIFF0007912602 000033.tif23128TIFF0007912602000034.tif28168TIFF0007912602000035.tif23128

[0150] RFX5 KO CAR T cells reduced HLA class I expression with a similar MFI (metabolic infiltration) as β2M KO CAR T cells, which reduced the host allogeneic response (Table 10). The table shows that RFX5 KO resulted in MHC class I knockdown (downregulation), while B2M KO resulted in MHC class I knockout (exclusion). On the other hand, similar TCR KO was observed in both TRAC+B2M KO cells and TRAC+RFX5 KO cells. MHC class I MFI was similar for both class I knockout and knockdown cells. However, efficient MHC class II KO was observed only in TRAC+RFX5 KO cells. TIFF0007912602000036.tif85128TIFF0007912602000037.tif16148

[0151] RFX5 knockout enhanced protection from host NK killing and reduced rejection from incompatible host CD8 T cells compared to β2M knockout (Tables 11A-B). Tables 11A-B show that RFX5 KO protected from host NK and reduced CD8 rejection. Table 11A: RFX5 KO improved protection from NK compared to B2M KO in 3 out of 3 donors. Table 11B: RFX5 KO reduced CD8 rejection to a similar level as B2M KO in 1 / 3 of incompatible donors and reduced CD8 rejection compared to unedited cells in 2 / 3 of incompatible donors. RFX5 KO cells showed the absence of MHC class II expression (Table 10), which is predicted to minimize the response by host CD4 T cells. TIFF0007912602000038.tif48146TIFF0007912602000039.tif41170

[0152] This example demonstrates that deletion of the transcription factor RFX5 enables the generation of CAR-T cells with low levels of MHC class I and MHC class II expression, which leads to reduced killing by MHC-incompatible CD8 T cells and NK cells. Example 2. Clinical trial of the allogeneic TRAC-RFX5 anti-CD19 / CD20 CAR

[0153] CD19 and CD20-targeted healthy donor allogeneic (HD Allo) products are being developed to treat cancer regardless of the patient's HLA type by utilizing zinc finger nucleases (ZFNs) to knock out genes. These products contain CD19 and CD20-targeted healthy donor T cells. Using ZFNs, the TRAC gene is disrupted to address the risk of graft-versus-host disease (GVHD), and the RFX5 gene is disrupted to reduce the expression of MHC classes I and II, minimizing host rejection.

[0154] Alternatively, the TRAC gene and the RFX5 gene can be disrupted by any of the gene editing approaches described herein or known in the art (e.g., via sgRNA and CRISPR / Cas9).

[0155] All modifications are designed to achieve the following essential product attributes: (i) an allogeneic anti-CD19 / CD20 CAR with disruption of the TRAC and B2M loci (allogeneic TRAC-B2M CD19 / CD20), and an in vivo activity profile equivalent to or improved compared to conventional, unedited anti-CD19 CAR T cells; (ii) minimization to zero risk of GVHD; and (iii) minimization of the risk of rejection by the patient's endogenous CD4 and CD8 T cells and NK cells, as demonstrated using in vitro assays. The following objectives are expected to be achieved. - Equivalent or improved in vivo activity compared to allogeneic TRAC / B2M KO CD19 / CD20 CAR-T cells, as demonstrated by tumor cell clearance. - Equivalent or improved CAR expression and cell proliferation dynamics compared to allogeneic TRAC-B2M KO CD19 / CD20 CAR-T cells (in vivo and in production). - Reduced in vitro incompatible NK cell rejection, as demonstrated by reduced NK cell killing by allogeneic CAR T cells compared to allogeneic TRAC-B2M KO CD19 / CD20 CAR-T cells, and - Reduced in vitro recognition of incompatible CD4 T cells and rejection of CD8 T cells compared to unedited anti-CD19 or anti-CD19 / 20 CAR-T cells, as evidenced by reduced killing and / or cytokine secretion. Example 3. Testing of RFX5 knockout CAR T cells

[0156] In this example, we evaluated the in vitro efficacy of research-grade clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-related protein 9 (Cas9) (CRISPR / Cas9) ribonucleoprotein targeting RFX5 gene deletions, as well as their ability to protect against T cell and NK cell-mediated rejection when used in allogeneic (MHC-incompatible) CAR-T cell products.

[0157] Methods: Healthy donor T cells were transduced with CLL-1 CAR (using a lentiviral vector (LLV)), followed by electroporation with CRISPR / Cas9 ribonucleoprotein having a single guide RNA (sgRNA) targeting TRAC (T cell receptor alpha constant region) and B2M (beta-2-microglobulin), or TRAC and RFX5. β2M knockout (KO) allo-CAR-T cells (i.e., TRAC+B2M KO, where both TRAC and B2M are knocked out), RFX5 KO allo-CAR-T cells (i.e., TRAC+RFX5 KO, where both TRAC and RFX5 are knocked out), unedited CAR-T cells (i.e., LVV), and untransduced control cells (i.e., NTD) were grown for 10 days, and CAR expression, TCRαβ, MHC class I, and MHC class II expression were examined on day 10. Cells were frozen on day 10 for all functional and allogeneic reactivity assays. Killing of edited and unedited CAR T cells by MHC-incompatible NK cells was measured.

[0158] Results: RFX5 knockout (KO) CAR-T cells possessed similar manufacturability and CAR functionality to β2M KO CAR-T cells. As shown in Tables 12A-C, RFX5 knockout (KO) allo-CAR-T cells exhibited better viability and similar proliferation rates compared to B2M KO allo-CAR-T cells. TIFF0007912602000040.tif35128TIFF0007912602000041.tif37128TIFF0007912602000042.tif42128

[0159] Furthermore, as shown in Table 13, RFX5 or B2M KO did not affect the expression of exogenous CAR molecules. TIFF0007912602000043.tif34139

[0160] Table 14 shows that RFX5 knockout resulted in MHC class I knockdown (downregulation). Meanwhile, similar TCR knockout was observed in both TRAC+B2M knockout cells and TRAC+RFX5 knockout cells. However, efficient MHC class II knockout was observed only in TRAC+RFX5 knockout cells. TIFF0007912602000044.tif85128

[0161] RFX5 knockout provided enhanced protection from host NK cell killing compared to β2M knockout (Table 15). Table 15: RFX5 KO improved protection from NK cells compared to B2M KO in 3 out of 3 donors. TIFF0007912602000045.tif40170

[0162] This example demonstrates that deletion of the transcription factor RFX5 enables the generation of CAR-T cells with low levels of MHC class I and MHC class II expression, which leads to reduced NK cell killing (compared to B2M KO cells). Example 4. Testing of RFX5 knockout CAR T cells

[0163] In this example, we evaluate the in vivo efficacy of RFX5 KO CAR T cells in mouse models of B-cell lymphoma and acute myeloid leukemia.

[0164] Methods: RFX5 KO CAR-T cells and corresponding controls generated by the methods described in Examples 1 and 3 will be used in in vivo studies. Female NSG mice (5 mice / group) aged 6-7 weeks will be evaluated. Two doses of CAR+ T cells will be tested: 1 × 10⁶ 6 or 2 × 10 6 CAR+ T cells / mouse and 5×10 6 or 10 x 10 6 CAR+ T cells / mouse. Tumor cells expressing the relevant antigen are intravenously transplanted into 6-7 week old female NSG mice via the lateral tail vein.

[0165] After tumor transplantation, the following groups of B-cell lymphoma or AML tumor-bearing mice will be evaluated: (1) no treatment, (2) treatment with non-transduction (NTD) T cells, (3) treatment with unedited CAR-T cells, (4) treatment with allogeneic RFX5 and TRAC KO CAR-T cells, and (5) treatment with allogeneic B2M and TRAC KO CAR-T cells.

[0166] Tumor killing is assessed by evaluating the effect of T cell products on tumor burden by bioluminescence (bioluminescence is positively correlated with tumor burden) and animal survival rate. RFX5 KO CAR-T cell proliferation dynamics and persistence are assessed by ex vivo analysis of blood samples taken 24 hours after CAR-T cell infusion, followed by weekly sampling to track CAR expression, TCR, MHC class I, MHC class II, and other relevant T cell phenotypic markers. * * *

[0167] While many embodiments have been described, it is clear that this disclosure and examples may provide other embodiments utilizing the compositions and methods described herein, or other embodiments encompassed by such compositions and methods. Therefore, it will be understood that the scope of the invention should be defined not by the embodiments presented as examples, but by what can be understood from this disclosure and the appended claims.

Claims

1. Isolated human T cells that have been modified to have (i) an inactivated or deficient endogenous gene for RFX5 (regulatory factor X5), (ii) an inactivated or deficient endogenous gene for TAC (T cell receptor alpha constant region), (iii) an inactivated or deficient endogenous gene for TAP1 (antigen processing-associated transporter 1), and (iv) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR), but in which the endogenous gene for T cell B2M (beta-2-microglobulin) has not been modified.

2. The T cells according to claim 1, wherein the endogenous genes of TAP2 (antigen peptide transporter 2) and CIITA (class II, major histocompatibility complex, transactivator) have not been manipulated.

3. The T cell according to claim 1, wherein the cell has normal B2M activity.

4. The T cell according to claim 1, wherein the CAR recognizes CD19 and / or CD20.

5. The T cell according to claim 1, wherein the CAR recognizes CLL-1.

6. The T cell according to claim 4, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 26 or 27.

7. The T cell according to claim 5, wherein the CAR comprises any of the amino acid sequences of SEQ ID NOs. 65-76 and 78-83.

8. The T cell according to claim 1, wherein the endogenous gene of RFX5 or TRAC is edited by CRISPR / Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, Cpf1, homologous recombination, single-stranded oligodeoxynucleotide (ssODN), or base editing.

9. A method for preparing T cells according to any one of claims 1 to 8, comprising manipulating T cells to knock out the endogenous genes RFX5, TRAC, and TAP1 and introduce exogenous polynucleotides.

10. Use of T cells according to any one of claims 1 to 8 in the manufacture of a pharmaceutical product for treating cancer in patients requiring treatment.

11. The use according to claim 10, wherein the T cells are not originally derived from the patient.

12. The use according to claim 10, wherein the cancer is lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin lymphoma, or non-Hodgkin lymphoma.

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