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 TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 314,848, filed February 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy created on February 22, 2023 is named K-1138-WO-PCT_SL.xml and is 147,560 bytes in size.

[0003] FIELD OF THEINVENTION The present disclosure relates to the field of cell therapy, and more specifically, to genetically engineered immune cells. [Background technology]

[0004] T cell therapy is based on human T cells that are enriched or engineered to target and kill cancer cells in patients. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct T cells to specific target cancer cells. Chimeric antigen receptors (CARs) containing binding domains that can interact with specific tumor antigens and engineered T cell receptors (TCRs) allow 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 may limit its clinical application to its full potential. Allogeneic CAR T therapy is an alternative strategy that overcomes the inherent limitations of autologous therapy and provides an "off-the-shelf" approach for clinical use.

[0006] In principle, allogeneic CAR T therapy uses T cells from healthy human donors that are subsequently genetically modified to confer specificity for tumor antigens. Furthermore, gene editing is also introduced to prevent graft-versus-host disease (GVHD) and rejection of allogeneic CAR T cells by the patient's immune system. GVHD is primarily due to interactions between T cell receptor (TCR) αβ proteins 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 bear incompatible major histocompatibility complex (MHC) class I molecules. Summary of the Invention

[0007] Efforts have been made to develop low immunogenic cells suitable for off-the-shelf use, particularly immune cells such as T cells and NK cells. In one 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 mismatched CD8 T cells in the host. However, it has been observed that B2M knockout CAR T cells are susceptible to killing by the host's NK cells, as the NK cells become stimulated to kill the allogeneic T cells that lack MHC class I activity.

[0008] The present disclosure, in various embodiments, provides a superior cell engineering approach to B2M knockout that can achieve minimal or no risk of GVHD, and minimal or no risk of CD4, CD8 and NK cell rejection, while retaining comparable or even improved therapeutic activity.

[0009] Thus, according to one embodiment of the present disclosure, allogeneic cells are provided that are genetically engineered to reduce MHC class I or MHC class II expression or activity. In some embodiments, MHC class I expression or activity is reduced, but not eliminated. In some embodiments, MHC class II expression and / or activity 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 compared to a corresponding unengineered immune cell. In some embodiments, the cells have at least 20% lower, or at least 40% lower MHC class II activity or expression compared to a corresponding unengineered immune cell. In some embodiments, the cells have at least 75% lower MHC class II activity or expression compared to a corresponding unengineered immune cell or a reference cell.

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

[0012] In some embodiments, the cell is a T cell or NK cell, or any other immune cell such as a monocyte or macrophage, or a cell derived from 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 comprises 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 of RFX5 (regulatory factor X5), TAP1 (transporter associated with antigen processing 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 in the cell. In some embodiments, the endogenous genes of TAP1, TAP2, and CIITA are not engineered.

[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 activity of B2M.

[0016] In some embodiments, inactivation is achieved by (a) editing of the endogenous gene, (b) expression of an inhibitory RNA, or (c) an inhibitor, preferably an antibody. In some embodiments, editing is by CRISPR / Cas9, zinc finger nucleases (ZFNs), TALENs, MegaTALs, meganucleases, Cpf1, homologous recombination, single-stranded oligodeoxynucleotides (ssODNs), or base editing.

[0017] Methods for the cells are also provided, including, but not limited to, those described herein. In some embodiments, CAR or TCR is introduced into the cells before editing genes such as RFX5. Also provided are methods of using the cells to treat disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition

[0019] In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are found throughout the specification.

[0020] As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive and includes both "or" and "and."

[0021] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B, A or B, A (single), and B (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: 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 (single), B (single), and C (single).

[0022] Unless specifically stated or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one or more standard deviations as per the practice of the art. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Additionally, particularly with respect to biological systems or processes, the term can mean a value of up to an order of magnitude or up to 5 times. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0023] "Administering" refers to the physical introduction of an agent, such as an engineered T cell disclosed herein, into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and 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 routes of administration, for example, intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0024] The terms "activated" and "activation" refer to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In one embodiment, activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are proliferating. Signals generated through the TCR alone may be insufficient for full activation of T cells, and one or more secondary or costimulatory signals may also be required. Thus, T cell activation includes a primary stimulatory signal via the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation may be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0025] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, e.g., allogeneic T cell transplantation.

[0026] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may include at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are embedded in more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs can mediate the binding of the immunoglobulin 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 tetrameric agents of about 150 kD, composed 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 what is commonly referred to as a "Y-shaped" structure. 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 connected to each other to form a tetramer. Naturally produced antibodies are also glycosylated, for example on their CH2 domains.

[0027] An "antigen-binding molecule," "antigen-binding portion," "antigen-binding fragment," or "antibody fragment" refers to any molecule that contains an antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule 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, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease 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 selected from the group consisting of 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A-kinase anchoring 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 (an oncogene fusion protein consisting of the breakpoint cluster region (BCR) and the Abelson murine leukemia viral 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, 9-0-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 (also called CD2 subset 1, 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), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (EL F2M), endosialin, 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), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor alpha, folate receptor beta, 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, hexasaccharide moiety of GloboH glycoceramide (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 cellular 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 scatter 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 lRa), 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, kappa light chain, KCS1, kinase insert 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), legumain, 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, ​​locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGEA3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory factor (MIS) receptor type II, neuroblastoma-derived homolog of the v-myc avian myelocytomatosis viral oncogene (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, oncofetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin binding protein sp32 (OY-TES1), p53, p53 mutant, 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 cell, prostein, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), proteasome (prosome, macropain) subunit, beta type, receptor for advanced glycation end products (RAG E-1), RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), kidney ubiquitous 1 (RU1), kidney ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), stage-specific embryonic antigen 4- (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative reading 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, TnAg binds to 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 X antigen family, member 1A (XAGE1). Amino acid sequences that specifically bind to the antigen are known in the art or can be prepared using methods known in the art, and include, for example, immunoglobulins, variable regions of immunoglobulins (e.g., variable fragments ("Fv") or bivalent variable fragments ("Fab")), single-chain antibodies, and the like. 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) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of an avimer.

[0028] The terms "variable region" or "variable domain" are used interchangeably. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino-terminal 110-120 amino acids of the mature heavy chain and about 90-115 amino acids of the mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FR).

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

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

[0031] Many definitions of CDRs are in common use: 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 an analysis of available complex crystal structures.

[0032] "Antigen" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in humans or animals, including compositions (such as those containing tumor-specific proteins) that are injected or absorbed into humans or animals. Antigens react with the products of specific humoral or cellular immunity, including those elicited 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 to which the binding domain of a TCR or CAR contemplated herein is designed to bind. Those skilled in the art will readily appreciate 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 recombinantly expressed. Antigens may be specific to a particular tissue, such as cancer cells, or may be broadly expressed. Additionally, fragments of larger molecules may act as antigens. In one embodiment, the antigen is a tumor antigen. In a particular embodiment, the antigen is 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A-kinase anchoring 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 (an oncogene fusion protein consisting of the breakpoint cluster region (BCR) and the Abelson murine leukemia viral 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, 9-0-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 (also called CD2 subset 1, 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), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (EL F2M), endosialin, 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), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor alpha, folate receptor beta, 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, hexasaccharide moiety of GloboH glycoceramide (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 cellular 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 scatter 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 lRa), 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, kappa light chain, KCS1, kinase insert 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), legumain, 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, ​​locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGEA3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory factor (MIS) receptor type II, neuroblastoma-derived homolog of the v-myc avian myelocytomatosis viral oncogene (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, oncofetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin binding protein sp32 (OY-TES1), p53, p53 mutant, 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 cell, prostein, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), proteasome (prosome, macropain) subunit, beta type, receptor for advanced glycation end products (RAG E-1), RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), kidney ubiquitous 1 (RU1), kidney ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), stage-specific embryonic antigen 4- (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative reading 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, TnAg, 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 all or a fragment of vimentin, Wilms tumor protein (WT1), or X antigen family, member 1A (XAGE1). A "target" is any molecule bound by a binding motif, antigen binding system, CAR, or antigen binder, e.g., an antibody.

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

[0034] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to contain a binding motif and a means to activate an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination 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 comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells engineered to express a chimeric antigen receptor may be referred to as CAR T cells. "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that is understood to reside outside the cell membrane, in the extracellular space, when the polypeptide resides in the cell membrane.

[0035] The term "extracellular ligand-binding domain" as used herein refers to a ligand, e.g., an oligo- or polypeptide, capable of binding to a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that may act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0036] The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that moves the antigen binding domain away from the effector cell surface to allow for 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 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 the 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 anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. The transmembrane domain can be a CD3 zeta transmembrane domain, however, other transmembrane domains that can be used include those obtained 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 transmembrane domain of CD137. In certain embodiments, the transmembrane domain is synthetic, in which case it contains primarily 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 transducing the message of an effective CAR binding to a target antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to a CAR-bound target cell, or other cellular responses elicited by antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, aid or activity, including cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling domain" or "signaling domain", as used interchangeably herein, refers to a portion of a protein that transduces an effector function signal and directs a cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domains, also known as "signal transduction domains," are typically derived from portions of the human CD3 or FcRy chain.

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

[0040] Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present 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 term "costimulatory signaling domain" or "costimulatory domain" refers to a 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 an Fc receptor that provides a second signal required for 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), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Thus, although the present disclosure provides exemplary costimulatory domains derived from CD3 zeta and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and proliferation of T cells expressing a CAR receptor. The intracellular signaling and costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0042] Although scFv-based CARs engineered to contain signaling domains from CD3 or FcR gamma 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 costimulatory signals. Other CARs containing binding domains, hinges, transmembrane and signaling domains derived from CD3 zeta or FcR gamma, together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134 and CD278), can more effectively direct anti-tumor activity and increase cytokine secretion, lytic activity, 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] A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, including, but not limited to, proliferation and / or up-regulation or down-regulation of key molecules.

[0044] "Costimulatory ligands" include molecules on antigen-presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of a costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, and differentiation. Costimulatory ligands induce signals, for example, by binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules, in addition to the primary signal provided by the stimulatory molecule. Examples of costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to the Toll ligand receptor, 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), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MHC class I chain-related protein B), and the like. B, MICB), OX40 ligand, PD-L2, or programmed death (PD) L1.Examples of costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to 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 cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, a "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.Costimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, 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, CDS, 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 la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[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 have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar 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 a framework region of the antibody or antigen-binding molecule thereof can be replaced with an amino acid residue having a similar side chain. In general, two sequences are generally considered to be "substantially similar" if they contain conservative 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 "non-polar" side chains. Substitution of another amino acid of the same type may be considered a conservative substitution. Exemplary amino acid classifications are summarized in Table 1 below. TIFF2025507619000001.tif137128

[0047] "T cell receptor" or "TCR" refers to an antigen-recognition molecule present on the surface of a T cell. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to give rise to a wide variety of TCR proteins.

[0048] The term "heterologous" means derived from any source other than the naturally occurring sequence. For example, the heterologous sequence included as part of the costimulatory protein is an amino acid that does not naturally occur as a wild-type human costimulatory protein, i.e., does not align with the wild-type human costimulatory protein. For example, a heterologous 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 relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods are known for calculating the percent identity between two provided polypeptide sequences. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then 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 percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account, optionally, the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0050] T cells for immunotherapy can 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 (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Additionally, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Further methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0051] A "patient" includes any human 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 a formally diagnosed disorder, subjects without a formally recognized disorder, subjects receiving medical attention, and subjects at risk for developing a disorder.

[0053] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or the benefits to that 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 not harmful to the recipient or the benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in the transport or transportation of a drug from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to the patient or the benefits to the recipient must outweigh any harmful effects. Some examples of materials which may function as pharma- ceutically 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 powdered tragacanth, malt, gelatin, talc, cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as 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 compatible 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 pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, the pharmaceutical compositions may be formulated for administration in solid or liquid form, including, but not limited to, forms adapted for oral administration, e.g., liquids (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue, parenteral administration, e.g., as sterile solutions or suspensions, or as 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, vaginally or rectally, e.g., as a pessary, cream, or foam, sublingually, ophthalmically, transdermally, or intranasally, to the lungs, and other mucosal surfaces.

[0055] The terms "reduce" and "decrease" are used interchangeably herein and refer to any change that is less than original. "Reduce" and "reduce" are relative terms, requiring a comparison of a before and after measurement. "Reduce" and "reduce" include complete depletion.

[0056] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, 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 historical reference or control, 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 similarity is sufficient to justify reliance on and / or comparison to the selected reference or control.

[0057] "Regulatory T cells" ("Treg", "Treg cells", or "Tregs") refer to a lineage of CD4+ T lymphocytes involved in controlling certain immune activities, such as responses to autoimmunity, allergy, and infection. Regulatory T cells can modulate the activity of T cell populations and can also affect certain innate immune system cell types. Tregs can be identified by expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically constitute approximately 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] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, e.g., an engineered CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability due to disease affliction. The ability of a therapeutic agent to promote regression of a disease can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or 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, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0060] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject or administration of an active agent to a subject with the goal of reversing, mitigating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical manifestation associated with a 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 treatment of a subject who does not show signs of the associated disease, disorder and / or condition and / or who shows only early signs of the disease, disorder and / or condition. In some embodiments, such treatment may be treatment of a subject who shows one or more definitive signs of the associated disease, disorder and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed as suffering from the associated disease, disorder and / or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated 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 replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors contain sequences that direct the expression of inserted genes to which they are operably linked. Such vectors may be referred to herein as "expression vectors." Standard techniques can be used for the manipulation of vectors, for example, as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d 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 a domain in a larger protein, that binds to DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through the coordination of a zinc ion. Thus, each zinc finger ZFP of a multi-finger contains a recognition helix region for binding to DNA within the scaffold. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. The term "zinc finger nuclease" includes a ZFN and a pair of ZFNs (the members of the pair are referred to as "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 that contains one or more TALE repeat domains / units. The repeat domains, each containing a repeat variable diresidue (RVD), are responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also called a "repeat") is typically 33-35 amino acids long and exhibits at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. TALE proteins can be designed to bind to target sites using canonical or non-canonical RVDs in the repeat unit. See, for example, U.S. Patent Nos. 8,586,526 and 9,458,205. Zinc finger and TALE DNA binding domains can be "engineered" to bind to a given nucleotide sequence, for example, via manipulation of the recognition helix region of a naturally occurring zinc finger protein (modification of one or more amino acids) or by manipulation of amino acids involved in DNA binding (the repeat variable diresidue or RVD region). Thus, engineered zinc finger proteins or TALE proteins are non-naturally occurring proteins. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. Designed proteins are non-naturally occurring proteins whose design / composition arises primarily from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms to process 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. Pat. Nos. 9,458,205, 8,586,526, 6,140,081, 6,453,242, and 6,534,261, and also WO 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536, and 03 / 016496. The term "TALEN" includes single TALENs and pairs of TALENs (wherein the members of the pair are referred to as "left and right" or "first and second" or a "pair") that dimerize to cleave a target gene.

[0064] The CRISPR / Cas (Clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) system has been considered the most powerful genome editing tool due to its unparalleled editing efficiency, convenience, and potential applications in living organisms. Directed by guide RNA (gRNA), Cas nucleases can generate DNA double-strand breaks (DSBs) at targeted genomic sites in various cells (both cell lines and cells from living organisms). These DSBs are then repaired by endogenous DNA repair systems that can be utilized to perform the desired genome editing.

[0065] Base editors (BEs) that integrate the CRISPR / Cas system with the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) cytosine deaminase family have been recently developed, which have significantly enhanced the efficiency of CRISPR / Cas9-mediated gene correction. Through fusion with Cas9 nickase (nCas9) or catalytically inactive Cas9 (dCas9), the cytosine (C) deamination activity of rat APOBEC1 (rA1) can be deliberately directed to target bases in the genome and catalyze the substitution of C to thymine (T) at these bases.

[0066] Prime editing (PE) is a genome editing technique that can modify the genome of an organism. Prime editing writes new genetic information directly into 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 a prime editing guide RNA (pegRNA) that can identify the target site and provide new genetic information to replace the target DNA nucleotide. Prime editing mediates targeted insertions, deletions, and base-to-base conversions without the need for double-strand breaks (DSBs) or donor DNA templates. RFX5 knockout cells

[0067] Allogeneic donor cells from healthy donors have the potential to provide ready-made cell products that can be applied as needed at a much lower cost than autologous cells. With the advancement of gene editing technology, attempts are being made to knock out or knock out specific genes to develop low immunogenic cells suitable for ready-made use.

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

[0069] The inventors developed an allogeneic anti-CD19 CAR T cell product in which portions of both the TCR alpha constant (TRAC) locus and B2M were deleted by zinc finger nucleases (ZFNs), resulting in reduced expression of the proteins on the cell surface. However, it was observed that these edited CAR T cells were sensitive to host NK cells, as they became stimulated and killed allogeneic T cells lacking MHC class I expression.

[0070] Therefore, the inventors searched for alternative gene editing approaches that are superior to B2M knockout and can achieve minimal or no risk of GVHD, and minimal or no risk of CD4, CD8 and NK cell rejection while retaining comparable or even improved therapeutic activity.

[0071] Thus, according to one embodiment of the present disclosure, allogeneic cells are provided that are genetically engineered to reduce MHC class I or MHC class II expression or activity. In some embodiments, MHC class I expression or activity is reduced, but not eliminated. In some embodiments, MHC class II expression and / or activity 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., a cell that has not been so engineered, e.g., a TRAC knockout only T cell). In one embodiment, MHC class I expression or activity is retained at a level of 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, MHC class I expression or activity is about 5% to 90%, 10% to 80%, 20% to 80%, 20% to 70%, 30% to 70%, 30% to 60%, 40% to 60%, 10% to 60%, 10% to 50%, 20% to 60%, 20% to 50%, 20% to 40%, 10% to 40%, or 10% to 30% compared to a reference allogeneic cell.

[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 a reference allogeneic cell. In one embodiment, MHC class II expression or activity is retained at a level of 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, MHC class II expression or activity is about 5% to 90%, 10% to 80%, 20% to 80%, 20% to 70%, 30% to 70%, 30% to 60%, 40% to 60%, 10% to 60%, 10% to 50%, 20% to 60%, 20% to 50%, 20% to 40%, 10% to 40%, or 10% to 30% compared to a reference allogeneic cell.

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

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

[0076] In some embodiments, MHC class I expression or activity is reduced or knocked down, while MHC class II expression or activity 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, genes associated with B2M, individual HLA molecules (e.g., HLA-A, -B, -C, -E, -G), TAP1, TAP2, and / or bare lymphocyte 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), genes associated with BLS II, and / or individual HLA molecules (e.g., HLA-DP, -DQ, -DR, -DiVI, -DO-alpha and beta chains).

[0079] Multiple candidate genes were investigated, including single knockouts of RFX5 (regulatory factor X5), TAP1 (transporter associated with antigen processing 1), TAP2 (antigen peptide transporter 2), and CIITA (class II, major histocompatibility complex, transactivator). It was discovered that RFX5 single 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 mismatched CD8 T cells, but did not promote killing by NK cells. Elimination of MHC class II expression also reduces responses by mismatched CD4 T cells. Importantly, CAR cells with RFX5 knockout had superior manufacturability and CAR functionality, as demonstrated in the experimental examples.

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

[0081] In some embodiments, the gene is selected from RFX5 (regulatory factor X5), TAP1 (transporter associated with antigen processing 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 are also fully or partially inactivated or inhibited. In some embodiments, only the expression or activity of RFX5 is reduced in the cell and no genetic changes are made to TAP1, TAP2, and CIITA.

[0082] In some embodiments, the endogenous gene for B2M (beta-2-microglobulin) in the cell is not manipulated, i.e., no gene editing is performed on the B2M locus and no inhibitors are introduced into the cell.

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

[0084] In some embodiments, the cells are derived from a healthy donor. In some embodiments, the cells are derived / 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. Techniques 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 instances, detectable RFX5 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% (e.g., reduced by 40%-90%, 40%-80%, or 50%-95%) compared to a control (the amount of RFX5 detected in a corresponding cell in which RFX5 is not inhibited).

[0087] In certain embodiments, 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% (e.g., reduced by 40%-90%, 40%-80%, or 50%-95%) compared to a control (the amount of TCR detected in a corresponding cell in which the TCR is not inhibited).

[0088] In certain embodiments, detectable B2M 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% (e.g., reduced by 40%-90%, 40%-80%, or 50%-95%) compared to a control (the amount of B2M detected in a corresponding cell in which B2M is not inhibited).

[0089] In certain embodiments, the reduction or elimination of gene expression occurs through direct inhibition of the gene (e.g., 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 occurs through indirect inhibition of the gene (e.g., knocking down or knocking out the RFX5 gene may reduce the expression or activity of an MHC class I molecule).

[0090] Percentage reduction and percent increase can be calculated by methods known in the art. As a non-limiting example, the percent reduction or decrease in expression or activity of a molecule in an edited cell (e.g., a cell containing RFX5 KO) relative to a reference or corresponding cell (e.g., a cell not containing RFX5 KO) can be calculated by subtracting the edited cell value from the reference / corresponding cell value, dividing the amount by the reference value, and then multiplying by 100 to obtain the percent reduction. If the percentage is negative, it may mean that there has been an increase rather than a decrease.

[0091] In some embodiments, gene expression or activity can be reduced using a suitable inhibitor, such as a small molecule inhibitor, an inhibitory RNA (e.g., siRNA, shRNA), or an antibody. 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 of RFX5 is reduced using, for example, a DNA binding domain coupled to a nuclease domain that specifically binds to a target site within the RFX5 gene and mediates a mutation at the target site, thereby reducing expression of functional RFX5. Any DNA binding domain can be used in the compositions and methods disclosed herein, including, but not limited to, a zinc finger DNA binding domain, a TALE DNA binding domain, a DNA binding portion of a CRISPR / Cas nuclease (sgRNA), or a DNA binding domain from a meganuclease.

[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 is 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. Engineering methods include, but are 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 containing three fingers typically recognize target sites containing 9 or 10 nucleotides. ZFPs containing four fingers typically recognize target sites containing 12-14 nucleotides, while ZFPs with six fingers can recognize target sites containing 18-21 nucleotides. ZFPs can also be fusion proteins containing one or more regulatory domains, which can be transcriptional activation domains or transcriptional repression domains.

[0095] In some embodiments, the DNA binding domain may be derived from a nuclease. For example, the recognition sequences of homing endonucleases and meganucleases are known, 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. Furthermore, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites.

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

[0097] In certain embodiments, the DNA binding domain is part of a CRISPR / Cas nuclease system that includes a single guide RNA (sgRNA) that binds to DNA. The CRISPR (clustered regularly interspaced short palindromic repeats) locus that encodes the RNA components of the system, and the cas (CRISPR-associated) locus that encodes proteins, constitute the genetic sequence of the CRISPR / Cas nuclease system. The CRISPR locus in a microbial host contains a combination of CRISPR-associated (Cas) genes and non-coding RNA elements 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 the present disclosure can be identified using CRISPR design tools. Exemplary sgRNA sequences are shown in Table 2. TIFF2025507619000002.tif75144

[0099] Type II CRISPR is one of the best-characterized systems and performs 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, tracrRNA hybridizes to the repeat region of pre-crRNA and mediates the processing of pre-crRNA into 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 next to the protospacer adjacent motif (PAM) that is further required for target recognition. Finally, Cas9 mediates the cleavage of the target DNA resulting in a double-strand break within the protospacer. The action of the CRISPR / Cas system involves 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 the associated proteins, and expression and processing of the array, followed by (iii) RNA-mediated interference with the foreign nucleic acid. Thus, in bacterial cells, several of the so-called "Cas" proteins are involved in the natural function of the CRISPR / Cas system and play a role in functions such as insertion of foreign DNA, etc.

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

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

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

[0103] In some embodiments, a CAR comprises an extracellular binding domain, including but not limited to, an extracellular domain of an antibody or antigen-binding fragment thereof, a tethered ligand, or a co-receptor, that specifically binds to a target antigen.

[0104] By way of non-limiting example, target antigens may include: HPV oncoproteins, including HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, alpha 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, 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, mesothelin, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TAG72, TEM, and VEGFRII, one or more hinge 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 a primary signaling domain 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 particular embodiment, the CAR binds to CD19. In a particular embodiment, the CAR binds to CD20. In a particular embodiment, the CAR comprises a first scFv that binds to CD19 and a second scFv that binds to CD20. Examples of CD19 or CD20 binding sequences are listed in Table 3. TIFF2025507619000003.tif221170TIFF2025507619000004.tif193170TIFF20255076190 00005.tif155170TIFF2025507619000006.tif253170TIFF2025507619000007.tif229170

[0105] In various embodiments, the binding motif may comprise a heavy chain variable domain of the disclosure (e.g., having at least 75% sequence identity to a heavy chain variable domain 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 a light chain variable domain 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., see Whitlow et al. Protein Eng. November 1993, 6(8):989-95). In various embodiments, the binding motif may include a leader or signal sequence, a heavy chain variable domain of the present disclosure (e.g., having at least 75% sequence identity to a heavy chain variable domain 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 present disclosure (e.g., having at least 75% sequence identity to a light chain variable domain 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] When an amino acid or nucleotide sequence of a binding motif comprising a heavy chain variable domain of the present disclosure and a light chain variable domain of the present disclosure is provided, the linker linking the two variable domains will be apparent from the sequence in view of this disclosure and the knowledge in the art. When an amino acid or nucleotide sequence of a binding motif comprising a heavy chain variable domain of the present disclosure and a light chain variable domain of the present disclosure is provided, the leader sequence will be apparent in view of this disclosure and the knowledge in the art. For the avoidance of doubt, the heavy chain variable domain and the light chain variable domain of the present disclosure may be present in any orientation, for example, an orientation in which the heavy chain variable domain is C-terminal to the light chain variable domain, or an orientation in which the heavy chain variable domain is N-terminal to the light chain variable domain. Exemplary anti-CD20 binding motifs are shown in Table 4. TIFF2025507619000008.tif208170TIFF2025507619000009.tif208170TIFF2025507619000010.tif221170TIFF2025507619000011.tif82170

[0107] Exemplary nucleotide sequences encoding the binding motif, hinge, and costimulatory domains are provided in Table 5. TIFF2025507619000012.tif255170TIFF2025507619000013.tif249170TIFF2025507619000014.tif249170 TIFF2025507619000015.tif245170TIFF2025507619000016.tif249170TIFF2025507619000017.tif233170 TIFF2025507619000018.tif233170TIFF2025507619000019.tif241170TIFF2025507619000020.tif237170 TIFF2025507619000021.tif233170TIFF2025507619000022.tif249170TIFF2025507619000023.tif249170

[0108] The hinge may be derived from natural or synthetic sources. In some embodiments, the antigen binding system of the present disclosure may include a hinge that is, from, or derived from (e.g., including 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 costimulator (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, activating NK cell receptor, or Toll ligand receptor, or a fragment or combination thereof. In certain embodiments, the CAR does not comprise a CD28 hinge.

[0109] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains include the alpha, beta, or zeta chains of the T cell receptor, 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, activated NK cell receptor 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 and ligands that bind to 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), signaling lymphocyte activation molecule (SLAM) protein, SLAM (SLAMF1, CD150;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, truncations, or combinations thereof (e.g., may include at least the transmembrane domain). In some embodiments, the transmembrane domain may be synthetic (and may include primarily hydrophobic residues, such as, for example, leucine and valine). In some embodiments, triplets of phenylalanine, tryptophan, and valine are included at both ends of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked or connected to the cytoplasmic domain. In some embodiments, a short oligo- or polypeptide linker (e.g., 2-10 amino acids in length) may form the link 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 that contain 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, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)).

[0111] CARs can include costimulatory signaling domains, for example, to enhance signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), 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 through the TCR alone may be insufficient for full activation of T cells, and secondary or costimulatory signals may increase activation. Thus, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., costimulatory signaling domains) that activate one or more immune cell effector functions (e.g., natural immune cell effector functions described herein). In some embodiments, a portion of such a costimulatory signaling domain may be used so long as that portion transduces an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or a fragment thereof). Non-limiting examples of such T cell co-receptors include 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 a ligand that binds CD83.

[0112] In certain embodiments, CARs contemplated herein may include linker residues between various domains, e.g., between the VH and VL domains, that are added for proper spacing conformation of the molecule. CARs contemplated herein may include one, two, three, four, or more linkers. In some embodiments, the linker is about 1 to about 25 amino acids in length, about 5 to about 20 amino acids, or about 10 to about 20 amino acids in length, or any intervening length of amino acids. In some embodiments, the linker is 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 amino acids in length.

[0113] In some embodiments, a CAR contemplated herein comprises an intracellular signaling domain. An "intracellular signaling domain" refers to a portion of a CAR that is involved in transducing an effective CAR message binding to a target antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors into a CAR-bound target cell or other cellular responses elicited by antigen binding to the extracellular CAR domain). In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs that contain 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, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)).In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, 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, 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, ligand binding to LFA-1, CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0114] Components of a CAR can be exchanged or "swapped" for equivalent components using routine techniques of biotechnology. In various embodiments, the disclosure provides a binding motif according to any one of SEQ ID NOs: 31-40, or a binding motif according to a sequence having at least 75% identity (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) to any one of SEQ ID NOs: 31-40, in combination (e.g., contiguously fused) with a hinge, and optionally further combined (e.g., contiguously fused) with a costimulatory domain. A few non-limiting examples thereof are provided in SEQ ID NOs: 42-64, or a sequence having at least 75% identity to any one of SEQ ID NOs: 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] A bicistronic CAR may comprise a first CAR sequence and a second CAR sequence, and is expressed as a single polypeptide comprising a cleavable linker between the first and second CAR. Examples of cleavable linkers include, but are not limited to, Furin-GSG-T2A (see, e.g., Chng et al. MAbs. 2015 Mar-Apr, 7(2):403-412 (incorporated herein by reference for cleavable linkers), see also Guedan et al. Mol Ther Methods Clin Dev. 2019 Mar 15, 12:145-156 (incorporated herein by reference for bicistronic CAR design)), 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, a porcine teschovirus (P2A), a CHYSEL sequence of a virus (T2A), or combinations, variants, and functional equivalents thereof.

[0116] Exemplary anti-CD20 / anti-CD19 bicistronic CARs may have or include the nucleotide and amino acid sequences set forth 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 can be a single polypeptide comprising a first binding motif and a second binding motif. An exemplary anti-CD20 / anti-CD19 bispecific CAR can have or comprise the nucleotide and amino acid sequences set forth in SEQ ID NOs:29 and 27, respectively, or a sequence having at least 75% identity (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) to SEQ ID NOs:29 and 27.

[0118] The present disclosure provides, inter alia, bispecific antibodies that bind CD20 and a second target antigen, e.g., CD19. The bispecific antibodies include antibodies having a first binding motif that binds a first target antigen and a second binding motif that binds a second target antigen. In some embodiments, the bispecific antibodies include an anti-CD20 binding motif of the present disclosure and an anti-CD19 binding motif of the present disclosure. In some embodiments, the bispecific antibodies include an anti-CD20 binding motif that includes an anti-CD20 heavy chain variable domain of the present disclosure and an anti-CD20 light chain variable domain of the present disclosure, and an anti-CD19 binding motif that includes an anti-CD19 heavy chain variable domain and an anti-CD19 light chain variable domain.

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

[0120] The present disclosure includes vectors comprising a nucleic acid of the present disclosure and / or vectors encoding a polypeptide of the present disclosure. In various embodiments, the present disclosure includes vectors comprising a nucleic acid encoding an anti-CD20 binding motif and / or an anti-CD19 binding motif provided herein. In various embodiments, the present disclosure includes vectors comprising a nucleic acid encoding an antibody provided herein, including but not limited to a nucleic acid encoding a binding motif molecule (e.g., an anti-CD20 binding motif or an anti-CD19 binding motif). In various embodiments, the present disclosure includes vectors comprising a nucleic acid encoding one or more antigen binding systems provided herein, including but not limited to a nucleic acid encoding a bicistronic or bispecific chimeric antigen receptor (e.g., a bicistronic and bispecific chimeric antigen receptor that binds CD20 and CD19).

[0121] In certain embodiments, the CAR of the present disclosure binds to CLL-1. Examples of CLL-1 CAR amino acid sequences are listed in Table 6. TIFF2025507619000024.tif250170TIFF2025507619000025.tif255169TIFF2025507619000026.tif250170

[0122] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence that is 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, a CAR or TCR of the present disclosure can 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 specialized function of a cell. Effector functions of T cells can be, for example, aiding or activities including cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and instructs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the 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). Libraries of TCRs can be screened for their selectivity for target antigens. In this way, native TCRs with high avidity and reactivity to target antigens can be selected, cloned, and subsequently introduced into T cell populations used for adoptive immunotherapy.

[0126] In one embodiment described herein, T cells are modified by introducing a polynucleotide encoding a subunit of a TCR capable of forming a TCR that confers specificity to the T cell for tumor cells expressing a target antigen. In some embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, so long as the subunit retains the ability to form a TCR that confers the transfected T cell the ability to home to the target cell and participates in immunologically relevant cytokine signaling. The TCR may also bind with high avidity to target cells displaying the relevant tumor associated peptide, and optionally mediate efficient killing of target cells presenting the relevant peptide in vivo.

[0127] The nucleic acid encoding the TCR can be isolated from its natural context in the (naturally occurring) chromosome of the T cell and can be incorporated into a suitable vector as described elsewhere herein. Both the nucleic acid and the vector containing them can be transferred into a cell, which can be a T cell. The modified T cell can then express one or more chains (and in some aspects two chains) of the TCR encoded by the transduced nucleic acid or nucleic acid. In some embodiments, the TCR is an exogenous TCR, since it is introduced into a T cell that does not normally express the introduced TCR. An aspect of the TCR is that it has high avidity for tumor antigens presented by the major histocompatibility complex (MHC) or similar immunological components. In contrast to the TCR, the CAR is engineered to bind to the target antigen in an MHC-independent manner.

[0128] The proteins encoded by the nucleic acids described herein can be expressed with additional polypeptides linked to the amino- or carboxyl-terminal portions of the α or β chains of the TCR, so long as the additional attached polypeptides do not interfere with the ability of the α or β chains to form a functional T cell receptor and MHC-dependent antigen recognition.

[0129] Antigens recognized by the TCRs contemplated herein include, but are not limited to, cancer antigens, including antigens of both hematological and solid tumor and virally induced cancers. Other exemplary antigens include HPV oncoproteins, including HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, alpha 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, These 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, mesothelin, Mucl, Muc16, NCAM, NKG2D ligand, NY-ES0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TAG72, TEM, and VEGFRII.

[0130] In some embodiments, a polynucleotide encoding a CAR or TCR is introduced into a cell after the cell has been engineered to reduce MHC class I and / or II expression or activity. In some embodiments, a polynucleotide encoding a CAR or TCR is introduced into a cell before the cell has been engineered to reduce MHC class I and / or II expression or activity. In some embodiments, the two rounds of engineering are performed at least one day apart (not on the same day or within 24 hours). treatment

[0131] The cells, e.g., allogeneic cells, of the present disclosure can be used to treat a variety of diseases and conditions, particularly cancer. In one embodiment, the 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's lymphoma, non-Hodgkin's lymphoma, and bladder cancer.

[0132] In some embodiments, the cells of the present disclosure may be used to treat bone marrow disorders including, but not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndromes (MDS), myeloproliferative disorders, myeloid neoplasms, myeloid sarcomas), blastic plasmacytoid dendritic cell neoplasms (BPDCN), or combinations thereof. Additional disorders include inflammatory and / or autoimmune disorders 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 lymphomas. In some embodiments, the cells of the disclosure may be used to treat diffuse large B cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B cell lymphoma, high-grade B cell lymphoma, and DLBCL arising from 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, e.g., a therapeutically effective amount, of a composition comprising the cells of the present disclosure. The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, and the appropriate dosage 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 the CAR or TCR molecule, such as CLL-1.

[0136] In other embodiments, methods are provided that include administering a therapeutically effective amount of the 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 to treat a patient at risk of developing cancer. Thus, the present disclosure provides a method for the treatment or prevention of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the modified T cells of the present disclosure.

[0137] CAR + / CAR-T + / TCR + The target dose of cells was approximately 1 × 10 6 ~about 2×10 10 Cells / kg (e.g., approximately 1 x 10 6 cells / kg, approximately 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 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 x 10 10 It will be understood that the dose may be in the range of 100 mg / kg (cells / kg). It will be understood that doses above and below this range may be appropriate for a particular subject.

[0138] Those skilled in the art will recognize that multiple administrations of the disclosed compositions may be required to affect the desired treatment. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

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

[0140] Methods for administering the cell compositions described herein include any method that is effective to result 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 ex vivo with a nucleic acid construct according to the present disclosure and returning the transduced cells to the subject.

[0141] Another embodiment described herein is a method of contacting a cancer cell with an engineered immune cell of the present disclosure, whereby cancer cell proliferation is inhibited or reduced.

[0142] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present 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 by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce similar results.

[0143] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present disclosure. In the event that any definitions or terms provided in a reference incorporated by reference differ from the terms and explanations provided herein, the terms and definitions shall control. The contents of all references cited throughout this application are expressly incorporated herein by reference. EXAMPLES

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

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

[0146] Methods: Healthy donor T cells were transduced (using a lentiviral vector (LLV)) with a CD19 / CD20 bicistronic CAR followed by electroporation of ZFNs targeting TRAC (T cell receptor alpha constant region) and RFX5. Edited and non-edited CAR T cells were expanded for 14 days and examined for expression of CAR, TCRαβ, MHC class I, and MHC class II on days 7 and 10. The majority of cells were frozen on day 10 for all functional and alloreactivity assays, while a small portion of cells were persisted until day 14 to confirm cell viability and maintained proliferation in similar edited cell conditions as non-edited cells throughout production. CAR functionality was assessed by co-culturing various E:T ratios of CAR-T cells with a CD19 / CD20 positive tumor target cell line (Raji WT) for cytotoxicity (24 and 48 h), proliferation (120 h), and cytokine secretion (24 h). CAR-T persistence and cytotoxic function were assessed by continuous stimulation of CAR-T cells with CD19 / CD20 positive Raji MHC KO tumor cells at an E:T ratio of 1:1 every 3-4 days. Furthermore, killing of edited and non-edited CAR T cells by MHC-mismatched CD8 T cells and NK cells was measured.

[0147] Results: RFX5 KO CAR-T cells had similar manufacturability and CAR functionality as β2M KO CAR-T cells. As shown in Table 7A-C, RFX5 knockout (KO) allogeneic (Allo) CAR-T cells had better viability and similar proliferation rates to B2M KO Allo CAR-T cells. TIFF2025507619000027.tif45128TIFF2025507619000028.tif37128TIFF2025507619000029.tif52128

[0148] Furthermore, as shown in Table 8, RFX5 or B2M KO did not affect the expression of exogenous CAR molecules. TIFF2025507619000030.tif101152

[0149] Tables 9A-C show that CD19 / CD20 B2M KO and RFX5 KO CAR-T had similar activity. TIFF2025507619000031.tif34169TIFF2025507619000032.tif28170TIFF2025507619 000033.tif23128TIFF2025507619000034.tif28168TIFF2025507619000035.tif23128

[0150] RFX5 KO CAR T cells reduced HLA class I expression with similar MFI as β2M KO CAR T cells, which reduced the host alloreactive response (Table 10). The table shows that RFX5 KO resulted in MHC class I knockdown (downregulation), whereas B2M KO resulted in MHC class I knockout (elimination). Meanwhile, similar TCR KO was observed in both TRAC+B2M KO 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 only observed in TRAC+RFX5 KO cells. TIFF2025507619000036.tif85128TIFF2025507619000037.tif16148

[0151] Knockout of RFX5 enhanced protection from host NK killing compared to knockout of β2M and reduced rejection from mismatched host CD8 T cells compared to non-edited CAR-T cells (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 levels similar to B2M KO in 1 / 3 mismatched donors and reduced CD8 rejection compared to unedited cells in 2 / 3 mismatched donors. RFX5 KO cells showed an absence of MHC class II expression (Table 10), which is predicted to minimize responses by host CD4 T cells. TIFF2025507619000038.tif48146TIFF2025507619000039.tif41170

[0152] This example demonstrated that deletion of the transcription factor RFX5 allowed the generation of CAR-T cells with low levels and the absence of MHC class I and MHC class II expression, leading to reduced killing by MHC-mismatched CD8 T cells and NK cells. Example 2. Clinical Trial of Allogeneic TRAC-RFX5 Anti-CD19 / CD20 CAR

[0153] A healthy donor-derived allogeneic (HD Allo) product directed against CD19 and CD20 is developed to treat cancer regardless of the patient's HLA type, utilizing zinc finger nucleases (ZFNs) to knock out the gene. The product contains healthy donor-derived T cells directed against CD19 and CD20. ZFNs are used to disrupt the TRAC gene to address the risk of graft-versus-host disease (GVHD) and to disrupt the RFX5 gene to reduce expression of MHC class I and II to minimize host rejection.

[0154] Alternatively, the TRAC and RFX5 genes 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) a similar or improved in vivo activity profile compared to allogeneic anti-CD19 / CD20 CAR with disruption of the TRAC and B2M loci (allogeneic TRAC-B2M CD19 / CD20) and conventional non-gene-edited anti-CD19 CAR T cells, (ii) minimized to zero risk of GVHD, and (iii) minimized risk of rejection by the patient's endogenous CD4 and CD8 T cells and NK cells as demonstrated using in vitro assays. It is expected that the following objectives will be achieved: - Similar or improved in vivo activity compared to allogeneic TRAC / B2M KO CD19 / CD20 CAR-T cells, as evidenced by tumor cell clearance; - Comparable or improved CAR expression and cell proliferation kinetics (in vivo and in manufacturing) compared to allogeneic TRAC-B2M KO CD19 / CD20 CAR-T cells; - Reduced in vitro mismatched NK cell rejection as evidenced by reduced NK cell killing of allogeneic CAR T cells compared to allogeneic TRAC-B2M KO CD19 / CD20 CAR-T cells; and - Reduced in vitro mismatched CD4 T cell recognition and CD8 T cell rejection compared to non-edited 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, the in vitro efficacy of a research-grade Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR / Cas9) ribonucleoprotein targeted to a deletion of the RFX5 gene and its ability to protect against T cell- and NK cell-mediated rejection when used in an allogeneic (MHC-mismatched) CAR-T cell product was evaluated.

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

[0158] Results: RFX5 KO CAR-T cells had similar manufacturability and CAR functionality as β2M KO CAR-T cells. As shown in Table 12A-C, RFX5 knockout (KO) allogeneic (Allo) CAR-T cells had better viability and similar proliferation rates to B2M KO Allo CAR-T cells. TIFF2025507619000040.tif35128TIFF2025507619000041.tif37128TIFF2025507619000042.tif42128

[0159] Furthermore, as shown in Table 13, RFX5 or B2M KO did not affect the expression of exogenous CAR molecules. TIFF2025507619000043.tif34139

[0160] Table 14 shows that RFX5 KO resulted in MHC class I knockdown (downregulation). Meanwhile, similar TCR KO was observed in both TRAC+B2M KO cells and TRAC+RFX5 KO cells. However, efficient MHC class II KO was only observed in TRAC+RFX5 KO cells. TIFF2025507619000044.tif85128

[0161] Knockout of RFX5 enhanced protection from host NK killing compared to knockout of β2M (Table 15). Table 15: RFX5 KO improved protection from NK compared to B2M KO in 3 out of 3 donors. TIFF2025507619000045.tif40170

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

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

[0164] Methods: RFX5 KO CAR-T cells and matched controls generated by the methods described in Examples 1 and 3 are used for in vivo studies. Female NSG mice aged 6-7 weeks (5 mice / group) are evaluated. Two doses of CAR+T cells are tested: 1x10 6 Or 2 x 10 6 of CAR+ T cells / mouse and 5 × 10 6 Or 10 x 10 6 CAR+ T cells / mouse. Tumor cells expressing the relevant antigen are implanted intravenously into 6-7 week old female NSG mice via the lateral tail vein.

[0165] After tumor implantation, the following groups of B cell lymphoma or AML tumor-bearing mice are evaluated: (1) no treatment, (2) treatment with non-transduced (NTD) T cells, (3) treatment with non-edited 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 the T cell product on tumor burden by bioluminescence (bioluminescence positively correlates with tumor burden) and animal survival. RFX5 KO CAR-T cell proliferation kinetics 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 a number of embodiments have been described, it is apparent that the disclosure and examples may provide other embodiments that utilize or are encompassed by the compositions and methods described herein. It will therefore be appreciated that the scope of the invention should be defined not by the embodiments represented by way of example, but by what can be understood from this disclosure and the appended claims.

Claims

1. An isolated human immune cell engineered to have (i) 10% to 80% less MHC class I activity or expression compared to a reference cell, (ii) at least 75% less MHC class II activity or expression compared to a reference cell, and (iii) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

2. The immune cell of claim 1 , which is a T cell or a NK cell.

3. 2. The immune cell of any one of the preceding claims, wherein the endogenous genes RFX5 (regulatory factor X5), TAP1 (transporter associated with antigen processing 1), TAP2 (antigen peptide transporter 2) or CIITA (class II, major histocompatibility complex, transactivator) are inactivated or deleted.

4. The immune cell of claim 3 , wherein both alleles of the endogenous gene are inactivated or deleted.

5. The immune cell of claim 3, wherein the endogenous RFX5 gene is inactivated or deleted.

6. The immune cell of claim 3, wherein the endogenous genes for TAP1, TAP2 and CIITA are not manipulated.

7. The immune cell according to any one of claims 1 to 6, wherein the endogenous gene for TRAC (T cell receptor alpha constant region) is further inactivated.

8. 2. The immune cell according to any one of the preceding claims, wherein the endogenous gene for B2M (beta-2-microglobulin) is not manipulated or the cell has normal activity of B2M.

9. The immune cell of claim 1 , wherein the CAR recognizes CD19 and / or CD20.

10. The immune cell of claim 1, wherein the CAR recognizes CLL-1.

11. The immune cell of claim 9, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 26 or 27, or a sequence having 90% sequence identity to SEQ ID NO: 26 or 27.

12. The immune cell of claim 10, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 65-76 or 78-83, or a sequence having 90% sequence identity to SEQ ID NO: 65-76 or 78-83.

13. The immune cell according to any one of claims 4 to 12, wherein inactivation is achieved by (a) editing of the endogenous gene, (b) expression of an inhibitory RNA, or (c) an inhibitor, preferably an antibody.

14. The immune cell of claim 13, wherein the editing is by CRISPR / Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, Cpf1, homologous recombination, single-stranded oligodeoxynucleotide (ssODN), or base editing.

15. The immune cell of claim 1, wherein the reference immune cell has not been edited to reduce expression of MHC class I or to reduce expression of MHC class II.

16. 1. A method for preparing allogeneic cells having reduced graft-versus-host disease (GVHD) or host rejection inducing activity, comprising engineering cells to reduce MHC class I expression or activity by 10% to 80% compared to a reference cell and reduce MHC class II expression or activity by at least 75% compared to a reference cell.

17. 17. The method of claim 16, wherein the expression or activity of RFX5 (regulatory factor X5), TAP1 (transporter associated with antigen processing 1), TAP2 (antigen peptide transporter 2), or CIITA (class II, major histocompatibility complex, transactivator) is reduced in the allogeneic cells.

18. 18. The method of claim 17, wherein both alleles of the endogenous gene are inactivated or deleted.

19. 19. The method of claim 17 or 18, wherein the expression or activity of RFX5 in the cell is reduced.

20. 17. The method of claim 16, wherein the endogenous genes for TAP1, TAP2 and CIITA are not manipulated.

21. 21. The method of any one of claims 16 to 20, further comprising reducing expression or activity of TRAC (T cell receptor alpha constant region) in said cells.

22. The method of any one of claims 16 to 21, wherein the endogenous gene for B2M (beta-2-microglobulin) in said cells is not manipulated.

23. The method of any one of claims 16 to 22, wherein the reduction is achieved by (a) gene editing, (b) expression of an inhibitory RNA, or (c) an inhibitor, preferably an antibody.

24. 24. The method of claim 23, wherein the editing is by CRISPR / Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease, Cpf1, homologous recombination, single-stranded oligodeoxynucleotide (ssODN), or base editing.

25. The method according to any one of claims 16 to 24, wherein the allogeneic cells are T cells or NK cells.

26. 17. The method of claim 16, further comprising introducing an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) into the allogeneic cells.

27. The method of claim 26, wherein the CAR recognizes CD19 and / or CD20.

28. 28. The method of claim 27, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 26 or 27, or an amino acid sequence having 90% identity to SEQ ID NO: 26 or 27.

29. The method of claim 26, wherein the CAR recognizes CLL-1.

30. 30. The method of claim 29, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 65-76 or 78-83, or a sequence having 90% sequence identity to SEQ ID NO: 65-76 or 78-83.

31. The method of any one of claims 26 to 30, wherein the CAR or TCR is introduced into the cell prior to gene editing.

32. A method for treating cancer in a patient in need of such treatment, comprising administering to said patient an immune cell according to any one of claims 1 to 15.

33. 33. The method of claim 32, wherein the immune cells are not naturally derived from the patient.

34. 33. The method of claim 32, wherein the cancer is lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

35. 16. A method of contacting a cancer cell with an immune cell according to any one of claims 1 to 15, wherein said cancer cell proliferation is inhibited or reduced.

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