CD19-targeting engineered immune cells and related methods

Abstract

Disclosed are engineered cells and anti-CD19 CAR-expressing engineered immune cells having inactivated immune checkpoint genes and armoring modifications, including the engineered HLA Class I expression and inhibition of native HLA Class II expression, as well as therapeutic compositions including the cells, therapeutic methods including administration of the cells, and companion diagnostic methods for the therapeutic compositions.

Description

Attorney Docket Number: CBI061.30CD19-TARGETING ENGINEERED IMMUNE CELLS AND RELATED METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 736,359, filed December 19, 2024, which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The invention relates to the field of oncology and more specifically, to cell therapy with genetically engineered tumor-targeting immune cells.REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing concurrently submitted herewith as file name CBI061_30_SL.xml, created on December 11, 2025, with a file size of 78,822 bytes, is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0004] CD19-targeting cell therapies such as autologous YESCARTA®, KYMRIAH® and BREYANZI® have brought dramatic improvement and even hopes for a cure to some but not all patients. In clinical studies YESCARTA® brought complete remission to nearly half of the treated patients (51% CR at 1 year and 50-54% CR at 2-year follow-up). In the allogeneic space, an allogeneic anti-CD19 CAR-T by Allogene Therapeutics produced complete response lasting longer than 6 months but only in 42% of patients. Such dramatic success in some patients underscores the need to improve CAR-T cell survival and potency so that a higher percentage of patients could achieve lasting remission. There is a need for next-generation engineered immune cells and anti-CD19 cell therapies that could bring higher response and cure rates than the existing therapeutic modalities.SUMMARY OF THE INVENITON

[0005] In one aspect, the present invention provides an engineered cell comprising: a genetic disruption of TRAC,' a genetic disruption of one or more immune checkpoint genes; reduced expression or absence of HLA Class I; reduced expression or absence of HLA Class II; and a polynucleotide encoding a fusion protein comprising B2M-HLA-E or B2M-HLA-E-HLA-G.

[0006] In some embodiments, the reduced expression or absence of HLA Class I is that of native HLA Class I. In some embodiments, the reduced expression or absence of HLA Class II is that of native HLA Class II.

[0007] In some embodiments, the engineered cell comprises: a genetic disruption of TRAC,'Attorney Docket Number: CBI061.30 a genetic disruption of one or more immune checkpoint genes; a genetic disruption of B2M,' reduced expression or absence of HLA Class II; and a polynucleotide encoding a fusion protein comprising B2M-HLA-E or B2M-HLA-E-HLA-G.

[0008] In some embodiments, the cell comprises a disruption of one or more immune checkpoint genes selected from PDCD1, CTLA-4, LAG-3, TIM-3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B.

[0009] In some embodiments, the reduced or absence of HLA Class II is by a disruption of one or more of genes selected from CIITA, RFXANK, RFX5, and RFXAP.

[0010] In some embodiments, the engineered cell comprises: a genetic disruption of TRAC,' a genetic disruption of PDCD1 ; a genetic disruption of TIGIT, a genetic disruption of B2M,' genetic disruption of CIITA, ' and a polynucleotide encoding a B2M-HLA-E fusion protein or B2M-HLA-E-HLA-G fusion protein.

[0011] In some embodiments of the engineered cell, the polynucleotide encoding the B2M-HLA-E fusion protein or the B2M-HLA-E-HLA-G fusion protein is inserted into the B2M, PDCD1, TIGIT, or CIITA locus.

[0012] In some embodiments, the engineered cell comprises the characteristics of: TRAC-; PDCD1-;TIGIT- ; B2M-; CIITA-; and positive for expression of B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein. In some embodiments, the engineered cell further comprises the characteristics of native HLA Class I- and / or native HLA Class II-.

[0013] In some embodiments, the engineered cell further comprises a polynucleotide encoding a heterologous protein. In some embodiments, the engineered cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the polynucleotide encoding the chimeric antigen receptor in the engineered cells is inserted into the TRAC, B2M, PDCD1, TIGIT, or CIITA locus.

[0014] Generally, the engineered cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is in vitro culture or ex vivo cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the immune cell is a T-cell, a natural killer cell (NK), a macrophage or a precursor thereof.Attorney Docket Number: CBI061.30

[0015] In another aspect, the present invention is directed to an engineered immune cell with the genetic disruption and expression characteristics herein, and also expressing a CD19-targeting chimeric antigen receptor (CAR) protein.

[0016] In some embodiments, the engineered immune cell comprises: a polynucleotide encoding a CD19-targeting chimeric antigen receptor (CAR) protein inserted into the T cell receptor alpha chain (TRAC) locus; a polynucleotide encoding a B2M-HLA-E fusion protein inserted into a B2M locus inhibition of one or more immune checkpoint; and inhibition of HLA Class II expression.

[0017] In some embodiments, the cell is a T cell, a natural killer (NK) cell, a macrophage or a precursor thereof. In some embodiments, the inhibition of one or more immune checkpoint comprises genomic or genetic disruption of one or more immune checkpoint genes selected from PDCD1, CTLA- 4, LAG-3, TIM-3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B. In some embodiments, genomic disruption is of PDCD1 and TIGIT.

[0018] In some embodiments, the inhibition of HLA Class II expression is via genomic or genetic disruption of one or more genes selected from CIITA, RFXANK, RFX5, and RFXAP. In some embodiments, the inhibition of HLA Class II expression is via genomic or genetic disruption of the CIITA gene.

[0019] In some embodiments, the sequence encoding the CAR is inserted into the TRAC locus within or near SEQ ID NO: 1. In some embodiments, polynucleotide encoding a B2M-HLA-E fusion protein or a B2M-HLA-E-HLA-G fusion protein is inserted into the B2Mlocus within or near SEQ ID NO: 2. In some embodiments, the PDCD1 locus is disrupted within or near SEQ ID NO: 3. In some embodiments, the TIGIT locus is disrupted within or near SEQ ID NO: 4. In some embodiments, the CIITA locus is disrupted within or near SEQ ID NO: 5.

[0020] In some embodiments, the CD19-targeting CAR comprises the scFv of FMC63, the 4-1BB costimulatory domain, the CD3^ activation domain, the CD8 hinge, and the CD8 transmembrane domain.

[0021] In a further aspect, the present invention is directed to a method of making the engineered immune cells described herein, the method comprising introducing into a cell one or more endonucleases capable of cleaving the TRAC locus, the B2M locus, one or more of PDCD1, CTLA-4, LAG-3, TIM-3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B locus, one or more of CIITA, RFXANK, RFX5, and RFXAP locus. In some embodiments, a method comprising introducing into a cell one or more endonucleases capable of cleaving the TRAC locus, the B2M locus, the PDCD1 locus, the TIGIT locus, and the CIITA locus. In some embodiments, the endonuclease is a CRISPRAttorney Docket Number: CBI061.30 endonuclease, and the method further comprises introducing into the cell nucleic acid targeting nucleic acids (NATNAs) capable of hybridizing to target sequences within the TRAC locus, the B2M locus, the PDCD1 locus, the TIGIT locus and the CIITA locus. In some embodiments, the CRISPR endonuclease is Casl2a and the NATNAs are SEQ ID NOs.: 6, 7, 8, 9, and 10. In some embodiments, the CRISPR endonuclease is selected from Cas9, Casl2a and CASCADE. In some embodiments, the CRISPR endonuclease and the NATNAs are introduced into the cell in the form of a nucleoprotein complex (NPC).

[0022] In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a ZFN-Fok I fusion, a transcription activator-like effector nuclease (TALEN), and a TALEN-Fok I fusion.

[0023] In some embodiments, the method further comprises introducing into a cell a vector construct comprising an expression cassette encoding the CD19-targeting CAR and an expression cassette encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein. In some embodiments, the vector construct comprises AAV6. In some embodiments, the vector construct comprising an expression cassette encoding the CD19-targeting CAR comprises SEQ ID NO: 11 or 18. In some embodiments, the vector construct comprising an expression cassette encoding the B2M-HLA-E fusion protein comprises SEQ ID NO: 17, or comprises SEQ ID NO: 12 or 16.

[0024] In one embodiment, the invention is a composition comprising a population of the engineered immune cells described herein and a pharmaceutically acceptable excipient, e.g., one or more of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, water, alcohols, polyols, glycerin, vegetable oils, phospholipids, surfactants, sugars, derivatized sugars, alditols, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, myoinositol, aldonic acid, esterified sugars, sugar polymers, monosaccharides, fructose, maltose, galactose, glucose, D- mannose, sorbose, disaccharides, lactose, sucrose, trehalose, cellobiose, polysaccharides, raffinose, melezitose, maltodextrins, dextrans, starches, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, and sodium phosphate. In some embodiments, the antimicrobial agent comprises one or more of benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, and thimerosal. In some embodiments, the composition further comprises an antioxidant selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, and sodium metabisulfite, and optionally a surfactant selected from polysorbates, sorbitan esters, lecithin, phosphatidylcholines, phosphatidylethanolamines, fatty acids, fatty acid esters and cholesterol, and further, optionally a freezing agent selected from 3% to 12% dimethylsulfoxide (DMSO) and 1% to 5% human albumin, and further optionally a preservative selected from one or more of methylparaben,Attorney Docket Number: CBI061.30 propylparaben, sodium benzoate, benzalkonium chloride, antioxidants, chelating agents, parabens, chlorobutanol, phenol, and sorbic acid.

[0025] In one embodiment, the invention is a method of inhibiting the growth or function of CD 19- expressing cells in a patient comprising administering to the patient the composition of CD-19-targeting engineered immune cells described herein. In some embodiments, the CD19-expressing cells are B cells. In some embodiments, the administering is selected from the group consisting of intravenous delivery, parenteral delivery, intramuscular delivery, subcutaneous delivery, intrathecal delivery, intratumor delivery, and intraperitoneal delivery. In some embodiments, the method further comprises administering a cytokine to the patient, e.g., IL-2, IL- 15 or IL-21.

[0026] In some embodiments, the method further comprises, prior to administering to the patient the composition comprising the engineered immune cells, preconditioning the patient with a lymphodepleting regimen. In some embodiments, lymphodepleting regimen comprises administration of agents comprising cyclophosphamide and / or fludarabine.

[0027] In some embodiments, the method further comprises prior to administering to the patient the composition comprising the engineered immune cells, applying to the immune cells a quality control measure comprising assessing one or more properties selected from presence of the CAR in the cellular genome, surface expression of the CAR, CD19-dependent lysis of CD19-expressing target cells, proliferation in the presence of CD19-expressing target cells, cytokine secretion in the presence of CD19-expressing target cells, disruption of one or more immune checkpoints, inhibition of the native HLA Class 1 expression, inhibition of the native HLA Class 2 expression, T cell exhaustion, and the presence of a memory cell phenotype.

[0028] In some embodiments, the presence of the CAR in the cellular genome is assessed by a method selected from nucleic acid hybridization, nucleic acid sequencing, polymerase chain reaction (PCR), quantitative PCR (qPCR), real-time PCR (rtPCR) and droplet digital PCR (ddPCR). In some embodiments, the surface expression of the CAR is assessed by flow cytometry, fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. In some embodiments, the surface expression of the CAR is assessed by flow cytometry or fluorescence-activated cell sorting (FACS) with an anti-Fab2 antibody.

[0029] In some embodiments, the immune cell population with the highest surface expression of the CAR is selected for administration to the patient. In some embodiments, the CD19-dependent lysis of CD19-expressing target cells is assessed by co-culturing the engineered immune cells with CD 19- expressing target cells at an effector:target ratio between about 0. 1 and about 10 and assessing target cell lysis, and the engineered immune cell population with the highest rate of lysis of CD19-expressing target cells is selected for administration to the patient. In some embodiments, the proliferation in theAttorney Docket Number: CBI061.30 presence of CD19-expressing target cells is assessed by co-culturing the engineered immune cells with CD19-expressing target cells, and engineered immune cell population with the highest rate of proliferation in the presence of CD19-expressing target cells is selected for administration to the patient. In some embodiments, the cytokine secretion in the presence of CD19-expressing target cells is assessed by quantifying one or more of gamma-interferon (IFNy), tumor necrosis factor alpha (TNFa), IL-2, IL-4, IL-6 in co-cultures of the engineered immune cells with CD19-expressing target cells, and the engineered immune cell population with the highest cytokine secretion in the presence of CD 19- expressing target cells is selected for administration to the patient. In some embodiments, the disruption of one or more immune checkpoints is assessed by sequencing one or more of the TIGIT locus or the PDCD1 locus or by assessing the presence of the TIGIT protein or the PD-1 protein, and the engineered immune cell population with the highest percentage of cells having the TIGIT or the PDCD1 gene disruption or lacking the TIGIT or the PD-1 protein is selected for administration to the patient. In some embodiments, the inhibition of the native HLA Class 1 expression is assessed by assessing the surface expression of HLA-A, HLA-B or HLA-C, and the engineered immune cell population with the lowest surface expression of HLA-A, HLA-B or HLA-C is selected for administration to the patient. In some embodiments, the inhibition of the native HLA Class 2 expression is assessed by assessing the surface expression of HLA-DR, HLA-DP or HLA-DQ, and the engineered immune cell population with the lowest surface expression of HLA-DR, HLA-DP or HLA-DQ is selected for administration to the patient.

[0030] In some embodiments, the celt exhaustion is assessed by measuring expression of one or more of PD-1, LAG-3, TIM-3, CTLA-4, and the BLIMP- 1 transcription factor, and the TOX transcription factor, and the engineered immune cell population with the lowest expression of PD-1, LAG-3, TIM-3, CTLA-4, and the BLIMP- 1 transcription factor is selected for administration to the patient. In some embodiments, the ceil exhaustion is assessed by measuring the rate of glycolysis, or oxidative phosphorylation, or a ratio of glycolysis to oxidative phosphorylation over time, and the engineered immune cell population with the lowest glycolysis, or the lowest ratio of glycolysis to oxidative phosphorylation over time is selected for administration to the patient.

[0031] In some embodiments, the memory phenotype is assessed by detecting expression of a combination of cell surface markers comprising CCR7, CD45RA, CD45RO, CD62L, and CD27, and the engineered immune cell population with the highest expression of the combination of CCR7, CD45RA, CD45RO, CD62L, and CD27 is selected for administration to the patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic diagram of the CD19-targeting CAR-T cells with immune cloaking and immune checkpoint disruption.Attorney Docket Number: CBI061.30

[0033] FIG. 2 is a diagram of the anti-CD19 CAR.

[0034] FIG. 3 shows in vitro antitumor activity of the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) against an MCL cell line.

[0035] FIG. 4 shows in vitro antitumor activity of the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) against a CLL cell line.

[0036] FIG. 5 shows in vitro proliferation of the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) in the presence of MCL cell line and IL2.

[0037] FIG. 6 shows in vitro cytokine secretion by the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) in the presence of the CLL cell line or the MCL cell line.

[0038] FIG. 7 shows results of an in vitro serial rechallenge (“serial killing”) assay with the CD 19- targeting CAR-T cells (anti-CD19 CAR, TRAC KO) and the MCL cell line.

[0039] FIG. 8 shows in vitro antitumor activity of the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) against primary tumor cells from five CLL patients.

[0040] FIG. 9 shows in vitro cytokine secretion by the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) in the presence of primary tumor cells from five CLL patients.

[0041] FIG. 10 shows in vivo tumor control by the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) in immunodeficient mice IV-injected with a human MCL tumor.

[0042] FIG. 11 shows in vivo tumor control by the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) in immunodeficient mice SC-injected with a human MCL tumor.

[0043] FIG. 12 shows TIGIT and PDCD1 gene expression in the CD 19 -targeting CAR-T cells (anti- CD19 CAR, TRAC KO) during a serial rechallenge with an MCL cell line.

[0044] FIG. 13 shows in vitro proliferation and anti -tumor cytotoxicity of the CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO with or without TIGIT and PDCD1 KO) during a serial rechallenge with an MCL cell line.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0045] The following definitions are provided to aid in understanding of the disclosure. Unless defined in this section, technical and scientific terms used in this disclosure have the meaning commonly understood by a person of ordinary skill in the art. See, e.g. , Sambrook et al., Molecular Cloning, A Laboratory Manual, 4thEd. Cold Spring Harbor Lab. Press (2012).Attorney Docket Number: CBI061.30

[0046] The term “activation” refers to the state of a T cell that includes one or both of cell proliferation and cytokine secretion by the cell.

[0047] The term “antibody” refers to an immunoglobulin molecule which specifically binds to an antigen. The term also refers to antibody fragments including Fv, Fab and F(ab)2, scFv and other forms described in, e.g., Antibodies: A Laboratory Manual, 2ndEd. Greenfield, E., ed., Cold Spring Harbor Lab. Press, N.Y. (2013).

[0048] The terms “the anti-CD19 (or CD19-targeting) CAR-T cell described herein” and “the antiCD 19 (or CD19-targeting) immune cell described herein” refer to the immune cells engineered to express an anti-CD19 CAR. In some embodiments, the immune cells are further engineered to disrupt expression of one or more genes selected from the group consisting of CIITA, PDCD1 and TIGIT and further engineered to lack expression of the native beta-2 microglobulin (B2M) and express a B2M- HLA-Class I fusion protein.

[0049] The term “co-stimulatory domain” refers to a part of a T cell receptor which is a binding partner that specifically binds a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell, proliferation, and cytokine secretion. Examples of co-stimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, and HVEM. Examples of co-stimulatory domains include CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0050] The term “therapeutic benefit” refers to an effect that improves the condition of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the tumor, or prevention of metastasis, or prolonging overall survival (OS) or progression free survival (PFS) of a subject with cancer.

[0051] The terms “pharmaceutically acceptable” and “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other deleterious reaction in a patient. For example, the pharmaceutically and pharmacologically acceptable preparations should meet the standards set forth by the FDA Office of Biological Standards.

[0052] The term “pharmaceutically acceptable carrier” and “excipient” refer to aqueous solvents (e.g., water, aqueous solutions of alcohols, saline solutions, sodium chloride, Ringer's solution, etc.), nonaqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters), as well as dispersion media, coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, stabilizers, binders, disintegration agents, lubricants, sweetening agents,Attorney Docket Number: CBI061.30 flavoring agents, and dyes. The concentration and pH of the various components in a pharmaceutical composition are adjusted according to well-known parameters for each component.

[0053] The term "domain" refers to one region in a polypeptide which is folded into a particular structure independently of other regions.

[0054] The term “effector function” refers to a specialized function of a differentiated cell, such as a NK cell.

[0055] The term “adoptive cell” refers to a cell that can be genetically modified for use in a cell therapy treatment. Examples of adoptive cells include T cells, macrophages, and natural killer (NK) cells.

[0056] The term “cell therapy” refers to the treatment of a disease or disorder that utilizes genetically modified cells. The term “adoptive cell therapy (ACT)” refers to a therapy that uses genetically modified adoptive cells. Examples of ACT include T cell therapies, CAR-T cells therapies, natural killer (NK) cell therapies, CAR-NK cell therapies and engineered macrophage cell therapies.

[0057] The term “lymphocyte” refers to a leukocyte that is part of the vertebrate immune system. Lymphocytes include T cells such as CD4+and / or CD8+cytotoxic T cells, a / p T cells, y / 8 T cells, and regulatory T cells. Lymphocytes also include natural killer (NK) cells, natural killer T (NKT) cells, cytokine induced killer (CIK) cells, and antigen presenting cells (APCs). Lymphocytes also include tumor infiltrating lymphocytes (TILs).

[0058] The terms “effective amount” and “therapeutically effective amount” of a composition such as a cell therapy composition, refer to a sufficient amount of the pharmaceutically active agent, or a composition thereof, to provide the desired physiological response in the patient to whom the active agent or the composition is administered.

[0059] The terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to polymers of amino acids, including natural and synthetic (unnatural) amino acids, as well as amino acids not found in naturally occurring proteins, e.g., peptidomimetics, and D optical isomers. A polypeptide may be branched or linear and be interrupted by non-amino acid residues. The terms also encompass amino acid polymers that have been modified through acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, cross-linking, or conjugation (e.g., with a label). The polypeptide need not include the full-length amino acid sequence of the reference molecule but can include only so much of the reference molecule as necessary for the polypeptide to retain its desired activity. Lor example, polypeptides comprising full-length proteins, fragments thereof, polypeptides with amino acid deletions, additions, and substitutions are encompassed by the terms “protein” and “polypeptide,” as long as the desired activity is retained. Lor example, polypeptides with 95%, 90%, 80%, or less ofAttorney Docket Number: CBI061.30 sequence identity with the reference polypeptide are included as long the desired activity is retained by the polypeptides.

[0060] The terms “CRISPR” (clustered regularly interspaced short palindromic repeats), “Cas” (CRISPR-associated protein) “CRISPR-Cas” and “CRISPR system” refer to the genome editing tool derived from prokaryotic organisms and comprising a nucleic acid guide molecule and a sequencespecific nucleic acid-guided endonuclease capable of cleaving a target nucleic acid strand at a site complementary to a sequence in the nucleic acid guide.

[0061] The term “NATNA” (nucleic acid targeting nucleic acid) refers to a nucleic acid guide molecule of the CRISPR system. NATNA may be comprised two nucleic acid targeting polynucleotides (“dual guide”) including a CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). NATNA may be comprised of a single nucleic acid targeting polynucleotide (“single guide”) comprising crRNA and tracrRNA connected by a fusion region (linker). The crRNA may comprise a targeting region and an activating region. The tracrRNA may comprise a region capable of hybridizing to the activating region of the crRNA. The term “targeting region” refers to a region that is capable of hybridizing to a sequence in a target nucleic acid. The term “activating region” refers to a region that interacts with a polypeptide, e.g., a CRISPR nuclease.Engineered Cells

[0062] In one aspect, the present invention provides an engineered cell for mitigating or reducing susceptibility of the cell to rejection by a host immune system, such as for allogenic cell therapy. In some further embodiments, the engineered cell also has reduced activity against host cells, for example when the engineered cell is an allogeneic immune cell.

[0063] In some embodiments, the engineered cell comprises: a genetic disruption of TRAC; a genetic disruption of one or more immune checkpoint genes; reduced expression or absence of HLA Class I; reduced expression or absence of HLA Class II; and a polynucleotide encoding a fusion protein comprising B2M-HLA-E or B2M-HLAE-HLA-G.

[0064] In the embodiments herein, the genetic disruption results in inactivation or inhibition of expression of the protein encoded by the gene or expression of the polynucleotide from the gene. In some embodiments, a reference to a gene includes the gene locus.

[0065] In some embodiments, the reduced expression or absence of HLA Class I is that of native HLA Class I. In some embodiments, the reduced expression or absence of HLA Class II is that of native HLA Class II.Attorney Docket Number: CBI061.30

[0066] In some embodiments, the engineered cell comprises: a genetic disruption of TRAC,' a genetic disruption of one or more immune checkpoint genes; a genetic disruption of B2M,' reduced expression or absence of HLA Class II; and a polynucleotide encoding a fusion protein comprising B2M-HLA-E or B2M-HLAE-HLA-G.

[0067] In some embodiments, the genetic disruption of one or more immune checkpoint genes is selected from PDCD1, CTLA-4, LAG-3, TIM-3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B.

[0068] In some embodiments, combinations of immune checkpoint genes are disrupted. In some embodiments, the genetic disruption of one or more immune checkpoint genes is PDCD1 and TIGIT.

[0069] In some embodiments, the reduced or absence of HLA Class II is from a genetic disruption of one or more genes encoding a protein selected from MHC class II transactivator (CIITA), regulatory factor -associated ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), and regulatory factor X associated protein (RFXAP). In some embodiments, the reduced or absence of HLA Class II is a genetic disruption of one or more of CIITA, RFXANK, RFX5, and RFXAP genes. In a preferred embodiment, the genetic disruption is of CIITA.

[0070] In some embodiments, the engineered cell comprises: a genetic disruption of TRAC,' a genetic disruption of PDCD1 ; a genetic disruption of TIGIT, a genetic disruption of B2M,' a genetic disruption of CIITA, ' and a polynucleotide encoding the B2M-HLA-E fusion protein or B2M-HLA-E-HLA-G fusion protein.

[0071] In some embodiments, a polynucleotide encoding the B2M-HLA-E fusion protein or the B2M- HLA-E-HLA-G fusion protein in the engineered cell is inserted into the B2M, PDCD1, TIGIT, or CIITA locus. In some embodiments, the polynucleotide encoding the B2M-HLA-E fusion protein or the B2M-HLA-E-HLA-G fusion protein in the engineered cell is inserted into the B2M locus.

[0072] In some embodiments, the engineered cell has the characteristics or phenotype of: TRAC ; PDCD1 ; TIGIT : B2M ; CIITA ; and B2M-HLA-E+or B2M-HLA-E-HLA-G+fusion protein. In the embodiments herein, positive expression of B2M-HLA-E fusion protein can be denoted as B2M-HLA- E+and positive expression of B2M-HLA-E-HLA-G fusion protein can be denoted as B2M-HLA-E- HLA-G+.Attorney Docket Number: CBI061.30

[0073] In some embodiments, the engineered cell further has the characteristics of native HLA Class 1" and / or native HLA Class IL.

[0074] In some embodiments, the encoded B2M-HLA-E fusion protein comprises the amino acid sequence of SEQ ID NO: 15, and the B2M-HLA-E-HLA-G fusion protein comprises the amino acid sequence of SEQ ID NO: 14.

[0075] In some embodiments, the polynucleotide encoding the B2M-HLA-E fusion protein comprises the polynucleotide sequence comprising SEQ ID NO: 17. In some embodiments, the polynucleotide encoding the B2M-HLA-E-HLA-G fusion comprises the polynucleotide sequence comprising SEQ ID NO: 12 or 16.

[0076] In some embodiments, the engineered cell further comprises a polynucleotide encoding a heterologous protein. In some embodiments, the engineered cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR).

[0077] In some embodiments, the polynucleotide encoding a heterologous protein is inserted into a gene targeted for genetic disruption in the engineered cell. In some embodiments, the polynucleotide encoding the heterologous protein is inserted into the TRAC, B2M, PDCD1, TIGIT, or CIITA locus.

[0078] In some embodiments, the polynucleotide encoding the chimeric antigen receptor is inserted into a gene targeted for genetic disruption in the engineered cell. In some embodiments, the polynucleotide encoding the chimeric antigen receptor is inserted into the TRAC, B2M, PDCD1, TIGIT, or CIITA locus.

[0079] In some embodiments, the polynucleotide encoding the chimeric antigen receptor is inserted into a genetic locus different from the insertion of the polynucleotide encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein.

[0080] In some embodiments, the engineered cell is generally a mammalian cell. In some embodiments, the engineered cell is an engineered rodent cell or human cell. In a preferred embodiment, the engineered cell is an engineered human cell.

[0081] In some embodiments, the engineered human cell is in vitro culture or ex vivo. In some embodiments, the human cell for engineering is ex vivo, i.e., obtained from a donor, as further described herein.

[0082] In some embodiments, the engineered cell is an immune cell. In some embodiments, the immune cell is a T-cell, a natural killer cell (NK), a macrophage or a precursor thereof. In a preferred embodiment, the engineered cell is a T-cell or NK cell. In some embodiments, the engineered cell is an engineered CD8+T-cell. In some embodiments, the engineered cell is a CD4+T-cell.Attorney Docket Number: CBI061.30

[0083] In some embodiments, the engineered cell is a population of cells. In some embodiments, the engineered cell is a population of cells having the characteristics described herein. In some embodiments, the population of cells is a population of immune cells. In some embodiments, the population of cells is a population of T-cells or a population of NK cells. In some embodiments, the population of cells is a population of CD8+and CD4+T-cells.

[0084] In some embodiments, the engineered cells can be made using the methods described herein, such as the method described for generating CD 19 CAR cells, where the CAR cell has the characteristics described herein. In some embodiments, the one or more genetic disruptions are made with a sequence specific nuclease. In some embodiments, the sequence-specific endonuclease is a TALEN, a Zinc-finger nuclease (ZFN), or a CRISPR endonuclease. In a preferred embodiment, the one or more genetic disruptions is by use of a CRISPR endonuclease, such as a CRISPR Class II Type II endonuclease or CRISPR Class II Type V endonuclease. Exemplary CRISPR endonuclease and corresponding guide polynucleotides for generating specific gene disruptions are described herein.

[0085] In some embodiments, the one or more genetic disruptions is done sequentially or simultaneously, for example by multiplexing. In some embodiments, the one or more genetic disruptions is done by a combination of sequential and simultaneous gene disruptions.

[0086] In some embodiments, the insertion of a polynucleotide encoding a heterologous protein, such as the polynucleotide encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein or a chimeric antigen receptor is done by providing the polynucleotide to the cell, where the polynucleotide is inserted into a cleaved genetic locus generated by a sequence specific endonuclease. In some embodiments, the insertion is by way of non-homologous end joining (NHEJ). In some embodiments, the insertion of the polynucleotide into a cleaved genetic locus is by way of homologous recombination (homology-directed repair or HR pathway).

[0087] In some embodiments, the polynucleotide encoding the heterologous protein is provided in a vector, which can include one or more control sequences for expression of the encoded heterologous protein, or one or more sequences for insertion into a cleaved genetic locus, e.g., homology arms. In some embodiments, the vector includes a promoter for expression of the heterologous protein. Exemplary promoters include, by way of example and not limitation, elongation factor -1 alpha (EFla), mouse stem cell virus (MSCV), cytomegalovirus (CMV), human phosphoglycerate kinase 1 (hPGK), RPBSA, and MND promoters.

[0088] In some embodiments, the vector does not include a promoter, and the expression of the encoded heterologous protein is by an endogenous promoter and related control sequences at the genetic locus-insertion site.Attorney Docket Number: CBI061.30

[0089] In some embodiments, the polynucleotide encoding a heterologous protein is provided in a viral vector (e.g., vectors based on lentivirus, adeno-associated virus, adenovirus, Herpes Simplex virus, etc.).

[0090] In some embodiments, the polynucleotide encoding the heterologous protein is delivered to the cell in nanoparticles, particularly lipid nanoparticles (LNP). Exemplary nanoparticles, including LNPs, for delivery into cells, are described in U.S. Patent No. 8,754,062; U.S. Patent No. 10,703,789; U.S. Patent No. 11,026,894; U.S. patent publication 20240189340; International patent publications W02023056033, W02010021865, and WO2023031394; all of which are incorporated by reference herein.Chimeric Antigen Receptors (CAR) and Cells Thereof

[0091] In a further aspect, the present invention provides a CD19 chimeric antigen receptor (CD19 CAR) cell having the genetic disruptions and protein expression characteristics described herein. In a clinical study, ZUMA-1 YESCARTA® resulted in an objective response rate (ORR) of 72% and complete remission (CR) rate of 51% at 1 year and 50-54% at 2-year follow-up in patients with relapsed or refractory cancers. Allogene Therapeutics reported at the American Society for Clinical Oncology (ASCO) meeting in 2023 that the allogeneic off-the-shelf anti-CD19 CAR-T clinical candidate ALLO-501 / ALLO-501A produced complete response lasting longer than 6 months in 42% of patients with some remaining in remission longer than 31 months.

[0092] Yet for many patients, CAR-T cell therapies are ineffective. For example, Schuster, et al., Chimeric antigen receptor T cells in refractory B cell lymphomas, 2018, NEJM 377(26):2545 have found that with autologous CD 19 -targeting T cell treatments, only 57% of lymphoma patients (FL and DLBCL) had complete response at 28.6 months. The authors noted that patients who had a response to the CD19-targeting CAR-T treatments tended to express lower levels immune checkpoint genes PD-L1, PD-1, LAG-3 and TIM=3. (Id., page 7). Another group also observed that remission was induced only in a subset of CLL patients treated with anti-CD19 CAR-T and those subjects possessed a population of CD27+PD-UCD8+CAR-T cells expressing high levels of the IL-6 receptor. (Fraietta et al., Determinants of response and resistance to CD 19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia, 2018, Nature Medicine, 24:563). Yet another group found that TIGIT, LAG3, and CD96 were the predominant checkpoint molecules expressed on exhausted T cells and CTLs, while only TIGIT expression was significantly increased after relapse in CD 19 CAR-T- treated MCL patients. (Jiang, et al., TIGIT is the central player in T cell suppression associated with CAR-T relapse in mantle cell lymphoma, 2022, Molecular Cancer 21: 185.) Another study found that the presence of CD8+CD19-targeting CAR-T cells with enhanced mitochondrial biogenesis correlating to expansion and persistence is associated with complete response in CLL patients (van Bruggen, et al.,Attorney Docket Number: CBI061.30Chronic lymphocytic leukemia cells impair mitochondrial fitness in CD8+ T cells and impede CAR-T cell efficacy, Blood, 2019, 134(1):44.

[0093] Another cause of poor persistence of allogeneic cells may be an attack by the patient’s immune system. Although patients undergo lymphodepletion prior to receiving allogeneic cells, patient’s T cells and natural killer (NK) cells are known to recover quickly. Eliminating HLA Class I expression on the surface of allogeneic cells should shield them from host’s T cell attack. However, under the “missing self’ hypothesis, cells lacking any HLA Class I surface proteins are targeted for elimination by NK cells via the NKG2D receptor. A method whereby native HLA Class I expression is eliminated by disrupting expression of the beta-2 microglobulin (B2M) gene, and at the same time, a non-polymorphic HLA Class I gene such as for example, HLA-E or HLA-G is expressed in an artificial fusion with B2M has been used in CAR-T cells, see, e.g., Inf 1 Patent Application Publication No. WO2024107646 and Int’l Patent Application Ser. No. PCT / US2023075395 respectively.

[0094] The present disclosure provides methods and compositions for treatment of B cell malignancies and autoimmune disease with B cell involvement with CD19-targeting immune cells engineered to express anti-CD19 CAR and further engineered to disrupt immune checkpoint and yet further engineered to avoid attack by the patient’s T cells and natural killer (NK) cells.

[0095] In some embodiments, the present invention comprises adoptive cells and the use of adoptive cells in cellular immunotherapy. Adoptive cells of the instant invention are cells that have undergone genome engineering. The cell types include lymphocytes, such as T cells and CAR-T cells. Other cell types such as natural killer (NK) cells and macrophages are also contemplated within the scope of the invention.

[0096] In some embodiments, the CD 19 -targeting engineered immune cells comprise three or more genome edits. In some embodiments, at least two of the three or more genome edits comprise a simultaneous gene disruption and gene insertion whereby a gene is disrupted by insertion of an expression cassette for a different gene.

[0097] In some embodiments, the engineered immune cell comprises: a polynucleotide encoding a CD19-targeting chimeric antigen receptor (CAR) protein; a genetic disruption of T cell receptor alpha chain (TRAC) gene locus; a polynucleotide encoding a fusion protein comprising B2M-HLA-E or B2M-HLA-E-HLA-G; inhibition of one or more immune checkpoints; and inhibition of HLA Class II expression.

[0098] In some embodiments, the inhibition of one or more immune checkpoints comprises genomic disruption of one or more immune checkpoint genes selected from PDCD1, CTLA-4, LAG-3, TIM-3,Attorney Docket Number: CBI061.30BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B. In some embodiments, the inhibition of one or more immune checkpoints comprises genetic disruption of PDCD1 and TIGIT gene.

[0099] In some embodiments, the inhibition of HLA Class II expression is a genetic disruption of one or more of genes selected from CIITA, RFXANK, RFX5, and RFXAP. In some embodiments, the inhibition of HLA Class II expression is via a genomic or genetic disruption of the CIITA gene.

[0100] In some embodiments, the CD 19 -targeting engineered immune cells comprise seven genome edits consisting of disruption of genes B2M, CIITA, PDCD1, TIGIT and TRAC, and insertion of an expression cassette encoding a chimeric antigen receptor (CAR) and insertion of an expression cassette encoding a protein comprising B2M-HLA-E fusion or a protein comprising a B2M-HLA-E-HLA-G fusion.

[0101] In some embodiments, the polynucleotide encoding the CD 19 CAR is inserted into B2M, CIITA, PDCD1, TIGIT or TRAC gene locus. In some embodiments, the polynucleotide encoding the CD19 CAR is inserted in the TRAC gene locus. In some embodiments, the insertion into the specified gene locus is through use of a sequence specific endonuclease to generate a cleaved site in the specified genetic locus for insertion of the polynucleotide. In one embodiment, insertion of the polynucleotide encoding the CD 19 CAR into a specified gene locus, for example an exon of the gene, results in genetic disruption of the gene.

[0102] In some embodiments, insertion of the polynucleotide, e.g., an expression cassette, encoding the fusion protein comprising B2M-HLA-E or B2M-HLA-E-HLA-G is inserted into B2M, CIITA, PDCD1, TIGIT or TRAC gene locus. In some embodiments, the polynucleotide encoding the B2M- HLA-E or B2M-HLA-E-HLA-G fusion protein is inserted in the B2M gene locus. In some embodiments, the polynucleotide encoding the CD 19 CAR is inserted into a genetic locus different from that of the polynucleotide encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein. In one embodiment, insertion of the polynucleotide encoding the fusion protein into a specified gene locus, for example an exon of the gene, results in genetic disruption of the gene.

[0103] In some embodiments, the engineered immune cell comprises: a polynucleotide encoding a CD 19 CAR; a genetic disruption of TRAC,' a genetic disruption of B2M,' a genetic disruption of PDCDT, a genetic disruption of TIGIT, a genetic disruption of CIITA, ' and a polynucleotide encoding a B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein. In some embodiments, the polynucleotide encoding a CD 19 CAR is inserted into the TRAC gene locus, and the polynucleotide encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein is inserted into the B2M gene locus.

[0104] In some embodiments, the CD 19 targeting CAR in the engineered immune cell comprises an amino acid sequence of SEQ ID NO: 13.Attorney Docket Number: CBI061.30

[0105] In some embodiments, the polynucleotide sequence encoding the CD 19 targeting CAR comprises a polynucleotide sequence comprising SEQ ID NO: 11 or 18. In some embodiments, the polynucleotide sequence encoding the CD 19 targeting CAR is provided as a transgene or in an expression cassette or vector.

[0106] In some embodiments, encoded B2M-HLA-E fusion protein in the engineered immune cell comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the encoded B2M-HLA- E-HLA-G fusion protein in the engineered immune cell comprises an amino acid sequence of SEQ ID NO: 14.

[0107] In some embodiments, the polynucleotide encoding the B2M-HLA-E fusion protein comprises a polynucleotide sequence comprising SEQ ID NO: 17. In some embodiments, the polynucleotide encoding the B2M-HLA-E-HLA-G fusion protein comprises a polynucleotide sequence comprising SEQ ID NO: 12 or 16. In some embodiments, the polynucleotide sequence encoding the B2M-HLA-E fusion protein, or the B2M-HLA-E-HLA-G fusion protein, is provided as a transgene or in an expression cassette or vector.

[0108] In some embodiments, the engineered immune cell has the following characteristics or phenotype: CD19 CAR+; TRAC ; B2M ; PDCD1 ; TIGIT : CIITA ; and fusion protein B2M-HLA-E+or B2M-HLA-E-HLA-G+.

[0109] The cells of the instant invention are allogeneic cells, i.e., cells isolated from a donor individual, i.e., a healthy human donor of either gender, where the donor individual is allogeneic to the intended host for the engineered cells. In some embodiments, the healthy donor is any age. In some embodiments, the healthy donor is 65 years old or less, 60 years old or less, 55 years old or less, 50 years old or less, 45 years old or less, 40 years or less, 35 years old or less, 30 years or less, or 25 years old or less. In some embodiments, the donor is 15 to 65 years old, 15 to 60 years old, 15 to 55 years old, 15 to 50 years old, 15 to 45 years old, 15 to 40 years old, 15 to 35 years old, 15 to 30 years old, or 15 to 25 years old. In some embodiments, the donor is 18 to 30 years old.

[0110] Generally, the healthy donor has no detectable infection with a pathogen, such as a viral, bacterial, or fungal infection. For example, the donor has no detectable infection with Human Immunodeficiency Virus, HAV, HBV, HCV, Treponema pallidum, HLTV-I / II, Trypanosoma cruzi, CMV, HSV-1 / 2, human herpes virus 6 / 7 / 8, EBV, and Parvovirus. The assays used are those accepted in the art or government-approved diagnostics for such pathogens.[OHl] In some embodiments, the cells are isolated from a healthy donor using standard techniques. For example, lymphocytes can be isolated from blood, or from lymphoid organs such as the thymus, bone marrow, lymph nodes, and mucosal-associated lymphoid tissues (MALT). Techniques for isolating lymphocytes from such tissues are well known in the art, see, e.g., Smith, J.W., ApheresisAttorney Docket Number: CBI061.30 techniques and cellular immunomodulation, 1997, Ther. Apher. 1:203-206. In some embodiments, isolated lymphocytes are characterized in terms of specificity, frequency and function. In some embodiments, the isolated lymphocyte population is enriched for specific subsets of T cells, such as CD4+, CD8+, CD25+, or CD62L+. See, e.g., Wang etal.,Mol. Therapy - Oncolytics, 2016, 3: 16015. In some embodiments, after isolation, the lymphocytes are activated in order to promote proliferation and differentiation into specialized lymphocytes. For example, T cells can be activated using soluble CD3 / CD28 activators, or magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies.

[0112] In some embodiments, a quality control measure or characterization step is applied to the isolated lymphocytes. In some embodiments, the quality control measure includes determining the percentage in the composition of CD4+, CD8+, CD25+, or CD62L+ cells, or cells expressing any combination of the above markers by flow cytometry.

[0113] The present invention comprises allogeneic engineered immune cells. In some embodiments, the cells described herein are genetically modified to express a chimeric antigen receptor (CAR). In some embodiments, the cells are further engineered to express an HLA Class I molecule. In some embodiments, the cells are further engineered to disrupt one or more immune checkpoint genes. In some embodiments, the cells are further engineered to affect immune cloaking by disrupt cell surface expression of HLA Class I and Class II proteins via disrupting certain HLA-related genes. In some embodiments, the cells described herein are genetically modified to express a B2M-HLA Class I gene fusion. These multiple genome engineering steps can occur sequentially in any order or can occur simultaneously.

[0114] A typical chimeric antigen receptor (CAR) comprises an extracellular domain comprising an antigen binding region, a transmembrane domain and one or more intracellular activation (costimulatory) domains. In some embodiments, the CAR also comprises a hinge domain. In some embodiments, the CAR also comprises a leader peptide (also known as a signal peptide) directing the CAR to the cell membrane. The signal peptide is cleaved off during the transport of the CAR through the endoplasmic reticulum. In some embodiments, the anti-CD19 CAR expression cassette contains an EFla promoter, FMC63 scFv, 4-1BB costimulatory domain, CD3^ activation domain, and CD8 secretion peptide, hinge and transmembrane domains (FIG. 2).

[0115] The CAR disclosed herein comprises an extracellular domain comprising an antigen binding region targeting CD 19. In some embodiments, the antigen binding region is derived from an antibody. In some embodiments, the antigen binding region is derived from a monoclonal antibody. In some embodiments, the antigen binding region comprises a single-chain variable fragment (scFv). An scFv comprises a variable region of an antibody light chain (VL) linked to a variable region of an antibody heavy chain (VH). In some embodiments, the VL is linked to the VH via a peptide linker.Attorney Docket Number: CBI061.30

[0116] A peptide linker generally comprises from about 5 to about 40 amino acids. The linker can be a naturally occurring sequence or an engineered sequence. For example, in some embodiments, the linker is derived from a human protein, e.g., an immunoglobulin selected from IgG, IgA, I IgD, IgE, or IgM. In some embodiments, the linker comprises 5-40 amino acids from the CHI, CH2, or CH3 domain of an immunoglobulin heavy chain. In some embodiments, the linker is a glycine and serine rich linker having the sequence (GxSy)n. Additional linker examples and sequences are disclosed in the U.S. Patent No. 5,525,491 Serine-rich peptide linkers, U.S. Patent No. 5,482,858 Polypeptide linkers for production of biosynthetic proteins, and a publication W02014087010 Improved polypeptides directed against IgE. In some embodiments, the peptide linker comprises one or more units or repeats of G3S (SEQ ID NO: 19) or G4S (SEQ ID NO: 20).

[0117] In some embodiments, the CAR comprises the scFv “FMC63” whose sequence is disclosed in Nicholson et al., Construction and characterization of a functional CD19 specific single chain Fv fragment for immunotherapy ofB lineage leukaemia and lymphoma, Mol. Immunol., 1997, 34: 1157.

[0118] In some embodiments, the CAR also comprises a hinge domain and the hinge domain is derived from CD8 or CD28 proteins.

[0119] In some embodiments, the CAR comprises a signal peptide (a signal sequence) that enables trafficking of the CAR to the cell membrane. In some embodiments, the signal sequence comprises a CD28 signal sequence. In some embodiments, the signal sequence consists essentially of a CD28 signal sequence.

[0120] In some embodiments, the transmembrane domain of the CAR is derived from a membrane - bound or transmembrane protein. In some embodiments, the transmembrane domain is derived from the same protein as the co-stimulatory domains described below. For example, the transmembrane domain of the CAR may be the transmembrane domain of a T cell receptor alpha-chain or beta-chain, a CD3- zeta chain, CD28, CD3-epsilon chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, ICOS, CD 154, DNAM1, NKp44, NKp46, NKG2D, 2B4, or GITR. In some embodiments, the transmembrane domain is the CD 8 a transmembrane domain. In some embodiments, the transmembrane domain is the CD28 transmembrane domain.

[0121] The cytoplasmic or intracellular signaling domain also referred to as the co-stimulatory domain of a CAR is responsible for activation of one or more effector functions of the immune cell expressing the CAR. In some embodiments, the co-stimulatory domain of the CAR comprises a part of or the entire sequence of the TCR ^ chain, CD3 chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, ICOS / CD278, IL-2RP / CD122, IL-2Ra / CD132, DAP10, DAP12, DNAM1, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, CD40 or a combination thereof. In some embodiments, the co-stimulatory domain of the CAR consists of a CD28 co-stimulatory domain. In some embodiments, the coAttorney Docket Number: CBI061.30 stimulatory domain of the CAR consists of a 4-1BB co-stimulatory domain. In some embodiments, the co-stimulatory domain of the CAR consists of a CD3s co-stimulatory domain. In some embodiments, the co-stimulatory domain of the CAR is a combination of domains. In some embodiments, the co- stimulatory domain of the CAR consists of a CD3s and a CD28 co-stimulatory domains. In some embodiments, the co-stimulatory domain of the CAR consists of a CD28 and a IL36y co-stimulatory domains. In some embodiments, the CAR comprises a P2A peptide cleavage site. In some embodiments, the cytoplasmic domain comprises a 4- IBB co-stimulatory domain and a CD3 C, chain.

[0122] In some embodiments, the CAR comprises FMC63 scFv, 4-1BB costimulatory domain, CD3^ activation domain, and CD8 secretion peptide (cleaved from the mature CAR present in the cell membrane), CD 8 hinge and CD 8 transmembrane domains (FIG. 2).

[0123] In some embodiments, the CAR is fully human or is humanized to reduce immunogenicity in human patients. In some embodiments, the CAR sequence is optimized for codon usage in human cells.

[0124] The nucleic acid encoding the CAR may be introduced into a cell as a genomic DNA sequence or a cDNA sequence. The cDNA sequence comprises an open reading frame for the translation of the protein (e.g., CAR) and in some embodiments, the cDNA further comprises untranslated elements that improve for example, the stability or the rate of translation of the CAR mRNA. In some embodiments, the CAR-encoding expression cassette is introduced into a cell via an AAV6-based construct (SEQ ID NO: 11).

[0125] In some embodiments, the CD 19 -targeting engineered immune cells comprise three or more genome edits. In some embodiments, at one of the three or more genome edits comprise a simultaneous gene disruption and gene insertion whereby a gene is disrupted by insertion of an expression cassette for a different gene. In some embodiments, the CD19-targeting engineered immune cells comprise five genome edits. In some embodiments, the CD19-targeting engineered immune cells comprise five genome edits of which two genome edits comprise a simultaneous gene disruption and gene insertion. Although the genome edits are referred hereto as “the first,” “the second,” etc., one of skill in the art would appreciate that this terminology does not reflect the chronological order of the genome edits. It is contemplated that the edits can be performed in any order and two or more and up to all five genome edits can be performed simultaneously.

[0126] In some embodiments, the first genome edit comprises insertion of the chimeric antigen receptor (CAR) coding sequence. In some embodiments, the first genome edit comprises simultaneous disruption of the TRAC gene and insertion of the CAR into the TRAC locus. In some embodiments, the polynucleotide encoding the CD 19 CAR is inserted into an exon of the TRAC locus. In some embodiments, the polynucleotide encoding the CD 19 CAR (e.g., a transgene) is inserted into an exon of the TRAC locus in either the forward or reverse orientation (e.g., relative to the 5’ to 3’- direction of theAttorney Docket Number: CBI061.30 endogenous TRAC gene). In some other embodiments, the polynucleotide encoding the CD 19 CAR is inserted into an exon of PDCD1 , TIGIT, B2M, or CIITA loci, in either the forward or reverse orientation. In some embodiments, the CAR transgene contains an EFl a promoter, MND promoter, or alternative suitable promoters. In some embodiments, the CAR transgene utilizes the endogenous promoter of the target gene.

[0127] In some embodiments, the CAR coding sequence is inserted into the cellular genome into the endogenous T cell receptor alpha chain (TRAC) gene. In some embodiments, the CAR is inserted into the TRAC locus on chromosome 14 in the target sequence shown in Table 1. In some embodiments, the TRAC locus is targeted by a CRISPR-Cas endonuclease. In some embodiments, the endonuclease comprises Casl2a and a guide polynucleotide has the sequence shown in Table 1. In Table 1, “r” prior to the nucleotide designation (e.g., rA) indicates a ribonucleotide. The absence of “r” prior to the nucleotide designation (e.g., A) indicates a deoxyribonucleotide.

[0128] In some embodiments, the second genome edit comprises insertion of the B2M-HLA-E coding sequence. In some embodiments, the second genome edit comprises simultaneous disruption of the B2M gene and insertion of the polynucleotide encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein (e.g., a transgene) into the B2M locus. In some embodiments, the polynucleotideAttorney Docket Number: CBI061.30 encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein is inserted into an exon of the B2M locus. In some embodiments, the polynucleotide encoding the CD 19 CAR is inserted into an exon of the B2M locus in either the forward or reverse orientation (e.g. , relative to the 5 ’ to 3 direction of the endogenous B2M gene). In some embodiments, the B2M-HLA-E or B2M-HLA-E-HLA-G transgene contains an EFla promoter, MND promoter, or alternative suitable promoters. In some embodiments, the fusion protein encoding transgene utilizes the endogenous promoter of the B2M gene.

[0129] In some embodiments, the CD19-targeting engineered immune cells comprise a genome modification resulting in armoring of the cells against an attack by the immune system of a recipient (host) of the allogeneic immune cells (immune cells derived from a donor). In some embodiments, the armoring modification comprises protection from recognition by the cytotoxic T cells of the host. Cytotoxic T cells recognize MHC Class I antigens. An MHC Class I molecule is a cell surface molecule comprised of beta-2 microglobulin (B2M) associated with heavy chains of HLA Class I proteins (the class consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G). The native B2M-HLA Class I complex on the surface of the allogeneic cell may be recognized by cytotoxic CD8+T cells of the host and, if HLA Class I molecule is recognized as non-self, the allogeneic cell is killed by the T cells. In some embodiments, the CD19-targeting engineered immune cells of the invention comprise an armoring genomic modification comprising a disruption of the B2M gene and therefore, disruption of the entire MHC Class I cell surface-bound complex. This disruption eliminates the MHC Class I antigen recognition that normally stimulates a cytotoxic T cell attack.

[0130] In some embodiments, the armoring genome modification in the CD19-targeting engineered immune cells comprises a disruption of the B2M gene and an insertion of a coding sequence for a protein comprising a fusion between a minimally polymorphic or non-polymorphic HLA Class I protein selected from HLA-E, HLA-G, HLA-F and combinations (fusions) thereof and the beta-2 - microglobulin (B2M) peptide, see, e.g., Gomalusse et al., HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells, Nat. Biotechnol., 2017, 35:765-772. In some embodiment, the fusion is between HLA-E and B2M. In some embodiments, the fusion between HLA- E and B2M further comprises HLA-G.

[0131] In the embodiments herein, the descriptions of B2M fusion to any combinations of HLA-E, HLA-G, and HLA-F is not to be construed as representing the specific order of the fusion components. For example, a reference to a “B2M-HLA-E-HLA-G fusion protein” can have the portions of the HLA- E and HLA-G in different ordering (from the amino to carboxy direction) to produce the intended physiological effect of the fusion construct.

[0132] In some embodiments, the B2M-HLA-E (and optional HLA-G) fusion is inserted into the target sequence in the B2M gene shown in Table 1. In some embodiments, the B2M locus is targeted byAttorney Docket Number: CBI061.30 a CRISPR-Cas endonuclease. In some embodiments, the endonuclease comprises Casl2a and a guide polynucleotide has sequence shown in Table 1.

[0133] In some embodiments, the B2M-HLA-E fusion expression cassette is introduced into a cell via an AAV6-based vector construct (SEQ ID NO: 12).

[0134] In some embodiments, the armoring genome modification in the CD 19 -targeting engineered immune cells comprises disruption of the CIITA gene. Class II major histocompatibility complex transactivator (CIITA) gene encodes the C2TA protein which is a positive regulator and a “master control factor” of MHC Class II (in humans HLA Class II) genes. Inhibiting or eliminating transcription of the master regulator gene results in reduced expression of the HLA Class II genes capable of activating the patient’s immune system to react against allogeneic CAR-T cells.

[0135] Insertion of the B2M-HLA-E fusion into the B2M locus described herein and optionally, inactivation of the CIITA gene may provide an in vivo survival advantage to T cells (including CAR-T cells) comprising the fusion compared to T cells or CAR-T cells not having the fusion or compared to T cells or CAR-T cells having the wild-type B2M locus and optionally, the wild-type CIITA locus.

[0136] In some embodiments, survival advantage may be assessed by coculturing the T cells or CAR- T cells having the B2M-HLA-E fusion inserted into the B2M locus with natural killer (NK) cells. In some embodiments, a control experiment includes coculturing the T cells or CAR-T cells having wildtype B2M locus, or genetically disrupted B2M locus with no B2M-HLA-E fusion transgene inserted, with natural killer (NK) cells. In some embodiments, survival advantage may be assessed by coculturing the T cells or CAR-T cells with the CIITA gene disruption with non-HLA matched cytotoxic and / or helper T cells and / or PBMCs. In some embodiments, a control experiment includes coculturing the T cells or CAR-T cells having wild-type CIITA locus with non-HLA matched cytotoxic T cells. The percentage or number of live or dead T cells or CAR-T cells in the coculture is assessed. The percentage or number of live or dead T cells or CAR-T cells in the coculture is assessed. In some embodiments, survival advantage due to the armoring genome modifications is assessed by comparing the percentage or number of live or dead T cells or CAR-T cells in the wild-type and genome-modified cell cocultures. In some embodiments, the percentage of specific lysis is calculated using the aforementioned parameters. In some embodiments, the activation signature of T cells and / or NK cells is evaluated by single or co-expression of CD25 and CD69.

[0137] In some embodiments, the genome modification comprises or further comprises transcriptionally silencing or disrupting of one or more immune checkpoint genes. In some embodiments, the immune checkpoint gene is selected from the group consisting of PD-1 (encoded by the PDCDI gene), CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4. InAttorney Docket Number: CBI061.30 some embodiments, the CD19-targeting engineered immune cells comprise genomic disruption of the checkpoint genes PDCD1 and TIGIT.

[0138] In some embodiments, the silenced or disrupted immune checkpoint gene is PDCD1. Programmed cell death protein 1 (PD-1, encoded by the gene PDCDP), also known as CD279, is a cell surface receptor that plays an important role in downregulating the immune system, and promoting selftolerance by suppressing T cell inflammatory activity. PD-1 binds to its cognate ligand, “programmed death-ligand 1,” also known as PD-L1, CD274, and B7 homolog 1 (B7-H1). PD-1 guards against autoimmunity through a dual mechanism of promoting programmed cell death (apoptosis) in antigenspecific T cells in lymph nodes, while simultaneously reducing apoptosis in anti-inflammatory, suppressive T cells (regulatory T cells). Through these mechanisms, PD-1 binding of PD-L1 inhibits the immune system, thus preventing autoimmune disorders, but also prevents the immune system from killing cancer cells. Accordingly, mutating or knocking out production of PD- 1 (e.g., by disrupting the PDCD1 gene) can be beneficial in T cell therapies.

[0139] Inhibiting expression of PD-1 by disrupting the PDCD1 gene as described herein results in increased antitumor activity of the CD 19 -targeting engineered immune cells as compared to T cells or CAR-T cells having a wild-type PDCD1. Without being bound by a particular theory, inventors attribute the increased antitumor activity at least in part to reduced inhibition by PD-1 ligand PD-L1 expressed by the tumor.

[0140] In some embodiments, the silenced or disrupted immune checkpoint gene is TIGIT. T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is expressed by activated CD8+T and CD4+T cells, natural killer (NK) cells, regulatory T cells (Tregs), and follicular T helper cells. High levels of TIGIT expression in CAR-T cells are associated with poor response to CAR-T cell therapy. Jackson et al., Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD 19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma, Cancer Discov., 2022, 12(8): 1886.

[0141] In some embodiments, two or more immune checkpoint genes are disrupted. In some embodiments, two or more immune checkpoint genes are PDCD1 and TIGIT.Endonucleases

[0142] In some embodiments, the gene disruption is accomplished using an endonuclease that specifically cleaves nucleic acid strands within a target sequence of the gene to be disrupted.

[0143] The strand cleavage by the sequence -specific endonuclease results in nucleic acid strand breaks that may be repaired by non-homologous end joining (NHEJ). NHEJ is an imperfect repair process that may result in direct re-ligation but more often, results in deletion, insertion, or substitution of one or more nucleotides in the target sequence. Such deletions, insertions, or substitutions of one or moreAttorney Docket Number: CBI061.30 nucleotides in the target sequence may result in missense or nonsense mutations in the protein coding sequence and eliminate production of any protein or cause production of a non-functional protein.

[0144] In some embodiments, one or more of the coding sequences described herein are introduced into the genome of the cell with the aid of a sequence -specific endonuclease. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease encoded by the CRISPR locus. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus is found many prokaryotic genomes and provides resistance to invasion of foreign nucleic acids. Structure, nomenclature and classification of CRISPR loci are reviewed in Koonin et al., Discovery of diverse CRISP R-Cas systems and expansion of the genome editing toolbox. Biochemistry, 2023, 62:3465.

[0145] Briefly, a typical CRISPR locus includes a number of short repeats regularly interspaced with spacers. The CRISPR locus also includes coding sequences for CRISPR-associated (Cas) genes. A spacer-repeat sequence unit encodes a CRISPR RNA (crRNA). In vivo, a mature crRNAs are processed from a polycistronic transcript referred to as pre-crRNA or pre-crRNA array. The repeats in the pre-crRNA array are recognized by Cas-encoded proteins that bind to and cleave the repeats liberating mature crRNAs. CRISPR systems perform cleavage of a target nucleic acid wherein Cas proteins and crRNA form a CRISPR ribonucleoproteins (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell) and the Cas nuclease proteins cleave the target nucleic acid.

[0146] Type I CRISPR systems include means for processing the pre-crRNA array that include a multi-protein complex called Cascade (CRISPR-associated complex for antiviral defense) comprised of subunits CasA, B, C, D and E. The Cascade-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. The bound nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of target nucleic acid.

[0147] Type II CRISPR systems include a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a crRNA repeat in the pre-crRNA array and recruits endogenous RNaselll to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with a nuclease, e.g., Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease, for target nucleic acid cleavage.

[0148] Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre-crRNA array with the help of one or more CRISPR polymerase-like proteins.

[0149] Type VI CRISPR systems comprise a different set of Cas-like genes, including Csfl, Csf2, Csf3 and Csf4 which are distant homologues of Cas genes in Type I-III CRISPR systems.Attorney Docket Number: CBI061.30

[0150] Type V CRISPR systems are classified into several different subtypes, including, e.g., N- , V- B, V-C, V-D, V-E, V-F, V-G, V-H, V-I, V-J, V-K and V-U. See, e.g., Koonin etal., Biochemistry, 2023, 62:3465; and Pausch et al., Science, 2020, 369(6501):333-337. The V-A subtype encodes the Casl2a protein (formerly known as Cpfl). Casl2a has a RuvC-like nuclease domain that is homologous to the respective domain of Cas9 but lacks the HNH nuclease domain that is present in Cas9 proteins. Type V systems can comprise a single crRNA sufficient for targeting of the Casl2 to a target site, or a crRNA-tracrRNA guide pair for targeting of the Casl2 to a target site.

[0151] In some embodiments, the CRISPR endonuclease, e.g., Cas9 or Casl2a, is fused to a nuclear localization sequence for efficient delivery of the CRISPR endonuclease, such as in a complex with the NATNA, to the nuclease. In some embodiments, the nuclear localization sequence is fused to the amino terminus, carboxy terminus, or both the amino and carboxy terminus. In some embodiments, 2 or more nuclear localization sequences are used.

[0152] CRISPR endonucleases require a nucleic acid targeting nucleic acid (NATNA) also known as guide RNAs. The endonuclease is capable of forming a ribonucleoprotein complex (RNP) with one or more guide RNAs. In some embodiments, the endonuclease is a Type II CRISPR endonuclease and NATNA comprises tracrRNA and crRNA.

[0153] In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, a NATNA can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some instances, a nucleic acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order.

[0154] In some embodiments, the guide nucleic acid-targeting nucleic acid can comprise a single guide NATNA. The NATNA comprises a spacer sequence which can be engineered to hybridize to the target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, NATNA can have a spacer extension and a tracrRNA extension. These elements can include elements that can contribute to stability of NATNA. The CRISPR repeat and the tracrRNA sequence can interact, to form a base-paired, double-stranded structure. The structure can facilitate binding of the endonuclease to the NATNA.

[0155] In some embodiments, the single guide NATNA comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge facilitates recruitment of the endonuclease to the NATNA. The bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence. The last pairedAttorney Docket Number: CBI061.30 nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA. The mid-tracrRNA can comprise one or more additional hairpins.

[0156] In some embodiments, the NATNA can comprise a double guide nucleic acid structure. The double guide NATNA comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The double guide NATNA does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNA sequence comprises a 5' tracrRNA sequence and the double guide NATNAs can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.

[0157] In some embodiments, NATNA is an engineered guide RNA comprising one or more DNA residues (CRISPR hybrid RNA-DNA or chRDNA). In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNA for use with a Type II CRISPR system may be composed of two strands forming a secondary structure that includes an activating region composed of an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. Other chRDNA may be a single guide D(R)NA for use with a Type II CRISPR system comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. For example, the targeting region may comprise DNA or a mixture of DNA and RNA, and an activating region may comprise RNA or a mixture of DNA and RNA.

[0158] In some embodiments, CRISPR Type V systems described in the International Application Pub. No. WO2022086846 (DNA-containing polynucleotides and guides for CRISPR Type V systems, and methods of making and using the same) are used. In some embodiments, the CRISPR hybrid RNA- DNA (chRDNA) comprises a targeting region capable of hybridizing to a desired locus in the genome is located 5’ of the activating region capable of interacting with the CRISPR endonuclease. In some embodiments, Casl2a chRDNA sequences listed in Table 1 are used.

[0159] In some embodiments, the CRISPR system comprises a nucleic acid-guided endonuclease and nucleic acid-targeting nucleic acid (NATNA) guides (e.g., a CRISPR guide RNAs selected from tracrRNA, crRNA or a single guide RNA incorporating the elements of the tracrRNA and crRNA in a single molecule). In some embodiments, the components of the CRISPR system are introduced into the cells (e.g., T cell, NK cell, a macrophage or a precursor of said cell types) in the form of nucleic acids. In some embodiments, the components of the CRISPR system are introduced into the cells in the form of DNA coding for the nucleic acid-guided endonuclease and NATNA guides. In some embodiments,Attorney Docket Number: CBI061.30 the gene coding for the nucleic acid-guided endonuclease (e.g., a CRISPR endonuclease selected from Cas9 and Casl2a) is inserted into a plasmid capable of propagating in the target cell. In some embodiments, the gene coding for the NATNA guides is inserted into a plasmid capable of propagating in the target cell.

[0160] In some embodiments, the nucleic acid-guided endonuclease and NATNA guides are introduced into the target cells (e.g., T cell, NK cell, a macrophage or a precursor of said cell types) in the form of RNA, e.g., the mRNA coding for the nucleic acid-guided endonuclease along with the NATNA guides.

[0161] In some embodiments, the nucleic acid-guided endonuclease and the NATNA guides are introduced into the target cells (e.g., T cell, NK cell, a macrophage or a precursor of said cell types) as a preassembled nucleoprotein complex. In some embodiments, the nucleic acid-guided endonuclease and the NATNA guides are introduced into the target cells via any combination of different means, e.g., the endonuclease is introduced as DNA via a plasmid containing the gene encoding the endonuclease while the guides are introduced as RNA (or RNA containing DNA nucleotides or other chemical modifications).

[0162] In some embodiments, the nucleic acids encoding the nucleic acid-guided endonuclease and NATNA guides are introduced into the cells via electroporation.

[0163] In some embodiments, the nucleic acids coding for the nucleic acid-guided endonuclease are introduced into cells in the form of mRNA via electroporation or viral pseudo-transduction as described e.g., in the U.S. patent No. 10,584,352.

[0164] In some embodiments, the endonuclease used to introduce one or more of the genetic modifications described herein (e.g., gene inactivation or insertion of the CAR-coding sequences, armoring sequences such as B2M-HLA protein fusions) into the genome of a cell is a restriction endonuclease, e.g., a Type II restriction endonuclease.

[0165] In some embodiments, the endonuclease used to introduce one or more of the genetic modifications described herein is a catalytically inactive CRISPR endonuclease (e.g., catalytically inactive Cas9 or Casl2a) conjugated to the cleavage domain of the restriction endonuclease Fok I. (see e.g., Guilinger, J. P., et al., Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nature biotechnology, 2014, 32(6), 577-582.

[0166] In some embodiments the endonuclease used to introduce one or more of the genetic modifications described herein is a zinc finger nuclease (ZFN), or a ZFN-Fok I fusion. In such embodiments, the target sequence is about 22-52 bases long and comprises a pair of ZFN recognition sequences, each 9-18 nucleotides long, separated by a spacer, which is 4-18 nucleotides long. (See e.g. .,Attorney Docket Number: CBI061.30Kim Y.G., et al., Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci USA., 1996, 93(3): 1156-1160.

[0167] In some embodiments, the endonuclease used to introduce one or more of the genetic modifications described herein is a transcription activator-like effector nuclease (TALEN), or a TALEN-Fok I fusion. In such embodiments, the target sequence is about 48-85 nucleotides long and comprises a pair of TALEN recognition sequences, each 18-30 bases long, separated by a spacer, which is 12-25 bases long. (See, e.g., Christian M. et al., Targeting DNA double-strand breaks with TAL effector nucleases, Genetics, 2010, 186 (2): 757-61.

[0168] In some embodiments, the sequence-specific endonuclease is selected from a rare-cutting restriction enzyme, a TALEN, a Zinc-finger nuclease (ZFN) and a CRISPR endonuclease.

[0169] In some embodiments, the TRAC locus is cleaved within the target sequence shown in Table 1. In some embodiments, the TRAC locus is targeted by a CRISPR-Cas endonuclease (e.g., Casl2a) and a guide polynucleotide (NATNA) “TRAC chRDNA” having the sequence shown in Table 1.

[0170] In some embodiments, the B2M gene is cleaved within the target sequence shown in Table 1. In some embodiments, the B2M locus is targeted by a CRISPR-Cas endonuclease (e.g., Casl2a) and a guide polynucleotide (NATNA) “B2M chRDNA” having the sequence shown in Table 1.

[0171] In some embodiments, the PDCD1 gene is disrupted by cleavage of the PDCD1 locus within the target sequence shown in Table 1. In some embodiments, the PDCD1 locus is targeted by a CRISPR-Cas endonuclease (e.g., Casl2a) and a guide polynucleotide (NATNA) “PDCD1 chRDNA” having the sequence shown in Table 1.

[0172] In some embodiments, the TIGIT gene is disrupted by cleavage of the TIGIT locus within the target sequence shown in Table 1. In some embodiments, the TIGIT locus is targeted by a CRISPR-Cas endonuclease (e.g., Casl2a) and a guide polynucleotide (NATNA) “TIGIT chRDNA” having the sequence shown in Table 1.

[0173] In some embodiments, the CIITA gene is disrupted by cleavage of the CIITA locus within the target sequence shown in Table 1. In some embodiments, the CIITA locus is targeted by a CRISPR-Cas endonuclease (e.g., Casl2a) and a guide polynucleotide (NATNA) “CIITA chRDNA” having the sequence shown in Table 1.

[0174] In some embodiments, the invention comprises a step of introducing an engineered coding sequence for an engineered protein into a cell. In some embodiments, the engineered protein is an antiCD 19 chimeric antigen receptor (CAR). In some embodiments, the engineered protein is a fusion between beta-2 microglobulin (B2M) and an HLA Class I protein such as HLA-E, HLA-G or a combination of HLA-E and HLA-G. In some embodiments, the nucleic acid encoding the engineeredAttorney Docket Number: CBI061.30 protein is introduced into a target cell where expression of the protein is desired. In some embodiments, the introduced nucleic acid is selected from an expression vector containing the coding sequence, an mRNA encoding the protein, and a delivery vector containing the coding sequence to be inserted into the cellular genome. In some embodiments, the target cells are contacted with the nucleic acid encoding the protein in vitro, in vivo or ex vivo.

[0175] In some embodiments, protein-coding nucleic acid sequences (e.g., CAR-coding sequences or sequences coding for the immune -cloaking B2M-HLA fusion protein) are introduced into a cell such as T cell, NK cell, a macrophage or a precursor of said cell types. In some embodiments, the “naked” nucleic acids are introduced into lymphocytes by electroporation as described in, e.g., U.S. Patent No. 6,410,319.

[0176] In some embodiments, the vector used to deliver the protein-coding nucleic acid is a viral vector (e.g., a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector). Suitable vectors are non-replicating in the target cells. In some embodiments, the vector is selected from or designed based on AAV, SV40, EBV, HSV, or BPV. The vector incorporates the protein expression sequences. In some embodiments, the expression sequences are codon-optimized for expression in mammalian cells. In some embodiments, the vector also incorporates regulatory sequences including transcriptional activator binding sequences, transcriptional repressor binding sequences, enhancers, introns, and the like. In some embodiments, the viral vector supplies a constitutive or an inducible promoter. In some embodiments, the promoter is selected from EFla, PGK1, MND, Ubc, CAG, CaMKIIa, and P-Actin promoter. In some embodiments, the promoter is selected from the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, the -interferon promoter, the hsp70 promoter and EF-la promoter. In some embodiments, the promoter is the EF-la promoter. In some embodiments, the viral vector supplies a transcription terminator. In some embodiments, the vector is a plasmid selected from a prokaryotic plasmid, a eukaryotic plasmid, and a shuttle plasmid.

[0177] In some embodiments, the engineered gene is expressed in a eukaryotic cell, such as a mammalian or human T cell, NK cell, a macrophage or a precursor of said cell types, and the vector is a plasmid comprising a eukaryotic promoter active in the desired cell type, a secretion signal, a polyadenylation signal, a stop codon, and, optionally, one or more regulatory elements such as enhancer elements.

[0178] In some embodiments, the expression vector comprises polyadenylation signals. In some embodiments, the polyadenylation sites are SV-40 polyadenylation signals.Attorney Docket Number: CBI061.30

[0179] In some embodiments, the coding sequence of the engineered protein is introduced into the cells via an AAV vector (e.g., AAV6) or any other suitable viral vector capable of delivering an adequate payload. In some embodiments, to facilitate homologous recombination (homology-directed repair or HR pathway), the coding sequence is joined to homology arms comprising sequences capable of hybridizing to genomic sequences located 5’ (upstream) and 3’ (downstream) of the cleavage site or the desired insertion site in the genome. In some embodiments, the homology arms are about 500 bp long. See Eyquem J., et al., Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection, Nature, 2017, 543: 113-117.

[0180] In some embodiments, the sequence coding for the CAR (“CAR expression cassette”) is flanked by the homology arms capable of hybridizing to the TRAC locus. In some embodiments, the sequence coding for the B2M-HLA-E fusion protein (“B2M-HLA-E expression cassette”) is flanked by the homology arms capable of hybridizing to the B2M locus. The expression cassette flanked by homology arms is cloned into a viral vector plasmid. The plasmid is used to package the sequences into a virus.

[0181] In some embodiments, the CAR expression cassette flanked by TRAC homology arms is delivered via an AAV6-based construct comprising SEQ ID NO: 11. In some embodiments, the B2M- HLA-E fusion expression cassette flanked by 2A7 homology arms is delivered via an AAV6-based construct comprising SEQ ID NO: 12.

[0182] In some embodiment, the cells such as T cells, NK cells, macrophages or precursors of said cell types are contacted with a viral vector so that the genetic material delivered by the vector is integrated into the genome of the target cell and then expressed in the cell or on the cell surface. Transduced and transfected cells can be tested to confirm transgene expression using methods well known in the art such as fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. For example, the cells can be tested by staining or by flow cytometry with antibodies specific for the engineered gene.

[0183] The present invention involves manipulating nucleic acids, including genomic DNA and plasmid DNA that were isolated or extracted from a sample. Methods of nucleic acid extraction are well known in the art. See J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, N.Y.). A variety of reagent and kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples, including products from BD Biosciences (San Jose, Cal.), Clontech (TaKaRa Bio ); Epicentre Technologies (Madison, Wise.); Gentra Systems, (Minneapolis, Minn.); Qiagen (Valencia, Cal.); Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Cal.); KAPA Biosystems (Roche Sequencing Solutions, Pleasanton, Cal.) and more.Attorney Docket Number: CBI061.30

[0184] In some embodiments, the invention involves intermediate purification or separation steps for nucleic acids, e.g., to remove unused reactants from the DNA. The purification or separation may be performed by a size selection method selected from gel electrophoresis, affinity chromatography and size exclusion chromatography. In some embodiments, size selection can be performed using Solid Phase Reversible Immobilization (SPRI) technology from Beckman Coulter (Brea, Cal.).

[0185] In some embodiments, a quality control measure assessing one or more properties of the CD 19- targeting engineered immune cells is applied to the cells prior to administering the cells to a patient.

[0186] In some embodiments, the assessed property is the presence of the CAR in the cellular genome. The presence of the CAR in the cellular genome may be assessed by a method selected from nucleic acid hybridization, nucleic acid sequencing and specific amplification including polymerase chain reaction (PCR), quantitative PCR (qPCR), real-time PCR (rtPCR) and droplet digital PCR (ddPCR). In some embodiments, the presence of the CAR in the cellular genome is assessed by ddPCR with amplification primers specific for one or both CAR insertion sites.

[0187] In some embodiments, the assessed property is surface expression of the CAR. The surface expression of the CAR may be assessed by fluorescence-activated cell sorting (FACS), microfluidics- based screening, ELISA, or Western blot. In some embodiments, the surface expression of the CAR is assessed by flow cytometry with an anti-FAB2 antibody. In some embodiments, the CAR-T cell population with the highest surface expression of the CAR is selected for administration to a patient.

[0188] In some embodiments, the fraction of cells harboring the CAR in the genome or the fraction of cells expressing the CAR on the cell surface is used to determine the total number of cells constituting a therapeutically effective dose.

[0189] In some embodiments, the properties of the CD19-targeting engineered immune cells are assessed in vitro and are selected from antigen-dependent lysis of antigen-expressing target cells (antigen-specific lysis); proliferation in the presence of antigen-expressing target cells (antigendependent proliferation); and cytokine secretion in the presence of antigen-expressing target cells, cell exhaustion and the presence of a memory cell phenotype.

[0190] In some embodiments, the in vitro assessment of the CD19-targeting engineered immune cells utilizes target cells or target cell lines. In some embodiments, the target cells are tumor cells selected from primary tumor cells and established tumor cell lines. In some embodiments, the tumor cells are known to express the specific antigen for the CAR-T cell, i.e., the tumor cells express CD19 recognized by the anti-CD19 CAR-T cells. In some embodiments, the tumor cell lines represent likely clinical targets for the anti-CD19 CAR-T cells such as JeKo-1 cell line (MCL) or JVM 13 cell line (CLL). In some embodiments, a control cell line, identical to the test cell line but lacking the CD 19 antigen on theAttorney Docket Number: CBI061.30 cell membrane is used. In some embodiments, the control cell line harbors an inactivated gene coding for CD19 (a CD19 KO cell line) or a cell line known to lack surface expression of CD19.

[0191] In some embodiments, the assessed property of the CD19-targeting engineered immune cells is antigen-dependent lysis of antigen-harboring target cells. The antigen-dependent cell lysis may be assessed by co-culturing the population comprising the engineered anti-CD19 CAR-T cells described herein (effector cells or effectors) with CD 19 expressing target cells (targets). The co-culture may be established at different effector:target ration (E:T ratios). In some embodiments, the E:T ratios are in the range of about 0. 1 and about 10. In some embodiments, two or more E:T ratios in the selected range are evaluated. In some embodiments, cell lysis is detected by labeling target cells with cell permeant stable fluorescent dyes (e.g., CellTrace™ Violet (CTV), ThermoFisher Scientific, Carlsbad, Cal.). The fraction of live target cells was determined by incorporation of the viability dye by effector cells. In some embodiments, a control experiment measures lysis of target cells lacking the antigen.

[0192] In some embodiments, the CD19-targeting engineered immune cell population effecting the highest percentage of specific target cell lysis is selected for administration to a patient. In some embodiments, the cell population effecting a high percentage of specific target cell lysis but having low non-specific target cell lysis is selected for administration to a patient.

[0193] In some embodiments, the assessed property of the CD19-targeting engineered immune cells is antigen-dependent proliferation of the cells. Proliferation may be assessed by co-culturing a population comprising engineered anti-CD19 CAR-T cells (effectors, E) with a CD19-expressing target cells (targets, T). In some embodiments, the co-culture is at E:T ratio of about 1. In some embodiments, cell proliferation is detected by labeling CAR-T cells with cell permeant stable fluorescent dyes (e.g., CellTrace™ Violet) and measuring dye dilution within the CAR-T cell population. In some embodiments, the CAR-T cell population exhibiting the highest rate of proliferation in the presence of target cells is selected for administration to a patient.

[0194] In some embodiments, the assessed property is cytokine secretion by the CD19-targeting engineered immune cells. In some embodiments, secretion of one or more cytokines is assessed. The one or more cytokines are selected from gamma-interferon (IFNy), tumor necrosis factor alpha (TNFa), IL-2, IL-4, IL-6, and non-cytokine molecules Granzyme A, Granzyme B, and perforin. Cytokine secretion may be assessed by co-culturing a population comprising the engineered anti-CD19 CAR-T cells (effectors, E) with CD19-expressing target cells (targets, T). In some embodiments, the co-culture is at E:T ratio of about 1. In some embodiments, the cytokines in the co-culture supernatant can be detected or quantitatively detected by an antibody-based or antibody conjugate-based assay such as Western blotting or ELISA and similar secondary antibody-based methods with colorimetric or fluorescent detection methods.Attorney Docket Number: CBI061.30

[0195] In some embodiments, the assessed property of the CD19-targeting engineered immune cells is T cell exhaustion. T cell exhaustion is characterized by expression of one or more of LAG-3, TIM-3, CTLA-4, the BLIMP- 1 transcription factor, and the TOX transcription factor. The expression of the one or more of the LAG-3, TIM-3, CTLA-4, BLIMP-1, and TOX may be assessed by assessing quantitatively or qualitatively, the presence of one or more of the above proteins or the mRNA encoding one or more of the above proteins. T cell exhaustion is also characterized by decreased metabolic fitness which may be assessed by measuring the rate of glycolysis or oxidative phosphorylation (mitochondrial respiration) or a ratio of glycolysis to oxidative phosphorylation over time.

[0196] The presence and amount of mRNA of the LAG-3, TIM-3, CTLA-4, BLIMP- 1, and TOX genes in the CD19-targeting engineered immune cells may be assessed by a method selected from nucleic acid hybridization, nucleic acid sequencing and specific amplification including reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), real-time RT- PCR (rtRT-PCR) and droplet digital RT-PCR (ddRT-PCR). In some embodiments, T cell exhaustion is assessed by assessing the presence and optionally, the amount of the one or more of the LAG-3, TIM-3, CTLA-4, BLIMP- 1, and TOX mRNAs is assessed by ddPCR with amplification primers specific for the mRNA being assessed.

[0197] The presence and amount of the one or more of the LAG-3, TIM-3, CTLA-4, BLIMP-1, and TOX proteins in the CD19-targeting engineered immune cells may be assessed by a method selected from flow cytometry inducing fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. In some embodiments, T cell exhaustion is assessed by assessing the presence and optionally, the amount of the one or more of the LAG-3, TIM-3, CTLA-4, BLIMP- 1 and TOX proteins by flow cytometry or FACS with an antibody or antibodies directed against said proteins.

[0198] The rate of glycolysis in the CD19-targeting engineered immune cells may be assessed by measuring mitochondrial respiration and glycolysis in the cells. In some embodiments, T cell exhaustion is assessed by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the cells by measuring the concentration of dissolved oxygen and free protons in the extracellular medium. Commercial analyzers of OCR and ECAR are available (e.g., from Agilent Technologies, Santa Clara, Cal.).

[0199] In some embodiments, the CD19-targeting engineered immune cell population with the lowest expression of exhaustion markers are selected for administration to a patient. In some embodiments, the cells with the lowest rate of glycolysis or the lowest ratio of glycolysis to mitochondrial respiration are selected for administration to a patient.Attorney Docket Number: CBI061.30

[0200] In some embodiments, the assessed property of the CD19-targeting engineered immune cells is T cell memory phenotype. The effector cell memory phenotype is characterized by the combination of cell surface markers comprising CCR7 CD45RA CD45RO CD62L CD27 . In some embodiments, the T cell memory phenotype is assessed by flow cytometry or FACS with antibodies directed against CCR7, CD45RA, CD45RO, CD62L, and CD27.

[0201] In some embodiments, the properties of the CD19-targeting engineered immune cells are assessed in vivo and are selected from affecting characteristics of experimental animals carrying target tumor cells. In some embodiments, the target cells are tumor cells known to express CD19 and experimental animals are mice engrafted with the tumor cells prior to being administered a dose of the CD19-targeting engineered immune cells. In some embodiments, the experimental animals are NGS mice engrafted with the JeKo-1 or the JVM 13 tumor cells. In some embodiments, the assessment of the CD19-targeting engineered immune cells comprises monitoring body weight, overall survival, and tumor burden of the mice engrafted with the tumor cells and administered a dose of the CD19-targeting engineered immune cells.

[0202] In some embodiments, the animals are engrafted with a fluorescently labeled tumor cells and tumor burden is assessed by measuring in vivo fluorescence (other mouse measurements).

[0203] In some embodiments, the CD19-targeting engineered immune cell population is selected for inclusion into the therapeutic composition described herein based on the properties detected during the assessment. In some embodiments, the invention comprises compositions including CD19-targeting engineered immune cells exhibiting an anti-tumor property. In some embodiments, the invention comprises compositions including the CD19-targeting engineered immune cells assessed for having one or more satisfactory property or a satisfactory level of one or more of the parameters selected from the group consisting of: the presence of the anti-CD19 CAR in the cellular genome, surface expression of the anti-CD19 CAR, antigen-dependent cytotoxicity, antigen-dependent proliferation, cytokine secretion, expression of T cell exhaustion markers, metabolic profile, expression of T cell memory markers, genomic disruption of one or more immune checkpoint genes, reduced transcription of one or more immune checkpoint genes, or the absence of a detectable transcript from one or more immune checkpoint genes, immune cloaking via disruption of one or more HLA Class I and HLA Class II related genes, reduced surface presentation of HLA Class I and HLA Class II proteins but with cell surface expression of a B2M-HLA Class I gene fusion.Compositions and Treatment

[0204] Once produced and (optionally) assessed for the desired properties, the CD19-targeting engineered immune cells can be formulated into compositions for delivery to a human subject to be treated. The compositions include the engineered lymphocytes, and one or more pharmaceuticallyAttorney Docket Number: CBI061.30 acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0205] In some embodiments, the composition further comprises an antimicrobial agent for preventing or deterring microbial growth. In some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0206] In some embodiments, the composition further comprises an antioxidant added to prevent the deterioration of the lymphocytes. In some embodiments, the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0207] In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates, sorbitan esters, lipids, such as phospholipids (lecithin and other phosphatidylcholines), phosphatidylethanolamines, fatty acids and fatty esters; steroids, such as cholesterol.

[0208] In some embodiments, the composition further comprises a freezing agent such as 3% to 12% dimethylsulfoxide (DMSO) or 1% to 5% human albumin.

[0209] The number of CAR-T cells in the composition will vary depending on a number of factors but will optimally comprise a therapeutically effective dose per vial. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the CAR-T cellcontaining composition in order to determine which amount produces a clinically desired endpoint.

[0210] In some embodiments, the total number of cells in the dose is adjusted based on the percentage of CAR-expressing cells among all the cells in the cell composition. In some embodiments, the totalAttorney Docket Number: CBI061.30 number of cells administered is multiplied by 100 / N where N is the percentage of CAR-expressing cells in the cell composition. The multiplication yields the total number of cells that must be administered to the patient in order to administer the desired number of CAR-expressing cells.

[0211] In some embodiments, the invention is a method of treating, preventing, or ameliorating a disease associated with expression of CD 19 comprising administering a population of immune cells (CAR-T cells, CAR-expressing macrophages or CAR-NK cells) expressing the anti-CD19 CAR inserted into the TRAC gene, lacking expression of one or more of the genes selected from the group consisting of CIITA, PDCD1 and TIGIT and further comprising a B2M-HLA-E fusion gene inserted into the B2M gene.

[0212] In some embodiments, the population of immune cells administered to a patient has been assessed for having a satisfactory property or a satisfactory level of a parameter selected from one or more of: the presence of the CAR in the cellular genome, surface expression of the CAR, antigendependent cytotoxicity, antigen-dependent proliferation, cytokine secretion, reduced cell surface expression of native HLA Class I and Class II proteins, surface expression of B2M-HLA Class I fusion, expression of T cell exhaustion markers, metabolic profde and expression of T cell memory markers.

[0213] In some embodiments, the diseases or conditions that can be treated by the CD19-targeting engineered immune cells of the disclosure include various B cell malignancies characterized by expression of CD19 on the tumor cell surface. These include, but are not limited to, chronic lymphocytic leukemia, small lymphocytic leukemia, Richter transformation, mantel cell lymphoma, diffuse large B cell lymphoma, large B cell lymphoma, follicular lymphoma, transformed follicular lymphoma, high grade B cell lymphoma, primary mediastinal B cell lymphoma, marginal zone lymphoma, transformed marginal zone lymphoma, Burkitt lymphoma, Hairy cell leukemia, and acute lymphocytic leukemia.

[0214] In some embodiments, the diseases or conditions that can be treated by the anti-CD19 immune cells of the disclosure include autoimmune diseases characterized by B cell involvement, such as the presence of auto-reactive B cells. These include, but are not limited to, lupus nephritis, extrarenal lupus (Systemic lupus erythematosus), multiple sclerosis, rheumatoid arthritis, type I diabetes, systemic sclerosis, idiopathic inflammatory myopathies, stiff person syndrome, Anti-Neutrophil Cytoplasmic antibody (ANCA)-associated vasculitis, dermatomyositis, or polymyositis.

[0215] In some embodiments, the invention comprises a method of administering to a subject or patient a therapeutically effective number of CD19-targeting engineered immune cells described herein. In some embodiments, the immune cells are pre-activated and expanded prior to administration. In some embodiments, the administration of the immune cells according to the invention results in treating, preventing, or ameliorating the disease or condition in the subject or patient. In someAttorney Docket Number: CBI061.30 embodiments, the disease or disorder is selected from cancers or tumors and autoimmune conditions that can be treated by administration CD19-targeting engineered immune cells described herein.

[0216] A pharmaceutical composition comprising the CD19-targeting engineered immune cells described herein can be delivered via various routes and delivery methods such as local or systemic delivery, including parenteral delivery, intramuscular, intravenous, subcutaneous, intrathecal or intradermal delivery.

[0217] In some embodiments, the composition of the present invention is administered to a subject who has been preconditioned with an immunodepleting (e.g., lymphodepleting) therapy. In some embodiments, preconditioning is with lymphodepleting agents, including combinations of cyclosporine and / or fludarabine.

[0218] In some embodiments, the composition or formulation for administering to the patient is a pharmaceutical composition or formulation which permits the biological activity of an active ingredient and contains only non-toxic additional components such as pharmaceutically acceptable carriers. In some embodiments, pharmaceutically acceptable carriers include buffers, excipients, stabilizers, and preservatives.

[0219] In some embodiments, a preservative is used. In some embodiments, the preservative comprises one or more of methylparaben, propylparaben, sodium benzoate, benzalkonium chloride, antioxidants, chelating agents, parabens, chlorobutanol, phenol, and sorbic acid. In some embodiments, the preservative is present at about 0.0001% to about 2% by weight of the total composition.

[0220] In some embodiments, a carrier is used. In some embodiments, the carrier comprises a buffer, antioxidants including ascorbic acid and methionine; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0221] In some embodiments, the carrier comprises a buffer. In some embodiments, the buffer comprises citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, the buffer is present at about 0.001% to about 4% by weight of the total composition.

[0222] In some embodiments, the pharmaceutical composition delivery systems such that the delivery of the composition occurs overtime. In such embodiments the pharmaceutical composition comprises release-timing components. In some embodiments, the pharmaceutical composition comprises aluminum monostearate or gelatin. In some embodiments, the pharmaceutical composition comprisesAttorney Docket Number: CBI061.30 semipermeable matrices of solid hydrophobic polymers. In some embodiments, the matrices are in the form of fdms or microcapsules.

[0223] In some embodiments, the pharmaceutical composition comprises a sterile liquid such as an isotonic aqueous solution, suspension, emulsion, dispersions, or viscous composition, which may be buffered to a selected pH. In some embodiments, the pharmaceutical composition is a sterile injectable solution prepared by incorporating the cells in a solvent such as sterile water, physiological saline, or solutions or glucose, dextrose, or the like. In some embodiments, the pharmaceutical composition further comprises dispersing, or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.

[0224] In some embodiments, the CD 19 -targeting engineered immune cell described herein is administered in an amount sufficient to treat the disease or indication. In some embodiments, the dosage of the of the CD19-targeting engineered immune cell ranges from about 40 x 106CAR+cells / dose to 360 x 106CAR+cells / dose. In some embodiments, the CD19-targeting engineered immune cells are administered in a single dose. In some embodiments, the CD 19 -targeting engineered immune cells are administered in 2 or more doses.

[0225] In some embodiments, CD19-targeting engineered immune cells described herein are coadministered with cytokines. In some embodiments, the cytokines are selected from IL-2, IL- 15 and IL- 21. In some embodiments, the cytokines are administered at a dose per kg of body weight of a human that is equivalent to 10 ng / mouse for IL-15, 100,000 units / mouse for IL-2, and 10 pg / mouse for IL-21.

[0226] In some embodiments, the patients are assessed or selected for treatment by a diagnostic test to determine whether the patient is likely to benefit from treatment with the anti-CD19 immune cells described herein or not likely to benefit from the treatment. In some embodiments, the diagnostic test is administered prior to the treatment and is used to selecting or recommending the patient for the treatment.

[0227] In some embodiments, the invention comprises a method of treatment with the anti-CD19 immune cells described herein comprising a step of measuring expression of CD 19 in the cells of the tumor.

[0228] In some embodiments, the test is qualitative, i.e., detects the presence or absence of CD 19 expression (absence including any expression of CD19 below the level of detection). In some embodiments, the test is quantitative, i.e., detects the level of CD19 expression. In some embodiments, expression of CD 19 on the surface of the cells of the tumor is measured. In some embodiments, expression of CD 19 in the cells of the tumor are measured. In some embodiments, the samples forAttorney Docket Number: CBI061.30 assessment of CD 19 expression include blood samples, tissue samples, or histological samples, e.g., paraffin embedded tissue.

[0229] In some embodiments, the patient is selected for treatment with the CD 19 -targeting engineered immune cells described herein if CD 19 expression is detected and the patient is advised against the treatment with the anti-CD19 immune cells described herein if CD19 expression is not detected. In some embodiments, the patient is selected for treatment with the anti-CD19 immune cells described herein if CD 19 expression is high and the patient is advised against the treatment with the anti-CD19 immune cells described herein if CD 19 expression is low.

[0230] In some embodiments, a threshold of CD 19 expression is established. In some embodiments, the threshold is equal to a top quantile in the population, such as the top half, top quartile, top 10% and so on. One of skill in the art is able to evaluate responses to treatment with the anti-CD19 immune cells described herein in patients with various levels of CD 19 expression and determine which quantile is a threshold for CD 19 expression indicating the likelihood of a positive response to treatment with the anti-CD19 immune cells described herein.

[0231] In some embodiments, the patient is selected for treatment with the anti-CD19 immune cells described herein if CD 19 expression is at or above the threshold. In some embodiments, the patient is advised against the treatment with the anti-CD19 immune cells described herein if CD 19 expression is below the threshold.

[0232] Methods of quantitatively detecting protein expression in the cell, including on the cell surface are known in the art. Such methods include for example, immunohistochemistry, flow cytometry and enzyme-linked immunosorbent assay (ELISA). Anti -human CD 19 antibodies are available from multiple vendors including ThermoFisher Scientific, Miltenyi Biotech, BioLegend, BD Biosciences, Sony Biotechnology and more.

[0233] In some embodiments, expression of CD 19 in the cells of the tumor is measured by a method detecting the mRNA encoding the CD 19 protein. Such methods include for example, Northern blotting, fluorescent in-situ hybridization (FISH), and quantitative reverse -transcription polymerase chain reaction (qRT-PCR). In a preferred embodiment, the expression of CD 19 in cells of the tumor are measured by qRT-PCR.

[0234] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.EXAMPLESExample 1Anti-CD19 CAR-T cellsAttorney Docket Number: CBI061.30

[0235] For transfection, T cells were isolated from cryopreserved PBMCs (STEMCELL Technologies, Vancouver, Canada) using the RoboSep-S instrument and the EasySep Human T cell Isolation Kit (STEMCELL Technologies) and activated for 3 days in the presence of anti-CD3 / CD28 Dynabeads (ThermoFisher Scientific, San Jose, Cal.) in ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies) supplemented with 5% CTS immune cell serum replacement (ThermoFisher Scientific) and 100 IU rhIL-2 / mL (PeproTech, ThermoFisher Scientific). Beads were removed and the cells were expanded for 24 hours with rhIL-2 prior to nucleofection.

[0236] Genomic targets and corresponding CRISPR hybrid RNA-DNA (chRDNA) guides are listed in Table 1. The Casl2a-guide nucleoprotein complexes (NPCs) were formed by combining chRDNA guides with Casl2a protein at a ratio 1:3 Casl2a:guides in PlasmaLyte (Baxter Medical Supplies, Hayward, Cal.) mixed with water for 10 minutes at 4°C. The T cells were prepared in MaxCyte EPB1 electroporation buffer (MaxCyte, Inc., Rockville, Md.) at a concentration of 7.5 x 107cells / mL and incubated with NPC solution taking up 10% of cell volume for an additional 10 minutes at 4°C. T cells were nucleofected using the MaxCyte GTx electroporation system (MaxCyte).Attorney Docket Number: CBI061.30

[0237] The anti-CD19 CAR was delivered to the T cells via AAV6 carrying the CAR expression cassette flanked by TRAC homology arms (SEQ ID NO: 11). In some instances, genomic disruption of TRAC and / or insertion of the CAR is performed prior to T cell activation with anti-CD3 / anti-CD28 antibodies or beads. Alternative versions of this plasmid sequence may contain a longer linker region between the EFla promoter and the Kozak sequence and / or swapped left and right AAV ITRs. The anti-CD19 CAR expression cassette contains an EFla promoter, FMC63 scFv, 4- IBB costimulatory domain, CD3^ activation domain, and CD8 secretion peptide, hinge and transmembrane domains (FIG. 2). The AAV6 construct was added to the T cells at a multiplicity of infection (MOI) of 105- 4 x 105. Virus was removed 24 hours later and the cells were expanded until cryopreservation ~7-9 days later. Genetic knockouts were evaluated by next-generation sequencing (NGS) analysis of the targeted region using the NextSeq platform (Illumina, San Diego, Cal.). Genetic insertion of the CAR into the TRAC locus was evaluated by digital droplet PCR (ddPCR). TCR disruption was also evaluated by flow cytometry with a TCRaP-specific antibody IP26. CAR surface expression was evaluated using PE- labelled anti-FMC63 antibody (ACROBiosystems, Newark, Del.).Example 2Antigen-specific in vitro antitumor activity of CD 19 -targeting CAR-T cells

[0238] This example demonstrates anti -tumor activity of CD 19 targeting CAR-T cells against mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) cell lines.

[0239] CD19+JeKo-1 (MCL, ATCC CRL-2006) or JVM13 (CLL, ATCC CRL-2003) cells were labelled with Cell Trace Violet1 1(CTV). Anti-CD19 CAR-T cells (Example 1) were cocultured with CTV-labelled JeKo-1 or JVM13 cells at various effectortarget (E:T) ratios in triplicate for 24 or 48 hours. TRAC KO (no CAR inserted) T cells were used as negative control. Propidium iodide uptake was evaluated via flow cytometry to quantify target cell death and specific lysis was calculated relative to target only controls (0: 1 E:T). FIG. 3 shows in vitro antitumor activity against JeKo-1 (MCL) and FIG. 4 shows in vitro antitumor activity against JVM 13 (CLL) determined as specific lysis at 24hr and 48hr. Area under the curve (AUC) was calculated to represent overall specific lysis across all E:T ratios at each timepoint.Example 3Antigen-dependent proliferation of CD19-targeting CAR-T cells

[0240] TRAC KO or anti-CD19 CAR-T cells (Example 1) were labelled with Cell Trace Violet™ (CTV). CTV-labelled T cells were cultured for up to 72hrs in X-VIVO™ complete media (X-VIVO™ base media with 9% CTS serum replacement (Lonza Biologies, Hayward, Cal.), lx GlutaMAX, lOmM HEPES, lx antibiotic / antimycotic) with or without supplementation with 100 lU / mL rhIL-2 (PeproTech, ThermoFiusher Sceintific), or CD19+JeKo-1 cells. Proliferation was evaluated byAttorney Docket Number: CBI061.30 comparing mean fluorescence intensities (MFIs) of CTV relative to a 0 hr control, generating fold dye dilution (FDD) values. Results are shown in FIG. 5: in the absence of targets and IL-2, neither TRAC KO (light grey) or anti-CD19 CAR-T cells (black) proliferate as indicated by the left graph. In the absence of targets but in the presence of IL-2, the anti-CD19 CAR-T cells moderately proliferate. In the presence of CD19+targets, the anti-CD19 CAR-T cells proliferate, indicating a high FDD value relative to the TRAC KO negative control.Example 4Antigen-dependent cytokine secretion by CD19-targeting CAR-T cells.

[0241] TRAC KO or anti-CD19 CAR-T cells (Example 1) were cocultured with JeKo-1 or JVM13 cells at a 1 : 1 ratio for 24 hrs. Supernatant was collected and analyzed for presence of cytokines and cytolytic granules using custom multiplex ProcartaPlex™ kits (Thermo Fisher Scientific, San Jose, Cal.) and manufacturer-provided protocols. Results are shown in FIG. 6: secretion by TRAC KO negative control T cells (light grey bars) is shown relative to that of anti-CD19 CAR-T cells (black bars). Across all pro-inflammatory analytes, anti-CD19 CAR-T cells show CD19+target cell-mediated secretion.Example 5Serial rechallenge of CD19-targeting CAR-T cells with CD19+cells

[0242] CD19-targeting CAR-T cells along with a negative TRAC KO control (Example 1) were thawed and recovered in IL-2 containing media for 24 hours prior to the first stimulation. T cells were then plated in cocultures at different effector: target ratios with the CD19+Jekol-GFP:ffLuc target cell line. Every 72-96 hours, specific lysis was quantified via luciferase-based luminescence readings relative to a target cell only control. Following analyte measurement at each round, residual T cells were then restimulated with fresh JeKol-GFP:ffLuc target cells. Serial rechallenge was continued for five total rounds of stimulation. Results are shown in FIG. 7 as % specific lysis and Area Under the Curve (AUC). The AUC analyses were conducted to quantify overall specific lysis across all E:T ratios at each round and summed to generate a cumulative AUC value.Example 6Antitumor activity against primary tumor cells from CLL patients

[0243] Donor-derived CLL B cells were isolated from whole blood of CLL patients and cryopreserved. The CLL B cells were evaluated for CD 19 expression via flow cytometry. Antigen density was assessed by BD Quantibrite™ beads (Beckton Dickinson, Franklin Lakes, N.J.). The CD 19 antigen densities on the surface of the cells of these donors ranged from 103to 104mol / cell. The CLL B cells were labelled with Cell Trace Violet1 1(CTV). Anti-CD19 CAR-T cells or TRAC KO negativeAttorney Docket Number: CBI061.30 control T cells (Example 1) were cocultured with CTV-labelled B-CLL cells at various effectortarget (E:T) ratios in triplicate for 48 hours. Propidium iodide uptake was evaluated via flow cytometry to quantify target cell death and specific lysis was calculated relative to target only controls (0: 1 E:T). Results are shown in FIG. 8 as % specific lysis of primary tumor cells.Example 7Cytokine secretion in the presence of primary tumor cells from CLL patients

[0244] TRAC KO T cells or anti-CD19 CAR-T cells (Example 1) were cocultured with CLL B cells from five donors (Example 6) at a 1 : 1 ratio for 24 hours. Supernatant was collected and analyzed for presence of cytokines and cytolytic granules using custom multiplex ProcartaPlex™ kits (ThermoFisher Scientific, San Jose, Cal.) and manufacturer-provided protocols. Results are shown in FIG. 9. Lower limits of detection (LLOD) are indicated for each analyte.Example 8In vivo tumor control by CD 19 -targeting CAR-T cells

[0245] A. Intravenous delivery of tumor cells

[0246] JeKo-l-GFP:ffLuc target cells were injected intravenously (IV) at 5 x 105cells / animal in PBS. Seven days post-JeKo-1 engraftment, anti-CD19 CAR-T cells or TRAC KO T cells (Example 1) were dosed IV at 107CAR+T cells / animal in X-VIVO™ medium (Lonza Biologies, Hayward, Cal.). The CAR+percentage for the anti-CD19 CAR-T cells was 52.8%. A vehicle only control (X-VIVO1 7medium) and TRAC KO control were used as negative control. Bioluminescence imaging (BLI) was conducted 2x / week using the IVIS Spectrum System (Revvity, Inc., Waltham, Mass.) to assess changes in tumor size. Results are shown in FIG. 10. Individual BLI curves (n=6-8 mice / group) are shown as well as the average BLI per treatment group. A Kruskal-Wallis test was used and indicated no significant differences in BLI of TRAC KO-treated mice relative to vehicle but significant reduction in BLI by anti-CD19 CAR-T cell-treated mice (pO.OOOl). Area under the curve (AUC) analyses from day 0 through day 22 BLIs were conducted, and a Kruskal-Wallis test was used to find a significant difference in AUC only in anti-CD19 CAR-T treated mice (p<0.0001). Animals were euthanized after losing >20% of their body weight compared to day 0 or after developing paralysis. Kaplan -Meier survival curves at any time (Mantel-Cox log -rank test) were developed (FIG. 10).

[0247] B. Subcutaneous delivery of tumor cells.

[0248] JeKo-l-GFP:ffLuc target cells were delivered subcutaneously (SC) at 5 x 105cells / animal in a 1: 1 mix of PBS: Cultrex. Eight days post-JeKo-1 engraftment, anti-CD19 CAR-T cells (Example 1) were dosed intravenously at 107CAR+T cells / animal in X-VIVO™ medium. The CAR percentage for the anti-CD19 CAR+T cells was 52.8%. A vehicle only (X-VIVO™) control and a TRAC KO controlAttorney Docket Number: CBI061.30 were used as negative controls. Tumor volume was assessed by caliper measurement 2x / week. Results are shown in FIG. 11. Individual tumor growth curves (n=6-8 mice / group) are shown as well as the average tumor volume per treatment group. Average tumor volume at day 28 was compared using a Kruskal -Wallis test and indicated a significant difference in day 28 tumor volume only in anti-CD19 CAR-T treated mice (p=0.0066).Example 9Immune checkpoint gene expression

[0249] The CD19-targeting CAR-T cells (anti-CD19 CAR, TRAC KO) (Example 1) were subjected to a serial rechallenge assay with CD19+Jekol-GFP:ffLuc target cell line essentially as described in Example 5. Expression of TIGIT and PDCD1 at the 1:8 effector: target ratio was assessed by flow cytometry with antibodies A15153G and EH12.1 respectively. Results are shown in FIG. 12.Example 10TIGIT and PDCD1 -deficient CD19-targeting CAR-T cells

[0250] Genome editing of CAR-T cells with Casl2a and chRDNAs was performed essentially as described in Example 1 utilizing the TIGIT and PDCD1 -specific chRDNAs and TIGIT and PDCD1 genomic targets listed in Table 1. In some instances, genomic disruption of PDCD1 and / or TIGIT is performed prior to T cell activation with anti-CD3 / anti-CD28 antibodies or beads. Genomic knockouts were confirmed by NGS as described in Example 1. Protein disruption was also evaluated by flow cytometry as described in Example 9.Example 11Antigen-dependent proliferation of TIGIT and PDCD1 KO CD19-targeting CAR-T cells during a serial rechallenge

[0251] PDCD1 and TIGIT YQ anti-CD19 CAR-T cells (Example 10) were thawed and recovered in IL-2 containing media for 48 hours prior to the first stimulation. T cells were plated in a scaled-up serial rechallenge assay with CD19+Jekol-GFP:ffLuc target cells in flasks at an E:T ratio of 1:8 or 1:2. Every 72-96 hours, cultures were counted and evaluated for GFP+target cell counts and CAR+T cell counts via flow cytometry. Results are shown in FIG. 13 (target cell counts and CAR-T cell counts). Data are shown for the first two rounds of restimulation. Target cell counts (top graphs) of anti-CD19 CAR-T cells (black) at both E:T ratios were compared to that of PD-l / TIGIT KO anti-CD19 CAR-T cells (light grey).Example 12Immune-cloaked CD19-targeting CAR-T cells with CIITA KO, B2M KO, and B2M-HLA-E overexpression (prophetic)Attorney Docket Number: CBI061.30

[0252] Genome editing of CAR-T cells with Casl2a and chRDNAs is performed essentially as described in Examples 1 and 10 utilizing the CUT A and 52A -specific chRDNAs and CUT A and B2M genomic targets listed in Table 1. Additionally, site-specific insertion of a B2M-HLA-E fusion protein into the B2M locus is performed using AAV6 carrying the B2M-HLA-E expression cassette flanked by 2 / V homology arms (SEQ ID NO: 12) essentially as described in Example 1 but at an MOI of 2.5 x 105. In some instances, genomic disruption of TRAC and / or CIITA and / or B2M and insertion of the B2M-HLA-E fusion cassette is performed prior to T cell activation with anti-CD3 / anti-CD28 antibodies or beads. Genomic knockouts of B2M and CIITA are confirmed by NGS as described in Example 1. Genomic insertion of the B2M-HLA-E fusion into the B2M locus is evaluated by ddPCR as described in Example 1. Successful immune cloaking via the B2M and CIITA disruption in CAR-T cells is further evaluated by flow cytometric staining (antibodies listed in Table 2) of HLA class I molecules (HLA-A, B, C) and HLA class II molecules (HLA-DR, DP, DQ) respectively. Expression of the B2M-HLA-E fusion in CAR-T cells is evaluated by flow cytometric staining of B2M and HLA-E proteins (antibodies listed in Table 2).

[0253] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

[0254] For all purposes, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.Attorney Docket Number: CBI061.30APPENDIX

[0255] LIST OF SEQ ID NOs. AND SEQUENCES IN THE SEQUENCE LISTINGAtorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Attorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Attorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Attorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Attorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30Atorney Docket Number: CBI061.30

Claims

Attorney Docket Number: CBI061.30CLAIMSWhat is claimed is:

1. An engineered cell comprising: a genetic disruption of TRAC,' a genetic disruption of one or more immune checkpoint genes; a genetic disruption of B2M,' reduced expression or absence of HLA Class II; and a polynucleotide encoding a fusion protein comprising B2M-HLA-E or B2M-HLAE-HLA-G.

2. The engineered cell of claim 1, wherein the genetic disruption of one or more immune checkpoint genes is selected from PDCDI, CTLA-4, LAGS, TIMS, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B.

3. The engineered cell of claim 2, wherein the genetic disruption of one or more immune checkpoint genes is PDCD1 and TIGIT.

4. The engineered cell of any one of claims 1-3, wherein the reduced or absence of HLA Class II is a genetic disruption of one or more of genes selected from CITTA, RFXANK, RFX5, and RFXAP.

5. The engineered cell of claim 4, wherein the genetic disruption is of CITTA .

6. The engineered cell of any one of claims 1-5, wherein the engineered cell comprises: a genetic disruption of TRAC,' a genetic disruption of PDCD1 ; a genetic disruption of TIGIT, a genetic disruption of B2M,' a genetic disruption of CITTA,, and a polynucleotide encoding the B2M-HLA-E fusion protein or B2M-HLA-E-HLA-G fusion protein.

7. The engineered cell of any one of claims 1-6, wherein the polynucleotide encoding the B2M-HLA-E fusion protein or the B2M-HLA-E-HLA-G fusion protein is inserted into the B2M, PDCD1, TIGIT, or CIITA locus.

8. The engineered cell of any one of claims 1-7, wherein the encoded B2M-HLA-E fusion protein comprises the amino acid sequence of SEQ ID NO: 14, and the B2M-HLA-E-HLA-G fusion protein comprises the amino acid sequence of SEQ ID NO: 15.

9. The engineered cell of any one of clams 1-8, wherein the polynucleotide encoding the B2M-HLA-E fusion protein comprises the polynucleotide sequence comprising SEQ ID NO: 17, andAttorney Docket Number: CBI061.30 wherein the polynucleotide encoding the B2M-HLA-E-HLA-G fusion comprises the polynucleotide sequence comprising SEQ ID NO: 12 or 16.

10. The engineered cell of any one of claims 1-9, having the characteristics of:TRAC ;PDCD1TIGIT :B2MCIITA and positive for expression of B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein.

11. The engineered cell of claim 10, further having the characteristics of native HLA Class 1" and / or native HLA Class II .

12. The engineered cell of any one of claims 1-11, wherein the engineered cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR).

13. The engineered cell of any one of claims 1-12, wherein the polynucleotide encoding the chimeric antigen receptor is inserted into the TRAC, B2M, PDCD1, TIGIT, or CIITA locus.

14. The engineered cell of any one of claims 1-13, wherein the cell is a rodent or human cell.

15. The engineered cell of any one of claims 1-14, wherein the cell comprises an immune cell.

16. The engineered cell of claim 15, wherein the immune cell is selected from a T cell, a natural killer (NK) cell, a macrophage and precursors thereof.

17. The engineered cell of any one of claims 1-16 wherein the cell is a population of the engineered cells.

18. An engineered immune cell comprising: a polynucleotide encoding a CD19-targeting chimeric antigen receptor (CAR) protein inserted into the T cell receptor alpha chain (TRAC) gene locus; a polynucleotide encoding a B2M-HLA-E fusion protein inserted into the B2M gene locus; inhibition of one or more immune checkpoints; and inhibition of HLA Class II expression.

19. The engineered immune cell of claim 18, selected from the group consisting of a T cell, a natural killer (NK) cell, a macrophage and precursors thereof.Attorney Docket Number: CBI061.3020. The engineered immune cell of claim 19 or 20, wherein the inhibition of one or more immune checkpoint comprises genomic disruption of one or more immune checkpoint genes selected from PDCD1, CTLA-4, LAGS, TIMS, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B.

21. The engineered immune cell of claim 20, comprising genetic disruption of PDCD1 gene and TIGIT gene.

22. The engineered immune cell of claim 21, wherein the PDCD1 gene is disrupted within or near SEQ ID NO: 3.

23. The engineered immune cell of claim 21, wherein the TIGIT gene is disrupted within or near SEQ ID NO: 4.

24. The engineered immune cell of any one of claims 18-23, wherein the inhibition of HLA Class II expression is via genomic disruption of the CIITA gene.

25. The engineered immune cell of claim 24, wherein the CIITA gene is disrupted within or near SEQ ID NO: 5.

26. The engineered immune cell of any one of claims 18-25, wherein the sequence encoding the CAR is inserted into the TRAC gene within or near SEQ ID NO: 1.

27. The engineered immune cell of any one of claims 18-26, wherein the sequence encoding the B2M-HLA-E fusion is inserted into the B2M gene within or near SEQ ID NO: 2.

28. The engineered immune cell of any one of claims 18-27, wherein the CD19-targeting CAR comprises a FMC63 scFv, a 4-1BB costimulatory domain, a CD3^ activation domain, a CD8 hinge, and a CD 8 transmembrane domain.

29. The engineered immune cell of any one of claims 18-28, comprising: a polynucleotide encoding a CD 19 CAR; a genetic disruption of TRAC,' a genetic disruption of B2M,' a genetic disruption of PDCD1 ; a genetic disruption of TIGIT, a genetic disruption of CIITA, ' and a polynucleotide encoding a B2M-HLA-E fusion protein or B2M-HLA-E-HLA-G fusion protein.

30. The engineered immune cell of any one of claims 18-29, wherein the encoded CD19 CAR comprises the amino acid sequence of SEQ ID NO: 13.Attorney Docket Number: CBI061.3031. The engineered immune cell of any one of claims 18-30, wherein the polynucleotide encoding the CD 19 CAR comprises the polynucleotide sequence comprising SEQ ID NO: 11 or SEQ ID NO: 18.

32. The engineered immune cell of any one of claims 18-31, wherein the encoded B2M- HLA-E fusion protein comprises the amino acid sequence of SEQ ID NO: 15, and the encoded B2M- HLA-E-HLA-G fusion protein comprises the amino acid sequence of SEQ ID NO: 14.

33. The engineered immune cell of any one of claims 18-32, wherein the polynucleotide encoding the B2M-HLA-E fusion protein comprises the polynucleotide sequence comprising SEQ ID NO: 17, and the polynucleotide encoding the B2M-HLA-E-HLA-G fusion protein comprises the polynucleotide sequence comprising SEQ ID NO: 12 or 16.

34. The engineered immune cell of any one of claims 18-29, having the characteristics of: CD19 CAR+;TRAC ;B2MPDCD1TIGIT :CIITA and fusion protein B2M-HLA-E+or B2M-HLA-E-HLA-G+.

35. A method of making the engineered immune cell of any one of claims 18-34, the method comprising introducing into a cell one or more endonucleases capable of cleaving the TRAC locus, the B2M locus, the PDCD1 locus, the TIGIT locus, and the CIITA locus.

36. The method of claim 35, wherein the endonuclease is a CRISPR endonuclease and further comprising introducing into the cell nucleic acid targeting nucleic acids (NATNAs) capable of hybridizing to target sequences within the TRAC locus, the B2M locus, the PDCD1 locus, the TIGIT locus and the CIITA locus.

37. The method of claim 36, wherein the CRISPR endonuclease is Casl2a and the NATNAs are SEQ ID NOs.: 6, 7, 8, 9, and 10.

38. The method of claim 36, wherein the CRISPR endonuclease is selected from Cas9, Casl2a, and CASCADE.

39. The method of any one of claims 36-38, wherein the CRISPR endonuclease and the NATNAs are introduced into the cell in the form of a nucleoprotein complex (NPC).Attorney Docket Number: CBI061.3040. The method of claim 35, wherein the endonuclease is selected from a zinc finger nuclease (ZFN), a ZFN-Fok I fusion, a transcription activator-like effector nuclease (TALEN), and a TALEN-Fok I fusion.

41. The method of any one of claims 35-40, further comprising introducing into a cell a vector construct comprising an expression cassette encoding the CD19-targeting CAR and an expression cassette encoding the B2M-HLA-E or B2M-HLA-E-HLA-G fusion protein.

42. The method of claim 41, wherein the vector construct comprises AAV6.

43. The method of claim 42, wherein the vector construct comprising an expression cassette encoding the CD19-targeting CAR comprises SEQ ID NO: 11 or 18.

44. The method of claim 42, wherein the vector construct comprising an expression cassette encoding the B2M-HLA-E fusion protein comprises SEQ ID NO: 17, and wherein the vector construct comprising an expression cassette encoding the B2M-HLA-E-HLA-G fusion protein comprises SEQ ID NO: 12 or 16.

45. A composition comprising a population of the engineered immune cells of any one of claims 18-34, and a pharmaceutically acceptable excipient.

46. The composition of claim 45, further comprising a freezing agent selected from 3% to 12% dimethylsulfoxide (DMSO) and l% to 5% human albumin.

47. A method of inhibiting the growth or function of CD19-expressing cells in a patient comprising, administering to a patient in need thereof an effective amount of the CD19-targeting CAR engineered immune cells of any one of claims 18-34, or the composition of claim 45 or 46.

48. The method of claim 47, wherein the CD19-expressing cells are B cells.

49. The method of claim 47 or 48, wherein the administering is selected from intravenous delivery, parenteral delivery, intramuscular delivery, subcutaneous delivery, intrathecal delivery, intratumoral delivery, or intraperitoneal delivery.

50. The method of any one of claims 47-49, further comprising administering a cytokine to the patient.

51. The method of claim 50, wherein the cytokine is selected from IL-2, IL- 15 and IL-21.

52. The method of claim 47, further comprising, prior to administering to the patient the engineered immune cells or composition thereof, preconditioning the patient with lymphodepleting agents comprising cyclophosphamide and / or fludarabine.Attorney Docket Number: CBI061.3053. The method of claim 47, further comprising, prior to administering to the patient the composition comprising the engineered immune cells, applying to the immune cells a quality control measure comprising assessing one or more properties selected from: presence of the CAR in the cellular genome, surface expression of the CAR,CD19-dependent lysis of CD19-expressing target cells, proliferation in the presence of CD19-expressing target cells, cytokine secretion in the presence of CD19-expressing target cells, disruption of one or more immune checkpoints, inhibition of the native HLA Class 1 expression, inhibition of the native HLA Class 2 expression,T cell exhaustion, and the presence of a memory cell phenotype.

54. The method of claim 53, wherein the engineered immune cell population with expression of the CCR7, CD45RA, CD45RO, CD62L, and CD27 is selected for administration to the patient.

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