Engineered cells with reduced gene expression to mitigate immune cell recognition

Abstract

Provided herein are engineered immune cells and populations thereof for administration to a subject to treat cancer (e.g., solid or liquid tumors) and other conditions. In particular, the cells are engineered to functionally express a reduced level of one or more of CD48, CD58, ICAM-1, RFX5, NLRC5, TAP2, [beta] 2m, TRAC, RFXAP, CIITA, and RFXANK. The cells are optionally further engineered to express one or more additional proteins, such as antigen binding proteins (e.g., chimeric antigen receptor (CAR) or T cell receptor) and / or CD70 binding proteins, to target tumor cells or other damaged cells in the subject, and / or to express other genes at reduced levels. Also provided are methods of making and using the engineered cells, compositions and kits comprising the engineered cells, and methods of treatment by administering the cells and the compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 393,350 filed on July 29, 2022 and U.S. Provisional Application No. 63 / 509,136 filed on June 20, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing, which is electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on July 13, 2023, named AT-053_03WO_SL.xml, and is 98,186 bytes in size. Technical Field

[0005] The present disclosure generally relates to the use of engineered immune cells (eg, T cells) for therapeutic applications. Background Art

[0006] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens has shown promise as a new approach to treating cancer (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). Immune cells can be genetically modified to express chimeric antigen receptors (CARs), i.e., fusion proteins comprising an antigen recognition portion and a T cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993)). Immune cells containing CARs, such as CAR-T cells (CAR-T), are engineered to confer antigen specificity while maintaining or enhancing their ability to recognize and kill target cells.

[0007] However, the production of autologous cell therapy modified by CAR is expensive, requiring weeks of process and quality testing, and produces products of different effectiveness according to the initial quality and quantity of the patient-specific T cells used. Allogeneic CAR-modified cell therapy—wherein cells from healthy donors are modified to express CAR and then administered to multiple patients—is expected to be a cheaper and more robust product than autologous therapy, which can be delivered immediately when needed (see, for example, Graham et al., Cells 2018, 7, 155; doi: 10.3390 / cells7100155). In addition, allogeneic therapy can be selected based on the desired product characteristics (e.g., gene editing efficiency, integration site, no harmful off-target gene editing, haplotype, etc.), and promotes more complex cell engineering (e.g., multi-gene editing improves effectiveness, persistence, homing, etc.). The main obstacle to realizing allogeneic CAR-modified cell therapy is the potential rejection of the patient's (host) immune system to the product (based on donor cells).

[0008] Killer lymphocytes such as CD8+T cells and natural killer (NK) cells identify and kill cancer cells, virus-infected cells, and foreign cells (including allogeneic cells) that are different from themselves. The main determinant of self-discrimination and non-self-discrimination is the major histocompatibility complex (MHC) molecules expressed on the surface of all nucleated cells. Each MHC class I molecule is a non-covalent trimeric complex of highly multi-allelic MHC class I heavy chains (the most common of which is HLA-A2), constant β2 microglobulin (β2m), and presenting peptides, which are derived from proteins expressed internally by proteolysis. In general, MHC I molecules are loaded with peptides representing the entire diversity of proteins expressed within the cell, enabling cancer cells and infected cells to 'announce' their predicament to the immune system. CD8+T cells recognize these non-self peptides through the T cell receptor (TCR) unique to each newborn T cell, and initiate killing based on the recognition of non-self peptide-MHC (see, for example, Z. et al. Nature 320, 232–238 (1986). T cell recognition of non-self is complemented by NK cell recognition of “loss of self”: inhibitory receptors on the surface of NK cells enable NK cell-mediated killing in the absence of MHC (see, e.g., K. et al., Nature 319, 675–678 (1986); see Figures 1 and 2 of PCT / US2022 / 14393 Figure 3, which is incorporated herein by reference in its entirety). Experimental ablation of MHC class I presentation—achieved by CRISPR / Cas9-mediated genomic knockout (KO) of the β2m gene encoding the universal β2m component of MHC class I—results in potent NK activation and selective killing of MHC-deficient T cells in the context of autologous and alloreactive responses (see PCT / US2022 / 14393). Figure 2 , which is incorporated herein by reference in its entirety).

[0009] Although allogeneic cell therapy has advantages over autologous cell therapy, allogeneic cells face potential rejection by host or recipient immune system cells that are reactive with T and NK epitope determinants on the surface of allogeneic cell products that are different from the host. Methods for avoiding allogeneic therapeutic cell rejection by downregulating or eliminating the expression of HLA molecules on the surface of allogeneic cells have been described (see, e.g., Lanza, R. et al., Engineering universal cells that evade immune detection. Nat. Rev. Immunol., December 2019; 19(12): 723-733, electronic publication on August 15, 2019).

[0010] Ablation of MHC class I molecules, for example by deletion of the β-2 microglobulin gene (β2m), prevents recognition and rejection by CD8 T cells, but the absence of MHC I is a strong signal for NK cell reactivity, which can lead to acute rejection of these cells. Downregulation of MHC molecules by knocking down β2m expression or inactivating genes involved in antigen processing and presentation can potentially avoid NK cell reactivity and reduce but not completely eliminate rejection by cytotoxic CD8 T cells. These modifications, typically achieved using gene editing tools or shRNA technology, can be combined with exogenous overexpression of NK cell and / or T cell inhibitory proteins (e.g., CD47 or PDL1), which can further reduce the degree of rejection but is not completely effective (Deuse et al., The SIRPα–CD47 immune checkpoint in NK cells. J Exp Med (2021) 218(3):e20200839; Han et al., Generation of hypoimmunogenic human pluripotent stem cells. PNAS, April 30, 2019, 116(21):10441-10446).

[0011] The present disclosure provides the advantages of improved allogeneic therapy, which provides increased persistence of administered cells regardless of the recipient's natural defenses. Summary of the invention

[0012] Provided herein are immune cells that have been engineered, such as genetically engineered, to mitigate rejection by a host or recipient into which the cells have been introduced, methods of mitigating rejection and / or recognition by the host or recipient's immune system, such as T cells and / or NK cells, compositions and populations comprising these engineered cells, and methods of using these engineered cells to treat cancer in a patient.

[0013] In one aspect, the present disclosure provides an engineered immune cell, such as a CAR T cell, or an engineered immune cell colony comprising an engineered immune cell, wherein the engineered immune cells include one or more functionally weakened or reduced genomic modifications of the expression of one or more targets described herein. In one embodiment, the engineered immune cell includes a genomic modification, which functionally weakens or reduces (i) RFX5 and / or NLRC5 and (ii) CD58 expression relative to a cell without the genomic modification. In another embodiment, the genomic modification includes (i) RFX5 and / or NLRC5 and / or (ii) CD58 knocking down and / or knocking out. In another embodiment, the genomic modification is included in one or more modifications at the locus of (i) RFX5 and / or NLRC5 and (ii) CD58. In one embodiment, the genomic modification is included in a deletion or insertion at the locus of (i) RFX5 and / or NLRC5 and (ii) CD58. In other embodiments, the genome modification is selected from the group consisting of: (i) insertion of one or more nucleotides, (ii) insertion of a polynucleotide sequence encoding a protein, (iii) deletion of one or more nucleotides, and (iv) substitution of one or more nucleotides. In other embodiments, the genome modification is introduced by a gene editing technology selected from TALEN, zinc finger, Cas-CLOVER and CRISPR / Cas system.

[0014] In one embodiment, the one or more genomic modifications are at the genomic location of one or more genes (corresponding to one or more targets as described herein), or at other locations within the genome other than at the location of the one or more genes (corresponding to one or more targets as described herein), such that the modifications functionally attenuate or reduce the expression of the one or more genes (corresponding to one or more targets as described herein).

[0015] In one embodiment, the genomic modification comprises the insertion of an RNA interference sequence. In another embodiment, the RNA interference sequence is a shRNA sequence, a siRNA sequence, or a miRNA sequence. In other embodiments, the RNA interference sequence comprises a sequence complementary to (i) RFX5 and / or NLRC5 and / or (ii) CD58 gene sequences.

[0016] In another embodiment, the engineered immune cell also includes a polynucleotide sequence encoding an antigen binding protein and / or a CD70 binding protein. In one embodiment, the antigen binding protein is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In another embodiment, the cell is further engineered to include one or more genome modifications, which functionally weaken or reduce one or more of TAP2, β2m, TRAC, CIITA, RFXAP, RFXANK, ICAM-1 and CD48 relative to unengineered cells.

[0017] In other embodiments, compared with immune cells that do not comprise the genomic modification, the engineered immune cell or the engineered immune cell colony comprising the engineered immune cell has improved persistence and / or improved resistance to alloreactive immune cell rejection. In one embodiment, alloreactive immune cell rejection includes rejection mediated by alloreactive T cells and / or rejection mediated by alloreactive natural killer (NK) cells. In other embodiments, the increased persistence is determined by mixed lymphocyte reaction (MLR) assay and / or is determined. In one embodiment, the improved resistance to alloreactive immune cell rejection is determined by MLR assay and / or is determined.

[0018] In other embodiments, the engineered immune cell comprises one or more genomic modifications that functionally weaken or reduce the expression of RFX5 and CD58. In one embodiment, the engineered immune cell comprises one or more genomic modifications that functionally weaken or reduce the expression of NLRC5 and CD58. In another embodiment, the engineered immune cell comprises one or more genomic modifications that functionally weaken or reduce the expression of RFX5, NLRC5 and CD58. In other embodiments, β2m is functionally expressed at a reduced level in the engineered immune cell. In another embodiment, the engineered immune cell comprises an unmodified β2m gene, and / or wherein β2m is not functionally expressed at a reduced level in the engineered immune cell.

[0019] In another embodiment, the engineered immune cell exhibits: (i) reduced levels of expression of MHC class I proteins or complexes at the cell surface, and / or (ii) reduced levels of expression of MHC class II proteins or complexes at the cell surface.

[0020] In one embodiment, the antigen binding protein is CAR. In other embodiments, the engineered immune cell expresses antigen binding proteins and / or CD70 binding proteins. In another embodiment, the polynucleotide sequence encoding the antigen binding protein and / or CD70 binding protein is located in the destroyed CD58, RFX5, NLRC5, ICAM-1, CD48, TAP2, β2m, TRAC, CIITA, RFXAP or RFXANK locus.

[0021] In other embodiments, the engineered immune cell further comprises one or more genomic modifications of the endogenous TCRα gene. In one embodiment, the engineered immune cell further comprises one or more genomic modifications of the endogenous CD52 gene.

[0022] In one other embodiment, the engineered immune cell is or is obtained from an immune cell of a healthy volunteer, is obtained from a patient, or is obtained from an induced pluripotent stem cell (iPSC). In other embodiments, the engineered immune cell is not a natural killer (NK) cell, or is not an NK cell obtained from a healthy volunteer or a patient. In another embodiment, the engineered immune cell is not obtained from an iPSC.

[0023] In one embodiment, the engineered immune cell or one or more engineered immune cells in the engineered immune cell population express or functionally express a CD70 binding protein. In other embodiments, the engineered immune cell comprises a polynucleotide sequence encoding a CD70 binding protein. In another embodiment, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In one embodiment, the CD70 binding domain constitutes a CD70 antibody, or a CD70 receptor or a CD70 binding fragment thereof. In other embodiments, the CD70 binding domain constitutes an anti-CD70 antibody, optionally the anti-CD70 antibody is a scFv. In another embodiment, the CD70 binding protein further comprises a hinge domain, optionally the hinge domain comprises a CD8 hinge. In one embodiment, the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of: a CD3z signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain or variants thereof. In another embodiment, the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a co-stimulatory domain. In other embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain. In another embodiment, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain. In other embodiments, the one or more intracellular domains comprise an amino acid sequence of one or more of SEQ ID NO: 1, 7-14, 17-31, 32-58, 59-70, or 89-90. In another embodiment, the CD70 binding protein does not include an intracellular signaling domain.

[0024] In one other embodiment, the engineered immune cell or one or more engineered immune cells in the engineered immune cell colony include unmodified β2m, RFX5, NLRC5, CIITA and TAP2 genes. In another embodiment, the expression of one or more of β2m, RFX5, NLRC5, CIITA and TAP2 is not functionally impaired or reduced in one or more engineered immune cells in the engineered immune cell or engineered immune cell colony. In one embodiment, the one or more genome modifications do not include genome modifications of one or more of β2m, RFX5, NLRC5, CIITA and TAP2.

[0025] In one aspect, the present disclosure provides an engineered immune cell population comprising one or more engineered immune cells described herein. In one embodiment, the population is characterized as having no more than 50% of the engineered immune cells functionally expressing (i) RFX5 and / or NLRC5 and (ii) CD58. In other embodiments, no more than 50% of the engineered immune cells functionally express RFX5 and CD58; or no more than 50% of the engineered immune cells functionally express NLRC5 and CD58; or no more than 50% of the engineered immune cells functionally express RFX5, NLRC5 and CD58. In other embodiments, no more than 50% of the engineered immune cells also functionally express a) any one or more of CD48, ICAM-1, TAP2, β2m, TRAC, CIITA, RFXAP and RFXANK; or b) only one of CD48 and ICAM-1; or c) both CD48 and ICAM-1.

[0026] In one embodiment, the engineered immune cell population comprises engineered immune cells, wherein at least 1% of the engineered immune cells functionally express (i) RFX5 and / or NLRC5 and (ii) CD58 at a level no greater than 50% of the expression level in unengineered immune cells. In another embodiment, at least 1% of the engineered immune cells functionally express RFX5 and CD58 at a level no greater than 50% of the expression level in unengineered immune cells. In one embodiment, at least 1% of the engineered immune cells functionally express NLRC5 and CD58 at a level no greater than 50% of the expression level in unengineered immune cells. In another embodiment, at least 1% of the engineered immune cells functionally express RFX5, NLRC5, and CD58 at a level no greater than 50% of the expression level in unengineered immune cells. In additional embodiments, at least 1% of the engineered immune cells functionally express a) any one or more of CD48, ICAM-1, TAP2, β2m, TRAC, CIITA, RFXAP and RFXANK; or b) only one of CD48 and ICAM-1; or c) both CD48 and ICAM-1, with expression levels no greater than 50% of the expression levels in non-engineered immune cells.

[0027] In another embodiment, the colony comprises cells with improved persistence and / or improved resistance to alloreactive immune cell rejection compared to unengineered immune cells. In another embodiment, the improved resistance is for alloreactive T cell-mediated rejection and / or alloreactive natural killer cell (NK)-mediated rejection. In one embodiment, the increased persistence is determined and / or determined by mixed lymphocyte reaction (MLR), and / or wherein the improved resistance to alloreactive immune cell rejection is determined and / or determined by MLR.

[0028] In another embodiment, the engineered immune cell colony comprises engineered immune cells, wherein at least 50% of the engineered immune cells exhibit reduced levels of MHC class I protein or complex expression at the cell surface. In another embodiment, the engineered immune cell colony comprises at least 10% engineered T cells, at least 20% engineered T cells, at least 30% engineered T cells, at least 40% engineered T cells, at least 50% engineered T cells, at least 75% engineered T cells, or at least 90% engineered T cells. In another embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 90% of the engineered immune cells further express antigen binding proteins or CD70 binding proteins. In another embodiment, the antigen binding protein is CAR or TCR.

[0029] In other embodiments, nucleic acids encoding antigen binding proteins (e.g., CAR or TCR) and / or CD70 binding proteins are inserted into disrupted CD58, RFX5, NLRC5, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA, and RFXANK loci. In another embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 90% of the engineered cells also include one or more genomic modifications of one or more of the endogenous TCRα gene and the endogenous CD52 gene. In one other embodiment, at least 10%, 20%, 30%, 40%, 50%, 75% or 90% of the engineered immune cells further express one or more proteins selected from the group consisting of: HLA-E, HLA-E single chain trimer, HLA-G or HLA-G single chain trimer, UL18 or UL18 single chain trimer, HLA-A2 and HLA-A2 single chain trimer.

[0030] In other embodiments, the functional expression level of one or more of TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK is measured by determining the surface expression level of HLA, β2 microglobulin (B2M) or both HLA and B2M on the surface of engineered immune cells, or measured by flow cytometry. In one embodiment, the functional expression level of one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of one or more of CD48, CD58 and ICAM-1 on the surface of engineered immune cells, or measured by flow cytometry.

[0031] In one other aspect, the present disclosure provides a method for preparing an engineered immune cell or an engineered immune cell colony as described herein. In one embodiment, the method includes a step of modifying the genome of an engineered immune cell. In another embodiment, the method also includes generating an engineered immune cell comprising a genome modification. In another embodiment, a TALEN, zinc finger, Cas-CLOVER or CRISPR / Cas system is used to modify the genome of an engineered immune cell.

[0032] In another aspect, the disclosure provides a pharmaceutical composition comprising an engineered immune cell or a cell colony comprising an engineered immune cell. In one embodiment, the pharmaceutical composition also includes at least one pharmaceutically acceptable carrier or excipient. In another embodiment, the engineered immune cell or one or more engineered immune cells of the cell colony (i) further express one or more proteins selected from the group consisting of the following items: HLA-E, HLA-E single chain trimer, HLA-G, HLA-G single chain trimer, UL18, UL18 single chain trimer, HLA-A2, HLA-A2 single chain trimer and human cytomegalovirus (HCMV) US11, and / or (ii) is further engineered to not express or express any one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFXANK, RFXAP and RFX5 at a reduced level.

[0033] In another aspect, the disclosure provides a method for treating a patient's condition or illness. In one embodiment, the method comprises administering to a subject an engineered immune cell, a cell colony comprising the engineered immune cell, or a pharmaceutical composition comprising the engineered immune cell or a cell colony comprising the engineered immune cell. In another embodiment, the condition is a solid tumor or a liquid tumor. In other embodiments, the condition is cancer, an autoimmune disorder, or an infection, as further described herein.

[0034] In another aspect, the present disclosure provides a method for improving (i) persistence of engineered immune cells described herein or (ii) resistance to rejection of alloreactive immune cells. In one embodiment, the engineered immune cells are allogeneic engineered immune cells. In other embodiments, the method includes modifying allogeneic immune cells to introduce one or more genome modifications to provide allogeneic engineered immune cells, which functionally weaken or reduce (i) RFX5 and / or NLRC5 and (ii) CD58 expression. In another embodiment, the method includes administering to a subject a step of allogeneic engineered immune cells. In one embodiment, the genome modification includes (i) RFX5 and / or NLRC5 and (ii) CD58 knockdown and / or knockout. In other embodiments, the genome modification is included in one or more modifications at the locus of (i) RFX5 and / or NLRC5 and (ii) CD58. In another embodiment, the genome modification is included in the deletion or insertion at the locus of (i) RFX5 and / or NLRC5 and (ii) CD58. In other embodiments, the genome modification is selected from the group consisting of: (i) insertion of one or more nucleotides, (ii) insertion of a protein-encoding sequence, (iii) deletion of one or more nucleotides, and (iv) substitution of one or more nucleotides. In one embodiment, the genome modification is introduced by a gene editing technology selected from TALEN, zinc finger, Cas-CLOVER and CRISPR / Cas system.

[0035] In another embodiment, the genome modification includes the insertion of an RNA interference sequence. In another embodiment, the interfering sequence is a shRNA sequence, a siRNA sequence or a miRNA sequence. In other embodiments, the interfering sequence includes a sequence complementary to (i) RFX5 and / or NLRC5 and (ii) CD58 gene sequences. In some embodiments, allogeneic engineered immune cells also include polynucleotide sequences encoding antigen binding proteins (e.g., CAR or TCR) and / or CD70 binding proteins. In another embodiment, allogeneic engineered immune cells are further engineered to include one or more genome modifications, which are functionally weakened or reduced relative to cells without modification. One or more of TAP2, β2m, TRAC, CIITA, RFXAP, RFXANK, ICAM-1 and CD48 expression. In other embodiments, genome modification functionally weakens or reduces expression to about 50% or less of the corresponding level in cells without genome modification. In one embodiment, allogeneic engineered immune cells have improved persistence and / or improved resistance to rejection of alloreactive immune cells compared to allogeneic unengineered immune cells. In other embodiments, the improved resistance is to rejection mediated by alloreactive T cells and / or rejection mediated by alloreactive natural killer cells (NK). In one embodiment, the increased persistence is determined and / or determined by a mixed lymphocyte reaction (MLR) assay, and / or wherein the improved resistance to rejection of alloreactive immune cells is determined and / or determined by a MLR assay.

[0036] In another embodiment, the method comprises functionally impairing or reducing the expression of RFX5 and CD58, or NLRC5 and CD58, or RFX5, NLRC5 and CD58. In another embodiment, the degree of reduction in the expression level of (i) RFX5 and / or NLRC5 and (ii) CD58 is determined relative to the expression level of (i) RFX5 and / or NLRC5 and (ii) CD58 in cells of the same type that have not been modified, respectively.

[0037] In another embodiment, the functional expression level of one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of CD48 protein, CD58 protein or ICAM-1 protein on the surface of the engineered immune cells. In another embodiment, the functional expression level of RFX5 and / or NLRC5 is measured by determining the surface expression level of HLA, β2 microglobulin (B2M) or both HLA and B2M on the surface of the engineered immune cells. In one embodiment, the surface expression level is measured by flow cytometry. In other embodiments, the method further includes one or more genome modifications of one or more of the TCRα gene and the CD52 gene. In one embodiment, the allogeneic engineered immune cells include an unmodified β2m gene, or wherein β2m is not functionally expressed at a reduced level in the allogeneic engineered immune cells.

[0038] In other embodiments, the immune cells of the present disclosure are engineered to functionally express reduced levels of a

[0039] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0040] Only one of CD48, CD58 and ICAM-1;

[0041] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0042] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0043] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0044] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0045] In another embodiment, an immune cell engineered to functionally express one or more targets as described herein at reduced levels relative to a corresponding cell that has not been so engineered comprises one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to a cell without the one or more genomic modifications.

[0046] In another embodiment, the engineered cells (i) have an unmodified β2m gene, (ii) functionally express normal levels of β2m and / or (iii) are not engineered to functionally express reduced levels of β2m.

[0047] The engineered cells can be further engineered to enhance resistance to rejection and / or provide a therapeutic effect, for example, the cells can be engineered to contain or express additional proteins, for example, antigen binding proteins such as chimeric antigen receptors (CARs) and / or T cell receptors, wherein the antigen binding proteins or T cell receptors enable the engineered immune cells to target tumor cells and / or other undesirable (e.g., disease state) cells expressing cognate antigens.

[0048] Thus, the present disclosure provides a method of increasing the persistence of allogeneic cells in a recipient, the method comprising engineering the cells to functionally express a reduced level of a

[0049] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0050] Only one of CD48, CD58 and ICAM-1;

[0051] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0052] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0053] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0054] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0055] In another embodiment, a method of engineering cells to functionally express one or more targets as described herein at reduced levels relative to corresponding cells that have not been so engineered comprises introducing into the cells one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to cells without the one or more genomic modifications.

[0056] In another embodiment, the method does not include engineering the cell to functionally express reduced levels of β2m.

[0057] In one aspect, the present disclosure provides an engineered immune cell that functionally expresses at reduced levels relative to non-engineered cells.

[0058] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0059] Only one of CD48, CD58 and ICAM-1;

[0060] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0061] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0062] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0063] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0064] In another embodiment, an engineered immune cell that functionally expresses one or more targets as described herein at reduced levels relative to a corresponding cell that has not been so engineered comprises one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to a cell without the one or more genomic modifications.

[0065] In another embodiment, the engineered cell (i) has an unmodified β2m gene, (ii) functionally expresses normal levels of β2m and / or (iii) is not engineered to functionally express reduced levels of β2m. In some embodiments, the reduction in expression level relative to the expression level of a corresponding but unengineered immune cell is 0%, such as when both chromosomal copies of the gene are knocked out, or 50% (i.e., 50% of the level in an unengineered control immune cell), such as when one of the two chromosomal copies of the gene is knocked out and the expression of the other chromosomal copy of the gene is not compensatorily increased. In some embodiments, the cell expresses

[0066] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0067] Only one of CD48, CD58 and ICAM-1;

[0068] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0069] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0070] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0071] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0072] The expression level is no greater than 90%, no greater than 75%, no greater than 50%, no greater than 25%, or no greater than 10% of the expression level in unengineered immune cells.

[0073] In another embodiment, a cell that expresses one or more targets at a level no greater than 90%, no greater than 75%, no greater than 50%, no greater than 25%, or no greater than 10% of the expression level in a non-engineered immune cell comprises one or more genomic modifications that functionally impair or reduce expression of the one or more targets as described herein relative to a cell without the one or more genomic modifications.

[0074] In another embodiment, the cell (i) has an unmodified β2m gene, (ii) functionally expresses normal levels of β2m and / or (iii) has not been engineered to functionally express reduced levels of β2m.

[0075] In some embodiments, in an engineered immune cell

[0076] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0077] Only one of CD48, CD58 and ICAM-1;

[0078] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0079] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0080] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0081] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0082] The expression level is any value between 0% and 90% of the level in the control cell without corresponding engineering for the corresponding gene. In some embodiments, the expression level in the engineered cell is, for example, 10% to 90%, 25% to 90%, 25% to 75%, 10% to 50%, 25% to 50%, 50% to 90%, or 50% to 75% of the level in the control cell. In some embodiments, a reduced expression level other than 0% or 50% is obtained when, for example, only one chromosomal copy of a gene is knocked out and compensatory mechanisms result in increased expression levels of the remaining chromosomal copy, or the reduction in expression is achieved by methods other than gene knockout, such as known knockdown methods, e.g., those employing any of a variety of RNA-based techniques (e.g., antisense RNA, miRNA, siRNA; see, e.g., Lam et al., Mol. Ther. - Nucleic Acids 4:e252 (2015), doi: 10.1038 / mtna.2015.23; Sridharan and Gogtay, Brit. J. Clin. Pharmacol. 82:659-72 (2016)). In another embodiment, an engineered immune cell that expresses one or more targets as described herein at a level between 0% and 90% of the level in a control cell that has not been correspondingly engineered comprises one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0083] In some embodiments, the engineered immune cells disclosed herein exhibit reduced levels of MHC class I protein or complex expression at the cell surface relative to a suitable control. In various embodiments, the cell is a T cell, such as a human T cell. In some embodiments, the cell comprises a mutation in the TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP and RFXANK loci or genes and / or a disruption in the TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP and RFXANK loci or genes, the mutation and / or disruption resulting in reduced functional expression of the disrupted loci or genes. In other embodiments, the cell may comprise additional mutations in one or more of the CD48, CD58 and ICAM-1 loci or genes and / or disruption in one or more of the CD48, CD58 and ICAM-1 loci or genes, the mutation and / or disruption resulting in reduced functional expression of the disrupted loci or genes. In another embodiment, the cell may comprise mutations in the following loci or genes

[0084] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0085] NLRC5 and CD58, RFX5 and CD58, RFX5 and CD48, NLRC5 and CD48, RFX5 and ICAM-1, NLRC5 and ICAM-1;

[0086] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0087] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; or (vi) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0088] These mutations result in reduced functional expression of the disrupted locus or gene. In another embodiment, the cell (i) does not contain a mutation in the β2m locus or gene, (ii) has an unmodified β2m gene, (iii) functionally expresses normal levels of β2m and / or (iv) is not engineered to functionally express reduced levels of β2m.

[0089] In another embodiment, a cell comprising a mutation in a locus or gene of one or more targets as described herein that results in reduced functional expression of the disrupted locus or gene comprises one or more genomic modifications that functionally impair or reduce expression of the one or more targets relative to a cell without the one or more genomic modifications.

[0090] In one embodiment, the mutation or disruption is introduced by any one or combination of gene mutation or gene editing techniques, including but not limited to known homologous recombination techniques and the use of meganucleases, TALENs, zinc fingers, shRNAs, Cas-CLOVERs and CRISPR / Cas systems (e.g., Cas9, Cas12 and MAD7) or other systems such as base editing systems or primer editing systems. In some embodiments, the cell is a non-human cell, such as a primate cell or a non-primate mammalian cell. In some embodiments, the cell is a human cell.

[0091] In various embodiments, the engineered immune cells further express antigen binding proteins, for example, engineered immune cells include nucleic acids encoding antigen binding proteins. In one embodiment, the antigen binding protein is a chimeric antigen receptor (CAR). In one embodiment, nucleic acids encoding antigen binding proteins (eg, CAR) are inserted into destroyed CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK loci, or inserted into the loci, thereby destroying the loci. In various embodiments, the antigen binding protein is a T cell receptor (TCR). In one embodiment, nucleic acids encoding TCR are inserted into destroyed CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK loci, or inserted into the loci, thereby destroying the loci. In one embodiment, the engineered immune cell further comprises one or more genomic modifications, such as modifications of endogenous loci, such as modifications of one or more of the following: an endogenous CD70 gene, an endogenous TCRα gene, and an endogenous CD52 gene. In various embodiments, the one or more genomic modifications result in a reduction or deletion of the functional expression of the gene containing the modification.

[0092] In another embodiment, the engineered immune cell that further expresses an antigen binding protein (e.g., CAR) comprises one or more genomic modifications that functionally attenuate or reduce the expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0093] The engineered immune cell can be derived from a cell from any source in each source. The engineered immune cell can be prepared from or derived from a cell, such as a stem cell or an immune cell from a person other than the person who will use the engineered immune cell, such as a donor (e.g., a healthy volunteer) other than a recipient, or can be prepared from or derived from a cell, such as a stem cell or an immune cell from a person (recipient) who will use the engineered immune cell, or can be derived from one or more induced pluripotent stem cells (iPSC). In one embodiment, the immune cell is an immune cell obtained from a healthy volunteer, obtained from a patient or derived from iPSC.

[0094] In another aspect, the engineered cell may further comprise a polynucleotide encoding a CD70 binding protein and / or may functionally express a CD70 binding protein. In one embodiment, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In some embodiments, the CD70 binding domain constitutes a CD70 antibody, or a CD70 receptor or a CD70 binding fragment thereof. In other embodiments, the CD70 binding domain constitutes an anti-CD70 antibody, and optionally the anti-CD70 antibody is a scFv. In one embodiment, the CD70 binding protein further comprises a hinge domain, and optionally the hinge domain comprises a CD8 hinge. In one other embodiment, the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of the following items: a CD3z signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain or a variant thereof. In another embodiment, the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a co-stimulatory domain. In an additional embodiment, the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain. In other embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain. In one embodiment, the one or more intracellular domains comprise an amino acid sequence of one or more of SEQ ID NO: 1, 7-14, 17-70 or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0095] On the other hand, the present disclosure provides a method for preparing an engineered immune cell disclosed herein. In one embodiment, the method includes using any gene editing technology, such as TALEN, zinc finger, Cas-CLOVER and CRISPR / Cas system, and / or using any known gene knockdown method, such as those using any of various RNA-based technologies (e.g., shRNA / antisense RNA, miRNA, siRNA; see, for example, Lam et al., "Molecular Therapy-Nucleic Acids (Mol.Ther.Nucleic Acids)" 4:e252(2015), doi:10.1038 / mtna.2015.23; Sridharan and Gogtay, "British Journal of Clinical Pharmacology (Brit.J.Clin.Pharmacol)". 82:659-72(2016)), to reduce the following functional expression relative to unengineered immune cells:

[0096] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0097] Only one of CD48, CD58 and ICAM-1;

[0098] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0099] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0100] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0101] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5. In another embodiment, the method does not include genetically editing the cells to functionally express reduced levels of β2m.

[0102] In another embodiment, a method of using gene editing technology to prepare an engineered immune cell to reduce the functional expression of one or more targets as described herein relative to a corresponding immune cell that has not been so engineered comprises introducing one or more genomic modifications into the immune cell that functionally attenuates or reduces the expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0103] In one embodiment, the method includes or further includes introducing a nucleic acid encoding an antigen binding protein (e.g., CAR or TCR) into an engineered immune cell. In an embodiment, the method includes or further includes introducing one or more genomic modifications of one or more of the endogenous TCRα gene and the endogenous CD52 gene into the genome of the engineered immune cell. In an embodiment, the one or more genomic modifications all or partly destroy and / or prevent the functional expression of one or more of the endogenous TCRα gene and the endogenous CD52 gene.

[0104] In various embodiments of the present disclosure, the functional expression level of any one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is measured by determining the surface expression level of one or more HLA proteins (such as HLA-A or HLA-B proteins) or β2 microglobulin (β2M) or one or more HLA proteins and β2M on the surface of engineered immune cells, or measured by flow cytometry. In various embodiments of the present disclosure, the functional expression level of any one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of each cell surface protein on the surface of engineered immune cells, such as one or more of CD48, CD58 or ICAM-1 proteins, or measured by flow cytometry. In some embodiments, the expression of any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK in the engineered immune cells of the present disclosure is determined by measuring the extent to which the engineered immune cells survive in the presence of effector cells (e.g., T cells) compared to the extent to which non-correspondingly engineered but otherwise comparable (e.g., identical) immune cells survive under the same conditions.

[0105] In some embodiments, an immune cell that has been engineered to functionally express reduced levels of

[0106] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0107] Only one of CD48, CD58 and ICAM-1;

[0108] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0109] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0110] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0111] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0112] The invention is further engineered (a) to express one or more proteins selected from the group consisting of HLA-E, HLA-E single chain trimer, HLA-G, HLA-G single chain trimer, UL18, UL18 single chain trimer, HLA-A2, HLA-A2 single chain trimer, human cytomegalovirus (HCMV) US11, and / or (b) to express reduced levels of

[0113] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0114] Only one of CD48, CD58 and ICAM-1;

[0115] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0116] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0117] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0118] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0119] This is achieved by knockout or knockdown as described herein.

[0120] In another embodiment, an immune cell that has been engineered to functionally express one or more targets as described herein at reduced levels relative to a corresponding cell that has not been so engineered comprises one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to a cell without the one or more genomic modifications.

[0121] In another aspect, the present disclosure provides an engineered immune cell population comprising the engineered immune cells provided herein. In one embodiment, the engineered immune cell population comprises 10 4 Up to 10 10 In various embodiments, the engineered immune cell population comprises 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 The engineered immune cells provided herein.

[0122] In another aspect, the disclosure provides engineered immune cell populations in which no more than, for example, 75% of the cells functionally express

[0123] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0124] Only one of CD48, CD58 and ICAM-1;

[0125] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0126] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0127] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0128] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0129] In another embodiment, the cells of the population functionally express β2m at normal levels, or do not express β2m at reduced levels.

[0130] In another embodiment, comprising 10 4 Up to 10 10The engineered immune cell population of a plurality of engineered immune cells comprises engineered immune cells containing one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0131] In another aspect, the disclosure provides a population of engineered immune cells wherein at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or 100% of the engineered immune cells functionally express at reduced levels a

[0132] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0133] Only one of CD48, CD58 and ICAM-1;

[0134] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0135] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0136] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0137] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0138] The reduced level can be a level no greater than 90%, no greater than 75%, no greater than 50%, no greater than 25%, or no greater than 10% of the expression level in an unengineered immune cell. Specifically, the reduced level can be a level no greater than 50% of the expression level in an unengineered immune cell. In another embodiment, the engineered immune cell population functionally expresses β2m at a normal level or does not functionally express β2m at a reduced level.

[0139] In another embodiment, a population of engineered immune cells wherein at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or 100% of the engineered immune cells functionally express one or more targets as described herein at reduced levels comprises engineered immune cells containing one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0140] In some embodiments, relative to an appropriate control (e.g., an expression level in a corresponding immune cell that has not been engineered), the reduced expression level is 0%, such as when both chromosome copies of a gene are knocked out (e.g., by using a method of a TALEN, zinc finger, Cas-CLOVER and / or CRISPR / Cas system), or 50%, such as when one chromosome copy of the two chromosome copies of a gene is knocked out and the expression of the other chromosome copy of the gene is not compensatory. In some embodiments, relative to the expression level of an unengineered immune cell, the reduced expression level is between 0% and 90%, or between any two intermediate values ​​between 0% and 90%, such as between 10% and 90%, between 25% and 90%, between 25% and 75%, between 10% and 50%, between 25% and 50%, between 50% and 90%, and between 50% and 75%. Values ​​within one or more of such intermediate ranges may be obtained when, for example, only one chromosomal copy of a gene is knocked out and compensatory mechanisms result in increased expression levels of the remaining chromosomal copy, or reduction in expression may be achieved by methods other than gene knockout, such as known knockdown methods, e.g., those employing any of a variety of RNA-based techniques (e.g., shRNA antisense RNA, miRNA, siRNA; see, e.g., Lam et al., Mol. Ther. - Nucleic Acids 4:e252 (2015), doi: 10.1038 / mtna.2015.23; Sridharan and Gogtay, Brit. J. Clin. Pharmacol. 82:659-72 (2016)). In some embodiments of the engineered immune cell populations disclosed herein, some or all of the engineered cells, e.g., 5%-10%, 10%-25%, 25%-50%, 50%-90%, or 90%-100%, exhibit reduced levels of expression of MHC class I proteins and / or MHC class II proteins or complexes at the cell surface relative to a suitable control.

[0141] In various embodiments, the engineered immune cell population as disclosed herein or the immune cell population comprising engineered immune cells include at least 10% engineered T cells, at least 20% engineered T cells, at least 30% engineered T cells, at least 40% engineered T cells, at least 50% engineered T cells, at least 75% engineered T cells, at least 90% engineered T cells or 100% engineered T cells. Also provided herein is a cell population in which at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90% or 100% are engineered immune cells, such as engineered T cells disclosed herein.

[0142] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 90% of the engineered cells in the population further express antigen binding proteins. In some embodiments, the antigen binding protein is a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encoding CAR is inserted into a destroyed CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK locus, and / or such insertions will destroy the locus. In some embodiments, the antigen binding protein is a T cell receptor (TCR). In some embodiments, the nucleic acid encoding TCR is inserted into a destroyed CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK locus, and / or such insertions will destroy the locus.

[0143] In certain embodiments of the engineered immune cell populations disclosed herein, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 90% of the engineered cells further comprise one or more genomic modifications of one or more of an endogenous TCRα gene and an endogenous CD52 gene.

[0144] In one embodiment, the engineered immune cell population is derived from one or more immune cells obtained from a human, e.g., from a human other than the human to whom the cells will be administered, e.g., from a donor other than the recipient or from a healthy volunteer, or from one or more immune cells obtained from a patient (e.g., the human to whom the cells will be administered), or from one or more iPSCs.

[0145] In another aspect, the engineered cell population may further comprise a polynucleotide encoding a CD70 binding protein and / or may functionally express a CD70 binding protein. In one embodiment, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In some embodiments, the CD70 binding domain constitutes a CD70 antibody, or a CD70 receptor or a CD70 binding fragment thereof. In other embodiments, the CD70 binding domain constitutes an anti-CD70 antibody, and optionally the anti-CD70 antibody is a scFv. In one embodiment, the CD70 binding protein further comprises a hinge domain, and optionally the hinge domain comprises a CD8 hinge. In another embodiment, the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of the following items: a CD3z signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain or a variant thereof. In another embodiment, the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a co-stimulatory domain. In an additional embodiment, the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain. In other embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain. In one embodiment, the one or more intracellular domains comprise an amino acid sequence of one or more of SEQ ID NO: 1, 7-14, 17-70 or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0146] In another aspect, the present disclosure provides a method for preparing an engineered immune cell population as described herein, wherein the method comprises using a gene editing technology, such as a gene editing technology selected from the group consisting of TALEN, zinc finger, Cas-CLOVER and CRISPR / Cas system, to reduce the functional expression of

[0147] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0148] Only one of CD48, CD58 and ICAM-1;

[0149] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0150] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0151] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0152] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0153] In one embodiment, the method does not include the use of gene editing techniques, such as gene editing techniques that reduce the functional expression of β2m.

[0154] In another embodiment, a method of using gene editing technology to prepare an engineered immune cell population as described herein to reduce the functional expression of one or more targets as described herein relative to corresponding immune cells that have not been so engineered includes introducing one or more genomic modifications into the immune cells that functionally attenuate or reduce the expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0155] In another aspect, the present disclosure provides a method for determining or measuring the functional expression level of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and / or RFXANK in cells of an engineered immune cell population disclosed herein, wherein the functional expression level is measured by determining the surface expression level of HLA protein, β2 microglobulin (B2M) or both HLA protein and B2M on the surface of the engineered immune cell, and / or measured by flow cytometry. In various embodiments of the present disclosure, the functional expression level of any one or more of CD48, CD58 and ICAM-1 in cells of an engineered immune cell population, wherein the functional expression level is measured by determining the surface expression level of each cell surface protein on the surface of the engineered immune cell, such as one or more of CD48, CD58 or ICAM-1 proteins, or measured by flow cytometry.

[0156] In various embodiments of the engineered immune cells described herein and the engineered immune cell populations disclosed herein, the engineered immune cell is, or one or more engineered immune cells of the population, e.g., at least 10%, 20%, 30%, 40%, 50%, 75%, 90% or 100% of the engineered immune cells of the population are further engineered by any method described herein or by any other method known to those of ordinary skill in the art (e.g., further engineered by any method disclosed herein or by any other method known to those of ordinary skill in the art) to express one or more proteins selected from the group consisting of: HLA-E, HLA-E single chain trimer, HLA-G, HLA-G single chain trimer, UL18, UL18 single chain trimer, HLA-A2, HLA-A2 single chain trimer, and human cytomegalovirus (HCMV) US11.

[0157] In another aspect, a method of treating a patient's condition comprises administering to the patient: an engineered immune cell, an engineered immune cell population, or a pharmaceutical composition comprising an engineered cell or an engineered immune cell population, wherein the engineered cell and / or the engineered cells of the population further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein. In one embodiment, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In some embodiments, the CD70 binding domain constitutes a CD70 antibody, or a CD70 receptor or a CD70 binding fragment thereof. In other embodiments, the CD70 binding domain constitutes an anti-CD70 antibody, optionally the anti-CD70 antibody is a scFv. In one embodiment, the CD70 binding protein further comprises a hinge domain, optionally the hinge domain comprises a CD8 hinge. In one other embodiment, the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of: CD3z signaling domain, CD3d signaling domain, CD3g signaling domain, CD3e signaling domain, CD28 signaling domain, CD2 signaling domain, OX40 signaling domain and 4-1BB signaling domain or variants thereof. In another embodiment, the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a co-stimulatory domain. In another embodiment, the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain. In other embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain. In one embodiment, the one or more intracellular domains comprise an amino acid sequence of one or more of SEQ ID NO: 1, 7-14, 17-70 or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0158] In another aspect, the present disclosure provides a pharmaceutical composition comprising an engineered immune cell as disclosed herein, wherein the composition further comprises one or more pharmaceutically acceptable carriers or excipients. In another aspect, the present disclosure provides a pharmaceutical composition comprising an engineered immune cell population as disclosed herein, wherein the composition further comprises one or more pharmaceutically acceptable carriers or excipients. In various embodiments of the composition, the engineered immune cell or one or more engineered immune cells of the population, for example, at least 10%, 20%, 30%, 40%, 50%, 75%, 90% or 100% of the engineered immune cells of the population, express one or more proteins selected from the group consisting of HLA-E, HLA-E single chain trimer, HLA-G, HLA-G single chain trimer, UL18, UL18 single chain trimer, HLA-A2, HLA-A2 single chain trimer and human cytomegalovirus (HCMV) US11, and / or do not express or express at a reduced level any one or more of CIITA, RFXANK, RFXAP and RFX5, as achieved by knockout or knockdown as described herein.

[0159] In another aspect, the engineered cells of the pharmaceutical composition further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein. In one embodiment, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In some embodiments, the CD70 binding domain constitutes a CD70 antibody, or a CD70 receptor or a CD70 binding fragment thereof. In other embodiments, the CD70 binding domain constitutes an anti-CD70 antibody, and optionally the anti-CD70 antibody is a scFv. In one embodiment, the CD70 binding protein further comprises a hinge domain, and optionally the hinge domain comprises a CD8 hinge. In one other embodiment, the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of the following items: a CD3z signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain or a variant thereof. In another embodiment, the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a co-stimulatory domain. In an additional embodiment, the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain. In other embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain. In one embodiment, the one or more intracellular domains comprise an amino acid sequence of one or more of SEQ ID NO: 1, 7-14, 17-70 or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0160] In another embodiment, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients and an engineered cell, e.g., an engineered immune cell as described herein, wherein the engineered cell comprises one or more genomic modifications that functionally attenuate or reduce the expression of one or more targets as described herein relative to a cell without the one or more genomic modifications.

[0161] In one aspect, an engineered immune cell as disclosed herein, a population of engineered immune cells as disclosed herein, or a pharmaceutical composition for use as a medicament is provided.

[0162] In another aspect, the present disclosure provides a method of treating a condition in a patient, the method comprising administering to the patient an engineered immune cell as disclosed herein, an engineered immune cell population as disclosed herein, or a pharmaceutical composition as disclosed herein. In one embodiment, the condition is selected from the group consisting of a solid tumor and a liquid tumor.

[0163] In another aspect, the present disclosure provides a method for reducing the surface expression level of MHC class I proteins in engineered immune cells to about 75% or less of the expression level of MHC class I proteins in unengineered immune cells, the method reducing the functional expression level of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP and RFXANK, for example, to about 75% or less of the expression level in unengineered immune cells. In another embodiment, the method further includes reducing the functional expression level of one or more of CD48, CD58 and ICAM-1, for example, to about 75% or less of the expression level in unengineered immune cells. In another embodiment, the method includes reducing the functional expression level of the following items

[0164] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0165] NLRC5 and CD58, RFX5 and CD58, RFX5 and CD48, NLRC5 and CD48, RFX5 and ICAM-1, NLRC5 and ICAM-1;

[0166] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0167] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; or (vi) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0168] For example, reduced to about 75% or less of the expression level in non-engineered immune cells.

[0169] In another embodiment, the surface expression level of MHC class I protein in an engineered immune cell is reduced by reducing the functional expression of one or more targets as described herein, in some embodiments to about 75% or less of the expression level of MHC class I protein in a non-engineered immune cell, comprising introducing one or more genomic modifications into the immune cell that functionally attenuates or reduces the expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0170] In another embodiment, the engineered immune cell functionally expresses β2m at normal levels or does not functionally express β2m at reduced levels. In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein.

[0171] In one embodiment, the engineered immune cells disclosed herein exhibit reduced levels of expression of MHC class I proteins or complexes at the cell surface relative to a suitable control.

[0172] In one embodiment, the engineered immune cells disclosed herein are engineered T cells. In certain embodiments, the engineered immune cells disclosed herein, such as engineered T cells disclosed herein, further express additional proteins, such as proteins encoded by exogenous DNA or proteins expressed by further engineering of the cell. In some embodiments, additional proteins are antigen binding proteins and / or CD70 binding proteins. In some embodiments, the antigen binding proteins are chimeric antigen receptors (CAR). In some embodiments, nucleic acids encoding additional proteins, such as antigen binding proteins (e.g., CAR) and / or CD70 binding proteins are introduced into cells by the methods described herein. In some embodiments, a nucleic acid is introduced into the disrupted CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK locus, or the CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK locus is disrupted by inserting a nucleic acid encoding an additional protein, such as an antigen binding protein (e.g., CAR) and / or a CD70 binding protein.

[0173] In one embodiment of the methods disclosed herein, the antigen binding protein is a T cell receptor (TCR) component, such as TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma) or TCRδ (TCR delta). In one embodiment, a nucleic acid encoding a TCR component is inserted into a disrupted CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK locus, or a CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK locus is disrupted by inserting a nucleic acid encoding a TCR component.

[0174] In another embodiment of the method disclosed herein, the engineered immune cell further comprises one or more genomic modifications (e.g., knockout, deletion, knockdown, insertion) of one or more of the endogenous TCRα gene and the endogenous CD52 gene. In some embodiments, the genomic modification partially or completely eliminates the functional expression of the modified gene. In another embodiment of the method disclosed herein, the immune cell is obtained from a person, for example, from a donor other than the person to whom the cell will be administered, for example, from an immune cell of a healthy volunteer, or from an immune cell of a patient (e.g., a person to whom the cell will be administered), or derived from iPSC.

[0175] In another aspect, the present disclosure provides a method for preparing an engineered immune cell population disclosed herein, wherein the method comprises using a gene editing technology, such as a gene editing technology selected from the group consisting of TALEN, zinc finger, shRNA, Cas-CLOVER and CRISPR / Cas system, to reduce the functional expression of the following in a cell, such as an immune cell:

[0176] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0177] Only one of CD48, CD58 and ICAM-1;

[0178] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0179] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0180] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0181] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0182] In another embodiment, the immune cell (before or after gene editing) further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein. In another embodiment, the method does not include gene editing of β2m in a cell (e.g., an immune cell).

[0183] In another embodiment, the gene editing technology introduces mutations into the following loci

[0184] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0185] Only one of CD48, CD58 and ICAM-1;

[0186] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0187] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0188] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0189] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0190] In another embodiment, a method of preparing an engineered immune cell population as described herein by introducing mutations into a locus or one or more targets to reduce the functional expression of one or more targets as described herein relative to corresponding immune cells that have not been so engineered comprises introducing one or more genomic modifications into the immune cells that functionally attenuate or reduce the expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0191] In another embodiment, the gene editing technology does not introduce mutations into the β2m locus. In one embodiment, the mutation is any one or more of the following: insertion, such as insertion of one or more nucleotides or base pairs, such as insertion of a protein-encoding sequence; deletion of one or more nucleotides or base pairs; and substitution of one or more nucleotides or base pairs.

[0192] In one embodiment of the method disclosed herein, the functional expression level of any one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is measured by determining the surface expression level of HLA protein, β2 microglobulin (B2M) or both HLA and B2M on the surface of engineered immune cells, or measured by flow cytometry. In various embodiments of the present disclosure, the functional expression level of any one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of each cell surface protein on the surface of engineered immune cells, such as one or more of CD48, CD58 or ICAM-1 proteins, or measured by flow cytometry. In one embodiment, the reduction degree of the surface expression level of the following items

[0193] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0194] Only one of CD48, CD58 and ICAM-1;

[0195] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0196] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0197] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0198] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0199] It is determined relative to the corresponding expression level in cells of the same type that have not been gene-edited.

[0200] In the embodiments of the methods disclosed herein, the engineered immune cells express additional proteins, which may be any desired proteins, including antigen binding proteins or CD70 binding proteins (as further described herein). In one embodiment, the antigen binding proteins include CAR. In some embodiments, the nucleic acid encoding CAR (and optionally CD70 binding proteins) is inserted into the destroyed CD58, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK loci, or such insertions will destroy the loci. In another embodiment, the antigen binding proteins are T cell receptor (TCR) components. In one embodiment, the nucleic acid encoding TCR is inserted into the destroyed CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP or RFXANK loci, or such insertions will destroy the loci.

[0201] In one embodiment, the method comprises reducing the functional expression level of the disrupted locus. In one embodiment, the method comprises reducing the functional expression level of

[0202] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0203] Only one of CD48, CD58 and ICAM-1;

[0204] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0205] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0206] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0207] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0208] In another embodiment, the method of reducing the functional expression of a disrupted locus by reducing the functional expression level of one or more targets as described herein comprises introducing one or more genomic modifications into the immune cells that functionally attenuate or reduce the expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0209] In another embodiment, the method does not comprise reducing the functional expression level of β2m.

[0210] In certain embodiments, the method further comprises introducing one or more genomic modifications of one or more of the genes encoding TCR components (e.g., TCRα gene) and the CD52 gene into the engineered immune cell.

[0211] In another aspect, the present disclosure provides a method for reducing the diversity of peptides presented on the cell surface of, for example, MHC class I, the method comprising reducing the functional expression level of any one or more of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK, in some embodiments, the level is reduced to about 90% or less (in other words, reduced by at least about 10%, for example, reduced to a level of about 90 or less compared to a control level of 100) or reduced to about 75% or less of comparable cells that have not been changed accordingly. In addition to reducing peptide diversity, the methods disclosed herein may also include simultaneously downregulating or eliminating certain cell surface receptors, which are known to play a role in immune cell adhesion and activation of one or more of the immune synapses, such as CD48, CD58, and ICAM-1. In various embodiments, as described herein, reducing the functional expression level of one or more genes includes using a gene editing technique selected from a group consisting of TALEN, zinc fingers, Cas-CLOVER, and CRISPR / Cas systems (including, for example, Cas9, Cas12, and MAD7). In another embodiment, the method comprises reducing the functional expression level of

[0212] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0213] NLRC5 and CD58, RFX5 and CD58, RFX5 and CD48, NLRC5 and CD48, RFX5 and ICAM-1, NLRC5 and ICAM-1;

[0214] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0215] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0216] In another embodiment, a method of reducing the diversity of peptides presented on the cell surface and simultaneously downregulating or eliminating certain cell surface receptors known to play a role in immune cell adhesion and activation of the immune synapse by reducing the functional expression level of one or more genes / targets as described herein comprises introducing one or more genomic modifications into the immune cell that functionally impairs or reduces the expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0217] In another embodiment, the method does not comprise reducing the functional expression of β2m.

[0218] In one embodiment, the reduction degree of the expression level of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is determined relative to the corresponding expression level in the same type of cells that have not been genetically edited. In one embodiment, the functional expression level of one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is measured by determining the surface expression level of HLA, β2 microglobulin (B2M) or both HLA and B2M on the surface of engineered immune cells. In various embodiments of the present disclosure, the reduction degree of the expression level of any one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of each cell surface protein on the surface of engineered immune cells, such as CD48, CD58 or ICAM-1 protein, or measured by flow cytometry.

[0219] In another aspect, the present disclosure provides a method of reducing T cell-mediated allogeneic cell killing, the method comprising reducing the functional expression level of the following in an engineered immune cell, such as an engineered T cell:

[0220] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0221] Only one of CD48, CD58 and ICAM-1;

[0222] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0223] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0224] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0225] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0226] In another embodiment, the method of reducing T cell-mediated allogeneic cell killing does not comprise reducing the functional expression level of β2m.

[0227] In another embodiment, a method of reducing T cell-mediated allogeneic cell killing by reducing the functional expression level of one or more targets as described herein in an engineered immune cell (such as an engineered T cell) comprises introducing one or more genomic modifications into an immune cell (such as a T cell) that functionally attenuates or reduces the expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0228] In some embodiments, the method reduces the expression level, relative to the expression level of unengineered immune cells, such as when both chromosome copies of the gene are knocked out, the reduced expression level is 0%, or, for example, when one chromosome copy of the two chromosome copies of the gene is knocked out and the expression of the other chromosome copy of the gene is not compensatory. Increase, the reduced expression level is 50%. In some embodiments, the method reduces the expression level, relative to the expression level of unengineered immune cells, the reduced expression level is between 0% and 90%, or between any two intermediate values ​​between 0% and 90%, such as between 10% and 90%, between 25% and 90%, between 25% and 75%, between 10% and 50%, between 25% and 50%, between 50% and 90%, and between 50% and 75%.

[0229] In some embodiments, the functional expression level of

[0230] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0231] Only one of CD48, CD58 and ICAM-1;

[0232] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0233] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0234] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0235] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0236] Included are the use of gene editing technologies, such as any one or more of TALEN, zinc fingers, Cas-CLOVER and / or CRISPR / Cas systems, and / or any one or more known knockout methods, such as those employing any of a variety of RNA-based technologies (e.g., shRNA, antisense RNA, miRNA, siRNA).

[0237] In another embodiment, the method comprises reducing the functional expression level of

[0238] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0239] Only one of CD48, CD58 and ICAM-1;

[0240] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0241] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0242] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0243] ·CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0244] In another embodiment, a method for reducing the functional expression level of one or more targets as described herein in an immune cell by using gene editing technology and / or any one or more known knockdown methods comprises introducing one or more genomic modifications into the immune cell that functionally impairs or reduces the expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0245] In another embodiment, the method does not comprise reducing the functional expression level of β2m.

[0246] In one embodiment, the extent of reduction in expression level of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is determined relative to the corresponding expression level in cells of the same type that have not been so altered and / or manipulated.

[0247] In one embodiment of the method for reducing T cell-mediated allogeneic cell killing disclosed herein, the functional expression level of one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is measured by determining the surface expression level of HLA, β2 microglobulin (B2M) or both HLA and B2M on the surface of engineered immune cells. In one embodiment, the surface expression level of one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is measured by flow cytometry. In various embodiments of the present disclosure, the functional expression level of any one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of each cell surface protein on the surface of engineered immune cells, such as CD48, CD58 or ICAM-1 protein, or measured by flow cytometry. In one embodiment, the method comprises introducing one or more genomic modifications of one or more of a gene encoding a TCR component (e.g., a TCRα gene) and a CD52 gene. In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein.

[0248] In some embodiments of the methods disclosed herein for reducing T cell-mediated allogeneic cell killing, the functional expression levels of the following in the engineered immune cells of the present disclosure are:

[0249] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0250] Only one of CD48, CD58 and ICAM-1;

[0251] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0252] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0253] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0254] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0255] The extent to which the engineered immune cells survive in the presence of effector cells (e.g., T cells) is measured as compared to the extent to which unengineered but otherwise comparable (e.g., identical) immune cells survive under the same conditions. In another embodiment, the engineered immune cells further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0256] In one aspect, the present disclosure provides an engineered immune cell that functionally expresses at reduced levels

[0257] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0258] Only one of CD48, CD58 and ICAM-1;

[0259] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0260] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0261] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0262] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0263] In another embodiment, an engineered cell that functionally expresses one or more targets as described herein at reduced levels comprises one or more genomic modifications that functionally attenuate or reduce expression of the one or more targets relative to an immune cell without the one or more genomic modifications.

[0264] In another embodiment, the engineered immune cell does not functionally express β2m at a reduced level. In various embodiments, the cell exhibits reduced expression of MHC class I proteins or complexes at the cell surface, reduced expression of MHC class II proteins or complexes at the cell surface, or reduced expression of MHC class I proteins or complexes at the cell surface and reduced expression of MHC class II proteins or complexes at the cell surface. In various embodiments, the cell is a T cell. In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein.

[0265] In various embodiments, the engineered immune cells disclosed herein further express other proteins. In some embodiments, other proteins are antigen binding proteins and / or CD70 binding proteins. In some embodiments, the antigen binding proteins are chimeric antigen receptors (CAR). In some embodiments, the antigen binding proteins are T cell receptors (TCR). In certain embodiments, the engineered immune cells include nucleic acids encoding other proteins. In some embodiments, nucleic acids encoding other proteins are located in destroyed CD48, CD58, ICAM-1, TAP2, NLRC5, CIITA, RFX5, RFXANK, β2m or RFXAP loci, and / or cause or produce destruction in such loci. In another embodiment, nucleic acids encoding other proteins are not located in destroyed β2m loci and / or do not cause or produce destruction in such loci.

[0266] In various embodiments, the engineered immune cells disclosed herein include or further comprise one or more genomic modifications of one or more of an endogenous TCRα (TCRα or TCR alpha) gene and an endogenous CD52 gene.

[0267] In various embodiments, the engineered immune cells disclosed herein are or are derived from immune cells obtained from healthy volunteers or patients, or are derived from iPSCs.

[0268] In one aspect, the present disclosure provides a method for preparing an engineered immune cell disclosed herein, the method comprising using a gene editing technology selected from the group consisting of TALEN, zinc finger, Cas-CLOVER and CRISPR / Cas system to reduce the functional expression of

[0269] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0270] Only one of CD48, CD58 and ICAM-1;

[0271] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0272] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0273] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0274] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0275] In another embodiment, a method of preparing engineered immune cells using gene editing technology to reduce the functional expression of one or more targets as described herein comprises introducing one or more genomic modifications into the immune cells that functionally attenuate or reduce the expression of the one or more targets relative to immune cells without the one or more genomic modifications.

[0276] In another embodiment, the method does not comprise using gene editing technology to reduce the functional expression of β2m. BRIEF DESCRIPTION OF THE DRAWINGS

[0277] Figure 1A The different components of the immune synapse are depicted (adapted from Huppa, J., Davis, T-cell-antigen recognition and the immunological synapse, Nat Rev Immunol 3, 973–983 (2003)).

[0278] Figure 1B A model of immune escape of CD58-deficient tumor cells has been described (see Challa-Malladi, M. et al., Combined genetic inactivation of β2-Microglobulin and CD58 reveals frequent escape from immune recognition in diffuse large B cell lymphoma). Figure 8 Cancer Cell. 2011 Dec 13;20(6):728-40, epub 2011 Dec 1)

[0279] Figure 1C Depicted is how MHC-I expression controls the balance between T cell and NK cell rejection. Normal MHC-I expression triggers T cell rejection (left), while the absence of MHC-I triggers NK cell rejection via "loss of self" elimination (right).

[0280] Figure 1D Depicting the use of CD70 binding proteins expressed on CAR T cells against alloreactive host (e.g., patient) immune cells. CD70 binding proteins (also known as dagger proteins) bind to CD70 polypeptides expressed on host immune cells, leading to their elimination. CAR T cells express another molecule, such as CD58, at reduced levels compared to unengineered cells, thereby providing further protection for the CAR T cells against host immune responses.

[0281] Figure 1E-1F Shown are the results of T cell MLR (E) and NK cell MLR (F) assays using cell populations with certain knockouts.

[0282] Figure 2 Shown are gene editing efficiencies of cell populations with certain knockouts.

[0283] Figure 3 Shown are the results of T cell MLR assays using cell populations with certain knockouts.

[0284] Figure 4 Shown are the results of PMBC MLR assays using cell populations with certain knockouts.

[0285] Figure 5A-Figure 5BShown are the results of a primed T MLR assay performed to test the effectiveness of CAR T cells against allogeneic T cell rejection. Figure 5C-5D The results of further sensitized T MLR assay (C) and NK cell MLR assay (D) are shown. Figure 5E Shown are the results of PBMC MLR assay. Fig. 5F It is shown that some CAR T cells with various knockouts are able to reduce the expansion of host immune cells (left panel: host CD8+ T cells; right panel: host CD4+ T cells). Figure 5G It was shown that some CAR T cells with various knockouts were able to reduce the expansion of host NK cells. Figure 5H The cytotoxicity of CAR T cells with different knockouts is shown.

[0286] Figure 6A-6B Shown are the results of a NK cell MLR assay performed to test the susceptibility of CAR T cells to allogeneic NK cell rejection. Figure 6C Demonstrated that CAR T cells did not exhibit IL-2-independent growth.

[0287] Figure 7 The gene editing efficiency of different CAR T cell populations is shown.

[0288] Figure 8 Expansion of different CAR T cell populations with different knockouts is shown.

[0289] Fig. 9 Shown are the results of cytotoxicity assays of different CAR T cells with certain knockouts.

[0290] Fig.10 Shown are the results of sensitized T cell MLR assays of different engineered (e.g., gene-edited) CAR T cells.

[0291] Fig.11 Shown are the results of PBMC MLR assays of different engineered (e.g., gene-edited) CAR T cells.

[0292] Figure 12A-12B Shown are the results of host immune cell expansion in MLR assays performed with different engineered (e.g., gene-edited) CAR T cells (A: host CD8+ T cells and CD4+ T cells; B: NK cells).

[0293] Figures 13A-13D Shown are the results of different assays using CD19 CAR / CD70 binding protein / CD58 knockout cells. Fig.13A Shown are the results of PBMC MLR assay. Figure 13B-Figure 13CResults of host cell expansion assays in the MLR assay are shown (B: host CD4+ and CD8+ T cells; C: host NK cells). Fig.13D Results of cytotoxicity assays are shown. DETAILED DESCRIPTION

[0294] The present disclosure provides a gene editing strategy for providing therapeutic allogeneic cell products, which do not cause or cause rejection of the recipient's immune system to a reduced extent. This allows the cell product to last longer in the recipient, and thus promotes and / or improves the therapeutic effect. The strategy of the present invention involves downregulating or eliminating certain cell surface receptors, which are known to play a role in immune cell adhesion and activation of one or more of the immune synapses, such as CD48, CD58 and ICAM-1. This strategy can be further supplemented by further downregulating or eliminating genes encoding molecules involved in HLA expression, so that the diversity of peptides presented by allogeneic cell products can be minimized. This is achieved by introducing one or more genomic modifications (e.g., genomic knockouts or knockouts low) of one or more of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP and RFXANK, the products of which play a role in the peptide presentation of cells. In one embodiment, genomic modifications (e.g., genomic knockouts or knockouts low) are directed to

[0295] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0296] NLRC5 and CD58, RFX5 and CD58, RFX5 and CD48, NLRC5 and CD48, RFX5 and ICAM-1, NLRC5 and ICAM-1;

[0297] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0298] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; or (vi) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0299] In one embodiment, the one or more genomic modifications are at the genomic location of one or more genes (corresponding to one or more targets as described herein), or at other locations within the genome other than at the location of the one or more genes (corresponding to one or more targets as described herein), such that the modifications functionally attenuate or reduce the expression of the one or more genes (corresponding to one or more targets as described herein).

[0300] When an immune cell comprises at least one modification, it can be referred to as an engineered immune cell. Thus, the engineered immune cells of the present disclosure can be referred to as immune cells comprising a genomic modification.

[0301] In one aspect, engineered immune cells and colonies comprising engineered immune cells demonstrate improved ability to resist alloreactive immune cell elimination. In one embodiment, improved ability to resist elimination is demonstrated by improved survival in the presence of alloreactive immune cells such as PBMC, T cells and / or natural killer (NK) cells. For example, the improvement in survival can be shown via one or more mixed lymphocyte (MLR) assays as described herein (including in the Examples section).

[0302] In another aspect, engineered immune cells and colonies comprising engineered immune cells show improved persistence in allogeneic subjects or HLA mismatch subjects. In one embodiment, the persistence of the cells or colonies is improved by improving the ability to remain viable after adoptive transfer into allogeneic subjects or HLA mismatch subjects. As described herein, the cells and colonies comprising the cells are characterized by improved ability to resist elimination of alloreactive immune cells. Without being bound by theory, after being administered to allogeneic subjects or HLA mismatch subjects, it is expected that cells and colonies with such improved abilities will also show improved persistence.

[0303] One aspect of the present invention relates to engineered cells (e.g., engineered immune cells, such as CAR T cells) encoding genes that are functionally reduced (or not expressed) in cell surface molecules that can be recognized and subsequently killed by recipient (host) immune cells, and methods for preparing and using such engineered cells. Certain cell surface molecules, including but not limited to CD58 and CD2, CD48 and CD2 and / or ICAM-1 and LFA-1, interact to provide appropriate cell adhesion and activation of immune cells (Dustin, ML The immunological synapse, Cancer Immunology Research. November 2014; 2(11): 1023-33). In addition, loss of CD58 has been associated with resistance of tumor cells to T cell-mediated killing and immune evasion (Frangieh, CJ et al., Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat Genet. 2021 Mar;53(3):332-341, epub 2021 Mar 1; Challa-Malladi, M. et al., Combined genetic inactivation of β2-Microglobulin and CD58 reveals frequent escape from immune recognition in diffuse large B cell lymphoma. Cancer Cell. Cell. 2011 Dec 13;20(6):728-40, epub 2011 Dec 1). For example, B cell lymphoma cells presenting tumor-associated antigens on HLA class I molecules can be recognized and subsequently killed by CD8 T cells (see Figure 1B Above - From Challa-Malladi, M. et al. Figure 8). Tumor cells often downregulate the expression of HLA class I. However, HLA class I is a ligand for natural killer (NK) cell inhibitory responses such as KIR (killer cell immunoglobulin-like receptors), which transduce signals to balance activation signals from receptors such as CD2 (see, e.g., Jaeger, BN, Vivier, E., Natural Killer Cell Tolerance: Control by Self or Self-Control? Cold Spring Harb Perspect Biol. 2012 Mar; 4(3): a007229–a007229). Therefore, the loss of cell surface expression of HLA class I renders tumor cells susceptible to NK cell killing, which is achieved via activation of CD2 by the ligand CD58 expressed on tumor cells (see Figure 1B Middle figure - from Challa-Malladi, M. et al. Figure 8 The simultaneous loss of HLA class I and CD58 can confer protection against T-cell and NK-cell killing, thereby leading to immune evasion of tumor cells (see Figure 1B Figure below - from Challa-Malladi, M. et al. Figure 8 ). In engineered cells from a donor cell population (e.g., allogeneic engineered immune cells, such as CAR T cells), functionally reduced expression (or absence of expression) of certain cell surface molecules, such as CD58, CD48, and ICAM-1, is a potential way to reduce recognition and subsequent killing of engineered cells by host T cells and NK cells after infusion of the engineered cells into a patient (i.e., an individual from whom the donor cell population is not derived).

[0304] The present invention provides engineered cells (e.g., engineered immune cells, such as CAR T cells) with reduced functional expression (or no functional expression) of certain genes encoding cell surface molecules that act at immune synapses, wherein the engineered cells can be part of an allogeneic cell transplant administered to a patient in need (i.e., a host or recipient of a transplant), which avoids or can avoid rejection by the patient's immune cells. Modifications of engineered cells that produce reduced (or no expression) functional expression of certain genes, such as gene editing modifications, do not require exogenous expression of proteins, which is advantageous. For example, due to the limited capacity of gene delivery vectors (e.g., vectors for delivering CAR T and other CAR T cell enhancers such as cytokines), gene editing methods that focus on introducing one or more exogenous genes into engineered cells may be challenging. On the contrary, the disclosure of the present invention includes methods for modifying engineered cells using gene editing techniques, such as CRISPR-Cas9, TALEN, etc., as described herein, during the manufacturing process to inactivate or downregulate one or more genes encoding cell surface receptors (e.g., one or more of CD48, CD58, and ICAM-1). As described herein, such modifications may also include gene editing to downregulate or eliminate the expression of genes involved in HLA molecules. In another embodiment, as described herein, the engineered cells (e.g., engineered immune cells, such as CAR T cells) further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein.

[0305] In one other aspect of the disclosure, the gene editing target is CD48 (also known as lymphocyte activation molecule 2 or SLAMF2), an immunoglobulin-like receptor that interacts with CD2 to facilitate the formation of immune synapses between T cells and antigen presenting cells. In other aspects, the gene editing target is CD58 (also known as lymphocyte function antigen 3 or LFA-3), a co-stimulatory receptor that interacts with its natural ligand CD2 and also facilitates the formation of immune synapses. In another aspect, the gene editing target is intercellular adhesion molecule 1 or ICAM-1 (also known as CD54), which (i) is a cell surface glycoprotein known for its role in stabilizing cell-cell interactions, (ii) is expressed in immune cells, and (iii) is a ligand for the LFA-1 receptor on leukocytes.

[0306] In another aspect of the present disclosure, the gene editing target is the TAP2 component of the transporter (TAP) associated with antigen processing. The main pathway for loading peptides on MHC class I molecules is TAP-dependent: peptides produced by the proteasome (or IFN-γ-inducible immunoproteasome) are imported into the endoplasmic reticulum (ER) through TAP and then loaded on MHC class I. A few peptides, mainly derived from signal peptides, are loaded through alternative TAP and proteasome-independent pathways after the signal sequence is cut by the ER signal peptide peptidase (SPP). Compared with the significant (10 to 100-fold) reduction in surface β2m in β2m KO cells, knocking out TAP2 moderately reduced surface β2m (reduced by 2 times after KO cell selection) (see PCT / US2022 / 14393) Figure 4 A, which is incorporated herein by reference in its entirety).

[0307] In another aspect of the present disclosure, gene editing genes can be molecules involved in the transcriptional regulation of HLA-I and HLA-II molecules. HLA-I and HLA-II molecules are strictly regulated at the transcriptional level by similar key cis-regulatory elements: W / S, X1, X2 and Y box motifs. The regulatory factor X (RFX) hybrid consists of RFX5, RFXAP and RFXANK and binds to the X1 box. The X2 box is occupied by the CREB / ATF1 family transcription factors, and the Y box is bound by the NF-Y protein. In addition, two members of the receptor (NLR) family containing nucleotide binding domains and leucine-rich repeat sequences, NLRC5 and CIITA, are necessary for the formation of the HLA enhancer complex to promote HLA-I and HLA-II transcription, respectively. NLRC5 and CIITA do not bind directly to DNA, but require the help of other subunits of the enhancer to dock (Meissner, TB et al., NLR family member NLRC5 is a transcriptional regulator of MHC class I genes. Proc. Natl. Acad. Sci. USA 107, 13794–13799 (2010); Meissner, TB et al., NLRC5 Cooperates with the RFX Transcription Factor Complex To Induce MHC Class I Gene Expression. J. Immunol. 188, 4951–4958 (2012)). Specifically, NLRC5 associates with RFXANK through its ankyrin repeat sequence.

[0308] In one embodiment, the gene editing target is a member of the nucleotide binding domain and leucine-rich repeat-containing receptor (NLR) family, called NLR caspase recruitment domain-containing protein 5 (NLRC5). CRISPR / Cas9-mediated knockout of NLRC5 caused a 2.5-fold decrease in surface β2m levels (see PCT / US2022 / 14393). Figure 4 B, which is incorporated herein by reference in its entirety). In another embodiment, the gene editing target is RFX5, which, along with RFXAP and RFXANK / B, is part of the RFX complex associated with the X1 box motif, as described herein.

[0309] The gene editing strategy disclosed herein surprisingly fully reduces peptide display, thereby reducing cell death caused by the recipient's T cell response, while not reducing peptide display too much, thereby causing killing of the recipient's NK cells. Therefore, the strategy provided herein represents a significant advance in allogeneic CAR-T therapy and other allogeneic cell therapies. The gene editing strategy provided herein confers an additional advantage, namely that the NLRC5 knockout effect that inhibits MHC class I presentation should occur in the presence or absence of IFN-γ. The upregulation of MHC class I induced by IFN-γ depends on NLRC5, and this finding supports this conclusion.

[0310] In one other aspect of the present invention, the gene editing target of the engineered cell can be one or more genes encoding one or more molecules, which have a role in one or more cellular pathways associated with the rejection of host or recipient immune cells, and these immune cells are reactive with T cells and NK cell epitope determinants on the surface of allogeneic cell products different from the host. In one embodiment, the gene editing target of the engineered cell can be one or more genes encoding the following substances: i) cell surface receptors known to play a role in immune cell adhesion and activation at the immune synapse (e.g., one or more of CD48, CD58, and ICAM-1) and / or ii) transcription factors or regulatory factors of HLA-I and HLA-II molecules (e.g., RFX5, NLRC5, CIITA, RFXAP, and RFXANK). The method for producing engineered cells with reduced or eliminated expression of one or more genes described herein can focus only on genes encoding molecules that have an effect at the immune synapse, but can also be supplemented by reducing or eliminating the expression of genes encoding molecules that act primarily as transcription factors for HLA-I and / or HLA-II molecules.

[0311] General Technology

[0312] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the following literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Thayer and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (JP Cellis, ed., 1998) Academic Press; Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-1998) John Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (John Wiley & Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 1994); Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press), 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995). For example, gene editing techniques using TALEN, CRISPR / Cas9 and megaTAL nucleases are within the skill of the art and are fully explained in the literature, such as T. Gaj et al., Genome-Editing Technologies: Principles and Applications, Cold Spring Harb Perspect Biol 2016;8:a023754 and citations therein.

[0313] definition

[0314] As used herein, "autologous" means a cell, cell line, or population of cells obtained from a subject for use in treating the subject.

[0315] As used herein, "allogeneic" means a cell or population of cells that is not obtained from the subject but rather is obtained from a donor for treating the subject.

[0316] As used herein, the term "endogenous" refers to any material that originates or originates from within an organism, cell, tissue or system.

[0317] As used herein, the term "exogenous" refers to any material introduced or generated from outside an organism, cell, tissue or system.

[0318] As used herein, "immune cell" refers to a cell of hematopoietic origin that is functionally involved in the initiation and / or execution of an innate and / or adaptive immune response. Examples of immune cells include T cells, such as α / βT cells and γ / δT cells, regulatory T (Treg) cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0319] As used herein, the term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0320] As used herein, "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0321] As described herein, the engineered immune cells of the present disclosure express at a reduced level, for example, functionally express one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK, and optionally further comprise additional features. For example, they can functionally express antigen binding proteins from exogenous nucleic acids encoding antigen binding proteins introduced into cells by the techniques described herein. In one embodiment, the engineered immune cells of the present disclosure functionally express a first antigen binding protein such as CAR, and / or a second protein such as CD70 binding protein. The engineered cells may also include genomic modifications that reduce or eliminate the functional expression of genes, such as mutations at endogenous genes such as TCR a and / or CD52, and / or they may express one or more additional proteins from exogenous nucleic acids encoding antigen binding proteins introduced into cells by the techniques described herein. As described herein, the engineered immune cells of the present disclosure may be derived from cells, such as immune cells obtained from various sources, such as prepared by the cells.

[0322] As used herein, "functionally expressing" a gene means that the gene is expressed, and expression produces a functional gene final product. For example, if a gene encodes a protein, then if the expression of the gene ultimately produces a functional protein, the cell functionally expresses the gene. Therefore, for example, if a gene is not transcribed, or the expression of the gene ultimately produces an untranslated RNA, or translation only produces a non-functional protein, such as a protein that is not correctly folded or is not transported to its site of action (e.g., a membrane for a membrane-bound protein), the gene is not functionally expressed. Functional expression can be measured directly (e.g., by measuring the gene product itself) or indirectly (e.g., by measuring the effect of the gene product).

[0323] As used herein, "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one nucleic acid sequence is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is able to affect the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

[0324] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to a nucleic acid sequence to be transcribed.

[0325] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence that is capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. It will be appreciated by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions.

[0326] In any of the vectors disclosed herein, the vector optionally includes a promoter disclosed herein.

[0327] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the present disclosure.

[0328] As used herein, the term "extracellular ligand binding domain" refers to an oligopeptide or polypeptide capable of binding a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, an extracellular ligand binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a specific disease state. The term "stem domain" is used herein to refer to any oligopeptide or polypeptide that acts to connect a transmembrane domain to an extracellular ligand binding domain. Specifically, the stem domain is used to provide greater flexibility and accessibility to the extracellular ligand binding domain.

[0329] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector signaling functions and directs the cell to perform a specialized function.

[0330] As used herein, "costimulatory molecules" refer to cognate binding partners that specifically bind to costimulatory ligands on T cells, thereby mediating the costimulatory response of cells, such as but not limited to proliferation. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc.

[0331] "Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory signal molecule on a T cell, thereby providing a signal in addition to the primary signal provided by, for example, the binding of a TCR / CD3 complex to an MHC molecule loaded with a peptide, which mediates T cell responses, including but not limited to proliferation activation, differentiation, etc. Co-stimulatory ligands may include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1 CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also specifically encompass antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0332] "Antibody" is an immunoglobulin molecule that can specifically bind to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term not only covers complete polyclonal or monoclonal antibodies, but also covers its antigen binding fragments (such as Fab, Fab', F(ab')2 and Fv), and any other modified configurations of immunoglobulin molecules including antigen recognition sites, such as including but not limited to single chain (scFv) and domain antibodies (such as including shark and camel antibodies), and fusion proteins including antibodies. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or its subclass), and antibodies do not need to have any specific class. Immunoglobulins can be assigned to different classes according to the antibody amino acid sequence of the constant region of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0333] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed in the term "antigen-binding fragment" of an antibody include Fab; Fab'; F(ab')2; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments (see, e.g., Ward et al., Nature 341:544-546, 1989) and individual complementary determining regions (CDRs).

[0334] The term "specific binding" to a target is used to refer to an antibody, antibody conjugate or polypeptide, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration and / or with greater affinity than an alternative cell or substance. An antibody "specifically binds" to a target if it binds to it with greater affinity, avidity, more easily and / or with a longer duration than it binds to other substances. It should also be understood by reading this definition that, for example, an antibody (or part or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding.

[0335] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions). The CDRs in each chain are tightly bound together by the FRs and, together with the CDRs in the other chain, contribute to the formation of the antigen binding site of the antibody. There are several techniques for determining CDRs, such as methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, MD); an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948), the Chothia system (i.e., Chothia and Lesk, J. Mol. Biol. (1987) 196(4):901-917). As used herein, CDRs may refer to CDRs defined by either approach or by a combination of both approaches.

[0336] The "CDR" of a variable domain is an amino acid residue within the variable region that is identified according to the definitions of Kabat, Chothia, accumulation of both Kabat and Chothia, AbM, contact and / or conformational definitions, or any CDR determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Protein Sequences of Immunological Significance, 5th Edition, Public Health Service, NIH, Washington DC. The position of the CDR can also be identified as the structural loop structure originally described by Chothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other methods of CDR identification include the "AbM definition," which is a compromise between Kabat and Chothia and is derived using the AbM antibody modeling software (now Oxford Molecular) from Oxford Molecular. ) derived, or based on the "contact definition" of CDRs based on observed antigen contacts, which is described in MacCallum et al., Journal of Molecular Biology, 262: 732-745, 1996. In another method referred to herein as the "conformational definition" of CDRs, the positions of CDRs can be identified as residues that make enthalpic contributions to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283: 1 156-1 166, 2008. Other CDR boundary definitions may still not strictly follow one of the above methods, but will nevertheless overlap with at least a portion of the Kabat CDRs, but in view of the prediction or experimental discovery that a particular residue or residue group or even the entire CDR does not significantly affect antigen binding, the boundary definition may be shortened or lengthened. As used herein, CDRs may refer to CDRs defined by any method known in the art, including a combination of methods. The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extension, AbM, contact, AHo, and / or conformational definitions.

[0337] The antibodies disclosed herein can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques or other techniques well known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA et al., J. Molecular Biology, 373(4): 924-40, 2007).

[0338] As known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs or any substrate that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembling the chain. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotides can be further modified after polymerization, such as by combining with a labeling component. Other types of modifications include, for example, "capping", substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramides, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing side groups such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those with modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl group usually present in sugar can be replaced by phosphonate group, phosphate group, protected by standard protecting groups or activated to prepare other bonds relative to other nucleotides, or can be conjugated to a solid support. 5' and 3' terminal OH can be partially phosphorylated or replaced with amine or organic end-capping groups with 1 to 20 carbon atoms. Other hydroxyls can also be derived into standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose commonly known in the art, including for example 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose), pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs (such as methyl riboside). One or more phosphodiester bonds can be replaced by alternative linking groups. These replaceable linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("disulfide"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formalate"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl or an aralkyl group.Not all bonds in a polynucleotide need to be identical.The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0339] As used herein, "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. When exogenous or heterologous RNA or DNA has been introduced into a cell, the cell has been "transfected" by such RNA or DNA. When the transfected RNA or DNA affects a phenotypic change, the cell has been "transformed" by the exogenous or heterologous RNA or DNA. The transforming RNA or DNA can be integrated (covalently linked) into the chromosomal DNA that makes up the genome of the cell.

[0340] As used herein, "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragment are referred to as "transgenic" or "recombinant" or "transformed" organisms.

[0341] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, yet more preferably at least 98% pure, and most preferably at least 99% pure. The term "competition" as used herein with respect to antibodies means that the first antibody or its antigen-binding fragment (or portion) binds to the epitope in a manner very similar to the binding of the second antibody or its antigen-binding portion, such that the binding result of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. An alternative, in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, may be, but is not necessarily, the case. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, in the case where each antibody detectably inhibits the binding of other antibodies to their cognate epitopes or ligands, whether to the same, greater or lesser extent, the antibodies are considered to "cross-compete" with each other to bind to their corresponding epitopes. The present disclosure encompasses competing antibodies and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), the skilled artisan will understand, based on the teachings provided herein, that such competing and / or cross-competing antibodies are contemplated and can be used in the methods disclosed herein.

[0342] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing (or destroying) the proliferation of tumors or cancer cells, inhibiting the metastasis of tumor cells, shrinking or reducing the size of a tumor, alleviating a disease (e.g., cancer), alleviating symptoms caused by a disease (e.g., cancer), improving the quality of life of a person suffering from a disease (e.g., cancer), reducing the dose of other drugs required to treat a disease (e.g., cancer), delaying the progression of a disease (e.g., cancer), curing a disease (e.g., cancer) and / or prolonging the survival of a subject suffering from a disease (e.g., cancer).

[0343] "Improvement" refers to the alleviation or improvement of one or more symptoms compared to no treatment. "Improvement" also includes shortening or reducing the duration of symptoms. As used herein, the "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For preventive purposes, the beneficial or desired results include eliminating or reducing the risk of a disease (including biochemical, histological and / or behavioral symptoms of the disease, complications of the disease and intermediate pathological phenotypes that occur during the development of the disease), reducing its severity or delaying its onset. For therapeutic purposes, the beneficial or desired results include clinical results, such as a reduction or improvement in the incidence of one or more symptoms of various diseases or conditions (such as cancer), reducing the dose of other drugs required for the treatment of the disease, enhancing the effect of another drug and / or delaying the progression of the disease. The effective dose can be administered once or multiple times. For the purposes of this disclosure, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly complete a preventive or therapeutic treatment. As understood in a clinical context, an effective dose of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of administration of one or more therapeutic agents, and an individual agent may be considered to be given in an effective amount if the desired result can be achieved or accomplished in combination with one or more other agents.

[0344] As used herein, a "subject" is any mammal, such as a human or a monkey. Mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats. In an exemplary embodiment, the subject is a human. In an exemplary embodiment, the subject is a monkey, such as a cynomolgus monkey.

[0345] As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0346] As used herein, "pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that allows the active ingredient to retain biological activity when combined with the ingredient and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions of the present disclosure including such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st ed. Mack Publishing Co., 2005).

[0347] As used herein, "allogeneic reactivity" refers to the ability of T cells to recognize MHC complexes not encountered during thymic development. Alloreactivity manifests clinically as host versus graft rejection and graft versus host disease.

[0348] References herein to "about" a value or parameter include (and describe) embodiments that refer to the value or parameter itself plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. For example, descriptions referring to "about X" include descriptions of "X". Numerical ranges include the numbers defining the range.

[0349] It should be understood that when an embodiment is described with the language "comprising" anywhere herein, otherwise similar embodiments described according to "consisting of" and / or "consisting essentially of" are also provided.

[0350] Where aspects or embodiments of the present disclosure are described according to Markush groups or other alternative groupings, the present disclosure encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group lacking one or more of the group members. The present disclosure also contemplates explicitly excluding one or more of any group members in the disclosed and / or claimed embodiments.

[0351] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present specification (including definitions) shall prevail. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of the stated integer or groups of integers, but not the exclusion of any other integer or groups of integers. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0352] Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0353] An "antigen binding protein" includes one or more antigen binding domains. As used herein, "antigen binding domain" means any polypeptide that binds to a specified target antigen. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease or involved in a viral or bacterial antigen.

[0354] Antigen binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term "immunologically functional fragment" (or "fragment") of an antigen binding domain is a species of an antigen binding domain that includes a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to a target antigen (however the portion is obtained or synthesized). Such fragments have biological activity because the fragments bind to the target antigen and can compete with other antigen binding domains comprising intact antibodies for binding to a given epitope.

[0355] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity determining regions ("CDRs"), diabodies (heavy chain variable domains and light chain variable domains are located on the same polypeptide, connected by a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprising two antigen binding sites), multispecific antigen binding domains, and single-chain antibodies. These fragments may be derived from any mammalian source, including, but not limited to, humans, mice, rats, camels, or rabbits. As will be appreciated by those skilled in the art, antigen binding domains may include non-protein components.

[0356] The variable region usually exhibits the same general structure of a relatively conservative framework region (FR) joined by three hypervariable regions (CDRs). The CDRs from the two chains of each pair are usually aligned by a framework region that can bind to a specific epitope. From the N-terminus to the C-terminus, both the light chain variable region and the heavy chain variable region usually include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are usually referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are usually referred to as LC CDR1, CDR2, and CDR3.

[0357] In some embodiments, the antigen binding domain includes one or more complementary binding regions (CDRs) present in the full-length light or heavy chains of an antibody, and in some embodiments includes a single heavy and / or light chain or a portion thereof. These fragments can be produced by recombinant DNA technology or can be produced by enzyme or chemical cleavage of the antigen binding domain (comprising a complete antibody).

[0358] In some embodiments, the antigen binding domain is an antibody or a fragment thereof, comprising one or more of its complementary determining regions (CDRs). In some embodiments, the antigen binding domain is a single chain variable fragment (scFv), comprising light chain CDRs: CDR1, CDR2, and CDR3, and heavy chain CDRs: CDR1, CDR2, and CDR3.

[0359] The assignment of amino acids to each of the framework, CDRs and variable domains is generally made according to the following numbering schemes: Kabat numbering (see, e.g., Kabat et al., Sequences of Proteins of Immunological Significance, 5th ed., NIH Publication No. 91-3242, Bethesda, MD 1991), Chothia numbering (see, e.g., Chothia and Lesk, (1987), J. Mol. Biol. 196:901-917; Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948; Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tramontano et al., (1990) J. Mol. Biol. 215(1):175-82; and U.S. Pat. No. 7,709,226), contact numbering, AbM scheme (Antibody Modeling Program, Oxford Molecular Biology, Inc. Molecular)) or AHo system (Honneger and Pluckthun, Journal of Molecular Biology (2001) 309(3):657-70).

[0360] In some embodiments, the antigen binding domain is a recombinant antigen receptor. As used herein, the term "recombinant antigen receptor" refers to a non-naturally occurring surface receptor, which includes an extracellular antigen binding domain or an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptor (CAR) is well known in the art. CAR is a fusion protein including an antigen recognition portion, a transmembrane domain, and a T cell activation domain (see, e.g., Eshhar et al., Proceedings of the National Academy of Sciences of the United States of America, 90 (2): 720-724 (1993)).

[0361] In some embodiments, the intracellular domain of the recombinant antigen receptor includes a co-stimulatory domain and an ITAM-containing domain. In some embodiments, the intracellular domain of the recombinant antigen receptor includes an intracellular protein or a functional variant thereof (e.g., truncated, inserted, deleted or substituted).

[0362] As used herein, the term "extracellular ligand binding domain" or "extracellular antigen binding domain" refers to a polypeptide that can bind to a ligand or antigen or can interact with a cell surface molecule, such as a ligand or surface antigen. For example, an extracellular ligand binding domain or antigen binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a specific disease state (e.g., a tumor-specific antigen). In some embodiments, the antigen binding domain includes an antigen binding fragment or antigen binding portion of an antibody or an antibody. In some embodiments, the antigen binding domain includes Fv or scFv, Fab or scFab, F(ab')2 or scF(ab')2, Fd, a monomer, an affibody, a camel antibody, a VHH antibody, a single domain antibody or a darpin. In some embodiments, the ligand binding domain includes a partner of a binding pair, such as a ligand that binds to a surface receptor, or an extracellular domain of a surface receptor that binds to a ligand.

[0363] The term "stem domain" or "hinge domain" is used interchangeably herein to refer to any polypeptide that functions to connect a transmembrane domain to an extracellular ligand-binding domain. In particular, the stem domain is often used to provide greater flexibility and accessibility to the extracellular ligand-binding domain.

[0364] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector signaling functions and directs the cell to perform a specialized function.

[0365] Carrier

[0366] Expression vectors and methods for administering polynucleotide compositions are known in the art and are further described herein.

[0367] In another aspect, the present disclosure provides a method of making any of the polynucleotides described herein.

[0368] The present disclosure also encompasses polynucleotides complementary to any such sequence. The polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be a DNA (genomic, cDNA or synthetic) or RNA molecule. The RNA molecule comprises an HnRNA molecule containing introns and corresponding to a DNA molecule in a one-to-one manner, and an mRNA molecule that does not contain introns. Additional coding or non-coding sequences may but need not be present in the polynucleotide of the present disclosure, and the polynucleotide may but need not be connected to other molecules and / or carrier materials.

[0369] Polynucleotides may include native sequences (i.e., endogenous sequences encoding antibodies or portions thereof), or may include variants of such sequences. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions, such that the immunoreactivity of the encoded polypeptide is not diminished relative to the natural immunoreactive molecule. The effect of the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. Variants preferably exhibit at least about 70% identity to the polynucleotide sequence encoding the natural antibody or portion thereof, more preferably at least about 80% identity, more preferably at least about 90% identity, and most preferably at least about 95% identity. If the sequences of nucleotides or amino acids in the two sequences are identical when the maximum correspondence is aligned as described below, the two polynucleotide or polypeptide sequences are referred to as "identical". Comparison between two sequences is generally performed by comparing sequences on a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, typically about 30 to about 75 or about 40 to about 50 contiguous positions in which a sequence can be compared to a reference sequence having the same number of contiguous positions after the two sequences are optimally aligned.

[0370] Sequences can be optimally aligned for comparison using the Megalign program in the Lasergene bioinformatics software suite (DNASTAR, Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, "A model of evolutionary change in proteins - a matrix for detecting long-range relationships," in Dayhoff, MO (ed.), Protein Sequence and Structure Atlas, Volume 5, Supplement 3, pp. 345-358, National Biomedical Research Foundation, Washington, DC; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645, Methods in Enzymology, Volume 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, Computer Applications in the Biological Sciences (CABIOS), 5: 151-153; Myers, EW and Muller, J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645, Methods in Enzymology, Volume 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, Computer Applications in the Biological Sciences (CABIOS), 5: 151-153; W., 1988, "Computer Applications in Biological Sciences", 4:11-17; Robinson, ED, 1971, "Combine Theor." 1 1:105; Santou, N., Nes, M., 1987, "Mol. Biol. Evolution", 4:406-425; Sneath, PHA and Sokal, RR, 1973, "Numerical Classification - Principles and Practice of Numerical Classification", Freeman Press, St. Francis, California; Wilbur, WJ and Lipman, DJ, 1983, "Proceedings of the National Academy of Sciences of the United States of America", 80:726-730.

[0371] In some embodiments, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) of 20 percent or less, typically 5 to 15 percent or 10 to 12 percent, compared to a reference sequence (the reference sequence does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to derive the number of matched positions; dividing the number of matched positions by the total number of positions in the reference sequence (i.e., window size); and multiplying the result by 100 to derive the percentage of sequence identity.

[0372] Variants may also or alternatively be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions with a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0373] Suitable "moderately stringent conditions" include pre-washing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C-65°C, 5×SSC; followed by washing twice with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS at 65°C for 20 minutes.

[0374] As used herein, "high stringency conditions" or "high stringency conditions" refer to those that: (1) employ low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) employ a denaturing agent such as formamide during hybridization, for example, 50% (v / v) formamide with 750 mM sodium chloride, 75 mM sodium citrate at pH 6.5 with 0.1% bovine serum albumin / 0.1% polysucrose / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer; or (3) employ 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.5) at 42°C. 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate, washes in 0.2x SSC (sodium chloride / sodium citrate) at 42° C. and in 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1x SSC containing EDTA at 55° C. The skilled artisan will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

[0375] Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. However, the disclosure specifically contemplates polynucleotides that vary due to differences in codon usage. Further, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the disclosure. Alleles are endogenous genes that change due to one or more mutations of nucleotides, such as deletions, additions and / or substitutions. The resulting mRNA and protein may, but need not, have a changed structure or function. Standard techniques (such as hybridization, amplification and / or database sequence comparisons) can be used to identify alleles.

[0376] Can use chemical synthesis, recombination method or PCR to obtain polynucleotide of the present disclosure.The method of chemical polynucleotide synthesis is well-known in the art and does not need to be described in detail in this article.Those skilled in the art can use the sequence provided by this article and commercial DNA synthesizer to produce the desired DNA sequence.

[0377] In order to prepare polynucleotides using recombinant methods, the polynucleotides including the desired sequence can be inserted into a suitable vector, and then the vector can be introduced into a suitable host cell for replication and amplification, as further described herein. The polynucleotides can be inserted into a host cell by any means known in the art. Exogenous polynucleotides are introduced to transform cells by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotides amplified in this way can be separated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0378] Alternatively, PCR allows the replication of a DNA sequence. PCR techniques are well known in the art and are described, for example, in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 and in PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston, 1994.

[0379] RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, for example, as described above by Sambrook et al., 1989.

[0380] Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected may be different according to the host cell to be used, useful cloning vectors will usually have the ability of self-replication, can have a single target for a specific restriction endonuclease and / or can carry the gene of the marker that can be used to select the clone containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (for example, pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers, such as BioRad, Strategene and Invitrogen.

[0381] An expression vector is typically a replicable polynucleotide construct containing a polynucleotide according to the present disclosure. This means that the expression vector must be replicable in a host cell as an episome or as a component of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and PCT publications disclosed in WO 87 / 04462. Vector components may typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation start sites, and stop codons, are also typically required.

[0382] The vector containing the polynucleotide of interest can be introduced into the host cell by any of a variety of appropriate means, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0383] The polynucleotide encoding the antigen binding protein (e.g., CAR) may be present in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfecting an insect host cell, or a plasmid or viral vector such as a lentivirus for transfecting a mammalian host cell). In some embodiments, the polynucleotide or vector may include a nucleic acid sequence encoding a ribosome skipping sequence, such as, but not limited to, a sequence encoding a 2A peptide. In the absence of a peptide bond between two amino acids encoded by a codon, the 2A peptide identified in the aphthous virus subgroup of a picornavirus causes the ribosome to "jump" from one codon to the next codon (see, e.g., Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008). "Codon" means three nucleotides on mRNA (or on the sense strand of a DNA molecule), which are translated into an amino acid residue by the ribosome. Therefore, when the polypeptide is separated by the 2A oligopeptide sequence in the frame, the two polypeptides can be synthesized from a single continuous open reading frame in the mRNA. Such ribosome skipping mechanisms are well known in the art and are known to be used by several vectors for expressing several proteins encoded by a single messenger RNA.

[0384] In order to guide the transmembrane polypeptide into the secretory pathway of the host cell, in some embodiments, a secretion signal sequence (also referred to as a leader sequence, pre-prosequence or pre-sequence) is provided in the polynucleotide sequence or the vector sequence. The secretion signal sequence is operably connected to the transmembrane nucleic acid sequence, that is, the two sequences are connected in the correct reading frame and are positioned to guide the newly synthesized polypeptide into the secretory pathway of the host cell. The secretion signal sequence is usually located at the 5' of the nucleic acid sequence encoding the polypeptide of interest, but some secretion signal sequences may be located at other places of the nucleic acid sequence of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, there may be considerable sequence variation in these polynucleotide molecules. In some embodiments, the nucleic acid sequence of the present disclosure is directed to expression in mammalian cells, preferably codon optimized for expression in human cells. Codon optimization refers to exchanging codons that are typically rare in highly expressed genes of a given species with codons that are typically common in highly expressed genes of such species in the sequence of interest, such codons encoding the same amino acid as the codon being exchanged.

[0385] Methods for preparing engineered cells

[0386] Provided herein is a method for preparing engineered cells for immunotherapy, such as engineered immune cells. In some embodiments, the method includes introducing an antigen binding protein (e.g., CAR) into one or more immune cells, or introducing a polynucleotide encoding an antigen binding protein (e.g., CAR), and amplifying the cell. In some embodiments, the disclosure relates to a method for engineering immune cells, the method including: providing immune cells, and expressing at least one antigen binding protein (e.g., CAR) on the cell surface. In some embodiments, the method includes: transfecting cells with at least one polynucleotide encoding an antigen binding protein (e.g., CAR), and expressing the at least one polynucleotide in the cell.

[0387] In some embodiments, the polynucleotides encoding antigen binding proteins (e.g., CAR) are present in one or more expression vectors for stable expression in cells. In some embodiments, the polynucleotides are present in viral vectors for stable expression in cells. In some embodiments, the viral vector can be, for example, a lentiviral vector or an adenoviral vector.

[0388] In some embodiments, the polynucleotide encoding a polypeptide according to the present disclosure can be an mRNA that is directly introduced into a cell, for example, by electroporation. In some embodiments, the CytoPulse technology can be used to transiently permeabilize living cells to deliver materials into the cell. Parameters can be modified to determine conditions for high transfection efficiency and minimal mortality.

[0389] Also provided herein are methods for transfecting immune cells (e.g., T cells). Generally, any conventional method known to those of ordinary skill in the art can be used, such as introducing any one of RNA, DNA, or protein into cells by electroporation. See, for example, Luft and Ketteler, Journal of Biomolecular Screening 20(8):932(2015)(DOI:10.1177 / 1087057115579638). In some embodiments, the method includes: contacting T cells with RNA and applying an agile pulse sequence to the T cells, wherein the agile pulse sequence consists of: (a) electric pulses with a voltage ranging from about 2250 to 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval of about 0.2 to 10 ms between the electric pulses of steps (a) and (b); (d) electric pulses with a voltage ranging from about 2250 to 3000 V per centimeter, and a pulse width of about 100 ms and a pulse interval of about 100 ms between the electric pulse of step (b) and the first electric pulse of step (c); and (e) four electric pulses with a voltage of about 325 V, and a pulse width of about 0.2 ms and a pulse interval of 2 ms between each of the four electric pulses. In some embodiments, a method of transfecting a T cell comprises contacting the T cell with an RNA and applying an agile pulse sequence to the T cell, the agile pulse sequence comprising: (a) an electric pulse having a voltage of about 1600, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) and a pulse width of about 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ms between the electric pulses of steps (a) and (b). (d) a voltage ranging from about 2250 to 3000 V per centimeter, for example, one electric pulse of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V per centimeter, and a pulse width of 100 ms and a pulse interval of 100 ms between the electric pulse of step (b) and the first electric pulse of step (c); and (e) 4 electric pulses with a voltage of about 325 V, and a pulse width of about 0.2 ms and a pulse interval of about 2 ms between each of the 4 electric pulses. Any value included in the value range described above is disclosed in the present application. The electroporation medium can be any suitable medium known in the art. In some embodiments, the conductivity of the electroporation medium ranges from about 0.01 to about 1.0 millisiemens.

[0390] In some embodiments, the method may further include the following steps: by inactivating at least one gene of expression such as but not limited to TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP and RFXANK, components of TCR, targets of immunosuppressants, HLA genes and / or immune checkpoint proteins (such as, for example, PDCD1 or CTLA-4) or reducing the expression level of at least one gene to genetically engineer the cell. Gene inactivation is intended to prevent the gene of interest from being expressed in the form of functional protein. In some embodiments, the gene to be inactivated is one or more of the genes selected from the group consisting of the following, such as but not limited to NLRC5, TAP2, TCRα, TCRβ, β2-microglobulin ("β2m" or β2m), CD52, CIITA, RFX5, RFXAP, RFXANK, GR, deoxycytidine kinase (DCK), PD-1 and CTLA-4. In some embodiments, the method comprises inactivating or reducing the expression level of one or more genes by introducing into the cell a rare-cutting endonuclease that is capable of selectively inactivating genes by selective DNA cleavage. In some embodiments, the rare-cutting endonuclease can be, for example, a transcription activator-like effector nuclease (TALE-nuclease or ), megaTAL nuclease or Cas9 endonuclease.

[0391] In another aspect, the step of genetically modifying or engineering immune cells (e.g., T cells) may include: modifying immune cells (e.g., T cells) by inactivating at least one gene expressing an immunosuppressant target, and optionally amplifying cells in the presence of an immunosuppressant. An immunosuppressant is an agent that suppresses immune function through a number of mechanisms of action. An immunosuppressant can reduce the degree and / or intensity of an immune response. Non-limiting examples of immunosuppressants include calcineurin inhibitors, rapamycin targets, interleukin-2 α-chain blockers, inosine monophosphate dehydrogenase inhibitors, dihydrofolate reductase inhibitors, corticosteroids, and immunosuppressive antimetabolites. Some cytotoxic immunosuppressants work by inhibiting DNA synthesis. Other cytotoxic immunosuppressants can work by activating T cells or by inhibiting the activation of helper cells. According to the method disclosed herein, it is allowed to confer, for example, T cell immunosuppression resistance to perform immunotherapy by inactivating the target of the immunosuppressant in T cells. As non-limiting examples, the target of the immunosuppressant may be a receptor for the immunosuppressant, such as, but not limited to, CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclophilin family gene members.

[0392] Provided herein are compositions and methods for expressing antigen binding proteins (e.g., CAR) and / or CD70 binding proteins (as described herein) and for lowering the functional expression of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK. In other embodiments, the functional expression is lowered by (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1. In another embodiment, the functional expression is lowered by NLRC5 and CD58, RFX5 and CD58, RFX5 and CD48, NLRC5 and CD48, RFX5 and ICAM-1, or NLRC5 and ICAM-1. In another embodiment, the functional expression is lowered by only one of CD48, CD58 and ICAM-1. In another embodiment, the downregulation of functional expression is (i) CD58, NLRC5 and RFX5, (ii) CD48, NLRC5 and RFX5, (iii) ICAM-1, NLRC5 and RFX5, (iv) CD58, ICAM-1 and RFX5, (v) CD48, ICAM-1 and RFX5, (vi) CD58, CD48 and RFX5, (vii) CD58, ICAM-1 and NLRC5, (viii) CD48, ICAM-1 and NLRC5, or (ix) CD58, CD48 and NLRC5. Also provided are uses of such compositions for improving the functional activity of immune cells (e.g., T cells, such as CAR-T cells) and methods for improving the functional activity of immune cells (e.g., T cells, such as CAR-T cells). The methods and compositions provided herein can be used to improve the in vivo persistence and therapeutic efficacy of engineered immune cells (e.g., engineered T cells, such as CAR-T cells).

[0393] Engineered cells

[0394] Engineered cells, such as engineered immune cells, for example, engineered T cells provided herein can express antigen binding proteins, such as chimeric antigen receptors (CARs), and / or CD70 binding proteins, and express any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK at a level no greater than 75%, no greater than 50%, no greater than 25%, or no greater than 10% of the expression level in unengineered immune cells. Advantageously, the engineered immune cells provided herein exhibit improved in vivo persistence and / or increased resistance to rejection by the recipient's immune system relative to unengineered cells.

[0395] In some embodiments, engineered cells, such as engineered immune cells, include CAR colonies, each CAR includes different extracellular antigen binding domains. In some embodiments, engineered cells, such as engineered immune cells, include CAR colonies, each CAR includes the same extracellular antigen binding domains.

[0396] In one embodiment, the engineered cells, such as engineered immune cells, are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes (Treg), helper T lymphocytes, tumor infiltrating lymphocytes (TIL)), natural killer T cells (NKT), TCR expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, engineered cells, such as engineered immune cells, can be derived from CD4+T lymphocytes or CD8+T lymphocytes. In another embodiment, the engineered cells can be derived from a T lymphocyte population containing CD4+T lymphocytes and CD8+T lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are γδT cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some exemplary embodiments, the engineered immune cells are natural killer (NK) cells.

[0397] In some embodiments, engineered cells, such as engineered immune cells, can be derived from, for example but not limited to, stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0398] In some embodiments, engineered cells, such as engineered immune cells, are obtained from peripheral blood or prepared from peripheral blood. In some embodiments, engineered cells are obtained from peripheral blood mononuclear cells (PBMC) or prepared therefrom. In some embodiments, engineered cells are obtained from bone marrow or prepared therefrom. In some embodiments, engineered cells are obtained from cord blood or prepared therefrom. In some embodiments, cells are human cells. Representative human cells are CD34+ cells.

[0399] In some embodiments, cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., a gene gun), lipofection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplex.

[0400] Any immune cell capable of expressing heterologous DNA can be used for the purpose of expressing an antigen binding protein, such as a CAR of interest, and further engineered to express reduced levels of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK.

[0401] In some embodiments, immune cells such as T cells provided herein are further modified, such as genetically modified, to express one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK at reduced levels relative to comparable cells that have not been so modified. For example, immune cells can be genetically modified to knock out all or part of one or more of the CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK loci so that the corresponding functional proteins are not expressed on the cell surface, such as by making a portion or all of the genomic DNA containing the complete coding sequence of the locus and / or the genomic DNA containing the transcriptional control elements and / or promoters and / or activation elements of the locus missing and / or by introducing insertions, deletions, or substitutions that prevent the production of functional proteins. In addition, immune cells can be genetically modified to knock out all or part of the following loci

[0402] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0403] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, or both NLRC5 and ICAM-1;

[0404] Only one of CD48, CD58 and ICAM-1; or

[0405] (i) CD58, NLRC5 and RFX5, (ii) CD48, NLRC5 and RFX5, (iii) ICAM-1, NLRC5 and RFX5, (iv) CD58, ICAM-1 and RFX5, (v) CD48, ICAM-1 and RFX5, (vi) CD58, CD48 and RFX5, (vii) CD58, ICAM-1 and NLRC5, (viii) CD48, ICAM-1 and NLRC5, or (ix) CD58, CD48 and NLRC5,

[0406] The corresponding functional protein is not expressed on the cell surface, for example, by deleting part or all of the genomic DNA containing the complete coding sequence of the locus and / or the genomic DNA containing the transcriptional control elements and / or promoter and / or activation elements of the locus and / or by introducing insertions, deletions or substitutions that prevent the production of functional proteins.

[0407] In one embodiment, the one or more genomic modifications are at the genomic location of one or more genes (corresponding to one or more targets as described herein), or at other locations within the genome other than at the location of the one or more genes (corresponding to one or more targets as described herein), such that the modifications functionally attenuate or reduce the expression of the one or more genes (corresponding to one or more targets as described herein).

[0408] In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein. In some embodiments, the CD70 binding protein provided herein comprises an extracellular domain (e.g., a single-chain variable fragment (scFv)) and a transmembrane domain. In some embodiments, the CD70 binding protein provided herein comprises an extracellular ligand binding domain (e.g., scFv), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CD70 binding protein includes one or more intracellular signaling domains selected from the group consisting of: CD3ζ signaling domain, CD3δ signaling domain, CD3γ signaling domain, CD3ε signaling domain, CD28 signaling domain, CD2 signaling domain, OX40 signaling domain, and 4-1BB signaling domain or a variant thereof. In another embodiment, the CD70 binding protein comprises an intracellular signaling domain, which comprises a CD3ζ signaling domain. In some embodiments, the CD70 binding protein is a CAR.

[0409] In some embodiments, the intracellular signaling domain comprises an amino acid sequence of one or more of SEQ ID NO: 1, 7-14, 17-70 or 89-90. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of one or more of SEQ ID NO: 7, 89, 8, 90, 12, 11, 61, 62, 64 or 65. In some embodiments, the CD70 binding protein comprises a CD3 ζ or CD3 γ signaling domain or a variant thereof and does not include a co-stimulatory domain. In some embodiments, the CD70 binding protein comprises a 4-1BB signaling domain or a variant thereof and does not include a CD3 signaling domain. In some embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3 ζ signaling domain. In some embodiments, the CD70 binding protein does not include an intracellular signaling domain. Different intracellular signaling domains or combinations thereof can confer different signaling strengths, which can contribute to T cell proliferation, potency, survival, persistence and / or resistance to host immune cell rejection. Described herein are CD70 binding proteins that do not contain or contain one or more intracellular signaling domains. In another embodiment, the CD70 binding protein comprises an scFv having an amino acid sequence as shown in SEQ ID NO: 82 or 85. In another embodiment, the CD70 binding protein comprises an amino acid sequence as shown in SEQ ID NO: 86. Any of SEQ ID NOs: 1, 7-14, 17-70 or 89-90 may comprise one or more substitutions, insertions or deletions. These sequences may contain 5, 4, 3, 2 or fewer substitutions, insertions or deletions. Polypeptides comprising such substitutions, insertions or deletions may retain their activity. For example, the intracellular signaling domain retains the ability to transduce the relevant signal, and the CD70 binding protein retains the ability to specifically bind to CD70.

[0410] In some embodiments, engineered immune cells that have been engineered to functionally express reduced levels of one or more targets described herein (relative to corresponding cells that have not been so engineered) and that have been further engineered to express CD70 binding proteins may exhibit varying levels of persistence and / or resistance to host immune cell rejection and, when administered to a patient, may be suitable for in vivo lymphocyte depletion. In some embodiments, engineered immune cells that functionally express the CD70 binding proteins described herein may inhibit the proliferation and / or activity of host immune cells to varying degrees when administered to a patient, which may allow fine-tuning of the depth of in vivo lymphocyte depletion. For example, engineered immune cells that functionally express CD70 binding proteins that exhibit long-term expansion and / or inhibition of host immune cell proliferation or activity in, for example, an MLR assay may be used for long-term lymphocyte depletion. Conversely, when less complete or less thorough lymphocyte depletion is desired, engineered immune cells that functionally express CD70 binding proteins that exhibit less long-term expansion and / or inhibition of host immune cells in the same or similar assay may be used.

[0411] In one aspect, the reduction in expression level can be a knockout or knockdown of expression relative to a comparable cell that has not been modified. Different knockdown methods may be appropriate, such as those employing any of a variety of RNA-based technologies (e.g., short hairpin RNA (shRNA), antisense RNA, microRNA (miRNA), small (or short) interfering RNA (siRNA); see, e.g., Van Hoeck et al., Biomaterials, Vol. 286, July 2022, 121510, ISSN 0142-9612; Lam et al., Mol. Ther. -Nucleic Acids 4:e252 (2015), doi: 10.1038 / mtna.2015.23; Sridharan and Gogtay, Brit. J. Clin. Pharmacol. 82:659-72 (2016), and U.S. Pat. No. 9,556,433 to Krause et al., which are incorporated herein by reference in their entirety). RNA-based agents, such as RNA interference molecules, can be delivered to cells, such as immune cells, so that one or more genes are knocked down. In one embodiment, the RNA-based agent can be configured to target the one or more genes or to target the one or more genes. In another embodiment, the RNA interference molecule comprises an RNA interference sequence, which comprises one or more sequences complementary to the one or more target genes. Alternatively, a polynucleotide comprising one or more RNA interference sequences for one or more target genes can be inserted into the genome. In one embodiment, the insertion can occur at a location in the genome that is not the location of the one or more target genes. In one embodiment, the RNA-based agent is configured to target or to target

[0412] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0413] Only one of CD48, CD58 and ICAM-1;

[0414] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0415] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0416] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0417] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0418] In another embodiment, the RNA-based agent is not configured to target β2m or does not target β2m.

[0419] In some embodiments, in immune cells, such as T cells of the present disclosure, the functional expression levels of

[0420] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0421] Only one of CD48, CD58 and ICAM-1;

[0422] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0423] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0424] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0425] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0426] The functional expression level of β2m may be reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to the functional expression level in comparable cells that have not been engineered to reduce the corresponding expression level. In another embodiment, the functional expression level of β2m is not reduced.

[0427] One or more antigen binding proteins (e.g., one or more CARs) and / or CD70 binding proteins can be synthesized in situ in cells after polynucleotide constructs encoding these proteins are introduced into cells. Alternatively, antigen binding proteins (e.g., CARs) and / or CD70 binding proteins can be produced extracellularly and then introduced into cells. The method of introducing a polynucleotide construct into a cell is known in the art. In some embodiments, a stable transformation method can be used to integrate a polynucleotide construct into the genome of a cell. In other embodiments, a transient transformation method can be used to transiently express a polynucleotide construct and a polynucleotide construct that is not integrated into the genome of a cell. In other embodiments, a viral-mediated method can be used. Polynucleotides can be introduced into cells by any suitable means, such as recombinant viral vectors (e.g., retroviruses, including lentiviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to microinjection, electroporation, or particle bombardment. The polynucleotide can be contained in a vector, such as a plasmid vector or a viral vector.

[0428] In some embodiments, the engineered immune cells (e.g., T cells) of the present disclosure may comprise at least one antigen binding protein (e.g., CAR) and / or CD70 binding protein. The engineered immune cells (e.g., T cells) are further modified, for example, genetically engineered to express reduced levels of

[0429] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0430] Only one of CD48, CD58 and ICAM-1;

[0431] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0432] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0433] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5;

[0434] (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0435] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0436] In one embodiment, the engineered immune cells (e.g., T cells) are not further modified to express reduced levels of β2m. In another embodiment, the engineered immune cells (such as CAR T cells) further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein as described herein.

[0437] In one other embodiment, the present disclosure provides an engineered immune cell, such as a CAR T cell, or an engineered immune cell colony comprising an engineered immune cell, wherein the engineered immune cells include one or more functionally weakened or reduced genome modifications of the expression of one or more targets described herein. In one embodiment, the one or more genome modifications are in the genome position of one or more genes (corresponding to one or more targets as described herein), or are in other positions within the genome, but not in the position of the one or more genes (corresponding to one or more targets as described herein), so that these modifications functionally weaken or reduce the expression of the one or more genes (corresponding to one or more targets as described herein).

[0438] In some embodiments, the engineered immune cells (e.g., engineered T cells) can comprise two or more different antigen binding proteins (e.g., two or more different CARs) and / or CD70 binding proteins, each CAR comprising a different extracellular ligand binding domain.

[0439] In some embodiments of engineered immune cells such as T cells provided herein, the CAR expressed by the cell may include an extracellular ligand binding domain (e.g., a single-chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain are in one polypeptide, i.e., in a single chain. Multi-chain CAR and polypeptides are also provided herein. In some embodiments, the multi-chain CAR includes: a first polypeptide containing a transmembrane domain and at least one extracellular ligand binding domain, and a second polypeptide containing a transmembrane domain and at least one intracellular signaling domain, wherein the polypeptides are assembled together to form a multi-chain CAR. In another embodiment, the engineered immune cell also includes or functionally expresses a CD70 binding protein, as described herein.

[0440] The extracellular ligand binding domain specifically binds to the target of interest. The extracellular ligand binding domain can specifically bind to a tumor antigen. For example, the extracellular ligand binding domain can specifically bind to a tumor antigen selected from a carcinoembryonic antigen, an overexpressed antigen, a tissue-restricted antigen, a testicular cancer antigen, or a tumor virus antigen. Tumor antigens may be associated with liquid tumors. In some embodiments, the target of interest can be any molecule of interest, including, for example, but not limited to BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin -18A2 or claudin 18 isoform 2), DLL3 (delta-like protein 3, Drosophila delta homolog 3, δ3), Muc17, Muc3, Muc3, Muc16, FAPα (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), RNF43 (E3 ubiquitin protein ligase RNF43, RING finger protein 43), specifically including the human form of any one of the exemplary targets listed.

[0441] In some embodiments, the antigen binding domain specifically binds to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY-ESO1, HER2 (ERBB2), CAIX (carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, occludin-18.2 (occludin-18A2 or occludin-18 isoform 2), PSCA, DLL3 (delta-like protein 3, Drosophila delta homolog 3, delta 3), Mud 7 (mucin 17, Muc3, Muc3), FAPα (fibroblast activation protein α), Ly6G6D (lymphocyte antigen 6 complex site protein G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).CARs and / or antibodies targeting antigens are disclosed, for example, in the following: BCMA-WO201616630, WO 2020150339, WO 2019196713, WO2016014565, WO 2017025038; MUC16: US9,169,328, WO 2016149368, WO 2020023888; EGFRvIII: WO 2017125830, WO 2016016341; Flt3: WO 2018222935, WO 2020010284, WO2017173410; CD20: WO 2018145649, WO 2020010235, WO 2020123691; CD38: WO 2017025323; CD70: WO 2019152742, WO 2018152181; CD33: WO 2016014576; CD133: WO 2018072025; CS1: WO 2019030240; ROR1: WO 2016115559; CD19: WO 2002077029, US11,077,144; sealin: WO2018006882, WO 2021008463; DLL3: WO 2020180591; WT1: US20160152725A1, US7622119B2; CD23: US6011138A, CN1568198A; CD30: US10815301B2, US10808035B2; PRAME: US20180148503A1, WO 2020186204A1; LIV1: US20200231699A1; NKG2D: WO 2021179353A1, US20210269501A1; FAPα: US20200246383A1, US20210115102A1; PSMA: US20210277141A1, WO2020108646A1; MSLN: CN109680002A, CN109628492A.

[0442] In some embodiments, the extracellular ligand binding domain includes an scFv, which includes a light chain variable (VL) region and a heavy chain variable (VH) region of a target antigen-specific monoclonal antibody connected by a flexible linker. Single-chain variable region fragments are prepared by connecting light chain and / or heavy chain variable regions using a short connecting peptide (Bird et al., Science 242: 423-426, 1988). An example of a connecting peptide is a GS linker having an amino acid sequence (GGGGS) 3 (SEQ ID NO: 72), which bridges about 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of another variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). Generally, the linker can be a short flexible polypeptide and preferably comprises about 20 or less amino acid residues. The linker can then be modified for additional functions, such as attaching drugs or attaching to a solid phase carrier. Single-chain variants can be produced by recombination or synthesis. For the synthetic production of scFv, an automatic synthesizer can be used. For the recombinant production of scFv, a suitable plasmid or other vector containing a polynucleotide encoding the scFv can be introduced into a suitable host cell (e.g., a eukaryotic cell such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell such as Escherichia coli). The polynucleotide encoding the scFv of interest can be prepared by conventional operations such as ligation of the polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0443] According to the intracellular signaling domain of the CAR disclosed herein, it is responsible for generating intracellular signaling after the extracellular ligand binding domain binds to the target, thereby causing the activation and immune response of immune cells. The intracellular signaling domain can activate at least one normal effector function of immune cells expressing CAR. For example, the effector function of T cells can be cytolytic activity or auxiliary activity (including the secretion of cytokines).

[0444] In some embodiments, the intracellular signaling domain used in CAR can be, for example, but not limited to, cytoplasmic sequences of T cell receptors and co-receptors, and any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities, the T cell receptors and co-receptors act synergistically after antigen receptor engagement to initiate signal transduction. The intracellular signaling domain includes two different types of cytoplasmic signaling sequences: cytoplasmic signaling sequences that initiate antigen-dependent primary activation, and cytoplasmic signaling sequences that act in an antigen-independent manner to provide secondary or costimulatory signals. The main cytoplasmic signaling sequence can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. ITAM is a well-defined signaling motif found in the cytoplasmic tail region of various receptors that act as binding sites for syk / zap70-like tyrosine kinases. Examples of ITAM used in the present disclosure may include those derived from TCR zeta, FcR γ, FcR β, FcR ε, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d as non-limiting examples. In some embodiments, the intracellular signaling domain of CAR may include a CD3 zeta signaling domain. In some embodiments, the intracellular signaling domain of CAR of the present disclosure includes the domain of a costimulatory molecule.

[0445] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure includes a portion of a co-stimulatory molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133) and CD28 (NP_006130 and isoforms thereof).

[0446] CAR is expressed on the surface membrane of the cell. Therefore, CAR may include a transmembrane domain. Suitable transmembrane domains of CAR disclosed herein have the following capabilities: (a) expressed on the cell surface, such as expressed on the surface of immune cells, such as, for example, but not limited to, lymphocytes (e.g., T cells) or natural killer (NK) cells; and (b) interacting with the ligand binding domain and the intracellular signaling domain to instruct the cellular response of immune cells to predetermined target cells. The transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain may be derived from any membrane-bound protein or transmembrane protein. As a non-limiting example, a transmembrane polypeptide may be a domain of a T cell receptor, such as α, β, γ or δ; a polypeptide constituting a CD3 complex; an IL-2 receptor such as p55 (α chain), p75 (β chain or γ chain); an Fc receptor, specifically a subunit chain of Fcγ receptor III; or a CD protein. Alternatively, the transmembrane domain may be synthetic and may mainly include hydrophobic residues, such as leucine and valine. In some embodiments, the membrane-spanning domain is derived from human CD8 α chain (e.g., NP_001139345.1). The membrane-spanning domain may also include a stem domain between the extracellular ligand binding domain and the membrane-spanning domain. The stem domain may include up to 300 amino acids, such as 10 to 100 amino acids or 25 to 50 amino acids. The stem region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region derived from CD8, CD4 or CD28, or all or part of an antibody constant region. Alternatively, the stem domain may be a synthetic sequence corresponding to a naturally occurring stem sequence, or may be a fully synthetic stem sequence. In some embodiments, the stem domain is a part of human CD8 α chain (e.g., NP_001139345 and its isotype). In another specific embodiment, the membrane-spanning domain includes a part of human CD8 α chain. In some embodiments, the CAR disclosed herein may include an extracellular ligand binding domain, a CD3ζ signaling domain, and a 4-1BB signaling domain that specifically binds to BCMA, a CD8α human stem domain, and a transmembrane domain. In some embodiments, CAR can be introduced into immune cells as a transgene via a vector such as a plasmid vector. In some embodiments, a vector such as a plasmid vector may also contain, for example, a selection marker for identifying and / or selecting cells that accept the vector.

[0447] After the polynucleotide encoding the CAR and / or CD70 binding protein polypeptide is introduced into the cell, the CAR and / or CD70 binding protein polypeptide can be synthesized in situ in the cell. Alternatively, the CAR and / or CD70 binding protein polypeptide can be produced outside the cell and then introduced into the cell. The method of introducing a polynucleotide construct into a cell is known in the art. In some embodiments, a stable transformation method can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used to transiently express a polynucleotide construct and a polynucleotide construct that is not integrated into the genome of the cell. In other embodiments, a viral-mediated method can be used. The polynucleotide can be introduced into the cell by any suitable means, such as a recombinant viral vector (e.g., a retrovirus (e.g., a lentivirus), an adenovirus), a liposome, etc. The transient transformation method includes, for example, but not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide can be contained in a vector, such as a plasmid vector or a viral vector.

[0448] Also provided herein are immune cells, such as T cells, such as isolated T cells obtained according to any of the methods described herein. Any immune cell capable of expressing heterologous DNA can be used for the purpose of expressing an antigen binding protein (e.g., a CAR of interest) and / or a CD70 binding protein, and further engineered to express reduced levels of

[0449] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0450] Only one of CD48, CD58 and ICAM-1;

[0451] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0452] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0453] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0454] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5.

[0455] In some embodiments, immune cells are T cells. In some embodiments, immune cells can be derived from, for example, but not limited to stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. The separated cells can also be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells or T cells selected from the group consisting of: inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. In some embodiments, cells can be derived from the group consisting of CD4+T lymphocytes and CD8+T lymphocytes. In another embodiment, engineered cells can be derived from a T lymphocyte colony containing CD4+T lymphocytes and CD8+T lymphocytes.

[0456] In some embodiments, the immune cell (e.g., T cell, such as an isolated T cell) is further modified, for example, by genetic engineering as described herein (e.g., using, for example, TALEN, CRISPR / Cas9, or megaTAL nucleases to partially or completely delete or destroy

[0457] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0458] Only one of CD48, CD58 and ICAM-1;

[0459] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0460] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0461] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0462] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0463] In another embodiment, the immune cells are not further modified, for example, genetically engineered by the methods described herein (e.g., using known gene editing techniques such as TALEN, CRISPR / Cas9 or megaTAL nucleases to partially or completely delete or destroy the β2m locus). In another embodiment, the immune cells (e.g., engineered immune cells such as CAR T cells) further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0464] In another embodiment, the disclosure provides a modified or engineered immune cell comprising one or more genomic modifications that functionally weaken or reduce the expression of one or more targets described herein. In one embodiment, the one or more genomic modifications are at the genomic position of one or more genes (corresponding to one or more targets as described herein), or at other positions within the genome, but not at the position of the one or more genes (corresponding to one or more targets as described herein), so that these modifications functionally weaken or reduce the expression of the one or more genes (corresponding to one or more targets as described herein).

[0465] The engineered immune cells provided herein may include one or more mimicking epitope sequences that can sort cells to enrich engineered cells as described herein, e.g., express antigen binding proteins, and / or provide a safety switch mechanism to inactivate immune cells after the cells are administered to a patient or recipient, e.g., to limit the cell population of side effects. Such mimicking epitope sequences and their use in cell sorting and as safety switches are known in the art and are described, e.g., in US2018 / 0002435, which is incorporated herein by reference in its entirety.

[0466] Before amplification and genetic modification, cell sources can be obtained from a subject by a variety of non-limiting methods. Cells can be obtained from a plurality of non-limiting sources, and the non-limiting sources include peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from healthy donors, from subjects diagnosed with cancer or from subjects diagnosed with infection. In some embodiments, cells can be part of a mixed cell colony presenting different phenotypic characteristics.

[0467] PBMC can be directly used for genetic modification with immune cells (e.g., CAR or TCR) using methods as described herein. In certain embodiments, after separation of PBMC, T lymphocytes can be further separated, and before or after genetic modification and / or amplification, cytotoxic and helper T lymphocytes can be sorted into primary, memory and effector T cell subsets.

[0468] In some embodiments, CD8+ cells are further sorted into primary cells, stem cell memory cells, central memory cells, and effector cells by identifying the unique cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, CD8+T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin.

[0469] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with unique cell surface antigens.

[0470] Also provided herein are cell lines obtained from modified, e.g., transformed or engineered immune cells, e.g., engineered T cells, according to any of the methods described herein. In some embodiments, the engineered immune cells (e.g., engineered T cells) according to the present disclosure comprise polynucleotides encoding antigen binding proteins (e.g., CARs) and / or CD70 binding proteins, and are further modified or engineered (e.g., genetically modified) to express (e.g., functionally express) at reduced levels.

[0471] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0472] Only one of CD48, CD58 and ICAM-1;

[0473] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0474] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0475] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0476] ·(i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5. In another embodiment, the engineered immune cell is not further modified or engineered (e.g., genetically modified) to express (e.g., functionally express) β2m. In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein.

[0477] In a specific embodiment, a T cell comprising a genome modification is provided, the genome modification functionally weakens or reduces the expression of RFX5 and CD58 relative to a T cell without genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes or CD8+T lymphocytes.

[0478] In another specific embodiment, a T cell comprising a genome modification is provided, which functionally weakens or reduces the expression of NLRC5 and CD58 relative to a T cell without a genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0479] In another specific embodiment, a T cell comprising a genome modification is provided, the genome modification functionally weakens or reduces the expression of RFX5 and ICAM-1 relative to a T cell without genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0480] In another specific embodiment, a T cell comprising a genome modification is provided, which functionally weakens or reduces the expression of NLRC5 and ICAM-1 relative to a T cell without a genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0481] In another specific embodiment, a T cell comprising a genome modification is provided, the genome modification functionally weakens or reduces the expression of CD58 and ICAM-1 relative to a T cell without genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0482] In another specific embodiment, a T cell comprising a genome modification is provided, which functionally weakens or reduces the expression of RFX5, NLRC5 and CD58 relative to a T cell without a genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0483] In another specific embodiment, a T cell comprising a genome modification is provided, which functionally weakens or reduces the expression of RFX5, NLRC5 and ICAM-1 relative to a T cell without a genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0484] In another specific embodiment, a T cell comprising a genome modification is provided, which functionally weakens or reduces the expression of RFX5, CD58 and ICAM-1 relative to a T cell without genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0485] In another specific embodiment, a T cell comprising a genome modification is provided, which functionally weakens or reduces the expression of NLRC5, CD58 and ICAM-1 relative to a T cell without a genome modification, wherein the T cell further comprises: a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally reducing or eliminating CD52 expression, and reducing or eliminating endogenous TCR expression. The T cell can be an allogeneic T cell associated with the subject to be treated. The T cell can be derived from a healthy donor. The T cell can be derived from a group consisting of PBMC, CD4+T lymphocytes and CD8+T lymphocytes.

[0486] Methods involving engineered immune cells

[0487] Immune cells (e.g., T cells) of the present disclosure can be activated and expanded before or after cell modification using methods as generally described, for example, but not limited to, methods in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005. Immune cells (e.g., T cells) can be expanded in vitro or in vivo. In general, immune cells of the present disclosure can be expanded, for example, by contacting with agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of immune cells to generate activation signals for the cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectin-like phytohemagglutinin (PHA) can be used to generate activation signals for immune cells (e.g., T cells).

[0488] In some embodiments, a T cell population can be stimulated in vitro by contact with, for example, an anti-CD3 antibody or an antigen binding fragment thereof or an anti-CD2 antibody immobilized on a surface or with a protein kinase C activator (e.g., bryostatin) conjugated to a calcium ionophore. In order to co-stimulate auxiliary molecules on the surface of T cells, a ligand that binds to the auxiliary molecule is used. For example, under conditions suitable for stimulating the proliferation of T cells, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody. Conditions suitable for T cell culture include an appropriate culture medium (e.g., minimal essential medium, RPMI medium 1640 or X-VIVO TM 5 (Lonza), the medium may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFβ and TNF or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may comprise RPMI 1640 (as described herein), AIM V, DMEM, MEM, α-MEM, F-12, X-VIVO TM 10. X-VIVOTM 15 and X-VIVO TM 20. OpTmizer TM , and with the addition of amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in an amount sufficient to grow and expand T cells. Antibiotics (such as penicillin and streptomycin) are included only in experimental cultures and not in cell cultures to be infused into individuals. Target cells are maintained under conditions required to support growth, such as an appropriate temperature (such as 37° C.) and an atmosphere (such as air plus 5% CO2). Immune cells (such as T cells) exposed to different stimulation times may exhibit different

[0489] In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo, for example in the blood of a subject after the cells are administered to the subject.

[0490] Compositions and populations comprising engineered immune cells

[0491] In another aspect, the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising any cell of the present disclosure. In some embodiments, the composition comprises a T cell containing a polynucleotide encoding an antigen binding protein (e.g., CAR) and / or a CD70 binding protein. The cell is further engineered to express a reduced level of

[0492] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0493] Only one of CD48, CD58 and ICAM-1;

[0494] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0495] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0496] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0497] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0498] Its level is reduced relative to comparable cells that have not been engineered to functionally express reduced or altered levels of the corresponding protein, or the composition comprises a cell population containing the engineered immune cells (e.g., T cells) of the present disclosure, and one or more pharmaceutically acceptable carriers or excipients. In another embodiment, the cell is not further engineered to express reduced levels of β2m. In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein.

[0499] In one embodiment, the engineered cells described herein, such as engineered immune cells, e.g., CAR T cells, comprise one or more genomic modifications that functionally weaken or reduce the expression of one or more targets described herein. In one embodiment, the one or more genomic modifications are in the genomic position of one or more genes, or are in other positions within the genome, but not in the position of the one or more genes, so that these modifications functionally weaken or reduce the expression of the one or more genes.

[0500] In some embodiments, primary cells isolated from a donor are engineered as described herein to provide a cell population, wherein a subpopulation of the resulting cells (e.g., a ratio less than 100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) includes all desired modifications. Such resulting populations, including mixtures of cells containing all modifications and cells that do not contain all modifications, can be used in the methods of treatment disclosed herein and for preparing compositions disclosed herein. Alternatively, the cell population ("starting population") can be manipulated by known methods (e.g., cell sorting and / or amplification of cells with desired modifications) to provide enrichment for those cells containing one or more desired modifications (e.g., enrichment for cells expressing a desired antigen binding protein, and / or enrichment for cells expressing the following at reduced levels relative to comparable cells that have not been engineered in terms of the expression level of the corresponding protein).

[0501] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0502] Only one of CD48, CD58 and ICAM-1;

[0503] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0504] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0505] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0506] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5), i.e., a cell population that contains a higher percentage of such modified or engineered cells than the starting population. The enriched cell population may contain a higher percentage of such modified or engineered cells than the starting population. In another embodiment, the enriched population does not contain cells that express β2m at a reduced level. In another embodiment, the enriched population comprises one or more engineered immune cells that further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0507] Then, enrichment of modified cell colonies can be used in the methods of treatment of the present invention, and for preparing compositions of the present invention, for example. In some embodiments, the enriched cell colonies contain or contain at least, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of cells with one or more modifications. In other embodiments, the cell ratio in the enriched cell colonies including one or more modifications is at least 30% higher than the cell ratio in the starting cell colony including the desired modification.

[0508] Treatment

[0509] In one aspect, the present disclosure provides a method of evading an immune response in a subject during an immune cell therapy. In one embodiment, the immune cell therapy comprises administering to the subject allogeneic immune cells, wherein the allogeneic immune cells are engineered immune cells. In other embodiments, the engineered immune cells are engineered to functionally express a reduced level of

[0510] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK;

[0511] only one of CD48, CD58, and ICAM-1;

[0512] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1;

[0513] both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0514] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; (x) CD48, CD58 and ICAM-1; or

[0515] (i) CD58, ICAM-1, RFX5 and NLRC5; (ii) CD48, ICAM-1, RFX5, NLRC5; (iii) CD48, CD58, RFX5 and NLRC5; (iv) CD48, CD58, ICAM-1 and RFX5; (v) CD48, CD58, ICAM-1 and NLRC5; (vi) β2m, CD58, CD48 and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5 and NLRC5, wherein the engineered immune cells are (i) less susceptible to rejection by an alloreactive immune response of the subject, (ii) less susceptible to rejection by an alloreactive immune response of the subject's CD8+ and / or CD4+ T cells, and / or (iii) have improved ability to coexist with the subject's immune cells compared to non-engineered immune cells. In another embodiment, the engineered immune cell is further characterized as having in vivo CAR T efficacy and one or more of (i), (ii), and (iii). In another embodiment, the engineered immune cell further comprises a polynucleotide encoding a CD70 binding protein and / or functionally expresses a CD70 binding protein, as described herein.

[0516] Immune cells, e.g., engineered immune cells, such as T cells obtained by the above methods, or cell lines derived from such immune cells or T cells, can be used to treat a condition or disorder of a subject, or can be used as a medicament. In some embodiments, such methods and / or medicaments can be used to treat a condition or disorder, such as a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immune disease, or an aging-related disease. In some embodiments, the cancer can be selected from the group consisting of: gastric cancer, sarcoma, lymphoma (including non-Hodgkin's lymphoma), leukemia, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. In some embodiments, the subject is a previously treated adult subject with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian cancer, sarcoma, or relapsed or refractory classical Hodgkin's lymphoma (cHL).

[0517] In some embodiments, immune cells (e.g., T cells according to the present disclosure) or cell lines derived from immune cells (e.g., engineered T cells) can be used to manufacture a medicament for treating a condition or disorder in a subject in need thereof. In some embodiments, the condition or disorder can be, for example, cancer, an autoimmune disorder, or an infection.

[0518] Also provided herein is a method for treating a subject. In some embodiments, the method includes administering or providing an immune cell (e.g., engineered T cell) of the present disclosure to a subject in need. In some embodiments, the method includes administering an immune cell (e.g., T cell) of the present disclosure to a subject in need.

[0519] In some embodiments, immune cells of the present disclosure, such as engineered T cells, can undergo robust in vivo cell expansion and can last for an extended amount of time. The treatment methods disclosed herein can be ameliorative, curative, or preventive. The methods disclosed herein can be part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. The present disclosure is particularly suitable for allogeneic immunotherapy. Standard protocols can be used to convert donor-provided immune cells (e.g., engineered T cells) into non-alloreactive cells and propagate as needed to produce, for example, CAR-T cells that can be administered to one or several subjects. Such CAR-T cell therapies can be used as allogeneic ALLO CAR T TM Therapeutic products may be prepared.

[0520] On the other hand, the present disclosure provides a method of inhibiting tumor growth or progression in a subject with a tumor, the method comprising administering to the subject an effective amount of engineered immune cells as described herein, such as engineered T cells. On the other hand, the present disclosure provides a method of inhibiting or preventing metastasis of cancer cells in a subject, the method comprising administering to a subject in need an effective amount of engineered immune cells as described herein, such as engineered T cells. On the other hand, the present disclosure provides a method of inducing tumor regression in a subject with a tumor, the method comprising administering to the subject an effective amount of engineered immune cells as described herein, such as engineered T cells.

[0521] In some embodiments, the immune cells (eg, T cells) provided herein can be administered parenterally to a subject. In some embodiments, the subject is a human.

[0522] In some embodiments, the method may further include administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is, for example, crizotinib, palbociclib, anti-CTLA4 antibody, anti-4-1BB antibody, PD-1 antibody or PD-L1 antibody.

[0523] Also provided herein is the use of any of the immune cells (eg, T cells) provided herein in the preparation of a medicament for treating cancer in a subject in need thereof or inhibiting tumor growth or progression.

[0524] In certain embodiments, the functional expression levels of the following in the engineered immune cells of the present disclosure are

[0525] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0526] Only one of CD48, CD58 and ICAM-1;

[0527] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0528] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0529] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0530] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5

[0531] The functional expression level of any other gene that is knocked down or knocked out according to the present disclosure is reduced by or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to the corresponding expression level in a comparable engineered immune cell that has not been so genetically modified.

[0532] In another embodiment, the functional expression level of β2m is not reduced in the engineered immune cells of the present disclosure. In another embodiment, the engineered immune cells further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0533] The expression level can be determined by any known method such as FACS or MAC. In some embodiments, the engineered immune cells disclosed herein functionally express

[0534] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0535] Only one of CD48, CD58 and ICAM-1;

[0536] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0537] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0538] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0539] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0540] Or any other gene that is knocked down or knocked out according to the present disclosure, the expression level is no greater than 75%, no greater than 50%, no greater than 25%, no greater than 10% or its level is 0% of the expression level of the non-engineered immune cells (otherwise identical to the engineered immune cells, e.g., comprising the same components as the engineered immune cells). In another embodiment, the engineered immune cells do not functionally express β2m at a reduced level relative to the expression level of β2m in the non-engineered immune cells (identical to the engineered immune cells, e.g., comprising the same components as the engineered immune cells). In another embodiment, the engineered immune cells further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0541] In some embodiments, both alleles of a gene are knocked out so that the gene expression level of the engineered immune cells disclosed herein is 0% of the gene expression level of the corresponding unengineered cells. In some embodiments, one of the two alleles of a gene is knocked out so that the gene expression level of the engineered immune cells disclosed herein is 50% or about 50% of the gene expression level of the corresponding unengineered cells (for example, if the compensatory mechanism causes the expression of the remaining allele to be higher than the normal level). As described herein, if, for example, expression is reduced by some means other than knocking out, a medium expression level can be observed.

[0542] In some embodiments, the expression level of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK in the engineered cells of the present disclosure, or any other gene whose expression level is manipulated according to the present disclosure, can be measured directly by measuring the gene products and characteristics of the cells using standard techniques known to those skilled in the art (e.g., RT-qPCR, nucleic acid sequencing, antibody staining, or some combination of techniques). In some embodiments, the functional expression level of one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK is measured by determining the surface expression level of one or more HLA proteins (such as HLA-A or HLA-B proteins) or β2 microglobulin (B2M) or both B2M and one or more HLA proteins on the surface of engineered immune cells using standard techniques known in the art (e.g., flow cytometry). In various embodiments of the present disclosure, the functional expression level of any one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of each cell surface protein on the surface of the engineered immune cell, such as one or more of CD48, CD58 or ICAM-1 proteins, or measured by flow cytometry. These measurements can be compared with the corresponding measurements made on comparable cells that have not been engineered to reduce the corresponding functional expression levels. In a cell population including the engineered cells of the present invention (e.g., engineered immune cells), a combined sample of the measured material (e.g., RNA or protein or cell) will reflect the fact that some cells do not express the gene of interest, two alleles have been knocked out, for example, some cells express 50% or about 50% of the gene of interest, only one allele has been knocked out, and if the population includes unengineered cells, some cells express the gene of interest at normal levels.

[0543] The functional expression level of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK expression in the engineered immune cells of the present disclosure can also be determined, for example, by measuring the extent to which the engineered immune cells survive in the presence of effector cells (e.g., T cells or NK cells) compared to the extent to which unengineered but otherwise comparable (e.g., identical) immune cells survive under the same conditions. See, e.g., Figure 1D-1E and Example 1.

[0544] In some embodiments, administration of engineered immune cells disclosed herein (e.g., engineered T cells), or administration of cell populations comprising such engineered immune cells (e.g., engineered T cells), reduces host rejection of the administered cells or cell populations relative to comparable but unengineered cells or comparable populations that do not comprise such engineered cells. In some embodiments, administration of engineered immune cells (e.g., engineered T cells) disclosed herein, comprising antigen binding proteins (e.g., CARs) and / or CD70 binding proteins and wherein the expression levels (e.g., functional expression levels) of the following are reduced:

[0545] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0546] Only one of CD48, CD58 and ICAM-1;

[0547] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0548] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0549] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5;

[0550] (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0551] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0552] Or administering a cell population comprising such engineered immune cells (e.g., engineered T cells), wherein administration reduces host rejection of the administered cells or cell population relative to comparable but unengineered cells or a population not comprising such engineered cells. In another embodiment, the expression level (e.g., functional expression level) of β2m is not reduced. In another embodiment, the engineered immune cells further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0553] For example, such administration reduces host rejection by 1% to 99%, such as 5% to 95%, 10% to 90%, 50% to 90%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, compared to host rejection of the same cells but not engineered to express the corresponding protein at a reduced level. In some embodiments, host rejection is reduced by more than 90%.

[0554] In some embodiments, an engineered immune cell (e.g., T cell) of the present disclosure is administered, which comprises an antigen binding protein (e.g., CAR) and / or a CD70 binding protein and wherein the functional expression level of:

[0555] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0556] Only one of CD48, CD58 and ICAM-1;

[0557] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0558] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0559] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5;

[0560] (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0561] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0562] Or administering a cell population comprising such immune cells (e.g., T cells), wherein administration enhances or improves the persistence of the cells and / or increases the persistence of the cells compared to the persistence of the same cells but not engineered to express reduced levels of the corresponding protein. In another embodiment, the functional expression level of β2m is not reduced. In another embodiment, the immune cells further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0563] In some embodiments, persistence increases by, for example, 1 to 7 days, 1 to 12 weeks (for example, 1 to 4 weeks, 4 to 8 weeks or 8 to 12 weeks), or increases by 1 to 12 months, or increases a specific time length falling within these ranges. In some embodiments, the difference in persistence is measured by comparing the half-life of the cells used in a colony or composition, wherein, for example, the half-life increases by, for example, 1 to 7 days, 1 to 12 weeks (for example, 1 to 4 weeks, 4 to 8 weeks or 8 to 12 weeks), or increases by 1 to 12 months, or increases a specific time length falling within these ranges. In some embodiments, the difference in persistence is measured by comparing the time length of the cells used that can be detected after administration. In some embodiments, by comparing engineered cells and unengineered cells in the presence of, for example, immune cells such as T cells or NK cells, for example, the survival rate of about 72 hours, 5 days, 7 days or 13 days after mixing, the improvement of persistence is measured in vitro. In some embodiments, in such in vitro assays, when measuring, the engineered cells that survive are about 1.5 to 10 times of unengineered cells. In some embodiments, in such in vitro assays, the engineered immune cells survived about 1.5 to 10 times more than the non-engineered immune cells when measured. The extent of improvement in persistence or survival of the engineered immune cells described herein depends in part on the extent to which the functional expression level of one or more targets is reduced, and additionally but optionally on the expression level of CD70 in the co-incubated (e.g., "attack" or host) immune cells.

[0564] In some embodiments, the reduction of host rejection and / or the increase in the persistence of the cells as disclosed herein administered are determined by any of various techniques known to those of ordinary skill in the art. In some embodiments, any of the following or a combination thereof is used: flow cytometry, PCR (e.g., quantitative PCR) and in vitro co-incubation with patient tumor material or a model tumor cell line expressing an antigen targeted by CAR-T cells. In some embodiments, qPCR is used to assess the number of CAR T cells with and without knockout of interest to determine the extent to which knockout provides a survival advantage.

[0565] In some embodiments, treatment may be combined with one or more therapies for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiation therapy.

[0566] In some embodiments, treatment can be administered to subjects undergoing immunosuppressive therapy. In fact, the present disclosure can rely on cells or cell populations that are resistant to at least one immunosuppressant due to gene inactivation of receptors encoding such immunosuppressants. In this regard, immunosuppressive therapy can facilitate the selection and expansion of T cells according to the present disclosure in a subject.

[0567] Administration of cells or cell populations according to the present disclosure can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are administered by intravenous injection.

[0568] In some embodiments, administration of cells or cell populations according to the present disclosure may include administration of, for example, about 10 3 or 10 4 to about 10 9 cells, including all integer values ​​of the number of cells within these ranges. In some embodiments, administration of cells or cell populations may include administration of about 10 cells per kg body weight. 5 to about 10 6 cells, including all integer values ​​within these ranges, or 0.1 × 10 cells / kg body weight 6 Up to 5×10 6 The engineered immune cells of the present invention, or a total of 0.1×10 8 Up to 5×10 8 Engineered immune cells. Cells or cell colonies can be administered in one or more doses. In some embodiments, an effective amount of cells can be administered as a single dose. In some embodiments, an effective amount of cells can be administered as more than one dose over a period of time. The administration time is within the discretion of the attending physician and depends on the clinical condition of the subject. Cells or cell colonies can be obtained from any source, such as a blood bank or a donor. Although individual needs are different, the determination of the optimal range of the effective amount of a given cell type for a specific disease or condition is within the technical scope of the art. An effective amount means the amount that provides a therapeutic or preventive benefit. The dosage administered will depend on the age, health and weight of the recipient, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the desired effect. In some embodiments, an effective amount of cells or a composition including those cells is administered parenterally. In some embodiments, administration can be intravenous administration. In some embodiments, administration can be performed directly by injection into the tumor.

[0569] In some embodiments of the present disclosure, cells are administered to a subject in conjunction with (e.g., prior to, concurrently with, or after) any number of related therapeutic modalities, including, but not limited to, treatment with agents such as monoclonal antibody therapy, CCR2 antagonists (e.g., INC-8761), antiviral therapy, cidofovir and interleukin 2, cytarabine (also known as ARA-C), or nataliziimab treatment for MS subjects or efaliztimab treatment for psoriasis subjects or other treatments for PML subjects. In some embodiments, BCMA-specific CAR-T cells are administered to a subject in combination with one or more of the following: anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, anti-PD-L1 antibodies (e.g., avelumab, atezolizumab, or durvalumab), anti-OX40 antibodies, anti-4-1BB antibodies (e.g., Utolimumab), anti-MCSF antibodies, anti-GITR antibodies, and / or anti-TIGIT antibodies. In additional embodiments, the immune cells (e.g., T cells) of the present disclosure can be administered in combination with chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies, or other immune clearing agents (such as CAMPATH (alendinib)). These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Henderson, Naya, et al. Immunology. 1991 Jul;73(3):316–321; Liu, Albers, et al. Biochemistry. 1992 Apr 28;31(16):3896-901; Bierer, Hollander, et al. Curr Opin. Immunol. 1993 Jul;73(3):316–321; Liu, Albers, et al. Biochemistry. 1992 Apr 28;31(16):3896-901; Bierer, Hollander, et al. Curr Opin. Immunol. 1993 Jul;73(3):316–321; Liu, Albers, et al. Biochemistry. 1992 Apr 28;31(16):3896-901; Bierer, Hollander, et al. Curr Opin. Immunol. 1993 Jul;73(3):316–321; Liu, Albers, et al. Biochemistry. 1992 Apr 28;31(16):3896-901; Bierer, Hollander, et al. Curr Opin. Immunol. 1993 Jul;73(3):316–321; Liu, Albers, et al. Opin Immunol. (1993 October; 5(5):763-73). In one embodiment, the immune cells (e.g., T cells) of the present disclosure can be administered to a subject who has previously been administered an immunosuppressant, wherein the immunosuppressant causes lymphocyte depletion in the subject, thereby allowing transplantation of the immune cells.However, over time, the subject's immune system will rebuild and recover from lymphodepletion (see, e.g., Tees et al., Safety and PK / PD of ALLO-647, an anti-CD52 antibody, with fludarabine (Flu) / cyclophosphamide (Cy) for lymphodepletion in the setting of allogeneic CAR-T cell therapy. J. Clin. Oncology, Vol. 39, No. 15_Supplement 2021). Therefore, the engineered immune cells of the present disclosure provide further protection for the subject's immune system at different times of the treatment regimen. In one embodiment, the engineered immune cells coexist with the subject's immune system and actively engage the subject's immune system while avoiding rejection.

[0570] In additional embodiments, the cell compositions of the present disclosure are administered to a subject in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as CAMPATH. In some embodiments, the cell compositions of the present disclosure are administered after B cell ablation therapy (e.g., an agent reactive with CD20, e.g., Rituxan). For example, in one embodiment, a subject may undergo standard treatment, wherein high-dose chemotherapy is followed by a peripheral blood stem cell transplant. In certain embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the present disclosure. In some embodiments, the expanded cells are administered before or after surgery.

[0571] Reagent test kit

[0572] The disclosure also provides a kit for the method of the present invention. The kit of the present disclosure includes one or more containers, and the one or more containers include a composition of the present disclosure or an immune cell of the present disclosure, such as a T cell or a cell colony including an immune cell of the present disclosure (e.g., an engineered T cell). In various embodiments, immune cells (e.g., T cells) include one or more polynucleotides encoding antigen binding proteins (e.g., CAR) as described herein, and are further engineered to express reduced levels of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK as described herein One or more. In another embodiment, immune cells are further engineered to express reduced levels of

[0573] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0574] Only one of CD48, CD58 and ICAM-1;

[0575] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0576] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0577] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0578] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0579] As described herein. In another embodiment, the immune cells are not further engineered to express reduced levels of β2m.

[0580] The kit further includes instructions for use according to any of the methods of the present disclosure described herein. Typically, these instructions include a description of the administration of the composition, immune cell (eg, T cell) or cell population for the therapeutic treatment described above.

[0581] Instructions for use of kit components typically include information about dosages, dosing regimens, and routes of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0582] The kit of the present disclosure adopts suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. It is also contemplated that packaging used in combination with a specific device (e.g., an inhaler, a nasal application device (e.g., a nebulizer), or an infusion device (e.g., a micropump)). The kit can have a sterile access port (e.g., the container can be an intravenous bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile inlet port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). According to the present disclosure, at least one active agent in the composition is an immune cell (e.g., a T cell). The container can also include a second pharmaceutically active agent.

[0583] The kit may optionally provide additional components such as buffers, and interpretative information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0584] Sorting and removal methods

[0585] In some embodiments, a method for in vitro sorting of an immune cell population is provided, wherein a subpopulation of the immune cell population comprises a gene engineered to express at reduced levels as described herein:

[0586] Any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP and RFXANK;

[0587] Only one of CD48, CD58 and ICAM-1;

[0588] (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48 and ICAM-1;

[0589] · both NLRC5 and CD58, both RFX5 and CD58, both RFX5 and CD48, both NLRC5 and CD48, both RFX5 and ICAM-1, both NLRC5 and ICAM-1, both CD58 and ICAM-1, both CD58 and CD48, or both CD48 and ICAM-1;

[0590] (i) CD58, NLRC5 and RFX5; (ii) CD48, NLRC5 and RFX5; (iii) ICAM-1, NLRC5 and RFX5; (iv) CD58, ICAM-1 and RFX5; (v) CD48, ICAM-1 and RFX5; (vi) CD58, CD48 and RFX5; (vii) CD58, ICAM-1 and NLRC5; (viii) CD48, ICAM-1 and NLRC5; or (ix) CD58, CD48 and NLRC5; or

[0591] (i) CD58, ICAM-1, RFX5, and NLRC5; (ii) CD48, ICAM-1, RFX5, and NLRC5; (iii) CD48, CD58, RFX5, and NLRC5; (iv) CD48, CD58, ICAM-1, and RFX5; (v) CD48, CD58, ICAM-1, and NLRC5; (vi) β2m, CD58, CD48, and ICAM-1; or (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5,

[0592] and / or immune cells expressing antigen binding proteins, such as CARs.

[0593] In various embodiments, the method includes contacting the immune cell population with a monoclonal antibody specific for an epitope (e.g., a mimetic epitope, such as those provided in US2018 / 0002435) that is unique to the engineered cells (e.g., an epitope of an antigen binding protein or a mimetic epitope incorporated into an antigen binding protein), and selecting immune cells that bind to the monoclonal antibody to obtain a cell population enriched in engineered immune cells that express the antigen binding protein.

[0594] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be accomplished by fluorescence activated cell sorting (FACS).

[0595] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to magnetic particles. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be accomplished by magnetic activated cell sorting (MACS).

[0596] In some embodiments, the mAb used in the method of sorting immune cells expressing an antigen binding protein (e.g., CAR) is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10.

[0597] In some embodiments, the CAR-expressing immune cell population obtained when using the method described above for in vitro sorting of CAR-expressing immune cells includes at least 70%, 75%, 80%, 85%, 90%, 95% CAR-expressing immune cells. In some embodiments, the CAR-expressing immune cell population obtained when using the method for in vitro sorting of CAR-expressing immune cells includes at least 85% of CAR-expressing immune cells.

[0598] In some embodiments, the immune cell colonies expressing CAR obtained when using the method described above for in vitro sorting of immune cells expressing CAR are compared with the initial (unsorted) cell colonies, showing enhanced in vitro cytotoxic activity. In some embodiments, the in vitro cytotoxic activity increases by 10%, 20%, 30% or 50%. In some embodiments, the immune cells are T cells.

[0599] The CAR-expressing immune cells to be administered to the recipient can be enriched in vitro from the source population. The method of expanding the source population can include using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting to select cells expressing antigens such as the CD34 antigen.

[0600] Flow cytometry can be used to quantify specific cell types within a cell population. Typically, flow cytometry is a method used to quantify components or structural features of cells, primarily by optical means. Since different cell types can be distinguished by quantitative structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0601] Flow cytometry analysis involves two main steps: 1) labeling selected cell types with one or more labeled markers, and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling cell types comprises binding a labeled antibody to a marker expressed by a specific cell type. The antibody can be labeled directly with a fluorescent compound or indirectly using, for example, a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0602] In some embodiments, the method for sorting T cells expressing CAR is magnetic activated cell sorting (MACS). Magnetic activated cell sorting (MACS) is a method for separating various cell populations according to the surface antigens (CD molecules) of various cell populations by using superparamagnetic nanoparticles and columns. MACS can be used to obtain pure cell populations. Cells in single cell suspensions can be magnetically labeled with microbeads. The sample is applied to a column consisting of ferromagnetic spheres, which is covered with a cell-friendly coating that allows rapid and gentle separation of cells. Unlabeled cells are retained in the column by magnetically labeled cells. The effluent can be collected as an unlabeled cell fraction. After the washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0603] Detailed protocols for purifying specific cell populations (such as T cells) can be found in Basu S et al. (2010). (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cellsorting. Journal of Visualized Experiments (J. Vis. Exp.) (41): 1546).

[0604] Table 1: Other exemplary sequences

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611] Example

[0612] Example 1: Protective effect of CD58 knockout (KO) + RFX5 or NLRC5 KO in non-CAR T cells.

[0613] Primed T and natural killer (NK) mixed lymphocyte reaction (MLR) assays were used to test the protective effect of CD58 KO as a single modification (single KO) to enhance survival or to provide further survival benefit of RFX5 KO or NLRC5 KO against host rejection.

[0614] PBMCs from healthy human donors expressing HLA-A2 (HLA-A2+) were used as allogeneic effectors (hosts), and PBMCs from healthy donors not expressing HLA-A2 (HLA-A2-) were used to generate target cells (grafts). Gene editing was performed to generate graft T cells with the following KOs via CRISPR / Cas9: TRAC KO, TRAC / CD58 KO, TRAC / β2mKO, TRAC / RFX5 KO, TRAC / NLRC5 KO, TRAC / β2m / CD58 KO, TRAC / RFX5 / CD58KO, and TRAC / NLRC5 / CD58 KO. After 1 week of in vitro expansion, target cells were purified to remove any cells expressing TCRα / β. The levels of specific gene knockout or MHC-I and MHC-II knockdown were evaluated via FACS.

[0615] Alloreactive T cells are considered to be the main mediators of allogeneic rejection. Therefore, a sensitized alloreactive T cell mixed lymphocyte reaction (MLR) was performed using gene-edited cells as target cells to evaluate the protective effect of different knockouts on the survival of sensitized alloreactive T cells. To generate sensitized effector T cells, allogeneic PBMCs from HLA-A2+ donors were co-cultured with irradiated PBMCs or pan T cells isolated from graft donors for 1 week to promote the growth and expansion of alloreactive T cells. Effector pan T cells were then purified and co-incubated with gene-edited graft T cells in R10+20IU / mL IL-2 at a 1:1 ratio for 2 days. The absolute number of surviving graft T cells was analyzed by gating for live HLA-A2-TCRαβ-CD4+CD8+ and desired gene editing (e.g., CD58 KO) to determine the extent of killing ( Figure 1E ).

[0616] like Figure 1E As shown, CD58 KO enhanced the survival of RFX5 KO cells or NLRC5 KO cells compared with single RFX5 KO or single NLRC5 KO.

[0617] In the NK MLR assay, human NK cells were isolated from freshly isolated HLA-A2+ human PBMCs via MACS purification (Miltenyi, human pan NK cell isolation kit, catalog number 130-092-657). In a 96-well plate, 20,000 graft T cells were inoculated with 20,000 or 100,000 host NK cells and cultured in R10+1000IU / mL IL-2 for 2 days. By flow cytometry, absolute counts were used to determine the survival of graft T cells by gating live HLA-A2-TCRαβ-CD56-CD4+CD8+ and desired gene editing (e.g., CD58 KO).

[0618] like Figure 1F As shown, CD58 KO enhanced the survival of RFX5KO cells or NLRC5 KO cells compared to single RFX5 KO cells or single NLRC5 KO cells. Single CD58 KO did not affect graft cell survival in NK MLR. Similar trends were observed for effector cell: target cell ratio (E:T) of 1:1 and E:T=5:1.

[0619] Example 2: Protective effect of CD48 KO or ICAM-1 KO + RFX5 KO in non-CAR T cell transplants

[0620] Pan T cells from HLA-A2 donors were used for target cell generation. Gene editing was performed to generate target cells with the following KOs via CRISPR / Cas9: TRAC KO, TRAC / RFX5 KO, TRAC / CD48 KO, TRAC / ICAM-1KO, TRAC / RFX5 / CD48 KO, and TRAC / RFX5 / ICAM-1KO. After 2 weeks of in vitro expansion, targets were then purified to remove any cells expressing TCRα / β. The levels of specific gene knockout or MHC-I and MHC-II knockdown were evaluated via FACS.

[0621] like Figure 2 As shown, high gene editing efficiencies were observed for all groups (based on the average of two donors). On day 13 of production, more than 80% of cells had the desired gene edits. Although RFX5 KO was used in this set of tests, NLRC5 KO and β2m KO were not used.

[0622] The protective effect of ICAM-1 or CD48 KO on enhancing the survival of RFX5 KO against allogeneic T cell rejection was tested using the primed T cell MLR assay as described in Example 1. Figure 3As shown, ICAM-1 KO slightly enhanced the survival of RFX5 KO compared to CD48 KO as shown in the primed T cell MLR assay. As a single KO, ICAM-1 KO enhanced target cell survival compared to control grafts (TRACKO). The survival of CD48 KO was similar to that of control grafts.

[0623] The protective effect of ICAM-1 KO further enhanced the survival of RFX5 KO against host PBMC rejection compared to CD48 KO was tested in a PBMC MLR assay. The survival of gene-edited graft cells in the presence of allogeneic PBMC donors (n=3) was assessed. The effector:target (E:T) ratio used was 10:1. After 9 days of co-culture, the survival of graft cells was quantified by FACS. Figure 4 As shown, ICAM-1 KO further enhanced the survival of RFX5 KO compared with CD48 KO as shown in the PBMC MLR assay.

[0624] Example 3: Protective effect of CD58 KO or ICAM-1 KO + RFX5 KO using CAR T cell transplantation

[0625] Sensitized T MLR assay was used to test CD58 KO as a single modification (single KO) to enhance CAR T cell survival or provide the protective effect of RFX5 KO or NLRC5 KO against further survival benefits of T cell rejection.CD19 CAR T cells (CD19 FMC63) engineered by healthy donor cells to express turbo domains (turbodomain), turbo domains are a constitutively active chimeric cytokine receptor (CACCR), including TpoR domain variants (478-582; H499L; S505N; W515K) and intracellular IL2Rb domains (339-379, 393-433, 518-551) (see Lin et al., US US2021-0260118A1, which is incorporated herein by reference in its entirety). In the present embodiment, these CAR T cells will be referred to as CD19 CAR T cells hereinafter.As described below, the expression of CAR and turbo domains is controlled by EF1α or PGK promoters.

[0626] CD19 CAR T cells were modified to introduce one or more knockouts and then used as target cells. Specifically, CD19 CAR T cells were gene-edited via CRISPR / Cas9 to provide the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / CD58 KO, TRAC / RFX5 KO, TRAC / NLRC5 KO, TRAC / β2m / CD58 KO, TRAC / CD58 / RFX5 KO, and TRAC / CD58 / NLRC5 KO.

[0627] for Figure 5A - Experimental data presented in Figure 5J, CD19 CAR T cells were engineered with the EF1α promoter controlling expression of the CAR and Turbine domains.

[0628] Sensitized T MLR assay was performed to test the effectiveness of gene-edited CD19 CAR T cells (as target cells) against allogeneic T cell rejection. Data were merged from 4 graft / host donor pairs: 1 graft and 4 hosts. In the presence of 20 IU / mL of IL-2, cells were cultured at a ratio of effector: target (E: T) = 1: 1 for 2 days. In some cases, β2m KO Raji tumor cells expressing GFP were used to activate CAR T cells. The survival rate of graft CAR T cells was assessed by FACS and gated as live GFP-HLA-A2-TCRαβ-CD4+CD8+CAR+ and desired gene editing (e.g., CD58 KO).

[0629] like Figure 5A As shown, single β2mKO, single CD58 KO, single NLRC5 KO, and single RFX5 KO all enhanced graft cell survival compared to control CAR T grafts (TRAC KO). In addition, when CD19 CAR T cells were not activated (without Raji tumor cells), CD58 KO further enhanced survival compared to single NLRC5 KO or single RFX5 KO CAR T cells. Activated CD19 CAR T cells (in the presence of Raji tumor cells) survived better than unactivated CAR T cells (without Raji tumor cells). All cryptic KO graft cells, such as CD58 KO, had relatively comparable survival, and all had significantly better survival than control grafts (TRAC KO).

[0630] The protective effect of CD58 KO or ICAM-1 KO as a single modification (single KO) to enhance CAR T cell survival or to provide further survival benefit of RFX5 KO against T cell rejection was tested using the primed T MLR assay.

[0631] CD19 CAR T cells were gene-edited to provide the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / CD58 KO, TRAC / ICAM-1KO, TRAC / β2m / CD58 KO, TRAC / β2m / ICAM-1KO, TRAC / RFX5 / CD58 KO, and TRAC / RFX5 / ICAM-1KO.

[0632] Sensitized T MLR assays were performed to test the effectiveness of gene-edited CD19 CAR T cells against allogeneic T cell rejection. The data shown are the average values ​​of three technical replicates from a representative graft / host pair of 3 unique pairs. In the presence of 20 IU / mL of IL-2, cells were cultured at a ratio of effector: target = 1: 1 for 2 days. CAR T cells were activated using β2m KO Raji tumor cells expressing GFP. The survival rate of transplant CAR T cells was assessed by FACS and gated as live GFP-HLA-A2-TCRαβ-CD4+CD8+CAR+ and desired gene editing (e.g., CD58 KO).

[0633] like Figure 5B As shown, compared with the control (TRAC KO), single β2m KO, single RFX5KO, single CD58 KO and single ICAM-1KO all enhance graft CAR T cell survival. CD58 KO provides a slightly increased benefit to the survival of β2m KO, while ICAM-1KO does not. Compared with RFX5 single KO grafts, both CD58 KO and ICAM-1KO provide a greatly increased survival benefit for RFX5 KO CART cells.

[0634] CD19 CAR T cells (expressing the turbine domain as described above) were engineered to introduce different knockout combinations of B2M, RFX5, CD58, and NLRC5 and then used as graft cells in primed T MLR assays, NK cell MLR assays, and PBMC MLR assays. Specifically, CD19 CAR T cells were gene edited via CRISPR / Cas9 to provide the following knockouts: TRACKO, TRAC / CD58 KO, TRAC / ICAM-1KO, TRAC / β2m KO, TRAC / β2m / CD58KO, TRAC / β2m / ICAM-1KO, TRAC / RFX5 KO, TRAC / RFX5 / CD58 KO, and TRAC / RFX5 / ICAM-1KO.

[0635] For the primed T MLR assay, the ratio of host:graft:Raji tumor cells (WT Raj i cells with B2M knockout) was 1:1:0.25. Primed host T cells were enriched 2-fold using the Miltenyi Pan T isolation kit. Readings were performed 2 days later. Figure 5C As shown, CD58KO and ICAM-1KO as single modifications or in combination with RFX5 knockout, resulted in improved CAR T cell survival in T cell MLR assays (each dot represents a unique allogeneic host / graft pair). The combination of RFX5, CD58, and ICAM-1 was found to significantly mitigate T cell rejection compared to knockout of RFX5 alone. Furthermore, CD58 / RFX5 double knockout was found to specifically mitigate T cell rejection to almost the same extent as B2M knockout (KO) cells alone.

[0636] For the primed NK MLR assay, a 10:1:0.25 ratio of NK cells: graft: Raji tumor cells (WT Raj i cells) was used. Frozen NK cells were thawed for use. Readings were performed 2 days later (1,000 IU / ml IL-2). Figure 5D As shown, as expected, it was found that B2M knockout cells were sensitive to NK cell killing (each point represents a graft / NK pair). Compared with B2M knockout, CD58 and ICAM-1 knockout cells, alone or in combination with other knockout targets, i.e., B2M and RFX5, did not show any additional NK cell rejection. These results indicate that RFX / CD58 or ICAM-1 double knockout may alleviate T cell rejection to the same extent as B2M KO, but will not cause NK cell rejection of the same magnitude, and may alleviate the rejection of T cells and NK cells.

[0637] These assays analyzed graft survival after co-culture with primed T cells or primed NK cells. CD58 and ICAM 1 knockout was found to reduce T cell-mediated rejection both as single knockouts and in the RFX5 KO background. In NK MLRs, CD58 and ICAM-1 knockout was not found to have a deleterious effect on graft survival ( Figure 5C-5D ).

[0638] For PBMC MLR assay, a 10:1 host:transplant cell ratio was used for 10 days of co-culture. PBMCs from 3 different donors were depleted of B cells prior to co-culture. Readings were performed after 10 days. Figure 5E As shown, CD58 knockout and ICAM-1 knockout as single modifications or in combination with RFX5 knockout improve graft survival (each point represents a unique allogeneic host / graft pair - graft, 3 host donors). Fig. 5FAs shown, it was found that adding CD58 knockout with B2M knockout or RFX5 knockout reduced host immune cell expansion in 2 out of 3 donors (left panel - host CD8+ cells; right panel - host CD4+ cells). Figure 5G As shown, host NK cells expanded to a higher extent when co-cultured with transplant cells having B2M knockout either alone or in combination with other knockouts. Addition of CD58 knockout alone or in combination with RFX5 knockout was found to reduce expansion.

[0639] In another PBMC MLR assay using CD19 CAR T cells, the effects of different knockouts on different immune cell subsets were evaluated. B cells from PBMCs from 3 different donors were depleted before co-culture. A 10:1 host: graft cell ratio was used for 10 days of culture. Readings were taken after 10 days. TRAC / CD58 KO and TRAC / ICAM-1KO results were measured on separate plates (error bars were removed for ease of visual observation).

[0640] like Figure 5H As shown in Figure 5A, when co-cultured with transplant cells with different knockouts and knockout combinations, host CD8+ cell expansion was low. For example, compared with controls (FMC63; TRAC KO transplants), adding RFX5 / CD58 combined knockouts reduced expansion. As shown in Figure 5I, when co-cultured with transplant cells with different knockouts and knockout combinations, host CD4+ cell expansion was low. For example, compared with controls (FMC63; TRAC KO transplants), adding RFX5 / CD58 combined knockouts reduced expansion.

[0641] Use CD19 CAR T cells to target cells, such as Raji WT cells or Raji CD19 低 Cells were assayed for in vitro long-term killing at an E:T ratio of 8:1. As shown in Figure 5J, CD19 CAR T cells containing different knockouts exhibited cytotoxic activity against Raji WT target cells. Control CAR T cells with TRAC knockout alone exhibited effective killing compared to most other CAR T cells with additional knockouts. Surprisingly, CAR T cells with TRAC / CD58 knockout conferred similar or slightly better killing compared to control CAR T cells. When Raji CD19 低 Similar results were obtained when cells were used as target cells.

[0642] Example 4: Protective effect of CD58 KO or ICAM-1 KO + RFX5 KO against NK cell rejection

[0643] The CD19 CAR T cells (expressing the turbine domain) described in Example 3 were modified to introduce one or more knockouts and then used as target cells. As described below, the expression of the CAR and turbine domain was controlled by the EF1α or PGK promoter.

[0644] Specifically, CD19 CAR T cells were gene-edited via CRISPR / Cas9 to provide the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / CD58 KO, TRAC / ICAM-1KO, β2m / CD58 KO, β2m / ICAM-1KO, RFX5 / CD58 KO, and RFX5 / ICAM-1KO.

[0645] for Fig. 6A The experiments presented used the EF1α promoter to control the expression of the CAR and turbine domains. Figure 6B The experiments presented use the PGK promoter to control the expression of the CAR and Turbine domains.

[0646] The NK MLR assay was used to test the survival of gene-edited CD19 CAR T cell grafts against allogeneic NK rejection. Data were combined from 3 graft / host donor pairs: 1 graft and 3 hosts. NK cells isolated from frozen PBMCs were co-cultured with gene-edited CD19 CAR T cells for 2 days in the presence of 1,000 IU / mL IL-2. CD19 CAR T cells were activated using Raji tumor cells expressing GFP. The ratio of NK cells: graft: Raji cells was 10:1:0.25. The survival of the graft CAR T cells was assessed by FACS and gated as live GFP-HLA-A2-TCRαβ-CD56-CD4+CD8+CAR+ and desired gene editing (e.g., CD58 KO).

[0647] like Fig. 6A As shown, single β2m KO caused substantial NK rejection, resulting in low survival, while RFX5 single KO caused some NK rejection, but to a lesser extent. Single CD58 KO or ICAM-1 KO had similar survival compared to control grafts (TRAC KO). CD58 KO slightly rescued β2m KO from NK rejection. CD58 KO or ICAM-1 did not show substantial rescue of RFX5 KO survival against NK rejection.

[0648] In another NK cell MLR, fresh NK cells were used. The ratio of NK cells: graft: Raji target cells was 1:1:0.25. NK cells were co-cultured with gene-edited CD19 CAR T cells in the presence of 1,000 IU / ml IL-2 for 2 days. Figure 6B As shown, CD58 knockout and ICAM-1 knockout did not induce NK cell rejection (each dot represents a graft / NK pair). In addition, CAR T knockout cells were not found to exhibit IL-2-independent growth ( Figure 6C ).

[0649] Table 4.1: sgRNA sequence list

[0650]

[0651]

[0652] Example 4.1 - Cytotoxic properties of CAR T cell knockout

[0653] The CD19 CAR T cells (expressing the turbine domain) described in Example 3 were modified to introduce one or more knockouts. The CD19 CAR T cells were engineered from pan T cells from two HLA-A2 donors. As described below, the expression of the CAR and turbine domains was controlled by the EF1α or PGK promoter.

[0654] Specifically, CD19 CAR T cells were gene-edited via CRISPR / Cas9 to provide the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / CD58 KO, TRAC / ICAM-1KO, TRAC / RFX5 / CD58KO, TRAC / RFX5 / ICAM-1KO, and TRAC / CD58 / ICAM-1KO.

[0655] for Figure 7 - Experimental data presented in Figure 12, CD19 CAR T cells were engineered with a PGK promoter for controlling expression of the CAR and Turbine domains.

[0656] TRAC knockout was common to all edited cells. After 16 days of in vitro expansion, the gene-edited cells were purified to remove any cells expressing TCRα / β. The level of specific gene knockout or MHC-I and MHC-II knockdown was evaluated by flow cytometry.

[0657] like Figure 7As shown, high gene editing efficiency was observed for all groups (based on the average of two donors). On day 16 of production, more than 50% of cells had the desired gene edits. For all knockout conditions listed above, 1×10 6 cells and then expanded. Figure 8 As shown, the expansion of edited cells was not affected by any of the various knockouts compared to the TRAC knockout control.

[0658] Use CD19 CAR T cells with various knockouts to target cells, such as Raji WT cells or Raji CD19 低 Cells were used for in vitro long-term killing assays at an E:T ratio of 8:1. Fig. 9 As shown, various CAR T cells containing different knockouts show cytotoxic activity for Raji WT target cells.It is found that all CAR T knockout cells show equal or higher cytotoxicity compared with control CAR T cells (only TRAC knockout).CAR T cells are co-cultured with 8:1 effector:target ratios of Raji cells expressing luciferase-GFP.Every 2-3 days, half of the cells are passaged to fresh Raji cells.Then Bright-glo reagent (Promega) is used to determine the remaining half of the cells for target cell killing.

[0659] A series of sensitized T MLR assays were performed using several gene-edited CD19 CAR T cells with various knockouts. Certainly. After 7 days of co-culture with WT Raji cells with B2M knockout irradiated graft pan T cells, sensitized host T cells were isolated twice using the Miltenyi Pan T Isolation Kit. A 1:1:0.25 ratio of T cell host:graft:Raji (B2M knockout) was used and readings were obtained on day 2. Fig.10 As shown, CD58 knockout was found to be effective in alleviating T cell rejection as a single knockout or in combination with other knockouts (each dot represents one graft / host pair).

[0660] A PBMC MLR assay was also performed. A 10:1 host:graft cell ratio was used for 10 days of culture. Prior to co-culture, PBMCs from the donor were depleted of B cells. Readings were taken on days 3, 7, and 9. Fig.11Shown in Figure 2, at the 7th day, the transplant CAR T cells with various knockouts show a survival advantage that is superior to control and B2M knockouts.Combination knockout (e.g., RFX5 / CD58, RFX5 / ICAM-1 and CD58 / ICAM-1) shows better anti-rejection survival rate than single knockout (e.g., RFX5, CD58 and ICAM-1).CD58 knockout cells show the best survival rate in single knockout transplant cells.CD58 knockout and RFX5 knockout or ICAM-1 knockout combination show best anti-rejection survival rate (data not shown) at the 9th day.In addition, compared with other single knockouts, single CD58 knockout shows best survival rate (data not shown) at the 9th day.

[0661] Evaluate the ability of different gene-edited CAR T cells to affect the expansion of host immune cells. Figure 12A-12B As shown, CAR T cells were found to reduce the expansion of host CD8+ and host CD4+ cells (A). B2M knockout CAR T cells were found to be the least effective in reducing the expansion of host NK cells compared to other knockout cells (B).

[0662] In addition, CD19 CAR T cells were engineered with the EF1α promoter for controlling the expression of the CAR and turbine domains. These cells were also tested for their cytotoxic capacity and found to exhibit similar cytotoxic capacity (data not shown).

[0663] Example 4.2 - CD19 CAR / CD70 binding protein T cells with CD58 knockout

[0664] Transduction of the LVV construct into PBMCs produces engineered cells with randomly integrated transgenes in the host cell genome. Transgenes can be introduced into cells by site-specific integration (SSI) into a predetermined locus to ensure consistency of the transgene insertion site in the genome and to limit the number of integration events. Site-specific integration assisted by adeno-associated virus vectors (AAV) can also express more than one gene while maintaining high transduction efficiency.

[0665] Three different constructs were delivered using an AAV-assisted SSI method to produce three different types of engineered cells for these experiments: 1) CD19 CAR constructs, 2) CD19CAR / CD70 binding protein constructs, and 3) CD70CAR constructs. For construct 2), the CD70 binding protein (or domain) is derived from anti-CD70 antibody clone 4F11 in the form of scFv and CD3ζ signaling domain (4F11z). In each case, the TRAC locus was targeted for integration of the construct. Three different PBMC donors were used. The expression of each construct is driven by the PGK promoter. Further gene editing was performed via CRISPR / Cas9 in 1) and 2) to produce graft CAR T cells with certain knockouts: only TRAC knockout, TRAC / B2M knockout, TRAC / CD58 knockout. After 1 week of in vitro expansion, the target cells were purified to remove any cells expressing TCRα / β. The levels of specific gene knockout or MHC-I and MHC-II knockdown were evaluated by flow cytometry.PBMCs from healthy human donors expressing HLA-A2 (HLA-A2+) were used as allogeneic effectors (hosts), while engineered cells were used as graft CAR T cells.

[0666] PBMC MLR assay was performed. A 10:1 host:graft cell ratio was used for 13 days of culture. PBMCs from 3 different donors were depleted of B cells prior to co-culture. Readings were taken on day 13. Fig.13A As shown, CD58 knockout was found to enhance the survival of CD19CAR / CD70 binding protein CAR T cells (each symbol represents 1 of 9 unique graft / host pairs; each symbol shape represents a unique graft).

[0667] After 9 days of co-culture, the ability of the engineered CAR T cells to influence the expansion of host immune cells was evaluated. Fig. 13B As shown in Figure 2, it was found that CD19 CAR / CD70 binding protein CAR T cells reduced the expansion of host CD8+ T cells or depleted host CD8+ T cells. Similar results were observed for host CD4+ T cells. Fig. 13C As shown, it was observed that CD19 CAR / CD70 binding protein CAR T cells prevented host NK cell expansion.

[0668] In vitro long-term killing assays were performed using various CAR T knockout cells against target Raji WT cells at an E:T ratio of 4:1. Fig.13DAs shown, the introduction of CD58 knockout did not reduce the cytotoxic activity of CAR T cells. CAR T cells were co-cultured with Raji cells expressing luciferase-GFP at an effector: target ratio of 8: 1. Every 2-3 days, half of the cells were passaged to fresh Raji cells. The remaining half of the cells were then used to determine target cell killing using Bright-glo reagent (Promega).

[0669] General solution

[0670] Generation of target cells (Figure 1)Primary human T cells were isolated from frozen healthy donor peripheral blood mononuclear cells (PBMCs) using magnetic activated cell sorting (MACS) negative selection (Miltenyi Biotec, human pan T cell isolation kit, catalog number 130-096-535) and activated with 1:100 (v:v) T cell TransAct (Miltenyi Biotec, catalog number 130-111-160) + 100 IU / mL IL-2 (Miltenyi Biotec, catalog number 130-097-746) in R10 (RPMI-1640 + 10% FBS + 25 mM HEPES + sodium pyruvate + non-essential amino acids). After 2 days, T cells were gene edited using the Neon transfection system (Invitrogen). In brief, the ribonucleoprotein (RNP) complex is produced by mixing the cas9 enzyme (IDT, catalog number 1081059) and sgRNA at a 2: 1 molar ratio at room temperature for 10 minutes. If two sgRNAs are used, cas9 is incubated at a 1: 1: 1 ratio (sgRNA1: sgRNA2: cas9). If three sgRNAs are used, cas9 is incubated at a 0.67: 0.67: 0.67: 1 ratio (sgRNA1: sgRNA2: sgRNA3: cas9). The cells were pulsed a total of 3 times at 1600V, 10ms width, and immediately recovered in R10 medium supplemented with 100IU / mL IL-2+10ng / mL IL-7 (Miltenyi Biotec, catalog number 130-095-363). The edited T cells were incubated at 37°C, 5% CO2. Fresh R10 containing 100 IU / mL IL-2 was added to the cells the next day. KO efficiency was assessed by flow cytometry 5-7 days after gene editing. KO efficiency was assessed in multiple ways: for TRAC, CD3 / TCRab expression was assessed; for β2m, NLRC5, and RFX5, anti-β2m or anti-HLA antibodies were used. Anti-CD58 antibody was used to assess CD58 expression. TRACKO cells were purified using MACS negative selection (Stem Cell Technologies, EasySep Human TCRα / β Depletion Kit, Catalog No. 17847) according to the manufacturer's recommendations. Purified T cells were used immediately or frozen in 5e6 cell aliquots in 90% FBS+10% DMSO.

[0671] Target cell production (except Figure 1E-1F All images exceptPrimary human T cells were isolated from frozen healthy donor peripheral blood mononuclear cells (PBMCs) using the EasySep Human T Cell Isolation Kit (Stem Cell Technology, catalog number 17951). The isolated T cells were activated with 1:100 (v:v) T cell TransAct (Miltenyi Biotec, catalog number 130-111-160) + 100 IU / mL IL-2 (Miltenyi Biotec, catalog number 130-097-746) in X-Vivo 15 medium (Lonza, catalog number 04-418Q) + 5% human AB serum (Gemini, catalog number 100-318). After 2 days, T cells were gene edited using the Nucleofector 4D system (Lonza). In brief, the ribonucleoprotein (RNP) complex is produced by mixing cas9 enzyme (IDT, catalog number 1081059) and sgRNA at a 1: 1 molar ratio at room temperature for 10 minutes. If two sgRNAs are used, cas9 is incubated at a ratio of 0.5: 0.5: 1 (sgRNA1: sgRNA2: cas9). If three sgRNAs are used, cas9 is incubated at a ratio of 0.3: 0.3: 0.3: 1 (sgRNA1: sgRNA2: sgRNA3: cas9). After electroporation, cells are immediately recovered in X-Vivo15 culture medium + 5% human AB serum and 100IU / mL IL-2. In some cases (Fig. 5-Fig. 6), electroporated T cells are transduced with adenovirus to express CD19 CAR (FMC63 TurboCAR). TCR depletion (Stem Cell Technology, EasySep Human TCRα / β Depletion Kit, Catalog No. 17847) is usually performed on days 14 to 16. TCR-depleted T cells are cryopreserved and thawed for further analysis later. Cell expansion during production is tracked by counting live cells with a Vi-CELL counter (Beckman Coulter). The amplification multiple is calculated by comparing the live cell counts at different time points to day 2 (gene editing time). CD19 CAR T cells are identified by anti-idiotypic antibodies (Acro Biosystems, Catalog No. FM3HPY53). KO efficiency is also assessed at multiple time points throughout the cell production process via flow cytometry. KO efficiency is assessed in a variety of ways: for TRAC, CD3 / TCRab expression is...

Claims

1. An engineered immune cell comprising a genomic modification that functionally impairs or reduces the expression of (i) RFX5 and / or NLRC5 and (ii) CD58 relative to cells without the genomic modification.

2. The engineered immune cell of claim 1, wherein the genomic modification comprises knockdown and / or knockout of (i) RFX5 and / or NLRC5 and / or (ii) CD58.

3. An engineered immune cell as described in claim 1 or 2, wherein the genomic modification comprises one or more modifications at the loci of (i) RFX5 and / or NLRC5 and (ii) CD58.

4. The engineered immune cell of any one of claims 1 to 3, wherein the genomic modification comprises a deletion or insertion at the loci of (i) RFX5 and / or NLRC5 and (ii) CD58.

5. The engineered immune cell of any one of claims 1 to 4, wherein the genomic modification is selected from the group consisting of: (i) insertion of one or more nucleotides, (ii) insertion of a polynucleotide sequence encoding a protein, (iii) deletion of one or more nucleotides, and (iv) substitution of one or more nucleotides.

6. The engineered immune cell of any one of claims 1 to 5, wherein the genomic modification is introduced by a gene editing technology selected from TALEN, zinc finger, Cas-CLOVER and CRISPR / Cas system.

7. The engineered immune cell of claim 1, wherein the genome modification comprises the insertion of an RNA interference sequence.

8. The engineered immune cell of claim 7, wherein the RNA interference sequence is a shRNA sequence, a siRNA sequence or a miRNA sequence.

9. The engineered immune cell of claim 7 or 8, wherein the RNA interference sequence comprises a sequence complementary to (i) RFX5 and / or NLRC5 and / or (ii) CD58 gene sequences.

10. The engineered immune cell of any one of claims 1 to 9, further comprising a polynucleotide sequence encoding an antigen binding protein and / or a CD70 binding protein.

11. The engineered immune cell of claim 10, wherein the antigen binding protein is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

12. The engineered immune cell of any one of claims 1 to 11, wherein the cell is further engineered to comprise one or more genomic modifications that functionally attenuate or reduce the expression of one or more of ICAM-1, TAP2, β2m, TRAC, CIITA, RFXAP, RFXANK, and CD48 relative to unengineered cells.

13. The engineered immune cell of any one of claims 1 to 12, wherein the engineered immune cell has improved persistence and / or improved resistance to alloreactive immune cell rejection compared to an immune cell not comprising the genomic modification.

14. The engineered immune cell of claim 13, wherein the alloreactive immune cell rejection comprises alloreactive T cell-mediated rejection and / or alloreactive natural killer (NK) cell-mediated rejection.

15. The engineered immune cell of any one of claims 1 to 14, comprising one or more genomic modifications that functionally attenuate or reduce the expression of RFX5 and CD58.

16. The engineered immune cell of any one of claims 1 to 14, comprising one or more genomic modifications that functionally attenuate or reduce the expression of NLRC5 and CD58.

17. The engineered immune cell of any one of claims 1 to 14, comprising one or more genomic modifications that functionally attenuate or reduce the expression of RFX5, NLRC5, and CD58.

18. The engineered immune cell of any one of claims 1 to 17, wherein β2m is functionally expressed at a reduced level.

19. The engineered immune cell of any one of claims 1 to 17, comprising an unmodified β2m gene, or wherein β2m is not functionally expressed at a reduced level.

20. The engineered immune cell of claim 13, wherein the increased persistence is determinable and / or determined by a mixed lymphocyte reaction (MLR) assay.

21. The engineered immune cell of claim 13, wherein the improved resistance to alloreactive immune cell rejection is determinable and / or determined by an MLR assay.

22. The engineered immune cell of any one of claims 1 to 21, wherein the engineered immune cell exhibits: (i) reduced levels of MHC class I protein or complex expression at the cell surface, (ii) reduced levels of MHC class II protein or complex expression at the cell surface, or (iii) reduced levels of expression of MHC class I proteins or complexes at the surface of said cells, and reduced levels of expression of MHC class II proteins or complexes at the surface of said cells.

23. The engineered immune cell of claim 10 or 11, wherein the antigen binding protein is a CAR.

24. The engineered immune cell of claim 10 or 11, which expresses the antigen binding protein and / or the CD70 binding protein.

25. The engineered immune cell of claim 10, wherein the polynucleotide sequence encoding the antigen binding protein and / or the CD70 binding protein is located within a disrupted CD58, RFX5, NLRC5, ICAM-1, CD48, TAP2, β2m, TRAC, CIITA, RFXAP or RFXANK locus.

26. The engineered immune cell of any one of claims 1 to 25, wherein the engineered immune cell further comprises one or more genomic modifications of an endogenous TCRα gene.

27. The engineered immune cell of claim 26, wherein the engineered immune cell further comprises one or more genomic modifications of an endogenous CD52 gene.

28. The engineered immune cell of any one of claims 1 to 27, wherein the engineered immune cell is or is obtained from an immune cell of a healthy volunteer, from a patient, or from an induced pluripotent stem cell (iPSC).

29. The engineered immune cell of any one of claims 1 to 27, wherein the engineered immune cell is not a natural killer (NK) cell, or is not a NK cell obtained from a healthy volunteer or patient.

30. The engineered immune cell of any one of claims 1 to 27, wherein the engineered immune cell is not obtained from iPSC.

31. An engineered immune cell population comprising the engineered cells of any one of claims 1 to 30, wherein no more than 50% of the engineered immune cells functionally express (i) RFX5 and / or NLRC5 and (ii) CD58.

32. The population of claim 31, wherein no more than 50% of the engineered immune cells functionally express RFX5 and CD58.

33. The population of claim 31, wherein no more than 50% of the engineered immune cells functionally express NLRC5 and CD58.

34. The population of claim 31, wherein no more than 50% of the engineered immune cells functionally express RFX5, NLRC5, and CD58.

35. A population as described in any one of claims 31 to 34, wherein no more than 50% of the engineered immune cells also functionally express a) any one or more of ICAM-1, CD48, TAP2, β2m, TRAC, CIITA, RFXAP and RFXANK; or b) only one of ICAM-1 and CD48; or c) Both CD48 and ICAM-1.

36. A colony as described in any one of claims 31 to 35, wherein the engineered immune cells of the engineered immune cell population have improved persistence and / or improved resistance to alloreactive immune cell rejection compared to non-engineered immune cells, Optionally wherein the improved resistance is to alloreactive T cell-mediated rejection and / or alloreactive Natural Killer cell (NK)-mediated rejection, and Optionally wherein said increased persistence is determinable and / or determined by a mixed lymphocyte reaction (MLR) assay, and / or wherein said improved resistance to alloreactive immune cell rejection is determinable and / or determined by a MLR assay.

37. An engineered immune cell population comprising the engineered immune cells of any one of claims 1 to 30, wherein at least 1% of the engineered immune cells functionally express (i) RFX5 and / or NLRC5 and (ii) CD58 at a level no greater than 50% of the expression level in unengineered immune cells.

38. The population of claim 37, wherein at least 1% of the engineered immune cells functionally express RFX5 and CD58 at a level no greater than 50% of the expression level in non-engineered immune cells.

39. The population of claim 37, wherein at least 1% of the engineered immune cells functionally express NLRC5 and CD58 at a level no greater than 50% of the expression level in non-engineered immune cells.

40. The population of claim 37, wherein at least 1% of the engineered immune cells functionally express RFX5, NLRC5, and CD58 at a level no greater than 50% of the expression level in non-engineered immune cells.

41. The population of claim 37, wherein at least 1% of the engineered immune cells functionally express a) any one or more of ICAM-1, CD48, TAP2, β2m, TRAC, CIITA, RFXAP, and RFXANK; or b) only one of ICAM-1 and CD48; or c) Both CD48 and ICAM-1, The expression level is no greater than 50% of the expression level in non-engineered immune cells.

42. A colony as described in any one of claims 37 to 41, wherein the engineered immune cells of the engineered immune cell population have improved persistence and / or improved resistance to alloreactive immune cell rejection compared to non-engineered immune cells, Optionally wherein the improved resistance is to alloreactive T cell-mediated rejection and / or alloreactive Natural Killer cell (NK)-mediated rejection, and Optionally wherein said increased persistence is determinable and / or determined by a mixed lymphocyte reaction (MLR) assay, and / or wherein said improved resistance to alloreactive immune cell rejection is determinable and / or determined by a MLR assay.

43. The engineered immune cell population of any one of claims 31 to 42, wherein at least 50% of the engineered immune cells exhibit reduced levels of MHC class I protein or complex expression at the cell surface.

44. An engineered immune cell colony as described in any one of claims 31 to 43, wherein the engineered immune cell colony comprises at least 10% engineered T cells, at least 20% engineered T cells, at least 30% engineered T cells, at least 40% engineered T cells, at least 50% engineered T cells, at least 75% engineered T cells, or at least 90% engineered T cells.

45. An engineered immune cell population as described in any one of claims 31 to 44, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 90% of the engineered immune cells further express an antigen binding protein or a CD70 binding protein.

46. ​​An engineered immune cell population as described in claim 45, wherein the antigen binding protein is a CAR or a TCR.

47. The engineered immune cell population of claim 45, wherein the nucleic acid encoding the antigen binding protein and / or the CD70 binding protein is inserted into the disrupted CD58, RFX5, NLRC5, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK loci.

48. An engineered immune cell population as described in claim 46, wherein the nucleic acid encoding the CAR is inserted into the destroyed CD58, RFX5, NLRC5, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK loci.

49. An engineered immune cell population as described in claim 46, wherein the nucleic acid encoding the TCR is inserted into a disrupted locus, and the disrupted locus is not a disrupted CD58, RFX5, NLRC5, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK locus.

50. The engineered immune cell population of any one of claims 31 to 49, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 90% of the engineered cells further comprise one or more genomic modifications of one or more of an endogenous TCRα gene and an endogenous CD52 gene.

51. An engineered immune cell population as described in any one of claims 31 to 50, wherein at least 10%, 20%, 30%, 40%, 50%, 75% or 90% of the engineered immune cells further express one or more proteins selected from the group consisting of: HLA-E, HLA-E single chain trimer, HLA-G or HLA-G single chain trimer, UL18 or UL18 single chain trimer, HLA-A2 and HLA-A2 single chain trimer.

52. An engineered immune cell colony as described in any one of claims 31 to 51, wherein the functional expression level of one or more of TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA and RFXANK is measured by determining the surface expression level of HLA, β2 microglobulin (B2M) or both HLA and B2M on the surface of the engineered immune cells, or measured by flow cytometry.

53. An engineered immune cell population as described in any one of claims 31 to 51, wherein the functional expression level of one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of one or more of CD48, CD58 and ICAM-1 on the surface of the engineered immune cells, or is measured by flow cytometry.

54. A method for preparing an engineered immune cell as described in any one of claims 1 to 30 or a population as described in any one of claims 31 to 53, the method comprising a) modifying the genome of the engineered immune cell, and b) producing said engineered immune cell comprising the genomic modification.

55. The method of claim 54, wherein the genome of the engineered immune cell is modified using TALEN, zinc fingers, Cas-CLOVER or CRISPR / Cas system.

56. A pharmaceutical composition comprising an engineered immune cell as described in any one of claims 1 to 30, an engineered immune cell population as described in any one of claims 31 to 53, or an engineered immune cell prepared by the method of claim 54 or 55, and further comprising at least one pharmaceutically acceptable carrier or excipient, and optionally Wherein the engineered immune cell or one or more cells of the population (i) further express one or more proteins selected from the group consisting of: HLA-E, HLA-E single chain trimer, HLA-G, HLA-G single chain trimer, UL18, UL18 single chain trimer, HLA-A2, HLA-A2 single chain trimer and human cytomegalovirus (HCMV) US11, and / or (ii) are further engineered to not express or express at a reduced level any one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFXANK, RFXAP and RFX5.

57. A method of treating a condition in a subject, the method comprising administering to the subject: An engineered immune cell as described in any one of claims 1 to 30, an engineered immune cell population as described in any one of claims 31 to 53, an engineered immune cell prepared by the method of claim 54 or 55, or a pharmaceutical composition as described in claim 56.

58. The method of claim 57, wherein the condition is a solid tumor or a liquid tumor.

59. A method of improving (i) persistence or (ii) resistance to rejection of alloreactive immune cells of allogeneic engineered immune cells, the method comprising a) modifying an allogeneic immune cell to introduce one or more genomic modifications that functionally attenuate or reduce the expression of (i) RFX5 and / or NLRC5 and (ii) CD58 to provide an allogeneic engineered immune cell; and b) administering the allogeneic engineered immune cells to a subject.

60. The method of claim 59, wherein the genomic modification comprises knockdown and / or knockout of (i) RFX5 and / or NLRC5 and / or (ii) CD58.

61. The method of claim 59 or 60, wherein the genomic modification comprises one or more modifications at the loci of (i) RFX5 and / or NLRC5 and (ii) CD58.

62. The method of any one of claims 59 to 61, wherein the genomic modification comprises a deletion or insertion at the locus of (i) RFX5 and / or NLRC5 and (ii) CD58.

63. The method of any one of claims 59 to 62, wherein the genome modification is selected from the group consisting of: (i) insertion of one or more nucleotides, (ii) insertion of a protein encoding sequence, (iii) deletion of one or more nucleotides, and (iv) substitution of one or more nucleotides.

64. The method of any one of claims 59 to 63, wherein the genome modification is introduced by a gene editing technology selected from TALEN, zinc fingers, Cas-CLOVER, and CRISPR / Cas systems.

65. The method of claim 59, wherein the genome modification comprises insertion of an RNA interference sequence.

66. The method of claim 65, wherein the interfering sequence is a shRNA sequence, a siRNA sequence or a miRNA sequence.

67. The method of claim 65 or 66, wherein the interfering sequence comprises a sequence complementary to (i) RFX5 and / or NLRC5 and (ii) CD58 gene sequences.

68. The method of any one of claims 59 to 67, wherein the allogeneic engineered immune cells further comprise a polynucleotide sequence encoding an antigen binding protein and / or a CD70 binding protein.

69. The method of claim 68, wherein the antigen binding protein is a CAR or a TCR.

70. The method of any one of claims 59 to 69, wherein the allogeneic engineered immune cells are further engineered to comprise one or more genomic modifications that functionally impair or reduce the expression of one or more of ICAM-1, TAP2, β2m, TRAC, CIITA, RFXAP, RFXANK, and CD48 relative to cells without the modifications.

71. The method of any one of claims 59 to 70, wherein the genomic modification functionally attenuates or reduces expression to about 50% or less of the corresponding level in a cell without the genomic modification.

72. The method of any one of claims 59 to 71, wherein the allogeneic engineered immune cells have improved persistence and / or improved resistance to rejection of alloreactive immune cells compared to allogeneic non-engineered immune cells.

73. The method of any one of claims 59 to 72, wherein the improved resistance is to alloreactive T cell-mediated rejection and / or alloreactive natural killer cell (NK)-mediated rejection.

74. The method of any one of claims 59 to 73, wherein the increased persistence is determinable and / or determined by a mixed lymphocyte reaction (MLR) assay, and / or wherein the improved resistance to alloreactive immune cell rejection is determinable and / or determined by a MLR assay.

75. The method of any one of claims 59 to 74, wherein the method comprises functionally attenuating or reducing the expression of RFX5 and CD58.

76. The method of any one of claims 59 to 74, wherein the method comprises functionally attenuating or reducing the expression of NLRC5 and CD58.

77. The method of any one of claims 59 to 74, wherein the method comprises functionally attenuating or reducing the expression of RFX5, NLRC5 and CD58.

78. The method of any one of claims 59 to 77, wherein the extent of reduction in the expression level of (i) RFX5 and / or NLRC5 and (ii) CD58 is determined relative to the expression level of (i) RFX5 and / or NLRC5 and (ii) CD58, respectively, in cells of the same type that have not been modified.

79. The method of any one of claims 59 to 78, wherein the functional expression level of one or more of CD48, CD58 and ICAM-1 is measured by determining the surface expression level of CD48 protein, CD58 protein or ICAM-1 protein on the surface of the engineered immune cells.

80. The method of any one of claims 59 to 79, wherein the functional expression level of RFX5 and / or NLRC5 is measured by determining the surface expression level of HLA, β2 microglobulin (B2M), or both HLA and B2M on the surface of the engineered immune cells.

81. The method of claim 79 or 80, wherein the surface expression level is measured by flow cytometry.

82. The method of any one of claims 59 to 81, wherein the method further comprises introducing one or more genomic modifications of one or more of the TCRα gene and the CD52 gene.

83. The method of any one of claims 59 to 82, wherein the allogeneic engineered immune cells comprise an unmodified β2m gene, or wherein β2m is not functionally expressed at a reduced level in the allogeneic engineered immune cells.

84. The engineered immune cell of any one of claims 20 to 30, comprising an unmodified β2m gene, or wherein β2m is not functionally expressed at a reduced level in the engineered immune cell.

85. The engineered immune cell of any one of claims 1 to 30, wherein the expression of one or more of β2m, RFX5, NLRC5, CIITA, and TAP2 is not functionally impaired or reduced.

86. The engineered immune cell colony of any one of claims 31 to 53, wherein the engineered immune cell colony does not comprise genomic modification of one or more of β2m, RFX5, NLRC5, CIITA, or TAP2 genes.

87. The engineered immune cell colony of any one of claims 31 to 53, wherein the expression of one or more of β2m, RFX5, NLRC5, CIITA, or TAP2 is not functionally impaired or reduced.

88. The method of claim 54 or 55, wherein the genomic modification does not comprise genomic modification of β2m.

89. The method of claim 54 or 55, wherein the genomic modification does not comprise genomic modification of one or more of β2m, RFX5, NLRC5, CIITA, and TAP2.

90. The method of any one of claims 59 to 83, wherein expression of β2m is not functionally impaired or reduced in the engineered immune cells.

91. The method of any one of claims 59 to 83, wherein the genomic modification does not comprise genomic modification of β2m.

92. The method of any one of claims 59 to 83, wherein the genomic modification does not comprise genomic modification of one or more of β2m, RFX5, NLRC5, CIITA, and TAP2.

93. The engineered immune cell of claim 68 or 69, wherein the engineered immune cell expresses the CD70 binding protein.

94. The engineered immune cell of any one of claims 11 to 30, further comprising a polynucleotide sequence encoding a CD70 binding protein.

95. The engineered cell of claim 94, wherein the engineered cell expresses the CD70 binding protein.

96. The engineered cell of any one of claims 1 to 30 and 84 to 85, or the population of any one of claims 31 to 53 and 86 to 87, or the method of any one of claims 54 to 55, 57 to 83 and 88 to 92, wherein the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain.

97. The engineered cell, population or method of claim 96, wherein the CD70 binding domain constitutes a CD70 antibody, or a CD70 receptor or a CD70 binding fragment thereof.

98. The engineered cell, population or method of claim 96 or 97, wherein the CD70 binding domain constitutes an anti-CD70 antibody, optionally the anti-CD70 antibody is a scFv.

99. The engineered cell, population or method of any one of claims 96 to 98, wherein the CD70 binding protein further comprises a hinge domain, optionally wherein the hinge domain comprises a CD8 hinge.

100. The engineered cell, population or method of any one of claims 96 to 99, wherein the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of a CD3ze signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain, or variants thereof.

101. The engineered cell, population or method of any one of claims 96 to 100, wherein the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a co-stimulatory domain.

102. The engineered cell, population or method of any one of claims 96 to 101, wherein the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain.

103. The engineered cell, population or method of any one of claims 96 to 98, wherein the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain.

104. The engineered immune cell of any one of claims 109 to 101, wherein the one or more intracellular domains comprise the amino acid sequence of one or more of SEQ ID NOs: 1, 7-14, 17-70, or 89-90.

105. The engineered cell, population or method of any one of claims 96 to 102, wherein the CD70 binding protein does not comprise an intracellular signaling domain.

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