Engineered cells with reduced gene expression to reduce 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. The cells are engineered to functionally express low levels of one or more of CD48, CD58, ICAM-1, RFX5, NLRC5, TAP2, β2m, TRAC, RFXAP, CIITA, and RFXANK. Optionally, the cells are further engineered to express one or more additional proteins, such as an antigen binding protein (e.g., a chimeric antigen receptor (CAR) or T cell receptor) and / or a CD70 binding protein, to target tumor cells or other damaged cells in the subject, and / or to express other genes at low levels. Also provided are methods of making and using the engineered cells, compositions, and kits comprising the same, as well as methods of treatment by administering the cells and compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 393,350, filed July 29, 2022, and U.S. Provisional Patent Application No. 63 / 509,136, filed June 20, 2023, the contents of both applications being incorporated herein by reference in their entireties.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy, created on July 13, 2023, is named AT-053_03WO_SL.xml and is 98,186 bytes in size.

[0003] The present disclosure relates generally to the use of engineered immune cells (e.g., T cells) for use in therapeutic applications. [Background technology]

[0004] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens has shown promise as a new approach to cancer treatment (see, for example, 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), fusion proteins composed of an antigen-recognition moiety and a T-cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724(1993)). CAR-containing immune cells, such as CAR-T cells (CAR-T), are engineered to have antigen specificity while retaining or enhancing their ability to recognize and kill target cells.

[0005] However, producing CAR-modified autologous cell therapies is expensive, requiring several weeks of processing and quality testing, and product efficacy varies depending on the initial quality and quantity of patient-specific T cells used. Allogeneic CAR-modified cell therapies involve modifying cells from healthy donors to express CARs and then administering them to multiple patients. They can be delivered immediately on demand, potentially resulting in cheaper and more stable products than autologous cell therapies (see, for example, Graham et al., Cells 2018, 7, 155; doi:10.3390 / cells7100155). Furthermore, allogeneic therapies allow for selection based on desired product characteristics (e.g., gene editing efficiency, integration site, lack of deleterious off-target gene edits, haplotype, etc.) and facilitate more sophisticated cell engineering (e.g., multiple gene edits to improve efficacy, persistence, homing, etc.). A major hurdle to implementing allogeneic CAR-modified cell therapies is the potential for rejection of the (donor cell-based) product by the patient's (host's) immune system.

[0006] Killer lymphocytes, such as CD8+ T cells and natural killer (NK) cells, identify and kill non-self, cancerous, virus-infected, and foreign cells (including allogeneic cells). The central determinant of self-non-self differentiation is the major histocompatibility complex (MHC) molecule, which is expressed on the surface of all nucleated cells. Each MHC class I molecule is a non-covalent trimeric complex of a highly multiallelic MHC class I heavy chain (the most common of which is HLA-A2), a uniform β2-microglobulin (β2m), and peptides presented by proteolysis of endogenously expressed proteins. Collectively, MHC class I molecules are loaded with peptides representing the diversity of proteins expressed within the cell, thereby "announcing" the dysfunction of cancerous and infected cells to immune cells. CD8+ T cells recognize these non-self peptides using T cell receptors (TCRs) unique to each developmental stage of T cells, and initiate killing following non-self peptide-MHC recognition (see, e.g., Dembic, Z. et al., Nature 320, 232-238 (1986)). T cell recognition of non-self is complemented by NK cell recognition of "self loss." That is, NK cell-mediated killing in the absence of MHC is achieved by inhibitory receptors on the surface of NK cells (see, e.g., K. Karre et al., Nature 319, 675-678 (1986)). See Figures 1 and 3 of PCT / US2022 / 14393, which are incorporated herein by reference in their entirety). Experimental ablation of MHC class I presentation was achieved via CRISPR / Cas9-mediated genomic knockout (KO) of the β2m gene, which encodes the universal β2m component of MHC class I. This resulted in potent NK activation and selective killing of MHC-deficient T cells in both autoreactive and alloreactive settings (see Figure 2 of PCT / US2022 / 14393, which is incorporated herein by reference in its entirety).

[0007] Although allogeneic cell therapy offers many advantages over autologous cell therapy, allogeneic cells face the problem of potential rejection by host or recipient immune system cells that react with T and NK epitope determinants on the surface of the allogeneic cell product that differ from those of the host. Approaches have been reported that avoid rejection of allogeneic therapy cells by downregulating or disabling the expression of HLA molecules on the surface of allogeneic cells (see, e.g., Lanza, R. et al. Engineering universal cells that evade immune detection. Nat. Rev. Immunol. 2019 Dec;19(12):723-733, Epub 2019 Aug 15).

[0008] Removal of MHC class I molecules, for example by deleting the beta-2 microglobulin gene (β2m), can prevent recognition and rejection by CD8 T cells. However, the absence of MHC class I is a strong signal for NK cell reactivity and can lead to acute rejection by these cells. Downregulation of MHC molecules by knocking down β2m expression or inactivating genes involved in antigen processing and presentation can avoid NK cell responses and reduce, but not completely eliminate, rejection by cytotoxic CD8 T cells. These modifications are typically achieved through the use of gene editing tools or shRNA technology and can be combined with exogenous overexpression of NK cell and / or T cell inhibitory proteins (e.g., CD47 or PDL1). Furthermore, although it can reduce the degree of rejection, it is still 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).

[0009] The present disclosure provides the benefit of improving allogeneic therapy by increasing the persistence of administered cells despite the recipient's natural defenses. Summary of the Invention

[0010] Provided herein are immune cells that have been engineered, e.g., by genetic engineering, to reduce rejection by a host or recipient into which the cells have been introduced, methods for reducing rejection and / or recognition by the host or recipient's immune system, e.g., T cells and / or NK cells, compositions and populations comprising the engineered cells, and methods of using the same to treat cancer in a patient.

[0011] In one aspect, the present disclosure provides engineered immune cells, e.g., CAR T cells, or populations of engineered immune cells, including engineered immune cells, that contain one or more genomic modifications that functionally impair or reduce expression of one or more targets described herein. In one embodiment, the engineered immune cells contain genomic modifications that functionally impair or reduce expression of (i) RFX5 and / or NLRC5, and (ii) CD58, compared to cells without the genomic modifications. In another embodiment, the genomic modifications include knockdown and / or knockout of (i) RFX5 and / or NLRC5, and / or (ii) CD58. In another embodiment, the genomic modifications include one or more modifications at the gene loci for (i) RFX5 and / or NLRC5, and (ii) CD58. In one embodiment, the genomic modifications include deletions or insertions at the gene loci for (i) RFX5 and / or NLRC5, and (ii) CD58. In other embodiments, the genomic modification is selected from the group consisting of (i) an insertion of one or more nucleotides, (ii) an insertion of a polynucleotide sequence encoding a protein, (iii) a deletion of one or more nucleotides, and (iv) a substitution of one or more nucleotides. In additional embodiments, the genomic modification is introduced by a gene editing technique selected from TALEN, zinc finger, Cas-CLOVER, and CRISPR / Cas systems.

[0012] 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 described herein) or at another location within the genome that is not at the location of the one or more genes (corresponding to one or more targets described herein), such that the modifications functionally impair or reduce expression of the one or more genes (corresponding to one or more targets described herein).

[0013] In one embodiment, the genome modification comprises the insertion of an RNA interference sequence. In another embodiment, the RNA interference sequence is an shRNA sequence, an siRNA sequence, or an miRNA sequence. In another embodiment, the RNA interference sequence comprises a sequence that is complementary to the gene sequence of (i) RFX5 and / or NLRC5, and / or (ii) CD58.

[0014] In further embodiments, the engineered immune cells further comprise 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 cells are further engineered to contain one or more genomic modifications that functionally impair or reduce expression of one or more of TAP2, β2m, TRAC, CIITA, RFXAP, RFXANK, ICAM-1, and CD48 compared to unengineered cells.

[0015] In additional embodiments, the engineered immune cells, or a population of engineered immune cells comprising the engineered immune cells, have improved persistence and / or improved resistance to alloreactive immune cell rejection compared to immune cells that do not comprise a genomic modification. In one embodiment, the alloreactive immune cell rejection comprises alloreactive T cell-mediated rejection and / or alloreactive natural killer (NK) cell-mediated rejection. In other embodiments, the increased persistence can be determined and / or is determined by a mixed lymphocyte reaction (MLR) assay. In one embodiment, the improved resistance to alloreactive immune cell rejection can be determined and / or is determined by an MLR assay.

[0016] In other embodiments, the engineered immune cells comprise one or more genomic modifications that functionally impair or reduce expression of RFX5 and CD58. In one embodiment, the engineered immune cells comprise one or more genomic modifications that functionally impair or reduce expression of NLRC5 and CD58. In another embodiment, the engineered immune cells comprise one or more genomic modifications that functionally impair or reduce expression of RFX5, NLRC5, and CD58. In other embodiments, β2m is functionally expressed at low levels in the engineered immune cells. In additional embodiments, the engineered immune cells comprise an unmodified β2m gene and / or β2m is not functionally expressed at low levels in the engineered immune cells.

[0017] In further embodiments, the engineered immune cells exhibit (i) low levels of MHC class I protein or MHC class I complex expression on the cell surface, and (ii) low levels of MHC class II protein or MHC class II complex expression on the cell surface.

[0018] In one embodiment, the antigen binding protein is a CAR. In another embodiment, the engineered immune cells express an antigen binding protein and / or a CD70 binding protein. In a further embodiment, the polynucleotide sequence encoding the antigen binding protein and / or CD70 binding protein is located within a disrupted locus of CD58, RFX5, NLRC5, ICAM-1, CD48, TAP2, β2m, TRAC, CIITA, RFXAP, or RFXANK.

[0019] In other embodiments, the engineered immune cells further comprise one or more genomic modifications of the endogenous TCRa gene. In one embodiment, the engineered immune cells further comprise one or more genomic modifications of the endogenous CD52 gene.

[0020] In one other embodiment, the engineered immune cells are immune cells of a healthy volunteer, or are obtained from immune cells of a healthy volunteer, are obtained from a patient, or are obtained from induced pluripotent stem cells (iPSCs). In another embodiment, the engineered immune cells are not natural killer (NK) cells, or are not obtained from NK cells of a healthy volunteer or patient. In a further embodiment, the engineered immune cells are not obtained from iPSCs.

[0021] In one embodiment, the engineered immune cell, or one or more engineered immune cells in the population of engineered immune cells, express or functionally express a CD70 binding protein. In another embodiment, the engineered immune cell comprises a polynucleotide sequence encoding a CD70 binding protein. In a further embodiment, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In one embodiment, the CD70 binding domain comprises a CD70 antibody or a receptor for CD70, or a CD70-binding fragment thereof. In another embodiment, the CD70 binding domain comprises an anti-CD70 antibody, optionally wherein the anti-CD70 antibody is an scFv. In another embodiment, the CD70 binding protein further comprises a hinge domain, optionally wherein 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 a further embodiment, the CD70 binding protein comprises a CD3z signaling domain or a CD3g signaling domain and does not comprise a costimulatory domain. In another embodiment, 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 another embodiment, the one or more intracellular domains comprise one or more amino acid sequences of SEQ ID NOs: 1, 7-14, 17-31, 32-58, 59-70, or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0022] In one alternative embodiment, the engineered immune cell, or one or more engineered immune cells in the population of engineered immune cells, comprise unmodified β2m, RFX5, NLRC5, CIITA, and TAP2 genes. In another embodiment, expression of one or more of β2m, RFX5, NLRC5, CIITA, and TAP2 is not functionally impaired or functionally reduced in the engineered immune cell, or one or more engineered immune cells in the population of engineered immune cells. In one embodiment, the one or more genomic modifications do not include genomic modifications of one or more of β2m, RFX5, NLRC5, CIITA, and TAP2.

[0023] In one aspect, the disclosure provides a population of engineered immune cells comprising one or more of the engineered immune cells described herein. In one embodiment, the population is characterized in that 50% or less of the engineered immune cells functionally express (i) RFX5 and / or NLRC5, and (ii) CD58. In other embodiments, 50% or less of the engineered immune cells functionally express RFX5 and CD58, or 50% or less of the engineered immune cells functionally express NLRC5 and CD58, or 50% or less of the engineered immune cells functionally express RFX5, NLRC5, and CD58. In other embodiments, 50% or less of the engineered immune cells further functionally express any one or more of: a) CD48, ICAM-1, TAP2, β2m, TRAC, CIITA, RFXAP, and RFXANK; b) only one of CD48 and ICAM-1; or c) both CD48 and ICAM-1.

[0024] In one embodiment, the population of engineered immune cells comprises engineered immune cells, wherein at least 1% of the engineered immune cells functionally express (i) RFX5 and / or NLRC5, and (ii) CD58, at levels that are 50% or less of the expression levels in non-engineered immune cells. In another embodiment, at least 1% of the engineered immune cells functionally express RFX5 and CD58 at levels that are 50% or less of the expression levels in non-engineered immune cells. In one embodiment, at least 1% of the engineered immune cells functionally express NLRC5 and CD58 at levels that are 50% or less of the expression levels in non-engineered immune cells. In another embodiment, at least 1% of the engineered immune cells functionally express RFX5, NLRC5, and CD58 at levels that are 50% or less of the expression levels in non-engineered 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, at levels that are 50% or less than the expression levels in non-engineered immune cells.

[0025] In a further embodiment, the population comprises improved persistence and / or improved resistance to alloreactive immune cell rejection compared to unmanipulated immune cells. In another embodiment, the improved resistance is to alloreactive T cell-mediated rejection and / or alloreactive natural killer (NK)-mediated rejection. In one embodiment, the increased persistence can be and / or is determined by a mixed lymphocyte reaction (MLR) assay, and / or the improved resistance to alloreactive immune cell rejection can be and / or is determined by a MLR assay.

[0026] In another embodiment, the population of engineered immune cells comprises engineered immune cells, wherein at least 50% of the engineered immune cells exhibit reduced levels of cell surface MHC class I protein or MHC class I complex expression. In another embodiment, the population of engineered immune cells 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 an antigen-binding protein or a CD70-binding protein. In a further embodiment, the antigen-binding protein is a CAR or a TCR.

[0027] In other embodiments, nucleic acids encoding antigen binding proteins (e.g., CAR or TCR) and / or CD70 binding proteins are inserted into the disrupted loci of CD58, RFX5, NLRC5, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA, and RFXANK. In further 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 further comprise one or more genomic modifications of one or more of the endogenous TCRa 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.

[0028] In other embodiments, the level of functional expression of one or more of TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA, and RFXANK is measured by determining the surface expression level of HLA, beta 2 microglobulin (B2M), or both HLA and B2M on the surface of the engineered immune cells, or by flow cytometry. In one embodiment, the level of functional expression 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 by flow cytometry.

[0029] In another aspect, the present disclosure provides a method for producing an engineered immune cell or a population of engineered immune cells described herein. In one embodiment, the method comprises modifying the genome of the engineered immune cell. In another embodiment, the method further comprises generating an engineered immune cell comprising the genome modification. In a further embodiment, the genome of the engineered immune cell is modified using TALEN, zinc finger, Cas-CLOVER, or CRISPR / Cas systems.

[0030] In another aspect, the present disclosure provides a pharmaceutical composition comprising the engineered immune cells, or a cell population comprising the engineered immune cells. In one embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier or excipient. In another embodiment, the engineered immune cells, or one or more of the engineered immune cells of the cell population, are further engineered to (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) not express or express at low levels any one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFXANK, RFXAP, and RFX5.

[0031] In an additional aspect, the present disclosure provides a method of treating a condition or disorder in a patient. In one embodiment, the method comprises administering to a subject engineered immune cells, a cell population comprising engineered immune cells, or a pharmaceutical composition comprising engineered immune cells or a cell population comprising engineered immune cells. In a further embodiment, the condition is a solid tumor or a liquid tumor. In other embodiments, the disorder is cancer, an autoimmune disorder, or an infectious disease, as further described herein.

[0032] In another aspect, the present disclosure provides methods for improving (i) the persistence or (ii) resistance to alloreactive immune cell rejection of engineered immune cells described herein. In one embodiment, the engineered immune cells are allogeneic engineered immune cells. In another embodiment, the method includes modifying allogeneic immune cells to introduce one or more genomic modifications that functionally impair or reduce expression of (i) RFX5 and / or NLRC5, and (ii) CD58, to provide allogeneic engineered immune cells. In a further embodiment, the method includes administering the allogeneic engineered immune cells to a subject. In one embodiment, the genomic modifications include knockdown and / or knockout of (i) RFX5 and / or NLRC5, and (ii) CD58. In another embodiment, the genomic modifications include one or more modifications at the gene loci for (i) RFX5 and / or NLRC5, and (ii) CD58. In further embodiments, the genomic modification comprises a deletion or insertion at the gene locus of (i) RFX5 and / or NLRC5, and (ii) CD58. In other embodiments, the genomic modification is selected from the group consisting of (i) an insertion of one or more nucleotides, (ii) an insertion of a protein-encoding sequence, (iii) a deletion of one or more nucleotides, and (iv) a substitution of one or more nucleotides. In one embodiment, the genomic modification is introduced by a gene editing technique selected from TALEN, zinc finger, Cas-CLOVER, and CRISPR / Cas systems.

[0033] In additional embodiments, the genomic modification comprises the insertion of an RNA interference sequence. In further embodiments, the interference sequence is an shRNA sequence, an siRNA sequence, or an miRNA sequence. In other embodiments, the interference sequence comprises a sequence complementary to the gene sequences of (i) RFX5 and / or NLRC5, and (ii) CD58. In some embodiments, the allogeneic engineered immune cells further comprise a polynucleotide sequence encoding an antigen-binding protein (e.g., a CAR or TCR) and / or a CD70-binding protein. In further embodiments, the allogeneic engineered immune cells are further engineered to contain one or more genomic modifications that functionally impair or reduce expression of one or more of TAP2, β2m, TRAC, CIITA, RFXAP, RFXANK, ICAM-1, and CD48 compared to cells that do not contain the modification. In other embodiments, the genomic modification functionally impairs or reduces expression to about 50% or less of the corresponding level in cells that do not contain the genomic modification. In one embodiment, the allogeneic engineered immune cells exhibit improved persistence and / or improved resistance to alloreactive immune cell rejection compared to allogeneic non-engineered immune cells. In another embodiment, the improved resistance is to alloreactive T cell-mediated rejection and / or alloreactive natural killer (NK)-mediated rejection. In one embodiment, the increased persistence can be and / or is determined by a mixed lymphocyte reaction (MLR) assay, and / or the improved resistance to alloreactive immune cell rejection can be and / or is determined by a MLR assay.

[0034] In a further embodiment, the method comprises functionally impairing or reducing the expression of RFX5 and CD58, or NLRC5 and CD58, or RFX5, NLRC5 and CD58. In an additional embodiment, the extent of reduction in the expression levels of (i) RFX5 and / or NLRC5, and (ii) CD58, is determined compared to the expression levels of (i) RFX5 and / or NLRC5, and (ii) CD58, respectively, in unmodified cells of the same type.

[0035] In a further embodiment, the level of functional expression of one or more of CD48, CD58, and ICAM-1 is measured by determining the surface expression levels of CD48 protein, CD58 protein, and ICAM-1 protein on the surface of the engineered immune cells. In another embodiment, the level of functional expression of RFX5 and / or NLRC5 is measured by determining the surface expression levels of HLA, beta-2 microglobulin (B2M), or both HLA and B2M on the surface of the engineered immune cells. In one embodiment, the surface expression levels are measured by flow cytometry. In another embodiment, the method further comprises introducing one or more genomic modifications of one or more of the TCRa gene and the CD52 gene. In one embodiment, the allogeneic engineered immune cells comprise an unmodified β2m gene, or β2m is not functionally expressed at low levels in the allogeneic engineered immune cells.

[0036] In other embodiments, the immune cells of the present disclosure exhibit reduced levels of IL-1 expression compared to corresponding cells that have not been so engineered. · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0037] In additional embodiments, immune cells engineered to functionally express one or more targets described herein at a lower level relative to corresponding cells that have not been so engineered comprise one or more genomic modifications that functionally impair or reduce expression of the one or more targets relative to cells that do not comprise the one or more genomic modifications.

[0038] In another embodiment, the engineered cells (i) have an unmodified β2m gene, (ii) functionally express normal levels of β2m, and / or (iii) have not been engineered to functionally express low levels of β2m.

[0039] The engineered cells can be further engineered to enhance resistance to rejection and / or to provide a therapeutic effect. For example, the cells can be engineered to contain or express additional proteins, e.g., antigen binding proteins such as chimeric antigen receptors (CARs) and / or T cell receptors, where the antigen binding proteins or T cell receptors target the engineered immune cells to tumor cells expressing the cognate antigen and / or other unwanted cells, e.g., cells in a disease state.

[0040] Thus, the present disclosure provides methods for increasing the persistence of allogeneic cells in a recipient, the methods comprising: providing cells with a low level of cytotoxicity compared to unmanipulated cells; · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0041] In additional embodiments, the method of engineering a cell to functionally express one or more targets described herein at a lower level compared to a corresponding cell that has not been so engineered comprises introducing into the cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to a cell that does not contain the one or more genomic modifications.

[0042] In another embodiment, the method does not include engineering the cells to functionally express low levels of β2m.

[0043] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, at a lower level compared to unengineered cells.

[0044] In additional embodiments, engineered immune cells that functionally express one or more targets described herein at a lower level compared to corresponding cells that have not been so engineered comprise one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to cells that do not contain the one or more genomic modifications.

[0045] In another embodiment, the engineered cells (i) have an unaltered β2m gene, (ii) functionally express normal levels of β2m, and / or (iii) have not been engineered to functionally express low levels of β2m. In some embodiments, the low level of expression, as compared to the expression level in a corresponding, unengineered immune cell, is, for example, 0% when both chromosomal copies of the gene are knocked out, or 50% (i.e., 50% of the level in an unengineered control immune cell) when, for example, one of the two chromosomal copies of the gene is knocked out without a compensatory increase in expression of the other chromosomal copy of the gene. In some embodiments, the cells are · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, The expression level is 90% or less, 75% or less, 50% or less, 25% or less, or 10% or less of the expression level in unengineered immune cells.

[0046] In additional embodiments, cells that express one or more targets at levels that are 90% or less, 75% or less, 50% or less, 25% or less, or 10% or less of the expression level in unengineered immune cells comprise one or more genomic modifications that functionally impair or reduce expression of one or more targets described herein compared to cells that do not comprise the one or more genomic modifications.

[0047] In another embodiment, the cells (i) have an unmodified β2m gene, (ii) functionally express normal levels of β2m, and / or (iii) have not been engineered to functionally express low levels of β2m.

[0048] In some embodiments, in engineered immune cells · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 the expression levels of (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,

[0049] For the corresponding gene, the expression level in the engineered cells may be any value between 0% and 90% of the level in the corresponding unengineered control cells. In some embodiments, the expression level in the engineered cells may be, 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 cells. In some embodiments, a low level of expression other than 0% or 50% is obtained, for example, when only one chromosomal copy of a gene is knocked out and a compensatory mechanism results in an increase in the expression level of the remaining chromosomal copy, or when the reduction in expression is achieved by methods other than gene knockout, such as known knockdown methods, for example, by methods employing any of a variety of RNA-based technologies (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 additional embodiments, engineered immune cells, wherein the expression level of one or more targets described herein is between 0% and 90% of the level in corresponding unengineered control cells, contain one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to immune cells that do not contain the one or more genomic modifications.

[0050] In some embodiments, the engineered immune cells disclosed herein exhibit reduced cell surface expression of MHC class I proteins or MHC class I complexes compared to a suitable control. In various embodiments, the cells are T cells, e.g., human T cells. In some embodiments, the cells contain mutations in the TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK loci or genes, and / or contain disruptions in the TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK loci or genes, resulting in reduced functional expression of the disrupted loci or genes. In other embodiments, the cells may contain additional mutations in one or more of the CD48, CD58, and ICAM-1 loci or genes, and / or contain disruptions in one or more of the CD48, CD58, and ICAM-1 loci or genes, resulting in reduced functional expression of the disrupted loci or genes. In another embodiment, the cells are (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, The cell may contain a mutation in the β2m locus or gene, resulting 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 unaltered β2m gene, (iii) functionally expresses normal levels of β2m, and / or (iv) has not been engineered to functionally express low levels of β2m.

[0051] In additional embodiments, a cell containing a mutation in one or more target loci or genes described herein, thereby resulting in reduced functional expression of the disrupted loci or genes, contains one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to a cell that does not have the one or more genomic modifications.

[0052] In some embodiments, the mutation or disruption is introduced by any one or a combination of gene mutation or gene editing techniques, including, but not limited to, known homologous recombination techniques, and techniques employing any one or more of meganucleases, TALENs, zinc fingers, shRNAs, Cas-CLOVER, and CRISPR / Cas systems (e.g., Cas9, Cas12, and MAD7), or other systems such as base editing systems or prime editing systems. In some embodiments, the cell is a non-human cell, e.g., a primate cell or a non-primate mammalian cell. In some embodiments, the cell is a human cell.

[0053] In various embodiments, the engineered immune cells further express an antigen binding protein, e.g., the engineered immune cells comprise a nucleic acid encoding the antigen binding protein. In one embodiment, the antigen binding protein is a chimeric antigen receptor (CAR). In certain embodiments, a nucleic acid encoding an antigen binding protein, such as a CAR, is inserted into or inserted into a disrupted locus of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, or RFXANK, disrupting the locus. In various embodiments, the antigen binding protein is a T cell receptor (TCR). In certain embodiments, a nucleic acid encoding a TCR is inserted into or inserted into a disrupted locus of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, or RFXANK, disrupting the locus. In certain embodiments, the engineered immune cells further comprise one or more genomic modifications, e.g., modifications of an endogenous gene locus, such as one or more of the endogenous CD70 gene, the endogenous TCRa gene, and the endogenous CD52 gene. In various embodiments, the one or more genetic modifications result in reduced or eliminated functional expression of the gene(s) comprising the modification(s).

[0054] In additional embodiments, the engineered immune cells that further express an antigen binding protein, e.g., a CAR, comprise one or more genomic modifications that functionally impair or reduce expression of one or more targets compared to immune cells that do not have the one or more genomic modifications.

[0055] The engineered immune cells can be derived from cells from any of a variety of sources. The engineered immune cells can be prepared or derived from cells, e.g., stem cells or immune cells, from a human other than the human to whom the engineered immune cells are administered, e.g., a donor other than the recipient (e.g., a healthy volunteer), or can be prepared or derived from cells, e.g., stem cells or immune cells, from the human to whom the engineered immune cells are administered (the recipient), or can be derived from one or more induced pluripotent stem cells (iPSCs). In certain embodiments, the immune cells are immune cells obtained from a healthy volunteer, obtained from a patient, or derived from iPSCs.

[0056] In another aspect, the engineered cells 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 comprises a CD70 antibody or a receptor for CD70, or a CD70-binding fragment thereof. In other embodiments, the CD70 binding domain comprises an anti-CD70 antibody, optionally the anti-CD70 antibody is an 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 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 signaling domain or a CD3g signaling domain, but does not comprise a costimulatory domain. In an additional embodiment, the CD70 binding protein comprises a 4-1BB signaling domain, but 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 one or more amino acid sequences of SEQ ID NOs: 1, 7-14, 17-70, or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0057] In another aspect, the present disclosure provides methods for producing the engineered immune cells disclosed herein. In certain embodiments, the methods include the use of any gene editing technology, such as, for example, TALEN, zinc finger, Cas-CLOVER, and CRISPR / Cas systems, and / or any known gene knockdown method, employing, for example, any of a variety of RNA-based technologies (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)), · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, compared to unmanipulated immune cells. In another embodiment, the method does not include gene editing of the cells to functionally express low levels of β2m.

[0058] In additional embodiments, methods of generating engineered immune cells using gene editing techniques to reduce functional expression of one or more targets described herein, relative to corresponding immune cells that have not been so engineered, include introducing into an immune cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets, relative to an immune cell that does not include the one or more genomic modifications.

[0059] In some embodiments, the method includes, or further includes, introducing into the engineered immune cell a nucleic acid encoding an antigen-binding protein, such as, for example, a CAR or TCR. In some embodiments, the method includes, or further includes, introducing into the genome of the engineered immune cell one or more genomic modifications of one or more of the endogenous TCRa gene and the endogenous CD52 gene. In some embodiments, the one or more genomic modifications completely or partially disrupt and / or prevent functional expression of one or more of the endogenous TCRa gene and the endogenous CD52 gene.

[0060] In various embodiments of the present disclosure, the level of functional expression 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 protein or HLA-B protein, or the surface expression level of beta-2 microglobulin (B2M), or the surface expression level of both one or more HLA proteins and B2M, on the surface of the engineered immune cells, or by flow cytometry. In various embodiments of the present disclosure, the level of functional expression of any one or more of CD48, CD58, and ICAM-1 is measured by determining the surface expression level of each cell surface protein, such as one or more of CD48, CD58, or ICAM-1 proteins, on the surface of the engineered immune cells, or by flow cytometry. In some embodiments, expression of any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK in engineered immune cells of the disclosure is analyzed 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 correspondingly unengineered but otherwise comparable, e.g., identical, immune cells survive under the same conditions.

[0061] In some embodiments, a lower level of ATP is produced compared to a corresponding cell that has not been so engineered. · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. (a) expressing 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 (b) expressing low levels of · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, This is achieved by knockout or knockdown as described herein.

[0062] In additional embodiments, immune cells engineered to functionally express one or more targets described herein at a lower level relative to corresponding cells that have not been so engineered comprise one or more genomic modifications that functionally impair or reduce expression of the one or more targets relative to cells that do not comprise the one or more genomic modifications.

[0063] In another aspect, the present disclosure provides a population of engineered immune cells comprising the engineered immune cells provided herein. In one embodiment, the population of engineered immune cells comprises the engineered immune cells provided herein. 4 ~10 10 In various embodiments, the population of engineered immune cells comprises 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 The present invention also includes the engineered immune cells provided herein.

[0064] In another aspect, the present disclosure provides a population of engineered immune cells, wherein, for example, 75% or less of the cells are: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0065] In another embodiment, the cells of the population functionally express β2m at normal levels or do not functionally express β2m at low levels.

[0066] In additional embodiments, 10 4 ~10 10 A population of engineered immune cells comprising an individual engineered immune cell includes engineered immune cells that contain one or more genomic modifications that functionally impair or reduce expression of one or more targets compared to immune cells that do not contain the one or more genomic modifications.

[0067] In another aspect, the present 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 are · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. The low level can be 90% or less, 75% or less, 50% or less, 25% or less, or 10% or less of the expression level in unengineered immune cells. In particular, the low level can be 50% or less of the expression level in unengineered immune cells. In another embodiment, the engineered immune cell population functionally expresses β2m at normal levels or does not functionally express β2m at low levels.

[0068] In additional embodiments, 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 described herein at a low level, comprises engineered immune cells that contain one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to immune cells that do not contain the one or more genomic modifications.

[0069] In some embodiments, a low level of expression, as compared to an appropriate control, e.g., expression levels in corresponding non-engineered immune cells, can be, for example, 0% when both chromosomal copies of a gene are knocked out (e.g., by methods using TALEN, zinc finger, Cas-CLOVER, and / or CRISPR / Cas systems), or 50% when, for example, one of two chromosomal copies of a gene is knocked out without a compensatory increase in expression of the other chromosomal copy of the gene. In some embodiments, a low level of expression, as compared to expression levels in non-engineered immune cells, can be 0%-90% or any two values between 0%-90%, e.g., 10%-90%, 25%-90%, 25%-75%, 10%-50%, 25%-50%, 50%-90%, and 50%-75%. Values within one or more of such intermediate ranges can be obtained, for example, when only one chromosomal copy of a gene is knocked out and compensatory mechanisms result in an increase in the expression level of the remaining chromosomal copy, or when the reduction in expression is achieved by methods other than gene knockout, such as known knockdown methods, for example, by methods employing any of a variety of RNA-based technologies (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 populations of engineered immune cells disclosed herein, some or all of the engineered cells, e.g., 5-10%, 10-25%, 25-50%, 50-90%, or 90-100%, exhibit low levels of expression of MHC class I proteins or complexes and / or MHC class II proteins or complexes on the cell surface compared to a suitable control.

[0070] In various embodiments, a population of engineered immune cells disclosed herein, or a population of immune cells comprising engineered immune cells, 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, at least 90% engineered T cells, or 100% engineered T cells. Also provided herein are cell populations, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, or 100% of which are engineered immune cells, e.g., engineered T cells disclosed herein.

[0071] 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 of the population further express an antigen binding protein. In some embodiments, the antigen binding protein is a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encoding the CAR is inserted into, and / or such insertion disrupts, a disrupted locus in CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, or RFXANK. In some embodiments, the antigen binding protein is a T cell receptor (TCR). In some embodiments, the nucleic acid encoding the TCR is inserted into, and / or such insertion disrupts a disrupted locus in CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, or RFXANK.

[0072] In certain embodiments of the populations of engineered immune cells 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 the endogenous TCRa gene and the endogenous CD52 gene.

[0073] In certain embodiments, the population of engineered immune cells is derived from one or more immune cells obtained from a human, e.g., obtained from a human other than the human to whom they are administered, e.g., obtained from a donor other than the recipient, or obtained from a healthy volunteer, or derived from one or more immune cells obtained from a patient, e.g., the human to whom they are administered, or derived from one or more iPSCs.

[0074] 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 comprises a CD70 antibody or a receptor for CD70, or a CD70-binding fragment thereof. In other embodiments, the CD70 binding domain comprises an anti-CD70 antibody, optionally the anti-CD70 antibody is an 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 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 signaling domain or a CD3g signaling domain, but does not comprise a costimulatory domain. In an additional embodiment, the CD70 binding protein comprises a 4-1BB signaling domain, but 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 one or more amino acid sequences of SEQ ID NOs: 1, 7-14, 17-70, or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0075] In another aspect, the present disclosure provides a method of making a population of engineered immune cells as described herein, wherein the method comprises using a gene editing technology, e.g., a gene editing technology selected from the group consisting of TALEN, zinc finger, Cas-CLOVER, and CRISPR / Cas system, to: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. In one embodiment, the method does not involve the use of gene editing techniques, for example, gene editing techniques to reduce functional expression of β2m.

[0076] In additional embodiments, methods of generating a population of engineered immune cells described herein using gene editing techniques to reduce functional expression of one or more targets described herein compared to corresponding immune cells that have not been so engineered include introducing into the immune cells one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to immune cells that do not include the one or more genomic modifications.

[0077] In another aspect, the present disclosure provides methods for determining or measuring the level of functional expression of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and / or RFXANK in cells of a population of engineered immune cells disclosed herein, wherein the level of functional expression is measured by determining the surface expression level of an HLA protein, beta-2 microglobulin (B2M), or both an HLA protein and B2M on the surface of the engineered immune cells, and / or by flow cytometry. In various embodiments of the present disclosure, the level of functional expression of any one or more of CD48, CD58, and ICAM-1 in cells of a population of engineered immune cells is measured by determining the surface expression level of each cell surface protein, e.g., one or more of CD48, CD58, or ICAM-1 proteins, on the surface of the engineered immune cells, or by flow cytometry.

[0078] In various embodiments of the engineered immune cells described herein, and in various embodiments of the populations of engineered immune cells disclosed herein, the engineered immune cells, or one or more of the 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 (e.g., by any method disclosed herein or by any other method known to one of 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, by any method described herein or by any other method known to one of skill in the art.

[0079] In another aspect, a method of treating a condition in a patient comprises administering to the patient engineered immune cells, a population of engineered immune cells, or a pharmaceutical composition comprising engineered immune cells or comprising a population of engineered immune cells, wherein the engineered cells 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 comprises a CD70 antibody or a receptor for CD70, or a CD70-binding fragment thereof. In other embodiments, the CD70 binding domain comprises an anti-CD70 antibody, optionally wherein the anti-CD70 antibody is an scFv. In one embodiment, the CD70 binding protein further comprises a hinge domain, optionally wherein the hinge domain comprises a CD8 hinge. In one alternative 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 signaling domain or a CD3g signaling domain and does not comprise a costimulatory domain. In an additional embodiment, 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 one embodiment, the one or more intracellular domains comprise one or more amino acid sequences of SEQ ID NOs: 1, 7-14, 17-70, or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0080] In another aspect, the present disclosure provides a pharmaceutical composition comprising an engineered immune cell 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 a population of engineered immune cells disclosed herein, wherein the composition further comprises one or more pharmaceutically acceptable carriers or excipients. In various embodiments of the composition, the engineered immune cells, or one or more of the 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, 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 low levels of any one or more of CIITA, RFXANK, RFXAP, and RFX5, as achieved by knockout or knockdown as described herein.

[0081] In another aspect, the engineered cells of the pharmaceutical composition further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express the 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 comprises a CD70 antibody or a receptor for CD70, or a CD70-binding fragment thereof. In other embodiments, the CD70 binding domain comprises an anti-CD70 antibody, optionally the anti-CD70 antibody is an 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 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 signaling domain or a CD3g signaling domain, but does not comprise a costimulatory domain. In an additional embodiment, the CD70 binding protein comprises a 4-1BB signaling domain, but 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 one or more amino acid sequences of SEQ ID NOs: 1, 7-14, 17-70, or 89-90. In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain.

[0082] In additional embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients and an engineered cell, e.g., an engineered immune cell described herein, wherein the engineered cell comprises one or more genomic modifications that functionally impair or reduce expression of one or more targets described herein compared to a cell that does not comprise the one or more genomic modifications.

[0083] In another aspect, there is provided an engineered immune cell disclosed herein, a population of engineered immune cells disclosed herein, or a pharmaceutical composition for use as a medicament.

[0084] In another aspect, the present disclosure provides a method of treating a condition in a patient, comprising administering to the patient an engineered immune cell disclosed herein, a population of engineered immune cells disclosed herein, or a pharmaceutical composition disclosed herein, hi one embodiment, the condition is selected from the group consisting of solid tumors and liquid tumors.

[0085] In another aspect, the disclosure provides methods for reducing the surface expression level of MHC class I proteins in engineered immune cells, in some embodiments to about 75% or less of the expression level of MHC class I proteins in non-engineered immune cells, the methods comprising reducing the functional expression levels of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK, e.g., to about 75% or less of the expression level in non-engineered immune cells. In another embodiment, the methods further comprise reducing the functional expression level of one or more of CD48, CD58, and ICAM-1, e.g., to about 75% or less of the expression level in non-engineered immune cells. In another embodiment, the methods comprise: (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, The method includes reducing the functional expression level of the IL-1 gene to about 75% or less of the expression level of an unengineered immune cell.

[0086] In additional embodiments, the method of reducing the surface expression level of MHC class I proteins in engineered immune cells by reducing the functional expression level of one or more targets described herein, in some embodiments to about 75% of the expression level of MHC class I proteins in unengineered immune cells, comprises introducing into the immune cells one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to immune cells that do not contain the one or more genomic modifications.

[0087] In another embodiment, the engineered immune cells functionally express β2m at normal levels or do not functionally express β2m at low levels, hi 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.

[0088] In certain embodiments, the engineered immune cells disclosed herein exhibit low levels of cell surface MHC class I protein or MHC class I complex expression compared to a suitable control.

[0089] In certain embodiments, the engineered immune cells disclosed herein are engineered T cells. In certain embodiments, the engineered immune cells disclosed herein, e.g., the engineered T cells disclosed herein, further express an additional protein, such as, for example, a protein encoded by exogenous DNA or a protein whose expression is brought about by further manipulation of the cell. In some embodiments, the additional protein is an antigen binding protein and / or a CD70 binding protein. In some embodiments, the antigen binding protein is a chimeric antigen receptor (CAR). In some embodiments, a nucleic acid encoding the additional protein, e.g., an antigen binding protein, e.g., a CAR and / or a CD70 binding protein, is introduced into the cell by the methods described herein. In some embodiments, a nucleic acid is introduced into a 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 insertion of a nucleic acid encoding an additional protein, e.g., an antigen binding protein, e.g., a CAR and / or a CD70 binding protein.

[0090] In one embodiment of the methods disclosed herein, the antigen binding protein is a component of a T cell receptor (TCR), e.g., TCR α (TCR alpha), TCR β (TCR beta), TCR γ (TCR gamma), or TCR δ (TCR delta). In certain embodiments, a nucleic acid encoding a component of a TCR is inserted into a 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 the insertion of a nucleic acid encoding a component of a TCR.

[0091] In further embodiments of the methods disclosed herein, the engineered immune cells further comprise one or more genomic modifications (e.g., knockout, deletion, knockdown, insertion) of one or more of the endogenous TCR alpha gene and the endogenous CD52 gene. In some embodiments, the genomic modification results in partial or complete loss of functional expression of the modified gene. In further embodiments of the methods disclosed herein, the immune cells are immune cells obtained from a human, e.g., from a non-human donor to whom the cells are administered, e.g., a healthy volunteer, or immune cells obtained from a patient, e.g., the human to whom the cells are administered, or immune cells derived from iPSCs.

[0092] In another aspect, the present disclosure provides a method of making a population of engineered immune cells as disclosed herein, wherein the method comprises using a gene editing technology, e.g., a gene editing technology selected from the group consisting of TALEN, zinc finger, shRNA, Cas-CLOVER, and CRISPR / Cas system, to: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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 in a cell, such as an immune cell.

[0093] In another embodiment, the immune cell (before or after gene editing) further comprises a polynucleotide encoding and / or further 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.

[0094] In another embodiment, the gene editing technique comprises: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0095] In additional embodiments, methods of generating a population of engineered immune cells described herein using gene editing techniques to reduce the functional expression of one or more targets described herein, relative to a corresponding immune cell that has not been so engineered, by introducing mutations into a genetic locus or the one or more targets, include introducing into the immune cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets, relative to an immune cell that does not comprise the one or more genomic modifications.

[0096] In another embodiment, the gene editing technique does not introduce a mutation into the β2m locus. In one embodiment, the mutation is any one or more of an insertion, e.g., an insertion of one or more nucleotides or base pairs, e.g., an insertion of a sequence encoding a protein, a deletion of one or more nucleotides or base pairs, and a substitution of one or more nucleotides or base pairs.

[0097] In certain embodiments of the methods disclosed herein, the level of functional expression of any one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK is measured by determining the surface expression level of an HLA protein, beta 2 microglobulin (B2M), or both HLA and B2M on the surface of the engineered immune cells, or by flow cytometry. In various embodiments of the present disclosure, the level of functional expression of any one or more of CD48, CD58, and ICAM-1 is measured by determining the surface expression level of each cell surface protein, e.g., one or more of CD48, CD58, or ICAM-1 proteins, on the surface of the engineered immune cells, or by flow cytometry. In certain embodiments, · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 the degree of reduction in the surface expression levels of (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, This is determined relative to the corresponding expression level in cells of the same type that have not been gene-edited.

[0098] In certain embodiments of the methods disclosed herein, the engineered immune cells express an additional protein, which can be any desired protein, including an antigen-binding protein or a CD70-binding protein (as described in detail herein). In certain embodiments, the antigen-binding protein comprises a CAR. In certain embodiments, a nucleic acid encoding a CAR (and optionally a CD70-binding protein) is inserted into, and / or such insertion disrupts, a disrupted locus in CD58, CD48, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, or RFXANK. In another embodiment, the antigen-binding protein is a component of a T cell receptor (TCR). In certain embodiments, a nucleic acid encoding a TCR is inserted into, and / or such insertion disrupts, a disrupted locus in CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, or RFXANK.

[0099] In some embodiments, the method comprises reducing the level of functional expression of the disrupted locus. · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0100] In additional embodiments, the methods of reducing the functional expression of one or more targets described herein, thereby reducing the functional expression of a disrupted locus, comprise introducing into an immune cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to an immune cell that does not comprise the one or more genomic modifications.

[0101] In another embodiment, the method does not include reducing the level of functional expression of β2m.

[0102] In certain embodiments, the method further comprises introducing into the engineered immune cells one or more genomic modifications in one or more of the genes encoding components of the TCR, e.g., the TCRa and CD52 genes.

[0103] In another aspect, the disclosure provides methods for reducing peptide diversity presented on cell surfaces, e.g., by MHC class I, comprising reducing the level of functional expression of any one or more of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK, and in some embodiments, reducing this level to about 90% or less (i.e., at least about a 10% reduction, e.g., to a level of about 90 or less compared to a control level of 100), or to about 75% or less of comparable cells without the corresponding alteration. In addition to reducing peptide diversity, the methods disclosed herein can also include the simultaneous downregulation or loss of one or more specific cell surface receptors known to play important roles in immune cell adhesion and activation at the immune synapse, e.g., CD48, CD58, and ICAM-1. In various embodiments, reducing the level of functional expression of one or more genes described herein comprises the use of a gene editing technology selected from the group consisting of TALEN, zinc finger, Cas-CLOVER, and CRISPR / Cas systems (e.g., including Cas9, Cas12, and MAD7). (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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 (vii) CD48, CD58, ICAM-1, RFX5, and NLRC5.

[0104] In additional embodiments, methods for reducing the level of functional expression of one or more genes / targets described herein, thereby reducing the diversity of peptides presented on the cell surface, and simultaneously downregulating or eliminating specific cell surface receptors known to play important roles in immune cell adhesion and activation at the immune synapse, comprise introducing into the immune cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to an immune cell that does not contain the one or more genomic modifications.

[0105] In another embodiment, the method does not include reducing functional expression of β2m.

[0106] In some embodiments, the degree of reduction in expression levels 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 levels in cells of the same type that are not gene-edited. In some embodiments, the level of functional expression of one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK is measured by determining the surface expression levels of HLA, beta-2 microglobulin (B2M), or both HLA and B2M on the surface of the engineered immune cells. In various embodiments of the present disclosure, the degree of reduction in expression levels of one or more of CD48, CD58, and ICAM-1 is measured by, for example, determining the surface expression levels of each cell surface protein, such as one or more of CD48, CD58, or ICAM-1 proteins, on the surface of the engineered immune cells, or by flow cytometry.

[0107] In another aspect, the disclosure provides a method for reducing T cell-mediated killing of allogeneic cells, the method comprising: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. In another embodiment, the method of decreasing T cell-mediated killing of allogeneic cells does not include reducing the level of functional expression of β2m.

[0108] In additional embodiments, a method for reducing T cell-mediated killing of allogeneic cells by reducing the level of functional expression of one or more targets described herein in an engineered immune cell, e.g., an engineered T cell, comprises introducing one or more genomic modifications into an immune cell, e.g., a T cell, that functionally impairs or reduces expression of the one or more targets compared to an immune cell that does not include the one or more genomic modifications.

[0109] In some embodiments, the methods result in a reduced level of expression, relative to expression levels in unengineered immune cells, where the reduced level is, for example, 0% when both chromosomal copies of a gene are knocked out, or, for example, 50% when one of two chromosomal copies of a gene is knocked out without a compensatory increase in expression of the other chromosomal copy of the gene. In some embodiments, the methods result in a reduced level of expression, relative to expression levels in unengineered immune cells, where the reduced level is 0% to 90% or any two values intermediate between 0% and 90%, for example, 10% to 90%, 25% to 90%, 25% to 75%, 10% to 50%, 25% to 50%, 50% to 90%, and 50% to 75%.

[0110] In some embodiments, · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 Reducing the level of functional expression of (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, This includes the use of gene editing techniques, such as any one or more of TALEN, zinc finger, Cas-CLOVER, and / or CRISPR / Cas systems, and / or any one or more known knockdown methods, such as methods employing any of a variety of RNA-based technologies (e.g., shRNA, antisense RNA, miRNA, siRNA).

[0111] In another embodiment, the method comprises: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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] In additional embodiments, the method of reducing the level of functional expression of one or more targets described herein in an immune cell by using gene editing techniques and / or any one or more known knockdown methods comprises introducing into the immune cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to an immune cell that does not comprise the one or more genomic modifications.

[0113] In another embodiment, the method does not include reducing the level of functional expression of β2m.

[0114] In one embodiment, the degree of reduction in 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 cells of the same type that have not been so altered and / or manipulated.

[0115] In one embodiment of the methods of reducing T cell-mediated killing of allogeneic cells 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, beta-2 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, such as one or more of CD48, CD58, or ICAM-1 proteins, on the surface of engineered immune cells, or by flow cytometry. In one embodiment, the method comprises introducing one or more genomic modifications into one or more of the genes encoding components of the TCR, e.g., the TCRa gene and the CD52 gene. In another embodiment, the engineered immune cell further comprises a polynucleotide encoding and / or functionally expresses a CD70 binding protein, as described herein.

[0116] In some embodiments of the methods of reducing T cell-mediated killing of allogeneic cells disclosed herein, in the engineered immune cells of the disclosure: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 the levels of functional expression of (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, The engineered immune cells are analyzed by measuring the extent to which they survive in the presence of effector cells (e.g., T cells) compared to the extent to which non-engineered 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 and / or functionally express a CD70 binding protein, as described herein.

[0117] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0118] In additional embodiments, engineered cells that functionally express one or more targets described herein at low levels comprise one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to immune cells that do not comprise the one or more genomic modifications.

[0119] In another embodiment, the engineered immune cells do not functionally express β2m at low levels. In various embodiments, the cells exhibit low levels of MHC class I protein or MHC class I complex expression on the cell surface, low levels of MHC class II protein or MHC class II complex expression on the cell surface, or low levels of MHC class I protein or MHC class I complex expression on the cell surface and low levels of MHC class II protein or MHC class II complex expression on the cell surface. In various embodiments, the cells are T 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.

[0120] In various embodiments, the engineered immune cells disclosed herein further express an additional protein. In some embodiments, the additional protein is an antigen binding protein and / or a CD70 binding protein. In some embodiments, the antigen binding protein is a chimeric antigen receptor (CAR). In some embodiments, the antigen binding protein is a T cell receptor (TCR). In certain embodiments, the engineered immune cells comprise a nucleic acid encoding the additional protein. In some embodiments, the nucleic acid encoding the additional protein is located within a disrupted CD48, CD58, ICAM-1, TAP2, NLRC5, CIITA, RFX5, RFXANK, β2m, or RFXAP locus and / or causes or results in a disruption within such locus. In other embodiments, the nucleic acid encoding the additional protein is not located within a disrupted β2m locus and / or does not cause or result in a disruption within such locus.

[0121] In various embodiments, the engineered immune cells disclosed herein comprise, or further comprise, one or more genomic modifications of one or more of the endogenous TCRa (TCRα or TCR alpha) gene and the endogenous CD52 gene.

[0122] 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.

[0123] In one aspect, the present disclosure provides a method of making an engineered immune cell as 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: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0124] In additional embodiments, methods of generating engineered immune cells using gene editing techniques to reduce functional expression of one or more targets described herein include introducing into an immune cell one or more genomic modifications that functionally impair or reduce expression of the one or more targets compared to an immune cell that does not include the one or more genomic modifications.

[0125] In another embodiment, the method does not include the use of gene editing techniques to reduce functional expression of β2m. [Brief explanation of the drawings]

[0126] [Figure 1A] Figure 1A shows the various components of the immune synapse (adapted from Huppa, J., Davis, MT-cell-antigen recognition and the immunological synapse, Nat Rev Immunol 3, 973-983 (2003)).

[0127] [Figure 1B] Figure 1B shows a model of immune escape by CD58-deficient tumor cells (see Figure 8 in 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. 2011 Dec 13;20(6):728-40, Epub 2011 Dec 1).

[0128] [Figure 1C] Figure 1C shows how MHC-I expression controls the balance between T cell and NK cell rejection. Normal MHC-I expression induces T cell rejection (left panel), whereas its absence induces NK cell rejection via "missing self" elimination (right panel).

[0129] [Figure 1D]Figure ID shows the use of a CD70 binding protein expressed on CAR T cells against alloreactive host (e.g., patient) immune cells. The CD70 binding protein (also called a Dagger protein) binds to the CD70 polypeptide expressed on the host's immune cells, resulting in their elimination. CAR T cells express lower levels of additional molecules, such as CD58, compared to unengineered cells, thereby providing the CAR T cells with additional protection against the host's immune response.

[0130] [Figure 1E] 1E-1F show the results of a T cell MLR assay (E) and an NK cell MLR assay (F) using cell populations with specific knockouts. [Figure 1F] Same as above.

[0131] [Figure 2] Figure 2 shows the efficiency of gene editing of cell populations carrying specific knockouts.

[0132] [Figure 3] FIG. 3 shows the results of a T cell MLR assay using cell populations with specific knockouts.

[0133] [Figure 4] FIG. 4 shows the results of a PMBC MLR assay using cell populations with specific knockouts.

[0134] [Figure 5A] Figures 5A-5B show the results of a primed T MLR assay performed to test the efficacy of CAR T cells against T cell allo-rejection. [Figure 5B] Same as above. [Figure 5C] Figures 5C-5D show the results of additional primed T MLR assays (C) and NK cell MLR assays (D). [Figure 5D] Same as above. [Figure 5E] FIG. 5E shows the results of the PBMC MLR assay. [Figure 5F] Figure 5F shows that several CAR T cells with various knockouts were able to reduce the expansion of host immune cells (left panel: host CD8+ T cells; right panel: host CD4+ T cells). [Figure 5G] Figure 5G shows that several CAR T cells with various knockouts were able to reduce the expansion of host NK cells. [Figure 5H] Figure 5H shows the cytotoxicity of CAR T cells with various knockouts.

[0135] [Figure 6A] Figures 6A-6B show the results of an NK cell MLR assay performed to test the susceptibility of CAR T cells to NK cell allorejection. [Figure 6B] Same as above. [Figure 6C] Figure 6C shows that CAR T cells do not exhibit IL-2-independent proliferation.

[0136] [Figure 7] Figure 7 shows gene editing efficiency for different CAR T cell populations.

[0137] [Figure 8] Figure 8 shows the expansion of different CAR T cell populations with different knockouts.

[0138] [Figure 9] Figure 9 shows the results of cytotoxicity assays for various CAR T cells with specific knockouts.

[0139] [Figure 10] Figure 10 shows the results of a primed T cell MLR assay of differently engineered (e.g., gene-edited) CAR T cells.

[0140] [Figure 11] Figure 11 shows the results of a PBMC MLR assay of differently engineered (e.g., gene-edited) CAR T cells.

[0141] [Figure 12A] Figures 12A-12B show the results of host immune cell expansion (A: host CD8+ T cells and CD4+ T cells; B: NK cells) in an MLR assay using differently engineered (e.g., gene-edited) CAR T cells. [Figure 12B] Same as above.

[0142] [Figure 13A] Figures 13A-13D show the results of various assays using CD19 CAR / CD70 binding protein / CD58 knockout cells. Figure 13A shows the results of a PBMC MLR assay. [Figure 13B] 13B-13C show the results of the host cell expansion assay in the MLR assay (B: host CD4+ and CD8+ T cells, C: host NK cells). [Figure 13C] Same as above. [Figure 13D] FIG. 13D shows the results of the cytotoxicity assay. DETAILED DESCRIPTION OF THE INVENTION

[0143] The present disclosure provides a gene editing strategy for providing therapeutic allogeneic cell products that do not induce or induce only a low level of rejection by the recipient's immune system. This allows the cell product to persist longer in the recipient, thereby promoting and / or improving therapeutic efficacy. This strategy involves downregulating or eliminating one or more specific cell surface receptors, such as CD48, CD58, and ICAM-1, which are known to play important roles in immune cell adhesion and activation at the immune synapse. This strategy can be further complemented by downregulating or eliminating genes encoding molecules involved in HLA expression, thereby minimizing the diversity of peptides presented by the allogeneic cell product. This strategy is achieved by introducing one or more genomic modifications (e.g., genomic knockout or knockdown) of one or more of TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK, which function in cellular peptide presentation. In one embodiment, the genomic modifications (e.g., genomic knockout or knockdown) are: (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (i) directed against CD58, ICAM-1, RFX5, and NLRC5; (ii) against CD48, ICAM-1, RFX5, and NLRC5; (iii) against CD48, CD58, RFX5, and NLRC5; (iv) against CD48, CD58, ICAM-1, and RFX5; (v) against CD48, CD58, ICAM-1, and NLRC5; or (vi) against CD48, CD58, ICAM-1, RFX5, and NLRC5.

[0144] 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 described herein) or at another location within the genome that is not at the location of the one or more genes (corresponding to one or more targets described herein), such that the modifications functionally impair or reduce expression of the one or more genes (corresponding to one or more targets described herein).

[0145] Immune cells may be referred to as engineered immune cells when they contain at least one modification. Thus, the engineered immune cells of the present disclosure may be referred to as immune cells that contain a genomic modification.

[0146] In one aspect, the engineered immune cells, and populations comprising them, exhibit an improved ability to resist elimination by alloreactive immune cells. In one embodiment, the improved ability to resist elimination is demonstrated by an improved ability to survive in the presence of alloreactive immune cells, such as PBMCs, T cells, and / or natural killer (NK) cells. For example, improved viability can be demonstrated by one or more mixed lymphocyte (MLR) assays described herein, including in the Examples section.

[0147] In another aspect, the engineered immune cells and populations comprising them show improved persistence in allogeneic or HLA-mismatched subjects. In one embodiment, the improved persistence of cells or populations is demonstrated by an improved ability to maintain survival after adoptive transfer into allogeneic or HLA-mismatched subjects. As described herein, cells and populations comprising them are characterized by an improved ability to resist elimination by alloreactive immune cells. Without being bound by theory, it is predicted that cells and populations with improved ability will also show improved persistence after administration to allogeneic or HLA-mismatched subjects.

[0148] One aspect of the present invention relates to engineered cells (e.g., engineered immune cells such as CAR T cells) that have functionally reduced (or absent) expression of genes encoding cell surface molecules that can be recognized and subsequently killed by recipient (host) immune cells, and methods of making 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 result in proper cell adhesion and activation of immune cells (Dustin, M.L. The immunological synapse, Cancer Immunol Res. 2014 Nov;2(11):1023-33). Furthermore, loss of CD58 has been associated with tumor cell resistance 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. 2011 Dec 13;20(6):728-40, Epub 2011 Dec 1). For example, B cell lymphoma cells displaying tumor-associated antigens on HLA class I molecules can be recognized and subsequently killed by CD8 T cells (see Figure 1B, top panel, from Figure 8 in Challa-Malladi, M et al.). Tumor cells often downregulate the expression of HLA class I. However, HLA class I is a ligand for the inhibitory response of natural killer (NK) cells, e.g., KIR (killer cell immunoglobulin-like receptor).KIRs transduce signals counterbalancing 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). Consequently, loss of HLA class I cell surface expression renders tumor cells vulnerable to NK cell killing via CD2 activation by the tumor cell-expressed ligand CD58 (see the middle panel of Figure 1B from Figure 8 in Challa-Malladi, M et al.). Simultaneous loss of HLA class I and CD58 confers protection from both T cell and NK cell killing, thereby resulting in immune evasion by tumor cells (see the bottom panel of Figure 1B from Figure 8 in Challa-Malladi, M et al.). The functional reduction (or absence) of expression of certain cell surface molecules, such as CD58, CD48, and ICAM-1, in engineered cells derived from a donor cell population (e.g., allogeneic engineered immune cells such as CAR T cells) is a promising method for reducing the recognition and subsequent killing of the engineered cells by host T cells and NK cells following infusion into a patient (i.e., an individual who is not the source of the donor cell population).

[0149] The present invention provides engineered cells (e.g., engineered immune cells, such as CAR T cells) with reduced (or absent) functional expression of specific genes encoding cell surface molecules that play important roles in the immune synapse, which can be part of an allogeneic cell transplant administered to a patient (i.e., the host, or transplant recipient) in need thereof, avoiding or circumventing rejection by the patient's immune cells. Modifications, such as gene editing, to generate engineered cells with reduced (or absent) functional expression of specific genes do not require exogenous protein expression, which is advantageous. For example, gene editing approaches focused on introducing one or more exogenous genes into engineered cells can be challenging due to the limited capabilities of gene delivery vectors, such as those used to deliver CAR T cells and other enhancers of CAR T cells, such as cytokines. Rather, the present disclosure includes methods of modifying engineered cells during the manufacturing process using gene editing techniques described herein, e.g., CRISPR-Cas9, TALEN, etc., to inactivate or downregulate one or more genes encoding cell surface receptors (e.g., one or more of CD48, CD58, and ICAM-1). Such modifications may further include gene editing to downregulate or eliminate expression of genes involved in HLA molecules, as described herein. In another embodiment, the engineered cells (e.g., engineered immune cells such as CAR T cells) further comprise a polynucleotide encoding and / or functionally express a CD70 binding protein, as described herein.

[0150] In one other embodiment of the present disclosure, the target of gene editing is CD48 (also known as lymphocytic activation molecule 2 or SLAMF2), an immunoglobulin-like receptor that interacts with CD2 to contribute to the formation of an immune synapse between T cells and antigen-presenting cells. In another embodiment, the target of gene editing is CD58 (also known as lymphocyte-function antigen 3 or LFA-3), a costimulatory receptor that interacts with its natural ligand, CD2, and also contributes to the formation of an immune synapse. In an additional embodiment, the target of gene editing is Intercellular Adhesion Molecule 1 or ICAM-1 (also known as CD54), which (i) is a cell surface glycoprotein known to function in stabilizing cell-cell interactions, (ii) is expressed on immune cells, and (iii) is a ligand for the LFA-1 receptor on leukocytes.

[0151] In another embodiment of the present disclosure, the gene editing target is the TAP2 component of the transporter associated with antigen processing (TAP). The primary pathway by which MHC class I molecules carry peptides is TAP-dependent. Peptides generated by the proteasome (or IFN-γ-induced immunoproteasome) are transported to the endoplasmic reticulum (ER) via TAP and then loaded onto MHC class I molecules. A minority of peptides substantially derived from signal peptides are loaded via an alternative TAP- and proteasome-independent pathway after signal sequence cleavage by ER-resident signal peptide peptidase (SPP). Knockout of TAP2 results in a modest reduction in surface β2m (2-fold reduction after selection of KO cells) compared to a significant reduction (10- to 100-fold) in surface β2m in β2m KO cells (see Figure 4A of PCT / US2022 / 14393, which is incorporated by reference in its entirety).

[0152] In another embodiment of the present disclosure, gene editing targets can be molecules involved in regulating the transcription of HLA-I and HLA-II molecules. HLA-I and HLA-II molecules are tightly regulated at the transcriptional level by similar key cis-regulatory elements: W / S, X1, X2, and Y-box motifs. The regulatory factor X (RFX) heterocomplex, composed of RFX5, RFXAP, and RFXANK, binds to the X1-box. The X2-box is occupied by CREB / ATF1 family transcription factors, and the Y-box is bound by NF-Y proteins. Furthermore, two members of the nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) family, NLRC5 and CIITA, are required for the formation of the HLA enhanceosome complex, which promotes the transcription of HLA-I and HLA-II, respectively. NLRC5 and CIITA do not bind directly to DNA; rather, docking requires assistance from other subunits of the enhanceosome (Meissner, T.B. 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, T.B. 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 via its ankyrin repeats.

[0153] In one embodiment, the target of gene editing is a member of the nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) family, called NLR caspase recruitment domain containing 5 (NLRC5). CRISPR / Cas9-mediated knockout of NLRC5 reduces surface β2m levels by 2.5-fold (see Figure 4B of PCT / US2022 / 14393, which is incorporated herein by reference in its entirety). In another embodiment, the target of gene editing is RFX5, which, as described herein, is part of the RFX complex that associates with RFXAP and RFXANK / B at an X1 box motif.

[0154] The gene editing strategy disclosed herein surprisingly reduces peptide presentation enough to reduce cell death caused by recipient's T cell response, while at the same time not reducing peptide presentation enough to induce killing by recipient's NK cell.Therefore, the strategy provided herein represents a significant advance in allogeneic CAR-T therapy and other allogeneic cell therapy.The gene editing strategy provided herein provides an additional advantage that the NLRC5 knockout effect of suppressing MHC class I presentation should occur under the presence or absence of IFN-γ.This conclusion is supported by the finding that IFN-γ-induced MHC class I upregulation depends on NLRC5.

[0155] In another aspect of the invention, the target of gene editing in the engineered cells can be one or more genes encoding one or more molecules that function in one or more cellular pathways associated with rejection by host or recipient immune cells that react with T cell epitope determinants or NK cell epitope determinants on the surface of allogeneic cell products distinct from the host. In one embodiment, the target of gene editing in the engineered cells can be one or more genes encoding i) cell surface receptors known to play important roles 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 regulators of HLA-I and HLA-II molecules (e.g., RFX5, NLRC5, CIITA, RFXAP, and RFXANK). The methods described herein for generating engineered cells with reduced or lost expression of one or more genes may focus solely on genes encoding molecules that function in the immune synapse, but may also be supplemented with reduced or lost expression of genes encoding molecules that play a key role as transcription factors for HLA-I and / or HLA-II molecules.

[0156] general technology 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 described in Molecular Cloning: A Laboratory Manual, second 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 (JECellis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-1998) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller 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 (JEColigan et al., eds.)., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. 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, 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 described fully in such references as T. Gaj et al. This is explained thoroughly in references such as [End Page 111] et al., Genome-Editing Technologies: Principles and Applications, Cold Spring Harb Perspect Biol 2016;8:a023754, and citations therein.

[0157] definition As used herein, "autologous" means a cell, cell line, or cell population obtained from a subject that is used to treat said subject.

[0158] As used herein, "allogeneic" means that the cells or cell population used to treat a subject are not derived from said subject, but rather from a donor.

[0159] As used herein, the term "endogenous" refers to any material that is produced from or within an organism, cell, tissue, or system.

[0160] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue, or system.

[0161] As used herein, "immune cells" refer to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Examples of immune cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, regulatory T (Treg) cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0162] As used herein, the term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0163] As used herein, "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all those 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) that incorporate the recombinant polynucleotide.

[0164] The engineered immune cells of the present disclosure express, e.g., functionally express, one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK at reduced levels as described herein, and optionally further comprise additional features. For example, they can functionally express an antigen binding protein from an exogenous nucleic acid encoding the antigen binding protein introduced into the cells by the techniques described herein. In one embodiment, the engineered immune cells of the present disclosure functionally express a first antigen binding protein, e.g., a CAR, and / or a second protein, e.g., a CD70-binding protein. The engineered cells may further comprise genomic modifications, such as mutations in endogenous genes, such as TCRa and / or CD52, that reduce or eliminate functional expression of the gene, and / or the engineered cells can express one or more additional proteins from an exogenous nucleic acid encoding the antigen binding protein introduced into the cells by the techniques described herein. As described herein, the engineered immune cells of the present disclosure can be derived, eg, prepared, from cells, eg, immune cells obtained from a variety of sources.

[0165] As used herein, "functionally express" a gene means that the gene is expressed, and its expression results in a functional gene end product.For example, if the gene encodes a protein, the cell will functionally express the gene if the expression of the gene ultimately produces a properly functioning protein.Therefore, if the gene is not transcribed, or if the expression of the gene ultimately produces RNA that is not translated, or if the translation only produces a non-functional protein, for example, if the protein is not properly folded or is not transported to its site of action (for example, the membrane for membrane-bound proteins), then the gene will not be functionally expressed.Functional expression can be measured directly (for example, by assaying the gene product itself) or indirectly (for example, by assaying the effect of the gene product).

[0166] 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 is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).

[0167] As used herein, "expression control sequence" refers to 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. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0168] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence that can control the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. It is understood by those skilled in the art that different promoters can induce 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.

[0169] In any of the vectors of the present disclosure, the vector optionally comprises a promoter as disclosed herein.

[0170] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the present disclosure.

[0171] As used herein, the term "extracellular ligand-binding domain" refers to an oligopeptide or polypeptide capable of binding to a ligand. Preferably, the domain is capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. The term "stalk domain" is used herein to refer to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk domain is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain.

[0172] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector signals and induces the cell to carry out a specialized function.

[0173] As used herein, a "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, 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 ligands that specifically bind to 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 CD83, etc.

[0174] A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory signal molecule on a T cell, thereby providing a signal that mediates T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, engagement of the TCR / CD3 complex with a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1 BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAMs), 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 B7-H3. Costimulatory ligands also include, among others, CD27, CD28, 4-1 These include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83.

[0175] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, and Fv), as well as any other modified configuration of an immunoglobulin molecule containing an antigen recognition site, including, but not limited to, single-chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising antibodies. Antibodies include antibodies of any class (or subclass thereof), such as IgG, IgA, or IgM; an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG1, and IgG2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0176] 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 fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, an Fd fragment consisting of the VH and CH1 domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a single-domain antibody (dAb) fragment (see, e.g., Ward et al., Nature 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0177] An antibody, antibody conjugate, or polypeptide that "specifically binds" to a target is a term well understood in the art, 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 longer duration, and / or with higher affinity than with another cell or substance. An antibody "specifically binds" to a target if it binds with higher affinity, avidity, more readily, and / or for longer duration than it binds to other substances. By reading this definition, it is also understood that, for example, an antibody (or moiety or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding.

[0178] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held in close proximity together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are several techniques for determining CDRs, such as approaches based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)), and approaches based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948), and the Chothia system (i.e., Chothia and Lesk, J. Mol. Biol. (1987) 196(4):901-917). As used herein, CDR may refer to CDRs defined by either approach or a combination of both approaches.

[0179] The "CDRs" of a variable domain are amino acid residues within the variable region identified according to the Kabat, Chothia, or both Kabat and Chothia pool, AbM, contact, and / or structural definitions, or any method of CDR determination known in the art. Antibody CDRs can be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The locations of CDRs may also be identified as structural loop structures as originally described by Chothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the "AbM definition," a compromise between Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts, as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the "structural definition" of CDRs, the positions of CDRs may be identified as residues that contribute enthalpic ionically to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1 156-1 166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but may nonetheless be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding, but overlap with at least a portion of a Kabat CDR. As used herein, CDR may refer to a CDR defined by any approach known in the art, including a combination of approaches.The methods used herein can utilize CDRs defined according to any of these approaches. In any given embodiment containing two or more CDRs, the CDRs can be defined according to any of the Kabat, Chothia, extended, AbM, contact, AHo, and / or structural definitions.

[0180] The antibodies of the present disclosure 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 readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45:1628-50, 1999, and Fellouse, FA, et al, J. Mol. Biol., 373(4):924-40, 2007).

[0181] As known in the art, "polynucleotide" or "nucleic acid," when used interchangeably herein, refers to a chain of nucleotides of any length, including 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 a chain by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "cap" substitutions with one or more analogs of naturally occurring nucleotides; internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralens, etc.); those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylators; those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide for additional linkage to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amine or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0182] As used herein, "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell is "transfected" by exogenous or heterologous RNA or DNA when such RNA or DNA is introduced into the cell. A cell is "transformed" by exogenous or heterologous RNA or DNA when the transfected RNA or DNA affects a phenotypic change. The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA that constitutes the cell's genome.

[0183] 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.

[0184] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure. The term "compete," when used herein with respect to antibodies, means that a first antibody, or antigen-binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding portion thereof, such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to binding of the first antibody in the absence of the second antibody. The alternative possibility that binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody can, but need not, be the case. That is, a first antibody can inhibit binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will understand, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0185] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing (or destroying) tumor or cancer cell proliferation, inhibiting metastasis of tumor cells, shrinking or reducing tumor size, remission of a disease (e.g., cancer), reducing 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 dosage of other drugs needed 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 with a disease (e.g., cancer).

[0186] "Ameliorating" refers to the alleviation or improvement of one or more symptoms compared to no treatment. "Ameliorating" also includes a shortening or reduction in the duration of symptoms. As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to produce any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifesting during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing or ameliorating the incidence of one or more symptoms of various diseases or conditions (e.g., cancer), reducing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the progression of the disease. An effective dosage may be administered in one or more administrations. For purposes of this disclosure, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dosage 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 dosage" can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0187] As used herein, a "subject" is any mammal, e.g., a human or a monkey. Mammals include, but are not limited to, farm animals, sport 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, e.g., a cynomolgus monkey.

[0188] As used herein, "vector" refers to a construct that can deliver and preferably express one or more target genes or sequences into 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 producer cells.

[0189] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. A preferred diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal (0.9%) saline. Compositions of the present disclosure containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy, 21st Ed., Mack Publishing, 2005).

[0190] As used herein, "allo-reactivity" refers to the ability of T cells to recognize MHC complexes not present during thymic development. Alloreactivity manifests clinically as host-versus-graft rejection and graft-versus-host disease.

[0191] References herein to "about" in connection with a value or parameter include (and describe) embodiments that are directed to plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of the value or parameter itself. For example, a reference to "about X" includes the reference to "X." Numeric ranges are inclusive of the numbers defining the range.

[0192] Wherever embodiments are described herein with the term "comprising," it is understood that otherwise similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0193] When aspects or embodiments of the present disclosure are described in terms of a Markush group or other alternative grouping, the present disclosure encompasses not only the entire group listed as a whole, but also each individual member of the group and all possible subgroups of the main group, as well as the absence of one or more of the group members in the main group. The present disclosure also envisions the explicit exclusion of one or more of any of the group members in the disclosed and / or claimed embodiments.

[0194] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise required by context, the singular shall include the plural, and the plural shall include the singular.

[0195] Although exemplary methods and materials are described herein, 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 not intended to be limiting.

[0196] An "antigen binding protein" comprises 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 to a viral or bacterial antigen.

[0197] Antigen-binding domains include, but are not limited to, immunologically functional fragments of antibody binding regions. The term "immunologically functional fragment" (or "fragment") of an antigen-binding domain refers to a species of antigen-binding domain that contains a portion of an antibody (regardless of how the portion is obtained or synthesized) 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. Such fragments are biologically active in that they can bind to the target antigen and compete with other antigen-binding domains, including intact antibodies, for binding to a given epitope.

[0198] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (such as Fab', F(ab')2), one or more complementarity-determining regions ("CDRs"), diabodies (a light chain variable domain and a heavy chain variable domain on the same polypeptide linked via 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, human, mouse, rat, camelid, or rabbit. As will be appreciated by those skilled in the art, antigen-binding domains may comprise non-proteinaceous components.

[0199] Variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (CDRs). The CDRs of the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the N-terminus to the C-terminus, both light chain variable regions and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are typically referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are typically referred to as LC CDR1, CDR2, and CDR3.

[0200] In some embodiments, an antigen-binding domain comprises one or more complementary binding regions (CDRs) present in a full-length light or heavy chain of an antibody, and in some embodiments, comprises a single heavy and / or light chain or a portion thereof. These fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the antigen-binding domain comprising an intact antibody.

[0201] In some embodiments, the antigen-binding domain is an antibody or fragment thereof, comprising one or more of its complementarity-determining regions (CDRs), hi 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.

[0202] The assignment of amino acids to each of the framework, CDR, and variable domains is typically based on Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda, Md. 1991), Chothia numbering (see, e.g., Chothia & 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, the AbM scheme (Antibody Modeling program, Oxford The numbering scheme follows that of the AHo system (Honneger and Pluckthun, J Mol Biol (2001) 309(3):657-70).

[0203] In some embodiments, the antigen-binding domain is a recombinant antigen receptor. As used herein, the term "recombinant antigen receptor" broadly refers to a non-naturally occurring surface receptor that 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 receptors (CARs) are well known in the art. A CAR is a fusion protein that includes an antigen-recognition portion, a transmembrane domain, and a T cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724(1993)).

[0204] In some embodiments, the intracellular domain of the recombinant antigen receptor comprises a costimulatory domain and an ITAM-containing domain, hi some embodiments, the intracellular domain of the recombinant antigen receptor comprises an intracellular protein or a functional variant thereof (e.g., a truncation, insertion, deletion, or substitution).

[0205] As used herein, the term "extracellular ligand-binding domain" or "extracellular antigen-binding domain" refers to a polypeptide capable of binding to a ligand or antigen or interacting with a cell surface molecule such as a ligand or surface antigen. For example, an extracellular ligand-binding domain or antigen-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state, e.g., a tumor-specific antigen. In some embodiments, the antigen-binding domain comprises an antibody, or an antigen-binding fragment or portion of an antibody. In some embodiments, the antigen-binding domain comprises an Fv or scFv, Fab or scFab, F(ab')2 or scF(ab')2, Fd, a monobody, an affibody, a camelid antibody, a VHH antibody, a single-domain antibody, or a DARPin. In some embodiments, the ligand-binding domain comprises a ligand that binds to a surface receptor, or a binding pair partner such as the ectodomain of a surface receptor that binds to the ligand.

[0206] The terms "stalk domain" or "hinge domain" are used interchangeably herein to refer to any polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the stalk domain is often used to provide additional flexibility and accessibility to the extracellular ligand-binding domain.

[0207] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector signals and induces the cell to carry out a specialized function.

[0208] vector Expression vectors and methods for administration of polynucleotide compositions are known in the art and are further described herein.

[0209] In another aspect, the disclosure provides a method of making any of the polynucleotides described herein.

[0210] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond one-to-one to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences can, but need not, be present within the polynucleotides of the present disclosure, and polynucleotides can, but need not, be linked to other molecules and / or supporting materials.

[0211] A polynucleotide can comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or can comprise a variant of such a sequence. A polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. A variant preferably exhibits at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity to a polynucleotide sequence encoding a native antibody or a portion thereof. Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over 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, usually 30 to about 75, or 40 to about 50, contiguous positions within which a sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned.

[0212] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 1 1:105, Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425, Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA, Wilbur, WJand. Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0213] In some embodiments, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence within the comparison window can contain 20 percent or less, typically 5-15 percent, or 10-12 percent, additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain 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 occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percent sequence identity.

[0214] 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 to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0215] Preferred "moderately stringent conditions" include a pre-wash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization overnight in 5x SSC at 50-65°C, followed by two washes at 65°C for 20 minutes each in 2x, 0.5x, and 0.2x SSC containing 0.1% SDS.

[0216] As used herein, "highly stringent conditions" or "high stringency conditions" refers to (1) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C, and (2) the use of a denaturing agent such as formamide during hybridization, e.g., 50% (v / v) formamide in 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5), 750 mM sodium chloride, 75 mM sodium citrate. or (3) 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes in 0.2x SSC (sodium chloride / sodium citrate) at 42°C and 50% formamide at 55°C, followed by a high stringency wash in 0.1x SSC containing EDTA at 55°C. Those skilled in the art will know how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

[0217] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present disclosure. Additionally, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes that are altered as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions. The resulting mRNAs and proteins can, but need not, have altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and / or database sequence comparison.

[0218] The polynucleotide of the present disclosure can be obtained by chemical synthesis, recombinant method or PCR.The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail herein.Those skilled in the art can use the sequence provided herein and commercially available DNA synthesizers to produce desired DNA sequence.

[0219] To prepare a polynucleotide using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, as further described herein, and the vector can then be introduced into a suitable host cell for replication and amplification. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. Such amplified polynucleotide can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al., 1989.

[0220] Alternatively, PCR allows for the reproduction of DNA sequences. PCR technology is well known in the art and is described, for example, in U.S. Patent 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., Birkauer Press, Boston, 1994.

[0221] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as described in Sambrook et al., 1989 (see above).

[0222] Suitable cloning vectors can be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary depending on the host cell intended for use, useful cloning vectors generally have the ability to autonomously replicate, may have a single target for a particular restriction endonuclease, and / or may carry a marker gene that may be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0223] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the present disclosure. It is implied that an expression vector must be replicable in a host cell either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, and retroviruses, cosmids, and expression vectors disclosed in International Publication WO 87 / 04462. Vector components generally may include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements are also usually required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0224] A vector containing a polynucleotide of interest can be introduced into a host cell by any of a number of suitable means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, microprojectile bombardment, lipofection, and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of introduction vector or polynucleotide often depends on the characteristics of the host cell.

[0225] A polynucleotide encoding an antigen-binding protein (e.g., a CAR) can be present in an expression cassette or expression vector (e.g., a plasmid for introduction into bacterial host cells, or a viral vector such as a baculovirus vector for transfection of insect host cells, or a plasmid or viral vector such as a lentivirus for transfection of mammalian host cells). In some embodiments, the polynucleotide or vector can include a nucleic acid sequence encoding a ribosomal skipping sequence, such as, but not limited to, a sequence encoding a 2A peptide. The 2A peptide, identified in the aphthovirus subgroup of picornaviruses, causes the ribosome to "skip" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (see, e.g., Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008). A "codon" refers to three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated into a single amino acid residue by the ribosome. Thus, two polypeptides can be synthesized from a single adjacent open reading frame within an mRNA if the polypeptides are separated by an in-frame 2A oligopeptide sequence. Such ribosomal skipping mechanisms are well known in the art and are known to be used by some vectors for the expression of several proteins encoded by a single messenger RNA.

[0226] To direct a transmembrane polypeptide into the secretory pathway of a host cell, in some embodiments, a secretory signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are generally positioned 5' to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences can be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). Those skilled in the art will recognize that considerable sequence variation is possible among these polynucleotide molecules given the degeneracy of the genetic code. In some embodiments, the nucleic acid sequences of the present disclosure are codon-optimized for expression in mammalian cells, preferably human cells. Codon optimization refers to the replacement in a sequence of interest of codons that are generally rare in highly expressed genes of a given species with codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acid as the codons being replaced.

[0227] Methods for preparing engineered cells Also provided herein are methods for preparing engineered cells, e.g., engineered immune cells, for use in immunotherapy. In some embodiments, the methods involve introducing an antigen-binding protein (e.g., a CAR) into one or more immune cells, or introducing a polynucleotide encoding an antigen-binding protein (e.g., a CAR), and growing the cells. In some embodiments, the present disclosure relates to methods for engineering immune cells, the methods involving providing immune cells and expressing at least one antigen-binding protein (e.g., a CAR) on the surface of the cells. In some embodiments, the methods involve transfecting the cells with at least one polynucleotide encoding the antigen-binding protein (e.g., a CAR) and expressing the at least one polynucleotide in the cells.

[0228] In some embodiments, the polynucleotide encoding the antigen binding protein (e.g., CAR) is present in one or more expression vectors for stable expression in cells. In some embodiments, the polynucleotide is present in a viral vector for stable expression in cells. In some embodiments, the viral vector may be, for example, a lentiviral vector or an adenoviral vector.

[0229] In some embodiments, the polynucleotide encoding the polypeptide according to the present disclosure can be mRNA, which is directly introduced into cells, for example, by electroporation. In some embodiments, CytoPulse technology can be used to transiently permeabilize live cells to deliver materials into cells. Parameters can be modified to determine conditions for high transfection efficiency with minimal mortality.

[0230] Also provided herein is a method for transfecting immune cells, such as T cells.Generally, any conventional method known to those skilled in the art can be used, such as by electroporation to introduce RNA, DNA or protein into cells.For example, see Luft and Ketteler, J. Biomolecular Screening 20(8):932(2015) (DOI:10.1177 / 1087057115579638). In some embodiments, the method includes contacting a T cell with RNA and applying to the T cell an agile pulse sequence consisting of: (a) an electrical pulse having a voltage range of about 2250-3000 V / centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval of about 0.2-10 ms between the electrical pulses of steps (a) and (b); (d) an electrical pulse having a voltage range of about 2250-3000 V / centimeter, with a pulse width of about 100 ms and a pulse interval of about 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) four electrical pulses having a voltage of about 325 V, with a pulse width of about 0.2 ms and a pulse interval of 2 ms between each of the four electrical pulses.In some embodiments, the method of transfecting a T cell includes contacting the T cell with RNA, applying to the T cell (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 / centimeter, (b) a pulse width of 0.1 ms, (c) and a pulse interval 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 pulse width of 100 ms, and applying to the T cell an electric pulse of step (b). and applying an agile pulse sequence including one electrical pulse having a voltage range of about 2250 to 3000 V / cm, e.g., 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V / cm, with a 100 ms pulse interval between the first electrical pulse and the pulse in step (c), and (e) four electrical pulses having a voltage of about 325 V, with a pulse width of about 0.2 ms and a pulse interval of about 2 ms between each of the four electrical pulses. Any value within the above range is disclosed herein. The electroporation medium can be any suitable medium known in the art. In some embodiments, the electroporation medium has a conductivity ranging from about 0.01 to about 1.0 millisiemens.

[0231] In some embodiments, the method can further include genetically engineering the cells by inactivating or reducing the expression level of at least one gene expressing, for example, but not limited to, TAP2, NLRC5, β2m, CIITA, RFX5, RFXAP, and RFXANK, components of TCRs, targets of immunosuppressants, HLA genes, and / or immune checkpoint proteins such as, for example, PDCD1 or CTLA-4. By inactivating a gene, it is meant that the gene of interest is not expressed in the form of a functional protein. In some embodiments, the inactivated gene is one or more genes selected from the group consisting of, for example, 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 includes inactivating or reducing the expression level of one or more genes by introducing into the cell a rare-cutting endonuclease that can selectively inactivate 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 TALEN®), a megaTAL nuclease, or a Cas9 endonuclease.

[0232] In another embodiment, the step of genetically modifying or engineering immune cells, e.g., T cells, can include modifying the immune cells, e.g., T cells, by inactivating at least one gene expressing the target of an immunosuppressant and optionally expanding the cells in the presence of the immunosuppressant. Immunosuppressants are drugs that suppress immune function through one of several mechanisms of action. Immunosuppressants can reduce the extent and / or voracity of an immune response. Non-limiting examples of immunosuppressants include calcineurin inhibitors, target of rapamycin, interleukin-2 alpha chain blockers, inhibitors of inosine monophosphate dehydrogenase, inhibitors of dihydrofolate reductase, corticosteroids, and immunosuppressant antimetabolites. Some cytotoxic immunosuppressants act by inhibiting DNA synthesis. Other cytotoxic immunosuppressants may act through T cell activation or by inhibiting helper cell activation. The method of the present disclosure allows for the conferring of resistance to immunosuppressants to T cells for immunotherapy, for example, by inactivating the target of the immunosuppressant in the T cells. As non-limiting examples, the target of an 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.

[0233] Provided herein are compositions and methods for downregulating the expression of an antigen-binding protein, such as a CAR, and / or a CD70-binding protein (as described herein), in conjunction with downregulating 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 downregulation of functional expression is downregulation of the functional expression of (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1. In another embodiment, the downregulation of functional expression is downregulation of the functional expression of 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. In another embodiment, the downregulation of functional expression is downregulation of the functional expression of only one of CD48, CD58, and ICAM-1. In additional embodiments, the downregulation of functional expression is downregulation of the functional expression of (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 compositions 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 are useful for improving the in vivo persistence and therapeutic efficacy of engineered immune cells, e.g., engineered T cells, such as CAR-T cells.

[0234] Engineered cells Engineered cells, e.g., engineered immune cells, e.g., engineered T cells provided herein, express an antigen binding protein, e.g., a chimeric antigen receptor (CAR) and / or a CD70 binding protein, and express any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK at levels that are 75% or less, 50% or less, 25% or less, or 10% or less of the expression levels 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 compared to unengineered cells.

[0235] In some embodiments, engineered cells, such as engineered immune cells, comprise a population of CARs, each CAR comprising a different extracellular antigen-binding domain. In some embodiments, engineered cells, such as engineered immune cells, comprise a population of CARs, each CAR comprising the same extracellular binding domain.

[0236] In one embodiment, the engineered cells, e.g., engineered immune cells, are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes (Tregs), helper T lymphocytes, tumor-infiltrating lymphocytes (TILs)), natural killer T cells (NKTs), TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the engineered cells, e.g., engineered immune cells, may be derived from CD4+ T lymphocytes or CD8+ T lymphocytes. In another embodiment, the engineered cells may be derived from a population of T lymphocytes 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 gamma delta T cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some exemplary embodiments, the engineered immune cells are natural killer (NK) cells.

[0237] In some embodiments, the engineered cells, such as engineered immune cells, may be derived from, for example, but not limited to, stem cells, which may be adult stem cells, non-human embryonic stem cells, more particularly 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.

[0238] In some embodiments, the engineered cells, e.g., engineered immune cells, are obtained or prepared from peripheral blood. In some embodiments, the engineered cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the engineered cells are obtained or prepared from bone marrow. In some embodiments, the engineered cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells. Exemplary human cells are CD34+ cells.

[0239] 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., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplexes.

[0240] Any immune cell capable of expressing heterologous DNA can be used to express an antigen binding protein of interest (e.g., a CAR), and can also be engineered to have low levels of expression of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK.

[0241] In some embodiments, immune cells, such as T cells provided herein, are further modified, e.g., genetically engineered to express one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK at lower levels compared to comparable cells that have not been so engineered. For example, immune cells can be genetically engineered 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, preventing expression of the corresponding functional protein at the cell surface, e.g., by deleting genomic DNA containing part or all of the entire coding sequence of the locus and / or genomic DNA containing the transcriptional regulatory and / or promoter and / or activation elements of the locus, and / or by introducing insertion, deletion, or substitution mutations that inhibit production of the functional protein. Additionally, immune cells can be engineered to: (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; Only one of CD48, CD58, and ICAM-1; or genetically engineered to knock out all or part of the loci for (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; The corresponding functional protein can be prevented from being expressed at the cell surface, for example, by deleting genomic DNA containing some or all of the entire coding sequence of the locus, and / or genomic DNA containing the transcriptional regulatory and / or promoter and / or activation elements of the locus, and / or by introducing insertion, deletion or substitution mutations that prevent production of the functional protein.

[0242] 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 described herein) or at another location within the genome that is not at the location of the one or more genes (corresponding to one or more targets described herein), such that the modifications functionally impair or reduce expression of the one or more genes (corresponding to one or more targets described herein).

[0243] 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 proteins provided herein comprise an extracellular domain (e.g., a single-chain variable fragment (scFv)) and a transmembrane domain. In some embodiments, the CD70 binding proteins provided herein comprise an extracellular ligand-binding domain (e.g., an scFv), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CD70 binding protein comprises one or more intracellular signaling domains selected from the group consisting of a CD3ζ signaling domain, a CD3δ signaling domain, a CD3γ signaling domain, a CD3ε 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 one other embodiment, the CD70 binding protein comprises an intracellular signaling domain comprising a CD3ζ signaling domain. In some embodiments, the CD70 binding protein is a CAR.

[0244] In some embodiments, the intracellular signaling domain comprises one or more amino acid sequences of SEQ ID NOs: 1, 7-14, 17-70, or 89-90. In some embodiments, the intracellular signaling domain comprises one or more amino acid sequences of SEQ ID NOs: 7, 89, 8, 90, 12, 11, 61, 62, 64, or 65. In some embodiments, the CD70 binding protein comprises a CD3ζ or CD3γ signaling domain, or a variant thereof, and does not comprise a costimulatory domain. In some embodiments, the CD70 binding protein comprises a 4-1BB signaling domain, or a variant thereof, and does not comprise a CD3 signaling domain. In some embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3ζ signaling domain. In some embodiments, the CD70 binding protein does not comprise an intracellular signaling domain. Different intracellular signaling domains, or combinations thereof, can confer different signaling strengths that 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 comprise one or more intracellular signaling domains. In another embodiment, the CD70 binding protein comprises an scFv having the amino acid sequence set forth as SEQ ID NO: 82 or 85. In one other embodiment, the CD70 binding protein comprises the amino acid sequence set forth as SEQ ID NO: 86. Any of SEQ ID NOs: 1, 7-14, 17-70, or 89-90 may contain one or more substitutions, insertions, or deletions. These sequences may contain five, four, three, two, or fewer substitutions, insertions, or deletions. Polypeptides containing 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.

[0245] In some embodiments, engineered immune cells engineered to functionally express one or more targets described herein at lower levels (compared to corresponding cells not so engineered) and further engineered to express a CD70 binding protein may exhibit varying levels of persistence and / or resistance to rejection by host immune cells and may be suitable for use when administered to a patient for in vivo lymphodepletion. In some embodiments, engineered immune cells functionally expressing a CD70 binding protein described herein may inhibit host immune cell proliferation and / or activity to different degrees, thereby allowing for fine-tuning of the depth of in vivo lymphodepletion when administered to a patient. For example, engineered immune cells functionally expressing a CD70 binding protein that demonstrate long-term amplification and / or inhibition of host immune cell proliferation or activity in an MLR assay may be used for long-term lymphodepletion. In contrast, engineered immune cells that functionally express CD70-binding proteins, which result in less long-term expansion and / or inhibition of host immune cells in the same or similar assays, can be used when less complete or less thorough lymphodepletion is desired.

[0246] In one embodiment, the reduced level of expression compared to comparable unmodified cells can be a knockout or knockdown of expression. Various knockdown methods can be suitable, including those using various RNA-based technologies (e.g., short hairpin RNA (shRNA), antisense RNA, microRNA (miRNA), and small interfering RNA (siRNA). See, for example, 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 See Gogtay, Brit. J. Clin. Pharmacol. 82:659-72 (2016), and U.S. Patent No. 9,556,433 to Krause et al. RNA-based reagents, such as RNA interference molecules, can be delivered to cells, such as immune cells, to knock down one or more genes. In one embodiment, the RNA-based reagent can be configured to target one or more genes or to be targeted by one or more genes. In another embodiment, the RNA interference molecule comprises an RNA interference sequence, which comprises one or more sequences complementary to one or more target genes. Alternatively, a polynucleotide comprising one or more RNA interference sequences directed to one or more target genes can be inserted into a genome. In one embodiment, the insertion can be at a location in the genome that is not the location of the one or more target genes. In one embodiment, the RNA-based reagent can be · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. In another embodiment, the RNA-based reagent is not configured to target or is not targeted to β2m.

[0247] In some embodiments, in immune cells such as T cells of the present disclosure: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 the levels of functional expression of (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, The level of functional expression 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% compared to the level of functional expression in an equivalent cell that has not been engineered to reduce the corresponding expression level. In another embodiment, the functional expression level of β2m is not reduced.

[0248] One or more antigen binding proteins, e.g., one or more CAR and / or CD70 binding proteins, can be synthesized in situ within a cell after introducing a polynucleotide construct encoding the protein into the cell. Alternatively, the antigen binding protein, e.g., CAR and / or CD70 binding protein, can be produced outside the cell and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods 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, viral-mediated methods can be used. The polynucleotide can be introduced into the cell by any suitable means, such as, for example, a recombinant viral vector (e.g., retrovirus (including lentivirus), adenovirus), liposome, etc. Transient transformation methods include, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide can be contained in a vector, such as, for example, a plasmid vector or a viral vector.

[0249] In some embodiments, engineered immune cells, such as, for example, T cells, of the present disclosure can comprise at least one antigen binding protein, e.g., a CAR and / or a CD70 binding protein. Engineered immune cells, e.g., T cells, can be used to express low levels of · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. In one embodiment, the engineered immune cells, e.g., T cells, are not further modified to express low levels of β2m. In another embodiment, the engineered immune cells, e.g., CAR T cells, further comprise a polynucleotide encoding and / or functionally express a CD70 binding protein, as described herein.

[0250] In one other aspect, the present disclosure provides engineered immune cells, e.g., CAR T cells, or populations of engineered immune cells, including engineered immune cells, that comprise one or more genomic modifications that functionally impair or reduce expression of one or more targets described herein. 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 described herein) or at another location within the genome that is not at the location of the one or more genes (corresponding to one or more targets described herein), such that the modifications functionally impair or reduce expression of the one or more genes (corresponding to one or more targets described herein).

[0251] In some embodiments, an engineered immune cell, such as, for example, an engineered T cell, may comprise two or more different antigen binding proteins, e.g., two or more different CARs, each CAR comprising a different extracellular ligand binding domain and / or CD70 binding protein.

[0252] In some embodiments of the engineered immune cells, e.g., T cells, provided herein, the CAR expressed by the cell may comprise 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, transmembrane domain, and intracellular signaling domain are in one polypeptide, i.e., in a single chain. Multi-chain CARs and polypeptides are also provided herein. In some embodiments, the multi-chain CAR contains a first polypeptide comprising a transmembrane domain and at least one extracellular ligand-binding domain, and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, where the polypeptides assemble together to form the multi-chain CAR. In another embodiment, the engineered immune cell further comprises or functionally expresses a CD70 binding protein, as described herein.

[0253] The extracellular ligand-binding domain specifically binds to a target of interest. The extracellular ligand-binding domain may specifically bind to a tumor antigen. For example, the extracellular ligand-binding domain may specifically bind to a tumor antigen selected from an oncofetal antigen, an overexpressed antigen, a tissue-restricted antigen, a cancer-testis antigen, or a cancer viral antigen. The tumor antigen may be associated with a liquid tumor. In some embodiments, the target of interest may be any molecule of interest, such as, 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 Claudin18 isoform. 2), DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Muc17, Muc3, Muc3, Muc16, FAP alpha (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 of the exemplary targets listed.

[0254] In some embodiments, the antigen binding domain is selected from the group consisting of 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, Claudin-18.2 (Claudin-18A2, or Claudin 18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta 3), Mud 7 (mucin 17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus 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: BCMA - WO2016 / 16630, WO2020 / 150339, WO2019 / 196713, WO2016 / 014565, WO2017 / 025038; MUC16: US9,169,328, WO2016 / 149368, WO2020 / 023888; EGFRvIII: WO2017 / 125830, WO2016 / 016341; Flt3: WO 2018 / 222935, WO2020 / 010284, WO2017 / 173410;CD20:WO2018 / 145649, WO2020 / 010235, WO2020 / 123691;CD38:WO2017 / 0 25323;CD70:WO2019 / 152742, WO2018 / 152181;CD33:WO2016 / 014576;CD133:WO2018 / 072025;CS1:WO2019 / 030240;ROR1: WO2016 / 115559;CD19:WO2002 / 077029, US11,077,144;Claudin:WO2018 / 006882, WO2021 / 008463;DLL3:WO2020 / 180591 ;WT1:US2016 / 0152725A1, US7622119B2;CD23:US6011138A, CN1568198A;CD30:US10815301B2, US10808035B2;PRAME:US2 018 / 0148503A1, WO2020 / 186204A1; LIV1: US2020 / 0231699A1; NKG2D: WO2021 / 179353A1, US2021 / 0269501A1; FAP alpha: US2020 / 0246383A1, US2021 / 0115102A1; PSMA: US2021 / 0277141A1, WO2020 / 108646A1; MSLN: CN109680002A, CN109628492A.

[0255] In some embodiments, the extracellular ligand-binding domain comprises an scFv containing the light chain variable (VL) and heavy chain variable (VH) regions of a monoclonal antibody specific for a target antigen, connected by a flexible linker. Single-chain variable region fragments are generated by linking the light and / or heavy chain variable regions using a short linking peptide (Bird et al., Science 242:423-426, 1988). One example of a linking peptide is the GS linker, which has the amino acid sequence (GGGGS)3 (SEQ ID NO: 72), bridging the carboxy terminus of one variable region to the amino terminus of the other variable region by approximately 3.5 nm. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). In general, linkers can be short, flexible polypeptides, preferably consisting of approximately 20 amino acid residues or less. Furthermore, linkers can be modified for additional functions, such as the attachment of drugs or solid supports. Single-chain variants can be produced recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of scFv, a suitable plasmid or other vector containing a polynucleotide encoding scFv can be introduced into a suitable host cell, such as a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, or a prokaryotic cell, such as E. coli. The polynucleotide encoding the desired scFv can be produced by routine manipulation, such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0256] The intracellular signaling domain of the CAR of the present disclosure is involved in intracellular signaling after the extracellular ligand-binding domain binds to the target, resulting in immune cell activation and immune response. The intracellular signaling domain has the ability to activate at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity including cytokine secretion.

[0257] In some embodiments, intracellular signaling domains for use in CARs may be, for example, but not limited to, the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same function. The intracellular signaling domain contains two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation, and sequences that act in an antigen-independent manner to generate secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs used in the present disclosure can include, by way of non-limiting example, ITAMs derived from ΤCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of a CAR may contain a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a domain of a costimulatory molecule.

[0258] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a portion of a costimulatory molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133) and CD28 (NP_006130 and its isoforms).

[0259] CARs are expressed on the surface membrane of cells. Therefore, CARs can contain a transmembrane domain. A suitable transmembrane domain for a CAR disclosed herein has (a) the ability to be expressed on the surface of a cell, for example, an immune cell, for example, but not limited to, a lymphocyte cell (e.g., T cell) or natural killer (NK) cell, and (b) the ability to interact with a ligand binding domain and an intracellular signaling domain to induce a cellular response of the immune cell against a predetermined target cell. The transmembrane domain can be derived from either a natural or synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As a non-limiting example, the transmembrane polypeptide can be a domain of a T cell receptor, such as the α, β, γ, or δ polypeptide that constitutes the CD3 complex, an IL-2 receptor, for example, p55 (α chain), p75 (β chain or γ chain), an Fc receptor, particularly a subunit chain of Fcγ receptor III, or a CD protein. Alternatively, the transmembrane domain can be synthetic and mainly comprise hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8 α chain (e.g., NP_001139345.1). The transmembrane domain may further comprise a stalk domain between the extracellular ligand-binding domain and the transmembrane domain. The stalk domain can comprise up to 300 amino acids, e.g., 10-100 amino acids or 25-50 amino acids. The stalk region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the stalk domain may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence. In some embodiments, the stalk domain is a portion of the human CD8 α chain (e.g., NP_001139345 and its isoforms). In another specific embodiment, the transmembrane domain comprises a portion of the human CD8 α chain.In some embodiments, the CARs disclosed herein may comprise an extracellular ligand-binding domain that specifically binds to BCMA, a CD8α human stalk and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. In some embodiments, the CAR may be introduced into immune cells as a transgene via a vector (e.g., a plasmid vector). In some embodiments, the vector, e.g., a plasmid vector, may also contain a selection marker, e.g., that provides for identification and / or selection of cells that have received the vector.

[0260] The CAR and / or CD70 binding protein polypeptide can be synthesized in situ within the cell after introducing a polynucleotide encoding the CAR and / or CD70 binding protein polypeptide into the cell. Alternatively, the CAR and / or CD70 binding protein polypeptide can be produced outside the cell and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods 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, viral-mediated methods can be used. The polynucleotide can be introduced into the cell by any suitable means, such as, for example, a recombinant viral vector (e.g., a retrovirus (e.g., a lentivirus), an adenovirus), a liposome, or the like. Transient transformation methods include, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide can be contained in a vector, such as, for example, a plasmid vector or a viral vector.

[0261] Also provided herein are immune cells, e.g., T cells, such as isolated T cells obtained according to any one of the methods described herein. Any immune cell capable of expressing heterologous DNA may be used, for example, to express an antigen binding protein, such as a CAR of interest, and / or a CD70 binding protein, and further, to express low levels of the antigen binding protein, such as a CD70 binding protein. · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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.

[0262] In some embodiments, the immune cells are T cells. In some embodiments, the immune cells can be derived from, for example, but not limited to, stem cells. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly, 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. Exemplary human cells are CD34+ cells. The isolated 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, the cells can originate from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In another embodiment, the engineered cells can be derived from a population of T lymphocytes containing CD4+ T lymphocytes and CD8+ T lymphocytes.

[0263] In some embodiments, immune cells, e.g., T cells, such as isolated T cells, are isolated from, e.g., T cells, using methods described herein (e.g., nucleotide sequences encoding the following gene loci: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 further modified, e.g., genetically engineered, by known gene editing techniques, e.g., employing TALEN, CRISPR / Cas9, or megaTAL nucleases, to partially or completely delete or disrupt (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, The immune cells express the corresponding protein at a lower level compared to comparable cells that have not been engineered to express the corresponding protein at a lower or altered level. In another embodiment, the immune cells are further modified, for example, genetically engineered, by a method described herein (such as known gene editing techniques employing TALEN, CRISPR / Cas9, or megaTAL nuclease to partially or completely delete or disrupt 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.

[0264] In one other aspect, the present disclosure provides modified or engineered immune cells comprising one or more genomic modifications that functionally impair or reduce expression of one or more targets described herein. 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 described herein) or at another location within the genome that is not at the location of the one or more genes (corresponding to one or more targets described herein), such that the modifications functionally impair or reduce expression of the one or more genes (corresponding to one or more targets described herein).

[0265] The engineered immune cells provided herein may comprise one or more mimotope sequences that allow for sorting of cells to enrich for a population of engineered cells as described herein, e.g., a population of cells expressing an antigen-binding protein, and / or provide a safety switch mechanism for inactivating the immune cells after the cells have been administered to a patient or recipient, e.g., to limit adverse effects. Such mimotope sequences and their use in cell sorting and as safety switches are known in the art and are described, for example, in US2018 / 0002435, which is incorporated herein by reference in its entirety.

[0266] Prior to expansion and genetic modification, cell sources can be obtained from subjects through a variety of non-limiting methods. Cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. 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 a healthy donor, a subject diagnosed with cancer, or a subject diagnosed with an infectious disease. In some embodiments, cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0267] PBMCs can be used directly for genetic modification with immune cells (such as CARs or TCRs) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0268] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and are 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 positive for granzyme B and perforin.

[0269] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with distinctive cell surface antigens.

[0270] Also provided herein are cell lines obtained from immune cells, e.g., engineered T cells, that have been modified, e.g., transformed or engineered, according to any of the methods described herein. In some embodiments, the engineered immune cells, e.g., T cells engineered according to the present disclosure, encode an antigen binding protein, e.g., a CAR, and / or a CD70 binding protein, e.g., at low levels. · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. In another embodiment, the engineered immune cells are further modified, e.g., genetically modified to express, e.g., functionally express β2m, or are not engineered. In another embodiment, the engineered immune cells further comprise a polynucleotide encoding and / or functionally express a CD70 binding protein, as described herein.

[0271] In certain embodiments, T cells are provided that contain genomic modifications that functionally impair or reduce expression of RFX5 and CD58 compared to T cells that do not contain the genomic modifications, wherein the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or absent expression of CD52, and low or absent expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, or CD8+ T-lymphocytes.

[0272] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce expression of NLRC5 and CD58 compared to T cells that do not contain the genomic modifications, wherein the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or absent expression of CD52, and low or absent expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0273] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce expression of RFX5 and ICAM-1 compared to T cells that do not contain the genomic modifications, in which case the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0274] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce the expression of NLRC5 and ICAM-1 compared to T cells that do not contain the genomic modifications, in which case the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0275] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce expression of CD58 and ICAM-1 compared to T cells that do not contain the genomic modifications, in which case the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0276] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce the expression of RFX5, NLRC5, and CD58 compared to T cells that do not contain the genomic modifications, wherein the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0277] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce the expression of RFX5, NLRC5, and ICAM-1 compared to T cells that do not contain the genomic modifications, wherein the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0278] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce the expression of RFX5, CD58, and ICAM-1 compared to T cells that do not contain the genomic modifications, wherein the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0279] In another embodiment, T cells are provided that contain genomic modifications that functionally impair or reduce the expression of NLRC5, CD58, and ICAM-1 compared to T cells that do not contain the genomic modifications, in which case the T cells also contain a CAR capable of specifically binding to a tumor antigen, optionally a CD70-specific CAR, optionally low or no expression of CD52, and low or no expression of an endogenous TCR. The T cells can be allogeneic to the subject being treated. The T cells can be derived from a healthy donor. The T cells can be derived from the group consisting of PBMCs, CD4+ T-lymphocytes, and CD8+ T-lymphocytes.

[0280] Methods Involving Engineered Immune Cells Immune cells, e.g., T cells, of the present disclosure can be prepared using methods described in, for example, but not limited to, U.S. Patent Nos. 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, 7,172,8 The cells can be activated and expanded before or after modification using methods generally described in US Patent Application Publication No. 20060121005, US Patent No. 69, US Patent No. 7,232,566, US Patent No. 7,175,843, US Patent No. 5,883,223, US Patent No. 6,905,874, US Patent No. 6,797,514, US Patent No. 6,867,041, and US Patent Application Publication No. 20060121005. Immune cells, such as T cells, can be expanded in vitro or in vivo. Generally, the immune cells of the present disclosure can be expanded by contacting with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the immune cells, for example, to generate an activation signal for the cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to generate activation signals for immune cells, e.g., T cells.

[0281] In some embodiments, a population of T cells may be stimulated in vitro, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Co-stimulation of accessory molecules on the surface of T cells is achieved using a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium, RPMI Medium 1640, or X-VIVO™ 5 (Lonza)) that can contain factors necessary for growth and survival, including serum (e.g., fetal bovine 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 of skill in the art. Other additives for cell growth include, but are not limited to, detergents, Plasmanate®, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The culture medium can include RPMI 1640 (described herein), AIM V, DMEM, MEM, α-MEM, F-12, X-VIVO™ 10, X-VIVO™ 15, and X-VIVO™ 20, OpTmizer™, with additional amino acids, sodium pyruvate, and vitamins. It can be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines sufficient for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). Immune cells, e.g., T cells, exposed to various stimulation times can exhibit different characteristics.

[0282] In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0283] Compositions and populations comprising engineered immune cells In another aspect, the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising any of the cells of the present disclosure. In some embodiments, the composition comprises a T cell comprising a polynucleotide encoding an antigen binding protein, e.g., a CAR, and / or a CD70 binding protein. The cell may be a T cell that expresses a low level of · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, which have been further engineered to express lower levels compared to comparable cells that have not been engineered to functionally express lower or altered levels of the corresponding proteins, or a population of cells comprising engineered immune cells, e.g., T cells of the present disclosure, and further engineered to include one or more pharmaceutically acceptable carriers or excipients. In another embodiment, the cells are not further engineered to express low levels of β2m. In another embodiment, the engineered immune cells further comprise a polynucleotide encoding a CD70 binding protein, as described herein, and / or functionally express a CD70 binding protein.

[0284] In one other embodiment, an engineered cell, such as an engineered immune cell, e.g., a CAR T cell, described herein, comprises one or more genomic modifications that functionally impair or reduce expression of one or more targets described herein. In one embodiment, the one or more genomic modifications are at the genomic location of one or more genes or at another location within the genome but not at the location of one or more genes, such that the modifications functionally impair or reduce expression of one or more genes.

[0285] In some embodiments, primary cells isolated from a donor are manipulated as described herein to provide a cell population in which a subpopulation of the resulting cells (e.g., a proportion less than 100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) contains all of the desired modifications. Such a resulting population, including a mixture of cells that contain and do not contain all of the modifications, can be used in the therapeutic methods of the disclosure and to prepare the compositions of the disclosure. Alternatively, this cell population (hereinafter the "starting population") can be enriched for cells that contain one or more of the desired modifications (e.g., enriched for cells that express a desired antigen binding protein, and / or · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, at lower levels relative to comparable cells that have not been engineered, can be engineered by known methods, e.g., sorting and / or amplifying cells having the desired modifications, to provide a population of cells that contains a higher percentage of such modified or engineered cells than the starting population. The enriched cell population contains a higher proportion of such modified or engineered cells than the starting population. In another embodiment, the enriched population does not contain cells that express β2m at low levels. In another embodiment, the enriched population contains one or more engineered immune cells that further comprise a polynucleotide encoding a CD70 binding protein and / or that functionally express a CD70 binding protein, as described herein.

[0286] The enriched population of modified cells can then be used in the therapeutic methods of the present disclosure, and, for example, to prepare the compositions of the present disclosure. In some embodiments, the enriched cell population contains, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or at least that percentage, of cells having one or more of the modifications. In other embodiments, the percentage of cells in the enriched cell population that contain one or more of the modifications is at least 30% higher than the percentage of cells in the starting population of cells that contain the desired modifications.

[0287] Treatment method In one aspect, the present disclosure provides a method of avoiding an immune response in a subject during the course of immune cell therapy. In one embodiment, the immune cell therapy comprises administering allogeneic immune cells to a subject, wherein the allogeneic immune cells are engineered immune cells. In another embodiment, the engineered immune cells are engineered to express low levels of any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; only one of CD48, CD58, and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; 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; (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 (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, In this case, the engineered immune cells (i) are less susceptible to rejection mediated by the subject's alloreactive immune response, (ii) are less susceptible to rejection mediated by the subject's CD8+ T cell and / or CD4+ T cell alloreactive immune response, 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 cells are further characterized as having in vivo CAR T efficacy simultaneously with one or more of (i), (ii), and (iii). In another embodiment, the engineered immune cells further comprise a polynucleotide encoding a CD70 binding protein described herein and / or functionally express a CD70 binding protein.

[0288] 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 in a subject or can be used as a pharmaceutical. In some embodiments, such methods and / or pharmaceuticals can be used to treat a condition or disorder, such as, for example, a viral disease, a bacterial disease, cancer, an inflammatory disease, an immune disorder, or an age-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 gland 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 human subject with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian cancer, sarcoma, or relapsed or refractory classical Hodgkin lymphoma (cHL).

[0289] In some embodiments, immune cells, e.g., T cells, or cell lines derived from immune cells, e.g., engineered T cells, according to the present disclosure may be used in the manufacture of a medicament for the treatment of a condition or disorder in a subject in need thereof. In some embodiments, the condition or disorder may be, for example, cancer, an autoimmune disorder, or an infectious disease.

[0290] Also provided herein are methods for treating a subject. In some embodiments, the methods comprise administering or providing immune cells, e.g., engineered T cells, of the present disclosure to a subject in need thereof. In some embodiments, the methods comprise administering immune cells, e.g., T cells, of the present disclosure to a subject in need thereof.

[0291] In some embodiments, the immune cells, e.g., engineered T cells, of the present disclosure can undergo stable in vivo cell expansion and can persist for long periods of time. The therapeutic methods of the present disclosure can be ameliorative, curative, or preventative. The methods of the present disclosure can be part of either autoimmune therapy or allogeneic immunotherapeutic treatment. The present disclosure is particularly suitable for allogeneic immunotherapy. Donated immune cells, e.g., engineered T cells, can be transformed into non-alloreactive cells using standard protocols and regenerated as needed, thereby producing CAR-T cells that can be administered to, for example, one or more subjects. Such CAR-T cell therapy can be made available as an allogeneic ALLO CAR T™ therapeutic product.

[0292] In another aspect, the present disclosure provides a method of inhibiting tumor growth or progression in a subject having a tumor, the method comprising administering to the subject an effective amount of engineered immune cells, e.g., the engineered T cells described herein. In another aspect, 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 thereof an effective amount of engineered immune cells, e.g., the engineered T cells described herein. In another aspect, the present disclosure provides a method of inducing tumor regression in a subject having a tumor, the method comprising administering to the subject an effective amount of engineered immune cells, e.g., the engineered T cells described herein.

[0293] In some embodiments, the immune cells provided herein, e.g., T cells, can be administered parenterally to a subject. In some embodiments, the subject is a human.

[0294] In some embodiments, the method can further include administering an effective amount of a second therapeutic agent, such as crizotinib, palbociclib, an anti-CTLA4 antibody, an anti-4-1 BB antibody, a PD-1 antibody, or a PD-L1 antibody.

[0295] Also provided is the use of any of the immune cells, e.g., T cells, provided herein in the manufacture of a medicament for treating cancer or inhibiting tumor growth or progression in a subject in need thereof.

[0296] In certain embodiments, · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, Or the functional expression level of any other gene that is knocked down or knocked out in accordance with the present disclosure is reduced by about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to the corresponding expression level in an equivalent but not so genetically modified engineered immune cell. In another embodiment, the functional expression level of β2m in the engineered immune cells of the present disclosure is not reduced, hi 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.

[0297] The expression level may be determined by any known method, such as FACS or MAC. In some embodiments, the engineered immune cells disclosed herein are · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, Alternatively, any gene knocked down or knocked out according to the present disclosure is functionally expressed at no more than 75%, no more than 50%, no more than 25%, no more than 10%, or no more than 0% of the expression level in 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 compared to the expression level of β2m in non-engineered immune cells that are 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 further comprise a polynucleotide encoding a CD70 binding protein and / or functionally express a CD70 binding protein, as described herein.

[0298] In some embodiments, both alleles of a gene are knocked out, resulting in an expression level of the gene in an engineered immune cell disclosed herein that is 0% of that in a corresponding unengineered cell. In some embodiments, one of the two alleles of a gene is knocked out, resulting in an expression level of the gene in an engineered immune cell disclosed herein that is 50% or about 50% of that in a corresponding unengineered cell (e.g., where a compensatory mechanism results in greater than normal expression of the remaining allele). As described herein, for example, intermediate expression levels may be observed when expression is reduced by some means other than knockout.

[0299] In some embodiments, the expression levels of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA, and RFXANK, or any other gene whose expression level is engineered according to the present disclosure, in engineered cells can be measured directly by assaying the cells for gene products and their properties using standard techniques known to those of skill in the art (e.g., RT-qPCR, nucleic acid sequencing, antibody staining, or any combination of techniques). In some embodiments, the level of functional expression 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 protein or HLA-B protein, or the surface expression level of beta-2 microglobulin (B2M), or the surface expression level of both B2M and one or more HLA proteins, on the surface of the engineered immune cells by standard techniques known in the art, such as flow cytometry. In various embodiments of the present disclosure, the level of functional expression of any one or more of CD48, CD58, and ICAM-1 is measured by, for example, determining the surface expression level of each cell surface protein, such as one or more of CD48, CD58, or ICAM-1 proteins, on the surface of engineered immune cells, or by flow cytometry. These measurements can be compared to corresponding measurements made on comparable cells that have not been engineered to reduce the corresponding functional expression level. In a cell population containing engineered cells, e.g., engineered immune cells of the present invention, a pooled sample of material (e.g., RNA or protein or cells) measured will reflect the fact that some cells do not express the gene of interest and have both alleles knocked out, e.g., some cells express the gene of interest at or about 50%; if only one allele has been knocked out and the population includes unengineered cells, some cells express normal levels of the gene of interest.

[0300] The functional expression level of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK in engineered immune cells of the present disclosure can also be analyzed 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 non-engineered but otherwise comparable, e.g., identical, immune cells survive under the same conditions (see, e.g., Figures 1D-1E and Example 1).

[0301] In some embodiments, administering engineered immune cells, e.g., engineered T cells as disclosed herein, or a cell population comprising such engineered immune cells, e.g., engineered T cells, results in a reduced host rejection of the administered cells or cell population compared to comparable, but unengineered, cells or a comparable population not comprising such engineered cells. In some embodiments, administering engineered immune cells, e.g., engineered T cells of the present disclosure, comprising an antigen binding protein, e.g., a CAR, and / or a CD70 binding protein, in which · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 the expression levels, such as the functional expression levels, of (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, or Administering a cell population comprising such engineered immune cells, e.g., engineered T cells, reduces the host's rejection of the administered cells or cell population compared 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 described herein and / or functionally express a CD70-binding protein.

[0302] For example, such administration reduces host rejection by 1% to 99%, e.g., 5% to 95%, 10% to 90%, 50% to 90%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to host rejection of identical cells but not engineered to express the corresponding protein at low levels. In some embodiments, host rejection is reduced by more than 90%.

[0303] In some embodiments, administering engineered immune cells, such as T cells of the present disclosure, comprising an antigen binding protein, e.g., a CAR, and / or a CD70 binding protein, wherein: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 the levels of functional expression of (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 are reduced, or administering a cell population comprising immune cells, e.g., T cells, whereby the administration improves or enhances and / or increases the persistence of the cells compared to the persistence of identical cells that have not been engineered to express a low level 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 described herein and / or functionally express a CD70 binding protein.

[0304] In some embodiments, persistence is increased by, e.g., 1-7 days, 1-12 weeks (e.g., 1-4 weeks, 4-8 weeks, or 8-12 weeks), or 1-12 months, or a specific length of time within these ranges. In some embodiments, the difference in persistence is measured by comparing the half-life of the administered cells in the population or composition, e.g., the half-life is increased by, e.g., 1-7 days, 1-12 weeks (e.g., 1-4 weeks, 4-8 weeks, or 8-12 weeks), or 1-12 months, or a specific length of time within these ranges. In some embodiments, the difference in persistence is measured by comparing the length of time the administered cells can be detected after administration. In some embodiments, the improvement in persistence is measured in vitro, e.g., by comparing the survival of engineered and unengineered cells in the presence of immune cells, such as T cells or NK cells, e.g., at about 72 hours, 5 days, 7 days, or 13 days after mixing. In some embodiments, the engineered cells survive about 1.5-10 times longer than non-engineered cells when measured in such in vitro assays. In some embodiments, the engineered immune cells survive about 1.5-10 times longer than non-engineered immune cells when measured in such in vitro assays. The extent of improved 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 co-incubated (e.g., "attacking" or host) immune cells.

[0305] In some embodiments, the reduced host rejection and / or increased persistence of the administered cells disclosed herein is determined by any of a variety of techniques known to those skilled in the art. In some embodiments, any one or a combination of flow cytometry, PCR (e.g., quantitative PCR), and ex vivo co-incubation with patient tumor material or with a model tumor cell line expressing the antigen targeted by the CAR-T cells is used. In some embodiments, qPCR is used to assess the number of CAR T cells with and without a knockout of interest to determine the extent to which the knockout provides a survival advantage.

[0306] In some embodiments, the treatment may be in combination with one or more therapies for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0307] In some embodiments, the treatment may be administered to a subject undergoing immunosuppressive therapy. Indeed, the present disclosure may rely on a cell or cell population that has been made resistant to at least one immunosuppressant due to inactivation of a gene encoding a receptor for such an immunosuppressant. In this aspect, the immunosuppressive therapy may assist in the selection and expansion of T cells according to the present disclosure in the subject.

[0308] Administration of cells or cell populations according to the present disclosure can be by any convenient method, including aerosol inhalation, injection, ingestion, infusion, injection, implantation, or transplantation. The compositions described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously or intralymphatically, or intraperitoneally. In one embodiment, the cell composition of the present disclosure is administered by intravenous injection.

[0309] In some embodiments, administration of cells or cell populations according to the present disclosure may be at a dose of, for example, about 10 per kg of body weight. 3 Or 10 4 pieces ~ about 10 9 In some embodiments, the administration of cells or cell populations is at least about 10 per kg of body weight, including all integer values within those ranges. 5 ~about 10 6 cells (including all integer cell numbers within this range), or 0.1 x 10 engineered immune cells of the invention per kg of body weight 6 ~5×10 6 or 0.1 × 10 total engineered immune cells 8 ~5×10 8 The administration of the cells or cell population may include administration of one or more doses. In some embodiments, an effective amount of cells may be administered as a single dose. In some embodiments, an effective amount of cells may be administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the subject. The cells or cell population may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount refers to an amount that provides a therapeutic or prophylactic benefit. The dosage administered depends on the age, health, and weight of the recipient, the type of concomitant 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 comprising the cells is administered parenterally. In some embodiments, administration may be intravenous. In some embodiments, administration may be by injection directly into the tumor.

[0310] In some embodiments of the present disclosure, the cells are administered to a subject in conjunction with (e.g., before, concurrently with, or after) any number of relevant 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 subjects with MS, or efaliztimab treatment for subjects with psoriasis, or other treatments for subjects with PML. In some embodiments, the BCMA-specific CAR-T cells are administered to the subject in combination with one or more of an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody (e.g., avelumab, atezolizumab, or durvalumab), an anti-OX40 antibody, an anti-4-1 BB antibody (e.g., utomirumab), an anti-MCSF antibody, an anti-GITR antibody, and / or an anti-TIGIT antibody. In further embodiments, the immune cells, e.g., T cells, of the present disclosure may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH (alemtuzumab), anti-CD3 antibodies, or other antibody therapies, cytoxan, fludarabine, cyclophosphamide, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and / or irradiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important in growth factor-induced signal transduction (rapamycin) (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 Oct;5(5):763-73).In one embodiment, immune cells, e.g., T cells, of the present disclosure can be administered to a subject previously treated with an immunosuppressant that lymphodepletes the subject, thereby allowing immune cell engraftment. However, over time, the subject's immune system reconstitutes and recovers 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, Issue 15_suppl 2021). Thus, engineered immune cells of the present disclosure provide additional immune protection to the subject at various points during the treatment regimen. In one embodiment, the engineered immune cells coexist with and actively participate in the subject's immune system, avoiding rejection.

[0311] In further embodiments, the cell compositions of the present disclosure are administered to a subject in conjunction with (e.g., before, concurrently with, or after) T-cell ablative therapy using bone marrow transplantation, chemotherapy 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 ablative therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, the subject can receive standard treatment using high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, 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.

[0312] kit The present disclosure also provides kits for use in the methods. The kits of the present disclosure include one or more containers containing a composition of the present disclosure, or immune cells of the present disclosure, e.g., T cells, or a cell population comprising immune cells of the present disclosure, e.g., engineered T cells. In various embodiments, the immune cells (e.g., T cells) contain one or more polynucleotides encoding an antigen binding protein, such as a CAR described herein, and are further engineered to express low levels of one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP, CIITA, and RFXANK, as described herein. In another embodiment, the immune cells are engineered to express low levels of a CAR, as described herein. · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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. In another embodiment, the immune cells are not further engineered to express low levels of β2m.

[0313] The kit further comprises instructions for use with any of the disclosed methods described herein. Generally, these instructions include instructions for administering the compositions, immune cells, e.g., T cells, or cell populations for the therapeutic treatment described above.

[0314] The instructions for use of the kit components generally include information regarding dosages, administration schedules, and routes of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions supplied with kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0315] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices such as minipumps, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an immune cell, e.g., T cell, according to the present disclosure. The container may further contain a second pharmaceutically active agent.

[0316] Kits can optionally be provided with additional components, such as buffers and interpretive information. Typically, kits include a container and a label or package insert on or associated with the container.

[0317] Sorting and depletion methods In some embodiments, a method of sorting a population of immune cells in vitro is provided, wherein a subset of the population of immune cells is selected from: · any one or more of CD48, CD58, ICAM-1, TAP2, NLRC5, β2m, TRAC, CIITA, RFX5, RFXAP, and RFXANK; ·Only one of CD48, CD58 and ICAM-1; (i) one or both of NLRC5 and RFX5, and (ii) one or more of CD58, CD48, and ICAM-1; · 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; (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 (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, and / or immune cells engineered as described herein to express an antigen-binding protein, such as a CAR.

[0318] In various embodiments, the method comprises contacting a population of immune cells with a monoclonal antibody specific for an epitope (e.g., a mimotope such as those described in US2018 / 0002435) unique to the engineered cells (e.g., an epitope of an antigen binding protein, or a mimotope incorporated into the antigen binding protein), and selecting immune cells that bind the monoclonal antibody to obtain a cell population enriched for engineered immune cells that express the antigen binding protein.

[0319] In some embodiments, the epitope-specific monoclonal antibody is optionally conjugated to a fluorophore, in which case the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0320] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a magnetic particle, in which case the step of selecting cells that bind to the monoclonal antibody can be performed by magnetic-activated cell sorting (MACS).

[0321] In some embodiments, the mAb used in the method for sorting immune cells expressing an antigen binding protein (such as a CAR) is alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab, The mAb is selected from vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab. In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10.

[0322] In some embodiments, the population of CAR-expressing immune cells obtained when using the methods for in vitro sorting of CAR-expressing immune cells described above comprises at least 70%, 75%, 80%, 85%, 90%, 95% CAR-expressing immune cells. In some embodiments, the population of CAR-expressing immune cells obtained when using the methods for in vitro sorting of CAR-expressing immune cells comprises at least 85% CAR-expressing immune cells.

[0323] In some embodiments, the population of CAR-expressing immune cells obtained when using the above-described in vitro sorting method for CAR-expressing immune cells exhibits increased in vitro cytotoxic activity compared to the initial (unsorted) cell population. In some embodiments, said cytotoxic activity is increased by 10%, 20%, 30%, or 50% in vitro. In some embodiments, the immune cells are T cells.

[0324] The CAR-expressing immune cells administered to the recipient can be enriched in vitro from a source population. Methods for expanding the source population can include selecting cells that express an antigen, such as the CD34 antigen, using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[0325] Flow cytometry can be used to quantify specific cell types in a cell population.Generally, flow cytometry is a method for quantifying cellular components or structural features mainly by optical means.By quantifying structural features, different cell types can be distinguished, so that flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0326] Flow cytometry analysis involves two major steps: 1) labeling a selected cell type with one or more markers, and 2) determining the number of labeled cells relative to the total number of cells in a population. In some embodiments, the method of labeling a cell type involves binding a labeled antibody to a marker expressed by a particular cell type. The antibody can be directly labeled with a fluorescent compound or indirectly labeled, for example, using a fluorescently labeled secondary antibody that recognizes the first antibody.

[0327] In some embodiments, the method used to sort CAR-expressing T cells is magnetically activated cell sorting (MACS). Magnetically activated cell sorting (MACS) is a method for separating various cell populations according to their surface antigens (CD molecules) by using superparamagnetic nanoparticles and columns. Using MACS, pure cell populations can be obtained. Cells in single-cell suspension can be magnetically labeled with microbeads. The sample is applied to a column consisting of ferromagnetic spheres, covered with a cell-friendly coating, allowing for rapid and gentle cell separation. Unlabeled cells pass through, while magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0328] Detailed protocols for the purification of 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 populations by fluorescence activated cell sorting (FACS). J Vis Exp. (41): 1546). [Table 1] TIFF2025525779000002.tif203157 TIFF2025525779000003.tif187151 TIFF2025525779000004.tif197150 TIFF2025525779000005.tif187149 TIFF2025525779000006.tif171149 TIFF2025525779000007.tif130160 TIFF2025525779000008.tif218163 [Example]

[0329] Example 1. Protective effect of CD58 knockout (KO) + RFX5 or NLRC5 KO in non-CAR T cells. The protective effect of CD58 KO against host rejection, either to enhance survival in the case of a single modification (single KO) or to provide an additional survival benefit with RFX5 KO or NLRC5 KO, was tested using primed T and natural killer (NK) mixed lymphocyte reaction (MLR) assays.

[0330] PBMCs from healthy human donors expressing HLA-A2 (HLA-A2+) were used as allogeneic effector (host) cells. PBMCs from healthy donors not expressing HLA-A2 (HLA-A2-) were used to generate target cells (grafts). CRISPR / Cas9-mediated gene editing was performed to generate transplanted T cells with the following knockouts: TRAC KO, TRAC / CD58 KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / NLRC5 KO, TRAC / β2m / CD58 KO, TRAC / RFX5 / CD58 KO, and TRAC / NLRC5 / CD58 KO. After 1 week of in vitro expansion, target cells were purified to remove all TCR α / β-expressing cells. The level of specific gene knockout or MHC-I and MHC-II knockdown was assessed via FACS.

[0331] Alloreactive T cells are thought to be the primary mediators of allogeneic rejection. Therefore, we performed a primed alloreactive T cell mixed lymphocyte reaction (MLR) using gene-edited cells as target cells to evaluate the protective effect of various knockouts on the survival of primed alloreactive T cells. To generate primed effector T cells, allogeneic PBMCs from HLA-A2+ donors were co-cultured with irradiated PBMCs or pan T cells isolated from the transplant donor for one week to promote the derivation and expansion of alloreactive T cells. Effector pan T cells were then purified and co-incubated with the gene-edited transplanted T cells at a 1:1 ratio for two days in R10 + 20 IU / mL IL-2. The extent of killing was determined by analyzing the absolute number of surviving engrafted T cells by gating on live HLA-A2-TCRαβ- CD4+ CD8+ cells with the desired gene edit (e.g., CD58 KO) (Figure 1E).

[0332] As shown in Figure 1E , CD58 KO enhanced the survival of RFX5 KO or NLRC5 KO cells compared with RFX5 KO alone or NLRC5 KO alone.

[0333] For the NK MLR assay, human NK cells were isolated from freshly isolated HLA-A2+ human PBMCs by MACS purification (Miltenyi human pan NK cell isolation kit, catalog number 130-092-657). 20,000 transplanted T cells were seeded with 20,000 or 100,000 host NK cells in a 96-well plate and cultured in R10 + 1000 IU / mL IL-2 for 2 days. Absolute counts were used to determine the viability of transplanted T cells by flow cytometry, gating on live HLA-A2-TCRαβ-CD56-CD4+CD8+ cells with the desired gene edit (e.g., CD58 KO).

[0334] As shown in Figure 1E, CD58 KO enhanced the survival of RFX5 KO and NLRC5 KO cells compared with RFX5 KO or NLRC5 KO single cells. CD58 KO did not affect the survival of transplanted cells in the NK MLR. Similar trends were observed for both effector:target cell ratios (E:T) of 1:1 and E:T = 5:1.

[0335] Example 2. Protective effect of CD48 KO or ICAM-1 KO + RFX5 KO in non-CAR T cell transplantation Pan T cells from an HLA-A2 donor were used to generate target cells. CRISPR / Cas9-mediated gene editing was performed to generate target cells with the following knockouts: TRAC KO, TRAC / RFX5 KO, TRAC / CD48 KO, TRAC / ICAM-1 KO, TRAC / RFX5 / CD48 KO, and TRAC / RFX5 / ICAM-1 KO. After 2 weeks of in vitro expansion, targets were purified to remove all TCR α / β-expressing cells. The level of specific gene knockout or MHC-I and MHC-II knockdown was assessed via FACS.

[0336] As shown in Figure 2, high gene editing efficiency was observed for all groups (based on the average of two human donors). At day 13 after generation, over 80% of cells had the desired gene edits. In this test setup, RFX5 KO was used, but NLRC5 KO or β2m KO were not used.

[0337] The protective effect of ICAM-1 or CD48 KO on enhancing the survival of RFX5 KO against allogeneic T cell rejection was tested using a primed T cell MLR assay as described in Example 1. As shown in Figure 3, ICAM-1 KO could slightly enhance 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 transplants (TRAC KO). The survival of CD48 KO was similar to that of control transplants.

[0338] The protective effect of ICAM-1 KO to further enhance the survival of RFX5 KO cells against host PBMC rejection compared to CD48 KO was tested using a PBMC MLR assay. The survival of gene-edited transplanted cells in the presence of allogeneic PBMC donors (n=3) was evaluated. The effector:target (E:T) ratio used was 10:1. Transplant cell survival was quantified by FACS after 9 days of coculture. As shown in Figure 4, ICAM-1 KO further enhanced the survival of RFX5 KO cells compared to CD48 KO cells, as demonstrated by the PBMC MLR assay.

[0339] Example 3. Protective effect of CD58 KO or ICAM-1 KO + RFX5 KO using CAR T cell transfer The protective effect of CD58 KO against T cell rejection, either to enhance CAR T cell survival with a single modification (single KO) or to provide the additional survival benefit of RFX5 KO or NLRC5 KO, was tested using a primed TMLR assay. CD19 CAR T cells (CD19 FMC63) expressing a turbodomain constitutively active chimeric cytokine receptor (CACCR) containing TpoR domain variants (478-582; H499L; S505N; W515K) and intracellular IL2Rb domains (339-379, 393-433, 518-551) (see Lin et al., US 2021-0260118 A1, incorporated herein by reference in its entirety), were engineered from healthy donor cells. These CAR T cells are hereafter referred to as CD19 CAR T cells. Expression of the CAR and turbodomain was under the control of either the EF1alpha promoter or the PGK promoter, as described below.

[0340] CD19 CAR T cells were engineered to introduce one or more knockouts and then used as target cells. Specifically, CD19 CAR T cells underwent CRISPR / Cas9-mediated gene editing 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.

[0341] For the experimental data presented in Figures 5A-J, CD19 CAR T cells were engineered to carry the EF1alpha promoter to control the expression of the CAR and turbodomain.

[0342] A primed T cell MLR assay was performed to test the efficacy of gene-edited CD19 CAR T cells (target cells) against allogeneic T cell rejection. Data were pooled from four transplant / host-donor pairs: one transplant and four hosts. Cells were cultured at a 1:1 effector:target (E:T) ratio in the presence of 20 IU / mL IL-2 for two days. In some cases, CAR T cells were activated using GFP-expressing β2m KO Raji tumor cells. Survival of transplanted CAR T cells was assessed by FACS, gating on live GFP-HLA-A2-TCRαβ-CD4+CD8+CAR+ cells with the desired gene edit (e.g., CD58 KO).

[0343] As shown in Figure 5A, β2m single KO, CD58 single KO, NLRC5 single KO, and RFX5 single KO all enhanced the survival of transplanted cells compared to control CAR T transplants (TRAC KO). Furthermore, when CD19 CAR T cells were not activated (no Raji tumor cells), CD58 KO further enhanced survival compared to NLRC5 single KO or RFX5 single KO CAR T cells. Activated CD19 CAR T cells (in the presence of Raji tumor cells) survived better than non-activated CAR T cells (no Raji tumor cells). All cloaking KO transplanted cells, e.g., CD58 KO, survived relatively equally, and all survived significantly better than control transplants (TRAC KO).

[0344] The protective effect of CD58 KO or ICAM-1 KO against T cell rejection to enhance CAR T cell survival in the case of a single modification (single KO) or to provide an additional survival benefit of RFX5 KO was tested using a primed T MLR assay.

[0345] CD19 CAR T cells were gene-edited to obtain the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / CD58 KO, TRAC / ICAM-1 KO, TRAC / β2m / CD58 KO, TRAC / β2m / ICAM-1 KO, TRAC / RFX5 / CD58 KO, and TRAC / RFX5 / ICAM-1 KO.

[0346] A primed T MLR assay was performed to test the efficacy of gene-edited CD19 CAR T cells against allogeneic T cell rejection. Data shown are the average of technical triplicates for one representative transplant / host pair from three unique pairs. Cells were cultured at a 1:1 effector:target ratio in the presence of 20 IU / mL IL-2 for two days. CAR T cells were activated using GFP-expressing β2m KO Raji tumor cells. Survival of transplanted CAR T cells was assessed by FACS, gating on live GFP-HLA-A2-TCRαβ-CD4+CD8+CAR+ cells with the desired gene edit (e.g., CD58 KO).

[0347] As shown in Figure 5B, β2m single KO, RFX5 single KO, CD58 single KO, and ICAM-1 single KO all enhanced the survival of transplanted CAR T cells compared to the control (TRAC KO). CD58 KO provided a small additional benefit to the survival of β2m KO, but ICAM-1 KO did not. Both CD58 KO and ICAM-1 KO provided a substantial additional benefit to the survival of RFX5 KO CAR T cells compared to RFX5 single KO transplants.

[0348] CD19 CAR T cells (expressing turbodomains as described above) were engineered to incorporate various knockout combinations of B2M, RFX5, CD58, and NLRC5, and then used as transplant cells in primed T MLR assays, NK cell MLR assays, and PBMC MLR assays. Specifically, CD19 CAR T cells were subjected to CRISPR / Cas9-mediated gene editing to provide the following knockouts: TRAC KO, TRAC / CD58 KO, TRAC / ICAM-1 KO, TRAC / β2m KO, TRAC / β2m / CD58 KO, TRAC / β2m / ICAM-1 KO, TRAC / RFX5 KO, TRAC / RFX5 / CD58 KO, and TRAC / RFX5 / ICAM-1 KO.

[0349] For the primed T MLR assay, a 1:1:0.25 ratio of host:graft:Raji tumor cells (WT Raji cells with B2M knockout) was used. Primed T cells were enriched 2-fold using the Miltenyi Pan T Isolation Kit. Readout was performed after 2 days. As shown in Figure 5C, CD58 KO and ICAM-1 KO, either as single modifications or in combination with RFX5 knockout, improved CAR T cell survival in the T cell MLR assay (each point represents one unique allogeneic host / graft pair). The combination of RFX5, CD58, and ICAM-1 was found to significantly reduce T cell rejection compared to RFX5 knockout alone. Furthermore, double knockout of CD58 / RFX5 was found to specifically reduce T cell rejection to nearly the same extent as B2M knockout (KO) cells alone.

[0350] For the primed NK MLR assay, a ratio of NK cells:graft:Raji tumor cells (WT Raji cells) was used at a ratio of 10:1:0.25. Frozen NK cells were thawed and used. Readout was performed 2 days later (1,000 IU / ml IL-2). As shown in Figure 5D, B2M knockout cells were found to be susceptible to NK cell killing, as expected (each dot represents one graft / NK pair). Knockout of CD58 and ICAM-1, either alone or in combination with other knockout targets, i.e., B2M or RFX5, did not exhibit any additional NK cell rejection compared to B2M knockout. These results suggest that double knockout of RFX / CD58 or ICAM-1 may potentially attenuate both T cell and NK cell rejection, potentially reducing T cell rejection to the same extent as B2M KO, without inducing the same degree of NK cell rejection.

[0351] These assays analyzed the survival of transplanted cells after coculture with primed T cells or primed NK cells. Knockout of CD58 and ICAM-1, both as single knockouts and in an RFX5 KO background, was found to reduce T cell-mediated rejection. In the NK MLR, knockout of CD58 and ICAM-1 was not found to have a detrimental effect on transplanted cell survival (Figure 5C-D).

[0352] For the PBMC MLR assay, a 10:1 ratio of host:transplant cells was used for 10 days of coculture. PBMCs from three different donors were B cell depleted prior to coculture. Readout was performed after 10 days. As shown in Figure 5E, CD58 knockout and ICAM-1 knockout, either as single modifications or in combination with RFX5 knockout, improved the survival of transplanted cells (each point represents one unique allogeneic host / transplant pair—transplant, three host-donors). In Figure 5F, adding CD58 knockout together with either B2M knockout or RFX5 knockout was found to reduce host immune cell expansion in two of the three donors (left panel—host CD8+ cells, right panel—host CD4+ cells). In Figure 5G, host NK cells expanded more when cocultured with transplanted cells bearing B2M knockout alone or in combination with other knockouts. Addition of CD58 knockout, alone or in combination with RFX5 knockout, was found to reduce expansion.

[0353] Another PBMC MLR assay using CD19 CAR T cells assessed the effect of various knockouts on various immune cell subsets. PBMCs from three different donors were B cell depleted and then co-cultured. A 10:1 ratio of host:transplant cells was used for 10 days of culture. Readout was performed after 10 days. TRAC / CD58 KO and TRAC / ICAM-1 KO results were measured on separate plates (error bars removed for easier visualization).

[0354] As shown in Figure 5H, host CD8+ cell expansion was reduced when cocultured with transplanted cells containing various knockouts and knockout combinations. For example, adding the RFX5 / CD58 knockout combination reduced expansion compared to the control (FMC63, TRAC KO transplant). As shown in Figure 5I, host CD4+ cell expansion was reduced when cocultured with transplanted cells containing various knockouts and knockout combinations. For example, adding the RFX5 / CD58 knockout combination reduced expansion compared to the control (FMC63, TRAC KO transplant).

[0355] In vitro long-term killing assays can be performed using, for example, Raji WT cells or Raji CD19 low The experiments were performed using CD19 CAR T cells against target cells such as Raji cells at an E:T ratio of 8:1. As shown in Figure 5J, CD19 CAR T cells containing various knockouts exhibited cytotoxic activity against Raji WT target cells. Control CAR T cells with only a TRAC knockout exhibited effective killing compared to many of the other CAR T cells with additional knockouts. Surprisingly, CAR T cells with a TRAC / CD58 knockout produced similar or slightly better killing than the control CAR T cells. Raji CD19 low Similar results were obtained when cells were used as target cells.

[0356] Example 4. Protective effect of CD58 KO or ICAM-1 KO + RFX5 KO against NK cell rejection CD19 CAR T cells (expressing a turbodomain) described in Example 3 were modified to introduce one or more knockouts and then used as target cells. Expression of the CAR and turbodomain was under the control of either the EF1alpha promoter or the PGK promoter, as described below.

[0357] Specifically, CD19 CAR T cells were subjected to CRISPR / Cas9-mediated gene editing to provide the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / CD58 KO, TRAC / ICAM-1 KO, β2m / CD58 KO, β2m / ICAM-1 KO, RFX5 / CD58 KO, and RFX5 / ICAM-1 KO.

[0358] For the experiment shown in Figure 6A, the EF1alpha promoter was used to control the expression of CAR and turbodomains. For the experiment shown in Figure 6B, the PGK promoter was used to control the expression of CAR and turbodomains.

[0359] We used an NK MLR assay to test the survival of gene-edited CD19 CAR T-transplanted cells against allogeneic NK rejection. Data were pooled from three transplant / host-donor pairs: one transplant and three hosts. NK cells isolated from frozen PBMCs were co-cultured with gene-edited CD19 CAR T cells for two days in the presence of 1,000 IU / mL IL-2. CD19 CAR T cells were activated using GFP-expressing Raji tumor cells. The ratio of NK cells:transplant:Raji cells was 10:1:0.25. Survival of transplanted CAR T cells was assessed by FACS and gated as live GFP-HLA-A2-TCRαβ-CD56-CD4+CD8+CAR+ cells with the desired gene edit (e.g., CD58 KO).

[0360] As shown in Figure 6A, β2m single KO resulted in significant NK rejection, resulting in poor survival, whereas RFX5 single KO resulted in some NK rejection, but to a lesser extent. CD58 single KO or ICAM-1 single KO resulted in similar survival rates compared to control grafts (TRAC KO). CD58 KO slightly rescued β2m KO from NK rejection. CD58 KO or ICAM-1 did not significantly rescue RFX5 KO from NK rejection.

[0361] In another NK cell MLR, fresh NK cells were used. The NK cell:transplant:Raji target cell ratio 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 of IL-2 for 2 days. As shown in Figure 6B, CD58 knockout and ICAM-1 knockout did not result in NK cell rejection (each dot represents one transplant / NK pair). Furthermore, CAR T knockout cells were found not to exhibit IL-2-independent proliferation (Figure 6C). [Table 2] Example 4.1 - Cytotoxicity of CAR T Cell Knockouts

[0362] CD19 CAR T cells (expressing a turbo domain) described in Example 3 were modified to introduce one or more knockouts. CD19 CAR T cells were engineered from pan T cells derived from two HLA-A2- donors. Expression of the CAR and turbo domain was under the control of either the EF1alpha promoter or the PGK promoter, as described below.

[0363] Specifically, CD19 CAR T cells underwent CRISPR / Cas9-mediated gene editing to provide the following knockouts: TRAC KO, TRAC / β2m KO, TRAC / RFX5 KO, TRAC / CD58 KO, TRAC / ICAM-1 KO, TRAC / RFX5 / CD58 KO, TRAC / RFX5 / ICAM-1 KO, and TRAC / CD58 / ICAM-1 KO.

[0364] For the experimental data presented in Figures 7-12, CD19 CAR T cells were engineered to carry the PGK promoter to control the expression of the CAR and turbodomain.

[0365] The TRAC knockout was common to all edited cells. After 16 days of in vitro expansion, the gene-edited cells were purified to remove all TCR α / β-expressing cells. The level of specific gene knockout, or MHC-I and MHC-II knockdown, was assessed via flow cytometry.

[0366] As shown in Figure 7, high gene editing efficiency was observed for all groups (based on the average of two human donors). At day 16 after generation, more than 50% of the cells had the desired gene editing. For all of the knockout conditions described above, 1 x 10 6 The cells were edited and then expanded. As shown in Figure 8, expansion of the edited cells was not affected by any of the various knockouts compared to the TRAC knockout control.

[0367] In vitro long-term killing assays can be performed using, for example, Raji WT cells or Raji CD19 low CD19 CAR T cells with various knockouts were used at an E:T ratio of 8:1 against target cells such as Raji cells. As shown in Figure 9, various CAR T cells containing various knockouts exhibited cytotoxic activity against Raji WT target cells. All CAR T knockout cells were found to exhibit comparable or superior cytotoxicity compared to control CAR T cells (TRAC knockout only). CAR T cells were co-cultured with luciferase-GFP-expressing Raji cells at an effector-to-target ratio of 8:1. Half of the cells were passaged onto fresh Raji cells every 2–3 days. The remaining half of the cells were then used to determine target cell killing using Bright-glo reagent (Promega).

[0368] A series of primed T MLR assays were performed using several gene-edited CD19 CAR T cells with various knockouts.Primed host T cells were isolated twice using the Miltenyi Pan T Isolation Kit after 7 days of co-culture with WT Raji cells carrying B2M knockout, and pan T transplant cells were irradiated. T cell host:transplant:Raji (B2M knockout) ratios were used at 1:1:0.25, and readouts were obtained on day 2. As shown in Figure 10, CD58 knockout, either as a single knockout or in combination with other knockouts, was found to effectively alleviate T cell rejection (each dot represents one graft / host pair).

[0369] A PBMC MLR assay was also performed. A 10:1 ratio of host:transplant cells was used for 10 days of culture. PBMCs from the donor were B cell-depleted before coculture. Readouts were performed on days 3, 7, and 9. As shown in Figure 11, CAR T transplant cells with various knockouts demonstrated a survival advantage over control and B2M knockouts at day 7. Knockout combinations (e.g., RFX5 / CD58, RFX5 / ICAM-1, and CD58 / ICAM-1) demonstrated superior survival against rejection compared with single knockouts (e.g., RFX5, CD58, and ICAM-1). CD58 knockout cells demonstrated the best survival among single knockout transplant cells. CD58 knockout combined with RFX5 knockout or ICAM-1 knockout demonstrated the best survival against rejection at day 9 (data not shown). Furthermore, the single CD58 knockout showed the best survival at day 9 compared to the other single knockouts (data not shown).

[0370] Various gene-edited CAR T cells were evaluated for their ability to affect host immune cell expansion. As shown in Figures 12A-12B, CAR T cells were found to reduce the expansion of host CD8+ and CD4+ cells (A). B2M knockout CAR T cells were found to be the least effective at reducing the expansion of host NK cells compared to the other knockout cells (B).

[0371] Furthermore, CD19 CAR T cells were engineered to carry the EF1alpha promoter to control expression of the CAR and turbo domains, and these cells were also tested for cytotoxicity and found to exhibit similar cytotoxicity (data not shown).

[0372] Example 4.2 - CD19 CAR / CD70 binding protein T cells with CD58 knockout Transduction of PBMCs with LVV constructs resulted in engineered cells with randomly integrated transgenes in the host cell genome. Transgenes are introduced into cells by site-specific integration (SSI) within a defined locus, ensuring uniformity of transgene insertion sites within the genome and limiting the number of integration events. For example, site-specific integration with adeno-associated viral vectors (AAV) can also maintain high transduction efficiency while allowing expression of multiple genes.

[0373] Three different constructs were delivered using AAV-mediated SSI to generate three different types of engineered cells for use in these experiments: 1) a CD19 CAR construct, 2) a CD19 CAR / CD70 binding protein construct, and 3) a CD70 CAR construct. For construct 2), the CD70 binding protein (or domain) was derived from the anti-CD70 antibody clone 4F11 in scFv form and the CD3ζ signaling domain (4F11z). In both cases, the TRAC locus was targeted for construct integration. Three different PBMC donors were used. Expression of each construct was driven by the PGK promoter. Further gene editing was performed using CRISPR / Cas9 in constructs 1) and 2) to generate CAR T-transplanted cells with the following specific knockouts: TRAC knockout only, TRAC / B2M knockout, and TRAC / CD58 knockout. After one week of in vitro expansion, the target cells were purified to remove all TCR α / β-expressing cells. The level of specific gene knockout or MHC-I and MHC-II knockdown was assessed by flow cytometry. PBMCs from healthy human donors expressing HLA-A2 (HLA-A2+) were used as allogeneic effector (host) cells, while engineered cells were used as CAR T-transplant cells.

[0374] A PBMC MLR assay was performed. A host:transplant cell ratio of 10:1 was used for 13 days of culture. PBMCs from three different donors were B cell depleted prior to co-culture. Readout was performed on day 13. As shown in Figure 13A, CD58 knockout was found to enhance the survival of CD19 CAR / CD70 binding protein CAR T cells (each symbol represents one of nine unique transplant / host pairs, and the shape of each symbol represents a unique transplant).

[0375] After 9 days in co-culture, the engineered CAR T cells were evaluated for their ability to affect the expansion of host immune cells. As shown in Figure 13B, CD19 CAR / CD70 binding protein CAR T cells were found to reduce or deplete the expansion of host CD8+ T cells. Similar results were observed for host CD4+ T cells. As shown in Figure 13C, CD19 CAR / CD70 binding protein CAR T cells were observed to inhibit the expansion of host NK cells.

[0376] In vitro long-term killing assays were performed using various CAR T knockout cells at an effector:target ratio of 4:1 against target Raji WT cells. As shown in Figure 13D, introduction of CD58 knockout did not reduce the cytotoxic activity of CAR T cells. CAR T cells were cocultured with luciferase-GFP-expressing Raji cells at an effector:target ratio of 8:1. Half of the cells were passaged onto fresh Raji cells every 2–3 days. The remaining half of the cells were then used to determine target cell killing using Bright-glo reagent (Promega).

[0377] Common Protocols Preparation of target cells (refer to 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 Human Pan T Cell Isolation Kit, catalog no. 130-096-535) and activated with 1:100 (v:v) T Cell TransAct (Miltenyi, catalog no. 130-111-160) + 100 IU / mL IL-2 (Miltenyi, catalog no. 130-097-746) in R10 (RPMI-1640 + 10% FBS + 25 mM HEPES + sodium pyruvate + non-essential amino acids). Two days later, T cells were gene-edited using the Neon Transfection System (Invitrogen). Briefly, ribonucleoprotein (RNP) complexes were generated by mixing cas9 enzyme (IDT, catalog no. 1081059) and sgRNAs at a 2:1 molar ratio for 10 minutes at room temperature. When two sgRNAs were used, cas9 was used at a 1:1:1 ratio (sgRNA1:sgRNA2:cas9). When three sgRNAs were used, cas9 was used at a 0.67:0.67:0.67:1 ratio (sgRNA1:sgRNA2:sgRNA3:cas9). Cells were pulsed three times at 1600V for 10 ms and immediately harvested in R10 medium supplemented with 100 IU / mL IL-2 and 10 ng / mL IL-7 (Miltenyi, catalog no. 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. Five to seven days after gene editing, KO efficiency was assessed via flow cytometry. KO efficiency was assessed using various methods: for TRAC, CD3 / TCRab expression was assessed; for β2m, NLRC5, and RFX5, anti-β2m or anti-HLA antibodies were used; and CD58 expression was assessed using an anti-CD58 antibody.TRAC KO cells were purified using MACS negative selection according to the manufacturer's recommendations (Stem Cell Technologies, EasySep human TCR alpha / beta depletion kit, catalog number 17847). Purified T cells were used immediately or frozen in 5e6 cell aliquots in 90% FBS + 10% DMSO.

[0378] Preparation of target cells (all figures except Figure 1E-F)Primary human T cells were isolated from frozen peripheral blood mononuclear cells (PBMCs) from healthy donors using the EasySep Human T cell Isolation Kit (StemCell Technologies, Catalog No. 17951). Isolated T cells were activated with T cell TransAct (Miltenyi, Catalog No. 130-111-160) at 1:100 (v:v) + 100 IU / mL IL-2 (Miltenyi, Catalog No. 130-097-746) in X-Vivo 15 medium (Lonza, Catalog No. 04-418Q) + 5% human AB serum (Gemini, Catalog No. 100-318). Two days later, T cells were gene-edited using the Nucleofector 4D system (Lonza). Briefly, ribonucleoprotein (RNP) complexes were generated by mixing cas9 enzyme (IDT, catalog no. 1081059) and sgRNAs at a 1:1 molar ratio for 10 minutes at room temperature. When two sgRNAs were used, cas9 was used at a 0.5:0.5:1 ratio (sgRNA1:sgRNA2:cas9). When three sgRNAs were used, cas9 was used at a 0.3:0.3:0.3:1 ratio (sgRNA1:sgRNA2:sgRNA3:cas9). Immediately after electroporation, cells were recovered in X-Vivo 15 medium plus 5% human AB serum and 100 IU / mL IL-2. In some cases (Figures 5-6), electroporated T cells were transduced with adenovirus for expression of a CD19 CAR (FMC63 TurboCAR). TCR depletion (Stem Cell Technologies, EasySep Human TCR Alpha / Beta Depletion Kit, Catalog No. 17847) was typically performed between days 14 and 16. TCR-depleted T cells were cryopreserved and thawed for additional assays at later time points. Cell proliferation during production was tracked by counting viable cells with a Vi-CELL counter (Beckman Coulter). Fold expansion was calculated by comparing the number of viable cells at various time points with day 2 (the time of gene editing).CD19 CAR T cells were identified using an anti-idiotypic antibody (Acro Biosystems, catalog number FM3HPY53). KO efficiency was also assessed by flow cytometry across cell products at multiple time points. KO efficiency was assessed using various methods: for TRAC, CD3 / TCRab expression was assessed; for β2m, NLRC5, and RFX5, anti-HLA-ABC antibodies were used; and for CD58, CD48, and ICAM-1, anti-CD58, anti-CD48, and anti-ICAM-1 antibodies, respectively, were used to assess KO efficiency. TRAC KO cells were purified using MACS negative selection according to the manufacturer's recommendations (Stem Cell Technologies, EasySep human TCR alpha / beta depletion kit, catalog number 17847). Purified T cells were frozen in 5e6 cell aliquots in CryoStor CS5 medium (Stem Cell Technologies, catalog number 07933).

[0379] Primed T MLR (Figure 1E-F, 3 and 5)To promote the expansion of alloreactive T cell clones, HLA-A2+ human PBMCs were primed with irradiated PBMCs or pan T cells from the donor (HLA-A2-) used to generate the target T cells described above. Briefly, target PBMCs were irradiated at 30 Gy and co-cultured with host PBMCs at a 1:1 ratio in R10 + 20 IU / mL IL-2 + 10 ng / mL IL-7 + 10 ng / mL IL-15 (Miltenyi, Catalog No. 130-095-765) for 4 days. The medium was replaced with R10 without cytokines, and the cells were cultured for an additional 3 days. Pan T cells were then isolated using MACS negative selection (Miltenyi, Human Pan T Cell Isolation Kit, Catalog No. 130-096-535) according to the manufacturer's recommendations. 20,000 target T cells were seeded with 20,000 primed host T cells in 96-well plates and cultured in R10 + 20 IU / mL IL-2 at 37°C and 5% CO2 for 2 days. In some cases (Figure 5), 5,000 GFP-expressing β2m KO Raji tumor cells were added to the co-culture to activate the CD19 CAR T-transplanted cells. Survival of the transplanted T cells was determined by flow cytometry using absolute counts by gating on live GFP-HLA-A2-TCRαβ- CD4+ CD8+ (CAR+) cells with the desired gene edit (e.g., CD58 KO).

[0380] NK MLR (Figures 1F and 6)Figure 1B: Human NK cells were isolated from freshly isolated human HLA-A2+ PBMCs via MACS purification (Miltenyi Human Pan NK Cell Isolation Kit, catalog number 130-092-657). In a 96-well plate, 20,000 HLA-A2-transplanted T cells were seeded with 20,000 or 100,000 host NK cells at an effector:target ratio of 1:1 or 5:1 and cultured in R10 + 1000 IU / mL IL-2 at 37°C, 5% CO2 for 2 days. Absolute counts were us...

Claims

1. A CAR-T cell having a genome modification introduced by a CRISPR-Cas system containing sgRNA, wherein the genome modification functionally impairs or reduces the expression of RFX5 and CD58 compared to a CAR-T cell without the genome modification. The CAR-T cell wherein the sgRNA for RFX5 contains the sequence of SEQ ID NO: 94, and the sgRNA for CD58 contains the sequence of SEQ ID NO:

95.

2. A CAR-T cell having a genome modification, wherein the genome modification functionally impairs or reduces the expression of RFX5 and CD58 compared to a CAR-T cell without the genome modification. The CAR-T cells described above, wherein the genome modification of RFX5 is located in a sequence targeted by an sgRNA sequence containing the sequence of SEQ ID NO: 94, and the genome modification of CD58 is located in a sequence targeted by an sgRNA sequence containing the sequence of SEQ ID NO:

95.

3. The CAR-T cells according to claim 1 or 2, wherein the genome modification includes knockout of RFX5 and CD58.

4. The CAR-T cell according to claim 1 or 2, wherein 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.

5. The CAR-T cell according to claim 1 or 2, further comprising a polynucleotide sequence encoding an antigen-binding protein and / or a CD70-binding protein.

6. The CAR-T cells according to claim 1 or 2, wherein the CAR-T cells have improved persistence and / or improved resistance to rejection by alloreactive immune cells compared to CAR-T cells that do not have the genome modification.

7. The CAR-T cell according to claim 5, wherein the rejection reaction of the alloreactive immune cells includes alloreactive T cell-mediated rejection and / or alloreactive natural killer (NK) cell-mediated rejection.

8. CAR-T cells according to claim 1 or 2, further comprising one or more genomic modifications that functionally impair or reduce the expression of NLRC5.

9. CAR-T cells according to claim 1 or 2, wherein β2-microglobulin (β2m) is functionally expressed at a low level.

10. CAR-T cells according to claim 1 or 2, comprising an unmodified β2m gene, or in which β2m is not functionally expressed at a low level.

11. The aforementioned CAR-T cells are (i) Expression of low levels of MHC class I protein or MHC class I complex on the cell surface, (ii) Low levels of MHC class II protein or MHC class II complex expression on the cell surface, or (iii) CAR-T cells according to claim 1 or 2, which exhibit low levels of MHC class I protein or MHC class I complex expression on the cell surface and low levels of MHC class II protein or MHC class II complex expression on the cell surface.

12. The CAR-T cells according to claim 1 or 2, further comprising one or more genomic modifications of the endogenous TCRa gene, and optionally further comprising one or more genomic modifications of the endogenous CD52 gene.

13. The CAR-T cells according to claim 1 or 2, wherein the CAR-T cells are immune cells from a healthy volunteer.

14. A method for producing CAR-T cells according to claim 1 or 2, a) Modifying the genome of CAR-T cells using a CRISPR-Cas system containing sgRNA, where the sgRNA for RFX5 contains sequence number 94, the sgRNA for CD58 contains the sequence of sequence number 95, and b) The method comprising producing the CAR-T cells containing genome modifications.

15. A pharmaceutical composition comprising CAR-T cells according to claim 1 or 2, further comprising at least one pharmaceutically acceptable carrier or excipient, Optionally, the CAR-T cells are, (i) one or more proteins selected from the group consisting of HLA-E, HLA-E monochain trimer, HLA-G, HLA-G monochain trimer, UL18, UL18 monochain trimer, HLA-A2, HLA-A2 monochain trimer, and human cytomegalovirus (HCMV) US11 may be further expressed, and / or (ii) The pharmaceutical composition which may be further modified to not express or to express at a low level one or more of TAP2, NLRC5, β2m, TRAC, CIITA, RFXANK, and RFXAP.

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