Membrane-anchored NK cell inhibitory receptor engagers

Abstract

Some aspects relate to engineered cells expressing synthetic natural killer (NK) cell inhibitory receptor (e.g., CD161, NKG2A) engagers. Some aspects relate to methods of producing engineered cells expressing a synthetic NK cell inhibitory receptor engager. Some aspects relate to uses of cells expressing synthetic NK cell inhibitory receptor engagers.

Description

[0001] MEMBRANE- ANCHORED NK CELL INHIBITORY RECEPTOR ENGAGERS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 692,869, filed September 10, 2024; U.S. Provisional Application No. 63 / 752,558, filed January 31, 2025; U.S. Provisional Application No. 63 / 802,449, filed May 8, 2025; U.S. Provisional Application No. 63 / 821,012, filed June 10, 2025; U.S. Provisional Application No. 63 / 838,395, filed July 3, 2025; and U.S. Provisional Application No. 63 / 805,770, filed May 14, 2025, the contents of each of which are incorporated by reference herein in their entirety

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (G097170045 WOOO-SEQ-NTJ.xml; Size: 395,795 bytes and Date of Creation: September 9, 2025) are herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] .Allogeneic cell therapy, in which T cells are derived from healthy donors for modification and transfer into a recipient, is at risk of attack by the recipient’s immune system. Deletion of human leukocyte antigen (HLA) on allogeneic cells mitigates T cell-mediated rejection, but transferred cells are still at risk of killing by recipient natural killer (NK) cells. To attenuate the risk of NK cell-mediated killing, allogeneic T cells may be engineered to express HLA-E on the cell surface.

[0008] SUMMARY

[0009] Autologous cell therapy has significant disadvantages in terms of logistical complexity, high manufacturing costs, potential cell degradation, and longer turnaround times, all due to their personalized nature, which can delay treatment and reduce efficacy. Patient- specific risk factors, such as age and comorbidities, can also negatively impact the quality and potential of the cells collected for treatment. In contrast, an off-the-shelf allogeneic cell therapy approach offers significant benefits over autologous therapies, such as drastically reduced cost and off-the-shelf availability, but suffers from poor durability due to host rejection of the foreign graft. To achieve durable and effective allogeneic cell therapies, host rejection of the allogeneic product must be addressed, which remains a significant barrier in the allogeneic therapy field. The first step for effective immune evasion engineering has been to eliminate HLA expression on the allogeneic cells. Mismatched HLA is the dominant antigen targeted by T cell and antibody- mediated responses in allograft rejection. HLA class I and II elimination via KO of B2M and CIITA, respectively, is now commonplace in the field with readily available gene engineering tools and resulting cells reliably evade HLA-targeted rejection mechanisms. While avoiding T cell and antibody-mediated responses through eliminating HLA expression has been relatively straightforward, the remaining challenge in the field is the ensuing natural killer (NK) cell- mediated rejection, which is normally suppressed by HLA class I expression. Robust NK cell inhibition combined with HLA KO has significant potential to generate allogeneic cells with long term durability. However, despite significant efforts to identify suitable NK receptor targets and develop NK cell inhibitory receptor ligands of these targets, consistent and durable survival of allogeneic cells in vivo had previously not been achieved without immunosuppressant drugs. The present inventions address this long-felt need in the art for technology to generate allogeneic cell therapies with durable persistence, and consequently solve the problem of providing persistent, allogeneic, hypoimmune cells. Some aspects first disclosed herein relate to synthetic natural killer (NK) cell inhibitory receptor engagers that suppress NK cell activation, which are useful for the preparation of cells for allogeneic cell therapy. One option for reducing NK cell-mediated killing is to further modify allogeneic cells to express HLA-E on the cell surface. HLA-E interacts with the NK group 2A (NKG2A) receptor expressed on NK cells to inhibit NK cell activity. However, HLA-E also interacts with the NK group 2C (NKG2C) receptor, the ligation of which activates NK cells. Thus, expression of HLA-E on allogeneic cells can increase their susceptibility to NK cell- mediated killing by some NK cell subsets and therefore may not be sufficient to uniformly protect allogeneic cells from recipient NK cells. Successfully identifying NK cell receptors suitable for targeting by ligands (such as engagers) to inhibit NK cell activity without NKG2C+ NK cell activation as occurs in the context of HLA-E expression has been one of the major challenges in protecting allogeneic cells from NK cell-mediated rejection. Accordingly, some aspects disclosed herein relate to NK cell inhibitory receptor engagers that inhibit NK cells without inducing activation of NKG2C+ NK cell subsets. Another major challenge arises from the difficulty in design of synthetic ligands, particularly engagers, as inhibition of a given target NK cell receptor by a synthetic targeting moiety, particularly a cell surface protein, is not trivial. For cell surface- anchored engagers, challenges in design arise partly through the presence of multiple engager components, including a targeting moiety (to bind the NK cell surface receptor), transmembrane domain (to anchor the engager on the allogeneic cell surface), and elements connecting the targeting moiety to the transmembrane domain (to provide suitable distance between the allogeneic cell surface and the targeting moiety for interaction with NK cells). In a complete engager, the varying structures that may be used in each component affect several biochemical features of the engager, including (1) targeting moiety affinity for the target NK cell surface receptor; (2) length; (3) flexibility; and. (4) expression level on the allogeneic cell surface. These features in turn affect the ability of the engager to successfully limit NK cell-mediated killing and protect allogeneic cells.

[0010] The effects of features such as affinity for the target NK cell surface receptor, engager length, engager flexibility, and engager expression level (and interactions between them) on NK cell activity are not readily predictable. As Example 14 reports, seemingly minor changes in VH- VL orientation, the linker connecting VH and VL regions in an scFv, and transmembrane domain caused significant changes in protection. On the background of one NKG2A engager having a. monalizumab-based scFv, a CD8a hinge, and PDGFRA transmembrane domain, protection was reduced by each of (i) reversal of a VH-linker-VL orientation to VL-linker-VH; (ii) replacement of a Whitlow 218 linker with a (648)3 linker; and replacement of a. PDGFRA transmembrane domain ’with a CD8a transmembrane domain. This Example also reports that a previously published NKG2A engager design was ineffective at protecting cells from killing despite having an apparently suitable arrangement of scFv components, hinge, and transmembrane domain, underscoring the effects of these factors on engager function. Additionally, features that were previously reported to have similar effects in one context (e.g., E56.A and R94A substitutions affecting scFv affinity) were shown to have striking differences in a 7-day measure of durable protection. Even where a given element may appear to be a selection from available options, then, the effect of any one choice (e.g., VF1-VL orientation in an scFv) on the inhibitory activity of a complete engager is not trivial. Nor can a given engager design be considered a simple combination of features each performing a predictable and recognized function, given the unpredictability associated with combining multiple unpredictable features in a single protein. Engineered T regulatory cells (EngTregs), which constitutively express the Tree lineage transcription factor forkhead box protein 3 (FOXP3), have previously been created for the treatment of autoimmune disease, such as type I diabetes (T1D). Transduction of EngTregs with constructs encoding synthetic NK cell engagers has been found not to interfere with the expression of genes that promote the Treg phenotype, indicating that cells expressing such NK cell engagers exhibit potential for use in allogeneic cell therapy.

[0011] Synthetic NK cell engagers may be expressed from any suitable locus.

[0012] Moreover, where cells are edited to express two or more additional components that allow inducible proliferation of edited cells (e.g., CISC components), coding sequences for the additional components may be separated across multiple donor templates due to limitations on donor template size (e.g., AAV packaging limits). In such instances, linking expression of a coding sequence for at least one of the additional components to expression of the NK cell engager and FOXP3 limits the potential for induced proliferation (e.g., by provision of a ligand for chemically induced dimerization) to select for cells that do not express both the NK cell engager and FOXP3. Expression of a. first CISC component, NK cell engager, and first coding exon of FOXP3 from the FOXP3 locus, combined with expression of another CISC component in a different locus, therefore allows positive selection for NK cell engager-expressing Tregs by chemically induced dimerization and e.g., IL-2R signal transduction. Such linkage of NK cell engager and FOXP3 expression to produce NK cell engager-expressing Tregs, optionally with positive and / or negative selection through chemically induced IL-2R signaling or rapamycin exposure, respectively, allows production of Treg populations with improved purity for allogeneic use.

[0013] Accordingly, some aspects relate to a synthetic CD161 engager comprising:

[0014] (i) a CD161 -binding domain that specifically binds CD161; and

[0015] (ii) a transmembrane domain.

[0016] In some embodiments, the CD161-binding domain is a CD 161 -specific antibody or fragment thereof. In some embodiments, the CD161 -binding domain is a single-chain variable fragment (scFv) of a CDI61-specific antibody.

[0017] In some embodiments, the CD161-binding domain is a fragment of an LET! extracellular domain. In some embodiments, the CD161-binding domain and the transmembrane domain are connected by a linker. In some embodiments, the CD161-binding domain and the transmembrane domain are connected by hinge.

[0018] In some embodiments, the transmembrane domain is a type II transmembrane domain. In some embodiments, the type II transmembrane domain is a transmembrane domain of SMIM1 having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the synthetic CD161 engager comprises a SMIM1 transmembrane anchor having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 46.

[0019] In some embodiments, the type II transmembrane domain has less than 90% identity to a transmembrane domain of human LLT1. In some embodiments, the type II transmembrane domain has at least 1 substitution, deletion, and / or insertion relative to a transmembrane domain of human LLT1 having SEQ ID NO: 45.

[0020] In some embodiments, the transmembrane domain is a type I transmembrane domain, wherein:

[0021] (i) the C-terminus of the CD 161 -binding domain is covalently linked to the N-terminus of the type I transmembrane domain; and

[0022] (ii) the N-terminus of the CD 161 -binding domain is covalently linked to a signal peptide derived from a type I transmembrane protein.

[0023] In some embodiments, the synthetic CD161 engager does not comprise a cytoplasmic signaling domain.

[0024] In some embodiments, the synthetic CD161 engager does not comprise a cytoplasmic region with more than 10 amino acids. In some embodiments, the synthetic CD 161 engager comprises a cytoplasmic region. In some embodiments, the cytoplasmic region comprises no more than 10 amino acids.

[0025] Some aspects relate to a synthetic NKG2A engager comprising: (i) single-chain variable fragment (scFv) of an NKG2A-specific antibody; and (ii) a transmembrane domain. In some embodiments, scFv comprises: (i) an hCDRl comprising the amino acid sequence of SEQ ID NO: 73; (ii) an hCDR2 comprising the amino acid sequence of SEQ ID NO: 74; (iii) an hCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (iv) an 1CDR1 comprising the amino acid sequence of SEQ ID NO: 76; (v) an 1CDR2 comprising the amino acid sequence of SEQ ID NO: 77; and (vi) an 1CDR3 comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the scFv comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the C-terminus of the VH region is connected to the N-terminus of the VL region by a linker. In some embodiments, the linker is a Whitlow linker. In some embodiments, the Whitlow linker comprises the amino acid sequence of SEQ ID NO: 29.

[0026] In some embodiments, the synthetic NKG2A engager further comprises a hinge connecting the scFv and the transmembrane domain. In some embodiments, the hinge comprises a hinge of CD8a or CD28, or an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD8a, CD28, or PDGFRA, or an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 40.

[0027] In some embodiments, the synthetic NKG2A engager does not comprise a cytoplasmic signaling domain. In some embodiments, the synthetic NKG2A engager does not comprise a cytoplasmic region with more than 10 amino acids. In some embodiments, wherein the synthetic NKG2A engager comprises a cytoplasmic region. In some embodiments, the cytoplasmic region comprises no more than 10 amino acids.

[0028] Some aspects relate to a synthetic NKG2A engager comprising, in N- to C-terminal order: (a) an scFv comprising (i) a VH region comprising the amino acid sequence of SEQ ID NO: 71 with an E56A substitution, (ii) a Whitlow linker, and (hi) a VL region comprising the amino acid sequence of SEQ ID NO: 72, (b) a CD8a hinge; and (c)a PDGFRA transmembrane domain. In some embodiments, the CD8a hinge comprises an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the synthetic NKG2A engager does not comprise a cytoplasmic region with more than 10 amino acids. Some aspects relate to a genetically modified cell comprising the synthetic CD161 engager or synthetic NKG2A engager. Some aspects relate to a genetically modified cell comprising a nucleic acid, the nucleic acid comprising a nucleotide sequence encoding the synthetic CD161 engager or synthetic NKG2A engager. In some embodiments, the nucleic acid is a chromosome, wherein the chromosome further comprises a heterologous promoter operably linked to the nucleotide sequence encoding the synthetic CD161 engager or NKG2A engager. In some embodiments, the nucleotide sequence encoding the synthetic CD161 engager or NKG2A engager is in a ^2M locus, wherein the cell does not express a class I MHC protein on its surface. In some embodiments, the nucleotide sequence encoding the synthetic CD161 engager or NKG2A engager is in a CIITA locus, wherein the cell does not express a class II MHC protein on its surface. In some embodiments, the nucleotide sequence encoding the synthetic CD161 engager or NKG2A engager is in a TRAC or TRBC locus, wherein the cell does not express a T cell receptor (TCR) on its surface. In some embodiments, the nucleotide sequence encoding the synthetic CD161 engager or synthetic NKG2A engager is codon-optimized for expression in a human cell. Some aspects relate to a genetically modified cell that constitutively expresses LLT1 or a fragment thereof that comprises a CD161-binding domain and a transmembrane domain. Some aspects relate to a genetically modified cell that constitutively expresses a supraphysiologic level of LLT1 or a fragment thereof that comprises a CD161-binding domain. Some aspects relate to a genetically modified cell comprising, in a genomic nucleic acid comprising a nucleotide sequence encoding LLT1 or a fragment thereof that comprises a CD161- binding domain, a heterologous promoter operably linked to the nucleotide sequence encoding LLT1 or fragment thereof. In some embodiments, the heterologous promoter is a constitutive promoter. In some embodiments, the constitutive promoter is an MND promoter, an EF-1, promoter, or a PGK promoter. In some embodiments, the constitutive promoter is an MND promoter. In some embodiments, the heterologous promoter is an inducible promoter. In some embodiments, the nucleotide sequence encoding LLT1 or the fragment thereof is codon-optimized for expression in a human cell. In some embodiments, the nucleotide sequence encodes a fragment of LLT1 that comprises: (i) a deletion of one or more N-terminal amino acids relative to SEQ ID NO: 45^^ and / or (ii) a deletion of one or more C-terminal amino acids relative to SEQ ID NO: 45. Some aspects relate to a nucleic acid encoding the synthetic CD161 engager or synthetic NKG2A engager. In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA), a messenger ribonucleic acid (mRNA), a circular ribonucleic acid (circRNA), or a self-amplifying ribonucleic acid (saRNA). Some aspects relate to a lipid nanoparticle comprising the nucleic acid. Some aspects relate to a viral vector encoding the synthetic CD161 engager or synthetic NKG2A engager. In some embodiments, the viral vector is a lentiviral vector or adeno-associated viral vector. Some aspects relate to a system comprising: (i) the nucleic acid, the lipid nanoparticle, or ^'^^^^^^^^^^^^^^^^^^^^*^^+^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^(^^'^^^^^^^^^^^ In some embodiments, the nuclease is a meganuclease, TALEN, zinc finger nuclease, or RNA-guided DNA endonuclease. In some embodiments, the nuclease is an RNA-guided DNA endonuclease, and wherein the system further comprises a guide RNA (gRNA) or a nucleic acid encoding the gRNA. In some embodiments, the RNA-guided DNA endonuclease is Cas9. Some aspects relate to a method comprising delivering the system to a cell, thereby producing a genetically modified cell. In some embodiments of genetically modified cells, the genetically modified cell is: (i) ^^^^^^^^^^^^ (ii) ^^'^^^^^^^^^^^^^^^^^^ (iii) ^^^ ^^'^^ ^^^^^^^^^ (iv) ^^#^^^^^^ (v) a CD4+ T ^^^^^^^^^<^^ (vi) a CD8+ T cell. In some embodiments, the genetically modified cell is a regulatory T cell (Treg). In some embodiments, the genetically modified cell is a FOXP3+ regulatory T cell (Treg). Some aspects relate to a genetically modified regulatory T cell (Treg) comprising a nucleic acid, the nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a synthetic NK cell inhibitory receptor engager, the synthetic NK cell inhibitory receptor engager comprising: (i) a NK cell inhibitory receptor-binding domain that specifically ^^^^^^^^^^^^^)^^^^^^^^'^^^^^^ ^^^^^^^^^^^^^^ (ii) a transmembrane domain. In some embodiments, the NK cell inhibitory receptor is selected from the group consisting of CD200R1, CD300A, CD96, HAVCR2, KIR2DL1, KIR2DL2, KIR2DL3, K1R2DL5, KIR3DL1 , K1R3DL2, K1R3DL3, KLRG1, LAG-3, LAIR1, LILRB1, LILRB3,

[0029] NKG2A, PD-1, RTN4R, Siglec-3, Siglec-7, Siglec-9, SIRPa, and T1GIT. In some embodiments, the NK cell inhibitory receptor is NKG2A. In some embodiments, the NK cell inhibitory receptor-binding domain comprises monalizumab or a fragment thereof. In some embodiments, the NK cell inhibitory receptor-binding domain comprises: (i) an hCDRl comprising the amino acid sequence of SEQ ID NO: 73; (ii) an 11CDR2 comprising the amino acid sequence of SEQ ID NO: 74; (ii) an hCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (ii) an 1CDR1 comprising the amino acid sequence of SEQ ID NO: 76; (ii) an 1CDR2 comprising the amino acid sequence of SEQ ID NO: 77; and (ii) an 1CDR3 comprising the amino acid sequence of SEQ ID NO: 78.

[0030] In some embodiments, the genetically modified cell comprises a heterologous promoter located in a genomic nucleic acid: (i) downstream from a Treg-specific demethylated region (TSDR); and (ii) upstream from a first coding exon of an endogenous FOXP3 gene.

[0031] In some embodiments, the genetically modified cell comprises: (i) a nucleic acid encoding a first component of a chemically induced signaling complex (CISC), the first CISC component comprising: (a) a first extracellular domain comprising an FK506-binding protein domain that binds rapamycin; (b) a. first transmembrane domain; and (c) a first cytoplasmic domain comprising an intracellular signaling domain of a first cytokine receptor; and (ii) a nucleic acid encoding a second CISC component, the second CISC component comprising: (a) a. second extracellular domain comprising an FKBP-rapamycin-binding (ERB) domain; (b) a second transmembrane domain, and (c) a second cytoplasmic domain comprising an intracellular signaling domain of a second cytokine receptor, wherein the first and second CISC components dimerize in the presence of rapamycin. In some embodiments, the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin- 2 receptor gamma (IL- 2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin- 2 receptor beta (IL-2Rp) transmembrane domain, and an IL-2R0 cytoplasmic domain.

[0032] In some embodiments, the genetically modified cell comprises a nucleic acid encoding a soluble FRB domain, wherein SEQ ID NO: 3 has at least 90% sequence identity to the amino acid sequence of the soluble FRB domain. Some aspects relate to a method comprising administering the genetically modified cell to a subject in need thereof. In some embodiments, the method is a method for treating or preventing an autoimmune disease, allergic disease, inflammatory disease, or adverse effect of transplantation in the subject.

[0033] Some aspects relate to genetically modified cells for use in a method of treating or preventing an autoimmune disease, allergic disease, or inflammatory disease in a subject.

[0034] In some embodiments, the autoimmune disease is selected from the group consisting of from type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early onset rheumatoid arthritis, ankylosing spondylitis, immune- mediated pregnancy loss, immune-mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn’s disease, inflammatory bowel disease (IBD), ulcerative colitis, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren’s syndrome, and celiac disease.

[0035] In some embodiments, the allergic disease is selected from the group consisting of allergic asthma, steroid-resistant asthma, atopic dermatitis, celiac disease, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis.

[0036] In some embodiments, the inflammatory disease is selected from the group consisting of stroke, myocardial infarction, acute swelling, severe wounding, muscle injuries, burn injuries, traumatic brain injury, acute respiratory distress syndrome (ARDS), pancreatic islet cell transplantation, asthma, hepatitis, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, Still’s disease, uveitis, ulcerative colitis, graft-versus-host disease (GvHD), tolerance induction for transplantation, transplant rejection, and sepsis.

[0037] In some embodiments, the genetically modified cell is allogeneic to the subject.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. I is an exemplary schematic of a natural killer (NK) cell engager.

[0040] FIGs. 2A-2D relate to the correlation between expression of two construct components, a an NK cell engager containing a Whitlow linker, and low-affinity nerve growth factor receptor (LNGFR) selectable marker, in cells transduced with varying constructs (FIG. 2A: untransduced control, Alexa-Fluor-( AF)647 fluorescence minus one (FMO) control, LV80366, LV80367; FIG. 2B: LV80358, LV80359, LV80368, LV80369; FIG. 2C: LV80360, LV80361, LV80370, LV80371; FIG. 21): LV80363, LV80364, LV80365, LV80372, LV80373, LV80374). FIGs. 3A-3D relate to the correlation between binding labeled recombinant human natural killer cell protein group 2A (rhNKG2A) and LNGFR marker expression in cells transduced with varying constructs (FIG. 3A: untransduced control, AF647 FMO control, LV80366, LV80367; FIG. 3B: LV80358, LV80359, LV80368, LV80369; FIG. 3C: LV 80360, LV80361, LV80370, LV80371 ; FIG. 3D: LV80363, LV80364, LV80365, LV80372, LV80373, LV80374).

[0041] FIGs. 4A-4B relate binding labeled rhNKG2A and expression of an NK cell engager (evidenced by Whitlow linker staining) in cells transduced with varying constructs. FIG. 4A show's the correlation between transduced cells binding labeled rhNKG2A with NK cell engager expression as geometric MFI (gMFI). FIG. 4B show's gMFI of Whitlow' linker labeling (top) and gMFI of labeling with rhNKG2A (bottom).

[0042] FIGs. 5A-5E relate to protection of cells from different donors transduced with varying constructs (FIG. 5A: untransduced control, LV80358, LV80359, LV80360, LV80361, LV80363; FIG. SB: untransduced control, LV80364, LV80365, LV80366, LV80367, LV80368; FIG. 5C: untransduced control, LV80369, LV80370, LV80371, LV80372, LV80373) against killing by NK cells. FIG. 5D show's the percent of transduced cells protected from NK cell-mediated killing. FIG. 5E show's a lack of correlation between binding labeled rhNKG2 A and protection of transduced cells against NK cell-mediated killing.

[0043] FIGs. 6A-6B relate to the percentage of transduced cells protected against killing by NKG2A+ NK cell-dominant responders (FIG. 6A) and NKG2C+ NK cell-high responders (FIG. 6B).

[0044] FIGs. 7A-7C relate to protection of transduced cells against total peripheral blood mononuclear cells (PBMCs). FIG. 7 A shows the percentage of transduced cells from two different donors killed by PBMCs. FIGs. 7B-7C show' the percentage of transduced cells protected against killing by NKG2A+ NK cell-dominant responders (FIG. 7B) and NKG2C+ NK cell-high responders (FIG. 7C).

[0045] FIGs. 8A-8B relate to the correlation betw'een protection against killing by NK cell populations and protection against whole PBMC populations for transduced cells from two different donors (FIG. 8A, donor 1; FIG. 8B, donor 2).

[0046] FIGs. 9A-9H relate to phenotypes of target cells transduced with varying constructs, as assessed by expression of related surface markers. FIG. 9 A show's untransduced cells (control) and cells transduced with LV80358, LV80359, or LV80360 and labeled for a 2A linker (y-axis, top), CD25 (y-axis, bottom), and the Treg lineage transcription factor FOXP3 (x-axis). FIG.9B shows cells transduced with LV80361, LV80381, LV80382, LV80383, or LV80384 and labeled for a 2A linker (y-axis, top), CD25 (y-axis, bottom), and FOXP3 (x-axis). FIG.9C shows untransduced cells and cells transduced with LV80358, LV80359, or LV80360 and labeled for a Whitlow linker (y-axis, top), ?2M (y-axis, bottom), and LNGFR (x-axis). FIG. 9D shows cells transduced with LV80361, LV80381, LV80382, LV80383, or LV80384 and labeled for a Whitlow linker (y-axis, top), ?2M (y-axis, bottom), and LNGFR (x-axis). FIG. 9E shows cells transduced with LV80401, LV80402, LV80403, or LV80404 and labeled for a 2A linker (y-axis, top), CD25 (y-axis, bottom), and FOXP3 (x-axis). FIG.9F shows cells transduced with LV80405, LV80406, LV80407, LV80400, or LV80390 and labeled for a 2A linker (y-axis, top), CD25 (y-axis, bottom), and FOXP3 (x-axis). FIG. 9G shows cells transduced with LV80401, LV80402, LV80403, or LV80404 and labeled for a Whitlow linker (y-axis, top), ?2M (y-axis, bottom), and LNGFR (x-axis). FIG.9H shows cells transduced with LV80405, LV80406, LV80407. LV80400, or LV80390 and labeled for a Whitlow linker (y-axis, top), ?2M (y-axis, bottom), and LNGFR (x-axis). FIG.9I shows untransduced cells and cells transduced with LV80358, LV80359, LV80360, LV80401, LV80402, LV80403, or LV80404 and labeled with rhNKG2A and for LNGFR. FIG.9J shows cells transduced with LV80361, LV80381, LV80382, KV80383, LV80384, LV80405, LV80406, LV80407, LV80400, or LV80390 labeled with rhNKG2A and for LNGFR. FIG.9K shows untransduced cells and cells transduced with LV80358, LV80359, LV80360, LV80401, LV80402, LV80403, or LV80404 labeled with rhNKG2C and for LNGFR. FIG.9L shows cells transduced with LV80361, LV80381, LV80382, LV80383, LV80384, LV80405, LV80406, LV80407, LV80400, or LV80390 and labeled with rhNKG2C and for LNGFR. FIGs.10A-10B relate to the relationship between Whitlow linker expression and binding to labeled rhNKG2A and / or rhNKG2C. FIG. 10A shows staining for Whitlow and by labeled rhNKG2A and rhNKG2C on cells transduced with various constructs. FIG.10B shows the correlation between Whitlow staining and binding labeled rhNKG2A. FIGs.11A-11B relate to phenotyping of PBMC responders. FIG. 11A shows the percentage of PBMCs from 10 donors that were NK cells and the percentage of NK cells that were NKG2A+, NKG2C+, and CD161+. FIG. 11B shows the percentage of different cell populations within PBMCs from 10 different donors.

[0047] FIGs. 12A-12E relate to protection of cells against NK cell-mediated killing, among cell populations from 10 different donors transduced with various constructs and incubated with PBMCs. FIGs. 12A-12B show the percentage of untransduced cells and cells from two sets of 5 donors (FIG. 12A, first donor set; FIG. 12B, second donor set) transduced with LV80358, LV80359, LV80360, LV80361 , LV80382, or LV80383 killed by NK cells. FIGs. 12C-12D show the percentage of untransduced cells and cells from two sets of 5 donors (FIG. 12C, first donor set; FIG. 12D, second donor set) transduced with LV80384, LV80401, LV80402, LV80403, LV80404. LV80405, or LV80406 killed by NK cells. FIG. 12E show's untransduced cells and cells from 10 different donors transduced with LV 80407, LV80400, or LV80390.

[0048] FIG. 13 shows the relationship between killing of P2MV" target cells and the percentage of different NK subsets in PBMC populations from 10 different donors.

[0049] FIGs. 14A-14B relate to protection of transduced cells against killing by NKG2C+ NK cells. FIG. 14A show's the percent of cells protected against killing by NKG2C+ NK cells (y- axis), after transduction with various constructs (x-axis). FIG. 14B demonstrates that cells transduced with L.V80390 or LV80360 were better protected against NKG2C+ NK cells when they were present in higher percentages compared to cells transduced with LV80400 or LV80402.

[0050] FIG. 15 shows Whitlow linker expression, intracellular LLT1 expression, surface LLT1 expression, and binding to labeled rhCDlbl by cells transduced with various constructs.

[0051] FIG. 16 shows the percentage of cells protected against killing by PBMCs, grouped by construct transduced into cell populations.

[0052] FIGs. 17A-17C relate to correlates of protection. FIGs. 17A-17B show the correlation between protection against killing by NK cells and percentage of NKG2C+ NK cells (FIG. 17A) or CD161+ NK cells (FIG. 17B) in PBMCs. FIG. 17C shows the correlation between protection and intracellular LLT1 expression in transduced cells.

[0053] FIGs. 18A-18B are representative flow plots of LNGFR (x-axis) expression at days 0, 3, and 7 post-transduction in HLA+ cells transduced with LV80361 and cultured with autologous cells (FIG. 18A) or allogeneic cells (FIG. 18B). Cultures without target cells were used as controls. FIGs.19A-19D relate to target persistence (“% of targets”) after 7 days of co-culture of cells transduced with various engager constructs (each line represents a different construct) and PBMCs from different donors (donor number indicated on the top of each line graph). FIG.19A shows results of all constructs. FIG.19B shows cells transduced with LV20003 (no engager), LV80432 (CD47), LV80400 (HLA-E), and no-target controls. FIG. 19C shows cells transduced with LV20003 (no engager), LV80385 (LLT1), LV80376 (CD161 engager), and no-target controls. FIG.19D shows cells transduced with LV20003 (no engager), LV80360 (NKG2A engager), LV80405 (NKG2A engager, low affinity), LV80390 (CD300A engager), and no-target controls. Transduced cells were obtained from a single donor. FIGs.20A-20G show target persistence (“% of targets”) at day 7 post-culture compared to day 0. Each line represents a different donor. FIG.20A shows HLA+ target cells that were not expressing an engager. FIG.20B shows HLA KO cells that do not express an engager. FIG.20C shows HLA KO cells expressing a parental NKG2A engager. FIG. 20D shows HLA KO cells expressing CD47. FIG. 20E shows HLA KO cells expressing a low-affinity NKG2A engager. FIG.20F shows cells expressing HLA-E but that are otherwise HLA KO. FIG.20G HLA KO cells expressing a CD300A engager. FIGs.21A-21B summarize endpoint target persistence (“% of targets”) of experiments shown in FIGs. 19A-20G. Each of FIG.21A and FIG.21B represent an independent technical replicate. FIGs.22A-22D relate to target persistence (“% of targets”, FIG. 22A and FIG.22B^^ “normalized count of targets”, FIG. 22C and FIG.22D) after 8 days (FIG.22B and FIG.22D) of co-culture of cells transduced with various engager constructs (x-axis) and PBMCs derived from different donors (indicated by the legend). Day 0 of culture is shown for comparison (FIG. 22A and FIG.22C). Transduced cells were obtained from a single donor, different from the experiments shown in FIGs.18A-21B. FIGs.23A-23E show target persistence (“% of targets”) at day 0 and day 8 of co-culture of PBMCs derived from various donors (indicated by the legends) and cells that were not engineered to express engagers (FIG.23A), cells engineered to express a parental NKG2A engager (FIG.23B), cells engineered to express a low-affinity NKG2A engager (FIG. 23C), cells engineered to express HLA-E (FIG. 23D), and cells engineered to express a CD300A engager (FIG.23E). Engineered cells were obtained from the same donor as in the experiments shown in FIGs. 22A-22D. FIG.24 shows representative flow plots of HLA KO or HLA-replete EngTregs transduced with LV80227 (LNGFR-Qluc, no engager). FIGs.25A-25D relate to persistence of target cells as a percentage (FIGs.25A and 25C) or normalized count (FIGs.25B and 24D) over time, at effector:target ratios of 30:1 (FIGs. 25A-25B) or 60:1 (FIGs. 25C-25D). FIGs.26A-26C relate to percentage of NK cell responders in cultures of EngTregs derived from donor 3589 transduced with LV80227 and co-cultured with autologous (FIG. 26A) PBMCs or allogeneic (FIG.26B^^^^^^^^!;!^^^FIG.26C, donor 4792) PBMCs. FIGs.27A-27H are representative flow plots of HLA class II, B2M, LLT1, LNGFR, Whitlow, and CD47 staining on EngTregs transduced with LV80227 (FIG. 27A^^.A^D^^FIG. 27B, HLA-), LV80360 (FIG.27C), LV80385 (FIG. 27D), LV80432 (FIG. 27E), LV80400 (FIG. 27F), or LV80390 (FIG.27G). FIGs.28A-28C relate to the protection of various engagers, measured as the percentage of targets killed (FIG.28A), or the percentage of protection (FIG.28B^^FIG.28C, CD300A engager not shown). FIGs.29A-29B relate to the protection of various engagers, measured as the percentage of dead targets (FIG. 29A) or the percentage of protection (normalized, FIG. 29B). FIG.30 shows the percentage of protection across various constructs. FIGs.31A-31D are representative flow plots of TCRVb13.6, FOXP3, CD25, CTLA4, LLT1, LNGFR, and Whitlow staining of untransduced EngTregs (FIGs.31A and 31B) or EngTregs transduced with constructs bearing an NKG2A engager (FIG.31C) or an LLT1 engager (FIG.31D). FIG.32 shows representative HLA class II and B2M expression on cells of FIGs.31A- 31D. FIGs.33A-33B relate to the protection of LV80360 and LV80385, measured as the percentage of targets killed (FIG.33A) or the percentage of protection (normalized, FIG.33B). FIGs.34A-34D show the percentage of EngTregs, transduced with LV80360 or LV80385 and co-cultured with CFSE-labeled K562 / DR4 cells at a 2:1 effector:target ratio in the presence of various amounts of IGRP305peptide or in the presence of anti-CD3 / anti-CD28 ImmunoCult (positive control), expressing CD69 (FIG. 34A), LAP (FIG. 34B), CD137 (FIG. 34C), and GARP (FIG. 34D). FIGs.35A-35B relate to the protection of NKG2A engager constructs, LLT1 constructs, and HLA-E constructs, measured as a percentage of target cells killed (FIG.35A) or percentage of protection (FIG.35B). FIG.36A shows an experimental design in which immunodeficient mice transgenic for human IL-15 (NOD.Cg-PrkdcscidIl2rgtm1WjlTg(IL15)1Sz / SzJ, Common Name: NSG-Tg(Hu- IL15), RRID: IMSR_JAX:030890) were injected with 20 million PBMCs and 2 million HLA KO EngTreg, HLA KO EngTreg with NKG2A engager (LV80405), or no EngTreg. Peripheral blood was collected on day 0 post-infusion, day 14, and day 28. Animals were sacrificed on day 28 and the additional tissues listed were also analyzed. Frequency and numbers of EngTreg were quantified based on B2M (HLA class I) expression on day 0 and day 14 (FIGs. 36B–36E). Representative plots for day 14 are shown in FIG.36F. Extent of humanization is shown by human CD45+ cell counts (FIG.36G). FIG.37A shows a quadruple-editing approach for producing an EngTreg by editing of the FOXP3, TRAC, B2M, and CIITA loci. The edited FOXP3 locus includes an MND promoter operably linked to nucleotide sequences encoding an FRB-IL2R? CISC component, NKG2A engager, naked FRB domain, and the first coding exon of the FOXP3 gene downstream from the TSDR. The edited TRAC locus includes an MND promoter operably linked to an FKBP-IL2R> CISC component and antigen-specific receptor, such as a CAR or TCR. The edited B2M and CIITA loci do not express functional B2M or CIITA, and so edited cells do not express MHC-I or MHC-II on their surface. FIG. 37B shows evasion of host T cells and antibodies resulting from knockout of HLA expression, and inhibition of host NK cells through surface expression of an NK cell inhibitory receptor (e.g., NKG2A) engager. FIG.38A shows an experimental design in which PBMCs were co-incubated with B2M KO EngTregs, and after 1 day of co-culture, the extent of B2M KO EngTreg killing was assayed. PBMCs were also analyzed to characterize the immune cell subsets therein. FIG. 38B shows regression analysis of proportional immune cell subset representation against the extent of B2M EngTreg killing. FIG.38C shows regression analysis of the percentage of NKG2A+cells among NK cells against the extent of B2M EngTreg killing. FIGs.39A–39C show designs and results of experiments in which purified NK cells from four different donors were incubated with B2M KO EngTregs (control cells) and B2M KO EngTregs expressing an LNGFR tag (FIG.39A), HLA-E (FIG. 39B), or NKG2A engager (LV80360, FIG. 39C). Within each group, left bars represent control B2M KO EngTregs labeled with CellTrace Violet (CTV), and right bars represent B2M KO EngTregs expressing test proteins labeled with carboxyfluorescein succinimidyl ester (CFSE). FIG.40A shows a dual-editing approach for producing an EngTreg by editing of FOXP3 and TRAC loci, as described in Uenishi et al., JCI Insight.2024. 9(6):e171844. The edited FOXP3 locus includes an MND promoter operably linked to nucleotide sequences encoding an FRB-IL2R? CISC component, naked FRB domain, and the first coding exon of the FOXP3 gene downstream from the TSDR. The edited TRAC locus includes an MND promoter operably linked to an FKBP-IL2R> CISC component and TCR specific to an IGRP305peptide. IGRP305-specific EngTregs were further edited to knockout B2M and CIITA expression (HLA KO), with or without lentivirus-mediated expression of NKG2A engager (LV80360). FIG.40B shows expression of B2M and MHC-II in control, HLA KO, and HLA KO + NKG2A engager cells, and expression of NKG2A engager as detected by anti-Whitlow antibody. FIG.40C shows relative protection of HLA KO EngTregs by NKG2A engager expression. FIG.40D shows expression of Treg functional markers LAP and GARP in response to cognate peptide or vehicle control. FIG.41A shows a dual-editing approach for producing an EngTreg. The edited FOXP3 locus includes an MND promoter operably linked to nucleotide sequences encoding an FRB- IL2R? CISC component, NK cell inhibitor, naked FRB domain, and the first coding exon of the FOXP3 gene downstream from the TSDR. The edited TRAC locus includes an MND promoter operably linked to nucleotide sequences encoding an FKBP-IL2R> CISC component and heterologous TCR components. FIG.41B shows representative flow cytometry analysis of EngTregs at start of co-culture (left) and after 7 days of co-culture (right) with PBMC populations of different donors. FIG.41C shows the change in EngTreg abundance, in proportion (top) and absolute numbers (bottom), from start of co-culture (Day 0) to end of co- culture (Day 7). Groups indicate co-culture with PBMCs from different donors. FIGs.42A-42E show results from an in vivo study in which mice were humanized with CD34+ hematopoietic stem cells and then administered allogeneic EngTregs () and then AP21967 (Takara Bio) 3x per week, every other week. Peripheral blood was collected on day 0 post-EngTreg infusion and approximately every ten days after. Samples were then screened for percentage of hCD45+ cells (FIG.42A), absolute number of LNGFR+ cells among human CD3+CD14-CD19-CD45+ cells (FIG.42B), the percentage of NK cells, measured by human CD56+ of the CD3-CD19-CD14-CD45+ population (FIG.42C), the percentage of NKG2A expression among NK cells (FIG.42D), and NKG2A expression level as measured by geometric mean fluorescence intensity (gMFI) among NKG2A+ NK cells (FIG.42E). FIG.43 is a schematic illustration of tissue EngTregs having an MND promoter in the FOXP3 gene to drive constitutive, high level expression and overexpressing ST2, which activates expression of key chemokine receptors and mediates homing to sites of inflammation, increases expression of receptors associated with Treg function (ST2, TNFR2, and CD25), enhances uptake of inflammatory cytokines (IL-33, TNFa, and IL-2), and increases pro-repair cytokines including osteopontin and IL-13. FIGs.44A-44B demonstrate that the hypo-immune approach can be incorporated with EngTregs expressing specific TCR and / or CAR constructs. EngTregs comprising a synthetic NK cell inhibitory receptor engager (e.g., NKG2A engager) and a T cell receptor that binds an IGRP305–324 peptide were synthesized and it was found that HLAs were readily knocked out (FIG. 44A) and the NK inhibitor was readily expressed (FIG. 44B). FIGs.45A-45B illustrate that the addition of the NK inhibitor protects HLA knockout EngTregs comprising a T cell receptor that binds an IGRP305–324 peptide from NK rejection (FIG. 45A) and does not interfere with activation of the HLA knockout EngTreg (FIG. 45B). FIG.46 provides sequences of NK cell inhibitory receptor engagers. FIGs.47 and 48 provide exemplary sequences of a FOXP3 locus donor template. FIGs.49 and 50 provide exemplary sequences of a TRAC locus donor template. FIG.51 provides exemplary guide RNA (gRNA) spacer sequences. FIG.52 provides exemplary T cell receptor (TCR) sequences. FIG.53 provides exemplary IL-33 and IL-18 receptor component sequences. FIG.54 provides exemplary anti-CD19 chimeric antigen receptor component sequences. FIG.55 shows an allogeneic EngTreg for treatment of type 1 diabetes (T1D) that avoids rejection in an allogeneic setting through B2M and CIITA knockout (KO) to ablate HLA class I and II expression, respectively, combined with expression of an NKG2A engager for natural killer cell inhibition. AlloEngTreg-TID leverages the established biology of AutoEngTreg-TID, while enabling use beyond autologous settings.

[0054] FIGs. 56A-56L show the results of the study described in Example 13. Peripheral blood was sampled approximately every 10 days and infusion products were detected by LNGFR positivity. FIGs. 56A-56D show test article persistence and NK cell compartment characterization in blood. FIGs. 56E-56H show human immune cell compartment characterization in blood. FIGs. 56I-56J show persistence in spleen (FIG. 561) and bone marrow (FIG. 56J) at study end or humane endpoint.

[0055] FIGs. 57A-57C show' protection by NKG2A engagers during 7-day co-culture with PBMCs, as described in Example 14. FIG. 57A show's results with NKG2A engagers having a parental monalizumab-based scFv, with varying (i) arrangements of VH-VL regions in the scFv; (ri) linkers in the scFv; and (iii) transmembrane domains. FIG. 57B compares the effects of affinity-affecting substitutions. FIG. 57C compares NKG2A engager 80405 with representative proteins described by Gracell or Zhang et al.

[0056] FIG. 58 shows strength of NKG2A binding by cells expressing NKG2A engagers 80360 (parental rnonalizumab scFv) or 80405 (80360 with E56A substitution).

[0057] BRIEF DESCRIPTION OF THE SEQUENCES

[0058] SEQ ID NOs: 1—118 relate to CISC components, linkers, hinges, transmembrane domains, LLTI, and antibodies targeting NKG2A or CD161 and portions thereof.

[0059] SEQ ID NOs: 121-178 relate to NK cell inhibitory receptor engagers.

[0060] SEQ ID NOs: 201-225 relate to FOXP3 locus donor templates.

[0061] SEQ ID NOs: 226-250 relate to TRAC locus donor templates.

[0062] SEQ ID NOs: 251-300 relate to guide RNA (gRNA) spacer sequences.

[0063] SEQ ID NOs: 301-310 relate to T cell receptor (TCR) amino acid sequences.

[0064] SEQ ID NOs: 311—314 relate to IL-33 and IL-18 receptor amino acid sequences.

[0065] SEQ ID NOs: 315-447 relate to anti-CD19 chimeric antigen receptor (CAR) amino acid sequences.

[0066] DETAILED DESCRIPTION

[0067] Some aspects relate to synthetic engagers of NK cell inhibitory receptors, which include

[0068] CD161, NKG2A, and CD300A. Without wishing to be bound by a particular theory, it is posited that engagement of inhibitory receptors on NK cells mitigates the risk of rejection of allogeneic cells having reduced expression of class I major histocompatibility complex (MHC) proteins on their surface. Specific engagement of certain receptors, such as NKG2A, addresses the potential of HLA-E to cause activation through NKG2C engagement, which limits the effectiveness of HLA-E-based approaches for preventing NK cell-mediated rejection. Other specific receptors, such as CD161 or CD300A, may be engaged, alternatively or additionally, to target different NK cell populations or transduce different inhibitory signals than NKG2A. A synthetic NK cell inhibitory receptor engager may be a protein or polypeptide or comprise two or more polypeptides or proteins in association. Some aspects relate to synthetic engagers comprising antigen-binding domains that bind, preferentially bind, and / or specifically bind an NK cell surface marker (e.g., NKG2A, CD161, CD300A). Generally, an antigen-binding domain (e.g., scFv) that “recognizes” an antigen will bind that antigen in a mixture of different antigens. Generally, but not always, “binding” describes preferential binding. An antigen-binding domain is said to “preferentially bind” to a particular antigen if it reacts or associates more frequently, more rapidly, with a greater duration, and / or with a greater affinity for the particular antigen than to other antigens. For example, the antigen-binding domain of monalizumab preferentially binds NKG2A with greater affinity, avidity, more readily, and / or with greater duration than it binds other NK cell surface markers. In some embodiments, preferential binding to an antigen (e.g., NKG2A, CD161, CD300A) is relative to a different NK cell surface marker (e.g., CD56). An antigen-binding domain (e.g., scFv) is said to “specifically bind” to a particular NK cell surface marker if interaction between (a) the antigen-binding domain, and (b) the NK cell surface marker on the surface of an NK cell, is sufficient to induce signal transduction in the NK cell via the NK cell surface marker. The skilled artisan will understand that determining whether NK cell surface marker-mediated signal transduction occurs involves use of an NK cell expressing a number of NK cell surface marker units on its surface. It should be understood that an NK cell surface marker that preferentially binds or specifically binds to an NK cell surface marker may or may not also bind, to some extent, to a second antigen having a different amino acid sequence or structure from the NK cell surface marker. As such, it should be understood that “specifically binding” to a particular NK cell surface marker does not necessarily require exclusive binding (i.e., that the antigen-binding domain bind to no antigens other than the particular NK cell surface marker) (e.g., the antigen-binding domain may cross-react with one or more similar or related antigens). In some embodiments, specific binding to an NK cell surface marker (e.g., NKG2A, CD161, CD300A) excludes binding of different NK cell surface markers (e.g., CD56). CD161 engagers Some aspects relate to a synthetic CD161 engager. Some aspects relate to nucleic acids encoding a synthetic CD161 engager. Some aspects relate to engineered cells expressing a synthetic CD161 engager. Some aspects relate to engineered cells comprising a synthetic CD161 engager. Some aspects relate to engineered cells comprising a nucleic acid encoding a synthetic CD161 engager. A synthetic CD161 engager may comprise a CD161-binding domain and a transmembrane domain. In some embodiments, the synthetic CD161 engager comprises a linker connecting the CD161-binding domain and the transmembrane domain. In some embodiments, the synthetic CD161 engager comprises a hinge connecting the CD161-binding domain and the transmembrane domain. Any suitable synthetic CD161 engager that binds, preferentially binds, or specifically binds CD161 may be encoded by a nucleic acid and / or used in engineered cells (e.g., engineered T cells). In some embodiments, a synthetic CD161 engager comprises LLT1 lacking amino acids Met1 through Gly21 of SEQ ID NO: 45. In some embodiments, a synthetic CD161 engager consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, a synthetic CD161 engager consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 41. In some embodiments, a synthetic CD161 engager consists of an amino acid sequence that lacks amino acids Met1 through Gly21 of SEQ ID NO: 45 and has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 41. In some embodiments, a synthetic CD161 engager comprises an LLT1 extracellular domain, a CD8, hinge, and a CD8, transmembrane domain. In some embodiments, a synthetic CD161 engager comprises an LLT1 extracellular domain, a PDGFRA hinge, and a PDGFRA transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a truncated LLT1 extracellular domain lacking amino acids 1–71 of SEQ ID NO: 45, a CD8, hinge, and a CD8, transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a truncated LLT1 extracellular domain lacking amino acids 1–71 of SEQ ID NO: 45, a PDGFRA hinge, and a PDGFRA transmembrane domain. In some embodiments, a synthetic CD161 engager comprises an LLT1 extracellular domain, a G4S2 (SEQ ID NO: 25) linker, and a SMIM1 transmembrane anchor. In some embodiments, a synthetic CD161 engager comprises an LLT1 extracellular domain and a SMIM1 transmembrane anchor. In some embodiments, a synthetic CD161 engager comprises an LLT1 extracellular domain and a SMIM1 transmembrane anchor and does not comprise a linker or a hinge. In some embodiments, a synthetic CD161 engager comprises a truncated LLT1 extracellular domain lacking amino acids 1–71 of SEQ ID NO: 45, a G4S2linker (SEQ ID NO: 25), and a SMIM1 transmembrane anchor. In some embodiments, a synthetic CD161 engager comprises a truncated LLT1 extracellular domain lacking amino acids 1–71 of SEQ ID NO: 45 and a SMIM1 transmembrane anchor. In some embodiments, a synthetic CD161 engager comprises a truncated LLT1 extracellular domain lacking amino acids 1–71 of SEQ ID NO: 45 and a SMIM1 transmembrane anchor and does not comprise a linker or a hinge. In some embodiments, a synthetic CD161 engager comprises an LLT1 extracellular domain, a first G4S linker (SEQ ID NO: 24), a myc tag, a second G4S linker (SEQ ID NO: 24), and a SMIM1 transmembrane anchor. In some embodiments, a synthetic CD161 engager comprises LLT1 lacking amino acids Met1 through Leu38 of SEQ ID NO: 45. In some embodiments, a synthetic CD161 engager consists of the amino acid sequence of SEQ ID NO: 42. In some embodiments, a synthetic CD161 engager consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, a synthetic CD161 engager consists of an amino acid sequence that lacks amino acids Met1 through Leu38 of SEQ ID NO: 45 and has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, a synthetic CD161 engager comprises (i) an scFv of KWI, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of KW 1 , a Whitlow linker, and a VL region comprising 1CDR 1 , 1CDR2, and 1CDR3 of KW1; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic CD 161 engager comprises (i) an scFv of KWI, the scFv comprising, in N-to-C- terminal order: the VH region of KWI , a Whitlow linker, and the VL region of KWI ; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0069] In some embodiments, a synthetic CD 161 engager comprises (i) an scFv of KWI, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of KWI, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of KWI; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic CD 161 engager comprises (i) an scFv of KW I the scFv comprising, in N-to-C- terminal order: the VL region of KWI, a Whitlow linker, and the VH region of KWI; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0070] In some embodiments, a synthetic CD161 engager comprises (i) an scFv of KW17, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of KWI 7, a Whitlow linker, and a VL region comprising 1CDR1 , 1CDR2, and 1CDR3 of KW1 7; (ii) a CD8a hinge, and (iii) a. PDGFRA transmembrane domain. In some embodiments, a synthetic CD161 engager comprises (i) an scFv of KWI 7, the scFv comprising, in N-to-C- terminal order: the VH region of KW17, a Whitlow linker, and the VL region of KW17; (ii) a. CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0071] In some embodiments, a synthetic CD161 engager comprises (1) an scFv of KW 17, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of KW17, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of KW1 7; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic CD161 engager comprises (i) an scFv of KW17, the scFv comprising, in N-to-C- terminal order: the \,rL region of KWI 7, a Whitlow linker, and the VH region of KWI 7; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0072] CD 161 -binding domains

[0073] A synthetic CD161 engager may comprise any suitable CD161-binding domain. A

[0074] CD161-binding domain may be derived from any suitable source that allows binding of CD 161, such as an antibody reagent. The term “antibody reagent” encompasses any complete antibody, as well as any antigen-binding fragment of an antibody, including a camelid heavy chain antibody fragment, an Fv, a scFv, a Fab fragment, a F(ab’) fragment, a F(ab’) fragment, an IgG fragment, a single domain VHH, a bivalent VHH, complementarity determining regions (CDRs), or a domain antibody (dAb) fragment. Complete antibodies typically contain one variable heavy chain region (VH) and one variable light chain region (VL), each of which typically comprise three CDRs that confer specificity for an antigen. CDRs are linked together by framework regions (typically four framework regions), which help maintain the structure of the variable heavy and light chains but do not affect the antigen specificity. The amino acid residues that make up the CDRs and framework regions can be identified using established numbering conventions, including Kabat, Chothia, or IMGT. Antibodies can be from any source, such as rodents, primates, or humans, and include midibodies, chimeric antibodies, and humanized antibodies. Suitable methods of humanizing antibodies to improve their efficacy in humans include, for example, grafting non-human CDRs onto human antibody frameworks. In some embodiments, a CD161-binding domain comprises means for binding CD161. In some embodiments, a CD161-binding domain comprises binding means for preferentially binding CD161. In some embodiments, a CD161-binding domain comprises binding means for specifically binding CD161. In some embodiments, the CD161-binding domain comprises a CD161-specific antibody or antigen-binding fragment thereof. In some embodiments, the CD161-binding domain is a Fv, a scFv, a Fab fragment, a F(ab’) fragment, a F(ab’)2 fragment, an IgG, a camelid heavy chain antibody, a single domain VHH, or a bivalent VHH. In some embodiments, a synthetic CD161 engager comprises an Fv that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises an scFv that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises a Fab fragment that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises a F(ab’) fragment that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises a F(ab’)2 fragment that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises an IgG that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises a camelid heavy chain antibody that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises a single domain VHH that binds, preferentially binds, or specifically binds, CD161. In some embodiments, a synthetic CD161 engager comprises a bivalent VHH that binds, preferentially binds, or specifically binds, CD161. In some embodiments, the CD161-specific binding domain comprises a heavy chain variable (VH) region and a light chain variable region (VL), where the VH region comprises three heavy chain complementarity-determining regions (hCDRs: hCDR1, hCDR2, and hCDR3) and the VL region comprises three light chain complementarity-determining regions (lCDRs: lCDR1, lCDR2, and lCDR3). In some embodiments, the CD161-binding domain is derived from the antibody KW1. See US Patent No. 11,459,389, herein incorporated by reference to the extent it describes antibody KW1. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 103, hCDR2 comprises the amino acid sequence of SEQ ID NO: 104, hCDR3 comprises the amino acid sequence of SEQ ID NO: 105, lCDR1 comprises the amino acid sequence of SEQ ID NO: 106, lCDR2 comprises the amino acid sequence of SEQ ID NO: 107, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 101 and the VL region comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 101 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 102. In some embodiments, the CD161-binding domain is derived from the antibody KW17. See US Patent No. 11,459,389, herein incorporated by reference to the extent it describes antibody KW17. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 113, hCDR2 comprises the amino acid sequence of SEQ ID NO: 114, hCDR3 comprises the amino acid sequence of SEQ ID NO: 115, lCDR1 comprises the amino acid sequence of SEQ ID NO: 116, lCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 111 and the VL region comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 111 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 112. Other suitable CD161-specific antibodies from which a CD161-binding domain may be derived are described in US Publication No. US 2021 / 0122826 and PCT Publication No. WO 2023 / 028501, which are herein incorporated by reference to the extent they describe CD161- specific antibodies. Other CD161-specific antibodies from which a CD161-binding domain may be derived include commercially available antibodies DX12, HP-3G10, QA20B14, W18070C, and 702228. In some embodiments, the VH and VL domains are connected by a linker. In some embodiments, the CD161-specific binding domain comprises, in N-to-C-terminal order, VH- Linker-VL. In some embodiments, the CD161-specific binding domain comprises, in N-to-C- terminal order, VL-Linker-VH. In some embodiments, the linker connecting the VH and VL regions is a Whitlow linker. A non-limiting example of a Whitlow linker amino acid sequence is provided by SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the linker connecting the VH and VL regions is a glycine linker. Non-limiting examples of glycine linkers are provided by SEQ ID NOs: 24–28. In some embodiments, the CD161-binding domain comprises a fragment of an LLT1 extracellular domain. In some embodiments, the CD161-binding domain comprises amino acids 165–187 of UniProt Accession No. Q9UHP7-1. See, e.g., Kamishikiryo et al., J Biol Chem. 2011.286(27):23823–23830. In some embodiments, the CD161-binding domain consists of an amino acid sequence lacking an amino acid sequence that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids long and present in SEQ ID NO: 45. Linkers In some embodiments, a synthetic CD161 engager comprises a linker connecting the CD161-binding domain to the transmembrane domain. In some embodiments, the linker connecting the CD161-binding domain and the transmembrane domain is a Whitlow linker. In some embodiments, the Whitlow linker comprises an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the linker connecting the CD161-binding domain and the transmembrane domain is a glycine linker. The skilled artisan will appreciate that glycine linkers may also include one or more serine residues. Non-limiting examples of glycine linkers are provided by SEQ ID NOs: 24–28. In some embodiments, the linker connecting the CD161- binding domain and transmembrane domain is a glycine-serine linker, such as (G4S)2(SEQ ID NO: 25). In some embodiments, the linker connecting the CD161-binding domain and transmembrane domain comprises a first G4S linker (SEQ ID NO: 24), a myc tag, and a second G4S linker (SEQ ID NO: 24) (collectively SEQ ID NO: 28). In some embodiments, the linker connecting the CD161-binding domain and transmembrane domain comprises the amino acid sequence G4S (SEQ ID NO: 24). In some embodiments, the linker connecting the CD161- binding domain and transmembrane domain comprises the amino acid sequence G4S (SEQ ID NO: 24) repeated 2, 3, 4, or 5 times in series. In some embodiments, the linker connecting the CD161-binding domain and transmembrane domain comprises the amino acid sequence G4S (SEQ ID NO: 24) repeated 3 times in series. Hinges In some embodiments, a synthetic CD161 engager comprises a hinge (also called a spacer or stalk) connecting the CD161-binding domain to the transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a hinge derived from CD8,, CD28, CD137 (4-1BB), OX40, CD3G, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD140a, CD154, or ICOS. In some embodiments, a synthetic CD161 engager comprises a CD8, hinge. In some embodiments, a synthetic CD161 engager comprises a CD28 hinge. In some embodiments, a synthetic CD161 engager comprises a CD137 (4-1BB) hinge. In some embodiments, a synthetic CD161 engager comprises an OX40 hinge. In some embodiments, a synthetic CD161 engager comprises a CD3G hinge. In some embodiments, a synthetic CD161 engager comprises a CD45 hinge. In some embodiments, a synthetic CD161 engager comprises a CD4 hinge. In some embodiments, a synthetic CD161 engager comprises a CD5 hinge. In some embodiments, a synthetic CD161 engager comprises a CD9 hinge. In some embodiments, a synthetic CD161 engager comprises a CD9 hinge. In some embodiments, a synthetic CD161 engager comprises a CD16 hinge. In some embodiments, a synthetic CD161 engager comprises a CD22 hinge. In some embodiments, a synthetic CD161 engager comprises a CD33 hinge. In some embodiments, a synthetic CD161 engager comprises a CD37 hinge. In some embodiments, a synthetic CD161 engager comprises a CD64 hinge. In some embodiments, a synthetic CD161 engager comprises a CD80 hinge. In some embodiments, a synthetic CD161 engager comprises a CD86 hinge. In some embodiments, a synthetic CD161 engager comprises a CD134 hinge. In some embodiments, a synthetic CD161 engager comprises a CD140a hinge. In some embodiments, a synthetic CD161 engager comprises a CD154 hinge. In some embodiments, a synthetic CD161 engager comprises an ICOS hinge. In some embodiments, a synthetic CD161 engager comprises a CD8, hinge. In some embodiments, the CD8, hinge comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the CD8, hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 33. In some embodiments, the CD8, hinge comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the CD8, hinge consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, a synthetic CD161 engager comprises an IgG4 hinge. In some embodiments, the IgG4 hinge comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments, the IgG4 hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 34. In some embodiments, the IgG4 hinge comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, a synthetic CD161 engager comprises a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 35. In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 35. In some embodiments, the CD28 hinge comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 35. Transmembrane domains A synthetic CD161 engager may comprise any suitable transmembrane domain. The skilled artisan will appreciate that transmembrane proteins and transmembrane domains thereof may be classified as distinct types depending on their topology relative to the cell membrane. See, e.g., Goder & Spiess, FEBS Lett.2001.504(3):87–93. In some embodiments, a synthetic CD161 engager comprises a Type I transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a Type II transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a Type III transmembrane domain. Type I transmembrane domains are those of single-pass type I membrane proteins, which span the cell membrane once, with the source protein N-terminus on the extracellular side of the membrane and have their signal sequence removed. Type II transmembrane domains are those of single-pass type II membrane proteins, which span the membrane once, with the source protein N-terminus on the cytoplasmic side of the membrane. Generally, the transmembrane domain of a single-pass type II membrane protein is close to the N-terminus and functions as an anchor. Type III transmembrane domains are those of single-pass type III membrane proteins, which span the cell membrane once, with the source protein N-terminus on the extracellular side of the membrane and no signal sequence. Non-limiting examples of Type I transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9905. Non-limiting examples of Type II transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9906. Non-limiting examples of Type II transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9906. In some embodiments, a synthetic CD161 engager comprises a transmembrane domain derived from TCR,, TCR?, TCRG, CD3H, CD3G, CD28, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137 (4-1BB), CD154, or ICOS. In some embodiments, a synthetic CD161 engager comprises a TCR, transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a TCR? transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a TCRG transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD3H transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD3G transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD28 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD45 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD4 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD5 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD7 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD8 transmembrane domain (e.g., a CD8, transmembrane domain). In some embodiments, a synthetic CD161 engager comprises a CD9 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD16 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD22 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD33 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD37 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD41 transmembrane domain. In some embodiments a synthetic CD161 engager comprises a CD64 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD68 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD80 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD86 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD134 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD137 (4-1BB) transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD154 transmembrane domain. In some embodiments, a synthetic CD161 engager comprises an ICOS transmembrane domain. In some embodiments, a synthetic CD161 engager comprises a CD8, transmembrane domain. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain consists of the amino acid sequence of SEQ ID NO: 36. In some embodiments, a synthetic CD161 engager comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, a synthetic CD161 engager comprises a type II transmembrane domain having at least one substitution, amino acid insertion, or amino acid deletion relative to a transmembrane domain of human LLT1 provided by SEQ ID NO: 38. See amino acids 39–58 of UniProt Accession No. Q9UHP7. In some embodiments, the type II transmembrane domain comprises an amino acid sequence having at least 1 substitution relative to SEQ ID NO: 38. In some embodiments, the type II transmembrane domain comprises an amino acid sequence having at least 1 amino acid insertion relative to SEQ ID NO: 38. In some embodiments, the type II transmembrane domain comprises an amino acid sequence having at least 1 amino acid deletion relative to SEQ ID NO: 38. In some embodiments, the type II transmembrane domain comprises an amino acid sequence having at least 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO: 38. In some embodiments, the type II transmembrane domain comprises an amino acid sequence having at least 1, 2, 3, 4, or 5 amino acid insertions relative to SEQ ID NO: 38. In some embodiments, the type II transmembrane domain comprises an amino acid sequence having at least 1, 2, 3, 4, or 5 amino acid deletions relative to SEQ ID NO: 38. In some embodiments, the type II transmembrane domain comprises an amino acid sequence with 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less sequence identity to SEQ ID NO: 38. In some embodiments, a synthetic CD161 engager comprises a SMIM1 transmembrane domain. See amino acids 47–67 of UniProt Accession No. B2RUZ4. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 39. In some embodiments, a synthetic CD161 engager comprises a SMIM1 transmembrane anchor. See amino acids 1–78 of UniProt Accession No. B2RUZ4. The skilled artisan will appreciate that a SMIM1 transmembrane anchor comprising the amino acid sequence of SEQ ID NO: 46, as present in CD161 engagers of SEQ ID NOs: 128–132, for example, includes the full- length SMIM1 protein amino acid sequence of UniProt Accession No. B2RUZ4. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, a synthetic CD161 engager comprises a PDGFRA transmembrane domain. See amino acids 529–549 of UniProt Accession No. P16234. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain consists of the amino acid sequence of SEQ ID NO: 40. Cytoplasmic regions In some embodiments, a synthetic CD161 engager does not comprise a cytoplasmic signaling domain. Without wishing to be bound by a particular theory, it is expected that lack of a cytoplasmic signaling domain, such that engagement of CD161 by engineered cells expressing the synthetic CD161 engager does not transduce a signal in the engineered cell, allows protection from NK cell-mediated killing with limited or no change to other intracellular processes within the engineered cell. In some embodiments, a synthetic CD161 engager comprises fewer than 50, fewer than 45, fewer than 40, fewer than 35, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than CD161, fewer than 4, fewer than 3, fewer than 2, fewer than 1, or 0 cytoplasmic amino acids. In some embodiments, a synthetic CD161 engager comprises fewer than 10 cytoplasmic amino acids. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid long. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is 1–5, 5–10, 10–15, 15–20, 20–25, 25–30, 30–35, 35–40, 40–45, or 45–50 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is 2–10 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is 1–10, 2–10, 3–10, 4–10, 5–10, 6–10, 7–10, 8–10, or 9–10 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is 1–10 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 50 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 45 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 40 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 35 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 30 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 25 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 20 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 15 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 10 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 9 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 8 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 7 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 6 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 5 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 4 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 3 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 2 amino acids in length. In some embodiments, a synthetic CD161 engager comprises a cytoplasmic region that is no more than 1 amino acid in length. Cytoplasmic amino acids of a membrane-anchored protein may be determined using predicted topology, such as that generated by DeepTHMM. Hallgren et al., bioRxiv.2022. 04.08.487609, or the annotated topology of known proteins from which transmembrane and / or cytoplasmic regions are derived as provided by UniProt. Where a transmembrane domain of a synthetic CD161 engager has the same amino acid sequence as a naturally occurring transmembrane protein, the UniProt annotation controls in the event of a conflict DeepTHMM predicted topology and UniProt annotation. In some embodiments, a synthetic CD161 engager does not comprise a C-terminal KKXX motif. When present at the C-terminus of a type I transmembrane protein, the amino acid sequence KKXX-COOH facilitates endoplasmic reticulum retention or retrieval, thereby limiting cell surface expression. See, e.g., Vincent et al., J Biol Chem.1998. 273(2):950–C"5^^^^^^4'^^^ et al., Biotechnol Bioeng. 1999.65(2):160–169. Signal peptides In some embodiments, a synthetic CD161 engager comprises a signal peptide (also called a leader peptide). In some embodiments, the signal peptide is an IgK signal peptide, a CD8, signal peptide, a CD8? signal peptide, an IL-2R, signal peptide, an IL-2R? signal peptide, an IL- 2R> signal peptide, an IL-15R, signal peptide, a CD45 signal peptide, or a GM-CSFR, signal peptide. In some embodiments, a synthetic CD161 engager comprises an IgK signal peptide. In some embodiments, a synthetic CD161 engager comprises a CD8, signal peptide. In some embodiments, a synthetic CD161 engager comprises a CD8? signal peptide. In some embodiments, a synthetic CD161 engager comprises an IL-2R, signal peptide. In some embodiments, a synthetic CD161 engager comprises an IL-2R? signal peptide. In some embodiments, a synthetic CD161 engager comprises an IL-2R> signal peptide. In some embodiments, a synthetic CD161 engager comprises an IL-15R, signal peptide. In some embodiments, a synthetic CD161 engager comprises a CD45 signal peptide. In some embodiments, a synthetic CD161 engager comprises a GM-CSFR, signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 20. NKG2A engagers Some aspects relate to a synthetic NKG2A engager. Some aspects relate to nucleic acids encoding a synthetic NKG2A engager. Some aspects relate to engineered cells expressing a synthetic NKG2A engager. Some aspects relate to engineered cells comprising a synthetic NKG2A engager. Some aspects relate to engineered cells comprising a nucleic acid encoding a synthetic NKG2A engager. NKG2A has the CD antigen name CD159a, and an exemplary amino acid sequence is provided by UniProt Accession No. P26715. A synthetic NKG2A engager may comprise an NKG2A-binding domain and a transmembrane domain. In some embodiments, the synthetic NKG2A engager comprises a hinge connecting the NKG2A-binding domain and the transmembrane domain. In some embodiments, the synthetic NKG2A engager comprises a linker connecting the NKG2A-binding domain and the transmembrane domain. Any suitable synthetic NKG2A engager that binds, preferentially binds, or specifically binds NKG2A may be encoded by a nucleic acid and / or used in engineered cells (e.g., engineered T cells). In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z199, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of Z199, a Whitlow linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of ZI99; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a

[0075] 5 synthetic NKG2A engager comprises (i) an scFv of Z199, the scFv comprising, in N-to-C- terminal order: the VH region of Z199, a Whitlow linker, and the VL region of Z 199; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0076] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z199, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR30 of Z199, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and 11CDR3 of Z199; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z199, the scFv comprising, in N-to-C- terminal order: the VL region of Z i 99. a Whitlow linker, and the VH region of Z199; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. 5 In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of rnonalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of rnonalizumab, a Whitlow linker, and a VL. region comprising 1CDR1, 1CDR2, and 1CDR3 of rnonalizumab; (ii) a. CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of 0 rnonalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of rnonalizumab, a Whitlow linker, and the VL region of rnonalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane dom ain.

[0077] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of rnonalizumab, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1 ,5 1CDR2, and 1CDR3 of rnonalizumab, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of rnonalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of rnonalizumab, the scFv comprising, in N-to-C-terminal order: the VL region of rnonalizumab, a Whitlow linker, and the VH region of rnonalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA0 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and 11CDR3 of monalizumab, a GrS linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C- terminal order: the VH region of monalizumab, a G4S linker, and the VL region of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0078] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab, a G4S linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VL region of monalizumab, a G4S linker, and the VH region of monalizumab; (ii) a. CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0079] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of monalizumab, a Whitlow linker, and a VL. region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a. CD8a hinge, and (iii) a CD8a transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab, a Whitlow linker, and the VL region of monalizumab; (ii) a CD8a hinge, and (iii) a CD8a transmembrane dom ain.

[0080] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1 , 1CDR2, and 1CDR3 of monalizumab, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a CD80. transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VL region of monalizumab, a Whitlow linker, and the VH region of monalizumab; (ii) a CD8a hinge, and (ill) a CD8a transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of monalizumab; (ii ) a CD28 hinge, and (iii) a CD28 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VL region of monalizumab, a Whitlow linker, and the VII region of monalizumab; (ii) a CD28 hinge, and (iii) a CD28 transmembrane domain.

[0081] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and 11CDR3 of monalizumab, a Whitlow linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a CD28 hinge, and (iii) a CD28 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab, a Whitlow linker, and the VL. region of monalizumab; (ii) a CD28 hinge, and (iii) a CD28 transmembrane dom ain.

[0082] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z270, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of Z270, a Whitlow linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of Z270; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z270, the scFv comprising, in N-to-C- terminal order: the VH region of Z270, a Whitlow linker, and the VL region of Z270; (ii ) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0083] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z270, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of Z270, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of Z270; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of Z270, the scFv comprising, in N-to-C- terminal order: the VL region of Z270, a Whitlow linker, and the VH region of Z270; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of 1 B2, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of 1B2, a Whitlow linker, and a VL region comprising 1CDRI, 1CDR2, and 1CDR3 of 1B2; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of 1B2, the scFv comprising, in N-to-C- terminal order: the VH region of 1B2, a Whitlow7linker, and the VL region of 1B2; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0084] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of 1B2, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of 1B2, a Whitlow7linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of 1B2; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of 1B2, the scFv comprising, in N-to-C-terminal order: the VL region of 1B2, a Whitlow7linker, and the VH region of 1B2; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0085] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of NKG2A.9, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of NKG2A.9, a Whitlow linker, and a VL region comprising 1CDR1 , 1CDR2, and 1CDR3 of NKG2A.9; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of NKG2A.9, the scFv comprising, in N-to-C-terminal order: the VH region of NKG2A.9, a Whitlow linker, and the VL region of NKG2A.9; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0086] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of NKG2A.9, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of NKG2A.9, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of NKG2A.9; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of NKG2A.9, the scFv comprising, in N-to-C-terminal order: the VL region of NKG2A.9, a Whitlow linker, and the VH region of NKG2A.9; (ii) a CD8ot hinge, and (iii) a PDGFRA transmembrane domain.

[0087] In some embodiments, a synthetic NKG2A engager comprises (1) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDR 1 , hCDR2, and hCDR3 of monalizumab having an S31A substitution, a Whitlow7linker, and a VL region comprising ICDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab having an S31A substitution, a Whitlow linker, and the VL region of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0088] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDR 1 , hCDR2, and hCDR3 of monalizumab having a D98A substitution, a Whitlow linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises

[0089] (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab having a D98A substitution, a Whitlow linker, and the VL region of monalizumab;

[0090] (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0091] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a. VH region comprising hCDRl, hCDR2, and hCDR3 of monalizumab having an E56A substitution, a Whitlow linker, and a VL region comprising 1CDR1 , 1CDR2, and 1CDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab having an E56A substitution, a Whitlow linker, and the VL region of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0092] In some embodiments, a synthetic NKG2A engager comprises, in N- to C-terminal order: (a) an scFv comprising (i) a VH comprising the amino acid sequence of SEQ ID NO: 71 with an E56 substitution, a Whitlow linker, and a VL comprising the amino acid sequence of SEQ ID NO: 72, wherein the scFv comprises an E56A substitution; (b) a CD8a hinge; and (c) a. PDGFRA transmembrane domain. In some embodiments, the CD8a hinge comprises an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the synthetic NKG2A engager does not comprise a cytoplasmic region. In some embodiments, the synthetic NKG2A engager comprises a cytoplasmic region of no more than 50 amino acids.

[0093] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and 11CDR3 of monalizumab having an R94A substitution, a Whitlow linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab having an R94A substitution, a Whitlow linker, and the VL region of monalizumab; (h) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0094] In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and 11CDR3 of monalizumab having an F99A substitution, a Whitlow linker, and a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of monalizumab; (ii) a CD8a hinge, and (iii) a. PDGFRA transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises (i) an scFv of monalizumab, the scFv comprising, in N-to-C-terminal order: the VH region of monalizumab having an F99 A substitution, a Whitlow linker, and the VL region of monalizumab; (ii) a CD8a hinge, and (iii) a PDGFR A transmembrane domain.

[0095] In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 90%' sequence identity to any one of SEQ ID NOs: 143-16.3 and 171-178. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%; sequence identity to any one of SEQ ID NOs: 143-163 and 171-178. In some embodiments, a synthetic NKG2A engager comprises any one of SEQ ID NOs: 143-16.3 and 171-178. In some embodiments, a synthetic NKG2A engager consists of any one of SEQ ID NOs: 143-163 and 171-178.

[0096] In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 143. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 143. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 144. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 144. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 145. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 145. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 146. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 146. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 147. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 147. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 148. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 148. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 149. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 149. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 150. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 150. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 151. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 151. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 151. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 152. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 152. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 152. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 153. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 153. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 153. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 154. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 154. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 154. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 155. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 155. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 156. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 156. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 157. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 157. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 157. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 158. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 158. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 159. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 159. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 159. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 160. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 160. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 161. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 161. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 162. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 162. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 163. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 163. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 163. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 171. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 171. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 172. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 172. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 172. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 173. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 173. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 174. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 174. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 175. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 175. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 176. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 177. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 177. In some embodiments, a synthetic NKG2A engager comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity with the amino acid sequence of SEQ ID NO: 178. In some embodiments, a synthetic NKG2A engager comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, the synthetic NKG2A engager consists of the amino acid sequence of SEQ ID NO: 178. The skilled artisan will appreciate that SEQ ID NOs: 143–163 and 171–178 begin with a signal peptide, but other signal peptides are suitable for expression of synthetic NKG2A engagers having the same combination of scFv, hinge, and transmembrane domain. Accordingly, in some embodiments, percent identity is calculated using the portion of the amino acid sequence downstream from the signal peptide. NKG2A-binding domains A synthetic NKG2A engager may comprise any suitable NKG2A-binding domain. An NKG2A-binding domain may be derived from any suitable source that allows binding of NKG2A, such as an antibody reagent. The term “antibody reagent” encompasses any complete antibody, as well as any antigen-binding fragment of an antibody, including a camelid heavy chain antibody fragment, an Fv, a scFv, a Fab fragment, a F(ab’) fragment, a F(ab’) fragment, an IgG fragment, a single domain VHH, a bivalent VHH, complementarity determining regions (CDRs), or a domain antibody (dAb) fragment. Complete antibodies typically contain one variable heavy chain region (VH) and one variable light chain region (VL), each of which typically comprise three CDRs that confer specificity for an antigen. CDRs are linked together by framework regions (typically four framework regions), which help maintain the structure of the variable heavy and light chains but do not affect the antigen specificity. The amino acid residues that make up the CDRs and framework regions can be identified using established numbering conventions, including Kabat, Chothia, or IMGT. Antibodies can be from any source, such as rodents, primates, or humans, and include midibodies, chimeric antibodies, and humanized antibodies. Suitable methods of humanizing antibodies to improve their efficacy in humans include, for example, grafting non-human CDRs onto human antibody frameworks. In some embodiments, an NKG2A-binding domain comprises means for binding NKG2A. In some embodiments, an NKG2A-binding domain comprises binding means for preferentially binding NKG2A. In some embodiments, an NKG2A-binding domain comprises binding means for specifically binding NKG2A. In some embodiments, the NKG2A-binding domain comprises an NKG2A-specific antibody or antigen-binding fragment thereof. In some embodiments, the NKG2A-binding domain is a Fv, a scFv, a Fab fragment, a F(ab’) fragment, a F(ab’)2 fragment, an IgG, a camelid heavy chain antibody, a single domain VHH, or a bivalent VHH. In some embodiments, a synthetic NKG2A engager comprises an Fv that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises an scFv that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises a Fab fragment that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises a F(ab’) fragment that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises a F(ab’)2 fragment that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises an IgG that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises a camelid heavy chain antibody that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises a single domain VHH that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, a synthetic NKG2A engager comprises a bivalent VHH that binds, preferentially binds, or specifically binds NKG2A. In some embodiments, the NKG2A-specific binding domain comprises a heavy chain variable (VH) region and a light chain variable region (VL), where the VH region comprises three heavy chain complementarity-determining regions (hCDRs: hCDR1, hCDR2, and hCDR3) and the VL region comprises three light chain complementarity-determining regions (lCDRs: lDR1, lDR2, and lDR3). In some embodiments, the NKG2A-binding domain is derived from the antibody Z199. See PCT Publication No. WO 2009 / 092805, herein incorporated by reference to the extent it describes antibody Z199. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 53, hCDR2 comprises the amino acid sequence of SEQ ID NO: 54, hCDR3 comprises the amino acid sequence of SEQ ID NO: 55, lCDR1 comprises the amino acid sequence of SEQ ID NO: 56, lCDR2 comprises the amino acid sequence of SEQ ID NO: 57, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, (i) hCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 53, hCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 54, hCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 55, lCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 56, lCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 57, and lCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 51 and the VL region comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 51 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, the VH region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 51 and the VL region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 52. In some embodiments, the VH region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 51 and the VL region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 52. In some embodiments, the NKG2A-binding domain is derived from the antibody Z270. See US Patent No. 8,206,709, herein incorporated by reference to the extent it describes antibody Z270. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 63, hCDR2 comprises the amino acid sequence of SEQ ID NO: 64, hCDR3 comprises the amino acid sequence of SEQ ID NO: 65, lCDR1 comprises the amino acid sequence of SEQ ID NO: 66, lCDR2 comprises the amino acid sequence of SEQ ID NO: 67, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, (i) hCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 63, hCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 64, hCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 65, lCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 66, lCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 67, and lCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 68. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 61 and the VL region comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 61 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 62. In some embodiments, the VH region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 61 and the VL region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 62. In some embodiments, the VH region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 61 and the VL region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 62. In some embodiments, the NKG2A-binding domain comprises an alanine at one or more positions corresponding to S31, E56, R94, D98, and / or F99 of the VH region of antibody Z270. These residues are annotated in the Exemplary Sequences table below, and Figure 12 of US Patent No. 8,206,709. The skilled artisan will appreciate that S31, E56, R94, D98, and F99 are numbered using the Kabat numbering scheme. The skilled artisan will appreciate that S31, E56, R94, D98, and F99 are numbered using the Kabat numbering scheme, and correspond to the serine (S) at position 31 of SEQ ID NO: 71, the glutamate (E) at position 57 of SEQ ID NO: 71, the arginine (R) at position 98 of SEQ ID NO: 71, the aspartate (D) at position 108 of SEQ ID NO: 61, and the phenylalanine (F) at position 109 of SEQ ID NO: 71, respectively. In some embodiments, the NKG2A-binding domain is derived from the antibody monalizumab. See PCT Publication No. WO 2020 / 225552, herein incorporated by reference to the extent it describes monalizaumab. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 73, hCDR2 comprises the amino acid sequence of SEQ ID NO: 74, hCDR3 comprises the amino acid sequence of SEQ ID NO: 75, lCDR1 comprises the amino acid sequence of SEQ ID NO: 76, lCDR2 comprises the amino acid sequence of SEQ ID NO: 77, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, (i) hCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 73, hCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 74, hCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 75, lCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 76, lCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 77, and lCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 71 and the VL region comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 71 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, the VH region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 71 and the VL region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, the VH region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 71 and the VL region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 72. In some embodiments, the NKG2A-binding domain comprises an alanine at one or more positions corresponding to S31, E56, R94, D98, and / or F99 of the VH region of monalizumab. These residues are annotated in the Exemplary Sequences tables below, and Figure 12 of US Patent No. 8,206,709. The skilled artisan will appreciate that S31, E56, R94, D98, and F99 are numbered using the Kabat numbering scheme, and correspond to the serine (S) at position 31 of SEQ ID NO: 71, the glutamate (E) at position 57 of SEQ ID NO: 71, the arginine (R) at position 98 of SEQ ID NO: 71, the aspartate (D) at position 108 of SEQ ID NO: 71, and the phenylalanine (F) at position 109 of SEQ ID NO: 71, respectively. In some embodiments, the NKG2A-binding domain is derived from the antibody NKG2A.9. See US Patent No. 11,274,150, herein incorporated by reference to the extent it describes antibody NKG2A.9. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 83, hCDR2 comprises the amino acid sequence of SEQ ID NO: 84, hCDR3 comprises the amino acid sequence of SEQ ID NO: 85, lCDR1 comprises the amino acid sequence of SEQ ID NO: 86, lCDR2 comprises the amino acid sequence of SEQ ID NO: 87, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, (i) hCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 83, hCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 84, hCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 85, lCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 86, lCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 87, and lCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 81 and the VL region comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 81 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 82. In some embodiments, the VH region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 81 and the VL region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 82. In some embodiments, the VH region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 81 and the VL region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 82. In some embodiments, the NKG2A-binding domain is derived from the antibody 1B2. See PCT Publication No. WO 2023 / 114176, herein incorporated by reference to the extent it describes monalizaumab. In some embodiments, (i) hCDR1 comprises the amino acid sequence of SEQ ID NO: 93, hCDR2 comprises the amino acid sequence of SEQ ID NO: 94, hCDR3 comprises the amino acid sequence of SEQ ID NO: 95, lCDR1 comprises the amino acid sequence of SEQ ID NO: 96, lCDR2 comprises the amino acid sequence of SEQ ID NO: 97, and lCDR3 comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, (i) hCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 93, hCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 94, hCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 95, lCDR1 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 96, lCDR2 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 97, and lCDR3 comprises no more than 2 substitutions, amino acid insertions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 91 and the VL region comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the VH region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 91 and the VL region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 92. In some embodiments, the VH region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 91 and the VL region comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 92. In some embodiments, the VH region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 91 and the VL region comprises an amino acid sequence with no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 92. Other NKG2A-specific antibodies from which an NKG2A-binding domain may be derived are described in US Patent Nos.11,274,150 and 8,206,709 and PCT Publication No. WO 2023 / 114176, which are herein incorporated by reference to the extent they describe NKG2A- specific antibodies. Linkers In some embodiments, the VH and VL domains are connected by a linker. In some embodiments, the NKG2A-specific binding domain comprises, in N-to-C-terminal order, VH- Linker-VL. In some embodiments, the NKG2A-specific binding domain comprises, in N-to-C- terminal order, VL-Linker-VH. In some embodiments, the linker connecting the VH and VL regions is a Whitlow linker. A non-limiting example of a Whitlow linker amino acid sequence is provided by SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the linker connecting the VH and VL regions is a glycine linker. Non-limiting examples of glycine linkers are provided by SEQ ID NOs: 24–28. In some embodiments, the linker connecting the VH and VL regions is a glycine-serine linker, such as (G4S) (SEQ ID NO: 24) or (G4S)2 (SEQ ID NO: 25). A glycine-serine linker may comprise the amino acid sequence G4S repeated multiple times in series. In some embodiments, the linker comprises the amino acid sequence (G4S) (SEQ ID NO: 24) repeated 2, 3, 4, or 5 times in series. In some embodiments, the linker comprises the amino acid sequence (G4S) (SEQ ID NO: 24). In some embodiments, the linker comprises the amino acid sequence (G4S)2 (SEQ ID NO: 25). In some embodiments, the linker comprises the amino acid sequence (G4S) (SEQ ID NO: 24) repeated three times in series. In some embodiments, the linker connecting the VH and VL regions is 5 to 30 amino acids long. In some embodiments, the linker is 5-25, 5-20, 5-15, or 5-10 amino acids long. In some embodiments, the linker is 10-30, 15-30, 20-30, or 25-30 amino acids long. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids long. In some embodiments, the linker is 10-20 amino acids long. In some embodiments, the linker is 15-20 amino acids long. Hinges In some embodiments, a synthetic NKG2A engager comprises a hinge (also called a spacer or stalk) connecting the NKG2A-binding domain to the transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a hinge derived from CD8,, CD28, CD137 (4-1BB), OX40, CD3G, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD140a, CD154, or ICOS. In some embodiments, a synthetic NKG2A engager comprises a CD8, hinge. In some embodiments, a synthetic NKG2A engager comprises a CD28 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD137 (4-1BB) hinge. In some embodiments, a synthetic NKG2A engager comprises an OX40 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD3G hinge. In some embodiments, a synthetic NKG2A engager comprises a CD45 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD4 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD5 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD9 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD9 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD16 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD22 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD33 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD37 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD64 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD80 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD86 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD134 hinge. In some embodiments, a synthetic NKG2A engager comprises a CD140a hinge. In some embodiments, a synthetic NKG2A engager comprises a CD154 hinge. In some embodiments, a synthetic NKG2A engager comprises an ICOS hinge. In some embodiments, a synthetic NKG2A engager comprises a CD8, hinge. In some embodiments, the CD8, hinge comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the CD8, hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 33. In some embodiments, the CD8, hinge comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the CD8, hinge consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, a synthetic NKG2A engager comprises an IgG4 hinge. In some embodiments, the IgG4 hinge comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments, the IgG4 hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 34. In some embodiments, the IgG4 hinge comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, a synthetic NKG2A engager comprises a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 35. In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 35. In some embodiments, the CD28 hinge comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 35. Transmembrane domains A synthetic NKG2A engager may comprise any suitable transmembrane domain. The skilled artisan will appreciate that transmembrane proteins and transmembrane domains thereof may be classified as distinct types depending on their topology relative to the cell membrane. See, e.g., Goder & Spiess, FEBS Lett.2001.504(3):87–93. In some embodiments, a synthetic NKG2A engager comprises a Type I transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a Type II transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a Type III transmembrane domain. Non-limiting examples of Type I transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9905. Non-limiting examples of Type II transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9906. Non-limiting examples of Type II transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9906. In some embodiments, a synthetic NKG2A engager comprises a transmembrane domain derived from TCR,, TCR?, TCRG, CD3H, CD3G, CD28, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137 (4-1BB), CD154, or ICOS. In some embodiments, a synthetic NKG2A engager comprises a TCR, transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a TCR? transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a TCRG transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD3H transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD3G transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD28 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD45 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD4 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD5 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD7 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD8 transmembrane domain (e.g., a CD8, transmembrane domain). In some embodiments, a synthetic NKG2A engager comprises a CD9 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD16 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD22 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD33 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD37 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD41 transmembrane domain. In some embodiments a synthetic NKG2A engager comprises a CD64 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD68 transmembrane domain. In some embodiments a synthetic NKG2A engager comprises a CD80 transmembrane domain. In some embodiments a synthetic NKG2A engager comprises a CD86 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD134 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD137 (4-1BB) transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD154 transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises an ICOS transmembrane domain. In some embodiments, a synthetic NKG2A engager comprises a CD8, transmembrane domain. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain consists of the amino acid sequence of SEQ ID NO: 36. In some embodiments, a synthetic NKG2A engager comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, a synthetic NKG2A engager comprises a SMIM1 transmembrane domain. See amino acids 47–67 of UniProt Accession No. B2RUZ4. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 39. In some embodiments, a synthetic CD161 engager comprises a SMIM1 transmembrane anchor. See amino acids 1–78 of UniProt Accession No. B2RUZ4. The skilled artisan will appreciate that a SMIM1 transmembrane anchor comprising the amino acid sequence of SEQ ID NO: 46, as present in CD161 engagers of SEQ ID NOs: 128–132, for example, includes the full- length SMIM1 protein amino acid sequence of UniProt Accession No. B2RUZ4. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, a synthetic NKG2A engager comprises a PDGFRA transmembrane domain. See amino acids 529–549 of UniProt Accession No. P16234. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain consists of the amino acid sequence of SEQ ID NO: 40. Cytoplasmic regions In some embodiments, a synthetic NKG2A engager does not comprise a cytoplasmic signaling domain. Without wishing to be bound by a particular theory, it is expected that lack of a cytoplasmic signaling domain, such that engagement of NKG2A by engineered cells expressing the synthetic NKG2A engager does not transduce a signal in the engineered cell, allows protection from NK cell-mediated killing with limited or no change to other intracellular processes within the engineered cell. In some embodiments, a synthetic NKG2A engager comprises fewer than 50, fewer than 45, fewer than 40, fewer than 35, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than NKG2A, fewer than 4, fewer than 3, fewer than 2, fewer than 1, or 0 cytoplasmic amino acids. In some embodiments, a synthetic NKG2A engager comprises fewer than 10 cytoplasmic amino acids. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid long. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is 1–5, 5–10, 10–15, 15–20, 20–25, 25–30, 30–35, 35–40, 40–45, or 45–50 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is 2–10 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is 1–10, 2–10, 3–10, 4–10, 5–10, 6–10, 7–10, 8–10, or 9–10 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is 1–10 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 50 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 45 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 40 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 35 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 30 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 25 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 20 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 15 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 10 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 9 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 8 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 7 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 6 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 5 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 4 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 3 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 2 amino acids in length. In some embodiments, a synthetic NKG2A engager comprises a cytoplasmic region that is no more than 1 amino acid in length. Cytoplasmic amino acids of a membrane-anchored protein may be determined using predicted topology, such as that generated by DeepTHMM. Hallgren et al., bioRxiv.2022. 04.08.487609, or the annotated topology of known proteins from which transmembrane and / or cytoplasmic regions are derived as provided by UniProt. Where a transmembrane domain of a synthetic NKG2A engager has the same amino acid sequence as a naturally occurring transmembrane protein, the UniProt annotation controls in the event of a conflict DeepTHMM predicted topology and UniProt annotation. In some embodiments, a synthetic NKG2A engager does not comprise a C-terminal KKXX motif. When present at the C-terminus of a type I transmembrane protein, the amino acid sequence KKXX-COOH facilitates endoplasmic reticulum retention or retrieval, thereby limiting cell surface expression. See, e.g., Vincent et al., J Biol Chem.1998. 273(2):950–C"5^^^^^^4'^^^ et al., Biotechnol Bioeng. 1999.65(2):160–169. Signal peptides In some embodiments, a synthetic NKG2A engager comprises a signal peptide (also called a leader peptide). In some embodiments, the signal peptide is an IgK signal peptide, a CD8, signal peptide, a CD8? signal peptide, an IL-2R, signal peptide, an IL-2R? signal peptide, an IL-2R> signal peptide, an IL-15R, signal peptide, a CD45 signal peptide, or a GM-CSFR, signal peptide. In some embodiments, a synthetic NKG2A engager comprises an IgK signal peptide. In some embodiments, a synthetic NKG2A engager comprises a CD8, signal peptide. In some embodiments, a synthetic NKG2A engager comprises a CD8? signal peptide. In some embodiments, a synthetic NKG2A engager comprises an IL-2R, signal peptide. In some embodiments, a synthetic NKG2A engager comprises an IL-2R? signal peptide. In some embodiments, a synthetic NKG2A engager comprises an IL-2R> signal peptide. In some embodiments, a synthetic NKG2A engager comprises an IL-15R, signal peptide. In some embodiments, a synthetic NKG2A engager comprises a CD45 signal peptide. In some embodiments, a synthetic NKG2A engager comprises a GM-CSFR, signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 20. NK cell inhibitory receptor engagers Some aspects relate to a synthetic NK cell inhibitory receptor engager. Some aspects relate to nucleic acids encoding a synthetic NK cell inhibitory receptor engager. Some aspects relate to engineered cells expressing a synthetic NK cell inhibitory receptor engager. Some aspects relate to engineered cells comprising a synthetic NK cell inhibitory receptor engager. Some aspects relate to engineered cells comprising a nucleic acid encoding a synthetic NK cell inhibitory receptor engager. In some embodiments, the synthetic NK inhibitory receptor engager is a synthetic CD300A engager or a synthetic KIR2D2L engager. In some embodiments, the synthetic NK cell inhibitory receptor engager is a synthetic CD300A engager. In some embodiments, a synthetic CD300A engager comprises (i) an scFv of CD300A077, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDR1, hCDR2, and hCDR3 of CD300A077, a Whitlow linker, and a VL region comprising lCDR1, ^403^^^^^^^^403!^^^^40!^^^^;;^^*^^+^^^CD8, hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic CD300A engager comprises (i) an scFv of CD300A077, the scFv comprising, in N-to-C-terminal order: the VH region of CD300A077, a Whitlow linker, and the VL region of CD300A077^^*^^+^^^CD8, hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic CD300A engager comprises (i) an scFv of CD300A077, the scFv comprising, in N-to-C-terminal order: a VL region comprising lCDR1, lCDR2, and lCDR3 of CD300A077, a Whitlow linker, and a VH region comprising hCDR1, hCDR2, and h403!^^^^40!^^^^;;^^*^^+^^^CD8, hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic CD300A engager comprises (i) an scFv of CD300A077, the scFv comprising, in N-to-C-terminal order: the VL region of CD300A077, a Whitlow linker, and the VH region of CD300A077; (ii) a CD8a hinge, and (iii) a PDGFR.A transmembrane domain.

[0097] In some embodiments, a synthetic CD300A engager comprises (i) an scFv of 25A11, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1, 1CDR2, and 1CDR3 of 25A11, a Whitlow linker, and a VH region comprising hCDRl, hCDR2, and hCDR3 of 25 Al I ; (ii) a CD8a hinge, and (iii) a CD8a transmembrane domain. In some embodiments, a synthetic CD300A engager comprises (i) an scFv of 25A11, the scFv comprising, in N-to-C- terminal order: the VL region of 25 Al 1 , a Whitlow' linker, and the VH region of 25 Al 1 ; (ii) a CD8a hinge, and (iii) a CD8a transmembrane domain.

[0098] In some embodiments, the synthetic NK ceil inhibitory receptor engager is a synthetic KIR2D2L engager. In some embodiments, a synthetic KIR2D2L engager comprises (i) an scFv of lirilumab, the scFv comprising, in N-to-C-terminal order: a VH region comprising hCDRl, hCDR2, and hCDR3 of lirilumab, a Whitlow linker, and a VL. region comprising 1CDR 1, 1CDR2, and 1CDR3 of lirilumab; (ii) a CD8a hinge, and (iii) a PDGFR.A transmembrane domain. In some embodiments, a synthetic KJR2D2L engager comprises (i) an scFv of lirilumab, the scFv comprising, in N-to-C-terminal order: the VH region of lirilumab, a Whitlow linker, and the VL. region of lirilumab; (ii) a. CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0099] In some embodiments, a synthetic KIR2D2L engager comprises (i) an scFv of lirilumab, the scFv comprising, in N-to-C-terminal order: a VL region comprising 1CDR1 , 1CDR2, and 1CDR3 of lirilumab, a Whitlow linker, and a VH region comprising hCDRl , hCDR2, and hCDR3 of lirilumab; (ii) a. CD8a hinge, and (iii) a PDGFRA transmembrane domain. In some embodiments, a synthetic KIR2D2L engager comprises (i) an scFv of lirilumab, the scFv comprising, in N-to-C-terminal order: the V L region of lirilumab, a Whitlow linker, and the VH region of lirilumab; (ii) a CD8a hinge, and (iii) a PDGFRA transmembrane domain.

[0100] NK cell inhibitory receptors and binding domains thereto

[0101] A synthetic NK cell inhibitory receptor engager may comprise an NK cell inhibitory receptor-binding domain and a transmembrane domain. In some embodiments, the synthetic NK cell inhibitory receptor engager comprises a hinge connecting the NK cell inhibitory receptorbinding domain and the transmembrane domain. Any suitable synthetic NK cell inhibitory receptor engager that preferentially binds an NK cell inhibitory receptor may be encoded by a nucleic acid and / or used in engineered cells (e.g., engineered T cells). The binding domain may bind any suitable NK cell inhibitory receptor. In some embodiments, the NK cell inhibitory receptor is CD300A. CD300A has the CD antigen name CD300a, and an exemplary amino acid sequence is provided by UniProt Accession No. Q9UGN4. In some embodiments, the NK cell inhibitory receptor is CD96. CD96 has the CD antigen name CD96, and an exemplary amino acid sequence is provided by UniProt Accession No. P40200. In some embodiments, the NK cell inhibitory receptor is HAVCR2. HAVCR2 has the CD antigen name CD366, and an exemplary amino acid sequence is provided by UniProt Accession No. Q8TDQ0. In some embodiments, the NK cell inhibitory receptor is KIR2DL1. KIR2DL1 has the CD antigen name CD158a, and an exemplary amino acid sequence is provided by UniProt Accession No. P43626. In some embodiments, the NK cell inhibitory receptor is KIR2DL2. KIR2DL2 has the CD antigen name CD158b1, and an exemplary amino acid sequence is provided by UniProt Accession No. P43627. In some embodiments, the NK cell inhibitory receptor is KIR2DL3. KIR2DL3 has the CD antigen name CD158b2, and an exemplary amino acid sequence is provided by UniProt Accession No. P43628. In some embodiments, the NK cell inhibitory receptor is KIR2DL5. KIR2DL5 has the CD antigen name CD158f1, and an exemplary amino acid sequence is provided by UniProt Accession No. Q8N109. In some embodiments, the NK cell inhibitory receptor is KIR3DL1. KIR3DL1 has the CD antigen name CD158e, and an exemplary amino acid sequence is provided by UniProt Accession No. P43629. In some embodiments, the NK cell inhibitory receptor is KIR3DL2. KIR3DL2 has the CD antigen name CD158k, and an exemplary amino acid sequence is provided by UniProt Accession No. P43630. In some embodiments, the NK cell inhibitory receptor is KIR3DL3. KIR3DL3 has the CD antigen name CD158z, and an exemplary amino acid sequence is provided by UniProt Accession No. Q8N743. In some embodiments, the NK cell inhibitory receptor is KLRG1. KLRG1 has the CD antigen name CLEC15A, and an exemplary amino acid sequence is provided by UniProt Accession No. Q96E93. In some embodiments, the NK cell inhibitory receptor is LAG-3. LAG-3 has the CD antigen name CD223, and an exemplary amino acid sequence is provided by UniProt Accession No. P18627. In some embodiments, the NK cell inhibitory receptor is LAIR1. LAIR1 has the CD antigen name CD305, and an exemplary amino acid sequence is provided by UniProt Accession No. Q6GTX8. In some embodiments, the NK cell inhibitory receptor is LILRB1. LILRB1 has the CD antigen name CD85j, and an exemplary amino acid sequence is provided by UniProt Accession No. Q8NHL6. In some embodiments, the NK cell inhibitory receptor is LILRB3. LILRB3 has the CD antigen name CD85a, and an exemplary amino acid sequence is provided by UniProt Accession No. O75022. In some embodiments, the NK cell inhibitory receptor is PD-1. PD-1 has the CD antigen name CD279, and an exemplary amino acid sequence is provided by UniProt Accession No. Q15116. In some embodiments, the NK cell inhibitory receptor is RTN4R. An exemplary amino acid sequence of RTNR4 is provided by UniProt Accession No. Q9BZR6. In some embodiments, the NK cell inhibitory receptor is Siglec-3. Siglec-3 has the CD antigen name CD33, and an exemplary amino acid sequence is provided by UniProt Accession No. P20138. In some embodiments, the NK cell inhibitory receptor is Siglec-7. Siglec-7 has the CD antigen name CD328, and an exemplary amino acid sequence is provided by UniProt Accession No. Q9Y286. In some embodiments, the NK cell inhibitory receptor is Siglec-9. Siglec-9 has the CD antigen name CD329, and an exemplary amino acid sequence is provided by UniProt Accession No. Q9Y336. In some embodiments, the NK cell inhibitory receptor is SIRP,. SIRP, has the CD antigen name CD172a, and an exemplary amino acid sequence is provided by UniProt Accession No. P78324. In some embodiments, the NK cell inhibitory receptor is TIGIT. An exemplary amino acid sequence of TIGIT is provided by UniProt Accession No. Q495A1. A synthetic NK cell inhibitory receptor engager may comprise any suitable an NK cell inhibitory receptor-binding domain. An NK cell inhibitory receptor-binding domain may be derived from any suitable source that allows binding of an NK cell inhibitory receptor, such as an antibody reagent. The term “antibody reagent” encompasses any complete antibody, as well as any antigen-binding fragment of an antibody, including a camelid heavy chain antibody fragment, an Fv, a scFv, a Fab fragment, a F(ab’) fragment, a F(ab’) fragment, an IgG fragment, a single domain VHH, a bivalent VHH, complementarity determining regions (CDRs), or a domain antibody (dAb) fragment. Complete antibodies typically contain one variable heavy chain region (VH) and one variable light chain region (VL), each of which typically comprise three CDRs that confer specificity for an antigen. CDRs are linked together by framework regions (typically four framework regions), which help maintain the structure of the variable heavy and light chains but do not affect the antigen specificity. The amino acid residues that make up the CDRs and framework regions can be identified using established numbering conventions, including Kabat, Chothia, or IMGT. Antibodies can be from any source, such as rodents, primates, or humans, and include midibodies, chimeric antibodies, and humanized antibodies. Suitable methods of humanizing antibodies to improve their efficacy in humans include, for example, grafting non-human CDRs onto human antibody frameworks. In some embodiments, an NK cell inhibitory receptor-binding domain comprises means for binding the NK cell inhibitory receptor. In some embodiments, an NK cell inhibitory receptor-binding domain comprises binding means for preferentially binding NK cell inhibitory receptor. In some embodiments, an NK cell inhibitory receptor-binding domain comprises binding means for specifically binding the NK cell inhibitory receptor. In some embodiments, the NK cell inhibitory receptor-binding domain comprises an NK cell inhibitory receptor-specific antibody or antigen-binding fragment thereof. In some embodiments, the NK cell inhibitory receptor-binding domain is a Fv, a scFv, a Fab fragment, a F(ab’) fragment, a F(ab’)2 fragment, an IgG, a camelid heavy chain antibody, a single domain VHH, or a bivalent VHH. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an Fv that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an scFv that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a Fab fragment that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a F(ab’) fragment that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a F(ab’)2 fragment that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IgG that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a camelid heavy chain antibody that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a single domain VHH that binds an NK cell inhibitory receptor. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a bivalent VHH that binds an NK cell inhibitory receptor. In some embodiments, the NK cell inhibitory receptor-specific binding domain comprises a heavy chain variable (VH) region and a light chain variable region (VL), where the VH region comprises three heavy chain complementarity-determining regions (hCDRs: hCDR1, hCDR2, and hCDR3) and the VL region comprises three light chain complementarity-determining regions (lCDRs: lCDR1, lCDR2, and lCDR3). Linkers In some embodiments, the VH and VL domains are connected by a linker. In some embodiments, the NK cell inhibitory receptor-specific binding domain comprises, in N-to-C- terminal order, VH-Linker-VL. In some embodiments, the NK cell inhibitory receptor-specific binding domain comprises, in N-to-C-terminal order, VL-Linker-VH. In some embodiments, the linker connecting the VH and VL regions is a Whitlow linker. A non-limiting example of a Whitlow linker amino acid sequence is provided by SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of an amino acid sequence having no more than 1, 2, or 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 29. In some embodiments, the Whitlow linker comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the Whitlow linker consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the linker connecting the VH and VL regions is a glycine linker. Non-limiting examples of glycine linkers are provided by SEQ ID NOs: 24–28. In some embodiments, the linker connecting the VH and VL regions is a glycine-serine linker, such as (G4S) (SEQ ID NO: 24) or (G4S)2 (SEQ ID NO: 25). A glycine-serine linker may comprise the amino acid sequence G4S repeated multiple times in series. In some embodiments, the linker comprises the amino acid sequence (G4S) (SEQ ID NO: 24) repeated 2, 3, 4, or 5 times in series. In some embodiments, the linker comprises the amino acid sequence (G4S) (SEQ ID NO: 24). In some embodiments, the linker comprises the amino acid sequence (G4S)2(SEQ ID NO: 25). In some embodiments, the linker comprises the amino acid sequence (G4S) (SEQ ID NO: 24) repeated three times in series. In some embodiments, the linker connecting the VH and VL regions is 5 to 30 amino acids long. In some embodiments, the linker is 5-25, 5-20, 5-15, or 5-10 amino acids long. In some embodiments, the linker is 10-30, 15-30, 20-30, or 25-30 amino acids long. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids long. In some embodiments, the linker is 10-20 amino acids long. In some embodiments, the linker is 15-20 amino acids long. Hinges In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a hinge (also called a spacer or stalk) connecting the NK cell inhibitory receptor-binding domain to the transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a hinge derived from CD8,, CD28, CD137 (4-1BB), OX40, CD3G, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD140a, CD154, or ICOS. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD8, hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD28 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD137 (4-1BB) hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an OX40 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD3G hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD45 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD4 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD5 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD9 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD9 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD16 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD22 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD33 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD37 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD64 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD80 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD86 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD134 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD140a hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD154 hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an ICOS hinge. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD8, hinge. In some embodiments, the CD8, hinge comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the CD8, hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 33. In some embodiments, the CD8, hinge comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the CD8, hinge consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IgG4 hinge. In some embodiments, the IgG4 hinge comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments, the IgG4 hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 34. In some embodiments, the IgG4 hinge comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 35. In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 35. In some embodiments, the CD28 hinge comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 35. Transmembrane domains A synthetic NK cell inhibitory receptor engager may comprise any suitable transmembrane domain. The skilled artisan will appreciate that transmembrane proteins and transmembrane domains thereof may be classified as distinct types depending on their topology relative to the cell membrane. See, e.g., Goder & Spiess, FEBS Lett.2001.504(3):87–93. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a Type I transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a Type II transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a Type III transmembrane domain. Non-limiting examples of Type I transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9905. Non-limiting examples of Type II transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9906. Non-limiting examples of Type II transmembrane domains include transmembrane domains of proteins provided in the list of UniProt subcellular location SL-9906. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a transmembrane domain derived from TCR,, TCR?, TCRG, CD3H, CD3G, CD28, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137 (4-1BB), CD154, or ICOS. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a TCR, transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a TCR? transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a TCRG transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD3H transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD3G transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD28 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD45 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD4 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD5 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD7 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD8 transmembrane domain (e.g., a CD8, transmembrane domain). In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD9 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD16 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD22 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD33 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD37 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD41 transmembrane domain. In some embodiments a synthetic NK cell inhibitory receptor engager comprises a CD64 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD68 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD80 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD86 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD134 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD137 (4-1BB) transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD154 transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an ICOS transmembrane domain. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD8, transmembrane domain. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the CD8, transmembrane domain consists of the amino acid sequence of SEQ ID NO: 36. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a SMIM1 transmembrane domain. See amino acids 47–67 of UniProt Accession No. B2RUZ4. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the SMIM1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 39. In some embodiments, a synthetic CD161 engager comprises a SMIM1 transmembrane anchor. See amino acids 1–78 of UniProt Accession No. B2RUZ4. The skilled artisan will appreciate that a SMIM1 transmembrane anchor comprising the amino acid sequence of SEQ ID NO: 46, as present in CD161 engagers of SEQ ID NOs: 128–132, for example, includes the full- length SMIM1 protein amino acid sequence of UniProt Accession No. B2RUZ4. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, the SMIM1 transmembrane anchor consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a PDGFRA transmembrane domain. See amino acids 529–549 of UniProt Accession No. P16234. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence having no more than 1, no more than 2, or no more than 3 substitutions, amino acid insertions, and / or amino acid deletions relative to SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the PDGFRA transmembrane domain consists of the amino acid sequence of SEQ ID NO: 40. Cytoplasmic regions In some embodiments, a synthetic NK cell inhibitory receptor engager does not comprise a cytoplasmic signaling domain. Without wishing to be bound by a particular theory, it is expected that lack of a cytoplasmic signaling domain, such that engagement of NK cell inhibitory receptor by engineered cells expressing the synthetic NK cell inhibitory receptor engager does not transduce a signal in the engineered cell, allows protection from NK cell- mediated killing with limited or no change to other intracellular processes within the engineered cell. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises fewer than 50, fewer than 45, fewer than 40, fewer than 35, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than NK cell inhibitory receptor, fewer than 4, fewer than 3, fewer than 2, fewer than 1, or 0 cytoplasmic amino acids. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises fewer than 10 cytoplasmic amino acids. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid long. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is 1–5, 5–10, 10–15, 15–20, 20–25, 25–30, 30–35, 35–40, 40–45, or 45–50 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is 2–10 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is 1–10, 2–10, 3–10, 4–10, 5–10, 6–10, 7–10, 8–10, or 9–10 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is 1–10 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 50 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 45 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 40 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 35 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 30 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 25 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 20 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 15 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 10 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 9 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 8 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 7 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 6 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 5 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 4 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 3 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 2 amino acids in length. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a cytoplasmic region that is no more than 1 amino acid in length. Cytoplasmic amino acids of a membrane-anchored protein may be determined using predicted topology, such as that generated by DeepTHMM. Hallgren et al., bioRxiv.2022. 04.08.487609, or the annotated topology of known proteins from which transmembrane and / or cytoplasmic regions are derived as provided by UniProt. Where a transmembrane domain of a synthetic NK cell inhibitory receptor engager has the same amino acid sequence as a naturally occurring transmembrane protein, the UniProt annotation controls in the event of a conflict DeepTHMM predicted topology and UniProt annotation. In some embodiments, a synthetic NK cell inhibitory receptor engager does not comprise a C-terminal KKXX motif. When present at the C-terminus of a type I transmembrane protein, the amino acid sequence KKXX-COOH facilitates endoplasmic reticulum retention or retrieval, thereby limiting cell surface expression. See, e.g., Vincent et al., J Biol Chem.1998.273(2):950– C"5^^^^^^4'^^^et al., Biotechnol Bioeng.1999. 65(2):160–169. Signal peptides In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a signal peptide (also called a leader peptide). In some embodiments, the signal peptide is an IgK signal peptide, a CD8, signal peptide, a CD8? signal peptide, an IL-2R, signal peptide, an IL-2R? signal peptide, an IL-2R> signal peptide, an IL-15R, signal peptide, a CD45 signal peptide, or a GM-CSFR, signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IgK signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD8, signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD8? signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IL-2R, signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IL-2R? signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IL-2R> signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises an IL- 15R, signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a CD45 signal peptide. In some embodiments, a synthetic NK cell inhibitory receptor engager comprises a GM-CSFR, signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 20. Engineered cells Some aspects relate to engineered cells comprising a synthetic NK cell inhibitory receptor engager or one or more nucleic acids encoding a synthetic NK cell inhibitory receptor engager. As shown in the Examples, expression of a synthetic NK cell inhibitory receptor engager benefits cells by protecting them from NK cell-mediated killing. In some embodiments, an engineered cell comprises a nucleic acid (e.g., a nucleotide sequence) encoding a synthetic NK cell inhibitory receptor engager. In some embodiments, a nucleotide sequence encoding a synthetic NK cell inhibitory receptor engager is in a chromosome of the cell. In some embodiments, a nucleotide sequence encoding a synthetic NK cell inhibitory receptor engager is episomal. In some embodiments, a nucleotide sequence encoding a synthetic NK cell inhibitory receptor engager is present in a targeted locus of a chromosome of the cell. Example of suitable targeted loci are described in the section entitled “Targeted loci.” In some embodiments, a heterologous promoter is operably linked to the nucleotide sequence encoding the synthetic NK cell inhibitory receptor engager. In some embodiments, an engineered cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the stem cell is a CD34+ hematopoietic stem cell. Engineered stem cells may be matured in vitro to produce differentiated (e.g., Treg) cells suitable for administration, or administered to allow for in vivo development into differentiated (e.g., Treg) cells. Engineered stem cells may be matured into Treg cells, such as CD3+ Treg cells, CD4+ Treg cells, CD8+ Treg cells, NK-Treg cells, or combinations thereof. Engineered stem cells may be matured into multiple cell types including, without limitation, islet cells, hepatocytes, neurons, cardiomyocytes, retinal epithelial cells, and endothelial cells. In some embodiments, an engineered cell is a T cell. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In preferred embodiments, an engineered cell is a regulatory T cell (Treg). In some embodiments, the Treg is a FOXP3+CD25+ T cell. In some embodiments, the Treg is a Helios+ Treg. In some embodiments, the Treg expresses one or more Treg-associated markers selected from the group consisting of CTLA-4 (CD152), LAG-3 (CD223), GITR (CD357), CD27, LAP, GARP, CD39, CD73, TIGIT, TNFRII (CD120b), and neuropilin-1 (CD304). In some embodiments, the Treg produces one or more cytokines selected from the group consisting of IL- 10, TGF-?, and IL-35. In some embodiments, an engineered cell is an islet cell. In some embodiments, an engineered cell is an islet cell precursor. In some embodiments, an engineered cell is a hepatocyte. In some embodiments, an engineered cell is a hepatocyte precursor. In some embodiments, an engineered cell is a neuron. In some embodiments, an engineered cell is a neuron precursor. In some embodiments, an engineered cell is a cardiomyocyte. In some embodiments, an engineered cell is a cardiomyocyte precursor. In some embodiments, an engineered cell is a retinal epithelial cell. In some embodiments, an engineered cell is a retinal epithelial cell precursor. In some embodiments, an engineered cell is an endothelial cell. In some embodiments, an engineered cell is an endothelial cell precursor. In some embodiments, an engineered cell exhibits stabilized FOXP3 expression. Examples of suitable approaches for stabilizing FOXP3 expression are described in the section entitled “Stabilized FOXP3 expression.” In some embodiments, an engineered cell comprises first and second chemically inducible signaling complex (CISC) components that dimerize in the presence of a ligand to create a signaling-competent CISC and induce proliferation of the cell, or one or more nucleic acids encoding the CISC components. In some embodiments, the engineered cell comprises a third CISC component consisting of a soluble FRB domain polypeptide. Examples of suitable chemically inducible signaling complexes and their components are described in the section entitled “Chemically inducible signaling complex (CISC).” In some embodiments, an engineered cell has been modified to reduce expression of one or more major histocompatibility complex (MHC) proteins. Reduction of expression of class I and II MHC proteins is described, e.g., in Kagoya & Guo et al., Cancer Immunol Res.2020. 8(7):932–C!5^^^^^^0^^^^^J^.^^et al., Nat Biotechnol. 2019.37(3):252–258. In some embodiments, the engineered cell has been modified to reduce expression of ?2-microglobulin (B2M). In some embodiments, the engineered cell has been modified to reduce expression of class II transactivator (CIITA). In some embodiments, the engineered cell has been modified to reduce expression of both B2M and CIITA. In some embodiments, the engineered cell has been modified to reduce expression of RFX5, RFXAP, RFXANK, NF-Y, or IRF-1. Modifications to reduce expression of a protein (e.g., B2M or CIITA) include, without limitation, in-frame introduction of a stop codon (nonsense mutation) at an early position in the open reading frame (e.g., codon 20), removal of one or more exons encoding the gene product, modification of a promoter region to reduce transcription initiation, and insertion of a microRNA target site for a microRNA expressed by the engineered cell. As another example, a nucleotide sequence encoding an RNAi molecule (e.g., siRNA) specific to a nucleotide sequence encoding the targeted protein may be inserted into the cell genome. Persistence of engineered cells Some embodiments relate to persistence of engineered cells in vivo. Example 13, for example, reports persistence of engineered cells through the entire duration of a study with a predetermined 84-day endpoint. Persistence may be evaluated, e.g., by obtaining a biological sample from a subject and analyzing it for the presence of an engineered cell. Unless otherwise clear from context, description of engineered cells being detectable in a subject or a sample obtained from the subject is to be understood as disclosure of corresponding methods of obtaining samples and detecting engineered cells. Similarly, description of methods of detecting engineered cells is to be understood as disclosure of corresponding engineered cells that are detectable in methods so described. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue sample. The skilled artisan will appreciate that an engineered cell detected after administration may be a progeny cell of an engineered cell that was actually administered. In some embodiments, an engineered cell is detectable in a sample obtained from the subject at least 1 week after administration of the engineered cell. In some embodiments, the sample is obtained from the subject at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks after administration of the engineered cell. In some embodiments, an engineered cell is detectable in a sample obtained from the subject up to 1 week after administration of the engineered cell. In some embodiments, the sample is obtained from the subject up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks after administration of the engineered cell. In some embodiments, the sample is obtained from the subject 1–52, 1–48, 1–44, 1–40, 1–36, 1–32, 1–28, 1–24, 1–20, 1–16, 1–12, 1–8, or 1–4 weeks after administration of the engineered cell. In some embodiments, the sample is obtained from the subject 1–52, 2–52, 3– 52, 4–52, 5–52, 6–52, 7–52, 8–52, 9–52, 10–52, 11–52, or 12–52 weeks after administration of the engineered cell. In some embodiments, the sample is obtained from the subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks after administration of the engineered cell. In some embodiments, an engineered cell is detectable in a sample obtained from the subject at least 1 month after administration of the engineered cell. In some embodiments, the sample is obtained from the subject at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 months after administration of the engineered cell. In some embodiments, an engineered cell is detectable in a sample obtained from the subject up to 1 month after administration of the engineered cell. In some embodiments, the sample is obtained from the subject up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 months after administration of the engineered cell. In some embodiments, the sample is obtained from the subject 1–60, 1–48, 1–36, 1–24, 1–12, or 1–6 months after administration of the engineered cell. In some embodiments, the sample is obtained from the subject 1–60, 2–60, 3–60, 4–60, 5–60, 6–60, 7–60, 8–60, 9–60, 10– 60, 11–60, or 12–60 months after administration of the engineered cell. In some embodiments, the sample is obtained from the subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of the engineered cell. In some embodiments, the timing of obtaining one or more samples is calculated starting from administration of a first dose of the engineered cell (e.g., 1 week is 7 days after the day on which the first dose is administered). In some embodiments, the timing of obtaining one or more samples is calculated starting from administration of the last dose of a predefined dosing regimen (i.e., 1 week is 7 days after the day on which the last dose is administered). In some embodiments, the engineered cell is detectable over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks following administration of the engineered cell. In some embodiments, the engineered cell is detectable over a period of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks following administration of the engineered cell. In some embodiments, detection is over a period of 1–52, 1–48, 1–44, 1–40, 1–36, 1–32, 1–28, 1–24, 1–20, 1–16, 1–12, 1–8, or 1–4 weeks. In some embodiments, detection is over a period of 1–52, 2–52, 3–52, 4–52, 5–52, 6–52, 7–52, 8–52, 9–52, 10–52, 11–52, or 12–52 weeks. In some embodiments, detection is over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks. In some embodiments, the engineered cell is detectable over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 months following administration of the engineered cell. In some embodiments, the engineered cell is detectable over a period of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 months following administration of the engineered cell. In some embodiments, detection is over a period of 1–60, 1–48, 1–36, 1–24, 1–12, or 1–6 months. In some embodiments, detection is over a period of 1– 60, 2–60, 3–60, 4–60, 5–60, 6–60, 7–60, 8–60, 9–60, 10–60, 11–60, or 12–60 months. In some embodiments, detection is over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the duration of detection is calculated starting from administration of a first dose of the engineered cell. In some embodiments, the duration of detection is calculated starting from administration of a last dose of the engineered cell. In some embodiments, samples are obtained from the subject at regular intervals for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks, and analyzed for the presence of the engineered cell. In some embodiments, samples are obtained from the subject at regular intervals for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 months, and analyzed for the presence of the engineered cell. In some embodiments, samples are obtained from the subject at regular intervals for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks, and analyzed for the presence of the engineered cell. In some embodiments, samples are obtained from the subject at regular intervals for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 months, and analyzed for the presence of the engineered cell. In some embodiments, the samples are analyzed for the abundance of the engineered cell and / or frequency of the engineered cell among cells of the sample. In some embodiments, samples are collected within 6–10 days between successive samples (e.g., weekly). In some embodiments, samples are collected within 4–6 weeks between successive samples (e.g., monthly). In some embodiments, a method comprises measuring the abundance of the engineered cell in the sample and / or the frequency of the engineered cell among cells of sample. In some embodiments, abundance of the engineered cell is measured. In some embodiments, frequency of the engineered cell among cells of the sample is measured. In some embodiments, a method comprises (i) identifying the engineered cell as not having persisted in the subject, if the engineered cell is not detected in the sample, and (ii) administering to the subject one or more additional doses of the engineered cell. Engineering reagents and methods Some aspects relate to methods of producing engineered cells expressing a synthetic NK cell inhibitory receptor engager. Some aspects relate to reagents engineering cells to express a synthetic NK cell inhibitory receptor engager, including synthetic NK cell inhibitory receptor engager, nucleic acids encoding synthetic NK cell inhibitory receptor engager, and vectors and lipid delivery vehicles comprising the same. Some aspects relate to kits comprising one or more reagents for use in engineering cells to express a synthetic NK cell inhibitory receptor engager. Unless otherwise clear from context, description of methods for engineering cells will be understood to also apply, mutatis mutandis, to reagents and kits for engineering cells. Some aspects relate to engineered cells made using such a method, reagent, or kit. Cell types Embodiments of methods (and reagents or kits) for producing engineered cells (e.g., by in vitro or ex vivo contact with nucleic acids, and / or administration of nucleic acids, vectors, or lipid delivery vehicles to a subject for in vivo contact with cells) may use any suitable cell type as a starting material for, e.g., introduction of nucleic acids. The skilled artisan will appreciate that methods of manipulating CD4+ T cells can be applied, mutatis mutandis, to other cell types (e.g., CD8+ T cells or CD3+ T cells generally). In some embodiments, a method comprises engineering a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the stem cell is a CD34+ hematopoietic stem cell. Engineered stem cells may be matured in vitro to produce differentiated (e.g., Treg) cells suitable for administration, or administered to allow for in vivo development into differentiated (e.g., Treg) cells. Engineered stem cells may be matured into CD3+ Treg cells, CD4+ Treg cells, CD8+ Treg cells, NK-Treg cells, or combinations thereof. In some embodiments, a method comprises engineering a T cell. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, a method comprises engineering a Treg cell. In some embodiments, the Treg cell is a CD3+ Treg cell. In some embodiments, the Treg cell is a CD4+ Treg cell. In some embodiments, the Treg cell is a CD8+ Treg cell. In some embodiments, the T cell expresses a TCR, polypeptide and a TCR? polypeptide. In some embodiments, the T cell expresses a TCR> polypeptide and a TCRK polypeptide. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR). In some embodiments, the T cell is an NK-T cell. In some embodiments, a method comprises engineering a FOXP3+CD25+ Treg. In some embodiments, the Treg has been previously engineered (i.e., manipulated to alter its genetic, epigenetic, or phenotypic identity). In some embodiments, the Treg has been isolated from a biological sample (e.g., peripheral blood). In some embodiments, a method comprises engineering an induced pluripotent stem cell (iPSC). In some embodiments, engineering the iPSC comprises contacting the iPSC with a lentiviral vector. Transduction of iPSCs with lentiviral vectors is described, e.g., in Naujok et al., Methods Mol Biol.2016.1341:67–85. In some embodiments, engineering the iPSC comprises contacting the iPSC with an adeno-associated viral (AAV) vector. Transduction of iPSCs with AAV vectors is described, e.g., in Martin et al., Cell Stem Cell.2019.24(5):821–828.e5. In some embodiments, engineering an iPSC comprises inserting a nucleic acid into the iPSC genome by homology-directed repair. Gene-targeted engineering of iPSCs is described, e.g., in Singh et al., Sci Rep. 2024.14(1):9933. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the iPSC comprises contacting the iPSC with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method further comprises differentiating the engineered iPSC into another cell type. In some embodiments, a method comprises engineering an islet cell or islet cell precursor. In some embodiments, the method comprises engineering an islet cell. In some embodiments, engineering the islet cell or precursor comprises contacting the cell with a lentiviral vector. Transduction of islet cells with lentiviral vectors is described, e.g., in Kobinger et al., Hum Gene Ther.2004.15(2):211–219. In some embodiments, engineering the islet cell or precursor comprises contacting the cell with an adeno-associated viral (AAV) vector. Transduction of islet cells with AAV vectors is described, e.g., in Pekrun et al., JCI Insight. 2019. 4(22):el31610. In some embodiments, engineering the islet cell or precursor comprises inserting a nucleic acid into the cell genome by homology-directed repair. Gene-targeted engineering of islet cells is described, e.g., in Bevacqua et at, Nat Commun. 2021. 12(1):2397; and Gerace et at, Cell Rep Med. 2023. 4(1): 100879. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the islet cell or precursor comprises contacting the cell with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method comprises engineering an islet cell precursor and differentiating the engineered cell into an islet cell. Stem cell-derived islet cells are described, e.g., in Sackett et al., Transplant Int. 2022. 35:10817; and Gerace et at., Cell Rep Med. 2023. 4(l):100879.

[0102] In some embodiments, a method comprises engineering a hepatocyte or hepatocyte precursor. In some embodiments, the method comprises engineering a hepatocyte. In some embodiments, engineering the hepatocyte or precursor comprises contacting the cell with a lentiviral vector. Transduction of hepatocytes with lentiviral vectors is described, e.g., in Zamule et al., Chem Biol Interact. 20098. 173(3): 179—186. In some embodiments, engineering the hepatocyte or precursor comprises contacting the cell with an adeno-associated viral (AAV) vector. Transduction of hepatocytes with AAV vectors is described, e.g., in Dalwadi et al., Mol Ther. 2021. 29(10):2898-2909; and Shao et al , Gene Ther. 2019. 36(12):504-514. In some embodiments, engineering the hepatocyte or precursor comprises inserting a nucleic acid into the cell genome by homology-directed repair. Gene-targeted engineering of hepatocytes is described in Zabulica et al., Mol Ther. 2021. 29(5):P1903-1917; and de Giorgi et al., Set Transl Med. 2025. 17(785):eadk3920. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the hepatocyte or precursor comprises contacting the cell with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method comprises engineering a hepatocyte precursor and differentiating the engineered cell into a hepatocyte. Differentiation of stem cells into hepatocytes is described, e.g., in Blackford et al., Stem Cells Transl Med. 2019. 8(2): 124-137.

[0103] In some embodiments, a method comprises engineering a neuron or neuron precursor. In some embodiments, the method comprises engineering a neuron. In some embodiments, engineering the neuron or precursor comprises contacting the cell with a lentiviral vector. In some embodiments, engineering the neuron or precursor comprises contacting the cell with an adeno-associated viral (AAV) vector. In some embodiments, engineering the neuron or precursor comprises inserting a nucleic acid into the cell genome by homology-directed repair. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the neuron or precursor comprises contacting the cell with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method comprises engineering a neuron precursor and differentiating the engineered cell into a neuron. In some embodiments, a method comprises engineering a cardiomyocyte or cardiomyocyte precursor. In some embodiments, the method comprises engineering a cardiomyocyte. In some embodiments, engineering the cardiomyocyte or precursor comprises contacting the cell with a lentiviral vector. In some embodiments, engineering the cardiomyocyte or precursor comprises contacting the cell with an adeno-associated viral (AAV) vector. In some embodiments, engineering the cardiomyocyte or precursor comprises inserting a nucleic acid into the cell genome by homology-directed repair. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the cardiomyocyte or precursor comprises contacting the cell with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method comprises engineering a cardiomyocyte precursor and differentiating the engineered cell into a cardiomyocyte. In some embodiments, a method comprises engineering a retinal epithelial cell or retinal epithelial cell precursor. In some embodiments, the method comprises engineering a retinal epithelial cell. In some embodiments, engineering the retinal epithelial cell or precursor comprises contacting the cell with a lentiviral vector. In some embodiments, engineering the retinal epithelial cell or precursor comprises contacting the cell with an adeno-associated viral (AAV) vector. In some embodiments, engineering the retinal epithelial cell or precursor comprises inserting a nucleic acid into the cell genome by homology-directed repair. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the retinal epithelial cell or precursor comprises contacting the cell with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method comprises engineering a retinal epithelial cell precursor and differentiating the engineered cell into a retinal epithelial cell. In some embodiments, a method comprises engineering a endothelial cell or endothelial cell precursor. In some embodiments, the method comprises engineering an endothelial cell. In some embodiments, engineering the endothelial cell or precursor comprises contacting the cell with a lentiviral vector. In some embodiments, engineering the endothelial cell or precursor comprises contacting the cell with an adeno-associated viral (AAV) vector. In some embodiments, engineering the endothelial cell or precursor comprises inserting a nucleic acid into the cell genome by homology-directed repair. In some embodiments, the nucleic acid is inserted into an essential gene locus as described in the section entitled “Selection by Essential Gene Exon Knock-In (SLEEK).” In some embodiments, engineering the endothelial cell or precursor comprises contacting the cell with a lipid delivery vehicle comprising a nucleic acid. In some embodiments, the method comprises engineering an endothelial cell precursor and differentiating the engineered cell into an endothelial cell. In some embodiments, a method comprises engineering a human cell. In some embodiments, a method comprises engineering a population of cells. Populations of cells may be enriched for cells with a certain characteristic, such that a substantial fraction of cells of the population being engineered share that characteristic. In some embodiments, the population is a CD3+ T cell population. In some embodiments, the population is a CD4+ T cell population. In some embodiments, the population is a CD8+ T cell population. In some embodiments, the population is a Treg population. In some embodiments, the population is a stem cell population. In some embodiments, the population is an iPSC population. In some embodiments, the population is an islet cell or islet cell precursor population. In some embodiments, the population is a hepatocyte or hepatocyte precursor population. In some embodiments, the population is a neuron or neuron precursor population. In some embodiments, the population is a cardiomyocyte or cardiomyocyte precursor population. In some embodiments, the population is a retinal epithelial cell or retinal epithelial cell precursor population. In some embodiments, the population is an endothelial cell or endothelial cell precursor population. In some embodiments, a T cell or T cell population is activated before contact with a nucleic acid, vector, or lipid delivery vehicle. T cells may be activated, for example, by stimulation with agonists of CD3 and CD28 (e.g., anti-CD3 / CD28 antibodies, beads, or matrix). In some embodiments, a method comprises engineering a cell that has been isolated from a biological sample. A biological sample may be a sample from a subject (e.g., a human subject) or a composition produced in a lab (e.g., by cell culture). A biological sample obtained from a subject may be a liquid sample (e.g., blood or a fraction thereof, a bronchial lavage, cerebrospinal fluid, or urine), or a solid sample (e.g., a piece of tissue). In some embodiments, the cell has been isolated from peripheral blood. In some embodiments, the cell has been isolated from umbilical cord blood. Nucleic acids Some aspects relate to nucleic acids encoding a chimeric receptor. Some embodiments relate to engineered cells comprising a nucleic acid encoding a chimeric receptor. Some embodiments relate to lipid delivery vehicles comprising a nucleic acid encoding a chimeric receptor. Some embodiments relate to methods of engineering a cell by contacting the cell with a nucleic acid encoding a chimeric receptor. Embodiments contemplating use of a nucleic acid (e.g., donor template) may use any suitable nucleic acid. In some embodiments, a nucleic acid is a DNA. DNA is suitable, e.g., for expressing a protein episomally, or insertion into a cell chromosome. In some embodiments, the DNA is double-stranded DNA (dsDNA). In some embodiments, the DNA is single-stranded DNA. In some embodiments, the DNA is a linear DNA. In some embodiments, the DNA is a closed-ended DNA (ceDNA). In some embodiments, a nucleic acid is an RNA. RNA is suitable, e.g., for expressing a protein without insertion into a cell chromosome. In some embodiments, an RNA is a messenger RNA (mRNA). A “messenger RNA,” as used herein, is an RNA that encodes at least one protein or a fragment thereof and can be translated to produce an encoded protein (e.g., FOXP3) or fragment in vitro, in vivo, in situ, or ex vivo. mRNA comprises an open reading frame (ORF) encoding a protein or fragment thereof. In some embodiments, the mRNA further comprises a 5L untranslated region (UTR), a 3L UTR, a polyA tail, and a 5L cap or cap analog. In some embodiments, an RNA is a self-amplifying RNA (saRNA). A “self-amplifying RNA,” as used herein, is an RNA encoding one or more proteins that, individually or in conjunction, can replicate the self-amplifying RNA. In some embodiments, the proteins encoded by the self-amplifying RNA are non-structural proteins nsP1, nsP2, nsP3, and nsP4, which form an RNA-dependent RNA polymerase (RdRp), or replicase, that can replicate the self- amplifying RNA. By encoding proteins that can replicate the RNA, a self-amplifying RNA is capable of self-amplification in a cell. A self-amplifying RNA is also known as an RNA replicon.

[0104] In some embodiments, an RNA is a circular RNA. A “circular RNA,” as used herein, is an RNA with no 5' terminal nucleotide or 3' terminal nucleotide. Every nucleotide in a circular RNA is covalently bonded to both (1) a 5' adjacent nucleotide; and (2) a 3' adjacent nucleotide. In a circular RNA with a nucleotide sequence comprising every nucleotide of the circular RNA in 5'-to-3' order, the last nucleotide of the nucleotide sequence is covalently bound to the first nucleotide of the nucleotide sequence.

[0105] In some embodiments, a nucleic acid is comprised in a vector. In some embodiments, the vector is an artificial chromosome. In some embodiments, the vector is a bacterial artificial chromosome. In some embodiments, the vector is a human artificial chromosome. In some embodiments, the vector is a plasmid.

[0106] In some embodiments, a vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an adeno-associated viral (A AV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,

[0107] AAV7, AAV8, AAV9, AAV10, or AAV 11 vector. In some embodiments, the A AV vector is an

[0108] AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector.

[0109] The skilled artisan will appreciate that unless otherwise clear from context, embodiments contemplating use of a nucleic acid also contemplate use of a vector comprising the nucleic acid.

[0110] In some embodiments, a nucleic acid comprises 5' and 3' homology arms for insertion into a targeted locus. Such homology arms promote insertion of the nucleic acid into the genome at the targeted locus by homologous recombination, such as by homology-directed repair following nuclease-mediated cleavage. Examples of suitable loci that may be targeted by a DNA endonuclease and corresponding homology arms are described in the section entitled “Targeted loci.”

[0111] The homology arms may be the same length, have similar lengths (within 100 bp of each other), or different lengths. In some embodiments, one or both homology arms have a length of 100-2,000 bp, 200-2,000 bp, 400- 1,500 bp, or 500-1,000 bp. In some embodiments, one or both homology arms are about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, or about 2,000 bp. In some embodiments, both homology arms are 100-2,000 nucleotides in length. In some embodiments, both homology arms are 300–1,000 nucleotides in length. In some embodiments, both homology arms are 300–700 nucleotides in length. In some embodiments, both homology arms are 300–500 nucleotides in length. In some embodiments, both homology arms are 500–700 nucleotides in length. In some embodiments, both homology arms are 700–1,000 nucleotides in length. In some embodiments, a nucleic acid comprises a heterologous promoter and homology arms for insertion into a genomic FOXP3 locus of a cell to stabilize FOXP3 expression in the cell. Examples of suitable approaches for stabilizing FOXP3 expression are described in the section entitled “Stabilized FOXP3 expression.” In some embodiments, a nucleic acid encodes first and / or second chemically inducible signaling complex (CISC) components that dimerize in the presence of a ligand to create a signaling-competent CISC and induce proliferation of a cell. In some embodiments, a nucleic acid encodes a third CISC component consisting of a soluble FRB domain polypeptide. Examples of suitable chemically inducible signaling complexes and their components are described in the section entitled “Chemically inducible signaling complex (CISC).” Lipid delivery vehicles Nucleic acids (e.g., RNA or DNA) may be present in a lipid delivery vehicle. Lipid delivery vehicles are described, e.g., in Hou et al., Nat Rev Mater.2022.6:1078–1094, incorporated by reference herein for this purpose. In some embodiments, the lipid delivery vehicle is an exosome or extracellular vesicle. In some embodiments, the lipid delivery vehicle is a liposome. A “liposome,” as used herein, is a lipid particle comprising lipids arranged in one or more concentric lipid bilayers around a central region (e.g., an aqueous solution, suspension, or other aqueous composition). In some embodiments, the lipid delivery vehicle is a lipoplex. A “lipoplex,” as used herein,” is a lipid particle comprising a cationic liposome and a nucleic acid (e.g., mRNA). Lipoplexes can be formed by contacting a liposome comprising a cationic lipid with a nucleic acid. In some embodiments, the lipoplex comprises multiple concentric lipid bilayers, wherein each concentric bilayer is separated by one or more nucleic acids. In some embodiments, the central region of the lipoplex is an aqueous solution, suspension, or other aqueous composition. In some embodiments, the lipid delivery vehicle is a lipid nanoparticle (LNP). LNPs typically comprise ionizable lipid (e.g., ionizable amino lipid), non-cationic lipid (e.g., phospholipid), structural lipid (e.g., sterol), and PEG-modified lipid components, along with the nucleic acid cargo of interest.

[0112] DNA endonucleases

[0113] Embodiments contemplating engineering of cells may include a DNA endonuclease. A "DN A endonuclease,” as used herein, refers to an enzyme that cleaves a phosphodiester bond between two internal deoxyribonucleotides in a DNA molecule. DNA endonucleases are generally reviewed, e.g., in Certo & Morgan, Mol Ther. 2016. 24(3):422-429. Following introduction of a double-strand break (DSB), a donor template may be inserted into a targeted locus by homology-directed repair (HDR). See, e.g., Jasin & Rothstein, Cold Spring Harb Perspect Biol. 2013. 5(ll):a012740.

[0114] In some embodiments, the DNA endonuclease is an RNA-guided DNA endonuclease. In some embodiments, the RNA-guided DNA endonuclease is a Cas endonuclease. In some embodiments, the RNA-guided DNA endonuclease is a Cas9 endonuclease. In some embodiments, the RNA-guided DNA endonuclease is a Cpf 1 endonuclease.

[0115] Embodiments contemplating RNA-guided DNA endonucleases may further include a guide RNA. RNA-guided DNA endonucleases and guide RNAs are generally reviewed, e.g., in Pacesa et al., Cell. 2024. 187(5): 1076-1100. A guide RNA typically comprises a spacer sequence that is complementary to a. nucleotide sequence in a targeted locus, such that an RNA-guided DNA endonuclease, when complexed with the gRNA, localizes to the targeted locus and cleaves DNA at the targeted locus. A spacer sequence to facilitate cleavage at the targeted locus will depend on the RNA-guided DNA endonuclease — Cas9, for example, has a protospacer adjacent motif of NGG, whereas Casl2a has a protospacer adjacent motif of TTTV. In some embodiments a guide RNA is a single guide RNA (sgRNA). Whereas RNA-guided DNA endonucleases in nature are directed to a specific target nucleotide sequence (protospacer) by a complex including a crRNA and a tracrRNA, parts of which are hybridized to each other, with the crRNA comprising a spacer that hybridizes to the target nucleotide sequence (protospacer), an sgRNA is a single-stranded RNA comprising a spacer sequence that directs an RNA-guided DNA endonuclease to a protospacer and facilitates cleavage. sgRNAs are described, e.g., in Jinek et al.. Science. 2012. 337(6096):816— -821 ; and Jinek et al., eLife. 2013. 2:e00471. The skilled artisan will appreciate that a nucleotide sequence within an sgRNA may be hybridized to another nucleotide sequence within the sgRN A, such that the sgRNA is a single RNA strand with some degree of self-hybridization. In some embodiments, an RNA-guided DNA endonuclease and gRNA are delivered to a cell as a guide ribonucleoprotein (gRNP) complex. In some embodiments, one or both of an RNA-guided DNA endonuclease and gRNA are encoded by a nucleic acid. In some embodiments, the nucleic acid is an mRNA.

[0116] In some embodiments, the DNA endonuclease is a meganuclease. In some embodiments, the DNA endonuclease is a TALEN. In some embodiments, the DNA endonuclease is a zinc finger nuclease (ZFN).

[0117] Examples of suitable loci that may be targeted by a DNA endonuclease are described in the section entitled ‘"Targeted loci.”

[0118] Targeted, loci

[0119] Embodiments contemplating insertion of a nucleotide sequence at a targeted locus and / or DNA endonuclease-mediated cleavage at a targeted locus may target any suitable locus. Loci to be targeted for cleavage and nucleotide sequence insertion may depend on the context of the nucleotide sequence to be inserted. For instance, stabilized expression of an endogenous FOXP3 coding sequence may be achieved by targeted insertion of a promoter in a FOXP3 locus downstream from a TSDR and upstream from the first coding exon in the FOXP3 locus. As another example, disrupted expression of an essential gene may be achieved by targeted insertion of a stop codon in an essential gene locus, such as a GAPDH locus. As a further example, disruption of major histocompatibility complex proteins may be achieved by an insertion or deletion in a gene locus necessary for their expression, such as a B2M (class I MHC) and / or CIITA (class II MHC) locus. Reduction of expression of class I and II MHC proteins by targeting of B2M and CIITA loci is described, e.g., in Kagoya & Guo et al.. Cancer Immunol Res. 2020. 8(7):932— 936; and Deuse & Hu et al., Nat Biotec hnol. 2019. 37(3):252-258.

[0120] In some embodiments, a targeted locus is a FOXP3 locus. In some embodiments, the targeted locus is downstream from a TSDR, and upstream or within a first coding exon, of the FOXP3 locus.

[0121] In some embodiments, a targeted locus is a TCR subunit locus. In some embodiments, the TCR subunit locus is a CD3G, CD3D, CD3E, TRAC, TRBC, TRGC, or TRDC locus. In some embodiments, a targeted locus is a B2M, CIITA, RFXAP, RFX5, RFXANK, CD74, NLRC5, TAP1, TAP2, CALR, PDIA3, or TAPBP locus. In some embodiments, the targeted locus is a B2M or CIITA locus. In some embodiments, both B2M and CIITA loci are targeted. In some embodiments, a targeted locus is a safe harbor locus. In some embodiments, the safe harbor locus is an AAVS1 locus, a HIPP11 locus, or a ROSA26 locus. Cells, methods, and nucleic acids for expression of synthetic engagers Some aspects relate to engineered cells comprising a synthetic CD161 engager, synthetic NKG2A engager, and / or synthetic NK cell inhibitory engager. Some aspects relate to engineered cells comprising a nucleic acid encoding a synthetic CD161 engager, synthetic NKG2A engager, and / or synthetic NK cell inhibitory engager. Some aspects relate to methods of engineering cells to expressing a synthetic CD161 engager, synthetic NKG2A engager, and / or synthetic NK cell inhibitory engager. Some aspects relate to nucleic acids encoding a synthetic CD161 engager, synthetic NKG2A engager, and / or synthetic NK cell inhibitory engager. Cell types suitable for engineered cells or use in methods of engineering cells are described in the sections entitled “Cell types.” Any suitable nucleic acid may encode a synthetic engager. Non-limiting examples of nucleic acids include DNA, mRNA, self-amplifying RNA, and circular RNA. A nucleic acid may contain or encode an open reading frame (ORF) that lacks introns and encodes the synthetic engager or may contain or encode one or more introns. In some embodiments, a nucleotide sequence encoding a synthetic engager is in a chromosome of the cell. In some embodiments, a nucleic acid encoding a synthetic engager is episomal. In some embodiments, a nucleotide sequence is present at a specific locus of a chromosome of the cell. In some embodiments, a nucleotide sequence encoding a synthetic engager is present in a safe harbor locus. In some embodiments, a nucleic acid comprises a heterologous promoter operably linked to the nucleotide sequence encoding the synthetic engager. In some embodiments, the heterologous promoter is a constitutive promoter. In some embodiments, the constitutive promoter is an EF-1,, PGK, or MND promoter. In some embodiments, the constitutive promoter is an MND promoter. Other suitable constitutive promoters include SV40, CMV, UBC, SFFV, EFS, or CAGG promoters. In some embodiments, the heterologous promoter is an inducible promoter. In some embodiments, a method comprises contacting an engineered cell with an agent that induces transcription through the inducible promoter. In some embodiments, a method comprises contacting a cell with a DNA endonuclease that cleaves at a target site or nucleic acid encoding the DNA endonuclease. In some embodiments, a system comprises the DNA endonuclease. In some embodiments, the nuclease is an RNA-guided DNA endonuclease, and the method comprises contacting the cell with a guide RNA (gRNA) or nucleic acid encoding the gRNA, or the system comprises the gRNA or nucleic acid encoding the gRNA. In some embodiments, the RNA-guided DNA endonuclease and gRNA are present in a guide ribonucleoprotein (gRNP) complex. Nucleases and guide RNAs are described below in the section entitled “Nucleases and guide RNAs.” Any suitable nuclease may be used for targeted cleavage. In some embodiments, the RNA-guided DNA endonuclease is a Cas endonuclease. In some embodiments, the RNA-guided DNA endonuclease is a Cas9 endonuclease. In some embodiments, the DNA endonuclease is a TALEN. In some embodiments, the DNA endonuclease is a zinc finger nuclease (ZFN). In some embodiments, a nucleic acid is present in a vector. Any suitable vector may comprise the nucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an integrase-deficient lentiviral vector (IDLV). In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the AAV vector is an AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector. In some embodiments, a nucleic acid is present in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises the DNA endonuclease or a nucleic acid encoding the DNA endonuclease. In some embodiments, the lipid nanoparticle comprises a guide RNA or a nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the nuclease is an mRNA. In some embodiments, a nucleic acid comprises a 5L homology arm and a 3L homology arm, where the homology arms have homology to a target locus, such that homologous recombination or homology-directed repair causes the nucleic acid to be inserted into the target locus. Examples of target loci are described, for instance, in the sections entitled “MHC-deficient cells” and “Selection by Essential Gene Exon Knock-In (SLEEK).” The homology arms may be the same length, have similar lengths (within 100 bp of each other), or different lengths. In some embodiments, one or both homology arms have a length of 100–2,000 bp, 200–2,000 bp, 400–1,500 bp, or 500–1,000 bp. In some embodiments, one or both homology arms are about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, or about 2,000 bp. In some embodiments, both homology arms are 100-2,000 nucleotides in length. In some embodiments, both homology arms are 300–1,000 nucleotides in length. In some embodiments, both homology arms are 300–700 nucleotides in length. In some embodiments, both homology arms are 300–500 nucleotides in length. In some embodiments, both homology arms are 500–700 nucleotides in length. In some embodiments, both homology arms are 700–1,000 nucleotides in length. In some embodiments, the target locus is a FOXP3 locus. Exemplary homology arms for targeting a donor template to the FOXP3 locus are provided in FIGs.47–48 and as SEQ ID NOs: 202 and 224. In some embodiments, 5L and 3L homology arms have at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 202 and 224, respectively. In some embodiments, the target locus is a TRAC locus. Exemplary homology arms for targeting a donor template to the TRAC locus are provided in FIGs. 49–50 and as SEQ ID NOs: 227 and 245. In some embodiments, 5L and 3L homology arms have at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 227 and 245, respectively. In some embodiments, the target locus is a B2M locus. In some embodiments, the target locus is a CIITA locus. Cells, methods, and nucleic acids for LLT1 expression Some aspects relate to engineered cells constitutively expressing LLT1. Some aspects relate to methods of engineering cells to constitutively express LLT1. Some aspects relate to nucleic acids for constitutive expression of LLT1. Some aspects relate to engineered cells having upregulated expression of LLT1. Some aspects relate to methods of engineering cells to upregulate expression level of LLT1. Some aspects relate to nucleic acids for upregulated expression of LLT1. Some aspects relate to engineered cells expressing a supraphysiologic level of LLT1. Some aspects relate to methods of engineering cells to express a supraphysiologic level of LLT1. Some aspects relate to nucleic acids for supraphysiologic expression of LLT1. Increased expression (e.g., to a supraphysiologic level) of LLT1 may be accomplished through any suitable route, non-limiting examples of which include: introduction of a promoter (or enhancer or other regulatory element) to cause increased transcription of an endogenous LLT1 coding sequence and providing an exogenous nucleic acid encoding LLT1 (e.g., a DNA comprising an open reading frame, without introns, that encodes LLT1). Exogenous nucleic acids may include mRNA (which may be translated directly), self-amplifying RNA (which may produce multiple mRNAs within a cell), and / or DNA (which may be transcribed episomally, or inserted into a chromosome of an engineered cell). Cell types suitable for engineered cells or use in methods of engineering cells are described in the sections entitled “Cell types.” In some embodiments, an engineered cell comprises a heterologous promoter operably linked to an endogenous nucleotide sequence encoding LLT1. In some embodiments, a method comprises contacting a cell with a nucleic acid comprising a heterologous promoter, where the heterologous promoter is inserted into a chromosome of the cell such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding LLT1. In some embodiments, a nucleic acid comprises a heterologous promoter that is between two homology arms, where the two homology arms have homology to nucleotide sequences within an LLT1 locus, such that homologous recombination or homology-directed repair between the nucleic acid and LLT1 locus causes the heterologous promoter to become operably linked to an endogenous nucleotide sequence encoding LLT1. In some embodiments, an engineered cell comprises a nucleic acid comprising an exogenous nucleotide sequence encoding LLT1. In some embodiments, the exogenous nucleotide sequence encoding LLT1 is present on a chromosome of the engineered cell. In some embodiments, a method comprises contacting a cell with a nucleic acid comprising an exogenous nucleotide sequence encoding LLT1. In some embodiments, the nucleic acid comprising an exogenous nucleotide sequence encoding LLT1 is inserted into a chromosome of the cell genome. In some embodiments, a heterologous promoter is operably linked to the exogenous nucleotide sequence encoding LLT1. In some embodiments, the heterologous promoter is a constitutive promoter. In some embodiments, the constitutive promoter is an EF-1,, PGK, or MND promoter. Other suitable constitutive promoters include SV40, CMV, UBC, SFFV, EFS, or CAGG promoters. In some embodiments, the heterologous promoter is an inducible promoter. In some embodiments, a method comprises contacting an engineered cell with an agent that induces transcription through the inducible promoter. In some embodiments, the nucleic acid comprising the exogenous nucleotide sequence encoding LLT1 is expressed episomally. In some embodiments, the nucleic acid comprising the exogenous nucleotide sequence encoding LLT1 is an mRNA. In some embodiments, the nucleic acid comprising the exogenous nucleotide sequence encoding LLT1 is a self-amplifying RNA. In some embodiments, the nucleic acid comprising the exogenous nucleotide sequence encoding LLT1 is a circular RNA. MHC-deficient cells In some embodiments, an engineered cell is modified to reduce risk of transplant rejection and improve engraftment (e.g., modified to be MHC-deficient). An engineered cell modified to be MHC-deficient (e.g., hypoimmunogenic ) may be less prone to immune rejection (e.g., less prone to immune rejection by about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) relative to an engineered cell that is not modified to be MHC- deficient. Such MHC-deficient engineered cells can be used as “off-the-shelf” therapeutics in the treatment or amelioration of autoimmune diseases, allergic diseases, inflammatory diseases, or cancer. Modifications to confer MHC deficiency to an engineered cell may include modifications to the genome of the engineered cell to knock out or reduce expression of genes that promote an inflammatory immune response compared to an engineered cell that has not been modified to be MHC-deficient. Such genes may include genes encoding proteins that regulate the expression of major histocompatibility complex (MHC) molecules or human leukocyte antigen (HLA), such as ?2-microglobulin (?2M), NLR family CARD domain containing 5 (NLRC5), class II transactivator (CIITA), regulatory factor X5 (RFX5), RFXAP, RFXANK, nuclear transcription factor Y (NF-Y) complex proteins, and / or interferon regulatory factor 1 (IRF-1). An engineered cell may also be modified to have reduced or eliminated expression of one or more HLAs corresponding to MHC class I (MHC-I) or MHC class II (MHC-II), such as HLA-A, HLA-B, and / or HLA-C compared to an engineered cell that has not been modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of ?2M, NLRC5, CIITA, RFX5, RFXAP, RFXANK, NF-Y complex proteins, IRF-1, HLA-A, HLA-B, and / or HLA-C relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of ?2M relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of NLRC5 relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of CIITA relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of RFX5 relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of RFXAP relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be hypoimmunogenic has reduced expression of NF-Y complex proteins relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of IRF-1 relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of HLA-A relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of HLA-B relative to an engineered cell that is not modified to be MHC-deficient. In some embodiments, an engineered cell that has been modified to be MHC-deficient has reduced expression of HLA-C relative to an engineered cell that is not modified to be MHC-deficient. Modifications to reduce immunogenicity of an engineered cell may include modifications to the genome of the engineered cell to knock in or increase expression of proteins that promote immune tolerance compared to an engineered cell that has not been modified to be MHC- deficient. In some embodiments, a nucleic acid encoding a synthetic CD161 engager, synthetic NKG2A engager, or synthetic NK cell inhibitory receptor engager is inserted into a specific locus, thereby reducing expression of the gene encoded by the targeted locus. For example, a nucleic acid may be inserted into a specific locus to reduce or abrogate expression of one or more MHC molecules, thereby reducing peptide presentation to T cells. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a TRAC locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a TRBC1 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a TRBC2 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CD3E locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CD3G locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CD3D locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CD247 / CD3Z locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a ^2M locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CIITA locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a RFXAP locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a RFX5 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a RFXANK locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CD74 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a NLRC5 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a TAP1 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a TAP2 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a CALR locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a PDIA3 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic CD161 engager is inserted in a TAPBP locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a TRAC locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a TRBC1 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a TRBC2 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CD3E locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CD3G locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CD3D locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CD247 / CD3Z locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a ^2M locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CIITA locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a RFXAP locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a RFXANK locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CD74 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a NLRC5 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a TAP1 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a TAP2 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a CALR locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a PDIA3 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NKG2A engager is inserted in a TAPBP locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a TRAC locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a TRBC1 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a TRBC2 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CD3E locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CD3G locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CD3D locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CD247 / CD3Z locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a ^2M locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CIITA locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a RFXAP locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a RFX5 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a RFXANK locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CD74 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a NLRC5 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a TAP1 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a TAP2 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a CALR locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a PDIA3 locus of the cell. In some embodiments, the nucleic acid encoding the synthetic NK cell inhibitory receptor engager is inserted in a TAPBP locus of the cell. Promoters and uses thereof Embodiments of compositions, cells, nucleic acids, vectors, and methods that contemplate use of a promoter (e.g., heterologous promoter) may use any suitable promoter. A promoter is “operably linked” to a sequence if it initiates transcription of the operably linked sequence (e.g., by recruitment of RNA polymerase). The promoters of the first and second nucleic acids may be any suitable promoter. In some embodiments, the heterologous promoter in the introduced nucleic acid is active, promoting transcription of RNA, even under pro- inflammatory conditions. In some embodiments, the promoter is a constitutive promoter, which continuously promotes transcription of an operably linked sequence (e.g., RNA encoding a polypeptide) at a consistent rate. Constitutive promoters may be strong promoters, which promote transcription at a higher rate than an endogenous promoter, or weak promoters, which promote transcription at a lower rate than a strong or endogenous promoter. In some embodiments, the constitutive promoter is a strong promoter. In some embodiments, the constitutive promoter is an EF-1,, PGK, or MND promoter. In some embodiments, another suitable promoter, such as an SV40, CMV, UBC, or CAGG promoter, is used. In some embodiments, the constitutive promoter is an MND promoter. In some embodiments, the heterologous promoter is an inducible promoter. Inducible promoters promote transcription of an operably linked sequence in response to the presence of an activating signal, or the absence of a repressor signal. In some embodiments, the inducible promoter is inducible by a drug or steroid. In some embodiments, the promoter is a unidirectional promoter. In some embodiments, the unidirectional promoter promotes transcription of a nucleotide sequence 5’ to (upstream from) the promoter, but not of a sequence 3’ to (downstream from) the promoter. In some embodiments, the promoter promotes transcription of a nucleotide sequence 3’ to (downstream from) the promoter, but not of a sequence 5’ to (upstream from) the promoter. In some embodiments, a promoter is a ubiquitous promoter. In some embodiments, a ubiquitous promoter is Reverse Orientation Splice Acceptor 26 (ROSA26) promoter. In some embodiments, the promoters of the first and second nucleic acids delivered to the cell are different promoters. In other embodiments, the first and second nucleic acid both comprise the same promoter. In some embodiments, the first and second nucleic acid both comprise an MND promoter. In embodiments where the first and second nucleic acid both comprise the same promoter, the promoter sequences may be identical between both nucleic acids. Alternatively, the promoter sequence of the first nucleic acid may comprise one or more mutations (e.g., insertions, deletions, substitutions) relative to the promoter sequence of the second nucleic acid. In some embodiments, a STOP codon is present upstream or within the first five nucleotides of the promoter on the first nucleic acid for insertion into the TRAC locus. In some embodiments, a STOP codon is present upstream or within the first five nucleotides of the promoter on the second nucleic acid for insertion into the FOXP3 locus. The presence of a STOP codon upstream from, within, or overlapping with the first five nucleotides of the promoter is expected to terminate translation of mRNAs that may be transcribed from an endogenous promoter upstream in the modified TRAC or FOXP3 locus, thereby inhibiting expression of inserted coding sequences (e.g., encoding CISC components, heterologous TCRp or TCRa chains, or FoxP3) under control of the endogenous promoter. In some embodiments, the STOP codon is in-frame with one or more upstream START codons, such that mRN A produced following transcription from the endogenous upstream promoter is not translated past the STOP codon.

[0122] Cell types

[0123] Embodiments of methods for producing genetically modified ceils (e.g., by in vitro or ex vivo gene editing, and / or administration of compositions, vectors, or nucleic acids to a subject for in vivo editing) may use any suitable ceil type as a material for, e.g., introduction of nucleic acids, vectors, and / or compositions. It is to be understood that methods that comprise manipulation of CD4+ ceils can be applied to other types of cells (e.g., CD8+ celis). In some embodiments, the methods comprise editing an immune cell. Non-limiting examples of immune cells include B cells, T cells, ILCs, and NK cells. In some embodiments, the methods comprise editing CD3+ cells, thereby producing edited CD3+ cells, including CD4+ and CD8+ Treg cells. In some embodiments, the methods comprise editing CD4+ T cells, thereby producing CD4+ Treg cells. In some embodiments, the methods comprise editing CD8+ T cells, thereby producing CD8+ Treg cells. In some embodiments, the methods comprise editing NK-T cells, thereby producing NK-Treg cells.

[0124] In some embodiments, the methods comprise editing a. stern cell. In some embodiments, the methods comprise editing a pluripotent stem cell. In some embodiments, the methods comprise editing CD34+ hematopoietic stem cells (HSCs). In some embodiments, the methods comprise editing induced pluripotent stem cells (iPSCs). Edited stem cells may be matured in vitro to produce Treg cells, or administered to a subject to allow in vivo development into Treg cells. Edited stem cells may be matured into CD3+ Treg cells, CD4+ Treg cells, CD8+ Treg cells, NK-Treg cells, or a combination thereof.

[0125] In some embodiments, a method comprises editing a T cell. AT cell or T lymphocyte is an immune system cell that matures in the thymus and produces a T cell receptor (TCR), e.g., an antigen-specific heterodimeric cell surface receptor typically comprised of an a-p heterodimer or a y-8 heterodimer. T cells of a given clonality typically express only a single TCR clonotype that recognizes a specific antigenic epitope presented by a syngeneic antigen-presenting cell in the context of a major histocompatibility complex-encoded determinant. T cells can be naive ("TN"; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased or no expression of CD45RO as compared to TCM (described herein)), memory T cells (TM) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced, cytotoxic). I'M can be further divided into subsets of central memory T cells (TCM, expresses CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEM, express CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA). The term “effector T cells (TE)” refers to antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to TCM, and are positive for granzyme and perforin. Helper T cells (TH) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on the presence of other cells and signals. T ceils can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, for example, using antibodies that specifically recognize one or more T cell surface phenotypic markers, by affinity binding to antibodies, flow' cytometry, fluorescence activated cell sorting (FACS), or immunomagnetic bead selection.

[0126] Other examples of T ceils include regulatory T cells (Tregs, also known as suppressor T cells), such as CD4+ CD25+ (FoxP3+) regulatory T cells and Treg 17 cells, as well as Tri, Th3, CD8+CD28-, or Qa- i restricted T cells. In some embodiments, the cell is a CD3+, CD4+, and / or CD8+ T cell. In some embodiments, the cell is a CD3+ T cell. In some embodiments, the cell is a CD4+CD8“ T cell. In some embodiments, the Treg cell is a FoxP3+ Treg cell. In some embodiments, the Treg cell expresses CTLA-4, LAG-3, CD25, CD39, CD27, CD70, CD357 (GITR), neuropilin- 1, galectin-1, and / or IL-2Ra on its surface.

[0127] In some embodiments, the Treg is an engineered Treg; that is, a Treg that has been manipulated so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In one example, an engineered Treg is one that expresses a regulatory or immunosuppressive phenotype through modification, expression / over-expression, or manipulation of FOXP3. In some embodiments, the engineered Treg is a sorted engineered Treg. A sorted engineered Treg, as used herein, refers to a sorted Treg that has been engineered to express a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). In some embodiments, the Treg is not a sorted Treg, wherein a sorted Treg is isolated from a human sample based on the following cell surface markers: CD4+CD25hiCD127ioor CD4+CD25hiCD127loCD45RA+.

[0128] In some embodiments, a sorted Treg is engineered to upregulate expression of FOXP3. In some embodiments, a sorted T cell is engineered to upregulate expression of FOXP3. In some embodiments, a sorted T cell is engineered to stabilize FOXP3 expression. In some embodiments, a sorted T cell is engineered to express a CAR and to upregulate expression of FOXP3.

[0129] In some embodiments, the sorted Treg does not have a genetically modified FOXP3 locus. Ill some embodiments, the sorted Treg has not been genetically modified to alter FOXP3 expression. In some embodiments, the sorted Treg does not express an exogenous FOXP3 coding sequence. In some embodiments, the sorted Treg has not been genetically modified to express a chemically induced signaling complex (CISC). In some embodiments, the sorted Treg does not express proteins that dimerize in the presence of rapamycin or a rapalog resulting in IL- 2 signaling.

[0130] In some embodiments, the Treg is an induced Treg (iTreg) or a peripheral Treg (pTreg); as used herein, an iTreg and a pTreg refer to a stem cell that has been stimulated (e.g., in the presence of TGF-p and IL-2), in vitro and in vivo, respectively. In some embodiments, the engineered Treg comprises a Treg engineered to express FOXP3 and CISC (IL-2).

[0131] In some embodiments, the cell is a. human cell. In some embodiments, a cell as is isolated from a biological sample. A biological sample may be a sample from a subject (e.g., a human subject) or a composition produced in a lab (e.g., a culture of cells). A biological sample obtained from a subject make be a liquid sample (e.g., blood or a fraction thereof, a bronchial lavage, cerebrospinal fluid, or urine), or a solid sample (e.g., a. piece of tissue). In some embodiments, the cell is obtained from peripheral blood. In some embodiments, the cell is obtained from umbilical cord blood. In some embodiments, the cell is obtained by sorting cells of peripheral blood to obtain a desired cell population (e.g., CD3+ cells), and one or more cells of the sorted population are modified. In some embodiments, the cell is in a subject. In some embodiments, the cell is in vivo.

[0132] Embodiments of genetically modified cells may be any suitable cell type. In some embodiments, the cell is a T cell, a precursor T cell, or a hematopoietic stem cell. In some embodiments, the cell is an NK-T cell (e.g., a FoxP3 - NK-T cell or a FoxP3+ NK-T cell). In some embodiments, the cell is a regulatory B (Breg) cell (e.g., a FoxP3– B cell or a FoxP3+ B cell). In some embodiments, the cell is a CD4+ T cell (e.g., a FoxP3–CD4+ T cell or a FoxP3+CD4+ T cell) or a CD8+ T cell (e.g., a FoxP3–CD8+ T cell or a FoxP3+CD8+ T cell). In some embodiments, the cell is a CD25- T cell. In some embodiments, the cell is a regulatory T (Treg) cell. Non-limiting examples of Treg cells are Tr1, Th3, CD8+CD28-, and Qa-1 restricted T cells. In some embodiments, the Treg cell is a FoxP3+ Treg cell. In some embodiments, the Treg cell expresses CTLA-4, LAG-3, CD25, CD39, CD27, CD70, CD357 (GITR), neuropilin-1, galectin-1, and / or IL-2R, on its surface. In some embodiments, an engineered cell is modified to reduce expression of ?2M. In some embodiments, an engineered cell is ?2M–. In some embodiments, an engineered cell is modified to reduce expression of CIITA. In some embodiments, an engineered cell is CIITA–. In some embodiments, an engineered cell is modified to reduce expression of RFXAP. In some embodiments, an engineered cell is RFXAP–. In some embodiments, an engineered cell is modified to reduce expression of RFX5. In some embodiments, an engineered cell is RFX5–. In some embodiments, an engineered cell is modified to reduce expression of RFXANK. In some embodiments, an engineered cell is RFXANK–. In some embodiments, an engineered cell is modified to reduce expression of CD74. In some embodiments, an engineered cell is CD74–. In some embodiments, an engineered cell is modified to reduce expression of NLRC5. In some embodiments, an engineered cell is NLRC5–. In some embodiments, an engineered cell is modified to reduce expression of TAP1. In some embodiments, an engineered cell is TAP1–. In some embodiments, an engineered cell is modified to reduce expression of TAP2. In some embodiments, an engineered cell is TAP2–. In some embodiments, an engineered cell is modified to reduce expression of CALR. In some embodiments, an engineered cell is CALR–. In some embodiments, an engineered cell is modified to reduce expression of PDIA3. In some embodiments, an engineered cell is PDIA3–. In some embodiments, an engineered cell is modified to reduce expression of TAPBP. In some embodiments, an engineered cell is TAPBP–. Stabilized FOXP3 expression Some embodiments of methods of modifying cells comprise introducing a genetic modification in a cell that stabilizes expression of FOXP3. Similarly, some embodiments of genetically modified cells comprise a genetic modification that stabilizes or increases FOXP3 expression, relative to an unmodified cell. Additionally, some embodiments of nucleic acids and vectors stabilize FOXP3 expression in a cell. In some embodiments, an endogenous FOXP3 locus is modified in a cell, resulting in stabilized expression. For example, in some embodiments, a heterologous promoter is inserted within or downstream from a Treg-specific demethylated region (TSDR) in the genome, and upstream from a first coding exon of an endogenous FOXP3 coding sequence. In some embodiments, a promoter is inserted downstream from the TSDR, and within or upstream from the first coding exon of FOXP3. Insertion of a heterologous promoter in this manner bypasses endogenous regulation of FOXP3 by the TSDR, which can become methylated in inflammatory conditions, inhibiting transcription of the endogenous FOXP3 coding sequence from the endogenous FOXP3 promoter located upstream from the TSDR. Thus, such stabilized FOXP3 expression by heterologous promoter insertion allows stable FOXP3 expression even in inflammatory conditions, preventing transdifferentiation into a T effector cell. The heterologous promoter may be inserted at any position between the endogenous promoter and the first coding exon of the FOXP3 coding sequence. In some embodiments, the heterologous promoter is inserted 1–10,000, 10–1,000, 10–100, 10–5,000, 20–4,000, 30–3,000, 40–2,000, 50–1,000, 60–750, 70–500, 80–400, 90–300, 100–200, 1–1,000, 1,000–2,000, 2,000– 3,000, 3,000–4,000, 4,000–5,000, 5,000–6,000, 6,000–7,000, 7,000–8,000, 8,000–9,000, or 9,000–10,000 nucleotides downstream from the TSDR of FOXP3. In some embodiments, the heterologous promoter is inserted 1–10,000, 10–1,000, 10–100, 10–5,000, 20–4,000, 30–3,000, 40–2,000, 50–1,000, 60–750, 70–500, 80–400, 90–300, 100–200, 1–1,000, 1,000–2,000, 2,000– 3,000, 3,000–4,000, 4,000–5,000, 5,000–6,000, 6,000–7,000, 7,000–8,000, 8,000–9,000, or 9,000–10,000 nucleotides upstream from the first coding exon of the FOXP3 coding sequence. In some embodiments, the heterologous promoter is inserted into the first coding exon, such that a synthetic first coding exon is created, where the synthetic first coding exon differs from the endogenous first coding exon but still comprises a start codon that is in-frame with the FOXP3 coding sequence of downstream FOXP3 exons. In some embodiments, the heterologous promoter is inserted into the TSDR, such that the TSDR is modified and does not inhibit transcription of the endogenous FOXP3 coding sequence in inflammatory conditions. In some embodiments, the nucleic acid comprising a heterologous promoter is comprised on a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the AAV vector is an AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector. In some embodiments, a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding FOXP3 or a functional derivative thereof is introduced into the cell. Expression of a heterologous promoter and sequence encoding FOXP3 is useful, for example, for expressing functional FOXP3 in cells containing genomic mutations in the FOXP3 coding sequence (e.g., cells from subjects having IPEX syndrome). In some embodiments, the sequence encoding FOXP3 is a cDNA sequence that does not comprise an intron. The introduced nucleic acid may be integrated into the genome at a targeted locus (e.g., by homologous recombination), integrated in a non-targeted manner (e.g., by delivery on a lentiviral vector), or not integrated. In some embodiments, the nucleic acid comprises a 5L homology arm that is upstream from the promoter, and a 3L homology arm that is downstream from the nucleic acid sequence encoding FOXP3, and both homology arms have homology to a targeted locus in a genome. Such homology arms promote insertion of the nucleic acid into the genome at the targeted locus by homologous recombination. The homology arms may be the same length, have similar lengths (within 100 bp of each other), or different lengths. In some embodiments, one or both homology arms have a length of 200–2,000 bp, 400–1,500 bp, 500– 1,000 bp. In some embodiments, one or both homology arms are about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, or about 2,000 bp. In some embodiments, the nucleic acid is integrated at a FOXP3 locus in the genome. In some embodiments, the nucleic acid is integrated at a non-FOXP3 locus. In some embodiments, the targeted locus is a safe harbor locus. In some embodiments, the safe harbor locus is an AAVS1 locus, a HIPP11 locus, or a ROSA26 locus. In some embodiments, the nucleic acid is integrated at a TCR, (TRAC) locus. In some embodiments, the nucleic acid is integrated at a TCR? (TRBC) locus. In some embodiments, a nuclease capable of cleaving the genome at a targeted locus, or a nucleic acid encoding the nuclease (e.g., an mRNA) is introduced into the cell. Following delivery of the nuclease or transcription of the nuclease inside the cell, the nuclease introduces a double-stranded break at the targeted locus, thereby promoting integration of a donor template (e.g., nucleic acid comprising a promoter and sequence encoding FOXP3, or nucleic acid comprising a heterologous promoter for promoting transcription of an endogenous FOXP3 coding sequence) into the genome at the targeted locus by homology-directed repair. The nuclease may be any suitable nuclease, including a meganuclease, zinc finger nuclease, TALEN, or RNA-guided nuclease. In embodiments where an RNA-guided nuclease (or nucleic acid encoding an RNA-guided nuclease) is delivered, a guide RNA (or nucleic acid encoding a guide RNA) comprising a spacer sequence complementary to a genomic sequence at the targeted locus is introduced into the cell. A gRNA or nucleic acid encoding a gRNA may be introduced into the cell with the nuclease or nucleic acid encoding the nuclease, or introduced separately (e.g., in a separate vector or delivery vehicle). The RNA-guided nuclease may be any suitable RNA-guided nuclease, such as those described in the section entitled “Nucleases and guide RNAs.” In some embodiments, a nucleic acid comprising a heterologous promoter operably linked to a sequence encoding FOXP3 or a functional derivative thereof is present on a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the AAV vector is an AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is bacterial artificial chromosome. In some embodiments, the vector is human artificial chromosome. In some embodiments, the vector integrates into a chromosome of the genome, and RNA encoding FOXP3 is transcribed from the genome of the cell. In other embodiments, the vector does not integrate into a chromosome, and the sequence encoding FOXP3 is expressed episomally. The heterologous promoter inserted into the FOXP3 locus or operably linked to the FOXP3 coding sequence may be any suitable promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND, PGK, or EF-1, promoter. In some embodiments, the promoter is an MND promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is inducible by a drug or steroid. Chemically induced signaling complex (CISC) Some embodiments of the methods of modifying cells comprise introducing into the cell one or more nucleic acids that collectively comprise (1) a first nucleic acid sequence encoding a first chemically inducible signaling complex (CISC) component, and (2) a second nucleic acid sequence encoding a second chemically inducible signaling complex (CISC) component, each CISC component comprising (a) an extracellular binding domain that is capable of binding to a CISC inducer molecule, (b) a transmembrane domain, and (c) an intracellular signaling domain, such that binding of the first and second CISC components to the CISC inducer molecule results in dimerization of the CISC components and a signal transduction event in the cell. Similarly, some embodiments of cells comprise (1) a first nucleic acid sequence encoding a first chemically inducible signaling complex (CISC) component, and (2) a second nucleic acid sequence encoding a second chemically inducible signaling complex (CISC) component, each CISC component comprising (a) an extracellular binding domain that is capable of binding to a CISC inducer molecule, (b) a transmembrane domain, and (c) an intracellular signaling domain, such that binding of the first and second CISC components to the CISC inducer molecule results in dimerization of the CISC components and a signal transduction event in the cell. Additionally, some nucleic acids and vectors may comprise (1) a first nucleic acid sequence encoding a first chemically inducible signaling complex (CISC) component, and / or (2) a second nucleic acid sequence encoding a second chemically inducible signaling complex (CISC) component, each CISC component comprising (a) an extracellular binding domain that is capable of binding to a CISC inducer molecule, (b) a transmembrane domain, and (c) an intracellular signaling domain, such that binding of the first and second CISC components to the CISC inducer molecule results in dimerization of the CISC components and a signal transduction event in a cell. Expression of CISC components in a cell allows selective induction of signaling in a cell by manipulation of the presence and / or concentration of the CISC inducer molecule. Such controllable induction of signaling allows, for example, selective expansion of cells expressing both CISC components, where the signal transduction event results in proliferation of the cell. In some embodiments, where two nucleic acids, each encoding a different CISC component, are introduced into the cell, such selective expansion allows for selection of cells that contain both nucleic acids, as contacting a cell comprising only one CISC component would not induce dimerization with the absent second CISC component. Non-limiting examples of intracellular signaling domains include IL-2R? and IL-2R> cytoplasmic domains and functional derivatives thereof. In some embodiments, an intracellular signaling domain of one CISC component comprises an IL-2R? cytoplasmic domain or a functional derivative thereof, and an intracellular signaling domain of the other CISC component comprises an IL-2R> cytoplasmic signaling domain or a functional derivative thereof. In some embodiments, dimerization of the CISC components induces phosphorylation of JAK1, JAK3, and / or STAT5 in the cell. In some embodiments, dimerization of the CISC components induces proliferation of the cell. In some embodiments, the IL-2R? cytoplasmic domain comprises the amino acid sequence of embodiments, the IL-2R? cytoplasmic domain is truncated, relative to a wild-type IL-2R? cytoplasmic domain. An exemplary truncated IL-2R? cytoplasmic domain that retains signal transduction activity is described, for example, in Cook et al., Mol Ther.2023. S1525- 0016(23)00255-1. In some embodiments, the truncated IL-2R? domain comprises the amino acid sequence of AYLSLQELQ (SEQ ID NO: 23). In some embodiments, the IL-2R> cytoplasmic domain comprises the amino acid sequence of EGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 7). Non-limiting examples of transmembrane domains include IL-2R?, IL-2R>, erythropoietin (Epo), and thrombopoietin (Tpo) transmembrane domains. In some embodiments, the transmembrane domain of a CISC component is derived from the same protein as the intracellular signaling domain of the CISC component (e.g., a CISC component comprising an IL-2R? intracellular domain comprises an IL-2R? transmembrane domain). In some embodiments, one CISC component comprises an IL-2R? transmembrane domain, and the other CISC component comprises an IL-2R> transmembrane domain. In some embodiments, an IL- 2R? transmembrane domain comprises the amino acid sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 8). In some embodiments, an IL-2R> transmembrane domain comprises the amino acid sequence of VVISVGSMGLIISLLCVYFWL (SEQ ID NO: 9). Non-limiting examples of extracellular binding domains capable of binding a CISC inducer molecule include an FK506-binding protein (FKBP) domain and an FKBP-rapamycin- binding (FRB) domain. FKBP and FRB domains are capable of binding to rapamycin or rapalogs, such as those described below. In some embodiments, an extracellular binding domain of one CISC component comprises an FKBP domain, and an extracellular binding domain of the other CISC component comprises an FRB domain. In some embodiments, the CISC components form a heterodimer in the presence of the CISC inducer molecule. In some embodiments, an FKBP domain comprises an amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGE (SEQ ID NO: 4). In some embodiments, an FKBP domain comprises an amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 11) (Gly107). In some embodiments, an FKBP domain comprises an F36V substitution, at a position corresponding to F36 of SEQ ID NO: 4 or SEQ ID NO: 11. Such a substitution allows the FKBP domain to interact with derivatives of rapamycin, such as AP1903. Clackson et al., Proc Natl Acad Sci U S A.1998.95(18):10437–10442. In some embodiments, an FRB domain comprises an amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 12). In some embodiments, an FRB domain comprises a T74L substitution, at a position corresponding to T74 of SEQ ID NO: 12. This T74L substitution corresponds to a T2098L substitution in the FRB domain as present on full-length mTOR. In some embodiments, an FRB domain comprises a K71P substitution, at a position corresponding to K71 of SEQ ID NO: 12. In some embodiments, an FRB domain comprises a K71T or K71A substitution, at a position corresponding to K71 of SEQ ID NO: 12. In some embodiments, an FRB domain comprises a W77F substitution, at a position corresponding to W77 of SEQ ID NO: 12. In some embodiments, an FRB domain comprises a K71P substitution, a T74L substitution, and a W77F substitution. Such substitutions allow the FRB domain to interact with derivatives of rapamycin, such as AP21967. Stankunas et al., Mol Cell.2003. 12(6):1615–^5^^^^=^ ^^^^^^^^^^^4'^^^=^^^^^^^^5^^^!*^+&CC–107. In some embodiments, a soluble FRB domain comprises an amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 3). In some embodiments, the soluble FRB domain consists of the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 3). Each of the extracellular binding domains, transmembrane domains, and intracellular signaling domains of the CISC components may be connected to another domain of the same CISC component by a linker. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, such as glycines, or a number of amino acids, such as glycine, within a range defined by any two of the aforementioned numbers. In some embodiments, the glycine spacer comprises at least 3 glycines. An extracellular binding domain may be connected to a transmembrane domain by a hinge. A hinge refers to a domain that links the extracellular binding domain to the transmembrane domain and may confer flexibility to the extracellular binding domain. In some embodiments, the hinge positions the extracellular domain close to the plasma membrane to minimize the potential for recognition by antibodies or binding fragments thereof. In some embodiments, the extracellular binding domain is located N-terminal to the hinge. In some embodiments, the hinge may be natural or synthetic. In some embodiments, a CISC component comprises a portion of an IL-2R> extracellular domain between the extracellular binding domain and transmembrane domain. In some embodiments, the portion of the IL-2R> extracellular domain comprises the amino acid sequence of GSNTSKENPFLFALEA (SEQ ID NO: 15). In some embodiments, a CISC component comprises a portion of an IL-2R? extracellular domain between the extracellular binding domain and transmembrane domain. In some embodiments, the portion of the IL-2R? extracellular domain comprises the amino acid sequence of GKDT (SEQ ID NO: 14). In some embodiments, the portion of the IL-2R> extracellular domain comprises the amino acid sequence of ID NO: 16). In some embodiments, a CISC component comprises a portion of an IL-2R? extracellular domain between the extracellular binding domain and transmembrane domain. In some embodiments, the portion of the IL-2R? extracellular domain comprises the amino acid sequence of ) In some embodiments, the CISC inducer molecule is rapamycin or a rapalog. In some embodiments, the CISC inducer molecule is rapamycin. Non-limiting examples of rapalogs include everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, C16-(S)-7-methylindolerapamycin, AP21967, C16-(S)Butylsulfonamidorapamycin, AP23050, sodium mycophenolic acid, benidipine hydrochloride, AP1903, and AP23573, and metabolites or derivatives thereof. In some embodiments, a method comprises introducing into a cell a nucleic acid encoding a third CISC component that is capable of binding to the CISC inducer molecule. Such CISC components are useful, for example, for binding to the intracellular CISC inducer molecules (e.g., intracellular rapamycin), thereby preventing the bound CISC inducer molecule from interacting with other intracellular molecules or structures (e.g., preventing rapamycin from interacting with mTOR). In some embodiments, the third CISC component is a soluble protein that does not comprise a transmembrane domain. In some embodiments, the third CISC component comprises an intracellular FRB domain. In some embodiments, a third CISC component is a soluble protein comprising an FRB domain and lacking a transmembrane domain. Nucleic acids encoding a first, second, and / or third CISC component may be comprised in one or more vectors. In some embodiments, a nucleic acid encoding a first CISC component is present on a separate vector from a nucleic acid encoding the second CISC component. In some embodiments, a nucleic acid encoding the third CISC component is present on the same vector as a nucleic acid encoding the first or second CISC component. In other embodiments, a nucleic acid encoding the third CISC component is present on a distinct vector from nucleic acids encoding the first and / or second CISC components. In some embodiments, one or more vectors are viral vectors. In some embodiments, one or more vectors are lentiviral vectors. In some embodiments, one or more vectors are adeno-associated viral (AAV) vectors. In some embodiments, one or more AAV vectors is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, one or more AAV vectors are AAV5 vectors. In some embodiments, one or more AAV vectors are AAV6 vectors. In some embodiments, a CISC component comprises an amino acid sequence with at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, 2, 21, or 22. In some embodiments, one or more CISC components further comprise a signal peptide. The signal peptide may be any suitable signal peptide directs the translated CISC component to the cell membrane. Non-limiting examples of signal peptides include an LCN2 signal peptide (MPLGLLWLGLALLGALHAQA (SEQ ID NO: 18)), a CD8, signal peptide (MALPVTALLLPLALLLHAARPILWH (SEQ ID NO: 19)), and GM-CSFR, signal peptide (MLLLVTSLLLCELPHPAFLLI (SEQ ID NO: 20)). In some embodiments, one CISC component comprises an amino acid sequence with at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and the other CISC component comprises an amino acid sequence with at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. In some embodiments, each CISC component further comprises a signal peptide, which may have the same or different amino acid sequences. The signal peptides may be any suitable signal peptide that directs the translated CISC component to the cell membrane. In some embodiments, one or more CISC components comprise an LCN2 signal peptide. In some embodiments, one or more CISC components comprise a CD8, signal peptide. In some embodiments, one or more CISC components comprise a GM-CSFR, signal peptide. In some embodiments, both CISC components comprise an LCN2 signal peptide. In some embodiments, both CISC components comprise a CD8, signal peptide. In some embodiments, both CISC components comprise a GM- CSFR, signal peptide. In some embodiments, a third CISC component comprises an amino acid sequence with at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. In some embodiments, a third CISC component consists of an amino acid sequence with at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. In some embodiments, the third CISC component does not comprise a signal peptide. In some embodiments, the third CISC component does not comprise a transmembrane domain. Cells for response to tissue damage and neurodegenerative diseases Some embodiments relate to engineered cells comprising a synthetic NK cell inhibitory receptor engager (e.g., NKG2A engager) and one or more IL-33 or IL-18 receptor components (e.g., ST2, IL1RAP, IL-18R1, IL18RAP) and / or comprising nucleic acids encoding the same. Expression of such receptor components is described, e.g., in International Publication No. WO 2024 / 159138, which is incorporated by reference herein for this purpose. In some embodiments, the engineered cell is a regulatory T cell. In some embodiments, the engineered cell is a CD4+ T cell that stably expresses FOXP3. In some embodiments, the engineered cell is a cell that is capable of differentiating into a regulatory T cell. In some embodiments, the engineered cell is a stem cell. In some embodiments, the engineered cell is an induced pluripotent stem cell or hematopoietic stem cell. In some embodiments, an engineered cell expresses a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding domain, a hinge, a transmembrane domain, a primary signaling domain, and a costimulatory domain. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) of an antibody. In some embodiments, the hinge is a CD8, or CD28 hinge. In some embodiments, the transmembrane domain is a CD8, or CD28 transmembrane domain. In some embodiments, the primary signaling domain is a CD3G signaling domain. In some embodiments, the costimulatory domain is a CD28 or CD137 (4-1BB) signaling domain. In some embodiments, an engineered cell expresses a T cell receptor (TCR). In some embodiments, the TCR comprises a TCR, subunit and a TCR? subunit. In some embodiments, the TCR comprises a TCR> subunit and a TCRK subunit. The IL-33 signaling pathway, or IL-33 / ST2 axis, in Tregs enhances transforming growth factor (TGF)-?1-mediated differentiation of Treg cells and provides a signal for Treg-cell accumulation and maintenance in inflamed tissues. In particular, IL-33 binds to the ST2 / IL-1 receptor accessory protein (IL1RAP) heterodimer, recruiting MyD88 to its intracellular domain. MyD88 binding promotes FOXP3 and GATA3 expression, while also promoting Treg function and expansion through enhancing TGF-?1-mediated differentiation though a p38-dependent mechanism. Griesenauer and Paczesny, Front Immunol.2017. 8:475. MyD88 binding also recruits IL-1R-associated kinases (IRAK1, IRAK2, IRAK3, IRAK4) and TRAF6, leading to either the NF-PB or AP-1 (via MAPK) pathway being activated. Chang et al., Transl Perioper Pain Med.2016. 1(2):24–32. These signaling events promote a tissue repair phenotype in Tregs, which may promote tissue repair through multiple mechanisms including mediating tolerance to inflammation, negatively regulate potentially damaging inflammatory cells (e.g., neutrophils and macrophages), and produce growth factors and mediators including amphiregulin (AREG), keratinocyte growth factor (KGF), and / or TGF-?. Such Tregs stimulated by IL-33 exert cardioprotective effects, thereby mitigating the effects of atherosclerosis, through multiple mechanisms, such as reducing cell death of cardiomyocytes, expansion of M2 macrophages, a reparative macrophage subset, and inducing production of IL-5, which promotes generation of cardioprotective antioxidized low-density lipoprotein (ox-LDL) antibodies. See, e.g., Chen et al., Cell Physiol Biochem.2018.49(1):349-!"B^^@^^^^^^et al., J Exp Med.2008. 205:339-346. IL-33 signaling in neuronal macrophages (microglia) induces a similar protective response, including phagocytosis of extracellular matrix (regulating synapse remodeling), clearance of A? (mitigating neuroinflammation), and preserving oligodendrocytes and oligodendrocyte precursor cells, thereby mitigating the effects of ischemic injury in stroke. Xie et al., Stroke.2021. 52(6):2150-2161. Neuroprotective benefits of IL-33 in stroke are also associated with increased abundance of IL-10-producing Tregs. See, e.g., Zhang et al., JCI Insight.2018.3(18):e121560. Similarly, the IL-18 signaling pathway also results in activation of NF-PB and / or AP-1. IL-18 signals through its receptor, which comprises an interleukin 18 receptor 1 (IL-18R1 or IL- 18R,) chain, and an interleukin 18 receptor accessory protein (IL18RAP or IL-18R?) chain. Following the binding of IL-18 to IL-18R,, IL-18R? then binds to form a trimer. MyD88 binds to the Toll-IL-1 receptor (TIR) domain of IL-18R1 and IL18RAP. IRAK1 and IRAK4 are the bound via the death domain of MyD88. TRAF6 then binds IRAK1, resulting in the degradation of inhibitor of PB (IPB) and translocation of phosphorylated p65 / p50 NF-PB into the nucleus. The MAPK cascade comprising Extracellular Signal-regulated Kinase (ERK), c-jun N-terminal kinase (JNK), and p38 is also activated, which induces IFN-> production promotes cell proliferation. IL-18 stimulation also induces the phosphorylation and activation of phosphatidylinositol-3 kinase (PI3K) / Akt / S6 and mammalian target of rapamycin (mTOR). Accordingly, some embodiments relate to engineered cells having increased surface presence and / or expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP, or one or more functional derivatives thereof. ST2 is not expressed on many circulating T cell subsets (see, e.g., Griesenauer and Paczesny, Front Immunol.2017.8:475), and surface expression of each of IL- 18R and IL1RAP is also low or absent on circulating T cells. Upregulation of ST2, IL1RAP, IL- 18R1, and / or IL18RAP, expression in engineered Treg allows these engineered Tregs to also respond to IL-33 or IL-18, promoting tissue repair by the engineered Tregs. Tregs that stably express FOXP3 and also exhibit one or more of ST2, IL1RAP, IL-18R1 on their surface, having a resulting tissue reparative phenotype, are useful in multiple conditions characterized by inflammation and resulting tissue damage. Examples of such conditions include stroke, acute kidney injury, conditions associated with solid organ transplant, graft-versus-host disease (GvHD), fibrosis, acute lung injury, acute respiratory distress syndrome, and neurodegenerative diseases. These and other conditions in which the reparative properties of such Tregs are expected to be beneficial are described below in the section entitled “Methods of use.” In some embodiments, an engineered cell comprises ST2 or a nucleic acid encoding ST2. In some embodiments, the nucleic acid encoding ST2 comprises a heterologous promoter operably linked to a nucleotide sequence encoding ST2. In some embodiments, the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding ST2. In some embodiments, the nucleic acid encoding ST2 is exogenous or comprises an exogenous nucleotide sequence encoding ST2. In some embodiments, the ST2 comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 311 and retains the functional activity of wild- type ST2. In some embodiments, the engineered cell expresses ST2 constitutively. In some embodiments, the engineered cell expresses ST2 at a supraphysiologic level. In some embodiments, an engineered cell comprises IL1RAP or a nucleic acid encoding IL1RAP. In some embodiments, the nucleic acid encoding IL1RAP comprises a heterologous promoter operably linked to a nucleotide sequence encoding IL1RAP. In some embodiments, the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding IL1RAP. In some embodiments, the nucleic acid encoding IL1RAP is exogenous or comprises an exogenous nucleotide sequence encoding IL1RAP. In some embodiments, the IL1RAP comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 312 and retains the functional activity of wild-type IL1RAP. In some embodiments, the engineered cell expresses IL1RAP constitutively. In some embodiments, the engineered cell expresses IL1RAP at a supraphysiologic level. In some embodiments, an engineered cell comprises both ST2 and IL1RAP or nucleic acids encoding ST2 and IL1RAP. In some embodiments, an engineered cell comprises IL-18R1 or a nucleic acid encoding IL-18R1. In some embodiments, the nucleic acid encoding IL-18R1 comprises a heterologous promoter operably linked to a nucleotide sequence encoding IL-18R1. In some embodiments, the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding IL- 18R1. In some embodiments, the nucleic acid encoding IL-18R1 is exogenous or comprises an exogenous nucleotide sequence encoding IL-18R1. In some embodiments, the IL-18R1 comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 313 and retains the functional activity of wild-type IL-18R1. In some embodiments, the engineered cell expresses IL-18R1 constitutively. In some embodiments, the engineered cell expresses IL-18R1 at a supraphysiologic level. In some embodiments, an engineered cell comprises IL18RAP or a nucleic acid encoding IL18RAP. In some embodiments, the nucleic acid encoding IL18RAP comprises a heterologous promoter operably linked to a nucleotide sequence encoding IL18RAP. In some embodiments, the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding IL18RAP. In some embodiments, the nucleic acid encoding IL18RAP is exogenous or comprises an exogenous nucleotide sequence encoding IL18RAP. In some embodiments, the IL18RAP comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 314 and retains the functional activity of wild-type IL18RAP. In some embodiments, the engineered cell expresses IL18RAP constitutively. In some embodiments, the engineered cell expresses IL18RAP at a supraphysiologic level. Some embodiments relate to engineered cells that have been cultured in the presence of IL-33 and / or IL-18. Some embodiments relate to methods of culturing engineered cells in the presence of IL-33 and / or IL-18. Culturing cells expressing cognate receptors in the presence of IL-33 and / or IL-18 allows stimulation by these cytokines, leading to enrichment of tissue Treg markers including CCR5, CCR8, HLA-DR, CD71, Ki-67, CD39, and TIGIT. In some embodiments, an engineered cell that comprises or expresses ST2 and / or IL1RAP is cultured in the presence of IL-33. In some embodiments, an engineered cell that comprises or expresses IL-18R1 and / or IL18RAP is cultured in the presence of IL-18. In some embodiments, one or more additional cytokines are present. In some embodiments, one or more additional cytokines are selected from the group consisting of IL-2, IL-4, and TNF-,. In some embodiments, an engineered cell comprises or expresses first and second CISC components, and rapamycin or a rapalog is also present. In some embodiments, rapamycin is present. In some embodiments, the engineered cell is cultured in the presence of IL-33 and / or IL- 18 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the engineered cell is cultured in the presence of IL-33 and / or IL-18 for 1–5, 5–10, 10–15, or 15–20 days. In some embodiments, the engineered cell is cultured in the presence of IL-33 and / or IL-18 for 1–20, 1–15, 1–10, or 1–5 days. In some embodiments, the engineered cell is cultured in the presence of IL-33 and / or IL-18 for 5–20, 10–20, or 15–20 days. In some embodiments, the concentration of IL-33 is 0.1 to 500 ng / mL. In some embodiments, the concentration of IL-33 is 0.5 to 1.0 ng / mL, 1.0 to 10 ng / mL, 10 to 50 ng / mL, 50 to 100 ng / mL, 100 to 200 ng / mL, 200 to 300 ng / mL, 300 to 400 ng / mL, or 400 to 500 ng / mL. In some embodiments, the concentration of IL-33 is 0.5 to 450 ng / mL.0.5 to 400 ng / mL. 0.5 to 350 ng / mL. 0.5 to 300 ng / mL.0.5 to 250 ng / mL.0.5 to 200 ng / mL. 0.5 to 150 ng / mL.0.5 to 100 ng / mL. In some embodiments, the concentration of IL-33 is 1.0 to 500 ng / mL, 10 to 500 ng / mL, 50 to 500 ng / mL, 100 to 500 ng / mL, 150 to 500 ng / mL, 200 to 500 ng / mL, or 250 to 500 ng / mL. In some embodiments, the concentration of IL-18 is 0.1 to 500 ng / mL. In some embodiments, the concentration of IL-18 is 0.5 to 1.0 ng / mL, 1.0 to 10 ng / mL, 10 to 50 ng / mL, 50 to 100 ng / mL, 100 to 200 ng / mL, 200 to 300 ng / mL, 300 to 400 ng / mL, or 400 to 500 ng / mL. In some embodiments, the concentration of IL-18 is 0.5 to 450 ng / mL.0.5 to 400 ng / mL. 0.5 to 350 ng / mL. 0.5 to 300 ng / mL.0.5 to 250 ng / mL.0.5 to 200 ng / mL. 0.5 to 150 ng / mL.0.5 to 100 ng / mL. In some embodiments, the concentration of IL-18 is 1.0 to 500 ng / mL, 10 to 500 ng / mL, 50 to 500 ng / mL, 100 to 500 ng / mL, 150 to 500 ng / mL, 200 to 500 ng / mL, or 250 to 500 ng / mL. In some embodiments, the concentration of IL-2, IL-4, or TNF-, is 0.1 to 500 ng / mL. In some embodiments, the concentration of IL-2, IL-4, or TNF-, is 0.5 to 1.0 ng / mL, 1.0 to 10 ng / mL, 10 to 50 ng / mL, 50 to 100 ng / mL, 100 to 200 ng / mL, 200 to 300 ng / mL, 300 to 400 ng / mL, or 400 to 500 ng / mL. In some embodiments, the concentration of IL-2, IL-4, or TNF-, is 0.5 to 450 ng / mL. 0.5 to 400 ng / mL.0.5 to 350 ng / mL. 0.5 to 300 ng / mL. 0.5 to 250 ng / mL.0.5 to 200 ng / mL.0.5 to 150 ng / mL. 0.5 to 100 ng / mL. In some embodiments, the concentration of IL-2, IL-4, or TNF-, is 1.0 to 500 ng / mL, 10 to 500 ng / mL, 50 to 500 ng / mL, 100 to 500 ng / mL, 150 to 500 ng / mL, 200 to 500 ng / mL, or 250 to 500 ng / mL. In some embodiments, the concentration of rapamycin or the rapalog is 0.01 to 100 nM. In some embodiments, the concentration of rapamycin or the rapalog is 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6.0 nM, 6.5 nM, 7.0 nM, 7.5 nM, 8.0 nM, 8.5 nM, 9.0 nM, 9.5 nM, 10 nM, 11 nM, 12 nM...

Claims

1. CLAIMS What is claimed is:

1. A synthetic CD161 engager comprising:(i) a CD161-^^^^^^(^^^^^^^^^'^^^^^^^^^^^^^^ ^^^^^^^40^5^^^^^^(ii) a transmembrane domain.

2. The synthetic CD161 engager of claim 1, wherein the CD161-binding domain is aCD161-specific antibody or fragment thereof.

3. The synthetic CD161 engager of claim 1 or 2, wherein the CD161-binding domain is asingle-chain variable fragment (scFv) of a CD161-specific antibody.

4. The synthetic CD161 engager of claim 1, wherein the CD161-binding domain is afragment of an LLT1 extracellular domain.

5. The synthetic CD161 engager of any one of claims 1–4, wherein the CD161-bindingdomain and the transmembrane domain are connected by a linker.

6. The synthetic CD161 engager of any one of claims 1–4, wherein the CD161-bindingdomain and the transmembrane domain are connected by hinge.

7. The synthetic CD161 engager of any one of the preceding claims, wherein thetransmembrane domain is a type II transmembrane domain.

8. The synthetic CD161 engager of claim 7, wherein the type II transmembrane domain is atransmembrane domain of SMIM1 having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 39.

9. The synthetic CD161 engager of claim 8, wherein the synthetic CD 161 engager comprises a SM1M1 transmembrane anchor having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 46.

10. The synthetic CD161 engager of claim 7, wherein the type II transmembrane domain has less than 90% identity to a transmembrane domain of human LET I.

11. The synthetic CD 161 engager of claim 7 or 10, wherein the type II transmembrane domain has at least 1 substitution, deletion, and / or insertion relative to a transmembrane domain of human LLT1 having SEQ ID NO: 45.

12. The synthetic CD 161 engager of any one of claims 1-6, wherein the transmembrane domain is a type I transmembrane domain, wherein:(i) the C-terminus of the CD 161 -binding domain is covalently linked to the N- termmus of the type I transmembrane domain; and(ii) the N-tenninus of the CD161-binding domain is covalently linked to a signal peptide derived from a type I transmembrane protein.

13. The synthetic CD161 engager of any one of the preceding claims, wherein the synthetic CD161 engager does not comprise a cytoplasmic signaling domain.

14. The synthetic CD161 engager of any one of the preceding claims, wherein the synthetic CD161 engager does not comprise a cytoplasmic region with more than 10 amino acids.

15. The synthetic CD161 engager of any one of the preceding claims, wherein the synthetic CD 161 engager comprises a cytoplasmic region.

16. The synthetic CD161 engager of claim 15, wherein the cytoplasmic region comprises no more than 10 amino acids.

17. A synthetic NKG2A engager comprising:(i) single-chain variable fragment (scFv) of an NKG2A-^^^^^^^^^^^^^^^^ ^^^^^ (ii) a transmembrane domain.

18. The synthetic NKG2A engager of claim 17, wherein scFv comprises: (i) an hCDR1 comprising the amino acid sequence of SEQ ID NO: 73^ (ii) an hCDR2 comprising the amino acid sequence of SEQ ID NO: 74^ (iii) an hCDR3 comprising the amino acid sequence of SEQ ID NO: 75^ (iv) an lCDR1 comprising the amino acid sequence of SEQ ID NO: 76^ (v) an lCDR2 comprising the amino acid sequence of SEQ ID NO: 77^^^^^ (vi) an lCDR3 comprising the amino acid sequence of SEQ ID NO:

78.

19. The synthetic NKG2A engager of any one of claims 17–18, wherein the scFv comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the C-terminus of the VH region is connected to the N-terminus of the VL region by a linker.

20. The synthetic NKG2A engager of any one of claims 17–19, wherein the linker is a Whitlow linker.

21. The synthetic NKG2A engager of claim 20, wherein the Whitlow linker comprises the amino acid sequence of SEQ ID NO:

29.

22. The synthetic NKG2A engager of any one of claims 17–21, further comprising a hinge connecting the scFv and the transmembrane domain.

23. The synthetic NKG2A engager of claim 22, wherein the hinge comprises a hinge of CD8, or CD28, or an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO:

35.

24. The synthetic NKG2A engager of any one of claims 17–23, wherein the transmembrane domain comprises a transmembrane domain of CD8,, CD28, or PDGFRA, or an amino acidsequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 40.

25. Fhe synthetic NKG2A engager of any one of claims 17-24, wherein the synthetic NKG2A engager does not comprise a cytoplasmic signaling domain.

26. The synthetic NKG2A engager of any one of claims 17-24, wherein the synthetic NKG2A engager does not comprise a cytoplasmic region with more than 10 amino acids.

27. The synthetic NKG2A engager of any one of claims 17---24, wherein the synthetic NKG2A engager comprises a cytoplasmic region.

28. The synthetic NKG2A engager of claim 27, wherein the cytoplasmic region comprises no more than 10 amino acids.

29. A synthetic NKG2A engager comprising, in N- to C-terminal order:(a) an scFv comprising (i) a VH region comprising the amino acid sequence of SEQ ID NO: 71 with an E56A substitution, (ii) a linker, and (iii) a VL region comprising the amino acid sequence of SEQ ID NO: 72;(b) a CD8a hinge; and(c) a PDGFRA transmembrane domain.

30. The synthetic NKG2A engager of claim 29, wherein the CD8a hinge comprises an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 33.

31. The synthetic NKG2A engager of claim 29 or 30, wherein the PDGFRA transmembrane domain comprises an amino acid sequence having no more than 2 substitutions, amino acid insertions, and / or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 40.

32. The synthetic NKG2A engager of any one of claims 29-31, wherein the syntheticNKG2A engager does not comprise a cytoplasmic region with more than 10 amino acids.

33. The synthetic NKG2A engager of any one of claims 29-32, wherein the linker is aWhitlow linker.

34. The synthetic NKG2A engager of any one of claims 29-33, wherein the syntheticNKG2A engager does not comprise a C-terminal KKXX motif.

35. A genetically modified cell comprising the synthetic CD161 engager of any one of claims1–16 or synthetic NKG2A engager of any one of claims 17–34.

36. A genetically modified cell comprising a nucleic acid, the nucleic acid comprising anucleotide sequence encoding the synthetic CD161 engager of any one of claims 1–16 or synthetic NKG2A engager of any one of claims 17–34.

37. The genetically modified cell of claim 36, wherein the nucleic acid is a chromosome,wherein the chromosome further comprises a heterologous promoter operably linked to the nucleotide sequence encoding the synthetic CD161 engager or NKG2A engager.

38. The genetically modified cell of claim 36 or 37 wherein the nucleotide sequenceencoding the synthetic CD161 engager or NKG2A engager is in a ^2M locus, wherein the cell does not express a class I MHC protein on its surface.

39. The genetically modified cell of claim 36 or 37, wherein the nucleotide sequenceencoding the synthetic CD161 engager or NKG2A engager is in a CIITA locus, wherein the cell does not express a class II MHC protein on its surface.

40. The genetically modified cell of claim 36 or 37, wherein the nucleotide sequenceencoding the synthetic CD161 engager or NKG2A engager is in a TRAC or TRBC locus, wherein the cell does not express a T cell receptor (TCR) on its surface.

41. The genetically modified cell of any one of claims 36-40, wherein the nucleotide sequence encoding the synthetic CD161 engager or synthetic NKG2A engager is codon- optimized for expression in a human cell.

42. A genetically modified cell that constitutively expresses LLT1 or a fragment thereof that comprises a CD161-binding domain and a transmembrane domain.

43. A genetically modified cell that constitutively expresses a supraphysiologic level of LLT1 or a fragment thereof that comprises a CD161-binding domain.

44. A genetically modified cell comprising, in a genomic nucleic acid comprising a nucleotide sequence encoding LLT1 or a fragment thereof that comprises a CD161-binding domain, a heterologous promoter operably linked to the nucleotide sequence encoding LLT1 or fragment thereof.

45. The genetically modified cell of claim 44, wherein the heterologous promoter is a constitutive promoter.

46. The genetically modified cell of claim 45, wherein the constitutive promoter is an MND promoter, an EF-1, promoter, or a PGK promoter.

47. The genetically modified cell of claim 45 or 46, wherein the constitutive promoter is an MND promoter.

48. The genetically modified cell of claim 44, wherein the heterologous promoter is an inducible promoter.

49. The genetically modified cell of any one of claims 38–48, wherein the nucleotide sequence encoding LLT1 or the fragment thereof is codon-optimized for expression in a human cell.

50. Fhe genetically modified cell of any one of claims 44-49, wherein the nucleotide sequence encodes a fragment of LLT1 that comprises:(i) a deletion of one or more N -terminal amino acids relative to SEQ ID NO: 45; and / or(ii) a deletion of one or more C -terminal amino acids relative to SEQ ID NO: 45.

51. A nucleic acid encoding the synthetic CD 161 engager of any one of claims 1 -16 or synthetic NKG2A engager of any one of claims 17 -34.

52. The nucleic acid of claim 51, wherein the nucleic acid is a deoxyribonucleic acid (DNA), a messenger ribonucleic acid (mRNA), a circular ribonucleic acid (circRNA), or a selfamplifying ribonucleic acid (saRNA).

53. A lipid nanoparticle comprising the nucleic acid of claim 51 or 52.

54. A viral vector encoding the synthetic CD161 engager of any one of claims 1—16 or synthetic NKG2A engager of any one of claims 17-34.

55. The viral vector of claim 54, wherein the viral vector is a lentiviral vector or adeno- associated viral vector.

56. A system comprising: (i) the nucleic acid of claim 51 or 52, the lipid nanoparticle of claim 53, or the viral vector of claim 54 or 55; and (ii) a nuclease or nucleic acid encoding the nuclease.

57. The system of claim 56, wherein the nuclease is a meganuclease, TALEN, zinc finger nuclease, or RNA-guided DNA endonuclease.

58. The system of claim 56 or 57, wherein the nuclease is an RNA-guided DNAendonuclease, and wherein the system further comprises a guide RNA (gRNA) or a nucleic acid encoding the gRNA.

59. The system of claim 58, wherein the RNA-guided DNA endonuclease is Cas9.

60. A method comprising delivering the system of any one of claims 56-59 to a cell, therebyproducing a genetically modified cell.

61. The genetically modified cell of any one of claims 35-50, wherein the geneticallymodified cell is: (i) ^^^^^^^^^^^^(ii) ^^'^^^^^^^^^^^^^^^^^^(iii) ^^^ ^^'^^ ^^^^^^^^^(iv) ^^#^^^^^^(v) ^^40^D^#^^^^^^^^^^<^^(vi) a CD8+ T cell.

62. The genetically modified cell of any one of claims 35-50 or 61, wherein the geneticallymodified cell is a regulatory T cell (Treg).

63. The genetically modified cell of any one of claims 35-55, 61, or 62, wherein thegenetically modified cell is a FOXP3+ regulatory T cell (Treg).

64. A genetically modified regulatory T cell (Treg) comprising a nucleic acid, the nucleicacid comprising a promoter operably linked to a nucleotide sequence encoding a synthetic NK cell inhibitory receptor engager, the synthetic NK cell inhibitory receptor engager comprising: (i) a NK cell inhibitory receptor-binding domain that specifically binds to an NK cell^^'^^^^^^ ^^^^^^^^^^^^^^ (ii) a transmembrane domain.

65. The genetically modified Treg of claim 64, wherein the NK cell inhibitory receptor is selected from the group consisting of CD200R1, CD300A, CD96, HAVCR2, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KLRG1, LAG-3, LAIR1, LILRB1, LILRB3, NKG2A, PD-1, RTN4R, Siglec-3, Siglec-7, Siglec-9, SIRP,, and TIGIT.

66. The genetically modified Treg of claim 64 or 65, wherein the NK cell inhibitory receptor is NKG2A.

67. The genetically modified Treg of claim 66, wherein the NK cell inhibitory receptor- binding domain comprises monalizumab or a fragment thereof.

68. The genetically modified Treg of claim 66 or 67, wherein the NK cell inhibitory receptor- binding domain comprises: (i) an hCDR1 comprising the amino acid sequence of SEQ ID NO: 73^ (ii) an hCDR2 comprising the amino acid sequence of SEQ ID NO: 74^ (iii) an hCDR3 comprising the amino acid sequence of SEQ ID NO: 75^ (iv) an lCDR1 comprising the amino acid sequence of SEQ ID NO: 76^ (v) an lCDR2 comprising the amino acid sequence of SEQ ID NO: 77^^^^^ (vi) an lCDR3 comprising the amino acid sequence of SEQ ID NO:

78.

69. The genetically modified cell of any one of claims 35-50 or 61-68, wherein the genetically modified cell comprises a heterologous promoter located in a genomic nucleic acid: (i) downstream from a Treg-^^^^^^^^^^^^^^' ^^^^^^^^(^^^^*#^03+^^^^^ (ii) upstream from a first coding exon of an endogenous FOXP3 gene.

70. The genetically modified cell of any one of claims 35-50 or 61-69, wherein the genetically modified cell comprises: (i) a nucleic acid encoding a first component of a chemically induced signaling complex (CISC), the first CISC component comprising: (a) a first extracellular domain comprising an FK506-binding protein domain ^'^^^^^^^^^^^^^^ ^^^^(b) a first transmembrane domain; and(c) a first cytoplasmic domain comprising an intracellular signaling domain of a first cytokine receptor; and(ii) a nucleic acid encoding a second CISC component, the second CISC component comprising:(a) a second extracellular domain comprising an FKBP-rapamycin- binding(FRB) domain;(b) a second transmembrane domain; and(c) a second cytoplasmic domain comprising an intracellular signaling domain of a second cytokine receptor, wherein the first and second CISC components dimerize in the presence of rapamycin.

71. The genetically modified cell of claim 70, wherein the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin- 2 receptor beta (IL-2Rp) transmembrane domain, and an IL-2Rp cytoplasmic domain.

72. The genetically modified cell of claim 70 or 71, wherein the genetically modified cell comprises a nucleic acid encoding a soluble FRB domain, wherein SEQ ID NO: 3 has at least 90%' sequence identity to the amino acid sequence of the soluble FRB domain.

73. A method comprising administering the genetically modified cell of any one of claims 35-50 or 61-72 to a subject in need thereof.

74. The method of claim 73, wherein the method is a method for treating or preventing an autoimmune disease, allergic disease, inflammatory disease, or adverse effect of transplantation in the subject.

75. The genetically modified cell of any one of claims 35-50 or 61-72, for use in a method oftreating or preventing an autoimmune disease, allergic disease, or inflammatory disease in a subject.

76. The method of claim 74 or the genetically modified cell of claim 75, wherein the autoimmune disease is selected from the group consisting of from type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early onset rheumatoid arthritis, ankylosing spondylitis, immune-mediated pregnancy loss, immune- mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn’s disease, inflammatory bowel disease (IBD), ulcerative colitis, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjögren’s syndrome, and celiac disease.

77. The method of claim 74 or genetically modified cell of claim 75, wherein the allergicdisease is selected from the group consisting of allergic asthma, steroid-resistant asthma, atopic dermatitis, celiac disease, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis.

78. The method of claim 74 or genetically modified cell of claim 75, wherein theinflammatory disease is selected from the group consisting of stroke, myocardial infarction, acute swelling, severe wounding, muscle injuries, burn injuries, traumatic brain injury, acute respiratory distress syndrome (ARDS), pancreatic islet cell transplantation, asthma, hepatitis, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, Still’s disease, uveitis, ulcerative colitis, graft-versus-host disease (GvHD), tolerance induction for transplantation, transplant rejection, and sepsis.

79. The method of any one of claims 74 or 76-78, or the genetically modified cell of any oneof claims 75-78, wherein the genetically modified cell is allogeneic to the subject.

80. The method of any one of claims 74 or 76–79, or the genetically modified cell of any oneof claims 75–79, wherein the genetically modified cell is detectable in the subject for at least 1– 60 months after administration.

81. The method of any one of claims 74 or 76–79, or the genetically modified cell of any oneof claims 75–79, wherein the engineered cell is detectable in the subject for up to 1–60 months after administration.

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