Inhibiting natural killer cell cytotoxicity against cell therapies

Abstract

Provided herein are recombinant nucleic acids encoding one or more binding domains specific for CD300a and / or NKG2A, and vectors, engineered cells, and compositions comprising the recombinant nucleic acids. Also provided are methods of using the recombinant nucleic acids to enhance persistence of an engineered cell, such as by protecting against natural killer (NK) cell mediated killing of the engineered cell, methods of producing an engineered cell comprising the recombinant nucleic acids, and methods of treating a subject and / or improving a clinical outcome in a subject using the engineered cells.

Description

[0001] INHIBITING NATURAL KILLER CELL CYTOTOXICITY AGAINST CELL THERAPIES [1] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 3, 2024, is named “01335-0001-00PCT.xml” and is 521,178 bytes in size. FIELD OF THE INVENTION [2] The present disclosure provides recombinant nucleic acids encoding one or more binding domains specific for CD300a and / or NKG2A, and methods of using the recombinant nucleic acids to enhance persistence of an engineered cell, such as by protecting against natural killer (NK) cell mediated killing of the engineered cell. INTRODUCTION [3] Adoptive T cell immunotherapy is a rapidly growing field, in particular in cancer treatments. Chimeric antigen receptor (CAR) T cell therapy represents a breakthrough in the field of immuno- oncology. In general, CAR T cell engagement of, e.g., CD19- or CD20-expressing cancer cells, results in T cell activation, proliferation and secretion of inflammatory cytokines and chemokines resulting in tumor cell lysis. However, autologous CAR T cell therapy presents multiple challenges, such as time for manufacturing and need for interim therapies in progressing patients, wide variations in terms of quality and quantity of T cells, and difficulties in obtaining enough cells for redosing. “Off-the-shelf” allogeneic CAR T cells generated by differentiation of pluripotent stem cells (such as induced pluripotent stem cells or human embryonic stem cells) into T cells or derived from third-party donor T cells may provide solutions to these varied problems. For example, off- the-shelf allogenic CAR T cells can be expanded in high numbers prior to treatment and made quickly available to patients. This high-volume manufacturing allows for an opportunity to quickly and easily re-dose patients with a new off-the-shelf cell therapy without delays due to manufacturing or scale of production. Additionally, pretreatment manufacturing protocols allow for multiple edits, manipulations, or T cell receptor selection strategies that otherwise could be difficult to accommodate in the autologous setting. [4] However, allogeneic T cells possess foreign immunological identities that can lead to histocompatibility considerations such as graft-versus-host disease (GvHD) and rejection or low persistence of the allogeneic cells. For example, iPSC-derived T cells that have not undergone engineering to make the cells immuno-compatible or hypoimmunogenic cannot be applied to third- party patients due to the presence of endogenous T cell receptor (TCR) and / or human leukocyte antigen (HLA) mismatch. GvHD is believed to be largely driven by donor- or iPSC-derived T cell recognition of host peptide-+ / $^FRPSOH[HV^WKURXJK^WKH^Įȕ^7^FHOO^UHFHSWRU^FRPSOH[^^Įȕ7&5^^ Rejection is mainly driven by host NK cells, CD8+ T-cells, CD4+ T cells, and, to a lesser extent, by macrophages. In the context of CAR T-cell therapy, the relative contribution of these cell types to allograft rejection may vary depending on their absolute numbers and reconstitution kinetics following preconditioning regimen. Because, in many cases, host NK cells and host CD8+ T cells appear to recover their initial levels more quickly than CD4+ T cells, CD8+ T cells and NK cells may play a larger role in controlling the length of the allogeneic CAR T-cell therapeutic window by being the first and primary contributors to rejection. [5] Because of the complexity of the immune system, engineering hypoimmunogenic T cells to enable adoptive cell transfer in an allogeneic setting has been a challenge. Some strategies for preventing or reducing GvHD and increasing therapeutic cell persistence include engineering approaches such as knocking out or disrupting the native alpha beta T cell receptor by gene editing of the T cell receptor alpha constant (TRAC) locus or by insertion of the CAR transgene into the TRAC locus, incorporating a receptor to target activated alloreactive host T cells, or disrupting HLA expression on CAR T cells by knocking out beta 2 microglobulin ^ȕ^P^^and evading NK cell- mediated killing by expressing HLA class I histocompatibility antigen alpha chain E (HLA-E). To date, automated, scalable, and efficient manufacturing of therapeutic immune cells, along with effective mechanisms for protecting these cells against host rejection, remain significant challenges to widespread access to CAR T cells for patients. SUMMARY [6] The presently disclosed recombinant nucleic acids, vectors, engineered cells, and methods of using the recombinant nucleic acids, vectors, and engineered cells allow for development of, e.g., immune-evasive (hypoimmunogenic) universal CAR T-cells that can resist host CD8+ T cell and NK-cell cytotoxicity and can be compatible with adoptive cell transfer in an allogeneic setting. [7] The following exemplary embodiments are provided. [8] Embodiment 1 is a recombinant nucleic acid encoding a construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain. [9] Embodiment 2 is the recombinant nucleic acid of embodiment 1, wherein the CD300a binding domain comprises an antibody or a fragment thereof, a variable domain on a heavy chain (VHH) antibody, a cytokine, a ligand, or a peptide.

[0010] Embodiment 3 is the recombinant nucleic acid of embodiment 1 or embodiment 2, wherein the NKG2A binding domain comprises an antibody or a fragment thereof, a VHH, a cytokine, a ligand, or a peptide.

[0011] Embodiment 4 is the recombinant nucleic acid of embodiment 2 or embodiment 3, wherein (a) the antibody or a fragment thereof comprises a single chain variable fragment (scFv) or a VHH, or (b) the peptide is an adnectin or a design ankyrin repeat protein (DARPin).

[0012] Embodiment 5 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the VHH comprises the VH domain of a camelid heavy chain antibody.

[0013] Embodiment 6 is the recombinant nucleic acid of any one of the preceding embodiments, wherein the CD300a binding domain comprises a VHH (CD300a VHH) and / or the NKG2A binding domain comprises a VHH (NKG2A VHH).

[0014] Embodiment 7 is the recombinant nucleic acid of any one of the preceding embodiments, wherein the construct for inhibiting NK cell cytotoxicity comprises or consists of the CD300a binding domain, and the CD300a binding domain comprises a VHH (CD300a VHH).

[0015] Embodiment 8 is the recombinant nucleic acid of any one of embodiments 2-7, wherein the CD300a VHH comprises: (a) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising AAKPGEDVY (SEQ ID NO: 182); (b) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLSQFAS (SEQ ID NO: 183); (c) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPRSGWGL (SEQ ID NO: 184); (d) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKTRYES (SEQ ID NO: 185); (e) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NTRLAHGRDVLGGVAYDI (SEQ ID NO: 186); (f) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NTRRLGRSGDLVQDY (SEQ ID NO: 187); (g) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSRYY (SEQ ID NO: 175), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPDRDY (SEQ ID NO: 188); (h) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLPDVLPLEY (SEQ ID NO: 189); (i) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYW (SEQ ID NO: 176), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKVDGSYGIVTEL (SEQ ID NO: 190); (j) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising INGSGGST (SEQ IS NO: 180), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising HTRRSGTSMAMDV (SEQ ID NO: 191); (k) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKLTMVY (SEQ ID NO: 192); (l) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLTNEY (SEQ ID NO: 193); (m) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKVRPSYEY (SEQ ID NO: 194); (n) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSPYY (SEQ ID NO: 177), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VAKPGYEY (SEQ ID NO: 195); (o) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGRT (SEQ IS NO: 181), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPGEDVY (SEQ ID NO: 196); (p) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKSNMVY (SEQ ID NO: 197); (q) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising TTKVDGSYGIVTEL (SEQ ID NO: 198); or (r) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYW (SEQ ID NO: 176), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising INGSGGST (SEQ IS NO: 180), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising AAARDRERDY (SEQ ID NO: 199).

[0016] Embodiment 9 is the recombinant nucleic acid of any one of embodiments 2-8, wherein the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 155-172.

[0017] Embodiment 10 is the recombinant nucleic acid of any one of embodiments 2-9, wherein the CD300a VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 155- 172.

[0018] Embodiment 11 is the recombinant nucleic acid of any one of embodiments 2-10, wherein a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 137-154.

[0019] Embodiment 12 is the recombinant nucleic acid of any one of embodiments 2-11, wherein a nucleotide sequence encoding the CD300a VHH comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 137-154.

[0020] Embodiment 13 is the recombinant nucleic acid of embodiment 1, wherein the CD300a binding domain comprises a scFv and / or the NKG2A binding domain comprises a scFv.

[0021] Embodiment 14 is the recombinant nucleic acid of any one of embodiments 1-4 or 13, wherein: (a) the NKG2A binding domain comprises: (i) a NKG2A light chain variable region (NKG2A VL); and (ii) a NKG2A heavy chain variable region (NKG2A VH); and / or (b) the CD300a binding domain comprises: (i) a CD300a light chain variable region (CD300a VL); and (ii) a CD300a heavy chain variable region (CD300a VH).

[0022] Embodiment 15 is the recombinant nucleic acid of embodiment 14, wherein: (a) the NKG2A light chain variable region (NKG2A VL) comprises a VL CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RASENIYSYLA (SEQ ID NO: 98), a VL CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NAKTLAE (SEQ ID NO: 99) and a VL CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising QHHYGTPRT (SEQ ID NO: 100); (b) the NKG2A heavy chain variable region (NKG2A VH) comprises a VH CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising SYWMN (SEQ ID NO: 101), a VH CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RIDPYDSETHYAQKLQG (SEQ ID NO: 102), and a VH CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GGYDFDVGTLYWFFDV (SEQ ID NO: 103); (c) the CD300a light chain variable region (CD300a VL) comprises a VL CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RASQDISNYLN (SEQ ID NO: 104) a VL CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising TSRLHS (SEQ ID NO: 105), and a VL CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising QQGNTLPWT (SEQ ID NO: 106); and (d) the CD300a heavy chain variable region (CD300a VH) comprises a VH CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising SYWMQ (SEQ ID NO: 107), a VH CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising EIDPSDSYTNYNQKFKG (SEQ IS NO: 108), and a VH CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising WGMAYGTSSYWYFDV (SEQ ID NO: 109).

[0023] Embodiment 16 is the recombinant nucleic acid of embodiment 14 or embodiment 15, wherein (a) the NKG2A VL comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) the NKG2A VH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 3-8; (c) the CD300a VL comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 17; and (d) the CD300a VH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 18.

[0024] Embodiment 17 is the recombinant nucleic acid of any one of embodiments 14-16, wherein (a) the NKG2A VL comprises or consists of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) the NKG2A VH comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 3- 8; (c) the CD300a VL comprises or consists of the amino acid sequence of SEQ ID NO: 17; and

[0025] (d) the CD300a VH comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0026] Embodiment 18 is the recombinant nucleic acid of any one of embodiments 14-17, wherein (a) a nucleotide sequence encoding the NKG2A VL comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10; (b) a nucleotide sequence encoding the NKG2A VH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 11-16; (c) a nucleotide sequence encoding the CD300a VL comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 19; and (d) a nucleotide sequence encoding the CD300a VH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 20.

[0027] Embodiment 19 is the recombinant nucleic acid of the immediately preceding embodiment, wherein (a) the nucleotide sequence encoding the NKG2A VL comprises or consists of SEQ ID NO: 9 or SEQ ID NO: 10; (b) the nucleotide sequence encoding the NKG2A VH comprises or consists of any one of SEQ ID NOs: 11-16; (c) the nucleotide sequence encoding the CD300a VL comprises or consists of SEQ ID NO: 19; and (d) the nucleotide sequence encoding the CD300a VH comprises or consists of SEQ ID NO: 20.

[0028] Embodiment 20 is the recombinant nucleic acid of any one of the preceding embodiments, wherein (a) a nucleotide sequence encoding the NKG2A binding domain is codon optimized to reduce or prevent undesired recombination events; and / or (b) a nucleotide sequence encoding the CD300a binding domain is codon optimized to reduce or prevent undesired recombination events.

[0029] Embodiment 21 is the recombinant nucleic acid of any one of embodiments 14-20, wherein (a) the nucleotide sequence encoding the NKG2A VL is codon optimized to reduce or prevent undesired recombination events; (b) the nucleotide sequence encoding the NKG2A VH is codon optimized to reduce or prevent undesired recombination events; (c) the nucleotide sequence encoding the CD300a VL is codon optimized to reduce or prevent undesired recombination events; and / or (d) the nucleotide sequence encoding the CD300a VH is codon optimized to reduce or prevent undesired recombination events.

[0030] Embodiment 22 is the recombinant nucleic acid of embodiment 20 or embodiment 21, wherein the codon optimized nucleotide sequence encoding the NKG2A binding domain has at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 113.

[0031] Embodiment 23 is the recombinant nucleic acid of any one of embodiments 20-22, wherein the codon optimized nucleotide sequence encoding the NKG2A VH has at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 112.

[0032] Embodiment 24 is the recombinant nucleic acid of any one of embodiments 20-23, wherein the codon optimized nucleotide sequence encoding the NKG2A VL has at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 110.

[0033] Embodiment 25 is the recombinant nucleic acid of any one of embodiments 14-24, wherein the recombinant nucleic acid comprises a first linker, and wherein the first linker links the nucleic acid encoding the NKG2A VL and the nucleic acid encoding the NKG2A VH.

[0034] Embodiment 26 is the recombinant nucleic acid of any one of embodiments 14-25, wherein the recombinant nucleic acid comprises a second linker, and wherein the second linker links the nucleic acid encoding the CD300a VL and the nucleic acid encoding the CD300a VH.

[0035] Embodiment 27 is the recombinant nucleic acid of any one of embodiments 13-26, wherein (a) the NKG2A scFv comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 118; or (b) the NKG2A scFv comprises or consists of SEQ ID NO: 118.

[0036] Embodiment 28 is the recombinant nucleic acid of any one of embodiments 13-27, wherein (a) a nucleotide sequence encoding the NKG2A scFv comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 113 or 120; or (b) a nucleotide sequence encoding the NKG2A scFv comprises or consists of any one of SEQ ID NOs: 113 or 120.

[0037] Embodiment 29 is the recombinant nucleic acid of any one of embodiments 13-28, wherein (a) the CD300a scFv comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 119; or (b) the CD300a scFv comprises or consists of SEQ ID NO: 119.

[0038] Embodiment 30 is the recombinant nucleic acid of any one of embodiments 13-29, wherein (a) a nucleotide sequence encoding the CD300a scFv comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 121; or (b) a nucleotide sequence encoding the CD300a scFv comprises or consists of SEQ ID NO: 121.

[0039] Embodiment 31 is the recombinant nucleic acid of any one of embodiments 13-30, wherein: (a) the nucleotide sequence encoding the NKG2A VL is located 5’ of the nucleotide sequence encoding the NKG2A VH, or the nucleotide sequence encoding the NKG2A VL is located 3’ of the nucleotide sequence encoding the NKG2A VH; and (b) the nucleotide sequence encoding the CD300a VL is located 5’ of the nucleotide sequence encoding the CD300a VH, or the nucleotide sequence encoding the CD300a VL is located 3’ of the nucleotide sequence encoding the CD300a VH.

[0040] Embodiment 32 is the recombinant nucleic acid of any one of embodiments 13-31, wherein (a) the nucleotide sequence encoding the NKG2A scFv is located 3’ of the nucleotide sequence encoding the CD300a scFv or (b) the nucleotide sequence encoding the NKG2A scFv is located 5’ of the nucleotide sequence encoding the CD300a scFv.

[0041] Embodiment 33 is the recombinant nucleic acid of any one of embodiments 13-32, wherein the recombinant nucleic acid encodes a third linker, and wherein the third linker links the NKG2A scFv and the CD300a scFv.

[0042] Embodiment 34 is the recombinant nucleic acid of any one of embodiments 25-33, wherein the first linker, the second linker, and / or the third linker comprise a cleavable peptide, a glycine- serine linker, or a Whitlow / 218 linker.

[0043] Embodiment 35 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the glycine-serine linker comprises (Glym-Ser)n, wherein m is 3 to 6 and n is 1 to 10.

[0044] Embodiment 36 is the recombinant nucleic acid of the immediately preceding embodiment, wherein m=4 and n=5.

[0045] Embodiment 37 is the recombinant nucleic acid of embodiment 34, wherein the cleavable peptide is a self-cleaving peptide.

[0046] Embodiment 38 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the self-cleaving peptide is a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide, and wherein the self-cleaving peptide optionally comprises the amino acids glycine-serine- glycine at an N-terminus.

[0047] Embodiment 39 is the recombinant nucleic acid of any one of embodiments 25-38, wherein (a) the first linker, the second linker, and / or the third linker comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 29-33 and 122; or (b) the first linker, the second linker, and / or the third linker comprises or consists of any one of SEQ ID NOs: 29-32 and 122.

[0048] Embodiment 40 is the recombinant nucleic acid of any one of embodiments 25-38, wherein (a) a nucleotide sequence encoding the first linker, the second linker, and / or the third linker comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 34-37, 111, 115, and 116; or (b) a nucleotide sequence encoding the first linker, the second linker, and / or the third linker comprises or consists of any one of SEQ ID NOs:34-37, 111, 115, and 116.

[0049] Embodiment 41 is the recombinant nucleic acid of any one of the preceding embodiments, further encoding a signal peptide.

[0050] Embodiment 42 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the signal peptide is a cell surface expression signal peptide that directs the protein product of the recombinant nucleic acid to the surface of a cell.

[0051] Embodiment 43 is the recombinant nucleic acid of embodiment 41 or embodiment 42, wherein (a) the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 38; or (b) the signal peptide comprises or consists of SEQ ID NO: 38.

[0052] Embodiment 44 is the recombinant nucleic acid of any one of embodiments 41-43, wherein (a) a nucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 39-41; or (b) a nucleotide sequence encoding the signal peptide comprises or consists of any one of SEQ ID NOs: 39-41.

[0053] Embodiment 45 is the recombinant nucleic acid of any one of the preceding embodiments, wherein the nucleotide sequence encoding the signal peptide is located 5’ of the nucleotide sequence encoding the CD300a binding domain, 5’ of the nucleotide sequence encoding the NKG2A binding domain, or 5’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain.

[0054] Embodiment 46 is the recombinant nucleic acid of any one of the preceding embodiments, further encoding a spacer.

[0055] Embodiment 47 is the recombinant nucleic acid of the immediately preceding embodiment, wherein (a) the spacer comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 42; or (b) the spacer comprises or consists of SEQ ID NO: 42.

[0056] Embodiment 48 is the recombinant nucleic acid of any one of embodiments 46 or 47, wherein (a) a nucleotide sequence encoding the spacer comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 49; or (b) a nucleotide sequence encoding the spacer comprises or consists of SEQ ID NO: 49.

[0057] Embodiment 49 is the recombinant nucleic acid of embodiment 48, wherein the nucleotide sequence encoding the spacer is located (a) 3’ of the nucleotide sequence encoding the signal peptide; (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain; or (c) both (a) and (b).

[0058] Embodiment 50 is the recombinant nucleic acid of any one of the preceding embodiments, wherein the recombinant nucleic acid further encodes a hinge region or (b) the recombinant nucleic acid does not encode a hinge region.

[0059] Embodiment 51 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the hinge region comprises a CD8 hinge, an hlgG1 hinge, an hlgG2 hinge, an hlgG3 hinge, a FACD hinge, or any combination thereof.

[0060] Embodiment 52 is the recombinant nucleic acid of embodiment 50 or embodiment 51, wherein (a) the hinge region comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 43-48; or (b) the hinge region comprises or consists of any one of SEQ ID NOs: 43-48.

[0061] Embodiment 53 is the recombinant nucleic acid of any one of embodiments 50-52, wherein (a) a nucleotide sequence encoding the hinge region comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 50-55; or (b) a nucleotide sequence encoding the hinge region comprises or consists of any one of SEQ ID NOs: 50-55.

[0062] Embodiment 54 is the recombinant nucleic acid of embodiment 53, wherein the nucleotide sequence encoding the hinge region is located (a) 3’ of the nucleotide sequence encoding the signal peptide; (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain; or (c) both (a) and (b).

[0063] Embodiment 55 is the recombinant nucleic acid of any one of the preceding embodiments, further encoding a transmembrane domain.

[0064] Embodiment 56 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the transmembrane domain is a human transmembrane domain or a murine transmembrane domain.

[0065] Embodiment 57 is the recombinant nucleic acid of embodiment 55 or embodiment 56, wherein the transmembrane domain comprises or consists of a CD8, a CD80, an ITGA, an HLA- B57, a proCAR-4, a CD28, a KIR2DL1, a PDGFRB, or a CD86 transmembrane domain.

[0066] Embodiment 58 is the recombinant nucleic acid of any one of embodiments 55-57, wherein (a) the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 67-76 and 96; or (b) the transmembrane domain comprises or consists of any one of SEQ ID NOs: 67-76 and 96.

[0067] Embodiment 59 is the recombinant nucleic acid of any one of embodiments 55-58, wherein (a) a nucleotide sequence encoding the transmembrane domain comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 56-65; or (b) a nucleotide sequence encoding the transmembrane domain comprises or consists of any one of SEQ ID NOs: 56-65.

[0068] Embodiment 60 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the nucleotide sequence encoding the transmembrane domain is located (a) 3’ of the nucleotide sequence encoding the signal peptide, (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain, (c) 3’ of the nucleotide sequence encoding the spacer; (d) 3’ of the nucleotide sequence encoding the hinge region, or (e) any combination of (a)-(d).

[0069] Embodiment 61 is the recombinant nucleic acid of any one of the preceding embodiments, further encoding a cytoplasmic domain.

[0070] Embodiment 62 is the recombinant nucleic acid of the immediately preceding embodiment, wherein the cytoplasmic domain is a human cytoplasmic domain or a murine cytoplasmic domain.

[0071] Embodiment 63 is the recombinant nucleic acid of embodiment 61 or embodiment 62, wherein the cytoplasmic domain comprises or consists of a CD8v2, a CD8v1, a mCD80, a CD80, a CD86, or an HLA-B57 cytoplasmic domain.

[0072] Embodiment 64 is the recombinant nucleic acid of any one of embodiments 61-63, wherein (a) the cytoplasmic domain comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 83-88; or (b) the cytoplasmic domain comprises or consists of any one of SEQ ID NOs: 83-88.

[0073] Embodiment 65 is the recombinant nucleic acid of any one of embodiments 61-64, wherein (a) a nucleotide sequence encoding the cytoplasmic domain comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 77-82; or (b) a nucleotide sequence encoding the cytoplasmic domain comprises or consists of any one of SEQ ID NOs: 77-82.

[0074] Embodiment 66 is the recombinant nucleic acid of embodiment 65, wherein the nucleotide sequence encoding the cytoplasmic domain is located (a) 3’ of the nucleotide sequence encoding the signal peptide, (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain, (c) 3’ of the nucleotide sequence encoding the spacer; (d) 3’ of the nucleotide sequence encoding the hinge region, (e) 3’ of the nucleotide sequence encoding the transmembrane domain, or (f) any combination of (a)-(e).

[0075] Embodiment 67 is the recombinant nucleic acid of any one of the preceding embodiments, encoding (a) an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 220-237; or (b) an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 220-237.

[0076] Embodiment 68 is the recombinant nucleic acid of any one of the preceding embodiments, (a) comprising a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 202-219; or (b) comprising or consisting of any one of SEQ ID NOs: 202-219.

[0077] Embodiment 69 is the recombinant nucleic acid of any one of embodiments 1-66, encoding (a) an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22; or (b) an amino acid sequence comprising or consisting of SEQ ID NO: 21 or SEQ ID NO: 22.

[0078] Embodiment 70 is the recombinant nucleic acid of any one of embodiments 1-66 or 69, (a) comprising a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 24; or (b) comprising or consisting of SEQ ID NO: 23 or SEQ ID NO: 24.

[0079] Embodiment 71 is the recombinant nucleic acid of any one of embodiments 1-66, encoding (a) an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 25 or SEQ ID NO: 26; or (b) an amino acid sequence comprising or consisting of SEQ ID NO: 25 or SEQ ID NO: 26.

[0080] Embodiment 72 is the recombinant nucleic acid of any one of embodiments 1-66 or 71, (a) comprising a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 27 or SEQ ID NO: 28; or (b) comprising or consisting of SEQ ID NO: 27 or SEQ ID NO: 28.

[0081] Embodiment 73 is a vector comprising the recombinant nucleic acid of any one of the preceding embodiments.

[0082] Embodiment 74 is the vector of embodiment 73, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenoviral vector, an adeno associated viral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.

[0083] Embodiment 75 is the vector of any one of embodiments 73 or 74, wherein the recombinant nucleic acid is operably linked to a promoter.

[0084] Embodiment 76 is the vector of the immediately preceding embodiment, wherein the promoter is an EF1a promoter, a CAG promoter, a PGK promoter, or a CMV promoter.

[0085] Embodiment 77 is the vector of any one of embodiments 73-76, wherein a nucleic acid sequence in the vector further comprises a poly(A) sequence.

[0086] Embodiment 78 is the vector of the immediately preceding embodiment, wherein the poly(A) sequence comprises a bGH poly(A) signal.

[0087] Embodiment 79 is the vector of any one of embodiments 73-78, wherein a nucleic acid sequence in the vector further comprises a 3’UTR.

[0088] Embodiment 80 is the vector of any one of embodiments 73-79, wherein the vector integrates into a genome at an adeno-associated virus integration site 1 (AAVS1) of the genome.

[0089] Embodiment 81 is the vector of any one of embodiments 73-80, further comprising an AAVS1 right homology arm and an AAVS1 left homology arm.

[0090] Embodiment 82 is an engineered cell comprising a vector comprising a recombinant nucleic acid encoding a construct for inhibiting NK cell cytotoxicity, wherein the construct comprises a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain.

[0091] Embodiment 83 is an engineered cell expressing a recombinant nucleic acid encoding a construct for inhibiting NK cell cytotoxicity, wherein the construct comprises a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain.

[0092] Embodiment 84 is an engineered cell comprising a first vector and a second vector, wherein (a) the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting NK cell cytotoxicity comprising a NKG2A binding domain comprising: (i) a NKG2A light chain variable region (NKG2A VL); and (ii) a NKG2A heavy chain variable region (NKG2A VH); and (b) the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain comprising: (i) a CD300a light chain variable region (CD300a VL); and (ii) a CD300a heavy chain variable region (CD300a VH).

[0093] Embodiment 85 is an engineered cell comprising the recombinant nucleic acid of any one of embodiments 1-72, or the vector of any one of embodiments 73-81.

[0094] Embodiment 86 is the engineered cell of any one of embodiments 82-85, wherein the recombinant nucleic acid is expressed in the engineered cell.

[0095] Embodiment 87 is the engineered cell of any one of embodiments 82-86, wherein the vector is inserted into a safe harbor locus of at least one allele of the engineered cell.

[0096] Embodiment 88 is the engineered cell of the immediately preceding embodiment, wherein the safe harbor locus is an AAVS1 locus.

[0097] Embodiment 89 is the engineered cell of any one of embodiments 82-88, wherein the engineered cell is MHC class I deficient.

[0098] Embodiment 90 is the engineered cell of any one of embodiments 82-^^^^ZKHUHLQ^D^ȕ^^ microglobulin (B2M) gene locus of the engineered cell is disrupted.

[0099] Embodiment 91 is the engineered cell of any one of embodiments 82-90, wherein the UHFRPELQDQW^QXFOHLF^DFLG^RU^WKH^YHFWRU^LV^LQVHUWHG^LQWR^D^ȕ^^PLFURJOREXOLQ^^%^0^^JHQH^ORFXV^RI^WKH^ engineered cell.

[0100] Embodiment 92 is the engineered cell of any one of embodiments 82-91, wherein the engineered cell is a stem cell, a progenitor cell, a cell that has been differentiated from a stem cell, or a cell that has been differentiated from a progenitor cell.

[0101] Embodiment 93 is the engineered cell of the immediately preceding embodiment, wherein the stem cell is a pluripotent stem cell.

[0102] Embodiment 94 is the engineered cell of the immediately preceding embodiment, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0103] Embodiment 95 is the engineered cell of embodiment 92, wherein the progenitor cell is an early hematopoietic progenitor cell or a CD34+ progenitor cell.

[0104] Embodiment 96 is the engineered cell of embodiment 92, wherein the cell that has been differentiated from a stem cell or the cell that has been differentiated from a progenitor cell is an engineered T cell.

[0105] Embodiment 97 is the engineered cell of any one of embodiments 82-92, wherein the engineered cell is an engineered T cell.

[0106] Embodiment 98 is the engineered cell of any one of embodiments 82-92, wherein the engineered cell is an induced pluripotent stem cell that is subsequently differentiated into an engineered T cell.

[0107] Embodiment 99 is the engineered cell of any one of embodiments 96-98 wherein the engineered T cell is a chimeric antigen receptor (CAR) T cell.

[0108] Embodiment 100 is the engineered cell of the immediately preceding embodiment, wherein the CAR is a CD19 CAR, a BCMA CAR, and / or a CD20 CAR.

[0109] Embodiment 101 is the engineered cell of any one of embodiments 82-100, wherein the engineered cell is T cell receptor alpha constant (TRAC) deficient.

[0110] Embodiment 102 is the engineered cell of any one of embodiments 82-101, wherein a T cell receptor alpha constant (TRAC) locus is disrupted in the engineered cell.

[0111] Embodiment 103 is the engineered cell of any one of embodiments 99-102, wherein the CAR is inserted into a T cell receptor alpha constant (TRAC) locus in the engineered cell.

[0112] Embodiment 104 is the engineered cell of any one of embodiments 96, 97, or 99-103, wherein the engineered T cell is derived from a pluripotent stem cell or a CD34+ progenitor cell.

[0113] Embodiment 105 is the engineered cell of the immediately preceding embodiment, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0114] Embodiment 106 is the engineered cell of any one of embodiments 82-105, wherein the engineered cell is an autologous cell or an allogeneic cell.

[0115] Embodiment 107 is the engineered cell of any one of embodiments 82-105, wherein the engineered cell is from a donor or is derived from a stem cell of a donor, wherein the donor is not the subject.

[0116] Embodiment 108 is the engineered cell of any one of embodiments 82-107, wherein the engineered cell is a human cell.

[0117] Embodiment 109 is the engineered cell of any one of embodiments 82-108, wherein the engineered cell is hypoimmunogenic.

[0118] Embodiment 110 is the engineered cell of the immediately preceding embodiment, wherein the engineered cell is resistant to NK cell mediated cellular cytotoxicity.

[0119] Embodiment 111 is a composition comprising the recombinant nucleic acid of any one of embodiments 1-72, the vector of any one of embodiments 73-81, or the engineered cell of any one of embodiments 82-110, and one or more of a cell culture media and a buffer.

[0120] Embodiment 112 is a pharmaceutical composition comprising the engineered cell of any one of embodiments 82-110, and a pharmaceutically acceptable carrier.

[0121] Embodiment 113 is a method of producing an engineered T cell, the method comprising differentiating the engineered cell of any one of embodiments 82-95 or 105-91 into a T cell.

[0122] Embodiment 114 is a method of producing an engineered, hypoimmunogenic induced pluripotent stem cell (iPSC), the method comprising expressing in an iPSC a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing the engineered hypoimmunogenic iPSC.

[0123] Embodiment 115 is a method of producing an engineered, hypoimmunogenic induced pluripotent stem cell (iPSC), the method comprising expressing in an iPSC a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain, thereby producing the engineered hypoimmunogenic iPSC.

[0124] Embodiment 116 is a method of producing an engineered induced pluripotent stem cell (iPSC), the method comprising contacting an induced pluripotent stem cell (iPSC) with a first vector and a second vector, wherein the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting NK cell cytotoxicity comprising a NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, and wherein the contacting occurs under conditions whereby the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the iPSC, thereby producing the engineered iPSC.

[0125] Embodiment 117 is a method of producing an engineered hypoimmunogenic T cell, the method comprising: (a) expressing in an iPSC a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing an engineered hypoimmunogenic iPSC; and (b) differentiating the engineered hypoimmunogenic iPSC into an engineered T cell, thereby producing the engineered T cell.

[0126] Embodiment 118 is a method of producing an engineered hypoimmunogenic T cell, the method comprising expressing in a T cell a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing the engineered hypoimmunogenic T cell.

[0127] Embodiment 119 is a method of producing an engineered hypoimmunogenic T cell, the method comprising expressing in a T cell a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain, thereby producing the engineered hypoimmunogenic T cell.

[0128] Embodiment 120 is a method of producing an engineered T cell, the method comprising contacting a T cell with a first vector and a second vector, wherein the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting NK cell cytotoxicity comprising a NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, and wherein the contacting occurs under conditions whereby the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the T cell, thereby producing the engineered T cell.

[0129] Embodiment 121 is a method of inhibiting or reducing natural killer (NK) cell cytotoxicity to an engineered cell, the method comprising expressing in the engineered cell a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby inhibiting or reducing NK cell cytotoxicity to the engineered cell.

[0130] Embodiment 122 is a method of inhibiting or reducing natural killer (NK) cell cytotoxicity to an engineered cell, the method comprising expressing in the engineered cell a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain thereby inhibiting or reducing NK cell cytotoxicity to the engineered cell.

[0131] Embodiment 123 is a method of inhibiting or reducing natural killer (NK) cell cytotoxicity to a T cell, the method comprising: (a) expressing in an iPSC a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing an engineered iPSC; and (b) differentiating the engineered iPSC into a T cell, thereby inhibiting or reducing NK cell cytotoxicity to the T cell.

[0132] Embodiment 124 is a method of inhibiting or reducing natural killer (NK) cell cytotoxicity to a T cell, the method comprising expressing in a T cell a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby inhibiting or reducing NK cell cytotoxicity to the T cell.

[0133] Embodiment 125 is a method of inhibiting or reducing natural killer (NK) cell cytotoxicity to a T cell, the method comprising expressing in a T cell a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain, thereby producing the engineered iPSC.

[0134] Embodiment 126 is the method of any one of embodiments 94-125, further comprising expressing in the engineered iPSC, the iPSC, the engineered cell, or the T cell a chimeric antigen receptor (CAR).

[0135] Embodiment 127 is the method of the immediately preceding embodiment, wherein the CAR is a BCMA CAR, a CD19 CAR, and / or a CD20 CAR.

[0136] Embodiment 128 is the method of any one of embodiments 113-127, wherein the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell is T cell receptor alpha constant (TRAC) deficient.

[0137] Embodiment 129 is the method of any one of embodiments 113-128, wherein a T cell receptor alpha constant (TRAC) locus is disrupted in the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell.

[0138] Embodiment 130 is the method of any one of embodiments 113-129, wherein the CAR is inserted into a T cell receptor alpha constant (TRAC) locus in the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell.

[0139] Embodiment 131 is the method of any one of embodiments 113-130, wherein the engineered cell is obtained by differentiation of a human pluripotent stem cell.

[0140] Embodiment 132 is the method of any one of embodiments 113-131, wherein the CD300a binding domain comprises a single chain variable fragment (scFv), a VHH, a cytokine, a ligand, or a peptide.

[0141] Embodiment 133 is the method of any one of embodiments 113-132, wherein the NKG2A binding domain comprises an scFv, a VHH, a cytokine, a ligand, or a peptide.

[0142] Embodiment 134 is the method of embodiment 132 or embodiment 133, wherein (a) the VHH comprises the VH domain of a camelid heavy chain antibody, or (b) the peptide is an adnectin or a designed ankyrin repeat protein (DARPin).

[0143] Embodiment 135 is the method of any one of embodiments 113-134, wherein the CD300a binding domain comprises a VHH (CD300a VHH) and / or the NKG2A binding domain comprises a VHH (NKG2A VHH).

[0144] Embodiment 136 is the method of any one of embodiments 113-135, wherein the recombinant nucleic acid encodes the CD300a binding domain, and the CD300a binding domain comprises a VHH (CD300a VHH).

[0145] Embodiment 137 is the method of any one of embodiments 113-136, wherein the NKG2A binding domain comprises a scFv comprising a NKG2A light chain variable region (NKG2A VL); and a NKG2A heavy chain variable region (NKG2A VH).

[0146] Embodiment 138 is the method of any one of embodiments 113-137, wherein the CD300a binding domain comprises a scFv comprising a CD300a light chain variable region (CD300a VL); and a CD300a heavy chain variable region (CD300a VH).

[0147] Embodiment 139 is the method of any one of embodiments 113-138, wherein the recombinant nucleic acid comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain comprises the recombinant nucleic acid of any one of embodiments 1-72.

[0148] Embodiment 140 is the method of any one of embodiments 113-139, further comprising a vector comprising the recombinant nucleic acid comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain.

[0149] Embodiment 141 is the method of the immediately preceding embodiment, wherein the vector comprising the recombinant nucleic acid comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain comprises the vector of any one of embodiments 73-81.

[0150] Embodiment 142 is the method of any one of embodiments 114, 115, 117, 119, 121-124, or 127-141, wherein the expressing comprises contacting the engineered cell, the iPSC, or the T cell with the recombinant nucleic acid of any one of embodiments 1-59, or with the vector of any one of embodiments 60-68, under conditions whereby the recombinant nucleic acid is expressed in the iPSC.

[0151] Embodiment 143 is the method of any one of embodiments 116, 120, or 142, wherein the contacting comprises introducing the recombinant nucleic acid into the engineered cell, the iPSC, or the T cell using transfection, electroporation, transduction, or knock-in.

[0152] Embodiment 144 is the method of the immediately preceding embodiment, wherein (a) the transfection comprises contacting the engineered cell, the iPSC, or the T cell with a cationic polymer and the recombinant nucleic acid; (b) the transduction comprises contacting the engineered cell, the iPSC, or the T cell with a lentivirus comprising the recombinant nucleic acid; and / or (c) the knock-in comprises contacting the engineered cell, the iPSC, or the T cell with an adeno- associated virus comprising the recombinant nucleic acid.

[0153] Embodiment 145 is the method of any one of embodiments 113-144, wherein the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell is MHC class I deficient.

[0154] Embodiment 146 is the method of any one of embodiments 113-^^^^^ZKHUHLQ^D^ȕ^^ microglobulin (B2M) gene locus of the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell is disrupted.

[0155] Embodiment 147 is the method of any one of embodiments 113-146, wherein the UHFRPELQDQW^QXFOHLF^DFLG^RU^WKH^YHFWRU^LV^LQVHUWHG^LQWR^D^ȕ^^PLFURJOREXOLQ^^%^0^^JHQH^ORFXV^RI^WKH^ engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell.

[0156] Embodiment 148 is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject an effective amount of the engineered cell of any one of embodiments 82-110 or the pharmaceutical composition of embodiment 112.

[0157] Embodiment 149 is the method of the immediately preceding embodiment, wherein the disease or the condition is a cancer.

[0158] Embodiment 150 is a method of improving a clinical outcome in a subject undergoing a T cell therapy, comprising administering to the subject an effective amount of the engineered cell of any one of embodiments 82-110, or the pharmaceutical composition of embodiment 112.

[0159] Embodiment 151 is the method of the immediately preceding embodiment, wherein improving a clinical outcome comprises one or more of: (a) an inhibition or reduction in NK cell cytotoxicity against the engineered cell; (b) an increased clinical response to the T cell therapy in the subject, optionally compared to the same T cell therapy not comprising the recombinant nucleic acid or compared to a T cell therapy comprising T cells (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand; (c) an increased survival rate of the engineered cell in the subject; (d) an increased persistence of the engineered cell in the subject, optionally compared to the same T cell therapy not comprising the recombinant nucleic acid or compared to a T cell therapy comprising the engineered cell (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand; (e) an improved pharmacokinetic (PK) property and / or an improved pharmacodynamic (PD) property of the T cell therapy in the subject, optionally compared to the same T cell therapy not comprising the recombinant nucleic acid or compared to a T cell therapy comprising the engineered cell (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand; and (f) a reduction in cytolysis of the engineered cell in the subject, optionally wherein the cytolysis of the engineered cell is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%.

[0160] Embodiment 152 is the method of the immediately preceding embodiment, wherein the at least one alternative ligand is HLA-E, KIR3D, Lair1, Siglec7, CD107a, TIGIT, CD24, LILRB1, CD47, KIR2D, TIM3, HHLA2, or HLA-G.

[0161] Embodiment 153 is the method of the immediately preceding embodiment, wherein the HLA-E is an HLA-E single chain dimer or trimer.

[0162] Embodiment 154 is the method of any one of embodiments 151-153, wherein the same T cell therapy not comprising the recombinant nucleic acid or the T cell therapy comprising the engineered cell (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand further comprises reduced or eliminated expression of CD48, CD54, CD58, and / or CD155.

[0163] Embodiment 155 is the method of any one of embodiments 148-154, wherein the subject has or is at risk of having a disease or a condition that can benefit from the T cell therapy, optionally wherein the disease or the condition is a cancer.

[0164] Embodiment 156 is the method of any one of embodiments 148-155, wherein the cancer is a hematologic cancer selected from B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell- follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

[0165] Embodiment 157 is the method of any one of embodiments 148-155, wherein the cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma or retinoblastoma.

[0166] Embodiment 158 is the method of any one of embodiments 148 or 150-155, wherein the disease or disorder is an autoimmune disease or disorder.

[0167] Embodiment 159 is the method of the immediately preceding embodiment, wherein the autoimmune disease or disorder is myasthenia gravis, neuromyelitis optica spectrum disorder, Sjogren’s syndrome, scleroderma, immune nephritis, systemic lupus erythematosus, arthritis, an autoimmune-induced fibrotic condition, pemphigus vulgaris, multiple sclerosis, colitis, type 1 diabetes, graft-versus-host disease, atherosclerosis, or mucosal-dominant PV.

[0168] Embodiment 160 is the method of any one of embodiments 148-159, wherein the subject is a human.

[0169] Embodiment 161 is a kit comprising the recombinant nucleic acid of any one of embodiments 1-72, the vector of any one of embodiments 73-81, the engineered cell of any one of embodiments 82-110, the composition of embodiment 111, and / or the pharmaceutical composition of embodiment 112.

[0170] Embodiment 162 is the kit of the immediately preceding embodiment, further comprising one or more of: (a) one or more cells, optionally wherein the one or more cells are stem cells, T cells, and / or NK cells; (b) a cell culture medium; (c) a buffer; and (d) a pharmaceutically acceptable carrier.

[0171] The numbered items of Example 31 provide additional support for and descriptions of the embodiments herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] FIG.1 illustrates four exemplary, non-limiting recombinant nucleic acid designs described herein, including a promoter (e.g., EF1a), a CD300a TASR and / or a NKG2A TASR, and a linker (e.g., P2A) in exemplary designs comprising both a CD300a TASR and a NKG2A TASR.

[0173] FIGS.2A-2B illustrate MHC class I deficient T cell killing by NK cells. FIG. 2A shows phenotyping of wild-type, CD59 knockout, CIITA knockout, and B2M knockout cells using flow cytometry. FIG.2B shows the results of a NK cell cytotoxicity assay, wherein % T cell survival was lowest for B2M knockout cells.

[0174] FIGS.3A-3B illustrate the efficacy of two NKG2A construct designs as compared to the control HLA-E single chain trimer. FIG.3A shows a schematic of the HLA-E and NKG2A scFv mRNA constructs, and phenotyping of cells electroporated with the constructs, using flow cytometry. FIG.3B shows that both of the NKG2A scFv constructs provided protection against NK cell killing. The NKG2A construct comprising the CD8 transmembrane domain appeared to perform better than the NKG2A construct comprising IgG1B7.

[0175] FIGS.4A-4B illustrate that the efficacy of the NKG2A scFv construct comprising the CD8 transmembrane domain (referred to hereafter as the Z199 construct) was reproducible. FIG.4A shows a schematic of the HLA-E and NKG2A scFv mRNA constructs, and phenotyping of cells electroporated with the constructs, using flow cytometry. The negative control shown data displayed in this figure are the same as that shown in FIG.8A FIG. 4B shows that the Z199 construct provided some protection against NK cell killing. The HLA-E and GFP data shown in this figure are the same as that shown in FIG.8B.

[0176] FIGS.5A-5B illustrate that a humanized anti-NKG2A (clone Z270) scFv is stable and functional. FIG.5A shows phenotyping of cells electroporated with the Z199, humanized Z199, humanized Z270, and negative control constructs, using flow cytometry. FIG.5B shows that all of the NKG2A scFv constructs provided protection against NK cell killing.

[0177] FIGS.6A-6B illustrate that the efficacy of the NKG2A humanized Z270 construct was reproducible. FIG.6A shows phenotyping of cells electroporated with the Z270 or HLA-E constructs using flow cytometry. The negative control shown data displayed in this figure are the same as that shown in FIG.10A FIG. 6B shows that the humanized Z270 construct provided protection against NK cell killing.

[0178] FIGS.7A-7B illustrate the efficacy of two CD300a construct designs as compared to the control HLA-E single chain trimer. FIG.7A shows a schematic of the HLA-E and CD300a scFv mRNA constructs, and phenotyping of cells electroporated with the constructs, using flow cytometry. FIG.7B shows that both of the CD300a scFv constructs provided protection against NK cell killing. The CD300a construct comprising the CD8 transmembrane domain appeared to perform better than the CD300a construct comprising IgG1B7.

[0179] FIGS.8A-8B illustrate that the efficacy of the CD300a scFv construct comprising the CD8 transmembrane domain (referred to hereafter as the TX49 construct) was reproducible. FIG.8A shows a schematic of the CD300a scFv mRNA construct, and phenotyping of cells electroporated with the constructs, using flow cytometry. The negative control shown data displayed in this figure are the same as that shown in FIG.4A FIG.8B shows that the TX49 construct provided some protection against NK cell killing. The HLA-E and GFP data shown in this figure are the same as that shown in FIG. 4B.

[0180] FIGS.9A-9C illustrate successful humanization of the TX49 construct. FIG. 9A shows amino acid sequence alignments of the VH (top) and VL (bottom) regions of the mouse anti-human CD300a antibody clone TX49, along with two humanized versions named “humTX49_v1” and “humTX49_v2”. FIG. 9B shows phenotyping of cells electroporated with the constructs, using flow cytometry. FIG.9C shows that both humanized TX49 constructs (v1 and v2) provided protection against NK cell killing.

[0181] FIGS.10A-10B illustrate that the efficacy of the humanized TX49 construct v1 (hereinafter referred to as humTX49) was reproducible. FIG.10A shows phenotyping of cells electroporated with the humTX49 and HLA-E constructs, using flow cytometry. The negative control shown data displayed in this figure are the same as that shown in FIG. 6A. FIG.10B shows that the humTX49 construct provided some protection against NK cell killing.

[0182] FIGS.11A-11B illustrate that humTX49 synergizes with humZ270 to provide NK protection. FIG.11A shows phenotyping of cells electroporated with the humTX49, hum270, and / or HLA-E constructs, using flow cytometry. Left panel shows staining of the HLA-E construct electroporated or non-electroporated cells with HLA-E. Middle panel shows staining of the indicated electroporated cells with CD300a. Right panel shows staining of the indicated electroporated cells with NKG2A. FIG.11B shows that cells expressing both the humZ270 and humTX49 constructs were protected against NK cell killing comparably to cells expressing the HLA-E construct.

[0183] FIGS.12A-12C illustrate the efficacy of a bispecific humTX49-humZ270 construct for protecting engineered cells against NK cell cytotoxicity relative to HLA-E. FIG.12A shows a schematic of two candidate bispecific constructs. “humTX49-humZ270” comprises a human GM- CSF signal peptide (“GM-CSF SP”), followed by the humTX49 scFV (vL-Linker-vH orientation), a (G4S)5 linker, then the humZ270 scFV (vL-Linker-vH orientation), followed by a CD8 stalk and transmembrane region (“CD8 TM”), a T2A self-cleaving peptide, and GFP. The “humZ270- humTX49” construct is similar, except the two scFVs are swapped in order. FIG.12B shows phenotyping of cells electroporated with the constructs, using flow cytometry. FIG.12C shows that cells expressing either construct were protected against NK cell killing, with the humTX49- humZ270 construct providing protection comparable to that of HLA-E.

[0184] FIGS.13A-13B illustrate that agonism by TX49 (CD300a) and Z270 (NKG2A) was synergistic and more potent than that by HLA-E. FIGS.13A and 13B show superior protection against NK cell killing in cells expressing the humTX49-humZ270 construct as compared to HLA- E. Cells electroporated with equimolar amounts of humTX49 and humZ270 constructs separately were also protected against NK cell killing.

[0185] FIGS.14A-14C illustrate that physiological expression of the HLA-E, humZ270, and humZ270-humTX49 constructs at the AAVS1 locus in human primary T cells provide comparable protection against NK cell killing. FIG.14A shows that cells expressing HLA-E, humZ270, or humZ270-humTX49 constructs, but not cells expressing the humTX49 construct, were equivalently protected against NK cell killing. FIG.14B shows levels of NKG2A / NKG2C expression in donor NK cells. FIG.14C shows a diagram of the expression constructs, and phenotyping of cells electroporated with the constructs, using flow cytometry.

[0186] FIGS.15A-15D illustrate that the mCD80 transmembrane domain impacts hTX49 mediated NK inhibition, and that transmembrane homodimerization does not appear to impact function. FIG. 15A shows a schematic of the experimental set up. FIG.15B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG.15C shows expression levels as measured for each construct. FIG. 15D shows that cells expressing any of the constructs were protected against NK cell killing.

[0187] FIGS.16A-16D illustrate that the cytoplasmic tails of CD8a or mCD80 appear to enhance expression and function of hTX49. FIG.16A shows a schematic of the experimental set up. FIG. 16B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG. 16C shows expression levels as measured for each construct. FIG.16D shows that cells expressing any of the constructs (with the exception of the t8 construct, which did not comprise transmembrane or cytoplasmic domains) were protected against NK cell killing.

[0188] FIGS.17A-17D illustrate that reduced hinge size increases the functional potency of hTX49. FIG.17A shows a schematic of the experimental set up. FIG.17B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG. 17C shows expression levels as measured for each construct. FIG. 17D shows that cells expressing any of the constructs (with the exception of the t8 construct, which did not comprise transmembrane or cytoplasmic domains) were protected against NK cell killing.

[0189] FIGS.18A-18D illustrate that an optimized CD300a TASR design (t12) improves cell persistence over the original construct (v1). FIG. 18A shows a schematic of the experimental set up. FIG.18B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG.18C shows expression levels as measured for each construct, using flow cytometry. FIG.18D shows that cells expressing any of the constructs (with the t12 construct not comprising a hung region performing best) were protected against NK cell killing.

[0190] FIGS.19A-19D illustrate that an optimized CD300a TASR design (t12) shows protection against NKG2C+ / NKG2A- NK cell donors. FIG. 19A shows a schematic of the experimental set up. FIG.19B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG.19C shows expression levels as measured for each construct, using flow cytometry. FIG.19D shows that cells expressing both the NKG2A_v1 and CD300a_t12 constructs were protected against NK cell killing.

[0191] FIGS.20A-20D illustrate that mouse CD80 cytoplasmic domain enhanced expression and function of CD300a TASR irrespective of the transmembrane domain. FIG.20A shows a schematic of the experimental set up. FIG. 20B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG.20C shows expression levels as measured for each construct, using flow cytometry. FIG. 20D shows that cells expressing the V2, t12, t14, or t18 constructs comprising a mouse CD80 cytoplasmic domain were protected against NK cell killing.

[0192] FIGS.21A-21C illustrate that the mCD80 cytoplasmic domain outperformed hCD80 and hCD86 cytoplasmic domains in TASR expression. FIG. 21A shows a schematic of the experimental set up. FIG. 21B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG. 21C shows expression levels as measured for each construct, using flow cytometry.

[0193] FIGS.22A-22D illustrate that hCD80 and hCD86 cytoplasmic domains do not perform as well as mCD80. FIG.22A shows a schematic of the experimental set up. FIG.22B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG.22C shows expression levels as measured for each construct, using flow cytometry. FIG.22D shows that cells expressing t23, t24, and t29 constructs comprising a hCD80 cytoplasmic domain were not as protected against NK cell killing as cells expressing the t12 and v2 constructs comprising a mCD80 cytoplasmic domain.

[0194] FIGS.23A-23D illustrate that an optimized CD300a TASR with fully human extra-cellular domains (v2) showed enhanced functional potency over the original design (v1). FIG. 23A shows a schematic of the experimental set up. FIG.23B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG.23C shows expression levels as measured for each construct, using flow cytometry. FIG.23D shows that cells expressing the v2 construct were more protected against NK cell killing than cells expressing the v1 and t13 constructs.

[0195] FIGS.24A-24D illustrate a repeated experiment using additional NK cell donors and showing that optimized CD300a TASR with fully human extra-cellular domains (v2) enhanced functional potency over the original design (v1). FIG.24A shows a schematic of the experimental set up. FIG. 24B shows a schematic illustrating the CD300a TASR constructs used to electroporate T cells. FIG. 24C shows expression levels as measured for each construct, using flow cytometry. FIG. 24D shows that cells expressing the v2 construct were more protected against NK cell killing than cells expressing the v1 construct.

[0196] FIGS.25A-25D illustrate an antibody-dependent cellular cytotoxicity (ADCC) assay using rituximab, showing that NKG2A_v1, CD300a_v2, and HLA-E single chain trimer constructs are capable of reducing ADCC. FIG.25A shows a schematic of the experimental set up. FIG.25B shows a schematic illustrating the CD300a TASR and the NKG2A TASR used to electroporate T cells. FIG.25C shows expression levels of CD300a TASR, NKG2A TASR, HLA-E, and RQR8 on T cells electroporated with the indicated mRNA constructs, using flow cytometry. FIG.25D shows that the humTX49_v2 and humZ270_v1 constructs were capable of reducing ADCC.

[0197] FIGS.26A-26D illustrate that reduced affinity variants of an NKG2A TASR provided enhanced functional protection against NK cell killing. FIG.26A shows a schematic of the experimental set up. FIG. 26B shows a schematic illustrating the NGK2A TASR constructs (with amino acid mutations noted) used to electroporate T cells. FIG.26C shows expression levels as measured for each construct, using flow cytometry. FIG. 26D shows that cells expressing the mutated constructs that had reduced affinity for NKG2A were more protected against NK cell killing than cells expressing the non-mutated (wild-type) construct or the v1 construct.

[0198] FIGS.27A-27E illustrate that reduced affinity variants of an NKG2A TASR provided enhanced functional protection and reduced NKG2A downregulation on effector NK cells. FIG. 27A shows a schematic of the experimental set up. FIG.27B shows a schematic illustrating the NGK2A TASR constructs (with amino acid mutations noted) used to electroporate T cells. FIG. 27C shows expression of the NKG2A TASR on T cells electroporated with the indicated mRNA constructs. FIG.27D shows that cells expressing the mutated constructs that had reduced affinity for NKG2A were more protected against NK cell killing than cells expressing the v1 or v2 construct. FIG.27E shows a flow cytometry gating scheme showing that NK cells tend to downregulate NKG2A expression when exposed to T cells expressing NKG2A TASRs toward the end of the NK cytotoxicity assay.

[0199] FIGS.28A-C show that B2M KO rescues allogeneic T cells from T cell alloreactivity but introduces NK cell alloreactivity. Survival of wildtype (WT) and B2M knockout (KO) T cells from an HLA-A2+ donor challenged with NK and allo-T cell effectors from HLA-A2- donors was assessed. Allo-T cells contained the AHIII T cell receptor reactive to EMC7 peptide, ALWGFFPVL (SEQ ID NO: 200), presented by HLA-A2. FIG.28A shows survival curves of WT and B2M KO target T cells challenged with the indicated effectors. N = 1 technical replicate curves per condition. FIG.28B shows HLA-I expression of WT and B2M KO T cells by flow cytometry, gated live single cell lymphocytes. B2M KO specifically renders T cells susceptible to NK cell rejection. FIG. 28C shows NK challenge assay with multiple NK donors and multiple target T cell effectors. The bar graph represents a summary of IC50 values, where each data point represents a WT or B2M KO T cell from one T cell donor against the indicated NK cell donor. Groups were compared using the Mann Whitney U-Test.

[0200] FIGS.29A-C show discovery and assessment of CD300a TASR. FIG.29A shows screening of strategies to inhibit NK alloreactivity against B2M KO T cells. Ligands are expressed by mRNA electroporation, and knock-out performed by CRISPR / Cas9. Dotted line indicates no protection. N = 1 curve of T cell survival at 6-7 E:T ratios challenged with one NK cell donor. (See FIG.30) FIG.29B shows an NK challenge assay. The graph legend indicates the cloaking transgene integrated into the B2M locus of human primary T cells, challenged with one of three NK cell donors. Inset shows the NK cell phenotype by flow cytometry, gated CD3-CD56+. N=2 technical replicate curves per condition. FIG. 29C shows a competition assay of pooled T cells from FIG.29B, along with HLA-I+ control, all derived from one HLA-A2+ donor, against allo-T and NK cells from HLA-A2- donors. The frequency of the indicated T cell member after challenge with indicated NK and / or allo-T cell effector is shown. Allo-T contained a T cell receptor reactive against HA-2 peptide presented on HLA-A2. N = 3 technical replicates per condition.

[0201] FIGS.30A-C show functional screening and validation of NK cloaking strategies by mRNA electroporation and CRISPR KO of B2M KO T cells, related to FIG.29A.Cloaking ligands were expressed by mRNA electroporation (EP) and functionally validated. Each graph shown in FIG. 30A represents a single screen member. CRISPR KOs are in addition to B2M KO. The negative control was GFP mRNA for mRNA EP conditions or B2M KO only for the CRISPR KO screen. For each graph, the left inset plot shows the expression profile by flow cytometry, gated on single cell lymphocytes. “NC” indicates the negative control peak. For each graph, the right inset plot shows theNK challenge assay of the given screen member. FIGS.30C-D show validation of CD300a TASR V1 and NKG2A TASR V1 by mRNA titration. FIG.30B shows expression of NKG2A and CD300a TASR by flow cytometry on B2M KO T cells 1-day post electroporation (EP) with the given quantity of respective mRNA. FIG. 30C shows survival of B2M KO T cells electroporated CD300a or NKG2A TASR at the indicated mRNA dose as in FIG.30B, challenged with NK cells at a constant E:T ratio of 4.6. N = 3 technical replicates per condition.

[0202] FIGS.31A-D show that Mouse B7-1 (also interchangeably referred to herein as “mCD80” and “mB7-1”) domains enhance TASR expression. FIG.31A shows an experimental overview. TASRs were constructed with various transmembrane and cytoplasmic domains, in-vitro transcribed into mRNA, and then transiently transfected into primary T cells to test for expression. All constructs were bicistronic with GFP to enable normalized comparison between difference constructs. FIG.31B shows various TM+Cyt domains tested. CD8 TM is from the V1 scaffold. FIG.31C shows GFP versus TASR median fluorescence intensity (MFI) of a KIR2D specific TASR with the various TM+Cyt domains from FIG. 31B as assessed by flow cytometry, gated on single cell lymphocytes. mB7-1 provides higher TASR expression normalized to GFP translation. FIG.31D shows expression of KIR2D, NKG2A, and CD300a TASRs with either CD8TM or mCD80 TM+Cyt after mRNA EP as measured by flow cytometry, gated on single cell lymphocytes.

[0203] FIGS.32A-G show an inverse correlation of hinge length and functional potency of CD300a TASR but not NKG2A TASR, and further optimization of CD300a TASR (V2). FIGS. 32A-E show the effect of hinge length on function of CD300a TASR and NKG2A TASR. FIG. 32A shows the structures of CD300a TASR variants with different hinge domains with the indicated amino acid lengths and their expression level after mRNA EP into B2M KO T cells. FIG. 32B shows NK challenge assay of B2M KO T cells expressing the indicated CD300a TASR variants. N = 2 technical replicate curves per condition. FIG.32C shows correlation of NK protection from FIG.32B as defined by IC50 value with hinge length. Error bar indicates 95% confidence interval of IC50 value. FIG.32D shows structures of NKG2A TASR V1 and a no hinge variant, along with their expression level by mRNA EP into B2M KO T cells. FIG.32E shows NK challenge assay of B2M KO T cells expressing the indicated NKG2A TASR variant, N = 1 technical replicate curve per condition. FIGS.32F-G show further optimization of CD300a TASR. FIG.32F shows CD300a TASR V1 and two optimized variants tested for expression via mRNA EP of B2M KO T cells. FIG.32G shows NK challenge assay of B2M KO T cells expressing the indicated CD300a TASR variant. N = 1 technical replicate curve per condition.

[0204] FIGS.33A-B show an experimental overview and phenotype of non-viral CRISPR- mediated targeted integration of cloaking transgenes into human primary T cells, related to FIG. 29B. FIG. 33A shows a process for non-viral targeted integration and purification of T cells at the B2M and AAVS1 locus. Stimulated primary T cells were edited with CRISPR Cas9 at the indicated locus, along with linear double stranded DNA (dsDNA) homology repair template. For AAVS1 homology directed repair (HDR), the transgene encodes EF1a promoter, cloaking transgene, P2A self-cleavable peptide, RQR8 epitope tag, and BGH polyA sequence. For B2M HDR, the cloaking transgene is integrated at the start codon of the B2M gene under control of endogenous B2M promoter and followed by BGH polyA sequence. Edited T cells were further purified by magnetic bead-based enrichment using antibody to either the cloaking transgene or RQR8 epitope tag if present, and then subjected to 1-2 additional rounds of stimulation and expansion prior to cryopreservation. FIG.33B shows the phenotype of engineered T cells containing the indicated transgenes in the top label by flow cytometry, gated on single cell lymphocytes. Histograms shown in dotted lines indicate negative control T cells stained with the same markers.

[0205] FIGS.34A-F show that CD300a TASR outperforms CD47 in B2M KO T cells in additional model systems and with IL-2 activated NK cells. FIGS.34A-B show a comparison of CD300a TASR and CD47 via mRNA electroporation. FIG.34A shows flow cytometry phenotypes of T cells electroporated with the indicated mRNA. FIG.34B shows NK challenge assay of T cells in FIG.34A against four NK cell donors with either 2Adom or 2Cdom NK phenotype. N = 1 technical replicate curves per condition. FIGS.34C-D show a comparison of CD300a TASR and CD47 expressed from the AAVS1 loci of B2M KO T cells in both NK and PBMC challenge assays. FIG. 34C shows flow cytometry phenotypes of T cells containing the indicated cloaking transgene or GFP, gated single cell lymphocytes. FIG. 34D shows NK and PBMC challenge assay of T cells from with either NK or PBMC donors as indicated. N = 1 technical replicate curves per condition. FIG.34E shows the eIIHFW^RI^F\WRNLQH^FRQFHQWUDWLRQ^DQG^FXOWXUH^WLPH^RQ^WKH^H[SUHVVLRQ^RI^6,53Į^ on cultured human NK cells. Cryopreserved NK cells were thawed and cultured at the indicated concentrations of IL-2 or 10 ng / mL IL-15, used for the standard NK challenge assay, for 3 and 5 days. Monocytes served as positive staining control. NK cells were gated CD3-CD56+, Monocytes were gated CD14+FSChiSSChi. FIG.34F shows NK challenge assay with B2M KO T cells expressing the indicated cloaking transgene at B2M loci as in FIG. 29C. NK cells were cultured for the indicated duration with the indicated cytokine, during both initial culture as in FIG.34E and during the 20-hour co-culture. N = 2 technical replicate curves per condition.

[0206] FIGS.35A-B show that CD300a TASR outperforms TIM3 Engager. CD300a TASR with TIM3 engager were expressed in T cells via mRNA electroporation. Both ligands were bicstronic with GFP. FIG. 35A shows cloaking ligand expression assessed using both ligand-based staining and antibody-based staining by flow cytometry with the indicated mRNA, gated on single cell lymphocytes. FIG.35B shows NK challenge assay of T cells from FIG. 35A challenged with the four indicated NK cell donors. N = 2 technical replicate curves per condition.

[0207] FIGS.36A-F show a flow cytometry gating scheme and additional conditions for Allo-T + NK competition assay, related to FIG.29D. FIGS.36A-E show representative gating strategies and readouts for competition assays. All samples were resuspended and acquired at equal volumes. FIG.36A shows pre-gating on CD3+ single-cell lymphocytes. FIG.36B shows gating for target pool only with no effector cells. Labels designate the pool member. FIG.36C shows gating with NK effector challenge only. The gate indicated by the dashed lines highlights depleted population relative to FIG.36B. FIG.36D shows gating with allo-T cell effector challenge only. The gate indicated by the dashed lines and label highlights depleted population relative to FIG.36B FIG. 36E shows gating with allo-T and NK cell challenge, red gate highlights CD300a TASR V2 survival relative to other members and FIG. 36B. FIG. 36F shows additional NK and Allo-T challenge conditions as in FIG.29. Allo-TEMC7 contains the AHIII T cell receptor reactive to EMC7 peptide, ALWGFFPVL (SEQ ID NO: 200), presented by HLA-A2. NK and allo-T cells effectors are mixed 1:1. N = 3 technical replicates per condition.

[0208] FIGS.37A-E show that CD300a TASR universally protects against NK cells and enhances CAR-T functional potency. FIG.37A shows the study design and demographic overview of the 45 PBMC donors used. FIG. 37B shows a PBMC challenge assay. Each datapoint represents the IC50 value of a 7-point curve of T cell survival with the indicated cloaking ligand against PBMCs from one donor. N = 45 donors. Groups were compared using Wilcoxon matched pairs signed rank test. FIG.37C shows Association of PBMC donor demographics from FIG.37A with functional data from FIG.37B Groups were compared using the following statistical analyses: Kruskal-Wallis for Ethnicity, Mann-Whitney for age, gender, and CMV status. Y-axis represents ratio of IC50 between CD300a TASR and HLA-E cloaking ligand, with one indicating equal protection. FIG.37D shows the relationship between functional potency and adaptive NK cell frequency for CD300a TASR (left) and HLA-E (right). The dashed line represents linear fit of log-log transformed data. N = 45 PBMC donors. FIG.37E (bottom row) shows a B cell lysis assay for engineered CAR-T cell therapy containing the indicated cloaking transgene against the indicated PBMC donors. N = 2 technical replicate curves per condition. FIG. 37E (top row) shows phenotypes of NK cells from the respective PBMC donor.

[0209] FIGS.38A-D show a PBMC challenge assay and validation, related to FIGS. 37A-D. FIG. 38A shows the experimental overview of the PBMC challenge assay. PBMC phenotyping was performed post-thaw, and cloaked T cell phenotyping performed at time of co-culture. FIG.38B shows the co-culture plate map and gating scheme for fluorescent barcoding flow cytometry readout in the PBMC challenge assay. FIG.38C shows inter-assay variation of the PBMC challenge assay of one PBMC donor against one B2M KO T cell source. Experiments were performed on separate days. N = 1 technical curve per experiment. Right panel shows the coefficient of variation (CV) of the IC50 values. FIG.38D shows phenotypes of the three engineered T cells targets used in FIG. 37, gated on live single lymphocytes. Histograms shown in dotted lines indicate negative control. Label indicates cloaking transgene.

[0210] FIGS.39A-D show NK cell phenotyping of PBMC challenge assay, related to FIGS.37A- D. FIG. 39A shows a gating scheme for bulk NK cells and adaptive NK cell phenotype. FIG.39B shows representative expression of NK cell phenotyping markers from one PBMC donor. “NC” indicates CD300a fluorescence-minus-one negative control staining. In panels where “NC” is not indicated due to peak overlap, the negative control is the leftmost peak. FIG.39C shows the percentage of NK cells expressing the indicated marker with gating from FIG.39B. N = 45 PBMC donors. FIG.39D shows adaptive NK cell frequency gated as in FIG.39A between CMV seropositive (N = 21 donors) and seronegative donors (N = 24 donors), Mann Whitney U Test.

[0211] FIGS.40A-D show survival curves of 45 donor PBMC challenge assay, related to FIGS. 37A-D. Number indicates PBMC donor. Brackets indicate the CMV serostatus of the donor. N = 1 technical replicate curves per condition.

[0212] FIGS.41A-G show generation of TASR-expressing CAR-T cells by multiplexed non-viral HDR into primary T cells and use in B cell killing assay, related to FIG.37E. FIG.41A shows knock-in efficiency of T cells 4-days post-editing with CD300a TASR at B2M loci and anti-CD19 CAR at TRAC loci. Post-electroporation, T cells were plated into either standard media or media containing the indicated small molecules for 24 hours prior to exchange back into standard media. (right panel) fold enhancement in editing efficiency of single KI cells after small molecule treatment as in FIG.41A. N = 3 editing runs. FIG.41B shows the process overview for generation, purification, and expansion cloaked CAR-T cells. Stimulated T cells are edited in a single step with B2M and TRAC RNPs and linear dsDNA HDR templates encoding cloaking ligand and anti-CD19 CAR. The cloaking HDR template integrates at the start codon of B2M gene while the CAR integrates into TRAC in bicistronic format via 2A cleavable peptide. Both constructs code for BGH polyA tail. Cloak ligand expressing cells are enriched using an appropriate antibody by magnetic enrichment, followed by selection expansion of CAR expressing cells by addition of mitomycin-C treated Raji feeder cells. FIG.41C shows phenotypes of three engineered CAR-T cells by flow cytometry expressed the indicated cloaking transgene at B2M locus, gated live single cells. The same anti-CD19 CAR is used for all CAR-T cells. FIG.41D shows a co-culture plate map and fluorescent barcoding scheme. PBMCs are seeded at 250,000 per well, and then edited CAR-T cells are added to the indicated CAR-T:PBMC ratio. Every column represents one unique PBMC : CAR- T cell pair and cytotoxicity curve. At the end of co-culture, rows are barcoded by staining with unique combinations of fluorescent anti-CD45 antibody along with phenotyping antibodies. The plate is washed and then every column is pooled into one well and acquired on flow cytometry. FIG.41E shows a gating scheme for identification of B cells and barcode demultiplexing. FIG. 41F shows B cell counts in the absence of CAR-T cells. PBMCs are seeded at equal density in one column, fluorescently barcoded, and then counted on flow cytometry as in FIGS.41D-E. FIG.41G shows a comparison of B cell lysis measurements by conventional and fluorescent barcoding flow cytometry. RQR8-expressing CAR-T cells were co-cultured with PBMCs at the indicated ratios in four identical columns on the plate. Two columns underwent fluorescent barcoding flow cytometry as in FIGS.41D-E, while two other columns were directly stained with phenotyping antibodies and then acquired on flow cytometry without pooling. N = 2 technical replicates per kill curve.

[0213] FIGS.42A-B show CD300a TASR expressed in a model allogeneic anti-CD19 CAR-T cell exhibit enhanced protection from NK cells with no effect on CAR-mediated killing potency, related to FIG. 37E. FIG.42A shows cytotoxicity of indicated cloaked anti-CD19 CAR-T cells against CD19-expressing Raji cells. Negative control (“No CAR”) contains CD300a TASR integrated into the B2M loci without anti-CD19 CAR. N = 3 technical replicate curves per condition. FIG.42B shows NK challenge of cloaked anti-CD19 CAR-T cells containing the indicated cloaking transgene with a 2Adom and 2Cdom NK donor. N = 3 technical replicates curves per condition.

[0214] FIG.43 shows that CD300a cloaking (using a CD300a TASR) outperforms HLA-E across 45 donor samples in an NK challenge assay. Target DKO edited T cells without inhibitory factor, with HLA-E, or with the CD300a TASR were co-cultured for 72 hours with effector cells (PBMCs, including NK cells) from 45 different donors selected for diverse age, gender, ethnicity, and CMV status. The graph plots the IC50 values of the 3 different edited T cell conditions across 5 bins of NK donors based on the percentage of NKG2A- / NKG2C+ NK cells from a respective donor. Only T cells comprising the CD300a TASR were protected when challenged with NK cells across all 5 bins of donors.

[0215] FIG.44 shows that the B2M / CIITA KO + CD300a TASR outcompetes other cloaking configurations against T and NK cell alloreactivity. Similar to the experiment described in figure 29 and example 27, a pool of DKO T cells that have been engineered to express the identified transgene or wt cells is challenged with either T effectors, NK effectors, or NK and T effectors. Only target T cells comprising the CD300a TASR were protected when challenged with both T and NK cells.

[0216] FIGS.45A-C show that the CD300a TASR comprising a CD300a VHH can provide equal or better protection in comparison to a CD300a scFv TASR. FIG. 45A provides an experimental overview of the survival assay and schematics of the VHH and scFv CD300a TASRs that were tested for NK protection. FIG. 45B shows phenotypes of T cells that express the indicated CD300a TASRs 24 hours (D1) after electroporation with mRNA encoding the identified TASR or GFP (control). The amino acid sequences of the VHHs of CD300a TASRs 20-37 are provided in SEQ ID NOs: 155-172, respectively. FIG.45C shows survival of engineered T cells containing the indicated CD300a TASRs in an NK cell cytotoxicity assay. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS I. Definitions

[0217] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0218] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids, reference to “a cell” includes a plurality of cells, and the like.

[0219] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0220] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0221] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.

[0222] The term “or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.

[0223] As used herein, “antibody” refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof, and can be from natural or from recombinant sources, or from a library with random or intentionally designed CDR sequences in an antibody construct.

[0224] As used herein, “CDR” refers to the complementarity determining region amino acid sequences of an antibody (or antibody fragment) which are the hypervariable domains of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein may refer to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate). CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. The CDR sequences of antibodies can be determined by the Kabat numbering system (Kabat et al; (Sequences of proteins of Immunological Interest NIH, 1987); alternatively they can be determined using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273, 927-948), the contact definition method (MacCallum R. M., and Martin A. C. R. and Thornton J. M, (1996), Journal of Molecular Biology, 262 (5), 732-745) or any other established method for numbering the residues in an antibody and determining CDRs known to the skilled man in the art. Other numbering conventions for CDR sequences available to a skilled person include “AbM” (University of Bath) and “contact” (University College London) methods. The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the “minimum binding unit.” The minimum binding unit may be a sub-portion of a CDR.

[0225] As used herein, “antigen-binding domain” means the portion of an antibody that is capable of specifically binding to an antigen or epitope. One example of an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer of an antibody. Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin.

[0226] As used herein, “antibody fragment” or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc) variable (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), diabodies, minibodies, nanobodies (also known as variable domain on a heavy chain (VHH) antibodies, such as camelid VHH antibodies; See, e.g., Bever et al., Analytical and Bioanalytical Chemistry.408(22): 5985–6002 (2016)), and multi-specific antibodies formed from antibody fragments. Exemplary binding domains useful in the disclosed embodiments can also include a cytokine, a ligand, or a peptide (such as an adnectin or a designed ankyrin repeat protein (DARPin) (See, e.g., Rafiq et al., Nat Rev Clin Oncol.2020;17:147–167). In certain embodiments, an NKG2A binding domain or a CD300a binding domain provided herein is an antibody fragment. For a review of certain antibody fragments, see Hudson et al. Nat. Med.9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med.9:129-134 (2003). Single- domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No.6,248,516 B1). In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0227] As used herein, “VHH,” “VHH antibody,” or “nanobody” refers to the antigen binding fragment of heavy chain only antibodies (HcAb), which lack VL domains (See, e.g., Bever et al., Analytical and Bioanalytical Chemistry.408(22): 5985–6002 (2016)). Heavy chain only antibodies are produced in nature by camelids and sharks. A variable domain on a heavy chain (VHH) typically comprise a single polypeptide chain. Compared to mAbs and other antibody fragments, VHHs are typically smaller in size.

[0228] As used herein, “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0229] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0230] As used herein, “antibody heavy chain,” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0231] As used herein, “antibody light chain,” refers to the smaller of the two types of polypeptide FKDLQV^SUHVHQW^LQ^DQWLERG\^PROHFXOHV^LQ^WKHLU^QDWXUDOO\^RFFXUULQJ^FRQIRUPDWLRQV^^.DSSD^^³^´^^DQG^ ODPEGD^^³^´^^OLJKW^FKDLQV^UHIHU^WR^WKH^WZR^PDMRU^DQWLERG\^OLJKW^FKDLQ^LVRW\SHV^^

[0232] As used herein, “antigen” or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.

[0233] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0234] A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0235] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0236] With regard to the binding of an antibody or fragment thereof to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.

[0237] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0238] The term “allogeneic” refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0239] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. As used herein, “preventing” refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.

[0240] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. A “patient” is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein. In some embodiments, the subject has cancer, e.g., a cancer described herein.

[0241] The terms “cancer” or “tumor” as used herein refer to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, hematological cancers, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

[0242] Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and / or extranodal sites. Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin's lymphomas (B- NHLs). B-NHLs may be low-grade (or indolent), intermediate-grade (or aggressive) or high-grade (very aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate- grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom’s macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease. NHL may also include T-cell non-Hodgkin’s lymphoma s (T-NHLs), which include, but are not limited to T-cell non-Hodgkin’s lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.

[0243] Hematopoietic cancers also include Hodgkin's lymphoma (or disease) including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocyte depleted Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues which include hematopoietic cells referred herein to as “hematopoietic cell tissues” include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.

[0244] Exemplary solid cancers that may be treated using a method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, uterine cancer, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0245] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0246] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0247] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0248] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor.

[0249] The term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. An “antagonist” is an entity that binds to and antagonizes a receptor.

[0250] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a TFP of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.

[0251] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0252] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.

[0253] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0254] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0255] The term “expression” refers to the transcription and / or translation of a particular nucleotide sequence, such as driven by a promoter.

[0256] A “vector” and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene). Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell). Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector. Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct. Vectors can be single-stranded or double-stranded nucleic acid molecules, and can be linear or circular nucleic acid molecules. A donor nucleic acid used for gene editing methods employing zinc finger nuclease, TALEN or CRISPR / Cas can be a type of a vector. One type of vector is a “plasmid,” which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and / or translating the transgene. A viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome. Examples of viral vectors include retroviral, lentiviral, adenoviral, adeno- associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. A vector may be a transfer vector, i.e., a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like. A vector may be an expression vector, i.e., a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Further examples of regulatory sequences and other elements for expression are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res.23:3605-3606. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0257] A transgene is “operably linked” to a vector when there is linkage between the transgene and the vector to permit functioning or expression of the transgene sequences contained in the vector. In one embodiment, a transgene is “operably linked” to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.

[0258] The terms “transfected” or “transformed” or “transduced” or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell (e.g., an engineered cell described herein). A “transfected” or “transformed” or “transduced” host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene). The host cell includes the primary subject cell and its progeny.

[0259] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0260] The term “promoter” refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, that can initiate the specific transcription of a polynucleotide sequence. The term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. The term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. In some embodiments, a promoter is an endogenous promoter. In some embodiments, such as certain embodiments wherein a recombinant nucleic acid or vector disclosed herein is inserted into a gene locus (such as a B2M or TRAC locus) in a cell, the promoter is an endogenous B2M promoter or an endogenous TRAC promoter.

[0261] The term “linker,” as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond. The term “linker” as used in the context of a scFv can refer to a peptide linker comprising amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)5 or (Gly4Ser)4. In another embodiment, the linkers include multiple repeats of (GlySer), (Gly2Ser), or (Gly3Ser). Also included within the scope of the disclosure are linkers described in WO2012 / 138475 (incorporated herein by reference). In some instances, the linker sequence comprises (G4S)n, wherein n=3 to 6. In some instances, the linker sequence comprises GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 30). In another embodiment, the linker is the Whitlow / 218 linker, GSTSGSGKPGSGEGSTKG (SEQ ID NO: 31). A linker may also be used to link one or more binding domains (such as one or more scFvs) together, such as within a bispecific TASR as described herein. Such linkers can comprise the linkers described above and / or a cleavable peptide linker, such as those described elsewhere herein.

[0262] As used herein, “NKG2A” refers to a particular member of the NKG2 family that dimerizes with CD94 to form an inhibitory receptor (CD94 / NKG2A). The NKG2 family (also known as CD159) includes seven members: A, B, C, D, E F, and H. CD94 / NKG2 receptors are C-type lectin receptors which are expressed predominantly on the surface of NK cells and a subset of CD8+ T- lymphocytes. These receptors stimulate or inhibit cytotoxic activity of NK cells, therefore they are divided into activating and inhibitory receptors according to their function. NKG2A, -B, -C, -E and -H form heterodimers with CD94, linked by disulfide bonds, whereas NKG2D forms homodimers. Inhibitory molecule NKG2A and its splice variant NKG2B contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the intracellular part of the molecule. Activating molecules NKG2C and NKG2E and its splice variant NKG2H contain a positively charged residue in their transmembrane regions through which they interact with adaptor molecules containing ITAMs. Inhibitory NKG2 molecules containing ITIMs recruit the Src homology 2 domain containing phosphatases SHP-1 and SHP-2, which leads to the inhibition of cytotoxicity. Ligands of CD94 / NKG2 heterodimeric molecules include nonclassical MHC class I molecules, such as HLA-E in humans.

[0263] As used herein, “cluster of differentiation 300a” or “CD300a”, also known as CMRF-35H, inhibitory receptor protein (Irp60) and IgSF12, is a member of the CD300 glycoprotein family of cell surface proteins that regulate a diverse array of immune cell processes. CD300a is found on the surface of leukocytes including neutrophils, basophils, eosinophils, mast cells, monocytes, B lymphocytes, NK cells and dendritic cells, and regulates their proliferation, differentiation, apoptosis and immunity. CD300a is an inhibitory receptor that has three classical and one non- classical ITIM motifs in its cytoplasmic tail. The ITIMs are phosphorylated by Src-family kinases, resulting in recruitment of src homology 2 domain containing protein tyrosine phosphatases (SHPs) or SH2 domain-containing inositol phosphatase (SHIP). CD300a mainly regulates the function of leukocytes by recruiting SHP-1 phosphatase. Signaling through CD300a is complex and involves many downstream signaling components. For example, CD300a engagement by agonist monoclonal antibodies has been shown to inhibit IgE-dependent Ca2+mobilization and mediator release from mast cells, SCF-mediated mast cell activation, differentiation, and survival, and downregulate NK cell cytolytic activity. CD300a signaling is involved in allergy response, autoimmune disease and viral infection, and CD300a is considered a therapeutic target in these diseases.

[0264] As used herein, a “signal peptide” (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) is a short peptide (such as a peptide 10-50 amino acids in length) present at the N-terminus (or nonclassically at the C-terminus or internally) to direct a synthesized protein of interest to the surface of a cell (such as to the cell membrane). Thus, signal peptides function to prompt a cell to translocate the protein, e.g., to the cellular membrane. This signal sequence is sometimes cleaved off by the cell in the maturation of a polypeptide.

[0265] As used herein, an “engineered cell” or a “population of engineered cells” or related terms as used herein refer to a cell (or a population thereof) into which foreign (exogenous or transgene) nucleic acids have been introduced. The foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and / or polypeptide encoded by the foreign nucleic acid (transgene). An engineered cell (or a population thereof) can be a cultured cell or can be extracted from a subject. The engineered cell (or a population thereof) includes the primary subject cell and its progeny without regard for the number of passages. Engineered cells encompass progeny cells. In embodiments, an engineered cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express a recombinant nucleic acid as disclosed herein. In one example, the engineered cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding the desired recombinant nucleic acid described herein. Engineered cells and populations thereof can harbor an expression vector that is stably integrated into the host’s genome or can harbor an extrachromosomal expression vector. In embodiments, engineered cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.

[0266] As used herein, “hypoimmunogenic” and “hypoimmune” refer to a reduction in immunogenicity. A hypoimmunogenic cell (such as an engineered cell described herein that is hypoimmunogenic, such as a hypoimmunogenic iPSC) gives rise to a reduced immunological rejection response when transferred into an allogeneic host. For example, a hypoimmunogenic pluripotent cell is a pluripotent cell that retains its pluripotent characteristics and yet gives rise to a reduced immunological rejection response when transferred into an allogeneic host. A hypoimmunogenic cell described herein may induce a reduced immune response (such as compared to cell that is not hypoimmunogenic) or no immune response. Thus, “hypoimmunogenic” or “hypoimmune” refers to any amount of reduced or eliminated immune response when compared to the immune response of a parental (i.e., “wild-type”) cell prior to engineering (e.g., prior to introduction of a recombinant nucleic acid comprising an NKG2A binding domain, a CD300a binding domain, or both, as described herein). For example, relative to a wild-type cell, such a hypoimmunogenic cell may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% less prone to immune rejection (e.g., cytolysis) by a subject into which such cells are transplanted. In the context of an engineered cell (such as a pluripotent stem cell, as used herein, such as an iPSC), “wild-type” means a cell that that may comprise some nucleic acid changes, but did not undergo the gene editing procedures of the present disclosure (i.e., introduction of a recombinant nucleic acid comprising an NKG2A binding domain, a CD300a binding domain, or both, as described herein) to achieve hypoimmunogenicity.

[0267] As used herein, “stem cells” refers to cells capable of going through numerous cycles of cell division, maintaining an undifferentiated state, and having the capacity to differentiate into specialized cell types. Stem cells are further classified into three categories: totipotent, pluripotent, or multipotent somatic. A totipotent cell has the ability to form an entire organism (e.g., a fertilized egg). As used herein, “pluripotent stem cells” refers to stem cells that lack the ability to form extraembryonic tissue and are therefore unable to generate a fetus, but have the potential to differentiate into any of the three germ layers: endoderm (e.g., the stomach lining, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.) or ectoderm (e.g., epidermal tissues and nervous system tissues). Pluripotent stem cells encompass embryonic stem cells (ESCs). Exemplary human embryonic stem cell (hESC) lines include those made available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (as described in Cowan, C. A. et. al, New England J. Med.350: 13. (2004), incorporated by reference herein in its entirety). Pluripotent stem cells also encompass “induced pluripotent stem cells” (iPSCs), a type of pluripotent stem cell derived from a non-pluripotent cell, typically an adult somatic cell, by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins (through a process known as somatic cell “reprogramming”). Methods for the derivation of iPSCs by somatic cell reprogramming are known in the art and are further described in, e.g., Takahashi and Yamanaka, Cell 126 (4): 663–76 (2006); Yu et al., Science 324(5928):797-801 (2009); Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol.26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); each of which is incorporated by reference herein in their entirety.

[0268] As used herein, the terms “inhibiting” or “reducing,” such as in the context of inhibiting or reducing NK cell cytotoxicity to an engineered cell as disclosed herein, refer to any amount of reduced or eliminated NK cell cytotoxicity to the engineered cell when compared to levels of NK cell cytotoxicity toward a parental (i.e., “wild-type”) cell prior to the engineering (e.g., prior to introduction of a recombinant nucleic acid comprising an NKG2A binding domain, a CD300a binding domain, or both, as described herein). For example, NK cell cytotoxicity to an engineered cell is “reduced” or “inhibited” when it is decreased, relative to a wild-type cell, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

[0269] As used herein, the term “MHC class I” refers to a class of major histocompatibility complex (MHC) molecules that are displayed on the surface of nucleated cells and platelets in vertebrates. MHC class I molecules function as part of the adaptive immune system by binding fragments of cytosolic foreign (non-self) proteins and displaying these antigens on the cell surface for recognition of cells of the immune system, e.g., cytotoxic T cells. In humans, MHC is also referred to as human leukocyte antigen (HLA). Rejection of allogenic therapeutic cells (e.g., in GvHD), such as CAR T cells, is believed to be largely driven by donor- or iPSC-derived T cell recognition of host peptide-+ / $^FRPSOH[HV^WKURXJK^WKH^Įȕ^7^FHOO^UHFHSWRU^FRPSOH[^^Įȕ7&5^^ Rejection is mainly driven by host NK cells, CD8+ T-cells, CD4+ T cells, and, to a lesser extent, by macrophages. HLAs include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. HLA class I histocompatibility antigen alpha chain E (HLA-E) is a non-classical HLA molecule that plays a role in recognition by NK cells. Without being bound by a particular theory, using the heterodimeric receptor CD94 / NKG2A / B / C, NK cells recognize HLA-E molecules bound to antigen at the cell surface. CD94 / NKG2A or CD94 / NKG2B engagement results in an inhibitory effect on the cytotoxic activity of the NK cell, thereby preventing cell lysis, while simultaneously causing NK cell activation.

[0270] As used herein, the term “ȕ^^PLFURJOREXOLQ” (B2M) refers to a particular polypeptide component of MHC class I molecules. The B2M protein is encoded by the B2M gene. MHC class I PROHFXOHV^DUH^KHWHURGLPHUV^FRPSULVHG^RI^WZR^SRO\SHSWLGH^FKDLQV^^Į^DQG^ȕ^-microglobulin, that are QRQFRYDOHQWO\^OLQNHG^YLD^LQWHUDFWLRQ^RI^%^0^DQG^WKH^Į^^GRPDLQ^ Without being bound to a SDUWLFXODU^WKHRU\^^ȕ^^PLFURJOREXOLQ^LV^required for cell surface expression of MHC class I molecules and for stability of the peptide-binding groove. Absence of B2M expression leads to significant reductions in MHC class I molecules detectable on the cell surface.

[0271] As used herein, the term “T cell receptor alpha constant” (TRAC) refers to the constant region of the T cell receptor (TCR) alpha chain. The T cell receptor (TCR) is a membrane-anchored heterodimeric protein typically FRQVLVWLQJ^RI^WKH^KLJKO\^YDULDEOH^DOSKD^^Į^^DQG^EHWD^^ȕ^^FKDLQV^ (encoded by TRA and TRB, respectively) expressed as part of a complex with invariant CD3 chain molecules. TCRs are found on the surface of T cells, or T lymphocytes, and recognize antigens bound to MHC molecules. Removal of the endogenous TCR by targeting TRAC (e.g., through CAR transgene knock-in) has been used to address histocompatibility barriers associated with cells derived from unrelated donors.

[0272] As used herein, the term “deficient” can refer to reduced or eliminated expression and / or functionality of a particular gene product (e.g., as measured by RNA and / or protein detection methods, and / or by functional assays), such as an MHC class I molecule component (e.g., B2M), a TCR component (e.g., TRA, such as via disruption of the TRAC locus), or a molecule that regulates or controls MHC class II expression (such as a Class II major histocompatibility complex transactivator (CIITA) gene product). In this context, “deficient” refers to any amount of reduced or eliminated expression or functionality when compared to levels of the same gene product in a parental (i.e., “wild-type”) cell prior to the deficiency (e.g., prior to engineered disruption of the gene, such as by any suitable means described herein or known in the art). In some embodiments, in a cell that is deficient in a particular gene product, the gene product is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, relative to a wild-type cell. In some embodiments, in a cell that is deficient in a particular gene product, the gene product is reduced by 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 81-100%, 82- 100%, 83-100%, 84-100%, 85-100%, 86-100%, 87-100%, 88-100%, 89-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100%, or 100% relative to a wild-type cell. “Deficient” can also refer to a reduction or elimination of a detectable molecule in a cell, such as an MHC class I molecule or an MHC class II molecule. For example, a cell is MHC class I deficient when expression of one or more components of an MHC class I molecule (such as B2M) is reduced, relative to a wild-type cell, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, resulting in reduced or eliminated detectable MHC class I on a cell surface by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, relative to a wild- type cell. By way of another example, a cell is MHC class II deficient when expression of one or more components of an MHC class II molecule or of a molecule that regulates or controls expression of an MHC class II molecule (such as CIITA) is reduced, relative to a wild-type cell, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, resulting in reduced or eliminated detectable MHC class II on a cell surface by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, relative to a wild-type cell. By way of another example, a cell is TRAC deficient when expression of the TRAC locus is reduced, relative to a wild-type cell, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, in a cell that is deficient in a particular gene product, the gene product is not detectable, such as by PCR, immunohistochemical staining, ELISA, western blotting, or other suitable nucleic acid or protein detection methods. In some embodiments, a gene locus is “disrupted,” resulting in a cell that is deficient for the gene product encoded by the gene. For example, insertion of a recombinant nucleic acid or vector disclosed herein into a gene locus (e.g., a B2M and / or TRAC locus) can disrupt the locus, thereby reducing or eliminating expression of the gene at the locus. Methods of disrupting a gene locus are known in the art, such as, but not limited to, homologous recombination, CRISPR- Cas9, and TALENs. Further, disruption can be achieved with (e.g., through gene knock out methods) or without (e.g., through insertion of a nucleotide sequence into the gene locus) removal of all or a portion of the targeted gene locus. Thus, methods disclosed herein can include disrupting a gene locus (such as a B2M and / or TRAC locus) using any suitable means known in the art. II. Recombinant Nucleic Acids A. Overview

[0273] Rejection of allogenic therapeutic cells (e.g., in GvHD), such as CAR T cells, is believed to be largely driven by donor- or iPSC-derived T cell recognition of host peptide-HLA complexes WKURXJK^WKH^Įȕ^7^FHOO^UHFHSWRU^FRPSOH[^^Įȕ7&5^^^5HMHFWLRQ^LV^PDLQO\^GULYHQ^E\^KRVW^1.^FHOOV^^ CD8+ T-cells, CD4+ T cells, and, to a lesser extent, by macrophages. In the context of CAR T-cell therapy, the relative contribution of these cell types to allograft rejection may vary depending on their absolute numbers and reconstitution kinetics following preconditioning regimen. In three cohorts (acute lymphoblastic leukemia (ALL) and large B-cell malignancies), patients showed significant differences in CD8+ T cell, CD4+ T cell, and NK-cell reconstitution kinetics following autologous CD19 CAR T-cell treatment and a preconditioning regimen (cyclophosphamide and fludarabine). (Jo et al. Nat Commun. 2022;13(1):3453; Strati et al. Haematologica.2021;106(10); Wang et al. Int J Lab Hematol.2021;43: 250– 258; Wang et al. Blood. 2019;134(Supplement_1):1301) CD8+ T cells and NK cells recovered to their initial levels within 3–4 weeks, whereas CD4+ T cells showed a significantly slower recovery rate and, in some patients, had not returned to their initial levels even 1 to 2 years after treatment onset. Thus, CD8+ T cells and NK cells may play a larger role in controlling the length of the allogeneic CAR T-cell therapeutic window by being the first and primary contributors to rejection. NK cells are a key component of the innate immune system and influence adaptive immune responses, e.g., via cytokine secretion. The activity of NK cells is thought to be controlled by a balance of inhibitory and activating signals delivered via NK cell-surface receptors. (Crew et al. Molecular Immunology, 2005;42(1):1205-1214). Eliminating ligands for NK cell activation receptors on allogenic cells or increasing the level of ligands for inhibitory cell receptors could reduce or prevent host NK cell- mediated destruction of allogenic immune cell therapies.

[0274] There are two classes of NK cell inhibitory receptors: the immunoglobulin-like KIR and LIR receptors and the C-type lectin-like receptors, CD94 / NKG2 heterodimers. In humans, the ligands for the KIR receptor family members are the classical class I antigens, HLA-A, -B, and -C and the ligand for one LIR (LIR-2) is the nonclassical class I antigen HLA-G. The major ligand for CD94 / NKG2 receptors is the nonclassical class I antigen HLA-E. Among the NK cell inhibitory receptors, CD94 / NKG2A appears to be the most widely expressed on NK cells. (Crew et al. Molecular Immunology, 2005;42(1):1205-1214)

[0275] Although CD8+ T cells need to go through a clonal expansion to mount an efficient alloresponse, they may be the first subset to reject allogeneic CAR T cells. In the case of CD8+ T cell-mediated rejection, a primary approach has been to eliminate expression of HLA class I molecules on CAR T cells. For example, deletion of the conserved gene ȕ^P^completely removes surface expression of HLA class I. (Wang et al. Stem Cells Transl Med.2015;4(10):1234–1245). In this way, the inactivation of HLA class I at the surface of CAR T cells could efficiently blunt such rejection and offer an initial therapeutic window to eradicate cancer cells. However, while immunogenic recognition by CD8+ T cells is reduced by this approach, the complete loss of HLA class I molecules increases the risk that host NK cells will recognize and destroy the allogeneic CAR T cells (the so-called “missing self” response). Embedding an NK inhibitor within CAR T cells could further extend their persistence. NK cell mediated destruction of HLA-edited T cells has been proposed to be prevented by expression on CAR T cells of nonpolymorphic HLA molecules such as HLA-E that will bind inhibitory receptors on NK cells. (Gornalusse et al. Nat Biotechnol. 2017; 35(8):765–772)

[0276] HLA-E can inhibit host NK cell cytotoxic activity and thus reduce or prevent immune therapeutic cell lysis. Crew et al. (Molecular Immunology, 200542(1):1205-1214) found that expression of an a single chain trimer (SCT) of HLA-E consisting of (from N- to C-terminus) the OHDGHU^SHSWLGH^RI^KXPDQ^ȕ^P^^90$357 / , / ^^DQ^+ / $-E-binding peptide), a 15 amino acid linker, PDWXUH^KXPDQ^ȕ^P^^D^^^^DPLQR^DFLG^OLQNHU^^DQG^PDWXUH^+ / $-E heavy chain reduced NK cell- mediated rejection of porcine xenografts by providing an inhibitory ligand for human NK cells expressing CD94 / NKG2A. SCT HLA-E expression was subsequently found to reduce NK cell cytotoxicity in HLA class I-depleted pluripotent stem cells and in universal CAR T cells. (Gornalusse et al. Nat Biotechnol. 2017;35(8):765-772; Guo et al. Eur J Immunol.2021;51:2513– 2521; Jo et al. Nat Commun. 2022;13(1):3453) SCT HLA-E-expressing CAR T cells are presently in clinical trials for treating cancer patients.

[0277] However, SCT HLA-E expression in immune therapy cells presents several drawbacks. While this construct offers some level of protection against NK cells, SCT HLA-E expression makes the therapeutic cells more vulnerable to rejection by HLA-E-restricted CD8+ T cells. Further, SCT HLA-E expression is only effective against NKG2A-expressing NK cells. Additionally, HLA-E also binds the NKG2C activating receptor on NK cells, causing NK cell activation and subsequently increased rejection of therapeutic cells.

[0278] In contrast, the presently disclosed recombinant nucleic acids allow for development of, e.g., immune-evasive universal CAR T-cells that can evade host CD8+ T cell and NK-cell cytotoxicity and can be compatible with adoptive cell transfer in an allogeneic setting. In some embodiments, a disclosed recombinant nucleic acid encodes a construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain. In some embodiments, the recombinant nucleic acid comprises a CD300a binding domain and does not encode a NKG2A binding domain. In other embodiments, the recombinant nucleic acid comprises a NKG2A binding domain and does not encode a CD300a binding domain. In a particular embodiment, the CD300a binding domain and the NKG2A binding domain are comprised in the same nucleic acid. In another particular embodiment, the CD300a binding domain and the NKG2A binding domain are comprised in at least two different nucleic acids. A disclosed recombinant nucleic acid comprising both a CD300a binding domain and a NKG2A binding domain may encode the binding domains in any order. Thus, in some embodiments, the nucleotide sequence encoding the NKG2A binding domain is located 5’ of the nucleotide sequence encoding the CD300a binding domain. In other embodiments, the nucleotide sequence encoding the NKG2A binding domain is located 3’ of the nucleotide sequence encoding the CD300a binding domain. In embodiments comprising both a NKG2A binding domain and a CD300a binding domain encoded in the same recombinant nucleic acid, the recombinant nucleic acid may further comprise a linker (such as any suitable linker, such as a linker described herein) that links the NKG2A binding domain and the CD300a binding domain.

[0279] In some embodiments, the CD300a binding domain comprises an antibody or a fragment thereof, a VHH, a cytokine, a ligand, or a peptide. In some embodiments, the NKG2A binding domain comprises an antibody or a fragment thereof, a VHH, a cytokine, a ligand, or a peptide. In particular embodiments, (a) the antibody or a fragment thereof comprises a single chain variable fragment (scFv) or a VHH, or (b) the peptide is an adnectin or a design ankyrin repeat protein (DARPin). In other particular embodiments, the VHH comprises the VH domain of a camelid heavy chain antibody.

[0280] In some embodiments, the CD300a binding domain comprises a VHH (CD300a VHH) and / or the NKG2A binding domain comprises a VHH (NKG2A VHH). In particular embodiments, the construct for inhibiting NK cell cytotoxicity comprises or consists of the CD300a binding domain, and the CD300a binding domain comprises a VHH (CD300a VHH). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to the amino acid sequence of any one of SEQ ID NOs: 173-177. In certain embodiments, the CD300a VHH comprises a CDR2 comprising 0, 1, or 2 mutations relative to the amino acid sequence of any one of SEQ ID NOs: 178-181. In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to the amino acid sequence of any one of SEQ ID NOs: 182-199.

[0281] In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising AAKPGEDVY (SEQ ID NO: 182). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLSQFAS (SEQ ID NO: 183). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPRSGWGL (SEQ ID NO: 184). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKTRYES (SEQ ID NO: 185). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NTRLAHGRDVLGGVAYDI (SEQ ID NO: 186). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NTRRLGRSGDLVQDY (SEQ ID NO: 187). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSRYY (SEQ ID NO: 175), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPDRDY (SEQ ID NO: 188). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLPDVLPLEY (SEQ ID NO: 189). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYW (SEQ ID NO: 176), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKVDGSYGIVTEL (SEQ ID NO: 190). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising INGSGGST (SEQ IS NO: 180), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising HTRRSGTSMAMDV (SEQ ID NO: 191). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKLTMVY (SEQ ID NO: 192). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLTNEY (SEQ ID NO: 193). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKVRPSYEY (SEQ ID NO: 194). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSPYY (SEQ ID NO: 177), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VAKPGYEY (SEQ ID NO: 195). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGRT (SEQ IS NO: 181), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPGEDVY (SEQ ID NO: 196). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ IS NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKSNMVY (SEQ ID NO: 197). In some embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ IS NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising TTKVDGSYGIVTEL (SEQ ID NO: 198). In certain embodiments, the CD300a VHH comprises a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYW (SEQ ID NO: 176), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising INGSGGST (SEQ IS NO: 180), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising AAARDRERDY (SEQ ID NO: 199).

[0282] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 155-172. In particular embodiments, the CD300a VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 155-172. CD300a TASRs #20-37 disclosed herein (e.g., as shown in FIG.45) comprise the CD300a VHHs of SEQ ID NOs: 155-172, respectively. In SEQ ID NOs: 155-172 shown below, the CDR1 is shown with underlining, the CDR2 is shown with double underlining, and the CDR3 is shown with wavy underlining.

[0283] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 155.

[0284] SEQ ID NO: 155: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKPGEDVYWGQGTLVTVSS

[0285] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 156.

[0286] SEQ ID NO: 156: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLSQFASWGQGTLVTVSS

[0287] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 157.

[0288] SEQ ID NO: 157: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPRSGWGLWGQGTLVTVSS

[0289] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 158.

[0290] SEQ ID NO: 158: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKTRYESWGQGTLVTVSS

[0291] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 159.

[0292] SEQ ID NO: 159: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTRLAHGRDVLGGVAYDIWGQGT LVTVSS

[0293] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 160.

[0294] SEQ ID NO: 160: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTRRLGRSGDLVQDYWGQGTLVT VSS

[0295] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 161.

[0296] SEQ ID NO: 161: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPDRDYWGQGTLVTVSS

[0297] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 162.

[0298] SEQ ID NO: 162: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLPDVLPLEYWGQGTLVTVSS

[0299] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 163.

[0300] SEQ ID NO: 163: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKVDGSYGIVTELWGQGTLVTVS S

[0301] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 164.

[0302] SEQ ID NO: 164: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKQREWVSAINGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCHTRRSGTSMAMDVWGQGTLVTVS S

[0303] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 165.

[0304] SEQ ID NO: 165: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKLTMVYWGQGTLVTVSS

[0305] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 166.

[0306] SEQ ID NO: 166: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLTNEYWGQGTLVTVSS

[0307] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 167.

[0308] SEQ ID NO: 167: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKVRPSYEYWGQGTLVTVSS

[0309] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 168.

[0310] SEQ ID NO: 168: EVQLVESGGGLVQPGGSLRLSCAASGFTFSPYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVAKPGYEYWGQGTLVTVSS

[0311] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 169.

[0312] SEQ ID NO: 169: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGRTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPGEDVYWGQGTLVTVSS

[0313] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 170.

[0314] SEQ ID NO: 170: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKSNMVYWGQGTLVTVSS

[0315] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 171.

[0316] SEQ ID NO: 171: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTKVDGSYGIVTELWGQGTLVTVS S

[0317] In some embodiments, the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 172.

[0318] SEQ ID NO: 172: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKQREWVSAINGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARDRERDYWGQGTLVTVSS

[0319] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 137-154. In particular embodiments, a nucleotide sequence encoding the CD300a VHH comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 137-154.

[0320] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 137.

[0321] SEQ ID NO: 137: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG GCTAAGCCCGGCGAAGATGTGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0322] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 138.

[0323] SEQ ID NO: 138: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTAGTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTC ACTAAGCTCAGCCAGTTTGCGTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0324] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 139.

[0325] SEQ ID NO: 139: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTTCCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGCCGCGGTCAGGCTGGGGTCTCTGGGGCCAGGGAACCCTGGTCACCGTTTCAT CG

[0326] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 140.

[0327] SEQ ID NO: 140: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCTTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTTCCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCA ACGAAGACTCGCTACGAGTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0328] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 141.

[0329] SEQ ID NO: 141: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGATTATGCCATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACAGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTAAC ACCCGCCTTGCACACGGACGGGATGTGCTCGGGGGCGTGGCCTATGACATATGGGGCC AGGGAACCCTGGTCACCGTTTCATCG

[0330] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 142.

[0331] SEQ ID NO: 142: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGACTATGCTATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACGGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTAAC ACCCGCCGCCTGGGGAGATCAGGAGATTTGGTACAGGATTACTGGGGCCAGGGAACCC TGGTCACCGTTTCATCG

[0332] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 143.

[0333] SEQ ID NO: 143: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGCTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGCCGGACCGCGACTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0334] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 144.

[0335] SEQ ID NO: 144: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTC ACTAAGCTCCCCGACGTTCTGCCTCTGGAATACTGGGGCCAGGGAACCCTGGTCACCGT TTCATCG

[0336] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 145.

[0337] SEQ ID NO: 145: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCATATTGGATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACCGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCA ACGAAGGTGGACGGCAGCTACGGCATCGTTACAGAACTGTGGGGCCAGGGAACCCTG GTCACCGTTTCATCG

[0338] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 146.

[0339] SEQ ID NO: 146: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGACTATGCCATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAACGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTCAT ACCCGCCGGAGCGGTACGTCTATGGCCATGGACGTGTGGGGCCAGGGAACCCTGGTCA CCGTTTCATCG

[0340] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 147.

[0341] SEQ ID NO: 147: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCCTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAGCGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG ACCAAGCTGACTATGGTTTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0342] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 148.

[0343] SEQ ID NO: 148: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTC ACTAAGCTGACTAACGAATATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0344] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 149.

[0345] SEQ ID NO: 149: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGGTTCGCCCATCCTACGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATC G

[0346] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 150.

[0347] SEQ ID NO: 150: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCCTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTG GCGAAGCCGGGTTACGAATATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0348] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 151.

[0349] SEQ ID NO: 151: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCTTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTCGGACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGCCCGGCGAAGATGTGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0350] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 152.

[0351] SEQ ID NO: 152: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCCTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCTGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGTCAAATATGGTCTACTGGGGCCAGGGAACCCTGGTCACCGTTTCATCG

[0352] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 153.

[0353] SEQ ID NO: 153: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACTGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTACG ACGAAGGTGGACGGCAGCTACGGCATCGTTACAGAACTGTGGGGCCAGGGAACCCTG GTCACCGTTTCATCG

[0354] In some embodiments, a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 154.

[0355] SEQ ID NO: 154: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCTTATTGGATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAATGGTAGTGGTGGTTCCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCT GCTGCTAGAGATCGCGAACGGGACTATTGGGGCCAGGGAACCCTGGTCACCGTTTCAT CG

[0356] In some embodiments, a recombinant nucleic acid encodes a construct for inhibiting NK cell cytotoxicity comprising (a) a NKG2A binding domain comprising a NKG2A light chain variable region (NKG2A VL) and a NKG2A heavy chain variable region (NKG2A VH); and / or

[0357] (b) a CD300a binding domain comprising a CD300a light chain variable region (CD300a VL) and a CD300a heavy chain variable region (CD300a VH). In a particular embodiment, the recombinant nucleic acid encodes a NKG2A binding domain comprising a NKG2A VL and a NKG2A VH. In another particular embodiment, the recombinant nucleic acid encodes a CD300a binding domain comprising a CD300a VL and CD300a VH.

[0358] In certain embodiments, the NKG2A VL comprises a VL CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RASENIYSYLA (SEQ ID NO: 98), a VL CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NAKTLAE (SEQ ID NO: 99), and a VL CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising QHHYGTPRT (SEQ ID NO: 100). In certain embodiments, the NKG2A VH comprises a VH CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising SYWMN (SEQ ID NO: 101), a VH CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RIDPYDSETHYAQKLQG (SEQ ID NO: 102), and a VH CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GGYDFDVGTLYWFFDV (SEQ ID NO: 103). In some embodiments, the CD300a VL comprises a VL CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RASQDISNYLN (SEQ ID NO: 104), a VL CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising YTSRLHS (SEQ ID NO: 105), and a VL CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising QQGNTLPWT (SEQ ID NO: 106). In some embodiments, the CD300a VH comprises a VH CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising SYWMQ (SEQ ID NO: 107), a VH CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising EIDPSDSYTNYNQKFKG (SEQ ID NO: 108), and a VH CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising WGMAYGTSSYWYFDV (SEQ ID NO: 109).

[0359] In particular embodiments, the NKG2A VL comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In particular embodiments, the NKG2A VL comprises or consists of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0360] SEQ ID NO: 1 (hZ270_vL): DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIK

[0361] SEQ ID NO: 2 (hZ199_vL): EIVLTQSPATLSLSPGERATLSCSASSSVSSYIYWYQQKPGQAPRLLIYLTSNLASGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQWSGNPYTFGQGTKLEIK

[0362] In particular embodiments, a nucleotide sequence encoding the NKG2A VL comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10. In particular embodiments, the nucleotide sequence encoding the NKG2A VL comprises or consists of SEQ ID NO: 9 or SEQ ID NO: 10.

[0363] SEQ ID NO: 9 (encoding hZ270_vL): GACATCCAGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCGCGTCAC CATCACCTGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAGCAGAAG CCTGGGAAAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGGGCGTGC CATCCCGCTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCGTCGCTC CAGCCGGAAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCGTACCTT CGGCGGCGGCACCAAGGTGGAGATCAAG

[0364] SEQ ID NO: 10 (encoding hZ199_vL): GAAATTGTCCTCACCCAGTCCCCAGCGACCCTGAGCCTTAGCCCTGGGGAGCGGGCTA CTCTGTCCTGCTCTGCATCGTCGTCGGTGTCCAGCTATATCTATTGGTACCAGCAGAAG CCCGGACAGGCACCGCGCCTTTTGATCTACCTGACCAGCAACCTGGCTTCTGGCATCCC GGCCAGGTTCTCTGGCTCTGGGTCCGGCACCGACTTTACACTTACCATCTCTTCTCTGG AGCCAGAAGACTTCGCGGTCTACTACTGTCAACAGTGGTCTGGCAACCCCTATACCTTC GGCCAGGGCACCAAGCTGGAGATCAAG

[0365] In particular embodiments, the NKG2A VH comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 3-8. In particular embodiments, the NKG2A VH comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 3-8.

[0366] SEQ ID NO: 3 (hZ270_vH): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSET HYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFDVWG QGTTVTVSS

[0367] SEQ ID NO: 4 (hZ270_vH_D98A): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSET HYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYAFDVGTLYWFFDVWG QGTTVTVSS

[0368] SEQ ID NO: 5 (hZ270_vH_E56A): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSAT HYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFDVWG QGTTVTVSS

[0369] SEQ ID NO: 6 (hZ270_vH_R94A): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSET HYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAAGGYDFDVGTLYWFFDVWG QGTTVTVSS

[0370] SEQ ID NO: 7 (hZ270_vH_Y53A): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPADSET HYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFDVWG QGTTVTVSS

[0371] SEQ ID NO: 8 (hZ199_VH): EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSEISSGGSYTYY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGDYPRFFDVWGQGTTVTVSS

[0372] In particular embodiments, a nucleotide sequence encoding the NKG2A VH comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 11-16. In particular embodiments, the nucleotide sequence encoding the NKG2A VH comprises or consists of any one of SEQ ID NOs: 11-16.

[0373] SEQ ID NO: 11 (encoding hZ270_vH): CAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAG GTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACTGGGTCCGCCA GGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCTATGACTCCGAGACT CACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACCTCGACCTCCA CAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGTACTACTGTGCT CGTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTTCTTTGACGTGTGGGGCCA GGGCACCACCGTGACGGTTTCTTCT

[0374] SEQ ID NO: 12 (encoding hZ270_vH_D98A): CAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAG GTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACTGGGTCCGCCA GGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCTATGACTCCGAGACT CACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACCTCGACCTCCA CAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGTACTACTGTGCT CGTGGCGGCTACGCTTTCGACGTGGGCACTCTTTATTGGTTCTTTGACGTGTGGGGCCA GGGCACCACCGTGACGGTTTCTTCT

[0375] SEQ ID NO: 13 (encoding hZ270_vH_E56A): CAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAG GTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACTGGGTCCGCCA GGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCTATGACTCCGCTACT CACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACCTCGACCTCCA CAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGTACTACTGTGCT CGTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTTCTTTGACGTGTGGGGCCA GGGCACCACCGTGACGGTTTCTTCT

[0376] SEQ ID NO: 14 (encoding hZ270_vH_R94A): CAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAG GTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACTGGGTCCGCCA GGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCTATGACTCCGAGACT CACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACCTCGACCTCCA CAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGTACTACTGTGCT GCTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTTCTTTGACGTGTGGGGCCA GGGCACCACCGTGACGGTTTCTTCT

[0377] SEQ ID NO: 15 (encoding hZ270_vH_Y53A): CAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAG GTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACTGGGTCCGCCA GGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCGCTGACTCCGAGACT CACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACCTCGACCTCCA CAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGTACTACTGTGCT CGTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTTCTTTGACGTGTGGGGCCA GGGCACCACCGTGACGGTTTCTTCT

[0378] SEQ ID NO: 16 (encoding hZ199_VH): GAAGTACAACTTGTAGAGTCTGGAGGTGGGCTGGTGAAGCCTGGGGGTAGCCTGCGCC TTAGTTGCGCGGCATCAGGATTTACGTTCTCTAGCTATGCTATGTCATGGGTGCGACAG GCTCCCGGCAAAGGCCTCGAGTGGGTCAGCGAGATCAGCTCTGGTGGTAGCTACACCT ACTACGCGGACAGCGTTAAGGGCCGCTTCACCATTAGTCGCGACAACGCCAAGAACTC CCTGTACCTGCAGATGAATTCCCTACGTGCCGAGGACACCGCCGTGTACTACTGTGCTC GCCATGGTGATTACCCCCGATTTTTTGACGTGTGGGGCCAGGGAACTACGGTAACGGTT TCTTCT

[0379] In particular embodiments, the CD300a VL comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 17. In particular embodiments, the CD300a VL comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0380] SEQ ID NO: 17 (hTX49v1_vL): DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRF SGSGSGTDYTLTISNLQPEDFATYFCQQGNTLPWTFGQGTKVEIK

[0381] In particular embodiments, a nucleotide sequence encoding the CD300a VL comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: X. In particular embodiments, the nucleotide sequence encoding the CD300a VL comprises or consists of SEQ ID NO: 19.

[0382] SEQ ID NO: 19 (encoding hTX49v1_vL): GACATCCAGATGACCCAGAGCCCGTCCTCTCTGTCCGCCTCCGTGGGGGACAGGGTCA CCATCACCTGCCGTGCCTCACAGGATATCTCGAACTACCTCAATTGGTACCAGCAGAAG CCCGGCAAAGCCCCCAAGCTGCTCATCTATTACACCTCCCGCCTTCACTCTGGTGTGCC CTCTCGCTTCTCGGGTAGTGGCTCCGGAACAGACTACACTCTGACCATTAGCAACCTGC AGCCAGAGGACTTTGCAACTTACTTCTGTCAACAGGGCAACACGCTACCGTGGACCTTC GGCCAGGGCACCAAGGTGGAGATCAAG

[0383] In particular embodiments, the CD300a VH comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 18. In particular embodiments, the CD300a VH comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0384] SEQ ID NO: 18 (hTX49v1_vH): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMQWVRQAPGQGLEWIGEIDPSDSYTN YNQKFKGRATLTVDTSTSTTYMELSSLTSEDTAVYYCARWGMAYGTSSYWYFDVWGRG TLVTVSS

[0385] In particular embodiments, a nucleotide sequence encoding the CD300a VH comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 20. In particular embodiments, the nucleotide sequence encoding the CD300a VH comprises or consists of SEQ ID NO: 20.

[0386] SEQ ID NO: 20 (encoding hTX49v1_vH): CAGGTCCAACTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCCGGGGCATCCGTAAAG GTGTCATGCAAGGCGTCTGGCTACACGTTCACCAGCTATTGGATGCAGTGGGTCCGCCA GGCCCCTGGCCAGGGCCTGGAGTGGATCGGTGAAATTGACCCGTCCGACAGCTACACC AACTACAACCAGAAATTTAAAGGCCGCGCTACACTGACCGTGGATACTTCGACCTCCA CGACCTACATGGAGCTGTCTTCTCTCACTTCCGAGGACACCGCCGTGTACTACTGTGCT CGGTGGGGAATGGCCTACGGCACCAGCTCGTATTGGTACTTCGACGTGTGGGGCCGTG GTACTTTGGTTACGGTTTCTTCT

[0387] In some embodiments, all or a portion of a nucleic acid sequence encoding a NKG2A binding domain is codon optimized to reduce or prevent undesired recombination events. In some embodiments, all or a portion of a nucleic acid sequence encoding a CD300a binding domain is codon optimized to reduce or prevent undesired recombination events. In some embodiments, all or a portion of a nucleic acid sequence encoding a NKG2A VL, a NKG2A VH, a CD300a VL, and / or a CD300a VH is codon optimized, such as to reduce or prevent undesired recombination events. In some embodiments, codon optimization encompasses replacing one or more 3-nucleotide sequences encoding for a particular amino acid with a different 3-nucleotide sequence (such as a 3-nucleotide sequence that differs from the initial 3-nucleotide sequence at the first, second, and / or third nucleotide position) encoding for the same amino acid. For example, the 3-nucleotide sequences (codons) GGA, GGG, GGT, GGC(GGU) all encode the amino acid glycine and each may be replaced with one of the remaining three in a given nucleic acid sequence in a codon optimization process.

[0388] In some embodiments, a codon optimized nucleotide sequence encoding the NKG2A binding domain has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 113. In particular embodiments, the codon optimized nucleotide sequence encoding the NKG2A binding domain comprises or consists of SEQ ID NO: 113.

[0389] SEQ ID NO: 113 (encoding codon optimized humZ270vL + Whitlow / 218 Linker + humZ270vH): GACATCCAGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCGCGTCAC CATCACCTGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAGCAGAAG CCTGGGAAAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGGGCGTGC CATCCCGCTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCGTCGCTC CAGCCGGAAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCGTACCTT CGGCGGCGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAACCTGGT TCTGGGGAGGGCTCCACTAAGGGACAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTG AAGAAGCCGGGTGCTTCCGTAAAGGTGTCATGCAAAGCATCTGGCTACACGTTCACCA GTTACTGGATGAACTGGGTCCGCCAGGCCCCTGGTCAAGGACTGGAGTGGATGGGCAG GATCGACCCCTATGACTCCGAGACTCACTACGCGCAGAAGTTGCAGGGGCGGGTTACC ATGACGACCGACACCTCGACCTCCACAGCCTACATGGAACTGCGCTCGCTCCGCAGCG ATGACACCGCCGTGTACTACTGTGCTCGTGGCGGCTACGACTTCGACGTGGGCACTCTT TATTGGTTCTTTGACGTGTGGGGCCAGGGCACCACCGTGACGGTTTCTTCT

[0390] In some embodiments, a codon optimized nucleotide sequence encoding the NKG2A VL has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 110. In particular embodiments, the codon optimized nucleotide sequence encoding the NKG2A VL comprises or consists of SEQ ID NO: 110.

[0391] SEQ ID NO: 110: GACATCCAGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCGCGTCAC CATCACCTGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAGCAGAAG CCTGGGAAAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGGGCGTGC CATCCCGCTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCGTCGCTC CAGCCGGAAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCGTACCTT CGGCGGCGGCACCAAGGTGGAGATCAAG

[0392] In some embodiments, a codon optimized nucleotide sequence encoding the NKG2A VH has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 112. In particular embodiments, the codon optimized nucleotide sequence encoding the NKG2A VH comprises or consists of SEQ ID NO: 112.

[0393] SEQ ID NO: 112: CAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAG GTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACTGGGTCCGCCA GGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCTATGACTCCGAGACT CACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACCTCGACCTCCA CAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGTACTACTGTGCT CGTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTTCTTTGACGTGTGGGGCCA GGGCACCACCGTGACGGTTTCTTCT

[0394] In some embodiments, the recombinant nucleic acid comprises a first linker (such as a first linker described herein) that links the nucleic acid encoding a NKG2A VL and the nucleic acid encoding a NKG2A VH. In other examples, the recombinant nucleic acid comprises a first linker (such as a first linker described herein) that links the nucleic acid encoding a CD300a VL and the nucleic acid encoding a CD300a VH. In some embodiments wherein the recombinant nucleic acid encodes a NKG2A binding domain and a CD300a binding domain, the recombinant nucleic acid comprises a first linker (such as a first linker described herein) that links the nucleic acid encoding a NKG2A VL and the nucleic acid encoding a NKG2A VH, and a second linker (such as a second linker described herein) that links the nucleic acid encoding the CD300a VL and the nucleic acid encoding the CD300a VH.

[0395] An NKG2A binding domain or a CD300a binding domain of the present disclosure can comprise any suitable structure, such as, but not limited to, an antibody or a fragment thereof, or a VHH. Commercially available NKG2A antibodies include, but are not limited to, those produced by R&D Systems (human NKG2A / CD159a, Catalog #: MAB1059), Creative Biolabs (human anti- NKG2A recombinant antibody, scFv fragment (HPAB-1355-FY-S(P)) (CAT#: HPAB-1355-FY- S(P)); human anti-NKG2A recombinant antibody (HPAB-1355-FY) (CAT#: HPAB-1355-FY); human anti-NKG2A recombinant antibody; Fab fragment (HPAB-1355-FY-F(E)) (CAT#: HPAB- 1355-FY-F(E)); recombinant anti-human KLRC1 antibody scFv fragment (CAT#: MOB-604- S(P))), and Miltenyl Biotec (CD159a (NKG2A) Antibody, anti-human, REAfinity™, Catalog # 130-122-329). Commercially available CD300a antibodies include, but are not limited to, those produced by R&D Systems (human CD300a / LMIR1 antibody, Catalog #s: MAB2640, MAB26401) and ThermoFisher Scientific (CD300a monoclonal antibody (MEM-260), PE, Catalog # A15778; CD300a monoclonal antibody (7H8E4), Catalog # MA5-38479; CD300a monoclonal antibody (2F9C5), Catalog # 67242-1-IG). Exemplary binding domains useful in the disclosed embodiments can also include a cytokine, a ligand, or a peptide (such as an adnectin or a designed ankyrin repeat protein (DARPin) (See, e.g., Rafiq et al., Nat Rev Clin Oncol.2020;17:147–167).

[0396] In some embodiments wherein the NKG2A binding domain is an scFv, the NKG2A scFv comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 118. In particular embodiments, the NKG2A scFv comprises or consists of the amino acid sequence of SEQ ID NO: 118.

[0397] SEQ ID NO: 118: DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIKGSTSGSGKPGSGEGSTK GQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSE THYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFDVW GQGTTVTVSS

[0398] In some embodiments wherein the NKG2A binding domain is an scFv, a nucleotide sequence encoding the NKG2A scFv comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 113 or 120. In particular embodiments, the nucleotide sequence encoding the NKG2A scFv comprises or consists of any one of SEQ ID NOs: 113 or 120.

[0399] SEQ ID NO: 120: GACATCCAGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCGCGTCAC CATCACCTGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAGCAGAAG CCTGGGAAAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGGGCGTGC CATCCCGCTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCGTCGCTC CAGCCGGAAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCGTACCTT CGGCGGCGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAACCTGGT TCTGGGGAGGGCTCCACTAAGGGACAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTG AAGAAGCCGGGTGCTTCCGTAAAGGTGTCATGCAAAGCATCTGGCTACACGTTCACCA GTTACTGGATGAACTGGGTCCGCCAGGCCCCTGGTCAAGGACTGGAGTGGATGGGCAG GATCGACCCCTATGACTCCGAGACTCACTACGCGCAGAAGTTGCAGGGGCGGGTTACC ATGACGACCGACACCTCGACCTCCACAGCCTACATGGAACTGCGCTCGCTCCGCAGCG ATGACACCGCCGTGTACTACTGTGCTCGTGGCGGCTACGACTTCGACGTGGGCACTCTT TATTGGTTCTTTGACGTGTGGGGCCAGGGCACCACCGTGACGGTTTCTTCT

[0400] In some embodiments wherein the CD300a binding domain is an scFv, the CD300a scFv comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 119. In particular embodiments, the CD300a scFv comprises or consists of the amino acid sequence of SEQ ID NO: 119.

[0401] SEQ ID NO: 119: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRF SGSGSGTDYTLTISNLQPEDFATYFCQQGNTLPWTFGQGTKVEIKGSTSGSGKPGSGEGSTK GQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMQWVRQAPGQGLEWIGEIDPSDSYT NYNQKFKGRATLTVDTSTSTTYMELSSLTSEDTAVYYCARWGMAYGTSSYWYFDVWGR GTLVTVSS

[0402] In some embodiments wherein the CD300a binding domain is an scFv, a nucleotide sequence encoding the CD300a scFv comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 121. In particular embodiments, the nucleotide sequence encoding the CD300a scFv comprises or consists of SEQ ID NO: 121.

[0403] SEQ ID NO: 121: GACATCCAGATGACCCAGAGCCCGTCCTCTCTGTCCGCCTCCGTGGGGGACAGGGTCA CCATCACCTGCCGTGCCTCACAGGATATCTCGAACTACCTCAATTGGTACCAGCAGAAG CCCGGCAAAGCCCCCAAGCTGCTCATCTATTACACCTCCCGCCTTCACTCTGGTGTGCC CTCTCGCTTCTCGGGTAGTGGCTCCGGAACAGACTACACTCTGACCATTAGCAACCTGC AGCCAGAGGACTTTGCAACTTACTTCTGTCAACAGGGCAACACGCTACCGTGGACCTTC GGCCAGGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAACCTGGTT CTGGGGAGGGCTCCACTAAGGGACAGGTCCAACTGGTGCAGAGCGGCGCGGAGGTGA AGAAGCCCGGGGCATCCGTAAAGGTGTCATGCAAGGCGTCTGGCTACACGTTCACCAG CTATTGGATGCAGTGGGTCCGCCAGGCCCCTGGCCAGGGCCTGGAGTGGATCGGTGAA ATTGACCCGTCCGACAGCTACACCAACTACAACCAGAAATTTAAAGGCCGCGCTACAC TGACCGTGGATACTTCGACCTCCACGACCTACATGGAGCTGTCTTCTCTCACTTCCGAG GACACCGCCGTGTACTACTGTGCTCGGTGGGGAATGGCCTACGGCACCAGCTCGTATT GGTACTTCGACGTGTGGGGCCGTGGTACTTTGGTTACGGTTTCTTCT

[0404] A disclosed recombinant nucleic acid comprising a CD300a scFv and / or a NKG2A scFv may encode the NKG2A VL and VH and the CD300a VL and VH in any suitable order. Thus, in some embodiments, the nucleotide sequence encoding the NKG2A VL is located 5’ of the nucleotide sequence encoding the NKG2A VH. In other embodiments, the nucleotide sequence encoding the NKG2A VL is located 3’ of the nucleotide sequence encoding the NKG2A VH. Similarly, in some embodiments, the nucleotide sequence encoding the CD300a VL is located 5’ of the nucleotide sequence encoding the CD300a VH. In other embodiments, the nucleotide sequence encoding the CD300a VL is located 3’ of the nucleotide sequence encoding the CD300a VH. In some embodiments comprising both a CD300a scFv and a NKG2A scFv, the nucleotide sequence encoding the NKG2A scFv is located 3’ of the nucleotide sequence encoding the CD300a scFv. In other embodiments comprising both a CD300a scFv and a NKG2A scFv, the nucleotide sequence encoding the NKG2A scFv is located 5’ of the nucleotide sequence encoding the CD300a scFv. In some embodiments comprising both a CD300a scFv and a NKG2A scFv, the recombinant nucleic acid encodes a third linker that links the NKG2A scFv and the CD300a scFv. B. Linkers

[0405] Embodiments of a recombinant nucleic acid disclosed herein may include one or more linkers, such as 1, 2, 3, 4, 5, 6, 7, or more linkers, or no linker. As described herein, a disclosed recombinant nucleic acid (such as a recombinant nucleic acid that comprises a NKG2A binding domain and does not comprise a CD300a binding domain) can comprise a first linker that links a nucleic acid encoding a NKG2A VL and a nucleic acid encoding a NKG2A VH. In other embodiments, a disclosed recombinant nucleic acid (such as a recombinant nucleic acid that comprises a CD300a binding domain and does not comprise a NKG2A binding domain) can comprise a first linker that links the nucleic acid encoding a CD300a VL and the nucleic acid encoding a CD300a VH. In embodiments wherein the recombinant nucleic acid encodes a NKG2A binding domain and a CD300a binding domain, the recombinant nucleic acid can comprise a first linker that links the nucleic acid encoding a NKG2A VL and the nucleic acid encoding a NKG2A VH, and a second linker that links the nucleic acid encoding the CD300a VL and the nucleic acid encoding the CD300a VH. Such a “first linker” may be 5’ or 3’ of such a “second linker” in the recombinant nucleic acid, such that the NKG2A scFv is encoded 5’ or 3’ of the CD300a scFv in the recombinant nucleic acid. Further, embodiments of a disclosed recombinant nucleic acid that encodes a NKG2A binding domain (such as a NKG2A scFv) and a CD300a binding domain (such as a CD300a scFv) can comprise a third linker that links the NKG2A binding domain and the CD300a binding domain. In particular embodiments, a first linker, a second linker, and / or a third linker comprise a peptide linker (such as a cleavable peptide linker), a glycine-serine linker (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS, SEQ ID NO: 30), or a Whitlow / 218 linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 31). The first, second, and third linkers may be the same linker type, different linker types, or any combination thereof.

[0406] A first linker, a second linker, and / or a third linker may be any suitable linker. In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (-S-) and hydroxylamino (-O-N(H)-) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide, and ether groups. In certain embodiments, the linear group comprises one or more alkyl groups. In some embodiments, the one or more linkers comprise one or more amino acids (e.g., a peptide linker). Such linkers can be 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, 30, 35, 40 or more amino acids in length. An illustrative peptide linker has a length from 3 amino acids (AA) to 90 AA, from 3 AA to 80 AA, from 3 AA to 70 AA, from 3 AA to 60 AA, from 3 AA to 50 AA, from 3 AA to 40 AA, from 3 AA to 30 AA, from 3 AA to 20 AA, from 3 AA to 10 AA. For example, the linker can have a length from 3 AA to 5 AA, from 5 AA to 10 AA, from 10 AA to 15 AA, from 15 AA to 20 AA, from 20 AA to 25 AA, from 25 AA to 30 AA, from 30 AA to 35 AA, from 35 AA to 40 AA, from 40 AA to 50 AA, from 50 AA to 60 AA, from 60 AA to 70 AA, from 70 AA to 80 AA, from 80 AA to 90 AA, or from 90 AA to 100 AA. In some embodiments, the peptide linker is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in length.

[0407] A peptide linker can comprise any of a variety of sequences. For example, a peptide linker can comprise one or more serine (S), glycine (G), and / or threonine (T) residues. Glycine may impart flexibility in a given linker, whereas serine and / or threonine may improve solubility. In some embodiments, the linker is a glycine-serine linker. In particular embodiments, the glycine-serine linker comprises (Glym-Ser)n, where m is 1 to 10 (such as 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 7, 3 to 6, 3 to 5, or 3 to 4) and n is 1 to 10 (such as 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3). In specific, non-limiting embodiments, the first linker, the second linker, and / or the third linker is a glycine-serine linker that comprises (Glym-Ser)n, wherein m is 3 to 6 and n is 1 to 10. In other specific, non-liming embodiments, m=4 and n=5.

[0408] In some embodiments wherein the first, second, and / or third linker is a cleavable peptide, the first, second, and / or third linker is a self-cleaving peptide. In particular embodiments, the self- cleaving peptide is a T2A peptide (e.g., GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 32), a P2A peptide (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 33), an E2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 122), or an F2A peptide (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 29). In some embodiments, the self-cleaving peptide optionally comprises the amino acids glycine-serine-glycine (GSG) at an N-terminus.

[0409] In particular embodiments, the first linker, the second linker, and / or the third linker comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 29-33 and 122. In other particular embodiments, the first linker, the second linker, and / or the third linker comprises any one of SEQ ID NOs: 29-33 and 122. In other particular embodiments, the first linker, the second linker, and / or the third linker consists of any one of SEQ ID NOs: 29-33 and 122.

[0410] In some embodiments, a nucleotide sequence encoding the first linker, the second linker, and / or the third linker comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 34-37, 111, 115, or 116. In particular embodiments, a nucleotide sequence encoding the first linker, the second linker, and / or the third linker comprises any one of SEQ ID NOs: 34-37, 111, 115, or 116. In particular embodiments, a nucleotide sequence encoding the first linker, the second linker, and / or the third linker consists of any one of SEQ ID NOs: 34-37, 111, 115, or 116.

[0411] SEQ ID NO: 34 (encoding the glycine-serine linker of SEQ ID NO: 30): GGTGGAGGAGGTTCTGGTGGTGGAGGATCAGGAGGCGGTGGAAGCGGAGGTGGAGGA TCTGGTGGAGGTGGATCA

[0412] SEQ ID NO: 35 (encoding the Whitlow / 218 linker of SEQ ID NO: 31): GGATCGACCTCAGGCTCTGGTAAACCTGGTAGTGGGGAGGGCTCCACCAAGGGA

[0413] SEQ ID NO: 36 (encoding the T2A linker of SEQ ID NO: 32): GGTTCTGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCG GCCCT

[0414] SEQ ID NO: 37 (encoding the P2A linker of SEQ ID NO: 33): GGATCCGGAGCCACCAACTTTAGCCTGCTCAAACAAGCCGGCGACGTGGAAGAGAACC CCGGACCT

[0415] SEQ ID NO: 111 (a codon optimized sequence encoding the Whitlow / 218 linker): GGATCGACCTCAGGCTCTGGTAAACCTGGTTCTGGGGAGGGCTCCACTAAGGGA

[0416] SEQ ID NO: 115 (a codon optimized sequence encoding a T2A linker): GGTAGTGGCGAAGGCAGAGGCTCATTGCTCACTTGTGGCGATGTGGAGGAAAACCCTG GGCCT

[0417] SEQ ID NO: 116 (encoding a T2A linker): GGAAGCGGACAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCA ACCCTGGACCT C. Signal Peptides

[0418] In some embodiments, a disclosed recombinant nucleic acid comprises a signal peptide, wherein the signal peptide is a cell surface expression signal peptide that directs the protein product of the recombinant nucleic acid to the surface of a cell. Any suitable signal peptide that directs the protein product of the recombinant nucleic acid to the surface of a cell can be of use in the disclosed embodiments. In some embodiments, the signal peptide is a GMCSF signal peptide. In some such embodiments, the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to the amino acid sequence MVLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 38). In particular embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 38. In some embodiments of a disclosed recombinant nucleic acid, a nucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 39-41. In particular embodiments, a nucleotide sequence encoding the signal peptide comprises or consists of any one of SEQ ID NOs: 39-41. In other particular embodiments, a nucleotide sequence encoding the signal peptide comprises or consists of any one of SEQ ID NOs: 39-41.

[0419] SEQ ID NO: 39: ATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCTCATCCTGCGTTCCTGCTG ATTCCC

[0420] SEQ ID NO: 40 (a codon optimized sequence encoding the signal peptide of SEQ ID NO: 38): ATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCGCACCCGGCTTTTCTCTTG ATCCCC

[0421] SEQ ID NO: 41: ATGGTGCTCTTGGTGACTTCCCTTCTGCTGTGCGAGCTGCCCCACCCTGCGTTCCTGCTC ATCCCT

[0422] In some embodiments, the signal peptide is a CD8 signal peptide. In some such embodiments, the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 93). In particular embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 93. In some embodiments of a disclosed recombinant nucleic acid, a nucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 114. In particular embodiments, a nucleotide sequence encoding the signal peptide comprises SEQ ID NO: 114. In other particular embodiments, a nucleotide sequence encoding the signal peptide consists of SEQ ID NO: 114.

[0423] SEQ ID NO: 114 (a codon optimized sequence encoding the signal peptide of SEQ ID NO: 93): GCAGCCGCAACAACGACACCTGCCCCACGCCCGCCAACTCCAGCTCCAACGATTGCGT CCCAGCCCCTCAGCTTGAGACCGGAAGCCTGTCGACCCGCAGCTGGCGGGGCCGTCCA TACTCGCGGGCTGGACTTTGCGTGTGACATCTACATCTGGGCTCCTTTGGCAGGCACCT GCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAAC

[0424] In some embodiments, a nucleotide sequence encoding the signal peptide is located 5’ of a nucleotide sequence encoding a NKG2A binding. In some embodiments, a nucleotide sequence encoding the signal peptide is located 5’ of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, a nucleotide sequence encoding the signal peptide is located 5’ of both a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain. D. Hinge Regions

[0425] Embodiments of the disclosed recombinant nucleic acids may include one or more extracellular hinge regions, which may or may not share sequence homology with immunoglobulin hinge regions and which provide flexibility to protein products of the recombinant nucleic acids. In some embodiments, a hinge region separates a binding domain (such as a CD300a binding domain (such as a disclosed CD300a VHH or CD300a scFv) or a NKG2A binding domain (such as a disclosed NKG2A scFv)) from a transmembrane domain. In some embodiments, the disclosed recombinant nucleic acids do not encode a hinge region. In some embodiments, a hinge region comprises a CD8 hinge, an hlgG1 hinge, an hlgG2 hinge, an hlgG3 hinge, a FACD hinge, or any combination thereof. In some embodiments, the hinge region comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 43-48. In particular embodiments, the hinge region comprises any one of SEQ ID NOs: 43-48. In particular embodiments, the hinge region consists of any one of SEQ ID NOs: 43-48.

[0426] SEQ ID NO: 43 (spacer + hIgG1 hinge): AAATTTPEPKSCDKTHTCP

[0427] SEQ ID NO: 44 (spacer + hIgG2 hinge): AAATTTPERKCCVECPPCP

[0428] SEQ ID NO: 45 (spacer + hIgG3 hinge): AAATTTPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPP CPRCP

[0429] SEQ ID NO: 46 (IGHG hinge): AAATTTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVD

[0430] SEQ ID NO: 47 (spacer + CD8 hinge): AAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0431] SEQ ID NO: 48 (spacer + FACD): AAATTTPFACD

[0432] In some embodiments of a disclosed recombinant nucleic acid, a nucleotide sequence encoding the hinge region comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 50-55. In particular embodiments, a nucleotide sequence encoding the hinge region comprises any one of SEQ ID NOs: 50-55. In particular embodiments, a nucleotide sequence encoding the hinge region consists of any one of SEQ ID NOs: 50-55.

[0433] SEQ ID NO: 50 (encoding spacer + hIgG1 hinge): GCAGCCGCAACAACGACACCTGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCT

[0434] SEQ ID NO: 51 (encoding spacer + hIgG2 hinge): GCAGCCGCAACAACGACACCTGAACGAAAATGCTGTGTGGAATGCCCACCCTGTCCT

[0435] SEQ ID NO: 52 (encoding spacer + hIgG3 hinge): GCAGCCGCAACAACGACACCTGAACTGAAGACACCACTCGGCGACACTACACACACTT GCCCCAGGTGCCCTGAACCAAAGTCATGCGATACCCCTCCTCCATGTCCACGGTGCCCC GAACCAAAATCTTGTGACACACCTCCTCCATGCCCAAGATGTCCGGAGCCCAAATCAT GTGATACCCCACCTCCTTGCCCAAGGTGTCCA

[0436] SEQ ID NO: 53 (encoding IGHG hinge): GCAGCCGCAACAACGACAGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCTCCAT GTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAG GACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCC ACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGC CAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTG ACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACA AGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGA ACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCC CTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCA ATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTC ATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTG TTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGA GCCTGTCTCCTGGCAAAGTGGAC

[0437] SEQ ID NO: 54 (encoding spacer + CD8 hinge): GCAGCCGCAACAACGACACCTGCTCCTAGACCTCCAACACCAGCTCCAACGATTGCGT CCCAGCCTCTCAGCTTGAGACCGGAAGCCTGTCGACCCGCAGCTGGCGGAGCCGTCCA CACAAGAGGACTGGACTTTGCGTGTGAC

[0438] SEQ ID NO: 5 (encoding spacer + FACD): GCAGCCGCAACAACGACACCTTTTGCGTGTGAC

[0439] In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the hinge region is located 3’ of the nucleotide sequence encoding the signal peptide. In other embodiments, the nucleotide sequence encoding the hinge region is located 3’ of a nucleotide sequence encoding a NKG2A binding domain. In other embodiments, the nucleotide sequence encoding the hinge region is located 3’ of a nucleotide sequence encoding a CD300a binding domain. In other embodiments, the nucleotide sequence encoding the hinge region is located 3’ of a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located 3’ of the nucleotide sequence encoding the signal peptide, and 3’ of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located 3’ of the nucleotide sequence encoding the signal peptide, and 3’ of a nucleotide sequence encoding a NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located 3’ of the nucleotide sequence encoding the signal peptide, 3’ of a nucleotide sequence encoding a NKG2A binding domain, and 3’ of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located (a) 3’ of a nucleotide sequence encoding a signal peptide; (b) 3’ of a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain; or (c) both (a) and (b). E. Transmembrane Domains

[0440] Some embodiments of a disclosed recombinant nucleic acid comprise one or more transmembrane domains. Any suitable transmembrane domain can be of use in the present disclosure. In some embodiments, a transmembrane domain is a human transmembrane domain or a murine transmembrane domain. In particular embodiments, the transmembrane domain comprises or consists of a CD8, a CD80, an mCD80, an ITGA, an HLA-B57, a proCAR-4, a CD28, a KIR2DL1, a PDGFRB, or a CD86 transmembrane domain.

[0441] In some embodiments, the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 67-76 or 96. In particular embodiments, the transmembrane domain comprises any one of SEQ ID NOs: 67-76 or 96. In other particular embodiments, the transmembrane domain consists of any one of SEQ ID NOs: 67-76 or 96.

[0442] SEQ ID NO: 67 (a CD8 TM): IYIWAPLAGTCGVLLLSLVITLYCN

[0443] SEQ ID NO: 68 (CD28 TM): FWVLVVVGGVLACYSLLVTVAFIIFWVR

[0444] SEQ ID NO: 69 (mCD80 TM): PPEDPPDSKNTLVLFGAGFGAVITVVVIVVII

[0445] SEQ ID NO: 70 (KIR2DL1 TM): PRHLHILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQ

[0446] SEQ ID NO: 71 (PDGFRB TM): AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0447] SEQ ID NO: 72 (proCAR-4 TM): PFWLLVALLALLAVIAALLAAIFALLWVR

[0448] SEQ ID NO: 73 (ITGA TM): VPLWVILLSAFAGLLLLMLLILALW

[0449] SEQ ID NO: 74 (CD80 TM): KQEHFPDNLLPSWAITLISVNGIFVICCL

[0450] SEQ ID NO: 75 (CD86 TM): PPDHIPWITAVLPTVIICVMVFCLILW

[0451] SEQ ID NO: 76 (HLA-B57 TM): VGIVAGLAVLAVVVIGAVVAAVMCR

[0452] SEQ ID NO: 96 (another CD8 TM domain): PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCN

[0453] In some embodiments of a disclosed recombinant nucleic acid, a nucleotide sequence encoding the transmembrane domain comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 56-65. In particular embodiments, the nucleotide sequence encoding the transmembrane domain comprises any one of SEQ ID NOs: 56-65. In other particular embodiments, the nucleotide sequence encoding the transmembrane domain consists of any one of SEQ ID NOs: 56-65.

[0454] SEQ ID NO: 56 (encoding the CD8 TM of SEQ ID NO: 67): ATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTGCTGCTGTCCCTGGTGAT CACCCTGTACTGTAAC

[0455] SEQ ID NO: 57 (encoding CD28 TM): TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGT GGCCTTTATTATTTTCTGGGTGAGG

[0456] SEQ ID NO: 58 (encoding mCD80 TM): CCTCCTGAGGACCCTCCAGACAGCAAGAATACCCTGGTGCTGTTCGGAGCCGGATTCG GCGCTGTGATTACCGTGGTGGTCATCGTCGTGATCATC

[0457] SEQ ID NO: 59 (encoding KIR2DL1 TM): CCCCGACACCTGCACATTCTGATTGGGACCTCAGTGGTCATCATCCTCTTCATCCTCCTC TTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCGGTAATGGACCAA

[0458] SEQ ID NO: 60 (encoding PDGFRB TM): GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGG TGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCC TCATCATGCTTTGGCAGAAGAAGCCACGT

[0459] SEQ ID NO: 61 (encoding proCAR-4 TM): CCTTTTTGGCTTTTAGTCGCCTTATTGGCGCTGCTTGCTGTAATCGCCGCATTATTAGCA GCTATCTTTGCATTGCTGTGGGTGAGG

[0460] SEQ ID NO: 62 (encoding ITGA TM): GTGCCATTATGGGTCATCCTGCTGAGTGCTTTTGCCGGATTGTTGCTGTTAATGCTGCTC ATTTTAGCACTGTGG

[0461] SEQ ID NO: 63 (encoding CD80 TM): AAGCAGGAGCACTTCCCCGACAACCTGCTGCCTTCTTGGGCCATCACTCTCATCTCCGT GAATGGCATCTTCGTCATTTGTTGCCTG

[0462] SEQ ID NO: 64 (encoding CD86 TM): CCTCCCGACCATATCCCGTGGATTACAGCCGTGCTGCCAACCGTCATCATCTGCGTGAT GGTGTTCTGCCTGATTCTCTGG

[0463] SEQ ID NO: 65 (encoding HLA-B57 TM): GTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGC TGCTGTGATGTGTAGG

[0464] In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a signal peptide. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a spacer. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a hinge region. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a signal peptide, and 3’ of a nucleotide sequence encoding a CD300a binding domain. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a signal peptide, 3’ of a nucleotide sequence encoding a CD300a binding domain, and 3’ of a nucleotide sequence encoding a spacer. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a signal peptide, and 3’ of a nucleotide sequence encoding a NKG2A binding domain. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the transmembrane domain is located 3’ of a nucleotide sequence encoding a signal peptide, 3’ of a nucleotide sequence encoding a NKG2A binding domain, and 3’ of a nucleotide sequence encoding a spacer. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the transmembrane domain is located (a) 3’ of a nucleotide sequence encoding a signal peptide, (b) 3’ of a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain, (c) 3’ of a nucleotide sequence encoding a hinge region, or (d) any combination of (a)-(c). F. Cytoplasmic Domains

[0465] Embodiments of a disclosed recombinant nucleic acid can comprise one or more cytoplasmic domains. wherein the cytoplasmic domain is a human cytoplasmic domain or a murine cytoplasmic domain. Any suitable cytoplasmic domain can be of use in the disclosed embodiments. In some embodiments, the cytoplasmic domain comprises or consists of a CD8v2, a CD8v1, a mCD80, a CD80, a CD86, or an HLA-B57 cytoplasmic domain. In some embodiments, the cytoplasmic domain comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 83-88. In particular embodiments, the cytoplasmic domain comprises any one of SEQ ID NOs: 83-88. In other particular embodiments, the cytoplasmic domain consists of any one of SEQ ID NOs: 83-88.

[0466] SEQ ID NO: 83 (CD8v2 cyt): HRNRRRVCKCPRPVVKSGDKPSLSARYV

[0467] SEQ ID NO: 84 (CD8v1 cyt): HRNRRRVCKCPRPVV

[0468] SEQ ID NO: 85 (mCD80 cyt): KCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFL

[0469] SEQ ID NO: 86 (CD80 cyt): TYCFAPRCRERRRNERLRRESVRPV

[0470] SEQ ID NO: 87 (CD86 cyt): KWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF

[0471] SEQ ID NO: 88 (HLA-B57 cyt): RKSSGGKGGSYSQAACSDSAQGSDVSLTA

[0472] In some embodiments of a disclosed recombinant nucleic acid, a nucleotide sequence encoding the cytoplasmic domain comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 77-82. In particular embodiments, a nucleotide sequence encoding the cytoplasmic domain comprises any one of SEQ ID NOs: 77-82. In other particular embodiments, a nucleotide sequence encoding the cytoplasmic domain consists of any one of SEQ ID NOs: 77-82.

[0473] SEQ ID NO: 77 (encoding CD8v2 cyt): CATCGCAATAGGCGGAGAGTCTGCAAATGCCCTCGGCCTGTTGTCAAGTCAGGAGACA AGCCAAGCCTCAGCGCACGCTATGTC

[0474] SEQ ID NO: 78 (encoding CD8v1 cyt): CACCGGAACAGGCGGAGAGTGTGCAAGTGCCCTAGACCTGTGGTT

[0475] SEQ ID NO: 79 (encoding mCD80 cyt): AAGTGTTTCTGCAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACA AACAACAGCCTGACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0476] SEQ ID NO: 80 (encoding CD80 cyt): ACCTACTGCTTTGCTCCGAGGTGTCGAGAACGGCGTCGCAACGAGCGCCTGAGGAGAG AGTCTGTCCGCCCAGTG

[0477] SEQ ID NO: 81 (encoding CD86 cyt): AAATGGAAGAAGAAGAAGCGCCCCCGCAACAGCTACAAGTGCGGCACCAACACGATG GAGCGCGAGGAGAGCGAACAGACTAAGAAAAGGGAGAAGATCCACATCCCTGAGCGG TCCGACGAGGCTCAGCGTGTTTTCAAGTCGTCCAAGACCTCCTCTTGTGATAAATCTGA CACCTGCTTT

[0478] SEQ ID NO: 82: AGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTGCAGCGACAGT GCCCAGGGCTCTGATGTGTCTCTCACAGCT

[0479] In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the cytoplasmic domain is located 3’ of a nucleotide sequence encoding a signal peptide. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3’ of a nucleotide sequence encoding a NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3’ of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3’ of a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3’ of a nucleotide sequence encoding a hinge region. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3’ of a nucleotide sequence encoding a transmembrane domain. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the cytoplasmic domain is located (a) 3’ of a nucleotide sequence encoding a signal peptide, (b) 3’ of a nucleotide sequence encoding a NKG2A binding domain and of a nucleotide sequence encoding a CD300a binding domain, (c) 3’ of a nucleotide sequence encoding a hinge region, (d) 3’ of a nucleotide sequence encoding a transmembrane domain, or (e) any combination of (a)-(d). G. Exemplary Trans Antigen Signaling Receptors (TASRs)

[0480] The present disclosure discloses a novel class of genetic constructs, “TASRs” (Trans Antigen Signaling Receptors) that, when expressed on the surface of a mammalian cell, can enhance the persistence of said cell by reducing or preventing its rejection by, e.g., autologous NK cells. TASRs are modular constructs comprising the following domains or functional parts: 1) a signal peptide to present the molecule to the cell surface, 2) a binding domain that binds to an inhibitory receptor expressed on NK cells, for example a VHH (such as a CD300a VHH disclosed herein), or a single chain variable fragment (scFv) that comprises a variable light / heavy chain linked to a variable heavy / light chain by a linker, 3) an optional extracellular hinge region, 4) a transmembrane region, and / or 5) an optional cytoplasmic region. TASRs disclosed herein can bind inhibitory receptors on an NK cell, e.g., NKG2A or CD300a. The present disclosure and Examples provided herein demonstrate certain TASR designs having surprising efficacy in reducing or preventing immune therapy cell destruction by, e.g., NK cells, although the disclosure is not limited to the particular designs examined within the Examples section.

[0481] In some embodiments, a recombinant nucleic acid disclosed herein encodes one or more components of such a TASR. In some embodiments, the TASR comprises a NKG2A binding domain (an “NKG2A TASR”), such as any suitable NKG2A binding domain, such as an NKG2A binding domain described herein. In some embodiments, the TASR comprises a CD300a binding domain (a “CD300a TASR”), such as any suitable CD300a binding domain, such as a CD300a binding domain described herein. In some embodiments, the TASR is defined by the formula:

[0482] SP-VL-Linker-VH-Hinge-TM-Cyt or SP-VH-Linker-VL-Hinge-TM-Cyt; wherein:

[0483] “SP” represents an optional signal peptide,

[0484] “VL” represents a light chain variable region (such as a NKG2A VL or a CD300a VL)

[0485] “Linker” represents a linker;

[0486] “VH” represent a heavy chain variable region (such as a NKG2A VH or a CD300a VH);

[0487] “Spacer” represents a spacer;

[0488] “Hinge” represents an optional hinge region;

[0489] “TM” represents a transmembrane domain; and

[0490] “Cyt” represents an optional cytoplasmic domain.

[0491] In some embodiments, the TASR is defined by the formula:

[0492] SP-VL-Linker-VH-Spacer-TM-Cyt or SP-VH-Linker-VL-Spacer-TM-Cyt.

[0493] In some embodiments, the TASR is defined by the formula:

[0494] SP-VHH-Spacer-TM-Cyt (such as SP-CD300a VHH-Spacer-TM-Cyt).

[0495] In some embodiments, the TASR is defined by the formula:

[0496] SP-VHH-Hinge-TM-Cyt (such as SP-CD300a VHH-Hinge-TM-Cyt).

[0497] In some embodiments, the TASR is defined by the formula:

[0498] SP-VHH-TM-Cyt (such as SP-CD300a VHH-TM-Cyt).

[0499] In some embodiments of a disclosed nucleic acid, the recombinant nucleic acid encodes a spacer. In some embodiments, a spacer of a TASR disclosed herein comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 42 (AAATTTP). In particular embodiments, the spacer comprises or consists of SEQ ID NO: 42. In some embodiments, a recombinant nucleic acid encoding a spacer (of a TASR disclosed herein) comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 49 (GCAGCCGCAACAACGACACCT). In some embodiments, the nucleotide sequence encoding the spacer comprises or consists of SEQ ID NO: 49. In some embodiments of a disclosed recombinant nucleic acid, the nucleotide sequence encoding the spacer is located (a) 3’ of the nucleotide sequence encoding the signal peptide; (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain; or (c) both (a) and (b). In some embodiments wherein the TASR comprises a spacer, the TASR does not further comprise a hinge.

[0500] FIGS.1, 31, 32, and 45 provide diagrams of exemplary formulas for TASRs of use herein. A TASR described herein (such as a TASR of FIGS. 1, 31, 32, or 45) can comprise any suitable signal peptide, linker, spacer, hinge, transmembrane domain, or cytoplasmic domain, or combination thereof, such as any of the signal peptides, linkers, spacers, hinges, transmembrane domains, or cytoplasmic domains described in detail herein. In particular embodiments, the linker is a Whitlow / 218 linker as described herein (such as the linker of SEQ ID NO: 31).

[0501] In some embodiments, a CD300a TASR comprises a CD300a VHH, such as a CD300a VHH having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 155-172. In some embodiments, such a CD300a TASR further comprises a signal peptide that directs the TASR (such as a protein product of a disclosed recombinant nucleic acid encoding a TASR comprising a CD300a VHH) to the surface of a cell, such as any of the signal peptides disclosed herein. In some embodiments, the signal peptide is a CD8 signal peptide. In some embodiments, such a CD300a TASR further comprises a transmembrane domain, such as any suitable transmembrane domain, such as any transmembrane domain disclosed herein. In particular embodiments, the transmembrane domain is a CD8 transmembrane domain or a mCD80 transmembrane domain. In particular embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, such a CD300a TASR further comprises a cytoplasmic domain, such as any suitable cytoplasmic domain, such as any cytoplasmic domain disclosed herein. In particular embodiments, the transmembrane domain is a mCD80 cytoplasmic domain. In some embodiments, such a CD300a TASR further comprises a spacer, such as a spacer of SEQ ID NO: 42. In such embodiments, the spacer is located between the CD300a VHH and a transmembrane domain in the TASR. In particular embodiments, the hinge region is a CD8 hinge region. In some embodiments, such a CD300a TASR further comprises a hinge region, such as any of the hinge regions disclosed herein. In such embodiments, the hinge region is located between the CD300a VHH and a transmembrane domain in the TASR. In particular embodiments, the hinge region is a CD8 hinge region. In some embodiments, such a CD300a TASR does not comprise a hinge region.

[0502] In particular embodiments, a CD300a TASR (e.g., a CD300a TASR encoded by a disclosed recombinant nucleic acid) comprises a signal peptide, a CD300a VHH (such as a CD300a VHH having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs 155-172), a spacer, a transmembrane domain, and a cytoplasmic domain, optionally further comprising a hinge region separating the CD300a VHH or the spacer and the transmembrane domain. In particular embodiments, the CD300a TASR (e.g., as encoded by a disclosed recombinant nucleic acid) comprises a CD8 signal peptide (such as a CD8 signal peptide having at least 80%, at least 85%, 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 100% sequence identity to SEQ ID NO: 93), a CD300a VHH (such as a CD300a VHH having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs 155-172), a spacer having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 42, a CD8 transmembrane domain (such as a transmembrane domain having at least 80%, at least 85%, 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 100% sequence identity to SEQ ID NO: 67), and a mCD80 cytoplasmic domain (such as a cytoplasmic domain having at least 80%, at least 85%, 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 100% sequence identity to SEQ ID NO: 85). In a particular embodiment, the CD300a TASR (e.g., as encoded by a disclosed recombinant nucleic acid) comprises the CD8 signal peptide of SEQ ID NO: 93, a CD300a VHH of any one of SEQ ID NOs 155-172, the spacer of SEQ ID NO: 42, the CD8 transmembrane domain of SEQ ID NO: 67, and the mCD80 cytoplasmic domain of SEQ ID NO: 85.

[0503] In particular embodiments, a recombinant nucleic acid encoding a CD300a TASR encodes an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 220-237. In particular embodiments, the recombinant nucleic acid encoding the CD300a TASR encodes an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 220-237.

[0504] SEQ ID NO: 220: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKPGEDVYWGQGTLVTVSSAAA TTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQ TVFL

[0505] SEQ ID NO: 221: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLSQFASWGQGTLVTVSSAAAT TTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0506] SEQ ID NO: 222: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPRSGWGLWGQGTLVTVSSAA ATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAE QTVFL

[0507] SEQ ID NO: 223: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKTRYESWGQGTLVTVSSAAATT TPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0508] SEQ ID NO: 224: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTRLAHGRDVLGGVAYDIWGQGT LVTVSSAAATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLT FGPEEALAEQTVFL

[0509] SEQ ID NO: 225: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTRRLGRSGDLVQDYWGQGTLVT VSSAAATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPE EALAEQTVFL

[0510] SEQ ID NO: 226: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPDRDYWGQGTLVTVSSAAAT TTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0511] SEQ ID NO: 227: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLPDVLPLEYWGQGTLVTVSSA AATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEAL AEQTVFL

[0512] SEQ ID NO: 228: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKVDGSYGIVTELWGQGTLVTVS SAAATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEE ALAEQTVFL

[0513] SEQ ID NO: 229: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKQREWVSAINGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCHTRRSGTSMAMDVWGQGTLVTVS SAAATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEE ALAEQTVFL

[0514] SEQ ID NO: 230: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKLTMVYWGQGTLVTVSSAAAT TTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0515] SEQ ID NO: 231: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLTNEYWGQGTLVTVSSAAATT TPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0516] SEQ ID NO: 232: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKVRPSYEYWGQGTLVTVSSAA ATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAE QTVFL

[0517] SEQ ID NO: 233: EVQLVESGGGLVQPGGSLRLSCAASGFTFSPYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVAKPGYEYWGQGTLVTVSSAAAT TTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0518] SEQ ID NO: 234: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGRTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPGEDVYWGQGTLVTVSSAAA TTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQ TVFL

[0519] SEQ ID NO: 235: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKSNMVYWGQGTLVTVSSAAAT TTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQT VFL

[0520] SEQ ID NO: 236: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAITGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTKVDGSYGIVTELWGQGTLVTVS SAAATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEE ALAEQTVFL

[0521] SEQ ID NO: 237: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKQREWVSAINGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARDRERDYWGQGTLVTVSSA AATTTPIYIWAPLAGTCGVLLLSLVITLYCNKCFCKHRSCFRRNEASRETNNSLTFGPEEAL AEQTVFL

[0522] In particular embodiments, a recombinant nucleic acid encoding a CD300a TASR has at least 80%, at least 85%, 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%, or at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 202-219. In particular embodiments, the recombinant nucleic acid encoding the CD300a TASR comprises or consists of the nucleic acid sequence of any one of SEQ ID NOs: 202-219.

[0523] SEQ ID NO: 202: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG GCTAAGCCCGGCGAAGATGTGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGG CAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGT GCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAA GCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCC CGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0524] SEQ ID NO: 203: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTAGTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTC ACTAAGCTCAGCCAGTTTGCGTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGC AGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTG CTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAG CTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCC GAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0525] SEQ ID NO: 204: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTTCCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGCCGCGGTCAGGCTGGGGTCTCTGGGGCCAGGGAACCCTGGTCACCGTTTCAT CGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGG GGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACA GAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCG GCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0526] SEQ ID NO: 205: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCTTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTTCCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCA ACGAAGACTCGCTACGAGTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGCAG CCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTG CTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAGCTG CTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGA AGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0527] SEQ ID NO: 206: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGATTATGCCATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACAGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTAAC ACCCGCCTTGCACACGGACGGGATGTGCTCGGGGGCGTGGCCTATGACATATGGGGCC AGGGAACCCTGGTCACCGTTTCATCGGCAGCCGCAACAACGACACCTATCTACATCTG GGCTCCTTTGGCAGGCACCTGCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACT GTAACAAGTGTTTCTGCAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGA GACAAACAACAGCCTGACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTT CTG

[0528] SEQ ID NO: 207: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGACTATGCTATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACGGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTAAC ACCCGCCGCCTGGGGAGATCAGGAGATTTGGTACAGGATTACTGGGGCCAGGGAACCC TGGTCACCGTTTCATCGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTG GCAGGCACCTGCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTG TTTCTGCAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAA CAGCCTGACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0529] SEQ ID NO: 208: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGCTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGCCGGACCGCGACTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGCAG CCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTG CTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAGCTG CTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGA AGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0530] SEQ ID NO: 209: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTC ACTAAGCTCCCCGACGTTCTGCCTCTGGAATACTGGGGCCAGGGAACCCTGGTCACCGT TTCATCGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCT GCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAG CACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACA TTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0531] SEQ ID NO: 210: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCATATTGGATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACCGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCA ACGAAGGTGGACGGCAGCTACGGCATCGTTACAGAACTGTGGGGCCAGGGAACCCTG GTCACCGTTTCATCGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGC AGGCACCTGCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTT TCTGCAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACA GCCTGACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0532] SEQ ID NO: 211: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGACTATGCCATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAACGGTAGTGGTGGTTCTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTCAT ACCCGCCGGAGCGGTACGTCTATGGCCATGGACGTGTGGGGCCAGGGAACCCTGGTCA CCGTTTCATCGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGC ACCTGCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTG CAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCT GACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0533] SEQ ID NO: 212: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCCTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAGCGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG ACCAAGCTGACTATGGTTTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGCAGC CGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTGC TGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAGCTGC TTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGAA GAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0534] SEQ ID NO: 213: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCCGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTC ACTAAGCTGACTAACGAATATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGCAG CCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTG CTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAGCTG CTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGA AGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0535] SEQ ID NO: 214: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGGTTCGCCCATCCTACGAGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATC GGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGG GTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAG AAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGG CCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0536] SEQ ID NO: 215: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCCTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCAGGTAGTGGTGGTTCAACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTG GCGAAGCCGGGTTACGAATATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGCAG CCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTG CTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAGCTG CTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGA AGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0537] SEQ ID NO: 216: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCTTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTCGGACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGCCCGGCGAAGATGTGTATTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGG CAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGT GCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAA GCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCC CGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0538] SEQ ID NO: 217: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCCTATTACATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTTCTGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGTT ACTAAGTCAAATATGGTCTACTGGGGCCAGGGAACCCTGGTCACCGTTTCATCGGCAG CCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTG CTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAAGCTG CTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGA AGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0539] SEQ ID NO: 218: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTACTGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTACG ACGAAGGTGGACGGCAGCTACGGCATCGTTACAGAACTGTGGGGCCAGGGAACCCTG GTCACCGTTTCATCGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGC AGGCACCTGCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTT TCTGCAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACA GCCTGACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0540] SEQ ID NO: 219: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC TCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTCTTATTGGATGAGCTGGGTCCGCCAG GCTCCAGGGAAGCAGCGTGAGTGGGTCTCAGCTATTAATGGTAGTGGTGGTTCCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACAC GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCT GCTGCTAGAGATCGCGAACGGGACTATTGGGGCCAGGGAACCCTGGTCACCGTTTCAT CGGCAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGG GGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACA GAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCG GCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0541] In some embodiments, an NKG2A TASR comprises a NKG2A VL and a NKG2A VH, such as any NKG2A VL and any NKG2A VH disclosed herein (such as comprised in a NKG2A scFv disclosed herein). In particular embodiments, a recombinant nucleic acid encoding a NKG2A TASR encodes an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 21. In particular embodiments, the recombinant nucleic acid encoding the NKG2A TASR encodes an amino acid sequence comprising or consisting of SEQ ID NO: 21.

[0542] SEQ ID NO: 21: MVLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPG KAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTK VEIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWV RQAPGQGLEWMGRIDPYDSETHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARGGYDFDVGTLYWFFDVWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN

[0543] In particular embodiments, the recombinant nucleic acid encoding a NKG2A TASR comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 23. In other particular embodiments, the recombinant nucleic acid encoding the NKG2A TASR comprises or consists of SEQ ID NO: 23.

[0544] SEQ ID NO: 23: ATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCGCACCCGGCTTTTCTCTTG ATCCCCGACATCCAGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCG CGTCACCATCACCTGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAG CAGAAGCCTGGGAAAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGG GCGTGCCATCCCGCTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCG TCGCTCCAGCCGGAAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCG TACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAA CCTGGTTCTGGGGAGGGCTCCACTAAGGGACAGGTCCAGCTGGTGCAGAGCGGCGCGG AGGTGAAGAAGCCGGGTGCTTCCGTAAAGGTGTCATGCAAAGCATCTGGCTACACGTT CACCAGTTACTGGATGAACTGGGTCCGCCAGGCCCCTGGTCAAGGACTGGAGTGGATG GGCAGGATCGACCCCTATGACTCCGAGACTCACTACGCGCAGAAGTTGCAGGGGCGGG TTACCATGACGACCGACACCTCGACCTCCACAGCCTACATGGAACTGCGCTCGCTCCGC AGCGATGACACCGCCGTGTACTACTGTGCTCGTGGCGGCTACGACTTCGACGTGGGCA CTCTTTATTGGTTCTTTGACGTGTGGGGCCAGGGCACCACCGTGACGGTTTCTTCTGCA GCCGCAACAACGACACCTGCCCCACGCCCGCCAACTCCAGCTCCAACGATTGCGTCCC AGCCCCTCAGCTTGAGACCGGAAGCCTGTCGACCCGCAGCTGGCGGGGCCGTCCATAC TCGCGGGCTGGACTTTGCGTGTGACATCTACATCTGGGCTCCTTTGGCAGGCACCTGCG GGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAAC

[0545] In some embodiments, a CD300a TASR comprises a CD300a VL and a CD300a VH, such as any CD300a VL and any CD300a VH disclosed herein (such as comprised in a CD300a scFv disclosed herein). In particular embodiments, the recombinant nucleic acid encoding a CD300a TASR encodes an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 22. In particular embodiments the recombinant nucleic acid encoding a CD300a TASR encodes an amino acid sequence comprising or consisting of SEQ ID NO: 22.

[0546] SEQ ID NO: 22: ATMVLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ KPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISNLQPEDFATYFCQQGNTLPWTFGQ GTKVEIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMQ WVRQAPGQGLEWIGEIDPSDSYTNYNQKFKGRATLTVDTSTSTTYMELSSLTSEDTAVYYC ARWGMAYGTSSYWYFDVWGRGTLVTVSSAAATTTPIYIWAPLAGTCGVLLLSLVITLYCN KCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFL

[0547] In particular embodiments, the recombinant nucleic acid encoding a CD300a TASR comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 24. In other particular embodiments, the recombinant nucleic acid encoding the CD300a TASR comprises or consists of SEQ ID NO: 24.

[0548] SEQ ID NO: 24: GCCACCATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCTCATCCTGCGTTC CTGCTGATTCCCGACATCCAGATGACCCAGAGCCCGTCCTCTCTGTCCGCCTCCGTGGG GGACAGGGTCACCATCACCTGCCGTGCCTCACAGGATATCTCGAACTACCTCAATTGGT ACCAGCAGAAGCCCGGCAAAGCCCCCAAGCTGCTCATCTATTACACCTCCCGCCTTCAC TCTGGTGTGCCCTCTCGCTTCTCGGGTAGTGGCTCCGGAACAGACTACACTCTGACCAT TAGCAACCTGCAGCCAGAGGACTTTGCAACTTACTTCTGTCAACAGGGCAACACGCTA CCGTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTG GTAAACCTGGTAGTGGGGAGGGCTCCACCAAGGGACAGGTCCAACTGGTGCAGAGCG GCGCGGAGGTGAAGAAGCCCGGGGCATCCGTAAAGGTGTCATGCAAGGCGTCTGGCTA CACGTTCACCAGCTATTGGATGCAGTGGGTCCGCCAGGCCCCTGGCCAGGGCCTGGAG TGGATCGGTGAAATTGACCCGTCCGACAGCTACACCAACTACAACCAGAAATTTAAAG GCCGCGCTACACTGACCGTGGATACTTCGACCTCCACGACCTACATGGAGCTGTCTTCT CTCACTTCCGAGGACACCGCCGTGTACTACTGTGCTCGGTGGGGAATGGCCTACGGCA CCAGCTCGTATTGGTACTTCGACGTGTGGGGCCGTGGTACTTTGGTTACGGTTTCTTCTG CAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGT GCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAA GCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCC CGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG H. Exemplary Bispecific TASRs

[0549] In some embodiments, a TASR disclosed herein is a bispecific TASR, wherein the bispecific TASR comprises two or more binding domains, and accordingly, may bind two or more inhibitory receptors on an NK cell, e.g., both NKG2A and CD300a. Such bispecific TASRs can comprise two scFvs, such as a first scFv that specifically binds a first inhibitory receptor expressed on NK cells (such as CD300a), and a second scFv that specifically binds a second inhibitory receptor expressed on NK cells (such as NKG2A). In some embodiments, a bispecific TASR is defined by the formula:

[0550] SP-VL1-Linker1-VH1-Linker3-VL2-Linker2-VH2-Hinge-TM-Cyt; wherein:

[0551] “SP” represents an optional signal peptide,

[0552] “VL1” represents a first light chain variable region (such as a NKG2A VL or a CD300a VL)

[0553] “Linker1” represents a first linker;

[0554] “VH1” represent a first heavy chain variable region (such as a NKG2A VH or a CD300a VH);

[0555] “Linker3” represents a third linker;

[0556] “VL2” represents a second light chain variable region (such as a NKG2A VL or a CD300a VL)

[0557] “Linker2” represents a second linker;

[0558] “VH2” represent a second heavy chain variable region (such as a NKG2A VH or a CD300a VH);

[0559] “Hinge” represents an optional hinge region;

[0560] “TM” represents a transmembrane domain; and

[0561] “Cyt” represents an optional cytoplasmic domain.

[0562] A bispecific TASR disclosed herein may alternatively be defined by any one of the following formulas: SP-VH1-Linker1-VL1-Linker3-VL2-Linker2-VH2-Hinge-TM-Cyt; SP-VL1- Linker1-VH1-Linker3-VH2-Linker2-VL2-Hinge-TM-Cyt; or SP-VH1-Linker1-VL1-Linker3-VH2- Linker2-VL2-Hinge-TM-Cyt. FIG. 1 provides a diagram of exemplary formulas for bispecific TASRs of use herein. A bispecific TASR described herein (such as a bispecific TASR of FIG. 1) can comprise any suitable signal peptide, linker, hinge, transmembrane domain, or cytoplasmic domain, such as an of the signal peptides, linkers, hinges, transmembrane domains, or cytoplasmic domains described in detail herein. In particular embodiments, the third linker comprises a cleavable peptide linker, such as a T2A linker described herein (such as the T2A linker of SEQ ID NO: 32), or a P2A linker described herein (such as the P2A linker of SEQ ID NO: 33).

[0563] In some embodiments of the disclosed bispecific TASRs, VL1-Linker1-VH1 (or VH1-L1- VL1) is a CD300a scFv (such as comprising a CD300a VL, a linker, and a CD300a VH disclosed herein) or is a NKG2A scFv (such as comprising a NKG2A VL, a linker, and a NKG2A VH disclosed herein). Similarly, in some embodiments, VL2-Linker2-VH2 (or VH2-Linker-VL2) is a CD300a scFv (such as a CD300a scFv disclosed herein, such as a CD300a scFv comprising a CD300a VL, a linker, and a CD300a VH disclosed herein) or is a NKG2A scFv (such as a NKG2A scFv disclosed herein, such as a NKG2A scFv comprising a NKG2A VL, a linker, and a NKG2A VH disclosed herein). In some embodiments, if VL1-Linker1-VH1 (or VH1-L1-VL1) is a CD300a scFv, then VL2-Linker2-VH2 (or VH2-Linker-VL2) is a NKG2A scFv. In other embodiments, if VL1-Linker1-VH1 (or VH1-L1-VL1) is a NKG2A scFv, then VL2-Linker2-VH2 (or VH2-Linker- VL2) is a CD300a scFv.

[0564] In some embodiments, the nucleotide sequence of the NKG2A VL, the NKG2A VH, the CD300a VL, and / or the CD300a VH of a disclosed recombinant nucleic acid encoding a bispecific TASR is codon optimized, such as to reduce or prevent undesired recombination events. In some embodiments, a codon optimized nucleotide sequence encoding an NKG2A scFv of the bispecific TASR has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 113. In particular embodiments, the codon optimized nucleotide sequence encoding the NKG2A scFv comprises or consists of SEQ ID NO: 113.

[0565] In some embodiments, a codon optimized nucleotide sequence encoding the NKG2A VL of the bispecific TASR has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 110. In particular embodiments, the codon optimized nucleotide sequence encoding the NKG2A VL comprises or consists of SEQ ID NO: 110.

[0566] In some embodiments, a codon optimized nucleotide sequence encoding the NKG2A VH of the bispecific TASR has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 112. In particular embodiments, the codon optimized nucleotide sequence encoding the NKG2A VH comprises or consists of SEQ ID NO: 112.

[0567] In particular embodiments, a recombinant nucleic acid encoding a bispecific TASR encodes an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 25 or SEQ ID NO: 26. In particular embodiments, the recombinant nucleic acid encoding the NKG2A TASR encodes an amino acid sequence comprising or consisting of SEQ ID NO: 25 or SEQ ID NO: 26.

[0568] SEQ ID NO: 25 (the CD300a TASR of SEQ ID NO: 22 linked to the NKG2A TASR of SEQ ID NO: 21 via the T2A linker of SEQ ID NO: 32): ATMVLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ KPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISNLQPEDFATYFCQQGNTLPWTFGQ GTKVEIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMQ WVRQAPGQGLEWIGEIDPSDSYTNYNQKFKGRATLTVDTSTSTTYMELSSLTSEDTAVYYC ARWGMAYGTSSYWYFDVWGRGTLVTVSSAAATTTPIYIWAPLAGTCGVLLLSLVITLYCN KCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFLGSGEGRGSLLTCGDVEENPGPMV LLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKA PKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEI KGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQA PGQGLEWMGRIDPYDSETHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARG GYDFDVGTLYWFFDVWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN

[0569] SEQ ID NO: 26: (the NKG2A TASR of SEQ ID NO: 21 linked to the CD300a TASR of SEQ ID NO: 22 via the T2A linker of SEQ ID NO: 32): MVLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPG KAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTK VEIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWV RQAPGQGLEWMGRIDPYDSETHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARGGYDFDVGTLYWFFDVWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNGSGEGRGSLLTCGDVEENPG PMVLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKP GKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGT KVEIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNW VRQAPGQGLEWMGRIDPYDSETHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYY CARGGYDFDVGTLYWFFDVWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN

[0570] In particular embodiments, the recombinant nucleic acid encoding the bispecific TASR comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 27 or SEQ ID NO: 28. In other particular embodiments, the recombinant nucleic acid encoding the bispecific TASR comprises or consists of SEQ ID NO: 27 or SEQ ID NO: 28.

[0571] SEQ ID NO: 27 (encoding the bispecific TASR of SEQ ID NO: 25): GCCACCATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCTCATCCTGCGTTC CTGCTGATTCCCGACATCCAGATGACCCAGAGCCCGTCCTCTCTGTCCGCCTCCGTGGG GGACAGGGTCACCATCACCTGCCGTGCCTCACAGGATATCTCGAACTACCTCAATTGGT ACCAGCAGAAGCCCGGCAAAGCCCCCAAGCTGCTCATCTATTACACCTCCCGCCTTCAC TCTGGTGTGCCCTCTCGCTTCTCGGGTAGTGGCTCCGGAACAGACTACACTCTGACCAT TAGCAACCTGCAGCCAGAGGACTTTGCAACTTACTTCTGTCAACAGGGCAACACGCTA CCGTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTG GTAAACCTGGTAGTGGGGAGGGCTCCACCAAGGGACAGGTCCAACTGGTGCAGAGCG GCGCGGAGGTGAAGAAGCCCGGGGCATCCGTAAAGGTGTCATGCAAGGCGTCTGGCTA CACGTTCACCAGCTATTGGATGCAGTGGGTCCGCCAGGCCCCTGGCCAGGGCCTGGAG TGGATCGGTGAAATTGACCCGTCCGACAGCTACACCAACTACAACCAGAAATTTAAAG GCCGCGCTACACTGACCGTGGATACTTCGACCTCCACGACCTACATGGAGCTGTCTTCT CTCACTTCCGAGGACACCGCCGTGTACTACTGTGCTCGGTGGGGAATGGCCTACGGCA CCAGCTCGTATTGGTACTTCGACGTGTGGGGCCGTGGTACTTTGGTTACGGTTTCTTCTG CAGCCGCAACAACGACACCTATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGT GCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACAAGTGTTTCTGCAAGCACAGAA GCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCC CGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTGGGTTCTGGTGAGGGCAGAGGAAGT CTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGTGCTCTTGGTGACTTC CCTTTTGCTGTGCGAGCTGCCGCACCCGGCTTTTCTCTTGATCCCCGACATCCAGATGA CCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCGCGTCACCATCACCTGCCGG GCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAGCAGAAGCCTGGGAAAGCTC CTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGGGCGTGCCATCCCGCTTTTCA GGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCGTCGCTCCAGCCGGAAGACTT CGCCACCTACTACTGTCAACACCATTACGGCACTCCGCGTACCTTCGGCGGCGGCACCA AGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAACCTGGTTCTGGGGAGGGCTC CACTAAGGGACAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGCCGGGTGC TTCCGTAAAGGTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGGATGAACT GGGTCCGCCAGGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACCCCTATGA CTCCGAGACTCACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGACCGACACC TCGACCTCCACAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACCGCCGTGT ACTACTGTGCTCGTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTTCTTTGAC GTGTGGGGCCAGGGCACCACCGTGACGGTTTCTTCTGCAGCCGCAACAACGACACCTG CCCCACGCCCGCCAACTCCAGCTCCAACGATTGCGTCCCAGCCCCTCAGCTTGAGACCG GAAGCCTGTCGACCCGCAGCTGGCGGGGCCGTCCATACTCGCGGGCTGGACTTTGCGT GTGACATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTGCTGCTGTCCCTG GTGATCACCCTGTACTGTAAC

[0572] SEQ ID NO: 28 (encoding the bispecific TASR of SEQ ID NO: 26): ATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCGCACCCGGCTTTTCTCTTG ATCCCCGACATCCAGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCG CGTCACCATCACCTGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAG CAGAAGCCTGGGAAAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGG GCGTGCCATCCCGCTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCG TCGCTCCAGCCGGAAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCG TACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAA CCTGGTTCTGGGGAGGGCTCCACTAAGGGACAGGTCCAGCTGGTGCAGAGCGGCGCGG AGGTGAAGAAGCCGGGTGCTTCCGTAAAGGTGTCATGCAAAGCATCTGGCTACACGTT CACCAGTTACTGGATGAACTGGGTCCGCCAGGCCCCTGGTCAAGGACTGGAGTGGATG GGCAGGATCGACCCCTATGACTCCGAGACTCACTACGCGCAGAAGTTGCAGGGGCGGG TTACCATGACGACCGACACCTCGACCTCCACAGCCTACATGGAACTGCGCTCGCTCCGC AGCGATGACACCGCCGTGTACTACTGTGCTCGTGGCGGCTACGACTTCGACGTGGGCA CTCTTTATTGGTTCTTTGACGTGTGGGGCCAGGGCACCACCGTGACGGTTTCTTCTGCA GCCGCAACAACGACACCTGCCCCACGCCCGCCAACTCCAGCTCCAACGATTGCGTCCC AGCCCCTCAGCTTGAGACCGGAAGCCTGTCGACCCGCAGCTGGCGGGGCCGTCCATAC TCGCGGGCTGGACTTTGCGTGTGACATCTACATCTGGGCTCCTTTGGCAGGCACCTGCG GGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAACGGTTCTGGTGAGGGCAGA GGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGTGCTCTTGG TGACTTCCCTTTTGCTGTGCGAGCTGCCGCACCCGGCTTTTCTCTTGATCCCCGACATCC AGATGACCCAGAGCCCCTCTTCTCTTTCCGCCTCCGTGGGGGACCGCGTCACCATCACC TGCCGGGCATCCGAGAACATCTATAGCTACCTGGCGTGGTACCAGCAGAAGCCTGGGA AAGCTCCTAAGCTGCTGATTTACAACGCCAAGACTCTAGCCGAGGGCGTGCCATCCCG CTTTTCAGGTAGTGGCTCCGGTACAGATTTCACCCTGACGATCTCGTCGCTCCAGCCGG AAGACTTCGCCACCTACTACTGTCAACACCATTACGGCACTCCGCGTACCTTCGGCGGC GGCACCAAGGTGGAGATCAAGGGATCGACCTCAGGCTCTGGTAAACCTGGTTCTGGGG AGGGCTCCACTAAGGGACAGGTCCAGCTGGTGCAGAGCGGCGCGGAGGTGAAGAAGC CGGGTGCTTCCGTAAAGGTGTCATGCAAAGCATCTGGCTACACGTTCACCAGTTACTGG ATGAACTGGGTCCGCCAGGCCCCTGGTCAAGGACTGGAGTGGATGGGCAGGATCGACC CCTATGACTCCGAGACTCACTACGCGCAGAAGTTGCAGGGGCGGGTTACCATGACGAC CGACACCTCGACCTCCACAGCCTACATGGAACTGCGCTCGCTCCGCAGCGATGACACC GCCGTGTACTACTGTGCTCGTGGCGGCTACGACTTCGACGTGGGCACTCTTTATTGGTT CTTTGACGTGTGGGGCCAGGGCACCACCGTGACGGTTTCTTCTGCAGCCGCAACAACG ACACCTGCCCCACGCCCGCCAACTCCAGCTCCAACGATTGCGTCCCAGCCCCTCAGCTT GAGACCGGAAGCCTGTCGACCCGCAGCTGGCGGGGCCGTCCATACTCGCGGGCTGGAC TTTGCGTGTGACATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTGCTGCT GTCCCTGGTGATCACCCTGTACTGTAAC III. Vectors

[0573] The present disclosure provides vectors comprising a disclosed recombinant nucleic acid, such as vectors comprising one or more NKG2A and / or CD300a binding domains, such as a CD300a VHH or one or more NKG2A and / or CD300a scFvs as disclosed herein. A vector of the present disclosure can comprise, e.g., an NKG2A binding domain (such as an NKG2A scFv) and / or a CD300a binding domain (such as a CD300a VHH or a CD300a scFv), or a NKG2A TASR and / or a CD300a TASR (such as a CD300a TASR comprising a CD300a VHH, or a bispecific TASR comprising both a NKG2A scFv and a CD300a scFv as described herein).

[0574] A vector useful in the present embodiments can be, for example, a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenoviral vector, an adeno associated viral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector. In particular embodiments of a vector, the recombinant nucleic acid is operably linked to a promoter. In specific, non-limiting embodiments, the promoter is an EF1a promoter, a CAG promoter, a PGK promoter, or a CMV promoter. In some embodiments, the promoter is an EF1a promoter and comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 92.

[0575] SEQ ID NO: 92 (encoding an EF1a promoter): GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG GGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGC CTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGA AGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGT TGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCC TGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACG CTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCG AGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGC CGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAG GCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCT GCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCC ACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGT ACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTA GGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTG AAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTG GATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGG TGTCGTGA

[0576] A recombinant nucleic acid herein can also include other regulatory elements, i.e., transcriptional and translational control sequences, such as enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a coding sequence and / or regulate translation of an encoded polypeptide (such as a TASR or a bispecific TASR described herein). Exemplary regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0577] In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) sequence. In particular embodiments, the poly(A) sequence comprises a bGH poly(A) signal, such as a bGH poly(A) signal encoded by a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 89.

[0578] SEQ ID NO: 89 (encoding a bGH poly(A) signal): CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGG ATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0579] In some embodiments, a nucleic acid sequence in the vector further comprises a 3’UTR.

[0580] In particular embodiments, a vector comprising a disclosed recombinant nucleic acid integrates into a genome at an adeno-associated virus integration site 1 (AAVS1) of the genome. Originally described as a major hotspot for adeno-associated virus (AAV) integration, intron 1 of the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19 is referred to the AAVS1 locus (Oceguera-Yanez et al., Methods.2016;101:43-55). This locus allows stable, long-term transgene expression in many cell types, including embryonic stem cells. Thus, in some embodiments, a vector disclosed herein comprises an AAVS1 right homology arm and an AAVS1 left homology arm. In some embodiments, a nucleic acid encoding the AAVS1 left homology arm comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 90.

[0581] SEQ ID NO: 90 (encoding an AAVS1 left homology arm): GCCGTGACGTCAGCACGCCGGGCGGGGACCGGGAGATCCTTGGGGCGGTGGGGGGCC AGCGGCAGTTCCCGGCGGCCCCCGGGGCGGGCGGGCGGGCGGGTGGTGGCGGCGGTT GGGGCTCGGCGCTCGCTCGCTCGCTGGGCGGGCGGGCGGTGCGATGTCCGGAGAGGAT GGCCCGGCGGCTGGCCCGGGGGCGGCGGCGGCGGCTGCCCGGGAGCGGCGACGGGAG CAGCTGCGGCAGTGGGGGGCGCGGGCGGGCGCCGAGCCTGGCCCCGGAGAGCGCCGC GCCCGCACCGTCCGCTTCGAGCGCGCCGCCGAGTTCCTGGCGGCCTGTGCGGGCGGCG ACCTGGACGAGGCGCGTCTGATGCTGCGCGCCGCCGACCCTGGCCCCGGCGCCGAGCT CGACCCCGCCGCGCCGCCGCCCGCCCGCGCCGTGCTGGACTCCACCAACGCCGACGGT ATCAGCGCCCTGCACCAGGTCAGCGCCCCCCGCCCGGCGTCTCCCGGGGCCAGGTCCA CCCTCTGCTGCGCCACCTGGGGCATCCTCCTTCCCCGTTGCCAGTCTCGATCCGCCCCGT CGTTCCTGGCCCTGGGCTTTGCCACCCTATGCTGACACCCCGTCCCAGTCCCCCTTACC ATTCCCCTTCGACCACCCCACTTCCGAATTGGAGCCGCTTCAACTGGCCCTGGGCTTAG CCACTCTGTGCTGACCACTCTGCCCCAGGCCTCCTTACCATTCCCCTT

[0582] In some embodiments, a nucleic acid encoding the AAVS1 right homology arm comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 91.

[0583] SEQ ID NO: 91 (encoding an AAVS1 right homology arm): GACCTACTCTCTTCCGCATTGGAGTCGCTTTAACTGGCCCTGGCTTTGGCAGCCTGTGCT GACCCATGCAGTCCTCCTTACCATCCCTCCCTCGACTTCCCCTCTTCCGATGTTGAGCCC CTCCAGCCGGTCCTGGACTTTGTCTCCTTCCCTGCCCTGCCCTCTCCTGAACCTGAGCCA GCTCCCATAGCTCAGTCTGGTCTATCTGCCTGGCCCTGGCCATTGTCACTTTGCGCTGCC CTCCTCTCGCCCCCGAGTGCCCTTGCTGTGCCGCCGGAACTCTGCCCTCTAACGCTGCC GTCTCTCTCCTGAGTCCGGACCACTTTGAGCTCTACTGGCTTCTGCGCCGCCTCTGGCCC ACTGTTTCCCCTTCCCAGGCAGGTCCTGCTTTCTCTGACCTGCATTCTCTCCCCTGGGCC TGTGCCGCTTTCTGTCTGCAGCTTGTGGCCTGGGTCACCTCTACGGCTGGCCCAGATCC TTCCCTGCCGCCTCCTTCAGGTTCCGTCTTCCTCCACTCCCTCTTCCCCTTGCTCTCTGCT GTGTTGCTGCCCAAGGATGCTCTTTCCGGAGCACTTCCTTCTCGGCGCTGCACCACGTG ATGTCCTCTGAGCGGATCCTCCCCGTGTCTGGGTCCTCTCCGGGCATCTCTCCTCCCTCA CCCAACCCCATGCCGTCTTCACTCGCTGGGTTCCCTTTTCCTTCTCCTTCTGGGGCCTGT GCCATCTCTCGTTTCTTAGGATGGCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTC CCCTTCTTGTAGGCCTGCATCA IV. Engineered Cells

[0584] Some embodiments of the present disclosure comprise an engineered (e.g., genetically modified) cell, such as an engineered human cell. In some embodiments, the cell is an allogeneic cell. An engineered cell refers to a cell (or progeny of a cell) comprising an engineered genetic modification, e.g., a cell that has been contacted with a gene editing system and genetically modified by the gene editing system. The terms “engineered cell” and “genetically modified cell” are used interchangeably throughout. The engineered human cell may be any of the exemplary cell types disclosed herein.

[0585] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a pluripotent stem cell such as an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC). In some embodiments, the engineered cell is selected from a stem cell, a progenitor cell, or a cell that has been differentiated from a stem cell or a progenitor cell. In some embodiments, the engineered cell is a pluripotent stem cell that is subsequently differentiated into an immune cell. In some embodiments, the cell is an immune cell. As used herein, “immune cell” refers to a cell of the immune system, including e.g., a lymphocyte (e.g., T cell, B cell, natural killer cell (“NK cell”, and NKT cell, or iNKT cell)), monocyte, macrophage, mast cell, dendritic cell, or granulocyte (e.g., neutrophil, eosinophil, and basophil). In some embodiments, the cell is a primary immune cell. In some embodiments, the cell is a pluripotent stem cell-derived immune cell. In some embodiments, the immune system cell may be selected from CD3+, CD4+and CD8+T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC). In some embodiments, the immune cell is allogeneic.

[0586] In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a NK cell. In some embodiments, the cell is a macrophage. In some embodiments, the lymphocyte is allogeneic.

[0587] As used herein, a T cell can be defined as a cell that expresses a T cell receptor (“TCR” or ³Įȕ^7&5´^RU^³ȖG TCR”), however in some embodiments, the TCR of a T cell may be genetically modified to reduce or delete its expression (e.g., by genetic modification to the TRAC or TRBC genes), therefore expression of the protein CD3 may be used as a marker to identify a T cell by standard flow cytometry methods. CD3 is a multi-subunit signaling complex that associates with the TCR. Thus, a T cell may be referred to as CD3+. In some embodiments, a T cell is a cell that expresses a CD3+ marker and either a CD4+ or CD8+ marker. In some embodiments, the T cell is allogeneic.

[0588] In some embodiments, the T cell expresses the glycoprotein CD8 and therefore is CD8+ by standard flow cytometry methods and may be referred to as a “cytotoxic” T cell. In some embodiments, the T cell expresses the glycoprotein CD4 and therefore is CD4+ by standard flow cytometry methods and may be referred to as a “helper” T cell. CD4+ T cells can differentiate into subsets and may be referred to as a Th1 cell, Th2 cell, Th9 cell, Th17 cell, Th22 cell, T regulatory (“Treg”) cell, or T follicular helper cells (“Tfh”). Each CD4+ subset releases specific cytokines that can have either proinflammatory or anti-inflammatory functions, survival or protective functions. A T cell may be isolated from a subject by CD4+ or CD8+ selection methods.

[0589] In some embodiments, the T cell is a memory T cell. In the body, a memory T cell has encountered antigen. A memory T cell can be located in the secondary lymphoid organs (central memory T cells) or in recently infected tissue (effector memory T cells). A memory T cell may be a CD8+ T cell. A memory T cell may be a CD4+ T cell.

[0590] As used herein, a “central memory T cell” can be defined as an antigen-experienced T cell, and for example, may express CD62L and CD45RO. A central memory T cell may be detected as CD62L+ and CD45RO+, e.g. by flow cytometry. A central memory T cell also expresses CCR7, therefore may be detected as CCR7+ by standard flow cytometry methods.

[0591] As used herein, an “early stem-cell memory T cell” (or “Tscm”) can be defined as a T cell that expresses CD27 and CD45RA, and therefore is CD27+ and CD45RA+ by standard flow cytometry methods. A Tscm does not express the CD45 isoform CD45RO, therefore a Tscm will further be CD45RO- if stained for this isoform by standard flow cytometry methods. A CD45RO- CD27+ cell is therefore also an early stem-cell memory T cell. Tscm cells further express CD62L and CCR7, therefore may be detected as CD62L+ and CCR7+ by standard flow cytometry methods. Early stem-cell memory T cells have been shown to correlate with increased persistence and therapeutic efficacy of cell therapy products.

[0592] In some embodiments, the cell is a B cell. As used herein, a “B cell” can be defined as a cell that expresses CD19 or CD20, or B cell mature antigen (“BCMA”), and therefore a B cell is CD19+, or CD20+, or BCMA+ by standard flow cytometry methods. A B cell is further negative for CD3 and CD56 by standard flow cytometry methods. The B cell may be a plasma cell. The B cell may be a memory B cell. The B cell may be a naïve B cell. The B cell may be IgM+ or may have a class-switched B cell receptor (e.g., IgG+, or IgA+). In some embodiments, the B cell is allogeneic.

[0593] In some embodiments, the cell is a mononuclear cell, such as from bone marrow or peripheral blood. In some embodiments, the cell is a peripheral blood mononuclear cell (“PBMC”). In some embodiments, the cell is a PBMC, e.g. a lymphocyte or monocyte. In some embodiments, the cell is a peripheral blood lymphocyte (“PBL”). In some embodiments, the mononuclear cell is allogeneic.

[0594] In some embodiments, the engineered cell is a stem cell, a progenitor cell, or a primary. Stem cells may be broadly defined as cells capable of going through numerous cycles of cell division, maintaining an undifferentiated state and having the capacity to differentiate into specialized cell types (Tesche et al., Stem Cells Int. 2010;2010:824876). They may go through asymmetric division wherein the stem cell creates a copy of itself and a daughter cell that is capable of differentiation. Stem cells are further classified into three categories: totipotent, pluripotent, or multipotent somatic. A totipotent cell has the ability to form an entire organism (e.g., a fertilized egg). Pluripotent stem cells lack the ability to form extraembryonic tissue and are therefore unable to generate a fetus, but can give rise to any cell type from the three germ cell layers (i.e., endoderm, mesoderm, or ectoderm). Examples of pluripotent stem cells include human embryonic stem cells (hESC) and induced pluripotent stem cells (ipSC). Adult stem cells or mesenchymal stem cells (MSCs) are examples of multipotent stem cells that are isolated from mature tissues and differentiate into other tissue types. Multipotent somatic stem cells are capable of differentiating into a variety of closely related cells within a tissue, but lack the ability to differentiate into other tissues. A much-studied area of multipotent somatic stem cells is the hematopoietic stem cell (HSC) which generates daughter cells that in turn differentiate into all subpopulations of hematopoietic cells (e.g., red blood cells, platelets, etc.).

[0595] Accordingly, stem cells useful herein can include pluripotent stem cells (PSCs); induced pluripotent stem cells (iPSCs); embryonic stem cells (ESCs); mesenchymal stem cells (MSCs, e.g., isolated from bone marrow (BM), peripheral blood (PB), placenta, umbilical cord (UC) or adipose); hematopoietic stem cells (HSCs; e.g. isolated from BM or UC); neural stem cells (NSCs); tissue specific progenitor stem cells (TSPSCs); and limbal stem cells (LSCs). Progenitor and primary cells include mononuclear cells (MNCs, e.g., isolated from BM or PB); endothelial progenitor cells (EPCs, e.g. isolated from BM, PB, and UC); neural progenitor cells (NPCs); and tissue-specific primary cells or cells derived therefrom (TSCs) including chondrocytes, myocytes, and keratinocytes. Cells for organ or tissue transplantations such as islet cells, cardiomyocytes, thyroid cells, thymocytes, neuronal cells, skin cells, and retinal cells are also included.

[0596] In some embodiments, the human cell is isolated from a human subject. In some embodiments, the cell is isolated from human donor PBMCs or leukopaks.

[0597] In some embodiments, the cell is from a cell line. In some embodiments, the cell line is derived from a human subject. In some embodiments, the cell is from a cell bank. In some embodiments, the cell is genetically modified and then transferred into a cell bank. In some embodiments the cell is removed from a subject, genetically modified ex vivo, and transferred into a cell bank. In some embodiments, a genetically modified population of cells is transferred into a cell bank. In some embodiments, a genetically modified population of immune cells is transferred into a cell bank. In some embodiments, a genetically modified population of immune cells comprising a first and second subpopulations, wherein the first and second sub-populations have at least one common genetic modification and at least one different genetic modification are transferred into a cell bank.

[0598] A variety of methods for introducing nucleic acids (such as a recombinant nucleic acid disclosed herein) into cells are known in the art, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent).

[0599] In some embodiments, a recombinant nucleic acid is introduced into a cell (e.g., to produce an engineered cell) using a CRISPR / Cas system; zinc finger nuclease (ZFN) system; or a transcription activator-like effector nuclease (TALEN) system. Generally, the gene editing systems involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick (e.g., a single strand break, ori SSB) in a target DNA sequence. Cleavage or nicking can occur through the use of specific nucleases such as engineered ZFN, TALENs, or using the CRISPR / Cas system with an engineered guide RNA to guide specific cleavage or nicking of a target DNA sequence. Further, targeted nuc...

Claims

WHAT IS CLAIMED IS:

1. A recombinant nucleic acid encoding a construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain.

2. The recombinant nucleic acid of claim 1, wherein the CD300a binding domain comprises an antibody or a fragment thereof, a variable domain on a heavy chain (VHH) antibody, a cytokine, a ligand, or a peptide.

3. The recombinant nucleic acid of claim 1 or claim 2, wherein the NKG2A binding domain comprises an antibody or a fragment thereof, a VHH, a cytokine, a ligand, or a peptide.

4. The recombinant nucleic acid of claim 2 or claim 3, wherein (a) the antibody or a fragment thereof comprises a single chain variable fragment (scFv) or a VHH, or (b) the peptide is an adnectin or a design ankyrin repeat protein (DARPin).

5. The recombinant nucleic acid of the immediately preceding claim, wherein the VHH comprises the VH domain of a camelid heavy chain antibody.

6. The recombinant nucleic acid of any one of the preceding claims, wherein the CD300a binding domain comprises a VHH (CD300a VHH) and / or the NKG2A binding domain comprises a VHH (NKG2A VHH).

7. The recombinant nucleic acid of any one of the preceding claims, wherein the construct for inhibiting NK cell cytotoxicity comprises or consists of the CD300a binding domain, and the CD300a binding domain comprises a VHH (CD300a VHH).

8. The recombinant nucleic acid of any one of claims 2-7, wherein the CD300a VHH comprises: (a) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and aCDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising AAKPGEDVY (SEQ ID NO: 182); (b) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLSQFAS (SEQ ID NO: 183); (c) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPRSGWGL (SEQ ID NO: 184); (d) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKTRYES (SEQ ID NO: 185); (e) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ ID NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NTRLAHGRDVLGGVAYDI (SEQ ID NO: 186); (f) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ ID NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NTRRLGRSGDLVQDY (SEQ ID NO: 187); (g) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSRYY (SEQ ID NO: 175), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPDRDY (SEQ ID NO: 188);(h) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLPDVLPLEY (SEQ ID NO: 189); (i) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYW (SEQ ID NO: 176), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ ID NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKVDGSYGIVTEL (SEQ ID NO: 190); (j) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSDYA (SEQ ID NO: 174), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising INGSGGST (SEQ ID NO: 180), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising HTRRSGTSMAMDV (SEQ ID NO: 191); (k) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ATKLTMVY (SEQ ID NO: 192); (l) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKLTNEY (SEQ ID NO: 193); (m) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKVRPSYEY (SEQ ID NO: 194); (n) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSPYY (SEQ ID NO: 177), a CDR2 comprising 0, 1, or 2 mutationsrelative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VAKPGYEY (SEQ ID NO: 195); (o) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGRT (SEQ ID NO: 181), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKPGEDVY (SEQ ID NO: 196); (p) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ISGSGGST (SEQ ID NO: 178), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising VTKSNMVY (SEQ ID NO: 197); (q) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYY (SEQ ID NO: 173), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising ITGSGGST (SEQ ID NO: 179), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising TTKVDGSYGIVTEL (SEQ ID NO: 198); or (r) a CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GFTFSSYW (SEQ ID NO: 176), a CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising INGSGGST (SEQ ID NO: 180), and a CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising AAARDRERDY (SEQ ID NO: 199).

9. The recombinant nucleic acid of any one of claims 2-8, wherein the CD300a VHH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 155- 172.

10. The recombinant nucleic acid of any one of claims 2-9, wherein the CD300a VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 155-172.

11. The recombinant nucleic acid of any one of claims 2-10, wherein a nucleotide sequence encoding the CD300a VHH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 137-154.

12. The recombinant nucleic acid of any one of claims 2-11, wherein a nucleotide sequence encoding the CD300a VHH comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 137-154.

13. The recombinant nucleic acid of claim 1, wherein the CD300a binding domain comprises a scFv and / or the NKG2A binding domain comprises a scFv.

14. The recombinant nucleic acid of any one of claims 1-4 or 13, wherein: (a) the NKG2A binding domain comprises: (i) a NKG2A light chain variable region (NKG2A VL); and (ii) a NKG2A heavy chain variable region (NKG2A VH); and / or (b) the CD300a binding domain comprises: (i) a CD300a light chain variable region (CD300a VL); and (ii) a CD300a heavy chain variable region (CD300a VH).

15. The recombinant nucleic acid of claim 14, wherein: (a) the NKG2A light chain variable region (NKG2A VL) comprises a VL CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RASENIYSYLA (SEQ ID NO: 98), a VL CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising NAKTLAE (SEQ ID NO: 99) and a VL CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising QHHYGTPRT (SEQ ID NO: 100); (b) the NKG2A heavy chain variable region (NKG2A VH) comprises a VH CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising SYWMN (SEQ ID NO: 101), a VH CDR2 comprising 0, 1, or 2 mutations relative to an amino acidsequence comprising RIDPYDSETHYAQKLQG (SEQ ID NO: 102), and a VH CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising GGYDFDVGTLYWFFDV (SEQ ID NO: 103); (c) the CD300a light chain variable region (CD300a VL) comprises a VL CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising RASQDISNYLN (SEQ ID NO: 104) a VL CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising TSRLHS (SEQ ID NO: 105), and a VL CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising QQGNTLPWT (SEQ ID NO: 106); and (d) the CD300a heavy chain variable region (CD300a VH) comprises a VH CDR1 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising SYWMQ (SEQ ID NO: 107), a VH CDR2 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising EIDPSDSYTNYNQKFKG (SEQ ID NO: 108), and a VH CDR3 comprising 0, 1, or 2 mutations relative to an amino acid sequence comprising WGMAYGTSSYWYFDV (SEQ ID NO: 109).

16. The recombinant nucleic acid of claim 14 or claim 15, wherein (a) the NKG2A VL comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) the NKG2A VH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 3-8; (c) the CD300a VL comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 17; and (d) the CD300a VH comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 18.

17. The recombinant nucleic acid of any one of claims 14-16, wherein (a) the NKG2A VL comprises or consists of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) the NKG2A VH comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 3-8; (c) the CD300a VL comprises or consists of the amino acid sequence of SEQ ID NO: 17; and (d) the CD300a VH comprises or consists of the amino acid sequence of SEQ ID NO:

18.

18. The recombinant nucleic acid of any one of claims 14-17, wherein (a) a nucleotide sequence encoding the NKG2A VL comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10; (b) a nucleotide sequence encoding the NKG2A VH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 11-16; (c) a nucleotide sequence encoding the CD300a VL comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 19; and (d) a nucleotide sequence encoding the CD300a VH comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO:

20.

19. The recombinant nucleic acid of the immediately preceding claim, wherein (a) the nucleotide sequence encoding the NKG2A VL comprises or consists of SEQ ID NO: 9 or SEQ ID NO: 10; (b) the nucleotide sequence encoding the NKG2A VH comprises or consists of any one of SEQ ID NOs: 11-16;(c) the nucleotide sequence encoding the CD300a VL comprises or consists of SEQ ID NO: 19; and (d) the nucleotide sequence encoding the CD300a VH comprises or consists of SEQ ID NO:

20.

20. The recombinant nucleic acid of any one of the preceding claims, wherein (a) a nucleotide sequence encoding the NKG2A binding domain is codon optimized to reduce or prevent undesired recombination events; and / or (b) a nucleotide sequence encoding the CD300a binding domain is codon optimized to reduce or prevent undesired recombination events.

21. The recombinant nucleic acid of any one of claims 14-20, wherein (a) the nucleotide sequence encoding the NKG2A VL is codon optimized to reduce or prevent undesired recombination events; (b) the nucleotide sequence encoding the NKG2A VH is codon optimized to reduce or prevent undesired recombination events; (c) the nucleotide sequence encoding the CD300a VL is codon optimized to reduce or prevent undesired recombination events; and / or (d) the nucleotide sequence encoding the CD300a VH is codon optimized to reduce or prevent undesired recombination events.

22. The recombinant nucleic acid of claim 20 or claim 21, wherein the codon optimized nucleotide sequence encoding the NKG2A binding domain has at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO:

113.

23. The recombinant nucleic acid of any one of claims 20-22, wherein the codon optimized nucleotide sequence encoding the NKG2A VH has at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 112.

24. The recombinant nucleic acid of any one of claims 20-23, wherein the codon optimized nucleotide sequence encoding the NKG2A VL has at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO:

110.

25. The recombinant nucleic acid of any one of claims 14-24, wherein the recombinant nucleic acid comprises a first linker, and wherein the first linker links the nucleic acid encoding the NKG2A VL and the nucleic acid encoding the NKG2A VH.

26. The recombinant nucleic acid of any one of claims 14-25, wherein the recombinant nucleic acid comprises a second linker, and wherein the second linker links the nucleic acid encoding the CD300a VL and the nucleic acid encoding the CD300a VH.

27. The recombinant nucleic acid of any one of claims 13-26, wherein (a) the NKG2A scFv comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 118; or (b) the NKG2A scFv comprises or consists of SEQ ID NO:

118.

28. The recombinant nucleic acid of any one of claims 13-27, wherein (a) a nucleotide sequence encoding the NKG2A scFv comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 113 or 120; or (b) a nucleotide sequence encoding the NKG2A scFv comprises or consists of any one of SEQ ID NOs: 113 or 120.

29. The recombinant nucleic acid of any one of claims 13-28, wherein (a) the CD300a scFv comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 119; or (b) the CD300a scFv comprises or consists of SEQ ID NO: 119.

30. The recombinant nucleic acid of any one of claims 13-29, wherein (a) a nucleotide sequence encoding the CD300a scFv comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 121; or (b) a nucleotide sequence encoding the CD300a scFv comprises or consists of SEQ ID NO:

121.

31. The recombinant nucleic acid of any one of claims 13-30, wherein: (a) the nucleotide sequence encoding the NKG2A VL is located 5’ of the nucleotide sequence encoding the NKG2A VH, or the nucleotide sequence encoding the NKG2A VL is located 3’ of the nucleotide sequence encoding the NKG2A VH; and (b) the nucleotide sequence encoding the CD300a VL is located 5’ of the nucleotide sequence encoding the CD300a VH, or the nucleotide sequence encoding the CD300a VL is located 3’ of the nucleotide sequence encoding the CD300a VH.

32. The recombinant nucleic acid of any one of claims 13-31, wherein (a) the nucleotide sequence encoding the NKG2A scFv is located 3’ of the nucleotide sequence encoding the CD300a scFv or (b) the nucleotide sequence encoding the NKG2A scFv is located 5’ of the nucleotide sequence encoding the CD300a scFv.

33. The recombinant nucleic acid of any one of claims 13-32, wherein the recombinant nucleic acid encodes a third linker, and wherein the third linker links the NKG2A scFv and the CD300a scFv.

34. The recombinant nucleic acid of any one of claims 25-33, wherein the first linker, the second linker, and / or the third linker comprise a cleavable peptide, a glycine-serine linker, or a Whitlow / 218 linker.

35. The recombinant nucleic acid of the immediately preceding claim, wherein the glycine- serine linker comprises (Glym-Ser)n, wherein m is 3 to 6 and n is 1 to 10.

36. The recombinant nucleic acid of the immediately preceding claim, wherein m=4 and n=5.

37. The recombinant nucleic acid of claim 34, wherein the cleavable peptide is a self- cleaving peptide.

38. The recombinant nucleic acid of the immediately preceding claim, wherein the self- cleaving peptide is a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide, and wherein the self-cleaving peptide optionally comprises the amino acids glycine- serine-glycine at an N-terminus.

39. The recombinant nucleic acid of any one of claims 25-38, wherein (a) the first linker, the second linker, and / or the third linker comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 29-33 and 122; or (b) the first linker, the second linker, and / or the third linker comprises or consists of any one of SEQ ID NOs: 29-32 and 122.

40. The recombinant nucleic acid of any one of claims 25-38, wherein (a) a nucleotide sequence encoding the first linker, the second linker, and / or the third linker comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 34-37, 111, 115, and 116; or (b) a nucleotide sequence encoding the first linker, the second linker, and / or the third linker comprises or consists of any one of SEQ ID NOs:34-37, 111, 115, and 116.

41. The recombinant nucleic acid of any one of the preceding claims, further encoding a signal peptide.

42. The recombinant nucleic acid of the immediately preceding claim, wherein the signal peptide is a cell surface expression signal peptide that directs the protein product of the recombinant nucleic acid to the surface of a cell.

43. The recombinant nucleic acid of claim 41 or claim 42, wherein (a) the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 38; or (b) the signal peptide comprises or consists of SEQ ID NO:

38.

44. The recombinant nucleic acid of any one of claims 41-43, wherein (a) a nucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 39-41; or (b) a nucleotide sequence encoding the signal peptide comprises or consists of any one of SEQ ID NOs: 39-41.

45. The recombinant nucleic acid of any one of the preceding claims, wherein the nucleotide sequence encoding the signal peptide is located 5’ of the nucleotide sequence encoding the CD300a binding domain, 5’ of the nucleotide sequence encoding the NKG2A binding domain, or 5’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain.

46. The recombinant nucleic acid of any one of the preceding claims, further encoding a spacer.

47. The recombinant nucleic acid of the immediately preceding claim, wherein (a) the spacer comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 42; or (b) the spacer comprises or consists of SEQ ID NO: 42.

48. The recombinant nucleic acid of any one of claims 46 or 47, wherein (a) a nucleotide sequence encoding the spacer comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 49; or (b) a nucleotide sequence encoding the spacer comprises or consists of SEQ ID NO:

49.

49. The recombinant nucleic acid of claim 48, wherein the nucleotide sequence encoding the spacer is located (a) 3’ of the nucleotide sequence encoding the signal peptide; (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain; or (c) both (a) and (b).

50. The recombinant nucleic acid of any one of the preceding claims, wherein the recombinant nucleic acid further encodes a hinge region or (b) the recombinant nucleic acid does not encode a hinge region.

51. The recombinant nucleic acid of the immediately preceding claim, wherein the hinge region comprises a CD8 hinge, an hlgG1 hinge, an hlgG2 hinge, an hlgG3 hinge, a FACD hinge, or any combination thereof.

52. The recombinant nucleic acid of claim 50 or claim 51, wherein (a) the hinge region comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 43-48; or (b) the hinge region comprises or consists of any one of SEQ ID NOs: 43-48.

53. The recombinant nucleic acid of any one of claims 50-52, wherein (a) a nucleotide sequence encoding the hinge region comprises a nucleotide sequencehaving at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 50-55; or (b) a nucleotide sequence encoding the hinge region comprises or consists of any one of SEQ ID NOs: 50-55.

54. The recombinant nucleic acid of claim 53, wherein the nucleotide sequence encoding the hinge region is located (a) 3’ of the nucleotide sequence encoding the signal peptide; (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain; or (c) both (a) and (b).

55. The recombinant nucleic acid of any one of the preceding claims, further encoding a transmembrane domain.

56. The recombinant nucleic acid of the immediately preceding claim, wherein the transmembrane domain is a human transmembrane domain or a murine transmembrane domain.

57. The recombinant nucleic acid of claim 55 or claim 56, wherein the transmembrane domain comprises or consists of a CD8, a CD80, an ITGA, an HLA-B57, a proCAR-4, a CD28, a KIR2DL1, a PDGFRB, or a CD86 transmembrane domain.

58. The recombinant nucleic acid of any one of claims 55-57, wherein (a) the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 67-76 and 96; or (b) the transmembrane domain comprises or consists of any one of SEQ ID NOs: 67-76 and 96.

59. The recombinant nucleic acid of any one of claims 55-58, wherein (a) a nucleotide sequence encoding the transmembrane domain comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 56-65; or (b) a nucleotide sequence encoding the transmembrane domain comprises or consists of any one of SEQ ID NOs: 56-65.

60. The recombinant nucleic acid of the immediately preceding claim, wherein the nucleotide sequence encoding the transmembrane domain is located (a) 3’ of the nucleotide sequence encoding the signal peptide, (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain, (c) 3’ of the nucleotide sequence encoding the spacer; (d) 3’ of the nucleotide sequence encoding the hinge region, or (e) any combination of (a)-(d).

61. The recombinant nucleic acid of any one of the preceding claims, further encoding a cytoplasmic domain.

62. The recombinant nucleic acid of the immediately preceding claim, wherein the cytoplasmic domain is a human cytoplasmic domain or a murine cytoplasmic domain.

63. The recombinant nucleic acid of claim 61 or claim 62, wherein the cytoplasmic domain comprises or consists of a CD8v2, a CD8v1, a mCD80, a CD80, a CD86, or an HLA-B57 cytoplasmic domain.

64. The recombinant nucleic acid of any one of claims 61-63, wherein (a) the cytoplasmic domain comprises an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one ofSEQ ID NOs: 83-88; or (b) the cytoplasmic domain comprises or consists of any one of SEQ ID NOs: 83-88.

65. The recombinant nucleic acid of any one of claims 61-64, wherein (a) a nucleotide sequence encoding the cytoplasmic domain comprises a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 77-82; or (b) a nucleotide sequence encoding the cytoplasmic domain comprises or consists of any one of SEQ ID NOs: 77-82.

66. The recombinant nucleic acid of claim 65, wherein the nucleotide sequence encoding the cytoplasmic domain is located (a) 3’ of the nucleotide sequence encoding the signal peptide, (b) 3’ of the nucleotide sequence encoding the CD300a binding domain, 3’ of the nucleotide sequence encoding the NKG2A binding domain, or 3’ of the nucleotide sequence encoding the NKG2A binding domain and of the nucleotide sequence encoding the CD300a binding domain, (c) 3’ of the nucleotide sequence encoding the spacer; (d) 3’ of the nucleotide sequence encoding the hinge region, (e) 3’ of the nucleotide sequence encoding the transmembrane domain, or (f) any combination of (a)-(e).

67. The recombinant nucleic acid of any one of the preceding claims, encoding (a) an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 220-237; or (b) an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 220- 237.

68. The recombinant nucleic acid of any one of the preceding claims, (a) comprising a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 202-219; or (b) comprising or consisting of any one of SEQ ID NOs: 202-219.

69. The recombinant nucleic acid of any one of claims 1-66, encoding (a) an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22; or (b) an amino acid sequence comprising or consisting of SEQ ID NO: 21 or SEQ ID NO:

22.

70. The recombinant nucleic acid of any one of claims 1-66 or 69, (a) comprising a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 24; or (b) comprising or consisting of SEQ ID NO: 23 or SEQ ID NO:

24.

71. The recombinant nucleic acid of any one of claims 1-66, encoding (a) an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 25 or SEQ ID NO: 26; or (b) an amino acid sequence comprising or consisting of SEQ ID NO: 25 or SEQ ID NO:

26.

72. The recombinant nucleic acid of any one of claims 1-66 or 71, (a) comprising a nucleotide sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to SEQ ID NO: 27 or SEQ ID NO: 28; or (b) comprising or consisting of SEQ ID NO: 27 or SEQ ID NO:

28.

73. A vector comprising the recombinant nucleic acid of any one of the preceding claims.

74. The vector of claim 73, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenoviral vector, an adeno associated viral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.

75. The vector of any one of claims 73 or 74, wherein the recombinant nucleic acid is operably linked to a promoter.

76. The vector of the immediately preceding claim, wherein the promoter is an EF1a promoter, a CAG promoter, a PGK promoter, or a CMV promoter.

77. The vector of any one of claims 73-76, wherein a nucleic acid sequence in the vector further comprises a poly(A) sequence.

78. The vector of the immediately preceding claim, wherein the poly(A) sequence comprises a bGH poly(A) signal.

79. The vector of any one of claims 73-78, wherein a nucleic acid sequence in the vector further comprises a 3’UTR.

80. The vector of any one of claims 73-79, wherein the vector integrates into a genome at an adeno-associated virus integration site 1 (AAVS1) of the genome.

81. The vector of any one of claims 73-80, further comprising an AAVS1 right homology arm and an AAVS1 left homology arm.

82. An engineered cell comprising a vector comprising a recombinant nucleic acid encoding a construct for inhibiting NK cell cytotoxicity, wherein the construct comprises a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain.

83. An engineered cell expressing a recombinant nucleic acid encoding a construct for inhibiting NK cell cytotoxicity, wherein the construct comprises a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain.

84. An engineered cell comprising a first vector and a second vector, wherein (a) the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting NK cell cytotoxicity comprising a NKG2A binding domain comprising: (i) a NKG2A light chain variable region (NKG2A VL); and (ii) a NKG2A heavy chain variable region (NKG2A VH); and (b) the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain comprising: (i) a CD300a light chain variable region (CD300a VL); and (ii) a CD300a heavy chain variable region (CD300a VH).

85. An engineered cell comprising the recombinant nucleic acid of any one of claims 1-72, or the vector of any one of claims 73-81.

86. The engineered cell of any one of claims 82-85, wherein the recombinant nucleic acid is expressed in the engineered cell.

87. The engineered cell of any one of claims 82-86, wherein the vector is inserted into a safe harbor locus of at least one allele of the engineered cell.

88. The engineered cell of the immediately preceding claim, wherein the safe harbor locus is an AAVS1 locus.

89. The engineered cell of any one of claims 82-88, wherein the engineered cell is MHC class I deficient.

90. The engineered cell of any one of claims 82-89, ZKHUHLQ^D^ȕ^^PLFURJOREXOLQ^^%^0^^JHQH^ locus of the engineered cell is disrupted.

91. The engineered cell of any one of claims 82-90, wherein the recombinant nucleic acid or the vector is LQVHUWHG^LQWR^D^ȕ^^PLFURJOREXOLQ^^%^0^^JHQH^ORFXV^RI^WKH^HQJLQHHUHG^FHOO^92. The engineered cell of any one of claims 82-91, wherein the engineered cell is a stem cell, a progenitor cell, a cell that has been differentiated from a stem cell, or a cell that has been differentiated from a progenitor cell.

93. The engineered cell of the immediately preceding claim, wherein the stem cell is a pluripotent stem cell.

94. The engineered cell of the immediately preceding claim, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

95. The engineered cell of claim 92, wherein the progenitor cell is an early hematopoietic progenitor cell or a CD34+ progenitor cell.

96. The engineered cell of claim 92, wherein the cell that has been differentiated from a stem cell or the cell that has been differentiated from a progenitor cell is an engineered T cell.

97. The engineered cell of any one of claims 82-92, wherein the engineered cell is an engineered T cell.

98. The engineered cell of any one of claims 82-92, wherein the engineered cell is an induced pluripotent stem cell that is subsequently differentiated into an engineered T cell.

99. The engineered cell of any one of claims 96-98 wherein the engineered T cell is a chimeric antigen receptor (CAR) T cell.

100. The engineered cell of the immediately preceding claim, wherein the CAR is a CD19 CAR, a BCMA CAR, and / or a CD20 CAR.

101. The engineered cell of any one of claims 82-100, wherein the engineered cell is T cell receptor alpha constant (TRAC) deficient.

102. The engineered cell of any one of claims 82-101, wherein a T cell receptor alpha constant(TRAC) locus is disrupted in the engineered cell.

103. The engineered cell of any one of claims 99-102, wherein the CAR is inserted into a T cell receptor alpha constant (TRAC) locus in the engineered cell.

104. The engineered cell of any one of claims 96, 97, or 99-103, wherein the engineered T cell is derived from a pluripotent stem cell or a CD34+ progenitor cell.

105. The engineered cell of the immediately preceding claim, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

106. The engineered cell of any one of claims 82-105, wherein the engineered cell is an autologous cell or an allogeneic cell.

107. The engineered cell of any one of claims 82-105, wherein the engineered cell is from a donor or is derived from a stem cell of a donor, wherein the donor is not the subject.

108. The engineered cell of any one of claims 82-107, wherein the engineered cell is a human cell.

109. The engineered cell of any one of claims 82-108, wherein the engineered cell is hypoimmunogenic.

110. The engineered cell of the immediately preceding claim, wherein the engineered cell is resistant to NK cell mediated cellular cytotoxicity.

111. A composition comprising the recombinant nucleic acid of any one of claims 1-72, the vector of any one of claims 73-81, or the engineered cell of any one of claims 82-110, and one or more of a cell culture media and a buffer.

112. A pharmaceutical composition comprising the engineered cell of any one of claims 82- 110, and a pharmaceutically acceptable carrier.

113. A method of producing an engineered T cell, the method comprising differentiating the engineered cell of any one of claims 82-95 or 105-91 into a T cell.

114. A method of producing an engineered, hypoimmunogenic induced pluripotent stem cell (iPSC), the method comprising expressing in an iPSC a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing the engineered hypoimmunogenic iPSC.

115. A method of producing an engineered, hypoimmunogenic induced pluripotent stem cell (iPSC), the method comprising expressing in an iPSC a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain, thereby producing the engineered hypoimmunogenic iPSC.

116. A method of producing an engineered induced pluripotent stem cell (iPSC), the method comprising contacting an induced pluripotent stem cell (iPSC) with a first vector and a second vector, wherein the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting NK cell cytotoxicity comprising a NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, and wherein the contacting occurs under conditions whereby the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the iPSC, thereby producing the engineered iPSC.

117. A method of producing an engineered hypoimmunogenic T cell, the method comprising: (a) expressing in an iPSC a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing an engineered hypoimmunogenic iPSC; and (b) differentiating the engineered hypoimmunogenic iPSC into an engineered T cell, thereby producing the engineered T cell.

118. A method of producing an engineered hypoimmunogenic T cell, the method comprising expressing in a T cell a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing the engineered hypoimmunogenic T cell.

119. A method of producing an engineered hypoimmunogenic T cell, the method comprising expressing in a T cell a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain, thereby producing the engineered hypoimmunogenic T cell.

120. A method of producing an engineered T cell, the method comprising contacting a T cell with a first vector and a second vector, wherein the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting NK cell cytotoxicity comprising a NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting NK cell cytotoxicity comprising a CD300a binding domain, and wherein the contacting occurs under conditions whereby the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the T cell, thereby producing the engineered T cell.

121. A method of inhibiting or reducing natural killer (NK) cell cytotoxicity to an engineered cell, the method comprising expressing in the engineered cell a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby inhibiting or reducing NK cell cytotoxicity to the engineered cell.

122. A method of inhibiting or reducing natural killer (NK) cell cytotoxicity to an engineered cell, the method comprising expressing in the engineered cell a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain thereby inhibiting or reducing NK cell cytotoxicity to the engineered cell.

123. A method of inhibiting or reducing natural killer (NK) cell cytotoxicity to a T cell, themethod comprising: (a) expressing in an iPSC a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby producing an engineered iPSC; and (b) differentiating the engineered iPSC into a T cell, thereby inhibiting or reducing NK cell cytotoxicity to the T cell.

124. A method of inhibiting or reducing natural killer (NK) cell cytotoxicity to a T cell, the method comprising expressing in a T cell a recombinant nucleic acid encoding a CD300a binding domain, a NKG2A binding domain, or both a NKG2A binding domain and a CD300a binding domain, thereby inhibiting or reducing NK cell cytotoxicity to the T cell.

125. A method of inhibiting or reducing natural killer (NK) cell cytotoxicity to a T cell, the method comprising expressing in a T cell a first recombinant nucleic acid comprising a CD300a binding domain and a second recombinant nucleic acid comprising a NKG2A binding domain, thereby producing the engineered iPSC.

126. The method of any one of claims 94-125, further comprising expressing in the engineered iPSC, the iPSC, the engineered cell, or the T cell a chimeric antigen receptor (CAR).

127. The method of the immediately preceding claim, wherein the CAR is a BCMA CAR, a CD19 CAR, and / or a CD20 CAR.

128. The method of any one of claims 113-127, wherein the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell is T cell receptor alpha constant (TRAC) deficient.

129. The method of any one of claims 113-128, wherein a T cell receptor alpha constant (TRAC) locus is disrupted in the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or thehypoimmunogenic engineered T cell.

130. The method of any one of claims 113-129, wherein the CAR is inserted into a T cell receptor alpha constant (TRAC) locus in the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell.

131. The method of any one of claims 113-130, wherein the engineered cell is obtained by differentiation of a human pluripotent stem cell.

132. The method of any one of claims 113-131, wherein the CD300a binding domain comprises a single chain variable fragment (scFv), a VHH, a cytokine, a ligand, or a peptide.

133. The method of any one of claims 113-132, wherein the NKG2A binding domain comprises an scFv, a VHH, a cytokine, a ligand, or a peptide.

134. The method of claim 132 or claim 133, wherein (a) the VHH comprises the VH domain of a camelid heavy chain antibody, or (b) the peptide is an adnectin or a designed ankyrin repeat protein (DARPin).

135. The method of any one of claims 113-134, wherein the CD300a binding domain comprises a VHH (CD300a VHH) and / or the NKG2A binding domain comprises a VHH (NKG2A VHH).

136. The method of any one of claims 113-135, wherein the recombinant nucleic acid encodes the CD300a binding domain, and the CD300a binding domain comprises a VHH (CD300a VHH).

137. The method of any one of claims 113-136, wherein the NKG2A binding domain comprises a scFv comprising a NKG2A light chain variable region (NKG2A VL); and a NKG2A heavy chain variable region (NKG2A VH).

138. The method of any one of claims 113-137, wherein the CD300a binding domain comprises a scFv comprising a CD300a light chain variable region (CD300a VL); and a CD300a heavy chain variable region (CD300a VH).

139. The method of any one of claims 113-138, wherein the recombinant nucleic acid comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain comprises the recombinant nucleic acid of any one of claims 1-72.

140. The method of any one of claims 113-139, further comprising a vector comprising the recombinant nucleic acid comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain.

141. The method of the immediately preceding claim, wherein the vector comprising the recombinant nucleic acid comprising a CD300a binding domain, a NKG2A binding domain, or both a CD300a binding domain and a NKG2A binding domain comprises the vector of any one of claims 73-81.

142. The method of any one of claims 114, 115, 117, 119, 121-124, or 127-141, wherein the expressing comprises contacting the engineered cell, the iPSC, or the T cell with the recombinant nucleic acid of any one of claims 1-59, or with the vector of any one of claims 60-68, under conditions whereby the recombinant nucleic acid is expressed in the iPSC.

143. The method of any one of claims 116, 120, or 142, wherein the contacting comprises introducing the recombinant nucleic acid into the engineered cell, the iPSC, or the T cell using transfection, electroporation, transduction, or knock-in.

144. The method of the immediately preceding claim, wherein (a) the transfection comprises contacting the engineered cell, the iPSC, or the T cell with a cationic polymer and the recombinant nucleic acid;(b) the transduction comprises contacting the engineered cell, the iPSC, or the T cell with a lentivirus comprising the recombinant nucleic acid; and / or (c) the knock-in comprises contacting the engineered cell, the iPSC, or the T cell with an adeno-associated virus comprising the recombinant nucleic acid.

145. The method of any one of claims 113-144, wherein the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell is MHC class I deficient.

146. The method of any one of claims 113-145, ZKHUHLQ^D^ȕ^^PLFURJOREXOLQ^^%^0^^JHQH^ORFXV^ of the engineered cell, the iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell is disrupted.

147. The method of any one of claims 113-146, wherein the recombinant nucleic acid or the vector is LQVHUWHG^LQWR^D^ȕ^^PLFURJOREXOLQ^^%^0^^JHQH^ORFXV^RI^WKH^HQJLQHHUHG^FHOO^^WKH^ iPSC, the engineered iPSC, the engineered hypoimmunogenic iPSC, the T cell, the engineered T cell, or the hypoimmunogenic engineered T cell.

148. A method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject an effective amount of the engineered cell of any one of claims 82-110 or the pharmaceutical composition of claim 112.

149. The method of the immediately preceding claim, wherein the disease or the condition is a cancer.

150. A method of improving a clinical outcome in a subject undergoing a T cell therapy, comprising administering to the subject an effective amount of the engineered cell of any one of claims 82-110, or the pharmaceutical composition of claim 112.

151. The method of the immediately preceding claim, wherein improving a clinical outcome comprises one or more of:(a) an inhibition or reduction in NK cell cytotoxicity against the engineered cell; (b) an increased clinical response to the T cell therapy in the subject, optionally compared to the same T cell therapy not comprising the recombinant nucleic acid or compared to a T cell therapy comprising T cells (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand; (c) an increased survival rate of the engineered cell in the subject; (d) an increased persistence of the engineered cell in the subject, optionally compared to the same T cell therapy not comprising the recombinant nucleic acid or compared to a T cell therapy comprising the engineered cell (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand; (e) an improved pharmacokinetic (PK) property and / or an improved pharmacodynamic (PD) property of the T cell therapy in the subject, optionally compared to the same T cell therapy not comprising the recombinant nucleic acid or compared to a T cell therapy comprising the engineered cell (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand; and (f) a reduction in cytolysis of the engineered cell in the subject, optionally wherein the cytolysis of the engineered cell is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%.

152. The method of the immediately preceding claim, wherein the at least one alternative ligand is HLA-E, KIR3D, Lair1, Siglec7, CD107a, TIGIT, CD24, LILRB1, CD47, KIR2D, TIM3, HHLA2, or HLA-G.

153. The method of the immediately preceding claim, wherein the HLA-E is an HLA-E single chain dimer or trimer.

154. The method of any one of claims 151-153, wherein the same T cell therapy not comprising the recombinant nucleic acid or the T cell therapy comprising the engineered cell (1) not comprising the recombinant nucleic acid and (2) engineered to express at least one alternative ligand further comprises reduced or eliminated expression of CD48, CD54, CD58, and / or CD155.

155. The method of any one of claims 148-154, wherein the subject has or is at risk of having a disease or a condition that can benefit from the T cell therapy, optionally wherein the disease or the condition is a cancer.

156. The method of any one of claims 148-155, wherein the cancer is a hematologic cancer selected from B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

157. The method of any one of claims 148-155, wherein the cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma or retinoblastoma.

158. The method of any one of claims 148 or 150-155, wherein the disease or disorder is anautoimmune disease or disorder.

159. The method of the immediately preceding claim, wherein the autoimmune disease or disorder is myasthenia gravis, neuromyelitis optica spectrum disorder, Sjogren’s syndrome, scleroderma, immune nephritis, systemic lupus erythematosus, arthritis, an autoimmune-induced fibrotic condition, pemphigus vulgaris, multiple sclerosis, colitis, type 1 diabetes, graft-versus-host disease, atherosclerosis, or mucosal-dominant PV.

160. The method of any one of claims 148-159, wherein the subject is a human.

161. A kit comprising the recombinant nucleic acid of any one of claims 1-72, the vector of any one of claims 73-81, the engineered cell of any one of claims 82-110, the composition of claim 111, and / or the pharmaceutical composition of claim 112.

162. The kit of the immediately preceding claim, further comprising one or more of: (a) one or more cells, optionally wherein the one or more cells are stem cells, T cells, and / or NK cells; (b) a cell culture medium; (c) a buffer; and (d) a pharmaceutically acceptable carrier.

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