Inhibition of the cytotoxicity of natural killer cells in cell therapy.

Abstract

Recombinant nucleic acids encoding one or more binding domains specific to CD300a and / or NKG2A, as well as vectors, engineered cells, and compositions containing the recombinant nucleic acids, are provided herein. Also provided are methods for enhancing the persistence of engineered cells by using the recombinant nucleic acids, for example, by preventing natural killer (NK) cell-mediated killing of the engineered cells, methods for producing engineered cells containing the recombinant nucleic acids, and methods for treating subjects and / or improving clinical outcomes in subjects using the engineered cells.

Description

[Technical Field]

[0001] This application includes a sequence listing, which is submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on 3 April 2024, is named "01335-0001-00PCT.xml" and is 521,178 bytes in size.

[0002] This disclosure provides recombinant nucleic acids encoding one or more binding domains specific to CD300a and / or NKG2A, and a method for enhancing the persistence of engineered cells by using the recombinant nucleic acids, for example, by preventing natural killer (NK) cell-mediated killing of the engineered cells. [Background technology]

[0003] Adoptive T-cell immunotherapy is a rapidly growing field, particularly in cancer treatment. Chimeric antigen receptor (CAR) T-cell therapy is a breakthrough in the field of immuno-oncology. Generally, CAR T-cell engagement with, for example, CD19 or CD20-expressing cancer cells leads to T-cell activation, proliferation, and secretion of inflammatory cytokines and chemokines, resulting in tumor cell lysis. However, autologous CAR T-cell therapy presents several challenges, including the time required for production and the need for interim therapy in patients with ongoing disease, wide variability in the quality and quantity of T cells, and the difficulty of obtaining enough cells for re-administration. "Ready-to-use" allogeneic CAR T-cells, generated by the differentiation of pluripotent stem cells (e.g., induced pluripotent stem cells or human embryonic stem cells) into T-cells or derived from third-party donor T-cells, may offer solutions to these diverse challenges. For example, ready-to-use allogeneic CAR T-cells can be proliferated to large numbers before treatment, making them readily available to patients. This mass production offers the opportunity to rapidly, easily, and without delay re-administer new, readily available cell therapies to patients due to manufacturing or production scale. In addition, the pre-treatment manufacturing protocol enables multiple editing, manipulation, or T cell receptor selection strategies that might otherwise be difficult to adapt in an in-house environment.

[0004] However, allogeneic T cells possess exogenous immunological properties that may lead to histocompatibility considerations such as graft-versus-host disease (GvHD) and rejection or low persistence of allogeneic cells. For example, iPSC-derived T cells that have not been manipulated to be immunocompatible 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) mismatches. GvHD is thought to be primarily driven by the recognition of host peptide-HLA complexes via the αβ T cell receptor complex (αβTCR) by T cells derived from donors or iPSCs. Rejection is primarily driven by host NK cells, CD8+ T cells, CD4+ T cells, and, to a lesser extent, macrophages. In the context of CAR T cell therapy, the relative contribution of these cell types to allogeneic graft rejection may vary depending on their absolute number and rearrangement dynamics after the preconditioning regimen. In many cases, host NK cells and host CD8+ T cells are thought to recover their initial levels more rapidly than CD4+ T cells; therefore, CD8+ T cells and NK cells may play a greater role in controlling the length of the allogeneic CAR T cell therapeutic time range by becoming the first major contributor to rejection.

[0005] Due to the complexity of the immune system, manipulating low immunogenic T cells to enable adoptive cell transfer in an allogeneic environment has been a challenge. Several strategies to prevent or reduce GvHD and increase the persistence of therapeutic cells include manipulative techniques such as knocking out or disrupting the native alpha-beta T cell receptor by gene editing of the T cell receptor alpha-stationary (TRAC) locus or by inserting CAR transgenes into the TRAC locus, incorporating the receptor into targeted activated alloreactive host T cells, or disrupting HLA expression on CAR T cells by knocking out beta-2 microglobulin (β2m) and expressing HLA class I histocompatibility antigen alpha chain E (HLA-E) to evade NK cell-mediated killing. To date, the automated, scalable, and efficient production of therapeutic immune cells with effective mechanisms to protect these cells from host rejection remains a major challenge in expanding patient use of CAR T cells. [Overview of the Initiative]

[0006] The recombinant nucleic acids, vectors, engineered cells, and methods using the recombinant nucleic acids, vectors, and engineered cells of this disclosure enable the development of immune-evading (low immunogenic), versatile CAR T cells that can, for example, exhibit resistance to the cytotoxicity of host CD8+ T cells and NK cells and are suitable for adoptive cell transfer in an allogeneic environment.

[0007] The following exemplary embodiments are provided.

[0008] Embodiment 1 is a recombinant nucleic acid encoding a construct for inhibiting the cytotoxicity of NK cells, comprising a CD300a-binding domain, an NKG2A-binding domain, or both a CD300a-binding domain and an NKG2A-binding domain.

[0009] Embodiment 2 is a recombinant nucleic acid as described in Embodiment 1, wherein the CD300a binding domain comprises an antibody or a fragment thereof, a variable domain (VHH) on a heavy chain antibody, a cytokine, a ligand, or a peptide.

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

[0011] Embodiment 4 is a recombinant nucleic acid according to Embodiment 2 or Embodiment 3, wherein (a) the antibody or fragment thereof contains a single-stranded variable fragment (scFv) or VHH, or (b) the peptide is adnectin or designed ankyrin repeat protein (DARPin).

[0012] Embodiment 5 is the recombinant nucleic acid described in Embodiment 4, wherein VHH contains the VH domain of a camelid heavy chain antibody.

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

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

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

[0016] Embodiment 9 is the recombinant nucleic acid according to any one of Embodiments 2 to 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 to 172.

[0017] Embodiment 1 is the recombinant nucleic acid according to any one of Embodiments 2 to 9, wherein the CD300a VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 155 to 172.

[0018] Embodiment 11 is a recombinant nucleic acid according to any one of Embodiments 2 to 10, wherein the nucleotide sequence encoding CD300a VHH includes 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 with any one of SEQ ID NOs. 137 to 154.

[0019] Embodiment 12 is a recombinant nucleic acid according to any one of Embodiments 2 to 11, wherein the nucleotide sequence encoding CD300a VHH includes or consists of any one of the nucleotide sequences of SEQ ID NOs. 137 to 154.

[0020] Embodiment 13 is the recombinant nucleic acid according to Embodiment 1, wherein the CD300a binding domain contains scFv and / or the NKG2A binding domain contains scFv.

[0021] Embodiment 14 is, (a) The NKG2A binding domain is (i) NKG2A light chain variable region (NKG2A VL), and (ii) NKG2A heavy chain variable region (NKG2A VH) Including, and / or (b) The CD300a binding domain is (i) CD300a light chain variable region (CD300a VL), and (ii) CD300a heavy chain variable region (CD300a VH) A recombinant nucleic acid according to any one of embodiments 1 to 4 or 13, including the above.

[0022] Embodiment 15 is, (a) The NKG2A light chain variable region (NKG2A VL) includes VL CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RASENIYSYLA (SEQ ID NO: 98), VL CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing NAKTLAE (SEQ ID NO: 99), and VL CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing QHHYGTPRT (SEQ ID NO: 100), (b) The NKG2A heavy chain variable region (NKG2A VH) includes VH CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing SYWMN (SEQ ID NO: 101), VH CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RIDPYDSETHYAQKLQG (SEQ ID NO: 102), and VH CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GGYDFDVGTLYWFFDV (SEQ ID NO: 103), (c) The CD300a light chain variable region (CD300a VL) includes VL CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RASQDISNYLN (SEQ ID NO: 104), VL CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing TSRLHS (SEQ ID NO: 105), and VL CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing QQGNTLPWT (SEQ ID NO: 106). (d) The CD300a heavy chain variable region (CD300a VH) includes VH CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing SYWMQ (SEQ ID NO: 107), VH CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing EIDPSSDSYTNYNQKFKG (SEQ ID NO: 108), and VH CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing WGMAYGTSSYWYFDV (SEQ ID NO: 109). This is the recombinant nucleic acid described in Embodiment 14.

[0023] Embodiment 16 is, (a) NKG2A VL contains 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 with SEQ ID NO: 1 or SEQ ID NO: 2, (b) NKG2A VH contains 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 with any one of SEQ ID NOs: (c) CD300a VL contains 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 with respect to SEQ ID NO: 17. (d) CD300a VH contains 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 with SEQ ID NO: 18. This is a recombinant nucleic acid as described in Embodiment 14 or Embodiment 15.

[0024] Embodiment 17 is, (a) NKG2A VL contains or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, (b) NKG2A VH contains or consists of one of the amino acid sequences of sequence numbers 3-8, (c) CD300a VL contains or consists of the amino acid sequence of SEQ ID NO: 17,

[0025] (d) CD300a VH contains or consists of the amino acid sequence of SEQ ID NO: 18, This is a recombinant nucleic acid as described in any one of embodiments 14 to 16.

[0026] Embodiment 18 is, (a) The nucleotide sequence encoding NKG2A VL includes 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 with SEQ ID NO: 9 or SEQ ID NO: 10, (b) The nucleotide sequence encoding NKG2A VH includes 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 with any one of sequence numbers 11-16, (c) The nucleotide sequence encoding CD300a VL includes 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 with respect to SEQ ID NO: 19. (d) The nucleotide sequence encoding CD300a VH includes 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 with respect to SEQ ID NO: 20. This is a recombinant nucleic acid as described in any one of Embodiments 14 to 17.

[0027] Embodiment 19 is, (a) The nucleotide sequence encoding NKG2A VL contains or consists of SEQ ID NO: 9 or SEQ ID NO: 10, (b) The nucleotide sequence encoding NKG2A VH contains or consists of one of sequence numbers 11-16, (c) The nucleotide sequence encoding CD300a VL contains or consists of SEQ ID NO: 19, (d) The nucleotide sequence encoding CD300a VH contains or consists of SEQ ID NO: 20 This is a recombinant nucleic acid as described in Embodiment 18.

[0028] Embodiment 20 is, (a) The nucleotide sequence encoding the NKG2A binding domain is codon-optimized to reduce or prevent undesirable recombination events, and / or (b) The nucleotide sequence encoding the CD300a binding domain is codon-optimized to reduce or prevent undesirable recombination events. This is a recombinant nucleic acid described in any one of the prior embodiments.

[0029] Embodiment 21 is, (a) The nucleotide sequence encoding NKG2A VL is codon-optimized to reduce or prevent undesirable recombination events. (b) The nucleotide sequence encoding NKG2A VH is codon-optimized to reduce or prevent undesirable recombination events. (c) The nucleotide sequence encoding CD300a VL is codon-optimized to reduce or prevent undesirable recombination events, and / or (d) The nucleotide sequence encoding CD300a VH is codon-optimized to reduce or prevent undesirable recombination events. This is a recombinant nucleic acid as described in any one of Embodiments 14 to 20.

[0030] Embodiment 22 is a recombinant nucleic acid according to 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 with respect to SEQ ID NO: 113.

[0031] Embodiment 23 is a recombinant nucleic acid according to any one of Embodiments 20 to 22, wherein the codon-optimized nucleotide sequence encoding 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 with respect to SEQ ID NO: 112.

[0032] Embodiment 24 is a recombinant nucleic acid according to any one of Embodiments 20 to 23, wherein the codon-optimized nucleotide sequence encoding 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 with respect to SEQ ID NO: 110.

[0033] Embodiment 25 is a recombinant nucleic acid according to any one of Embodiments 14 to 24, wherein the recombinant nucleic acid includes a first linker, and the first linker links the nucleic acid encoding NKG2A VL and the nucleic acid encoding NKG2A VH.

[0034] Embodiment 26 is a recombinant nucleic acid according to any one of Embodiments 14 to 25, wherein the recombinant nucleic acid includes a second linker, the second linker linking the nucleic acid encoding CD300a VL and the nucleic acid encoding CD300a VH.

[0035] Embodiment 27 is, (a) NKG2A scFv contains an amino acid sequence that 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 with SEQ ID NO: 118, or (b) NKG2A scFv contains or consists of sequence number 118, This is a recombinant nucleic acid as described in any one of embodiments 13 to 26.

[0036] Embodiment 28 is, (a) The nucleotide sequence encoding NKG2A scFv contains 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 with either SEQ ID NO: 113 or 120, or (b) The nucleotide sequence encoding NKG2A scFv contains or consists of either sequence number 113 or 120. This is a recombinant nucleic acid as described in any one of Embodiments 13 to 27.

[0037] Embodiment 29 is, (a) CD300a scFv contains an amino acid sequence that 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 with SEQ ID NO: 119, or (b) CD300a scFv contains or consists of sequence number 119, This is a recombinant nucleic acid as described in any one of Embodiments 13 to 28.

[0038] Embodiment 30 is, (a) The nucleotide sequence encoding CD300a scFv contains 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 with respect to SEQ ID NO: 121, or (b) The nucleotide sequence encoding CD300a scFv contains or consists of SEQ ID NO: 121, This is a recombinant nucleic acid as described in any one of Embodiments 13 to 29.

[0039] Embodiment 31 is, (a) The nucleotide sequence encoding NKG2A VL is located 5' to the nucleotide sequence encoding NKG2A VH, or the nucleotide sequence encoding NKG2A VL is located 3' to the nucleotide sequence encoding NKG2A VH, (b) The nucleotide sequence encoding CD300a VL is located 5' to the nucleotide sequence encoding CD300a VH, or the nucleotide sequence encoding CD300a VL is located 3' to the nucleotide sequence encoding CD300a VH. This is a recombinant nucleic acid as described in any one of embodiments 13 to 30.

[0040] Embodiment 32 is a recombinant nucleic acid according to any one of Embodiments 13 to 31, wherein (a) the nucleotide sequence encoding NKG2A scFv is located on the 3' side of the nucleotide sequence encoding CD300a scFv, or (b) the nucleotide sequence encoding NKG2A scFv is located on the 5' side of the nucleotide sequence encoding CD300a scFv.

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

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

[0043] Embodiment 35 is a glycine-serine linker (Gly m -Ser) n This is a recombinant nucleic acid according to Embodiment 34, comprising (where m is 3 to 6 and n is 1 to 10).

[0044] Embodiment 36 is the recombinant nucleic acid described in Embodiment 35, wherein m=4 and n=5.

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

[0046] Embodiment 38 is a recombinant nucleic acid according to Embodiment 37, wherein the self-cleaving peptide is a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide, and the self-cleaving peptide optionally contains the amino acid glycine-serine-glycine at its N-terminus.

[0047] Embodiment 39 is, (a) The first linker, the second linker, and / or the third linker contain 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 with any one of SEQ ID NOs. 29-33 and 122, or (b) The first linker, the second linker, and / or the third linker include or consist of one of sequence numbers 29-32 and 122, This is a recombinant nucleic acid as described in any one of embodiments 25 to 38.

[0048] Embodiment 40 is, (a) The nucleotide sequences encoding the first linker, the second linker, and / or the third linker contain nucleotide sequences 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 with any one of SEQ ID NOs. 34-37, 111, 115, and 116, or (b) The nucleotide sequences encoding the first linker, the second linker, and / or the third linker include or consist of one of the sequence numbers 34-37, 111, 115, and 116. This is a recombinant nucleic acid as described in any one of embodiments 25 to 38.

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

[0050] Embodiment 42 is the recombinant nucleic acid according to Embodiment 41, wherein the signal peptide is a cell surface expression signal peptide that induces the protein product of the recombinant nucleic acid to the cell surface.

[0051] Embodiment 43 is, (a) The signal peptide contains 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 with SEQ ID NO: 38, or (b) The signal peptide contains or consists of SEQ ID NO: 38. This is a recombinant nucleic acid as described in Embodiment 41 or Embodiment 42.

[0052] Embodiment 44 is, (a) The nucleotide sequence encoding the signal peptide contains 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 with any one of SEQ ID NOs. 39-41, or (b) The nucleotide sequence encoding the signal peptide contains or consists of one of the sequence numbers 39-41, This is a recombinant nucleic acid as described in any one of embodiments 41 to 43.

[0053] Embodiment 45 is a recombinant nucleic acid according to any one of the prior embodiments, wherein the nucleotide sequence encoding the signal peptide is located at the 5' end of the nucleotide sequence encoding the CD300a binding domain, at the 5' end of the nucleotide sequence encoding the NKG2A binding domain, or at the 5' end of both the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain.

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

[0055] Embodiment 47 is, (a) The spacer contains 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 with respect to SEQ ID NO: 42, or (b) The spacer includes or consists of Sequence ID No. 42, This is the recombinant nucleic acid described in Embodiment 46.

[0056] Embodiment 48 is, (a) The nucleotide sequence encoding the spacer contains 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 with respect to SEQ ID NO: 49, or (b) The nucleotide sequence encoding the spacer contains or consists of SEQ ID NO: 49 This is a recombinant nucleic acid as described in Embodiment 46 or 47.

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

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

[0059] Embodiment 51 is a recombinant nucleic acid according to Embodiment 50, wherein the hinge region includes a CD8 hinge, an hIgG1 hinge, an hIgG2 hinge, an hIgG3 hinge, an FACD hinge, or any combination thereof.

[0060] Embodiment 52 is, (a) The hinge region contains 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 with any one of sequence numbers 43-48, or (b) The hinge region includes or consists of one of sequence numbers 43-48, This is a recombinant nucleic acid as described in Embodiment 50 or Embodiment 51.

[0061] Embodiment 53 is, (a) The nucleotide sequence encoding the hinge region contains 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 with any one of SEQ ID NOs. 50-55, or (b) The nucleotide sequence encoding the hinge region contains or consists of one of sequence numbers 50-55, This is a recombinant nucleic acid as described in any one of embodiments 50 to 52.

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

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

[0064] Embodiment 56 is the recombinant nucleic acid according to Embodiment 55, wherein the transmembrane domain is a human transmembrane domain or a mouse transmembrane domain.

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

[0066] Embodiment 58 is, (a) The transmembrane domain contains 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 with any one of SEQ ID NOs. 67-76 and 96, or (b) The transmembrane domain contains or consists of one of sequence numbers 67-76 and 96, This is a recombinant nucleic acid according to any one of embodiments 55 to 57.

[0067] Embodiment 59 is, (a) The nucleotide sequence encoding the transmembrane domain contains 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 with any one of SEQ ID NOs. 56-65, or (b) The nucleotide sequence encoding the transmembrane domain contains or consists of one of the sequence numbers 56-65, This is a recombinant nucleic acid as described in any one of embodiments 55 to 58.

[0068] Embodiment 60 is a recombinant nucleic acid according to Embodiment 59, wherein the nucleotide sequence encoding the transmembrane domain is located at (a) the 3' end of the nucleotide sequence encoding the signal peptide, (b) the 3' end of the nucleotide sequence encoding the CD300a binding domain, the 3' end of the nucleotide sequence encoding the NKG2A binding domain, or the 3' end of the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain, (c) the 3' end of the nucleotide sequence encoding the spacer, (d) the 3' end of the nucleotide sequence encoding the hinge region, or (e) any combination of (a) to (d).

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

[0070] Embodiment 62 is the recombinant nucleic acid according to Embodiment 61, wherein the cytoplasmic domain is a human cytoplasmic domain or a mouse cytoplasmic domain.

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

[0072] Embodiment 64 is, (a) The cytoplasmic domain contains 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 with any one of SEQ ID NOs. 83-88, or (b) The cytoplasmic domain contains or consists of one of sequence numbers 83-88, This is a recombinant nucleic acid as described in any one of embodiments 61 to 63.

[0073] Embodiment 65 is, (a) The nucleotide sequence encoding the cytoplasmic domain contains 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 with any one of SEQ ID NOs. 77-82, or (b) The nucleotide sequence encoding the cytoplasmic domain contains or consists of one of sequence numbers 77-82, This is a recombinant nucleic acid as described in any one of embodiments 61 to 64.

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

[0075] Embodiment 67 is, (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 with any one of sequence numbers 220-237, or (b) an amino acid sequence containing or consisting of one of sequence numbers 220-237 This is a recombinant nucleic acid described in any one of the prior embodiments, which encodes [the specified character].

[0076] Embodiment 68 is, (a) 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 sequence numbers 202-219, or (b) containing or consisting of one of sequence numbers 202-219 This is a recombinant nucleic acid described in any one of the prior embodiments.

[0077] Embodiment 69 is, (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 with SEQ ID NO: 21 or SEQ ID NO: 22, or (b) an amino acid sequence containing or consisting of SEQ ID NO: 21 or SEQ ID NO: 22 This is a recombinant nucleic acid according to any one of embodiments 1 to 66, which codes for [the specified character].

[0078] Embodiment 70 is, (a) 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 with respect to SEQ ID NO: 23 or SEQ ID NO: 24, or (b) containing or consisting of sequence number 23 or sequence number 24, This is a recombinant nucleic acid as described in any one of Embodiments 1 to 66 or 69.

[0079] Embodiment 71 is, (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 with SEQ ID NO: 25 or SEQ ID NO: 26, or (b) an amino acid sequence containing or consisting of SEQ ID NO: 25 or SEQ ID NO: 26 This is a recombinant nucleic acid according to any one of embodiments 1 to 66, which codes for [the specified character].

[0080] Embodiment 72 is, (a) 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 with respect to SEQ ID NO: 27 or SEQ ID NO: 28, or (b) containing or consisting of sequence number 27 or sequence number 28, This is a recombinant nucleic acid according to any one of embodiments 1 to 66 or 71.

[0081] Embodiment 73 is a vector containing recombinant nucleic acid as described in any one of the prior embodiments.

[0082] Embodiment 74 is a vector according to Embodiment 73, which is a DNA vector, RNA vector, plasmid, lentiviral vector, adenovirus vector, adeno-associated virus vector, Rous sarcoma virus (RSV) vector, or retroviral vector.

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

[0084] Embodiment 76 is the vector according to Embodiment 75, wherein the promoter is the EF1a promoter, the CAG promoter, the PGK promoter, or the CMV promoter.

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

[0086] Embodiment 78 is the vector according to Embodiment 77, wherein the poly(A) sequence includes a bGH poly(A) signal.

[0087] Embodiment 79 is a vector according to any one of Embodiments 73 to 78, wherein the nucleic acid sequence in the vector further comprises a 3'UTR.

[0088] Embodiment 80 is a vector according to any one of Embodiments 73 to 79, which is incorporated into the genome at adeno-associated virus integration site 1 (AAVS1) of the genome.

[0089] Embodiment 81 is a vector according to any one of embodiments 73 to 80, further including an AAVS1 right homology arm and an AAVS1 left homology arm.

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

[0092] Embodiment 84 is an engineered cell comprising a first vector and a second vector, (a) The first vector is (i) NKG2A light chain variable region (NKG2A VL), and (ii) NKG2A heavy chain variable region (NKG2A VH) It comprises a first recombinant nucleic acid encoding a first construct for inhibiting the cytotoxicity of NK cells, which includes an NKG2A binding domain, (b) The second vector is (i) CD300a light chain variable region (CD300a VL), and (ii) CD300a heavy chain variable region (CD300a VH) The manipulated cells contain a second recombinant nucleic acid encoding a second construct for inhibiting the cytotoxicity of NK cells, which includes a CD300a-binding domain.

[0093] Embodiment 85 is an engineered cell comprising a recombinant nucleic acid as described in any one of Embodiments 1 to 72 or a vector as described in any one of Embodiments 73 to 81.

[0094] Embodiment 86 is an engineered cell according to any one of Embodiments 82 to 85, in which recombinant nucleic acid is expressed in the engineered cell.

[0095] Embodiment 87 is an engineered cell according to any one of Embodiments 82 to 86, wherein the vector is inserted into the safe harbor locus of at least one allele of the engineered cell.

[0096] Embodiment 88 is the manipulated cell described in Embodiment 87, wherein the safe harbor locus is the AAVS1 locus.

[0097] Embodiment 89 is an engineered cell according to any one of Embodiments 82 to 88, which is MHC class I deficient.

[0098] Embodiment 90 is an engineered cell according to any one of Embodiments 82 to 89, wherein the β2 microglobulin (B2M) gene locus of the engineered cell is disrupted.

[0099] Embodiment 91 is an engineered cell according to any one of Embodiments 82 to 90, wherein a recombinant nucleic acid or vector is inserted into the β2 microglobulin (B2M) gene locus of the engineered cell.

[0100] Embodiment 92 is an engineered cell according to any one of Embodiments 82 to 91, which is a stem cell, a progenitor cell, a cell differentiated from a stem cell, or a cell differentiated from a progenitor cell.

[0101] Embodiment 93 is the manipulated cells described in Embodiment 92, wherein the stem cells are pluripotent stem cells.

[0102] Embodiment 94 is the manipulated cell described in Embodiment 93, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0103] Embodiment 95 is the manipulated cells described in Embodiment 92, wherein the progenitor cells are early hematopoietic progenitor cells or CD34+ progenitor cells.

[0104] Embodiment 96 is the manipulated cell described in Embodiment 92, wherein the manipulated T cell is a cell differentiated from a stem cell or a cell differentiated from a progenitor cell.

[0105] Embodiment 97 is an engineered cell, which is an engineered T cell, as described in any one of Embodiments 82 to 92.

[0106] Embodiment 98 is an engineered cell according to any one of Embodiments 82 to 92, which is an induced pluripotent stem cell that later differentiates into engineered T cells.

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

[0108] Embodiment 100 is the manipulated cell described in Embodiment 99, wherein the CAR is a CD19 CAR, a BCMA CAR, and / or a CD20 CAR.

[0109] Embodiment 101 is an engineered cell according to any one of Embodiments 82 to 100, which is deficient in T cell receptor alpha (TRAC).

[0110] Embodiment 102 is an engineered cell according to any one of Embodiments 82 to 101, wherein the T cell receptor alpha constant (TRAC) locus is disrupted in the engineered cell.

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

[0112] Embodiment 104 is an engineered cell according to any one of Embodiments 96, 97, or 99 to 103, wherein the engineered T cells are derived from pluripotent stem cells or CD34+ progenitor cells.

[0113] Embodiment 105 is the manipulated cell described in Embodiment 104, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0114] Embodiment 106 is an engineered cell according to any one of Embodiments 82 to 105, which is either an autologous cell or an allogeneic cell.

[0115] Embodiment 107 is an engineered cell according to any one of Embodiments 82 to 105, wherein the engineered cells are donor-derived or derived from donor stem cells, and the donor is not the target.

[0116] Embodiment 108 is a human cell, which is an engineered cell according to any one of Embodiments 82 to 107.

[0117] Embodiment 109 is a manipulated cell according to any one of Embodiments 82 to 108, which is low immunogenic.

[0118] Embodiment 110 is the manipulated cells described in Embodiment 109, which are resistant to NK cell-mediated cytotoxicity.

[0119] Embodiment 111 is a composition comprising a recombinant nucleic acid according to any one of Embodiments 1 to 72, a vector according to any one of Embodiments 73 to 81, or an engineered cell according to any one of Embodiments 82 to 110, and one or more of a cell culture medium and a buffer.

[0120] Embodiment 112 is a pharmaceutical composition comprising the manipulated cells described in any one of Embodiments 82 to 110 and a pharmaceutically acceptable carrier.

[0121] Embodiment 113 is a method for producing engineered T cells, comprising differentiating the engineered cells described in any one of Embodiments 82 to 95 or 105 to 91 into T cells.

[0122] Embodiment 114 is a method for producing engineered low immunogenic induced pluripotent stem cells (iPSCs), comprising expressing a recombinant nucleic acid encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in iPSCs, thereby producing engineered low immunogenic iPSCs.

[0123] Embodiment 115 is a method for producing manipulated low immunogenic induced pluripotent stem cells (iPSCs), comprising expressing a first recombinant nucleic acid containing a CD300a binding domain and a second recombinant nucleic acid containing an NKG2A binding domain in iPSCs, thereby producing manipulated low immunogenic iPSCs.

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

[0125] Embodiment 117 is a method for producing manipulated low immunogenic T cells, (a) Expressing recombinant nucleic acids encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in iPSCs, thereby creating manipulated low-immunogenic iPSCs, (b) Differentiating engineered low immunogenic iPSCs into engineered T cells, thereby generating engineered T cells. This method includes [something].

[0126] Embodiment 118 is a method for producing engineered low immunogenic T cells, comprising expressing recombinant nucleic acids encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in T cells, thereby producing engineered low immunogenic T cells.

[0127] Embodiment 119 is a method for producing engineered low immunogenic T cells, comprising expressing a first recombinant nucleic acid containing a CD300a binding domain and a second recombinant nucleic acid containing an NKG2A binding domain in T cells, thereby producing engineered low immunogenic T cells.

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

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

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

[0131] Embodiment 123 is a method for inhibiting or reducing the cytotoxicity of natural killer (NK) cells against T cells, (a) Expressing recombinant nucleic acids encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in iPSCs, thereby creating manipulated iPSCs, (b) Differentiating the manipulated iPSCs into T cells, thereby inhibiting or reducing the cytotoxicity of NK cells against T cells. This method includes [something].

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

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

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

[0135] Embodiment 127 is the method of Embodiment 126, wherein the CARs are BCMA CAR, CD19 CAR, and / or CD20 CAR.

[0136] Embodiment 128 is the method according to any one of Embodiments 113 to 127, wherein the engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells are T cell receptor alpha constant (TRAC) deficient.

[0137] Embodiment 129 is a method according to any one of Embodiments 113 to 128, wherein the T cell receptor alpha constant (TRAC) locus is disrupted in engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells.

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

[0139] Embodiment 131 is the method according to any one of Embodiments 113 to 130, wherein the manipulated cells are obtained by differentiation of human pluripotent stem cells.

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

[0141] Embodiment 133 is the method according to any one of Embodiments 113 to 132, wherein the NKG2A binding domain comprises scFv, VHH, cytokine, ligand, or peptide.

[0142] Embodiment 134 is, The method according to Embodiment 132 or Embodiment 133, wherein (a) VHH contains the VH domain of a camelid heavy chain antibody, or (b) the peptide is adonectin or a programmed ankyrin repeat protein (DARPin).

[0143] Embodiment 135 is the method according to any one of Embodiments 113 to 134, wherein the CD300a binding domain includes VHH(CD300a VHH) and / or the NKG2A binding domain includes VHH(NKG2A VHH).

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

[0145] Embodiment 137 is the method according to any one of Embodiments 113 to 136, wherein the NKG2A binding domain includes an scFv comprising an NKG2A light chain variable region (NKG2A VL) and an NKG2A heavy chain variable region (NKG2A VH).

[0146] Embodiment 138 is the method according to any one of Embodiments 113 to 137, wherein the CD300a binding domain includes an scFv comprising a CD300a light chain variable region (CD300a VL) and a CD300a heavy chain variable region (CD300a VH).

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

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

[0149] Embodiment 141 is the method of Embodiment 140, wherein the vector comprising a recombinant nucleic acid containing a CD300a-binding domain, an NKG2A-binding domain, or both a CD300a-binding domain and an NKG2A-binding domain comprises the vector described in any one of Embodiments 73 to 81.

[0150] Embodiment 142 is a method according to any one of Embodiments 114, 115, 117, 119, 121 to 124, or 127 to 141, wherein expression is achieved by contacting an engineered cell, iPSC, or T cell with a recombinant nucleic acid described in any one of Embodiments 1 to 59, or a vector described in any one of Embodiments 60 to 68, under conditions in which the recombinant nucleic acid is expressed in the iPSC.

[0151] Embodiment 143 is a method according to any one of Embodiments 116, 120, or 142, wherein contact involves introducing recombinant nucleic acid into engineered cells, iPSCs, or T cells using transfection, electroporation, transduction, or knock-in.

[0152] Embodiment 144 is, (a) Transfection comprising contacting engineered cells, iPSCs, or T cells with a cationic polymer and recombinant nucleic acid, (b) Transduction involves contacting engineered cells, iPSCs, or T cells with a lentivirus containing recombinant nucleic acid, and / or (c) The knock-in procedure involves contacting the manipulated cells, iPSCs, or T cells with an adeno-associated virus containing recombinant nucleic acid. This is the method described in Embodiment 143.

[0153] Embodiment 145 is the method according to any one of Embodiments 113 to 144, wherein the engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells are MHC class I deficient.

[0154] Embodiment 146 is a method according to any one of Embodiments 113 to 145, wherein the β2 microglobulin (B2M) locus of the engineered cell, iPSC, engineered iPSC, engineered hypoimmunogenic iPSC, T cell, engineered T cell, or hypoimmunogenic engineered T cell is disrupted.

[0155] Embodiment 147 is a method according to any one of Embodiments 113 to 146, wherein a recombinant nucleic acid or vector is inserted into the β2 microglobulin (B2M) locus of an engineered cell, iPSC, engineered iPSC, engineered hypoimmunogenic iPSC, T cell, engineered T cell, or hypoimmunogenic engineered T cell.

[0156] Embodiment 148 is a method for treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of manipulated cells described in any one of Embodiments 82 to 110 or a pharmaceutical composition described in Embodiment 112.

[0157] Embodiment 149 is the method of Embodiment 148, wherein the disease or condition is cancer.

[0158] Embodiment 150 is a method for improving clinical outcomes in a subject receiving T-cell therapy, comprising administering to the subject an effective amount of manipulated cells described in any one of Embodiments 82 to 110, or the pharmaceutical composition described in Embodiment 112.

[0159] Embodiment 151 is capable of improving clinical outcomes. (a) Inhibition or reduction of NK cell cytotoxicity against manipulated cells, (b) Increased clinical response to T cell therapy in subjects compared to the same T cell therapy without recombinant nucleic acid, or compared to T cell therapy containing (1) T cells without recombinant nucleic acid and (2) T cells engineered to express at least one alternative ligand. (c) Increase in the viability of manipulated cells in the subject, (d) Increased persistence of engineered cells in the subject compared to the same T-cell therapy without recombinant nucleic acid, or compared to T-cell therapy containing engineered cells that (1) do not contain recombinant nucleic acid and (2) are engineered to express at least one alternative ligand. (e) Improvements in the pharmacokinetic (PK) and / or pharmacodynamic (PD) properties of T cell therapy in a subject, compared to the same T cell therapy without recombinant nucleic acid at random, or compared to T cell therapy containing engineered cells that (1) do not contain recombinant nucleic acid and (2) are engineered to express at least one alternative ligand, and (f) A reduction in cytolysis of manipulated cells in the subject, wherein the cytolysis of manipulated cells 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%. The method according to Embodiment 150, which includes one or more of the above.

[0160] Embodiment 152 is the method according to Embodiment 151, wherein 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 according to Embodiment 152, wherein the HLA-E is an HLA-E single-stranded dimer or trimer.

[0162] Embodiment 154 is the same T-cell therapy without recombinant nucleic acids, or the method according to any one of Embodiments 151 to 153, wherein the T-cell therapy comprising engineered cells that (1) do not contain recombinant nucleic acids and (2) are engineered to express at least one alternative ligand further comprises a reduction or loss of expression of CD48, CD54, CD58, and / or CD155.

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

[0164] Embodiment 156 describes cancer as B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell leukemia The method according to any one of Embodiments 148 to 155, wherein the hematological malignancy is selected from follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, or preleukemia.

[0165] Embodiment 157 describes cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, 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, and pheochromocytoma. The method according to any one of Embodiments 148 to 155, wherein the cancer is sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, seminomasm, bladder carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroneurioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0166] Embodiment 158 ​​is the method according to any one of Embodiments 148 or 150 to 155, wherein the disease or disorder is an autoimmune disease or disorder.

[0167] Embodiment 159 is the method according to Embodiment 158, wherein the autoimmune disease or disorder is myasthenia gravis, neuromyelitis optica spectrum disorder, Sjögren's syndrome, scleroderma, immunonephritis, systemic lupus erythematosus, arthritis, autoimmune-induced fibrosis, pemphigus vulgaris, multiple sclerosis, colitis, type 1 diabetes mellitus, graft-versus-host disease, atherosclerosis, or mucosal-dominant PV.

[0168] Embodiment 160 is a method according to any one of Embodiments 148 to 159, wherein the subject is a human.

[0169] Embodiment 161 is a kit comprising a recombinant nucleic acid according to any one of Embodiments 1 to 72, a vector according to any one of Embodiments 73 to 81, manipulated cells according to any one of Embodiments 82 to 110, a composition according to Embodiment 111, and / or a pharmaceutical composition according to Embodiment 112.

[0170] Embodiment 162 is, (a) One or more types of cells, which are optionally selected to be stem cells, T cells, and / or NK cells, (b) Cell culture medium, (c) buffer, and (d) Pharmaceutically acceptable carriers The kit according to Embodiment 161 further includes one or more of the following.

[0171] The numbered items in Example 31 provide further support and explanation for the embodiments described herein. [Brief explanation of the drawing]

[0172] [Figure 1] This figure shows four exemplary and non-limiting recombinant nucleic acid designs described herein, including a promoter (e.g., EF1a), a CD300a TASR and / or an NKG2A TASR, and, in the case of exemplary designs including both the CD300a TASR and the NKG2A TASR, a linker (e.g., P2A). [Figure 2-1] This figure shows the killing of MHC class I-deficient T cells by NK cells. Figure 2A shows the phenotypic analysis of wild-type, CD59 knockout, CIITA knockout, and B2M knockout cells using flow cytometry. Figure 2B shows the results of the cytotoxicity assay of NK cells, where T cell survival % was lowest in B2M knockout cells. [Figure 2-2] (As stated above.) [Figure 3-1] This figure shows the effectiveness of two NKG2A construct designs compared to a control, the HLA-E single-strand trimer. Figure 3A shows schematic diagrams of the HLA-E and NKG2A scFv mRNA constructs, as well as phenotypic analysis of cells into which the constructs were introduced by electroporation, using flow cytometry. Figure 3B shows that both NKG2A scFv constructs provided protection against NK cell killing. The NKG2A construct containing the CD8 transmembrane domain appeared to function better than the NKG2A construct containing IgG1B7. [Figure 3-2] (As stated above.) [Figure 4-1] This figure shows that the efficacy of the NKG2A scFv construct containing the CD8 transmembrane domain (hereinafter referred to as the Z199 construct) was reproducible. Figure 4A shows schematic diagrams of the HLA-E and NKG2A scFv mRNA constructs, as well as phenotypic analysis of cells into which the constructs were introduced by electroporation, using flow cytometry. The negative control data shown in this figure are the same as those shown in Figure 8A. Figure 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 those shown in Figure 8B. [Figure 4-2] (As stated above.) [Figure 5] This figure shows that the humanized anti-NKG2A (clone Z270) scFv is stable and functional. Figure 5A shows the phenotypic analysis of cells electroporated with Z199, humanized Z199, humanized Z270, and negative control constructs using flow cytometry. Figure 5B shows that all NKG2A scFv constructs provided protection against NK cell killing. [Figure 6-1] This figure shows that the efficacy of the NKG2A humanized Z270 construct was reproducible. Figure 6A shows phenotypic analysis of cells into which Z270 or HLA-E constructs were introduced by electroporation, using flow cytometry. The negative control data shown in this figure is the same as that shown in Figure 10A. Figure 6B shows that the humanized Z270 construct provided protection against NK cell killing. [Figure 6-2] (As stated above.) [Figure 7-1]This figure shows the effectiveness of two CD300a construct designs compared to a control HLA-E single-strand trimer. Figure 7A shows schematic diagrams of the HLA-E and CD300a scFv mRNA constructs, as well as phenotypic analysis of cells into which the constructs were introduced by electroporation, using flow cytometry. Figure 7B shows that both CD300a scFv constructs provided protection against NK cell killing. The CD300a construct containing the CD8 transmembrane domain appeared to function better than the CD300a construct containing IgG1B7. [Figure 7-2] (As stated above.) [Figure 8-1] This figure shows that the efficacy of the CD300a scFv construct containing the CD8 transmembrane domain (hereinafter referred to as the TX49 construct) was reproducible. Figure 8A shows a schematic diagram of the CD300a scFv mRNA construct and phenotypic analysis of cells into which the construct was introduced by electroporation, using flow cytometry. The negative control data shown in this figure are the same as those shown in Figure 4A. Figure 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 those shown in Figure 4B. [Figure 8-2] (As stated above.) [Figure 9-1] This figure shows the successful humanization of the TX49 construct. Figure 9A shows the amino acid sequence alignment of the VH (top) and VL (bottom) regions of the mouse anti-human CD300a antibody clone TX49 with two humanized versions named "humTX49_v1" and "humTX49_v2". Figure 9B shows the phenotypic analysis of cells into which the construct was introduced by electroporation, using flow cytometry. Figure 9C shows that both humanized TX49 constructs (v1 and v2) provided protection against NK cell killing. [Figure 9-2] (As stated above.) [Figure 9-3] (As stated above.) [Figure 10-1]This figure shows that the efficacy of the humanized TX49 construct v1 (hereinafter referred to as humTX49) was reproducible. Figure 10A shows the phenotypic analysis of cells into which humTX49 and HLA-E constructs were introduced by electroporation, using flow cytometry. The negative control data shown in this figure is the same as that shown in Figure 6A. Figure 10B shows that the humTX49 construct provided some protection against NK cell killing. [Figure 10-2] (As stated above.) [Figure 11-1] This figure shows that humTX49 acts synergistically with humZ270 to provide protection from NK cells. Figure 11A shows phenotypic analysis of cells electroporated with humTX49, humZ270, and / or HLA-E constructs using flow cytometry. The left panel shows staining for HLA-E in cells electroporated with or without HLA-E constructs. The center panel shows staining for CD300a in the shown electroporated cells. The right panel shows staining for NKG2A in the shown electroporated cells. Figure 11B shows that cells expressing both the humZ270 construct and the humTX49 construct were protected from NK cell killing to the same extent as cells expressing the HLA-E construct. [Figure 11-2] (As stated above.) [Figure 12-1]This figure shows the efficacy of the bispecific humTX49-humZ270 construct in protecting manipulated cells from NK cell cytotoxicity compared to HLA-E. Figure 12A shows schematic diagrams of two candidate bispecific constructs. "humTX49-humZ270" contains human GM-CSF signal peptide ("GM-CSF SP"), followed by humTX49 scFV (vL-linker-vH orientation), (G4S)5 linker, then humZ270 scFV (vL-linker-vH orientation), followed by CD8 stalk and transmembrane domain ("CD8 TM"), T2A autocleavage peptide, and GFP. The "humZ270-humTX49" construct is similar except that the order of the two scFVs is reversed. Figure 12B shows phenotypic analysis of cells into which the constructs were introduced by electroporation, using flow cytometry. Figure 12C shows that cells expressing either construct were protected against NK cell killing, and that the humTX49-humZ270 construct provided protection equivalent to that provided by HLA-E. [Figure 12-2] (As stated above.) [Figure 13-1] This figure shows that agonism by TX49 (CD300a) and Z270 (NKG2A) was synergistic and more potent than agonism by HLA-E. Figures 13A and 13B demonstrate superior protection against NK cell killing in cells expressing the humTX49-humZ270 construct compared to HLA-E. Cells into which equimolar amounts of humTX49 and humZ270 constructs were separately introduced by electroporation were also protected against NK cell killing. [Figure 13-2] (As stated above.) [Figure 14-1]This figure shows that physiological expression of HLA-E, humZ270, and humZ270-humTX49 constructs at the AAVS1 locus in human primary T cells provides equivalent protection against NK cell killing. Figure 14A shows that cells expressing HLA-E, humZ270, or humZ270-humTX49 constructs were equivalently protected against NK cell killing, while cells expressing the humTX49 construct were not. Figure 14B shows the levels of NKG2A / NKG2C expression in donor NK cells. Figure 14C shows the expression constructs and phenotypic analysis of cells into which the constructs were introduced by electroporation using flow cytometry. [Figure 14-2] (As stated above.) [Figure 14-3] (As stated above.) [Figure 15-1] This figure shows that the mCD80 transmembrane domain affects hTX49-mediated NK inhibition, and that transmembrane homodimerization does not appear to affect function. Figure 15A shows a schematic diagram of the experimental setup. Figure 15B shows a schematic diagram of the CD300a TASR constructs used to introduce into T cells by electroporation. Figure 15C shows the expression levels measured for each construct. Figure 15D shows that cells expressing any of the constructs were protected from NK cell killing. [Figure 15-2] (As stated above.) [Figure 16-1] This figure shows that the cytoplasmic tail of CD8a or mCD80 appears to enhance the expression and function of hTX49. Figure 16A shows a schematic diagram of the experimental setup. Figure 16B shows a schematic diagram of the CD300a TASR construct used to introduce into T cells by electroporation. Figure 16C shows the expression levels measured for each construct. Figure 16D shows that cells expressing any of the constructs (except the t8 construct, which does not contain either a transmembrane or cytoplasmic domain) were protected from NK cell killing. [Figure 16-2] (As stated above.) [Figure 17-1]This figure shows that reducing the hinge size increases the functional potency of hTX49. Figure 17A shows a schematic diagram of the experimental setup. Figure 17B shows a schematic diagram of the CD300a TASR constructs used to introduce into T cells by electroporation. Figure 17C shows the expression levels measured for each construct. Figure 17D shows that cells expressing any of the constructs (except the t8 construct, which does not contain either a transmembrane or cytoplasmic domain) were protected from NK cell killing. [Figure 17-2] (As stated above.) [Figure 17-3] (As stated above.) [Figure 18-1] This figure shows that the optimized CD300a TASR design (t12) improves cell persistence compared to the original construct (v1). Figure 18A shows a schematic diagram of the experimental setup. Figure 18B shows a schematic diagram of the CD300a TASR constructs used to introduce T cells by electroporation. Figure 18C shows the expression levels measured for each construct using flow cytometry. Figure 18D shows that cells expressing any of the constructs (the t12 construct, which did not include the hinge region, performed best) were protected against NK cell killing. [Figure 18-2] (As stated above.) [Figure 19-1] This figure shows that the optimized CD300a TASR design (t12) provides protection against NKG2C+ / NKG2A-NK cell donors. Figure 19A shows a schematic diagram of the experimental setup. Figure 19B shows a schematic diagram of the CD300a TASR constructs used to introduce T cells by electroporation. Figure 19C shows the expression levels measured for each construct using flow cytometry. Figure 19D shows that cells expressing both the NKG2A_v1 construct and the CD300a_t12 construct were protected against NK cell killing. [Figure 19-2] (As stated above.) [Figure 20-1]This figure shows that the mouse CD80 cytoplasmic domain enhanced the expression and function of CD300a TASR, regardless of the transmembrane domain. Figure 20A shows a schematic diagram of the experimental setup. Figure 20B shows a schematic diagram of the CD300a TASR constructs used to introduce into T cells by electroporation. Figure 20C shows the expression levels measured for each construct using flow cytometry. Figure 20D shows that cells expressing V2, t12, t14, or t18 constructs containing the mouse CD80 cytoplasmic domain were protected against NK cell killing. [Figure 20-2] (As stated above.) [Figure 21-1] This figure shows that the mCD80 cytoplasmic domain performed better than the hCD80 and hCD86 cytoplasmic domains in TASR expression. Figure 21A shows a schematic diagram of the experimental setup. Figure 21B shows a schematic diagram of the CD300a TASR construct used to introduce into T cells by electroporation. Figure 21C shows the expression levels measured for each construct using flow cytometry. [Figure 21-2] (As stated above.) [Figure 22-1] This figure shows that the hCD80 and hCD86 cytoplasmic domains do not function as well as mCD80. Figure 22A shows a schematic diagram of the experimental setup. Figure 22B shows a schematic diagram of the CD300a TASR constructs used to introduce into T cells by electroporation. Figure 22C shows the expression levels measured for each construct using flow cytometry. Figure 22D shows that cells expressing the t23, t24, and t29 constructs containing the hCD80 cytoplasmic domain were not as protected against NK cell killing as cells expressing the t12 and v2 constructs containing the mCD80 cytoplasmic domain. [Figure 22-2] (As stated above.) [Figure 22-3] (As stated above.) [Figure 23-1]This figure shows that the optimized CD300a TASR (v2) with a fully human extracellular domain exhibited enhanced functional efficacy compared to the original design (v1). Figure 23A shows a schematic diagram of the experimental setup. Figure 23B shows a schematic diagram of the CD300a TASR constructs used for electroporation into T cells. Figure 23C shows the expression levels measured for each construct using flow cytometry. Figure 23D shows that cells expressing the v2 construct were more protected against NK cell killing than cells expressing the v1 and t13 constructs. [Figure 23-2] (As stated above.) [Figure 24-1] This figure shows repeated experiments demonstrating that an optimized CD300a TASR (v2) with a fully human extracellular domain, using additional NK cell donors, exhibited enhanced functional efficacy compared to the original design (v1). Figure 24A shows a schematic diagram of the experimental setup. Figure 24B shows a schematic diagram of the CD300a TASR constructs used for electroporation into T cells. Figure 24C shows the expression levels measured for each construct using flow cytometry. Figure 24D shows that cells expressing the v2 construct were more protected against NK cell killing than cells expressing the v1 construct. [Figure 24-2] (As stated above.) [Figure 24-3] (As stated above.) [Figure 24-4] (As stated above.) [Figure 25-1]This figure shows a rituximab-assisted ADCC assay demonstrating that NKG2A_v1, CD300a_v2, and HLA-E single-strand trimer constructs can reduce antibody-dependent cell-mediated cytotoxicity (ADCC). Figure 25A shows a schematic diagram of the experimental setup. Figure 25B shows a schematic diagram of the CD300a TASR and NKG2A TASR used to introduce the mRNA constructs into T cells by electroporation. Figure 25C shows the expression levels of CD300a TASR, NKG2A TASR, HLA-E, and RQR8 on T cells introduced by electroporation using the indicated mRNA constructs, using flow cytometry. Figure 25D shows that the humTX49_v2 and humZ270_v1 constructs were able to reduce ADCC. [Figure 25-2] (As stated above.) [Figure 25-3] (As stated above.) [Figure 26-1] This figure shows that a variant of NKG2A TASR with reduced affinity resulted in enhanced functional protection against NK cell killing. Figure 26A shows a schematic diagram of the experimental setup. Figure 26B shows a schematic diagram of the NKG2A TASR constructs (with the described amino acid mutations) used to introduce into T cells by electroporation. Figure 26C shows the expression levels measured for each construct using flow cytometry. Figure 26D shows that cells expressing the mutated construct with reduced affinity for NKG2A were more protected against NK cell killing than cells expressing the unmutated (wild-type) construct or the v1 construct. [Figure 26-2] (As stated above.) [Figure 26-3] (As stated above.) [Figure 27-1]This figure shows that a low-affinity variant of NKG2A TASR resulted in enhanced functional protection and reduced downregulation of NKG2A on effector NK cells. Figure 27A shows a schematic diagram of the experimental setup. Figure 27B shows a schematic diagram of the NKG2A TASR construct (with the described amino acid mutation) used for electroporation into T cells. Figure 27C shows the expression of NKG2A TASR on T cells electroporated with the shown mRNA construct. Figure 27D shows that cells expressing the mutant construct with reduced affinity for NKG2A were more protected against NK cell killing than cells expressing the v1 or v2 construct. Figure 27E shows a flow cytometry gating scheme showing that NK cells tend to downregulate NKG2A expression towards the end of the NK cytotoxicity assay when exposed to T cells expressing NKG2A TASR. [Figure 27-2] (As stated above.) [Figure 27-3] (As stated above.) [Figure 27-4] (As stated above.) [Figure 27-5] (As stated above.) [Figure 28-1]This figure shows that B2M KO rescues allogeneic T cells from T cell alloreactivity but introduces NK cell alloreactivity. The viability of wild-type (WT) and B2M knockout (KO) T cells from HLA-A2+ donors, loaded with HLA-A2-donor-derived NK and allo-T cell effectors, was evaluated. The allo-T cells contained an AHIII T cell receptor reactive to the EMC7 peptide presented by HLA-A2, namely ALWGFFPVL (SEQ ID NO: 200). Figure 28A shows the survival curves of WT and B2M KO target T cells loaded with the indicated effectors. Technically repeated curves with N=1 per condition. Figure 28B shows HLA-I expression of WT and B2M KO T cells by flow cytometry, with lymphocytes gated as single viable cells. B2M KO specifically makes T cells sensitive to NK cell rejection. Figure 28C shows an NK loading assay using multiple NK donors and multiple targeted T cell effectors. The bar graph summarizes the IC50 values, with each data point representing a WT or B2M KO T cell from one T cell donor relative to the indicated NK cell donor. The Mann-Whitney U test was used to compare the groups. [Figure 28-2] (As stated above.) [Figure 28-3] (As stated above.) [Figure 29-1]This figure shows the discovery and evaluation of CD300a TASR. Figure 29A shows the screening of strategies to inhibit NK alloreactivity against B2M KO T cells. Ligands are expressed by mRNA electroporation, and knockout is performed by CRISPR / Cas9. Dotted lines indicate no protection. Curves of N=1 T cell viability at E:T ratios of 6-7 with one NK cell donor loaded. (See Figure 30) Figure 29B shows the NK loading assay. The legend in the graph shows cloaking transgenes integrated into the B2M locus of human primary T cells loaded with one of three NK cell donors. The inset shows NK cell phenotypes by flow cytometry, gated with CD3-CD56+. Technical repeat curves with N=2 per condition. Figure 29C shows a competitive assay of pooled T cells from Figure 29B against allo-T and NK cells derived from an HLA-A2-donor, with HLA-I+ controls, all derived from a single HLA-A2+ donor. The frequency of the indicated T cell members after loading of the indicated NK and / or allo-T cell effectors is shown. Allo-T cells contained T cell receptors reactive to the HA-2 peptide presented on HLA-A2. Technical replicates of N=3 per condition. [Figure 29-2] (As stated above.) [Figure 29-3] (As stated above.) [Figure 29-4] (As stated above.) [Figure 30-1]Figure 30A shows the functional screening and validation of NK cloaking strategies using mRNA electroporation and CRISPR knockout in B2M KO T cells. Cloaking ligands were expressed by mRNA electroporation (EP) and functionally validated. Each graph shown in Figure 30A represents a single screening member. CRISPR knockout was performed in addition to B2M knockout. The negative control was either GFP mRNA against mRNA EP conditions or B2M knockout against CRISPR knockout screening. For each graph, the inset on the left shows the expression profile by flow cytometry of a gated lymphocyte as a single cell. "NC" indicates the negative control peak. For each graph, the inset on the right shows the NK loading assay for a given screening member. Figures 30C-D show the validation of CD300a TASR V1 and NKG2A TASR V1 by mRNA titration. Figure 30B shows the expression of NKG2A and CD300a TASR by flow cytometry in B2M KO T cells one day after electroporation (EP) introduction of a given amount of each mRNA. Figure 30C shows the viability of B2M KO T cells electroporated with the same mRNA doses as shown in Figure 30B, with NK cells loaded at a constant E:T ratio of 4.6. Technical replicates were performed with N=3 per condition. [Figure 30-2] (As stated above.) [Figure 30-3] (As stated above.) [Figure 30-4] (As stated above.) [Figure 30-5] (As stated above.) [Figure 30-6] (As stated above.) [Figure 31-1]This figure shows that the mouse B7-1 domain (also interchangeably referred to herein as "mCD80" and "mB7-1") enhances TASR expression. Figure 31A outlines the experiment. TASR was constructed using various transmembrane and cytoplasmic domains, transcribed in vitro into mRNA, and then transiently transfected into primary T cells for expression testing. All constructs were GFP and bicistronic to allow normalized comparisons between the various constructs. Figure 31B shows the various TM+Cyt domains tested. The CD8 TM is derived from the V1 scaffold. Figure 31C shows the median GFP fluorescence intensity (MFI) and median TASR fluorescence intensity (MFI) of KIR2D-specific TASRs with the various TM+Cyt domains from Figure 31B, evaluated by flow cytometry with lymphocytes as single cells, gated. mB7-1 results in higher TASR expression, normalized to GFP translation. Figure 31D shows the expression of KIR2D, NKG2A, and CD300a TASR, each possessing either CD8TM or mCD80TM+Cyt, after gating of lymphocytes as single cells and measurement by flow cytometry. [Figure 31-2] (As stated above.) [Figure 32-1]Figure 32A-E shows an inverse correlation between hinge length and functional efficacy for CD300a TASR, but no inverse correlation for NKG2A TASR, and further optimization of CD300a TASR(V2). Figures 32A-E show the effect of hinge length on the function of CD300a TASR and NKG2A TASR. Figure 32A shows the structures of CD300a TASR variants with various hinge domains of the indicated amino acid lengths, and their expression levels after mRNA EP to B2M KO T cells. Figure 32B shows the NK loading assay of B2M KO T cells expressing the indicated CD300a TASR variants. Technical repeat curves with N=2 per condition. Figure 32C shows the correlation between protection from NK and hinge length in Figure 32B, defined by the IC50 value. Error bars indicate the 95% confidence interval of the IC50 value. Figure 32D shows the structures of NKG2A TASR V1 and the non-hinge variant, along with their expression levels mediated by mRNA EP in B2M KO T cells. Figure 32E shows the NK loading assay of B2M KO T cells expressing the indicated NKG2A TASR variant, with technical repeat curves of N=1 per condition. Figures 32F-G show further optimization of CD300a TASR. Figure 32F shows CD300a TASR V1 and two optimized variants tested for mRNA EP-mediated expression in B2M KO T cells. Figure 32G shows the NK loading assay of B2M KO T cells expressing the indicated CD300a TASR variant, with technical repeat curves of N=1 per condition. [Figure 32-2] (As stated above.) [Figure 32-3] (As stated above.) [Figure 33-1]Figure 29B shows an overview and phenotype of experiments involving the nonviral CRISPR-mediated targeted integration of cloaking transgenes into human primary T cells. Figure 33A shows the processes of nonviral targeted integration and T cell purification at the B2M and AAVS1 loci. Stimulated primary T cells were edited using CRISPR-Cas9 at the indicated loci, along with a linear double-stranded DNA (dsDNA) homology repair template. In the case of AAVS1 homologous recombination repair (HDR), the transgene encodes the EF1a promoter, cloaking transgene, P2A self-cleavable peptide, RQR8 epitope tag, and BGH polyA sequence. In the case of B2M HDR, the cloaking transgene is integrated at the start codon of the B2M gene under the control of the endogenous B2M promoter, followed by the BGH polyA sequence. The edited T cells were further purified by magnetic bead-based enrichment using antibodies against either the cloaking transgene or, if present, the RQR8 epitope tag, then subjected to an additional 1-2 rounds of stimulation and growth, and subsequently cryopreserved. Figure 33B shows the phenotype of the engineered T cells containing the transgenes indicated in the upper label, as flow cytometry of gated lymphocytes as single cells. The histogram, indicated by the dotted line, shows negative control T cells stained with the same markers. [Figure 33-2] (As stated above.) [Figure 34-1]This figure shows that CD300a TASR performs better than CD47 in additional model systems and in B2M KO T cells with IL-2 activated NK cells. Figures 34A-B show a comparison of CD300a TASR and CD47 via mRNA electroporation transfection. Figure 34A shows the flow cytometry phenotype of T cells transfected with the indicated mRNA by electroporation. Figure 34B shows the NK loading assay of T cells in Figure 34A for four NK cell donors with either 2Adom or 2Cdom NK phenotypes. Technical repeat curves with N=1 per condition. Figures 34C-D show a comparison of CD300a TASR and CD47 expressed from the AAVS1 locus of B2M KO T cells in both NK loading assays and PBMC loading assays. Figure 34C shows the flow cytometry phenotype of T cells containing the indicated cloaking transgene or GFP, with lymphocytes gated as single cells. Figure 34D shows NK and PBMC loading assays of T cells using either the indicated NK donor or PBMC donor. Technically repeated curves with N=1 per condition. Figure 34E shows the effect of cytokine concentration and culture time on SIRPα expression on cultured human NK cells. Cryopreserved NK cells were thawed and cultured for 3 and 5 days in the indicated concentrations of IL-2 or 10 ng / mL IL-15 used for standard NK loading assays. Monocytes served as a positive staining control. NK cells were gated with CD3-CD56+, and monocytes were gated with CD14+FSChiSSChi. Figure 34F shows an NK loading assay using B2M KO T cells expressing the indicated cloaking transgene at the B2M locus, similar to Figure 29C. NK cells were cultured for the indicated periods with the indicated cytokines, both during the initial culture and during the 20-hour co-culture, similar to Figure 34E. Technically repeated curves with N=2 per condition. [Figure 34-2] (As stated above.) [Figure 34-3] (As stated above.) [Figure 34-4] (As stated above.) [Figure 34-5] (As stated above.) [Figure 34-6] (As stated above.) [Figure 35-1] This figure shows that CD300a TASR performs better than the TIM3 engager. CD300a TASR, along with the TIM3 engager, was expressed in T cells via mRNA electroporation. Both ligands were GFP and bicistronic. Figure 35A shows cloaking ligand expression using the indicated mRNA, assessed by flow cytometry using both ligand-based and antibody-based staining, with lymphocytes as single cells being gated. Figure 35B shows the NK loading assay of T cells from Figure 35A, loaded with four indicated NK cell donors. Technically repeated curves with N=2 per condition. [Figure 35-2] (As stated above.) [Figure 36-1] Figure 36A-E shows the flow cytometry gating scheme and additional conditions for the allo-T + NK competitive assay, related to Figure 29. Figures 36A-E show representative gating strategies and readouts for the competitive assay. All samples were resuspended and obtained in equivolumes. Figure 36A shows pre-gating of CD3+ lymphocytes as single cells. Figure 36B shows gating for the target pool only, without effector cells. Labels indicate members of the pool. Figure 36C shows gating for NK effector loading only. Gates indicated by dashed lines highlight depleted populations compared to Figure 36B. Figure 36D shows gating for allo-T cell effector loading only. Gates indicated by dashed lines and labels highlight depleted populations compared to Figure 36B. Figure 36E shows gating for allo-T and NK cell loading, with red gates highlighting CD300a TASR V2 survival compared to other members and Figure 36B. Figure 36F shows additional NK and allo-T cell loading conditions similar to those in Figure 29. Allo-TEMC7 contains the AHIII T cell receptor reactive to the EMC7 peptide presented by HLA-A2, i.e., ALWGFFPVL (SEQ ID NO: 200). NK and allo-T cell effectors are mixed in a 1:1 ratio. N=3 technical replicates per condition. [Figure 36-2] (As described above.) [Figure 36-3] (As described above.) [Figure 36-4] (As described above.) [Figure 37-1] Figure showing that CD300a TASR universally protects against NK cells and enhances CAR-T functional potency. Figure 37A shows the study design and demographic overview of 45 PBMC donors used. Figure 37B shows the PBMC loading assay. Each data point represents the IC50 value of the 7-point curve of T cell survival using the indicated cloaking ligand against PBMCs from 1 donor. N = 45 donors. Wilcoxon signed-rank test with pairing was used to compare the groups. Figure 37C shows the relationship between the PBMC donor demographic information in Figure 37A and the functional data in Figure 37B. The following statistical analyses were used to compare the groups: Kruskal-Wallis for ethnicity, and Mann-Whitney for age, gender, and CMV status. The Y-axis represents the ratio of the IC50 of the CD300a TASR cloaking ligand to the IC50 of the HLA-E cloaking ligand, and 1 indicates equal protection. Figure 37D shows the relationship between the functional potency regarding CD300a TASR (left) and HLA-E (right) and the adaptive NK cell frequency. The dashed line represents the linear fitting of the log-transformed data. N = 45 PBMC donors. Figure 37E (lower panel) shows the B cell lysis assay of engineered CAR-T cell therapy containing the indicated cloaking transgene against the indicated PBMC donors. N = 2 technical replicate curves per condition. Figure 37E (upper panel) shows the phenotype of NK cells from each PBMC donor. [Figure 37-2] (As described above.) [Figure 37-3] (As described above.) [Figure 37-4] (As described above.) [Figure 37-5] (As described above.) [Figure 37-6] (As described above.) [Figure 38-1]FIGURES related to FIGS. 37A - D showing PBMC loading assays and validations. FIG. 38A shows an overview of an experiment of the PBMC loading assay. PBMC phenotype analysis was performed after thawing, and cloaking T cell phenotype analysis was performed at the time of co - culture. FIG. 38B shows a co - culture plate map and gating scheme for fluorescence barcoding flow cytometry readout in the PBMC loading assay. FIG. 38C shows the variation between assays of the PBMC loading assay of one PBMC donor against one B2M KO T cell source. Experiments were performed on separate days. Technical curve with N = 1 per experiment. The right panel shows the coefficient of variation (CV) of the IC50 values. FIG. 38D shows the phenotypes of three engineered T cell targets used in FIG. 37, with single live lymphocytes gated. The histogram shown by the dotted line represents the negative control. Labels indicate the cloaking transgene. [Figure 38-2] (As above.) [Figure 38-3] (As above.) [Figure 38-4] (As above.) [Figure 39-1] FIGURES related to FIGS. 37A - D showing NK cell phenotype analysis of the PBMC loading assay. FIG. 39A shows a gating scheme for bulk NK cell and adaptive NK cell phenotypes. FIG. 39B shows representative expression of markers for NK cell phenotype analysis from one PBMC donor. "NC" indicates CD300a fluorescence minus one negative control staining. In panels where "NC" is not shown due to peak overlap, the negative control is the left - most peak. FIG. 39C shows the percentage of NK cells expressing the indicated markers with respect to the gating of FIG. 39B. N = 45 PBMC donors. FIG. 39D shows the frequency of adaptive NK cells gated as in FIG. 39A for CMV antibody - positive donors (N = 21 donors) and CMV antibody - negative donors (N = 24 donors). Mann - Whitney U test. [Figure 39-2] (As above.) [Figure 39-3] (As above.) [Figure 39-4] (As above.) [Figure 40-1] Figures 37A-D show the survival curves of 45 donors from PBMC loading assays. The number indicates the PBMC donor. Parentheses indicate the donor's CMV antibody status. Each curve is a technically repeated curve with N=1 per condition. [Figure 40-2] (As stated above.) [Figure 40-3] (As stated above.) [Figure 40-4] (As stated above.) [Figure 41-1]Figure 37E relates to the generation of TASR-expressing CAR-T cells by multiple nonviral HDR to primary T cells and their use in a B cell killing assay. Figure 41A shows the knock-in efficiency of T cells 4 days after editing, with CD300a TASR at the B2M locus and anti-CD19 CAR at the TRAC locus. After electroporation, T cells were plated in either standard medium or medium containing the indicated small molecules, the latter medium being replaced with standard medium after 24 hours. (Right panel) Enhancement factor of editing efficiency of single KI cells after small molecule treatment similar to Figure 41A. Editing runs N=3. Figure 41B outlines the process for generating, purifying, and amplifying cloaked CAR-T cells. Stimulated T cells are edited in a single step using linear dsDNA HDR templates encoding B2M and TRAC RNPs, as well as cloaking ligands and anti-CD19 CAR. The cloaking HDR template is incorporated at the start codon of the B2M gene, and the CAR is incorporated into the TRAC in a bicistronic format via a 2A cleavable peptide. Both constructs encode the BGH poly-A tail. Cloak ligand-expressing cells are enriched by magnetic enrichment using appropriate antibodies, followed by selective enrichment of CAR-expressing cells by the addition of mitomycin C-treated Raji feeder cells. Figure 41C shows the phenotypes of three engineered CAR-T cells by flow cytometry, with single viable cells gated, indicating that the indicated cloaking transgene expressed at the B2M locus. The same anti-CD19 CAR is used for all CAR-T cells. Figure 41D shows the co-culture plate map and fluorescence barcoding scheme. PBMCs are seeded at 250,000 cells per well, and then the edited CAR-T cells are added at the indicated CAR-T:PBMC ratio. Each column represents one unique PBMC:CAR-T cell pair and cytotoxicity curve. At the end of the co-culture, rows are barcoded by staining using a proprietary combination of fluorescent anti-CD45 antibody and phenotypic analysis antibody. The plates are washed, and then each column is pooled into a single well and acquired by flow cytometry.Figure 41E shows a gating scheme for B cell identification and barcode-based demultiplexing. Figure 41F shows the number of B cells in the absence of CAR-T cells. Similar to Figures 41D-E, PBMCs were seeded at equal density in one column, fluorescently barcoded, and then counted by flow cytometry. Figure 41G shows a comparison of B cell lysis measurements by conventional flow cytometry and fluorescence barcode flow cytometry. RQR8-expressing CAR-T cells were co-cultured with PBMCs in the indicated ratio in four identical columns of a plate. Fluorescence barcode flow cytometry was performed on two columns as in Figures 41D-E, while the other two columns were directly stained with antibodies for phenotypic analysis and then acquired by flow cytometry without pooling. N=2 technical iterations per killing curve. [Figure 41-2] (As stated above.) [Figure 41-3] (As stated above.) [Figure 41-4] (As stated above.) [Figure 41-5] (As stated above.) [Figure 41-6] (As stated above.) [Figure 42-1] Figure 37E shows that CD300a TASR expressed in an allogeneic anti-CD19 CAR-T cell model enhances protection from NK cells without affecting CAR-mediated killing efficacy. Figure 42A shows the cytotoxicity of the indicated cloaked anti-CD19 CAR-T cells against CD19-expressing Raji cells. The negative control ("no CAR") contains CD300a TASR incorporated at the B2M locus without the anti-CD19 CAR. Technically repeated curves with N=3 per condition. Figure 42B shows NK loading against cloaked anti-CD19 CAR-T cells containing the indicated cloaking transgene, using 2Adom and 2Cdom NK donors. Technically repeated curves with N=3 per condition. [Figure 42-2] (As stated above.) [Figure 43]This figure shows that CD300a cloaking (using CD300a TASR) performed better than HLA-E in an NK loading assay across 45 donor samples. Targeted DKO edited T cells, either without inhibitors, with HLA-E, or with CD300a TASR, were co-cultured for 72 hours with effector cells (PBMCs containing NK cells) from 45 different donors selected considering diverse ages, sexes, ethnicities, and CMV status. The graph plots the IC50 values ​​of three different edited T cell conditions across five bins of NK donors, based on the percentage of NKG2A- / NKG2C+ NK cells from each donor. Across all five donor bins, only T cells containing CD300a TASR were protected when loaded with NK cells. [Figure 44] This figure shows that B2M / CIITA KO+CD300a TASR outperforms other cloaking configurations against T and NK cell alloreactivity. Similar to the experiments described in Figure 29 and Example 27, a pool of DKO T cells or wt cells engineered to express the identified transgenes was loaded with either a T effector, an NK effector, or both an NK effector and a T effector. When both T cells and NK cells were loaded, only target T cells containing CD300a TASR were protected. [Figure 45-1] This figure shows that CD300a TASR containing CD300a VHH can provide equal or better protection compared to CD300a scFv TASR. Figure 45A provides an overview of the survival assay experiment and schematic diagrams of VHH and scFv CD300a TASR tested for protection from NK cells. Figure 45B shows the phenotype of T cells expressing the indicated CD300a TASR 24 hours (day 1) after electroporation introduction of mRNA encoding the identified TASR or GFP (control). The amino acid sequences of CD300a TASR 20-37 VHH are provided in SEQ ID NOs. 155-172, respectively. Figure 45C shows the viability of engineered T cells containing the indicated CD300a TASR in an NK cell cytotoxicity assay. [Figure 45-2] (As stated above.) [Figure 45-3] (As stated above.) [Modes for carrying out the invention]

[0173] I. Definition Next, certain embodiments of the present invention will be described in detail. While the present invention is described with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to encompass all alternative forms, modifications, and equivalents that may be included in the present invention as defined by the appended claims.

[0174] Before describing this instruction in detail, it should be understood that this disclosure is not limited to any particular composition or process step, and is therefore diverse. It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise clearly indicated in the context. Thus, for example, a reference to “a nucleic acid” includes multiple nucleic acids, a reference to “a cell” includes multiple cells, and so on.

[0175] Numerical ranges include the numbers that define that range. Measured and measurable values ​​are understood to be approximations, taking into account significant figures and errors associated with the measurement. Furthermore, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting. It should be understood that both the general and detailed explanations above are merely illustrative and descriptive, and do not limit the teaching.

[0176] Unless otherwise specifically described in the above specification, embodiments in this specification that describe various components as "comprising" are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments in this specification that describe various components as "consisting of" are also contemplated as "comprising" or "consisting essentially of" the recited components, and embodiments in this specification that describe various components as "consisting essentially of" 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).

[0177] The section headings used in this specification are for the purpose of organization and should in no way be construed as limiting the disclosed subject matter. If any document or other material incorporated by reference conflicts with any explicit content of this specification, including definitions, this specification shall prevail.

[0178] The term "or" is used in an inclusive sense, i.e., synonymous with "and / or" unless the context requires otherwise.

[0179] As used herein, "antibody" refers to a protein that specifically binds to an antigen or a polypeptide sequence derived from an immunoglobulin molecule. An antibody can be a complete immunoglobulin of polyclonal or monoclonal origin, or a fragment thereof, and can be of natural origin, from a recombinant source, or from a library with randomly or deliberately designed CDR sequences present in an antibody construct.

[0180] As used herein, “CDR” refers to the complementarity-determining region amino acid sequence of an antibody (or antibody fragment), which is the hypervariable domain of the immunoglobulin heavy chain and light chain. The variable region of an immunoglobulin contains three heavy chain CDRs and three light chain CDRs (or CDR regions). Therefore, as used herein, “CDR” may refer to all three heavy chain CDRs or all three light chain CDRs (or, where appropriate, both all heavy chain CDRs and all light chain CDRs). The CDRs provide the majority of the contact residues for the antibody's binding 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 RM, and Martin ACR and Thornton J. M, (1996), Journal of Molecular Biology, 262 (5), 732-745), or any other established method known to those skilled in the art for numbering residues in antibodies and determining CDRs. Other conventional numbering methods for CDR sequences available to those skilled in the art include "AbM" (University of Bath) and "contact" (University College The London method is one example. The minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM, and contact methods to obtain the "minimum join unit". The minimum join unit may be a sub-part of the CDR.

[0181] As used herein, “antigen-binding domain” means a portion of an antibody that can specifically bind to an antigen or epitope. An example of an antigen-binding domain is the one formed by the VH-VL dimer of an antibody. Another example of an antigen-binding domain is the one formed by the diversification of a specific loop derived from the 10-fibronectin type III domain of adnectin.

[0182] As used herein, “antibody fragment” or “antibody-binding domain” refers to at least one portion of an antibody or its recombinant variant containing an antigen-binding domain sufficient to result in the recognition and specific binding of the antibody fragment to a target such as an antigen and its defined epitope, i.e., the antigen-determining variable region of a complete antibody. Examples of antibody fragments, but not limited to, include Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc) variable ("scFv") antibody fragments, linear antibodies, single-domain antibodies (abbreviated as "sdAb") (either VL or VH), diabodies, minibodies, nanobodies (variable domains (VHH) on heavy-chain antibodies, also known as variable domains on camelid VHH antibodies, for example; see, e.g., Bever et al., Analytical and Bioanalytical Chemistry. 408(22): 5985-6002 (2016)), and multispecific antibodies formed from antibody fragments. Useful exemplary binding domains in the disclosed embodiments include cytokines, ligands, or peptides (e.g., adnectin or engineered ankyrin repeat protein (DARPin) (see, e.g., Rafiq et al., Nat Rev Clin Oncol. 2020;17:147-167)). In certain embodiments, the NKG2A binding domain or CD300a binding domain provided herein is an antibody fragment. For a review of a particular antibody fragment, see, for example, Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315. See (1994). Also see International Publication No. 93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458.A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. 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). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516). In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, 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 includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, e.g., a monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass has changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0183] As used herein, “VHH,” “VHH antibody,” or “nanobody” refers to an antigen-binding fragment of a heavy-chain-only antibody (HcAb) lacking a VL domain (see, for example, Bever et al., Analytical and Bioanalytical Chemistry. 408(22): 5985-6002 (2016)). Heavy-chain-only antibodies are produced in nature by camelids and sharks. The variable domain (VHH) on the heavy chain typically contains a single polypeptide chain. Compared to mAbs and other antibody fragments, VHHs are typically smaller in size.

[0184] As used herein, "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are sequentially linked by a short, flexible polypeptide linker, and the scFv can be expressed as a single polypeptide chain, and the scFv retains the specificity of the complete antibody from which it is derived.

[0185] With respect to an antibody, the “heavy chain variable region” or “VH” (or, in the case of a single-domain antibody, e.g., a nanobody, “VHH”) refers to a heavy chain fragment containing three CDRs sandwiched between adjacent segments known as framework regions, these framework regions generally being more conserved than the CDRs and forming a scaffold supporting the CDRs. Unless otherwise specified, as used herein, the scFv may have the VL variable region and the VH variable region in either order, and for example, with respect to the N-terminus and C-terminus of a polypeptide, the scFv may contain VL-linker-VH or VH-linker-VL.

[0186] As used herein, "antibody heavy chain" refers to the larger of two polypeptide chains present in naturally occurring antibody molecules, which typically determine the class to which the antibody belongs.

[0187] As used herein, “antibody light chain” refers to the smaller of the two polypeptide chains present in naturally occurring antibody molecules in their three-dimensional structure. Kappa ("κ") and lambda ("λ") light chains refer to the two main antibody light chain isotypes.

[0188] As used herein, “antigen” or “Ag” refers to a molecule to which an antibody can specifically bind, or otherwise, to which an immune response is triggered. This immune response may involve either the production of antibodies, the activation of certain immunocompetent cells, or both.

[0189] The "humanized" form of a non-human antibody is a chimeric antibody that contains a minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which one or more CDR-derived residues are replaced by one or more CDR-derived residues from the non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, e.g., a mouse, rat, rabbit, chicken, or non-human primate antibody having the desired specificity, affinity, or biological effect. In some examples, selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. The humanized antibody may also contain residues not found in either the recipient or donor antibody. Such modifications may be made to further improve 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, which are incorporated in their entirety by reference.

[0190] A "human antibody" is an antibody that is produced by a human or human cell, or has an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from a non-human source that utilizes the human antibody repertoire or a human antibody coding sequence (e.g., obtained from a human source or newly designed). Specifically, human antibodies exclude humanized antibodies.

[0191] "Affinity" refers to the sum of the strength 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 otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of molecule X for its partner Y is given by the dissociation equilibrium constant (K). D It can be expressed by ). The dynamic components contributing 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) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0192] Regarding the binding of an antibody or fragment thereof to a target molecule, the terms "bound to," "specifically," "bound specifically to," "specific to," and "selectively to" a specific antigen (e.g., a polypeptide target) or epitope on a specific antigen mean binding that is distinctly different from nonspecific or nonselective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring the binding to the target molecule and comparing it to the binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics a recognized epitope on the target molecule. In this case, specific binding is indicated when the binding of the antibody to the target molecule is competitively inhibited by the control molecule.

[0193] The term "autologous" refers to any substance that originates from the same individual and is later reintroduced into that individual.

[0194] The term "allogeneic" refers to any substance originating from a different animal of the same species or a different patient in the individual into which the substance is introduced. Two or more individuals are said to be allogeneic if their genes are not identical at one or more loci. In some embodiments, allogeneic substances originating from individuals of the same species may be genetically distinct enough to interact antigenically.

[0195] The term “to treat” (and its variations such as “to treat” or “treatment”) refers to a clinical intervention in an attempt to alter the natural course of a disease or condition in an object requiring treatment. Treatment can be performed both for preventive purposes and during the course of a clinical condition. Desired effects of treatment include prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and remission or improvement of prognosis. As used herein, “prevention” refers to the prevention of a disease or condition in a patient, such as tumor formation. For example, if an individual at risk of developing a tumor or other form of cancer is treated using the methods of this disclosure and subsequently does not develop a tumor or other form of cancer, the disease has been prevented in that individual for at least a certain period of time.

[0196] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. “Patient” means a subject who has a disease, disorder, or condition, is at risk of developing one, or otherwise requires the compositions and methods provided herein. In some embodiments, the subject has cancer, for example, the cancer described herein.

[0197] The terms “cancer” or “tumor,” as used herein, refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers, described herein but not limited to, include hematological cancers, which include both lymphoid and myeloid malignancies. Examples of myeloid malignancies, but not limited to, include acute myeloid (or myeloid, or myeloid or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic, or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryocytic (or megakaryoblastic) leukemia. These leukemias are sometimes collectively referred to as acute myeloid (or myeloid, or myeloid) leukemia (AML). Myeloid malignancies also include, but are not limited to, chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV), as well as myeloproliferative disorders (MPD). Myeloid malignancies also include myelodysplastic syndromes (or MDS), sometimes referred to as refractory anemia (RA), refractory anemia with blast cell proliferation (RAEB), and refractory anemia with transitional blast cell proliferation (RAEBT), as well as myelofibrosis (MFS) with or without primary myelofibrosis.

[0198] Hematopoietic cancers also include lymphoid malignancies that may affect lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal areas. Lymphoid cancers include, but are not limited to, B-cell non-Hodgkin lymphoma (B-NHL). B-NHL can be low-grade (or slowly progressive), intermediate-grade (or progressive), or high-grade (very progressive). Slowly progressive B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL), such as nodal MZL, extranodal MZL, splenic MZL, and splenic MZL with choriolymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHL includes mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLBCL), follicular large cell lymphoma (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML), with or without leukemia. High-grade B-NHL includes Burkitt lymphoma (BL), Burkitt-like lymphoma, small non-incisional nuclear cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHL include immunoblastic lymphoma (or immunocytoma), primary exudative lymphoma, HIV-related (or AIDS-related) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. Other examples of B-cell malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenström macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoblastic (or lymphocytic or lymphoblastic) leukemia, and Castleman disease. Other examples of NHLs include, but are not limited to, T-cell non-Hodgkin lymphoma (NOS) T-cell non-Hodgkin lymphoma (T-NHL), not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphadenopathy (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome.

[0199] Hematopoietic cancers also include Hodgkin lymphomas (or diseases), including classical Hodgkin lymphoma, nodular sclerosing Hodgkin lymphoma, mixed-cell Hodgkin lymphoma, lymphocyte-predominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphopenic Hodgkin lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM), e.g., smoldering MM, monoclonal immunoglobulinemia of unknown significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of further hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes, and natural killer cells. Tissues containing hematopoietic cells, referred to herein as “hematopoietic cell tissue,” include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as the spleen, lymph nodes, lymphoid tissue associated with mucosa (e.g., intestinal lymphoid tissue), tonsils, Peyer’s patches, and appendix, as well as other mucosa, such as lymphoid tissue associated with the bronchial lining.

[0200] Examples of solid cancers that can be treated using the methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, stomach cancer, ovarian cancer, lung cancer, head cancer, thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, and skin melanoma. Examples of cancers that may be present include melanoma, colonic adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, invasive breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, uterine cancer, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, pancreatic endocrine or exocrine neoplasms, medullary thyroid carcinoma, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0201] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredients contained herein is made effective in treating a subject, and which does not contain any additional ingredients that would be unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0202] The terms “increase” and “activate” refer to increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or more than these.

[0203] The terms “reduce” and “inhibit” refer to a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or greater than that of the variable described.

[0204] The term "agonizing" refers to the activation of receptor signaling pathways that induce a biological response associated with receptor activation. An "agonist" is an entity that binds to a receptor and agonizes it.

[0205] The term "antagonizing" refers to the inhibition of receptor signaling, which inhibits the biological response associated with receptor activation. An "antagonist" is an entity that binds to a receptor and antagonizes it.

[0206] The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the TFP of this disclosure can be replaced with other amino acid residues from the same side chain family, and modifications to the TFP can be tested using the functional assays described herein.

[0207] The term "coding" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the 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 resulting biological properties. Thus, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand used as a template for the transcription of the gene or cDNA, may be said to code for a protein or other product of that gene or cDNA.

[0208] Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence. The phrase “nucleotide sequence encoding a protein or RNA” may also include introns if the nucleotide sequence encoding that protein may contain one or more introns in one version.

[0209] The term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0210] The term "exogenous" refers to any substance introduced from an organism, cell, tissue, or system, or produced outside of an organism, cell, tissue, or system.

[0211] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence, driven, for example, by a promoter.

[0212] As used herein, “vector” and related terms refer to a nucleic acid molecule (e.g., DNA or RNA) that can be operably ligated to foreign genetic material (e.g., a nucleic acid transgene). A vector can be used as a vehicle for introducing foreign genetic material into a cell (e.g., a host cell). A vector may contain at least one restriction endonuclease recognition sequence for insertion of a transgene into the vector. A vector may contain at least one gene sequence that confers a selectable feature, such as antibiotic resistance, or a selectable feature that aids in the selection of host cells that possess a vector-transgene construct. A vector can be a single-stranded or double-stranded nucleic acid molecule and can be linear or circular nucleic acid molecules. Donor nucleic acids used for gene editing methods using zinc finger nucleases, TALENs, or CRISPR / Cas can be a type of vector. One type of vector is a “plasmid,” which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be ligated to a transgene, replicate in a host cell, and transcribe and / or translate the transgene. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be linked to a transgene. The viral backbone sequence can be modified to be non-infectious but to retain the insertion of the viral backbone and the interconnected transgene into the host cell genome. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, papovaviruses, vaccinia viruses, herpes simplex viruses, and Epstein-Barr virus vectors. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) are integrated into the host cell genome upon introduction into the host cell and thereby replicate together with the host genome. A vector may also be a transfer vector, i.e., a composition containing isolated nucleic acid that can be used to deliver the isolated nucleic acid into the 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. Therefore, the term “transfer vector” includes autonomously replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors. A vector may also be an expression vector, i.e., a vector containing a recombinant polynucleotide with an expression regulatory sequence operatively linked to the nucleotide sequence to be expressed. An expression vector may contain sufficient cis-acting elements for expression, with other elements for expression being supplied by host cells or in an in vitro expression system. Further examples of regulatory sequences and other elements for expression are described, for example, in 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 known in the art, including cosmids incorporating recombinant polynucleotides, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0213] An introduced gene is "operably linked" to a vector if there is a linkage between the introduced gene and the vector that allows the introduced gene sequence contained in the vector to function or be expressed. In one embodiment, if a regulatory sequence affects the expression of the introduced gene (e.g., the level, timing, or location of expression), the introduced gene is "operably linked" to the regulatory sequence.

[0214] As used herein, the terms “transfected,” “transformed,” or “transduced,” or other related terms, refer to the process by which an exogenous nucleic acid (e.g., a transgene) is transferred to or introduced into a host cell (e.g., an engineered cell as described herein). A “transfected,” “transformed,” or “transduced” host cell is a cell that has been transfected with, transformed with, or transduced with an exogenous nucleic acid (transgene). Host cells include primary target cells and their progeny.

[0215] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which many types exist, commonly referred to in the art as proteins. Examples of “polypeptides” include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0216] The term “promoter” refers to a DNA sequence that can initiate the specific transcription of a polynucleotide sequence, either recognized by the cell’s transcriptional mechanism or introduced by a synthetic mechanism. The term “constitutive” promoter refers to a nucleotide sequence that, when operably ligated to a polynucleotide encoding or defining a gene product, causes the cell to produce that gene product under most or all physiological conditions. The term “inducible” promoter refers to a nucleotide sequence that, when operably ligated to a polynucleotide encoding or defining a gene product, causes the cell to produce that gene product only if a substantially promoter-corresponding inducer is present in the cell. In some embodiments, the promoter is an endogenous promoter. In some embodiments, such as certain embodiments in which the recombinant nucleic acid or vector disclosed herein is inserted into a gene locus (e.g., a B2M or TRAC locus) in a cell, the promoter is an endogenous B2M promoter or an endogenous TRAC promoter.

[0217] The term "linker," as used herein, refers to a chemical group or molecule that links two adjacent molecules or parts. Typically, a linker is located between or adjacent to two groups, molecules, or other parts and is connected to each other by a covalent bond. When used in the context of scFv, the term "linker" may refer to a peptide linker, including amino acids such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together. In one embodiment, a flexible polypeptide linker is a Gly / Ser linker comprising the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n (where n is a positive integer greater than or equal to 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, examples of a flexible polypeptide linker include, but are not limited to, (Gly4Ser)5 or (Gly4Ser)4. In another embodiment, the linker comprises multiple repetitions of (GlySer), (Gly2Ser), or (Gly3Ser). The linker described in International Publication No. 2012 / 138475 (incorporated herein by reference) is also included within the scope of this disclosure. In some examples, the linker sequence is (G4S) n (where n=3-6) is included. In some examples, the linker sequence includes GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 30). In another embodiment, the linker is the Whitlow / 218 linker, i.e., GTSTGSGKPGSGEGSTKG (SEQ ID NO: 31). The linker may also be used to link together one or more binding domains (e.g., one or more scFv) within a bispecific TASR, for example, as described herein. Such linkers may include the linkers described above and / or cleavable peptide linkers, for example, as described elsewhere herein.

[0218] As used herein, “NKG2A” refers to a specific 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. The CD94 / NKG2 receptor is a type C lectin receptor primarily expressed on the surface of NK cells and a subset of CD8+ T lymphocytes. These receptors stimulate or inhibit the cytotoxic activity of NK cells and are therefore divided into activating and inhibitory receptors according to their function. NKG2A, B, C, E, and H form heterodimers with CD94 by disulfide bonds, while NKG2D forms a homodimer. The inhibitory molecule NKG2A and its splice variant NKG2B contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular portion of the molecule. The activating molecules NKG2C and NKG2E, and the splice variant of NKG2E, NKG2H, contain positively charged residues in their transmembrane domains, through which they interact with adapter molecules containing ITAM. Inhibitory NKG2 molecules containing ITIM recruit Src homology 2-domain phosphatases SHP-1 and SHP-2, thereby inducing cytotoxic inhibition. Ligands for CD94 / NKG2 heterodimer molecules include non-classical MHC class I molecules such as human HLA-E.

[0219] As used herein, “differentiation antigen group 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 wide variety 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 having three classical ITIM motifs and one non-classical ITIM motif in its cytoplasmic tail. These ITIMs are phosphorylated by Src family kinases, resulting in the recruitment of src homology 2-domain-containing protein tyrosine phosphatase (SHP) or SH2-domain-containing inositol phosphatase (SHIP). CD300a primarily regulates leukocyte function by recruiting SHP-1 phosphatase. CD300a-mediated signaling is complex and involves many downstream signaling components. For example, CD300a engagement by agonist monoclonal antibodies leads to IgE-dependent Ca254. 2+ It has been shown to inhibit mast cell recruitment and mediator release, SCF-mediated mast cell activation, differentiation, and survival, and to downregulate the cytolytic activity of NK cells. CD300a signaling is involved in allergic responses, autoimmune diseases, and viral infections, and CD300a is considered a therapeutic target in these diseases.

[0220] As used herein, a “signal peptide” (which may also be referred to as a signal sequence, targeting signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide) is a short peptide (e.g., 10–50 amino acids long) located at the N-terminus (or non-classical, the C-terminus or interior) that guides a target synthetic protein to the cell surface (e.g., the cell membrane). Thus, the signal peptide functions to prompt the cell to move the protein, for example, to the cell membrane. This signal sequence may be cleaved by the cell during polypeptide maturation.

[0221] Where used herein, “engineered cells” or “population of engineered cells,” or any related terms used herein, refer to cells (or populations thereof) into which an exogenous (exogenous or transgene) nucleic acid has been introduced. The exogenous nucleic acid may include an expression vector operably linked to the transgene, and host cells may be used to express the nucleic acid and / or polypeptide encoded by the exogenous nucleic acid (transgene). Engineered cells (or populations thereof) may be cultured cells or may be extracted from a subject. Engineered cells (or populations thereof) include primary subject cells and their progeny, regardless of the number of passages. Engineered cells encompass progeny cells. In some embodiments, engineered cells refer to any cells (including their progeny) that have been modified, transfected, transduced, transformed, and / or manipulated by any means to express a recombinant nucleic acid disclosed herein. In one example, engineered cells (or populations thereof) may be into which an expression vector operably linked to a nucleic acid encoding a desired recombinant nucleic acid described herein has been introduced. The manipulated cells and their populations may possess expression vectors that are stably integrated into the host genome, or they may possess extrachromosomal expression vectors. In several embodiments, the manipulated cells and their populations may possess extrachromosomal vectors that are present after several cell divisions, or that are transiently present and lost after several cell divisions.

[0222] As used herein, “low immunogenicity” and “low immunity” refer to a reduction in immunogenicity. Low immunogenic cells (e.g., engineered cells described herein that are low immunogenic, e.g., low immunogenic iPSCs) will produce a reduced immunological rejection response when transferred to an allogeneic host. For example, low immunogenic pluripotent cells are pluripotent cells that, when transferred to an allogeneic host, retain their pluripotency but produce a reduced immunological rejection response. Low immunogenic cells described herein may induce a reduced immune response (e.g., compared to non-low immunogenic cells) or may not induce an immune response. Therefore, “low immunogenicity” or “low immunity” refers to any reduction or absence of immune response compared to the immune response of parental (i.e., “wild-type”) cells before manipulation (e.g., before the introduction of recombinant nucleic acids containing the NKG2A-binding domain, CD300a-binding domain, or both, as described herein). For example, compared to wild-type cells, such low immunogenic cells 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 likely to be subjected to immune rejection (e.g., cell lysis) by the target to which such cells are transplanted. In the context of engineered cells (e.g., pluripotent stem cells as used herein, e.g., iPSCs), “wild-type” means cells that may contain some nucleic acid alterations but have not undergone the gene editing procedures of this disclosure to achieve low immunogenicity (i.e., introduction of recombinant nucleic acids containing the NKG2A binding domain, CD300a binding domain, or both as described herein).

[0223] As used herein, “stem cell” refers to a cell that can undergo multiple cycles of cell division, maintain an undifferentiated state, and have the ability to differentiate into specialized cell types. Stem cells are further classified into three categories: totipotent, pluripotent, or multipotent. Totipotent cells have the ability to form an entire organism (e.g., a fertilized egg). As used herein, “pluripotent stem cell” refers to a stem cell that lacks the ability to form extraembryonic tissues and therefore cannot develop a fetus, but has the potential to differentiate into one of the three germ layers: endoderm (e.g., the inner wall of the stomach, the gastrointestinal tract, the lungs), mesoderm (e.g., muscle, bone, blood, genitourinary tissues), or ectoderm (e.g., epidermal tissue and nervous system tissue). Pluripotent stem cells include embryonic stem cells (ESCs). Examples of human embryonic stem cell (hESC) lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES Collection (described in their entirety in Cowan, CA et. al, New England J. Med. 350: 13. (2004), which are incorporated herein by reference). Pluripotent stem cells also include "induced pluripotent stem cells" (iPSCs), a type of pluripotent stem cell derived from non-pluripotent cells, typically adult somatic cells, 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 obtaining iPSCs by somatic cell reprogramming are known in the art and are further described, for example, in 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 herein by reference in its entirety.

[0224] As used herein, the terms “inhibit” or “reduce” in the context of inhibiting or reducing the cytotoxicity of NK cells against engineered cells as disclosed herein means any reduction or elimination of NK cell cytotoxicity against engineered cells by any amount compared to the level of NK cell cytotoxicity against parental (i.e., “wild-type”) cells before the engineerment (e.g., before the introduction of recombinant nucleic acids including the NKG2A-binding domain, CD300a-binding domain, or both as described herein). For example, if the cytotoxicity of NK cells against engineered cells is reduced, it means that it is “reduced” or “inhibited” 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% compared to wild-type cells.

[0225] As used herein, the term “MHC class I” refers to a class of major histocompatibility complex (MHC) molecules presented on the surface of nucleated cells and platelets in vertebrates. MHC class I molecules function as part of the adaptive immune system by binding to fragments of cytoplasmic exogenous (non-self) proteins and presenting these antigens on the cell surface for recognition by immune system cells, such as cytotoxic T cells. In humans, MHC is also referred to as human leukocyte antigen (HLA). Rejection of allogeneic therapeutic cells, such as CAR T cells (e.g., in GvHD), is thought to be primarily driven by the recognition of host peptide-HLA complexes via the αβ T cell receptor complex (αβTCR) by T cells derived from a donor or iPSC. Rejection is primarily driven by host NK cells, CD8+ T cells, CD4+ T cells, and, to a lesser extent, macrophages. Examples of HLA 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. Although not constrained by a specific theory, NK cells recognize antigen-bound HLA-E molecules on their cell surface using the heterodimeric receptor CD94 / NKG2A / B / C. CD94 / NKG2A or CD94 / NKG2B engagement results in an inhibitory effect on the cytotoxic activity of NK cells, thereby preventing cell lysis while simultaneously inducing NK cell activation.

[0226] As used herein, the term “β2-microglobulin” (B2M) refers to a specific polypeptide component of MHC class I molecules. The B2M protein is encoded by the B2M gene. MHC class I molecules are heterodimers composed of two polypeptide chains, namely α and β2-microglobulin, non-covalently linked via the interaction of B2M with the α3 domain. While not bound by any particular theory, β2-microglobulin is required for the cell surface expression of MHC class I molecules and the stability of the peptide bond groove. The absence of B2M expression results in a significant decrease in detectable MHC class I molecules on the cell surface.

[0227] 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 heterodimer protein typically consisting of highly variable alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively) expressed as part of a complex with an invariant CD3 chain molecule. The TCR is found on the surface of T cells, or T lymphocytes, and recognizes antigens bound to MHC molecules. Removal of endogenous TCRs by targeting the TRAC (e.g., via CAR transgene knock-in) has been used to address histocompatibility barriers associated with cells derived from unrelated donors.

[0228] As used herein, the term “deficiency” may mean a reduction or loss of expression and / or functionality of a particular gene product, such as an MHC class I molecular component (e.g., B2M), a TCR component (e.g., TRA, via disruption of the TRAC locus, etc.), or a molecule that modulates or controls MHC class II expression (e.g., the class II major histocompatibility complex transactivator (CIITA) gene product) (e.g., measured by RNA and / or protein detection methods and / or functional assays). In this context, “deficiency” means any reduction or loss of expression or functionality of the same gene product compared to the level in parental (i.e., “wild-type”) cells before the deficiency (e.g., before the gene disruption operation by any preferred means described herein or known in the art). In some embodiments, cells lacking a particular gene product show a reduction of 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% compared to wild-type cells. In some embodiments, cells lacking a particular gene product show a decrease of 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% compared to wild-type cells. "Defect" can also refer to a decrease or absence of detectable molecules in cells, such as MHC class I molecules or MHC class II molecules.For example, cells show a decrease in the expression of one or more components of MHC class I molecules (e.g., B2M) 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% compared to wild-type cells. In other words, cells are MHC class I deficient if the detectable MHC class I on the cell surface is reduced or absent 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% compared to wild-type cells. As another example, cells expressing one or more components of MHC class II molecules or molecules that regulate or control the expression of MHC class II molecules (e.g., CIITA) is 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 less than that of wild-type cells. A cell is MHC class II deficient if, as a result, detectable MHC class II on the cell surface is reduced or absent 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% compared to wild-type cells. As another example, cells are TRAC-deficient if TRAC locus expression 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% compared to wild-type cells.In some embodiments, cells lacking a specific gene product are undetectable by PCR, immunohistochemical staining, ELISA, Western blotting, or other suitable nucleic acid or protein detection methods. In some embodiments, a locus is "disrupted," resulting in cells lacking the gene product encoded by that gene. For example, insertion of recombinant nucleic acids or vectors disclosed herein into a locus (e.g., the B2M and / or TRAC locus) can disrupt the locus, thereby reducing or eliminating gene expression at that locus. Methods for disrupting loci, such as but not limited to homologous recombination, CRISPR-Cas9, and TALEN, are known in the art. Furthermore, disruption can be achieved with or without the removal of all or part of the targeted locus (e.g., via gene knockout methods) (e.g., via insertion of a nucleotide sequence into the locus). Thus, the methods disclosed herein may include disrupting a locus (e.g., the B2M and / or TRAC locus) using any suitable means known in the art.

[0229] II. Recombinant Nucleic Acids A. Overview Rejection of allogeneic therapeutic cells such as CAR T cells (e.g., in GvHD) is thought to be primarily driven by the recognition of host peptide-HLA complexes via the αβ T cell receptor complex (αβTCR) by T cells derived from donors or iPSCs. Rejection is primarily driven by host NK cells, CD8+ T cells, CD4+ T cells, and, to a lesser extent, macrophages. In the context of CAR T cell therapy, the relative contribution of these cell types to allogeneic graft rejection may vary depending on their absolute numbers and rearrangement dynamics after preconditioning regimens. In three cohorts (one acute lymphoblastic leukemia (ALL) cohort and two large B-cell malignancies), patients showed significant differences in CD8+ T cell, CD4+ T cell, and NK cell rearrangement dynamics after autologous CD19 CAR T cell treatment and preconditioning regimens (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 initial levels within 3-4 weeks, while CD4+ T cells showed a significantly slower recovery rate, and in some patients, they did not return to initial levels even 1-2 years after the start of treatment. Therefore, CD8+ T cells and NK cells may play a greater role in controlling the length of the allogeneic CAR T cell therapeutic time range by being the first major contributors to rejection. NK cells are an important component of the innate immune system and influence adaptive immune responses, for example, through cytokine secretion. NK cell activity is thought to be regulated by the balance between 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 activating receptors on allogeneic cells, or increasing levels of ligands for inhibitory cell receptors, may reduce or prevent host NK cell-mediated destruction in allogeneic immunotherapy.

[0230] There are two classes of NK cell suppressor receptors: immunoglobulin-like KIR and LIR receptors, and a type C lectin-like receptor which is a CD94 / NKG2 heterodimer. In humans, the ligands for KIR receptor family members are the classical class I antigens HLA-A, -B, and -C, while the ligand for one LIR (LIR-2) is the non-classical class I antigen HLA-G. The major ligand for the CD94 / NKG2 receptor is the non-classical class I antigen HLA-E. Of the NK cell suppressor receptors, CD94 / NKG2A is thought to be the most widely expressed on NK cells. (Crew et al. Molecular Immunology, 2005;42(1):1205-1214)

[0231] CD8+ T cells need to undergo clonal growth to initiate an efficient allo-response, but they can be the primary subset that rejects allogeneic CAR T cells. In cases of CD8+ T cell-mediated rejection, the primary approach has been to eliminate the expression of HLA class I molecules on CAR T cells. For example, deletion of the conserved gene β2m completely removes surface expression of HLA class I (Wang et al. Stem Cells Transl Med. 2015;4(10):1234-1245). Thus, inactivation of HLA class I on the surface of CAR T cells can efficiently mitigate such rejection, providing an early therapeutic window and potentially eradicating cancer cells. However, while this approach reduces immunogenicity recognition by CD8+ T cells, the complete loss of HLA class I molecules increases the risk of host NK cells recognizing and destroying allogeneic CAR T cells (the so-called "missing self" response). Embedding NK inhibitors into CAR T cells may further extend the persistence of CAR T cells. It has been proposed that NK cell-mediated disruption of HLA-edited T cells is prevented by the expression of non-polymorphic HLA molecules, such as HLA-E, that bind to inhibitory receptors on NK cells, on CAR T cells. (Gornalusse et al. Nat Biotechnol. 2017; 35(8):765-772)

[0232] HLA-E inhibits the cytotoxic activity of host NK cells and can therefore reduce or prevent the lysis of immunotherapeutic cells. Crew et al. (Molecular Immunology, 2005 42(1):1205-1214) found that expression of a single-stranded trimer (SCT) of HLA-E, consisting of human β2m leader peptide, VMAPRTLIL (HLA-E binding peptide), a 15-amino acid linker, mature human β2m, a 20-amino acid linker, and a mature HLA-E heavy chain (from N-terminus to C-terminus), reduced NK cell-mediated rejection of porcine xenografts by providing an inhibitory ligand for human NK cells expressing CD94 / NKG2A. Subsequently, SCT HLA-E expression was found to reduce the cytotoxicity of NK cells in HLA class I depleted pluripotent stem cells and general-purpose 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) Currently, SCT HLA-E expressing CAR T cells are undergoing clinical trials for the treatment of cancer patients.

[0233] However, SCT HLA-E expression in immunotherapy cells presents several drawbacks. While this construct provides some level of protection against NK cells, SCT HLA-E expression makes therapeutic cells more vulnerable to rejection by HLA-E-restricted CD8+ T cells. Furthermore, SCT HLA-E expression is only effective against NKG2A-expressing NK cells. In addition, HLA-E also binds to the NKG2C-activating receptor on NK cells, leading to NK cell activation and subsequent increased rejection of therapeutic cells.

[0234] In contrast, the recombinant nucleic acids of this disclosure enable the development of immune-evading, versatile CAR T cells that can, for example, evade the cytotoxicity of host CD8+ T cells and NK cells and are suitable for adoptive cell transfer in an allogeneic environment. In some embodiments, the disclosed recombinant nucleic acids encode constructs for inhibiting the cytotoxicity of NK cells, comprising a CD300a-binding domain, an NKG2A-binding domain, or both CD300a-binding and NKG2A-binding domains. In some embodiments, the recombinant nucleic acid comprises a CD300a-binding domain but does not encode an NKG2A-binding domain. In other embodiments, the recombinant nucleic acid comprises an NKG2A-binding domain but does not encode a CD300a-binding domain. In certain embodiments, the CD300a-binding domain and the NKG2A-binding domain are contained in the same nucleic acid. In another particular embodiment, the CD300a-binding domain and the NKG2A-binding domain are contained in at least two different nucleic acids. The disclosed recombinant nucleic acids, comprising both CD300a-binding and NKG2A-binding domains, may encode the binding domains in any order. Therefore, in some embodiments, the nucleotide sequence encoding the NKG2A binding domain is located at the 5' end of the nucleotide sequence encoding the CD300a binding domain. In other embodiments, the nucleotide sequence encoding the NKG2A binding domain is located at the 3' end of the nucleotide sequence encoding the CD300a binding domain. In embodiments that include both an NKG2A binding domain and a CD300a binding domain encoded in the same recombinant nucleic acid, the recombinant nucleic acid may further include a linker (e.g., any preferred linker, e.g., the linker described herein) that connects the NKG2A binding domain and the CD300a binding domain.

[0235] In some embodiments, the CD300a binding domain contains an antibody or fragment thereof, VHH, a cytokine, a ligand, or a peptide. In some embodiments, the NKG2A binding domain contains an antibody or fragment thereof, VHH, a cytokine, a ligand, or a peptide. In certain embodiments, (a) the antibody or fragment thereof contains a single-strand variable fragment (scFv) or VHH, or (b) the peptide is adnectin or engineered ankyrin repeat protein (DARPin). In other specific embodiments, the VHH contains the VH domain of a camelid heavy chain antibody.

[0236] In some embodiments, the CD300a binding domain comprises VHH(CD300a VHH), and / or the NKG2A binding domain comprises VHH(NKG2A VHH). In certain embodiments, a construct for inhibiting the cytotoxicity of NK cells comprises or consists of a CD300a binding domain, and the CD300a binding domain comprises VHH(CD300a VHH). In certain embodiments, CD300a VHH comprises a CDR1 containing 0, 1, or 2 mutations compared to any one amino acid sequence of SEQ ID NOs. 173-177. In certain embodiments, CD300a VHH comprises a CDR2 containing 0, 1, or 2 mutations compared to any one amino acid sequence of SEQ ID NOs. 178-181. In certain embodiments, CD300a VHH comprises a CDR1 containing 0, 1, or 2 mutations compared to any one amino acid sequence of SEQ ID NOs. 182-199.

[0237] In a particular embodiment, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing AAKPGEDVY (SEQ ID NO: 182). In some embodiments, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKPRSGWGL (SEQ ID NO: 184). In a particular embodiment, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ATKTRYES (SEQ ID NO: 185). In some embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSDYA (SEQ ID NO: 174), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ITGSGGST (SEQ ID NO: 179), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing NTRLAHGRDVLGGVAYDI (SEQ ID NO: 186).In certain embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSDYA (SEQ ID NO: 174), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ITGSGGST (SEQ ID NO: 179), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing NTRRLGRSGDLVQDY (SEQ ID NO: 187). In some embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSRYY (SEQ ID NO: 175), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKPDRDY (SEQ ID NO: 188). In certain embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKLPDVLPLEY (SEQ ID NO: 189). In some embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYW (SEQ ID NO: 176), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ITGSGGST (SEQ ID NO: 179), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ATKVDGSYGIVTEL (SEQ ID NO: 190). In a particular embodiment, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSDYA (SEQ ID NO: 174), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing INGSGGST (SEQ ID NO: 180), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing HTRRSGTSMAMDV (SEQ ID NO: 191).In some embodiments, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ATKLTMVY (SEQ ID NO: 192). In a particular embodiment, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKLTNEY (SEQ ID NO: 193). In some embodiments, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKVRPSYEY (SEQ ID NO: 194). In a particular embodiment, CD300a VHH comprises CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSPYY (SEQ ID NO: 177), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VAKPGYEY (SEQ ID NO: 195). In some embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGRT (SEQ ID NO: 181), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKPGEDVY (SEQ ID NO: 196).In certain embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ISGSGGST (SEQ ID NO: 178), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing VTKSNMVY (SEQ ID NO: 197). In some embodiments, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYY (SEQ ID NO: 173), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing ITGSGGST (SEQ ID NO: 179), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing TTKVDGSYGIVTEL (SEQ ID NO: 198). In a particular embodiment, CD300a VHH includes CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GFTFSSYW (SEQ ID NO: 176), CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing INGSGGST (SEQ ID NO: 180), and CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing AAAARDRERDY (SEQ ID NO: 199).

[0238] In some embodiments, CD300a VHH contains 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 certain embodiments, CD300a VHH contains or consists of any one of SEQ ID NOs. 155-172. CD300a TASR #20-37 disclosed herein (for example, shown in Figure 45) each contain CD300a VHH of SEQ ID NOs. 155-172. In SEQ ID NOs. 155-172 shown below, CDR1 is indicated by an underline, CDR2 by a double underline, and CDR3 by a wavy underline.

[0239] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 155.

[0240] Sequence ID 155:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKPGEDVYWGQGTLVTVSS

[0241] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 156.

[0242] Sequence ID 156:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLSQFASWGQGTLVTVSS

[0243] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 157.

[0244] Sequence ID 157:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPRSGWGLWGQGTLVTVSS

[0245] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 158.

[0246] Sequence ID 158:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKTRYESWGQGTLVTVSS

[0247] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 159.

[0248] Sequence ID 159:

[0249] [ka]

[0250] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 160.

[0251] Sequence ID 160:

[0252] [ka]

[0253] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 161.

[0254] Sequence ID 161:EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPDRDYWGQGTLVTVSS

[0255] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 162.

[0256] Sequence ID 162:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLPDVLPLEYWGQGTLVTVSS

[0257] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 163.

[0258] Sequence ID 163:

[0259] [ka]

[0260] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 164.

[0261] Sequence ID 164:

[0262] [ka]

[0263] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 165.

[0264] Sequence ID 165:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATKLTMVYWGQGTLVTVSS

[0265] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 166.

[0266] Sequence ID 166:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKLTNEYWGQGTLVTVSS

[0267] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 167.

[0268] Sequence ID 167:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKVRPSYEYWGQGTLVTVSS

[0269] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 168.

[0270] Sequence ID 168:EVQLVESGGGLVQPGGSLRLSCAASGFTFSPYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVAKPGYEYWGQGTLVTVSS

[0271] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 169.

[0272] Sequence ID 169:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKPGEDVYWGQGTLVTVSS

[0273] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 170.

[0274] Sequence ID 170:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKQREWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTKSNMVYWGQGTLVTVSS

[0275] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 171.

[0276] Sequence ID 171:

[0277] [ka]

[0278] In some embodiments, CD300a VHH contains 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 with respect to SEQ ID NO: 172.

[0279] Sequence ID 172:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKQREWVSAINGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARDRERDYWGQGTLVTVSS

[0280] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with any one of SEQ ID NOs. 137-154. In certain embodiments, the nucleotide sequence encoding CD300a VHH includes or consists of any one of SEQ ID NOs. 137-154.

[0281] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 137.

[0282] Sequence ID 137:

[0283] [ka]

[0284] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 138.

[0285] Sequence ID 138:

[0286] [ka]

[0287] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 139.

[0288] Sequence ID 139:

[0289] [ka]

[0290] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 140.

[0291] Sequence ID 140:

[0292] [ka]

[0293] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 141.

[0294] Sequence ID 141:

[0295] [ka]

[0296] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 142.

[0297] Sequence ID 142:

[0298] [ka]

[0299] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 143.

[0300] Sequence ID 143:

[0301] [ka]

[0302] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 144.

[0303] Sequence ID 144:

[0304] [ka]

[0305] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 145.

[0306] Sequence ID 145:

[0307] [ka]

[0308] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 146.

[0309] Sequence ID 146:

[0310] [ka]

[0311] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 147.

[0312] Sequence ID 147:

[0313] [ka]

[0314] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 148.

[0315] Sequence ID 148:

[0316] [ka]

[0317] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 149.

[0318] Sequence ID 149:

[0319] [ka]

[0320] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 150.

[0321] Sequence ID 150:

[0322] [ka]

[0323] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 151.

[0324] Sequence ID 151:

[0325] [ka]

[0326] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 152.

[0327] Sequence ID 152:

[0328] [ka]

[0329] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 153.

[0330] Sequence ID 153:

[0331] [ka]

[0332] In some embodiments, the nucleotide sequence encoding CD300a VHH includes 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 with respect to SEQ ID NO: 154.

[0333] Sequence ID 154:

[0334] [ka]

[0335] In some embodiments, the recombinant nucleic acid includes (a) an NKG2A-binding domain comprising an NKG2A light chain variable region (NKG2A VL) and an NKG2A heavy chain variable region (NKG2A VH), and / or

[0336] (b) Encoding a construct for inhibiting the cytotoxicity of NK cells, comprising a CD300a binding domain including 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 an NKG2A binding domain comprising NKG2A VL and NKG2A VH. In another particular embodiment, the recombinant nucleic acid encodes a CD300a binding domain comprising CD300a VL and CD300a VH.

[0337] In a particular embodiment, NKG2A VL includes VL CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RASENIYSYLA (SEQ ID NO: 98), VL CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing NAKTLAE (SEQ ID NO: 99), and VL CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing QHHYGTPRT (SEQ ID NO: 100). In a particular embodiment, NKG2A VH includes VH CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing SYWMN (SEQ ID NO: 101), VH CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RIDPYDSETHYAQKLQG (SEQ ID NO: 102), and VH CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing GGYDFDVGTLYWFFDV (SEQ ID NO: 103). In some embodiments, CD300a VL includes VL CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RASQDISNYLN (SEQ ID NO: 104), VL CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing YTSRLHS (SEQ ID NO: 105), and VL CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing QQGNTLPWT (SEQ ID NO: 106). In some embodiments, CD300a VH includes VH CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing SYWMQ (SEQ ID NO: 107), VH CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing EIDPSSDSYTNYNQKFKG (SEQ ID NO: 108), and VH CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing WGMAYGTSSYWYFDV (SEQ ID NO: 109).

[0338] In certain embodiments, NKG2A VL contains 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 with SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, NKG2A VL contains or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0339] Sequence ID 1 (hZ270_vL):DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIK

[0340] Sequence ID 2 (hZ199_vL):EIVLTQSPATLSLSPGERATLSCSASSSVSSYIYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSGNPYTFGQGTKLEIK

[0341] In certain embodiments, the nucleotide sequence encoding NKG2A VL includes 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 with respect to SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the nucleotide sequence encoding NKG2A VL includes or consists of SEQ ID NO: 9 or SEQ ID NO: 10.

[0342] Sequence ID 9 (encoding hZ270_vL):

[0343] [ka]

[0344] Sequence ID 10 (encoding hZ199_vL):

[0345] [ka]

[0346] In certain embodiments, NKG2A VH contains 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 with any one of SEQ ID NOs: 3 to 8. In certain embodiments, NKG2A VH contains or consists of any one of SEQ ID NOs: 3 to 8.

[0347] Sequence ID 3 (hZ270_vH):

[0348] [ka]

[0349] Sequence ID 4 (hZ270_vH_D98A):

[0350] [ka]

[0351] Sequence ID 5 (hZ270_vH_E56A):

[0352] [ka]

[0353] Sequence ID 6 (hZ270_vH_R94A):

[0354] [ka]

[0355] Sequence ID 7 (hZ270_vH_Y53A):

[0356] [ka]

[0357] Sequence ID 8(hZ199_VH):EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSEISSGGSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGDYPRFFDVWGQGTTVTVSS

[0358] In certain embodiments, the nucleotide sequence encoding NKG2A VH includes 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 with any one of SEQ ID NOs: 11-16. In certain embodiments, the nucleotide sequence encoding NKG2A VH includes or consists of any one of SEQ ID NOs: 11-16.

[0359] Sequence ID 11 (encoding hZ270_vH):

[0360] [ka]

[0361] Sequence ID 12 (encoding hZ270_vH_D98A):

[0362] [ka]

[0363] Sequence ID 13 (encoding hZ270_vH_E56A):

[0364] [ka]

[0365] Sequence ID 14 (encoding hZ270_vH_R94A):

[0366] [ka]

[0367] Sequence ID 15 (encoding hZ270_vH_Y53A):

[0368] [ka]

[0369] Sequence ID 16 (encoding hZ199_VH):

[0370] [ka]

[0371] In certain embodiments, CD300a VL contains 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 with respect to SEQ ID NO: 17. In certain embodiments, CD300a VL contains or consists of the amino acid sequence of SEQ ID NO: 17.

[0372] Sequence ID 17(hTX49v1_vL):DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISNLQPEDFATYFCQQGNTLPWTFGQGTKVEIK

[0373] In certain embodiments, the nucleotide sequence encoding CD300a VL includes 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 with respect to sequence number X. In certain embodiments, the nucleotide sequence encoding CD300a VL includes or consists of sequence number 19.

[0374] Sequence ID 19 (encoding hTX49v1_vL):

[0375] [ka]

[0376] In certain embodiments, CD300a VH contains 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 with respect to SEQ ID NO: 18. In certain embodiments, CD300a VH contains or consists of the amino acid sequence of SEQ ID NO: 18.

[0377] Sequence ID 18 (hTX49v1_vH):

[0378] [ka]

[0379] In certain embodiments, the nucleotide sequence encoding CD300a VH includes 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 with respect to SEQ ID NO: 20. In certain embodiments, the nucleotide sequence encoding CD300a VH includes or consists of SEQ ID NO: 20.

[0380] Sequence ID 20 (encoding hTX49v1_vH):

[0381] [ka]

[0382] In some embodiments, all or part of the nucleic acid sequence encoding the NKG2A binding domain is codon-optimized to reduce or prevent undesirable recombination events. In some embodiments, all or part of the nucleic acid sequence encoding the CD300a binding domain is codon-optimized to reduce or prevent undesirable recombination events. In some embodiments, all or part of the nucleic acid sequence encoding NKG2A VL, NKG2A VH, CD300a VL, and / or CD300a VH is codon-optimized, for example, to reduce or prevent undesirable recombination events. In some embodiments, codon optimization involves replacing a sequence of one or more 3-nucleotides encoding a particular amino acid with a sequence of different 3-nucleotides encoding the same amino acid (e.g., a sequence of the first 3 nucleotides and a sequence of 3 nucleotides different at the 1st, 2nd, and / or 3rd nucleotide positions). For example, the 3-nucleotide sequences (codons) GGA, GGG, GGT, and GGC (GGU) all encode the amino acid glycine, and each can be replaced by one of the remaining three during the codon optimization process in a given nucleic acid sequence.

[0383] In some embodiments, the 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 with respect to SEQ ID NO: 113. In certain embodiments, the codon-optimized nucleotide sequence encoding the NKG2A binding domain includes or consists of SEQ ID NO: 113.

[0384] Sequence ID 113 (encoding codon-optimized humZ270vL + Whitlow / 218 linker + humZ270vH):

[0385] [ka]

[0386] In some embodiments, the codon-optimized nucleotide sequence encoding 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 with respect to SEQ ID NO: 110. In certain embodiments, the codon-optimized nucleotide sequence encoding NKG2A VL includes or consists of SEQ ID NO: 110.

[0387] Sequence ID 110:

[0388] [ka]

[0389] In some embodiments, the codon-optimized nucleotide sequence encoding 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 with respect to SEQ ID NO: 112. In certain embodiments, the codon-optimized nucleotide sequence encoding NKG2A VH includes or consists of SEQ ID NO: 112.

[0390] Sequence ID 112:

[0391] [ka]

[0392] In some embodiments, the recombinant nucleic acid includes a first linker (e.g., the first linker described herein) that links the nucleic acid encoding NKG2A VL to the nucleic acid encoding NKG2A VH. In other embodiments, the recombinant nucleic acid includes a first linker (e.g., the first linker described herein) that links the nucleic acid encoding CD300a VL to the nucleic acid encoding CD300a VH. In some embodiments where the recombinant nucleic acid encodes an NKG2A binding domain and a CD300a binding domain, the recombinant nucleic acid includes a first linker (e.g., the first linker described herein) that links the nucleic acid encoding NKG2A VL to the nucleic acid encoding NKG2A VH, and a second linker (e.g., the second linker described herein) that links the nucleic acid encoding CD300a VL to the nucleic acid encoding CD300a VH.

[0393] The NKG2A-binding domain or CD300a-binding domain of this disclosure may include any suitable structure, for example, an antibody or a fragment thereof, or VHH, but is not limited to these. Commercially available NKG2A antibodies include, but are not limited to, those from R&D Systems (human NKG2A / CD159a, catalog number: MAB1059), Creative Biolabs (human anti-NKG2A recombinant antibody, scFv fragment (HPAB-1355-FY-S(P)) (catalog number: HPAB-1355-FY-S(P)); human anti-NKG2A recombinant antibody (HPAB-1355-FY) (catalog number: HPAB-1355-FY); human anti-NKG2A recombinant antibody; Fab fragment (HPAB-1355-FY-F(E)) (catalog number: HPAB-1355-FY-F(E)); recombinant anti-human KLRC1 antibody scFv fragment (catalog number: MOB-604-S(P))), and Miltenyl Examples include those produced by Biotec (CD159a (NKG2A) antibody, anti-human, REAfinity®, catalog number 130-122-329). Commercially available CD300a antibodies include, but are not limited to, those produced by R&D Systems (human CD300a / LMIR1 antibody, catalog numbers: MAB2640, MAB26401) and ThermoFisher Scientific (CD300a monoclonal antibody (MEM-260), PE, catalog number A15778; CD300a monoclonal antibody (7H8E4), catalog number MA5-38479; CD300a monoclonal antibody (2F9C5), catalog number 67242-1-IG). Examples of useful binding domains in the disclosed embodiments include cytokines, ligands, or peptides (e.g., adnectin or engineered ankyrin repeat protein (DARPin) (see, for example, Rafiq et al., Nat Rev Clin Oncol. 2020;17:147-167)).

[0394] In some embodiments where the NKG2A binding domain is scFv, the NKG2A scFv contains 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 with respect to SEQ ID NO: 118. In certain embodiments, the NKG2A scFv contains or consists of the amino acid sequence of SEQ ID NO: 118.

[0395] Sequence ID 118:

[0396] [ka]

[0397] In some embodiments where the NKG2A binding domain is scFv, the nucleotide sequence encoding 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 with respect to either SEQ ID NO: 113 or 120. In certain embodiments, the nucleotide sequence encoding NKG2A scFv comprises or consists of either SEQ ID NO: 113 or 120.

[0398] Sequence ID 120:

[0399] [ka]

[0400] In some embodiments where the CD300a binding domain is an scFv, the CD300a scFv contains 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 with respect to SEQ ID NO: 119. In certain embodiments, the CD300a scFv contains or consists of the amino acid sequence of SEQ ID NO: 119.

[0401] Sequence ID 119:

[0402] [ka]

[0403] In some embodiments where the CD300a binding domain is scFv, the nucleotide sequence encoding 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 with respect to SEQ ID NO: 121. In certain embodiments, the nucleotide sequence encoding CD300a scFv comprises or consists of SEQ ID NO: 121.

[0404] Sequence ID 121:

[0405] [ka]

[0406] The disclosed recombinant nucleic acids, including CD300a scFv and / or NKG2A scFv, can encode NKG2A VL and VH, as well as CD300a VL and VH, in any preferred order. Therefore, in some embodiments, the nucleotide sequence encoding NKG2A VL is located 5' to the nucleotide sequence encoding NKG2A VH. In other embodiments, the nucleotide sequence encoding NKG2A VL is located 3' to the nucleotide sequence encoding NKG2A VH. Similarly, in some embodiments, the nucleotide sequence encoding CD300a VL is located 5' to the nucleotide sequence encoding CD300a VH. In other embodiments, the nucleotide sequence encoding CD300a VL is located 3' to the nucleotide sequence encoding CD300a VH. In some embodiments, including both CD300a scFv and NKG2A scFv, the nucleotide sequence encoding NKG2A scFv is located 3' to the nucleotide sequence encoding CD300a scFv. In other embodiments including both CD300a scFv and NKG2A scFv, the nucleotide sequence encoding NKG2A scFv is located at the 5' end of the nucleotide sequence encoding CD300a scFv. In some embodiments including both CD300a scFv and NKG2A scFv, the recombinant nucleic acid encodes a third linker that ligates NKG2A scFv and CD300a scFv.

[0407] B. Linker Embodiments of recombinant nucleic acids disclosed herein may include one or more linkers, for example, one, two, three, four, five, six, seven, or more, or may not include any linkers. As described herein, a disclosed recombinant nucleic acid (e.g., a recombinant nucleic acid including an NKG2A binding domain but not a CD300a binding domain) may include a first linker that links the nucleic acid encoding NKG2A VL to the nucleic acid encoding NKG2A VH. In other embodiments, a disclosed recombinant nucleic acid (e.g., a recombinant nucleic acid including a CD300a binding domain but not an NKG2A binding domain) may include a first linker that links the nucleic acid encoding CD300a VL to the nucleic acid encoding CD300a VH. In embodiments in which recombinant nucleic acids encode an NKG2A-binding domain and a CD300a-binding domain, the recombinant nucleic acid may include a first linker linking the nucleic acid encoding NKG2A VL to the nucleic acid encoding NKG2A VH, and a second linker linking the nucleic acid encoding CD300a VL to the nucleic acid encoding CD300a VH. Such a “first linker” may be on the 5’ or 3’ side of such a “second linker” in the recombinant nucleic acid, such that NKG2A scFv is encoded on the 5’ or 3’ side of CD300a scFv in the recombinant nucleic acid. Furthermore, embodiments of the disclosed recombinant nucleic acid encoding an NKG2A-binding domain (e.g., NKG2A scFv) and a CD300a-binding domain (e.g., CD300a scFv) may include a third linker linking the NKG2A-binding domain and the CD300a-binding domain. In certain embodiments, the first linker, the second linker, and / or the third linker include a peptide linker (e.g., 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 species, different linker species, or any combination thereof.

[0408] The first linker, the second linker, and / or the third linker may be any preferred linker. In certain embodiments, the linker comprises at least a linear group comprising a group selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (-S-), and hydroxylamino (-ON(H)-) groups. In certain embodiments, the linear group comprises a group selected from alkyl, amide, and ether groups. In certain embodiments, the linear group comprises one or more alkyl groups. In some embodiments, one or more linkers comprise one or more amino acids (e.g., a peptide linker). Such linkers may have amino acid lengths of 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 longer. Exemplary peptide linkers have lengths of 3 amino acids (AA) to 90AA, 3AA to 80AA, 3AA to 70AA, 3AA to 60AA, 3AA to 50AA, 3AA to 40AA, 3AA to 30AA, 3AA to 20AA, and 3AA to 10AA. For example, linkers may have lengths of 3AA to 5AA, 5AA to 10AA, 10AA to 15AA, 15AA to 20AA, 20AA to 25AA, 25AA to 30AA, 30AA to 35AA, 35AA to 40AA, 40AA to 50AA, 50AA to 60AA, 60AA to 70AA, 70AA to 80AA, 80AA to 90AA, or 90AA to 100AA. In some embodiments, the peptide linker has a length of 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.

[0409] The peptide linker can include any of a variety of sequences. For example, the peptide linker can include one or more serine (S), glycine (G), and / or threonine (T) residues. Glycine can impart flexibility to a given linker, and serine and / or threonine can improve solubility. In some embodiments, the linker is a glycine-serine linker. In certain embodiments, the glycine-serine linker is (Gly m -Ser) n (where m is 1 to 10 (e.g., 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 (e.g., 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 and non-limiting embodiments, the first linker, the second linker, and / or the third linker is a glycine-serine linker that includes (Gly m -Ser) n (where m is 3 to 6 and n is 1 to 10). In other specific and non-limiting embodiments, m = 4 and n = 5.

[0410] In some embodiments where the first, second, and / or third linker is a cleavable peptide, the first, second, and / or third linker is a self-cleaving peptide. In certain 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 includes the amino acid glycine-serine-glycine (GSG) at the N-terminus.

[0411] In certain embodiments, the first linker, the second linker, and / or the third linker include 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 with respect to any one of SEQ ID NOs. 29-33 and 122. In other certain embodiments, the first linker, the second linker, and / or the third linker include any one of SEQ ID NOs. 29-33 and 122. In other certain embodiments, the first linker, the second linker, and / or the third linker consist of any one of SEQ ID NOs. 29-33 and 122.

[0412] In some embodiments, the nucleotide sequences encoding the first linker, the second linker, and / or the third linker include nucleotide sequences 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 with any one of SEQ ID NOs: 34-37, 111, 115, or 116. In certain embodiments, the nucleotide sequences encoding the first linker, the second linker, and / or the third linker include any one of SEQ ID NOs: 34-37, 111, 115, or 116. In certain embodiments, the nucleotide sequences encoding the first linker, the second linker, and / or the third linker consist of any one of SEQ ID NOs: 34-37, 111, 115, or 116.

[0413] Sequence ID 34 (encoding the glycine-serine linker in Sequence ID 30): GGTGGAGGAGGTTCTGGTGGTGGAGGATCAGGAGGCGGTGGAAGCGGAGGTGGAGGATCTGGTGGAGGTGGATCA

[0414] Sequence ID 35 (coding the Whitlow / 218 linker in Sequence ID 31): GGATCGACCTCAGGCTCTGGTAAACCTGGTAGTGGGGAGGGCTCCACCAAGGGA

[0415] Sequence ID 36 (encoding the T2A linker in Sequence ID 32): GGTTCTGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0416] Sequence ID 37 (encoding the P2A linker in Sequence ID 33): GGATCCGGAGCCACCAACTTTAGCCTGCTCAAACAAGCCGGCGACGTGGAAGAGAACCCCGGACCT

[0417] Sequence ID 111 (Codon-optimized sequence encoding the Whitlow / 218 linker): GGATCGACCTCAGGCTCTGGTAAACCTGGTTCTGGGGAGGGCTCCACTAAGGGA

[0418] Sequence ID 115 (codon-optimized sequence encoding the T2A linker): GGTAGTGGCGAAGGCAGAGGCTCATTGCTCACTTGTGGCGATGTGGAGGAAAACCCTGGGCCT

[0419] Sequence ID 116 (codes the T2A linker): GGAAGCGGACAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAACCCTGGACCT

[0420] C. signal peptide In some embodiments, the disclosed recombinant nucleic acid comprises a signal peptide, which is a cell surface expression signal peptide that induces the recombinant nucleic acid protein product to the cell surface. Any preferred signal peptide that induces the recombinant nucleic acid protein product to the cell surface may be useful 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 certain embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 38. In some embodiments of the disclosed recombinant nucleic acids, the 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 with any one of SEQ ID NOs. 39-41. In certain embodiments, the nucleotide sequence encoding the signal peptide comprises or consists of any one of SEQ ID NOs. 39-41. In other specific embodiments, the nucleotide sequence encoding the signal peptide comprises or consists of any one of SEQ ID NOs. 39-41.

[0421] Sequence ID 39: ATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCTCATCCTGCGTTCCTGCTGATTCCC

[0422] SEQ ID NO: 40 (codon-optimized sequence encoding the signal peptide of SEQ ID NO: 38): ATGGTGCTCTTGGTGACTTCCCTTTTGCTGTGCGAGCTGCCGCACCCGGCTTTTCTCTTGATCCCC

[0423] Sequence ID 41:ATGGTGCTCTTGGTGACTTCCCTTCTGCTGTGCGAGCTGCCCCACCCTGCGTTCCTGCTCATCCCT

[0424] 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 certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments of the disclosed recombinant nucleic acid, the 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 certain embodiments, the nucleotide sequence encoding the signal peptide comprises SEQ ID NO: 114. In other specific embodiments, the nucleotide sequence encoding the signal peptide consists of SEQ ID NO: 114.

[0425] SEQ ID NO: 114 (codon-optimized sequence encoding the signal peptide of SEQ ID NO: 93):

[0426] [ka]

[0427] In some embodiments, the nucleotide sequence encoding the signal peptide is located 5' to the nucleotide sequence encoding the NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the signal peptide is located 5' to the nucleotide sequence encoding the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the signal peptide is located 5' to both the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain.

[0428] D. Hinge region Embodiments of the recombinant nucleic acids disclosed may include one or more extracellular hinge regions, which may or may not share sequence homology with immunoglobulin hinge regions, providing flexibility to the recombinant nucleic acid protein product. In some embodiments, the hinge region separates a binding domain (e.g., a CD300a binding domain (e.g., the disclosed CD300a VHH or CD300a scFv) or an NKG2A binding domain (e.g., the disclosed NKG2A scFv)) from a transmembrane domain. In some embodiments, the disclosed recombinant nucleic acids do not encode a hinge region. In some embodiments, the hinge region includes a CD8 hinge, an hIgG1 hinge, an hIgG2 hinge, an hIgG3 hinge, an FACD hinge, or any combination thereof. In some embodiments, the hinge region contains 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 with respect to any one of sequence numbers 43-48. In certain embodiments, the hinge region contains any one of sequence numbers 43-48. In certain embodiments, the hinge region consists of any one of sequence numbers 43-48.

[0429] Sequence ID 43 (Spacer + hIgG1 Hinge): AAATTTPEPKSCDKTHTCP

[0430] Sequence ID 44 (Spacer + hIgG2 Hinge): AAATTTPERKCCVECPPCP

[0431] Sequence ID 45 (Spacer + hIgG3 Hinge): AAATTTPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

[0432] Sequence ID 46 (IGHG hinge):

[0433] [ka]

[0434] Sequence ID 47 (Spacer + CD8 Hinge): AAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0435] Sequence ID 48 (Spacer + FACD): AAATTTPFACD

[0436] In some embodiments of the disclosed recombinant nucleic acids, the 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 with any one of SEQ ID NOs. 50-55. In certain embodiments, the nucleotide sequence encoding the hinge region comprises any one of SEQ ID NOs. 50-55. In certain embodiments, the nucleotide sequence encoding the hinge region comprises any one of SEQ ID NOs. 50-55.

[0437] Sequence ID 50 (encoding spacer + hIgG1 hinge): GCAGCCGCAACAACGACACCTGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCT

[0438] Sequence ID 51 (encoding spacer + hIgG2 hinge): GCAGCCGCAACAACGACACCTGAACGAAAATGCTGTGTGGAATGCCCACCCTGTCCT

[0439] Sequence ID 52 (encoding spacer + hIgG3 hinge):

[0440] [ka]

[0441] Sequence ID 53 (encoding the IGGH hinge):

[0442] [ka]

[0443] Sequence ID 54 (coding spacer + CD8 hinge):

[0444] [ka]

[0445] Sequence ID 5 (encoding spacer + FACD): GCAGCCGCAACAACGACACCTTTTGCGTGTGAC

[0446] In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the signal peptide. In other embodiments, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the NKG2A binding domain. In other embodiments, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the CD300a binding domain. In other embodiments, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the signal peptide and 3' to the nucleotide sequence encoding the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the signal peptide and 3' to the nucleotide sequence encoding the NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located 3' to the nucleotide sequence encoding the signal peptide, 3' to the nucleotide sequence encoding the NKG2A binding domain, and 3' to the nucleotide sequence encoding the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the hinge region is located (a) 3' to the nucleotide sequence encoding the signal peptide, (b) 3' to the nucleotide sequences encoding the NKG2A binding domain and the CD300a binding domain, or (c) both (a) and (b).

[0447] E. Transmembrane domain Some embodiments of the recombinant nucleic acids disclosed include one or more transmembrane domains. Any suitable transmembrane domain may be useful in this disclosure. In some embodiments, the transmembrane domain is a human transmembrane domain or a mouse transmembrane domain. In certain embodiments, the transmembrane domain includes or consists of the CD8, CD80, mCD80, ITGA, HLA-B57, proCAR-4, CD28, KIR2DL1, PDGFRB, or CD86 transmembrane domain.

[0448] 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 with any one of SEQ ID NOs. 67-76 or 96. In certain embodiments, the transmembrane domain comprises any one of SEQ ID NOs. 67-76 or 96. In other specific embodiments, the transmembrane domain comprises any one of SEQ ID NOs. 67-76 or 96.

[0449] Sequence ID 67 (CD8 TM): IYIWAPLAGTCGVLLLSLVITLYCN

[0450] Sequence ID 68 (CD28 TM):FWVLVVVGGVLACYSLLVTVAFIIFWVR

[0451] Sequence ID 69 (mCD80 TM): PPEDPPDSKNTLVLFGAGFGAVITVVVIVVII

[0452] Sequence ID 70 (KIR2DL1 TM):PRHLHILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQ

[0453] Sequence ID 71 (PDGFRB TM): AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0454] Sequence ID 72 (proCAR-4™): PFWLLVALLALLAVIAALLAAIFALLWVR

[0455] Sequence ID 73 (ITGA™): VPLWVILLSAFAGLLLLMLLILALW

[0456] Sequence ID 74 (CD80™): KQEHFPDNLLPSWAITLISVNGIFVICCL

[0457] Sequence ID 75 (CD86 TM): PPDHIPWITAVLPTVIICVMVFCLILW

[0458] Sequence ID 76 (HLA-B57™):VGIVAGLAVLAVVVIGAVVAAVMCR

[0459] Sequence ID 96 (another CD8™ domain): PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN

[0460] In some embodiments of the disclosed recombinant nucleic acids, the 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 with any one of SEQ ID NOs. 56-65. In certain embodiments, the nucleotide sequence encoding the transmembrane domain comprises any one of SEQ ID NOs. 56-65. In other specific embodiments, the nucleotide sequence encoding the transmembrane domain comprises any one of SEQ ID NOs. 56-65.

[0461] Sequence ID 56 (encoding the CD8™ of Sequence ID 67): ATCTACATCTGGGCTCCTTTGGCAGGCACCTGCGGGGTGCTGCTGCTGTCCCTGGTGATCACCCTGTACTGTAAC

[0462] Sequence ID 57 (coded CD28™): TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG

[0463] Sequence ID 58 (encoding mCD80™): CCTCCTGAGGACCCTCCAGACAGCAAGAATACCCTGGTGCTGTTCGGAGCCGGATTCGGCGCTGTGATTACCGTGGTGGTCATCGTCGTGATCATC

[0464] Sequence ID 59 (encoding KIR2DL1™): CCCCGACACCTGCACATTCTGATTGGGACCTCAGTGGTCATCATCCTCTTCATCCTCCTCTTCTTTCTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCGGTAATGGACCAA

[0465] Sequence ID 60 (encoding PDGFRB™):

[0466] [ka]

[0467] Sequence ID 61 (encoding proCAR-4™): CCTTTTTGGCTTTTAGTCGCCTTATTGGCGCTGCTTGCTGTAATCGCCGCATTATTAGCAGCTATCTTTGCATTGCTGTGGGTGAGG

[0468] Sequence ID 62 (coding ITGA™): GTGCCATTATGGGTCATCCTGCTGAGTGCTTTTGCCGGATTGTTGCTGTTAATGCTGCTCATTTTAGCACTGTGG

[0469] Sequence ID 63 (encoding CD80™): AAGCAGGAGCACTTCCCCGACAACCTGCTGCCTTCTTGGGCCATCACTCTCATCTCCGTGAATGGCATCTTCGTCATTTGTTGCCTG

[0470] Sequence ID 64 (encoding CD86™): CCTCCCGACCATATCCCGTGGATTACAGCCGTGCTGCCAACCGTCATCATCTGCGTGATGGTGTTCTGCCTGATTCTCTGG

[0471] Sequence ID 65 (encoding HLA-B57™): GTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGCTGCTGTGATGTGTAGG

[0472] In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the signal peptide. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequences encoding the NKG2A binding domain and the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the spacer. In some embodiments, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the hinge region. In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the signal peptide and 3' to the nucleotide sequence encoding the CD300a binding domain. In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the signal peptide, 3' to the nucleotide sequence encoding the CD300a binding domain, and 3' to the nucleotide sequence encoding the spacer. In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the signal peptide and 3' to the nucleotide sequence encoding the NKG2A binding domain. In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the transmembrane domain is located 3' to the nucleotide sequence encoding the signal peptide, 3' to the nucleotide sequence encoding the NKG2A binding domain, and 3' to the nucleotide sequence encoding the spacer.In some embodiments of the recombinant nucleic acids disclosed, the nucleotide sequence encoding the transmembrane domain is located at (a) the 3' end of the nucleotide sequence encoding the signal peptide, (b) the 3' end of the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain, (c) the 3' end of the nucleotide sequence encoding the hinge region, or (d) any combination of (a) to (c).

[0473] F. Cytoplasmic Domain Embodiments of recombinant nucleic acids disclosed may comprise one or more cytoplasmic domains, where the cytoplasmic domain is a human cytoplasmic domain or a mouse cytoplasmic domain. Any preferred cytoplasmic domain may be useful in the disclosed embodiments. In some embodiments, the cytoplasmic domain comprises or comprises the CD8v2, CD8v1, mCD80, CD80, CD86, or 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 certain embodiments, the cytoplasmic domain comprises any one of SEQ ID NOs. 83-88. In other specific embodiments, the cytoplasmic domain comprises any one of SEQ ID NOs. 83-88.

[0474] Sequence ID 83 (CD8v2 cyt):HRNRRRVCKCPRPVVKSGDKPSLSARYV

[0475] Sequence ID 84 (CD8v1 cyt):HRNRRRVCKCPRPVV

[0476] Sequence ID 85 (mCD80 cyt): KCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFL

[0477] Sequence ID 86 (CD80 cyt): TYCFAPRCRERRRNERLRRESVRPV

[0478] Sequence ID 87 (CD86 cyt): KWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF

[0479] Sequence ID 88 (HLA-B57 cyt):RKSSGGKGGSYSQAACSDSAQGSDVSLTA

[0480] In some embodiments of the disclosed recombinant nucleic acids, the 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 with any one of SEQ ID NOs. 77–82. In certain embodiments, the nucleotide sequence encoding the cytoplasmic domain comprises any one of SEQ ID NOs. 77–82. In other specific embodiments, the nucleotide sequence encoding the cytoplasmic domain comprises any one of SEQ ID NOs. 77–82.

[0481] Sequence ID 77 (encoding CD8v2 cyt): CATCGCAATAGGCGGAGAGTCTGCAAATGCCCTCGGCCTGTTGTCAAGTCAGGAGACAAGCCAAGCCTCAGCGCACGCTATGTC

[0482] Sequence ID 78 (encoding CD8v1 cyt): CACCGGAACAGGCGGAGAGTGTGCAAGTGCCCTAGACCTGTGGTT

[0483] Sequence ID 79 (encoding mCD80 cyt): AAGTGTTTCTGCAAGCACAGAAGCTGCTTCCGGCGGAACGAGGCCAGCAGAGAGACAAACAACAGCCTGACATTCGGCCCCGAAGAGGCCCTGGCTGAGCAGACAGTTTTTCTG

[0484] Sequence ID 80 (encoding CD80 cyt): ACCTACTGCTTTGCTCCGAGGTGTCGAGAACGGCGTCGCAACGAGCGCCTGAGGAGAGAGTCTGTCCGCCCAGTG

[0485] Sequence ID 81 (encoding CD86 cyt):

[0486] [ka]

[0487] SEQ ID NO: 82:AGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTGCAGCGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCT

[0488] In some embodiments of the disclosed recombinant nucleic acids, the nucleotide sequence encoding the cytoplasmic domain is located 3' to the nucleotide sequence encoding the signal peptide. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3' to the nucleotide sequence encoding the NKG2A binding domain. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3' to the nucleotide sequence encoding the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3' to the nucleotide sequences encoding the NKG2A binding domain and the CD300a binding domain. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3' to the nucleotide sequence encoding the hinge region. In some embodiments, the nucleotide sequence encoding the cytoplasmic domain is located 3' to the nucleotide sequence encoding the transmembrane domain. In some embodiments of the recombinant nucleic acids disclosed, the nucleotide sequence encoding the cytoplasmic domain is located at (a) the 3' end of the nucleotide sequence encoding the signal peptide, (b) the 3' end of the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain, (c) the 3' end of the nucleotide sequence encoding the hinge region, (d) the 3' end of the nucleotide sequence encoding the transmembrane domain, or (e) any combination of (a) to (d).

[0489] G. Exemplary trans-antigen signaling receptors (TASRs) This disclosure discloses a novel class of gene constructs called "TASRs" (trans-antigen signaling receptors) that, when expressed on the surface of mammalian cells, can enhance the persistence of such cells by reducing or preventing, for example, rejection of such cells by autologous NK cells. A TASR is a modular construct comprising the following domains or functional parts: 1) a signal peptide for presenting a molecule on the cell surface; 2) a binding domain that binds to an inhibitory receptor expressed on NK cells, e.g., VHH (e.g., CD300a VHH as disclosed herein), or a single-stranded variable fragment (scFv) containing 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. The TASRs disclosed herein can bind to inhibitory receptors on NK cells, e.g., NKG2A or CD300a. The Disclosure and the Examples Provided herein demonstrate certain TASR designs that have remarkable efficacy in reducing or preventing immunotherapy cell destruction by NK cells, for example, but the Disclosure is not limited to the specific designs discussed in the Examples section.

[0490] In some embodiments, the recombinant nucleic acids disclosed herein encode one or more components of such a TASR. In some embodiments, the TASR includes an NKG2A binding domain ("NKG2A TASR"), for example, any preferred NKG2A binding domain, for example, the NKG2A binding domain described herein. In some embodiments, the TASR includes a CD300a binding domain ("CD300a TASR"), for example, any preferred CD300a binding domain, for example, the CD300a binding domain described herein. In some embodiments, the TASR is defined by the following formula:

[0491] SP-VL-linker-VH-hinge-TM-Cyt or SP-VH-linker-VL-hinge-TM-Cyt (in the formula,

[0492] "SP" stands for arbitrarily selected signal peptide.

[0493] "VL" represents the light chain variable region (e.g., NKG2A VL or CD300a VL),

[0494] "Linker" refers to a linker.

[0495] "VH" represents the heavy chain variable region (e.g., NKG2A VH or CD300a VH),

[0496] "Spacer" refers to a spacer,

[0497] "Hinge" refers to an optional hinge area.

[0498] "TM" represents the transmembrane domain,

[0499] ("Cyt" represents an optional cytoplasmic domain.)

[0500] In some embodiments, TASR is defined by the following formula:

[0501] SP-VL-linker-VH-spacer-TM-Cyt or SP-VH-linker-VL-spacer-TM-Cyt.

[0502] In some embodiments, TASR is defined by the following formula:

[0503] SP-VHH-Spacer-TM-Cyt (for example, SP-CD300a VHH-Spacer-TM-Cyt).

[0504] In some embodiments, TASR is defined by the following formula:

[0505] SP-VHH-Hinge-TM-Cyt (for example, SP-CD300a VHH-Hinge-TM-Cyt).

[0506] In some embodiments, TASR is defined by the following formula:

[0507] SP-VHH-TM-Cyt (for example, SP-CD300a VHH-TM-Cyt).

[0508] In some embodiments of the disclosed nucleic acids, the recombinant nucleic acid encodes a spacer. In some embodiments, the spacer of the 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 with respect to SEQ ID NO: 42 (AAATTTP). In certain embodiments, the spacer comprises or consists of SEQ ID NO: 42. In some embodiments, the recombinant nucleic acid encoding the spacer (of the 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 with respect 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 the disclosed recombinant nucleic acids, the nucleotide sequence encoding the spacer is located (a) at the 3' end of the nucleotide sequence encoding the signal peptide, (b) at the 3' end of the nucleotide sequence encoding the CD300a binding domain, at the 3' end of the nucleotide sequence encoding the NKG2A binding domain, or at the 3' end of the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain, or (c) at both (a) and (b). In some embodiments in which the TASR includes a spacer, the TASR further does not include a hinge.

[0509] Figures 1, 31, 32, and 45 illustrate exemplary formulas of useful TASRs as described herein. The TASRs described herein (e.g., the TASRs in Figures 1, 31, 32, or 45) may include any suitable signal peptide, linker, spacer, hinge, transmembrane domain, or cytoplasmic domain, or any combination thereof, for example, any of the signal peptides, linkers, spacers, hinges, transmembrane domains, or cytoplasmic domains described in detail herein. In certain embodiments, the linker is the Whitlow / 218 linker described herein (e.g., the linker in SEQ ID NO: 31).

[0510] In some embodiments, the CD300a TASR comprises 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 the CD300a VHHs, e.g., SEQ ID NOs: 155-172. In some embodiments, such a CD300a TASR further comprises a signal peptide, e.g., one of the signal peptides disclosed herein, that induces the TASR (e.g., a protein product of a disclosed recombinant nucleic acid encoding a TASR containing CD300a VHH) to the surface of a cell. In some embodiments, the signal peptide is a CD8 signal peptide. In some embodiments, such a CD300a TASR further comprises a transmembrane domain, e.g., any preferred transmembrane domain, e.g., any transmembrane domain disclosed herein. In certain embodiments, the transmembrane domain is a CD8 transmembrane domain or an mCD80 transmembrane domain. In certain embodiments, the transmembrane domain is the CD8 transmembrane domain. In some embodiments, such a CD300a TASR further includes a cytoplasmic domain, for example any preferred cytoplasmic domain, for example any cytoplasmic domain disclosed herein. In certain embodiments, the transmembrane domain is the mCD80 cytoplasmic domain. In some embodiments, such a CD300a TASR further includes a spacer, for example the spacer of SEQ ID NO: 42. In such embodiments, the spacer is located in the TASR between the CD300a VHH and the transmembrane domain. In certain embodiments, the hinge region is the CD8 hinge region. In some embodiments, such a CD300a TASR further includes a hinge region, for example any of the hinge regions disclosed herein. In such embodiments, the hinge region is located in the TASR between the CD300a VHH and the transmembrane domain. In certain embodiments, the hinge region is the CD8 hinge region.In some embodiments, such a CD300a TASR does not include a hinge region.

[0511] In certain embodiments, the CD300a TASR (e.g., the CD300a TASR encoded by the disclosed recombinant nucleic acid) comprises a signal peptide, a CD300a VHH (e.g., 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 spacer from the transmembrane domain.In certain embodiments, CD300a TASR (e.g., encoded by the disclosed recombinant nucleic acid) is a CD8 signal peptide (e.g., 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, for example, SEQ ID NO: 93), CD300a VHH (e.g., a CD300a 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, for example, any one of SEQ ID NOs: 155-172). Spacers 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 sequence number 42, CD8 transmembrane domains (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least to sequence number 67) The CD300a TASR (e.g., encoded by the disclosed recombinant nucleic acid) comprises a transmembrane domain having 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and an mCD80 cytoplasmic domain (e.g., 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 certain embodiments, the CD300a TASR (e.g., encoded by the disclosed recombinant nucleic acid) comprises the CD8 signal peptide of SEQ ID NO: 93, one of the CD300a VHH 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.

[0512] In certain embodiments, the recombinant nucleic acid encoding 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 with respect to any one of the amino acid sequences of SEQ ID NOs. 220-237. In certain embodiments, the recombinant nucleic acid encoding CD300a TASR encodes an amino acid sequence containing or consisting of any one of the amino acid sequences of SEQ ID NOs. 220-237.

[0513] Sequence ID 220:

[0514] [ka]

[0515] Sequence ID 221:

[0516] [ka]

[0517] Sequence ID 222:

[0518] [ka]

[0519] Sequence ID 223:

[0520] [ka]

[0521] Sequence ID 224:

[0522] [ka]

[0523] Sequence ID 225:

[0524] [ka]

[0525] Sequence ID 226:

[0526] [ka]

[0527] Sequence ID 227:

[0528] [ka]

[0529] Sequence ID 228:

[0530] [ka]

[0531] Sequence ID 229:

[0532] [ka]

[0533] Sequence ID 230:

[0534] [ka]

[0535] Sequence ID 231:

[0536] [ka]

[0537] Sequence ID 232:

[0538] [ka]

[0539] Sequence ID 233:

[0540] [ka]

[0541] Sequence ID 234:

[0542] [ka]

[0543] Sequence ID 235:

[0544] [ka]

[0545] Sequence ID 236:

[0546] [ka]

[0547] Sequence ID 237:

[0548] [ka]

[0549] In certain embodiments, the recombinant nucleic acid encoding CD300a TASR has sequence identity of 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% with respect to any one nucleic acid sequence of sequence numbers 202 to 219. In certain embodiments, the recombinant nucleic acid encoding CD300a TASR contains or consists of any one nucleic acid sequence of sequence numbers 202 to 219.

[0550] Sequence ID 202:

[0551] [ka]

[0552] Sequence ID 203:

[0553] [ka]

[0554] Sequence ID 204:

[0555] [ka]

[0556] Sequence ID 205:

[0557] [ka]

[0558] Sequence ID 206:

[0559] [ka]

[0560] Sequence ID 207:

[0561] [ka]

[0562] Sequence ID 208:

[0563] [ka]

[0564] Sequence ID 209:

[0565] [ka]

[0566] Sequence ID 210:

[0567] [ka]

[0568] Sequence ID 211:

[0569] [ka]

[0570] Sequence ID 212:

[0571] [ka]

[0572] Sequence ID 213:

[0573] [ka]

[0574] Sequence ID 214:

[0575] [ka]

[0576] Sequence ID 215:

[0577] [ka]

[0578] Sequence ID 216:

[0579] [ka]

[0580] Sequence ID 217:

[0581] [ka]

[0582] Sequence ID 218:

[0583] [ka]

[0584] Sequence ID 219:

[0585] [ka]

[0586] In some embodiments, NKG2A TASR includes NKG2A VL and NKG2A VH, for example, any NKG2A VL and any NKG2A VH disclosed herein (for example, included in NKG2A scFv disclosed herein). In certain embodiments, the recombinant nucleic acid encoding 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 with respect to SEQ ID NO: 21. In certain embodiments, the recombinant nucleic acid encoding NKG2A TASR encodes an amino acid sequence comprising or consisting of SEQ ID NO: 21.

[0587] Sequence ID 21:

[0588] [ka]

[0589] In certain embodiments, the recombinant nucleic acid encoding 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 with respect to SEQ ID NO: 23. In other specific embodiments, the recombinant nucleic acid encoding NKG2A TASR comprises or consists of SEQ ID NO: 23.

[0590] Sequence ID 23:

[0591] [ka]

[0592] In some embodiments, CD300a TASR comprises CD300a VL and CD300a VH, for example, any CD300a VL and any CD300a VH disclosed herein (for example, included in CD300a scFv disclosed herein). In certain embodiments, the recombinant nucleic acid encoding 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 with respect to SEQ ID NO: 22. In certain embodiments, the recombinant nucleic acid encoding CD300a TASR encodes an amino acid sequence comprising or consisting of SEQ ID NO: 22.

[0593] Sequence ID 22:

[0594] [ka]

[0595] In certain embodiments, the recombinant nucleic acid encoding 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 with respect to SEQ ID NO: 24. In other specific embodiments, the recombinant nucleic acid encoding CD300a TASR comprises or consists of SEQ ID NO: 24.

[0596] Sequence ID 24:

[0597] [ka]

[0598] H. Exemplary bispecific TASR In some embodiments, the TASRs disclosed herein are bispecific TASRs, which comprise two or more binding domains and are therefore capable of binding to two or more inhibitory receptors on NK cells, e.g., both NKG2A and CD300a. Such a bispecific TASR may include two scFvs, e.g., a first scFv that specifically binds to a first inhibitory receptor expressed on NK cells (e.g., CD300a) and a second scFv that specifically binds to a second inhibitory receptor expressed on NK cells (e.g., NKG2A). In some embodiments, a bispecific TASR is defined by the following formula:

[0599] SP-VL1-Linker1-VH1-Linker3-VL2-Linker2-VH2-Hinge-TM-Cyt(in the formula,

[0600] "SP" stands for arbitrarily selected signal peptide.

[0601] "VL1" represents the first light chain variable region (e.g., NKG2A VL or CD300a VL),

[0602] "Linker 1" refers to the first linker.

[0603] "VH1" represents the first heavy chain variable region (e.g., NKG2A VH or CD300a VH),

[0604] "Linker 3" refers to the third linker.

[0605] "VL2" represents the second light chain variable region (e.g., NKG2A VL or CD300a VL),

[0606] "Linker 2" refers to the second linker.

[0607] "VH2" represents the second heavy chain variable region (e.g., NKG2A VH or CD300a VH),

[0608] "Hinge" refers to an optional hinge area.

[0609] "TM" represents the transmembrane domain,

[0610] ("Cyt" represents an optional cytoplasmic domain.)

[0611] Alternatively, the bispecific TASRs disclosed herein may 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. Figure 1 shows a diagram of an exemplary formula for a useful bispecific TASR in this specification. The bispecific TASRs described herein (e.g., the bispecific TASR in Figure 1) may include any suitable signal peptide, linker, hinge, transmembrane domain, or cytoplasmic domain, e.g., any of the signal peptides, linkers, hinges, transmembrane domains, or cytoplasmic domains described in detail herein. In certain embodiments, the third linker includes a cleavable peptide linker, for example, a T2A linker as described herein (e.g., the T2A linker of SEQ ID NO: 32), or a P2A linker as described herein (e.g., the P2A linker of SEQ ID NO: 33).

[0612] In some embodiments of the disclosed dual-specificity TASR, VL1-linker1-VH1 (or VH1-L1-VL1) is either CD300a scFv (including, for example, CD300a VL, linker, and CD300a VH as disclosed herein) or NKG2A scFv (including, for example, NKG2A VL, linker, and NKG2A VH as disclosed herein). Similarly, in some embodiments, VL2-linker2-VH2 (or VH2-linker-VL2) is either CD300a scFv (e.g., CD300a scFv disclosed herein, e.g., CD300a scFv including CD300a VL, linker, and CD300a VH disclosed herein) or NKG2A scFv (e.g., NKG2A scFv disclosed herein, e.g., NKG2A scFv including NKG2A VL, linker, and NKG2A VH disclosed herein). In some embodiments, if VL1-linker1-VH1 (or VH1-L1-VL1) is CD300a scFv, then VL2-linker2-VH2 (or VH2-linker-VL2) is NKG2A scFv. In another embodiment, if VL1-linker1-VH1 (or VH1-L1-VL1) is NKG2A scFv, then VL2-linker2-VH2 (or VH2-linker-VL2) is CD300a scFv.

[0613] In some embodiments, the nucleotide sequences of the disclosed recombinant nucleic acids encoding bispecificity TASR, NKG2A VL, NKG2A VH, CD300a VL, and / or CD300a VH, are codon-optimized to reduce or prevent undesirable recombination events, for example. In some embodiments, the codon-optimized nucleotide sequences encoding bispecificity TASR, NKG2A scFv, have 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 with respect to SEQ ID NO: 113. In certain embodiments, the codon-optimized nucleotide sequences encoding NKG2A scFv include or consist of SEQ ID NO: 113.

[0614] In some embodiments, the codon-optimized nucleotide sequence encoding the bispecific TASR 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 with respect to SEQ ID NO: 110. In certain embodiments, the codon-optimized nucleotide sequence encoding the NKG2A VL includes or consists of SEQ ID NO: 110.

[0615] In some embodiments, the codon-optimized nucleotide sequence encoding the bispecific TASR 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 with respect to SEQ ID NO: 112. In certain embodiments, the codon-optimized nucleotide sequence encoding NKG2A VH includes or consists of SEQ ID NO: 112.

[0616] In certain embodiments, the recombinant nucleic acid encoding the bispecificity 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 with respect to SEQ ID NO: 25 or SEQ ID NO: 26. In certain 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.

[0617] Sequence ID 25 (CD300a TASR of Sequence ID 22 linked to NKG2A TASR of Sequence ID 21 via T2A linker of Sequence ID 32):

[0618] [ka]

[0619] Sequence ID 26: (NKG2A TASR of Sequence ID 21 linked to CD300a TASR of Sequence ID 22 via T2A linker of Sequence ID 32):

[0620] [ka]

[0621] In certain embodiments, the recombinant nucleic acid encoding the bispecificity 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 with respect to SEQ ID NO: 27 or SEQ ID NO: 28. In other specific embodiments, the recombinant nucleic acid encoding the bispecificity TASR comprises or consists of SEQ ID NO: 27 or SEQ ID NO: 28.

[0622] Sequence ID 27 (encoding the bispecificity TASR of Sequence ID 25):

[0623] [ka]

[0624] [ka]

[0625] Sequence ID 28 (encoding the bispecificity TASR of Sequence ID 26):

[0626] [ka]

[0627] [ka]

[0628] III. Vectors This disclosure provides vectors comprising the disclosed recombinant nucleic acids, for example, vectors comprising one or more NKG2A and / or CD300a binding domains, for example, CD300a VHH as disclosed herein, or one or more NKG2A and / or CD300a scFv. The vectors of this disclosure may include, for example, an NKG2A binding domain (e.g., NKG2A scFv) and / or a CD300a binding domain (e.g., CD300a VHH or CD300a scFv), or an NKG2A TASR and / or CD300a TASR (e.g., a CD300a TASR containing CD300a VHH, or a bispecific TASR containing both NKG2A scFv and CD300a scFv as described herein).

[0629] Vectors useful in this embodiment may be, for example, DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, Roussarcoma virus (RSV) vectors, or retroviral vectors. In certain embodiments of the vector, recombinant nucleic acids are operably ligated to a promoter. In specific and 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 includes 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 with respect to SEQ ID NO: 92.

[0630] Sequence ID 92 (encoding the EF1a promoter):

[0631] [ka]

[0632] Recombinant nucleic acids as used herein may also include other regulatory elements that enable and / or regulate the transcription of coding sequences and / or regulate the translation of encoded polypeptides (e.g., TASRs or bispecific TASRs as described herein), namely transcriptional and translational regulatory sequences, such as enhancers, polyadenylation signals, terminators, proteolytic signals, etc. Exemplary regulatory sequences are well known in the art and are described, for example, in Goddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0633] In some embodiments, the nucleic acid sequence in the vector further comprises a poly(A) sequence. In certain embodiments, the poly(A) sequence comprises 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, for example, SEQ ID NO: 89.

[0634] Sequence ID 89 (encoding the bGH poly(A) signal):

[0635] [ka]

[0636] In some embodiments, the nucleic acid sequence in the vector further includes a 3'UTR.

[0637] In certain embodiments, the vector containing the recombinant nucleic acid disclosed is integrated into the genome at adeno-associated virus integration site 1 (AAVS1) of the genome. Intron 1 of the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene on human chromosome 19 is referred to as the AAVS1 locus, as first described as a major hotspot for adeno-associated virus (AAV) integration (Oceguera-Yanez et al., Methods. 2016;101:43-55). This locus enables stable and long-term transgene expression in many cell types, including embryonic stem cells. Therefore, in some embodiments, the vector disclosed herein includes an AAVS1 right homology arm and an AAVS1 left homology arm. In some embodiments, the nucleic acid encoding the AAVS1 left-side 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 with respect to SEQ ID NO: 90.

[0638] Sequence ID 90 (coding the left homology arm of AAVS1):

[0639] [ka]

[0640] In some embodiments, the nucleic acid encoding the AAVS1 right-side 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 with respect to SEQ ID NO: 91.

[0641] Sequence ID 91 (coding the right-side homology arm of AAVS1):

[0642] [ka]

[0643] IV. Manipulated Cells Some embodiments of this disclosure include engineered (e.g., genetically modified) cells, such as engineered human cells. In some embodiments, the cells are allogeneic cells. Engineered cells refer to cells (or offspring of cells) that include engineered genetic modifications, such as cells that have come into contact with a gene editing system and have been genetically modified by that gene editing system. The terms “engineered cells” and “genetically modified cells” are used interchangeably throughout. Engineered human cells may be any of the exemplary cell types disclosed herein.

[0644] In some embodiments, the cells are stem cells. In some embodiments, the cells are pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs). In some embodiments, the manipulated cells are selected from stem cells, progenitor cells, or cells differentiated from stem cells or progenitor cells. In some embodiments, the manipulated cells are pluripotent stem cells that later differentiate into immune cells. In some embodiments, the cells are immune cells. As used herein, “immune cells” refers to cells of the immune system, including, for example, lymphocytes (e.g., T cells, B cells, natural killer cells ("NK cells", and NKT cells, or iNKT cells)), monocytes, macrophages, mast cells, dendritic cells, or granulocytes (e.g., neutrophils, eosinophils, and basophils). In some embodiments, the cells are primary immune cells. In some embodiments, the cells are pluripotent stem cell-derived immune cells. In some embodiments, the immune system cells are CD3 + CD4 + and CD8 + The immune cells can be selected from T cells, regulatory T cells (Treg), B cells, NK cells, and dendritic cells (DCs). In some embodiments, the immune cells are allogeneic.

[0645] In some embodiments, the cells are lymphocytes. In some embodiments, the cells are adaptive immune cells. In some embodiments, the cells are T cells. In some embodiments, the cells are B cells. In some embodiments, the cells are NK cells. In some embodiments, the cells are macrophages. In some embodiments, the lymphocytes are allogeneic.

[0646] As used herein, T cells can be defined as cells expressing a T cell receptor ("TCR," "αβTCR," or "γδTCR"). In some embodiments, the TCR on T cells may be genetically modified (e.g., by genetic modification of the TRAC or TRBC gene) to reduce or delete its expression, and therefore, the expression of the protein CD3 may be used as a marker for identifying T cells by standard flow cytometry methods. CD3 is a multi-subunit signaling complex that associates with the TCR. Therefore, T cells may be referred to as CD3+. In some embodiments, T cells are cells expressing the CD3+ marker and either the CD4+ marker or the CD8+ marker. In some embodiments, T cells are allogeneic.

[0647] In some embodiments, T cells express the glycoprotein CD8 and are therefore CD8+ by standard flow cytometry methods, and may be referred to as “cytotoxic” T cells. In some embodiments, T cells express the glycoprotein CD4 and are therefore CD4+ by standard flow cytometry methods, and may be referred to as “helper” T cells. CD4+ T cells can differentiate into several subsets, which may be referred to as Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, regulatory T ("Treg") cells, or follicular helper T cells ("Tfh"). Each CD4+ subset releases specific cytokines that may have either pro-inflammatory or anti-inflammatory, survival, or protective functions. T cells can be isolated from a subject by CD4+ or CD8+ selection methods.

[0648] In some embodiments, T cells are memory T cells. In the body, memory T cells have encountered an antigen before. Memory T cells may be present in secondary lymphoid organs (central memory T cells) or in recently infected tissues (effector memory T cells). Memory T cells may be CD8+ T cells. Memory T cells may be CD4+ T cells.

[0649] As used herein, “central memory T cells” can be defined as antigen-experienced T cells that may express, for example, CD62L and CD45RO. Central memory T cells can be detected, for example, by flow cytometry as CD62L+ and CD45RO+. Central memory T cells also express CCR7 and can therefore be detected as CCR7+ by standard flow cytometry methods.

[0650] As used herein, “early stem cell memory T cells” (or “Tscm”) can be defined as T cells expressing CD27 and CD45RA, and are therefore CD27+ and CD45RA+ by standard flow cytometry methods. Tscm do not express CD45RO, which is a CD45 isoform, and therefore, if stained for this isoform by standard flow cytometry methods, Tscm will also be CD45RO-. Thus, CD45RO-CD27+ cells are also early stem cell memory T cells. Tscm cells further express CD62L and CCR7, and can therefore 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 formulations.

[0651] In some embodiments, the cells are B cells. As used herein, “B cells” can be defined as cells expressing CD19, or CD20, or B cell maturation antigen (“BCMA”), and therefore B cells are CD19+, or CD20+, or BCMA+ by standard flow cytometry methods. B cells are further negative for CD3 and CD56 by standard flow cytometry methods. B cells may be plasma cells. B cells may be memory B cells. B cells may be naive B cells. B cells may be IgM+ or may have a class-switched B cell receptor (e.g., IgG+, or IgA+). In some embodiments, B cells are allogeneic.

[0652] In some embodiments, the cells are mononuclear cells derived from, for example, bone marrow or peripheral blood. In some embodiments, the cells are peripheral blood mononuclear cells ("PBMCs"). In some embodiments, the cells are PBMCs, for example, lymphocytes or monocytes. In some embodiments, the cells are peripheral blood lymphocytes ("PBLs"). In some embodiments, the mononuclear cells are allogeneic.

[0653] In some embodiments, the manipulated cells are stem cells, progenitor cells, or primary cells. Stem cells can be broadly defined as cells that can undergo multiple cycles of cell division, maintain an undifferentiated state, and have the ability to differentiate into specialized cell types (Tesche et al., Stem Cells Int. 2010;2010:824876). Stem cells may undergo asymmetric division, producing copies of themselves and daughter cells that can differentiate. Stem cells are further classified into three categories: totipotent, pluripotent, or multipotent. Totipotent cells have the ability to form an entire organism (e.g., a fertilized egg). Pluripotent stem cells lack the ability to form extraembryonic tissues and therefore cannot develop a fetus, but can give rise to any cell type derived from the three embryonic cell layers (i.e., endoderm, mesoderm, or ectoderm). Examples of pluripotent stem cells include human embryonic stem cells (hESCs) and induced pluripotent stem cells (ipSCs). Adult stem cells, or mesenchymal stem cells (MSCs), are examples of pluripotent stem cells that are isolated from mature tissues and differentiate into other tissue types. Pluripotent stem cells can differentiate into various closely related cells within a tissue but lack the ability to differentiate into other tissues. A well-studied area of ​​pluripotent stem cells is hematopoietic stem cells (HSCs), which produce daughter cells that differentiate into all subpopulations of hematopoietic cells (e.g., red blood cells, platelets, etc.).

[0654] Therefore, useful stem cells in this specification 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 tissue), 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, muscle cells, and keratinocytes. Cells for organ or tissue transplantation, e.g., islet cells, cardiomyocytes, thyroid cells, thymocytes, neuronal cells, skin cells, and retinal cells are also included.

[0655] In some embodiments, human cells are isolated from human subjects. In some embodiments, cells are isolated from human donor PBMCs or leukopaks.

[0656] In some embodiments, the cells are derived from a cell line. In some embodiments, the cell line is derived from a human subject. In some embodiments, the cells are derived from a cell bank. In some embodiments, the cells are genetically modified and then transferred to a cell bank. In some embodiments, the cells are taken from a subject, genetically modified ex vivo, and transferred to a cell bank. In some embodiments, a population of genetically modified cells is transferred to a cell bank. In some embodiments, a population of genetically modified immune cells is transferred to a cell bank. In some embodiments, a population of genetically modified immune cells comprising first and second subpopulations, wherein the first and second subpopulations have at least one common genetic modification and at least one distinct genetic modification, is transferred to a cell bank.

[0657] Various methods for introducing nucleic acids (e.g., recombinant nucleic acids disclosed herein) into cells are known in the art, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; nonviral transfection; particulate guns; lipofection; and infection (e.g., when the vector is an infectious agent).

[0658] In some embodiments, recombinant nucleic acids are introduced into cells (e.g., to create engineered cells) using a CRISPR / Cas system, a zinc finger nuclease (ZFN) system, or a transcription activator-like effector nuclease (TALEN) system. Generally, gene editing systems involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks, ori SSBs) on target DNA sequences. Cleavage or nicking can be performed via the use of engineered ZFNs, TALENs, or other specific nucleases, or using a CRISPR / Cas system with engineered guide RNA that induces specific cleavage or nicking of the target DNA sequence. Furthermore, targeted nucleases are under development based on the Argonaut system (for example, derived from T. thermophilus, known as "TtAgo"; see Swarts et al (2014) Nature 507(7491): 258-261), which may also have potential use in gene editing and gene therapy.

[0659] In some embodiments, the gene editing system is a TALEN system. Activator-like effector nucleases (TALENs) are restriction enzymes that can be manipulated to cleave specific sequences of DNA. They are created by fusing a TAL effector DNA-binding domain with a DNA-cleaving domain (a nuclease that cleaves DNA strands). Activator-like effectors (TALEs) can be manipulated to bind to a desired DNA sequence to facilitate DNA cleavage at a specific location. Restriction enzymes can be introduced into cells for use in gene editing or for in situ gene editing, a technique known as gene editing using manipulated nucleases. Such methods and compositions for use therein are known in the art. See, for example, International Publication Nos. 2019147805, 2014040370, and 2018073393, whose entire contents are incorporated herein.

[0660] In some embodiments, the gene editing system is a zinc finger system. A zinc finger nuclease (ZFN) is an artificial restriction enzyme produced by fusing a zinc finger DNA-binding domain with a DNA-cleavage domain. The zinc finger domain can be manipulated to target a specific desired DNA sequence, enabling the zinc finger nuclease to target a unique sequence within a complex genome. A nonspecific cleavage domain derived from the IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs. The cleavage is repaired by an endogenous DNA repair mechanism, which allows the ZFN to precisely modify the genome of higher organisms. Such methods and compositions for use therein are known in the art. See, for example, International Publication No. 2011091324, whose entire contents are incorporated herein.

[0661] In some embodiments, the gene editing system is a CRISPR / Cas system comprising, for example, a guide sequence and a CRISPR guide RNA containing an RNA-inducing DNA binder.

[0662] In some embodiments, the manipulated cells disclosed contain a chimeric antigen receptor (e.g., the disclosed cells are manipulated to express a CAR). Exemplary CAR constructs are described, for example, in Fresnak AD, et al. Nat Rev Cancer. 2016;16(9):566-81, which is incorporated by reference in whole with respect to teaching these CAR models. For example, a CAR may be a TRUCK, a general-purpose CAR, a self-driven CAR, an armored CAR, a self-destructive CAR, a condition-dependent CAR, a labeled CAR, a TenCAR, a dual CAR, or a sCAR. A TRUCK (a T cell redirected for universal cytokine killing) co-expresses a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression may be constitutive or induced by T cell activation. Local production of pro-inflammatory cytokines targeted by CAR specificity can recruit endogenous immune cells to the tumor site and enhance the antitumor response. Universal allogeneic CAR T cells can be engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby reducing or preventing graft-versus-host disease (GVHD) or rejection. Self-driven CARs co-express the CAR with a chemokine receptor that binds to a tumor ligand, thereby enhancing tumor homing. CAR T cells engineered to be resistant to immunosuppression (enhanced CARs) can be genetically modified using immune checkpoint switch receptors to no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)), or they can be administered with monoclonal antibodies that block immune checkpoint signaling. Self-destructive CARs can be designed using electroporated RNA that encodes the CAR. Alternatively, T-cell-induced apoptosis may be achieved based on a system of human caspase-9 activation by ganciclovir, which binds to thymidine kinase, or, more recently, by low-molecular-weight dimer-inducing compounds, in genetically modified lymphocytes.Condition-dependent CAR T cells are initially refractory or switched off until a small molecule that completes the circuit is added, which enables complete transduction of both signal 1 and signal 2, thereby activating the CAR T cells. Alternatively, T cells can be engineered to express adapter-specific receptors that have affinity for a subsequently administered secondary antibody targeting a target antigen. Labeled CAR T cells express a CAR and a tumor epitope to which an existing monoclonal antibody agent binds. In situations of intolerable adverse effects, administration of a monoclonal antibody eliminates CAR T cells and alleviates symptoms without further off-tumor effects. Tandem CAR (TanCAR) T cells express a single CAR consisting of two linked single-stranded variable fragments (scFv) with different affinities, fused to an intracellular costimulatory domain and a CD3 domain. TanCAR T cell activation is achieved only when the target cell co-expresses both targets. Dual-CAR T cells express two distinct CARs with different ligand-binding targets, one containing only the CD3 domain and the other containing only the costimulatory domain. Dual-CAR T cell activation requires co-expression of both targets on the tumor. Safety CARs (sCARs) contain an extracellular scFv fused with an intracellular repressive domain. sCAR T cells co-expressing a standard CAR are activated only when they encounter target cells that have the standard CAR target but lack the sCAR target.

[0663] The antigen-recognition domain of a disclosed CAR may be an scFv or an antibody fragment. However, many alternative forms exist. Antigen-recognition domains derived from native T cell receptor (TCR) alpha and beta single-strands have been described, as have simple external domains (e.g., the CD4 external domain that recognizes HIV-infected cells) and more specialized recognition components, such as linked cytokines (which trigger recognition by cells with cytokine receptors). In practice, almost anything that binds to a given target with high affinity can be used as an antigen-recognition region. The internal domain is a crucial part of the CAR that, after antigen recognition signals the immune effector cell, activates at least one of the normal effector functions of the immune effector cell. The effector function of the T cell may be cytolytic activity or helper activity, including cytokine secretion. Thus, the internal domain may include the “intracellular signaling domain” of the T cell receptor (TCR) and optional co-receptors. Typically, the entire intracellular signaling domain may be used, but often it is not necessary to use the entire chain. When a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain, insofar as it transmits effector functional signals.

[0664] Cytoplasmic signaling sequences that act stimulily and modulate the primary activation of the TCR complex may contain signaling motifs known as immune receptor tyrosine-based activation motifs (ITAMs). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from CD8, CD3ζ, CD35, CD3y, CD3ε, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcyRIy, FceRip (FCERIB), and FceRIy (FCERIG).

[0665] In some embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3Q (TCR zeta, GenBank accession number BAG36664.1). CD3 zeta (T cell receptor T3 zeta chain) or CD247 (CD3Q chain, also known as differentiation antigen group 247), a T cell surface glycoprotein, is a protein encoded by the CD247 gene in humans.

[0666] First-generation CARs typically possessed an intracellular domain derived from the CD3 chain, the primary signaling molecule from the endogenous TCR. Second-generation CARs provide further signaling to T cells by adding intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD27, CD28, 4-1BB, ICOS) to the CAR's internal domain. Preclinical studies have shown that second-generation CAR designs improve the antitumor activity of T cells. More recent third-generation CARs combine multiple signaling domains to further amplify efficacy. T cells transplanted with these CARs have demonstrated improved growth, activation, persistence, and tumor eradication efficiency, independent of costimulatory receptor / ligand interactions. (Imai C, et al. Leukemia 2004 18:676-84, Maher J, et al. Nat Biotechnol 2002 20:70-5)

[0667] For example, the internal domain of a CAR can be designed to include a CD3 signaling domain alone or in combination with any other desired cytoplasmic domain useful in the context of the CAR. For instance, the cytoplasmic domain of a CAR may include a CD3 chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Therefore, while CAR is primarily exemplified with CD28 as a co-stimulus signaling element, other co-stimulus elements may be used alone or in combination with other co-stimulus signaling elements.

[0668] In some embodiments, the manipulated cells of the Disclosure comprise a vector containing recombinant nucleic acids encoding constructs for inhibiting the cytotoxicity of NK cells as disclosed herein. In certain embodiments, the construct comprises an NKG2A binding domain, a CD300a binding domain (e.g., CD300a VHH as disclosed herein), or both an NKG2A binding domain and a CD300a binding domain.

[0669] In some embodiments, the manipulated cells disclosed express recombinant nucleic acids (e.g., recombinant nucleic acids disclosed herein) encoding a construct for inhibiting the cytotoxicity of NK cells, wherein the construct comprises an NKG2A-binding domain, a CD300a-binding domain (e.g., CD300a VHH disclosed herein), or both an NKG2A-binding domain and a CD300a-binding domain.

[0670] In a particular embodiment, the manipulated cell comprises a first vector and a second vector, wherein (a) the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting the cytotoxicity of NK cells comprising an NKG2A binding domain including (i) an NKG2A light chain variable region (NKG2A VL) and (ii) an NKG2A heavy chain variable region (NKG2A VH); and (b) the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting the cytotoxicity of NK cells comprising a CD300a binding domain including (i) a CD300a light chain variable region (CD300a VL) and (ii) a CD300a heavy chain variable region (CD300a VH).

[0671] In some embodiments of the engineered cells, a vector containing the recombinant nucleic acid disclosed herein is inserted into the safe harbor locus of at least one allele of the engineered cells. In certain embodiments, the safe harbor locus is the AAVS1 locus.

[0672] In any of the embodiments of the manipulated cells disclosed herein, the manipulated cells may be deficient in MHC class I, TRAC, and / or MHC class II. In some embodiments, the manipulated cells are deficient in MHC class I. In some embodiments, the manipulated cells are deficient in MHC class II. In some embodiments, the manipulated cells are deficient in TRAC. In some embodiments, the manipulated cells are deficient in both MHC class I and MHC class II. In some embodiments, the manipulated cells are deficient in both MHC class I and TRAC. In some embodiments, the manipulated cells are deficient in both MHC class II and TRAC. In some embodiments, the manipulated cells are deficient in MHC class I, MHC class II, and TRAC.

[0673] In some embodiments, the manipulated cells are MHC class I deficient. In some embodiments, in MHC class I deficient cells, the MHC class I molecular functionality (e.g., the amount of MHC class I molecules detectable on the cell surface) 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% compared to wild-type cells that are not MHC class I deficient. In some embodiments, in MHC class I-deficient cells, the MHC class I molecular functionality (e.g., the amount of MHC class I molecules detectable on the cell surface) is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, and 81% compared to wild-type cells that are not MHC class I-deficient. It is down by ~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%.

[0674] In some embodiments of the engineered cells disclosed, the β2 microglobulin (B2M) locus of the engineered cells is disrupted. In certain embodiments, the recombinant nucleic acid or vector disclosed herein is inserted into the B2M locus of the engineered cells. In some embodiments, in cells that are MHC class I deficient due to B2M locus disruption, B2M expression and / or functionality (e.g., functionality of the B2M 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% compared to wild-type cells without B2M locus disruption. In some embodiments, in cells that are MHC class I deficient due to B2M locus disruption, B2M expression and / or functionality (e.g., functionality of the B2M gene product) is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, compared to wild-type cells without B2M locus disruption. It is decreasing by 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%.

[0675] In some embodiments in which the disclosed recombinant nucleic acid or vector is inserted into the B2M locus of a cell, the expression of the recombinant nucleic acid or vector is driven by the cell's endogenous B2M promoter.

[0676] In some embodiments, the manipulated cells are MHC class II deficient. In some embodiments, in MHC class II deficient cells, the MHC class II molecular functionality (e.g., the amount of MHC class II molecules detectable on the cell surface) 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% compared to wild-type cells that are not MHC class II deficient. In some embodiments, in MHC class II-deficient cells, the functionality of MHC class II molecules (e.g., the amount of MHC class II molecules detectable on the cell surface) is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, compared to wild-type cells that are not MHC class II-deficient. It is decreasing by 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%.

[0677] In some embodiments of the manipulated cells disclosed, the CIITA locus of the manipulated cells is disrupted. In some embodiments, in cells that are MHC class II deficient due to CIITA locus disruption, CIITA expression and / or functionality (e.g., functionality of the CIITA 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% compared to wild-type cells that do not have CIITA locus disruption. In some embodiments, in cells that are MHC class II deficient due to CIITA locus disruption, CIITA expression and / or functionality (e.g., functionality of the CIITA gene product) is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80- It is decreasing by 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%.

[0678] In certain embodiments, the manipulated cells are stem cells, progenitor cells, cells differentiated from stem cells, or cells differentiated from progenitor cells. For example, stem cells may be pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs). In certain embodiments, the cells are progenitor cells, such as early hematopoietic progenitor cells or CD34+ progenitor cells. In some embodiments, the manipulated cells are cells differentiated from stem cells. In other embodiments, the manipulated cells are cells differentiated from progenitor cells. In some embodiments, the manipulated cells are manipulated T cells. In certain embodiments, manipulated cells differentiated from progenitor cells are manipulated T cells. In other specific embodiments, the manipulated cells are induced pluripotent stem cells that later differentiate into T cells.

[0679] As described above, the manipulated T cells may be chimeric antigen receptor (CAR) T cells. In specific and non-limiting embodiments, the CARs are BCMA CARs, CD19 CARs, and / or CD20 CARs.

[0680] In some embodiments, the manipulated cells are T cell receptor alpha stationary (TRAC) deficient. In some embodiments, the TRAC locus is disrupted in the manipulated cells. In certain embodiments, a CAR is inserted into the TRAC locus in the manipulated cells. In some embodiments, in cells that are TRAC deficient (e.g., due to disruption of the TRAC gene locus), TRAC expression and / or functionality (e.g., functionality of the TRAC 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% compared to wild-type cells that are not TRAC deficient. In some embodiments, in cells that are TRAC-deficient (e.g., due to TRAC locus disruption), TRAC expression and / or functionality (e.g., functionality of the TRAC gene product) is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, and 80-100% compared to wild-type cells that are not TRAC-deficient. The decrease is 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%. In some embodiments in which the CAR is inserted into the cell's TRAC locus, CAR expression is driven by the cell's endogenous TRAC promoter.

[0681] In certain embodiments, the manipulated T cells are derived from pluripotent stem cells or CD34+ progenitor cells. In some such embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

[0682] In some embodiments, the manipulated cells are autologous cells. In other embodiments, the manipulated cells are allogeneic cells. In certain embodiments, the manipulated cells are donor-derived or derived from donor stem cells, and the donor is not the subject.

[0683] In certain embodiments, the manipulated cells are human cells. In some embodiments, the manipulated cells are low immunogenic. Low immunogenicity may result from the introduction of recombinant nucleic acids disclosed herein (i.e., recombinant nucleic acids comprising the NKG2A binding domain, the CD300a binding domain (e.g., CD300a VHH disclosed herein), or both) into the manipulated cells. For example, compared to wild-type cells, such low immunogenic cells may tend to result in 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 99% or more of immune rejection by the subjects to whom such cells are transplanted. In certain embodiments, low immunogenicity engineered cells (for example, engineered cells expressing recombinant nucleic acids including the NKG2A binding domain described herein, the CD300a binding domain (e.g., CD300a VHH disclosed herein), or both) are resistant to NK cell-mediated cytotoxicity.

[0684] V. Composition Disclosed herein are compositions comprising a disclosed recombinant nucleic acid, a vector containing the disclosed recombinant nucleic acid, and / or engineered cells containing the disclosed recombinant nucleic acid. Such compositions comprise one or more of a cell culture medium and a buffer.

[0685] A pharmaceutical composition comprising the manipulated cells described in any one of the claims further comprises a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier useful herein is a chemical component to which the manipulated cells disclosed herein may be combined, and which, after combination, can be used to administer the manipulated cells to a target requiring them. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, Ringer's lactate, ordinary sucrose, ordinary glucose, and salt solutions (e.g., Ringer's solution). Such preparations may be sterilized and, if desired, mixed with adjuvants that do not react adversely with the manipulated cells disclosed herein, such as stabilizers, emulsifiers, salts to affect osmotic pressure, and / or buffers. Those skilled in the art will recognize that other pharmaceutically acceptable carriers are useful herein.

[0686] VI. Method A. Overview The methods of this disclosure, using the recombinant nucleic acids, vectors, and engineered cells disclosed herein, enable the development of, for example, low immunogenic iPSCs and / or low immunogenic T cells (e.g., CAR T cells) that may be resistant to the cytotoxicity of host CD8+ T cells and NK cells and suitable for adoptive cell transfer in an allogeneic environment.

[0687] B. Methods for producing manipulated cells This disclosure provides methods for producing engineered cells, such as engineered iPSCs, which are differentiated into various cell types, for example, for transplantation into a subsequent target. As will be understood by those skilled in the art, methods for differentiation using known techniques depend on the desired cell type. For example, engineered cells may be differentiated in suspension and then placed in a gel matrix form, such as Matrigel, gelatin, or fibrin / thrombin form, to promote cell viability. An exemplary method for differentiating pluripotent stem cells into CD34+ progenitor cells and further into T cells is described, for example, in U.S. Provisional Application No. 63 / 483,814, filed February 8, 2023, the entirety of which is incorporated herein by reference. Differentiation is generally assayed by evaluating the presence of cell-specific markers, as is known in the art.

[0688] General techniques for recombinant nucleic acid manipulation are described, for example, by Sambrook et al. in Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2nd ed., 1989, which are incorporated herein by reference in their entirety, or by F. Ausubel et al. in Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987), and their periodically updated versions. In some embodiments, the nucleic acid (e.g., DNA) containing the recombinant nucleic acid disclosed is operably ligated to an expression vector containing one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include transcriptional promoters, optional operator sequences that control transcription, sequences encoding suitable mRNA-ribosome binding sites, and sequences that control transcriptional and translational termination. The expression vector may include origins of replication that confer replication ability in host cells. The expression vector may include genes that confer selectivity that facilitates recognition of transgenic host cells (e.g., transformants). Expression vector constructs can be introduced into cells using methods appropriate to those cells. Various methods for introducing nucleic acids into cells are known in the art, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; nonviral transfection; particulate guns; lipofection; and infection (for example, when the vector is an infectious agent).

[0689] In some embodiments, a method for producing engineered T cells includes differentiating engineered cells containing recombinant nucleic acids disclosed herein into T cells. In some embodiments, a method for producing engineered low immunogenic induced pluripotent stem cells (iPSCs) includes expressing recombinant nucleic acids encoding an NKG2A binding domain, a CD300a binding domain (e.g., CD300a VHH disclosed herein), or both an NKG2A binding domain and a CD300a binding domain in iPSCs, thereby producing engineered low immunogenic iPSCs. In other embodiments, a method for producing engineered low immunogenic induced pluripotent stem cells (iPSCs) includes expressing a first recombinant nucleic acid containing an NKG2A binding domain and a second recombinant nucleic acid containing a CD300a binding domain in iPSCs, thereby producing engineered low immunogenic iPSCs.

[0690] In a particular embodiment, a method for producing an engineered iPSC comprises contacting an 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 the cytotoxicity of NK cells, comprising an NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting the cytotoxicity of NK cells, comprising a CD300a binding domain, and the contact is carried out under conditions in which the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the iPSC, thereby producing an engineered iPSC.

[0691] Some embodiments of a method for producing engineered hypoimmunogenic T cells include (a) expressing recombinant nucleic acid encoding a CD300a-binding domain (e.g., CD300a VHH as disclosed herein), an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in iPSCs to produce engineered hypoimmunogenic iPSCs, and (b) differentiating the engineered hypoimmunogenic iPSCs into engineered T cells to produce engineered T cells. In some embodiments, a method for producing engineered hypoimmunogenic T cells includes expressing recombinant nucleic acid encoding a CD300a-binding domain (e.g., CD300a VHH as disclosed herein), an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in T cells to produce engineered hypoimmunogenic T cells. In another embodiment, the method involves expressing a first recombinant nucleic acid containing an NKG2A binding domain and a second recombinant nucleic acid containing a CD300a binding domain in T cells, thereby producing engineered low-immunogenic T cells.

[0692] Some embodiments of a method for producing engineered T cells include contacting T cells with a first vector and a second vector, wherein the first vector comprises a first recombinant nucleic acid encoding a first construct for inhibiting the cytotoxicity of NK cells, comprising an NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting the cytotoxicity of NK cells, comprising a CD300a binding domain, and the contact is carried out under conditions in which the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the T cells, thereby producing engineered T cells.

[0693] In certain embodiments, the method comprises expressing a chimeric antigen receptor (CAR) in an engineered iPSC, an engineered cell, or a T cell. In certain embodiments, the CAR is a BCMA CAR, a CD19 CAR, and / or a CD20 CAR.

[0694] In some embodiments of the disclosed methods, the engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells are T cell receptor alpha stationary (TRAC) deficient. In some such embodiments, the TRAC locus is disrupted in the engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells. In certain embodiments, a CAR (e.g., CD19 CAR, CD20 CAR, or BCMA CAR) is inserted into the TRAC locus in the engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells. In some embodiments, in engineered cells, iPSCs, engineered iPSCs, engineered hypoimmunogenic iPSCs, T cells, engineered T cells, or hypoimmunogenic engineered T cells that are TRAC-deficient (e.g., due to TRAC locus disruption), TRAC expression and / or functionality (e.g., functionality of the TRAC 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% compared to wild-type cells that are not TRAC-deficient.In some embodiments, in engineered cells, iPSCs, engineered low immunogenic iPSCs, T cells, engineered T cells, or low immunogenic engineered T cells that are TRAC-deficient (e.g., due to TRAC locus disruption), TRAC expression and / or functionality (e.g., functionality of the TRAC gene product) is 10-100%, 20-100%, 30-100%, 40-100%, compared to wild-type cells that are not TRAC-deficient. It has decreased by 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%.

[0695] In some embodiments, where the CAR is inserted into the TRAC locus of engineered cells, iPSCs, engineered low immunogenic iPSCs, T cells, engineered T cells, or low immunogenic engineered T cells, the expression of the CAR is d...

Claims

1. Recombinant nucleic acids encoding constructs for inhibiting the cytotoxicity of NK cells, comprising a CD300a-binding domain, an NKG2A-binding domain, or both CD300a-binding domains and NKG2A-binding domains.

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

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

4. (a) the antibody or fragment thereof comprises a single-stranded variable fragment (scFv) or VHH, or (b) the peptide is adnectin or a designed ankyrin repeat protein (DARPin), according to claim 2 or 3.

5. The recombinant nucleic acid according to claim 4, wherein the VHH comprises the VH domain of a camelid heavy chain antibody.

6. The recombinant nucleic acid according to any one of claims 1 to 5, wherein the CD300a binding domain comprises VHH (CD300a VHH) and / or the NKG2A binding domain comprises VHH (NKG2A VHH).

7. The recombinant nucleic acid according to any one of claims 1 to 6, wherein the construct for inhibiting the cytotoxicity of NK cells comprises or consists of the CD300a-binding domain, and the CD300a-binding domain comprises VHH (CD300a VHH).

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

9. The recombinant nucleic acid according to any one of claims 2 to 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 with respect to any one of sequence numbers 155 to 172.

10. The recombinant nucleic acid according to any one of claims 2 to 9, wherein the CD300a VHH comprises or consists of one of the amino acid sequences of sequence numbers 155 to 172.

11. The recombinant nucleic acid according to any one of claims 2 to 10, wherein the nucleotide sequence encoding CD300a VHH includes 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 with respect to any one of sequence numbers 137 to 154.

12. The recombinant nucleic acid according to any one of claims 2 to 11, wherein the nucleotide sequence encoding CD300a VHH includes or consists of any one of the nucleotide sequences of sequence numbers 137 to 154.

13. The recombinant nucleic acid according to claim 1, wherein the CD300a binding domain contains scFv and / or the NKG2A binding domain contains scFv.

14. (a) The NKG2A binding domain is (i) NKG2A light chain variable region (NKG2A VL), and (ii) NKG2A heavy chain variable region (NKG2A VH) including, and / or (b) The CD300a binding domain is (i) CD300a light chain variable region (CD300a VL), and (ii) CD300a heavy chain variable region (CD300a VH) Recombinant nucleic acid according to any one of claims 1 to 4 or 13, comprising:

15. (a) The NKG2A light chain variable region (NKG2A VL) includes VL CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing RASENIYSYLA (SEQ ID NO: 98), VL CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing NAKTLAE (SEQ ID NO: 99), and VL CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing QHHYGTPRT (SEQ ID NO: 100), (b) The NKG2A heavy chain variable region (NKG2A VH) includes VH CDR1 having 0, 1, or 2 mutations compared to the amino acid sequence containing SYWMN (SEQ ID NO: 101), VH CDR2 having 0, 1, or 2 mutations compared to the amino acid sequence containing RIDPYDSETHYAQKLQG (SEQ ID NO: 102), and VH CDR3 having 0, 1, or 2 mutations compared to the amino acid sequence containing GGYDFDVGTLYWFFDV (SEQ ID NO: 103), (c) The CD300a light chain variable region (CD300a VL) includes VL CDR1 which contains 0, 1, or 2 mutations compared to the amino acid sequence containing RASQDISNYLN (SEQ ID NO: 104), VL CDR2 which contains 0, 1, or 2 mutations compared to the amino acid sequence containing TSRLHS (SEQ ID NO: 105), and VL CDR3 which contains 0, 1, or 2 mutations compared to the amino acid sequence containing QQGNTLPWT (SEQ ID NO: 106), (d) The CD300a heavy chain variable region (CD300a VH) includes VH CDR1 containing 0, 1, or 2 mutations compared to the amino acid sequence containing SYWMQ (SEQ ID NO: 107), VH CDR2 containing 0, 1, or 2 mutations compared to the amino acid sequence containing EIDPSDSYTNYNQKFKG (SEQ ID NO: 108), and VH CDR3 containing 0, 1, or 2 mutations compared to the amino acid sequence containing WGMAYGTSSYWYFDV (SEQ ID NO: 109). Recombinant nucleic acid according to claim 14.

16. (a) The NKG2A VL contains 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 with 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 with any one of SEQ ID NOs: (c) CD300a VL contains 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 with respect to Sequence ID No.

17. (d) CD300a VH contains 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 with respect to SEQ ID NO:

18. Recombinant nucleic acid according to claim 14 or claim 15.

17. (a) The NKG2A VL contains or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, (b) The NKG2A VH contains or consists of any one of the amino acid sequences of SEQ ID NOs: 3 to 8. (c) CD300a VL contains or consists of the amino acid sequence of SEQ ID NO: 17, (d) CD300a VH contains or consists of the amino acid sequence of Sequence ID No.

18. Recombinant nucleic acid according to any one of claims 14 to 16.

18. (a) The nucleotide sequence encoding NKG2A VL includes 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 with respect to SEQ ID NO: 9 or SEQ ID NO: 10, (b) The nucleotide sequence encoding NKG2A VH includes 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 with any one of SEQ ID NOs: (c) The nucleotide sequence encoding CD300a VL includes 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 with respect to SEQ ID NO:

19. (d) The nucleotide sequence encoding CD300a VH includes 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 with respect to SEQ ID NO:

20. Recombinant nucleic acid according to any one of claims 14 to 17.

19. (a) The nucleotide sequence encoding NKG2A VL includes or consists of SEQ ID NO: 9 or SEQ ID NO: 10, (b) The nucleotide sequence encoding NKG2A VH includes or consists of any one of sequence numbers 11 to 16, (c) The nucleotide sequence encoding CD300a VL includes or consists of SEQ ID NO: 19, (d) The nucleotide sequence encoding CD300a VH includes or consists of SEQ ID NO: 20 Recombinant nucleic acid according to claim 18.

20. (a) The nucleotide sequence encoding the NKG2A binding domain is codon-optimized to reduce or prevent undesirable recombination events, and / or (b) The nucleotide sequence encoding the CD300a binding domain is codon-optimized to reduce or prevent undesirable recombination events. Recombinant nucleic acid according to any one of claims 1 to 19.

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

22. The recombinant nucleic acid according to claim 20 or 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 with respect to SEQ ID NO:

113.

23. The recombinant nucleic acid according to any one of claims 20 to 22, wherein the codon-optimized nucleotide sequence encoding 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 with respect to SEQ ID NO:

112.

24. The recombinant nucleic acid according to any one of claims 20 to 23, wherein the codon-optimized nucleotide sequence encoding 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 with respect to SEQ ID NO:

110.

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

26. The recombinant nucleic acid according to any one of claims 14 to 25, wherein the recombinant nucleic acid includes a second linker, the second linker linking the nucleic acid encoding CD300a VL and the nucleic acid encoding CD300a VH.

27. (a) The NKG2A scFv contains 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 with SEQ ID NO: 118, or (b) The NKG2A scFv includes or consists of sequence number 118, Recombinant nucleic acid according to any one of claims 13 to 26.

28. (a) The nucleotide sequence encoding NKG2A scFv contains 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 with either SEQ ID NO: 113 or 120, or (b) The nucleotide sequence encoding NKG2A scFv includes or consists of either SEQ ID NO: 113 or 120, Recombinant nucleic acid according to any one of claims 13 to 27.

29. (a) CD300a scFv contains 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 with SEQ ID NO: 119, or (b) CD300a scFv includes or consists of sequence number 119, Recombinant nucleic acid according to any one of claims 13 to 28.

30. (a) The nucleotide sequence encoding CD300a scFv contains 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 with respect to SEQ ID NO: 121, or (b) The nucleotide sequence encoding CD300a scFv includes or consists of SEQ ID NO: 121, Recombinant nucleic acid according to any one of claims 13 to 29.

31. (a) The nucleotide sequence encoding NKG2A VL is located on the 5' side of the nucleotide sequence encoding NKG2A VH, or the nucleotide sequence encoding NKG2A VL is located on the 3' side of the nucleotide sequence encoding NKG2A VH, (b) The nucleotide sequence encoding CD300a VL is located on the 5' side of the nucleotide sequence encoding CD300a VH, or the nucleotide sequence encoding CD300a VL is located on the 3' side of the nucleotide sequence encoding CD300a VH. Recombinant nucleic acid according to any one of claims 13 to 30.

32. (a) The nucleotide sequence encoding NKG2A scFv is located on the 3' side of the nucleotide sequence encoding CD300a scFv, or (b) The nucleotide sequence encoding NKG2A scFv is located on the 5' side of the nucleotide sequence encoding CD300a scFv, according to any one of claims 13 to 31.

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

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

35. The glycine-serine linker is (Gly ｍ -Ser) ｎ Recombinant nucleic acid according to claim 34, comprising (where m is 3 to 6 and n is 1 to 10).

36. The recombinant nucleic acid according to claim 35, wherein m = 4 and n = 5.

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

38. The recombinant nucleic acid according to claim 37, wherein the self-cleaving peptide is a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide, and the self-cleaving peptide optionally contains the amino acid glycine-serine-glycine at its N-terminus.

39. (a) The first linker, the second linker, and / or the third linker contain 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 with any one of SEQ ID NOs. 29-33 and 122, or (b) The first linker, the second linker, and / or the third linker include or consist of any one of sequence numbers 29-32 and 122, Recombinant nucleic acid according to any one of claims 25 to 38.

40. (a) The nucleotide sequences encoding the first linker, the second linker, and / or the third linker include 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 with any one of SEQ ID NOs. 34-37, 111, 115, and 116, or (b) The nucleotide sequences encoding the first linker, the second linker, and / or the third linker include or consist of one of sequence numbers 34-37, 111, 115, and 116. Recombinant nucleic acid according to any one of claims 25 to 38.

41. A recombinant nucleic acid according to any one of claims 1 to 40, further encoding a signal peptide.

42. The recombinant nucleic acid according to claim 41, wherein the signal peptide is a cell surface expression signal peptide that induces the protein product of the recombinant nucleic acid to the cell surface.

43. (a) The signal peptide contains 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 with SEQ ID NO: 38, or (b) The signal peptide comprises or consists of SEQ ID NO: 38 Recombinant nucleic acid according to claim 41 or claim 42.

44. (a) The nucleotide sequence encoding the signal peptide contains 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 with any one of SEQ ID NOs: 39 to 41, or (b) The nucleotide sequence encoding the signal peptide contains or consists of one of the sequence numbers 39 to 41, Recombinant nucleic acid according to any one of claims 41 to 43.

45. The recombinant nucleic acid according to any one of claims 1 to 44, wherein the nucleotide sequence encoding the signal peptide is located on the 5' side of the nucleotide sequence encoding the CD300a binding domain, on the 5' side of the nucleotide sequence encoding the NKG2A binding domain, or on the 5' side of the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain.

46. Recombinant nucleic acid according to any one of claims 1 to 45, further encoding a spacer.

47. (a) The spacer contains 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 with respect to Sequence ID No. 42, or (b) The spacer includes or consists of sequence number 42, Recombinant nucleic acid according to claim 46.

48. (a) The nucleotide sequence encoding the spacer contains 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 with respect to SEQ ID NO: 49, or (b) The nucleotide sequence encoding the spacer includes or consists of Sequence ID No. 49, Recombinant nucleic acid according to claim 46 or 47.

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

50. A recombinant nucleic acid according to any one of claims 1 to 49, further encoding a hinge region, or (b) not encoding a hinge region.

51. The recombinant nucleic acid according to claim 50, wherein the hinge region includes a CD8 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, a FACD hinge, or any combination thereof.

52. (a) The hinge region contains 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 with any one of sequence numbers 43 to 48, or (b) The hinge region includes or consists of any one of sequence numbers 43 to 48, Recombinant nucleic acid according to claim 50 or claim 51.

53. (a) The nucleotide sequence encoding the hinge region contains 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 with any one of SEQ ID NOs. 50 to 55, or (b) The nucleotide sequence encoding the hinge region includes or consists of one of sequence numbers 50 to 55, Recombinant nucleic acid according to any one of claims 50 to 52.

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

55. A recombinant nucleic acid according to any one of claims 1 to 54, further encoding a transmembrane domain.

56. The recombinant nucleic acid according to claim 55, wherein the transmembrane domain is a human transmembrane domain or a mouse transmembrane domain.

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

58. (a) The transmembrane domain contains 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 with any one of SEQ ID NOs. 67-76 and 96, or (b) The transmembrane domain comprises or consists of one of sequence numbers 67-76 and 96, Recombinant nucleic acid according to any one of claims 55 to 57.

59. (a) The nucleotide sequence encoding the transmembrane domain contains 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 with any one of SEQ ID NOs. 56 to 65, or (b) The nucleotide sequence encoding the transmembrane domain includes or consists of one of sequence numbers 56 to 65, Recombinant nucleic acid according to any one of claims 55 to 58.

60. The recombinant nucleic acid according to claim 59, wherein the nucleotide sequence encoding the transmembrane domain is located at (a) the 3' side of the nucleotide sequence encoding the signal peptide, (b) the 3' side of the nucleotide sequence encoding the CD300a binding domain, the 3' side of the nucleotide sequence encoding the NKG2A binding domain, or the 3' side of the nucleotide sequence encoding the NKG2A binding domain and the nucleotide sequence encoding the CD300a binding domain, (c) the 3' side of the nucleotide sequence encoding the spacer, (d) the 3' side of the nucleotide sequence encoding the hinge region, or (e) any combination of (a) to (d).

61. Recombinant nucleic acid according to any one of claims 1 to 60, further encoding a cytoplasmic domain.

62. The recombinant nucleic acid according to claim 61, wherein the cytoplasmic domain is a human cytoplasmic domain or a mouse cytoplasmic domain.

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

64. (a) The cytoplasmic domain contains 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 with any one of SEQ ID NOs: 83 to 88, or (b) The cytoplasmic domain contains or consists of one of sequence numbers 83 to 88, Recombinant nucleic acid according to any one of claims 61 to 63.

65. (a) The nucleotide sequence encoding the cytoplasmic domain contains 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 with any one of SEQ ID NOs. 77 to 82, or (b) The nucleotide sequence encoding the cytoplasmic domain includes or consists of one of sequence numbers 77 to 82, Recombinant nucleic acid according to any one of claims 61 to 64.

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

67. (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 with any one of sequence numbers 220 to 237, or (b) an amino acid sequence containing or consisting of one of sequence numbers 220 to 237 A recombinant nucleic acid according to any one of claims 1 to 66, which codes for

68. (a) 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 with any one of sequence numbers 202 to 219, or (b) containing or consisting of any one of sequence numbers 202 to 219 Recombinant nucleic acid according to any one of claims 1 to 67.

69. (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 with SEQ ID NO: 21 or SEQ ID NO: 22, or (b) an amino acid sequence containing or consisting of SEQ ID NO: 21 or SEQ ID NO: 22 A recombinant nucleic acid according to any one of claims 1 to 66, which codes for

70. (a) 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 with respect to SEQ ID NO: 23 or SEQ ID NO: 24, or (b) containing or consisting of Sequence ID No. 23 or Sequence ID No. 24, Recombinant nucleic acid according to any one of claims 1 to 66 or 69.

71. (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 with SEQ ID NO: 25 or SEQ ID NO: 26, or (b) an amino acid sequence containing or consisting of SEQ ID NO: 25 or SEQ ID NO: 26 A recombinant nucleic acid according to any one of claims 1 to 66, which codes for

72. (a) 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 with respect to SEQ ID NO: 27 or SEQ ID NO: 28, or (b) containing or consisting of Sequence ID No. 27 or Sequence ID No. 28, Recombinant nucleic acid according to any one of claims 1 to 66 or 71.

73. A vector comprising a recombinant nucleic acid according to any one of claims 1 to 72.

74. The vector according to claim 73, which is a DNA vector, RNA vector, plasmid, lentiviral vector, adenovirus vector, adeno-associated virus vector, Rous sarcoma virus (RSV) vector, or retroviral vector.

75. The vector according to claim 73 or 74, wherein the recombinant nucleic acid is operably linked to a promoter.

76. The vector according to claim 75, wherein the promoter is the EF1a promoter, the CAG promoter, the PGK promoter, or the CMV promoter.

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

78. The vector according to claim 77, wherein the poly(A) sequence includes a bGH poly(A) signal.

79. The vector according to any one of claims 73 to 78, further comprising a 3'UTR in the nucleic acid sequence of the vector.

80. The vector according to any one of claims 73 to 79, which is incorporated into the genome at the adeno-associated virus integration site 1 (AAVS1) of the genome.

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

82. Engineered cells comprising a vector containing a recombinant nucleic acid encoding a construct for inhibiting the cytotoxicity of NK cells, wherein the construct comprises a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain.

83. Engineered cells expressing recombinant nucleic acids encoding constructs for inhibiting the cytotoxicity of NK cells, wherein the constructs comprise a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain.

84. A manipulated cell containing a first vector and a second vector, (a) The first vector is (i) NKG2A light chain variable region (NKG2A VL), and (ii) NKG2A heavy chain variable region (NKG2A VH) It comprises a first recombinant nucleic acid encoding a first construct for inhibiting the cytotoxicity of NK cells, which includes an NKG2A binding domain, (b) The second vector is (i) CD300a light chain variable region (CD300a VL), and (ii) CD300a heavy chain variable region (CD300a VH) A second recombinant nucleic acid comprising a CD300a-binding domain containing a second construct for inhibiting the cytotoxicity of NK cells, Manipulated cells.

85. A manipulated cell comprising a recombinant nucleic acid according to any one of claims 1 to 72 or a vector according to any one of claims 73 to 81.

86. The manipulated cell according to any one of claims 82 to 85, wherein the recombinant nucleic acid is expressed in the manipulated cell.

87. The manipulated cell according to any one of claims 82 to 86, wherein the vector is inserted into the safe harbor locus of at least one allele of the manipulated cell.

88. The manipulated cell according to claim 87, wherein the safe harbor locus is the AAVS1 locus.

89. The manipulated cells according to any one of claims 82 to 88, which are MHC class I deficient.

90. The manipulated cell according to any one of claims 82 to 89, wherein the β2 microglobulin (B2M) gene locus of the manipulated cell is disrupted.

91. The engineered cell according to any one of claims 82 to 90, wherein the recombinant nucleic acid or the vector is inserted into the β2 microglobulin (B2M) locus of the engineered cell.

92. The manipulated cell according to any one of claims 82 to 91, which is a stem cell, a progenitor cell, a cell differentiated from a stem cell, or a cell differentiated from a progenitor cell.

93. The manipulated cell according to claim 92, wherein the stem cell is a pluripotent stem cell.

94. The manipulated cells according to claim 93, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

95. The manipulated cell according to claim 92, wherein the progenitor cell is an early hematopoietic progenitor cell or a CD34+ progenitor cell.

96. The manipulated cell according to claim 92, wherein the cell differentiated from the stem cell or the cell differentiated from the progenitor cell is a manipulated T cell.

97. The manipulated cell according to any one of claims 82 to 92, which is a manipulated T cell.

98. The manipulated cell according to any one of claims 82 to 92, which is an induced pluripotent stem cell that later differentiates into manipulated T cells.

99. The manipulated cell according to any one of claims 96 to 98, wherein the manipulated T cell is a chimeric antigen receptor (CAR) T cell.

100. The manipulated cell according to claim 99, wherein the CAR is a CD19 CAR, a BCMA CAR, and / or a CD20 CAR.

101. The manipulated cell according to any one of claims 82 to 100, which is deficient in T cell receptor alpha constant (TRAC).

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

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

104. The manipulated cells according to any one of claims 96, 97, or 99 to 103, wherein the manipulated T cells are derived from pluripotent stem cells or CD34+ progenitor cells.

105. The manipulated cells according to claim 104, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

106. The manipulated cell according to any one of claims 82 to 105, which is an autologous cell or an allogeneic cell.

107. The manipulated cells according to any one of claims 82 to 105, wherein the manipulated cells are derived from a donor or from the stem cells of a donor, and the donor is not the target.

108. The manipulated cell according to any one of claims 82 to 107, which is a human cell.

109. The manipulated cells according to any one of claims 82 to 108, which are low immunogenic.

110. The manipulated cells according to claim 109, which are resistant to NK cell-mediated cytotoxicity.

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

112. A pharmaceutical composition comprising manipulated cells according to any one of claims 82 to 110 and a pharmaceutically acceptable carrier.

113. A method for producing manipulated T cells, comprising differentiating the manipulated cells according to any one of claims 82 to 95 or 105 to 91 into T cells.

114. A method for producing manipulated low immunogenic induced pluripotent stem cells (iPSCs), comprising expressing a recombinant nucleic acid encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in the iPSCs, thereby producing the manipulated low immunogenic iPSCs.

115. A method for producing manipulated low immunogenic induced pluripotent stem cells (iPSCs), comprising expressing a first recombinant nucleic acid containing a CD300a binding domain and a second recombinant nucleic acid containing an NKG2A binding domain in the iPSCs, thereby producing the manipulated low immunogenic iPSCs.

116. A method for producing engineered induced pluripotent stem cells (iPSCs), comprising contacting an engineered 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 the cytotoxicity of NK cells comprising an NKG2A binding domain, and the second vector comprises a second recombinant nucleic acid encoding a second construct for inhibiting the cytotoxicity of NK cells comprising a CD300a binding domain, and the contact is performed under conditions in which the first recombinant nucleic acid and the second recombinant nucleic acid are expressed in the iPSC, thereby producing the engineered iPSC.

117. A method for producing manipulated low immunogenic T cells, (a) Expressing recombinant nucleic acids encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in iPSCs, thereby creating manipulated low-immunogenic iPSCs, (b) Differentiating the manipulated low immunogenic iPSCs into manipulated T cells, thereby producing the manipulated T cells. A method that includes this.

118. A method for producing manipulated low immunogenic T cells, comprising expressing a recombinant nucleic acid encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in T cells, thereby producing the manipulated low immunogenic T cells.

119. A method for producing manipulated low immunogenic T cells, comprising expressing a first recombinant nucleic acid containing a CD300a binding domain and a second recombinant nucleic acid containing an NKG2A binding domain in T cells, thereby producing the manipulated low immunogenic T cells.

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

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

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

123. A method for inhibiting or reducing the cytotoxicity of natural killer (NK) cells against T cells, (a) Expressing recombinant nucleic acids encoding a CD300a-binding domain, an NKG2A-binding domain, or both an NKG2A-binding domain and a CD300a-binding domain in iPSCs, thereby creating manipulated iPSCs, (b) Differentiating the manipulated iPSCs into T cells, thereby inhibiting or reducing the cytotoxicity of NK cells against the T cells. A method that includes this.

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

125. A method for inhibiting or reducing the cytotoxicity of natural killer (NK) cells against T cells, comprising expressing a first recombinant nucleic acid containing a CD300a binding domain and a second recombinant nucleic acid containing an NKG2A binding domain in T cells, thereby producing the manipulated iPSC.

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

127. The method according to claim 126, wherein the CAR is a BCMA CAR, a CD19 CAR, and / or a CD20 CAR.

128. The method according to any one of claims 113 to 127, wherein the manipulated cells, iPSCs, manipulated iPSCs, manipulated hypoimmunogenic iPSCs, T cells, manipulated T cells, or hypoimmunogenic manipulated T cells are T cell receptor alpha constant (TRAC) deficient.

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

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

131. The method according to any one of claims 113 to 130, wherein the manipulated cells are obtained by differentiation of human pluripotent stem cells.

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

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

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

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

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

137. The method according to any one of claims 113 to 136, wherein the NKG2A binding domain includes an scFv comprising an NKG2A light chain variable region (NKG2A VL) and an NKG2A heavy chain variable region (NKG2A VH).

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

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

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

141. The method according to claim 140, wherein the vector comprising the recombinant nucleic acid comprising a CD300a-binding domain, an NKG2A-binding domain, or both a CD300a-binding domain and an NKG2A-binding domain comprises the vector according to any one of claims 73 to 81.

142. The method according to any one of claims 114, 115, 117, 119, 121 to 124, or 127 to 141, wherein expressing the manipulated cells, iPSCs, or T cells is performed by contacting them with a recombinant nucleic acid according to any one of claims 1 to 59, or a vector according to any one of claims 60 to 68, under conditions that the recombinant nucleic acid is expressed in the iPSCs.

143. The method according to any one of claims 116, 120, or 142, wherein the contact includes introducing the recombinant nucleic acid into the manipulated cells, iPSCs, or T cells using transfection, electroporation, transduction, or knock-in.

144. (a) The transfection comprises contacting the manipulated cells, iPSCs, or T cells with a cationic polymer and recombinant nucleic acid, (b) The transduction involves contacting the manipulated cells, iPSCs, or T cells with a lentivirus containing the recombinant nucleic acid, and / or (c) The knock-in includes contacting the manipulated cells, iPSCs, or T cells with an adeno-associated virus containing the recombinant nucleic acid, The method according to claim 143.

145. The method according to any one of claims 113 to 144, wherein the manipulated cells, iPSCs, manipulated iPSCs, manipulated hypoimmunogenic iPSCs, T cells, manipulated T cells, or hypoimmunogenic manipulated T cells are MHC class I deficient.

146. The method according to any one of claims 113 to 145, wherein the β2 microglobulin (B2M) locus of the manipulated cell, the iPSC, the manipulated iPSC, the manipulated hypoimmunogenic iPSC, the T cell, the manipulated T cell, or the hypoimmunogenic manipulated T cell is disrupted.

147. The method according to any one of claims 113 to 146, wherein the recombinant nucleic acid or the vector is inserted into the β2 microglobulin (B2M) 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.

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

149. The method according to claim 148, wherein the disease or condition is cancer.

150. A method for improving clinical outcomes in a subject receiving T-cell therapy, comprising administering to the subject an effective amount of manipulated cells according to any one of claims 82 to 110, or the pharmaceutical composition according to claim 112.

151. Improving clinical outcomes is (a) Inhibition or reduction of NK cell cytotoxicity against the manipulated cells, (b) an increase in the clinical response to the T cell therapy in the subjects compared to the same T cell therapy which does not contain the recombinant nucleic acid, or compared to a T cell therapy which contains T cells which (1) do not contain the recombinant nucleic acid and (2) are engineered to express at least one alternative ligand. (c) Increase in the viability of the manipulated cells in the subject, (d) Increased persistence of the manipulated cells in the subject compared to the same T cell therapy that optionally does not contain the recombinant nucleic acid, or compared to T cell therapy that contains the manipulated cells which (1) do not contain the recombinant nucleic acid and (2) are manipulated to express at least one alternative ligand. (e) Improvements in the pharmacokinetic (PK) and / or pharmacodynamic (PD) properties of the T cell therapy in the subject, compared to the same T cell therapy that optionally does not contain the recombinant nucleic acid, or compared to a T cell therapy comprising the engineered cells that (1) do not contain the recombinant nucleic acid and (2) are engineered to express at least one alternative ligand, and (f) A reduction in the cytolysis of the manipulated cells in the subject, wherein the cytolysis of the manipulated cells 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%. The method according to claim 150, comprising one or more of the above.

152. The method according to claim 151, 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 according to claim 152, wherein the HLA-E is an HLA-E single-stranded dimer or trimer.

154. The method according to any one of claims 151 to 153, wherein the T cell therapy comprises (1) the recombinant nucleic acid and (2) the engineered cells which are engineered to express at least one alternative ligand, and further comprises a reduction or loss of expression of CD48, CD54, CD58, and / or CD155.

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

156. The aforementioned cancers include B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, and small cell follicular lymphoma. The method according to any one of claims 148 to 155, wherein the hematological malignancy is selected from leukemia, large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, or preleukemia.

157. The aforementioned cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, 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, and pheochromocytoma. The method according to any one of claims 148 to 155, wherein the cancer is sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, seminomasm, bladder carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroneurioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

158. The method according to any one of claims 148 or 150 to 155, wherein the disease or disorder is an autoimmune disease or disorder.

159. The method according to claim 158, wherein the autoimmune disease or disorder is myasthenia gravis, neuromyelitis optica spectrum disorder, Sjögren's syndrome, scleroderma, immunonephritis, systemic lupus erythematosus, arthritis, autoimmune-induced fibrosis, pemphigus vulgaris, multiple sclerosis, colitis, type 1 diabetes mellitus, graft-versus-host disease, atherosclerosis, or mucosal-dominant PV.

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

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

162. (a) One or more types of cells, which are optionally selected to be stem cells, T cells, and / or NK cells, (b) Cell culture medium, (c) Buffer, and (d) Pharmacologically acceptable carriers The kit according to claim 161, further comprising one or more of the following.

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