Chimeric adaptor polypeptides

Abstract

Aspects of the disclosure include compositions and methods for the treatment of a wide variety of diseases / conditions using engineered host cells. In some embodiments, the engineered host cells comprise a chimeric adaptor (CAD) polypeptide comprising DAP10. The CAD polypeptide comprises substitution mutations and / or additional protein domains that interact with associated receptors to enhance cell survival and proliferation of host cells and promote cell killing activity in non-host cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 272,613, filed October 27, 2021.

[0002] Field of Disclosure The present disclosure relates generally to cellular immunotherapy, and in particular to chimeric adaptor polypeptides that associate with specific receptor(s) of various cell types to improve cell survival, proliferation, signaling, and the like. [Background technology]

[0003] Adoptive cell therapy has undergone almost continuous iterations for over 30 years, from early days focused on basal lymphokine activation and / or tumor infiltration, to more recent strategies to engineer immune cells to express genetically engineered antigen receptors such as chimeric antigen receptors (CARs). However, although there have been some hints and indications of therapeutic potential with the various approaches to date, myriad problems remain.

[0004] One issue relates to the problem of low or lost expression of the target of the adoptive cell therapy (i.e., antigen escape). Specifically, a common mechanism of resistance to adoptive (or natural) cell therapy is the emergence of cell types (e.g., tumors) with loss or downregulation of the target antigen. Such loss or downregulation can lead to reduced efficacy of the adoptive (or natural) cell response (Majzner RG and Mackall, CL. (2018) Cancer Discovery, 8(10):1219-26).

[0005] Another related problem is the downregulation of naturally occurring receptor(s) that can recognize ligands that are specifically present on cells related to a particular disease, which can also lead to the ineffectiveness of cellular responses to various disease conditions.As a representative example, NKG2D is an activating immune receptor found on human natural killer (NK) cells, CD8+αβT cells, and γδT cells, which regulates both innate and adaptive immune responses.Natural ligands of NKG2D include MICA and MICB, and some UL16 binding proteins (Bauer S, et al. (1999) Science, vol. 285 5428: 727-729; Burgess SJ, et al. (2008) Immunol Res, 2008, 40 (1): 18-34). In humans, NKG2D ligands are not expressed on normal cells, but are widely expressed at various levels on transformed or virally infected cancer cells (see, for example, Bauer S, et al. (1999) Science, vol. 285 5428: 727-729; Burgess SJ, et al. (2008) Immunol Res, 40 (1): 18-34; Baugh R, et al. (2020) Cancers 12 (12): 3827). Expression of NKG2D ligands on the tumor cell surface activates NKG2D to activate NK cells and costimulate effector T cells, thereby sensitizing tumor cells to immune cell-mediated destruction. Thus, the NKG2D receptor and its ligands are targeted targets for cancer immunotherapy.

[0006] Unfortunately, NKG2D can be downregulated when it is needed most. For example, tumor-derived transforming growth factor-β (TGF-β) downregulates NKG2D, thereby downregulating NK cell and CD8 + This may reduce tumor cell killing by the cells (see, e.g., Crane, C., et al. (2010) Neuro-Oncology, 12(1):7-13, and Dasgupta, S., et al. (2005) Journal of Immunol, 175:5541-50). In turn, this is associated with poor prognosis for tumor treatment.

[0007] NKG2D is mentioned as an example, inter alia, to illustrate the need in the art for compositions and methods that can improve, for example, immune cell survival and proliferation, prevent downregulation of endogenous receptors (e.g., NKG2D), and compensate for immune escape of antigens that are targets of, for example, adoptive immunotherapy approaches. Such compositions and methods would improve the prognosis of patients undergoing adoptive immunotherapy.

[0008] Disclosure Summary The present invention addresses the above-mentioned shortcomings of the prior art through chimeric adaptor (CAD) constructs and polypeptides comprising human DAP10, and methods of their use. As specifically described and demonstrated for the first time herein, the subject CAD constructs and polypeptides can improve the stability of receptors capable of recognizing target antigens on various cell surfaces, promote a favorable balance of cell signaling pathway(s) upon receptor-target binding, and / or improve the functional properties (e.g., enhanced cytolysis, proliferation, survival, and / or costimulatory properties) elicited upon binding of the receptor to various ligands (e.g., NKG2D).

[0009] In one aspect, the invention provides an isolated nucleic acid encoding a chimeric adaptor (CAD) polypeptide, the CAD polypeptide comprising a DAP10 domain and at least one costimulatory signaling domain, the CAD polypeptide specifically lacking an extracellular domain comprising a functional extracellular receptor and / or ligand binding domain. In a preferred embodiment, the DAP10 domain comprises a human DAP10 amino acid sequence.

[0010] In embodiments, the at least one costimulatory domain comprises or is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD3C, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD70, CD80, CD83, CD86, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), FcR, LAT, NKD2C, SLP76, TRIM, and ZAP70, or a combination thereof. In embodiments, the at least one costimulatory domain is 4-1BB. In embodiments, the at least one costimulatory domain is CD28.

[0011] In embodiments, the CAD polypeptide further comprises at least one intracellular signaling domain, the at least one intracellular signaling domain selected from or consisting of the group comprising CD3zeta, DAP12, LFA-1, and CD3t, or a combination thereof. In embodiments, the at least one signaling domain is CD3zeta. In embodiments, the at least one costimulatory domain is 4-1BB and the at least one intracellular signaling domain is CD3zeta. In embodiments, the CAD polypeptide comprises, from N-terminus to C-terminus, a DAP10 domain, a 4-1BB costimulatory domain followed by a CD3zeta intracellular signaling domain.

[0012] In embodiments, a CAD polypeptide comprises, from N-terminus to C-terminus, a DAP10 domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain. In embodiments, a CAD polypeptide comprises, from N-terminus to C-terminus, a DAP10 domain, a 4-1BB costimulatory domain, followed by a CD28 costimulatory domain, followed by a CD3 zeta intracellular signaling domain. In embodiments, a CAD polypeptide comprises, from N-terminus to C-terminus, a DAP10 domain, a 4-1BB costimulatory domain, followed by a CD28 costimulatory domain, followed by a CD3 zeta intracellular signaling domain.

[0013] In embodiments, the DAP10 domain comprises a human DAP10 amino acid sequence having an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1. In embodiments, the human DAP10 amino acid sequence comprises a mutated human DAP10 amino acid sequence. In embodiments, the mutated human DAP10 amino acid sequence comprises an amino acid substitution at a position corresponding to K84 and / or Y86. In embodiments, the amino acid substitution at position K84 comprises a K84R substitution. In embodiments, the amino acid substitution at position Y86 comprises a Y86F substitution.

[0014] In embodiments, the isolated nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO:60, or SEQ ID NO:62, or SEQ ID NO:64, or SEQ ID NO:66, or SEQ ID NO:68, or SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:82, or SEQ ID NO:90.

[0015] In embodiments, the isolated nucleic acid is operably linked to a regulatable promoter. In embodiments, the isolated nucleic acid further encodes a cytokine. In embodiments, the cytokine is selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, IL-23. In embodiments, the isolated nucleic acid further encodes a marker protein. In embodiments, the marker protein is selected from the group consisting of truncated CD19, CD20 (rituximab recognition domain), truncated EGFR, and LNGFR.

[0016] In another aspect, the invention provides an expression vector comprising any of the above-described isolated nucleic acids.

[0017] In another aspect, the invention provides a chimeric adapter (CAD) polypeptide encoded by any of the above-described isolated nucleic acids or expression vectors.

[0018] In another aspect, the present invention provides a mammalian cell comprising any of the above-mentioned expression vectors or CAD polypeptides, wherein the mammalian cell expresses at least one receptor associated with DAP10. In an embodiment, the at least one receptor associated with DAP10 is endogenous, exogenous, or overexpressed. In an exemplary embodiment, the receptor is NKG2D. In an embodiment, the mammalian cell is an immune cell, and preferably, the immune cell is a cytotoxic cell.

[0019] In another aspect, the present invention provides a method for activating immune cells, the method comprises expressing the CAD polypeptide of the present invention in immune cells, the immune cells express at least one receptor related to DAP10, and the activation occurs in response to the receptor binding to the corresponding target molecule.In an embodiment, the receptor is endogenous, exogenous, or overexpressed.In an exemplary embodiment, the receptor is NKG2D.In an embodiment, the immune cells or a plurality thereof are introduced into a subject in need thereof, and the activation occurs in the subject.

[0020] In a further aspect, the invention provides the use of a mammalian cell or a plurality of cells of the invention in the preparation of a medicament for treating a subject with a condition, which reduces at least one symptom or sign of said condition in the subject.

[0021] In yet another aspect, there is provided a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a plurality of the mammalian cells of the invention. Other features, objects and advantages will be apparent from the following disclosure.

[0022] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0023] [Figure 1A] 1 is a schematic diagram showing an exemplary chimeric DAP10 adaptor polypeptide of the present disclosure in combination with a receptor that it binds. In the diagram shown, the receptor is NKG2D. A chimeric DAP10 adaptor polypeptide with K84R and Y86F modifications is shown. [Figure 1B] 1 is a schematic diagram showing an exemplary chimeric DAP10 adaptor polypeptide of the present disclosure in combination with a receptor that it binds. In the diagram shown, the receptor is NKG2D. A chimeric DAP10 adaptor polypeptide with K84R and Y86F modifications and a C-terminal fusion with the CD3ζ signaling domain are shown. [Figure 1C] 1 is a schematic diagram showing an exemplary chimeric DAP10 adaptor polypeptide of the present disclosure in combination with a receptor that it binds. In the diagram shown, the receptor is NKG2D. A chimeric DAP10 adaptor polypeptide with K84R and Y86F modifications and a C-terminal fusion containing the 4-1BB costimulatory domain are shown. [Figure 1D] 1 is a schematic diagram showing an exemplary chimeric DAP10 adaptor polypeptide of the present disclosure in combination with a receptor that it binds. In the diagram shown, the receptor is NKG2D. A chimeric DAP10 adaptor polypeptide with K84R and Y86F modifications and a C-terminal fusion containing both the 4-1BB costimulatory domain and the CD3 zeta signaling domain are shown. [Figure 2A] Graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the disclosure expressed in V51 cells and tested against PLC / PRF / 5 cells compared to tumor cells alone and a control chimeric antigen receptor (CAR) construct that was also expressed in V51 cells. [Figure 2B] Graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the disclosure expressed in V51 cells and tested against PLC / PRF / 5 cells compared to tumor cells alone and a control chimeric antigen receptor (CAR) construct that was also expressed in V51 cells. [Figure 2C]Graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the disclosure expressed in V51 cells and tested against PLC / PRF / 5 cells compared to tumor cells alone and a control chimeric antigen receptor (CAR) construct that was also expressed in V51 cells. [Figure 2D] FIG. 2C is a plot showing the extent of Vδ1 cell proliferation observed in the assays of FIGS. 2A-2C. [Figure 2E] FIG. 2C is a plot showing the extent of Vδ1 cell proliferation observed in the assays of FIGS. 2A-2C. [Figure 2F] FIG. 2C is a plot showing the extent of Vδ1 cell proliferation observed in the assays of FIGS. 2A-2C. [Figure 3A] Graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the disclosure expressed in V51 cells and tested against HepG2 cells, compared to tumor cells alone and a control CAR construct that was also expressed in V51 cells. [Figure 3B] Graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the disclosure expressed in V51 cells and tested against HepG2 cells, compared to tumor cells alone and a control CAR construct that was also expressed in V51 cells. [Figure 3C] Graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the disclosure expressed in V51 cells and tested against HepG2 cells, compared to tumor cells alone and a control CAR construct that was also expressed in V51 cells. [Figure 3D] FIG. 3 is a plot showing the extent of Vδ1 cell proliferation observed in the assays of FIGS. 3A-3C. [Figure 3E] FIG. 3 is a plot showing the extent of Vδ1 cell proliferation observed in the assays of FIGS. 3A-3C. [Figure 3F] FIG. 3 is a plot showing the extent of Vδ1 cell proliferation observed in the assays of FIGS. 3A-3C. [Figure 4]Graph showing viability of V51 cells transduced with various chimeric DAP10 adaptor constructs of the present disclosure after 5 days of co-culture with PLC / PRF / 5 cells. [Figure 5A] Graph showing robust in vivo tumor growth mediated by V51 cells transduced with selected chimeric DAP10 adaptor polypeptides of the present disclosure. [Figure 5B] Graph showing robust in vivo tumor growth mediated by V51 cells transduced with selected chimeric DAP10 adaptor polypeptides of the present disclosure. [Figure 6] 1 is a graph showing V51 cells transduced with various DAP10 adaptor polypeptides of the present disclosure and NKG2D expression levels after co-culture of the transduced cells with PLC cells. The highest NKG2D expression levels were observed in cells transduced with DAP10 adaptor polypeptides containing K84R, Y86F, the 4-1BB costimulatory domain, and the CD3 zeta signaling domain, respectively. [Figure 7] Western blot of CAD protein visualized with anti-DAP10 and anti-CD3ζ antibodies, showing that DAP10 CAD expression is similar between different lots of Vδ1 cells. [Figure 8A] Graphs showing that the cytotoxic activity of DAP10 CAD is mediated by NKG2D. CAD+ (Figures 8A-8B) or chimeric antigen receptor (CAR)+Vδ1 cells (Figures 8C-8D) were preincubated with various dilutions of anti-NKG2D antibody (clone 1D11) or isotype control (1 μg / ml to 0.01 ng / ml) before co-culture with luciferase-labeled target cells (PLC / PRF / 5 or HL60). Target cell killing was assessed 18 hours later by measuring the luciferase signal. NKG2D-mediated cytotoxicity can be assessed by comparing the % cytotoxicity with isotype preincubation with the % cytotoxicity with NKG2D antibody preincubation. [Figure 8B]Graphs showing that the cytotoxic activity of DAP10 CAD is mediated by NKG2D. CAD+ (Figures 8A-8B) or chimeric antigen receptor (CAR)+Vδ1 cells (Figures 8C-8D) were preincubated with various dilutions of anti-NKG2D antibody (clone 1D11) or isotype control (1 μg / ml to 0.01 ng / ml) before co-culture with luciferase-labeled target cells (PLC / PRF / 5 or HL60). Target cell killing was assessed 18 hours later by measuring the luciferase signal. NKG2D-mediated cytotoxicity can be assessed by comparing the % cytotoxicity with isotype preincubation with the % cytotoxicity with NKG2D antibody preincubation. [Figure 8C] Graphs showing that the cytotoxic activity of DAP10 CAD is mediated by NKG2D. CAD+ (Figures 8A-8B) or chimeric antigen receptor (CAR)+Vδ1 cells (Figures 8C-8D) were preincubated with various dilutions of anti-NKG2D antibody (clone 1D11) or isotype control (1 μg / ml to 0.01 ng / ml) before co-culture with luciferase-labeled target cells (PLC / PRF / 5 or HL60). Target cell killing was assessed 18 hours later by measuring the luciferase signal. NKG2D-mediated cytotoxicity can be assessed by comparing the % cytotoxicity with isotype preincubation with the % cytotoxicity with NKG2D antibody preincubation. [Figure 8D]Graphs showing that the cytotoxic activity of DAP10 CAD is mediated by NKG2D. CAD+ (Figures 8A-8B) or chimeric antigen receptor (CAR)+Vδ1 cells (Figures 8C-8D) were preincubated with various dilutions of anti-NKG2D antibody (clone 1D11) or isotype control (1 μg / ml to 0.01 ng / ml) before co-culture with luciferase-labeled target cells (PLC / PRF / 5 or HL60). Target cell killing was assessed 18 hours later by measuring the luciferase signal. NKG2D-mediated cytotoxicity can be assessed by comparing the % cytotoxicity with isotype preincubation with the % cytotoxicity with NKG2D antibody preincubation. [Figure 9] AC show that the DAP10 CAD of the present disclosure has a consistent molecular activation signature. Data were obtained from Nanostring analysis after stimulation from multiple donors and cell lines. [Figure 10A] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10B] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10C]Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10D] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10E] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10F]Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10G] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. The graphs shown in Figures 10A-10G represent the cytotoxic activity rate (%) of V51 cells transduced with DAP10CAD. In an 18-hour short-term cytotoxicity assay, DAP10 CAD+V51 cells or controls were co-cultured with various luciferase-expressing target cell lines at various E:T ratios (1:6-10:1). [Figure 10H] 1 is a table showing that the target cell lines tested contain a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns. Data are presented as fold change in mean fluorescence intensity (MFI) of NKG2D ligand relative to relevant isotype control. [Figure 10I] FIG. 10C is a series of graphs showing the raw data used to populate the tables shown in FIG. 10H. [Figure 10J] FIG. 10C is a series of graphs showing the raw data used to populate the tables shown in FIG. 10H. [Figure 11A]Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 11B] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 11C] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 11D]Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 11E] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 11F] Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 11G]Figure 10 shows that V51 cells transduced with DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Graph showing cytotoxicity index of V51 cells transduced with DAP10 CAD at different effector:target ratios compared to controls. Target cell lines tested include a wide range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figures 10H-10J). [Figure 12A] Graph showing that the cytotoxic activity of V51 cells transduced with DAP10 CAD is comparable between different lots of V51 cells and DAP10 CAD. The 120 hour cytotoxicity assay is shown in FIG. 12A, where the target cells were PLC / PRF / 5 cells. The % reduction in cytotoxicity of tumors alone compared to treatment using the final time point of the assay in FIG. 12A is shown in FIG. 12B. [Figure 12B] Graph showing that the cytotoxic activity of V51 cells transduced with DAP10 CAD is comparable between different lots of V51 cells and DAP10 CAD. The 120 hour cytotoxicity assay is shown in FIG. 12A, where the target cells were PLC / PRF / 5 cells. The % reduction in cytotoxicity of tumors alone compared to treatment using the final time point of the assay in FIG. 12A is shown in FIG. 12B. [Figure 12C] FIG. 1 shows a comparison of cytotoxicity of Vδ1 cells transduced with either DAP10.6, DAP10.16, or DAP10.17 constructs. Data obtained for each construct is an aggregate of three donors. PLC / PRF / 5 cells were used as target cells for the 120 hour cytotoxicity assay. Controls included DAP10.0 and PLC / PRF / 5 only. In this particular assay using a stringent E:T ratio, DAP10.6 showed improved cytotoxicity compared to DAP10.16 and DAP10.17. [Figure 12D]Graphs showing donor dependency of three different DAP10 constructs (DAP10.6, FIG. 12D; DAP10.16, FIG. 12E; DAP10.17, FIG. 12F) as measured by cytotoxicity index in a 120 hour co-culture cytotoxicity assay. DAP10 constructs were transduced into V51 cells and target cells were PLC / PRF / 5 cells. Controls included untransduced V51 cells from the same three different donors. [Figure 12E] Graphs showing donor dependency of three different DAP10 constructs (DAP10.6, FIG. 12D; DAP10.16, FIG. 12E; DAP10.17, FIG. 12F) as measured by cytotoxicity index in a 120 hour co-culture cytotoxicity assay. DAP10 constructs were transduced into V51 cells and target cells were PLC / PRF / 5 cells. Controls included untransduced V51 cells from the same three different donors. [Figure 12F] Graphs showing donor dependency of three different DAP10 constructs (DAP10.6, FIG. 12D; DAP10.16, FIG. 12E; DAP10.17, FIG. 12F) as measured by cytotoxicity index in a 120 hour co-culture cytotoxicity assay. DAP10 constructs were transduced into V51 cells and target cells were PLC / PRF / 5 cells. Controls included untransduced V51 cells from the same three different donors. [Figure 13A] Figure 13 shows that DAP10 CAD stimulation results in a multifunctional cytokine profile that is a function of different DAP10 CAD constructs and target cell types. Cytokine profiles as a function of DAP10 CAD and target cells are shown in Figure 13A. [Figure 13B] Figure 13 shows that DAP10 CAD stimulation results in a multifunctional cytokine profile that is a function of different DAP10 CAD constructs and target cell types. Cytokine profiles as a function of DAP10 CAD and target cells are shown in Figure 13A. Figure 13B is a graph showing interferon gamma induction as a function of DAP10 CAD and target cell type. [Figure 13C] FIG. 13 is a plot showing interferon gamma secretion levels after co-culture with DAP10 CAD+Vδ1 cells alone (effector) and various target cells. [Figure 13D] FIG. 13 is a plot showing interferon gamma secretion levels after co-culture with DAP10 CAD+Vδ1 cells alone (effector) and various target cells. [Figure 13E] FIG. 13 is a plot showing interferon gamma secretion levels after co-culture with DAP10 CAD+Vδ1 cells alone (effector) and various target cells. [Figure 13F] FIG. 13 is a plot showing interferon gamma secretion levels after co-culture with DAP10 CAD+Vδ1 cells alone (effector) and various target cells. [Figure 14] Cytokine profile of V51 cells transduced with DAP10 CAD in the presence or absence of target cells compared to that of V51 cells transduced with chimeric antigen receptor (CAR). [Figure 15] Figure 1 shows that the DAP10 CAD of the present disclosure promotes the expansion of V51 cells from multiple donors. Figure 1 shows plots showing donor dependency for the expansion of V51 cells obtained from two different donors (SCT29 and SCT46) transduced with either DAP10.6, DAP10.16 or DAP10.17 based on co-culture experiments with PLC / PRF / 5 cells. Controls included previously tested V51 cells obtained from a different donor (SCT06) and transduced with DAP10.6 (positive control) and V51 cells obtained from two different donors (SCT29 and SCT46) and transduced with DAP10.0 (negative control). [Figure 16A] 16B shows in vivo tumor control in a mouse model of V51 cells transduced with DAP10 CAD. A graph comparing in vivo tumor control of DAP10.6 and DAP10 CAD containing an altered ("1XX") CD3ζ intracellular signaling domain (DAP10.16). A schematic of the experimental procedure is shown in FIG. 16B. [Figure 16B] FIG. 13 shows in vivo tumor control in a mouse model of Vδ1 cells transduced with DAP10 CAD. [Figure 17A] Figure 1 shows that the anti-tumor activity of V51 cells transduced with DAP10 CAD of the present disclosure exhibits anti-tumor activity with similar kinetics as CARV51 cells.Figure 1 shows a comparison of in vivo tumor growth kinetics of DAP10 CAD+V51 cells compared to CARV51 cells in the HCT-15 mouse xenograft model. [Figure 17B] 4 shows that the anti-tumor activity of V51 cells transduced with the DAP10 CAD of the present disclosure exhibits anti-tumor activity with similar kinetics as CARV51 cells. Graph of quantification of tumor volume at day 27. [Figure 17C] 17A-17B show that the anti-tumor activity of V51 cells transduced with the DAP10 CAD of the present disclosure exhibits anti-tumor activity with similar kinetics as CARV51 cells. [Figure 18A] 1 shows that V51 cells transduced with the DAP10 CAD of the present disclosure proliferate in tumor tissue in vivo in a mouse model.FIG. 2 shows flow cytometry plots showing that proliferation of DAP10 CAD-transduced V51 cells is specific to tumor tissue. [Figure 18B] Figure 1 shows that V51 cells transduced with the DAP10 CAD of the present invention grow in tumor tissue in vivo in a mouse model.Graph showing the progression of growth in tumor tissue over 14 days. [Figure 18C] 1 shows that V51 cells transduced with the DAP10 CAD of the present invention proliferate in tumor tissue in vivo in a mouse model. Graph showing quantification of V51 cells in tumor tissue or other tissues taken 4, 7 and 14 days after treatment. [Figure 18D]We show that V51 cells transduced with the DAP10 CAD of the present invention grow in tumor tissue in vivo in a mouse model. The experimental procedure used to obtain the data shown in Figures 18A-18C is shown diagrammatically in Figure 18D. [Figure 19] 1 shows that treatment of mice with V51 cells transduced with the DAP10 CAD of the present disclosure is not associated with significant changes in body weight. [Figure 20] A and B show that V51 cells transduced with the DAP10 CAD of the present disclosure target tumor cells while sparing non-tumor cells. V51 cells transduced with the DAP10 CAD of the present disclosure significantly reduce the viability of THP1 cells compared to controls, as shown in the graph in Figure 20A, and do not target healthy PBMCs, as shown in the graph in Figure 20B. [Figure 21] FIG. 13 is a graph showing fold expansion of V51 cells in small scale shake flask expansion of 6 different donors. Fold expansion was measured on day 14. Data compares V51 cells transduced with DAP10.6 and DAP10.16 CAD. [Figure 22A] 22A-22C are graphs showing the proliferation rate of V51 cells transduced with the lead DAP10 CAD of the present disclosure (DAP10.6, DAP10.16, DAP10.17) compared to the control. Data in FIG. 22A corresponds to V51 cells obtained from the first donor (SCT06), data in FIG. 22B corresponds to V51 cells obtained from the second donor (SCT29), and data in FIG. 22C corresponds to V51 cells obtained from the third donor (SCT45). For each of FIG. 22A-22C, the V51 cell percentage (%) was measured as a function of proliferation time (days). [Figure 22B]22A-22C are graphs showing the proliferation rate of V51 cells transduced with the lead DAP10 CAD of the present disclosure (DAP10.6, DAP10.16, DAP10.17) compared to the control. Data in FIG. 22A corresponds to V51 cells obtained from the first donor (SCT06), data in FIG. 22B corresponds to V51 cells obtained from the second donor (SCT29), and data in FIG. 22C corresponds to V51 cells obtained from the third donor (SCT45). For each of FIG. 22A-22C, the V51 cell percentage (%) was measured as a function of proliferation time (days). [Figure 22C] 22A-22C are graphs showing the proliferation rate of V51 cells transduced with the lead DAP10 CAD of the present disclosure (DAP10.6, DAP10.16, DAP10.17) compared to the control. Data in FIG. 22A corresponds to V51 cells obtained from the first donor (SCT06), data in FIG. 22B corresponds to V51 cells obtained from the second donor (SCT29), and data in FIG. 22C corresponds to V51 cells obtained from the third donor (SCT45). For each of FIG. 22A-22C, the V51 cell percentage (%) was measured as a function of proliferation time (days). [Figure 23A] FIG. 1 shows a schematic diagram depicting the process of generating "off-the-shelf" allogeneic DAP10 CAD Vδ1 cells. [Figure 23B] Graph showing independent proliferation of V51 cells transduced with preferred DAP10 CAD constructs of the present disclosure. V51 cells used in the experiments shown were obtained from three different donors (SCT06, SCT29, SCT45), respectively. [Figure 23C] Graph showing independent proliferation of V51 cells transduced with preferred DAP10 CAD constructs of the present disclosure. V51 cells used in the experiments shown were obtained from three different donors (SCT06, SCT29, SCT45), respectively. [Figure 23D]Graph showing independent proliferation of V51 cells transduced with preferred DAP10 CAD constructs of the present disclosure. V51 cells used in the experiments shown were obtained from three different donors (SCT06, SCT29, SCT45), respectively. [Figure 23E] 23A-23F are graphs showing that ex vivo culture of V51 cells results in a significant fold expansion (Figure 23E) and strong DAP10 CAD transduction (Figure 23F). Data in Figure 23E are expressed as V51 fold expansion and data in Figure 23F are expressed as %DAP10 CAD of V51 cells. [Figure 23F] 23A-23F are graphs showing that ex vivo culture of V51 cells results in a significant fold expansion (Figure 23E) and strong DAP10 CAD transduction (Figure 23F). Data in Figure 23E are expressed as V51 fold expansion and data in Figure 23F are expressed as %DAP10 CAD of V51 cells. [Figure 23G] FIG. 1 shows plots depicting the cellular composition of V51, V52, αβ, and NK cells over time, expressed as % culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention provides chimeric adapters, i.e., CADs, which generally comprise an endodomain comprising a DAP10 domain and at least one co-stimulatory domain, and optionally further comprise at least one intracellular signaling domain, but specifically lack an extracellular domain comprising a ligand-binding domain. Thus, in some embodiments, the CAD polypeptides of the present invention may further comprise a transmembrane domain and / or an extracellular spacer domain, but specifically lack a functional extracellular receptor and / or a ligand-binding domain. In contrast, the prior art typically used DAP10 as a component of CAR or NKG2D fusion chimeras. See, e.g., Zhao et al., OncoImmunology. 2019;8(1):e1509173; Lynch et al., 2017, Immunol 152:472; US2020 / 0308248; WO / 2018 / 183385; CN109096404; CN111995689. The CAD polypeptides of the invention are distinct from those found in nature and generally comprise at least two polypeptide domains that are not naturally associated with each other, and optionally further comprise additional advantageous signaling domains and mutations as detailed herein.

[0025] The CAD polypeptide of the present invention preferably comprises a DAP10 domain comprising human DAP10, optionally comprising one or more substitution, deletion and / or addition mutations. For example, the DAP10 domain may have a Y86F and / or K84R mutation.

[0026] A "costimulatory domain" in the context of a CAD polypeptide of the present disclosure promotes cell proliferation, cell survival, and memory cell development of cytotoxic cells expressing a chimeric adaptor. The CAD polypeptide of the present invention may comprise one or more co-stimulatory domains selected from the co-stimulatory domains of proteins of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dapl0, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOSLIGHT, NKG2C, B7-H3, or combinations thereof. When a CAD comprises multiple co-stimulatory domains, these domains may be arranged in tandem, optionally separated by linkers. The co-stimulatory domain is an intracellular domain that may be located between the DAP10 domain and any intracellular signaling domain in the CAD.

[0027] In embodiments, the costimulatory domain includes the costimulatory domains of CD28, CD27, ICOS, 4-1BB, OX40, and CD40L. The term "costimulatory domain" as used herein encompasses any modifications thereof, examples of which are described in U.S. Patent Application No. 20200129554, U.S. Patent Application No. 20200317777, WO2019010383, Li, W., et al., (2020) Immunity 53:456-470; and Li, G., et al., (2017) J Immunol 198(1 Supplement):198.4, the contents of each of which are incorporated herein in their entirety.

[0028] An "intracellular signaling domain" in the context of the CAD polypeptides of the present disclosure transmits effector function signals and directs the cytotoxic cell to perform its specialized function, i.e., to damage and / or destroy a target cell. Examples of suitable intracellular signaling domains include, for example, the zeta chain of the T cell receptor complex or its homologues, such as the eta chain, FcsRly and β chains, MB1 (Iga) chain, B29 (Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling (e.g., CD2, CD5, and CD28). Specifically, the intracellular signaling domain may be human CD3 zeta chain, FcγRIII, FcsRI, the cytoplasmic tail of the Fc receptor, immunoreceptor tyrosine-based activation motifs (ITAMs) with cytoplasmic receptors, and combinations thereof.

[0029] The intracellular signaling domain may include several types of intracellular signaling domains of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins, including CD3, B7 family costimulatory factors, and tumor necrosis factor receptor (TNFR) superfamily receptors (Park et al., “Are all chimeric antigen receptors created equal?” J Clin Oncol., vol. 33, pp. 651-653, 2015). Additional intracellular signaling domains include signaling domains used by NK cells and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immunol., vol. 6, p. 195, 2015), such as the signaling domain of NKp30 (B7-H6) (Zhang et al., "An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo," J Immunol., vol. 189, pp. 2290-2299, 2012), and DAP12 (Topfer et al., "DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol., vol. 194, pp. 3201-3212, 2015), NKG2D, NKp44, NKp46, DAP10, and CD3z.In addition, intracellular signaling domains also include signaling domains of human immunoglobulin receptors containing immune receptor tyrosine-based activation motifs (ITAMs), such as FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, and FcRL5 (Gillis et al., “Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies,” Front Immunol., vol. 5, p. 254, 2014).

[0030] In embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In exemplary embodiments, the intracellular signaling domain in a CAD comprises the cytoplasmic signaling domain of human CD3ζ. The term "intracellular signaling domain" as used herein also encompasses any modifications thereof, examples of which are described in U.S. Patent Application No. 2020 / 0317777, as well as Combadiere, B., et al., (1996) J Exp Med 183(5):2109-17; Lowin-Kropf B., et al., (1998) J Cell Biol 140(4):861-871; Ardouin L., et al., (1999) Immunity 10(4):409-20; Liu H. and Vignali DAA., (1999) J Immunol 163:599-602; Kersh EN., et al., J Exp Med(1999)190(11):1627-36; Chae WJ., et al., (2004) Int Immunol 16(9):1225-36;Becker,AM.,et al.,(2007)J Immunol 178(7):4120-8;Methi T.,et al.,(2007)Eur J Immunol 37(9):2539-48;Baudouin SJ.,et al.,(2008)Mol Biol Cell 19(6):2444-56;Zhao Y.,et al.,(2009)J Immunol 183(9):5563-74;Kochenderfer JN.,et al.,(2010)Blood 116(19):3875-86;Bridgeman JS.,et al.,(2014)Clin Exp Immunol 175(2):258-67;Long AH.,et al.,(2015)Nat Med 21(6):581-90;Hwang S.,et al.,(2015)Nat Commun 6:6982;WO2019126748;Feucht J.,et al.,(2019)Nat Med 25(1):82-88;Roda-Navarro,P., and Reyburn, H. T., (2009) J Biol Chem 284(24):16463-16472; Giurisato, E., et al., (2007) Mol Cell Biol 27(24):8583-8599; and Wu, J., et al., (2000) J Exp Med 192(7):1059-1068, the contents of each of which are incorporated herein in their entirety.

[0031] In embodiments, two or more components of the CAD of the present invention may be separated by one or more linkers. The linker is an oligo- or polypeptide region of about 1-100 amino acids in length. In some embodiments, the linker may be, for example, 5-12 amino acids in length, 5-15 amino acids in length, or 5-20 amino acids in length. The linker may be composed of flexible residues such as glycine and serine so that adjacent protein domains are free to move relative to one another. Longer linkers, for example, linkers of more than 100 amino acids, may be used in conjunction with alternative embodiments of the present invention, and may be selected, for example, so that the two adjacent domains do not sterically interfere. Examples of linkers that may be used in the present invention include, but are not limited to, 2A-like linkers, or 2A linkers (e.g., T2A), including their functional equivalents.

[0032] In an exemplary embodiment, a chimeric DAP10-4-1BB adaptor polypeptide is provided that includes a DAP10 domain and a 4-1BB costimulatory domain. In another exemplary embodiment, a chimeric DAP10-CD28 adaptor polypeptide is provided that includes a DAP10 domain and a CD28 costimulatory domain. In another exemplary embodiment, a chimeric DAP10-4-1BB-CD3ζ adaptor polypeptide is provided that includes a DAP10 domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. In another exemplary embodiment, a chimeric DAP10-CD28-CD3ζ adaptor polypeptide is provided that includes a DAP10 domain, a CD28 costimulatory domain, and a CD3ζ intracellular signaling domain. In yet another exemplary embodiment, a chimeric DAP10-4-1BB-CD28-CD3ζ adaptor polypeptide is provided that includes a DAP10 domain, a 4-1BB costimulatory domain, a CD28 costimulatory domain, and a CD3ζ intracellular signaling domain.

[0033] The chimeric adaptor polypeptide of the present invention may optionally further comprise a transmembrane domain. The transmembrane domain of the CAD is a region capable of spanning the cell membrane of the cytotoxic cell. The transmembrane domain is selected from the transmembrane regions of transmembrane proteins, such as type I transmembrane proteins, artificial hydrophobic sequences, or combinations thereof. Suitable examples of transmembrane domains include the transmembrane regions of the alpha, beta, or gamma chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. The synthetic transmembrane domain may comprise a triad of phenylalanine, tryptophan, and valine. Optionally, a short oligo- or polypeptide linker (preferably 2-10 amino acids in length) may form the bond between the transmembrane domain of the CAD and the intracellular signaling domain. A glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0034] The chimeric adapter polypeptide of the present invention may optionally further comprise an extracellular spacer domain. The extracellular spacer domain of a CAD is usually a hydrophilic region located between the ligand binding domain (not present in the present invention) and the transmembrane domain. In some embodiments, this domain promotes proper protein folding of the CAD. The extracellular spacer domain may comprise a domain selected from an Fc fragment of an antibody, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include the CD8a hinge, an artificial spacer composed of three glycines (Gly), possibly as small polypeptides, and the CH1 and CH3 domains of IgG (e.g., human IgG4).

[0035] definition For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that a set forth definition conflicts with any document incorporated herein by reference, the set forth below shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0036] As used herein, "about" when referring to a measurable value, e.g., amount, temporal duration, etc., is meant to encompass variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, as appropriate for carrying out the disclosed methods.

[0037] As used herein, "any" or "optionally" means that a particular limitation, event, circumstance, etc. may occur, but need not occur, and includes cases where the limitation, event, or circumstance occurs and cases where the limitation, event, or circumstance does not occur.

[0038] As used herein, the term "DAP10" refers to a transmembrane adaptor protein present in mammalian lymphoid and myeloid cells, the exact sequence of which may vary slightly between species, isoforms, and individuals. Other names for DAP10 recognized in the art include hematopoietic cell signaling substrate (HCST), DNAX-activating protein 10, membrane protein DAP10, transmembrane adaptor protein KAP10 (KAP10), and PIK3AP. For example, in humans, DAP10 refers to a protein represented by the major polypeptide sequence UnitProt Q9UBK5 and NCBI accession numbers NP_055081.1 and AF072845, although different isoforms and variants may exist. The name DAP10 may refer to multiple proteins with related structures and polypeptide sequences from various species, i.e., protein members of the DAP10 protein family that have high sequence identity with human DAP10 (SEQ ID NO: 1), although one of skill in the art would be able to identify human DAP10-related proteins in mammals, even if they differ from the sequences referenced herein.

[0039] The term "host cell" as used herein refers to a cell type that is selected to express a CAD polypeptide of the present disclosure. In an embodiment, the host cell endogenously expresses at least one receptor associated with DAP10, and thus expresses a chimeric adapter polypeptide of the present disclosure. In an embodiment, the host cell is engineered to express at least one receptor associated with DAP10, and thus a chimeric adapter polypeptide of the present disclosure. Exemplary host cells may include, but are not limited to, a wide variety of immune cells, including, in particular, cytotoxic cells, preferred examples of which are disclosed herein (e.g., γδ T cells, αβ T cells, NK cells, NKT cells, B cells, neutrophils, monocytes / macrophages). It is also within the scope of the present disclosure that "host cells" may include non-immune cells, such as, but not limited to, stem cells (e.g., embryonic stem cells, hematopoietic stem cells, stromal stem cells, induced pluripotent stem cells, etc.).

[0040] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses or is expressing CD3 (CD3+) and the T cell receptor (TCR+). T cells play a central role in cell-mediated immunity. T cells that are "expressing" CD3 and TCR are engineered to have no CD3 and / or TCR cell surface expression.

[0041] The term "γδ T cells (gamma delta T cells)" as used herein refers to a subset of T cells expressing a distinct T cell receptor (TCR), i.e., γδ TCR, on their surface, composed of one γ chain and one δ chain. The term "γδ T cells" specifically includes all subsets of γδ T cells (including, but not limited to, Vδ1, Vδ2, and Vδ3 γδ T cells, as well as naive, effector memory, central memory, and terminally differentiated γδ T cells). As a further example, the term "γδ T cells" includes Vγ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells. In some embodiments, γδ T cells are Vδ1 - , Vδ2 - , or Vδ1 - and Vδ2 -Compositions and methods for making and using engineered and non-engineered γδ T-cells and / or subtypes thereof include, but are not limited to, those described in US2016 / 0175358; WO2017 / 197347; US9499788; US2018 / 0169147; US9907820; US2018 / 0125889, and US2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including compositions and methods for making and using engineered and non-engineered γδ T-cells and / or subtypes thereof. The present application further contemplates T cells, or other engineered leukocytes or lymphocytes, that express one γ chain or one δ chain, optionally in combination with a second polypeptide, to form a functional TCR. Such engineered leukocytes or lymphocytes expressing one gamma chain or one delta chain may be used in the methods described herein or present in the compositions described herein.

[0042] The γδ T cells described herein can be δ1, δ2, δ3, or δ4 γδ T cells, or a combination thereof. In some cases, the γδ T cells are predominantly (greater than 50%), mostly (greater than 90%), essentially all, or entirely δ2 γδ T cells. In some cases, the γδ T cells are predominantly (greater than 50%), mostly (greater than 90%), essentially all, or entirely δ1 γδ T cells. In some cases, the γδ T cells are predominantly (greater than 50%), mostly (greater than 90%), essentially all, or entirely δ3 γδ T cells.

[0043] As described herein, T cells for use in γδ can be obtained from allogeneic or autologous donors. γδ T cells can be partially or completely purified or expanded ex vivo without purification. Methods and compositions for ex vivo expansion include, but are not limited to, those described in WO2017 / 197347. Expansion can be performed before or after, or before and after, the chimeric adapter polypeptide of the present disclosure is introduced into the γδ T cell(s). Other additional or alternative expansion methods include the use of artificial antigen presenting cells (aAPCs), aminobisphosphonates, cytokine cocktails, and feeder cells (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0044] The term "αβ T cell" as used herein refers to a T cell that expresses the α and β chains of the TCR as part of a complex with a CD3 chain molecule. Each α and β chain contains one variable domain and one constant domain. αβ T cells primarily recognize peptide antigens presented by major histocompatibility complex (MHC) class I and class II molecules, with most of the receptor diversity contained within the third complementarity determining region (CDR3) of the TCR α and β chains.

[0045] As used herein, the term "natural killer (NK) cells" refers to CD56 cells that play a key role in immunity against viruses and immune surveillance of tumors and constitute a key cell subset of the innate immune system. + CD3 -Refers to granular lymphocytes (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). NK cells express a highly diverse repertoire of inhibitory and activating receptors on the cell surface, which control the immune response. NK cells can kill mutant and infected cells by releasing perforin and granzymes, or by using effector molecules of the tumor necrosis factor (TNF) family (e.g., TNF, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand, which induce apoptosis in target cells). In addition, upon activation, NK cells rapidly produce chemokines and cytokines (e.g., interferon (IFN)-γ, GM-CSF, and IL-10), which recruit and affect the function of host hematopoietic and non-hematopoietic cells. Cytotoxic CD8 + Unlike T lymphocytes, NK cells can exert cytotoxicity against tumor cells without the need for prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are considered to be fairly safe effector cells, as they can avoid the potentially fatal complications of cytokine storm (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and off-target tumor effects.

[0046] NK cells can be obtained from allogeneic or autologous donors. NK cells can be partially or completely purified or expanded ex vivo without purification. Methods and compositions for ex vivo expansion include, but are not limited to, those described in: Becker et al., (2016) Cancer Immunol. Immunother. 65(4): 477-84. Expansion can be performed before or after or before and after the chimeric DAP10 adaptor polypeptide is introduced into the NK cells. Briefly, expansion of NK cells can include the use of modified feeder cells, cytokine cocktails (e.g., IL-2, IL-15), and / or aAPCs (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0047] In some examples, placental hematopoietic stem cell derived natural killer (PNK) cells or immortalized cell lines (e.g., NK-92) can be modified to express the chimeric adapter polypeptides of the present disclosure. In other examples, NK cells that can be used to engineer expression of the chimeric adapter polypeptides of the present disclosure can be differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). As used herein, the term "natural killer T (NKT) cells" refers to T lineage cells that share morphological and functional characteristics with both T cells and NK cells. NKT cells are rapid responders of the innate immune system and mediate potent immunoregulatory and effector functions in a variety of disease settings. Recognition of ligands in NKT cells results in the rapid secretion of proinflammatory cytokines (e.g., IFN-γ and TNF-α) and anti-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13). This can be achieved, for example, by directly targeting tumor cells and indirectly modulating antitumor responses through the release of various cytokines or by modifying the TME to enhance immune responses against cancer, etc. After activation, NKT cells can immediately start secreting cytokines without first differentiating into effector cells. Due to their rapidity of response, NKT cells play a key role in the first line of natural defense against several types of bacterial and viral infections. In addition, many of the cytokines secreted by NKT cells have a strong effect on the differentiation and function of αβT cells, linking NKT cells to adaptive defense. NKT cells bridge the adaptive immune system with the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. NKT cells can be obtained from allogeneic or autologous donors. NKT cells can be partially or completely purified or expanded ex vivo without purification.Briefly, NKT cells can be expanded by the use of ex vivo IL-2 and / or a monoclonal antibody specific for the TCR α chain CDR3 loop (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1):45-59).

[0048] As used herein, the term "γδ natural killer T cells" or "γδ NKT cells" refers to iPSC-derived cells that express γδ TCR and NK receptors but lack expression of characteristic γδ T cell markers (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59). These cells have been shown to have anti-tumor activity against a number of cancer cell lines, but not normal cells, and exhibited more potent killing than donor-derived γδ T cells or donor-derived NK cells (Zeng J et al., (2019) PLoS ONE 14(5): e0216815). In embodiments herein, chimeric adaptor polypeptides can be expressed in γδ NKT cells, which are used according to the methods disclosed herein.

[0049] As used herein, the term "myeloid cells" refers to a subgroup of leukocytes represented by granulocytes, monocytes, macrophages, and dendritic cells (DCs). They circulate through the blood and lymphatic systems and are rapidly recruited to sites of tissue injury and infection via various chemokine receptors. Within tissues, they are activated for phagocytosis and secretion of inflammatory cytokines, thereby playing an important role in protective immunity. Myeloid cells can also be found in tissues at steady state, where they control development, homeostasis, and tissue repair.

[0050] As used herein, the term "macrophage" refers to highly plastic innate cells that contain functional and phenotypic signatures that can be molded in response to various stimuli. Macrophage polarization is broadly classified into two distinct states: M1 phenotype (classically activated), which responds to factors such as lipopolysaccharide (LPS) or IFN-γ, or M2 phenotype, which responds to cytokines such as IL-4, IL-5, and IL-13. An example of an M1-like macrophage expresses iNOS and inflammatory cytokines such as TNF-α, IL1-β, IL-6, IL-12, and IL-23. An example of an M2 macrophage shows increased expression of CD209, CD200R, CD1a, and CD1b in humans, suggesting that it is involved in wound healing and anti-tumor responses. The ability of macrophages to infiltrate and be reprogrammed into solid tumors and the anti-tumor effects associated with switching to an M1 phenotype are relevant to the present disclosure with respect to engineered macrophages expressing the chimeric adaptor polypeptides described herein. For example, in a mouse ovarian cancer model, it has been shown that inhibiting NK-κB signaling can reprogram macrophages into anti-tumor M1 phenotype cells capable of producing nitric oxide and inducing IL-12-dependent NK-mediated anti-tumor effects (Zhang F et al., (2019) Nat Commun 10:3974).

[0051] Macrophages can be obtained / derived from allogeneic or autologous donors. Macrophages can be partially or completely purified, or cultured ex vivo without purification (see, for example, Davies JQ and Gordon A (2005) Methods Mol Biol 290:105016). In some embodiments, the present disclosure encompasses hESC-derived macrophages (Karlsson, KR et al., (2008) Exp Hematol 36:1167-1175), or iPSC-derived macrophages (Takata K. et al., (2017) Immunity 47:183-198).

[0052] The term "NKG2D receptor" as used herein refers to a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D functions as a major activating receptor, and ligand binding induces cytotoxicity and cytokine production. NKG2D provides costimulation through the related adaptor molecule DAP10, which recruits phosphatidylinositol 3 kinase. In mice, NKG2D is also associated with DAP12, which recruits protein tyrosine kinase. NKG2D is encoded by the KLRK1 gene in the NK gene complex (NKC), located on chromosome 6 in mice and chromosome 12 in humans. In humans, NKG2D is expressed by NK cells, γδ T cells and CD8+αβ T cells, as well as CD4+ T cells under certain pathological conditions (Stanjanovic A., et al. (2018) Front. Immunol. 23:1-15). In mice, NKG2D is expressed by NK cells, NK1.1+ T cells, γδ T cells, activated CD8+ αβ T cells, and activated macrophages. The full-length human NKG2G amino acid sequence is set forth herein as SEQ ID NO:95, the amino acid sequence of the transmembrane domain of human NKG2D is set forth herein as SEQ ID NO:96, and the amino acid sequence of the transmembrane and extracellular ligand-binding domain of NKG2D is set forth herein as SEQ ID NO:97.

[0053] As used herein, the term "recombinant mammalian cell" refers to a cell or cell line derived from a mammal that contains at least one alteration made using genetic engineering techniques. In some embodiments, the "recombinant mammalian cell" is a γδ T cell or NK cell or NKT cell or αβ T cell, etc., that contains a nucleic acid construct encoding a chimeric DAP10 adaptor polypeptide. The "recombinant mammalian cell" can be obtained from any mammal, e.g., human, rodent, etc.

[0054] As used herein, the term "TCR" or "T cell receptor" refers to dimeric heterologous cell surface signaling proteins that form αβ or γδ receptors, or a combination thereof. αβTCRs recognize antigens presented by MHC molecules, whereas γδTCRs can recognize antigens independent of MHC presentation.

[0055] The term "MHC" (major histocompatibility complex) refers to a subset of genes that code for cell surface antigen-presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC or HLA are used interchangeably.

[0056] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded only in the full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded in a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0057] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be a complete immunoglobulin obtained from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is usually a tetramer of immunoglobulin molecules. The antibodies of the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), antibody compositions with polyepitopic specificity, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), diabodies, single domain antibodies (sdAbs), Fvs, Fabs and F(abs), so long as they exhibit the desired biological or immunological activity, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY: Harlow et al., 1989, In; Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Nat Acad. Sci. USA 85:5879-5883: Bird et al., 1988, Science 242:423-426).

[0058] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specific binding to an immunoglobulin or receptor (e.g., a T-cell receptor). Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipid or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0059] As used herein, an "alteration" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the change results from a modification of the sequence that includes the amino acid residue / position. An "alteration" of an amino acid residue / position is synonymous with a "mutation" of the amino acid residue / position. For example, representative modifications include the substitution of a residue (or the position) with another amino acid (e.g., conservative or non-conservative substitution), the insertion of one or more amino acids, and the deletion of one or more amino acids. An "amino acid substitution" or variant thereof refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid residue. Generally, and preferably, the modification results in a change in at least one physico-biochemical activity of the mutant polypeptide compared to the polypeptide that includes the starting (or "wild type") amino acid sequence. Thus, a "altered" amino acid sequence as referred to herein includes an amino acid sequence in which one or more amino acids have been mutated and / or any number of amino acids have been inserted and / or any number of amino acids have been deleted.

[0060] As used herein, the term "endogenous" refers to a substance and / or process that originates from within a system, including, but not limited to, an organism, tissue, or cell. For example, in the context of the present disclosure, "endogenous" refers to a nucleic acid molecule or polypeptide that is normally expressed within a cell or tissue.

[0061] Conversely, the term "exogenous" as used herein refers to a substance and / or process originating from outside a system, including, but not limited to, an organism, tissue, or cell. In particular, "exogenous" in the context of the present disclosure refers to a nucleic acid molecule or polypeptide that does not naturally occur within a cell. Thus, the term "exogenous" encompasses any foreign or heterologous recombinant nucleic acid molecule or polypeptide expressed within a cell, including exogenous nucleic acids that have a sequence that differs from the native endogenous counterpart. As is well known in the art, these exogenous sequences can be introduced into the cell itself or its precursor cells by genetic engineering, and can optionally be linked to alternative control sequences, such as non-native promoters or secretion sequences.

[0062] The term "overexpression" as used herein refers to expression at a level that exceeds the endogenous expression level of the subject nucleic acid or polypeptide in a cell or tissue. In an exemplary embodiment, a receptor of interest (e.g., NKG2D) can be overexpressed in a host cell, and the expression level of the receptor is higher than the naturally occurring expression level of the same receptor. The method for overexpressing a nucleic acid or polypeptide of interest is not particularly limited and is discussed in more detail herein, for example, a polypeptide (e.g., NKG2D) can be overexpressed by transferring a corresponding nucleic acid using the same or different expression vector as that encoding a CAD polypeptide. The expression vector is not particularly limited as long as it can be used for genetic engineering. For example, any of a plasmid vector, a virus vector, a cosmid vector, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and other non-plasmid vectors can be used.

[0063] The term "anti-tumor effect" as used herein refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested primarily by the ability of the polynucleotides and cells of the present invention in preventing the development of tumors.

[0064] As used herein, the term "autologous" refers to any material that originates from an individual, which is later to be reintroduced into the same individual.

[0065] As used herein, the term "allogeneic" refers to a material derived from an animal that is subsequently introduced into another animal of the same species.

[0066] The term "syngeneic" as used herein refers to material that is genetically similar or identical, and therefore immunologically compatible, such that transplantation will not provoke an immune response.

[0067] As used herein, the term "agent" refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell (e.g., a cell engineered to express a chimeric adaptor polypeptide). An agent may include a therapeutic agent, a diagnostic agent, or a pharmaceutical agent. A therapeutic agent or pharmaceutical agent, alone or together with additional agents, induces a desired response (e.g., inducing a therapeutic or prophylactic effect when administered to a subject, e.g., treating a subject suffering from cancer, viral infection (e.g., cytomegalovirus (CMV), influenza, hepatitis B, Epstein-Barr virus, adenovirus, etc.), bacterial infection (e.g., E. coli, Mycobacterium tuberculosis, etc.), rheumatoid arthritis (RA), or other disease / condition). Agents discussed herein may be referred to as modulators.

[0068] As used herein, the term "diagnosis" or "diagnosing" refers to the process of identifying a disease, such as cancer, through signs, symptoms, and / or the results of various tests. The conclusion reached through such a process is a diagnosis. Common forms of testing include blood tests, medical imaging, urine tests, biopsies, etc.

[0069] The term "therapeutically effective amount" or simply "effective amount" refers to an amount of an agent or composition (e.g., a composition containing an agent) that elicits the biological or medical response of a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of an agent or a composition containing an agent that is sufficient, when administered, to prevent or alleviate to some extent the manifestation of one or more signs or symptoms of the disorder or disease (e.g., hematological or solid tumor) being treated. The therapeutically effective amount will vary depending on the component, the disease and its severity, as well as the age, weight, etc., of the subject to be treated.

[0070] "Treating" a disease, as the term is used herein, means decreasing or reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0071] The term "reduce" as used herein means to reduce the quality, amount, or intensity of something. In one example, treatment (e.g., administration of a therapeutic agent of the present disclosure) reduces one or more signs or symptoms associated with a disease or condition, e.g., compared to the response in the absence of treatment. For example, administration of a therapeutic agent can result in an anti-tumor effect that reduces one or more signs or symptoms associated with cancer.

[0072] As used herein, the term "administration" means providing or giving to a subject, by any effective route, one or more agents, e.g., agents to treat one or more signs or symptoms associated with a condition / disorder or disease (including, but not limited to, cancer, viral infection, bacterial infection, etc.). Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0073] The term "pharmaceutical acceptable" as used herein refers to a substance (including, but not limited to, salts, carriers, or diluents) that does not inhibit the biological activity or properties of a compound and is relatively non-toxic. That is, the substance may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is included. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995) describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, e.g., one or more modulators. In general, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations may include injectable fluids that include pharmaceutical and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol) as vehicles. In addition to biologically neutral carriers, the pharmaceutical agent to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.

[0074] The term "cytokine" as used herein refers to a diverse group of soluble proteins and peptides released from cells that function as humoral regulators at nanomolar to picomolar concentrations and regulate the functional activities of individual cells and tissues under normal or pathological conditions. These proteins directly mediate interactions between cells and control processes occurring in the extracellular environment. Many growth factors and cytokines function as cell survival factors by preventing programmed cell death. Cytokines include both naturally occurring peptides and variants that retain full or partial biological activity.

[0075] "Encoding" refers to the inherent property of a particular nucleotide sequence of a polynucleotide (e.g., in a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to the gene results in the production of the protein within a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0076] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from the existing materials of its natural state. An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as a host cell.

[0077] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns.

[0078] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0079] The term "specifically binds" as used herein with respect to cell surface receptors refers to a receptor that recognizes a particular molecule / ligand but does not substantially recognize or bind other molecules in the sample. For example, a receptor that specifically binds to a certain type of molecule may also bind to one or more species of molecules. However, such cross-species reactivity in itself does not change the classification as specific. In another example, a receptor that specifically binds to a molecule may also bind to different allelic forms of the molecule. However, such cross-reactivity in itself does not change the classification as specific. In some cases, the terms "specific binding" or "specifically binds" can be used with respect to the interaction of a protein (or peptide) with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, a receptor recognizes and binds to a particular structure, rather than a general protein. If a receptor is specific for epitope "A", then in a reaction containing labeled "A" and the receptor, the presence of a molecule containing epitope A (or not, unlabeled A) will reduce the amount of labeled A that binds to the receptor.

[0080] In some embodiments, specific binding is at least about 1×10 -8 The binding can be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller K indicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0081] The term "cancer" as used herein refers to a physiological condition in mammals in which a cell population is characterized by unregulated cell proliferation. Neoplasia, malignancy, cancer, and tumor may be used interchangeably and refer to the abnormal proliferation of tissues or cells resulting in excessive cell division. The amount of tumor in an individual is the "tumor burden" which can be measured as tumor number, volume, or weight. A tumor that does not metastasize is called "benign". A tumor that can invade surrounding tissues and / or metastasize is called "malignant". A "non-cancerous tissue" is a tissue from the same organ in which a malignant tumor formed, but does not have the characteristic pathology of a tumor. Generally, a non-cancerous tissue appears histologically normal. A "normal tissue" is a tissue from an organ where the organ is not affected by cancer or another disease or disorder of that organ. A "cancer-free" subject has not been diagnosed with cancer in that organ and does not have detectable cancer.

[0082] Symptoms of cancer may include, but are not limited to, persistent cough or bloody saliva, change in bowel habits, bloody stool, unexplained anemia (low blood counts), breast lump or breast discharge, lump in a testicle, change in urination, blood in the urine, hoarseness, persistent lump or swollen gland, visible changes in a wart or mole, indigestion, difficulty swallowing, unusual vaginal bleeding or discharge, unexpected weight loss, night sweats or fever, persistent itching around the anus or genital area, sores that do not heal, headache, back pain, pelvic pain, and bloating, among others.

[0083] Hematological cancer is cancer that occurs in blood or bone marrow.Examples of hematological cancer (or hematopoietic cancer) include leukemia, for example acute leukemia (for example acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (for example chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, myelodysplasia.

[0084] A solid tumor is one that comprises a tumor mass derived from at least about 10 or at least about 100 tumor cells. A solid tumor can be a soft tissue tumor, a primary solid tumor, or a metastatic lesion.

[0085] Examples of solid tumors include sarcomas, adenocarcinomas, and carcinomas, such as those affecting various organ systems, such as liver, lung, breast, lymph, gastrointestinal (e.g., colon), genitourinary (e.g., kidney, urothelial cells), pancreas, prostate, and pharynx. Adenocarcinomas include malignant tumors, such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer. In one embodiment, the cancer is melanoma, such as advanced melanoma. In another embodiment, the cancer is glioma. Metastatic lesions of the above-mentioned cancers can also be treated or prevented using the methods and compositions of the present invention.

[0086] "Expression cassette" refers to a nucleic acid comprising an expression control sequence operably linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression, and other elements for expression can be supplied by a host cell or an in vitro expression system. An expression cassette can be a component of a vector, e.g., a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be within a host cell, e.g., an immune cell (e.g., γδ T cell). Ranges: Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, recitation of a range such as 1 to 6 is considered to have specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the broadness of the range.

[0087] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof refers to a nucleotide sequence identity (%) of nucleotide bases when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), for example, 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%, at least 99%, at least 99.5%, or 100% (as determined by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP, discussed below). A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0088] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share 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%, at least 99%, at least 99.5%, or 100% sequence identity when optimally aligned, such as with the GAP or BESTFIT programs using default gap weights. In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids with side chains that have similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.Alternatively, a conservative substitution is any change that has a PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256:1443-45, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0089] Sequence identity and / or similarity of polypeptides is usually measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared with FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the most overlapping regions between the query sequence and the search sequence (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm, which uses an affine gap search with a gap opening penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm for comparing the sequences disclosed herein with a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25: 3389-3402, each of which is incorporated herein by reference.

[0090] I. Introduction DNAX-activating protein of 10 kDa (DAP10) is an adaptor molecule associated with the cell surface cytotoxicity receptor natural killer group 2 member D (NKG2D). The NKG2D receptor is a type II transmembrane-anchored C-type lectin-like protein that belongs to the CD94 / NKG2 family of C-type lectin-like receptors (Houchins et al., (1991) J. Exp. Med. 173: 1017-1020). NKG2D is capable of binding a large number of highly diverse MHC class I-like self-molecules. These ligands are often poorly expressed on normal cells, but can be induced in damaged, transformed, or infected cells (Zingoni, A et al. (2018) Front. Immunol. 9 (476): 1-12). The ligands belong to the H60(ac), RAE(α-ε) and MULT1 families in mice and to the MIC (MICA and MICB) and ULBP (ULBP1-ULBP6) families in humans, with a repertoire that is more complex than in other species. In fact, MIC molecules are encoded by the most highly polymorphic human genes after classical HLA molecules (Eagle, RA and Trowsdale, J. (Nat. Rev. Immunol. (2007) 7(9):737-44).

[0091] NKG2D is an activating immune receptor that regulates both innate and adaptive immune responses. NKG2D is abundant in all NK cells, CD8 T cells, a subset of γδ T cells, and some autoreactive CD4 T cells. NKG2D acts together with other costimulatory molecules such as DAP10 to modify the strength and duration of antigen-specific responses via T cell receptors and affect the pattern of antitumor responses by T lymphocytes (see, for example, Maccalli C, et al. (2003) Eur. J. Immunol. 33 (7): 2033-43).

[0092] NKG2D lacks signaling motifs in the cytoplasmic domain. Thus, after ligand binding, NKG2D signaling and cell activation depend on the association of NKG2D with the DAP10 adaptor molecule, which promotes and stabilizes NKG2D surface membrane expression (Wu, J., et al., (1999) Science 285: 730-732). However, it is known, for example, that TGF-β can downregulate NKG2D (and NKG2DL) surface expression (Lazarova M and Steinle (2019) Front. Immunol 10 (2689): 1-11), and that TGF-β can significantly reduce DAP10 expression at both the mRNA and protein levels (Park, YP et al. (2011) Blood. 118: 3019-27; Lee, JC et al. (2011) Tumori 97: 350-7). It is therefore recognised herein that methodologies capable of modulating NKG2D and / or DAP10 expression and / or associated signalling pathways are of therapeutic interest.

[0093] In addition to NKG2D, DAP10 is known to associate with many other receptors. For example, Ly49H and Ly49D are co-immunoprecipitated with DAP10 from mouse NK cells and are shown to associate with DAP10 when co-transfected into 293T cells (Coudert JD et al. (2008) Blood 111:3571-3578). Co-transfection studies have also shown that DAP10 associates with human Sirp-b1 in transfected rat RBL-2H3 cells (Anfossi N et al. (2003) Eur. J. Immunol. 33:3514-3522). Similarly, human and mouse Siglec-15 (Angata T et al. (2007) Glycobiology 17:838-846) and Cd300lb (Yamanishi Y et al. (2008) Blood 111:688-698) have been shown to pair with DAP10 by co-transfection and co-immunoprecipitation. As mentioned above, certain DAP10-associated receptors (i.e., NKG2D) appear to recognize host-encoded molecules, e.g., carbohydrate and protein ligands, whereas other DAP10-associated receptors may directly recognize microbial ligands. As an example, the glycoprotein m157 encoded by mouse CMV is recognized by Ly49H (Lanier LL (2008) Nat. Rev. Immunol. 8(4):259-68; Smith HR et al. (2002) Proc. Natl. Acad. Sci. USA 99(13):8826-31). m157 is a GPI-anchored glycoprotein with homology to MHC class I that is presented on the surface of mouse CMV-infected cells, leading to Ly49H+ NK cell-mediated cytotoxicity and activation of cytokine production.

[0094] The above-mentioned receptors are intended to be illustrative, but not exhaustive, of the ability of DAP10 to pair with numerous receptors in addition to NKG2D, thereby regulating signal transduction. It should be understood that the nucleic acids, encoded polypeptides, cells, compositions, and methods of the present disclosure apply to any and all receptors with which DAP10 can partner.

[0095] II. General Methods Unless otherwise stated, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In particular, this disclosure utilizes routine techniques in the fields of recombinant genetics, immunology, and biochemistry. Basic texts that disclose the general terms of molecular biology and genetics include, for example, Lackie, Dictionary of Cell and Molecular Biology, Elsevier (5th ed.2013). Basic texts that disclose methods of recombinant genetics and molecular biology include, for example, Sambrook et al, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Press 4th Edition (Cold Spring Harbor, NY2012) and Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016). Basic texts disclosing general methods and terminology in biochemistry include, for example: Lehninger Principles of Biochemistry sixth edition, David L. Nelson and Michael M. Cox eds. WH Freeman (2012). Basic texts disclosing general methods and terminology in immunology include: Janeway's Immunobiology (Ninth Edition) by Kenneth M. Murphy and Casey Weaver (2017) Garland Science; Fundamental Immunology (Seventh Edition) by William E. Paul (2013) Lippincott, Williams and Wilkins.

[0096] DAP10 As mentioned above, the NKG2D receptor lacks signaling motifs, for example, in the cytoplasmic domain. Thus, the signaling function of the NKG2D receptor is closely linked to, among other things, its association with the adaptor molecule DAP10. The following reference to DAP10 is presented with respect to the NKG2D receptor for clarity and simplicity of presentation, but it should be understood that the teachings are broadly applicable to other receptors with which DAP10 can partner.

[0097] The human NKG2D receptor assembles with the DAP10 signaling dimer, with one NKG2D homodimer paired with a DAP10 dimer by the formation of two salt bridges between conserved transmembrane (TM) arginine residues (Garrity, D. et al. (2005) PNAS USA 102(21):7641-7646). The DAP10 dimer has a pair of aspartic acid residues near the center of the transmembrane (TM) domain, which interact with conserved arginines in the TM sequence of NKG2D to assemble with the DAP10 dimer. Thus, the NKG2D homodimer associates with the DAP10 adaptor molecule in its transmembrane domain to form a hexameric structure that can initiate a signaling cascade (see, e.g., Garrity et al (2005) supra).

[0098] As mentioned above, the DAP10 dimer is a disulfide-bonded homodimer. The amino acid sequence of the wild-type human DAP10 polypeptide is shown below as SEQ ID NO: 1: MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 1). See also Table 2, which lists a number of sequences relevant to this disclosure.

[0099] The DAP10 cytoplasmic domain contains a tyrosine-based motif (YINM) that includes residues 86-89 of SEQ ID NO:1. Upon tyrosine (Y86) phosphorylation, DAP10 can bind either the p85 subunit of phosphatidylinositol 3 kinase (PI3K, via YXXM) or the adaptor Grb2 (via YXNX). Because these two binding sites overlap, a single DAP10 chain binds either p85 or Grb2, but not both (Lanier LL. (2008) Nat. Immunol. 9(5):495-502). This YINM motif is similar to that of CD28, which couples with the immunoreceptor tyrosine-based activation motif (ITAM)-based TCR / CD3 complex in T cells to provide costimulatory signaling.

[0100] Recruitment of either the p85 subunit of PI3K or the adaptor protein Grb2 can both activate Vav1 and PLC-γ2, thereby upregulating Ca 2+ It is essential for mobilization and activation of cytotoxicity against cells (Upshaw JL et al. (2006) Nat. Immunol. 7(5):524-32).

[0101] DAP10 further contains a ubiquitination site encompassing the lysine at amino acid 84 of the DAP10 protein sequence (SEQ ID NO:1). Ligand stimulation of NKG2D on NK cells results in ubiquitination of DAP10, which is required for endocytosis and degradation of the NKG2D-DAP10 complex (see, e.g., Molfetta, R., et al. (2014) Eur. J. Immunol. 44, 2761-2770). Furthermore, ubiquitin-dependent receptor endocytosis has been shown to be required for activation of extracellular signal-regulated kinase (ERK) and NK cell function, such as secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. Thus, NKG2D-DAP10 endocytosis is a means to reduce cell surface receptor abundance and control signaling in cytotoxic lymphocytes.

[0102] Chimeric adapter constructs Aspects of the present disclosure include constructs comprising a nucleic acid encoding a chimeric adaptor polypeptide. In embodiments, the nucleic acid encodes a chimeric adaptor polypeptide comprising i) a human DAP10 amino acid sequence, and ii) one or more costimulatory domains (e.g., 4-1BB, OX40, ICOS, CD28), where the CAD polypeptide specifically lacks an extracellular domain. In embodiments, the CAD polypeptide may further comprise one or more intracellular signaling domains (e.g., CD3ζ) as described herein. In embodiments, the CAD polypeptide may also comprise one or more mutations, e.g., one or more mutations in DAP10 and / or one or more modifications (e.g., one or more mutations, additions, or deletions) in the costimulatory domain(s) and / or the intracellular signaling domain(s).

[0103] In embodiments, CAD polypeptides function to modulate and / or inhibit signaling through one or more receptors with which they are associated. In embodiments, CAD polypeptides are engineered with modulated / inhibited attributes (e.g., by one or more mutations) and / or added signaling attributes (e.g., by C-terminal fusions) and expressed in host cells to promote a favorable balance of signaling pathways upon receptor-target binding (e.g., NKG2D binding of an extracellular target ligand), which may help address issues of low or lost expression of the target of the primary TCR (i.e., antigen escape). The term "favorable balance" refers broadly to the introduction of a signaling cascade that acts to complement, substitute for, or modulate the primary DAP10 signaling cascade in a desired manner. Thus, the CAD polypeptides disclosed herein provide altered (e.g., improved) functional properties, including, but not limited to, altered (e.g., enhanced) cytolysis, proliferation, survival, and / or costimulatory properties induced by binding of DAP10 to a ligand of the partnering receptor (e.g., a widely expressed ligand of NKG2D). The exact composition of the CAD polypeptide can be designed for a given disease indication, in some examples, based on the specificity of other receptor(s) (e.g., TCR receptor(s)) present on the same cell and in combination with signaling components. As an example, it is within the scope of the present disclosure that immunosuppressive signals within the tumor microenvironment (TME) can inhibit anti-tumor T cell responses via inhibitory receptors on T cells, and that such inhibitory output can be switched to an immune stimulatory output through the use of CAD polypeptides. Other relevant examples can be found, for example, in: Guo, J., et al., (2021) Journal for ImmunoTherapy of Cancer 9:e002628.

[0104] In embodiments, CAD polypeptides may function, at least in part, to stabilize the cell surface receptor(s) with which they are associated (e.g., NKG2D). "Stabilizing" a cell surface receptor(s) disclosed herein means decreasing the rate at which the cell surface receptor is internalized or otherwise removed from the cell surface compared to the rate at which the cell surface receptor is otherwise removed under similar circumstances in the absence of a CAD polypeptide as described herein. Included within the scope of receptor stabilization as described herein is a positive feedback mechanism in which CAD polypeptide signaling leads to increased cell surface expression of endogenous DAP10 and the endogenous receptor(s) with which the CAD polypeptide is associated (e.g., NKG2D) (Wu, J., et al., (2000) Journal of Exp Med 192(7):1059-1068).

[0105] In embodiments, expression of a CAD polypeptide containing a heterologous signaling domain (e.g., IL2R, 4-1BB, CD27, etc.) that affects cell survival and proliferation, when combined with an endogenous receptor (e.g., endogenous TCR) on the same host cell (e.g., T cell or NK cell) that engages a target that has low levels of expression in normal tissue, allows for a logical gating strategy (e.g., "AND" gating) that may increase the stringency and / or potency of targeted attack. Thus, a host cell that has both a receptor that recognizes a primary target (e.g., a primary cancer target) and an appropriate CAD polypeptide may promote sustained survival, proliferation, and killing of target cells that express both the primary target and a ligand for a receptor associated with DAP10 (e.g., NKG2D). Examples of logic gating strategies applicable to the present disclosure can be found, for example, in: WO2019118518, WO2020154635, WO2020223445, WO2021035093, WO2019222642A1, WO2018236825A1, WO2019164979, and Chang, ZL, and Chen YY (2017) Trends Mol Med 23(5):430-450.

[0106] The present invention also provides a stably expressed CAD polypeptide. In an embodiment, the CAD polypeptide is expressed at a level substantially similar to that at which endogenous DAP10 is expressed in a host cell of interest. In an embodiment, the CAD polypeptide is expressed at a level higher than that at which endogenous DAP10 is expressed in a host cell of interest. For example, the CAD polypeptide may be expressed at a level 10% higher, or 10-20% higher, or 20-30% higher, or 30-40% higher, or 40-50% higher, or 50-60% higher, or 60-70% higher, or 70-80% higher, or 80-90% higher, or 90-100% higher, or even higher, e.g., 2-fold higher, or 3-fold higher, or 4-fold higher, or 5-fold higher, or 6-fold higher, or 7-fold higher, or 8-fold higher, or 9-fold higher, or 10-fold higher, or 20-fold higher, or 30-fold higher, or 40-fold higher, or 50-fold higher, or 100-fold higher than the corresponding level at which endogenous DAP10 is expressed. Thus, in embodiments, it may be understood that the CAD polypeptide of the present invention may compete with endogenous DAP10 for binding to a receptor (e.g., NKG2D) depending on the particular host cell type.

[0107] Aspects of the disclosure include nucleic acids encoding CAD polypeptides, and constructs / vectors containing such nucleic acids. Thus, described herein are nucleic acids encoding CAD polypeptides that incorporate selected mutations and signaling domains that modulate and / or add signaling properties that confer desired properties (e.g., including but not limited to, sustained survival, proliferation, and / or killing in host cells expressing the CAD polypeptide).

[0108] A. Mutation In an embodiment, the CAD polypeptide has one or both of the amino acid modifications at position K84 and / or position Y86 of SEQ ID NO:1. In an embodiment, K84 is modified to include another positively charged amino acid, e.g., K84R or K84H. However, it is within the scope of this disclosure that the modification at K84 may include other amino acid substitutions. In one embodiment, the modification is a K84R modification. In an embodiment, the CAD polypeptide having a K84R modification comprises SEQ ID NO:18. In an embodiment, Y86 is modified to another aromatic amino acid, e.g., Y86F or Y86W. However, it is within the scope of this disclosure that the modification at Y86 may include other amino acid substitutions. In one embodiment, the modification is a Y86F modification. In an embodiment, the CAD polypeptide having a Y86F modification comprises SEQ ID NO:19. In an embodiment, the CAD polypeptide having both a K84R modification and a Y86F modification comprises SEQ ID NO:20.

[0109] In an embodiment, the modification at position 86 of SEQ ID NO:1 reduces or eliminates binding of p85 / PI3K to the CAD polypeptide, which in turn reduces or eliminates PI3K / AKT / PKCθ signaling. Thus, the modification at position 86 may serve to functionally reduce or eliminate one or more of costimulation, calcium influx, and / or degranulation. In a preferred embodiment, the modification is Y86F.

[0110] In additional or alternative embodiments, a modification at position 86 of SEQ ID NO:1 reduces or eliminates binding of Grb2 to a CAD polypeptide, which in turn reduces or eliminates Vav1 / SLP-76 / PLCγ signaling. Thus, a modification at position 86 may serve to reduce or eliminate one or more of calcium influx and / or degranulation. In a preferred embodiment, the modification is Y86F.

[0111] In additional or alternative embodiments, the modification at position 84 of SEQ ID NO:1 reduces or completely inhibits the ubiquitination of the CAD polypeptide, which in turn reduces or completely prevents the internalization of the chimeric DAP10 adaptor polypeptide-endogenous receptor complex in the cell membrane of certain host cells (see, e.g., Quatrini, L., et al., (2015) Sci Signal 8 (400): ra108). In this way, by relying on the CAD polypeptide having at least K84 modification (e.g., K84R), the endogenous receptor associated with DAP10 (e.g., NKG2D) can be stabilized at the cell surface. Furthermore, the modification of K84 (e.g., K84R) can reduce or eliminate signaling (e.g., ERK1 / 2) that occurs on the way to lysosomal degradation. Reducing or eliminating signal transduction (e.g., ERK1 / 2) can serve to reduce or eliminate one or more of exhaustion, activation-induced cell death, and / or induction of cell cycle arrest upon overactivation, one or more of which might otherwise occur in the absence of a CAD polypeptide having a K84 modification (e.g., K84R).

[0112] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 57 of SEQ ID NO:1, e.g., a D57A modification, although amino acid modifications other than alanine are within the scope of the present disclosure. In embodiments, a chimeric DAP10 adaptor polypeptide having a D57A modification comprises SEQ ID NO:37. A modification at D57 may, in embodiments, serve to modify (e.g., reduce or abolish) stable interactions with KLRK1 (Wu, J., et al., (1999) Science 285(5428):730-2).

[0113] In additional or alternative embodiments, a CAD polypeptide may include a modification at position 88 of SEQ ID NO:1, e.g., an N88Q modification, although amino acid modifications other than glutamine are within the scope of the present disclosure. In embodiments, a CAD polypeptide having an N88Q modification comprises SEQ ID NO:38. The N88 modification, in embodiments, has minimal to no effect on interaction with PIK3R1 and may serve to modify (e.g., reduce) cell killing activity and / or interaction with GRB2 (Upshaw, JL., (2006) Nat Immunol 7:524-532).

[0114] In additional or alternative embodiments, a CAD polypeptide may include a modification at position 89 of SEQ ID NO:1, e.g., an M89Q modification, although amino acid modifications other than glutamine are within the scope of this disclosure. In embodiments, a CAD polypeptide having an M89Q modification includes SEQ ID NO:39. The M89Q modification, in embodiments, has minimal to no effect on interaction with GRB2 and may serve to modify (e.g., reduce) cell killing activity and / or interaction with PIK3R1 (Upshaw, JL., (2006) Nat Immunol 7:524-532).

[0115] The present disclosure can be understood to encompass chimeric adapter polypeptides having one or more, or each, of the above modifications.

[0116] B. Costimulatory and Signaling Domains In embodiments, the endodomain of a chimeric adapter polypeptide of the present disclosure comprises one or more costimulatory domains, the costimulatory domains comprising functional costimulatory signaling domains derived from, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, and the like. For example, it is within the scope of the present disclosure that the endodomain of a disclosed CAD polypeptide may comprise two, three, four or more costimulatory domains. It is also within the scope of the present disclosure that when multiple costimulatory domains are included, the costimulatory domains may be the same or different.In embodiments, the costimulatory domain is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19aCD27, CD28, CD29, C D30, CD40, CDS, CD49a, CD49D, CD49f, CD54(ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD 134(OX40), CD137(4-1BB), CD152(CTLA-4), CD160(BY55), CD162(SELPLG), CD244(2B4), CD270(HVEM), CD226(DNAM1), CD229(Ly9 ), CD278(ICOS), ICAM-1, LFA-1(CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, ​​L AT, GADS, LIGHT, HVEM(LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA 4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, and TRANCE / RANKL, or a combination thereof.

[0117] In some embodiments, the CAD construct encodes at least one 4-1BB costimulatory domain, and optionally encodes a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the costimulatory domains described above. In some embodiments, the construct encodes at least two 4-1BB costimulatory domains, or at least two 4-1BB costimulatory domains in combination with one, two, three, or four or more costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the costimulatory domains described above. In some embodiments, the 4-1BB costimulatory domain comprises SEQ ID NO:2 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL). In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least one, at least two, or at least three or more modifications of the amino acid sequence of SEQ ID NO:2. In embodiments, the 4-1BB costimulatory domain is substantially similar to a 4-1BB costimulatory domain comprising SEQ ID NO:2.

[0118] In some embodiments, the CAD construct encodes at least one CD27 costimulatory domain and optionally at least one second costimulatory domain selected from 4-1BB, ICOS, CD28, OX40, 2B4, and CD27 costimulatory domains, or any of the costimulatory domains described above. In some embodiments, the construct encodes at least one CD27 costimulatory domain and a 4-IBB costimulatory domain. In some embodiments, the construct encodes two CD27 costimulatory domains and at least one second costimulatory domain selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the CD27 costimulatory domain comprises SEQ ID NO:5 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP). In some embodiments, the CD27 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO:5. In embodiments, the CD27 costimulatory domain is substantially similar to a CD27 costimulatory domain comprising SEQ ID NO:5.

[0119] In some embodiments, the CAD construct encodes at least one CD28 costimulatory domain and optionally a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the costimulatory domains described above. In some embodiments, the CAD construct encodes at least two CD28 costimulatory domains, or at least two CD28 costimulatory domains in combination with one, two, three, or more costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the costimulatory domains described above. In some embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 40 (FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). In embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 41 (FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). SEQ ID NO:40 and SEQ ID NO:41 comprise three subdomains, YMNM, PRRP, and PYAP, that can regulate signal transduction pathways. In embodiments, the disclosed CAD polypeptides comprise one or more mutations or deletions of the subdomains (see, e.g., WO2019010383). In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41. In some embodiments, the CD28 costimulatory domain is substantially similar to a CD28 costimulatory domain comprising SEQ ID NO:40. In some embodiments, the CD28 costimulatory domain is substantially similar to a CD28 costimulatory domain comprising SEQ ID NO:41.

[0120] In some embodiments, the CAD construct encodes at least one ICOS costimulatory domain, and optionally encodes a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the costimulatory domains described above. In some embodiments, the CAD construct encodes at least two ICOS costimulatory domains, or at least two ICOS costimulatory domains in combination with one, two, three, or more costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the costimulatory domains described above. In some embodiments, the ICOS costimulatory domain comprises SEQ ID NO: 42. In some embodiments, the ICOS costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO: 42 (see, e.g., US20170209492). In some embodiments, the ICOS costimulatory domain is substantially similar to an ICOS costimulatory domain comprising SEQ ID NO: 42.

[0121] In some embodiments, the CAD construct encodes at least one OX40 costimulatory domain, and optionally encodes a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the costimulatory domains described above. In some embodiments, the CAD construct encodes at least two OX40 costimulatory domains, or at least two OX40 costimulatory domains in combination with one, two, three, or four or more costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the costimulatory domains described above. In some embodiments, the OX40 costimulatory domain comprises SEQ ID NO: 43 (RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI). In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO: 43. In some embodiments, the OX40 costimulatory domain is substantially similar to an OX40 costimulatory domain comprising SEQ ID NO:43.

[0122] In embodiments, one or more intracellular signaling domains are included in the chimeric adapter polypeptide of the invention. In embodiments, one or more intracellular signaling domains are in addition to one or more costimulatory domains. In embodiments, one or more intracellular signaling domains are included to increase proliferation, persistence, and / or cytotoxic activity of host cells (e.g., NK cells, NKT cells, γδ cells, etc.) having a CAD polypeptide disclosed herein. For example, in some embodiments, the intracellular signaling domain(s) include CD3ζ, repeated (e.g., 2-5) DAP10Y INM motifs, signaling domains (from LFA-1, DAP12, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, etc.). It is within the scope of the present disclosure that the endodomains of the disclosed chimeric adapter polypeptides can include multiple (e.g., 2, 3, 4, or more) intracellular signaling domains. When multiple intracellular signaling domains are included, the intracellular signaling domains may be the same or different.

[0123] In some embodiments, the intracellular signaling domain is or comprises a CD3 zeta signaling domain. In some embodiments, the CD3 zeta signaling domain is RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 3), or RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4), or RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 76) (also referred to herein as "1XX"). (See, e.g., US2020 / 0317777, the contents of which are incorporated by reference herein in their entirety.) Without being bound by theory, in some embodiments, the inclusion of a 1XX signaling domain may improve activation and / or survival of the engineered immune cells of the present disclosure by limiting overactivation.

[0124] In some embodiments, the CAD construct encodes one or more costimulatory domain(s) (e.g., a 4-1BB costimulatory domain) and one or more intracellular signaling domains (e.g., a CD3ζ signaling domain). In some embodiments, the CAD construct encodes at least one 4-1BB costimulatory domain, at least one CD28 domain, and at least one CD3ζ signaling domain. In other embodiments, the CAD construct encodes one or more first costimulatory domains (e.g., 4-1BB, CD28, OX40, ICOS) and one or more second costimulatory domains (e.g., 4-1BB, CD28, OX40, ICOS), and one or more intracellular signaling domains (e.g., CD3ζ). In embodiments, the CD3ζ signaling domain is downstream (C-terminal) of the costimulatory domain(s) (e.g., 4-1BB). In some embodiments, the CD3ζ signaling domain is upstream (N-terminal) of the costimulatory domain(s) (e.g., 4-1BB).

[0125] C. Cytokine Coexpression In additional embodiments, the CAD construct of the invention may also encode one or more multicistronic linker region(s) configured to facilitate translation of the CAD polypeptide and one or more soluble common gamma chain cytokines as separate polypeptides. In embodiments, the nucleic acid encoding the cytokine and associated linker region may be located at the 3' end of the isolated nucleic acid, or at the 5' end of the isolated nucleic acid, or, in some examples, at both the 5' and 3' ends of the isolated nucleic acid. The one or more soluble common gamma chain cytokines include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In embodiments, the linker region(s) may encode a self-cleaving and / or cleavage polypeptide sequence. In some examples, the self-cleavage sequence is a 2A self-cleavage sequence (e.g., T2A, P2A, E2A, F2A) that may induce ribosomal skipping during translation of the chimeric DAP10 adaptor polypeptide. In embodiments, the cleavage sequence is a Furin sequence. In some instances, the cleavage sequence (e.g., a furin cleavage sequence) is amino-terminal to the self-cleavage sequence. In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. In some embodiments, the multicistronic linker region comprises any one of SEQ ID NOs: 9-15, or the sequence of SEQ ID NO: 44. In embodiments, the addition of an optional linker, such as "GSG" or "SGSG", may improve cleavage efficiency. In this manner, one or more gamma chain cytokines contained therein may be released from the chimeric DAP10 adaptor polypeptide and secreted by the host cell.

[0126] For example, in some embodiments, the cleavage sequence is a P2A cleavage sequence of SGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 9). In some embodiments, the P2A cleavage sequence is a P2A cleavage sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 44). In some embodiments, the cleavage sequence is a Furin cleavage sequence of RAKR (SEQ ID NO: 10). In some embodiments, the cleavage sequence is a P2A + Furin cleavage (FP2A) sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 11).

[0127] In some embodiments, the cleavage sequence is or comprises a P2A cleavage sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 12). In some embodiments, the cleavage sequence is or comprises a F2A cleavage sequence of VKQTLNNFDLLKLAGDVESNPGP (SEQ ID NO: 13). In some embodiments, the cleavage sequence is or comprises an E2A cleavage sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14). In some embodiments, the cleavage sequence is or comprises a T2A cleavage sequence of EGRSLTCGDVEENPGP (SEQ ID NO: 15). In certain aspects, multiple self-cleavage sequences can be encoded at the carboxy terminus of the signaling and / or costimulatory domain and the amino terminus of the encoded secreted cytokine (e.g., a common gamma chain cytokine such as IL-15), preferably the multiple self-cleavage sequences are independently selected from the group consisting of a P2A cleavage sequence, a T2A cleavage sequence, an E2A cleavage sequence, and an F2A cleavage sequence. In certain aspects, one or more autocleaving sequences and one or more sequences that are cleaved by an endogenous protease are encoded in the constructs described herein, in certain embodiments, an endogenous protease recognition site is encoded amino-terminal to the autocleaving sequence.

[0128] In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by the following: (SEQ ID NO: 16).

[0129] Another exemplary internal ribosome entry site is encoded by: AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC (SEQ ID NO: 17).

[0130] Further suitable internal ribosome entry sites include, but are not limited to, those disclosed in Nucleic Acids Res. 2010 Jan;38(Database issue):D131-6.doi:10.1093 / nar / gkp981.Epub 2009 Nov 16; those described in iresite.org; those described in WO2018 / 215787; sequences described in GenBank accession No. KP019382.1; and IRES elements described in GenBank accession No. LT727339.1. Additional multicistronic linker regions containing cleavage self-cleaving and IRES elements are disclosed in US2018 / 0360992 and US8,865,467.

[0131] In some embodiments, the construct encodes an operably linked secretion signal (e.g., MALPVTALLLPLALLLHAARP (SEQ ID NO: 6)) to facilitate secretion of the C-terminal polypeptide, e.g., a cytokine. In some embodiments, the secretion signal is a secretion signal of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 7). In some embodiments, the construct encodes an operably linked secretion signal to facilitate secretion of a common gamma chain cytokine, e.g., IL-15, or an active fragment thereof, e.g., NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCELLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 8); other IL-15 sequences, including codon-optimized nucleic acid sequences encoding sIL15, are disclosed in WO2007 / 037780. Examples of common gamma chain cytokines include IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In some embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In some embodiments, the common gamma chain cytokine is IL-15. IL-15 sequences, including codon-optimized nucleic acid sequences encoding sIL15, are disclosed herein and in WO2007 / 037780.

[0132] Thus, a CAD construct of the present disclosure encodes a CAD polypeptide that includes at least one costimulatory domain and, optionally, one or more intracellular signaling domains. In embodiments, the CAD construct may encode one or more common gamma chain cytokines that are released from the CAD polypeptide (e.g., during translation). As discussed above in some embodiments, the CAD construct of the present disclosure may further include a mutated DAP10, e.g., DAP10 mutated at K84 and / or Y86, among others, of SEQ ID NO:1.

[0133] In embodiments, one or more costimulatory domains may be located 5' of one or more signaling domains. In embodiments, one or more costimulatory domains may be located 3' of one or more signaling domains. In some embodiments, one or more costimulatory domains may be located 5' of one or more signaling domains, and one or more costimulatory domains may be located 3' of one or more signaling domains. In some embodiments, one or more signaling domains may be located 5' of one or more costimulatory domains, and one or more signaling domains may be located 3' of one or more costimulatory domains. In some embodiments, the C-terminal fusion may comprise alternating one or more costimulatory domains and one or more signaling domains.

[0134] For reference, Figures 1A-1D show exemplary diagrams of various chimeric adapter polypeptides and their association with receptors (e.g., depicted as NKG2D). These representations are for illustrative purposes only and are not representative of all of the various permutations of chimeric adapter polypeptides disclosed herein. Figure 1A exemplarily shows a chimeric adapter polypeptide that includes both K84R and Y86F modifications. Figure 1B exemplarily shows a chimeric adapter polypeptide that includes both K84R and Y86F modifications in addition to a C-terminal fusion with CD3ζ. Figure 1C exemplarily shows a chimeric adapter polypeptide that includes both K84R and Y86F modifications in addition to a C-terminal fusion with 4-1BB. Figure 1D shows a chimeric adapter polypeptide that includes both K84R and Y86F modifications in addition to a C-terminal fusion with both 4-1BB and CD3ζ.

[0135] D. Marker co-expression In additional embodiments, the CAD construct of the present invention may encode one or more labels or markers, for example, to facilitate the ability to monitor CAD expression levels, and serve as internal controls. In some embodiments, the CAD construct may encode a fluorescent protein, examples of which include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), enhanced GFP (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), and the like. Other examples may include, but are not limited to, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, β-lactamase, luciferase, and the like.

[0136] In other embodiments, the CAD construct may encode a protein to be expressed on the cell surface to facilitate detection and / or isolation of cells expressing the protein, e.g., by fluorescence-activated cell sorting (FACS); or enrichment by positive selection using an antibody specific for the encoded protein (e.g., using the antibody to purify or enrich cell products on a column or device); or to allow in vivo binding of an antibody to the protein to enhance or remove activity, e.g., to facilitate removal of cells expressing the protein in a patient as a safety consideration. Exemplary proteins useful for these purposes include, e.g., CD19, CD20 (rituximab recognition domain), LNGFR (amino acid sequence set forth in SEQ ID NO: 100, encoded by SEQ ID NO: 101), truncated form of human epidermal growth factor receptor (EGFRt) (amino acid sequence set forth in SEQ ID NO: 74, encoded by SEQ ID NO: 75), and the like. As an example, in embodiments, a marker protein may be targeted with a clinical stage antibody, such that treatment of a patient with the antibody results in the elimination of cells containing isolated nucleic acid encoding a CAD polypeptide disclosed herein (Philip B, et al., (2014) Blood, 124(8): 1277-1287; Wang X, et al., (2011) Blood, 118(5): 1255-1263; Smith J, et al., (2015) Meeting Abstract, ASCO Annual Meeting I; 3069; Gouble A, et al., (2014) Blood, 124(21): 4689).

[0137] In embodiments, linker regions, examples of which are described herein, can be used to facilitate translation of a CAD polypeptide and a desired marker. For example, a Furin and P2A linker gene that facilitates CAD expression and a desired marker can be included in an isolated nucleic acid construct of the present disclosure. As discussed below with respect to Example 1, a Furin and P2A linker gene can be used to express a desired CAD polypeptide with a truncated CD19 that serves as a marker. Also exemplified herein are CAD polypeptides that include other markers (e.g., EGFRt). Such examples are intended to be illustrative and non-limiting.

[0138] Chimeric adapter expression As used herein, an isolated nucleic acid is intended to mean a DNA molecule that can be transformed or introduced into a host cell (e.g., a T cell, a NK cell, a NKT cell, etc.) and transcribed and translated to produce a product (e.g., a chimeric adapter polypeptide described herein). In the isolated nucleic acids of the invention, a promoter is operably linked to a nucleic acid sequence encoding a chimeric adapter polypeptide of the invention, i.e., positioned to promote transcription of messenger RNA from the DNA encoding the chimeric adapter polypeptide. The term "operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting them to function in their intended manner.

[0139] The promoter may be genomic or synthetically produced. Various promoters for use in host cells related to the present disclosure are well known in the art (e.g., the CD4 promoter disclosed below; Marodon, et al. (2003) Blood 101(9):3416-23). ​​The promoter may be constitutive or inducible, with induction being associated with a particular cell type or a particular level of maturation. Alternatively, a number of well-known viral promoters may also be suitable. Promoters of interest include the β-actin promoter, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen focus forming virus promoters. The promoter may or may not be associated with an enhancer, and the enhancer may be naturally associated with a particular promoter or associated with a different promoter. In embodiments, expression of the chimeric adapter polypeptide is under the control of an inducible promoter, for example, a promoter inducible by a molecule present in the tumor microenvironment (e.g., TGFβ).

[0140] The sequences of the open reading frames encoding the various segments of the chimeric adapter polypeptides of the present disclosure can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or combinations thereof.

[0141] In embodiments, for expression of the chimeric adapter polypeptides of the present invention, the naturally occurring or endogenous transcription initiation region of the nucleic acid sequence encoding the N-terminal component of DAP10 can be used to generate the chimeric adapter polypeptide in the host cell. Alternatively, an exogenous transcription initiation region that allows for constitutive or inducible expression can be used, and expression can optionally be controlled depending on the host cell, the desired expression level, the nature of the host cell, etc.

[0142] A termination region may be included that encodes a C-terminal component of the chimeric adapter polypeptide. Generally speaking, the source of the termination region is not believed to be critical to expression of the recombinant protein, and a variety of termination regions can be used without adversely affecting expression.

[0143] The isolated nucleic acid encoding the chimeric adapter polypeptide according to the invention can be prepared by conventional methods. The sequences (natural or synthetic) are isolated and manipulated, if necessary, to allow the appropriate joining of the various components. Thus, the various nucleic acid sequences encoding the various segments of the chimeric adapter polypeptide can be isolated, for example, by utilizing the polymerase chain reaction (PCR), using appropriate primers. If necessary, specific primers can be designed that result in the deletion of unnecessary parts of the nucleic acid sequence used as a template. Additionally or alternatively, restriction digests of the cloned genes can be used to generate the isolated nucleic acid constructs of the present disclosure. In either case, sequences can be selected to provide blunt-ended restriction sites or to have complementary overlaps to facilitate integration into various vectors. In examples, modifications of the nucleic acid sequence (e.g., to introduce one or more point mutations, insertions, or deletions) are performed. The modifications can include, for example, amino acid changes at positions Y86 and / or K84 of SEQ ID NO:1. Methods for introducing modifications into nucleic acid sequences are known in the art and can include the use of various commercially available kits (e.g., QuickChange Site Directed Mutagenesis Kit, Agilent, Santa Clara, Calif.).

[0144] Various manipulations for preparing isolated nucleic acids encoding the chimeric adapter polypeptides of the present disclosure can be performed in vitro. In certain embodiments, sequences encoding the chimeric adapter polypeptides are introduced into vectors for cloning and expression in suitable host cells using standard transformation or transfection methods. Thus, after each manipulation, the construct resulting from the ligation of the DNA sequences is cloned, the vector is isolated, and the sequences are screened to confirm that they encode the desired chimeric adapter polypeptide. Sequences can be screened by restriction analysis, sequencing, and the like.

[0145] It is contemplated that the isolated nucleic acid can be introduced into the host cell as naked DNA or in a suitable vector. Many suitable vectors are known to those skilled in the art of molecular biology, the choice of which depends on the desired function, and include plasmids, cosmids, viruses, bacteriophages, and other vectors conventionally used in genetic engineering. Methods well known to those skilled in the art can be used to construct various plasmids and vectors, see, for example, the procedures described in: Sambrook et al. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989), (1994). Alternatively, the polynucleotides and vectors of the present disclosure can be reconstituted into liposomes for delivery to target cells.

[0146] Methods for stably transfecting host cells by electroporation using naked DNA are known in the art (see, for example, U.S. Patent No. 6,410,319, which discloses transfection of T cells). Naked DNA generally refers to DNA encoding the chimeric adapter of the present invention contained within a plasmid expression vector in the proper orientation for expression. Advantageously, the use of naked DNA reduces the time required to generate host cells expressing the chimeric DAP10 adapter polypeptide of the present invention.

[0147] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the isolated nucleic acid encoding the chimeric adapter polypeptide of the present invention into a host cell. A vector suitable for use according to the methods of the present invention is one that does not replicate in T cells. A number of virus-based vectors are known, in which the copy number of the virus maintained in the cell is low enough to maintain cell viability. Exemplary vectors include the pFB-neo vector (STRATAGENE®) and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0148] Thus, in some embodiments, the isolated nucleic acid may be understood to be a circular nucleic acid. In some embodiments, the isolated nucleic acid is a vector, such as a plasmid vector, an adenovirus vector, an adeno-associated virus vector, a virus vector, a retrovirus vector (e.g., a gamma retrovirus vector), or a lentivirus vector. In some embodiments, the isolated nucleic acid, or a contiguous portion thereof, for example, containing a DAP10 sequence (e.g., at least a portion of SEQ ID NO:1, modified or unmodified) and one or more signaling domains and / or costimulatory domains, is integrated into the genome of a host γδ T host cell. In an exemplary embodiment, the isolated nucleic acid is a retrovirus vector.

[0149] host cell The chimeric adapter polypeptide of the present disclosure can be expressed in a wide variety of host cells via the corresponding chimeric nucleic acid construct. In embodiments, the host cell is a mammalian cell. In embodiments, the CAD polypeptide is expressed in a host cell type that exhibits endogenous expression of a receptor associated with DAP10. For example, the CAD polypeptide can be expressed in a host cell that expressed NKG2D. In embodiments, the CAD polypeptide is expressed in a host cell type that exhibits some endogenous expression of a receptor associated with DAP10 (e.g., NKG2D). In embodiments, the host cell type can also be engineered to express the same receptor (e.g., NKG2D), e.g., to increase the expression level of the receptor above the naturally occurring endogenous expression level. In embodiments, the CAD polypeptide is expressed in a cell type that does not exhibit endogenous expression of a receptor associated with DAP10, in which case the host cell is engineered to express such a receptor (e.g., expression of NKG2D in a cell type that does not otherwise express NKG2D).

[0150] In embodiments, expression of a CAD polypeptide of the present disclosure in a host cell results in the CAD polypeptide competing with endogenous cellular DAP10 (e.g., WTDAP10). Competition can redirect intracellular signaling through a receptor associated with DAP10 via a chimeric DAP10 adaptor polypeptide. To be effective in a desired outcome (e.g., killing of tumor cells), signaling need not be 100% redirected through a CAD polypeptide, although such percentages are within the scope of the present disclosure. Signaling through a CAD polypeptide may include a range of 20%-100% of signaling through DAP10, such as 90-100%, 80-100%, 70-100%, 60-100%, 50-100%, etc. As a representative example for illustrative purposes, a host cell in which 80% of signaling through a receptor associated with DAP10 is rerouted through a CAD polypeptide means that only 20% of such signaling is still through endogenous DAP10, but 80% of signaling is through the CAD polypeptide.

[0151] The host cells described herein can be used in adoptive cell transfer, for example, cryopreserved and preserved. In an embodiment, the host cells are preserved before engineering the cells to express a chimeric DAP10 adaptor polypeptide. In an embodiment, the cells are engineered to express a chimeric DAP10 adaptor polypeptide, and then the cells are preserved.

[0152] Preferred host cells for use with the chimeric DAP10 adaptor polypeptides of the present disclosure include immune cells. Such cells can be obtained from the subject to be treated (i.e., autologous) or immune cell lines or donor immune cells (allogeneic, syngeneic) can be used. Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells can be obtained from blood drawn from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be obtained by apheresis. In some embodiments, immune cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugation. Specific subpopulations of immune cells can be further separated by positive or negative selection techniques. For example, immune cells can be isolated using a combination of antibodies against surface markers specific to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time sufficient to positively select the desired immune cells. Alternatively, enrichment of immune cell populations can be achieved by negative selection using a combination of antibodies that target surface markers specific to the negatively selected cells. Other specific methods of separation and / or enrichment are disclosed herein.

[0153] In some embodiments, immune cells include any white blood cell involved in the body's defense against infections and foreign substances. For example, immune cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune cells related to the present disclosure may include, but are not limited to, αβ T cells, γδ T cells, NK cells, NKT cells, γδ NKT cells, B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, regulatory T cells, and the like. In embodiments, preferred immune cells include αβ T cells, γδ T cells, NK cells, NKT cells, γδ NKT cells, and in some examples, macrophages. In embodiments, immune cells related to the present disclosure include allogeneic cells, autologous cells, or syngeneic cells.

[0154] Aspects of the disclosure include immune cells that have in vitro or in vivo cytotoxic activity against blood or solid tumor cells that exhibit cell surface expression of tumor-associated antigens (TAA), virally infected cells that present virus-derived antigens, bacterial cells, etc. In embodiments, the cytotoxic activity is innate activity. In embodiments, immune cells that functionally express a CAD polypeptide exhibit a higher level of in vitro and / or in vivo cell killing activity than the level of cell killing activity in control immune cells that do not contain a CAD polypeptide of the disclosure.

[0155] In embodiments, cytotoxicity is significantly (greater than about 25%) enhanced or improved by the presence of the CAD polypeptide compared to cytotoxicity in the absence of the CAD polypeptide. In some cases, the cytotoxicity is at least partially, significantly (greater than about 25%), or entirely due to the presence of the CAD polypeptide.

[0156] In embodiments, engineered immune cells associated with the present disclosure may exhibit potent and / or sustained cell killing activity (e.g., tumor cells, virus-infected cells) through direct and / or indirect mechanisms. In some cases, the cell killing activity persists for at least about 6-120 days, or at least about 6-180 days, from initial contact with the target cell. In some cases, the cell killing activity of an immune cell disclosed herein engineered to express a CAD polypeptide or its progeny persists for at least about 6-120 days, or at least about 6-180 days, from initial contact with the target cell or from administration of an engineered immune cell disclosed herein. This sustained cell killing activity may be exerted in vitro, in vivo, or both in vitro and in vivo.

[0157] In embodiments, aspects of the disclosure include immune cells that proliferate in response to contact with cells that exhibit cell surface expression of a ligand recognized by a receptor associated with DAP10, specifically associated with a CAD polypeptide of the disclosure. One example of such a receptor is NKG2D, but the disclosure is not limited to CAD polypeptides that interact with NKG2D and may include other receptor(s) that are also associated with DAP10. In embodiments, the proliferation is at least partially, substantially (more than about 20%, or more than about 25%, or more than about 50%, or more than about 80%), or completely (e.g., 100%) due to the presence of a CAD polypeptide construct associated with a receptor (e.g., NKG2D) expressed on the host cell. In some cases, the immune cells exhibit higher levels of proliferation in vitro and / or in vivo compared to control immune cells (e.g., immune cells of the same type) that do not contain a CAD polypeptide. A higher level of proliferation may include a 20-50% increase, a 50-80% increase, an 80-100% increase, or a 2-fold increase, a 3-fold increase, a 4-fold increase, a 5-fold increase, a 5-10-fold increase, a 10-20-fold increase, or a greater than 20-fold increase, e.g., a 50-100-fold increase or more, compared to control immune cells lacking a CAD polypeptide described herein.

[0158] In some embodiments, immune cells engineered to contain a CAD polypeptide as described herein express and secrete, or persistently express and secrete, one or more proinflammatory cytokines, e.g., after contact with a cell expressing a ligand (e.g., NKG2D) that is recognized by a cell surface receptor on immune cells associated with DAP10. In embodiments, the expression and secretion associated with immune cells engineered to express a CAD polypeptide is at least partially, significantly (greater than about 20%, or greater than about 25%, or greater than about 50%, or greater than about 80%), or entirely due to the CAD polypeptide. In embodiments, expression and / or secretion is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 5-10-fold, 10-20-fold, or more than 20-fold, e.g., 50-100-fold or more, greater than expression and / or secretion otherwise observed in a control immune cell (e.g., an immune cell of the same type) that lacks expression of the CAD polypeptide.

[0159] In embodiments, the modified immune cells of the present disclosure may function to modify the cellular microenvironment (e.g., TME) to favor, for example, an anti-tumor response. For example, solid tumors may recruit suppressor cells, such as myeloid-derived suppressor cells (MDSCs), which may reinforce the suppressive TME. The frequency of circulating or intratumor MDSCs correlates with cancer stage, disease progression, and resistance to standard chemotherapy and radiation therapy. Since certain ligands (e.g., NKG2D ligands) are expressed at high levels on some solid tumors and tumor-infiltrating MDSCs, in embodiments, the modified immune cells of the present disclosure may be used to modify the TME to favor an anti-tumor response by reducing or removing suppressive molecules (e.g., TGF-β) in the TME. In embodiments, the engineered immune cells of the present disclosure are cytotoxic to MDSCs but are non-toxic to normal tissues expressing NKG2D ligands (see, e.g., Parihar, R., et al., (2019) Cancer Immunol Res 7(3):363-375). In some embodiments, the cell killing activity associated with, e.g., killing of MDSCs by the engineered host cells of the present disclosure reduces the suppressive effect of the TME. For example, the suppressive effect may be reduced by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more, compared to the suppressive effect of the TME in the absence of a host cell engineered to express a CAD polypeptide as described herein.

[0160] In embodiments, the modified immune cells of the present disclosure may function to reduce the growth and / or proliferation of target cells. For example, immune cells engineered to express a CAD polypeptide as described herein may reduce the growth and / or proliferation of target cells by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or any percentage therebetween, compared to the growth and / or proliferation of target cells in the absence of the engineered immune cells. In embodiments, the target cells express a cell surface ligand that is recognized by a receptor (e.g., NKG2D) on the cell surface of the modified immune cells, which receptor is associated, at least in part, with the CAD polypeptide.

[0161] Thus, in embodiments, a method of introducing into a cell comprising a CAD polypeptide is provided, comprising introducing into the cell an isolated nucleic acid encoding the CAD polypeptide such that the cell expresses the CAD polypeptide. In embodiments, the cell is an immune cell as described herein, e.g., an NK cell, an NKT cell, a gamma delta T cell, an alpha beta T cell, or a gamma delta NKT cell. In some embodiments, the method of making the cell further comprises introducing into the cell another isolated nucleic acid encoding a receptor capable of associating with the CAD polypeptide.

[0162] How to use In one aspect, the disclosure provides a method of modulating a signal transduced through a receptor of a host cell, the host cell comprising an immune cell engineered to express a CAD polypeptide described herein associated with the receptor, preferably the immune cell being cytotoxic. In an embodiment, the receptor is endogenous to and endogenously expressed in the immune cell. In additional or alternative embodiments, the receptor is expressed by introducing an isolated nucleic acid encoding the receptor into the immune cell.

[0163] In embodiments, the method of modulating a signal transduced through a receptor results in the stimulation of an immune cell and / or the activation of an immune cell.

[0164] In embodiments, a method of modulating a signal transduced through a receptor results in an increased level of proliferation of an immune cell compared to a level of proliferation of a control immune cell lacking a CAD polypeptide.

[0165] In embodiments, the method of modulating a signal transmitted through a receptor results in increased expression and secretion of one or more cytokines compared to the level of expression and secretion of one or more cytokines in a control immune cell lacking a CAD polypeptide.

[0166] In embodiments, modulating a signal transduced through a receptor includes routing at least a portion of the signal through a CAD polypeptide rather than through endogenous DAP10. In embodiments, the portion of the signal routed through the CAD polypeptide is 80% or more, e.g., 90-95% or more, e.g., 99% or 100%.

[0167] Treatment Method As described herein, pharmaceutical compositions comprising engineered host cells expressing a CAD polypeptide, and / or mixtures thereof, may be administered for prophylactic and / or therapeutic treatments. The mixture may comprise different types of host cells engineered to express the same or different CAD polypeptides, as described herein. For example, but not limited to, the mixture may comprise a population of NK cells expressing a first CAD polypeptide and a population of γδ cells engineered to express a second CAD polypeptide. As another example, but not limited to, the mixture may comprise a population of NK cells and a population of γδ cells, each engineered to express the same CAD polypeptide. As yet another example, the mixture may comprise a population of engineered host cells and may further comprise a non-engineered cell population. For example, but not limited to, the mixture may comprise a population of NK cells engineered to express a CAD polypeptide as described herein and another non-engineered cell population, e.g., NK cells, NKT cells, γδ cells, αβ cells, etc. In therapeutic applications, the composition may be administered to a subject already suffering from a disease or condition in an amount sufficient to reduce at least one sign or symptom associated with the disease or condition. In some embodiments, the amount is sufficient to cure a disease or condition.

[0168] The genetically engineered host cell population and / or mixtures thereof can also be administered to reduce the likelihood of developing, contracting, or worsening a condition. For therapeutic uses, the effective amount of a population of engineered host cells, non-engineered host cells, and / or mixtures thereof can vary based on the severity and course of the disease or condition, previous treatments, the subject's health status, weight, and / or response to various drugs, and / or the judgment of the treating physician.

[0169] In embodiments, one or more engineered host cell populations, non-engineered cells and / or mixtures thereof of the present disclosure can be used to treat a subject in need of treatment of a condition. Examples of such diseases include, but are not limited to, cancer, infectious diseases, and autoimmune diseases. The subject can be a human, a non-human primate (e.g., chimpanzee, and other ape and monkey species); livestock (e.g., cows, horses, sheep, goats, wild boars); domestic animals (e.g., rabbits, dogs, and cats); laboratory animals (e.g., rodents, such as rats, mice, and guinea pigs). The subject can be of any age. The subject can be, for example, an elderly person, an adult, an adolescent, a prepubescent, a child, a toddler, or an infant.

[0170] A method of treating a condition (e.g., a disease) in a subject may include administering to the subject a therapeutically effective amount of one or more engineered host cell populations (e.g., to express a CAD polypeptide), non-engineered cells, and / or mixtures thereof. The one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof may be administered in various regimens (e.g., timing, concentration, dosage, interval between treatments, and / or formulations). The subject may also be pre-conditioned, for example, with chemotherapy, radiation therapy, or a combination of both, before being administered a therapeutically effective amount of one or more Asian dust host cell populations, non-engineered cells, and / or mixtures thereof. As part of the treatment, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof may be administered to the subject in a first regimen, and the subject may be monitored to determine whether the treatment with the first regimen meets a predetermined therapeutic efficacy level. In some cases, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof may be administered to the subject in a second regimen based on information gathered from providing the first regimen to the subject.

[0171] In embodiments, a pharmaceutical composition comprising at least one host cell engineered to express a CAD polypeptide may be administered in a first regimen. The subject may be monitored, for example, by a health care provider (e.g., a treating physician or nurse). In some examples, the subject is monitored to determine or measure the effectiveness of the engineered host cell in treating the subject's condition. In some circumstances, the subject may also be monitored to determine the in vivo growth of the engineered host cell population in the subject. Another pharmaceutical composition comprising at least one host cell engineered to express a CAD polypeptide may be administered to the subject in a second regimen. The pharmaceutical composition administered in the second regimen may comprise the same type of host cell expressing the same CAD polypeptide as that administered to the subject in the first regimen. However, it is within the scope of this disclosure that the pharmaceutical composition administered in the second regimen may optionally comprise a different type of host cell expressing a different CAD polypeptide (e.g., a CAD polypeptide with a different mutation and / or costimulatory or signaling domain). In some instances, for example, if the first regimen proves effective, a second regimen is implemented (e.g., one administration may be sufficient to treat the condition). In some embodiments, the population of engineered host cells can be administered to a variety of subjects (e.g., the host cells have universal donor properties).

[0172] Therapeutically effective amounts of one or more engineered host cell populations (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof may be used to treat a variety of conditions. In some cases, therapeutically effective amounts of one or more engineered host cell populations (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof may be used in the treatment of cancer, including solid tumors and hematological malignancies. In some cases, therapeutically effective amounts of one or more engineered host cell populations (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof may be used to treat infections caused, for example, by pathogenic bacteria or viruses.

[0173] Treatment with one or more engineered host cell populations (expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof of the present invention may be provided to a subject before, during, and after clinical onset of a condition. Treatment may be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of a disease. Treatment may be provided to a subject more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years after clinical onset of a disease. Treatment may be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of a disease. Treatment may also include treating humans in clinical trials. Treatment may include administering to a subject a pharmaceutical composition comprising one or more engineered host cell populations (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof of the present disclosure.

[0174] In some cases, administration of one or more engineered host cell populations of the present disclosure (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof modulates the activity of endogenous lymphocytes in the subject's body. In some cases, administration of one or more engineered host cell populations of the present disclosure (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof results in the cytotoxic activation of another immune cell. In some cases, the other immune cell is a CD8+ T cell. In some cases, the other immune cell is a natural killer T cell. Other examples of other immune cells are also included in the present disclosure. In some cases, administration of one or more engineered host cell populations of the present disclosure (e.g., expressing a CAD polypeptide), non-engineered cells, and / or mixtures thereof suppresses a regulatory T cell. In some cases, the regulatory T cell is a Fox3+ Treg cell. In some cases, the regulatory T cell is a Fox3- Treg cell. Non-limiting examples of cells whose activity can be modulated by administration of one or more engineered host cell populations (e.g., expressing a CAD polypeptide) of the present disclosure, non-engineered cells, and / or mixtures thereof include hematopoietic stem cells; B cells; CD4+ cells; CD8+ cells; red blood cells; white blood cells; dendritic cells (e.g., dendritic antigen presenting cells); leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophil granulocytes; T helper cells; and T killer cells.

[0175] For example, but not limited to, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof having cytotoxic activity against blood or solid tumor cells, or virus-infected cells, or bacterial cells, can be administered to a subject in any order or simultaneously. At the same time, the engineered host cell(s) and / or mixtures of the present disclosure can be provided in a single unified form, such as an intravenous injection, or in multiple forms, such as multiple intravenous infusions, subcutaneous injections, or tablets. One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure can be packaged together or separately in a single package or multiple packages. One or all of the one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present invention can be administered in multiple doses. If not simultaneously, the interval between multiple doses can vary from about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the engineered host cells of the present disclosure can be grown in vivo within the subject's body after being administered to the subject. One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure can be frozen to provide cells for multiple treatments with the same cell preparation. One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, as well as pharmaceutical compositions comprising them, can be packaged as a kit. The kit may include instructions (e.g., written instructions) for use of one or more engineered host cell populations, non-engineered modified cells, and / or mixtures thereof, as well as compositions comprising them.

[0176] In some cases, a method of treating a subject in need thereof includes administering to the subject a therapeutically effective amount of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure, where the administration treats a particular condition (e.g., cancer, viral or bacterial infection, autoinflammatory disease). In some embodiments, a therapeutically effective amount of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, a therapeutically effective amount of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof is administered for at least 1 week. In some embodiments, a therapeutically effective amount of one or more engineered host cell populations of the present disclosure, non-engineered cells, and / or mixtures thereof is administered for at least two weeks.

[0177] One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the pharmaceutical composition containing the engineered host cell population can vary. For example, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof can be used as prophylactics and administered continuously to subjects prone to a condition or disease to reduce the likelihood of the onset of the disease or condition. The initial administration can be via any practical route, such as any route described herein, using any formulation described herein. In some examples, the administration of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure is intravenous administration. One or more administrations of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof can be administered as soon as possible after onset of a particular condition (e.g., hematological or solid cancer, viral infection, bacterial infection, autoimmune disease, etc.) for the period of time required to treat the disease / condition, e.g., from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. In some embodiments, one or more administrations of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof can be administered several years after onset of the disease / condition (e.g., cancer), and before or after other treatments.

[0178] In some embodiments, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure are administered simultaneously or sequentially with one or more methods for increasing common gamma chain cytokine(s). As used herein, "one or more methods for increasing common gamma chain cytokine(s)" refers to a method, or combination of methods, that alters the physiological condition of a subject to increase at least one common gamma chain cytokine level in the subject. In some embodiments, the method increases the level of one or more common gamma chain cytokine(s) selected from the group consisting of IL-2, IL-4, IL-7, and IL-15 in the subject. In some embodiments, the method includes lymphodepletion. In some embodiments, the method includes administering one or more common gamma chain cytokine(s) to the subject. In some cases, IL-2, IL-4, IL-7, and / or IL-15 are administered. In some embodiments, the method includes secreting the common gamma chain cytokine(s) from the administered engineered host cells. In some cases, IL-2, IL-4, IL-7, and / or IL-15 are secreted.

[0179] In some embodiments, the administration of one or more methods for elevating common gamma chain cytokine(s) includes lymphodepletion prior to the introduction of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure. In some embodiments, the administration of one or more methods for elevating common gamma chain cytokine(s) includes administering, simultaneously with or sequentially with the introduction of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or combinations thereof, of the introduced one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof. The amount of common gamma chain cytokine(s) administered can be an amount effective to increase proliferation, cytotoxic activity, persistence, or combinations thereof, of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof. Exemplary amounts of IL-15 include, but are not limited to, 0.01-10 μg / kg / dose of IL-15 every 24 hours. An exemplary amount of IL-2 is about 3×10 6 ~Approx. 22×10 6 For example, the dosing regimen for IL2 in RCC is 600,000 International Units / kg (0.037 mg / kg) administered intravenously over 15 minutes for up to 14 doses for 48 hours.

[0180] In some embodiments, administration of one or more methods of increasing common gamma chain cytokine(s) includes lymphodepletion prior to administration of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, and administering, simultaneously with or sequentially to the introduction of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof.

[0181] In some embodiments, the elevation of the common gamma chain cytokine(s) is achieved, at least in part, via engineered host cell(s), where the common gamma chain cytokine(s) is / are expressed from a CAD construct as disclosed herein. In such instances, it is within the scope of the present disclosure that one or more additional gamma chain cytokine(s) are additionally administered in a manner that elevates the additional gamma chain cytokine(s).

[0182] Dosage One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure may be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms include additional lymphocytes. In unit administration forms, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit dosages may take the form of packages containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be packaged in non-reclosable single-dose containers. Multi-dose reclosable containers may be used, for example, with or without preservatives. In some examples, the pharmaceutical composition does not include preservatives. Formulations for parenteral injection may be provided in unit dosage forms, for example, in ampoules, or in multi-dose containers containing preservatives.

[0183] One or more engineered host cell populations of the present disclosure, non-engineered cells, and / or mixtures thereof may be present in the composition in the following amounts: at least 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1×10 3 Cells, at least 2 x 10 3 Cells, at least 3 x 103 Cells, at least 4 x 10 3 Cells, at least 5 x 10 3 Cells, at least 6 x 10 3 Cells, at least 7 x 10 3 Cells, at least 8 x 10 3 Cells, at least 9 x 10 3 Cells, at least 1 x 10 4 Cells, at least 2 x 10 4 Cells, at least 3 x 10 4 Cells, at least 4 x 10 4 Cells, at least 5 x 10 4 Cells, at least 6 x 10 4 Cells, at least 7 x 10 4 Cells, at least 8 x 10 4 Cells, at least 9 x 10 4 Cells, at least 1 x 10 5 Cells, at least 2 x 10 5 Cells, at least 3 x 10 5 Cells, at least 4 x 10 5 Cells, at least 5 x 10 5 Cells, at least 6 x 10 5 Cells, at least 7 x 10 5 Cells, at least 8 x 10 5 Cells, at least 9 x 10 5 Cells, at least 1 x 10 6 Cells, at least 2 x 10 6 Cells, at least 3 x 10 6 Cells, at least 4 x 10 6 Cells, at least 5 x 10 6 Cells, at least 6 x 10 6 Cells, at least 7 x 10 6 Cells, at least 8 x 10 6 Cells, at least 9 x 10 6 Cells, at least 1 x 10 7 Cells, at least 2 x 10 7 Cells, at least 3 x 10 7 Cells, at least 4 x 10 7 Cells, at least 5 x 10 7 Cells, at least 6 x 10 7 Cells, at least 7 x 107 Cells, at least 8 x 10 7 Cells, at least 9 x 10 7 Cells, at least 1 x 10 8 Cells, at least 2 x 10 8 Cells, at least 3 x 10 8 Cells, at least 4 x 10 8 Cells, at least 5 x 10 8 Cells, at least 6 x 10 8 Cells, at least 7 x 10 8 Cells, at least 8 x 10 8 Cells, at least 9 x 10 8 Cells, at least 1 x 10 9 cells, or more.

[0184] A therapeutically effective amount of one or more engineered host cell populations, unengineered cells, and / or mixtures thereof of the present invention can be: from about 1 cell to about 10 cells, from about 1 cell to about 100 cells, from about 1 cell to about 10 cells, from about 1 cell to about 20 cells, from about 1 cell to about 30 cells, from about 1 cell to about 40 cells, from about 1 cell to about 50 cells, from about 1 cell to about 60 cells, from about 1 cell to about 70 cells, from about 1 cell to about 80 cells, from about 1 cell to about 90 cells, from about 1 cell to about 100 cells, from about 1 cell to about 1 x 10 3 Cells, about 1 cell ~ about 2 x 10 3 Cells, about 1 cell ~ about 3 x 10 3 Cells, about 1 cell ~ about 4 x 10 3 Cells, about 1 cell ~ about 5 x 10 3 Cells, about 1 cell ~ about 6 x 10 3 Cells, about 1 cell ~ about 7 x 10 3 Cells, about 1 cell ~ about 8 x 10 3 Cells, about 1 cell ~ about 9 x 10 3 Cells, about 1 cell ~ about 1 x 10 4 Cells, about 1 cell ~ about 2 x 10 4 Cells, about 1 cell ~ about 3 x 10 4 Cells, about 1 cell ~ about 4 x 10 4 Cells, about 1 cell ~ about 5 x 10 4 Cells, about 1 cell ~ about 6 x 10 4 Cells, about 1 cell ~ about 7 x 10 4 Cells, about 1 cell ~ about 8 x 10 4Cells, about 1 cell ~ about 9 x 10 4 Cells, about 1 cell ~ about 1 x 10 5 Cells, about 1 cell ~ about 2 x 10 5 Cells, about 1 cell ~ about 3 x 10 5 Cells, about 1 cell ~ about 4 x 10 5 Cells, about 1 cell ~ about 5 x 10 5 Cells, about 1 cell ~ about 6 x 10 5 Cells, about 1 cell ~ about 7 x 10 5 Cells, about 1 cell ~ about 8 x 10 5 Cells, about 1 cell ~ about 9 x 10 5 Cells, about 1 cell ~ about 1 x 10 6 Cells, about 1 cell ~ about 2 x 10 6 Cells, about 1 cell ~ about 3 x 10 6 Cells, about 1 cell ~ about 4 x 10 6 Cells, about 1 cell ~ about 5 x 10 6 Cells, about 1 cell ~ about 6 x 10 6 Cells, about 1 cell ~ about 7 x 10 6 Cells, about 1 cell ~ about 8 x 10 6 Cells, about 1 cell ~ about 9 x 10 6 Cells, about 1 cell ~ about 1 x 10 7 Cells, about 1 cell ~ about 2 x 10 7 Cells, about 1 cell ~ about 3 x 10 7 Cells, about 1 cell ~ about 4 x 10 7 Cells, about 1 cell ~ about 5 x 10 7 Cells, about 1 cell ~ about 6 x 10 7 Cells, about 1 cell ~ about 7 x 10 7 Cells, about 1 cell ~ about 8 x 10 7 Cells, about 1 cell ~ about 9 x 10 7 Cells, about 1 cell ~ about 1 x 10 8 Cells, about 1 cell ~ about 2 x 10 8 Cells, about 1 cell ~ about 3 x 10 8 Cells, about 1 cell ~ about 4 x 10 8 Cells, about 1 cell ~ about 5 x 10 8 Cells, about 1 cell ~ about 6 x 10 8 Cells, about 1 cell ~ about 7 x 10 8 Cells, about 1 cell ~ about 8 x 10 8 Cells, about 1 cell ~ about 9 x 10 8 cells, or about 1 cell to about 1 x 10 9 cell.

[0185] In some cases, a therapeutically effective dose of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present invention may be less than about 1×10 3 cells ~ approx. 2 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 3 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 7 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 1 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 2 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 4 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 7 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 1 x 10 5 cells, approximately 1 x 10 3 cells ~ approx. 2 x 10 5 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 5 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 5 cells, approximately 1 x 103 Cells ~ about 5×10 5 cells, about 1×10 3 Cells ~ about 6×10 5 cells, about 1×10 3 Cells ~ about 7×10 5 cells, about 1×10 3 Cells ~ about 8×10 5 cells, about 1×10 3 Cells ~ about 9×10 5 cells, about 1×10 3 Cells ~ about 1×10 6 cells, about 1×10 3 Cells ~ about 2×10 6 cells, about 1×10 3 Cells ~ about 3×10 6 cells, about 1×10 3 Cells ~ about 4×10 6 cells, about 1×10 3 Cells ~ about 5×10 6 cells, about 1×10 3 Cells ~ about 6×10 6 cells, about 1×10 3 Cells ~ about 7×10 6 cells, about 1×10 3 Cells ~ about 8×10 6 cells, about 1×10 3 Cells ~ about 9×10 6 cells, about 1×10 3 Cells ~ about 1×10 7 cells, about 1×10 3 Cells ~ about 2×10 7 cells, about 1×10 3 Cells ~ about 3×10 7 cells, about 1×10 3 Cells ~ about 4×10 7 cells, about 1×10 3 Cells ~ about 5×10 7 cells, about 1×10 3 Cells ~ about 6×10 7 cells, about 1×10 3 Cells ~ about 7×10 7 cells, about 1×10 3 Cells ~ about 8×10 7 cells, about 1×10 3 Cells ~ about 9×10 7 cells, about 1×10 3 Cells ~ about 1×10 8cells, approximately 1 x 10 3 cells ~ approx. 2 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 8 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 7 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 8 cells, or approximately 1 x 10 3 cells ~ approx. 1 x 10 9 cell.

[0186] In some cases, a therapeutically effective dose of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present invention may be less than about 1×10 6 cells ~ approx. 2 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 3 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 4 x 10 6 cells, approximately 1 x 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 6 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 7 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 8 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 9 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 1 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 2 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 3 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 4 x 10 7 cells, approximately 1 x 106 Cells ~ about 5×10 7 cells, about 1×10 6 Cells ~ about 6×10 7 cells, about 1×10 6 Cells ~ about 7×10 7 cells, about 1×10 6 Cells ~ about 8×10 7 cells, about 1×10 6 Cells ~ about 9×10 7 cells, about 1×10 6 Cells ~ about 1×10 8 cells, about 1×10 6 Cells ~ about 2×10 8 cells, about 1×10 6 Cells ~ about 3×10 8 cells, about 1×10 6 Cells ~ about 4×10 8 cells, about 1×10 6 Cells ~ about 5×10 8 cells, about 1×10 6 Cells ~ about 6×10 8 cells, about 1×10 6 Cells ~ about 7×10 8 cells, about 1×10 6 Cells ~ about 8×10 8 cells, about 1×10 6 Cells ~ about 9×10 8 cells, about 1×10 6 Cells ~ about 1×10 9 cells, about 1×10 6 Cells ~ about 2×10 9 cells, about 1×10 6 Cells ~ about 3×10 9 cells, about 1×10 6 Cells ~ about 4×10 9 cells, about 1×10 6 Cells ~ about 5×10 9 cells, about 1×10 6 Cells ~ about 6×10 9 cells, about 1×10 6 Cells ~ about 7×10 9 cells, about 1×10 6 Cells ~ about 8×10 9 cells, about 1×10 6 Cells ~ about 9×10 9 cells, about 1×10 7 Cells ~ about 1×10 9cells, approximately 1 x 10 7 cells ~ approx. 2 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 3 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 4 x 10 9 cells, approximately 1 x 10 7 Cells ~ approx. 5 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 6 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 7 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 8 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 9 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 1 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 2 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 3 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 4 x 10 9 cells, approximately 1 x 10 8 Cells ~ approx. 5 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 6 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 7 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 8 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 9 x 10 9 cells, or approximately 1 x 10 8 cells ~ approx. 1 x 10 10 cell.

[0187] keep In some embodiments, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the invention may be formulated in a freezing medium and placed in a cryogenic storage unit, such as a liquid nitrogen freezer (-195°C) or an ultra-low temperature freezer (-65°C, -80°C, or -120°C), for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freezing medium may contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate, and / or hydroethyl starch (HES), along with physiological pH buffers to maintain a pH of about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. In embodiments, the cryopreserved cells can be thawed and further treated, for example, by stimulation with antibodies, proteins, peptides, and / or cytokines as referred to herein. The cryopreserved cells can be thawed and genetically modified with viral vectors (e.g., retroviral and lentiviral vectors) or non-viral means (e.g., RNA, DNA, and proteins) as described herein. Alternatively, the host cells described herein can be expanded, genetically modified, and then cryopreserved, for example, optionally by the methods described herein.

[0188] Thus, the genetically engineered and / or non-genetically engineered cells disclosed herein can be stored in at least about 1, 5, 10, 100, 150, 200, 500 vials, at least about 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or at least about 10 10The cryopreserved cells can be cryopreserved to generate a cell bank in amounts of cells of 10 ... EXAMPLES

[0189] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0190] Example 1. Construction of DAP10 constructs DAP10 CAD constructs were constructed with 0 to 4 elements selected from the Y86F mutation, the K84R mutation, the 4-1BB costimulatory domain, and the CD3ζ signaling domain. The construction used the pSIN vector (Hariharan, MJ et al., (1998) Journal of Virology 72(2):950-958). Briefly, the pSIN vector backbone was fully synthesized by Genewiz® (South Plainfield, NJ) from sequences provided by EUFETS (Germany), a subsidiary of BioNTech (Germany). The base plasmid used in all constructs is called pL077pRetroSIN-GFP, which has a green fluorescent protein (GFP) cassette replaced with the gene of interest. The various constructs are shown in Table 1 below. For the following constructs, FP2A refers to the Furin and P2A linker genes, and CD19t refers to the truncated CD19 marker. SEQ ID NOs correspond to the amino acid sequences. Tables 3-15 below annotate the nucleic acid sequences encoding the amino acid sequences of the constructs shown in Table 1, and select sequences in Table 2. Table 1 shows DAP10 constructs that include CD19t, however other variations are within the scope of the present disclosure, such as similar DAP10 constructs incorporating EGFRt. Examples of such sequences are shown in Table 2. [Table 1]

[0191] Example 2. Tumor control of various DAP10 CAD constructs in the PLC / PRF / 5 assay Various DAP10 CAD constructs from Table 1 above were tested in the PLC / PRF / 5 cytolytic assay. Figure 2A shows that DAP10.0 (DAP10 wild type) and DAP10.13 (DAP10-K84R) do not show good tumor control in the PLC / PRF / 5 assay. Figure 2B shows some tumor control by the DAP10.3 (DAP10-CD3ζ) and DAP10.14 (DAP10-CD3ζ-K84R) constructs. Figure 2C shows that the DAP10 CAD constructs containing both the 4-1BB costimulatory domain and the CD3ζ signaling domain show good tumor control in the absence of additional mutations (i.e., DAP10.4), and adding only the Y86F mutation (i.e., DAP10.5) or only the K84R mutation (i.e., DAP10.15) does not significantly improve tumor control over the DAP10.4 construct. However, the DAP10 CAD construct DAP10.6, which incorporates the CD3ζ signaling domain, the 4-1BB costimulatory domain, and both mutations (i.e., Y86F and K84R), respectively, significantly improved tumor control (Figure 2C). Figures 2A-2C also show plots showing the cytotoxicity index of tumor control and tumor cells alone, respectively, tested with a positive control CAR.

[0192] The proliferation of Vδ1γδ cells was also tested under test conditions corresponding to Figures 2A-2C, and the results are shown in Figures 2D-2F, respectively. As shown, good proliferation was observed with most of the DAP10 CAD constructs.

[0193] Example 3. Tumor control of various DAP10 CAD constructs in the HepG2 assay Various DAP10 CAD constructs from Table 1 above were tested in the HepG2 assay. Figure 3A shows data obtained using the DAP10.0 (DAP10 wild type) and DAP10.13 (DAP10-K84R) constructs, Figure 3B shows data obtained using the DAP10.3 (DAP10-CD3ζ) and DAP10.14 (DAP10-CD3ζ-K84R) constructs, and Figure 3C shows data obtained using the DAP10.4, DAP10.5, DAP10.6, and DAP10.15 constructs. Figures 3A-3C also show plots showing the cytotoxicity index of tumor control and tumor cells alone, each tested with a positive control CAR.

[0194] Proliferation of V51γδ cells was also tested under test conditions corresponding to Figures 3A-3C, and the results are shown in Figures 3D-3F, respectively. In the Hep2G assay, good proliferation and cytotoxicity were observed with constructs containing both the CD3ζ and 4-1BB domains and at least the Y86F mutation (i.e., DAP10.15 and DAP10.6), with the DAP10.6 construct (containing CD3ζ, 4-1BB, Y86F, and K84R, respectively) showing the best performance in terms of cytotoxicity and V51γδ cell proliferation.

[0195] Example 4. Survival of V51 cells transduced with DAP10 constructs after tumor co-culture In this example, we assessed survival of V51 cells transduced with various DAP10 constructs from Table 1. Specifically, survival was assessed after 5 days of PLC / PRF / 5 co-culture. As shown in Figure 4, cells transduced with DAP10.6 showed the highest V51 survival after 5 days of co-culture with PLC / PRF / 5.

[0196] Example 5. In Vivo Tumor Control of DAP10 CAD Constructs in a Mouse Model In this example, DAP10 CAD constructs (DAP10.6 and DAP10.15, see Table 1) were tested for their efficacy in controlling the growth of PLC / PRF / 5 tumors in a mouse model. As shown in Figure 5A, in tumor-only bearing mice (Group A), the average tumor volume steadily increased over 35 days. 6 Similar results were observed in mice injected with V51 T cells (untransduced, group B). When mice were injected with V51 T cells transduced with DAP10.6 (group C) or DAP10.15 (group D), tumor growth was significantly reduced, with the strongest efficacy in terms of tumor control being observed for DAP10.6CAD. Figure 5B shows the data obtained in Figure 5A at day 35, analyzed with a Kruskal-Wallis test with Dunn's multiple comparisons and plotted as a function of tumor volume as indicated. For each group A-D in Figures 5A and 5B, N=5 and the number of tumor cells administered was 4e 6 It was.

[0197] Example 6. NKG2D expression in V51 T cells transduced with DAP10 CAD In this example, we show increased NKG2D expression levels in cells transduced with a specific DAP10 CAD (e.g., DAP10.6, see Table 1). Briefly, V51 T cells were transduced with either DAP10.6, DAP10.13, DAP10.15, DAP10.5, and control CARs, and then the transduced cells were co-cultured with PLC cells. V51 NKG2D expression levels were assessed using a combination of fluorescent labeling of NKG2D and FACS analysis. In Figure 6, expression levels of cells containing different DAP10 CADs or CAR controls are plotted as geometric mean fluorescence intensity (gMFI), showing that the DAP10.6CAD construct expressed in V51 T cells results in significantly higher NKG2D expression levels compared to other DAP10 CADs (10.13, 10.15, 10.5) and CAR controls. In particular, the DAP10.6CAD construct contains the K84R mutation, the Y86F mutation, the 4-1BB costimulatory domain, and the CD3ζ signaling domain, respectively. A smaller, yet statistically significant increase in NKG2D expression over the CAR control was observed in DAP10.15 (K84R+4-1BB+CD3ζ) and DAP10.5 (Y86F+4-1BB+CD3ζ).

[0198] Example 7. DAP10 CAD Expression In this example, we show that expression of DAP10 CAD is similar between different lots of V51 cells.

[0199] The DAP10.6.3 construct (SEQ ID NO: 75) contains a truncated EGFR as a marker, as compared to the DAP10.6 construct (SEQ ID NO: 29), which contains, for example, CD19. The DAP10 construct, referred to herein as DAP10.16 (SEQ ID NO: 77), is the same as the DAP10.6.3 construct, but contains a 1XX mutation in the CD3ζ signaling domain. Vδ1 cells transfected with DAP10.6 or DAP10.16 constructs were found to show substantially similar expression levels. CAD protein was directly detected by Western blot analysis using anti-DAP10 and anti-CD3ζ antibodies (n=2 donors) (Figure 7).

[0200] Example 8. Cytotoxic activity of DAP10 CAD is mediated by NKG2D This example shows that blocking NKG2D abolishes cytotoxic activity. Specifically, blocking NKG2D by use of NKG2D blocking antibodies abolished the cytotoxic activity of DAP10 CAD-expressing Vδ1 cells in three donors across two cell lines (Figures 8A-8B). The cytotoxic activity of control Vδ1 cells transfected with CAR was unaffected (Figures 8C-8D). In Figures 8A, 8C, PLC target cells (E:T ratio 5:1) were used. In Figures 8B, 8D, HL60 target cells (E:T ratio 2.5:1) were used. NKG2D blocking antibodies also had no effect on cells expressing the DAP10.0 construct (i.e., lacking the costimulatory and intracellular signaling domains) (data not shown). Example 9. DAP10 CAD molecular activation signature

[0201] This example shows a consistent DAP10 CAD activation signature across multiple donors and cell lines. In this example, Nanostring analysis (Nanostring Technologies, Seattle, WA) was performed after stimulation to evaluate molecular activation signatures. Cell lines used for stimulation included PLC (HCC), HL60, THP1 (AML), and HCT15 (CRC). The data shows a consistent activation signature mediated by interferon gamma, 4-1BB, and granzyme B. Figure 9A shows data for DAP10.6 versus natural control (DAP10.0), and Figure 9B shows data for DAP10.16 versus natural control (DAP10.0). No consistent detectable differences were observed between DAP10.6 and DAP10.16 (Figure 9C).

[0202] Example 10. Broad anti-cancer activity of Vδ1 cells transduced with DAP10 CAD This example shows that V51 cells transduced with the DAP10 CAD of the present disclosure exhibit anti-cancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns.

[0203] Figures 10A-10E are graphs showing % cytotoxic activity as a function of E:T ratio for V51 cells transduced with DAP10.0 compared to V51 cells transduced with DAP10.6 or cells not transduced with DAP10 CAD in an 18 hour assay. Target cell lines included HCT116 (Figure 10A), SKMEL5 (Figure 10B), MinoD2 (Figure 10C), ScaBER (Figure 10D), and RajiB4 (Figure 10E). Figures 10F-10G are graphs showing percent cytotoxicity for V51 cells transduced with DAP10.6 compared to natural control (V51 cells transduced with DAP10.0) or irrelevant CAR control in an 18 hour assay. Target cell lines for Figures 10F-10G were NCI-H1581 and NCI-H2172, respectively. As shown, cytotoxicity potency was significantly increased compared to controls. Figure 10H shows that the selected cell lines used in the assay represent a wide range of NKG2D ligand expression levels / patterns. To obtain the data in Figure 10H, various cancer cell lines as indicated, derived from various hematological and solid tumors, were assessed for NKG2D ligands by flow cytometry. The five antibodies used in the staining were detecting MICA / MICB, ULBP1, ULBP2 / 5 / 6, ULBP3, and ULBP4. Data are presented as fold change in mean fluorescence intensity (MFI) of NKG2D ligands relative to relevant isotype controls. Raw data is shown in Figures 10I-10J (cancer cell lines were stained in triplicate).

[0204] 11A-11G are graphs showing the cytotoxicity index as a function of target and effector co-culture time. Target cells included 22Rv1, Mino, HCT116, and HCT-15. Effectors included V51 cells transduced with DAP10CAR, CAR control, or non-transduced V51 cells. Enhanced cytotoxicity was observed when V51 cells were transduced with the DAP10 CAD of the present disclosure compared to controls. The degree of cytotoxicity was found to be cell line and E:T ratio dependent. The cell lines tested represent various levels / patterns of NKG2D ligand expression (see Figures 10H-10J). Effector cells were co-cultured with NucRed-expressing target cells at a submaximal E:T ratio of 5:1 or 1.5:1 depending on the cell line. The cytotoxicity index was calculated as the total area (mm2) of NucRed objects at each time point. 2 / well) divided by the time=0 value.

[0205] Example 11. Comparable cytotoxic activity across multiple lots of DAP10 CAD-transduced V51 cells This example shows that the cytotoxic activity of V51 cells transduced with DAP10 CAD is comparable for different lots of V51 cells and DAP10 CAD. Figure 12A is a graph showing the data of a representative 120 hour cytotoxicity assay. Target cells were PLC / PRF / 5. V51 cells used in the assay were transduced with DAP10.6, DAP10.16, DAP10 reference lot (i.e., positive control batch of expanded V51 cells), and DAP10.0 (control). Data for PLC / PRF / 5 cells alone (i.e., no co-culture with V51 cells) are also shown. Figure 12B is a graph showing the % reduction in cytotoxicity of tumors alone compared to tumors treated with V51 cells transduced with DAP10 CAD using the end time point of the 120 hour assay.

[0206] The cytotoxicity of DAP10.6 vs. DAP10.16 vs. DAP10.17 constructs incorporated into V51 cells was tested in a 120 hour cytotoxicity assay. V51 cells from three donors were tested with the DAP10 construct using PLC / PRF / 5 target cells. Each construct shown in Figure 12C is an aggregate of all three donors. The DAP10.6 construct showed slightly better average cytotoxicity than DAP10.16 and DAP10.17 in this assay using stringent E:T ratios.

[0207] The efficacy of DAP10.6 vs. DAP10.16 vs. DAP10.17 showed some donor dependency. Cytotoxicity index was measured in co-culture experiments using Vδ1 cells of three different donors (SCT06, SCT29, SCT46) transduced with DAP10.6, DAP10.16, or DAP10.17, or non-transduced cells of the same donors. Target cells in the co-culture experiments were PLC / PRF / 5 cells. Co-culture time was 120 h. As shown in Figures 12D-12E, DAP10.6 and DAP10.16 show very similar profiles. DAP10.17 was found to be the most donor-dependent (Figure 12F). A reference lot of Vδ1 cells transduced with DAP10.6 was used in all assays and served as a reproducible control (data not shown).

[0208] Example 12. Cytokine profile in response to DAP10 CAD stimulation This example shows that DAP10 CAD stimulation results in a polyfunctional cytokine profile.

[0209] FIG. 13A shows the cytokine profile as a function of different DAP10 constructs of the present disclosure. As shown in the figure, the degree of cytokine activation was cell line specific. Cell lines tested included only PLC, Mino, and T cells. Importantly, potentially problematic cytokines (e.g., IL-6 and IL-17) were not detected. FIG. 13B shows the interferon gamma induction (pg / ml / 1E) in PLC, Mino, and T cells alone for various DAP10 CADs (from various donors, e.g., SCT06, SCT46). 6 13C-13F are graphs showing interferon gamma secretion of CAD+V51 cells alone and after approximately 18 hours of co-culture with submaximal E:T ratios of PLC / PRF / 5, HL60, THP1, and PC3 target cells.

[0210] Example 13. Cytokine profile of DAP10 CAD compared to chimeric antigen receptor (CAR) This example shows a high similarity of cytokine profiles from the DAP10 CAD of the present disclosure compared to CAR (Figure 14). Conditions tested included V51 cells transduced with DAP10 CAD and PLC cells or alone (i.e., no target cells) and V51 cells transduced with CAR and target cells (HepG2, PLC, Raji) or alone (i.e., no target cells). Notably, V51 cells transduced with DAP10 CAD in the absence of target cells show less background cytokine secretion than V51 cells transduced with CAR in the absence of target cells.

[0211] Example 14. DAP10 CAD stimulation promotes proliferation across multiple donors This example shows that DAP10 CAD stimulation promotes the proliferation of V51 cells in all donors tested. Specifically, this example assessed the proliferation of V51 cells by co-culture with PLC / PRF / 5 (E:T ratio 5:1) after transduction with DAP10 CAD constructs of the present disclosure (DAP10.6, DAP10.16, DAP10.17). Controls included V51 cells transduced with DAP10 control batch (or DAP10.0). The less / slower proliferation of V51 cells transduced with DAP10.16 could be a result of the 1XX CD3ζ signaling domain. In this regard, enhanced regulation of activation / proliferation could be beneficial for the long-term efficacy / survival of engineered V51 cells by reducing overstimulation / exhaustion.

[0212] In the co-culture experiments, V51 cells obtained from two different donors (SCT29 and SCT46) were used. As shown in Figure 15, robust proliferation of DAP10.6 and DAP10.16 transduced V51 cells was observed for both donors, whereas proliferation for DAP10.17 transduced V51 cells was somewhat donor dependent.

[0213] Example 15.1XX In vivo tumor control by V51 cells transduced with DAP10 CAD containing the CD3ζ intracellular signaling domain This example demonstrates that incorporation of the 1XX CD3ζ intracellular signaling domain can improve tumor control in vivo.

[0214] In this example, the DAP10 CAD constructs DAP10.6 and DAP10.16 were tested for their efficacy in modulating PLC / PRF / 5 tumors grown in a mouse model. As shown in Figure 16A, tumor control in mice treated with V51 cells transduced with DAP10.16CAD (containing 1XX mutations in the CD3ζ intracellular signaling domain) is improved when compared directly with mice treated with V51 cells transduced with DAP10.6CAD (p=0.0079, Mann-Whitney test (two-tailed)). A schematic diagram of the experimental procedure used in this example is shown in Figure 16B.

[0215] Example 16. Comparison of in vivo antitumor activity of DAP10 CADV51 cells and CARV51 cells This example shows that DAP10 CAD+V51 cells exhibit antitumor activity with similar kinetics as CARV51 cells.

[0216] DAP10 CAD+Vδ1 cells (5e) compared to control CARVδ1 cells and tumor-only conditions in the HCT-15 mouse xenograft model 6 Cells / dose and 15e 6 In vivo tumor growth kinetics (cells / dose) is shown in Figure 17A. Tumor volumes quantified on day 27 are shown in Figure 17B. A schematic of the experimental procedure used in this example is shown in Figure 17C. Data are presented as mean ± SEM of 5 mice / group. Kruskal-Wallis test with Dunn's multiple comparisons was used to assess final statistical significance between complete cohorts of each treatment (ns = not significant).

[0217] Example 17. In vivo expansion, persistence, and targeting of engineered V51 cells This example shows that V51 cells transduced with the DAP10 CAD of the present disclosure proliferate in tumors in vivo in mouse models but not in other organs. In this example, two separate studies were performed. As shown in FIG. 18A, proliferation of DAP10.6-transduced V51 cells was observed in tumor tissue (subcutaneous PLC / PRF / 5 cells) but not in spleen, lung, liver, bone marrow, or blood at day 7 post-treatment. The V51 cells used in study 1 were obtained from a different donor than the V51 cells used in study 2. In FIG. 18A, the HuCD45+, V51+ population is shown. FIG. 18B is a graph quantification of engineered V51 cells per mg of tumor tissue at days 4, 7, and 14 (study 1) and days 7 and 14 (study 2) in each study. In each study, 5e 6 engineered Vδ1 cells were used. Of note, an increase in total Vδ1 cells in the tumor was observed throughout each study. Figure 18C is a graph showing quantification of Vδ1 cells in tumor tissues or other tissues (lung, liver, spleen, bone marrow, blood) harvested 4, 7, or 14 days after treatment assessed by flow cytometry, representing a cumulative analysis across two independent studies presented in this example. Figure 18D is a schematic showing the experimental procedure corresponding to this example for reference. No significant changes in body weight or acute clinical signs of toxicity or xenograft-versus-host disease (GvHD) were observed in treated mice (Figure 19). Data shown in Figure 19 are for n=4 independent efficacy studies.

[0218] V51 cells transduced with the DAP10 CAD of the present disclosure were found to efficiently target tumor cells while protecting normal cells (i.e., non-tumor cells). Specifically, a short-term cytotoxicity assay was adapted to an annexin / DAPI flow-based method for the analysis of primary cellular targets. Figure 20A is a graph showing that both V51 cells transduced with the DAP10 CAD of the present disclosure (DAP10.6, DAP10.16) and V51 cells transduced with NKG2D CAR significantly reduced the viability of THP1 cells compared to V51 cells transduced with THP1 alone and natural control (DAP10.0). Figure 20B is a graph showing the substantial lack of targeting of healthy PBMCs by V51 cells transduced with the DAP10 CAD of the present disclosure (DAP10.6, DAP10.16) as well as the natural control (V51 cells transduced with DAP10.0). Notably, V51 cells transduced with DAP10 CAD consistently showed lower PBMC targeting than the NKG2DCAR reference (Figure 20B).

[0219] Example 18. Small-scale donor screening This example shows that expansion of V51 cells transduced with DAP10 CAD containing CD3ζ 1XX modifications can be improved over a similar construct that does not include 1XX in the CD3ζ intracellular signaling domain.

[0220] In this example, Vδ1 cells from six donors (SE001, ARC007, HC45, DLS003, SE015, and SCT029) were tested in two small-scale shake flask expansion experiments. Specifically, Vδ1 cells from different donors were transduced with either DAP10.6 or DAP10.16. Across all donors, 50-135% more Vδ1 cells (from DAP10.16 transduced cells) were measured compared to DAP10.6 transduced cells (Figure 21). Expansion was measured on days 14 and 15.

[0221] Example 19. DAP10 CADVδ1 cell growth rate This example shows that there is a clear shift towards higher Vδ1 cell percentages in cultures transduced with the lead DAP10 CAD constructs (DAP10.6, DAP10.16, DAP10.17) compared to controls (DAP10.0, CAR control).

[0222] In this example, V51 cells from three different donors (SCT06, Figure 22A; SCT29, Figure 22B; SCT45, Figure 22C) were transduced with the lead DAP10 construct or a control and %V51 of total cells was expanded as a function of expansion time. As shown in each of Figures 22A-22C, a clear shift towards higher V51 percentage was observed with the lead DAP10 construct compared to the control.

[0223] Example 20. Expansion of cells transduced with DAP10 CAD constructs This example shows that V51 cells can be efficiently expanded and transduced with the DAP10 CAD of the present disclosure. Figure 23A shows a schematic diagram depicting the process of generating "off-the-shelf" allogeneic CAD V51 cells. Data showing independent expansion of lead DAP10 CAD constructs (DAP10.6, DAP10.16, DAP10.17) transduced into V51 cells obtained from three different donors (SCT06, SCT29, SCT45) are shown in Figures 23B, 23C, 23D, respectively. Independent expansion (blue vs. red) shows similar trends in the growth profile of the DAP10 CAD constructs. In two of the three donors, growth was found to be construct dependent.

[0224] A representative experiment shows that ex vivo culture of V51 cells results in significant fold expansion (Figure 23E) and strong DAP10 CAD transduction (Figure 23F) of V51 cells. Data in Figures 23E-23F were obtained from 12 independent cultures using PBMCs from 7 different donors. A series of graphs showing the cell composition (V51, V52, αβ, and NK cells) over time (day 0, before αβ T cell depletion, and after αβ T cell depletion) expressed as percent culture is shown in Figure 23G. Certain modifications and improvements will occur to those skilled in the art upon reading the above description. It should be understood that all such modifications and improvements are omitted herein for the sake of brevity and readability, but are properly included within the scope of the following claims. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11]

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Claims

1. An isolated nucleic acid encoding a chimeric adaptor (CAD) polypeptide, wherein the CAD polypeptide comprises a DAP10 domain comprising a human DAP10 amino acid sequence and at least one costimulatory domain, and wherein the CAD polypeptide specifically lacks an extracellular domain comprising a functional extracellular receptor and / or ligand binding domain.

2. 2. The isolated nucleic acid of claim 1, wherein the costimulatory domain is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD3C, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD70, CD80, CD83, CD86, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), FcR, LAT, NKD2C, SLP76, TRIM, and ZAP70, or a combination thereof.

3. 3. The isolated nucleic acid of claim 2, wherein the at least one costimulatory domain is 4-1BB.

4. The isolated nucleic acid of claim 2, wherein the at least one costimulatory domain is CD28.

5. 3. The isolated nucleic acid of claim 1, wherein the CAD polypeptide further comprises at least one intracellular signaling domain, and the at least one intracellular signaling domain is selected from CD3ζ, DAP12, LFA-1, and CD3t.

6. 6. The isolated nucleic acid of claim 5, wherein the at least one signaling domain is CD3ζ, and optionally, CD3ζ has the amino acid sequence set forth as SEQ ID NO:

76.

7. 6. The isolated nucleic acid of claim 5, wherein the at least one costimulatory domain is 4-1BB and the intracellular signaling domain is CD3ζ.

8. 8. The isolated nucleic acid of claim 7, wherein the CAD polypeptide comprises, from N-terminus to C-terminus, the DAP10 domain, a 4-1BB costimulatory domain, followed by a CD3ζ intracellular signaling domain.

9. 2. The isolated nucleic acid of claim 1, wherein the CAD polypeptide comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain.

10. 10. The isolated nucleic acid of claim 9, wherein the CAD polypeptide comprises, from N-terminus to C-terminus, the DAP10 domain, the 4-1BB costimulatory domain, followed by the CD28 costimulatory domain, followed by a CD3ζ intracellular signaling domain, optionally wherein CD3ζ has the amino acid sequence set forth in SEQ ID NO:

76.

11. 10. The isolated nucleic acid of claim 9, wherein the CAD polypeptide comprises, from N-terminus to C-terminus, the DAP10 domain, the CD28 costimulatory domain, followed by the 4-1BB costimulatory domain, followed by a CD3ζ signaling domain, optionally wherein CD3ζ has the amino acid sequence set forth as SEQ ID NO:

76.

12. 3. The isolated nucleic acid of claim 1 or 2, wherein the human DAP10 amino acid sequence comprises an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO:

1.

13. The isolated nucleic acid of claim 12 , wherein the human DAP10 amino acid sequence comprises a mutated human DAP10 amino acid sequence.

14. 14. The isolated nucleic acid of claim 13, wherein the mutant human DAP10 amino acid sequence comprises an amino acid substitution at a position corresponding to K84 and / or Y86.

15. 15. The isolated nucleic acid of claim 14, wherein the amino acid substitution at position K84 comprises a K84R substitution.

16. 15. The isolated nucleic acid of claim 14, wherein the amino acid substitution at position Y86 comprises a Y86F substitution.

17. 3. The isolated nucleic acid of claim 1 or 2, wherein the isolated nucleic acid is operably linked to a regulatable promoter.

18. The isolated nucleic acid of claim 1 or 2, wherein the isolated nucleic acid further encodes a cytokine.

19. 19. The isolated nucleic acid of claim 18, wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, and IL-23.

20. An expression vector comprising the isolated nucleic acid of claim 1 or 2.

21. A chimeric adapter (CAD) polypeptide encoded by the isolated nucleic acid of claim 1 or 2.

22. 21. A mammalian cell comprising the expression vector of claim 20, wherein the mammalian cell expresses at least one receptor related to DAP10.

23. 23. The mammalian cell of claim 22, wherein the at least one receptor related to DAP10 is endogenous.

24. 23. The mammalian cell of claim 22, wherein the at least one receptor related to DAP10 is overexpressed.

25. 23. The mammalian cell of claim 22, wherein the at least one receptor related to DAP10 is exogenous.

26. The mammalian cell of claim 22, wherein the receptor is NKG2D.

27. 23. The mammalian cell of claim 22, wherein the mammalian cell is an immune cell, preferably the immune cell is a cytotoxic cell.

28. 1. A method for activating immune cells, comprising: expressing the CAD polypeptide of claim 21 in said immune cells, wherein said immune cells express at least one receptor related to DAP10; wherein said activation occurs in response to said receptor engaging a corresponding target molecule.

29. 29. The method of claim 28, wherein the receptor is endogenous.

30. 29. The method of claim 28, wherein the receptor is exogenous.

31. 29. The method of claim 28, wherein the receptor is overexpressed.

32. 29. The method of claim 28, wherein the receptor is NKG2D.

33. 23. Use of a mammalian cell or a plurality thereof described in claim 22 in the preparation of a medicament for treating a subject with a condition, wherein the mammalian cell or a plurality thereof reduces at least one symptom or sign of the condition in the subject.

34. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a plurality of mammalian cells according to claim 22.

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