Compositions and methods comprising chimeric adaptor polypeptides

Abstract

Aspects of the present disclosure include compositions and methods for treating a variety of diseases / disorders using an engineered host cell, where the engineered host cell comprises a chimeric adaptor (CAD) polypeptide comprising DAP10 and at least one chimeric receptor. The CAD polypeptides may comprise substituted mutations and / or additional protein domains that function in binding to associated receptors to enhance cell survival and proliferation of the host cells and enhance cell killing activity of non-host cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 347,194, filed on May 31, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to cellular immunotherapy, and in particular to compositions and methods for improving cell survival, proliferation, signaling, and the like comprising mammalian cells comprising at least one chimeric adaptor polypeptide and one or more chimeric receptors. Background Art

[0004] Adoptive cell therapy has undergone more than three decades of near-continuous iteration, from early efforts focused on basic lymphokine activation and / or tumor infiltration to more recent strategies of engineering immune cells to express genetically engineered antigen receptors, such as chimeric antigen receptors (CARs). However, despite some signs along the way that various approaches have therapeutic potential, numerous questions remain.

[0005] One problem involves the problem of low expression or loss of the target of adoptive cell therapy (i.e., antigen escape). Specifically, a common mechanism of resistance to adoptive (or even innate) cell therapy is the presence of cell types (e.g., tumors) in which the target antigen is lost or downregulated. This loss or downregulation can lead to reduced efficacy of the adoptive (or innate) cell response (Majzner RG and Mackall, CL. (2018) Cancer Discovery, 8(10): 1219-26).

[0006] Another related issue is the downregulation of naturally occurring receptors that can recognize ligands that are specifically present on cells associated with a particular disease, which can also lead to ineffective cell responses to various disease conditions. As a representative example, NKG2D is an activating immune receptor that is present on human natural killer (NK) cells, CD8+αβT cells, and γδT cells, regulating innate and adoptive immune responses. The natural ligands of NKG2D include MICA and MICB and several 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 different levels on transformed or virus-infected cancer cells (see, e.g., 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 surface of tumor cells activates NK cells and co-stimulates effector T cells by binding to NKG2D, sensitizing tumor cells to immune cell-mediated destruction. Therefore, NKG2D receptors and their ligands are targets of interest for cancer immunotherapy.

[0007] Unfortunately, NKG2D may be downregulated when it is most needed. For example, tumor-derived tumor growth factor-β (TGF-β) can downregulate NKG2D, thereby reducing NK and CD8 + Cells kill tumor cells (see, for example, Crane, C. et al. (2010) Neuro-Oncology, 12(1):7–13; and Dasgupta, S. et al. (2005) Journal of Immunol, 175:5541-50). This in turn is associated with a poor prognosis in tumor treatment.

[0008] Taking NKG2D as an example, it is demonstrated that there is a need in the art for compositions and methods that can, among other things, improve, for example, immune cell survival and proliferation, prevent receptor downregulation, and compensate for immune escape of, for example, antigens that are targets of adoptive immunotherapy. Such compositions and methods will improve the prognosis of patients receiving adoptive immunotherapy. Summary of the invention

[0009] The present invention solves the above-mentioned shortcomings in the prior art by mammalian cells comprising a chimeric adaptor (CAD) polypeptide (comprising human DAP10) and at least one chimeric receptor and methods of use thereof, optionally wherein the CAD polypeptide is associated with at least one chimeric receptor. As first described and demonstrated herein, the subject CAD constructs and polypeptides can improve the stability of receptors (e.g., chimeric and / or non-chimeric) capable of recognizing target antigens on the surface of various cells, promote a good balance of cell signaling pathways upon receptor-target binding, and / or improve functional properties (e.g., enhanced cell lysis, proliferation, survival and / or co-stimulatory properties) caused by binding to various ligands of at least one chimeric receptor (e.g., NKG2D), and optionally to various ligands of non-chimeric receptors associated with the CAD polypeptide.

[0010] In one aspect, the invention provides a mammalian cell comprising a chimeric adaptor (CAD) polypeptide comprising at least one of a DAP10 domain, a co-stimulatory domain and / or an intracellular signaling domain, and specifically lacking an extracellular domain comprising a functional extracellular receptor and / or a ligand binding domain, wherein the mammalian cell further comprises at least one chimeric receptor comprising an extracellular targeting domain that specifically binds to a target antigen on a target cell. In a preferred embodiment, the DAP10 domain comprises a human DAP10 amino acid sequence.

[0011] In embodiments, the chimeric receptor comprises at least one of an intracellular signaling domain and / or a co-stimulatory domain.

[0012] In embodiments, the chimeric receptor associates with DAP10 and comprises at least one DAP10 interacting domain. In embodiments, the DAP10 interacting domain comprises the amino acid sequence set forth in SEQ ID NO:75, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity to SEQ ID NO:75.

[0013] In an embodiment, the target antigen on the target cell is selected from the group consisting of: CD20, TyrD, B7H6, CD3, CD19; CD123; CD22; CD30; CD70, CD171; CD6, CS-1 (also known as CD2 subset 1, Claudin 18.2, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44, CD44v6; CD44v7 / 8; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); IL-11Rα; mesothelin; interleukin 11 receptor alpha (IL-URa); prostate stem cell antigen (PSCA); proteinase serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); ErbB3, ErbB4, mucin 1, cell surface-related (MUC1); epidermal growth factor receptor (EGFR); neuroendocrine tumor suppressor protein (N-T); Cell adhesion molecule (NCAM); prostatic enzyme; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); IGF-II receptor; carbonic anhydrase IX (CAIX); proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; adrenergic type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe);Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); melanoma-associated antigen; o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD 179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); urokinase 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); MAGE-A4; MAGE-A9; ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survival; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytic neoplasm viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2);Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein-like (BORIS or brother of imprinted site regulator), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor Fc fragment (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);5T4, 8H9, ALCAM, B7-1 (CD80), B7-2 (CD86), B7-H4, B7-H6, B-human chorionic gonadotropin, CA-9, CA-125, CD133, CD138, CD23, CD25, CD34, CD4, CD40, CD56, CD8, c-Met, CSPG4, CMV-specific antigen, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), DLL3, disialylganglioside GD2, GD3, ductal epithelial mucin, EBV-specific antigen, EGP-2, EGP-40, endoglin, epithelial tumor antigen, fetal acetylcholine receptor, FBP, folate binding protein, folate receptor α, glioma-associated antigen, Glycosphingolipids, gp36, G250 / CAIX, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp41, HPV-specific antigens, KDR, kappa chain, insulin growth factor (IGF1)-1, ligands belonging to the human MIC family (MICA and MICB) and ligands belonging to the human ULBP family (ULBP1-ULBP6), LAGA-1a, Lewis Y, lambda chain, lectin-reactive AFP, L1 cell adhesion molecule, MAGE, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, MN-CA IX, MUC-1, MUC-16, NKG2D, NKG2D ligands, neutrophil elastase, Nkp30, carcinoembryonic antigen (h5T4) p53, prostate specific antigen (PSA), PSC1, prostate specific antigen protein, STEAP1, STEAP2, surface adhesion molecules, survival and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, Tem8, tumor stromal antigen, VEGF-A and immunoglobulin lambda-like polypeptide 1 (IGLL1). ;

[0014] In embodiments, at least one co-stimulatory domain of a CAD polypeptide and optionally at least one co-stimulatory domain of a chimeric receptor are 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. In preferred embodiments, at least one co-stimulatory domain of a CAD polypeptide is 4-1BB or CD28.

[0015] In an embodiment, the CAD polypeptide comprises at least one intracellular signaling domain. In an embodiment, the at least one intracellular signaling domain of the CAD polypeptide and optionally the at least one intracellular signaling domain of the chimeric receptor is selected from CD3ζ, DAP12, LFA-1, and a repeating (2-5) DAP10 YINM motif. In a preferred embodiment, the at least one intracellular signaling domain of the CAD polypeptide is CD3ζ.

[0016] In an embodiment, at least one co-stimulatory domain of the CAD polypeptide is 4-1BB, and at least one intracellular signaling domain of the CAD polypeptide is CD3ζ. In a preferred embodiment, the CAD polypeptide comprises a DAP10 domain, a 4-1BB co-stimulatory domain, followed by a CD3ζ intracellular signaling domain from N-terminus to C-terminus.

[0017] In an embodiment, at least one co-stimulatory domain of the CAD polypeptide is CD28, and at least one intracellular signaling domain of the CAD polypeptide is CD3ζ. In a preferred embodiment, the CAD polypeptide comprises, from N-terminus to C-terminus, a DAP10 domain, a CD28 co-stimulatory domain, followed by a CD3ζ intracellular signaling domain.

[0018] In an embodiment, the CAD polypeptide comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain. In an embodiment, the CAD polypeptide comprises a DAP10 domain, a 4-1BB co-stimulatory domain, a subsequent CD28 co-stimulatory domain, and a subsequent CD3ζ intracellular signaling domain from N-terminus to C-terminus. In another embodiment, the CAD polypeptide comprises a DAP10 domain, a CD28 co-stimulatory domain, a subsequent 4-1BB co-stimulatory domain, and a subsequent CD3ζ intracellular signaling domain from N-terminus to C-terminus.

[0019] In an embodiment, 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. In an embodiment, 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: 78. In an embodiment, 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: 81.

[0020] In an embodiment, the human DAP10 amino acid sequence comprises a mutated human DAP10 amino acid sequence. In some examples, the mutated human DAP10 amino acid sequence comprises an amino acid substitution at a position corresponding to K84 and / or Y86. In an embodiment, the amino acid substitution at position K84 comprises a K84R substitution. In an embodiment, the amino acid substitution at Y86 comprises a Y86F substitution.

[0021] In an embodiment, the CAD polypeptide is encoded by an isolated nucleic acid operably linked to a regulatable promoter, and the chimeric receptor is encoded by an isolated nucleic acid operably linked to a regulatable promoter. In one embodiment, the regulatable promoter operably linked to the nucleic acid encoding the CAD polypeptide is different from the regulatable promoter operably linked to the nucleic acid encoding the chimeric receptor. In one embodiment, the regulatable promoter operably linked to the nucleic acid encoding the CAD polypeptide is the same as the regulatable promoter operably linked to the nucleic acid encoding the chimeric receptor.

[0022] In embodiments, the isolated nucleic acid encoding the CAD polypeptide comprises the nucleic acid sequence shown 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 or SEQ ID NO:72.

[0023] In an embodiment, the isolated nucleic acid encoding the CAD polypeptide comprises at its N-terminus the signal peptide of SEQ ID NO: 45. In an embodiment, the isolated nucleic acid encoding the CAD polypeptide comprises at its N-terminus the signal peptide of SEQ ID NO: 93.

[0024] In an embodiment, the isolated nucleic acid encoding the CAD polypeptide encodes a cytokine. In an additional or alternative embodiment, the isolated nucleic acid encoding the chimeric receptor encodes a cytokine. In an embodiment where the isolated nucleic acid encoding the CAD polypeptide and the isolated nucleic acid encoding the chimeric receptor both encode cytokines, the cytokines may be different. In an embodiment, the cytokine is selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21 and IL-23. In an embodiment, the isolated nucleic acid also encodes a marker protein. In an embodiment, the isolated nucleic acid also encodes a marker protein. In an embodiment, the marker protein is selected from the group consisting of truncated CD19, CD20 (Rituxumab (Rituxumab) recognition domain), truncated EGFR and LNGFR.

[0025] In embodiments, wherein the chimeric receptor comprises an intracellular signaling domain, the CAD polypeptide comprises a co-stimulatory domain, and vice versa.

[0026] In embodiments, the mammalian cell further comprises at least one receptor associated with DAP10, wherein the at least one receptor is not at least one chimeric receptor. In embodiments, the at least one receptor associated with DAP10 is endogenous. In embodiments, the at least one receptor associated with DAP10 is exogenous or. In embodiments, the at least one receptor associated with DAP10 is overexpressed. In specific embodiments, the at least one receptor is selected from NKG2D, Ly49H, Ly49D, Sirp-b1, Siglec-15 and Cd3001b. In preferred embodiments, the at least one receptor is NKG2D.

[0027] In an embodiment, the mammalian cell is an immune cell, preferably wherein the immune cell is a cytotoxic cell.

[0028] In embodiments, a mammalian cell comprising a CAD polypeptide as disclosed herein and at least one chimeric receptor exhibits in vitro and / or in vivo killing activity against a target cell that exhibits cell surface expression of a target antigen recognized by the at least one chimeric receptor.

[0029] In embodiments, the target cell is a hematological tumor cell.

[0030] In embodiments, the target cell is a solid tumor cell.

[0031] In embodiments, the in vitro and / or in vivo killing activity is greater than the intrinsic level of in vitro and / or in vivo killing activity in a control mammalian cell lacking expression of at least one chimeric receptor and / or one or both of the CAD polypeptide.

[0032] In embodiments, the mammalian cell proliferates in response to contact with the target cell. In embodiments, the mammalian cell exhibits increased proliferation in response to contact with the target cell compared to a control mammalian cell lacking expression of one or both of the chimeric receptor and / or CAD polypeptide. In embodiments, the mammalian cell proliferates in a host organism comprising the target cell.

[0033] In embodiments, the mammalian cell expresses a pro-inflammatory cytokine in response to contact with the target cell. In embodiments, the pro-inflammatory cytokine comprises tumor necrosis factor alpha or interferon gamma.

[0034] In one aspect, the invention provides a plurality of mammalian cells comprising a CAD polypeptide as disclosed herein and at least one chimeric receptor. In embodiments, the plurality of mammalian cells comprises at least about 10 6 cells, at least 10 7 cells or at least 10 8 cells, preferably about 10 8 Up to 10 11 cells.

[0035] In one aspect, the invention provides a method of preparing a mammalian cell as disclosed herein, comprising transfecting a mammalian cell with a construct comprising an isolated nucleic acid encoding a CAD polypeptide and at least one isolated nucleic acid construct encoding at least one chimeric receptor.

[0036] In embodiments, the method comprises retroviral transduction.

[0037] In an embodiment, the method comprises ex vivo expansion of mammalian cells, wherein the ex vivo expansion is performed prior to and / or after transfection of an isolated nucleic acid encoding a CAD polypeptide and at least one construct encoding at least one chimeric receptor.

[0038] In one aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a mammalian cell, preferably a plurality of mammalian cells, more preferably at least about 10 6 cells, at least 10 7 cells or at least 10 8 cells, still more preferably about 10 8 Up to 10 11 cells.

[0039] In one aspect, the invention provides a method of activating a mammalian cell or a plurality of said mammalian cells comprising a CAD polypeptide as disclosed herein and at least one chimeric receptor, comprising contacting one or more target cells with the mammalian cell. In an embodiment, the mammalian cell is introduced into a subject in need thereof, wherein the activation occurs in vivo in the subject.

[0040] In one aspect, the present invention provides the use of a mammalian cell comprising a CAD polypeptide as disclosed herein and at least one chimeric receptor, or a plurality of said mammalian cells, or a pharmaceutical composition comprising said mammalian cells, in the preparation of a medicament for treating a subject suffering from a disease, wherein said mammalian cell alleviates at least one symptom or sign of said disease in the subject.

[0041] In one aspect, the present invention provides the use of a tumor cell killing effective amount of a mammalian cell or a plurality of said mammalian cells or a pharmaceutical composition comprising said mammalian cells in the preparation of a medicament for treating cancer in a subject in need thereof.

[0042] In one aspect, the present invention provides a method of killing tumor cells, the method comprising contacting tumor cells with a tumor cell killing effective amount of a mammalian cell, or a plurality of said mammalian cells, or a pharmaceutical composition comprising said mammalian cell. In an embodiment, the method comprises introducing a therapeutically effective amount of mammalian cells or pharmaceutical compositions into a host organism comprising tumor cells. In an embodiment, the method comprises introducing a therapeutically effective amount of mammalian cells or pharmaceutical compositions into a host organism comprising tumor cells, and administering simultaneously or sequentially one or more methods of increasing common chain γ chain cytokines.

[0043] In an embodiment, the method of administering one or more methods of elevating common γ chain cytokines comprises administering an amount of common γ chain cytokines effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced mammalian cells simultaneously or sequentially with the introduction of the mammalian cells, preferably wherein the method comprises administering IL-2, more preferably wherein the method comprises administering IL-15.

[0044] In embodiments, the one or more methods of elevating a common gamma chain cytokine comprise administering an amount of a common gamma chain cytokine effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced mammalian cells prior to and / or after introduction into the mammalian cells.

[0045] In embodiments, the one or more methods of elevating the common gamma chain cytokine comprises lymphodepletion prior to introduction into the mammalian cells.

[0046] In embodiments, the one or more methods of elevating common gamma chain cytokines comprise secretion of one or more common gamma chain cytokines from introduced mammalian cells.

[0047] In embodiments, the methods reduce in vivo tumor burden in a host organism, and / or increase average survival time of a host organism, compared to a control organism, wherein the control organism has not been treated with the mammalian cell or pharmaceutical composition.

[0048] Other features, objects and advantages will be apparent from the following disclosure.

[0049] Incorporated by Reference

[0050] 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

[0051] Figure 1A-Figure 1D is a schematic diagram of the binding of an exemplary chimeric DAP10 adaptor polypeptide of the present disclosure to a receptor to which it associates. In the depicted diagram, the receptor is NKG2D. Figure 1A A chimeric DAP10 adaptor polypeptide is depicted, which has K84R and Y86F modifications; Figure 1B Depicted is a chimeric DAP10 adaptor polypeptide with K84R and Y86F modifications and a C-terminal fusion comprising a CD3ζ signaling domain; Figure 1C depicts a chimeric DAP10 adaptor polypeptide having K84R and Y86F modifications and a C-terminal fusion comprising a 4-1BB costimulatory domain; and Figure 1D Depicted is a chimeric DAP10 adaptor polypeptide with K84R and Y86F modifications and a C-terminal fusion comprising a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.

[0052] Figure 2A-2C is a graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the present disclosure expressed in Vδ1 cells and tested against PLC / PRF / 5 cells compared to tumor cells alone and a control chimeric antigen receptor (CAR) construct also expressed in Vδ1 cells.

[0053] Figure 2D-2F It shows Figure 2A-2CGraph showing the extent of Vδ1 cell proliferation seen in an assay of .

[0054] Figure 3A-3C is a graph showing the cytotoxicity index of various chimeric DAP10 adaptor polypeptides of the present disclosure expressed in Vδ1 cells and tested against HepG2 cells, compared to tumor cells alone and a control CAR construct also expressed in Vδ1 cells.

[0055] Figure 3D-Figure 3F It shows Figure 3A-3C Graph showing the extent of Vδ1 cell proliferation seen in an assay of .

[0056] Figure 4 is a graph showing the survival of Vδ1 cells transduced with various chimeric DAP10 adaptor constructs of the present disclosure after 5 days of co-culture with PLC / PRF / 5 cells.

[0057] Figure 5A-Figure 5B is a graph illustrating robust in vivo tumor growth achieved via Vδ1 cells transduced with selected chimeric DAP10 adaptor polypeptides of the present disclosure.

[0058] Figure 6 is a graph illustrating NKG2D expression levels on Vδ1 cells transduced with various DAP10 adaptor polypeptides of the present disclosure and after co-culturing the transduced cells with PLC cells. The highest NKG2D expression levels were observed in cells transduced with DAP10 adaptor polypeptides comprising K84R, Y86F, 4-1BB co-stimulatory domains, and CD3ζ signaling domains.

[0059] Figure 7 is a Western blot of CAD protein visualized by anti-DAP10 and anti-CD3ζ antibodies, which demonstrates that DAP10CAD expression is similar in different batches of Vδ1 cells.

[0060] Figure 8A-8D is a graph illustrating that the cytotoxic activity of DAP10 CAD is mediated by NKG2D. Figure 8A-B ) or chimeric antigen receptor (CAR) + Vδ1 cells ( Figure 8C-Figure 8D ) were pre-incubated with different dilutions of anti-NKG2D antibody (clone 1D11) or isotype control (1 μg / ml-0.01 ng / ml) and then co-cultured with luciferase-labeled target cells (PLC / PRF / 5 or HL60). Target cell killing was assessed by measuring the luciferase signal after 18 hours. NKG2D-mediated cytotoxicity can be assessed by comparing the percentage of cytotoxicity of isotype pre-incubation with the percentage of cytotoxicity of NKG2D antibody pre-incubation.

[0061] Figure 9A-9C This demonstrates that the DAP10 CADs of the present disclosure have consistent molecular activation signatures. The data were obtained by nanostring analysis after stimulation of multiple donors and cell lines.

[0062] Figures 10A-10G It is demonstrated that Vδ1 cells transduced with the DAP10 CAD of the present disclosure exhibit anticancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in an 18-hour assay. Figures 10A-10G The graph shown in represents the percentage of cytotoxic activity of Vδ1 cells transduced with DAP10 CAD. In a short-term 18-hour cytotoxicity assay, DAP10 CAD+Vδ1 cells or controls were co-cultured with various luciferase-expressing target cell lines at different E:T ratios (1:6-10:1).

[0063] Fig. 10H 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 mean fluorescence intensity (MFI) of NKG2D ligands relative to the relevant isotype control.

[0064] Figure 10I-10J is a series of charts that show the Fig. 10H The raw data of the table is shown in .

[0065] Figures 11A-11G It is demonstrated that Vδ1 cells transduced with the DAP10 CAD of the present disclosure exhibit anticancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns in a 120 hour assay. Figures 11A-11G is a graph illustrating the cytotoxicity index of Vδ1 cells transduced with DAP10 CAD at different effector cell:target cell ratios compared to controls. The target cell lines tested encompass a broad range of NKG2D ligand (MICA / B, ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4) expression levels / patterns (see Figure 10H-10J ).

[0066] Figure 12A-12B is a graph showing that the cytotoxic activity of Vδ1 cells transduced with DAP10 CAD is comparable for different batches of Vδ1 cells and DAP10 CAD. Fig. 12A Shown is a 120 hour cytotoxicity assay where the target cells were PLC / PRF / 5 cells. Fig. 12B shows the relative use Fig. 12AThe final time point of the assay was the percent reduction in cytotoxicity of the treated tumors alone.

[0067] Fig. 12C is a graph showing a comparison of the cytotoxicity of Vδ1 cells transduced with DAP10.6, DAP10.16 or DAP10.17 constructs. The data obtained for each construct is an aggregate of 3 donors. PLC / PRF / 5 cells were used as target cells in a 120 hour cytotoxicity assay. Controls included DAP10.0 and PLC / PRF / 5 alone. Using a strict E:T ratio, DAP10.6 showed improved cytotoxicity compared to DAP10.16 and DAP10.17 in this particular assay.

[0068] Figure 12D-12F is a diagram illustrating three different DAP10 constructs (DAP10.6, Fig.12D ;DAP10.16, Fig.12E ;DAP10.17, Fig.12F ) as measured by the cytotoxicity index in a 120-hour co-culture cytotoxicity assay. The DAP10 construct was transduced into Vδ1 cells, and the target cells were PLC / PRF / 5 cells. The control group included non-transduced Vδ1 cells from the same three different donors.

[0069] Figure 13A-13B DAP10 CAD stimulation produces a multifunctional cytokine profile that varies with different DAP10CAD constructs and target cell types. Fig.13A Cytokine profiles that vary with DAP10 CAD and target cells are depicted. Fig. 13B is a graph showing the relationship between interferon-γ induction and DAP10 CAD and target cells.

[0070] Figure 13C-13F is a graph showing the levels of interferon-γ secreted by DAP10 CAD+Vδ1 cells (effector cells) alone and after co-culture with various target cells.

[0071] Fig.14 Exemplified are the cytokine profiles of Vδ1 cells transduced with DAP10 CAD compared to the cytokine profiles of Vδ1 cells transduced with a chimeric antigen receptor (CAR) in the presence or absence of target cells.

[0072] Fig.15 It is demonstrated that the DAP10 CAD of the present disclosure drives the proliferation of Vδ1 cells from multiple donors. Fig.15Shown is a graph illustrating donor dependence of proliferation of Vδ1 cells obtained from two different donors (SCT29 and SCT46) and transduced with DAP10.6, DAP10.16 or DAP10.17, depending on co-culture experiments using PLC / PRF / 5 cells. Controls included previously tested Vδ1 cells obtained from a different donor (SCT06) and transduced with DAP10.6 (positive control), and Vδ1 cells obtained from two different donors (SCT29 and SCT46) and transduced with DAP10.0 (negative control).

[0073] Figure 16A-16B In vivo tumor control of Vδ1 cells transduced with DAP10 CAD in a mouse model is exemplified. Fig.16A is a graph comparing the in vivo tumor control of DAP10.6 and DAP10CAD (DAP10.16) containing a modified ("1XX") CD3ζ intracellular signaling domain. A schematic diagram of the experimental procedure is in Fig. 16B Depicted in.

[0074] Figure 17A-17C It was demonstrated that the anti-tumor activity of Vδ1 cells transduced with the DAP10 CAD of the present disclosure exhibited anti-tumor activity with similar kinetics to that of CAR Vδ1 cells. Fig.17A is a graph showing the in vivo tumor growth kinetics of DAP10CAD+Vδ1 cells compared to CAR Vδ1 cells in the HCT-15 mouse xenograft model. Fig. 17B is a graph quantifying tumor volume at day 27. Fig. 17C is used to obtain Figure 17A-17B Schematic representation of the experimental procedure depicting the data.

[0075] Figures 18A-18D It is demonstrated that Vδ1 cells transduced with the DAP10 CAD of the present disclosure proliferate in vivo in tumor tissue in a mouse model. Fig.18A is a flow cytometry graph illustrating that the proliferation of Vδ1 cells transduced with DAP10 CAD is specific to tumor tissue, and Fig.18B is a graph showing the progression of tumor tissue proliferation over 14 days. Fig.18C is a graph showing the quantification of Vδ1 cells in tumor tissue or other tissues at 4, 7 and 14 days after treatment. Figures 18A-18C The experimental procedures for the data depicted in Fig.18D It is schematically shown in FIG.

[0076] Fig.19 It is demonstrated that treatment of mice with DAP10 CAD-transduced Vδ1 cells of the present disclosure is not associated with significant changes in body weight.

[0077] Figure 20A-20B It is demonstrated that Vδ1 cells transduced with the DAP10 CAD of the present disclosure target tumor cells while being harmless to non-tumor cells. Compared with the control, Vδ1 cells transduced with the DAP10 CAD of the present disclosure significantly reduced THP1 cell viability, such as Fig. 20A As depicted in the diagram, and does not target healthy PBMCs, such as Fig. 20B as shown in the chart.

[0078] Fig.21 is a graph showing fold expansion of Vδ1 cells from six different donors in small-scale shake flask expansion. Fold expansion was measured at day 14. The data compares Vδ1 cells transduced with DAP10.6 and DAP10.16 CAD.

[0079] Figure 22A-22C is a graph illustrating the expansion kinetics of Vδ1 cells transduced with leading DAP10 CADs of the present disclosure (DAP10.6, DAP10.16, DAP10.17) compared to controls. Fig.22A The data at correspond to Vδ1 cells obtained from the first donor (SCT06), Fig. 22B The data at correspond to Vδ1 cells obtained from a second donor (SCT29), and Fig. 22C The data at correspond to Vδ1 cells obtained from a third donor (SCT45). Figure 22A-22C For each, % Vδ1 cells were measured as a function of expansion time (days).

[0080] Fig.23A A schematic diagram is depicted representing the process used to generate "off-the-shelf" allogeneic DAP10CAD Vδ1 cells.

[0081] Figure 23B-Figure 23D is a graph showing independent expansion of Vδ1 cells transduced with a preferred DAP10 CAD construct of the present disclosure. The Vδ1 cells used in the depicted experiments were obtained from three different donors (SCT06, SCT29, SCT45), respectively.

[0082] Figure 23E-23F is a graph illustrating that ex vivo culture of Vδ1 cells resulted in a significant fold expansion ( Fig.23E ) and robust DAP10 CAD transduction ( Fig.23F ). Fig.23E The data are expressed as Vδ1 fold expansion, while Fig.23F Data are expressed as %DAP10 CAD of Vδ1 cells.

[0083] Figure 23GDepicted are graphs illustrating the cell composition of Vδ1 cells, Vδ2 cells, αβ cells, and NK cells over time, expressed as a percentage of the culture.

[0084] Figure 24A-Figure 24C is a diagram illustrating the use of Raji cells ( Fig.24A )、Mino ​​cells( Fig. 24B ) and MOLP-8 cells ( Fig.24C ) Results of restimulation assays on Vδ1γδ cells co-expressing CAR constructs and various DAP10 CAD constructs. DETAILED DESCRIPTION

[0085] The present invention provides mammalian cells and compositions thereof, comprising a chimeric adapter (CAD) polypeptide, the CAD polypeptide comprising at least one of a DAP10 domain, a co-stimulatory domain and / or an intracellular signaling domain, and specifically lacking an extracellular domain comprising a ligand binding domain, wherein the mammalian cell also expresses at least one chimeric receptor, the at least one chimeric receptor comprising an extracellular targeting domain that specifically binds to a target antigen on a target cell. In an embodiment, the at least one chimeric receptor comprises an extracellular targeting domain and a DAP10 interaction domain, optionally further comprising at least one co-stimulatory domain and / or at least one intracellular signaling domain. In an embodiment, the CAD polypeptide of the present invention may also comprise a transmembrane domain (e.g., SEQ ID NO: 79) and / or an extracellular domain (e.g., SEQ ID NO: 80 or other extracellular spacer domains), but specifically lacks a functional extracellular receptor and / or a ligand binding domain. In contrast, the prior art generally uses DAP10 as a component of a CAR or NKG2D fusion chimera. 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 and chimeric receptors of the present invention are significantly different from the CAD polypeptides found in nature, generally comprising at least two polypeptide domains that are not naturally linked together, and optionally further comprising additional favorable signaling domains and mutations as described in detail herein.

[0086] The CAD polypeptide of the present invention preferably comprises a DAP10 domain comprising human DAP10, wherein the DAP10 domain optionally comprises one or more substitution mutations, deletion mutations and / or addition mutations. For example, the DAP10 domain may have a Y86F mutation and / or a K84R mutation.

[0087] The CAD polypeptides of the present disclosure and the "costimulatory domain" in the context of chimeric receptors in some embodiments enhance cell proliferation, cell survival and memory cell development of cytotoxic cells expressing CAD polypeptides and / or chimeric receptors. The CAD polypeptides and / or chimeric receptors of the present invention may include one or more costimulatory domains, which are selected from the costimulatory domains of proteins in the following TNFR superfamily: CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3 or a combination thereof. If CAD or a chimeric receptor includes more than one costimulatory domain, these domains can be arranged in series, optionally separated by a linker. The costimulatory domain is an intracellular domain that may be located between the DAP10 domain and an optional intracellular signaling domain in CAD.

[0088] In embodiments, the costimulatory domain includes the costimulatory domain of CD28, CD27, ICOS, 4-1BB, OX40 and CD40L. As used herein, the term "costimulatory domain" also encompasses any modification 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 respective contents of the documents are incorporated herein in their entirety.

[0089] The "intracellular signaling domain" in the context of the CAD polypeptides of the present disclosure and, in some embodiments, chimeric receptors converts effector function signals and directs cytotoxic cells to perform their specialized functions, i.e., to injure and / or destroy target cells. Examples of suitable intracellular signaling domains include, for example, the ζ chain of the T cell receptor complex or any homolog thereof, such as the η chain, FcsRly and β chain, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 ζ chain, CD3 polypeptide (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28. In embodiments, the intracellular signaling domain of the CAD polypeptide and / or chimeric receptor can be a human CD3 ζ chain, FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine activation motif (ITAM), and combinations thereof.

[0090] 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 co-stimulatory receptors, 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). Other intracellular signaling domains include signaling domains used by NK 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, the intracellular signaling domain also includes the signaling domain of human immunoglobulin receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, and FcRL5 (Gillis et al., "Contribution of Human Fc.gamma.Rsto Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies," Front Immunol., Vol. 5, p. 254, 2014).

[0091] In embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In exemplary embodiments, the intracellular signaling domain in the CAD and / or chimeric receptor comprises a cytoplasmic signaling domain of human CD3 zeta.As used herein, the term "intracellular signaling domain" also encompasses any modifications thereof, examples of which are described in U.S. Patent Application No. 2020 / 0317777 and Combadière, 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, HT., (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.

[0092] In an embodiment, two or more components of the CAD polypeptide of the present invention and / or chimeric receptor can be separated by one or more joints. A joint is an oligopeptide or polypeptide region of about 1 to 100 amino acids in length. In some embodiments, the length of a joint can be, for example, 5 to 12 amino acids, 5 to 15 amino acids, or 5 to 20 amino acids. The joint can be composed of flexible residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer joints, such as joints with a length of more than 100 amino acids, can be used in conjunction with alternative embodiments of the present invention, and can be selected to, for example, ensure that two adjacent domains do not interfere with each other spatially. Examples of joints that can be used for the present invention include, but are not limited to, 2A joints (such as T2A), 2A-like joints, or their functional equivalents.

[0093] In one exemplary embodiment, a chimeric DAP10-4-1BB adapter polypeptide is provided, which comprises a DAP10 domain and a 4-1BB costimulatory domain. In another exemplary embodiment, a chimeric DAP10-CD28 adapter polypeptide is provided, which comprises a DAP10 domain and a CD28 costimulatory domain. In another exemplary embodiment, a chimeric DAP10-4-1BB-CD3ζ adapter polypeptide is provided, which comprises a DAP10 domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. In another exemplary embodiment, a chimeric DAP10-CD28-CD3ζ adapter polypeptide is provided, which comprises 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 adapter polypeptide is provided, which comprises a DAP10 domain, a 4-1BB costimulatory domain, a CD28 costimulatory domain, and a CD3ζ intracellular signaling domain.

[0094] The chimeric adaptor polypeptide of the present invention may also optionally include a transmembrane domain. The transmembrane domain of CAD is a region that can span the plasma membrane of cytotoxic cells. The transmembrane domain is selected from the transmembrane region of a transmembrane protein, such as a type I transmembrane protein, an artificial hydrophobic sequence, or a combination thereof. Suitable examples of transmembrane domains include transmembrane regions of α, β, or ζ chains of T cell receptors CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may include triplets of phenylalanine, tryptophan, and valine. Optionally, a short oligopeptide or polypeptide linker (preferably between 2 and 10 amino acids in length) may form a bond between the transmembrane domain of CAD and the intracellular signaling domain. Glycine-serine doublets provide a particularly suitable linker between a transmembrane domain and an intracellular signaling domain.

[0095] The chimeric adaptor polypeptide of the present invention may also optionally include an extracellular spacer domain. The extracellular spacer domain of CAD is a hydrophilic region that is typically located between the ligand binding domain (not present in the CAD polypeptide of the present invention) and the transmembrane domain. In some embodiments, this domain promotes the correct protein folding of CAD. The extracellular spacer domain may include a domain selected from the following: an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include a CD8a hinge, an artificial spacer made of a polypeptide that may be as small as three glycine (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0096] definition

[0097] For the purpose of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition shown conflicts with any document incorporated herein by reference, the definition set forth below shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0098] As used herein, "about" when referring to a measurable value such as an amount, duration, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

[0099] As used herein, "optional" or "optionally" means that a particular limitation, event, circumstance, etc. can but does not have to occur, and that the description includes instances where the limitation, event, or circumstance occurs and instances where it does not.

[0100] As used herein, the term "DAP10" refers to a transmembrane adaptor protein present in lymphoid and myeloid cells of mammals, the exact sequence of which may vary slightly from species to species, isoform to individual. Alternative names for DAP10 recognized in the art include hematopoietic cell signal transducer (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 primary polypeptide sequence UnitProtQ9UBK5 and NCBI accessions NP_055081.1 and AF072845; however, different isoforms and variants may exist. Although the name DAP10 may refer to a variety of proteins with related structures and polypeptide sequences from different species, for protein members of the DAP10 protein family with a high degree of sequence identity to human DAP10 (SEQ ID NO: 1), a technician will be able to identify human DAP10-related proteins in mammals, even if they are different from the sequences cited herein. Full-length DAP10 (SEQ ID NO: 1) can be processed in cells into a mature form (SEQ ID NO: 78) without the signal peptide SEQ ID NO: 93.

[0101] As used herein, the term "host cell" refers to a cell type selected to express the CAD polypeptide of the present disclosure and at least one chimeric receptor. The host cell may endogenously express at least one receptor associated with DAP10, and by extension express the chimeric adapter polypeptide of the present disclosure. The host cell may be engineered to express at least one chimeric receptor associated with DAP10, and by extension express the chimeric adapter polypeptide of the present disclosure. Exemplary host cells may include, but are not limited to, a variety of immune cells, particularly including cytotoxic cells, preferred examples of which are disclosed herein (e.g., γδT cells, αβT cells, NK cells, NKT cells, B cells, neutrophils, monocytes / macrophages). Within the scope of the present disclosure, "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.).

[0102] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses or has expressed CD3 (CD3+) and T cell receptor (TCR+). T cells play a central role in cell-mediated immunity. T cells that "express" CD3 and TCR are engineered to eliminate CD3 and / or TCR cell surface expression.

[0103] As used herein, the term "γδT cells" refers to a subset of T cells that express different T cell receptors (TCRs) (i.e., γδTCRs) on their surface, which are composed of one γ chain and one δ chain. The term "γδT cells" specifically includes all subsets of γδT cells, including but not limited to Vδ1, Vδ2, and Vδ3γδT cells, as well as initial, effector memory, central memory, and terminally differentiated γδT cells. As other examples, the term "γδT cells" includes Vδ4, Vδ5, Vδ7, and Vδ8γδT cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11γδT cells. In some embodiments, the γδT cell is Vδ1 - 、Vδ2 - or Vδ1 - and Vδ2 - Compositions and methods for preparing and using engineered and non-engineered γδT cells and / or subtypes thereof include, but are not limited to, those described in US2016 / 0175358; WO 2017 / 197347; US 9499788; US2018 / 0169147; US 9907820; US 2018 / 0125889 and US2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including the compositions and methods for preparing and using engineered and non-engineered γδT cells and / or subtypes thereof. The present application further contemplates expressing a Chain or a TM A T cell or other engineered leukocyte or lymphocyte expressing a ... lymphocyte chain, optionally in combination with a second polypeptide to form a functional TCR. Chain or a TM Such engineered leukocytes or lymphocytes containing such chains can be used in the methods described herein or be present in the compositions described herein.

[0104] The γδT cells described herein may be δ1, δ2, δ3, or δ4 γδT cells, or a combination thereof. In some cases, the γδT cells are primarily (>50%), substantially (>90%), substantially all, or completely δ2 γδT cells. In some cases, the γδT cells are primarily (>50%), substantially (>90%), substantially all, or completely δ1 γδT cells. In some cases, the γδT cells are primarily (>50%), substantially (>90%), substantially all, or completely δ3 γδT cells.

[0105] γδT cells for use as used herein may be obtained from allogeneic or autologous donors. γδT cells may be partially or completely purified, or not purified, and may be amplified ex vivo. Methods and compositions for ex vivo amplification include, but are not limited to, those described in WO 2017 / 197347. Amplification may be performed before or after, or before and after, the chimeric adaptor polypeptide of the present disclosure is introduced into γδT cells. Other additional or alternative amplification methods include the use of, for example, artificial antigen presenting cells (aAPCs), aminobisphosphonates, cytokine mixtures, and feeder cells (Cortés-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0106] As used herein, the term "αβT cells" refers to T cells that express the α and β chains of the TCR as part of a complex with a CD3 chain molecule. Each α and β chain contains a variable domain and a constant domain. αβT 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.

[0107] As used herein, the term "natural killer (NK) cells" refers to CD56 + CD3 - Granular lymphocytes, which play an important role in antiviral immunity and tumor immune surveillance, and constitute a key cell subset of the innate immune system (Godfrey J et al. Leuk Lymphoma 2012 53: 1666-1676). NK cells express a remarkably diverse repertoire of inhibitory and activating receptors on their cell surface that regulate their immune response. NK cells can kill transformed or infected cells by releasing perforins and granzymes or by using effector molecules of the tumor necrosis factor (TNF) family (such as TNF, TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand, which induce apoptosis of target cells). In addition, NK cells rapidly produce chemokines and cytokines after activation, including interferon (IFN)-γ, GM-CSF and IL-10, which recruit hematopoietic and non-hematopoietic cells of the host and affect their function. Unlike cytotoxic CD8 +Unlike T lymphocytes, NK cells can produce cytotoxicity against tumor cells without prior sensitization, and can also eliminate MHC-I negative cells (Narni-Mancinelli E et al. Int Immunol 2011 23:427-431). NK cells are considered to be quite safe effector cells because they can avoid cytokine storms (Morgan RA et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter D L et al. N Engl J Med 2011 365:725-733) and potentially fatal complications of on-target and off-tumor effects.

[0108] NK cells can be obtained from allogeneic or autologous donors. NK cells can be partially or completely purified, or not purified, and can be expanded in vitro. 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). Amplification can be performed before or after, or before and after, the introduction of chimeric DAP10 adapter polypeptides and / or chimeric receptors into NK cells. In short, but not limited to, the expansion of NK cells may include the use of engineered feeder cells, cytokine mixtures (e.g., IL-2, IL-15) and / or aAPC (Cortés-Selva, D et al., (2021) Trends Pharmacol Sci. 42 (1): 45-59).

[0109] In some embodiments, placental hematopoietic stem cell-derived natural killer (PNK) cells or immortalized cell lines (e.g., NK-92) can be engineered to express the chimeric adapter polypeptides disclosed herein. In other examples, NK cells that can be used to engineer the expression of the chimeric adapter polypeptides herein can be differentiated from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). As used herein, the term "natural killer T (NKT) cells" are T lineage cells with the same morphological and functional properties as T cells and NK cells. NKT cells are rapid responders of the innate immune system and mediate powerful immunomodulatory and effector functions in a variety of disease environments. Ligand recognition in NKT cells leads to rapid secretion of proinflammatory cytokines (such as IFN-γ and TNF-α) and anti-inflammatory cytokines (such as IL-4, IL-10, and IL-13), which enhance immune responses to, for example, cancer by directly targeting tumor cells and by indirectly regulating anti-tumor responses (by releasing different cytokines) or by changing TME. Upon activation, NKT cells can immediately begin cytokine secretion without first differentiating into effector cells. The rapidity of their response makes NKT cells play an important role in the first line of innate defense against some types of bacterial and viral infections. In addition, many cytokines secreted by NKT cells have a powerful 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 that 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 not purified, and can be expanded ex vivo. Briefly, NKT cells can be expanded through the use of ex vivo IL-2 and / or monoclonal antibodies specific for the TCR α chain CDR3 loop (Cortés-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1):45-59).

[0110] As used herein, the term "γδ natural killer T cells" or "γδ NKT cells" refers to iPSC-derived cells that express γδ TCRs and NK receptors but lack expression of hallmark γδ T cell markers (Cortés-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59). These cells have been shown to have anti-tumor activity against a large number of cancer cell lines, but not against normal cells, and exhibit 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 the embodiments herein, the chimeric adapter polypeptides and chimeric receptors can be expressed in γδ NKT cells for use according to the methods disclosed herein.

[0111] As used herein, the term "myeloid cells" refers to a subpopulation of leukocytes represented by granulocytes, monocytes, macrophages, and dendritic cells (DCs). They circulate through the blood and lymphatic system and are rapidly recruited to tissue damage and infection sites via various chemokine receptors. Within the tissue, they are activated to perform phagocytosis and secrete inflammatory cytokines, thereby playing an important role in protective immunity. Myeloid cells are also found in tissues under steady-state conditions, where they control development, homeostasis, and tissue repair.

[0112] As used herein, the term "macrophage" refers to an innate cell with a high degree of plasticity, whose functional and phenotypic characteristics can be formed in response to various stimuli. Macrophage polarization is roughly simplified into two different states, the M1 phenotype (classical activation) in response to factors such as lipopolysaccharide (LPS) or IFN-γ, or the M2 phenotype in response to cytokines such as IL-4, IL-5 and IL-13. Examples of M1-like macrophages express iNOS and proinflammatory cytokines such as TNF-α, IL1-β, IL-6, IL-12 and IL-23. Examples of M2 macrophages show increased expression of CD209, CD200R, CD1a and CD1b in humans and are associated with wound healing and anti-tumor responses. The ability of macrophages to infiltrate solid tumors and reprogram, as well as the anti-tumor effects associated with conversion to the M1 phenotype, makes macrophages relevant to the present disclosure in terms of engineered macrophages expressing the chimeric adapter polypeptides described herein. For example, it has been demonstrated that in a mouse ovarian cancer model, macrophages can be reprogrammed into anti-tumor M1 phenotype cells that are capable of producing nitric oxide and inducing IL-12-dependent NK-mediated anti-tumor effects by inhibiting NK-κB signaling (Zhang F et al., (2019) Nat Commun 10:3974).

[0113] Macrophages can be obtained from / derived from allogeneic or autologous donors. Macrophages can be partially or completely purified, or not purified, and can be cultured in vitro (see, e.g., Davies JQ and Gordon A (2005) Methods Mol Biol 290: 105016). In some embodiments, the disclosure encompasses macrophages derived from hESC (Karlsson, KR et al., (2008) Exp Hematol 36: 1167-1175) or iPSC-derived macrophages (Takata K. et al., (2017) Immunity 47: 183-198).

[0114] As used herein, the term "NKG2D receptor" refers to a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D acts as a major activating receptor, in which ligand binding triggers cytotoxicity and cytokine production. NKG2D provides co-stimulation through the related adapter molecule DAP10, which recruits phosphatidylinositol-3 kinase. In mice, NKG2D also associates with DAP12, which recruits protein tyrosine kinases. NKG2D is encoded by the KLRK1 gene, which is located in the NK gene complex (NKC), which is located in chromosome 6 in mice and chromosome 12 in humans. In humans, NKG2D is expressed by NK cells, γδT cells, CD8+αβT cells, and 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, γδT cells, activated CD8+αβT cells and activated macrophages. The full-length human NKG2G amino acid sequence is shown herein as SEQ ID NO: 74, the amino acid sequence of the human NKG2D transmembrane domain is shown herein as SEQ ID NO: 75, and the amino acid sequence of the transmembrane and extracellular ligand binding domains of NKG2D is shown herein as SEQ ID NO: 76.

[0115] As used herein, the term "recombinant mammalian cell" refers to a cell or cell line derived from a mammal, which comprises at least one change brought about by genetic engineering technology. In some embodiments, the "recombinant mammalian cell" is a γδT cell, or a NK cell, or a NKT cell, or an αβT cell, etc., which comprises a nucleic acid construct encoding a chimeric DAP10 adaptor polypeptide and / or a chimeric receptor, preferably a chimeric DAP10 adaptor polypeptide and a chimeric receptor. The "recombinant mammalian cell" can be derived from any mammal, such as, for example, a human, a rodent, etc.

[0116] As used herein, the term "TCR" or "T cell receptor" refers to a dimeric heterologous cell surface signaling protein that forms an α-β or γ-δ receptor or a combination thereof. αβTCR recognizes antigens presented by MHC molecules, while γδTCR can recognize antigens independent of MHC presentation.

[0117] The term "MHC" (major histocompatibility complex) refers to a subset of genes encoding cell surface antigen presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. In this article, the abbreviations MHC or HLA are used interchangeably.

[0118] As used herein, the term "antigen" or "Ag" is defined as a molecule that causes an immune response. This immune response may involve antibody production, or the activation of specific immune competent cells, or both. It will be understood by those skilled in the art that any macromolecule, including proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant DNA or genomic DNA. It will be understood by the technician that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response thus encodes the term "antigen" used herein. In addition, it will be understood by those skilled in the art that antigens do not necessarily have to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present disclosure includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to cause a desired immune response. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded by "genes" at all. It is obvious that antigens can be generated, synthesized, or can be derived from biological samples. Such biological samples can include but are not limited to tissue samples, tumor samples, cells or biological fluids.

[0119] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural source or a recombinant source, and can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibodies of the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with multiple epitope specificities, multivalent antibodies, multispecific antibodies formed by at least two intact antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity), diabodies, single domain antibodies (sdAb), as long as they exhibit the desired biological or immunological activity, single chain fragment variable (scFv), Fab and F(ab')2, 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).

[0120] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant that can specifically bind to an immunoglobulin or receptor (e.g., a T cell receptor). Epitope determinants are usually composed of active surface groups of molecules (such as amino acids, lipids or sugar side chains) and usually have specific three-dimensional structural characteristics and specific charge characteristics.

[0121] As used herein, "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue / position. "Modification" of an amino acid residue / position is synonymous with "mutation" of an amino acid residue / position. For example, typical modifications include replacing a residue (or at the position) with another amino acid (e.g., conservative or non-conservative substitution), inserting one or more amino acids, and deleting one or more amino acids. "Amino acid substitution" or its variants refers to replacing an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Generally and preferably, compared to a polypeptide comprising an initial (or "wild type") amino acid sequence, modification results in a change in at least one physical and biochemical activity of a variant polypeptide. Therefore, as referred to herein, "modified" amino acid sequences include amino acid sequences in which one or more amino acids have been mutated, and / or amino acid sequences in which any number of amino acids have been inserted, and / or amino acid sequences in which any number of amino acids have been deleted.

[0122] As used herein, the term "endogenous" refers to substances and / or processes originating 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 in a cell or tissue.

[0123] In contrast, as used herein, the term "exogenous" refers to substances and / or processes originating from outside the system, including but not limited to organisms, tissues or cells. Specifically, "exogenous" in the context of the present disclosure means nucleic acid molecules or polypeptides that do not naturally occur in cells. Therefore, the term "exogenous" encompasses any foreign or heterologous recombinant nucleic acid molecules or polypeptides expressed in cells, including exogenous nucleic acids with different sequences relative to their natural endogenous counterparts. As is well known in the art, these exogenous sequences can be introduced into the cell itself or its progenitor cells by genetic engineering, and can be optionally connected to alternative control sequences, such as non-natural promoters or secretory sequences.

[0124] As used herein, the term "overexpression" refers to an expression level that exceeds the endogenous expression level of a 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, wherein 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 an expression vector that is the same or different from an expression vector encoding a CAD polypeptide. There are no particular limitations on the expression vector, as long as the vector can be used for genetic engineering. For example, plasmid vectors, viral vectors, cosmid vectors, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), and any other non-plasmid vectors can be used.

[0125] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancerous conditions. An "anti-tumor effect" can also be manifested by the ability of the polynucleotides and cells of the present disclosure to initially prevent tumorigenesis.

[0126] As used herein, the term "autologous" is intended to refer to any material derived from an individual that is subsequently reintroduced into the same individual.

[0127] As used herein, the term "allogeneic" refers to material derived from an animal that is subsequently introduced into a different animal of the same species.

[0128] As used herein, the term "homologous" refers to material that is genetically similar or identical and therefore immunologically compatible, such that transplantation will not elicit an immune response.

[0129] 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, or other molecule of interest (e.g., 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 is an agent that is capable of inducing a desired response alone or with other agents (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject suffering from cancer, viral infection (e.g., cytomegalovirus (CMV), influenza, hepatitis B, Epstein-Barr virus (Epstein-Barr), adenovirus, etc.), bacterial infection (e.g., Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tuberculosis), etc.), rheumatoid arthritis (RA) or other diseases / disorders). The agents discussed herein may be referred to as modulators.

[0130] As used herein, the term "diagnosis" or "diagnosing" refers to the process of identifying a disease (such as cancer) through its signs, symptoms, and / or the results of various tests. The conclusion drawn through this process is the diagnosis. Commonly performed tests include blood tests, medical imaging, urine analysis, biopsy, etc.

[0131] The term "therapeutically effective amount" (or simply "effective amount") refers to the amount of an agent or composition (e.g., a composition comprising an agent) that will elicit a biological or medical response in a tissue, system, or subject that is sought by a researcher, veterinarian, medical doctor, or other clinician. The term "therapeutically effective amount" includes an amount of an agent or composition comprising an agent that, when administered, is sufficient to prevent the development of one or more signs or symptoms of the condition or disease being treated (e.g., a hematological tumor or a solid tumor) or to alleviate the signs or symptoms to some extent. The therapeutically effective amount will vary depending on the composition, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0132] As used herein, the term "treating" a disease means reducing or decreasing the frequency or severity of at least one sign or symptom of the disease or condition experienced by a subject.

[0133] As used herein, the term "reduce" means to reduce the quality, amount, or intensity of something. In one example, therapy (e.g., administration of a therapeutic agent of the present disclosure) reduces one or more signs or symptoms associated with a disease or disorder, such as compared to the response in the absence of treatment. For example, in an example, administration of a therapeutic agent can provide an anti-tumor effect that reduces one or more signs or symptoms associated with cancer.

[0134] As used herein, the term "administering" means providing or giving one or more agents to a subject by any effective route, such as an agent that treats 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 (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0135] As used herein, the term "pharmaceutically acceptable" refers to a material (including but not limited to salt, carrier or diluent) that does not eliminate the biological activity or properties of a compound, and is relatively nontoxic, i.e., the material can be applied to an individual without causing an undesirable biological effect or interacting with any component contained in the composition in a harmful manner. Pharmaceutically acceptable carriers (vehicles) that can be used for the present disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W.Martin, Mack Publishing Co., Easton, Pa., the 19th edition (1995) describes compositions and preparations suitable for drug delivery of one or more agents (such as one or more regulators). Generally speaking, the properties of the carrier depend on the specific mode of administration adopted. For example, parenteral preparations can include injectable fluids, which include pharmaceutically and physiologically acceptable fluids (such as water, saline, balanced salt solution, dextrose aqueous solution, glycerol, etc.) as vehicles. In addition to biologically neutral carriers, the pharmaceutical preparation 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.

[0136] As used herein, the term "cytokine" refers to a variety of soluble proteins and peptides released from cells that act as humoral regulators at nanomolar to picomolar concentrations and regulate the functional activity of individual cells and tissues under normal or pathological conditions. These proteins also directly mediate interactions between cells and regulate processes occurring in the extracellular environment. Many growth factors and cytokines act as cell survival factors by preventing programmed cell death. Cytokines include naturally occurring peptides and variants that retain all or part of their biological activity.

[0137] "Coding" refers to the specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA or mRNA) that serves as the intrinsic properties and resulting biological properties of the template for synthesizing other polymers and macromolecules with a limited nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a limited amino acid sequence in a biological process. Therefore, if the transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes the protein. The coding strand (whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a template for gene or cDNA transcription) can be referred to as encoding a protein or other product of the gene or cDNA.

[0138] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially pure form, or may exist in a non-natural environment such as, for example, a host cell.

[0139] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0140] 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.

[0141] As used herein, the term "specific binding" with respect to cell surface receptors means a receptor that recognizes a specific molecule / ligand but does not substantially recognize or bind to other molecules in the sample. For example, a receptor that specifically binds to a molecule from one species can also bind to the molecule from one or more species. However, this cross-species reactivity itself does not change a specific classification. In another example, a receptor that specifically binds to a molecule can also bind to different allelic forms of the molecule. However, this cross-reactivity itself does not change a specific classification. In some cases, the term "specific binding" or "specifically binding" can be used to refer to the interaction of a protein (or peptide) with a second chemical substance, meaning that the interaction depends on the presence of a specific structure (such as an antigenic determinant or epitope) on the chemical substance; for example, the receptor recognizes and binds to a specific structure rather than a common protein. If the receptor is specific for epitope "A", then in the reaction of labeled "A" and receptor, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the receptor.

[0142] In some embodiments, specific binding can be achieved by at least about 1×10 -8 The specific binding of a molecule may be characterized by an equilibrium dissociation constant of M or less (eg, a smaller KD indicates tighter 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.

[0143] As used herein, the term "cancer" refers to a physiological condition in mammals in which a cell population is characterized by uncontrolled cell growth. Neoplasia, malignant tumor, cancer and tumor are used interchangeably and refer to abnormal growth of tissues or cells due to excessive cell division. The amount of tumor in an individual is the "tumor burden", which can be measured by the number, volume or weight of the tumor. Tumors that do not metastasize are referred to as "benign". Tumors that invade surrounding tissues and / or can metastasize are referred to as "malignant". "Non-cancerous tissue" is tissue from the same organ that forms a malignant tumor, but does not have the characteristic pathology of the tumor. Generally speaking, non-cancerous tissue appears normal histologically. "Normal tissue" is tissue from an organ, wherein the organ is not affected by cancer or other diseases or conditions of the organ. "Cancer-free" subjects have not been diagnosed with cancer of the organ and do not suffer from detectable cancer.

[0144] Symptoms of cancer may include, but are not limited to, a persistent cough or blood in the saliva, changes in bowel habits, blood in the stool, unexplained anemia (low blood cell counts), breast lumps or breast discharge, testicular lumps, changes in urination, blood in the urine, hoarseness, persistent lumps or swollen glands, noticeable changes in warts or moles, indigestion, difficulty swallowing, unusual vaginal bleeding or discharge, unexpected weight loss, night sweats or fever, persistent itching in the anal or genital area, ulcers that do not heal, headaches, back pain, pelvic pain, and bloating.

[0145] Hematological cancer is a cancer that originates from the blood or bone marrow. Examples of hematological (or blood-borne) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0146] A solid tumor is a tumor that contains a tumor mass of 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.

[0147] Examples of solid tumors include, for example, sarcomas, adenocarcinomas, and carcinomas of various organ systems, such as those affecting the liver, lungs, breasts, lymph nodes, gastrointestinal tract (e.g., colon), urogenital tract (e.g., kidneys, urothelial cells), pancreas, prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small intestinal cancers, and esophageal cancers. In one embodiment, the cancer is a melanoma, such as an advanced melanoma. In another embodiment, the cancer is a glioma. Metastatic lesions of the above cancers can also be treated or prevented using the methods and compositions disclosed herein.

[0148] An "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 expression elements; other expression elements may be provided by the host cell or in an in vitro expression system. An expression cassette may be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome) or a virus (e.g., a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus). An expression cassette may be in a host cell such as a γδ T cell.

[0149] Scope: Throughout this disclosure, various aspects of the disclosure may be presented in range form. It should be understood that the description in range form is merely for convenience and brevity, and should not be interpreted as an immutable limitation on the scope of the disclosure. Therefore, the description of a range should be considered to have explicitly disclosed all possible subranges and individual numerical values ​​within the range. For example, a range description such as 1 to 6 should be considered to have explicitly 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 the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0150] When referring to nucleic acids or fragments thereof, the term "substantially identical" or "substantially identical" means that when optimally aligned with another nucleic acid (or the complementary strand of another nucleic acid), the nucleotide sequence identity is %, e.g., 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% of the nucleotide bases, as measured by any well-known sequence identity algorithm (such as FASTA, BLAST, or GAP), as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule encodes a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0151] When applied to polypeptides, the term "substantially similar" 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 by using the programs GAP or BESTFIT with default gap weights). In some aspects, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is substituted 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 change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitutions. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids with side chains of similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy 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 acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine. Alternatively, conservative substitutions are any changes with positive values ​​in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45 (incorporated herein by reference). "Moderately conservative" substitutions are any changes with non-negative values ​​in the PAM250 log-likelihood matrix.

[0152] The sequence identity and / or similarity of polypeptides are usually measured using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT, and the program can use default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different organism species), or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) utilizing default or recommended parameters. FASTA (such as FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlap region between query sequence and search sequence (Pearson (2000) the same). Sequences can also be compared using Smith-Waterman homology search algorithm using affine gap search with gap open penalty (gap open penalty) of 12, and gap extension penalty (gap extension penalty) of 2, BLOSUM matrix of 62. When comparing the sequences disclosed herein to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., 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.

[0153] I. Introduction

[0154] The 10kDa DNAX activating protein (DAP10) is a linker molecule associated with the cell surface cytotoxic receptor natural killer cell group 2 member D (NKG2D). The NKG2D receptor is a type II transmembrane anchored C-type lectin-like protein, which belongs to the CD94 / NKG2 family of C-type lectin-like receptors (Houchins et al., (1991) J. Exp. Med. 173: 1017-1020). NKG2D is able to bind to a large number of highly diverse MHC class I-like self molecules. These ligands are usually poorly expressed in normal cells, but can be induced on 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, and their repertoire is more complex than that of other species. In fact, MIC molecules are encoded by the most polymorphic human genes after classical HLA molecules (Eagle, RA and Trowsdale, J. (Nat. Rev. Immunol. (2007) 7 (9): 737-44).

[0155] NKG2D is an activating immune receptor that regulates innate and adoptive immune responses. NKG2D is present in large quantities in all NK cells, CD8 T cells, γδT cell subsets, and some autoreactive CD4 T cells. NKG2D acts with other co-stimulatory molecules (such as DAP10) to change the intensity and duration of T cell receptor-mediated antigen-specific responses and affect the anti-tumor response pattern of T lymphocytes (see, e.g., Maccalli C et al. (2003) Eur. J. Immunol. 33 (7): 2033-43).

[0156] NKG2D lacks a signaling motif in its cytoplasmic domain. Therefore, after ligand binding, NKG2D signal transduction and cell activation depend on the association of NKG2D with the DAP10 adapter molecule, which promotes and stabilizes NKG2D surface membrane expression (Wu, J. et al., (1999) Science 285: 730-732). However, it is known that, for example, TGF-β can mediate the downregulation of NKG2D (and NKG2DL) surface expression (Lazarova M and Steinle. (2019) Front. Immunol 10 (2689): 1-11), and TGF-β can significantly reduce DAP10 expression at mRNA and protein levels (Park, YP et al. (2011) Blood. 118: 3019-27; Lee, JC et al. (2011) Tumori 97: 350-7). Thus, it is recognized herein that methods capable of modulating NKG2D and / or DAP10 expression and / or related signaling pathways have therapeutic implications.

[0157] Human NKG2D receptors assemble with DAP10 signaling dimers, where one NKG2D homodimer pairs with the DAP10 dimer by forming two salt bridges between conserved transmembrane (TM) arginine residues (Garrity, D. et al. (2005) PNAS USA 102(21):7641-7646). The DAP10 dimer carries a pair of aspartate residues near the center of the transmembrane (TM) domain, and these residues interact with the conserved arginine in the TM sequence of NKG2D, thereby assembling 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).

[0158] The DAP10 dimer is a disulfide-linked 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 3, which lists a number of sequences relevant to the present disclosure.

[0159] The DAP10 cytoplasmic domain contains a tyrosine-based motif (YINM) comprising residues 86-89 of SEQ ID NO: 1. Upon phosphorylation of tyrosine (Y86), DAP10 is able to bind to the p85 subunit of phosphatidylinositol 3-kinase (PI3K, via YXXM) or the adapter Grb2 (via YXNX). Since these two binding sites overlap, a single DAP10 chain will bind to either p85 or Grb2, but not both (Lanier LL. (2008) Nat. Immunol. 9(5): 495-502). This YINM motif is similar to the motif in CD28, which provides costimulatory signaling with the TCR / CD3 complex based on the immunoreceptor tyrosine activation motif (ITAM) in T cells.

[0160] The recruitment of the p85 subunit of PI3K or the adaptor protein Grb2 can activate Vav1 and PLC-γ2, thus activating Ca 2+ Mobilization and cytotoxicity to cells are crucial (Upshaw JL et al. (2006) Nat. Immunol. 7(5):524-32).

[0161] DAP10 also contains a ubiquitination site that encompasses a lysine at amino acid 84 of the DAP10 protein sequence (SEQ ID NO: 1). Ligand stimulation of NKG2D on NK cells results in DAP10 ubiquitination, 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). In addition, ubiquitin-dependent receptor endocytosis has been shown to be required for activation of extracellular signal-regulated kinases (ERKs) and NK cell functions such as secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. Therefore, NKG2D-DAP10 endocytosis represents a way to reduce cell surface receptor abundance and control signaling in cytotoxic lymphocytes.

[0162] In addition to NKG2D, DAP10 is known to associate with a variety of other receptors. For example, Ly49H and Ly49D co-immunoprecipitate with DAP10 in mouse NK cells and have been shown to associate with DAP10 after co-transfection into 293T cells (Coudert JD et al. (2008) Blood 111: 3571-3578). Through co-transfection studies, DAP10 has also been shown to associate 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 Cd3001b (Yamanishi Y et al. (2008) Blood 111:688-698) have been shown to pair with DAP10 by co-transfection and co-immunoprecipitation. As described above, some DAP10-associated receptors (i.e., NKG2D) appear to recognize host-encoded molecules, including carbohydrate and protein ligands, but other DAP10-associated receptors can directly recognize microbial ligands. One example includes Ly49H recognizing the mouse CMV-encoded glycoprotein m157 (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 homologous to MHC class I, which is displayed on the surface of mouse CMV-infected cells, thereby activating Ly49H+ NK cell-mediated cytotoxicity and cytokine production.

[0163] The above receptors are intended to illustrate the ability of DAP10 to pair with a variety of receptors other than NKG2D and thus modulate signal transduction through the receptors, and are not intended to be exhaustive. It should be understood that for purposes of the present disclosure, nucleic acids, polypeptides encoded therefrom, cells, compositions, and methods are applicable to any and all receptors with which DAP10 is capable of cooperating. For example, embodiments herein encompass chimeric receptors comprising a DAP10 interacting domain, thereby conferring the ability of the CAD polypeptides of the present disclosure to modulate signal transduction through one or more chimeric receptors expressed in the same cell as the CAD polypeptide, as disclosed herein.

[0164] II. Compositions and Methods of the Invention

[0165] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In particular, the disclosure utilizes conventional techniques in recombinant genetics, immunology, and biochemistry. The basic texts disclosing the general terms in molecular biology and genetics include, for example, Lackie, Dictionary of Cell and Molecular Biology, Elsevier (5th edition 2013). The basic texts disclosing the methods in 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, NY 2012) and Current Protocols in Molecular Biology Vol. 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplementary Materials 1-115 (1987-2016). Basic texts that disclose general methods and terminology in biochemistry include, for example, Lehninger Principles of Biochemistry, 6th edition, David L. Nelson and Michael M. Cox, ed. WH Freeman (2012). Basic texts that disclose general methods and terminology in immunology include Janeway's Immunobiology (9th edition) by Kenneth M. Murphy and Casey Weaver (2017) Garland Science; Fundamental Immunology (7th edition) by William E. Paul (2013) Lippincott, Williams and Wilkins.

[0166] A. Chimeric Adaptor Constructs

[0167] Aspects of the present disclosure include constructs comprising nucleic acids encoding chimeric adapter polypeptides. In embodiments, nucleic acids encode chimeric adapter polypeptides comprising i) human DAP10 amino acid sequences and ii) one or more costimulatory domains (e.g., 4-1BB, OX40, ICOS, CD28), wherein the CAD polypeptide specifically lacks an extracellular domain. In embodiments, the CAD polypeptide may additionally or alternatively comprise one or more intracellular signaling domains as described herein (e.g., CD3ζ). In embodiments, the CAD polypeptide may also comprise one or more mutations, such as one or more mutations in DAP10 and / or one or more modifications in a costimulatory domain and / or an intracellular signaling domain (e.g., one or more mutations, additions or deletions).

[0168] In an embodiment, the CAD polypeptide is used to regulate and / or inhibit signal transduction through one or more receptors associated therewith. In an embodiment, the CAD polypeptide is designed to have a regulated / inhibitory property (e.g., by one or more mutations) and / or an added signal transduction property (e.g., by C-terminal fusion), and is expressed in a host cell to promote a favorable balance of signal transduction pathways during receptor-target binding (e.g., binding of NKG2D to an extracellular target ligand), which can be used to solve the problem of low or lost target expression of the primary TCR (i.e., antigen escape). The term "favorable balance" broadly refers to the introduction of a signal cascade that complements the primary DAP10 signal transduction cascade, replaces the primary DAP10 signal transduction cascade, or regulates the primary DAP10 signal transduction cascade in a desired manner. Therefore, the CAD polypeptide disclosed herein provides altered (e.g., improved) functional properties, including but not limited to altered (e.g., enhanced) cell lysis, proliferation, survival and / or co-stimulatory properties, which are caused when binding to a receptor ligand that cooperates with DAP10 (e.g., a widely expressed NKG2D ligand). The precise composition of the CAD polypeptide can be designed according to a given disease indication, and in some instances, can be designed based on the specificity and signaling component pairing with other receptors present on the same cell (e.g., TCR receptors and / or chimeric receptors as disclosed herein). As an illustrative example, immunosuppressive signals within the tumor microenvironment (TME) can inhibit anti-tumor T cell responses through inhibitory receptors on T cells, and within the scope of the present disclosure, such inhibitory output can be converted into immunostimulatory output via the use of CAD polypeptides. Other related examples can be found, for example, in Guo, J. et al., (2021) Journal for ImmunoTherapy of Cancer 9: e002628.

[0169] In an embodiment, the CAD polypeptide can be used, at least in part, to stabilize a cell surface receptor associated therewith (e.g., NKG2D and / or chimeric receptors as disclosed herein). As disclosed herein, "stabilizing" a cell surface receptor means reducing the rate at which the cell surface receptor is endocytosed or otherwise removed from the cell surface compared to the rate at which the cell surface receptor is otherwise removed in a similar situation in the absence of the CAD polypeptide as described herein. Included within the scope of receptor stabilization as described herein are positive feedback mechanisms by which CAD polypeptide signaling results in increased cell surface expression of receptors associated with endogenous DAP10 and CAD polypeptides (Wu, J. et al., (2000) Journal of Exp Med 192(7): 1059-1068).

[0170] The present disclosure also provides stably expressed CAD polypeptides. In embodiments, the expression level of the CAD polypeptide is substantially similar to the expression level of endogenous DAP10 in the host cell of interest. In embodiments, the expression level of the CAD polypeptide is higher than the expression level of endogenous DAP10 in the host cell of interest. For example, the expression level of the CAD polypeptide may be 10%, or 10-20%, or 20-30%, or 30-40%, or 40-50%, or 50-60%, or 60-70%, or 70-80%, or 80-90%, or 90-100%, or even higher, such as 2 times, or 3 times, or 4 times, or 5 times, or 6 times, or 7 times, or 8 times, or 9 times, or 10 times, or 20 times, or 30 times, or 40 times, or 50 times or 100 times higher than the corresponding expression level of endogenous DAP10. Thus, it will be appreciated that, in embodiments, CAD polypeptides of the invention may compete with endogenous DAP10 for binding to a receptor (eg, NKG2D), depending on the particular host cell type.

[0171] Aspects of the present disclosure include nucleic acids encoding CAD polypeptides, and constructs / vectors containing such nucleic acids. Thus, nucleic acids encoding CAD polypeptides are described herein, incorporating selective mutations and signaling domains that modulate and / or add signaling properties that confer desired properties, including, but not limited to, sustained survival, proliferation, and / or killing on host cells expressing the CAD polypeptides. In some embodiments, the nucleic acid encodes a CAD polypeptide comprising a signal peptide at its N-terminus. A signal peptide is an amino acid sequence in a protein (e.g., at the N-terminus of a protein) that is used to translocate a protein to a specific intracellular location (e.g., the endoplasmic reticulum for secretion). In some embodiments, the nucleic acid-encoded CAD comprises mature Dap10 (e.g., SEQ ID NO: 78) or a fragment thereof (e.g., SEQ ID NO: 79, 80, or 81), and a signal peptide SEQ ID NO: 93 at the N-terminus of mature Dap10 or a fragment thereof. In embodiments, the nucleic acid encoding CAD comprises mature Dapl0 (e.g., SEQ ID NO: 78) or a fragment thereof (e.g., SEQ ID NO: 79, 80, or 81), and a signal peptide at the N-terminus of mature Dapl0 or a fragment thereof of SEQ ID NO: 45. In some embodiments, the nucleic acid encoding CAD comprises mature Dapl0 (e.g., SEQ ID NO: 78) or a fragment thereof (e.g., SEQ ID NO: 79, 80, or 81), and a signal peptide comprising SEQ ID NO: 45 and SEQ ID NO: 93 (e.g., SEQ ID NO: 45 at the N-terminus of SEQ ID NO: 95).

[0172] 1. Mutation

[0173] In an embodiment, the CAD polypeptide carries one or two 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, such as K84R or K84H, although within the scope of the present disclosure, the modification at K84 may include other amino acid substitutions. In one embodiment, the modification is a K84R modification. In an embodiment, a CAD polypeptide with a K84R modification comprises SEQ ID NO: 18. In an embodiment, Y86 is modified to another aromatic amino acid, such as Y86F or Y86W, although within the scope of the present disclosure, the modification at Y86 may include other amino acid substitutions. In one embodiment, the modification is a Y86F modification. In an embodiment, a CAD polypeptide with a Y86F modification comprises SEQ ID NO: 19. In an embodiment, a CAD polypeptide with a K84R modification and a Y86F modification comprises SEQ ID NO: 20.

[0174] In an embodiment, the modification at position 86 of SEQ ID NO:1 reduces or eliminates the binding of p85 / PI3K to the CAD polypeptide, thereby reducing or eliminating PI3K / AKT / PKCθ signaling. Therefore, the modification at position 86 can be functionally used to reduce or eliminate one or more of co-stimulation, calcium flux and / or degranulation. In a preferred embodiment, the modification is Y86F.

[0175] In additional or alternative embodiments, the modification at position 86 of SEQ ID NO: 1 reduces or eliminates the binding of Grb2 to the CAD polypeptide, thereby reducing or eliminating Vav1 / SLP-76 / PLCγ signaling. Thus, the modification at position 86 can be used to reduce or eliminate one or more of calcium influx and / or degranulation. In a preferred embodiment, the modification is Y86F.

[0176] In additional or alternative embodiments, modification at position 84 of SEQ ID NO: 1 reduces or completely abolishes ubiquitination of the CAD polypeptide, which in turn reduces or completely prevents internalization of the chimeric DAP10 adaptor polypeptide-endogenous receptor complex at the cell membrane of a particular host cell (see, e.g., Quatrini, L. et al., (2015) Sci Signal 8(400):ra108). In this way, receptors associated with DAP10 (e.g., NKG2D and / or chimeric receptors disclosed herein) can be stabilized at the cell surface via reliance on a CAD polypeptide containing at least a K84 modification (e.g., K84R). In addition, modifications at K84 (e.g., K84R) can reduce or eliminate signaling (e.g., ERK1 / 2) that would otherwise occur en route to lysosomal degradation. Reduction or elimination of signaling (e.g., ERK1 / 2) can be used to reduce or eliminate one or more of: depletion, activation-induced cell death, and / or induction of cell cycle arrest upon overactivation, one or more of which can occur in the absence of a CAD polypeptide containing a K84 modification (e.g., K84R).

[0177] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 57 of SEQ ID NO: 1, such as a D57A modification, although modifications of amino acids other than alanine are also within the scope of the present disclosure. In embodiments, a chimeric DAP10 adaptor polypeptide having a D57A modification comprises SEQ ID NO: 37. In embodiments, modifications at D57 can be used to modify (e.g., reduce or eliminate) a stabilizing interaction with KLRK1 (Wu, J. et al., (1999) Science 285(5428):730-2).

[0178] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 88 of SEQ ID NO: 1, such as an N88Q modification, although modifications of amino acids other than glutamine are also within the scope of the present disclosure. In an embodiment, a CAD polypeptide having an N88Q modification comprises SEQ ID NO: 38. In an embodiment, the modification at N88 can be used to modify (e.g., reduce) cell killing activity and / or interaction with GRB2, while having little or no effect on the interaction with PIK3R1 (Upshaw, JL., (2006) Nat Immunol 7: 524-532).

[0179] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 89 of SEQ ID NO: 1, such as an M89Q modification, although modifications of amino acids other than glutamine are also within the scope of the present disclosure. In an embodiment, a CAD polypeptide having an M89Q modification comprises SEQ ID NO: 39. In an embodiment, the modification at M89Q can be used to modify (e.g., reduce) cell killing activity and / or interaction with PIK3R1, while having little or no effect on interaction with GRB2 (Upshaw, JL., (2006) Nat Immunol 7: 524-532).

[0180] It will be appreciated that the present disclosure encompasses chimeric adaptor polypeptides having any one or more, or each, of the above modifications.

[0181] 2. Co-stimulatory and signaling domains

[0182] In an embodiment, the intracellular domain of the chimeric adaptor polypeptide of the present disclosure comprises one or more costimulatory domains, wherein the costimulatory domain comprises a functional costimulatory signaling domain, and the functional costimulatory signaling domain is derived from, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, etc. For example, within the scope of the present disclosure, the intracellular domain of the disclosed CAD polypeptide may comprise 2, 3, 4 or more costimulatory domains. Also within the scope of the present disclosure, when more than one costimulatory domain is included, the costimulatory domains may be the same, or may be different.In embodiments, the costimulatory domain is derived from one or more of the following: 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, CD19aC D27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54(ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96(Tactile), CD100 (SEMA4D), CD103, CD134(OX40), CD137(4-1BB), CD152(CTLA-4), CD160(BY55), CD162(SELPLG), CD244(2B4), CD270(HVEM), CD2 26(DNAM1), CD229(Ly9), CD278(ICOS), ICAM-1, LFA-1(CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZA P70, GITR, BAFFR, LTBR, ​​LAT, GADS, LIGHT, HVEM(LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITG B2, ITGB7, NKG2C, NKG2D, IA4, 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 portions thereof, and combinations thereof.

[0183] In some embodiments, the CAD construct encodes at least one 4-1BB costimulatory domain, and optionally encodes a second costimulatory domain, which is selected from 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 costimulatory domains, or any of the above costimulatory domains. In some embodiments, the construct encodes at least two 4-1BB costimulatory domains, or a combination of at least two 4-1BB costimulatory domains and one, two, three, or four or more costimulatory domains, and the costimulatory domains are selected from 4-1BB, ICOS, CD28, OX40 and CD27, or any of the above costimulatory domains. In some embodiments, the 4-1BB costimulatory domain comprises SEQ ID NO: 2 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL). In some embodiments, the 4-1BB costimulatory domain comprises at least one, at least two or at least three or more modified amino acid sequences having an amino acid sequence of SEQ ID NO: 2. In an embodiment, the 4-1BB costimulatory domain is substantially similar to the 4-1BB costimulatory domain comprising SEQ ID NO:2.

[0184] In some embodiments, the CAD construct encodes at least one CD27 costimulatory domain, and optionally encodes at least one second costimulatory domain, the second costimulatory domain being selected from 4-1BB, ICOS, CD28, OX40, 2B4 and CD27 costimulatory domains, or any of the above costimulatory domains. 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 (QRRKYRSNKGESPV EPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP). In some embodiments, the CD27 costimulatory domain comprises at least one, at least two, at least three or more modified amino acid sequences having an amino acid sequence of SEQ ID NO: 5. In embodiments, the CD27 costimulatory domain is substantially similar to the CD27 costimulatory domain comprising SEQ ID NO:5.

[0185] In some embodiments, the CAD construct encodes at least one CD28 costimulatory domain, and optionally encodes a second costimulatory domain, which is selected from 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 costimulatory domains, or any of the above costimulatory domains. In some embodiments, the CAD construct encodes at least two CD28 costimulatory domains, or a combination of at least two CD28 costimulatory domains and one, two, three, or four or more costimulatory domains, and the costimulatory domains are selected from 4-1BB, ICOS, CD28, OX40 and CD27, or any of the above costimulatory domains. In some embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 40FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). In an embodiment, 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, which are capable of regulating signal transduction pathways. In an embodiment, the disclosed CAD polypeptide comprises a mutation or deletion of one or more of the subdomains (see, e.g., WO2019010383). In some embodiments, the CD28 costimulatory domain comprises at least one, at least two, at least three or more modified amino acid sequences having an amino acid sequence of SEQ ID NO: 40 or an amino acid sequence of SEQ ID NO: 41. In some embodiments, the CD28 costimulatory domain is substantially similar to the CD28 costimulatory domain comprising SEQ ID NO: 40. In some embodiments, the CD28 costimulatory domain is substantially similar to the CD28 costimulatory domain comprising SEQ ID NO: 41.

[0186] In some embodiments, the CAD construct encodes at least one ICOS costimulatory domain, and optionally encodes a second costimulatory domain, which is selected from 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 costimulatory domains or any of the above costimulatory domains. In some embodiments, the CAD construct encodes at least two ICOS costimulatory domains, or a combination of at least two ICOS costimulatory domains and one, two, three, or four or more costimulatory domains, and the costimulatory domains are selected from 4-1BB, ICOS, CD28, OX40 and CD27 or any of the above costimulatory domains. In some embodiments, the ICOS costimulatory domain includes SEQ ID NO: 42. In some embodiments, the ICOS costimulatory domain includes at least one, at least two, at least three or more modified amino acid sequences (see, e.g., US20170209492) of the amino acid sequence of SEQ ID NO: 42. In some embodiments, the ICOS costimulatory domain is substantially similar to the ICOS costimulatory domain comprising SEQ ID NO:42.

[0187] In some embodiments, the CAD construct encodes at least one OX40 costimulatory domain, and optionally encodes a second costimulatory domain, which is selected from 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 costimulatory domains, or any of the above costimulatory domains. In some embodiments, the CAD construct encodes at least two OX40 costimulatory domains, or a combination of at least two OX40 costimulatory domains and one, two, three, or four, or more costimulatory domains, and the costimulatory domains are selected from 4-1BB, ICOS, CD28, OX40 and CD27, or any of the above costimulatory domains. In some embodiments, the OX40 costimulatory domain comprises SEQ ID NO: 43 (RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI). In some embodiments, the OX40 costimulatory domain comprises at least one, at least two, at least three or more modified amino acid sequences having an amino acid sequence of SEQ ID NO: 43. In some embodiments, the OX40 costimulatory domain is substantially similar to the OX40 costimulatory domain comprising SEQ ID NO:43.

[0188] In an embodiment, the chimeric adapter polypeptide of the present invention includes one or more intracellular signaling domains. In an embodiment, one or more intracellular signaling domains are outside one or more costimulatory domains. In an embodiment, one or more intracellular signaling domains are included to increase the proliferation, persistence and / or cytotoxic activity of host cells (e.g., NK cells, NKT cells, γδ cells, etc.) containing CAD polypeptides as disclosed herein. For example, in some embodiments, the intracellular signaling domain includes CD3ζ, repeats (e.g., 2-5) DAP10 YINM motifs, derived from LFA-1, DAP12, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, etc. Signaling domains. Within the scope of the present disclosure, the intracellular domain of the disclosed chimeric adapter polypeptide may include multiple (e.g., 2, 3, 4 or more) intracellular signaling domains. Where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same, or may be different.

[0189] In some embodiments, the intracellular signaling domain is or comprises a CD3 zeta signaling domain. In some embodiments, the CD3 zeta signaling domain is or comprises RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:3), or RVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:4), or RVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO:5). ID NO: 82), also referred to herein as "1XX" (see, e.g., US2020 / 0317777, the contents of which are incorporated herein by reference in their entirety). Without being bound by theory, in some embodiments, inclusion of an 1XX signaling domain results in increased activation and / or survival of the modified immune cells of the present disclosure by limiting over-activation.

[0190] In some embodiments, the CAD construct encodes one or more costimulatory domains (e.g., 4-1BB costimulatory domain), and one or more intracellular signaling domains (e.g., CD3 ζ 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 CD3 ζ 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., CD3 ζ). In embodiments, the CD3 ζ signaling domain is located downstream (C-terminal) of the costimulatory domain (e.g., 4-1BB). In some embodiments, the CD3 ζ signaling domain is located upstream (N-terminal) of the costimulatory domain (e.g., 4-1BB).

[0191] 3. Cytokine Co-expression

[0192] In additional embodiments, the CAD construct of the present invention can also encode one or more polycistronic linker regions, which are configured to promote the translation of CAD polypeptides and one or more soluble common γ chain cytokines into separate polypeptides. In embodiments, the nucleic acid encoding the cytokine and the associated linker region can be located at the 3' end of the isolated nucleic acid, or at the 5' end of the isolated nucleic acid, or at the 5' end and 3' end of the isolated nucleic acid in some instances. One or more soluble common γ chain cytokines can 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 can encode self-cleavage and / or cleavage polypeptide sequences. In some instances, the self-cleavage sequence is a 2A self-cleavage sequence (e.g., T2A, P2A, E2A, F2A), which can induce ribosome jumping during the translation of the chimeric DAP10 adapter polypeptide. In embodiments, the cleavage sequence is a furin protease sequence. In some instances, the cleavage sequence (e.g., furin protease sequence) is the amino terminal of the self-cleavage sequence. In some embodiments, the polycistronic linker region encodes an internal ribosome entry site. In some embodiments, the polycistronic linker region comprises the sequence of any one of SEQ ID NO: 9-15 or SEQ ID NO: 44. In embodiments, the addition of an optional linker "GSG" or "SGSG" or the like can improve cleavage efficiency. In this way, the included one or more gamma chain cytokines can be released from the chimeric DAP10 adapter polypeptide and secreted by the host cell.

[0193] For example, in some embodiments, the cleavage sequence is the P2A cleavage sequence of SGSGATNFSLLKQAGD VEENPGP (SEQ ID NO: 9). In some embodiments, the P2A cleavage sequence is the P2A cleavage sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 44). In some embodiments, the cleavage sequence is the furin cleavage sequence RAKR (SEQ ID NO: 10). In some embodiments, the cleavage sequence is the P2A+furin cleavage (FP2A) sequence RAKRSGSGATNFSLLKQAG DVEE NPGP (SEQ ID NO: 11).

[0194] In some embodiments, the cleavage sequence is or comprises the P2A cleavage sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 12). In some embodiments, the cleavage sequence is or comprises the F2A cleavage sequence VKQTLNNFDLLKLAGDVESNPGP (SEQ ID NO: 13). In some embodiments, the cleavage sequence is or comprises the E2A cleavage sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14). In some embodiments, the cleavage sequence is or comprises the T2A cleavage sequence EGRSLLTCGDVEENPGP (SEQ ID NO: 15). In certain aspects, multiple self-cleavage sequences can be encoded at the carboxyl 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 wherein 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 a F2A cleavage sequence. In certain aspects, one or more self-cleavage sequences and one or more sequences cleaved by endogenous proteases are encoded in the constructs described herein. In certain embodiments, an endogenous protease recognition site is encoded at the amino terminus of the self-cleavage sequence.

[0195] In some embodiments, the polycistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by (SEQ ID NO: 16).

[0196] Another exemplary internal ribosome entry site is represented by AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGC CCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATT ACTGACCAC(SEQ ID NO:17) encoding.

[0197] Other 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 WO 2018 / 215787, the sequence described in GenBank Accession No. KP019382.1, and the IRES elements disclosed in GenBank Accession No. LT727339.1. Additional polycistronic linker regions, including cleavage, self-cleavage, and IRES elements, are disclosed in US2018 / 0360992 and US8,865,467.

[0198] In some embodiments, the construct encodes a secretion signal, e.g., (MALPVTALLL PLALLLHAARP (SEQ ID NO: 6)), which is operably linked to promote secretion of a C-terminal polypeptide, such as a cytokine. In some embodiments, the secretion signal is the secretion signal of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCF SAGLP KTEA (SEQ ID NO: 7). In some embodiments, the construct encodes a secretion signal, e.g., SEQ ID NO: 7, which is operably linked to promote secretion of a common γ chain cytokine, such as IL-15 or an active fragment thereof, e.g., NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCELLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV QMFINTS (SEQ ID NO: 8); other IL-15 sequences, including codon-optimized nucleic acid sequences encoding sIL15, are disclosed in WO 2007 / 037780. Exemplary common γ chain cytokines include IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In some embodiments, the common γ chain cytokine is selected from IL-2, IL-7, and IL-15. In some embodiments, the common γ chain cytokine is IL-15. IL-15 sequences (including codon-optimized nucleic acid sequences encoding sIL15) are disclosed herein and in WO 2007 / 037780.

[0199] Therefore, the CAD construct of the present disclosure encodes a CAD polypeptide comprising at least one co-stimulatory domain and optionally one or more intracellular signaling domains. In an embodiment, the CAD construct may encode one or more common γ chain cytokines released from the CAD polypeptide (e.g., during translation). As discussed above, in some embodiments, the CAD construct of the present disclosure may also include a mutated DAP10, such as a DAP10 mutated at K84 and / or Y86, etc., in SEQ ID NO:1.

[0200] In embodiments, one or more costimulatory domains may be located 5' to one or more signaling domains. In embodiments, one or more costimulatory domains may be located 3' to one or more signaling domains. In some embodiments, one or more costimulatory domains may be located 5' to one or more signaling domains, and additional one or more costimulatory domains may be located 3' to one or more signaling domains. In some embodiments, one or more signaling domains may be located 5' to one or more costimulatory domains, and additional one or more signaling domains may be located 3' to one or more costimulatory domains. In some embodiments, a C-terminal fusion may include alternating one or more costimulatory domains and one or more signaling domains.

[0201] For reference, Figure 1A-Figure 1D An exemplary representation of various chimeric adaptor polypeptides and their association with a receptor (e.g., exemplified as NKG2D) is depicted. This rendering is for illustrative purposes only and does not represent all of the various arrangements of chimeric adaptor polypeptides disclosed herein. Figure 1A Illustratively, a chimeric adaptor polypeptide is depicted comprising K84R and Y86F modifications. Figure 1B Illustratively depicted are chimeric adaptor polypeptides comprising K84R and Y86F modifications and a C-terminal fusion comprising CD3ζ. Figure 1C Illustratively depicted are chimeric adaptor polypeptides comprising K84R and Y86F modifications, and a C-terminal fusion comprising 4-1BB. Figure 1D Illustratively depicted are chimeric adaptor polypeptides comprising K84R and Y86F modifications and a C-terminal fusion of 4-1BB and CD3ζ.

[0202] 4. Marker Co-expression

[0203] In additional embodiments, the CAD constructs of the invention may encode one or more tags or markers, for example to facilitate the ability to monitor CAD expression levels, to serve as internal controls, etc. In some embodiments, the CAD constructs may encode fluorescent proteins, examples of which 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), etc. Other examples may include, but are not limited to, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, β-lactamase, luciferase, etc.

[0204] In other embodiments, the CAD construct can encode a protein expressed on the cell surface to facilitate detection and / or isolation of cells expressing the protein, such as via fluorescence activated cell sorting (FACS); or for enrichment by positive selection using antibodies specific for the encoded protein, such as using antibodies to purify or enrich cell products on a column or device; or for in vivo binding of antibodies to proteins to enhance or eliminate activity, such as to remove cells expressing the protein in the patient for safety reasons. Exemplary proteins that can be used for these purposes include, for example, CD19, CD20 (rituximab recognition domain), LNGFR (amino acid sequence as shown in SEQ ID NO: 89, encoded by SEQ ID NO: 90), a truncated form of human epidermal growth factor receptor (EGFRt) (amino acid sequence as shown in SEQ ID NO: 91, encoded by SEQ ID NO: 92), etc. By way of example, in embodiments, marker proteins can be targeted by clinical stage antibodies, wherein such treatment of patients with the antibodies results in elimination of cells containing CAD constructs and chimeric receptors as disclosed herein. See, e.g., 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.

[0205] In embodiments, the linker region (examples of which are described herein) can be used to promote translation of CAD polypeptides and desired markers. For example, furin and P2A linker genes can be included in the isolated nucleic acid constructs of the present disclosure to promote CAD expression and desired markers. As discussed below with respect to Example 1, furin and P2A linker genes can be used to express desired CAD polypeptides and truncated CD19 as markers. Such examples are meant to be illustrative and non-restrictive.

[0206] B. Chimeric Receptor Constructs

[0207] As disclosed herein, a chimeric receptor comprises a polypeptide comprising an extracellular targeting domain that specifically binds to a target antigen on a target cell and at least one additional domain that is not usually found with the extracellular targeting domain. For example, a chimeric receptor may comprise an extracellular targeting domain and at least one DAP10 interaction domain, wherein the extracellular targeting domain is heterologous to the DAP10 interaction domain. The chimeric receptor may also optionally comprise one or more additional domains (e.g., one or more costimulatory domains and / or one or more intracellular signaling domains). In another example, a chimeric receptor may comprise an extracellular targeting domain and a DAP10 interaction domain (e.g., NKG2D) that are usually found together, but wherein the chimeric receptor also comprises at least one other domain that is not usually found with the extracellular targeting domain and the DAP10 interaction domain (e.g., one or more costimulatory domains and / or one or more intracellular signaling domains). In yet another example, a chimeric receptor may comprise at least two domains that are not usually found together, and wherein the chimeric receptor does not comprise a DAP10 interaction domain. As a representative example, such a chimeric receptor may include an extracellular targeting domain, a transmembrane domain (e.g., a CD8αTM domain) and a cytoplasmic domain including one or more costimulatory domains and / or one or more intracellular signaling domains. With regard to costimulatory domains and / or intracellular signaling domains, it should be understood that the costimulatory domains and / or intracellular signaling domains that may be included in the chimeric receptor as disclosed herein include any one or more domains discussed above with respect to the CAD polypeptide. In an embodiment, the chimeric receptor may include an extracellular targeting domain, a transmembrane domain (e.g., a CD8αTM domain) and a cytoplasmic domain including one or more costimulatory domains but not including any intracellular signaling domain. For example, such a chimeric receptor can be a non-signaling CAR described in Ding et al., Cancer Res (2023) 83 (7 Supplement): 1777.

[0208] Furthermore, it should be understood that the chimeric receptor constructs of the present invention may also encode one or more polycistronic linker regions configured to facilitate translation of the chimeric receptor and one or more soluble common gamma chain cytokines into separate polypeptides, similar to the discussion above with respect to CAD polypeptides.

[0209] As described above, DAP10 associates with NKG2D by forming two salt bridges between two arginine residues in the NKG2D transmembrane domain and two aspartic acid residues in the DAP10 transmembrane domain (Garrity, D. et al. (2005) PNAS USA 102 (21): 7641-7646). Thus, in an embodiment, the DAP10 interaction domain comprises the amino acid sequence shown in SEQ ID NO: 75, which corresponds to the TM domain of human NKG2D. In an embodiment, the DAP10 interaction domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97% or 99% sequence identity with SEQ ID NO: 75. In an embodiment, the DAP10 interaction domain comprises an amino acid sequence comprising a portion of the amino acid sequence shown in SEQ ID NO: 75.

[0210] In embodiments where the chimeric receptor comprises a DAP10 interaction domain coupled to an extracellular targeting domain typically found with a DAP10 interaction domain, in embodiments, the chimeric receptor may comprise the amino acid sequence shown in SEQ ID NO: 76, or at least a portion of the amino acid sequence shown in SEQ ID NO: 74, and additional one or more costimulatory domains (e.g., 4-1BB, CD28, OX40, ICOS) and / or one or more intracellular signaling domains (e.g., CD3ζ). In some embodiments, the chimeric receptor may comprise at least the extracellular targeting domain and the DAP10 interaction domain of other receptors, which typically comprise an extracellular targeting domain and a DAP10 interaction domain, such as Ly49H, Ly49D, Sirp-b1, Siglec-15, Cd300lb, and the like. In some embodiments, when the chimeric receptor comprises a DAP10 interacting domain coupled to an extracellular targeting domain that is typically found with a DAP10 interacting domain, the chimeric receptor may comprise an amino acid sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 74, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 76. In exemplary embodiments, the chimeric receptor additionally comprises at least one intracellular signaling domain and / or at least one intracellular co-stimulatory domain.

[0211] In embodiments where the chimeric receptor comprises an extracellular targeting domain and at least one DAP10 interaction domain, wherein the extracellular targeting domain is heterologous to the DAP10 interaction domain, the DAP10 interaction domain may comprise an amino acid sequence corresponding to at least a portion of the TM domain of a receptor, which typically comprises an extracellular targeting domain and a DAP10 interaction domain. As a representative example, at least a portion of the DAP10 interaction domain of NKG2D (SEQ ID NO: 75) may be coupled to a heterologous extracellular targeting domain, wherein the resulting chimeric receptor optionally further comprises one or more co-stimulatory domains and / or one or more intracellular signaling domains. Other DAP10 interaction domains from other receptors (e.g., Ly49H, Ly49D, Sirp-b1, Siglec-15, Cd300lb) may be used alternatively.

[0212] The present disclosure also encompasses chimeric receptors lacking a DAP10 interaction domain. Such chimeric receptors can be designed to include a TM domain fused to the extracellular ligand binding domain of the chimeric receptor, but the TM domain does not interact with DAP10 (i.e., does not include a DAP10 interaction domain). In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the chimeric receptor is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0213] Transmembrane domains can be derived from natural or synthetic sources. In the case of natural sources, the domain can be derived from any membrane-bound protein or transmembrane protein. The transmembrane region used in particular in the present invention can be derived from (i.e., at least including the following transmembrane region) 4-1BB / CD137, activated NK cell receptors, immunoglobulins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3) , CD28, CD29, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD 8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC Class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments, truncations or combinations thereof.Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain.

[0214] In an embodiment, the transmembrane domain in the chimeric receptor lacking the DAP10 interaction domain is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 16 of U.S. Patent No. 9,102,760. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760.

[0215] In an embodiment, the chimeric receptor may include a joint between each domain, and the joint is added to obtain appropriate molecular spacing and conformation. For example, in one embodiment, there may be a joint between the binding domains VH or VL, and its length may be between 1 and 20 amino acids. In other embodiments, the length of the joint between any domains of the chimeric antigen receptor may be between 1 and 15 or 20 amino acids. In this regard, the length of the joint may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In a further embodiment, the length of the joint may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Also included herein are ranges including the numbers described herein, for example, joints with a length of 10-30 amino acids.

[0216] In certain embodiments, the linker suitable for chimeric receptors described herein is a flexible linker. Suitable linkers can be easily selected and can be any suitable different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0217] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine (GS)n polymers (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to be used as neutral tethers between domains of fusion proteins (such as the chimeric receptors described herein). Glycine can enter significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of ordinary skill will recognize that the design of a chimeric receptor may include a fully or partially flexible linker such that the linker may include a flexible linker and one or more portions that impart a less flexible structure to provide the desired chimeric receptor structure. Specific linkers include (G4S) n Linkers, wherein n=1-3, and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77). In some embodiments, the linker comprises 3-20 amino acids and an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77). The linker may be present between the LCVR and HCVR regions of the scFv fragment, between the variable region (such as the HCVR) and the hinge region (such as the CD8α hinge), or both. For example, the present disclosure provides a chimeric receptor comprising a (G4S)3 linker between the LCVR and the HCVR, and a (G4S)1 linker between the HCVR and the CD8α hinge. Although described herein in the context of chimeric receptors, it is also within the scope of the disclosure to use such linkers in the context of the CAD polypeptides described herein.

[0218] In some examples, the extracellular targeting domain of the chimeric receptor of the present disclosure may be followed by a "spacer" or "hinge", which refers to the region where the extracellular targeting domain is moved away from the surface of the effector cell to achieve appropriate cell / cell contact, target (i.e., ligand, antigen) binding, and activation in examples (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in the chimeric receptor as described herein is generally located between the transmembrane (TM) and the targeting domain. In the embodiments herein, at least a portion of the TM domain of the chimeric receptor includes a DAP10 interaction domain. In certain embodiments, the hinge region is an immunoglobulin hinge region and may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the chimeric receptors described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (such as CD8α, CD8β, CD4, CD28, CD28T, 4-1BB, and CD7), which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, the spacer domain may comprise a chemically induced dimer to control expression upon addition of a small molecule. In some embodiments, no spacer is used.

[0219] The extracellular targeting domain of the chimeric receptor as disclosed herein may include targeting domains derived from various receptors, including but not limited to pattern recognition receptors (PRRs), Toll-like receptors (TLRs), killer-activating receptors and killer-inhibiting receptors (KARs and KIRs, respectively), complement receptors, Fc receptors, B cell receptors, and T cell receptors. In some embodiments, the extracellular targeting domain may comprise a polypeptide derived from, for example, a monoclonal antibody, referred to herein as an antigen-binding fragment. Non-limiting examples of antigen-binding fragments derived from antibodies include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, scFv molecules, dAb fragments, and minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides, or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the expression "antigen-binding fragment" as used herein.

[0220] In some embodiments, the extracellular targeting domain specifically binds to the target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is selected from tumor-associated surface antigens, CD20, TyrD, B7H6, CD3, CD19; CD123; CD22; CD30; CD70, CD171; CD6, CS-1 (also known as CD2 subset 1, Claudin 18.2, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (ll) Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44, CD44v6; CD44v7 / 8; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); IL-11Rα; mesothelin; interleukin 11 receptor alpha (IL-URa); prostate stem cell antigen (PSCA); proteinase serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); ErbB3, ErbB4, mucin 1, cell surface associated (M UC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic enzyme; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); IGF-II receptor; carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase;Adrenergic type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); melanoma-associated antigen; o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD 179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); urokinase 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); MAGE-A4; MAGE-A9; ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutants; prostein; survival; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytic tumor viral oncogene neuroblastoma-derived homolog (MYCN);Ras homology family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein-like (brother of BORIS or imprinted site regulator), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchoring protein 4 (AKAP-4); synovial sarcoma, breakpoint X 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor Fc fragment (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);5T4, 8H9, ALCAM, B7-1 (CD80), B7-2 (CD86), B7-H4, B7-H6, B-human chorionic gonadotropin, CA-9, CA-125, CD133, CD138, CD23, CD25, CD34, CD4, CD40, CD56, CD8, c-Met, CSPG4, CMV-specific antigen, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), DLL3, disialylganglioside GD2, GD3, ductal epithelial mucin, EBV-specific antigen, EGP-2, EGP-40, endoglin, epithelial tumor antigen, fetal acetylcholine receptor, FBP, folate binding protein, folate receptor α, glioma-associated antigen, Glycosphingolipids, gp36, G250 / CAIX, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp41, HPV-specific antigens, KDR, kappa chain, insulin growth factor (IGF1)-1, ligands belonging to the human MIC family (MICA and MICB) and ligands belonging to the human ULBP family (ULBP1-ULBP6), LAGA-1a, Lewis Y, lambda chain, lectin-reactive AFP, L1 cell adhesion molecule, MAGE, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, MN-CA IX, MUC-1, MUC-16, NKG2D, NKG2D ligands, neutrophil elastase, Nkp30, carcinoembryonic antigen (h5T4) p53, prostate specific antigen (PSA), PSC1, prostate specific antigen protein, STEAP1, STEAP2, surface adhesion molecules, survival and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, Tem8, tumor stromal antigen, VEGF-A and immunoglobulin lambda-like polypeptide 1 (IGLL1). ;

[0221] In some additional or alternative embodiments, the target antigen is a viral antigen, such as the Epstein-Barr virus antigen EBVA and the human papillomavirus (HPV) antigens E6 and E7, viral antigens present on cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza virus, coronavirus, etc.

[0222] Other additional or alternative examples of antigens that can be targeted by the chimeric antigens of the present disclosure include, but are not limited to, the antigens described in WO2016 / 172583, WO 2017 / 062820, WO 2016 / 149254, WO 2017 / 149515, WO 2014 / 039523, WO2013 / 123061, WO 2016 / 166544, WO 2017 / 222593, and WO 2018 / 165228.

[0223] Targeting domains for specifically binding antigens such as the above-mentioned antigens are known, and it is understood that such targeting domains (e.g., monoclonal antibodies or fragments thereof, or at least one or more CDRs thereof) can be easily incorporated into the chimeric receptors of the present disclosure. For example, a targeting domain for use as disclosed herein may include or be derived from any targeting domain for a chimeric receptor described by Sadelain et al. (Cancer Discov. 3(4): 388-398(2013), see, e.g., Table 1 and references cited therein) (i.e., anti-α-folate receptor, anti-CAIX, anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD24, anti-CD30, anti-CD33, anti-CD38, anti-CD44v7 / 8, anti-CEA, anti-EGFRvIII, anti-EGP-2, anti-EGP-40, anti-EphA2, anti-erb-B2,3,4, anti-FBP, anti-fetal acetylcholine e receptor, anti-G D2 , Anti-G D3, anti-Her-2, anti-HMW-MAA, anti-IL-11Rα, anti-IL-13-α2, anti-KDR, anti-κ light chain, anti-Lewis Y, anti-L1 cell adhesion molecule, anti-MAGE-A1, anti-mesothelin, anti-MUC1, anti-MUC16, anti-NKG2D ligand, anti-NY-ESO-1 (157-165), anti-carcinoembryonic antigen, anti-PSCA, anti-PSMA, anti-ROR1, anti-IgE, anti-TAG-72, anti-VEGF-R2). In some embodiments, the known targeting domain may include or be derived from, but is not limited to, the domains described and / or cited in U.S. Patent Application No. 08 / 940,544 (e.g., anti-CD28). In some embodiments, the known targeting domain may include or be derived from, but is not limited to, the domains described and / or cited in WO 2016199141 (e.g., anti-TyrD, anti-WT1, anti-MAGE-A4, anti-MAGE-A9, anti-PAP). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in WO 2020 / 072536 and / or referenced therein (e.g., anti-BCMA, anti-CD20). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in WO 2020 / 072546, WO2014180306, and / or U.S. Provisional Patent Application No. 63 / 235093 and / or referenced therein (e.g., anti-GPC3, anti-TyrD). In some embodiments, the known targeting domain may include or be derived from, but is not limited to, the domains described in WO 2015123642 and / or referenced therein (e.g., anti-CD19, anti-CD4, anti-CD44v6, anti-CD45, anti-CD28, anti-CD3, anti-CD3e, anti-CD123, anti-CD138, anti-CD52, anti-CD56, anti-CD74, anti-CD30, anti-Gp75, anti-CD38, anti-CD33, anti-CD20, anti-Her-3, anti-ROR1, anti-c-Met, anti-c-Myc, anti-EGFR, anti-Dectin, anti-Ebola virus, anti-fungal antigen, anti-HERVK, anti-NY-ESO-1, anti-VEGF-R2, anti-TGF-bR2, anti-IgG4, anti-biotin, anti-CSI protein, anti-mesothelin, anti-phosphatidylserine). In some embodiments, the known targeting domain may include or be derived from, but is not limited to, the domains described in WO 2017 / 222593 and / or referenced therein (e.g., anti-CD19, anti-CD20, anti-CD22, anti-CD123, anti-CD33, anti-BAFF-R, anti-CD269, anti-CS-1, anti-CD45).In some embodiments, known targeting domains may include or be derived from, but are not limited to, WO Domains described in 2017 / 149515 and / or referenced therein (e.g., anti-mesothelin, anti-EGFRvIII, anti-claudin6, anti-GD2, anti-Tn, anti-sTn, anti-Tn-O-glycopeptide antigen, sTn-O-glycopeptide, anti-PSMA, anti-CD97, anti-CD44v6, anti-CEA, anti-EPCAM, anti-KIT, anti-CD171, anti-PSCA, anti-MAD-CT-2, anti-folate receptor α, anti-ERBB2, anti-MUC1, anti-EGFR, anti-NCAM, anti-CAIX, anti-Fos-related antigen I, anti-SSEA-4, anti-PDGFR-β, anti-ALK, anti-polysialic acid, anti-PLAC1, anti-GloboH, anti-NY-BR-1, anti-sperm protein 17, anti-TRP-2, anti-CYP1B1, anti-RAGE-1, anti-human telomerase transcriptase, anti-carboxylesterase, anti-mut hsp70-2, anti-MAD-CT-2, anti-CD123, anti-CD34, anti-Flt3, anti-CD33, anti-CLL-1, anti-CD19, anti-BCMA, anti-ROR1, anti-CD22, anti-CD20) or WO 2016 / 044605 (e.g., anti-mesothelin). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described and / or cited in WO 2017 / 025038 (e.g., anti-CD19, anti-CD20, anti-BCMA, anti-CD38). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described and / or cited in WO2019215500 (e.g., anti-CD19, anti-CD33, anti-CD70, anti-BCMA). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described and / or cited in WO 2016 / 016343 (e.g., anti-ROR1). In some embodiments, the known targeting domains may include or be derived from, but are not limited to, domains described in WO 2019 / 028051 and / or referenced therein (e.g., anti-CD19, anti-CD20, anti-CD33). In some embodiments, the known targeting domains may include or be derived from, but are not limited to, domains described in WO2012058460 or WO2016093878 (e.g., anti-CD70). In some embodiments, the known targeting domains may include or be derived from, but are not limited to, domains described in WO2013169691A1 and / or referenced therein (e.g., anti-B7-H6). In some embodiments, the known targeting domains may include or be derived from, but are not limited to, domains described in WO2016160620, WO2014100439, WO2013025779 and / or referenced therein (e.g., anti-B7-H4).In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in WO2019219089, WO2020200196, WO2020043044, WO2020147321 and / or referenced therein (e.g., anti-Claudin 18.2). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in WO 2016 / 090337, WO2017 / 096120, WO 2014 / 210064, U.S. Pat. No. 7,999,077, WO 2016 / 205520, U.S. Pat. No. 7,105,149, WO 2006 / 076691, WO 2010 / 114940, WO 2010 / 120561 and / or referenced therein (e.g., anti-FCRL5 / FCRH5 / CD307). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in and / or cited in U.S. Provisional Patent Application No. 63 / 411,988 (e.g., anti-B7H6). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in and / or cited in U.S. Provisional Patent Application No. 63 / 393,787 (e.g., anti-CD70). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in and / or cited in U.S. Provisional Patent Application No. 63 / 397,296 (e.g., anti-PSMA). In some embodiments, known targeting domains may include or be derived from, but are not limited to, domains described in Tran E. et al., Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia, J Exp Med., Jun. 3, 2013; 210(6): 1125-35 (e.g., anti-FAP).

[0224] In an embodiment, the chimeric receptor that does not comprise a DAP10 interaction domain comprises the amino acid sequence shown in SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 46 or SEQ ID NO: 48 of WO 2020 / 072536, wherein the chimeric receptor comprises an anti-CD20 extracellular targeting domain. In each of SEQ ID NOs: 8, 9, 10, 11, 12, 46 and 48 of WO 2020 / 072536, the chimeric receptor comprises a CD8αTM domain. Thus, in an embodiment, the CD8αTM domain can be replaced with a DAP10 interaction domain, such as a DAP10 interaction domain comprising the amino acid sequence shown in SEQ ID NO: 75 herein, to generate an anti-CD20 chimeric receptor associated with a CAD polypeptide of the present disclosure. Similar logic also applies to other chimeric receptors disclosed in WO 2020 / 072536.

[0225] In an embodiment, a chimeric receptor that does not comprise a DAP10 interaction domain comprises the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:22 of WO 2020 / 072546, wherein the chimeric receptor comprises an anti-GPC3 extracellular targeting domain. In each of SEQ ID NO:20 and 22 of WO 2020 / 072546, the chimeric receptor comprises a CD8αTM domain. Thus, in an embodiment, the CD8αTM domain can be replaced with a DAP10 interaction domain, such as a DAP10 interaction domain comprising the amino acid sequence shown in SEQ ID NO:75 herein, to produce an anti-GPC3 chimeric receptor associated with a CAD polypeptide of the present disclosure. Similar logic also applies to other chimeric receptors disclosed in WO 2020 / 072546.

[0226] Therefore, it should be understood that, in embodiments, chimeric receptors known in the art may be used in the methods of the present disclosure with or without modification. For example, but not limited to, any chimeric receptor listed in Table 1 of Sadelain et al. (Cancer Discov. 3(4): 388-398 (2013) may be used without modification, or may be modified in some way to incorporate a DAP10 interaction domain so that the chimeric receptor associates with DAP10 and, by inference, with the CAD polypeptide of the present disclosure. Of course, other modifications are also within the scope of the present disclosure, including but not limited to the incorporation of one or more additional or alternative intracellular domains (e.g., co-stimulatory and / or intracellular signaling domains). Similar logic applies to other chimeric receptors known in the art, such as, but not limited to, those disclosed in WO 2016 / 090337.

[0227] C. Expression of chimeric constructs

[0228] 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 or a chimeric receptor as described herein). In an isolated nucleic acid of the invention, a promoter is operably linked to a nucleic acid sequence encoding a chimeric adapter polypeptide or a chimeric receptor of the invention, i.e., their position promotes transcription of messenger RNA from the DNA encoding the chimeric adapter polypeptide or the chimeric receptor. The term "operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in their intended manner.

[0229] The promoter may be of genomic origin or synthetically produced. A variety of promoters used in host cells relevant to the present disclosure are well known in the art (e.g., CD4 promoter disclosed in Marodon et al. (2003) Blood 101(9):3416-23). ​​The promoter may be constitutive or inducible, wherein induction is associated with a particular cell type or a particular level of maturation. Alternatively, many well-known viral promoters may also be suitable. Promoters of interest include β-actin promoters, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen lesion formation virus promoters. The promoter may or may not be associated with an enhancer, wherein the enhancer may be naturally associated with a particular promoter or with a different promoter. In an embodiment, the expression of the chimeric adapter polypeptide is controlled by an inducible promoter (e.g., a promoter that can be induced by a molecule present in the tumor microenvironment (e.g., TGFβ)).

[0230] The open reading frame sequences encoding the various fragments of the chimeric adaptor polypeptides and chimeric receptors of the present disclosure can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or from a combination thereof.

[0231] In an embodiment, to express the chimeric adaptor polypeptide of the 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 produce the chimeric adaptor polypeptide in a host cell. Alternatively, an exogenous transcription initiation region that allows for constitutive or inducible expression can be used, wherein expression can optionally be controlled depending on the host cell, the desired expression level, the nature of the host cell, etc.

[0232] A termination region encoding a chimeric adaptor polypeptide or a C-terminal component of a chimeric receptor may be included. In general, the source of the termination region is not believed to be important for expression of the recombinant protein, and a variety of termination regions may be employed without adversely affecting expression.

[0233] The isolated nucleic acid encoding the chimeric adapter polypeptide or chimeric receptor according to the present invention can be prepared in a conventional manner. The sequence (natural or synthetic) is separated and manipulated as needed to allow each component to be correctly joined. Therefore, the various nucleic acid sequences encoding the various segments of the chimeric adapter polypeptide can be separated, for example, by using polymerase chain reaction (PCR), using appropriate primers. If necessary, specific primers can be designed, which lead to the deletion of undesirable parts in the nucleic acid sequence used as a template. Additionally or alternatively, restricted digestion of cloned genes can be used to generate isolated nucleic acid constructs disclosed herein. In either case, the sequence can be selected to provide a restriction site with a flat end, or the sequence can have a complementary overlap to facilitate incorporation into various vectors. In an example, the nucleic acid sequence is modified (for example, one or more point mutations, insertions or deletions are introduced). In one embodiment of the chimeric adapter polypeptide disclosed herein, for example, the modification can include 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 may include the use of various commercially available kits (eg, QuickChange Site-Directed Mutagenesis Kit, Agilent, Santa Clara, CA).

[0234] Various manipulations for preparing isolated nucleic acids encoding the chimeric adapter polypeptides or chimeric receptors of the present disclosure can be performed in vitro. In specific embodiments, sequences encoding the chimeric adapter polypeptides are introduced into vectors using standard transformation or transfection methods for cloning and expression in appropriate host cells. Thus, after each manipulation, the constructs generated by ligation of the DNA sequences are cloned, the vectors are isolated, and the sequences are screened to ensure that the sequences encode the desired chimeric adapter polypeptides. The sequences can be screened by restriction analysis, sequencing, and the like.

[0235] It is contemplated that the isolated nucleic acid can be introduced into a host cell as naked DNA or in a suitable vector. Many suitable vectors are known to molecular biologists, and the selection of the vector depends on the desired function and includes plasmids, cosmids, viruses, phages, and other vectors routinely 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, Sambrook et al. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989), (1994) described in the technology. Alternatively, the polynucleotides and vectors of the present disclosure can be reconstituted into liposomes for delivery to target cells.

[0236] Methods for stably transfecting host cells using naked DNA by electroporation are known in the art (see, e.g., U.S. Pat. No. 6,410,319, which discloses T cell transfection). Naked DNA generally refers to DNA encoding a chimeric adaptor polypeptide or chimeric receptor of the invention contained in a plasmid expression vector in an orientation suitable for expression. Advantageously, the use of naked DNA reduces the time required to generate host cells expressing the chimeric polypeptide of the invention.

[0237] 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 an isolated nucleic acid encoding a chimeric polypeptide of the invention into a host cell. Suitable vectors for use according to the methods of the invention are not replicable in T cells. A variety of viral-based vectors are known in which the number of viral copies maintained in the cell is sufficiently low to maintain cell viability. Illustrative vectors include the novel vector pFB and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0238] Therefore, it is understood that in some embodiments, the isolated nucleic acid is a circular nucleic acid. In some embodiments, the isolated nucleic acid is a vector, such as a plasmid vector, an adenoviral vector, an adeno-associated viral vector, a viral vector, a retroviral vector (e.g., a γ retroviral vector) or a lentiviral vector. In some embodiments, an isolated nucleic acid containing a DAP10 sequence (e.g., at least a portion of SEQ ID NO: 1, modified or unmodified) and one or more signaling and / or costimulatory domains or, for example, a continuous portion thereof is integrated into the genome of a host cell (such as a host γδT cell). In an exemplary embodiment, the isolated nucleic acid is a retroviral vector.

[0239] D.Host cells

[0240] The chimeric polypeptides including CAD polypeptides and chimeric receptors disclosed herein can be expressed in a variety of host cells via their corresponding chimeric nucleic acid constructs. 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 expressing NKG2D. In embodiments, the CAD polypeptide is expressed in a host cell type that exhibits endogenous expression of a receptor associated with DAP10 (e.g., NKG2D, Ly49H, Ly49D, Sirp-b1, Siglec-15, Cd300lb, etc.) at some level. In embodiments, the host cell type can also be engineered to express the same receptor (e.g., NKG2D), for example, to increase the expression level of the receptor, exceeding the naturally occurring endogenous expression level. In embodiments, CAD polypeptides are expressed in cell types that do not exhibit endogenous expression of a receptor that associates with DAP10, in which case the host cell is engineered to express such a receptor (e.g., expressing NKG2D in a cell type that does not otherwise express NKG2D, or expressing a chimeric receptor comprising a DAP10 interaction domain). In embodiments, CAD polypeptides are expressed in cell types that exhibit endogenous expression of a receptor that associates with DAP10 (e.g., NKG2D), wherein the cell is further engineered to express a chimeric receptor lacking a DAP10 interaction domain. In embodiments, CAD polypeptides are expressed in cell types that exhibit endogenous expression of a receptor that associates with DAP10 (e.g., NKG2D), wherein the cell is further engineered to express at least one chimeric receptor comprising a DAP10 interaction domain, the chimeric receptor comprising at least one extracellular targeting domain and a DAP10 interaction domain, optionally further comprising one or more co-stimulatory domains and / or one or more intracellular domains. In embodiments, the CAD polypeptide is expressed in a cell type that lacks endogenous expression of a receptor that associates with DAP10, and is engineered to express a non-chimeric receptor that associates with DAP10, and is further engineered to express a chimeric receptor, wherein in some instances the chimeric receptor comprises a DAP10 interacting domain, but in other instances lacks such a DAP10 interacting domain, and wherein the chimeric receptor comprises one or more costimulatory domains and / or one or more intracellular ligand binding domains.

[0241] In an embodiment, expression of a CAD polypeptide of the present disclosure in a host cell results in competition of the CAD polypeptide with endogenous cellular DAP10 (e.g., WT DAP10). Via competition, intracellular signaling through a receptor (e.g., endogenous, exogenous, or chimeric) associated with DAP10 can be redirected by a chimeric DAP10 adaptor polypeptide. In order to effectively achieve a desired outcome (e.g., tumor cell killing), signaling does not need to be 100% redirected by a CAD polypeptide, although such percentages are within the scope of the present disclosure. Signaling through a CAD polypeptide can include any value between 20% and 100% of signaling through DAP10, for example, between 90-100%, between 80-100%, between 70-100%, between 60-100%, between 50-100%, etc. As a representative example for illustrative purposes, in a host cell, 80% of signaling through a receptor associated with DAP10 is rerouted through the CAD polypeptide, meaning that only 20% of such signaling remains through endogenous DAP10, while 80% of signaling is through the CAD polypeptide.

[0242] Host cells as described herein may be stored, e.g., cryopreserved, for use in adoptive cell transfer. In embodiments, the host cells are stored prior to being engineered to express a CAD polypeptide and a chimeric receptor. In embodiments, the cells are engineered to express a chimeric DAP10 adaptor polypeptide prior to being stored.

[0243] Preferred host cells for use with the CAD polypeptides and chimeric receptors of the present disclosure include immune cells. Such cells can be obtained from the subject to be treated (i.e., autologous), or alternatively immune cell lines or donor immune cells (allogeneic, isogenic) can be used. Immune cells can be obtained from many 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. Any number of techniques known to those skilled in the art (such as Ficoll) can be used. TM Immune cells are obtained from blood collected from a subject by separation. For example, cells in the circulating blood of an individual can be obtained by apheresis. In some embodiments, immune cells are separated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL TMGradient centrifugation or by countercurrent centrifugal elutriation. Specific immune cell subsets can be further separated by positive or negative selection techniques. For example, immune cells can be separated using a combination of antibodies for surface markers specific to positively selected cells, for example, by incubating with antibody-conjugated beads for a time sufficient to positively select the desired immune cells. Alternatively, negative selection can also be performed using a combination of antibodies for surface markers specific to negatively selected cells, thereby achieving enrichment of immune cell populations. Other specific methods of separation and / or enrichment are disclosed herein.

[0244] In some embodiments, immune cells include any leukocytes involved in protecting the body from infectious diseases 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 αβT cells, γδT cells, NK cells, NKT cells, γδNKT cells, B cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, regulatory T cells, etc. In embodiments, preferred immune cells include αβT cells, γδT cells, NK cells, NKT cells, γδNKT cells, and / or macrophages in some examples. In embodiments, preferred immune cells include γδT cells. In embodiments, immune cells related to the present disclosure include allogeneic cells, autologous cells or isogenic cells.

[0245] Aspects of the present disclosure include immune cells that have in vitro or in vivo cytotoxic activity against blood tumors or solid tumor cells that display cell surface expression of tumor-associated antigens (TAAs), virus-infected cells that display virus-derived antigens, bacterial cells, etc. In embodiments, the cytotoxic activity is an innate activity. In embodiments, immune cells that functionally express a CAD polypeptide and at least one chimeric receptor exhibit a cell-killing activity that is higher than the level of in vitro and / or in vivo cell-killing activity in a control immune cell that does not contain a CAD polypeptide and / or at least one chimeric receptor of the present disclosure.

[0246] In embodiments, the cytotoxicity is significantly (> about 25%) enhanced or improved due to the presence of the CAD polypeptide and at least one chimeric receptor compared to the cytotoxicity in the absence of the CAD polypeptide and / or at least one chimeric receptor. In some cases, the cytotoxicity is at least partially, significantly (> about 25%), or completely due to the presence of the CAD polypeptide and at least one chimeric receptor.

[0247] In embodiments, the engineered immune cells associated with the present disclosure can exhibit robust and / or persistent cell killing activity (e.g., tumor cells, virus-infected cells) through direct and / or indirect mechanisms. In some cases, the cell killing activity lasts for at least about 6 days to 120 days, or at least about 6 days to 180 days, from the first contact with the target cell. In some cases, the cell killing activity of the immune cells or their progeny that are engineered to express the CAD polypeptide and at least one chimeric receptor disclosed herein lasts for at least about 6 days to 120 days, or at least about 6 days to 180 days, from the first contact with the target cell, or from the administration of the engineered immune cells disclosed herein. This sustained cell killing activity can be performed in vitro, in vivo, or in vitro and in vivo.

[0248] 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 that is recognized by a receptor that associates with DAP10 and that specifically associates with a CAD polypeptide of the disclosure. An example of such a receptor is NKG2D, although the disclosure is not limited to CAD polypeptides that interact with NKG2D, and may include other receptors that also associate with DAP10, such as chimeric receptors that are engineered to include a DAP10 interaction domain. In embodiments, proliferation is at least partially, significantly (> about 20%, or > about 25%, or > about 50%, or > about 80%), or completely (e.g., 100%) attributed to the presence of a CAD polypeptide construct that associates with a receptor expressed on a host cell (e.g., endogenous or exogenous NKG2D, i.e., a chimeric receptor that is engineered to include a DAP10 interaction domain). In some cases, the immune cells exhibit a higher level of proliferation in vitro and / or in vivo compared to a control immune cell (e.g., an immune cell of the same type) that does not comprise a CAD polypeptide and at least one chimeric receptor. The higher level of proliferation can include a 20-50% increase, a 50-80% increase, an 80-100% increase, or even a 2-fold increase, a 3-fold increase, a 4-fold increase, a 5-fold increase, a 5-fold increase, a 5-10-fold increase, a 10-20-fold increase, or even an increase greater than 20-fold, such as, for example, a 50-100-fold increase or more, compared to a control immune cell lacking a CAD polypeptide and / or at least one chimeric receptor as described herein.

[0249] In some embodiments, immune cells engineered to comprise a CAD polypeptide and at least one chimeric receptor described herein express and secrete, or continue to express and secrete, one or more proinflammatory cytokines, e.g., upon contact with a cell expressing a ligand that is recognized by a cell surface receptor on the immune cell that associates with DAP10. In embodiments, expression and secretion associated with immune cells engineered to express a CAD polypeptide and at least one chimeric receptor is at least partially, significantly (> about 20%, or > about 25%, or > about 50%, or > about 80%), or entirely attributable to one or both of the CAD polypeptide and / or at least one chimeric receptor. In embodiments, expression and / or secretion is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even greater than 100%, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 5-10-fold, 10-20-fold, or even greater than 20-fold, such as, for example, 50-100-fold or more, as compared to the expression and / or secretion observed in a control immune cell (e.g., an immune cell of the same type) that lacks expression of a CAD polypeptide and / or at least one chimeric receptor.

[0250] In embodiments, the engineered immune cells of the present disclosure can be used to change the cell microenvironment (e.g., TME), to facilitate, for example, anti-tumor response. For example, solid tumors can recruit suppressive cells, such as myeloid suppressor cells (MDSC), which can enhance suppressive TME. The frequency of circulating or intratumoral MDSC is related to cancer staging, disease progression, and resistance to standard chemotherapy and radiotherapy. Certain ligands (e.g., NKG2D ligands) are expressed at high levels on several solid tumors and tumor-infiltrating MDSC, so in embodiments, the engineered immune cells of the present disclosure can be used to change TME by reducing or eliminating inhibitory molecules (such as TGF-β) of TME, to facilitate anti-tumor response. In embodiments, the engineered immune cells of the present disclosure have cytotoxicity to MDSC, but are harmless (i.e., 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 of engineered host cells of the present disclosure, such as MDSC, reduces the inhibitory effect of the TME. For example, the inhibitory 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 inhibitory effect of the TME in the absence of host cells engineered to express CAD polypeptides and chimeric receptors as described herein.

[0251] In embodiments, the engineered immune cells of the present disclosure can be used to reduce the growth and / or proliferation of target cells. For example, compared to the growth and / or proliferation of target cells in the absence of engineered immune cells or the presence of engineered immune cells expressing CAD polypeptides or chimeric receptors but not both, the immune cells engineered to express CAD polypeptides as described herein and at least one chimeric receptor can 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 even about 100%, or any percentage therebetween. In embodiments, the target cell expresses at least one cell surface antigen recognized by at least one receptor (e.g., NKG2D chimeric receptor) on the cell surface of the engineered immune cell, wherein the receptor is at least partially associated with the CAD polypeptide.

[0252] In embodiments, the engineered immune cells of the present disclosure can be used to produce cytotoxicity to target cells. For example, compared to the amount of cell death in the absence of engineered immune cells or the presence of engineered immune cells expressing CAD polypeptides or chimeric receptors but not both, immune cells engineered to express CAD polypeptides as described herein and at least one chimeric receptor may cause an increase in the death of one or more target cell populations. The amount of cell death may be about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, or any percentage between the two, compared to the amount of cell death in the absence of engineered immune cells or the presence of engineered immune cells expressing CAD polypeptides or chimeric receptors but not both.

[0253] The expression of CAD polypeptides containing heterologous signaling domains (e.g., 4-1BB, OX40, CD28, ICOS, IL2R, CD27, etc.) that affect cell survival and proliferation implements a logical gating strategy (e.g., "AND" gating), which can increase the stringency and / or efficacy of target attack when paired with chimeric receptors on the same host cell (e.g., T cells or NK cells), where the endogenous receptors bind to targets that are expressed at lower levels in normal tissues. Therefore, host cells with chimeric receptors that recognize primary targets (e.g., major cancer targets, viral antigens, autoimmune antigens, etc.) and appropriate CAD polypeptides can promote the continued survival, proliferation, and killing of target cells that express primary targets and receptor (e.g., NKG2D) ligands that associate with DAP10. In such examples, the chimeric receptor may include a DAP10 interaction domain in an embodiment. Examples of logic gating strategies suitable for use in the present disclosure can be found in, for example, WO2019118518, WO2020154635, WO2020223445, WO2021035093, WO2019222642A1, WO2018236825A1, WO2019164979, and Chang, ZL and Chen YY (2017) Trends Mol Med 23(5):430-450).

[0254] Thus, in an embodiment, a host cell may comprise a CAD polypeptide comprising at least one of a DAP10 domain, a co-stimulatory domain or an intracellular domain, and specifically lacking an extracellular domain, wherein the host cell further expresses at least one chimeric receptor, the at least one chimeric receptor comprising at least an extracellular targeting domain, an intracellular signaling domain and / or a co-stimulatory domain, wherein when the chimeric receptor comprises an intracellular signaling domain, the CAD polypeptide comprises a co-stimulatory domain, and vice versa. In some embodiments, the chimeric receptor lacks a DAP10 interaction domain, wherein the CAD polypeptide interacts with a receptor containing a DAP10 interaction domain (e.g., NKG2D). In other embodiments, the chimeric receptor comprises a DAP-10 interaction domain. As an example, a CAD polypeptide may comprise at least one 4-1BB co-stimulatory domain, but lacks an intracellular signaling domain, wherein the chimeric receptor comprises at least one intracellular signaling domain (e.g., CD3ζ), and vice versa. As another example, the CAD polypeptide may include at least one CD28 co-stimulatory domain, or at least one OX40 co-stimulatory domain, or at least one ICOS co-stimulatory domain, but lacks an intracellular signaling domain, wherein the chimeric receptor includes at least one intracellular signaling domain (e.g., CD3ζ), or vice versa. In some embodiments, the CAD polypeptide may include at least two different co-stimulatory domains, such as a CAD polypeptide may include at least one 4-1BB and at least one CD28 (or OX40, or ICOS, etc.) co-stimulatory domain, but lacks an intracellular signaling domain, wherein the chimeric receptor includes at least one intracellular signaling domain (e.g., CD3ζ), optionally wherein the chimeric receptor includes at least two different intracellular signaling domains, or vice versa.

[0255] Thus, in an embodiment, a method for preparing a cell comprising a CAD polypeptide and at least one chimeric receptor is provided, the method comprising introducing an isolated nucleic acid encoding a CAD polypeptide and at least one isolated nucleic acid encoding at least one chimeric receptor into a cell, such that the cell expresses the CAD polypeptide and at least one chimeric receptor. In an embodiment, the cell is an immune cell as described herein, such as a NK cell, a NKT cell, a γδT cell, an αβT cell, or a γδNKT cell. In some embodiments, the method for preparing a cell further comprises introducing another isolated nucleic acid into the cell, the nucleic acid encoding a receptor capable of associating with a CAD polypeptide.

[0256] E. How to use

[0257] In one aspect, the present disclosure provides a method of modulating a signal transduced by at least one receptor of a host cell, the host cell comprising an immune cell engineered to express a CAD polypeptide as described herein and at least one chimeric receptor, and preferably wherein the immune cell is cytotoxic, more preferably wherein the immune cell is a γδT cell. In an embodiment, the receptor is endogenous to the immune cell and is endogenously expressed therein. In an additional or alternative embodiment, the receptor is expressed by introducing an isolated nucleic acid encoding the receptor into the immune cell. In an additional or alternative embodiment, the receptor is a chimeric receptor as described herein, and optionally includes a DAP10 interaction domain.

[0258] In embodiments, the method of modulating a signal transduced by at least one receptor results in stimulation of an immune cell and / or activation of an immune cell.

[0259] In embodiments, the method of modulating a signal transduced by at least one receptor results in an increase in the level of proliferation of an immune cell compared to the level of proliferation of a control immune cell lacking the CAD polypeptide.

[0260] In embodiments, the method of modulating signals transduced by a receptor results in increased expression and secretion of the one or more cytokines as compared to the expression and secretion levels of the one or more cytokines in a control immune cell lacking the CAD polypeptide and / or the chimeric receptor.

[0261] In embodiments, modulating a signal transduced through a receptor comprises directing at least a portion of the signal through a CAD polypeptide rather than endogenous DAP10. In embodiments, the portion of the signal directed through a CAD polypeptide is 80% or more, such as 90-95% or more, such as 99% or 100%.

[0262] F. Treatment Methods

[0263] As described herein, a pharmaceutical composition can be administered for preventive and / or therapeutic treatment, comprising an engineered host cell expressing a CAD polypeptide and at least one chimeric receptor and / or a mixture thereof. In a preferred embodiment, the pharmaceutical composition comprises γδT cells engineered to express a CAD polypeptide and at least one chimeric receptor. The mixture may comprise different types of host cells engineered to express the same or different CAD polypeptides and the same or different chimeric receptors as described herein. For example, but not limited to, the mixture may comprise a NK cell population expressing a first CAD polypeptide and a first chimeric receptor, and a γδ cell population engineered to express a second CAD polypeptide and a second chimeric receptor, wherein the first CAD polypeptide is different from the second CAD polypeptide, and wherein the first chimeric receptor is the same or different from the second chimeric receptor. As another example and not limitation, the mixture may comprise a NK cell population and a γδ cell population, each of which is engineered to express the same CAD polypeptide and the same or different chimeric receptor. As yet another example, the mixture may comprise an engineered host cell population, and may also comprise a non-engineered cell population. For example, but not limited to, the mixture may include a population of γδ cells or NK cells engineered to express a CAD polypeptide as described herein and at least one chimeric receptor, and another population of unengineered cells (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 disorder in an amount sufficient to reduce at least one sign or symptom associated with the disease or disorder. In some embodiments, the amount is sufficient to cure the disease or disorder.

[0264] The engineered host cell population and / or mixture thereof can also be administered to reduce the likelihood of developing a disease, contracting a disease, or worsening a disease. The effective amount of the engineered host cell population, non-engineered host cells, and / or mixture thereof for therapeutic use may vary depending on the severity and course of the disease or disorder, previous therapy, the health status, weight, and / or response to various drugs of the subject, and / or the judgment of the treating physician.

[0265] In embodiments, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof disclosed herein can be used to treat subjects in need of treatment of a disease. Examples of such diseases include, but are not limited to, cancer, infectious diseases, and autoimmune disorders. The subject can be a human, a non-human primate, such as a chimpanzee and other ape and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; experimental animals, including 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, a teenager, a pre-adolescent, a child, a toddler, an infant.

[0266] The method for treating the disease (e.g., pain) of a subject can include administering to the subject one or more engineered host cell groups (e.g., to express CAD polypeptides and at least one chimeric receptor), non-engineered cells and / or mixtures thereof of a therapeutically effective amount. One or more engineered host cell groups, non-engineered cells and / or mixtures thereof can be administered according to various schemes (e.g., time, concentration, dosage, treatment interval and / or formulation). Before receiving one or more engineered host cell groups, non-engineered cells and / or mixtures thereof of a therapeutically effective amount, the subject can also receive preconditioning such as chemotherapy, radiotherapy or a combination of the two. As part of treatment, one or more engineered host cell groups, non-engineered cells and / or mixtures thereof can be administered to the subject according to a first scheme, and the subject can be monitored to determine whether the treatment of the first scheme meets a given therapeutic efficacy level. In some cases, based on the information collected from providing the first scheme to the subject, one or more engineered host cell groups, non-engineered cells and / or mixtures thereof can be administered to the subject according to a second scheme.

[0267] In an embodiment, a pharmaceutical composition may be administered in a first regimen, the pharmaceutical composition comprising at least one host cell engineered to express a CAD polypeptide and at least one chimeric receptor. The subject may be monitored, for example, by a healthcare provider (e.g., a treating physician or nurse). In some instances, the subject is monitored to determine or measure the efficacy of the engineered host cell in treating the subject's illness. In some cases, the subject may also be monitored to determine the in vivo expansion of the engineered host cell population in the subject's body. Another pharmaceutical composition may be administered to the subject in a second regimen, the pharmaceutical composition comprising at least one host cell engineered to express a CAD polypeptide and at least one chimeric receptor. The pharmaceutical composition administered in the second regimen may comprise a host cell expressing the same CAD polypeptide and at least one chimeric receptor of the same type as that administered to the subject in the first regimen. However, within the scope of the present disclosure, the pharmaceutical composition administered in the second regimen may comprise different types of host cells, which optionally express different CAD polypeptides (e.g., CAD polypeptides with different mutations and / or co-stimulatory or signaling domains) and / or different chimeric receptors. In some instances, the second regimen is not performed, for example, when the first regimen is found to be effective (e.g., a single round of administration may be sufficient to treat the illness). In some embodiments, populations of engineered host cells can be administered to various subjects (eg, where the host cells have universal donor properties).

[0268] A therapeutically effective amount of one or more engineered host cell populations (e.g., expressing a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof can be used to treat a variety of disorders. In some cases, a therapeutically effective amount of one or more engineered host cell populations (e.g., expressing a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof can be used to treat cancer, including solid tumors and hematological malignancies. In some cases, a therapeutically effective amount of one or more engineered host cell populations (e.g., expressing a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof can be used to treat infectious diseases caused by, for example, pathogenic bacteria or viruses.

[0269] Treatment using one or more engineered host cell populations (e.g., expressing a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof of the present disclosure may be provided to a subject before, during, and after clinical onset of the disease. Treatment may be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years or longer after clinical onset of the disease. Treatment may be provided to a subject greater 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 or longer after clinical onset of the 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 the disease. Treatment may also include treating a person in a clinical trial. Treatment may include administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising one or more engineered host cell populations (e.g., expressing a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof.

[0270] In some cases, the use of one or more engineered host cell groups (e.g., expressing CAD polypeptides and at least one chimeric receptor), non-engineered cells and / or their mixtures disclosed herein can regulate the activity of endogenous lymphocytes in the subject. In some cases, the use of one or more engineered host cell groups (e.g., expressing CAD polypeptides and at least one chimeric receptor), non-engineered cells and / or their mixtures disclosed herein can cause the activation of the cytotoxicity of another immune cell. In some cases, another immune cell is a CD8+T cell. In some cases, another immune cell is a natural killer T cell. The present disclosure encompasses other examples of other immune cells. In some cases, the use of one or more engineered host cell groups (e.g., expressing CAD polypeptides and at least one chimeric receptor), non-engineered cells and / or their mixtures disclosed herein can inhibit regulatory T cells. In some cases, regulatory T cells are Fox3+Treg cells. In some cases, regulatory T cells are Fox3-Treg cells. Non-limiting examples of cells whose activity can be modulated by administering one or more engineered host cell populations (e.g., expressing a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof of the present disclosure include hematopoietic stem cells; B cells; CD4+ cells; CD8+ cells; erythrocytes; white blood cells; dendritic cells, including dendritic antigen-presenting cells; leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophils; helper T cells; and killer T cells.

[0271] One or more engineered host cell groups, non-engineered cells and / or their mixtures can be administered to a subject in any order or simultaneously, and they have cytotoxic activity for, for example, but not limited to, blood tumors or solid tumor cells, or virus-infected cells, or bacterial cells. If administered simultaneously, the engineered host cells of the present disclosure and / or their mixtures can be provided in a single, unified form (such as intravenous injection), or in a variety of forms (for example, as a variety of intravenous infusions, subcutaneous injections or pills). One or more engineered host cell groups, non-engineered cells and / or their mixtures of the present disclosure can be packaged together or packaged separately in a single package or multiple packages. One or all of the one or more engineered host cell groups, non-engineered cells and / or their mixtures of the present disclosure can be given in multiple doses. If not administered simultaneously, the time arrangement between multiple doses can vary up to about one week, one month, two months, three months, four months, five months, six months or about one year. In some cases, the engineered host cells of the present disclosure can be propagated in vivo in the body of the subject after being administered to the subject. One or more engineered host cell groups, 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 groups, non-engineered cells and / or mixtures thereof of the present disclosure and pharmaceutical compositions comprising them can be packaged as test kits. Test kits may include instructions for use (e.g., written instructions) for one or more engineered host cell groups, non-engineered cells and / or mixtures thereof and pharmaceutical compositions comprising them.

[0272] In some cases, the method for treating a subject in need includes administering to the subject a therapeutically effective amount of one or more engineered host cell groups, non-engineered cells and / or mixtures thereof of the present disclosure, wherein the administration treats a specific illness (e.g., cancer, viral or bacterial infection, autoinflammatory disease). In some embodiments, a therapeutically effective amount of one or more engineered host cell groups, non-engineered cells and / or mixtures thereof are 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 groups, non-engineered cells and / or mixtures thereof of the present disclosure are administered for at least one week. In some embodiments, a therapeutically effective amount of one or more engineered host cell groups, non-engineered cells and / or mixtures thereof of the present disclosure are administered for at least two weeks.

[0273] One or more engineered host cell groups, non-engineered cells and / or its mixture as described herein can be used before, during or after a disease or illness occurs, and the time arrangement of applying the pharmaceutical composition containing an engineered host cell group can vary. For example, one or more engineered host cell groups, non-engineered cells and / or its mixture can be used as a prophylactic, and can be continuously applied to a subject with an illness or disease tendency, so as to reduce the possibility of a disease or illness occurring. Initial application can be carried out via any practical approach, such as using any preparation as described herein to be carried out by any approach as described herein. In some instances, the application of one or more engineered host cell groups, non-engineered cells and / or its mixture of the present disclosure is intravenous administration. One or more doses of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof can be administered as soon as practicable after the onset of a particular disorder (e.g., hematological or solid cancers, viral infections, bacterial infections, autoimmune disorders, etc.), and for the length of time necessary to treat the disease / disorder, such as, for example, about 24 hours to about 48 hours, about 48 hours to about 1 week, about 1 week to about 2 weeks, about 2 weeks to about 1 month, about 1 month to about 3 months. In some embodiments, one or more doses of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof can be administered years after the onset of a disease / disorder (e.g., cancer) and before or after other treatments.

[0274] In some embodiments, one or more engineered host cell groups, non-engineered cells and / or mixtures thereof disclosed herein are administered simultaneously or sequentially with one or more methods of increasing shared γ chain cytokines. As used herein, "one or more methods of increasing shared γ chain cytokines" refers to a method or combination of methods for changing the physiological state of a subject so that at least one shared γ chain cytokine level in a subject is increased. In some embodiments, the method increases the level of one or more shared γ chain cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-15 and IL-21 of the subject. In some embodiments, the method includes lymphocyte depletion. In some embodiments, the method includes administering one or more shared γ chain cytokines to the subject. In some cases, IL-2, IL-4, IL-7, IL-15 and / or IL-21 are administered. In some embodiments, the method includes secreting shared γ chain cytokines from the administered engineered host cells. In some cases, IL-2, IL-4, IL-7, IL-15 and / or IL-21 are secreted.

[0275] In some embodiments, the method of administering one or more methods of increasing common γ chain cytokines includes lymphocyte depletion prior to introducing one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof disclosed herein. In some embodiments, the method of administering one or more methods of increasing common γ chain cytokines includes administering at the same time as introducing one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, or administering a certain amount of common γ chain cytokines in sequence, the amount being sufficient to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof introduced. The amount of common γ chain cytokines administered may be an amount that effectively increases the proliferation, cytotoxic activity, persistence, or a combination 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 to 10 μg / kg / dose of IL-15 per 24 hours. Exemplary amounts of IL-2 include, but are not limited to, about 3×10 every 8-48 hours. 6 About 22×10 6 For example, the dosing regimen for IL2 in RCC is 600,000 International Units / kg (0.037 mg / kg) IV for 48 hr, infused over 15 minutes, for a maximum of 14 doses.

[0276] In some embodiments, the method of administering one or more common gamma chain elevating cytokines comprises lymphocyte depletion prior to administering one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, and administering at the same time as introducing one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, or administering sequentially an amount of common gamma chain cytokines sufficient 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.

[0277] In some embodiments, the common γ chain cytokine is increased at least in part by engineering host cells, wherein the common γ chain cytokine is expressed by a CAD construct and / or a chimeric receptor construct as disclosed herein. In such an example, it is within the scope of the present disclosure to additionally administer one or more additional γ chain cytokines in a manner that increases the additional γ chain cytokine.

[0278] G. Dosage

[0279] One or more engineered host cell populations, non-engineered cells and / or mixtures thereof of the present disclosure can be formulated into unit dosage forms suitable for single administration of precise doses. In some cases, the unit dosage form contains additional lymphocytes. In the unit dosage form, the preparation is divided into unit doses containing an appropriate amount of one or more compounds. The unit dose can be a packaged form containing a discrete amount of preparation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. The aqueous suspension composition can be packaged in a single dose non-resealable container. For example, a multi-dose resealable container can be used in combination with a preservative or not. In some instances, the pharmaceutical composition does not include a preservative. Preparations for parenteral injection can be present in a unit dosage form together with a preservative, such as in an ampoule or in a multi-dose container.

[0280] The amount of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof present in the composition can be 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 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 x 10 3 cells, at least 2x 10 3 cells, at least 3x 10 3 cells, at least 4 x 10 3 cells, at least 5x 10 3 cells, at least 6 x 10 3 cells, at least 7x 10 3 cells, at least 8 x 10 3 cells, at least 9x10 3 cells, at least 1x 10 4 cells, at least 2x 10 4 cells, at least 3x10 4 cells, at least 4 x 10 4 cells, at least 5x 10 4 cells, at least 6 x 10 4 cells, at least 7x 10 4 cells, at least 8 x 10 4 cells, at least 9 x 10 4 cells, at least 1x 10 5 cells, at least 2x 10 5 cells, at least 3x 10 5cells, at least 4x10 5 cells, at least 5x 10 5 cells, at least 6 x 10 5 cells, at least 7x 10 5 cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1x 10 6 cells, at least 2x 10 6 cells, at least 3x 10 6 cells, at least 4 x 10 6 cells, at least 5x 10 6 cells, at least 6 x 10 6 cells, at least 7x 10 6 cells, at least 8x10 6 cells, at least 9 x 10 6 cells, at least 1x 10 7 cells, at least 2x 10 7 cells, at least 3x 10 7 cells, at least 4 x 10 7 cells, at least 5x 10 7 cells, at least 6 x 10 7 cells, at least 7x 10 7 cells, at least 8x10 7 cells, at least 9 x 10 7 cells, at least 1x 10 8 cells, at least 2x 10 8 cells, at least 3x10 8 cells, at least 4 x 10 8 cells, at least 5x 10 8 cells, at least 6 x 10 8 cells, at least 7x 10 8 cells, at least 8 x 10 8 cells, at least 9 x 10 8 cells, at least 1x 10 9 cells or more.

[0281] The therapeutically effective dose of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the invention can be about 1 cell to about 10 cells, about 1 cell to about 100 cells, about 1 cell to about 10 cells, about 1 cell to about 20 cells, about 1 cell to about 30 cells, about 1 cell to about 40 cells, about 1 cell to about 50 cells, about 1 cell to about 60 cells, about 1 cell to about 70 cells, about 1 cell to about 80 cells, about 1 cell to about 90 cells, about 1 cell to about 100 cells, about 1 cell to about 1 x 10 3 cells, about 1 cell to about 2 x 10 3 cells, about 1 cell to about 3 x 10 3 cells, about 1 cell to about 4 x 10 3 cells, about 1 cell to about 5 x 10 3 cells, about 1 cell to about 6 x 10 3 cells, about 1 cell to about 7 x 10 3 cells, about 1 cell to about 8 x 10 3 cells, about 1 cell to about 9 x 10 3 cells, about 1 cell to about 1x 10 4 cells, about 1 cell to about 2 x 10 4 cells, about 1 cell to about 3 x 10 4 cells, about 1 cell to about 4 x 10 4 cells, about 1 cell to about 5 x 10 4 cells, about 1 cell to about 6 x 10 4 cells, about 1 cell to about 7x10 4 cells, about 1 cell to about 8 x 10 4 cells, about 1 cell to about 9 x 10 4 cells, about 1 cell to about 1x 10 5 cells, about 1 cell to about 2 x 10 5 cells, about 1 cell to about 3 x 10 5 cells, about 1 cell to about 4 x 10 5 cells, about 1 cell to about 5 x 10 5 cells, about 1 cell to about 6 x 10 5 cells, about 1 cell to about 7 x 10 5 cells, about 1 cell to about 8 x 10 5 cells, about 1 cell to about 9 x 10 5 cells, about 1 cell to about 1x 106 cells, about 1 cell to about 2 x 10 6 cells, about 1 cell to about 3 x 10 6 cells, about 1 cell to about 4 x 10 6 cells, about 1 cell to about 5 x 10 6 cells, about 1 cell to about 6 x 10 6 cells, about 1 cell to about 7 x 10 6 cells, about 1 cell to about 8 x 10 6 cells, about 1 cell to about 9 x 10 6 cells, about 1 cell to about 1x 10 7 cells, about 1 cell to about 2 x 10 7 cells, about 1 cell to about 3x10 7 cells, about 1 cell to about 4 x 10 7 cells, about 1 cell to about 5 x 10 7 cells, about 1 cell to about 6 x 10 7 cells, about 1 cell to about 7 x 10 7 cells, about 1 cell to about 8 x 10 7 cells, about 1 cell to about 9 x 10 7 cells, about 1 cell to about 1x 10 8 cells, about 1 cell to about 2 x 10 8 cells, about 1 cell to about 3 x 10 8 cells, about 1 cell to about 4 x 10 8 cells, about 1 cell to about 5 x 10 8 cells, about 1 cell to about 6 x 10 8 cells, about 1 cell to about 7 x 10 8 cells, about 1 cell to about 8 x 10 8 cells, about 1 cell to about 9 x 10 8 cells, or about 1 cell to about 1 x 10 9 cells.

[0282] In some cases, a therapeutically effective dose of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the invention may be about 1 x 10 3 cells to about 2x 10 3 cells, about 1x 10 3 cells to about 3x10 3 cells, about 1x 10 3 cells to about 4 x 103 cells, about 1x 10 3 cells to about 5 x 10 3 cells, about 1x10 3 cells to about 6 x 10 3 cells, about 1x 10 3 cells to about 7 x 10 3 cells, about 1x 10 3 cells to about 8x10 3 cells, about 1x 10 cells to about 9x 10 3 cells, about 1x 10 cells to about 1x 10 4 cells, about 1x 10 3 cells to about 2x10 4 cells, about 1x 10 3 cells to about 3x 10 4 cells, about 1x 10 3 cells to about 4 x 10 4 cells, about 1x 10 3 cells to about 5 x 10 4 cells, about 1x 10 3 cells to about 6 x 10 4 cells, about 1x 10 3 cells to about 7 x 10 4 cells, about 1x 10 3 cells to about 8 x 10 4 cells, about 1x 10 3 cells to about 9 x 10 4 cells, about 1x 10 3 cells to about 1x 10 5 cells, about 1x 10 3 cells to about 2x10 5 cells, about 1x 10 3 cells to about 3x 10 5 cells, about 1x 10 3 cells to about 4 x 10 5 cells, about 1x 10 3 cells to about 5 x 10 5 cells, about 1x 10 3 cells to about 6 x 10 5 cells, about 1x 10 3 cells to about 7 x 10 5 cells, about 1x 10 3 cells to about 8 x 10 5cells, about 1x 10 3 cells to about 9 x 10 5 cells, about 1x 10 3 cells to about 1x 10 6 cells, about 1x 10 3 cells to about 2x10 6 cells, about 1x 10 3 cells to about 3x 10 6 cells, about 1x 10 3 cells to about 4x10 6 cells, about 1x 10 3 cells to about 5 x 10 6 cells, about 1x 10 3 cells to about 6 x 10 6 cells, about 1x10 3 cells to about 7 x 10 6 cells, about 1x 10 3 cells to about 8 x 10 6 cells, about 1x 10 3 cells to about 9x10 6 cells, about 1x 10 3 cells to about 1x 10 7 cells, about 1x 10 3 cells to about 2x10 7 cells, about 1x 10 3 cells to about 3x 10 7 cells, about 1x 10 3 cells to about 4 x 10 7 cells, about 1x 10 3 cells to about 5 x 10 7 cells, about 1x 10 3 cells to about 6 x 10 7 cells, about 1x 10 3 cells to about 7 x 10 7 cells, about 1x 10 3 cells to about 8 x 10 7 cells, about 1x 10 3 cells to about 9 x 10 7 cells, about 1x 10 3 cells to about 1x 10 8 cells, about 1x 10 3 cells to about 2x10 8 cells, about 1x 10 3 cells to about 3x 108 cells, about 1x 10 3 cells to about 4 x 10 8 cells, about 1x 10 3 cells to about 5 x 10 8 cells, about 1x 10 cells to about 6x 10 8 cells, about 1x 10 cells to about 7x 10 8 cells, about 1x 10 3 cells to about 8 x 10 8 cells, about 1x 10 3 cells to about 9 x 10 8 cells, or about 1x 10 3 cells to about 1x 10 9 cells.

[0283] In some cases, a therapeutically effective dose of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the invention may be about 1 x 10 6 cells to about 2x 10 6 cells, about 1x 10 6 cells to about 3x10 6 cells, about 1x 10 6 cells to about 4 x 10 6 cells, about 1x 10 6 cells to about 5 x 10 6 cells, about 1x10 6 cells to about 6 x 10 6 cells, about 1x 10 6 cells to about 7 x 10 6 cells, about 1x 10 6 cells to about 8x10 6 cells, about 1x 10 6 cells to about 9 x 10 6 cells, about 1x 10 6 cells to about 1x 10 7 cells, about 1x10 6 cells to about 2x10 7 cells, about 1x 10 6 cells to about 3x 10 7 cells, about 1x 10 6 cells to about 4x10 7 cells, about 1x 10 6 cells to about 5 x 10 7 cells, about 1x 10 6cells to about 6 x 10 7 cells, about 1x10 6 cells to about 7 x 10 7 cells, about 1x 10 6 cells to about 8 x 10 7 cells, about 1x 10 6 cells to about 9x10 7 cells, about 1x 10 6 cells to about 1x 10 8 cells, about 1x 10 6 cells to about 2x10 8 cells, about 1x 10 6 cells to about 3x 10 8 cells, about 1x 10 6 cells to about 4 x 10 8 cells, about 1x 10 6 cells to about 5 x 10 8 cells, about 1x 10 6 cells to about 6 x 10 8 cells, about 1x 10 6 cells to about 7 x 10 8 cells, about 1x 10 6 cells to about 8 x 10 8 cells, about 1x 10 6 cells to about 9 x 10 8 cells, about 1x 10 6 cells to about 1x 10 9 cells, about 1x 10 6 cells to about 2x10 9 cells, about 1x 10 6 cells to about 3x 10 9 cells, about 1x 10 6 cells to about 4 x 10 9 cells, about 1x 10 6 cells to about 5 x 10 9 cells, about 1x 10 6 cells to about 6 x 10 9 cells, about 1x 10 6 cells to about 7 x 10 9 cells, about 1x 10 6 cells to about 8 x 10 9 cells, about 1x 10 6 cells to about 9 x 10 9 cells, about 1x 107 cells to about 1x 10 9 cells, about 1x 10 7 cells to about 2x10 9 cells, about 1x 10 7 cells to about 3x 10 9 cells, about 1x 10 7 cells to about 4 x 10 9 cells, about 1x 10 7 cells to about 5 x 10 9 cells, about 1x 10 7 cells to about 6 x 10 9 cells, about 1x 10 7 cells to about 7 x 10 9 cells, about 1x 10 7 cells to about 8 x 10 9 cells, about 1x 10 7 cells to about 9 x 10 9 cells, about 1x 10 8 cells to about 1x 10 9 cells, about 1x 10 8 cells to about 2x10 9 cells, about 1x 10 8 cells to about 3x 10 9 cells, about 1x10 8 cells to about 4 x 10 9 cells, about 1x 10 8 cells to about 5 x 10 9 cells, about 1x 10 8 cells to about 6x10 9 cells, about 1x 10 8 cells to about 7 x 10 9 cells, about 1x 10 8 cells to about 8 x 10 9 cells, about 1x10 8 cells to about 9 x 10 9 cells, or about 1x 10 8 cells to about 1x 10 10 cells.

[0284] H. Preservatives

[0285] In some embodiments, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the invention can be formulated in a freezing medium and placed in a cryogenic storage device, 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, δ 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 hydroxyethyl starch (HES) and a physiological pH buffer to maintain the pH at 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, cryopreserved cells can be thawed and further processed, for example by stimulation with antibodies, proteins, peptides, and / or cytokines mentioned herein. Cryopreserved cells can be thawed and genetically modified with viral vectors (including retroviral and lentiviral vectors) or non-viral means (including RNA, DNA and proteins) as described herein. Alternatively, host cells as described herein can be, for example, optionally amplified by the methods described herein, genetically modified, and then cryopreserved.

[0286] Thus, genetically engineered and / or non-engineered cells as disclosed herein can be cryopreserved to generate a bank of cells in the amount of at least about 1, 5, 10, 100, 150, 200, 500 vials in a freezing medium for approximately at least 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or at least about 10 10In some embodiments, the cell bank of cryopreservation can retain its function, and can thaw, and optionally can activate / stimulate and / or amplify. In some aspects, the thawed cell can be stimulated and amplified in a suitable closed container (such as a cell culture bag and / or a bioreactor) to generate a large amount of cells as an allogeneic cell product. In other examples, the cell of cryopreservation includes an autologous cell product. The cell of cryopreservation can maintain its biological function at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months or at least about 60 months under low temperature storage conditions. In some aspects, preservative is not used in the preparation. In some embodiments, the cell of cryopreservation can be thawed and infused into multiple patients as an allogeneic ready-made cell product.

[0287] Example

[0288] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare 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 numerals used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0289] Example 1. Construction of DAP10 construct

[0290] The DAP10 CAD construct was constructed with 0-4 elements selected from the group consisting of the Y86F mutation, the K84R mutation, the 4-1BB co-stimulatory domain, and the CD3ζ signaling domain. For the construction, the pSIN vector (Hariharan, MJ et al., (1998) Journal of Virology 72(2):950-958) was used. In short, the pSIN vector backbone is composed entirely of (South Plainfield, NJ) was synthesized from sequences provided by EUFETS (Germany), a subsidiary of BioNTech (Germany). The base plasmid used for all constructs is called pL077 pRetroSIN-GFP, which has a green fluorescent protein (GFP) box replaced by a gene of interest. Various constructs are depicted in Table 1 below. With respect to the following constructs, FP2A refers to furin and P2A linker genes, and CD19t refers to truncated CD19 markers. SEQ ID corresponds to amino acid sequences. Tables 4-19 below annotate the nucleic acid sequences encoding the amino acid sequences of the constructs depicted in Table 1, and sequences are selected from Table 3. Although Table 1 depicts a DAP10 construct including CD19t, other variants are also within the scope of the present disclosure, such as similar DAP10 constructs incorporating EGFRt, etc. Table 3 provides examples of such sequences.

[0291]

[0292] Example 2. Tumor Control of Various DAP10 CAD Constructs in the PLC / PRF / 5 Assay

[0293] Various DAP10CAD constructs in Table 1 above were tested in a PLC / PRF / 5 cell lysis assay. Figure 2A It is illustrated that DAP10.0 (DAP10 wild-type) and DAP10.13 (DAP10-K84R) did not show good tumor control in the PLC / PRF / 5 assay. Figure 2B Tumor controlling amounts of DAP10.3 (DAP10-CD3ζ) and DAP10.14 (DAP10-CD3ζ-K84R) constructs are exemplified. Figure 2C It is illustrated that the DAP10 CAD construct comprising a 4-1BB co-stimulatory domain and a CD3ζ signaling domain exhibits good tumor control in the absence of additional mutations (ie, DAP10.4), and the addition of only the Y86F mutation (ie, DAP10.5) or only the K84R mutation (ie, DAP10.15) does not significantly further improve tumor control relative to the DAP10.4 construct. However, in the DAP10 CAD construct DAP10.6 incorporating each of the CD3ζ signaling domain, the 4-1BB co-stimulatory domain, and both mutations (ie, Y86F and K84R), tumor control was significantly improved ( Figure 2C ).exist Figure 2A-2C In each of the Figures, tumor control tested with a positive control CAR is also depicted, as well as a graph showing the cytotoxicity index of individual tumor cells.

[0294] Also for Figure 2A-2CThe corresponding test conditions were used to examine the proliferation of Vδ1γδ cells. Figure 2D-2F As shown in the figure, good proliferation was observed from most of the DAP10 CAD constructs.

[0295] Example 3. Tumor Control of Various DAP10 CAD Constructs in the HepG2 Assay

[0296] Various DAP10 CAD constructs in Table 1 above were tested in the HepG2 assay. Figure 3A Data obtained using DAP10.0 (DAP10 wild-type) and DAP10.13 (DAP10-K84R) constructs are exemplified. Figure 3B Data obtained using DAP10.3 (DAP10-CD3ζ) and DAP10.14 (DAP10-CD3ζ-K84R) constructs are exemplified, and Figure 3C Data obtained using DAP10.4, DAP10.5, DAP10.6 and DAP10.15 constructs are exemplified. Figure 3A-3C In each of the Figures, tumor control tested with a positive control CAR is also depicted, as well as a graph showing the cytotoxicity index of individual tumor cells.

[0297] Also for Figure 3A-3C The corresponding test conditions were used to examine the proliferation of Vδ1γδ cells. Figure 3D-Figure 3F In the Hep2G assay, good proliferation and cytotoxicity were observed in constructs comprising CD3ζ and 4-1BB domains and at least the Y86F mutation (i.e., DAP10.15 and DAP10.6), with the DAP10.6 construct (which comprises each of CD3ζ, 4-1BB, Y86F, and K84R) showing the best performance in terms of cytotoxicity and Vδ1γδ cell proliferation.

[0298] Example 4. Survival of Vδ1 cells transduced with DAP10 constructs after tumor co-culture

[0299] In this example, the survival of Vδ1 cells transduced with various DAP10 constructs of Table 1 was evaluated. Specifically, survival was evaluated after 5 days of PLC / PRF / 5 co-culture. Figure 4 As shown, cells transduced with DAP10.6 showed optimal Vδ1 survival after 5 days of co-culture with PLC / PRF / 5.

[0300] Example 5. In vivo tumor control of DAP10 CAD constructs in mouse models

[0301] In this example, the effectiveness of DAP10 CAD constructs (DAP10.6 and DAP10.15, see Table 1) in a mouse model under controlled PLC / PRF / 5 tumor growth was examined. Figure 5A As shown, in mice bearing tumors only (group A), the mean tumor volume increased steadily over 35 days. 6 Similar results were observed in mice injected with Vδ1 T cells transduced with DAP10.6 (group C) or DAP10.15 (group D). Tumor growth was significantly reduced when mice were injected with Vδ1 T cells transduced with DAP10.6 (group C) or DAP10.15 (group D), with the most robust efficacy observed for DAP10.6 CAD in tumor control. Figure 5B Shown on day 35 Figure 5A The data obtained in were analyzed by Kruskal-Wallistest and Dunn's multiple comparison and plotted as a function of tumor volume as shown in the figure. Figure 5A and 5B In each of the AD groups, N = 5, and the number of tumor cells administered was 4e 6 .

[0302] Example 6. NKG2D expression in Vδ1 T cells transduced with DAP10 CAD

[0303] This example demonstrates that NKG2D expression levels are increased on cells transduced with a specific DAP10 CAD (e.g., DAP10.6, see Table 1). Briefly, Vδ1 T cells were transduced with one of DAP10.6, DAP10.13, DAP10.15, DAP10.5, and a control CAR, and the transduced cells were then co-cultured with PLC cells. Vδ1 NKG2D expression levels were assessed using a combination of NKG2D fluorescent labeling and FACS analysis. Figure 6In the figure, the expression levels of cells containing different DAP10 CAD or CAR controls are plotted as geometric mean fluorescence intensity (gMFI), thereby illustrating that the DAP10.6 CAD construct expressed in Vδ1T cells leads to significantly higher NKG2D expression levels compared to other DAP10 CAD (10.13, 10.15, 10.5) and CAR controls. It is worth noting that the DAP10.6 CAD construct contains each of the K84R mutation, the Y86F mutation, the 4-1BB co-stimulatory domain, and the CD3ζ signaling domain. Compared with the CAR control, a smaller but still statistically significant increase in NKG2D expression was observed in DAP10.15 (K84R+4-1BB+CD3ζ) and DAP10.5 (Y86F+4-1BB+CD3ζ).

[0304] Example 7. DAP10 CAD expression

[0305] This example demonstrates that DAP10 CAD expression is similar in different batches of Vδ1 cells.

[0306] For example, the DAP10.6.3 construct (SEQ ID NO: 77) includes a truncated EGFR as a marker, compared to the DAP10.6 construct (SEQ ID NO: 29) which includes CD19. The DAP10 construct, referred to herein as DAP10.16 (SEQ ID NO: 96), is identical to the DAP10.6.3 construct but includes a 1XX mutation in the CD3ζ signaling domain. It was found that Vδ1 cells transfected with either the DAP10.6 or DAP10.16 constructs exhibited 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 ).

[0307] Example 8. Cytotoxic activity of DAP10 CAD is mediated by NKG2D

[0308] This example demonstrates that blocking NKG2D abolishes cytotoxic activity. Specifically, blocking NKG2D using an NKG2D blocking antibody abolished the cytotoxic activity of Vδ1 cells expressing DAP10 CAD in three donors in two cell lines ( Figure 8A-B The cytotoxic activity of control Vδ1 cells transfected with CAR was not affected ( Figure 8C-8D ).for Fig. 8A , 8C , using PLC target cells, the E:T ratio was 5:1. Figure 8B , 8D, using HL60 target cells, with an E:T ratio of 2.5: 1. NKG2D blocking antibodies also had no effect on cells expressing the DAP10.0 construct (ie, lacking the co-stimulatory and intracellular signaling domains) (data not shown).

[0309] Example 9. DAP10 CAD molecule activation characteristics

[0310] This embodiment demonstrates consistent DAP10 CAD activation features in multiple donors and cell lines. In this embodiment, nanostring analysis (Nanostring Technologies, Seattle, WA) was performed after stimulation to assess molecular activation features. Cell lines used for stimulation include PLC (HCC), HL60, THP1 (AML) and HCT15 (CRC). Data illustrate consistent activation features by interferon gamma, 4-1BB and granzyme B treatment. Fig. 9A Data for DAP10.6 versus innate control (DAP10.0) are depicted, and Fig. 9B Data for DAP10.16 and innate control (DAP10.0) are depicted. No consistently detectable differences were observed between DAP10.6 and DAP10.16 ( Fig. 9C ).

[0311] Example 10. Broad anticancer activity of Vδ1 cells transduced with DAP10 CAD

[0312] This example demonstrates that Vδ1 cells transduced with the DAP10 CAD of the present disclosure exhibit anticancer activity against various cancer types with a wide range of NKG2D ligand expression levels / patterns.

[0313] Figures 10A-10E is a graph illustrating the percentage of cytotoxic activity of Vδ1 cells transduced with DAP10.6 versus E:T ratio in an 18 hour assay compared to Vδ1 cells transduced with DAP10.0 or cells not transduced with DAP10 CAD. Target cell lines included HCT116 ( Fig. 10A )、SKMEL5( Fig. 10B )、Mino ​​D2( Fig. 10C )、ScaBER( Fig. 10D ) and Raji B4( Fig. 10E ). Figure 10F-10G is a graph showing the percent cytotoxicity of Vδ1 cells transduced with DAP10.6 compared to an innate control (Vδ1 cells transduced with DAP10.0) or an irrelevant CAR control in an 18 hour assay. Figure 10F-10GThe target cell lines were NCI-H1581 and NCI-H2172, respectively. As shown in the figure, the cytotoxicity was significantly increased relative to the control. Fig. 10H The cell lines selected for the assay are exemplified to represent a broad range of NKG2D ligand expression levels / patterns. Fig. 10H Data from the NKG2D ligands assay were performed by flow cytometry on the indicated different cancer cell lines derived from a variety of hematological and solid tumors. The five antibodies used for staining detected MICA / MICB, ULBP1, ULBP2 / 5 / 6, ULBP3, and ULBP4. Data are presented as the mean fluorescence intensity (MFI) of the NKG2D ligands relative to the relevant isotype control. The raw data are in Figure 10I-10J Shown in (cancer cell lines were stained in triplicate).

[0314] Figures 11A-11G is a graph illustrating the relationship between the cytotoxicity index and the co-culture time of target cells and effector cells. Target cells include 22Rv1, Mino, HCT116 and HCT-15. Effector cells include Vδ1 cells transduced with DAP10 CAR, CAR control or untransduced cells. Compared with the control, enhanced cytotoxicity was observed when Vδ1 cells were transduced with the DAP10 CAD of the present disclosure. It was found that the degree of cytotoxic potential depends on the cell line and the E:T ratio. The cell lines tested represent different levels / patterns of NKG2D ligand expression (see Figures 10H-10J Effector cells were co-cultured with target cells expressing NucRed at a submaximal E:T ratio of 5:1 or 1.5:1 (depending on the cell line). The cytotoxicity index was calculated by calculating the total NucRed object area (mm) at each time point. 2 It is calculated by dividing the value at time = 0.

[0315] Example 11. Comparable cytotoxic activity of multiple batches of Vδ1 cells transduced with DAP10 CAD

[0316] This example demonstrates that the cytotoxic activity of Vδ1 cells transduced with DAP10 CAD is comparable for different batches of Vδ1 cells and DAP10 CAD. Fig. 12A is a graph showing data from a representative 120-hour cytotoxicity assay. The target cells were PLC / PRF / 5. The Vδ1 cells used in the assay were transduced with DAP10.6, DAP10.16, DAP10 reference batch (i.e., positive control batch of amplified Vδ1 cells), and DAP10.0 (control). Data from PLC / PRF / 5 cells alone (i.e., not co-cultured with Vδ1 cells) are also shown. Fig. 12Bis a graph depicting the percent reduction in cytotoxicity of individual tumors relative to tumors treated with DAP10 CAD-transduced Vδ1 cells using the final time point of the 120 hour assay.

[0317] The cytotoxicity of DAP10.6, DAP10.16 and DAP10.17 constructs incorporated into Vδ1 cells was examined in a 120 hour cytotoxicity assay.Vδ1 cells from three donors were tested with the DAP10 construct using PLC / PRF / 5 target cells. Fig. 12C Each construct shown is the total of all three donors. Using stringent E:T ratios, the DAP10.6 construct exhibited slightly better average cytotoxicity than DAP10.16 and DAP10.17 in this assay.

[0318] The efficacy of DAP10.6, DAP10.16 and DAP10.17 showed some donor dependence. The cytotoxicity index was measured in co-culture experiments using Vδ1 cells transduced with DAP10.6, DAP10.16 or DAP10.17 from three different donors (SCT06, SCT29, SCT46) or untransduced cells from the same donors. The target cells in the co-culture experiments were PLC / PRF / 5 cells. The co-culture time was 120 hours. Figure 12D-12E As shown, DAP10.6 and DAP10.16 showed very similar characteristics. DAP10.17 was found to be most donor dependent ( Fig.12F A reference batch of Vδ1 cells transduced with DAP10.6 was used in all assays and served as a reproducible control (data not shown).

[0319] Example 12. Cytokine profiles corresponding to DAP10 CAD stimulation

[0320] This example demonstrates that DAP10 CAD stimulation produces a multifunctional cytokine profile.

[0321] Fig.13A The relationship between the cytokine profile and the different DAP10 constructs disclosed herein is illustrated. As shown, the extent of cytokine activation is cell line specific. The cell lines tested only included PLC, Mino and T cells. Importantly, no detection of potentially problematic cytokines (e.g., IL-6 and IL-17) was observed. Fig. 13B is a graph showing interferon gamma induction (pg / ml / 1E) in individual PLCs, Mino and T cells of different DAP10CAD (from different donors, e.g. SCT06, SCT 46). 6 CAD+ cells). Figure 13C-13Fis a representative experiment illustrating interferon-γ secretion by CAD+Vδ1 cells alone after co-culture with PLC / PRF / 5, HL60, THP1, and PC3 target cells, respectively, at submaximal E:T ratios for approximately 18 hours.

[0322] Example 13. Cytokine profile of DAP10 CAD compared to chimeric antigen receptor (CAR)

[0323] This example demonstrates that the cytokine profile of the DAP10 CAD disclosed herein is highly similar to that of CAR ( Fig.14 Conditions examined included Vδ1 cells transduced with DAP10 CAD plus PLC cells, or Vδ1 cells alone (i.e., minus target cells), and Vδ1 cells transduced with CAR plus target cells (HepG2, PLC, Raji), or Vδ1 cells alone (i.e., minus target cells). Notably, Vδ1 cells transduced with DAP10 CAD in the absence of target cells showed less background cytokine secretion than Vδ1 cells transduced with CAR in the absence of target cells.

[0324] Example 14. DAP10 CAD stimulation drives proliferation across multiple donors

[0325] This example demonstrates that DAP10 CAD stimulation drives Vδ1 cell proliferation in all tested donors. Specifically, for this example, Vδ1 cell proliferation was assessed by co-culturing with PLC / PRF / 5 (5:1 E:T ratio) after transduction of the DAP10 CAD constructs disclosed herein (DAP10.6, DAP10.16, DAP10.17). Controls included Vδ1 cells transduced with DAP10 control batch or DAP10.0. The less / slower proliferation of Vδ1 cells transduced with DAP10.16 may be due to the 1XXCD3ζ signaling domain. In this regard, enhanced regulation of activation / proliferation may be beneficial to the long-term efficacy / survival of engineered Vδ1 cells due to reduced over-simulation / exhaustion.

[0326] Vδ1 cells from two different donors (SCT29 and SCT46) were used in the co-culture experiments. Fig.15 As shown, robust proliferation of Vδ1 cells transduced with DAP10.6 and DAP10.16 was observed in both donors, whereas proliferation of Vδ1 cells transduced with DAP10.17 was slightly more donor dependent.

[0327] Example 15. In vivo tumor control of Vδ1 cells transduced with DAP10CAD containing the 1XX CD3ζ intracellular signaling domain

[0328] This example demonstrates that incorporation of the 1XX CD3ζ intracellular signaling domain can improve tumor control in vivo.

[0329] In this example, the effectiveness of the DAP10 CAD constructs DAP10.6 and DAP10.16 in a mouse model under controlled PLC / PRF / 5 tumor growth was examined. Fig.16A As shown, mice treated with Vδ1 cells transduced with DAP10.16 CAD (in which the CD3ζ intracellular signaling domain comprises a 1XX mutation) had improved tumor control compared to mice treated with Vδ1 cells transduced with DAP10.6 CAD (p=0.0079, Mann-Whitney test (two-tailed)). A schematic diagram of the experimental procedure used in this example is depicted in Fig. 16B middle.

[0330] Example 16. Comparison of in vivo anti-tumor activity of DAP10 CAD Vδ1 cells and CAR Vδ1 cells

[0331] This example demonstrates that DAP10 CAD+Vδ1 cells exhibit anti-tumor activity with similar kinetics to CAR Vδ1 cells.

[0332] Fig.17A DAP10 CAD+Vδ1 cells (5e 6 Cells / dose and 15e 6 cells / dose) in vivo tumor growth kinetics. Fig. 17B Tumor volumes quantified on day 27 are shown. A schematic diagram of the experimental procedures used in this example is depicted in Fig. 17C Data are shown as mean ± SEM of 5 mice / group. Final statistical significance between the complete cohorts for each treatment was assessed using the Kruskal-Wallis test with Dunn's multiple comparisons (ns = not significant).

[0333] Example 17. In vivo proliferation, persistence and targeting of engineered Vδ1 cells

[0334] This example demonstrates that Vδ1 cells transduced with the DAP10 CAD of the present disclosure proliferate in vivo in tumors in a mouse model, but not in other organs. For this example, two independent studies were performed. Fig.18AAs shown, proliferation of Vδ1 cells transduced with DAP10.6 was observed in tumor tissues (subcutaneous PLC / PRF / 5 cells) but not in spleen, lung, liver, bone marrow or blood on day 7 after treatment. The Vδ1 cells used in Study 1 were obtained from different donors than those used in Study 2. Fig.18A The HuCD45+, Vδ1+ population is shown. Fig.18B is a graph quantifying engineered Vδ1 cells per mg of tumor tissue in the corresponding studies at Day 4, Day 7, and Day 14 (Study 1) and Day 7 and Day 14 (Study 2). Each study used 5e 6 Notably, an increase in the total number of Vδ1 cells within the tumor was observed in each study. Fig.18C is a graph showing quantification of Vδ1 cells in tumor tissue or other tissues (lung, liver, spleen, bone marrow, blood) 4, 7 or 14 days after treatment as assessed by flow cytometry and represents a cumulative analysis of two independent studies presented in this Example. Fig.18D Schematic diagram of the experimental procedure corresponding to this example is provided for reference. No significant changes in body weight or acute clinical signs of toxicity or xenogeneic graft-versus-host disease (GvHD) were observed in the treated mice ( Fig.19 ). Fig.19 Data shown are from n=4 independent efficacy studies.

[0335] Vδ1 cells transduced with the DAP10 CAD of the present disclosure were found to effectively target tumor cells while being non-detrimental to normal (ie, non-tumor) cells. Specifically, short-term cytotoxicity assays employed an Annexin / DAPI flow-based approach to analyze primary cellular targets. Fig. 20A is a graph showing that Vδ1 cells transduced with the DAP10 CAD (DAP10.6, DAP10.16) of the present disclosure and Vδ1 cells transduced with NKG2D CAR significantly reduced THP1 cell viability compared to THP1 alone and Vδ1 cells transduced with an innate control (DAP10.0). Fig. 20B is a graph showing that Vδ1 cells transduced with the DAP10 CAD of the present disclosure (DAP10.6, DAP10.16) essentially lack targeting to healthy PBMCs, similar to the innate control (Vδ1 cells transduced with DAP10.0). Notably, Vδ1 cells transduced with DAP10 CAD consistently exhibited lower PBMC targeting than the NKG2D CAR reference ( Fig. 20B ).

[0336] Example 18. Small-scale donor screening

[0337] This example demonstrates that expansion of Vδ1 cells transduced with DAP10 CAD comprising CD3ζ 1XX modifications may be improved compared to similar constructs that do not comprise 1XX in the CD3ζ intracellular signaling domain.

[0338] For this example, Vδ1 cells from 6 donors (SE001, ARC007, HC45, DLS003, SE015, and SCT029) were tested in two experiments in small-scale shake flask expansion. Specifically, Vδ1 cells from different donors were transduced with either DAP10.6 or DAP10.16. Compared to DAP10.6 transduced cells from all donors, 50-135% more Vδ1 cells ( Fig.21 ). Expansion was measured on days 14 and 15.

[0339] Example 19. DAP10 CAD Vδ1 Cell Growth Kinetics

[0340] This example demonstrates that in culture, transduced with leading DAP10CAD constructs (DAP10.6, DAP10.16, DAP10.17) resulted in a significantly higher shift in the percentage of Vδ1 cells compared to controls (DAP10.0, CAR control).

[0341] For this example, 144 samples from three different donors (SCT06, Fig.22A ; SCT29, Fig. 22B ; SCT45, Fig. 22C ) were transduced with the leading DAP10 construct or control, and the % Vδ1 of total cells was expanded as a function of expansion time. Figure 22A-22C As shown in either of the , a significantly higher shift in the Vδ1 percentage was observed for the leading DAP10 construct compared to the control.

[0342] Example 20. Expansion of cells transduced with DAP10 CAD constructs

[0343] This example demonstrates that Vδ1 cells can be efficiently expanded and transduced with the DAP10CAD of the present disclosure. Fig.23A A schematic diagram representing the process for generating "off-the-shelf" allogeneic CAD Vδ1 cells is depicted. Fig. 23B , 23C, 23D depict data showing independent amplification of leading DAP10 CAD constructs (DAP10.6, DAP10.16, DAP10.17) transduced into Vδ1 cells obtained from three different donors (SCT06, SCT29, SCT45). Independent amplifications (blue and red) show similar trends in growth characteristics of DAP10 CAD constructs. In two of the three donors, growth was considered construct-dependent.

[0344] Representative experiment illustrates that in vitro culture of Vδ1 cells resulted in a significant fold expansion of Vδ1 cells ( Fig.23E ) and robust DAP10 CAD transduction ( Fig.23F ). Figure 23E-23F The data were obtained from 12 independent cultures using PBMCs from 7 different donors. Figure 23G Shown is a series of graphs showing changes in cell composition (Vδ1 cells, Vδ2 cells, αβ cells, and NK cells) over time (day 0, before αβ T cell depletion, and after αβ T cell depletion), expressed as a percentage of the culture.

[0345] Example 21 CAR-CAD co-expression construct

[0346] Vδ1 cells co-expressing CAR and Dap10 CAD were generated using the constructs in Table 2 below. Briefly, the CAR-DAP10 CAD construct was constructed with 0-4 elements selected from the group consisting of the Y86F mutation, the K84R mutation, the 4-1BB co-stimulatory domain, and the CD3ζ signaling domain. For the construction, pSIN was used. Briefly, the pSIN vector backbone consisted entirely of (South Plainfield, NJ) was synthesized from sequences provided by EUFETS (Germany), a subsidiary of BioNTech (Germany). The base plasmid used for all constructs is called pL077 pRetroSIN-GFP, which has a green fluorescent protein (GFP) box replaced by a gene of interest. Various constructs are depicted in Table 2 below. With respect to the following constructs, 3H7-5.1 refers to a CAR bound to CD20 (see, e.g., Nishimoto et al., Clin Transl Immunology. 2022; 11 (2): e1373), and P2A refers to a P2A cleavage sequence.

[0347]

[0348]

[0349] Vδ1γδ cells expressing the CAR-Dap10CAD construct were tested by an Incucyte-based restimulation assay of Raji, Mino, and MOLP-8 cell lines, which express the target of the CAR, but not the MOLP-8 cell line. Figure 24A-24C Results are shown for Raji, Mino and MOLP8 cell lines, respectively. Vδ1 cells transduced with the CAR-Dap10 CAD construct exhibited prolonged cytotoxic activity against targets with and without CAR antigen compared to non-transduced controls.

[0350] Those skilled in the art will think of certain modifications and improvements after reading the foregoing description. It should be understood that all such modifications and improvements have been deleted herein for the sake of brevity and readability, but they are fully within the scope of the following claims.

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[0405] Table 8: SEQ ID NO:30 annotation describe Nucleotide number Signal peptide 1-60 DAP10.6(DAP10-K84R-Y86F+4-1BB+CD3z) 61-804 DAP10-K84R-Y86F (mutations are bold and underlined) 61-339 4-1BB co-stimulatory domain 340-465 CD3z signaling domain 466-804 Furin P2A 805-885 Truncated CD19 886-1884

[0406] Table 9: SEQ ID NO:32 annotation describe Nucleotide number Signal peptide 1-60 DAP10.13 (DAP10-K84R) (mutation in bold and underlined) 61-339 Furin P2A 340-420 Truncated CD19 421-1419

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Claims

1. A mammalian cell comprising a chimeric adaptor (CAD) polypeptide comprising at least one of a DAP10 domain comprising a human DAP10 amino acid sequence, a co-stimulatory domain and / or an intracellular signaling domain, and specifically lacking an extracellular domain comprising a functional extracellular receptor and / or a ligand binding domain, wherein the mammalian cell further comprises at least one chimeric receptor comprising an extracellular targeting domain that specifically binds to a target antigen on a target cell.

2. The mammalian cell of claim 1, wherein the chimeric receptor comprises at least one of an intracellular signaling domain and / or a co-stimulatory domain.

3. The mammalian cell of claim 1 or claim 2, wherein the chimeric receptor comprises at least one DAP10 interacting domain. 4 . The mammalian cell of claim 3 , wherein the DAP10 interaction domain comprises the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence having at least 80%, 90% or 95% sequence identity to SEQ ID NO:

75.

5. The mammalian cell according to any one of claims 1-4, wherein the target antigen on the target cell is selected from the group consisting of: CD20, BCMA, GPC3, TyrD, FcRL5, B7H6, CD70, PSMA, CD19 and FAP.

6. The mammalian cell of any one of claims 1-5, wherein the CAD polypeptide comprises at least one co-stimulatory domain 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.

7. The mammalian cell of claim 6, wherein the at least one co-stimulatory domain of the CAD polypeptide is 4-1BB.

8. The mammalian cell of claim 6, wherein the at least one co-stimulatory domain of the CAD polypeptide is CD28.

9. The mammalian cell of any one of claims 1-8, wherein the CAD polypeptide comprises at least one intracellular signaling domain selected from the group consisting of CD3ζ, DAP12, LFA-1, and a repeat (2-5) DAP10 YINM motif.

10. The mammalian cell of claim 9, wherein the at least one intracellular signaling domain of the CAD polypeptide is CD3ζ, optionally wherein CD3ζ has the amino acid sequence shown in SEQ ID NO:

82.

11. A mammalian cell according to claim 9 or claim 10, wherein the at least one co-stimulatory domain of the CAD polypeptide is 4-1BB and the at least one intracellular signaling domain of the CAD polypeptide is CD3ζ.

12. The mammalian cell of claim 11, wherein the CAD polypeptide comprises, from N-terminus to C-terminus, the DAP10 domain, the 4-1BB co-stimulatory domain, followed by the CD3ζ intracellular signaling domain.

13. The mammalian cell of claim 6, wherein the CAD polypeptide comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain.

14. The mammalian cell of claim 13, wherein the CAD polypeptide comprises, from N-terminus to C-terminus, the DAP10 domain, the 4-1BB co-stimulatory domain, followed by the CD28 co-stimulatory domain, followed by a CD3ζ intracellular signaling domain, optionally wherein CD3ζ has an amino acid sequence as shown in SEQ ID NO:

82.

15. The mammalian cell of claim 13, wherein the CAD polypeptide comprises, from N-terminus to C-terminus, the DAP10 domain, the CD28 co-stimulatory domain, followed by the 4-1BB co-stimulatory domain, followed by a CD3ζ signaling domain, optionally wherein CD3ζ has the amino acid sequence shown in SEQ ID NO:

82.

16. The mammalian cell according to any one of claims 1 to 15, wherein the human DAP10 amino acid sequence comprises the following amino acid sequence: (i) having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 1; (ii) having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO:78; or (iii) has at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO:

81.

17. The mammalian cell according to any one of claims 1-16, wherein the human DAP10 amino acid sequence comprises a mutated human DAP10 amino acid sequence.

18. The mammalian cell according to claim 17, wherein the mutant human DAP10 amino acid sequence comprises an amino acid substitution at a position corresponding to K84 and / or Y86.

19. The mammalian cell of claim 18, wherein the amino acid substitution at position K84 comprises a K84R substitution.

20. The mammalian cell of claim 18 or claim 19, wherein the amino acid substitution at Y86 comprises a Y86F substitution.

21. The mammalian cell of any one of claims 1-20, wherein the CAD polypeptide is encoded by an isolated nucleic acid operably linked to a regulatable promoter; and Wherein the chimeric receptor is encoded by an isolated nucleic acid operably linked to a regulatable promoter.

22. The mammalian cell of claim 21, wherein the isolated nucleic acid encoding the CAD polypeptide encodes a cytokine, and / or wherein the isolated nucleic acid encoding the chimeric receptor encodes a cytokine.

23. The mammalian cell of claim 22, wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21 and IL-23.

24. The mammalian cell of claim 2, wherein when the chimeric receptor comprises an intracellular signaling domain, the CAD polypeptide comprises a co-stimulatory domain, and vice versa.

25. The mammalian cell of any one of claims 1-24, further comprising at least one receptor that associates with DAP10, wherein the at least one receptor is not the at least one chimeric receptor.

26. The mammalian cell of claim 25, wherein the at least one receptor is exogenous.

27. The mammalian cell of claim 26, wherein the exogenous receptor is overexpressed.

28. The mammalian cell of any one of claims 26-27, wherein the at least one exogenous receptor is selected from NKG2D, Ly49H, Ly49D, Sirp-b1, Siglec-15 and Cd3001b.

29. The mammalian cell of claim 28, wherein the at least one receptor is NKG2D.

30. The mammalian cell according to any one of claims 1-29, wherein the mammalian cell is an immune cell, preferably wherein the immune cell is a cytotoxic cell.

31. The mammalian cell of any one of claims 1-30, wherein the mammalian cell exhibits in vitro and / or in vivo killing activity against the target cell that exhibits cell surface expression of the target antigen.

32. The mammalian cell of claim 31, wherein the target cell is a hematological tumor cell.

33. The mammalian cell of claim 31, wherein the target cell is a solid tumor cell.

34. A mammalian cell according to any one of claims 31-33, wherein the in vitro and / or in vivo killing activity is greater than the intrinsic level of in vitro and / or in vivo killing activity in a control mammalian cell lacking expression of one or both of the chimeric receptor and / or the CAD polypeptide.

35. The mammalian cell of any one of claims 1-34, wherein the mammalian cell proliferates in response to contact with the target cell.

36. The mammalian cell of any one of claims 1-35, wherein the mammalian cell exhibits increased proliferation in response to contact with the target cell as compared to a control mammalian cell lacking expression of one or both of the chimeric receptor and / or the CAD polypeptide.

37. The mammalian cell of any one of claims 35-36, wherein the mammalian cell is propagated in a host organism comprising the target cell.

38. The mammalian cell of any one of claims 1-37, wherein the mammalian cell expresses a pro-inflammatory cytokine in response to contact with the target cell.

39. The mammalian cell of claim 38, wherein the pro-inflammatory cytokine comprises tumor necrosis factor alpha or interferon gamma.

40. A plurality of mammalian cells according to any one of claims 1-39.

41. The plurality of mammalian cells of claim 40, wherein the plurality of mammalian cells comprises at least about 10 6 cells, at least 10 7 cells or at least 10 8 cells, preferably about 10 8 Up to 10 11 cells.

42. A method of preparing a mammalian cell according to any one of claims 21-41, wherein the method comprises transfecting the mammalian cell with a construct comprising the isolated nucleic acid encoding the CAD polypeptide and at least one construct encoding the at least one chimeric receptor.

43. The method of claim 42, wherein the method comprises retroviral transduction.

44. The method of claim 42 or claim 43, wherein the method comprises ex vivo expansion of the mammalian cells, wherein the ex vivo expansion is performed before and / or after transfection of the isolated nucleic acid encoding the CAD polypeptide and at least one construct encoding the at least one chimeric receptor.

45. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a mammalian cell according to any one of claims 1-39 or a plurality of mammalian cells according to claim 40 or claim 41.

46. ​​A method of activating a mammalian cell according to any one of claims 1-39 or a plurality of mammalian cells according to claim 40 or claim 41, comprising contacting the target cell with the mammalian cell.

47. The method of claim 46, wherein the mammalian cell or cells are introduced into a subject in need thereof; and wherein said activation occurs in said subject.

48. Use of a mammalian cell according to any one of claims 1-39, a plurality of mammalian cells according to claim 40 or claim 41, or a pharmaceutical composition according to claim 45 in the preparation of a medicament for treating a subject having a disease, wherein the mammalian cell or the plurality of mammalian cells reduces at least one symptom or sign of the disease in the subject.

49. Use of a tumor cell killing effective amount of a mammalian cell according to any one of claims 31-34, or a plurality of mammalian cells according to claim 40 or claim 41, or a pharmaceutical composition according to claim 45 in the preparation of a medicament for treating cancer in a subject in need thereof.

50. A method for killing tumor cells, the method comprising contacting the tumor cells with a tumor cell killing effective amount of the mammalian cells according to any one of claims 31-34, the plurality of mammalian cells according to claim 40 or claim 41, or the pharmaceutical composition according to claim 45.

51. The method of claim 50, wherein the method comprises introducing a therapeutically effective amount of the mammalian cells or the pharmaceutical composition into a host organism comprising the tumor cells.

52. The method of claim 51, wherein the method comprises introducing the therapeutically effective amount of the mammalian cells or the pharmaceutical composition into the host organism comprising the tumor cells, and simultaneously or sequentially administering one or more methods of elevating common gamma chain cytokines.

53. The method of claim 52, wherein the method of administering one or more methods of elevating common γ chain cytokines comprises administering an amount of common γ chain cytokines effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced mammalian cells simultaneously or sequentially with the introduction of the mammalian cells, preferably wherein the method comprises administering IL-2, more preferably wherein the method comprises administering IL-15.

54. The method of claim 53, wherein the one or more methods of increasing common gamma chain cytokines comprise administering an amount of common gamma chain cytokines effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced mammalian cells prior to and / or after introduction of the mammalian cells or the pharmaceutical composition.

55. The method of any one of claims 52-54, wherein the one or more methods of elevating common gamma chain cytokines comprise lymphocyte depletion prior to introduction into the mammalian cells.

56. The method of any one of claims 52-55, wherein the one or more methods of increasing common gamma chain cytokines comprise secreting one or more common gamma chain cytokines from the introduced mammalian cells.

57. The method of any one of claims 51 to 56, wherein the method reduces the in vivo tumor burden in the host organism, and / or increases the average survival time of the host organism, compared to a control organism, wherein the control organism has not been treated with the mammalian cell or the pharmaceutical composition.

58. The method of any one of claims 50 to 57, wherein the method is a method of treating cancer in a subject in need thereof.

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