Compositions and Methods Comprising Chimeric Adapter Polypeptides

Abstract

Aspects of the present disclosure include compositions and methods for treating a variety of diseases / conditions in engineered host cells, the engineered host cells comprising a chimeric adapter (CAD) polypeptide comprising DAP10 and at least one chimeric receptor. The CAD polypeptide may include substitution mutations and / or additional protein domains that function in conjunction with the associated receptor to enhance cell survival and proliferation of the host cell and enhance cell killing activity of non-host cells.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 347,194, filed on May 31, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Field of the Disclosure The present disclosure generally relates to cellular immunotherapy, and in particular, to compositions and methods comprising mammalian cells comprising at least one chimeric adapter polypeptide and one or more chimeric receptors for improving cell survival, proliferation, signal transduction, etc.

Background Art

[0003] Adoptive immunocyte therapy has experienced continuous repetition for more than 30 years, from the early days focusing on basic lymphokine activation and / or tumor infiltration to the most recent strategies of genetically engineering these immune cells to express genetically engineered antigen receptors such as chimeric antigen receptors (CARs). However, although there have been some hints and indications about the therapeutic potential of various approaches so far, there are still numerous problems.

[0004] One problem relates to low or absent expression of the target of adoptive cell therapy (i.e., antigen escape). Specifically, a common mechanism of resistance to adoptive (or natural) cell therapy is the emergence of cell types (e.g., tumors) accompanied by loss or down - regulation of the target antigen. Such loss or down - regulation can lead to a reduction in the effectiveness of adoptive (or natural) cell responses (Majzner RG and Mackall, CL. (2018) Cancer Discovery, 8(10):1219 - 26).

[0005] Another related problem is the downregulation of naturally occurring receptor(s) that can recognize ligands specifically present on cells associated with a particular disease, which can also lead to the invalidation of the effects of cellular responses to various disease states. As a representative example, NKG2D is an activating immune receptor found on human natural killer (NK) cells, CD8+ αβ T cells, and γδ T cells, which regulates both 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 S.J., et al. (2008) Immunol Res, 2008, 40(1):18-34). In humans, NKG2D ligands are not expressed on normal cells but are widely expressed at various levels on transformed or virus-infected cancer cells (see, for example: Bauer S, et al. (1999) Science, vol. 285, 5428:727-729; Burgess S.J., et al. (2008) Immunol Res, 40(1):18-34; Baugh R, et al. (2020), Cancers, 12(12):3827). The expression of NKG2D ligands on the surface of tumor cells sensitizes tumor cells to immune cell-mediated destruction by activating NKG2D, activating NK cells, and co-stimulating effector T cells. Thus, the NKG2D receptor and its ligands are targets for the purposes of cancer immunotherapy.

[0006] However, unfortunately, NKG2D can be downregulated when it is most needed. For example, tumor-derived transforming growth factor-β (TGF-β) downregulates NKG2D, thereby reducing the killing of tumor cells by NK cells and CD8 + 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 for the treatment of tumors.

[0007] NKG2D is mentioned by way of example, inter alia, to illustrate that there is a need in the art for compositions and methods that can, for example, improve the survival and proliferation of immune cells, prevent downregulation of receptors, and compensate for immune evasion of antigens that are targets of adoptive immunotherapy approaches. Such compositions and methods would improve the prognosis of patients undergoing adoptive immunotherapy. SUMMARY OF THE INVENTION

[0008] The present invention addresses the above-mentioned drawbacks in the prior art by mammalian cells comprising both a chimeric adapter (CAD) polypeptide comprising human DAP10 and at least one chimeric receptor, optionally mammalian cells in which the CAD polypeptide associates with at least one chimeric receptor, and methods of using the same. As explicitly demonstrated for the first time herein, the subject CAD constructs and polypeptides improve the stability of receptors (e.g., chimeric and / or non-chimeric) capable of recognizing target antigens on various cell surfaces, promote a favorable balance of cell signaling pathway(s) upon engagement of the receptor with the target, and / or, upon engagement of at least one chimeric receptor with various ligands, and optionally, upon engagement of non-chimeric receptor(s) that associate with the CAD polypeptide (e.g., NKG2D) with various ligands, improve functional properties (e.g., enhanced cytolytic properties, proliferative properties, survival properties, and / or costimulatory properties) elicited.

[0009] In one aspect, the present invention provides mammalian cells comprising a chimeric adapter (CAD) polypeptide comprising a DAP10 domain and at least one of a costimulatory domain and / or an intracellular signaling domain, specifically lacking an extracellular domain comprising a functional extracellular receptor and / or ligand-binding domain, the mammalian cells further comprising 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.

[0010] In an embodiment, the chimeric receptor comprises at least one of an intracellular signaling domain and / or a co-stimulatory domain.

[0011] In an embodiment, the chimeric receptor associates with DAP10 and comprises at least one DAP10-interacting domain. In an embodiment, 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.

[0012] In the embodiments, the target antigens on the target cells are CD20, TyrD, B7H6, CD3, CD19; CD123; CD22; CD30; CD70, CD171; CD6, CS-1 (also called 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(1-4)bDGlcp(11)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((TnAg) 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 (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); IL-11R alpha; mesothelin; interleukin 11 receptor alpha (IL-URa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha receptor-type tyrosine protein kinase ERBB2 (Her2 / neu); ElbB3, ErbB4, mucin 1, cell surface-bound (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostate acid phosphatase (PAP); elongation factor 2 mutation (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 (prososome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gplOO);Cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Ab1) (bcr-ab1); Tyrosine kinase; Ephrin type-A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGrcp(11)Cer); Transglutaminase 5 (TGS5); High molecular weight melanoma-associated antigen (HMWMAA); Melanoma-associated antigen; o-Acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; 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; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexose portion of GloboH glycosphingolipid (GloboH); Breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); Adrenergic receptor beta-3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K (LY6K); Olfactory receptor 51E2 (OR51E2); TCR gamma alternative 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); Semen protein 17 (SPA17); X antigen family, member 1A (XAGE1) Angiopoietin-binding cell surface receptor 2 (Tie2); 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 variant, protein; Survivin; Telomerase;Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells (MelanA or MartI); rat sarcoma (Ras) variant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis 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 the imprinted site regulator), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK), kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPVE6); human papillomavirus E7 (HPVE7); intestinal carboxylesterase; heat shock protein 70-2 variant (mutant hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (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);Selected from the group consisting of 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, disialoganglioside 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-alpha, glioma-associated antigen, glycosphingolipid, gp36, G250 / CAIX, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp41, HPV-specific antigen, KDR, kappa chain, insulin growth factor (IGF1)-l, ligands belonging to MIC in humans (MICA and MICB) and ULBP (ULBP1-ULBP6) family, LAGA-la, LewisY, lambda chain, lectin-reactive AFP, L1 cell adhesion molecule, MAGE, major histocompatibility complex (MHC) molecule, tumor-specific peptide epitope of the presence of major histocompatibility complex (MHC) molecule, M-CSF, MN-CAIX, MUC-1, MUC-16, NKG2D, NKG2D ligand, neutrophil elastase, Nkp30, cancer fetal antigen (h5T4) p53, prostate-specific antigen (PSA), PSC1, prostate-specific antigen protein, STEAP1, STEAP2, surface adhesion molecule, survivin and telomerase, TAG-72, additional domain A (EDA) and additional domain B (EDB) of fibronectin and Al domain of tenascin-C (TnCAl), thyroglobulin, Tem8, tumor stromal antigen, VEGF-A, and immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0013] In embodiments, at least one co-stimulatory domain of the CAD polypeptide and optionally at least one co-stimulatory domain of the 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 combinations thereof. In preferred embodiments, at least one co-stimulatory domain of the CAD polypeptide is 4-1BB or CD28.

[0014] In embodiments, the CAD polypeptide comprises at least one intracellular signaling domain. In embodiments, at least one intracellular signaling domain of the CAD polypeptide and optionally at least one intracellular signaling domain of the chimeric receptor are selected from CD3ζ, DAP12, LFA-1, and the repeat (2-5) DAP10 YINM motif. In preferred embodiments, at least one intracellular signaling domain of the CAD polypeptide is CD3ζ.

[0015] In embodiments, 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 preferred embodiments, the CAD polypeptide comprises a DAP10 domain, a 4-1BB co-stimulatory domain, and a subsequent CD3ζ intracellular signaling domain, from N-terminus to C-terminus.

[0016] In embodiments, at least one co-stimulatory domain is CD28 and at least one intracellular signaling domain is CD3ζ. In embodiments, the CAD polypeptide comprises a DAP10 domain, a CD28 co-stimulatory domain, and a subsequent CD3ζ intracellular signaling domain, from N-terminus to C-terminus.

[0017] In embodiments, the CAD polypeptide comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain. In embodiments, the CAD polypeptide comprises, from the N-terminus to the C-terminus, a DAP10 domain, a 4-1BB co-stimulatory domain, followed by a CD28 co-stimulatory domain, and then followed by a CD3ζ intracellular signaling domain. In another embodiment, the CAD polypeptide comprises, from the N-terminus to the C-terminus, a DAP10 domain, a CD28 co-stimulatory domain, followed by a 4-1BB co-stimulatory domain, and then followed by a CD3ζ signaling domain.

[0018] In embodiments, 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 embodiments, 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 embodiments, 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.

[0019] In embodiments, 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 embodiments, the amino acid substitution at position K84 comprises a K84R substitution. In embodiments, the amino acid substitution at position Y86 comprises a Y86F substitution.

[0020] 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.

[0021] In an embodiment, 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.

[0022] In an embodiment, the isolated nucleic acid encoding the CAD polypeptide comprises signal peptide SEQ ID NO: 45 at its N-terminus. In an embodiment, the isolated nucleic acid encoding the CAD polypeptide comprises signal peptide SEQ ID NO: 93 at its N-terminus.

[0023] In embodiments, the isolated nucleic acid encoding the CAD polypeptide encodes a cytokine. In additional or alternative embodiments, the isolated nucleic acid encoding the chimeric receptor encodes a cytokine. In embodiments where both the isolated nucleic acid encoding the CAD polypeptide and the isolated nucleic acid encoding the chimeric receptor encode cytokines, the cytokines can be different. In embodiments, the cytokine(s) is selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, and IL-23. In embodiments, the isolated nucleic acid further encodes a marker protein. In embodiments, the isolated nucleic acid further encodes a marker protein. In embodiments, the marker protein is selected from the group consisting of truncated CD19, CD20 (rituximab recognition domain), truncated EGFR, and LNGFR.

[0024] In embodiments, when the chimeric receptor includes an intracellular signaling domain, the CAD polypeptide includes a co-stimulatory domain, and vice versa.

[0025] In embodiments, the mammalian cell further includes at least one receptor that associates with DAP10, and the at least one receptor is not at least one chimeric receptor. In embodiments, the at least one receptor that associates with DAP10 is endogenous. In embodiments, the at least one receptor that associates with DAP10 is exogenous. In embodiments, the at least one receptor that associates with DAP10 is overexpressed. In certain embodiments, the at least one receptor is selected from NKG2D, Ly49H, Ly49D, Sirp-b1, Siglec-15, and Cd300lb. In preferred embodiments, the at least one receptor is NKG2D.

[0026] In embodiments, the mammalian cell is an immune cell, and preferably, the immune cell is a cytotoxic cell.

[0027] In an embodiment, mammalian cells comprising both the CAD polypeptide disclosed herein and at least one chimeric receptor exhibit killing activity in vitro and / or in vivo against target cells that exhibit cell surface expression of a target antigen recognized by the at least one chimeric receptor.

[0028] In an embodiment, the target cells are hematological tumor cells.

[0029] In an embodiment, the target cells are solid tumor cells.

[0030] In an embodiment, the in vitro and / or in vivo killing activity is greater than the native level of in vitro and / or in vivo killing activity in control mammalian cells lacking expression of one or both of the at least one chimeric receptor and / or the CAD polypeptide.

[0031] In an embodiment, the mammalian cells proliferate in response to contact with the target cells. In an embodiment, the mammalian cells exhibit an increase in proliferation in response to contact with the target cells as compared to control mammalian cells lacking expression of one or both of the chimeric receptor and / or the CAD polypeptide. In an embodiment, the mammalian cells proliferate within a host organism containing the target cells.

[0032] In an embodiment, the mammalian cells express pro-inflammatory cytokines in response to contact with the target cells. In an embodiment, the pro-inflammatory cytokines include tumor necrosis factor alpha or interferon gamma.

[0033] In one aspect, the present invention provides a plurality of mammalian cells comprising a CAD polypeptide and at least one chimeric receptor as disclosed herein. In an embodiment, 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 to 10 11 cells.

[0034] In one aspect, the present invention provides a method of generating mammalian cells as disclosed herein, comprising transfecting mammalian cell(s) 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.

[0035] In embodiments, the method comprises retroviral transduction.

[0036] In embodiments, the method comprises in vitro expansion of the mammalian cell(s), which is performed before and / or after transfection with an isolated nucleic acid encoding a CAD polypeptide and at least one construct encoding at least one chimeric receptor.

[0037] In one aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and mammalian cells comprising the CAD polypeptide and at least one chimeric receptor disclosed herein, 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, even more preferably about 10 8 ~10 11 cells.

[0038] In one aspect, the present invention provides a method of activating mammalian cells comprising the CAD polypeptide and at least one chimeric receptor disclosed herein, or a plurality of said mammalian cells, comprising contacting one or more target cell(s) with the mammalian cell(s). In embodiments, the mammalian cell(s) is introduced into a subject in need thereof and the activation occurs in the subject.

[0039] In one aspect, the present invention provides for the use of mammalian cell(s) comprising a CAD polypeptide and at least one chimeric receptor as disclosed herein, or a plurality of said mammalian cells, or a pharmaceutical composition comprising said mammalian cell(s), in the preparation of a medicament for treating a subject in a state wherein the mammalian cell(s) reduces at least one symptom or sign of the state in the subject.

[0040] In one aspect, the present invention provides for the use of an amount of mammalian cell(s) comprising a CAD polypeptide and at least one chimeric receptor as disclosed herein, or a plurality of said mammalian cells, or a pharmaceutical composition comprising said mammalian cell(s), effective for killing tumor cells, in the preparation of a medicament for treating cancer in a subject in need thereof.

[0041] In one aspect, the present invention provides a method of killing tumor cells, the method comprising contacting the tumor cells with an amount of mammalian cell(s) comprising a CAD polypeptide and at least one chimeric receptor as disclosed herein, or a plurality of said mammalian cells, or a pharmaceutical composition comprising said mammalian cell(s), effective for killing tumor cells. In an embodiment, the method comprises introducing a therapeutically effective amount of mammalian cell(s) or pharmaceutical composition into a host organism comprising the tumor cells. In an embodiment, the method comprises performing one or more methods for increasing common gamma chain cytokine(s) simultaneously or sequentially with or subsequent to introducing a therapeutically effective amount of mammalian cell(s) or pharmaceutical composition into a host organism comprising the tumor cells.

[0042] In an embodiment, the one or more administration methods for increasing common gamma chain cytokine(s) comprise administering an amount of common gamma chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced mammalian cell(s) simultaneously or sequentially with or subsequent to introducing the mammalian cell(s). Preferably, the method comprises administering IL-2, and more preferably, the method comprises administering IL-15.

[0043] In embodiments, one or more methods of increasing common gamma chain cytokine(s) include administering an effective amount of common gamma chain cytokine(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced mammalian cell(s) before and / or after introducing the mammalian cell(s).

[0044] In embodiments, one or more methods of increasing common gamma chain cytokine(s) include lymphodepletion prior to introducing the mammalian cell(s).

[0045] In embodiments, one or more methods of increasing common gamma chain cytokine(s) include secretion of one or more common gamma chain cytokine(s) from the introduced mammalian cell(s).

[0046] In embodiments, the method decreases the in vivo tumor burden of the host organism and / or increases the mean survival time of the host organism compared to a control organism that is not treated with the mammalian cell(s) or pharmaceutical composition.

[0047] Other features, objects, and advantages will be apparent from the following disclosure.

[0048] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby 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

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[0050] The present invention provides mammalian cell(s) and compositions thereof comprising a chimeric adapter (CAD) polypeptide that comprises a DAP10 domain and at least one of a costimulatory domain and / or an intracellular signaling domain, and specifically lacks an extracellular domain comprising a ligand-binding domain. The mammalian cell(s) further express at least one chimeric receptor that comprises an extracellular targeting domain that specifically binds to a target antigen on a target cell. In embodiments, the at least one chimeric receptor comprises an extracellular targeting domain and a DAP10-interacting domain, and optionally further comprises at least one costimulatory domain and / or at least one intracellular signaling domain. In embodiments, the CAD polypeptide of the present invention may further comprise a transmembrane domain (e.g., SEQ ID NO: 79) and / or an extracellular domain (e.g., SEQ ID NO: 80 or another extracellular spacer domain), but specifically lacks a functional extracellular receptor and / or ligand-binding domain. In contrast, the prior art typically employed DAP10 as a component of a CAR or NKG2D fusion chimera. See, for example, Zhao et al., OncoImmunology. 2019;8(1):e1509173; Lynch et al., 2017, Immunol. 152:472; US2020 / 0308248; WO / 2018 / 183385; CN109096404; CN111995689. The CAD polypeptide and chimeric receptor of the present invention are clearly different from those found in nature and generally comprise at least two polypeptide domains that do not naturally bind to each other, and optionally further comprise additional advantageous signaling domains and mutations detailed herein.

[0051] The CAD polypeptide of the present invention preferably comprises a DAP10 domain comprising human DAP10, which 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.

[0052] The "costimulatory domain" in the context of the CAD polypeptide and, in embodiments, chimeric receptors of the present disclosure enhances cell proliferation, cell survival, and development of memory cells for cytotoxic cells expressing the CAD polypeptide and / or chimeric receptor. The CAD polypeptide and / or chimeric receptor of the present invention may comprise one or more costimulatory domains selected from the costimulatory domains of proteins of the 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 combinations thereof. If the CAD or chimeric receptor comprises more than one costimulatory domain, these domains may optionally be separated by a linker and arranged in tandem. The costimulatory domain is an intracellular domain that may be located between the DAP10 domain within the CAD and any intracellular signaling domain.

[0053] In embodiments, the costimulatory domain comprises the costimulatory domains of CD28, CD27, ICOS, 4-1BB, OX40, and CD40L. As used herein, the term "costimulatory domain" also encompasses any modifications thereof, examples of which are described in U.S. Patent Application No. 20200129554, U.S. Patent Application No. 20200317777, WO2019010383, Li, W., et al., (2020) Immunity, 53:456-470; and Li, G., et al., (2017) J. Immunol. 198(1 Supplement):198.4, the contents of each of which are incorporated herein by reference in their entirety.

[0054] The CAD polypeptide of the present disclosure, and in embodiments the "intracellular signaling domain" in the context of a chimeric receptor, transmits signals of effector function and directs cytotoxic cells to perform its special function, namely the function of damaging and / or destroying target cells. Examples of suitable intracellular signaling domains include, for example, the ζ chain of the T cell receptor complex or its homologues, such as the η chain, FcsRly chain and β chain, MB1 (Iga) chain, B29 (Ig) chain, etc., human CD3ζ chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transmission, such as CD2, CD5, and CD28. In embodiments, the intracellular signaling domain of the CAD polypeptide and / or chimeric receptor may be the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, immunoreceptor tyrosine-based activation motif (ITAM) having a cytoplasmic receptor, and combinations thereof.

[0055] Intracellular signaling domains may include intracellular signaling domains of some types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory factors, and tumor necrosis factor receptor (TNFR) superfamily receptors (Park et al., “Are all chimeric antigen receptors created equal?” J Clin Oncol., vol. 33, pp. 651 - 653, 2015). Additional intracellular signaling domains include signaling domains used by NK cells and NKT cells (Hermanson, et al., “Utilizing chimeric antigen receptors to direct natural killer cell activity,” Front Immunol., vol. 6, p. 195, 2015), for example, 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, as well as CD3z.Furthermore, the intracellular signaling domain also includes the signaling domains of immunoglobulin receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as those of human FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (Gillis et al., “Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies,” Front Immunol., vol. 5, p. 254, 2014).

[0056] In an embodiment, the intracellular signaling domain includes the cytoplasmic signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In an exemplary embodiment, the intracellular signaling domain in the CAD and / or chimeric receptor includes the cytoplasmic signaling domain of human CD3ζ. As used herein, the term "intracellular signaling domain" encompasses any modification thereof, examples of which are U.S. Patent Application No. 2020 / 0317777, as well as Combadiere, B., et al., (1996), J. Exp. Med. 183(5):2109-17; Lowin-Kropf B., et al., (1998), J. Cell Biol 140(4): 861-871; Ardouin L., et al., (1999), Immunity, 10(4):409-20; Liu H. and Vignali DAA., (1999), J. Immunol. 163:599-602; Kersh EN., et al., J. Exp. Med. (1999), 190(11):1627-36; Chae WJ., et al., (2004), Int. Immunol. 16(9):1225-36; Becker, AM., et al., (2007), J. Immunol. 178(7):4120-8; Methi T., et al., (2007), Eur. J. Immunol. 37(9):2539-48; Baudouin SJ., et al., (2008), Mol. Biol. Cell, 19(6):2444-56; Zhao Y., et al., (2009), J. Immunol. 183(9):5563-74; Kochenderfer JN. et al., (2010), Blood, 116(19):3875-86; Bridgeman JS., et al., (2014), Clin. Exp. Immunol. 175(2):258-67; Long AH., et al., (2015), Nat. Med. 21(6):581-90; Hwang S., et al., (2015), Nat. Commun. 6:6982; WO2019126748; Feucht J., et al., (2019), Nat Med.25(1):82-88; Roda-Navarro, P., and Reyburn, H. T., (2009), J. Biol. Chem. 284(24):16463-16472; Giurisato, E., et al., (2007), Mol. Cell Biol. 27(24):8583-8599; and Wu, J., et al., (2000), J. Exp. Med. 192(7):1059-1068, the contents of each of which are incorporated herein by reference in their entirety.

[0057] In embodiments, two or more components of the CAD polypeptides and / or chimeric receptors of the invention may be separated by one or more linkers. The linker is a region of an oligopeptide or polypeptide that is about 1 to 100 amino acids in length. In some embodiments, the linker may be, for example, 5 to 12 amino acids in length, 5 to 15 amino acids in length, or 5 to 20 amino acids in length. The linker may be composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely in concert with each other. Longer linkers, for example, linkers greater than 100 amino acids, may be used in connection with alternative embodiments of the invention and may be selected, for example, so that two adjacent domains do not sterically interfere. Examples of linkers that may be used in the present invention include 2A-like linkers, or 2A linkers (e.g., T2A) including functional equivalents thereof, but are not limited thereto.

[0058] In an exemplary embodiment, a chimeric DAP10-4-1BB adapter polypeptide comprising a DAP10 domain and a 4-1BB co-stimulatory domain is provided. In another exemplary embodiment, a chimeric DAP10-CD28 adapter polypeptide comprising a DAP10 domain and a CD28 co-stimulatory domain is provided. In another exemplary embodiment, a chimeric DAP10-4-1BB-CD3ζ adapter polypeptide comprising a DAP10 domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain is provided. In another exemplary embodiment, a chimeric DAP10-CD28-CD3ζ adapter polypeptide comprising a DAP10 domain, a CD28 co-stimulatory domain, and a CD3ζ intracellular signaling domain is provided. In yet another exemplary embodiment, a chimeric DAP10-4-1BB-CD28-CD3ζ adapter polypeptide comprising a DAP10 domain, a 4-1BB co-stimulatory domain, a CD28 co-stimulatory domain, and a CD3ζ intracellular signaling domain is provided.

[0059] The chimeric adapter polypeptides of the present invention may optionally further comprise a transmembrane domain. The transmembrane domain of the CAD is a region capable of spanning the cell membrane of a cytotoxic cell. The transmembrane domain is selected from transmembrane proteins, such as the transmembrane regions of type I transmembrane proteins, artificial hydrophobic sequences, or combinations thereof. Suitable examples of transmembrane domains include the transmembrane regions of the alpha, beta, or gamma chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. The synthetic transmembrane domain may comprise a triplet of phenylalanine, tryptophan, and valine. Optionally, a short oligopeptide or polypeptide linker, preferably 2 to 10 amino acids in length, may form a bond between the transmembrane domain and the intracellular signaling domain of the CAD. A pair of glycine-serine provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0060] The chimeric adapter polypeptide of the present invention may optionally further comprise an extracellular spacer domain. The extracellular spacer domain of CAD is a hydrophilic region that is normally located between the ligand-binding domain (which is not present in the CAD polypeptide of the present invention) and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding of CAD. The extracellular spacer domain may comprise a domain selected from 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 the CD8a hinge, three glycines (Gly), an artificial spacer composed of similarly sized polypeptides, and the CH1 and CH3 domains of IgG (e.g., human IgG4).

[0061] Definitions

[0062] For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural, and vice versa. If the definitions described conflict with the documents incorporated herein by reference, the definitions set forth below shall prevail. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0063] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, etc. means including a variation of ±20%, ±10%, more preferably ±5%, still more preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for performing the disclosed method.

[0064] As used herein, "optional" or "optionally" means that a particular limitation, event, situation, etc. may occur, but need not occur, and includes both the case where the limitation, event, or situation occurs and the case where it does not occur.

[0065] As used herein, the term "DAP10" refers to a transmembrane adapter protein present in mammalian lymphoid and myeloid cells, and its exact sequence may vary slightly by species, isoform, and individual. Aliases of DAP10 recognized in the art include hematopoietic cell signal transducer (HCST), DNAX-activating protein 10, membrane protein DAP10, transmembrane adapter protein KAP10 (KAP10), and PIK3AP. For example, in humans, DAP10 refers to the protein represented by the major polypeptide sequences UnitProt Q9UBK5 and NCBI accession numbers NP_055081.1 and AF072845, although different isoforms and variants may exist. The name DAP10 may refer to a protein member of the DAP10 protein family having high sequence identity with a plurality of proteins having related structures and polypeptide sequences from various species, i.e., human DAP10 (SEQ ID NO: 1), while those skilled in the art will be able to identify human DAP10-related proteins in mammals even if they differ from the sequences referenced herein. Full-length DAP10 (SEQ ID NO: 1) may be processed intracellularly into a mature form (SEQ ID NO: 78) that does not have the signal peptide sequence number 93.

[0066] As used herein, the term "host cell" refers to a cell type selected to express both the CAD polypeptide of the present disclosure and at least one chimeric receptor. The host cell may endogenously express DAP10 and thus at least one receptor that associates with the chimeric adapter polypeptide of the present disclosure. The host cell may be engineered to express DAP10 and thus at least one chimeric receptor that associates with the chimeric adapter polypeptide of the present disclosure. Exemplary host cells include, but are not limited to, a wide variety of immune cells, particularly cytotoxic cells, preferred examples of which are disclosed herein (e.g., γδ T cells, αβ T cells, NK cells, NKT cells, B cells, neutrophils, monocytes / macrophages). The "host cell" may also include non-immune cells, such as, but not limited to, stem cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, induced pluripotent stem cells, etc.) within the scope of the present disclosure.

[0067] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses, or is expressing, CD3 (CD3+) and a T cell receptor (TCR+). T cells play a central role in cell-mediated immunity. T cells that "are expressing" CD3 and TCR are engineered to remove CD3 and / or TCR cell surface expression.

[0068] As used herein, the term "γδ T cell (gamma delta T cell)" refers to a subset of T cells that express on their surface a distinct T cell receptor (TCR), namely a γδ TCR, which is composed of one γ chain and one δ chain. The term "γδ T cell" specifically includes all subsets of γδ T cells (including, but not limited to, Vδ1, Vδ2, and Vδ3 γδ T cells, as well as naive, effector memory, central memory, and terminally differentiated γδ T cells). As further examples, the term "γδ T cell" includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells. In some embodiments, the γδ T cells are Vδ1-, Vδ2-, or both Vδ1- and Vδ2-. Compositions and methods for generating and using engineered and non-engineered γδ T cells and / or their subtypes include, but are not limited to, those described below: US2016 / 0175358; WO2017 / 197347; US9499788; US2018 / 0169147; US9907820; US2018 / 0125889, and US2017 / 0196910 (the contents of each of which are incorporated by reference for all purposes, including the compositions and methods for generating and using engineered and non-engineered γδ T cells and / or their subtypes). This application further contemplates T cells, or other engineered leukocytes or lymphocytes, that express one γ chain or one δ chain and optionally form a functional TCR in combination with a second polypeptide. Such engineered leukocytes or lymphocytes that express one γ chain or one δ chain may be used in the methods described herein or may be present in the compositions described herein.

[0069] The γδ T cells described herein can be δ1, δ2, δ3, or δ4 γδ T cells, or combinations thereof. In some cases, the γδ T cells are mostly (more than 50%), in most cases (more than 90%), essentially all, or completely δ2 γδ T cells. In some cases, the γδ T cells are mostly (more than 50%), mostly (more than 90%), essentially all, or completely δ1 γδ T cells. In some cases, the γδ T cells are mostly (more than 50%), mostly (more than 90%), essentially all, or completely δ3 γδ T cells.

[0070] As described herein, T cells for use of γδ can be obtained from allogeneic or autologous donors. The γδ T cells can be partially or completely purified or can be expanded ex vivo without purification. Methods and compositions for ex vivo expansion include, but are not limited to, those described in WO2017 / 197347. Expansion can be carried out before, after, or before and after the chimeric adapter polypeptide of the present disclosure is introduced into the γδ T cell(s). Other additional or alternative expansion methods include the use of artificial antigen-presenting cells (aAPCs), aminobisphosphonates, cytokine cocktails, and feeder cells (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1):45-59).

[0071] As used herein, the term "αβ T cell" refers to a T cell that expresses the α and β chains of the TCR as part of a complex with the CD3 chain molecule. Each α and β chain contains one variable domain and one constant domain. αβ T cells mainly recognize peptide antigens presented by major histocompatibility complex (MHC) class I and class II molecules, and most of the receptor diversity is contained within the third complementarity-determining region (CDR3) of the TCR α and β chains.

[0072] As used herein, the term "natural killer (NK) cell" refers to CD56, which plays an important role in immunity against viruses and immune surveillance of tumors and constitutes an important cell subset of the innate immune system. + CD3 - It refers to granular lymphocytes (Godfrey J, et al. Leuk Lymphoma, 2012, 53:1666-1676). NK cells express a very diverse repertoire of inhibitory and activating receptors on their cell surface, which controls the immune response. NK cells can kill mutant and infected cells by releasing perforin and granzyme, or using effector molecules of the tumor necrosis factor (TNF) family (e.g., TNF that induces apoptosis in target cells, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand). Furthermore, upon activation, NK cells rapidly produce chemokines and cytokines (e.g., interferon (IFN)-γ, GM-CSF, and IL-10), which mobilize the host's hematopoietic and non-hematopoietic cells and affect their functions. Cytotoxic CD8 + Unlike T lymphocytes, NK cells can exert cytotoxicity against tumor cells without prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int. Immunol. 2011, 23:427-431). NK cells are considered to be relatively safe effector cells because they can avoid potentially fatal complications of cytokine storm (Morgan R.A, 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 off-target tumor effects.

[0073] NK cells can be obtained from allogeneic or autologous donors. NK cells can be partially or fully purified or expanded ex vivo without purification. Methods and compositions for ex vivo expansion include, but are not limited to, those described below: Becker et al., (2016) Cancer Immunol. Immunother. 65(4):477-84. Expansion may be performed before, after, or before and after the introduction of a chimeric DAP10 adapter polypeptide into the NK cells. Briefly and without limitation, expansion of NK cells can involve the use of engineered feeder cells, cytokine cocktails (e.g., IL-2, IL-15), and / or aAPCs (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1):45-59).

[0074] In some examples, placental hematopoietic stem cell-derived natural killer (PNK) cells or immortalized cell lines (e.g., NK-92) can be modified to express the chimeric adapter polypeptides of the present disclosure. In other examples, NK cells that can be used to manipulate the expression of the chimeric adapter polypeptides herein can be differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). As used herein, the term "natural killer T (NKT) cells" refers to T lineage cells that share morphological and functional characteristics with both T cells and NK cells. NKT cells are rapid responders of the innate immune system and mediate potent immunoregulatory and effector functions in various disease settings. Recognition of ligands in NKT cells results in the rapid secretion of pro-inflammatory cytokines (e.g., IFN-γ and TNF-α) as well as anti-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13). This can enhance the immune response against, for example, tumors by directly targeting tumor cells and indirectly modulating the anti-tumor response through the release of various cytokines, or by altering the TME. After activation, NKT cells can immediately initiate cytokine secretion without first differentiating into effector cells. Due to the speed of their response, NKT cells play an important role at the forefront of natural defense against some types of bacterial and viral infections. Furthermore, many of the cytokines secreted by NKT cells strongly influence the differentiation and function of αβ T 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 fully purified or expanded in vitro without purification.Briefly, NKT cells can be expanded by the use of ex vivo IL-2 and / or monoclonal antibodies specific for the TCRα chain CDR3 loop (Cortes-Selva, D et al., (2021), Trends Pharmacol. Sci. 42(1):45-59).

[0075] As used herein, the terms "gamma-delta natural killer T cells" or "γδ NKT cells" refer to iPSC-derived cells that express γδ TCR and NK receptors but lack the expression of characteristic γδ T cell markers (Cortes-Selva, D et al., (2021) Trends Pharmacol. Sci. 42(1):45-59). These cells are not normal cells and have been shown to have anti-tumor activity against a number of cancer cell lines, exhibiting more potent killing ability than donor-derived γδ T cells or donor-derived NK cells (Zeng J et al., (2019) PLoS ONE 14(5):e0216815). Chimeric adapter polypeptides and chimeric receptors can be used according to the methods disclosed herein and, in embodiments herein, can be expressed in γδ NKT cells.

[0076] As used herein, the term "myeloid cells" refers to a subset of white blood cells represented by granulocytes, monocytes, macrophages, and dendritic cells (DC). They circulate through the blood and lymphatic system and are rapidly mobilized to sites of tissue damage and infection via various chemokine receptors. In tissues, they are activated for phagocytosis and the secretion of inflammatory cytokines, thereby playing an important role in defensive immunity. Myeloid cells can also be found in steady-state tissues where they regulate development, homeostasis, and tissue repair.

[0077] As used herein, the term "macrophage" refers to highly plastic native cells that include functional and phenotypic signatures that can be shaped in response to various stimuli. Macrophage polarization is roughly classified into two different states: an M1 phenotype (classically activated) in response to factors such as lipopolysaccharide (LPS) or IFN-γ, or an M2 phenotype in response to cytokines such as IL-4, IL-5, and IL-13. An example of M1-like macrophages is the expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokines such as TNF-α, IL1-β, IL-6, IL-12, and IL-23. An example of M2 macrophages shows an increase in the expression of CD209, CD200R, CD1a, and CD1b in humans, suggesting their involvement in wound healing and anti-tumor responses. The ability of macrophages to infiltrate and reprogram solid tumors and the anti-tumor effects associated with switching to the M1 phenotype are relevant to the present disclosure with respect to engineered macrophages that express the chimeric adapter polypeptides described herein. For example, in a mouse ovarian cancer model, it has been shown that macrophages can be reprogrammed into anti-tumor M1 phenotype cells that produce nitric oxide and induce an IL-12-dependent NK-mediated anti-tumor effect by inhibiting NF-κB signaling (Zhang F et al., (2019), Nat. Commun. 10:3974).

[0078] Macrophages can be obtained / derived from allogeneic or autologous donors. Macrophages can also be partially or fully purified or cultured ex vivo without purification (see, for example: Davies JQ and Gordon A (2005) Methods Mol Biol 290:105016). In some embodiments, the present disclosure includes macrophages derived from hESCs (Karlsson, KR et al., (2008) Exp Hematol 36:1167-1175), or macrophages derived from iPSCs (Takata K. et al., (2017) Immunity 47:183-198).

[0079] As used herein, the term "NKG2D receptor" refers to a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D functions as a major activating receptor, and ligand binding induces cytotoxicity and cytokine production. NKG2D provides co-stimulation via the associated adapter molecule DAP10 that recruits phosphatidylinositol-3 kinase. In mice, NKG2D also associates with DAP12 that recruits protein tyrosine kinase. NKG2D is encoded by the KLRK1 gene in the NK gene complex (NKC) located on chromosome 6 in mice and chromosome 12 in humans. In humans, NKG2D is expressed by NK cells, γδ T cells and CD8+ αβ T cells, as well as CD4+ T cells under certain pathological conditions (Stanjanovic A., et al. (2018) Front. Immunol. 23:1-15). In mice, NKG2D is expressed by NK cells, NK1.1+ T cells, γδ T cells, activated CD8+ αβ T cells, and activated macrophages. The amino acid sequence of full-length human NKG2D is shown herein as SEQ ID NO: 74, the amino acid sequence of the transmembrane domain of human NKG2D 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.

[0080] As used herein, the term "recombinant mammalian cell" refers to a mammalian-derived cell or cell line that contains at least one modification brought about using genetic engineering techniques. In some embodiments, a "recombinant mammalian cell" is a γδ T cell, or an NK cell, or an NKT cell, or an αβ T cell, etc. that contains a nucleic acid construct encoding a chimeric DAP10 adapter polypeptide and / or a chimeric receptor, preferably both a chimeric DAP10 adapter polypeptide and a chimeric receptor. A "recombinant mammalian cell" can be derived from any mammal, such as a human, a rodent, etc.

[0081] As used herein, the term "TCR" or "T cell receptor" refers to a heterodimeric cell surface signaling protein that forms an αβ receptor, a γδ receptor, or a combination thereof. αβ TCR recognizes antigens presented by MHC molecules, while γδ TCR can recognize antigens independently of MHC presentation.

[0082] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encode cell surface antigen-presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC or HLA are used interchangeably.

[0083] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve the production of antibodies, the activation of specific immune competent cells, or both. Those skilled in the art will understand that any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as the term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. The present disclosure includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be produced, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0084] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be a complete immunoglobulin obtained from a natural or recombinant source and can be the immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies of the present invention can be present in a variety of forms, such as polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), antibody compositions having polyepitope specificity, multivalent antibodies, multispecific antibodies formed from at least two complete 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 variants (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, N.Y.; Houston et al., 1988, Proc. Nat. Acad. Sci. USA. 85:5879-5883; Bird et al., 1988, Science, 242:423-426).

[0085] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant that can specifically bind to an immunoglobulin or receptor, such as a T cell receptor. Epitope determinants are usually composed of the 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.

[0086] 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, where the change results from an alteration of the sequence containing the amino acid residue / position. "Alteration" of an amino acid residue / position is synonymous with "mutation" of the amino acid residue / position. For example, representative modifications include substitution of the residue (or at that position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more amino acids, and deletion of one or more amino acids. "Amino acid substitution" or variations thereof refers to replacing an existing amino acid residue within a given (starting) amino acid sequence with a different amino acid residue. Generally, preferably, the modification results in a change in at least one physicochemical activity of the mutant polypeptide compared to the polypeptide containing the starting (or "wild-type") amino acid sequence. Thus, an "modified" amino acid sequence as referred to herein includes an amino acid sequence in which one or more amino acids have been mutated and / or any number of amino acids have been inserted and / or any number of amino acids have been deleted.

[0087] As used herein, the term "endogenous" refers to substances and / or processes that originate 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 nucleic acid molecules or polypeptides that are normally expressed within a cell or tissue.

[0088] Conversely, as used herein, the term "exogenous" refers to substances and / or processes that originate from outside of a system, including but not limited to an organism, tissue, or cell. In particular, "exogenous" in the context of the present disclosure means a nucleic acid molecule or polypeptide that does not naturally exist within a cell. Thus, the term "exogenous" encompasses any foreign or heterologous recombinant nucleic acid molecule or polypeptide expressed within a cell, which includes exogenous nucleic acids having a sequence different from the naive endogenous counterpart. 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 optionally ligated to alternative regulatory sequences such as non-native promoters or secretion sequences.

[0089] As used herein, the term "overexpression" refers to expression at a level that exceeds the endogenous expression level of the subject nucleic acid or polypeptide in a cell or tissue. In an exemplary embodiment, a receptor of interest (e.g., NKG2D) can be overexpressed in a host cell, and the expression level of the receptor is higher than the naturally occurring expression level of the same receptor. Methods for overexpressing a nucleic acid or polypeptide of interest are not particularly limited and are discussed in more detail herein. For example, a polypeptide (e.g., NKG2D) can be overexpressed by introducing the corresponding nucleic acid using an expression vector that is the same as or different from the one encoding the CAD polypeptide. The expression vector is not particularly limited as long as it can be used in genetic engineering. For example, any of a plasmid vector, a viral vector, a cosmid vector, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and other non-plasmid vectors can be used.

[0090] As used herein, the term "anti-tumor effect" refers to a biological effect that can be demonstrated by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean survival, or an improvement in various physiological symptoms associated with the cancer condition. The "anti-tumor effect" can also be demonstrated by the ability of the polynucleotides and cells of the present invention in the prevention of tumor development.

[0091] As used herein, the term "autologous" refers to any substance derived from an individual, which will later be reintroduced into the same individual.

[0092] As used herein, the term "allogeneic" refers to a substance derived from an animal, which will later be introduced into another animal of the same species.

[0093] As used herein, the term "syngeneic" refers to a material that is genetically similar or identical and thus immunologically compatible and does not cause an immune response upon transplantation.

[0094] 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, molecule for other purposes, or cell (e.g., a cell engineered to express a chimeric adapter polypeptide). An agent can include a therapeutic, a diagnostic, or a pharmaceutical. A therapeutic or a pharmaceutical can, alone or together with additional agents, induce a desired response (e.g., induce a therapeutic or prophylactic effect when administered to a subject, e.g., for the treatment of a subject suffering from cancer, viral infection (e.g., cytomegalovirus (CMV), influenza, hepatitis B, Epstein - Barr virus, adenovirus, etc.), bacterial infection (e.g., Escherichia coli, Mycobacterium tuberculosis, etc.), rheumatoid arthritis (RA), or other diseases / conditions). The agents discussed herein may sometimes be referred to as modulators.

[0095] As used herein, the term "diagnosis" or "diagnosing" refers to the process of identifying a disease, such as cancer, based on signs, symptoms, and / or the results of various tests. The conclusion reached through such a process is a diagnosis. Forms of tests commonly performed include blood tests, medical imaging, urine tests, biopsies, and the like.

[0096] The term "therapeutically effective amount" or simply "effective amount" refers to the amount of an agent or composition (e.g., a composition containing an agent) that elicits a biological or medical response of a tissue, system, or subject that a researcher, veterinarian, physician, or other clinician is seeking. The term "therapeutically effective amount" includes the amount of an agent or composition containing an agent that, when administered, is sufficient to prevent or to some extent alleviate the manifestation of one or more signs or symptoms of the disorder or disease being treated (e.g., a hematological or solid tumor). The therapeutically effective amount will vary depending on the component, the disease and its severity, as well as the age, weight, and the like of the subject to be treated.

[0097] As used herein, "treatment" of a disease as a term means reducing or lessening the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0098] As used herein, the term "reduce" means to lower some quality, quantity, or intensity. In one example, a treatment (e.g., administration of a therapeutic agent of the present disclosure) reduces one or more signs or symptoms associated with a disease or condition, e.g., as compared to the response in the absence of treatment. For example, administration of a therapeutic agent can result in an antitumor effect that reduces one or more signs or symptoms associated with cancer.

[0099] As used herein, the term "administer" means to provide or give to a subject, by any effective route, one or more agents, e.g., agents that treat one or more signs or symptoms associated with a condition / disorder or disease (including, but not limited to, cancer, viral infection, bacterial infection, etc.). Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0100] As used herein, the term "pharmaceutically acceptable" refers to substances (including, but not limited to, salts, carriers, or diluents) that do not inhibit the biological activity or properties of a compound and are relatively non-toxic. That is, this substance can be administered to an individual without causing undesirable biological effects or interacting in a harmful way with any of the components of the composition in which the component is included. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for the delivery of one or more agents, such as one or more modulators. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations may include injectable solutions containing pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as a vehicle. In addition to biologically neutral carriers, the agent to be administered may contain trace amounts of non-toxic auxiliary substances (such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate).

[0101] As used herein, the term "cytokine" refers to a diverse group of soluble proteins and peptides released from cells, which function as humoral regulators at nanomolar to picomolar concentrations and regulate the functional activities of individual cells and tissues in normal or pathological states. These proteins directly mediate cell-cell interactions and control processes occurring in the extracellular environment. Many growth factors and cytokines function as cell survival factors by preventing programmed cell death. Cytokines include both naturally occurring peptides and variants that retain full or partial biological activity.

[0102] "Encoding" refers to the unique property of a specific nucleotide sequence of a polynucleotide (e.g., within a gene, cDNA, or mRNA) that functions as a template for the synthesis of other polymers and macromolecules in a biological process having defined biological properties with either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to the gene results in the production of the protein within a cell or other biological system. Both strands of the coding strand are such that their nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0103] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated", but the same nucleic acid or peptide is "isolated" if it has been partially or completely separated from the substances in its natural state. An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as within a host cell.

[0104] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" are degenerate versions of each other and include all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0105] Terms such as "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is human.

[0106] As used herein with respect to cell surface receptors, the term "specifically binds" means a receptor that recognizes a particular molecule / ligand and does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a certain molecule may also bind to one or more species of molecules. However, such cross-reactivity per se does not change the classification as a particular one. In another example, a receptor that specifically binds to a molecule may also bind to different allelic genotypes of the molecule. However, such cross-reactivity per se does not change the classification as a particular one. In some cases, the terms "specific binding" or "specifically binds" can be used with respect to the interaction of a protein (or peptide) with a second chemical species, meaning that the interaction depends on the presence of a particular structure on the chemical species (e.g., an antigenic determinant or epitope). For example, the receptor recognizes and binds not a general protein but a specific structure. If the receptor is specific for epitope "A", in a reaction containing the label "A" and the receptor, the amount of labeled A that binds to the receptor will be reduced if a molecule containing (or not containing, unlabeled) A of epitope A is present.

[0107] In some embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10 -8 M or less (e.g., the smaller the KD, the stronger the 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.

[0108] As used herein, the term "cancer" refers to a mammalian physiological state characterized by unregulated cell growth. Tumorigenesis, malignancy, cancer, and tumor may be used interchangeably and refer to the abnormal growth of tissues or cells that results in excessive cell division. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that can invade surrounding tissues and / or metastasize are called "malignant." "Non-cancerous tissue" is tissue from the same organ in which a malignant tumor has formed but that does not have the characteristic pathology of the tumor. Generally, non-cancerous tissue appears histologically normal. "Normal tissue" is tissue from an organ that has not been affected by cancer or another disease or disorder of that organ. A "cancer-free" subject has not been diagnosed with cancer of that organ and does not have detectable cancer.

[0109] The symptoms of cancer include, but are not limited to, persistent cough or blood-tinged sputum, changes in bowel habits, bloody stools, anemia of unknown origin (decrease in blood cell count), breast lumps or breast discharge, testicular lumps, changes in urination, blood in the urine, hoarseness, persistent lumps or swollen glands, obvious changes in moles or warts, indigestion, difficulty swallowing, abnormal vaginal bleeding or discharge, unexpected weight loss, night sweats or fever, persistent itching around the anus or genitals, non-healing sores, headaches, back pain, pelvic pain, and bloating.

[0110] Hematologic cancers are cancers that occur in the blood or bone marrow. Examples of hematologic cancers (or hematogenous cancers) include leukemia, such as acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, as well as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (low grade and high grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0111] A solid tumor is a tumor that comprises a tumor mass derived from at least about 10 or at least about 100 tumor cells. A solid tumor can be a soft tissue tumor, a primary solid tumor, or a metastatic lesion.

[0112] Examples of solid tumors include, for example, sarcomas, adenocarcinomas, and carcinomas that affect various organ systems, such as the liver, lung, breast, lymph, gastrointestinal (e.g., colon), genitourinary (e.g., kidney, urothelial cells), pancreas, prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colorectal cancers, rectal cancers, renal cell cancers, liver cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. In one embodiment, the cancer is melanoma, such as advanced melanoma. In another embodiment, the cancer is glioma. Metastatic lesions of the cancers described above can also be treated or prevented using the methods and compositions of the present invention.

[0113] "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. The expression cassette contains cis-acting elements sufficient for expression, and other elements for expression can be supplied by the host cell or in vitro expression system. The expression cassette can be a component of a vector, such as a cosmid, plasmid (e.g., naked or contained in liposomes), or virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). The expression cassette can be within a host cell, such as an immune cell (e.g., γδ T cell).

[0114] Scope: Throughout the present disclosure, various aspects of the disclosure can be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, a description of a range should be considered to specifically disclose not only the individual numerical values within that range but also all possible sub-ranges. For example, a description of a range such as 1 to 6 is considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0115] The terms "substantially identical" or "substantially the same" when referring to a nucleic acid or a fragment thereof mean that when optimally aligned with another nucleic acid (or the complementary strand of another nucleic acid), there is a nucleotide sequence identity (%) of nucleotide bases, 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% (determined by any well-known sequence identity algorithm considered hereinafter, e.g., FASTA, BLAST, or GAP). A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0116] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that when optimally aligned using, for example, the GAP or BESTFIT programs with default gap weights, 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. In some embodiments, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will not substantially change the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example: Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine, and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and, 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.Alternatively, a conservative substitution is any change having the PAM250 logarithmic likelihood matrix disclosed below: Gonnet et al. (1992) Science 256:1443-45 (incorporated herein by reference). A "moderately conservative" substitution is any change having a non-negative value in the PAM250 logarithmic likelihood matrix.

[0117] The sequence identity and / or similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutant proteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using the FASTA program of GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the most overlapping regions between a query sequence and a search sequence and the percent sequence identity (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm for comparing a database containing a large number of sequences from different organisms with the sequences disclosed herein is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example: Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402 (each of which is incorporated herein by reference).

[0118] I. Introduction

[0119] The 10 kDa DNAX-activating protein (DAP10) is an adapter molecule associated with the cell surface cytotoxic receptor natural killer group 2 member D (NKG2D). The NKG2D receptor is a type II transmembrane anchor-type 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 can bind to a large number of very diverse MHC class I-like self-molecules. These ligands are often not expressed on normal cells, but can be induced on damaged cells, transformed cells, or infected cells (Zingoni, A et al. (2018) Front. Immunol. 9(476):1-12). The ligands belong to the H60 (a-c), RAE (α-ε), and MULT1 families in mice and to the MIC (MICA and MICB) and ULBP (ULBP1-ULBP6) families in humans, and its repertoire is more complex than that of other species. In fact, the MIC molecules are encoded by the most highly polymorphic human genes after the classical HLA molecules (Eagle, R A and Trowsdale, J. (Nat. Rev. Immunol. (2007) 7(9):737-44).

[0120] NKG2D is an activating immune receptor that regulates both innate and adoptive immune responses. NKG2D is abundantly present in all NK cells, subsets of CD8 T cells, γδ T cells, and some autoreactive CD4 T cells. NKG2D acts together with other costimulatory molecules such as DAP10 to alter the intensity and duration of antigen-specific responses mediated by the T cell receptor and to influence the pattern of antitumor responses by T lymphocytes (see, for example: Maccalli C, et al. (2003) Eur. J. Immunol. 33(7):2033-43).

[0121] NKG2D lacks a signaling motif in its cytoplasmic domain. Thus, after ligand binding, NKG2D signaling and cell activation are dependent on the association of NKG2D with the DAP10 adapter molecule, which promotes and stabilizes NKG2D cell surface membrane expression (Wu, J., et al., (1999) Science 285:730-732). However, for example, TGF-β is capable of downregulating the cell surface expression of NKG2D (and NKG2DL) (Lazarova M and Steinle (2019) Front. Immunol 10(2689):1-11), and TGF-β is known to be able to significantly reduce DAP10 expression at both the mRNA and protein levels (Park, YP et al. (2011) Blood. 118:3019-27; Lee, JC et al. (2011) Tumori 97:350-7). Therefore, it is recognized herein that a methodology capable of modulating the expression and / or associated signaling pathways of NKG2D and / or DAP10 is of therapeutic interest.

[0122] The human NKG2D receptor associates with the DAP10 signaling dimer, and one NKG2D homodimer pairs with the DAP10 dimer by formation of two salt bridges between conserved transmembrane (TM) arginine residues (Garrity, D. et al. (2005) PNAS. USA, 102(21):7641-7646). The DAP10 dimer has a pair of aspartic acid residues near the center of the transmembrane (TM) domain, and these residues interact with conserved arginines within the TM sequence of NKG2D to assemble with the DAP10 dimer. Thus, the NKG2D homodimer can associate with the DAP10 adapter molecule within its transmembrane domain to form a hexameric structure capable of initiating a signaling cascade (see, for example, Garrity et al (2005) supra).

[0123] The DAP10 dimer is a disulfide-linked homodimer. The amino acid sequence of the wild-type human DAP10 polypeptide is shown 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.

[0124] The DAP10 cytoplasmic domain contains a tyrosine-based motif (YINM) that includes residues 86 - 89 of SEQ ID NO: 1. Upon phosphorylation of tyrosine (Y86), DAP10 can bind to either 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 can bind to either p85 or Grb2, but not both (Lanier LL. (2008) Nat. Immunol. 9(5):495 - 502). This YINM motif is similar to that of CD28, which provides co-stimulatory signaling in conjunction with the immunoreceptor tyrosine-based activation motif (ITAM)-based TCR / CD3 complex in T cells.

[0125] The recruitment of both the p85 subunit of PI3K or the adapter protein Grb2 can activate both Vav1 and PLC-γ2, thereby + being essential for Ca2 mobilization and activation of cytotoxicity against cells (Upshaw JL et al. (2006) Nat. Immunol. 7(5):524 - 32).

[0126] DAP10 further contains a ubiquitination site encompassing lysine at amino acid 84 of the DAP10 protein sequence (SEQ ID NO: 1). Ligation stimulation of NKG2D on NK cells results in ubiquitination of DAP10 required for endocytosis and degradation of the NKG2D-DAP10 complex (see, for example, Molfetta, R., et al. (2014) Eur. J. Immunol. 44, 2761-2770). Furthermore, ubiquitin-dependent receptor endocytosis has been shown to be required for activation of extracellular signal-regulated kinase (ERK) and for NK cell functions such as secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. Thus, endocytosis of NKG2D-DAP10 represents a means of reducing the abundance of cell surface receptors and similarly controlling signal transduction in cytotoxic lymphocytes.

[0127] In addition to NKG2D, DAP10 is known to associate with many other receptors. For example, Ly49H and Ly49D co-immunoprecipitate with DAP10 from mouse NK cells and are shown to be associated with DAP10 when co-transfected into 293T cells (Coudert JD et al. (2008) Blood 111:3571-3578). Co-transfection studies have also shown that DAP10 is associated with human Sirp-b1 in transfected rat RBL-2H3 cells (Anfossi N et al. (2003) Eur. J. Immunol. 33:3514-3522). Similarly, human and mouse Siglec-15 (Angata T et al. (2007) Glycobiology 17:838-846) and Cd300lb (Yamanishi Y et al. (2008) Blood 111:688-698) have been shown to pair with DAP10 by co-transfection and co-immunoprecipitation. As described above, certain DAP10-associated receptors (i.e., NKG2D) appear to recognize host-encoded molecules such as carbohydrate and protein ligands, while other DAP10-associated receptors may directly recognize microbial ligands. As an example, the glycoprotein m157 encoded by murine CMV is recognized by Ly49H (Lanier LL (2008) Nat. Rev. Immunol. 8(4):259-68; Smith HR et al. (2002) Proc. Natl. Acad. Sci. USA 99(13):8826-31). m157 is a GPI-anchored glycoprotein with homology to MHC class I, which is presented on the surface of murine CMV-infected cells and leads to the activation of Ly49H+ NK cell-mediated cytotoxicity and cytokine production.

[0128] The above receptors, in addition to NKG2D, are intended to exemplify, but not be comprehensive of, the ability of DAP10 to pair with a number of receptors and thereby regulate signaling. With respect to the present disclosure, it should be understood that the nucleic acids, polypeptides, cells, compositions, and methods are applicable to any and all receptors with which DAP10 can partner. For example, embodiments herein include chimeric receptors that include a DAP10-interacting domain, thereby conferring on the CAD polypeptides of the present disclosure the ability to regulate signaling via one or more chimeric receptors expressed in the same cell as the CAD polypeptide, as disclosed herein.

[0129] II. Compositions and Methods of the Invention

[0130] Unless otherwise indicated, the technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. In particular, the present disclosure utilizes routine techniques in the fields of recombinant genetics, immunology, and biochemistry. Basic texts that disclose general terms in molecular biology and genetics include, for example: Lackie, Dictionary of Cell and Molecular Biology, Elsevier (5th ed. 2013). Basic texts that disclose 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, N.Y. 2012) and Current Protocols in Molecular Biology Volumes 1 - 3, John Wiley & Sons, Inc. (1994 - 1998) and Supplements 1 - 115 (1987 - 2016). Basic texts that disclose general methods and terms in biochemistry include, for example: Lehninger Principles of Biochemistry sixth edition, David L. Nelson and Michael M. Cox eds. W.H. Freeman (2012). Basic texts that disclose general methods and terms in immunology include: Janeway’s Immunobiology (Ninth Edition) by Kenneth M. Murphy and Casey Weaver (2017) Garland Science; Fundamental Immunology (Seventh Edition) by William E. Paul (2013) Lippincott, Williams and Wilkins.

[0131] Chimeric adapter construct

[0132] Aspects of the present disclosure include constructs comprising nucleic acids encoding chimeric adapter polypeptides. In embodiments, the nucleic acid encodes a chimeric adapter polypeptide comprising i) a human DAP10 amino acid sequence, and ii) one or more co-stimulatory domains (e.g., 4-1BB, OX40, ICOS, CD28), and the CAD polypeptide lacks a particular extracellular domain. In embodiments, the CAD polypeptide can further or alternatively comprise one or more intracellular signaling domains (e.g., CD3ζ) as described herein. In embodiments, the CAD polypeptide can also comprise one or more mutations, e.g., one or more mutations in DAP10 and / or one or more modifications (e.g., one or more mutations, additions, or deletions) in the co-stimulatory domain(s) and / or intracellular signaling domain(s).

[0133] In embodiments, the CAD polypeptides function to modulate and / or suppress signaling via one or more receptors to which they are related. In embodiments, the CAD polypeptides are designed with modified (e.g., by one or more mutations) and / or additional signaling attributes (e.g., by C-terminal fusions), expressed in host cells, and promote a favorable balance of signaling pathways upon receptor-target binding (e.g., NKG2D binding of an extracellular target ligand), which can help address the problem that the expression of the target of the primary TCR is low or lost (i.e., antigen escape). The term "favorable balance" broadly refers to the introduction of a signaling cascade that acts to complement, substitute for, or modulate the major DAP10 signaling cascade in a desired manner. Thus, the CAD polypeptides disclosed herein provide modified (e.g., improved) functional properties, including but not limited to, altered (e.g., enhanced) cytolytic, proliferative, survival, and / or costimulatory properties induced upon binding of a ligand of a receptor that partners with DAP10 (e.g., a widely expressed ligand of NKG2D). The exact composition of the CAD polypeptide can be designed based on a given disease indication and, in some instances, on the specificity of other receptor(s) (s) present on the same cell (e.g., TCR receptor(s) and / or chimeric receptors as disclosed herein) and combination with signaling components. As one example, immunosuppressive signals within the tumor microenvironment (TME) may inhibit anti-tumor T cell responses via inhibitory receptors on T cells, and it is within the scope of the present disclosure that such inhibitory outcomes may be switched to immunostimulatory outcomes via the use of CAD polypeptides. Other relevant examples may be found, for example, in Guo, J., et al., (2021), Journal for ImmunoTherapy of Cancer. 9: e002628.

[0134] In embodiments, the CAD polypeptides may function, at least in part, to stabilize cell surface receptor(s) (e.g., NKG2D and / or chimeric receptors as disclosed herein) to which they associate. "Stabilizing" a cell surface receptor(s) as disclosed herein means decreasing the rate at which the cell surface receptor is internalized or otherwise removed from the cell surface as compared to the rate at which the cell surface receptor is removed in another manner under similar circumstances in the absence of the CAD polypeptides as described herein. Included within the scope of receptor stabilization as described herein is a positive feedback mechanism in which signaling by the CAD polypeptides results in an increase in the cell surface expression of the endogenous DAP10 and the receptor(s) to which the CAD polypeptide associates (Wu, J., et al., (2000), Journal of Exp. Med. 192(7):1059-1068).

[0135] The present disclosure also provides a CAD polypeptide that is stably expressed. In embodiments, the CAD polypeptide is expressed at a level substantially similar to the level at which endogenous DAP10 is expressed in the host cell of interest. In embodiments, the CAD polypeptide is expressed at a level higher than the level at which endogenous DAP10 is expressed in the host cell of interest. For example, the CAD polypeptide may be 10% higher, or 10-20% higher, or 20-30% higher, or 30-40% higher, or 40-50% higher, or 50-60% higher, or 60-70% higher, or 70-80% higher, or 80-90% higher, or 90-100% higher, or even higher, for example, 2-fold higher, or 3-fold higher, or 4-fold higher, or 5-fold higher, or 6-fold higher, or 7-fold higher, or 8-fold higher, or 9-fold higher, or 10-fold higher, or 20-fold higher, or 30-fold higher, or 40-fold higher, or 50-fold higher, or 100-fold higher than the corresponding level at which endogenous DAP10 is expressed. Thus, in embodiments, it can be understood that the CAD polypeptide of the present invention may compete with endogenous DAP10 that binds to a receptor (e.g., NKG2D) depending on the type of host cell.

[0136] Aspects of the present disclosure include nucleic acids encoding CAD polypeptides and constructs / vectors containing such nucleic acids. Accordingly, described herein are nucleic acids encoding a CAD polypeptide, for example, that modulate and / or add signaling properties that confer desired properties, including but not limited to sustained survival, proliferation, and / or killing in a host cell expressing the CAD polypeptide. In some embodiments, the nucleic acid encodes a CAD polypeptide that includes a signal peptide at its N-terminus. A signal peptide is a segment of the amino acid sequence in a protein (e.g., the N-terminus of a protein) for moving the protein to a specific intracellular location (e.g., the endoplasmic reticulum for secretion). In some embodiments, the nucleic acid encodes a CAD that includes 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 an embodiment, the nucleic acid encodes a CAD that includes 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: 45 at the N-terminus of mature DAP10 or a fragment thereof. In some embodiments, the nucleic acid encodes a CAD that includes 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 that includes both SEQ ID NO: 45 and SEQ ID NO: 93 (e.g., SEQ ID NO: 45 at the N-terminus of SEQ ID NO: 95).

[0137] 1. Mutation

[0138] In embodiments, the CAD polypeptide carries one or both of the amino acid modifications at position K84 and / or Y86 of SEQ ID NO: 1. In embodiments, it is within the scope of the present disclosure that the modification at K84 can include other amino acid substitutions, but K84 is modified to include another positively charged amino acid, for example, modified to K84R or K84H. In one embodiment, the modification is a K84R modification. In embodiments, the CAD polypeptide having a K84R modification includes SEQ ID NO: 18. In embodiments, Y86 is modified to another aromatic amino acid, for example, Y86F or Y86W. However, it is within the scope of the present disclosure that the modification of Y86 can include other amino acid substitutions. In one embodiment, the modification is a Y86F modification. In embodiments, the CAD polypeptide having a Y86F modification includes SEQ ID NO: 19. In embodiments, the CAD polypeptide having both a K84R modification and a Y86F modification includes SEQ ID NO: 20.

[0139] In embodiments, the modification at position 86 of SEQ ID NO: 1 reduces or eliminates the binding of p85 / PI3K to the CAD polypeptide, and then reduces or eliminates PI3K / AKT / PKCθ signaling. Accordingly, the modification at position 86 can help to functionally reduce or eliminate one or more of co-stimulation, calcium influx, and / or degranulation. In a preferred embodiment, the modification is Y86F.

[0140] 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, and then reduces or eliminates Vav1 / SLP-76 / PLCγ signaling. Accordingly, the modification at position 86 can help to reduce or eliminate one or more of calcium influx and / or degranulation. In a preferred embodiment, the modification is Y86F.

[0141] In additional or alternative embodiments, the modification at position 84 of SEQ ID NO: 1 reduces or completely abrogates ubiquitination of the CAD polypeptide, and then reduces or completely blocks the internalization of the chimeric DAP10 adapter 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, a receptor that associates with DAP10 (e.g., NKG2D and / or a chimeric receptor as disclosed herein) can be stabilized at the cell surface depending on the CAD polypeptide having at least an internal modification of K84 (e.g., K84R). Further, the modification of K84 (e.g., K84R) may reduce or eliminate signal transduction (e.g., ERK1 / 2) that occurs during lysosomal degradation. Reduction or elimination of signal transduction (e.g., ERK1 / 2) may help reduce or eliminate one or more of exhaustion, activation-induced cell death, and / or induction of cell cycle arrest upon overactivation, which otherwise may occur in the absence of a CAD polypeptide having a K84 modification (e.g., K84R).

[0142] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 57 of SEQ ID NO: 1, e.g., a D57A modification, although amino acid modifications other than alanine are within the scope of the present disclosure. In an embodiment, the chimeric DAP10 adapter polypeptide having a D57A modification includes SEQ ID NO: 37. The modification at D57 may, in an embodiment, serve to modify (e.g., reduce or abrogate) a stable interaction with KLRK1 (Wu, J., et al., (1999) Science, 285(5428):730-2).

[0143] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 88 of SEQ ID NO: 1, for example, an N88Q modification, although amino acid modifications other than glutamine are within the scope of the present disclosure. In embodiments, the CAD polypeptide having the N88Q modification includes SEQ ID NO: 38. The modification of N88 can, in embodiments, have minimal to no effect on the interaction with PIK3R1 and can serve to modify (e.g., reduce) the cell killing activity and / or the interaction with GRB2 (Upshaw, JL., (2006) Nat Immunol 7:524-532).

[0144] In additional or alternative embodiments, the CAD polypeptide may include a modification at position 89 of SEQ ID NO: 1, for example, an M89Q modification, although amino acid modifications other than glutamine are within the scope of the present disclosure. In embodiments, the CAD polypeptide having the M89Q modification includes SEQ ID NO: 39. The modification of M89 can, in embodiments, have minimal to no effect on the interaction with GRB2 and can serve to modify (e.g., reduce) the cell killing activity and / or the interaction with PIK3R1 (Upshaw, JL., (2006) Nat Immunol 7:524-532).

[0145] It may be understood that the present disclosure encompasses chimeric adapter polypeptides having one or more or each of the modifications mentioned above.

[0146] 2. Costimulatory domain and signaling domain

[0147] In an embodiment, the end domain of the chimeric adapter polypeptide of the present disclosure includes one or more co-stimulatory domains, and the co-stimulatory domain includes, for example, a functional co-stimulatory signaling domain derived from an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activated NK cell receptor, BTLA, a Toll ligand receptor, and the like. For example, it is within the scope of the present disclosure that the end domain of the disclosed CAD polypeptide may include two, three, four or more co-stimulatory domains. When multiple co-stimulatory domains are included, it is also within the scope of the present disclosure that the co-stimulatory domains may be the same or different.In an embodiment, the co-stimulatory domain is derived from one or more, or a part thereof, and combinations thereof, of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19aCD27, CD28, CD29, 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), CD226(DNAM1), CD229(Ly9), CD278(ICOS), ICAM-1, LFA-1(CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM(LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, 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.

[0148] In some embodiments, the CAD construct encodes at least one 4-1BB co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the co-stimulatory domains described above. In some embodiments, the construct encodes at least two 4-1BB co-stimulatory domains, or at least two 4-1BB co-stimulatory domains in combination with one, two, three, or more than four co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the co-stimulatory domains described above. In some embodiments, the 4-1BB co-stimulatory domain comprises SEQ ID NO: 2 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL). In some embodiments, the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least one, at least two, or at least three or more modifications of the amino acid sequence of SEQ ID NO: 2. In embodiments, the 4-1BB co-stimulatory domain is substantially similar to the 4-1BB co-stimulatory domain comprising SEQ ID NO: 2.

[0149] In some embodiments, the CAD construct encodes at least one CD27 co-stimulatory domain and optionally encodes at least one second co-stimulatory domain selected from 4-1BB, ICOS, CD28, OX40, 2B4, and the CD27 co-stimulatory domain, or any of the co-stimulatory domains described above. In some embodiments, the construct encodes at least one CD27 co-stimulatory domain and a 4-IBB co-stimulatory domain. In some embodiments, the construct encodes two CD27 co-stimulatory domains and at least one second co-stimulatory domain selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the CD27 co-stimulatory domain comprises SEQ ID NO: 5 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP). In some embodiments, the CD27 co-stimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO: 5. In embodiments, the CD27 co-stimulatory domain is substantially similar to the CD27 co-stimulatory domain comprising SEQ ID NO: 5.

[0150] In some embodiments, the CAD construct encodes at least one CD28 co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the co-stimulatory domains described above. In some embodiments, the CAD construct encodes at least two CD28 co-stimulatory domains, or at least two CD28 co-stimulatory domains in combination with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the co-stimulatory domains described above. In some embodiments, the CD28 co-stimulatory domain comprises SEQ ID NO: 40 (FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). In an embodiment, the CD28 co-stimulatory domain comprises SEQ ID NO: 41 (FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). SEQ ID NO: 40 and SEQ ID NO: 41 contain YMNM, PRRP, and PYAP, three subdomains that can regulate signal transduction pathways. In an embodiment, the disclosed CAD polypeptide comprises one or more mutations or deletions of the subdomains (see, e.g., WO2019010383). In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO: 40, or the amino acid sequence of SEQ ID NO: 41. In some embodiments, the CD28 co-stimulatory domain is substantially similar to the CD28 co-stimulatory domain comprising SEQ ID NO: 40. In some embodiments, the CD28 co-stimulatory domain is substantially similar to the CD28 co-stimulatory domain comprising SEQ ID NO: 41.

[0151] In some embodiments, the CAD construct encodes at least one ICOS co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from the 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the co-stimulatory domains described above. In some embodiments, the CAD construct encodes at least two ICOS co-stimulatory domains, or at least two ICOS co-stimulatory domains in combination with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the co-stimulatory domains described above. In some embodiments, the ICOS co-stimulatory domain comprises SEQ ID NO: 42. In some embodiments, the ICOS co-stimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO: 42 (see, e.g., US20170209492). In some embodiments, the ICOS co-stimulatory domain is substantially similar to the ICOS co-stimulatory domain comprising SEQ ID NO: 42.

[0152] In some embodiments, the CAD construct encodes at least one OX40 co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the co-stimulatory domains described above. In some embodiments, the CAD construct encodes at least two OX40 co-stimulatory domains, or at least two OX40 co-stimulatory domains in combination with one, two, three, or more than four co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the co-stimulatory domains described above. In some embodiments, the OX40 co-stimulatory domain comprises SEQ ID NO: 43 (RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI). In some embodiments, the OX40 co-stimulatory domain comprises an amino acid sequence having at least one, at least two, at least three, or more modifications of the amino acid sequence of SEQ ID NO: 43. In some embodiments, the OX40 co-stimulatory domain is substantially similar to the OX40 co-stimulatory domain comprising SEQ ID NO: 43.

[0153] In embodiments, one or more intracellular signaling domains are included in the chimeric adapter polypeptide of the invention. In embodiments, one or more intracellular signaling domains are added to one or more co-stimulatory domains. In embodiments, 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.) having the CAD polypeptides disclosed herein. For example, in some embodiments, the intracellular signaling domain(s) is CD3ζ, repeat (e.g., 2-5) DAP10 YINM motif; signaling domains derived from LFA-1, DAP12, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, etc. It is within the scope of the present disclosure that the endodomains of the disclosed chimeric adapter polypeptides can include multiple (e.g., two, three, four, or more) intracellular signaling domains. When multiple intracellular signaling domains are included, the intracellular signaling domains may be the same or different.

[0154] In some embodiments, the intracellular signaling domain is or comprises the CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 3), or RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4), or RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 82), also referred to herein as “1XX” (see, e.g., US2020 / 0317777, the contents of which are hereby incorporated by reference in their entirety), or comprises the same. Without being bound by theory, in some embodiments, including the 1XX signaling domain may lead to improved activation and / or survival of the engineered immune cells of the present disclosure by limiting overactivation.

[0155] In some embodiments, the CAD construct encodes one or more co-stimulatory domains (e.g., 4-1BB co-stimulatory domain) and one or more intracellular signaling domains (e.g., CD3ζ signaling domain). In some embodiments, the CAD construct encodes at least one 4-1BB co-stimulatory domain, at least one CD28 domain, and at least one CD3ζ signaling domain. In other embodiments, the CAD construct encodes one or more first co-stimulatory domains (e.g., 4-1BB, CD28, OX40, ICOS) and one or more second co-stimulatory domains (e.g., 4-1BB, CD28, OX40, ICOS), and one or more intracellular signaling domains (e.g., CD3ζ). In embodiments, the CD3ζ signaling domain is downstream (C-terminus) of the co-stimulatory domain(s) (e.g., 4-1BB). In some embodiments, the CD3ζ signaling domain is upstream (N-terminus) of the co-stimulatory domain(s) (e.g., 4-1BB).

[0156] 3. Co-expression of cytokines

[0157] In additional embodiments, the CAD constructs of the invention can also encode one or more multicistronic linker regions (s) configured to facilitate translation of the CAD polypeptide and one or more soluble common gamma chain cytokines as separate polypeptides. In embodiments, the nucleic acid encoding the cytokine and 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, in some examples, at both the 5' and 3' ends of the isolated nucleic acid. The one or more soluble common gamma chain cytokines include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In embodiments, the linker region(s) can encode a self-cleaving and / or cleaving polypeptide sequence. In some examples, the self-cleaving sequence is a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A) that can induce ribosome skipping during translation of the chimeric DAP10 adapter polypeptide. In embodiments, the cleaving sequence is a furin sequence. In some examples, the cleaving sequence (e.g., furin cleavage sequence) is at the amino terminus of the self-cleaving sequence. In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. In some embodiments, the multicistronic linker region comprises any one of SEQ ID NOs: 9-15, or the sequence of SEQ ID NO: 44. In embodiments, adding any linker such as "GSG" or "SGSG" can improve the cleavage efficiency. In this way, the one or more gamma chain cytokines included can be released from the chimeric DAP10 adapter polypeptide and secreted by the host cell.

[0158] For example, in some embodiments, the cleavage sequence is the P2A cleavage sequence of SGSGATNFSLLKQAGDVEENPGP (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 of RAKR (SEQ ID NO: 10). In some embodiments, the cleavage sequence is the P2A + Furin cleavage (FP2A) sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 11).

[0159] In some embodiments, the cleavage sequence is or comprises the P2A cleavage sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 12). In some embodiments, the cleavage sequence is or comprises the F2A cleavage sequence of VKQTLNNFDLLKLAGDVESNPGP (SEQ ID NO: 13). In some embodiments, the cleavage sequence is or comprises the E2A cleavage sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14). In some embodiments, the cleavage sequence is or comprises the T2A cleavage sequence of EGRSLLTCGDVEENPGP (SEQ ID NO: 15). In certain aspects, multiple self-cleaving sequences can be encoded at the carboxy terminus of the signaling and / or co-stimulatory domain and the amino terminus of the encoded secreted cytokine (e.g., a common gamma chain cytokine such as IL-15), preferably, the multiple self-cleaving sequences are independently selected from the group consisting of P2A cleavage sequences, T2A cleavage sequences, E2A cleavage sequences, and F2A cleavage sequences. In certain aspects, one or more sequences cleaved by one or more self-cleaving sequences and endogenous proteases are encoded in the constructs described herein. In certain embodiments, the endogenous protease recognition site is encoded at the amino terminus of the self-cleaving sequence.

[0160] In some embodiments, the polycistronic linker region encodes an internal ribosome entry site. Exemplary internal ribosome entry sites are encoded by: CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATA (SEQ ID NO: 16).

[0161] Another exemplary internal ribosome entry site is encoded below: AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC (SEQ ID NO: 17).

[0162] Further preferred internal ribosome entry sites include, for example, 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 at iresite.org; those described in WO2018 / 215787; the sequence described in GenBank accession No. KP019382.1; and the IRES element described in GenBank accession No. LT727339.1. Additional multicistronic linker regions containing cleavage self-cleaving and IRES elements are disclosed in US2018 / 0360992 and US8,865,467.

[0163] In some embodiments, the construct encodes a cytokine, for example, a secretion signal (e.g., MALPVTALLLPLALLLHAARP (SEQ ID NO: 6)) operably linked to promote secretion of the C-terminal polypeptide. In some embodiments, the secretion signal is the secretion signal of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 7). In some embodiments, the construct encodes a secretion signal operably linked to promote secretion of a common gamma chain cytokine, for example, IL-15 or an active fragment thereof, for example, NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCELLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 8); other IL-15 sequences comprising a codon-optimized nucleic acid sequence encoding sIL15 are disclosed in WO2007 / 037780. Examples of common gamma chain cytokines include IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In some embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In some embodiments, the common gamma chain cytokine is IL-15. IL-15 sequences comprising a codon-optimized nucleic acid sequence encoding sIL15 are disclosed herein and in WO2007 / 037780.

[0164] Accordingly, the CAD constructs of the present disclosure encode a CAD polypeptide comprising at least one co-stimulatory domain and optionally one or more intracellular signaling domains. In embodiments, the CAD construct may encode one or more common gamma chain cytokines that are released from the CAD polypeptide (e.g., during translation). As discussed above in some embodiments, the CAD constructs of the present disclosure may further comprise a mutant DAP10, for example, DAP10 mutated at K84 and / or Y86, particularly of SEQ ID NO: 1.

[0165] In embodiments, one or more co-stimulatory domains may be located 5' to one or more signaling domains. In embodiments, one or more co-stimulatory domains may be located 3' to one or more signaling domains. In some embodiments, one or more co-stimulatory domains may be located 5' to one or more signaling domains, and further, one or more co-stimulatory 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 co-stimulatory domains, and further, one or more signaling domains may be located 3' to one or more co-stimulatory domains. In some embodiments, the C-terminal fusion may include alternating one or more co-stimulatory domains and one or more signaling domains.

[0166] For reference, FIGS. 1A-1D show exemplary diagrams of the relevance of various chimeric adapter polypeptides and their receptors (e.g., shown as NKG2D). These representations are for illustrative purposes only and do not represent all of the various permutations of the chimeric adapter polypeptides disclosed herein. FIG. 1A illustratively shows a chimeric adapter polypeptide that includes both the K84R and Y86F modifications. FIG. 1B illustratively shows a chimeric adapter polypeptide that includes both the K84R and Y86F modifications in addition to a C-terminal fusion that includes CD3ζ. FIG. 1C illustratively shows a chimeric adapter polypeptide that includes both the K84R and Y86F modifications in addition to a C-terminal fusion that includes 4-1BB. FIG. 1D shows, by way of example, a chimeric adapter polypeptide that includes both the K84R and Y86F modifications in addition to a C-terminal fusion that includes both 4-1BB and CD3ζ.

[0167] 4. Co-expression of Markers

[0168] In additional embodiments, the CAD constructs of the invention can encode one or more labels or markers, for example, to assist in the ability to monitor CAD expression levels and can serve as internal controls, among other things. In some embodiments, the CAD constructs can encode a fluorescent protein, examples of which include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), enhanced GFP (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), and the like. Other examples can include, but are not limited to, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, β-lactamase, luciferase, and the like.

[0169] In other embodiments, the CAD construct can encode a protein expressed on the cell surface, for example, to facilitate detection and / or isolation of cells expressing said protein via fluorescence-activated cell sorting (FACS); or for enrichment by positive selection using an antibody specific for the encoded protein, for example, an antibody for purifying or concentrating cell products on a column or device; or for in vivo binding of an antibody to a protein to enhance or remove activity, for example, to facilitate removal of cells expressing the protein in a patient, taking into account safety. Exemplary proteins useful for these purposes include, for example, CD19, CD20 (rituximab recognition domain), LNGFR (amino acid sequence shown in SEQ ID NO: 89, encoded by SEQ ID NO: 90), truncated form of human epidermal growth factor receptor (EGFRt) (amino acid sequence shown in SEQ ID NO: 91, encoded by SEQ ID NO: 92), and the like. By way of example, in an embodiment, the marker protein can be targeted by a clinical-stage antibody, and such treatment of a patient with said antibody results in elimination of cells containing the CAD construct and chimeric receptor as disclosed herein. See, for example, 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.

[0170] In embodiments, the linker regions, examples of which are described herein, can be used to facilitate translation of the CAD polypeptide and the desired marker. For example, the Furin and P2A linker genes that facilitate CAD expression and the desired marker can be included in the isolated nucleic acid constructs of the present disclosure. As will be discussed below with respect to Example 1, the Furin and P2A linker genes can be used to express the desired CAD polypeptide together with the truncated CD19 that functions as a marker. Such examples are illustrative and non-limiting.

[0171] B. Chimeric Receptor Constructs

[0172] Chimeric receptors as disclosed herein include 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 normally found together with the extracellular targeting domain. For example, a chimeric receptor can include an extracellular targeting domain and at least one DAP10-interacting domain, where the extracellular targeting domain is heterologous to the DAP10-interacting domain. Optionally, the chimeric receptor can further include one or more additional domains (e.g., one or more co-stimulatory domains and / or one or more intracellular signaling domains). In another example, a chimeric receptor can include an extracellular targeting domain and a DAP10-interacting domain (e.g., NKG2D) that are normally found together, but the chimeric receptor further includes at least one other domain (e.g., one or more co-stimulatory domains and / or one or more intracellular signaling domains) that is not normally found together with the extracellular targeting domain and the DAP10-interacting domain. In yet another example, a chimeric receptor can include at least two domains that are not normally found together, and the chimeric receptor does not include a DAP10-interacting domain. As a representative example, such a chimeric receptor can include an extracellular targeting domain, a transmembrane domain (e.g., the CD8α TM domain), and a cytoplasmic domain that includes one or more co-stimulatory domains and / or one or more intracellular signaling domains. With respect to co-stimulatory domains and / or intracellular signaling domains, it should be understood that the co-stimulatory domains and / or intracellular signaling domains that can be included in a chimeric receptor as disclosed herein constitute any one or more of those domains discussed above with respect to the CAD polypeptide. In embodiments, the chimeric receptor can include an extracellular targeting domain, a transmembrane domain (e.g., the CD8α TM domain), and a cytoplasmic domain that includes one or more co-stimulatory domains but no intracellular signaling domains. For example, such a chimeric receptor can be a non-signaling CAR as described in Ding et al., Cancer Res. (2023), 83(7 Supplement):1777.

[0173] Furthermore, the chimeric receptor constructs of the invention can also be configured to encode one or more multi-cistronic linker regions (s) that facilitate the translation of the chimeric receptor and one or more soluble common gamma chain cytokines as separate polypeptides, similar to that discussed above with respect to the CAD polypeptide.

[0174] As described above, DAP10 associates with NKG2D through the formation of two salt bridges between two arginine residues of the NKG2D transmembrane domain and two aspartic acid residues of the DAP10 transmembrane domain (Garrity, D. et al. (2005) PNAS. USA. 102(21):7641-7646). Thus, in an embodiment, the DAP10-interacting domain comprises the amino acid sequence shown in SEQ ID NO: 75, said sequence corresponding to the TM domain of human NKG2D. In an embodiment, the DAP10-interacting domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 75. In an embodiment, the DAP10-interacting domain comprises an amino acid sequence comprising a portion of the amino acid sequence shown in SEQ ID NO: 75.

[0175] In embodiments where the chimeric receptor comprises a DAP10 - interacting domain linked to an extracellular targeting domain that is normally found together with the DAP10 - interacting domain, in certain embodiments, the chimeric receptor can comprise the amino acid sequence set forth in SEQ ID NO: 76, or at least a portion of the amino acid sequence represented by SEQ ID NO: 74, and further can comprise one or more co - stimulatory 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 can comprise at least the extracellular targeting domain and the DAP10 - interacting domain of another receptor that normally comprises an extracellular targeting domain and a DAP10 - interacting domain, such as Ly49H, Ly49D, Sirp - b1, Siglec - 15, Cd300lb, etc. In some embodiments where the chimeric receptor comprises a DAP10 - interacting domain linked to an extracellular targeting domain that is normally found together with the DAP10 - interacting domain, the chimeric receptor can 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 an exemplary embodiment, the chimeric receptor further comprises at least one intracellular signaling domain and / or at least one intracellular co - stimulatory domain.

[0176] In embodiments where the chimeric receptor comprises an extracellular targeting domain and at least one DAP10-interacting domain, when the extracellular targeting domain is heterologous to the DAP10-interacting domain, the DAP10-interacting domain can comprise an amino acid sequence corresponding to at least a portion of the TM domain of a receptor that normally comprises the extracellular targeting domain and the DAP10-interacting domain. As a representative example, at least a portion of the DAP10-interacting domain of NKG2D (SEQ ID NO: 75) can be linked to a heterologous extracellular targeting domain, and the resulting chimeric receptor optionally further comprises one or more co-stimulatory domains and / or one or more intracellular signaling domains. Other DAP10-interacting domains from other receptors (e.g., Ly49H, Ly49D, Sirp-b1, Siglec-15, Cd300lb) can be used instead.

[0177] Also included in the present disclosure are chimeric receptors lacking a DAP10-interacting domain. Such chimeric receptors comprise a TM domain fused to the extracellular ligand-binding domain of the chimeric receptor, but the TM domain can be designed not to interact with DAP10 (i.e., not to comprise a DAP10-interacting domain). In one embodiment, a transmembrane domain that naturally associates with one of the domains in the chimeric receptor is used. Optionally, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains binding to the transmembrane domains of the same or different surface membrane proteins and to minimize interaction with other members of the receptor complex.

[0178] The transmembrane domain may be derived from either a natural or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane regions particularly used in the present invention are 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BL4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, 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, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma 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 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A;It may be derived from Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncated forms, or combinations thereof (i.e., including at least their transmembrane region(s)). Alternatively, the transmembrane domain may be synthetic, in which case it will mainly contain hydrophobic residues such as leucine and valine. Preferably, the triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.;

[0179] In an embodiment, the transmembrane domain in a chimeric receptor lacking a DAP10-interacting domain is the transmembrane domain of CD8. In one embodiment, the CD8 transmembrane domain comprises the 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 the amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760.

[0180] In embodiments, the chimeric receptor may include a linker between various domains added for proper spacing and conformation of the molecules. For example, in one embodiment, a linker may be present between the binding domains VH or VL, which may be between 1 and 20 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be 1 to 15 or 20 amino acids in length. In this regard, the linker 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 length. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Ranges including the numbers described herein, for example, a linker 10 to 30 amino acids in length, are also included herein.

[0181] In certain embodiments, linkers suitable for use in the chimeric receptors described herein are flexible linkers. Suitable linkers can be readily selected and can be, for example, any of a suitable different length such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0182] 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. Since glycine and glycine-serine polymers are relatively unstructured, they may serve as neutral connectors between domains of fusion proteins such as the chimeric receptors described herein. Glycine utilizes much more φ-ψ space than alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of ordinary skill in the art will recognize that the design of a chimeric receptor can include all or partially flexible linkers such that the linker can include one or more moieties that impart a less flexible structure as well as a flexible linker to provide the desired chimeric receptor structure. Specific linkers include (G4S)n linkers (where n = 1-3), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77). In some embodiments, the linker comprises 3-20 amino acids and an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77). The linker can be present between the LCVR and HCVR regions of the scFv fragment, between the variable region (e.g., HCVR) and the hinge region (e.g., CD8α hinge), or both. For example, the present disclosure provides a chimeric receptor that includes a (G4S)3 linker between LCVR and HCVR and a (G4S)1 linker between HCVR and the CD8α hinge. While described herein in the context of chimeric receptors, the use of such linkers in the context of the CAD polypeptides described herein is also within the scope of the present disclosure.

[0183] In some instances, the extracellular targeting domain of the chimeric receptors of the present disclosure may be followed later by a "spacer" or "hinge", which refers to a region that moves the extracellular targeting domain away from the effector cell surface to enable appropriate cell / cell contact, target (i.e., ligand, antigen) binding, and in some instances activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region of the chimeric receptors described herein is generally between the transmembrane (TM) domain and the targeting domain. In embodiments herein, at least a portion of the TM domain of the chimeric receptor comprises a DAP10-interacting domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a mutated 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 I membrane proteins such as CD8 alpha, CD8 beta, CD4, CD28, CD28T, 4-1BB, and CD7, which may be wild-type hinge regions derived from these molecules or may be mutated. In some embodiments, the spacer domain may include a chemically inducible dimerizer that controls expression upon addition of a small molecule. In some embodiments, no spacer is used.

[0184] The extracellular targeting domains of chimeric receptors as disclosed herein can include targeting domains derived from a variety of receptors including, without limitation, pattern recognition receptors (PRRs), Toll-like receptors (TLRs), killer activating receptors and killer inhibitor receptors (KAR and KIR, respectively), complement receptors, Fc receptors, B cell receptors, and T cell receptors. In some embodiments, the extracellular targeting domain can include, for example, a polypeptide derived from a monoclonal antibody, which is 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 regions of an antibody (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the expression “antigen-binding fragment” as used herein.

[0185] In some embodiments, the extracellular targeting domain specifically binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is a tumor-associated surface antigen, CD20, TyrD, B7H6, CD3, CD19; CD123; CD22; CD30; CD70, CD171; CD6, CS-1 (also called 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(1-4)bDGlcp(11)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((TnAg) 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 (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); IL-11R alpha; mesothelin; interleukin 11 receptor alpha (IL-URa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha receptor-type tyrosine protein kinase ERBB2 (Her2 / neu); ElbB3, ErbB4, mucin 1, cell surface-bound (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostate acid phosphatase (PAP); elongation factor 2 mutation (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 (prososome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gpl00); breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr - abl) oncogene fusion protein; tyrosine kinase; Ephrin type - A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2 - 3)bDGalp(1 - 4)bDGrcp(11)Cer); transglutaminase 5 (TGS5); high - molecular - weight melanoma - associated antigen (HMWMAA); melanoma - associated antigen; o - acetyl - GD2 ganglioside (OAcGD2); folate receptor beta; 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; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta - specific 1 (PLAC1); hexasaccharide moiety of GloboH glycosphingolipid (GloboH); breast differentiation antigen (NY - BR - 1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein - coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative 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 (ETV6 - AML) located on chromosome 12p; semen protein 17 (SPA17); X - antigen family, member 1A (XAGE1); angiopoietin - binding cell - surface receptor 2 (Tie2); 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, protein; survivin; telomerase;Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells (MelanA or MartI); rat sarcoma (Ras) variant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene homolog derived from neuroblastoma (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 the imprinted site regulator), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK), kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPVE6); human papillomavirus E7 (HPVE7); intestinal carboxylesterase; heat shock protein 70-2 variant (mutant hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (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);Selected from 5T4, 8H9, ALCAM, B7-1 (CD80), B7-2 (CD86), B7-H4, B7-H6, β-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, disialoganglioside 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-alpha, glioma-associated antigen, glycosphingolipid, gp36, G250 / CAIX, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp41, HPV-specific antigen, KDR, kappa chain, insulin-like growth factor (IGF1)-l, ligands belonging to MIC in humans (MICA and MICB) and ULBP (ULBP1-ULBP6) family, LAGA-la, LewisY, lambda chain, lectin-reactive AFP, L1 cell adhesion molecule, MAGE, major histocompatibility complex (MHC) molecule, tumor-specific peptide epitope of the presence of major histocompatibility complex (MHC) molecule, M-CSF, MN-CAIX, MUC-1, MUC-16, NKG2D, NKG2D ligand, neutrophil elastase, Nkp30, cancer fetal antigen (h5T4) p53, prostate-specific antigen (PSA), PSC1, prostate-specific antigen protein, STEAP1, STEAP2, surface adhesion molecule, survivin and telomerase, TAG-72, additional domain A (EDA) and additional domain B (EDB) of fibronectin and Al domain of tenascin-C (TnCAl), thyroglobulin, Tem8, tumor stromal antigen, VEGF-A, and immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0186] In some additional or alternative embodiments, the target antigen is a viral antigen such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7, viral antigens present in cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza virus, coronavirus, and the like.

[0187] Without limitation to other additional or alternative examples of antigens that can be targeted by the chimeric antigens of the present disclosure, those described in WO2016 / 172583, WO2017 / 062820, WO2016 / 149254, WO2017 / 149515, WO2014 / 039523, WO2013 / 123061, WO2016 / 166544, WO2017 / 222593, and WO2018 / 165228 are included.

[0188] Targeting domains used to specifically bind to an antigen as discussed above are known, and it should be understood that such targeting domains (e.g., monoclonal antibodies or fragments thereof, or at least one or more of their CDRs) can be readily incorporated into the chimeric receptors of the present disclosure. For example, targeting domains for use as disclosed herein are exemplified for use in chimeric receptors as 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-GD2, anti-GD3, 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-cancer fetal antigen, anti-PSCA, anti-PSMA, anti-ROR1, anti-IgE, anti-TAG-72, anti-VEGF-R2), or can be derived therefrom. In some embodiments, known targeting domains can include, without limitation, those described in U.S. Patent Application No. 08 / 940,544 and / or those cited by reference therein (e.g., anti-CD28), or can be derived therefrom. In some embodiments, known targeting domains can include, without limitation, those described in WO2016199141 and / or those cited by reference therein (e.g., anti-TyrD, anti-WT1, anti-MAGE-A4, anti-MAGE-A9, anti-PAP), or can be derived therefrom.In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2020 / 072536 and / or those cited by reference therein (e.g., anti-BCMA, anti-CD20). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2020 / 072546, WO2014180306, and / or U.S. Provisional Patent Application No. 63 / 235093 and / or those cited by reference therein (e.g., anti-GPC3, anti-TyrD). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2015123642 and / or those cited by reference 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, known targeting domains can include, or be derived from, without limitation, those described in WO2017 / 222593 and / or those cited by reference 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 can include, without limitation, those described in WO2017 / 149515 and / or those cited by reference therein (e.g., anti-mesothelin, anti-EGFRvIII, anti-claudin 6, 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 alpha, anti-ERBB2, anti-MUC1, anti-EGFR, anti-NCAM, anti-CAIX, anti-Fos-related antigen I, anti-SSEA-4, anti-PDGFR-beta, 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 reverse 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 can be derived therefrom. In some embodiments, known targeting domains can include, without limitation, those described in WO2017 / 025038 and / or those cited by reference therein (e.g., anti-CD19, anti-CD20, anti-BCMA, anti-CD38), or can be derived therefrom. In some embodiments, known targeting domains can include, without limitation, those described in WO2019215500 and / or those cited by reference therein (e.g., anti-CD19, anti-CD33, anti-CD20, anti-BCMA), or can be derived therefrom. In some embodiments, known targeting domains can include, without limitation, those described in WO2016 / 016343 and / or those cited by reference therein (e.g., anti-ROR1), or can be derived therefrom.In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2019 / 028051 and / or those cited by reference therein (e.g., anti-CD19, anti-CD20, anti-CD33). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2012058460 or WO2016093878 (e.g., anti-CD70). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2013169691A1 (e.g., anti-B7-H6). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2016160620, WO2014100439, WO2013025779 and / or those cited by reference therein (e.g., anti-B7-H4). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2019219089, WO2020200196, WO2020043044, WO2020147321 and / or those cited by reference therein (e.g., anti-Claudin 18.2). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in WO2016 / 090337, WO2017 / 096120, WO2014 / 210064, U.S. Patent No. 7,999,077, WO2016 / 205520, U.S. Patent No. 7,105,149, WO2006 / 076691, WO2010 / 114940, WO2010 / 120561 and / or those cited by reference therein (e.g., anti-FCRL5 / FCRH5 / CD307).In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in U.S. Provisional Patent Application No. 63 / 411,988 and / or those cited therein by reference (e.g., anti-B7H6). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in U.S. Provisional Patent Application No. 63 / 393,787 and / or those cited therein by reference (e.g., anti-CD70). In some embodiments, known targeting domains can include, or be derived from, without limitation, those described in U.S. Provisional Patent Application No. 63 / 397,296 and / or those cited therein by reference (e.g., anti-PSMA). In some embodiments, known targeting domains can include, or be derived from, without limitation, those 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., 2013, Jun. 3; 210(6):1125-35 (e.g., anti-FAP).

[0189] In an embodiment, a chimeric receptor that does not contain a DAP10-interacting domain includes 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 WO2020 / 072536, and the chimeric receptor includes an anti-CD20 extracellular targeting domain. In each of SEQ ID NOs: 8, 9, 10, 11, 12, 46, and 48 of WO2020 / 072536, the chimeric receptor includes a CD8α TM domain. Thus, in an embodiment, the CD8α TM domain can be replaced with a DAP10-interacting domain, for example, a DAP10-interacting domain including the amino acid sequence shown in SEQ ID NO: 75 herein, to obtain an anti-CD20 chimeric receptor that associates with the CAD polypeptide of the present disclosure. The same logic similarly applies to other chimeric receptors disclosed in WO2020 / 072536.

[0190] In an embodiment, a chimeric receptor that does not contain a DAP10-interacting domain includes the amino acid sequence shown in SEQ ID NO: 20, or SEQ ID NO: 22 of WO2020 / 072546, and the chimeric receptor includes an anti-GPC3 extracellular targeting domain. In each of SEQ ID NOs: 20 and 22 of WO2020 / 072546, the chimeric receptor includes a CD8α TM domain. Thus, in an embodiment, the CD8α TM domain is replaced with a DAP10-interacting domain, for example, a DAP10-interacting domain including the amino acid sequence shown in SEQ ID NO: 75 herein, to obtain an anti-GPC3 chimeric receptor that associates with the CAD polypeptide of the present disclosure. The same logic similarly applies to other chimeric receptors disclosed in WO2020 / 072546.

[0191] Thus, in embodiments, it should be understood that chimeric receptors known in the art can be used in the methods of the present disclosure, with or without modification. For example, without limitation, any of the chimeric receptors listed in Table 1 of Sadelain et al. (Cancer Discov. 3(4):388-398 (2013)) can be used without modification, or can be modified by incorporating a DAP10-interacting domain such that the chimeric receptor associates with DAP10 and, by extrapolation, associates with the CAD polypeptides of the present disclosure. Of course, other modifications including, but not limited to, the incorporation of one or more additional or alternative intracellular domains (e.g., co-stimulatory domains and / or intracellular signaling domains) are within the scope of the present disclosure. The same logic applies equally to other chimeric receptors known in the art, for example, without limitation, those disclosed in WO2016 / 090337.

[0192] C. Expression of Chimeric Constructs

[0193] 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, NK cell, NKT cell, etc.) and transcribed and translated to produce a product (e.g., a chimeric adapter polypeptide or chimeric receptor as described herein). In the isolated nucleic acids of the present invention, a promoter is operably linked to a nucleic acid sequence encoding a chimeric adapter polypeptide or chimeric receptor of the present invention, i.e., is arranged to facilitate the transcription of messenger RNA from the DNA encoding the chimeric adapter polypeptide or chimeric receptor. The term "operably linked" refers to an arrangement of the components so described in a relationship that enables the components to function in their intended manner.

[0194] The promoter can be derived from the genome or be synthetically generated. Various promoters for use in host cells related to the present disclosure are well known in the art (e.g., the CD4 promoter disclosed by Marodon, et al. (2003) Blood, 101(9):3416-23). The promoter can be constitutive or inducible, and the induction is related to a specific cell type or a specific maturation level. Alternatively, a number of well-known viral promoters may also be suitable. Promoters of interest include the β-actin promoter, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen focus-forming virus promoters. The promoter may or may not be associated with an enhancer, and the enhancer may be naturally associated with a specific promoter or associated with a different promoter. In an embodiment, the expression of the chimeric adapter polypeptide is under the control of an inducible promoter, e.g., a promoter that can be induced by a molecule present in the tumor microenvironment (e.g., TGFβ).

[0195] The sequences of the open reading frames encoding the various segments of the chimeric adapter polypeptide or chimeric receptor of the present disclosure can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or a combination thereof.

[0196] In an embodiment, for the expression of the chimeric adapter polypeptide of the present invention, the naturally occurring or endogenous transcription start region of the nucleic acid sequence encoding the N-terminal component of DAP10 can be used to generate the chimeric adapter polypeptide in a host cell. Alternatively, an exogenous transcription start region that allows for constitutive or inducible expression can be used, and the expression can be optionally controlled according to the host cell, the desired expression level, the nature of the host cell, etc.

[0197] It can include a termination region encoding a component of the C-terminus of a chimeric adapter polypeptide or chimeric receptor. Generally speaking, the source of the termination region is considered unimportant for the expression of recombinant proteins, and a wide variety of termination regions can be employed without adversely affecting expression.

[0198] The isolated nucleic acid encoding the chimeric adapter polypeptide or chimeric receptor according to the present invention can be prepared by conventional methods. The sequence (natural or synthetic) is isolated and manipulated as appropriate so that the various components can be properly joined. Thus, the various nucleic acid sequences encoding the various segments of the chimeric adapter polypeptide can be isolated by using appropriate primers, for example, by utilizing the polymerase chain reaction (PCR). If desired, specific primers can be designed that result in the deletion of unwanted portions of the nucleic acid sequence used as a template. Additionally or alternatively, restriction digests of cloned genes can be used to generate the isolated nucleic acid constructs of the present disclosure. In either case, the sequences can be selected to provide blunt-ended restriction sites or to have complementary overlaps to facilitate integration into various vectors. In the examples, modifications of the nucleic acid sequence (e.g., introducing one or more point mutations, insertions, or deletions) are carried out. In one embodiment regarding the chimeric adapter polypeptide of the present disclosure, the modification can include, for example, amino acid changes at positions Y86 and / or K84 of SEQ ID NO: 1. Methods for introducing modifications into nucleic acid sequences are known in the art and can include the use of various commercially available kits (e.g., QuickChange Site Directed Mutagenesis Kit, Agilent, Santa Clara, CA).

[0199] Various operations for preparing an isolated nucleic acid encoding a chimeric adapter polypeptide or a chimeric receptor of the present disclosure can be performed in vitro. In certain embodiments, the sequence encoding the chimeric adapter polypeptide is introduced into a vector for cloning and expression in a suitable host cell using standard transformation or transfection methods. Thus, after each operation, the construct obtained from the ligation of the DNA sequences is cloned, the vector is isolated, and the sequence is screened to confirm that it encodes the desired chimeric adapter polypeptide. The sequence can be screened by restriction analysis, sequencing, and the like.

[0200] The isolated nucleic acid is thought to be capable of being introduced into a host cell as naked DNA or in a suitable vector. Many suitable vectors are known to those of ordinary skill in the art of molecular biology, and this selection depends on the desired function and includes plasmids, cosmids, viruses, bacteriophages, and other vectors conventionally used in genetic engineering. Methods well known to those of skill in the art can be used to construct various plasmids and vectors. See, for example, the techniques described below: Sambrook et al. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the present disclosure can be reconstituted into liposomes for delivery to target cells.

[0201] Methods for stably transfecting host cells by electroporation using naked DNA are known in the art (see, e.g., U.S. Patent No. 6,410,319, which discloses transfection of T cells). Naked DNA generally refers to DNA encoding the chimeric adapter polypeptide or chimeric receptor of the present invention that is contained within a plasmid expression vector in the appropriate orientation for expression. Advantageously, the use of naked DNA shortens the time required to generate host cells that express the chimeric polypeptide(s) of the present invention.

[0202] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce an isolated nucleic acid encoding the chimeric polypeptide of the present invention into a host cell. Vectors suitable for use in accordance with the methods of the present invention are those that do not replicate in T cells. A number of virus-based vectors are known, and the copy number of the virus maintained within the cell is low enough to maintain the viability of the cell. Exemplary vectors include the pFB-neo vector (STRATAGENE®) and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0203] Thus, in some embodiments, it can be appreciated that the isolated nucleic acid is a circular nucleic acid. In some embodiments, the isolated nucleic acid is a vector, e.g., a plasmid vector, an adenoviral vector, an adeno-associated viral vector, a viral vector, a retroviral vector (e.g., a gammaretroviral vector), or a lentiviral vector. In some embodiments, a continuous portion of the isolated nucleic acid, or, e.g., a DAP10 sequence (e.g., at least a portion of SEQ ID NO: 1, modified or unmodified), and one or more signaling domains and / or costimulatory domains, is integrated into the genome of the host γδT host cell. In an exemplary embodiment, the isolated nucleic acid is a retroviral vector.

[0204] D. Host Cells

[0205] The chimeric polypeptides of the present disclosure, including CAD polypeptides and chimeric receptors, may 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 that associates with DAP10. For example, the CAD polypeptide may be expressed in a host cell that expresses NKG2D. In embodiments, the CAD polypeptide is expressed in a host cell type that exhibits some endogenous expression of a receptor that associates with DAP10 (e.g., NKG2D, Ly49H, Ly49D, Sirp-b1, Siglec-15, Cd300lb, etc.). In embodiments, the host cell type can be engineered to express the same receptor (e.g., NKG2D) such that, for example, the expression level of the receptor is higher than the level of endogenous expression that naturally occurs. In embodiments, the CAD polypeptide is expressed in a cell type that does 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., expression of NKG2D in a cell type that does not express NKG2D, or expression of a chimeric receptor that includes a DAP10-interacting domain). In embodiments, the CAD polypeptide is expressed in a cell type that exhibits endogenous expression of a receptor that associates with DAP10 (e.g., NKG2D), and the cell is further engineered to express a chimeric receptor that lacks a DAP10-interacting domain. In embodiments, the CAD polypeptide is expressed in a cell type that exhibits endogenous expression of a receptor that associates with DAP10 (e.g., NKG2D), and the cell is further engineered to express at least one chimeric receptor that includes a DAP10-interacting domain, the chimeric receptor including at least an extracellular targeting domain and a DAP10-interacting domain, and optionally further including one or more costimulatory domains and / or one or more intracellular domains.In embodiments, the CAD polypeptide is expressed in cell types lacking endogenous expression of the receptor that associates with DAP10, engineered to express a non-chimeric receptor that associates with DAP10, and further engineered to express a chimeric receptor that, in some examples, includes a DAP10-interacting domain, while in other examples lacks such a DAP10-interacting domain, and wherein the chimeric receptor includes one or more co-stimulatory domains and / or one or more intracellular ligand-binding domains.

[0206] In embodiments, expression of the CAD polypeptide of the present disclosure in a host cell results in the CAD polypeptide competing with endogenous cellular DAP10 (e.g., WT DAP10). Due to the competition, intracellular signaling via the receptor that associates with DAP10 (e.g., endogenous, exogenous, chimeric) can be re-directed via the chimeric DAP10 adapter polypeptide. For the desired result (e.g., killing of tumor cells) to be effective, the signaling does not need to be 100% re-directed via the CAD polypeptide, but such a percentage is within the scope of the present disclosure. Signaling via the CAD polypeptide may include ranges from 20% to 100% of signaling via DAP10, such as 90 - 100%, 80 - 100%, 70 - 100%, 60 - 100%, 50 - 100%, etc. As a representative example for illustrative purposes, a host cell in which 80% of the signaling via the receptor associated with DAP10 is re-routed via the CAD polypeptide means that only 20% of such signaling still occurs via endogenous DAP10, while 80% of the signaling occurs via the CAD polypeptide.

[0207] The host cells described herein can be stored for use in adoptive cell transfer, e.g., cryopreserved. In embodiments, the host cells are stored prior to engineering the cells to express the CAD polypeptide and the chimeric receptor. In embodiments, the cells are engineered to express the chimeric DAP10 adapter polypeptide and then the cells are stored.

[0208] Preferred host cells for use with the CAD polypeptides and chimeric receptors of the present disclosure include immune cells. Such cells may be obtained from the subject to be treated (i.e., autologous), or alternatively, immune cell lines or donor immune cells (allogeneic, syngeneic) may be used. Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells can be obtained from blood collected from a subject using any number of techniques known to those of skill in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be obtained by apheresis. In some embodiments, immune cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation on a PERCOLL™ gradient or counterflow centrifugation. Specific subpopulations of immune cells can be further separated by positive or negative selection techniques. For example, immune cells can be isolated using a combination of antibodies to surface markers unique to the positively selected cells, for example, by incubating with antibody-conjugated beads for a time sufficient for positive selection of the desired immune cells. Alternatively, enrichment of an immune cell population can be achieved by negative selection using a combination of antibodies that target surface markers unique to the negatively selected cells. Other specific methods of separation and / or enrichment are disclosed herein.

[0209] In some embodiments, immune cells include any white blood cells involved in the body's defense against infections and foreign substances. For example, immune cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune cells related to the present disclosure can include, but are not limited to, αβ T cells, γδ T cells, NK cells, NKT cells, γδ NKT cells, B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, regulatory T cells, etc. In embodiments, preferred immune cells include αβ T cells, γδ T cells, NK cells, NKT cells, γδ NKT cells, and / or, in some examples, macrophages. In embodiments, preferred immune cells include γδ T cells. In embodiments, immune cells related to the present disclosure include allogeneic cells, autologous cells, or syngeneic cells.

[0210] Aspects of the present disclosure include immune cells that have cytotoxic activity in vitro or in vivo against blood or solid tumor cells that exhibit cell surface expression of tumor-associated antigens (TAAs), virus-infected cells that present virus-derived antigens, bacterial cells, etc. In embodiments, the cytotoxic activity is an activity that is inherently present. In embodiments, immune cells that functionally express both the CAD polypeptide and at least one chimeric receptor exhibit higher cytotoxic activity than the level of cytotoxic activity in vitro and / or in vivo in control immune cells that do not contain the CAD polypeptide and / or at least one chimeric receptor of the present disclosure.

[0211] In embodiments, the cytotoxicity is significantly (> about 25%) enhanced or improved by the presence of both the CAD polypeptide and at least one chimeric receptor as 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 both the CAD polypeptide and at least one chimeric receptor.

[0212] In embodiments, the engineered immune cells related to the present disclosure can exhibit potent and / or persistent cell killing activity (e.g., tumor cells, virus-infected cells) via direct and / or indirect mechanisms. In some cases, the cell killing activity persists 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 cells. In some cases, the cell killing activity of the immune cells or their progeny cells disclosed herein engineered to express a CAD polypeptide and at least one chimeric receptor persists 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 cells or from the administration of the engineered immune cells disclosed herein. This persistent cell killing activity may be demonstrated in vitro, in vivo, or both in vitro and in vivo.

[0213] In embodiments, aspects of the present disclosure include immune cells that associate with DAP10 and proliferate in response to contact with cells that exhibit cell surface expression of a ligand recognized by a receptor that associates, in particular, with a CAD polypeptide of the present disclosure. An example of such a receptor is NKG2D, but the present disclosure is not limited to CAD polypeptides that interact with NKG2D and can include other receptor(s) that also associate with DAP10, such as chimeric receptors engineered to include a DAP10-interacting domain. In embodiments, the proliferation is at least partially, significantly (more than about 20%, or more than about 25%, or more than about 50%, or more than about 80%), or completely (e.g., 100%) due to the presence of a CAD polypeptide construct that associates with a receptor (e.g., endogenous or exogenous NKG2D, a chimeric receptor engineered to include a DAP10-interacting domain) expressed on a host cell. In some cases, the immune cells exhibit even higher levels of proliferation in vitro and / or in vivo compared to control immune cells (e.g., immune cells of the same type) that do not contain both a CAD polypeptide and at least one chimeric receptor. The even higher levels of proliferation can include an increase of 20-50%, an increase of 50-80%, an increase of 80-100%, or a two-fold increase, a three-fold increase, a four-fold increase, a five-fold increase, a 5-10-fold increase, a 10-20-fold increase, or an increase greater than 20-fold, e.g., an increase of 50-100-fold or more, compared to control immune cells lacking a CAD polypeptide and / or at least one chimeric receptor described herein.

[0214] In some embodiments, immune cells engineered to contain both the CAD polypeptides and at least one chimeric receptor described herein express and secrete, or continuously express and secrete, one or more pro-inflammatory cytokines after contacting cells that express a ligand recognized by a cell surface receptor on the immune cell that associates with DAP10. In embodiments, the expression and secretion associated with immune cells engineered to express both the CAD polypeptide and at least one chimeric receptor is at least partially, significantly (more than about 20%, or more than about 25%, or more than about 50%, or more than about 80%), or completely due to one or both of the CAD polypeptide and / or at least one chimeric receptor. In embodiments, the expression and / or secretion is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or even 100% more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 5- to 10-fold, 10- to 20-fold, or even 20-fold more, e.g., 50- to 100-fold or more more than the expression and / or secretion observed in control immune cells (e.g., immune cells of the same type) lacking expression of the CAD polypeptide and / or at least one chimeric receptor.

[0215] In embodiments, the engineered immune cells of the present disclosure may function, for example, to alter the cellular microenvironment (e.g., TME) to be advantageous for an anti-tumor response. For example, solid tumors can recruit suppressive cells such as myeloid-derived suppressor cells (MDSCs), which can enhance the suppressive TME. The frequency of MDSCs in circulation or within tumors correlates with cancer stage, disease progression, and resistance to standard chemotherapy and radiotherapy. Since certain ligands (e.g., NKG2D ligands) are expressed at high levels on some solid tumors and tumor-infiltrating MDSCs, in embodiments, the modified immune cells of the present disclosure can be used to alter the TME to be advantageous for an anti-tumor response by reducing or removing suppressive molecules (e.g., TGF-β) of the TME. In embodiments, the modified immune cells of the present disclosure are cytotoxic to MDSCs but do not harm normal tissues expressing NKG2D ligands (i.e., are non-toxic) (see, for example: Parihar, R., et al., (2019) Cancer Immunol Res 7(3):363-375). In some embodiments, the cell killing activity associated with, for example, killing of MDSCs by the engineered host cells of the present disclosure reduces the suppressive effect of the TME. For example, the suppressive effect may be reduced by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more compared to the suppressive effect of the TME in the absence of host cells engineered to express a CAD polypeptide and a chimeric receptor as described herein.

[0216] In embodiments, the engineered immune cells of the present disclosure may function to reduce the growth and / or proliferation of target cells. For example, engineered immune cells engineered to express both a CAD polypeptide and at least one chimeric receptor as described herein may have reduced growth and / or proliferation of target cells compared to the growth and / or proliferation of target cells in the absence of the engineered immune cells, or in the presence of engineered immune cells that express either the CAD polypeptide or the chimeric receptor but not both, 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 cells express 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 cells, and the receptor associates at least partially with the CAD polypeptide.

[0217] In embodiments, the engineered immune cells of the present disclosure may function to exert cytotoxicity against target cells. For example, engineered immune cells engineered to express both a CAD polypeptide and at least one chimeric receptor as described herein may result in an increase in the death of a population of one or more target cells compared to the amount of cell death in the absence of the engineered immune cells, or in the presence of engineered immune cells that express either the CAD polypeptide or the chimeric receptor but not both. 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 therebetween, compared to the amount of cell death in the absence of the engineered immune cells, or in the presence of engineered immune cells that express either the CAD polypeptide or the chimeric receptor but not both.

[0218] Expression of a CAD polypeptide comprising a heterologous signaling domain (e.g., 4-1BB, OX40, CD28, ICOS, IL2R, CD27, etc.) that affects cell survival and proliferation, when paired with a chimeric receptor on the same host cell (e.g., T cell or NK cell) that engages a target having low-level expression in normal tissue, enables a logic gating strategy (e.g., "AND" gating) that can increase the stringency and / or efficacy of target attack. Thus, host cells having both a chimeric receptor that recognizes a primary target (e.g., a primary cancer target, a viral antigen, an autoimmune antigen, etc.) and an appropriate CAD polypeptide may promote the sustained survival, proliferation, and killing of target cells that express both the primary target(s) and a ligand for a receptor (e.g., NKG2D) that associates with DAP10. In such examples, the chimeric receptor can, in embodiments, comprise a DAP10-interacting domain. Examples of logic gating strategies applicable to the present disclosure can be found, for example, in WO2019118518, WO2020154635, WO2020223445, WO2021035093, WO2019222642A1, WO2018236825A1, WO2019164979, and Chang, ZL, and Chen YY, (2017), Trends Mol. Med. 23(5):430-450).

[0219] Thus, in embodiments, the host cell can comprise a CAD polypeptide that includes a DAP10 domain and at least one of a co-stimulatory domain or an intracellular domain, particularly lacking an extracellular domain, and the host cell further expresses at least one chimeric receptor that includes at least an extracellular targeting domain, an intracellular signaling domain, and / or a co-stimulatory domain. When the chimeric receptor includes an intracellular signaling domain, the CAD polypeptide includes a co-stimulatory domain, and vice versa. In some embodiments, the chimeric receptor lacks a DAP10-interacting domain, and the CAD polypeptide interacts with a receptor having a DAP10-interacting domain (e.g., NKG2D) internally. In other embodiments, the chimeric receptor includes a DAP-10 interacting domain. As an example, the CAD polypeptide can include at least one 4-1BB co-stimulatory domain but lacks an intracellular signaling domain, and the chimeric receptor includes at least one intracellular signaling domain (e.g., CD3ζ), and vice versa. As another example, the CAD polypeptide can 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, and the chimeric receptor includes at least one intracellular signaling domain (e.g., CD3ζ), and vice versa. In some embodiments, the CAD polypeptide can include at least two different co-stimulatory domains. For example, the CAD polypeptide can include co-stimulatory domains of at least one 4-1BB and at least one CD28 (or OX40, or ICOS, etc.), but lacks an intracellular signaling domain, and the chimeric receptor includes at least one intracellular signaling domain (e.g., CD3ζ), and optionally, the chimeric receptor includes at least two different intracellular signaling domains, and vice versa.

[0220] Accordingly, in an embodiment, there is provided a method of generating a cell comprising both a CAD polypeptide and at least one chimeric receptor, the method comprising introducing into the cell an isolated nucleic acid encoding a CAD polypeptide and at least one isolated nucleic acid encoding at least one chimeric receptor, such that the cell expresses both the CAD polypeptide and at least one chimeric receptor. In an embodiment, the cell is an immune cell described herein, such as, for example, an NK cell, an NKT cell, a γδ T cell, an αβ T cell, or a γδ NKT cell. In some embodiments, the method of generating the cell further comprises introducing into the cell another isolated nucleic acid encoding a receptor capable of associating with the CAD polypeptide.

[0221] E. Methods of Use

[0222] In one aspect, the present disclosure provides a method of modulating a signal transmitted through at least one receptor of a host cell, the host cell comprising an immune cell engineered to express both a CAD polypeptide and at least one chimeric receptor as described herein, preferably, the immune cell is cytotoxic, and more preferably, the immune cell is a γδ T cell. In an embodiment, the receptor is endogenous to the immune cell and is endogenously expressed therein. In additional or alternative embodiments, the receptor is expressed by introducing an isolated nucleic acid encoding the receptor into the immune cell. In additional or alternative embodiments, the receptor is a chimeric receptor as described herein and optionally comprises a DAP10-interacting domain.

[0223] In an embodiment, the method of modulating a signal transmitted through at least one receptor results in stimulation and / or activation of the immune cell.

[0224] In an embodiment, the method of modulating a signal transmitted through at least one receptor results in an increase in the proliferation level of the immune cell as compared to the proliferation level of a control immune cell lacking the CAD polypeptide.

[0225] In an embodiment, a method of modulating a signal transmitted through a receptor results in an increase in the expression and secretion of the one or more cytokines as compared to the levels of expression and secretion of the one or more cytokines in control immune cells lacking the CAD polypeptide and / or the chimeric receptor.

[0226] In an embodiment, modulating a signal transmitted through a receptor includes routing at least a portion of the signal through the CAD polypeptide, as opposed to endogenous DAP10. In an embodiment, a portion of the signal routed through the CAD polypeptide is 80% or more, for example, 90 - 95% or more, for example, 99% or 100%.

[0227] F. Methods of Treatment

[0228] As described herein, a pharmaceutical composition comprising an engineered host cell that expresses both a CAD polypeptide and at least one chimeric receptor, and / or a mixture thereof, may be administered for prophylactic treatment and / or therapeutic treatment. In a preferred embodiment, the pharmaceutical composition comprises γδ T cells engineered to express both a CAD polypeptide and at least one chimeric receptor. The mixture may, as described herein, comprise different types of host cells engineered to express the same or different CAD polypeptides and the same or different chimeric receptors. For example, and without limitation, the mixture may comprise a population of NK cells expressing a first CAD polypeptide and a first chimeric receptor and a population of γδ cells 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 the first chimeric receptor is the same as or different from the second chimeric receptor. As another example, and without limitation, the mixture may comprise a population of NK cells and a population of γδ cells, each engineered to express the same CAD polypeptide and the same or different chimeric receptors. As yet another example, the mixture may comprise a population of engineered host cells and, additionally, a population of non-engineered cells. For example, without limitation, the mixture may comprise a population of γδ cells or NK cells engineered to express both a CAD polypeptide and at least one chimeric receptor as described herein and a population of another non-engineered cell, such as NK cells, NKT cells, γδ cells, αβ cells, etc. In a therapeutic application, the composition may be administered to a subject already suffering from a disease or condition in an amount sufficient to reduce at least one sign or symptom associated with the disease or condition. In some embodiments, the amount is sufficient to cure the disease or condition.

[0229] The genetically engineered host cell population and / or mixtures thereof can also be administered to reduce the likelihood of onset, incidence, or worsening of a condition. The effective amount of a population of engineered host cells, non-engineered host cells, and / or mixtures thereof for therapeutic use can vary depending on the severity and course of the disease or condition, previous treatment, the health status, body weight, and / or response to various agents of the subject, and / or the judgment of the treating physician.

[0230] In embodiments, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure can be used to treat a subject in need of treatment of a condition. Examples of such diseases include, but are not limited to, cancer, infectious diseases, and autoimmune diseases. The subject can be a human, non-human primate, such as a chimpanzee, and other ape and monkey species; livestock, such as cows, horses, sheep, goats, pigs; companion animals, such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, etc. The subject can be of any age. The subject can be, for example, an elderly person, an adult, a youth, a pre-pubescent, a child, an infant, a neonate.

[0231] A method of treating a condition (e.g., a disease) in a subject may include administering to the subject a therapeutically effective amount of one or more populations of engineered host cells (e.g., engineered to express both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or a mixture thereof. The one or more populations of engineered host cells, non-engineered cells, and / or a mixture thereof can be administered according to various treatment regimens (e.g., timing, concentration, dosage, interval between treatments, and / or formulation). The subject can also be pretreated, for example, by chemotherapy, radiation therapy, or a combination of both, prior to being administered a therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and / or a mixture thereof. As part of the treatment, the one or more populations of engineered host cells, non-engineered cells, and / or a mixture thereof may be administered to the subject according to a first treatment regimen, and the subject may be monitored to determine whether treatment according to the first treatment regimen meets a given level of therapeutic efficacy. Optionally, the one or more populations of engineered host cells, non-engineered cells, and / or a mixture thereof may be administered to the subject according to a second treatment regimen based on information collected from providing the first treatment regimen to the subject.

[0232] In an embodiment, a pharmaceutical composition comprising at least one host cell engineered to express both a CAD polypeptide and at least one chimeric receptor may be administered in a first treatment regimen. The subject may be monitored, for example, by a healthcare provider (e.g., a treating physician or nurse). In some examples, the subject is monitored to determine or measure the effectiveness of the engineered host cells in the treatment of the subject's condition. In some situations, the subject may be monitored to determine the in vivo expansion of the engineered host cell population in the subject. Another pharmaceutical composition comprising at least one host cell engineered to express a CAD polypeptide and at least one chimeric receptor may be administered to the subject in a second treatment regimen. The pharmaceutical composition administered in the second treatment regimen may comprise the same type of host cells expressing the same CAD polypeptide and at least one chimeric receptor as those administered to the subject in the first treatment regimen. However, it is within the scope of the present disclosure that the pharmaceutical composition administered in the second treatment regimen may optionally comprise different CAD polypeptides (e.g., CAD polypeptides having different mutations and / or co-stimulatory or signaling domains) and / or different chimeric receptor(s), and thus comprise different types of host cells. In some examples, for instance, if the first treatment regimen is found to be effective, the second treatment regimen may not be implemented (e.g., a single administration may be sufficient to treat the condition). In some embodiments, the population of engineered host cells can be administered to various subjects (e.g., the host cells have universal donor characteristics).

[0233] A therapeutically effective amount of one or more engineered host cell populations (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof may be used to treat various pathologies. Optionally, a therapeutically effective amount of one or more engineered host cell populations (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof may be used to treat cancers, including solid tumors and hematological malignancies. Optionally, a therapeutically effective amount of one or more engineered host cell populations (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof may be used to treat, for example, infections caused by pathogenic bacteria or viruses.

[0234] Treatment with one or more engineered host cell populations (e.g., expressing both 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 the clinical onset of a pathology. Treatment may be provided to the subject 1 day, 1 week, 6 months, 12 months, or more than 2 years after the clinical onset of the disease. Treatment may be provided to the subject for a period of 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 more after the clinical onset. Treatment may be provided to the subject less than 1 day, less than 1 week, less than 1 month, less than 6 months, less than 12 months, or less than 2 years after the clinical onset. Treatment may also include treating humans in a clinical trial. Treatment can include administering to the subject a pharmaceutical composition comprising one or more engineered host cell populations (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof of the present disclosure.

[0235] In some cases, administration of one or more populations of engineered host cells of the disclosure (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof modulates the activity of endogenous lymphocytes in a subject. In some cases, administration of one or more populations of engineered host cells of the disclosure (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof results in activation of the cytotoxicity of other immune cells. In some cases, the other immune cells are CD8+ T cells. In some cases, the other immune cells are natural killer T cells. Other examples of other immune cells are also encompassed by the disclosure. In some cases, administration of one or more populations of engineered host cells of the disclosure (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof suppresses regulatory T cells. In some cases, the regulatory T cells are Fox3+ Treg cells. In some cases, the regulatory T cells are Fox3− Treg cells. Non-limiting examples of cells whose activity can be modulated by administration of one or more populations of engineered host cells of the disclosure (e.g., expressing both a CAD polypeptide and at least one chimeric receptor), non-engineered cells, and / or mixtures thereof include hematopoietic stem cells; B cells; CD4+ cells; CD8+ cells; erythrocytes; leukocytes; dendritic cells (e.g., dendritic antigen-presenting cells); leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophil granulocytes; T helper cells; and T killer cells.

[0236] For example, without limitation, one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof having cytotoxic activity against hematological tumor cells or solid tumor cells, or virus-infected cells, or bacterial cells can be administered to a subject in any order or simultaneously. Also, the engineered host cell(s) and / or mixture thereof of the present disclosure can be provided in a single unified form, such as by intravenous injection, or in multiple forms, such as by multiple intravenous infusions, subcutaneous injections, or as a pill. One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure can be packaged together or separately in a single package or multiple packages. One or all of the one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present invention can be administered in multiple divided doses. If not simultaneous, the timing between multiple administrations can vary from about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the engineered host cells of the present disclosure can proliferate in vivo within the subject after being administered to the subject. One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure can be frozen to provide cells for multiple treatments with the same cell preparation. One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, and pharmaceutical compositions containing them can be packaged as a kit. The kit may include one or more engineered host cell populations, non-engineered modified cells, and / or mixtures thereof, and instructions for use (e.g., written instructions) regarding the use of the compositions containing them.

[0237] In some cases, a method of treating a subject in need of a method comprises administering to the subject a therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof, wherein the administration treats a particular condition (e.g., cancer, viral, or bacterial infection, autoinflammatory disease). In some embodiments, the therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof is administered for at least 1 week. In some embodiments, the therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof of the present disclosure is administered for at least 2 weeks.

[0238] One or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of a pharmaceutical composition containing an engineered host cell population can vary. For example, one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof can be used as a prophylactic agent and administered continuously to a subject predisposed to a condition or disease to reduce the likelihood of onset of the disease or condition. The first administration can be via any practical route, such as any route described herein, using any formulation described herein. In some examples, administration of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof according to the disclosure is by intravenous administration. One or more administrations of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof can be as soon as possible after the onset of a particular condition (e.g., blood cancer or solid cancer, viral infection, bacterial infection, autoimmune disease, etc.), for the period required for treatment of the disease / condition, e.g., 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 administrations of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof can be administered years after the onset of a disease / condition (e.g., cancer) and before or after other treatments.

[0239] In some embodiments, one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof of the present disclosure are administered simultaneously or sequentially with one or more methods for increasing one or more common gamma chain cytokines. As used herein, "one or more methods for enhancing one or more common gamma chain cytokines" refers to a method, or combination of methods, that modifies the physiological state of a subject so as to increase the level of at least one common gamma chain cytokine in the subject. In some embodiments, the method increases the level of one or more common gamma chain cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-15, and IL-21 in the subject. In some embodiments, the method includes lymphodepletion. In some embodiments, the method includes administering to the subject one or more common gamma chain cytokines. Optionally, IL-2, IL-4, IL-7, IL-15, and / or IL-21 are administered. In some embodiments, the method includes secreting one or more common gamma chain cytokines from the administered engineered host cells. Optionally, IL-2, IL-4, IL-7, IL-15, and / or IL-21 are secreted.

[0240] In some embodiments, administering one or more methods for increasing common gamma chain cytokine(s) comprises lymphodepletion prior to introducing one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the present disclosure. In some embodiments, administering one or more methods for increasing common gamma chain cytokine(s) comprises administering an effective amount of common gamma chain cytokine(s) simultaneously with, or sequentially to, the introduction of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof to enhance the proliferation, cytotoxic activity, persistence, or combinations thereof of the introduced one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof. The amount of common gamma chain cytokine(s) administered can be an amount effective to enhance the proliferation, cytotoxic activity, persistence, or combinations thereof of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof. Exemplary amounts of IL-15 include, but are not limited to, 0.01 - 10 μg / kg / dose for IL-15 every 24 hours. Exemplary amounts of IL-2 include, but are not limited to, about 3×10 6 ~ about 22×10 6 units every 8 - 48 hours. For example, the IL2 administration treatment regimen in RCC was intravenous infusion of 600,000 international units / kg (0.037 mg / kg) over 15 minutes up to 14 times every 48 hours.

[0241] In some embodiments, performing one or more methods for increasing common gamma chain cytokine(s) comprises lymphodepletion prior to administering one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof, and administering an effective amount of common gamma chain cytokine(s) simultaneously with, or sequentially to, the introduction of one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof to enhance the proliferation, cytotoxic activity, persistence, or combinations thereof of the introduced one or more engineered host cell populations, non-engineered cells, and / or mixtures thereof.

[0242] In some embodiments, the elevation of common gamma chain cytokine(s) is achieved, at least in part, via the engineered host cell(s), and the common gamma chain cytokine(s) is expressed from a CAD construct and / or a chimeric receptor construct as disclosed herein. In such instances, it is within the scope of the disclosure that one or more additional gamma chain cytokine(s) may be further administered in a manner that elevates the additional gamma chain cytokine(s).

[0243] G. Dosage

[0244] One or more engineered host cell populations, non-engineered cells, and / or mixtures thereof of the disclosure may be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage form contains additional lymphocytes. In the unit dosage form, the formulation is divided into unit doses containing an appropriate amount of one or more compounds. The unit dosage can take the form of a package containing a separate amount of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampules. An aqueous suspension composition can be packaged in a non-resealable single-dose container. A resealable container for multiple administrations can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not contain a preservative. A formulation for parenteral injection can be provided, for example, in unit dosage form in an ampule, or in a multiple-dose container containing a preservative.

[0245] One or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof according to the present disclosure may be present in a composition in the following amounts: at least 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1×10 3 cells, at least 2×10 3 cells, at least 3×10 3 cells, at least 4×10 3 cells, at least 5×10 3 cells, at least 6×10 3 cells, at least 7×10 3 cells, at least 8×10 3 cells, at least 9×10 3 cells, at least 1×10 4 cells, at least 2×10 4 cells, at least 3×10 4 cells, at least 4×10 4 cells, at least 5×10 4 cells, at least 6×10 4 cells, at least 7×10 4 cells, at least 8×10 4 cells, at least 9×10 4 cells, at least 1×10 5 cells, at least 2×10 5 cells, at least 3×10 5 cells, at least 4×10 5 cells, at least 5×10 5 cells, at least 6×10 5 cells, at least 7×10 5 cells, at least 8×10 5 cells, at least 9×10 5 cells, at least 1×10 6 cells, at least 2×10 6 cells, at least 3×106 Cells, at least 4×10 6 Cells, at least 5×10 6 Cells, at least 6×10 6 Cells, at least 7×10 6 Cells, at least 8×10 6 Cells, at least 9×10 6 Cells, at least 1×10 7 Cells, at least 2×10 7 Cells, at least 3×10 7 Cells, at least 4×10 7 Cells, at least 5×10 7 Cells, at least 6×10 7 Cells, at least 7×10 7 Cells, at least 8×10 7 Cells, at least 9×10 7 Cells, at least 1×10 8 Cells, at least 2×10 8 Cells, at least 3×10 8 Cells, at least 4×10 8 Cells, at least 5×10 8 Cells, at least 6×10 8 Cells, at least 7×10 8 Cells, at least 8×10 8 Cells, at least 9×10 8 Cells, at least 1×10 9 Cells, or more.

[0246] A therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof of the present 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 1×10 3 Cells, about 1 cell to about 2×10 3 Cells, about 1 cell to about 3×10 3 Cells, about 1 cell to about 4×10 3 Cells, about 1 cell to about 5×103 Cells, about 1 cell to about 6×10 3 Cells, about 1 cell to about 7×10 3 Cells, about 1 cell to about 8×10 3 Cells, about 1 cell to about 9×10 3 Cells, about 1 cell to about 1×10 4 Cells, about 1 cell to about 2×10 4 Cells, about 1 cell to about 3×10 4 Cells, about 1 cell to about 4×10 4 Cells, about 1 cell to about 5×10 4 Cells, about 1 cell to about 6×10 4 Cells, about 1 cell to about 7×10 4 Cells, about 1 cell to about 8×10 4 Cells, about 1 cell to about 9×10 4 Cells, about 1 cell to about 1×10 5 Cells, about 1 cell to about 2×10 5 Cells, about 1 cell to about 3×10 5 Cells, about 1 cell to about 4×10 5 Cells, about 1 cell to about 5×10 5 Cells, about 1 cell to about 6×10 5 Cells, about 1 cell to about 7×10 5 Cells, about 1 cell to about 8×10 5 Cells, about 1 cell to about 9×10 5 Cells, about 1 cell to about 1×10 6 Cells, about 1 cell to about 2×10 6 Cells, about 1 cell to about 3×10 6 Cells, about 1 cell to about 4×10 6 Cells, about 1 cell to about 5×10 6 Cells, about 1 cell to about 6×10 6 Cells, about 1 cell to about 7×10 6 Cells, about 1 cell to about 8×10 6 Cells, about 1 cell to about 9×10 6 Cells, about 1 cell to about 1×10 7 Cells, about 1 cell to about 2×10 7 Cells, about 1 cell to about 3×10 7 Cells, about 1 cell to about 4×10 7 Cells, about 1 cell to about 5×10 7 Cells, about 1 cell to about 6×10 7 Cells, about 1 cell to about 7×10 7Cells, about 1 cell to about 8×10 7 Cells, about 1 cell to about 9×10 7 Cells, about 1 cell to about 1×10 8 Cells, about 1 cell to about 2×10 8 Cells, about 1 cell to about 3×10 8 Cells, about 1 cell to about 4×10 8 Cells, about 1 cell to about 5×10 8 Cells, about 1 cell to about 6×10 8 Cells, about 1 cell to about 7×10 8 Cells, about 1 cell to about 8×10 8 Cells, about 1 cell to about 9×10 8 Cells, or about 1 cell to about 1×10 9 Cells.

[0247] In some cases, a therapeutically effective dose of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof of the present invention is about 1×10 3 Cells to about 2×10 3 Cells, about 1×10 3 Cells to about 3×10 3 Cells, about 1×10 3 Cells to about 4×10 3 Cells, about 1×10 3 Cells to about 5×10 3 Cells, about 1×10 3 Cells to about 6×10 3 Cells, about 1×10 3 Cells to about 7×10 3 Cells, about 1×10 3 Cells to about 8×10 3 Cells, about 1×10 cells to about 9×10 3 Cells, about 1×10 cells to about 1×10 4 Cells, about 1×10 3 Cells to about 2×10 4 Cells, about 1×10 3 Cells to about 3×10 4 Cells, about 1×10 3 Cells to about 4×10 4 Cells, about 1×10 3 Cells to about 5×10 4 Cells, about 1×10 3 Cells to about 6×10 4 Cells, about 1×10 3Cells ~ about 7×10 4 Cells, about 1×10 3 Cells ~ about 8×10 4 Cells, about 1×10 3 Cells ~ about 9×10 4 Cells, about 1×10 3 Cells ~ about 1×10 5 Cells, about 1×10 3 Cells ~ about 2×10 5 Cells, about 1×10 3 Cells ~ about 3×10 5 Cells, about 1×10 3 Cells ~ about 4×10 5 Cells, about 1×10 3 Cells ~ about 5×10 5 Cells, about 1×10 3 Cells ~ about 6×10 5 Cells, about 1×10 3 Cells ~ about 7×10 5 Cells, about 1×10 3 Cells ~ about 8×10 5 Cells, about 1×10 3 Cells ~ about 9×10 5 Cells, about 1×10 3 Cells ~ about 1×10 6 Cells, about 1×10 3 Cells ~ about 2×10 6 Cells, about 1×10 3 Cells ~ about 3×10 6 Cells, about 1×10 3 Cells ~ about 4×10 6 Cells, about 1×10 3 Cells ~ about 5×10 6 Cells, about 1×10 3 Cells ~ about 6×10 6 Cells, about 1×10 3 Cells ~ about 7×10 6 Cells, about 1×10 3 Cells ~ about 8×10 6 Cells, about 1×10 3 Cells ~ about 9×10 6 Cells, about 1×10 3 Cells ~ about 1×10 7 Cells, about 1×10 3 Cells ~ about 2×10 7 Cells, about 1×10 3 Cells ~ about 3×10 7 Cells, about 1×103 Cells ~ about 4×10 7 Cells, about 1×10 3 Cells ~ about 5×10 7 Cells, about 1×10 3 Cells ~ about 6×10 7 Cells, about 1×10 3 Cells ~ about 7×10 7 Cells, about 1×10 3 Cells ~ about 8×10 7 Cells, about 1×10 3 Cells ~ about 9×10 7 Cells, about 1×10 3 Cells ~ about 1×10 8 Cells, about 1×10 3 Cells ~ about 2×10 8 Cells, about 1×10 3 Cells ~ about 3×10 8 Cells, about 1×10 3 Cells ~ about 4×10 8 Cells, about 1×10 3 Cells ~ about 5×10 8 Cells, about 1×10 Cells ~ about 6×10 8 Cells, about 1×10 Cells ~ about 7×10 8 Cells, about 1×10 3 Cells ~ about 8×10 8 Cells, about 1×10 3 Cells ~ about 9×10 8 Cells, or about 1×10 3 Cells ~ about 1×10 9 Cells can be.

[0248] In some cases, a therapeutically effective dose of one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof of the present invention is: about 1×10 6 Cells ~ about 2×10 6 Cells, about 1×10 6 Cells ~ about 3×10 6 Cells, about 1×10 6 Cells ~ about 4×10 6 Cells, about 1×10 6 Cells ~ about 5×10 6 Cells, about 1×10 6 Cells ~ about 6×10 6 Cells, about 1×10 6 Cells ~ about 7×10 6Cells, about 1×10 6 cells~about 8×10 6 Cells, about 1×10 6 cells~about 9×10 6 Cells, about 1×10 6 cells~about 1×10 7 Cells, about 1×10 6 cells~about 2×10 7 Cells, about 1×10 6 cells~about 3×10 7 Cells, about 1×10 6 cells~about 4×10 7 Cells, about 1×10 6 cells~about 5×10 7 Cells, about 1×10 6 cells~about 6×10 7 Cells, about 1×10 6 cells~about 7×10 7 Cells, about 1×10 6 cells~about 8×10 7 Cells, about 1×10 6 cells~about 9×10 7 Cells, about 1×10 6 cells~about 1×10 8 Cells, about 1×10 6 cells~about 2×10 8 Cells, about 1×10 6 cells~about 3×10 8 Cells, about 1×10 6 cells~about 4×10 8 Cells, about 1×10 6 cells~about 5×10 8 Cells, about 1×10 6 cells~about 6×10 8 Cells, about 1×10 6 cells~about 7×10 8 Cells, about 1×10 6 cells~about 8×10 8 Cells, about 1×10 6 cells~about 9×10 8 Cells, about 1×10 6 cells~about 1×10 9 Cells, about 1×10 6 cells~about 2×10 9 Cells, about 1×10 6 cells~about 3×10 9 Cells, about 1×10 6 cells~about 4×109 cells, about 1×10 6 cells~about 5×10 9 cells, about 1×10 6 cells~about 6×10 9 cells, about 1×10 6 cells~about 7×10 9 cells, about 1×10 6 cells~about 8×10 9 cells, about 1×10 6 cells~about 9×10 9 cells, about 1×10 7 cells~about 1×10 9 cells, about 1×10 7 cells~about 2×10 9 cells, about 1×10 7 cells~about 3×10 9 cells, about 1×10 7 cells~about 4×10 9 cells, about 1×10 7 cells~about 5×10 9 cells, about 1×10 7 cells~about 6×10 9 cells, about 1×10 7 cells~about 7×10 9 cells, about 1×10 7 cells~about 8×10 9 cells, about 1×10 7 cells~about 9×10 9 cells, about 1×10 8 cells~about 1×10 9 cells, about 1×10 8 cells~about 2×10 9 cells, about 1×10 8 cells~about 3×10 9 cells, about 1×10 8 cells~about 4×10 9 cells, about 1×10 8 cells~about 5×10 9 cells, about 1×10 8 cells~about 6×10 9 cells, about 1×10 8 cells~about 7×10 9 cells, about 1×10 8 cells~about 8×10 9 cells, about 1×10 8 cells~about 9×10 9 cells, or about 1×10 8Cells ~ about 1×10 10 can be cells.

[0249] H. Storage

[0250] In some embodiments, one or more populations of engineered host cells, non-engineered cells, and / or mixtures thereof of the invention may be formulated in a freezing medium and stored in a cryopreservation unit such as a liquid nitrogen freezer (-195°C) or an ultra-low temperature freezer (-65°C, -80°C, or -120°C) for long-term storage for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freezing medium may contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate and / or hydroxyethyl starch (HES) along with 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 as referred to herein. Cryopreserved cells can be thawed and genetically modified with viral vectors (e.g., retroviral vectors and lentiviral vectors) or non-viral means (e.g., RNA, DNA, and proteins) as described herein. Alternatively, the host cells described herein can be optionally grown, genetically modified, and then cryopreserved by the methods described herein.

[0251] Thus, the genetically engineered cells and / or non-genetically engineered cells disclosed herein can be at least about 1, 5, 10, 100, 150, 200, 500 vials per mL in a freezing medium, at least about 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、108 and 10 9 or at least about 10 10 of cells can be cryopreserved to generate a cell bank. The cryopreserved cell bank can retain functionality and can be thawed and optionally activated / stimulated and / or expanded. In some embodiments, the thawed cells can be stimulated and expanded in a suitable sealed container such as a cell culture bag and / or a bioreactor to generate large amounts of cells as an allogeneic cell product. In other examples, the cryopreserved cells include an autologous cell product. The cryopreserved cells can maintain biological function for 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 cryopreservation conditions. In some embodiments, preservatives are not used in the formulation. In some embodiments, the cryopreserved cells can be thawed and injected into multiple patients as an off-the-shelf allogeneic cell product. EXAMPLE

[0252] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention and are not intended to limit the scope that the inventors regard as their invention. Efforts have been made to ensure the accuracy with respect to the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0253] Example 1. Construction of the DAP10 construct

[0254] DAP10 CAD constructs were constructed with 0 to 4 elements selected from the Y86F mutation, K84R mutation, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. In the construction, the pSIN vector (Hariharan, MJ et al., (1998) Journal of Virology 72(2):950-958) was used. Briefly, the backbone of the pSIN vector was fully synthesized by Genewiz® (South Plainfield, NJ) from the sequences provided by EUFETS (Germany), a subsidiary of BioNTech (Germany). The basic plasmid used in all constructs is called pL077 pRetroSIN-GFP, which has a green fluorescent protein (GFP) cassette that is replaced by the gene of interest. Various constructs are shown in Table 1 below. For the following constructs, FP2A refers to the Furin and P2A linker genes, and CD19t refers to the truncated CD19 marker. The sequence numbers correspond to amino acid sequences. Tables 4 to 19 below annotate the nucleic acid sequences encoding the amino acid sequences of the constructs shown in Table 1 and the selected sequences in Table 3. Table 1 shows DAP10 constructs containing CD19t, but other variants, such as similar DAP10 constructs incorporating EGFRt, are also within the scope of the present disclosure. Examples of such sequences are presented in Table 3.

Table 1

[0255] Example 2. Tumor control of various DAP10 CAD constructs in the PLC / PRF / 5 assay

[0256] The various DAP10 CAD constructs of Table 1 above were tested in a PLC / PRF / 5 cell lysis assay. Figure 2A shows that DAP10.0 (DAP10 wild type) and DAP10.13 (DAP10-K84R) do not show good tumor control in the PLC / PRF / 5 assay. Figure 2B shows some tumor control by the DAP10.3 (DAP10-CD3ζ) and DAP10.14 (DAP10-CD3ζ-K84R) constructs. Figure 2C shows that the DAP10 CAD construct containing both the 4-1BB co-stimulatory domain and the CD3ζ signaling domain shows good tumor control when there are no additional mutations (i.e., DAP10.4), and adding only the Y86F mutation (i.e., DAP10.5) or only the K84R mutation (i.e., DAP10.15) does not significantly improve tumor control over the DAP10.4 construct. However, the DAP10 CAD construct DAP10.6, which incorporates the CD3ζ signaling domain, the 4-1BB co-stimulatory domain, and both mutations (i.e., Y86F and K84R), showed a significant improvement in tumor control (Figure 2C). Figures 2A-2C also showed plots indicating the tumor control tested with the positive control CAR and the cytotoxicity index of tumor cells alone, respectively.

[0257] The proliferation of Vδ1 γδ cells was also tested under the test conditions corresponding to Figures 2A-2C, and the results are shown in Figures 2D-2F, respectively. As shown, good proliferation was observed with most of the DAP10 CAD constructs.

[0258] Example 3. Tumor control of various DAP10 CAD constructs in the HepG2 assay

[0259] The various DAP10 CAD constructs in Table 1 above were tested in the HepG2 assay. Figure 3A shows data obtained using the DAP10.0 (DAP10 wild type) and DAP10.13 (DAP10-K84R) constructs, Figure 3B shows data obtained using the DAP10.3 (DAP10-CD3ζ) and DAP10.14 (DAP10-CD3ζ-K84R) constructs, and Figure 3C shows data obtained using the DAP10.4, DAP10.5, DAP10.6, and DAP10.15 constructs. Figures 3A-3C each also showed plots indicating tumor control tested with the positive control CAR and the cytotoxicity index of tumor cells alone.

[0260] The proliferation of Vδ1γδ cells was also tested under the test conditions corresponding to Figures 3A-3C, and the results are shown in Figures 3D-3F, respectively. In the Hep2G assay, good proliferation and cytotoxicity were observed with constructs containing both the CD3ζ domain and the 4-1BB domain, as well as at least the Y86F mutation (i.e., DAP10.15 and DAP10.6), which indicates that the DAP10.6 construct (containing CD3ζ, 4-1BB, Y86F, and K84R, respectively) showed the best performance with respect to cytotoxicity and Vδ1γδ cell proliferation.

[0261] Example 4. Survival of Vδ1 cells transduced with DAP10 constructs after tumor co-culture

[0262] In this example, the survival of Vδ1 cells transduced with various DAP10 constructs in Table 1 was evaluated. Specifically, survival was evaluated 5 days after co-culture with PLC / PRF / 5. As shown in Figure 4, the cells transduced with DAP10.6 showed the highest Vδ1 survival after 5 days of co-culture with PLC / PRF / 5.

[0263] Example 5. In vivo tumor control of DAP10 CAD constructs in a mouse model

[0264] In this example, the DAP10 CAD constructs (DAP10.6 and DAP10.15, see Table 1) were tested for their effectiveness in controlling the growth of PLC / PRF / 5 tumors in a mouse model. As shown in Figure 5A, in mice with only tumors (Group A), the average tumor volume increased steadily over 35 days. Similar results were observed in mice injected with Vδ1T cells carrying tumors (untransduced, Group B). When mice were injected with Vδ1T cells transduced with DAP10.6 (Group C) or DAP10.15 (Group D), tumor growth was significantly reduced, and the most potent effectiveness regarding tumor control was observed for DAP10.6 CAD. Figure 5B shows the data obtained in Figure 5A on day 35, analyzed by the Kruskal-Wallis test with Dunn's multiple comparisons and plotted as a function of tumor volume as shown. In each of Groups A - D in Figures 5A and 5B, N = 5, and the number of tumor cells administered was 4e 6 Similar results were observed in mice injected with Vδ1T cells carrying tumors (untransduced, Group B). When mice were injected with Vδ1T cells transduced with DAP10.6 (Group C) or DAP10.15 (Group D), tumor growth was significantly reduced, and the most potent effectiveness regarding tumor control was observed for DAP10.6 CAD. Figure 5B shows the data obtained in Figure 5A on day 35, analyzed by the Kruskal-Wallis test with Dunn's multiple comparisons and plotted as a function of tumor volume as shown. In each of Groups A - D in Figures 5A and 5B, N = 5, and the number of tumor cells administered was 6 as follows.

[0265] Example 6. Expression of NKG2D in Vδ1T cells transduced with DAP10 CAD

[0266] This example shows an increase in NKG2D expression levels in cells transfected with a specific DAP10 CAD (e.g., DAP10.6, see Table 1). Briefly, Vδ1 T cells were transfected with one of DAP10.6, DAP10.13, DAP10.15, DAP10.5, and a control CAR, and then the transfected cells were co-cultured with PLC cells. The expression level of Vδ1 NKG2D was evaluated using a fluorescent label for NKG2D in combination with FACS analysis. In Figure 6, the expression levels of cells containing different DAP10 CADs or CAR controls are plotted as geometric mean fluorescence intensity (gMFI), which shows that the DAP10.6 CAD construct expressed in Vδ1 T cells results in a significantly higher NKG2D expression level compared to other DAP10 CADs (10.13, 10.15, 10.5) and the CAR control. In particular, the DAP10.6 CAD construct contains the K84R mutation, the Y86F mutation, the 4-1BB co-stimulatory domain, and the CD3ζ signaling domain, respectively. In DAP10.15 (K84R + 4-1BB + CD3ζ) and DAP10.5 (Y86F + 4-1BB + CD3ζ), although there was less NKG2D expression exceeding the CAR control, a further statistically significant increase was observed.

[0267] Example 7. DAP10 CAD Expression

[0268] This example demonstrates that the expression of DAP10 CAD is similar among different lots of Vδ1 cells.

[0269] The DAP10.6.3 construct (SEQ ID NO: 77) includes, for example, a truncated form of EGFR as a marker, compared to the DAP10.6 construct (SEQ ID NO: 29) that includes CD19. The DAP10 construct herein referred to as DAP10.16 (SEQ ID NO: 96) is the same as the DAP10.6.3 construct but includes a 1XX mutation in the CD3ζ signaling domain. Vδ1 cells transfected with the DAP10.6 construct or the DAP10.16 construct were found to exhibit substantially similar expression levels. The CAD protein was directly detected by Western blot analysis using anti-DAP10 antibody and anti-CD3ζ antibody (n = 2 donors) (Figure 7).

[0270] Example 8. The cytotoxic activity of DAP10 CAD is mediated by NKG2D

[0271] This example shows that blocking NKG2D abolishes the cytotoxic activity. Specifically, blocking NKG2D via the use of an NKG2D-blocking antibody abrogated the cytotoxic activity of Vδ1 cells expressing DAP10 CAD in three donors across two cell lines (Figures 8A - 8B). The cytotoxic activity of control Vδ1 cells transfected with a CAR was not affected (Figures 8C - 8D). In Figures 8A, 8C, PLC target cells (E:T ratio 5:1) were used. In Figures 8B, 8D, HL60 target cells (E:T ratio 2.5:1) were used. The NKG2D inhibitory antibody also had no effect on cells expressing the DAP10.0 construct (i.e., lacking the costimulatory domain and the intracellular signaling domain) (data not shown).

[0272] Example 9. DAP10 CAD molecular activation signature

[0273] This example shows a consistent DAP10 CAD activation signature across multiple donors and cell lines. In this example, to evaluate the molecular activation signature, after stimulation, Nanostring analysis (Nanostring Technologies, Seattle, WA) was performed. The cell lines used for stimulation included PLC(HCC), HL60, THP1 (AML), and HCT15 (CRC). The data show a consistent activation signature that is dosed via interferon gamma, 4-1BB, and granzyme B. Figure 9A shows data for DAP10.6 versus the natural control (DAP10.0), and Figure 9B shows data for DAP10.16 versus the natural control (DAP10.0). No consistently detectable differences were observed between DAP10.6 and DAP10.16 (Figure 9C).

[0274] Example 10. Broad anti-cancer activity of Vδ1 cells transduced with DAP10 CAD

[0275] This example demonstrates that Vδ1 cells transduced with the disclosed DAP10 CAD exhibit anti-cancer activity against various cancer types with broad NKG2D ligand expression levels / patterns.

[0276] Figures 10A - 10E are graphs showing % cytotoxic activity as a function of the E:T ratio for Vδ1 cells transduced with DAP10.0 compared to Vδ1 cells transduced with DAP10.6 or untransduced cells in an 18 - hour assay. The target cell lines included HCT116 (Figure 10A), SKMEL5 (Figure 10B), MinoD2 (Figure 10C), ScaBER (Figure 10D), and RajiB4 (Figure 10E). Figures 10F - 10G are graphs showing the cytotoxicity rates of Vδ1 cells transduced with DAP10.6 compared to natural controls (Vδ1 cells transduced with DAP10.0) or irrelevant CAR controls in an 18 - hour assay. The target cell lines for Figures 10F - 10G were NCI - H1581 and NCI - H2172, respectively. As shown, the cytotoxic efficacy was significantly increased compared to the controls. Figure 10H shows that the selected cell lines used in the assay represent a wide range of NKG2D ligand expression levels / patterns. To obtain the data in Figure 10H, various cancer cell lines from different hematological malignancies and solid tumors were evaluated for NKG2D ligands by flow cytometry. The five antibodies used for staining detected MICA / MICB, ULBP1, ULBP2 / 5 / 6, ULBP3, and ULBP4. The data are presented as the fold change in the mean fluorescence intensity (MFI) of NKG2D ligands relative to the relevant isotype control. The raw data are shown in Figures 10I - 10J (the cancer cell lines were stained 3 times).

[0277] Figures 11A - 11G are graphs showing the cytotoxicity index as a function of the co - culture time of the target and effector cells. The target cells included 22Rv1, Mino, HCT116, and HCT - 15. The effector cells included DAP10 CAR, Vδ1 cells transduced with a CAR control, or non - transduced Vδ1 cells. When Vδ1 cells were transduced with the DAP10 CAD of the present disclosure, an enhancement of cytotoxicity was observed compared to the control. The degree of cytotoxicity was found to depend on the cell line and the E:T ratio. The cell lines tested represent various levels / patterns of NKG2D ligand expression (see Figures 10H - 10J). Effector cells were co - cultured with NucRed - expressing target cells at an E:T ratio below the maximum of 5:1 or 1.5:1, depending on the cell line. The cytotoxicity index was calculated by dividing the total area (mm 2 / well) of NucRed objects at each time point by the value at time = 0.

[0278] Example 11. Equivalent cytotoxic activity across multiple lots of Vδ1 cells transduced with DAP10 CAD

[0279] This example shows that the cytotoxic activity of Vδ1 cells transduced with DAP10 CAD is equivalent for different lots of Vδ1 cells and DAP10 CAD. Figure 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, a DAP10 reference lot (i.e., a positive control batch of proliferating Vδ1 cells), and DAP10.0 (control). Data for PLC / PRF / 5 cells alone (i.e., without co - culture with Vδ1 cells) are also shown. Figure 12B is a graph showing the percentage reduction in cytotoxicity of tumors alone compared to tumors treated with Vδ1 cells transduced with DAP10 CAD, using the final time point of the 120 - hour assay.

[0280] The cytotoxicity of the DAP10.6 vs. DAP10.16 vs. DAP10.17 constructs incorporated into Vδ1 cells was tested in a 120-hour cytotoxicity assay. Using PLC / PRF / 5 target cells, Vδ1 cells from three donors were tested with the DAP10 constructs. Each construct shown in Figure 12C is the aggregation of all three donors. In this assay using a stringent E:T ratio, the DAP10.6 construct showed slightly superior mean cytotoxicity compared to DAP10.16 and DAP10.17.

[0281] The efficacy of DAP10.6 vs. DAP10.16 vs. DAP10.17 showed some donor dependence. The cytotoxicity index was measured in co-culture experiments with Vδ1 cells from three different donors (SCT06, SCT29, SCT46) transduced with DAP10.6, DAP10.16, or DAP10.17, or untransduced cells from the same donor. The target cells for the co-culture experiments were PLC / PRF / 5 cells. The co-culture time was 120 hours. As shown in Figures 12D - 12E, DAP10.6 and DAP10.16 showed very similar profiles. DAP10.17 was found to be the most donor-dependent (Figure 12F). A reference lot of Vδ1 cells transduced with DAP10.6 was used in all assays and served as a reproducible control (data not shown).

[0282] Example 12. Cytokine Profile Corresponding to DAP10 CAD Stimulation

[0283] This example shows that DAP10 CAD stimulation results in a multifunctional cytokine profile.

[0284] Figure 13A shows the cytokine profile as a function of different DAP10 constructs of the present disclosure. As shown in the figure, the degree of cytokine activation was cell line-specific. The cell lines tested included only PLC, Mino, and T cells. Importantly, potentially problematic cytokines (e.g., IL-6 and IL-17) were not detected. Figure 13B is a graph showing interferon gamma induction (pg / ml / 1E 6 CAD+ cells) in PLC, Mino, and T cells alone for various DAP10 CADs (from various donors, e.g., SCT06, SCT46). Figures 13C - 13F are representative experiments showing interferon gamma secretion from CAD+Vδ1 cells alone and after approximately 18 hours of co-culture with PLC / PRF / 5, HL60, THP1, and PC3 target cells at a near-maximal E:T ratio.

[0285] Example 13. Cytokine Profile of DAP10 CAD Compared to Chimeric Antigen Receptor (CAR)

[0286] This example shows a high similarity in the cytokine profile from the DAP10 CAD of the present disclosure compared to CAR (Figure 14). The conditions examined included Vδ1 cells + PLC cells transduced with DAP10 CAD or Vδ1 cells alone transduced with DAP10 CAD (i.e., without target cells), and Vδ1 cells + target cells (HepG2, PLC, Raji) transduced with CAR or Vδ1 cells alone transduced with CAR (i.e., without 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.

[0287] Example 14. DAP10 CAD Stimulation Promotes Proliferation Among Multiple Donors

[0288] This example shows that DAP10 CAD stimulation promotes the proliferation of Vδ1 cells in all donors tested. Specifically, in this example, after transduction with the DAP10 CAD constructs (DAP10.6, DAP10.16, DAP10.17) of the present disclosure, the proliferation of Vδ1 cells was evaluated by co-culture with PLC / PRF / 5 (E:T ratio 5:1). The control included Vδ1 cells transduced with a DAP10 control batch (or DAP10.0). The low / slow proliferation of Vδ1 cells transduced with DAP10.16 may be the result of the 1XX CD3ζ signaling domain. In this regard, enhanced regulation of activation / proliferation may be beneficial for the long-term effectiveness / survival of engineered Vδ1 cells by reducing overstimulation / exhaustion.

[0289] In the co-culture experiment, Vδ1 cells obtained from two different donors (SCT29 and SCT46) were used. As shown in Figure 15, robust proliferation of Vδ1 cells transduced with DAP10.6 and DAP10.16 was observed for both donors, while the proliferation of Vδ1 cells transduced with DAP10.17 was somewhat donor-dependent.

[0290] Example 15. In Vivo Tumor Control by Vδ1 Cells Transduced with DAP10 CAD Containing the 1XX CD3ζ Intracellular Signaling Domain

[0291] This example shows that incorporating the 1XX CD3ζ intracellular signaling domain can improve tumor control in vivo.

[0292] In this example, the DAP10 CAD constructs DAP10.6 and DAP10.16 were tested for their effectiveness in modulating PLC / PRF / 5 tumors grown in a mouse model. As shown in Figure 16A, tumor control in mice treated with Vδ1 cells transduced with DAP10.16 CAD (containing a 1XX mutation in the CD3ζ intracellular signaling domain) was improved when directly compared to mice treated with Vδ1 cells transduced with DAP10.6 CAD (p = 0.0079, Mann-Whitney test (two-sided test)). A schematic diagram of the experimental procedure used in this example is shown in Figure 16B.

[0293] Example 16. Comparison of in vivo anti-tumor activities of DAP10 CAD Vδ1 cells and CAR Vδ1 cells

[0294] This example shows that DAP10 CAD + Vδ1 cells exhibit anti-tumor activity in a similar kinetics as CAR Vδ1 cells.

[0295] In an HCT-15 mouse xenograft model, the in vivo tumor growth kinetics of DAP10 CAD + Vδ1 cells (5e 6 cells / dose and 15e 6 cells / dose) compared to control CAR Vδ1 cells and tumor alone conditions are shown in Figure 17A. The tumor volumes quantified on day 27 are shown in Figure 17B. A schematic diagram of the experimental procedure used in this example is shown in Figure 17C. Data are shown as mean ± SEM of 5 mice / group. To evaluate the final statistical significance between the complete cohorts of each treatment, the Kruskal-Wallis test with Dunn's multiple comparison was used (ns = not significant).

[0296] Example 17. In vivo proliferation, persistence, and targeting of engineered Vδ1 cells

[0297] This example shows that Vδ1 cells transduced with DAP10 CAD of the present disclosure grow in tumors but not in other organs in a mouse model in vivo. In this example, two separate studies were conducted. As shown in FIG. 18A, the growth of Vδ1 cells transduced with DAP10.6 was observed in tumor tissue (subcutaneous PLC / PRF / 5 cells) on day 7 after treatment, but not in the spleen, lung, liver, bone marrow, or blood. The Vδ1 cells used in Study 1 were obtained from a different donor than the Vδ1 cells used in Study 2. In FIG. 18A, the HuCD45+, Vδ1+ population is shown. FIG. 18B is a graph quantifying engineered Vδ1 cells per mg of tumor tissue in each study at day 4, 7, and 14 (Study 1), and at day 7 and 14 (Study 2). In each study, 5e 6 engineered Vδ1 cells were used. Notably, an increase in total Vδ1 cells within the tumor was observed throughout each study. FIG. 18C is a graph showing the quantification of Vδ1 cells in tumor tissue or other tissues (lung, liver, spleen, bone marrow, blood) collected at 4, 7, or 14 days after treatment as evaluated by flow cytometry, representing a cumulative analysis across two independent studies presented in this example. FIG. 18D is a schematic diagram showing the experimental procedure corresponding to this example for reference. No significant change in body weight or acute clinical signs of toxicity or graft-versus-host disease (GvHD) were observed in the treated mice (FIG. 19). The data shown in FIG. 19 relate to an independent efficacy study with n = 4.

[0298] The Vδ1 cells transduced with DAP10 CAD of the present disclosure were found to efficiently target tumor cells while protecting normal cells (i.e., non-tumor cells). Specifically, a short-term cytotoxicity assay was adapted to an annexin / DAPI flow-based method for the analysis of primary cell targets. Figure 20A is a graph showing that both Vδ1 cells transduced with DAP10 CAD (DAP10.6, DAP10.16) of the present disclosure and Vδ1 cells transduced with NKG2D CAR significantly reduced the viability of THP1 cells compared to Vδ1 cells transduced with THP1 alone and a natural control (DAP10.0). Figure 20B is a graph showing that Vδ1 cells transduced with DAP10 CAD (DAP10.6, DAP10.16) of the present disclosure do not substantially target healthy PBMCs, similar to the natural control (Vδ1 cells transduced with DAP10.0). Notably, Vδ1 cells transduced with DAP10 CAD consistently showed lower PBMC targeting than the NKG2D CAR reference (Figure 20B).

[0299] Example 18. Small-Scale Donor Screening

[0300] This example shows that the proliferation of Vδ1 cells transduced with DAP10 CAD containing a CD3ζ 1XX modification can be improved compared to similar constructs without 1XX in the CD3ζ intracellular signaling domain.

[0301] In this example, Vδ1 cells from six donors (SE001, ARC007, HC45, DLS003, SE015, and SCT029) were tested in two experiments of small-scale shake flask proliferation. Specifically, Vδ1 cells from different donors were transduced with either DAP10.6 or DAP10.16. Across all donors, 50 - 135% more Vδ1 cells were measured from cells transduced with DAP10.16 compared to cells transduced with DAP10.6 (Figure 21). Proliferation was measured on days 14 and 15.

[0302] Example 19. Growth Rate of DAP10 CAD Vδ1 Cells

[0303] This example shows a clear shift to a high Vδ1 cell rate (%) in cultures transduced with lead DAP10 CAD constructs (DAP10.6, DAP10.16, DAP10.17) compared to the controls (DAP10.0, CAR control).

[0304] In this example, Vδ1 cells from three different donors (SCT06, Figure 22A, SCT29, Figure 22B, SCT45, Figure 22C) were transduced with the lead DAP10 constructs or controls, and the %Vδ1 of total cells was grown as a function of the growth time. As shown in each of Figures 22A - 22C, a clear shift to a higher Vδ1 percentage was observed with the lead DAP10 constructs compared to the controls.

[0305] Example 20. Proliferation of cells transduced with DAP10 CAD constructs

[0306] This example shows that Vδ1 cells can be efficiently grown and transduced with the DAP10 CAD of the present disclosure. Figure 23A shows a schematic diagram representing the process of generating "off - the - shelf" allogeneic CAD Vδ1 cells. Data showing independent expansions of lead DAP10 CAD constructs (DAP10.6, DAP10.16, DAP10.17) transduced into Vδ1 cells obtained from three different donors (SCT06, SCT29, SCT45) are shown in Figures 23B, 23C, and 23D respectively. Independent proliferation (blue vs. red) shows a similar trend in the growth profiles of the DAP10 CAD constructs. Growth was found to be construct - dependent in two out of the three donors.

[0307] Representative experiments show that ex vivo culture of Vδ1 cells results in significant expansion (Figure 23E) and robust transduction of DAP10 CAD into Vδ1 cells (Figure 23F). The data in Figures 23E - 23F were obtained from 12 independent cultures using PBMCs from 7 different donors. Shown in Figure 23G are a series of graphs showing the cell composition (Vδ1 cells, Vδ2 cells, αβ cells, NK cells) over time (day 0, before αβ T cell depletion, and after αβ T cell depletion), represented as a percentage of the culture.

[0308] Example 21. Constructs for Co - expression of CAR - CAD

[0309] Vδ1 cells co - expressing CAR and DAP10 CAD were generated using the constructs in Table 2 below. Briefly, the CAR - DAP10 CAD constructs were built with 0 - 4 elements selected from the Y86F mutation, K84R mutation, 4 - 1BB co - stimulatory domain, and CD3ζ signaling domain. pSIN was used for construction. Briefly, the backbone of the pSIN vector was fully synthesized by Genewiz® (South Plainfield, NJ) from sequences provided by EUFETS (Germany), a subsidiary of BioNTech (Germany). The basic plasmid used in all constructs is called pL077 pRetroSIN - GFP, which has a green fluorescent protein (GFP) cassette that is replaced by the gene of interest. Various constructs are shown in Table 2 below. For the following constructs, 3H7 - 5.1 refers to a CAR that binds to CD20 (see, for example, Nishimoto et al. Clin. Transl. Immunol. 2022;11(2):e1373), and P2A refers to the P2A cleavage sequence.

Table 2

[0310] Vδ1γδ cells expressing the CAR-Dap10 construct were examined in an Incucyte-based restimulation assay using the Raji, Mino, and MOLP-8 cell lines. In this assay, the Raji and Mino cell lines expressed the CAR target, while the MOLP-8 cell line did not. Figures 24A - 24C show the results for the Raji, Mino, and MOLP8 cell lines, respectively. Vδ1 cells transduced with the CAR-Dap10 construct showed sustained cytotoxic activity against the target, regardless of the presence or absence of the CAR antigen, compared to untransduced controls.

[0311] Upon reading the foregoing description, certain modifications and improvements will occur to those skilled in the art. For the sake of brevity and readability, all such modifications and improvements, although omitted herein, are to be properly included within the scope of the following claims.

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

Table 3-39

Table 3-40

Table 3-41

Table 3-42

Table 3-43

Table 3-44

Table 3-45

Table 3-46

Table 3-47

Table 3-48

Table 3-49

Table 3-50

Table 3-51

Table 3-52

Table 3-53

Table 3-54

Table 3-55

Table 3-56

Table 3-57

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Claims

1. A mammalian cell comprising a chimeric adapter (CAD) polypeptide comprising a DAP10 domain comprising a human DAP10 amino acid sequence and at least one of 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 chimeric receptor comprises at least one of an intracellular signaling domain and / or a costimulatory domain, and if the chimeric receptor comprises an intracellular signaling domain, the CAD polypeptide may also comprise a costimulatory domain, and vice versa; and / or, The mammalian cell according to claim 1, wherein the chimeric receptor comprises at least one DAP10-interaction domain, and the DAP10-interaction domain may comprise the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity with respect to SEQ ID NO:

75.

3. The mammalian cell according to claim 1, 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.

4. The CAD polypeptide contains at least one costimulatory domain 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, or The CAD polypeptide has at least one co-stimulatory domain that is 4-1BB or CD28. The mammalian cell according to claim 1.

5. The CAD polypeptide comprises at least one intracellular signaling domain selected from CD3ζ, DAP12, LFA-1, and repeat (2-5) DAP10 YINM motifs, or The at least one intracellular signaling domain of the CAD polypeptide is CD3ζ, and CD3ζ may have the amino acid sequence shown as SEQ ID NO: 82, and / or The CAD polypeptide has at least one co-stimulatory domain that is 4-1BB, and the CAD polypeptide has at least one intracellular signaling domain that is CD3ζ. The mammalian cell according to claim 1.

6. The mammalian cell according to claim 5, wherein the CAD polypeptide comprises, from the N-terminus to the C-terminus, the DAP10 domain, the 4-1BB costimulatory domain, and the CD3ζ intracellular signaling domain.

7. The CAD polypeptide comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain. The CAD polypeptide may comprise, from the N-terminus to the C-terminus, the DAP10 domain, the 4-1BB costimulatory domain, followed by the CD28 costimulatory domain, and further, the CD3ζ intracellular signaling domain, wherein CD3ζ may have the amino acid sequence shown in SEQ ID NO: 82; or, The CAD polypeptide may comprise, from the N-terminus to the C-terminus, the DAP10 domain, the CD28 costimulatory domain, the subsequent 4-1BB costimulatory domain, and further the CD3ζ signaling domain, and CD3ζ may have the amino acid sequence shown as SEQ ID NO:

82. The mammalian cell according to claim 4.

8. The amino acid sequence of the aforementioned human DAP10 is (i) Sequence identity of at least 90%, 95%, 97%, or 99% with respect to sequence number 1; (ii) Sequence identity of at least 90%, 95%, 97%, or 99% with respect to sequence number 78; or (iii) Sequence identity of at least 90%, 95%, 97%, or 99% for sequence number 81 It includes an amino acid sequence having; and / or, The amino acid sequence of the human DAP10 includes the amino acid sequence of the mutated human DAP10. The mammalian cell according to claim 1.

9. The mutated human DAP10 amino acid sequence includes amino acid substitutions at the positions corresponding to K84 and / or Y86. The amino acid substitution at the K84 position may include a K84R substitution, and / or, The amino acid substitution at Y86 may include the Y86F substitution. The mammalian cell according to claim 8.

10. The CAD polypeptide is encoded by an isolated nucleic acid to which an adjustable promoter is operably linked. The chimeric receptor is encoded by an isolated nucleic acid which is operably linked to a moduloable promoter, and The isolated nucleic acid encoding the CAD polypeptide encodes a cytokine, and / or the isolated nucleic acid encoding the chimeric receptor encodes a cytokine. The cytokine may be selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, and IL-23. The mammalian cell according to claim 1.

11. The present invention further comprises at least one receptor that associates with DAP10, wherein the at least one receptor is not the at least one chimeric receptor. The at least one of the receptors may be exogenous. The mammalian cell according to claim 1.

12. The exogenous receptor is overexpressed; and / or, The at least one exogenous receptor is selected from NKG2D, Ly49H, Ly49D, Sirp-b1, Siglec-15, and Cd300lb, preferably the at least one receptor is NKG2D. The mammalian cell according to claim 11.

13. a) The mammalian cells are immune cells, preferably cytotoxic cells; b) The mammalian cells exhibit in vitro and / or in vivo killing activity against the target cells that express the target antigen on their cell surface, i) The target cells may be hematological tumor cells or solid tumor cells, and / or ii) The in vitro and / or in vivo killing activity may be higher than the level of in vitro and / or in vivo killing activity inherent in control mammalian cells lacking expression of one or both of the chimeric receptor and / or the CAD polypeptide; c) The mammalian cells proliferate in response to contact with the target cells; and / or, The mammalian cells exhibit increased proliferation in response to contact with the target cells, compared to control mammalian cells lacking expression of one or both of the chimeric receptor and / or the CAD polypeptide. The mammalian cells may proliferate in a host organism containing the target cells, and / or, d) The mammalian cells express pro-inflammatory cytokines in response to contact with the target cells, and the pro-inflammatory cytokines may include tumor necrosis factor alpha or interferon gamma. The mammalian cell according to claim 1.

14. A plurality of mammalian cells according to any one of claims 1 to 13, A plurality of mammalian cells, wherein the plurality of mammalian cells may include at least about 10⁶ cells, at least 10⁷ cells, or at least 10⁸ cells, preferably about 10⁸ to 10¹¹ cells.

15. Mammalian cells according to any one of claims 1 to 13, or A plurality of mammalian cells according to any one of claims 1 to 13 A method for producing a CAD polypeptide, comprising transtransferring a mammalian cell(s) with a construct comprising an isolated nucleic acid encoding a CAD polypeptide and at least one construct encoding at least one chimeric receptor, The method described above may include retroviral transduction, and / or, The method may include the ex vivo proliferation of the mammalian cells(s), and the ex vivo proliferation may be carried out before and / or after the translocation of the isolated nucleic acid encoding the CAD polypeptide and at least one construct encoding at least one chimeric receptor. method.

16. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a mammalian cell according to any one of claims 1 to 13, or a plurality of mammalian cells according to any one of claims 1 to 13.

17. A method for activating a mammalian cell according to any one of claims 1 to 13, or a plurality of mammalian cells according to any one of claims 1 to 13, the method comprising bringing a target cell into contact with the mammalian cell.

18. The method according to claim 17, wherein the mammalian cells, or the plurality of mammalian cells, are introduced into a target requiring introduction, and the activation occurs in the target.

19. (i) Mammalian cell according to any one of claims 1 to 13, (ii) A plurality of mammalian cells according to any one of claims 1 to 13, or (iii) A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a mammalian cell according to any one of claims 1 to 13, or a plurality of mammalian cells according to any one of claims 1 to 13. of, Use in the preparation of pharmaceuticals for treating a subject having a certain condition, The use wherein the mammalian cells or the plurality of mammalian cells alleviate at least one symptom or sign of the condition in the subject.

20. (i) Mammalian cells according to any one of claims 1 to 13, or (ii) A plurality of mammalian cells according to any one of claims 1 to 13, or (iii) A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a mammalian cell according to any one of claims 1 to 13, or a plurality of mammalian cells according to any one of claims 1 to 13. The use of an effective amount for killing tumor cells in the preparation of a pharmaceutical for treating cancer in a subject requiring cancer treatment.

21. A medicine used to kill tumor cells, (i) Mammalian cells according to any one of claims 1 to 13, or (ii) A plurality of mammalian cells according to any one of claims 1 to 13, or (iii) A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a mammalian cell according to any one of claims 1 to 13, or a plurality of mammalian cells according to any one of claims 1 to 13, A pharmaceutical product comprising a therapeutically effective amount of the mammalian cells (or any number of cells) or the pharmaceutical composition being introduced into a host organism containing tumor cells.

22. The therapeutically effective amount of the mammalian cells (or more) or the pharmaceutical composition is introduced into the host organism, including the tumor cells. The pharmaceutical product according to claim 21, wherein one or more methods for increasing common gamma chain cytokines are performed simultaneously or sequentially.

23. The pharmaceutical product according to claim 22, wherein the method of increasing common gamma chain cytokines (or more) comprises administering, simultaneously with or subsequently to the introduction of mammalian cells (or more), an amount of common gamma chain cytokines (or more) effective in increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced mammalian cells (or more), preferably the method comprising administering IL-2, and more preferably the method comprising administering IL-15.

24. The pharmaceutical product according to claim 23, wherein the one or more methods for increasing common gamma chain cytokines (or more) include administering an amount of common gamma chain cytokines (or more) effective in increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced mammalian cells (or more) or the pharmaceutical composition before and / or after the introduction of the introduced mammalian cells (or more).

25. One or more methods for increasing common gamma chain cytokines include lymph depletion before the introduction of mammalian cells, and / or The one or more methods for increasing common gamma chain cytokines include the secretion of one or more common gamma chain cytokines from the introduced mammalian cells. The pharmaceutical product according to claim 22.

26. The pharmaceutical agent according to claim 21, wherein the pharmaceutical agent reduces the amount of tumor in vivo in the host organism compared to a control organism, and / or increases the average survival time of the host organism, and the control organism is not treated with the mammalian cells(s) or the pharmaceutical composition.

27. The pharmaceutical product according to claim 21, wherein the pharmaceutical product is for treating cancer in a person in need of treatment.

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