Enhancing immune cell innate and adaptive responses

Abstract

Provided herein is a recombinant polypeptide comprising: the transmembrane domain of FcεR1γ or CD3ζ, or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and / or CD16, a co-stimulatory domain, and one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Cells expressing the same and methods of treatment are also provided.

Description

SF2024-070-2 ENHANCINGIMMUNECELLINNATE ANDADAPTIVERESPONSESCROSS-REFERENCINGThis application claims the benefit of U.S. provisional application serial no. 63 / 565,216, filed on March 14, 2024, which application is incorporated by reference herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE LISTING XML FILE A Sequence Listing is provided herewith as a Sequence Listing XML, “UCSF- 785WO_SEQLIST” created on March 4, 2025, and having a size of 16,455 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. BACKGROUNDNatural killer (NK) cells are highly cytotoxic immune effectors, killing their targets in a non-specific manner. NK cells lack the potential to cause graft-versus-host disease (GVHD) and thus open opportunities to produce an off-the-shelf allogeneic product that could be readily available for immediate clinical use. Thus, the inherent qualities of NK cells make them promising candidates for immunotherapy. Strategies that improve the innate and / or adaptive anti-tumor activity of NK cells and T cells are of great clinical value. SUMMARY Provided herein is a recombinant polypeptide comprising: (i) the transmembrane domain of FcεR1γ or CD3ζ, or a variant of the transmembrane domain that binds to NKp30, NKp46 and / or CD16, (ii) a co-stimulatory domain, and (iii) one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Without wishing to be held to any specific theory, addition of a co-stimulatory domain to an adapter protein selected from FcεR1γ and CD3ζ in NK cells is believed to increase the expression and activation of NKp30, NKp46 (which are natural NK cell activating receptors) and CD16 (which is the Fc receptor and mediates antibody-dependent cellular cytotoxicity (ADCC)) that, in turn, dramatically increases the ability of those cells to kill cancer cells (see, Figs. 3A andSF2024-070-2 3B, for example). These interactions are mediated by the transmembrane domains of these proteins and, as such, are expected to occur in recombinant polypeptides that comprise a transmembrane domain of an adapter protein selected from FcεR1γ and CD3ζ, or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and / or CD16. The FcεR1γ and CD3ζ adapter proteins do not have an extracellular binding domain in their wild-type form and, as such, in several embodiments the recombinant peptide may not have an extracellular binding domain. In these embodiments, the NK cells or T cells may be activated via endogenous signaling pathways (which may increase the expression and activation of the NKp30, NKp46, and / or CD16 receptors). It has also been found that adding an extracellular binding domain to these recombinant polypeptides provides a potent way to activate NK cells or T cells when they bind to a specific antigen on a cancer cell. As such, in some embodiments, the recombinant protein may comprise an extracellular binding domain, e.g., a scFv or nanobody, that recognizes a cancer antigen. While the recombinant polypeptide has been reduced to practice using the transmembrane domain from FcεR1γ, the FcεR1γ ITAM and the co-stimulatory domain from 4- 1BB, variants of the transmembrane domain as well alternative transmembrane domains (such that from CD3ζ), co-stimulatory domains and ITAMs could be used since those domains / motifs are functionally similar. Cells expressing the recombinant polypeptide, methods of killing cancer cells and methods of treatment are also provided. Additional embodiments and other features, advantages and variations may become apparent in view of the description that follows below. BRIEFDESCRIPTION OF THEFIGURESThe skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. FIGS. 1A-1F. FcεR1γ41BBincreases NK cell NKp30 and NKp46 expression. FIG. 1A: Schematic representation of the FcɛR1γ41BB(FCRG41BB) design relative to FcɛR1γ (FCRG) native form. FIG. 1B: FcɛR1γ constructs were overexpressed in the FcɛR1γ-negative CD16- negative human natural killer cell line, NK92. FIG. 1C: Flow-cytometry histograms of FcɛR1γSF2024-070-2 or CD3ζ intracellular protein expression in the transduced NK92 cells (Parental: bottom, FCRG middle, or FCRG41BB: top). FIG. 1D: Flow-cytometry histograms of NKp30 or NKp46 protein surface expression in the transduced NK92 cells (Parental: bottom, FCRG: middle, or FCRG41BB: top). FIG. 1E: Flow-cytometry histograms of NKp30 or NKp46 protein surface expression in the transduced FCRG41BBpositive NK92 cells following FCER1G gene CRISPR-knockout. (Florescent-minus-one [FMO] control: bottom, Parental: second from bottom, FCRG41BB: middle, gRNA-1: second from top, gRNA-2: top). FIG. 1F: Flow-cytometry histograms of the inhibitory receptor NKG2A, the activating receptor NKG2D or NKp44, or the activation markers 41BB or TRAIL surface protein expression between parental (bottom), FCRG (middle), or FCRG41BB(top) NK92 cells. FIGS. 2A-2C. FcεR1γ41BBincreases NK cell NKp30 function. FIG. 2A: Top panels: schematic representation of the pro-B murine cell line, Ba / F3 (left), engineered to express B7H6 (activating NKp30 ligand) (middle) or both B7H6 and HLA-EHLA-G(inhibitory NKG2A ligand) (right). Lower panels: flow-cytometry dot-plot panels of the expression of B7H6 vs. HLA-E for cell lines corresponding to top panels. FIG. 2B: 5 hours lysis assay: NK92 cells were co-cultured with Ba / F3 cells at an effector to target (E:T) ratio of 1:1. (left to right: Ba / F3, Ba / F3-B7H6+, Ba / F3-B7H6+HLA-E+). FIG. 2C: 24 hours lysis assay: NK92 cells were co-cultured with Ba / F3 cells at an effector to target (E:T) ratio of 1:4, 1:8, or 1:16. (left to right: Ba / F3, Ba / F3-B7H6+, Ba / F3-B7H6+HLA-E+). Mean+ / SEM, unpaired t-test, one-tailed, *p < 0.05, **p < 0.001, ***p < 0.001. FIGS. 3A-3D. FcεR1γ41BBincreases NK cell NKp30 function against human cancer cells with lower B7H6 expression. FIG. 3A: Median binding values of NK cell receptors- human Fc fusions to the human cell line, K562. Mean ± SEM. FIG. 3B: Flow-cytometry histograms of B7H6 surface expression in Ba / F3 cells or K562 cells relative to FMO controls. K562 cells express lower B7H6 levels. FIG. 3C: 24 hours lysis assay: parental, FCRG, or FCRG41BBNK92 cells were co-cultured with K562 cells at an effector to target (E:T) ratio of 2:1 to 1:16. FIG. 3D: lysis assay of K562 B7H6-knockout cells by parental, FCRG, or FCRG41BBNK92 cells. Mean ± SEM, unpaired t-test, one-tailed, **p < 0.001, ***p < 0.001. Figs. 4A-4E. FcεR1γ41BBincreases NK cell CD16 expression and function. FIG. 4A:SF2024-070-2 CD16 was overexpressed in the CD16-negative NK92 cell line. Right panel: Flow-cytometry histograms of CD16 surface expression in NK92 cells. FIG. 4B: 5 hours CD107a assay: CD16+ NK92 cells or NK92 cells were stimulated with anti-NKp30, anti-NKp46, anti-CD16, or anti- NKG2D mIgG1 coated beads relative to mIgG1 control. Mean ± SEM, unpaired t-test, one- tailed, ***p < 0.001. FIG. 4C: Flow-cytometry histograms of NKp30 or CD16 in NK92 cells (parental: bottom, CD16+: second from bottom, CD16+ FCRG: second from top, or CD16+ FCRG41BB: top). FIG. 4D: Left panel: 5 hours CD107a assay where CD16+, CD16+ FCRG, or CD16+ FCRG41BBNK92 cells were stimulated with anti-CD16 beads. Right panel: negative control: stimulation of CD16 negative cells with anti-CD16 beads. Mean ± SEM, unpaired t-test, one-tailed, **p < 0.001, ***p < 0.001. FIG. 4E: Flow-cytometry histograms of human CD20 expression in Ba / F3 cells (top left). Schematic representation of lysis assay by ADCC using Rituximab (anti-hCD20, top right). 5 hours lysis assay: NK92 cells were co-cultured with Ba / F3- CD20 cells at an effector to target (E:T) ratio of 1:1 (bottom). Mean ± SEM, unpaired t-test, one- tailed, ***p < 0.001. FIGS. 5A, 5B. CD19ScFv-CD8hingefused to FcεR1γ41BBincreases NKp30 and CD16 expression. FIG. 5A: Schematic representation of CD19ScFvCD8Hingefused to FcεR1γ41BB(FCRG10; X1 ITAM) design relative to FcɛR1γ41BB(FCRG41BB,X1 ITAM) or CD19CAR41BB-CD3ζ(X3 ITAMs) design. A Myc-tag was integrated into the N-terminal of FCRG10 or CD19CAR41BB-CD3ζfor detection of surface expression. FIG. 5B: Top row: flow-cytometry histograms of Myc-tag, NKp30 or CD16 in CD16+ NK92 cells (parental: bottom, CD16+ FCRG41BB: second from bottom, CD16+ FCRG10: second from top, or CD16+ CD19CAR41BB-CD3ζ:top). Bottom row: flow-cytometry dot-plots of NKp30 or CD16 surface expression relative to Myc-tag expression. FIGS. 6A-6F. CD19ScFv-FcεRIγ41BBCAR improves NK cell function against HLA-E+target cells. FIG. 6A: Top row: schematic representation of Ba / F3 cells engineered to co- express human CD19, B7H6 and HLA-E. Bottom row: flow-cytometry dot-plots of B7H6 vs. HLA-E surface expression in Ba / F3 cells. FIG. 6B: Flow-cytometry histograms of human CD19 surface expression in Ba / F3 cells. FIGS. 6C-6F: 5 hours lysis assay: NK92 cells were co- cultured with Ba / F3 cells at an effector to target (E:T) ratio of 1:1. (Ba / F3 FIG. 6C), Ba / F3- CD19+ FIG. 6D), Ba / F3-B7H6+ FIG. 6E), Ba / F3-CD19+B7H6+HLA-E+ FIG. 6F)). Mean ±SF2024-070-2 SEM, unpaired t-test, one-tailed, ***p < 0.001, ****p < 0.0001. FIGS. 7A, 7B. CD19ScFv-FcεRIγ41BBCAR is functional in primary human NK cells. FIG. 7A) Flow-cytometry histograms of CD19ScFv-FcεRIγ41BBCAR surface expression in primary human NK cells. FIG. 7B: 5 hours lysis assay: CD19ScFv-FcεRIγ41BBCAR positive or non-transduced primary human NK cells were co-cultured with CD19+ Ba / F3 cells at an effector to target (E:T) ratio of 1:5. Mean ± SEM, unpaired t-test, one-tailed, **p < 0.01. FIG. 8A illustrates certain exemplary fusion proteins of the present disclosure. FIG. 8B shows sequence alignments of the transmembrane domains of various proteins. DETAILEDDESCRIPTIONBefore the methods and compositions of the present disclosure are described in greater detail, it is to be understood that the methods and compositions are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.SF2024-070-2 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and compositions are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. It is appreciated that certain features of the methods and compositions, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and featuresSF2024-070-2 which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Recombinant polypeptides Provided herein (and illustrated in FIG. 8A) is a recombinant polypeptide comprising: (i) the transmembrane domain of FcεR1γ of CD3ζ, or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and / or CD16, (ii) a co-stimulatory domain, and (iii) one or more immunoreceptor tyrosine-based activation motifs (ITAMs). As would be apparent, the co-stimulatory domain and the one or more immunoreceptor tyrosine-based activation motifs (ITAMs) are intracellular domains and, in these embodiments, may be in either order. The full-length sequence of the wild-type FcεR1γ (including the signal sequence, which is not part of the mature protein) is deposited at Genbank as accession numbers NM_004106.2 and EAW52623.1 (which deposit is incorporated by reference herein). FcεR1γ has a single ITAM. The wild type FcεR1γ transmembrane domain has the following amino acid sequence: LCYILDAILFLYGIVLTLLYCRLK; SEQ ID NO: 1. The full sequence of the wild-type CD3 zeta chain (referred to herein as CD3ζ) is deposited at Genbank as accession number XP_011508446.1 (which deposit is incorporated by reference herein). CD3ζ has three ITAMs. The wild type CD3ζ transmembrane domain has the following amino acid sequence: LCYLLDGILFIYGVILTALFLRVK (SEQ ID NO: 2). While many embodiments of the recombinant polypeptide comprise the wild-type transmembrane domain of FcεR1γ (i.e., SEQ ID NO: 1) or CD3ζ (i.e., SEQ ID NO: 2) in certain instances the recombinant polypeptide may comprise a variant of the wild-type transmembrane domain of FcεR1γ or CD3ζ defined by the following consensus sequence, which was generated by comparing the FcεR1γ, or CD3ζ transmembrane domains (which bind to the same or overlapping natural cytotoxicity receptors or CD16) and to the orthologs from other species (see FIG. 8B): L C Y X L D A / G I L F X Y G X X X T X L X / Y X / C R X K (SEQ ID NO: 3) where X is a hydrophobic amino acid, where the term hydrophobic amino acid in this disclosure refers to an amino acid selected from G, A, V, L, I, P, F, M and W. In SEQ ID NOS: 1 and 2 as well as the consensus sequence, the underlined “D” residueSF2024-070-2 (i.e., aspartic acid) is responsible binding with the opposing positively charged residue in the natural cytotoxicity receptors to which these proteins binds, e.g., NKp30 and NKp46; see Fig. 1 of Kruse et al (Immunology and Cell Biology 201492: 221–229). The underlined “C” residue (i.e., cysteine) forms a disulfide bond and should also potentially be retained, particularly for the formation of disulfide bonds. If the C is not retained, it may be any hydrophobic residue. In some embodiments, a variant sequence may comprise the conserved Cys at position 2, the conserved Asp at position 6, and up to 5 amino acid substitutions (e.g., 1, 2, 3, 4, or 5 substitutions) relative to SEQ ID NO: 1 or 2. For example, since this is a transmembrane domain, up to five of the hydrophobic residues can be substituted with another hydrophobic amino acid. In some embodiments, the recombinant polypeptide may lack an extracellular binding domain and, as such, the mature form of the recombinant polypeptide may have the same N- terminus as a wild-type adapter protein (which also lacks an extracellular binding domain). For example, the protein may have less than 50, less than 20, less than 10 or less than 5 amino acids that are extracellular (N-terminal to the transmembrane domain) and those amino acids do not form an extracellular binding domain. In other embodiments, the recombinant polypeptide may comprise an extracellular binding domain, e.g., a scFv or nanobody, that recognizes a cancer antigen. In these embodiments, the recombinant polypeptide may follow the general design of a chimeric antigen receptor (CAR). CARs can be designed in several ways (see, generally, e.g., Guedan et al, Methods and Clinical Development 201912: 145-156). CARs generally include an extracellular domain that contains an antigen binding domain such as a scFv or nanobody, a hinge, a transmembrane region, an ITAM and a co-stimulatory domain. In some embodiments, the recombinant polypeptide may comprise, in order from N- to C-, the extracellular binding domain, the transmembrane domain, the co-stimulatory domain and the one or more ITAMs. In some embodiments, the co-stimulatory domain may be the co-stimulatory domain of CD28, ICOS, CD27, 4‐1BB, OX40, CD40L, DNAM1 or 2B4 (as reviewed in Weinkove Clin Transl Immunology. 2019; 8: e1049, among others). These domains have been proposed for chimeric antigen receptors and can be readily used herein. In some embodiments, the co- stimulatory domain may be the 4-1BB co-stimulatory domain, as illustrated in the examples section of the present application. In some embodiments the present recombinant polypeptide may contain a single co-stimulatory domain. However, in other embodiments, the recombinantSF2024-070-2 polypeptide may contain multiple co-stimulatory domains (e.g., 2, 3, 4 or 5 co-stimulatory domains). In addition to the co-stimulatory domain(s) or alternatively to the co-stimulatory domain(s), the recombinant polypeptide may contain one or more other signaling domains (e.g., STAT3, STAT4, STAT5 signaling domains), gene regulatory domains such an NFAT regulatory domain, or sites for recruiting signaling proteins protein such as phospholipase C gamma 1 (PLCγ1), TNF receptor-associated factor associated factors (TRAFs), growth factor receptorbound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (GADS), Src homology region 2 domain-containing phosphatase (SHP-1), vav guanine nucleotide exchange factor 1 (Vav1), phosphatidylinositol-3-kinase (PI3K), lymphocyte-specific protein tyrosine kinase (Lck), and Pellino protein. See, e.g., Daniels et al (Science 2022378: 1194-1200) and Miao (Front Immunol. 2021: 12:687822. For example, the recombinant polypeptide could contain a truncated cytoplasmic domain from IL-2Rβ and / or a STAT3-binding tyrosine-X-X- glutamine motif, among many others. The recombinant polypeptide may contain 1, 2, 3, 4, or 5 or more ITAM motifs, wherein an ITAM conforms to the consensus sequence (YX1X2L / I)(X3)n(YX1X2L / I), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid (see, e.g., Chong et al Biochim Biophys Acta Gen Subj. 20221866: 130221). In some cases, the recombinant polypeptide may comprise two ITAMs or three ITAMs. In some embodiments, the one or more ITAMs may be independently selected from the ITAMs of CD3γ, CD3δ, CD3ε, CD3ζ, TYROBP (DAP12), DAP10, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD28, and CD72. In some embodiments, the one or more ITAMs may comprise the ITAM of FcεRIγ. In practice, any of these domains may be a variation of a wild-type sequence, e.g., a sequence that is at least 90%, 95%, or 98% identical to a sequence described in WO2014127261, for example. In some embodiments, the recombinant polypeptide may have a signaling domain from CD3ζ in which two of the three ITAM motifs (the second and third ITAM motifs) have been altered to be non-functional. More specifically, both tyrosine (Y) phosphorylation sites in the second and third ITAMs may be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated. This altered CD3ζ signaling domain is described in Feucht et al (Nat Med. 201925: 82-88). In any embodiment, the recombinant polypeptide may further comprise one or more other signaling motifs and / or protein binding motifs that are not already present in the adapter protein.SF2024-070-2 Nucleic acid and cells Also provided is a nucleic acid encoding the recombinant polypeptide. In some embodiments, the nucleic acid may be in a plasmid or viral vector such as a lentiviral or retroviral vector, and it may be operably linked to suitable promoter and, if necessary, terminator. In these embodiments, the coding sequence for the recombinant polypeptide may additionally comprise a signal sequence for targeting the protein to the plasma membrane. In any embodiment, the NK cell or T cell may be mammalian (e.g., mouse, human, primate). Human cells are often used, however. In any embodiment, the NK cell or T cell may be a primary NK or T cell, i.e., a cell obtained from peripheral blood, or a progenitor of the same (e.g., cell made by culturing primary NK cells or T cells in proliferation or expansion medium) and in any embodiment, the NK cell or T cell may be genetically modified to be allogeneic in a human host. NK cells or T cells may be obtained from any suitable source. For example, NK cells or T cells may be differentiated in vitro from a hematopoietic stem cell population, or NK cells or T cells may be obtained from a subject. NK cells or T cells may be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, NK cells or T cells may be derived from one or more NK cell or T cell lines available in the art. NK cells or T cells may also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation and / or apheresis. In any embodiment, the cells that are expanded can be primary NK cells or T cells. NK cells or T cells may be genetically modified, made from iPSCs, obtained from umbilical cord blood, etc. A method comprising genetically modifying an NK cell or T cell to express the recombinant polypeptide is also provided. As would be apparent, this method may be done ex vivo (on primary NK cells or T cells or their progenitors) or a cell line, etc. In one example, a nucleic acid encoding the recombinant polypeptide may be introduced into the NK cell or T cell. In other embodiments, a nucleic acid encoding the co-stimulatory domain may be knocked into an existing gene.SF2024-070-2 Populations of NK cells or T cells A population of the NK cells or T cells is also provided. In some embodiments, these cells may be present in vitro and may be progenitors of primary NK cells or T cells that have been genetically modified to express the recombinant protein. In some embodiments, NK cells or T cells may be genetically modified to be allogeneic in a human host, although this may be unnecessary in many cases. In these embodiments, the cells may be frozen. The population may comprise any number of the NK cells or T cells (e.g. 100,000-1 Bn cells). In some embodiments, the harvested cells may be cryopreserved, where the term “cryopreserved” refers to cells that have been preserved or maintained by cooling to low sub- zero temperatures, such as 77 K or -196 deg. C. (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. Useful methods of cryopreservation and thawing cryopreserved cells, as well as processes and reagents related thereto, include but are not limited to e.g., those described in U.S. Patent Nos. 10370638; 10159244; 9078430; 7604929; 6136525; and 5795711, the disclosures of which are incorporated herein by reference in their entirety. In contrast, the term “fresh”, as used herein with reference to cells, may refer to cells that have not been cryopreserved and, e.g., may have been directly obtained and / or used (e.g., transplanted, cultured, etc.) following collection from a subject or organ thereof. Harvested therapeutic cell populations produced by the methods as described herein and therapeutic or pharmaceutical compositions thereof may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and / or device. Such populations of cells and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g., where the therapeutic cells are present in a suitable diluent and / or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like.SF2024-070-2 In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means. In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgCl2, KCl, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions. A population may include a therapeutically effective amount of the cells. By “therapeutically effective amount” it is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to affect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations. A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a therapeutically effective amount of NK cells or T cells may be 100,000-200M of the NK cells or T cells.SF2024-070-2 The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like. Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration. The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation. A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM. In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinationsSF2024-070-2 thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v). Cell manufacture methods Natural killer or T cell therapies can be manufactured from a variety of sources: these include peripheral blood, either steady-state or taking advantage of apheresis performed to collect hematopoietic stem and progenitor cells mobilized with growth factors such as granulocyte colony-stimulating factor (G-CSF), bone marrow, cord blood, ES cells, iPS cells and NK cell lines or T cell lines. In some embodiments, the method may comprise expanding the NK cells or T cells ex vivo to produce expanded NK or T cells and harvesting the expanded NK or T cells to produce an NK or T cell population, where the term “harvesting” is intended to refer to a step in which the cells are removed from the container(s) / bioreactor(s) in which the cells were expanded. In some embodiments, the cells may be concentrated if desired, e.g., by centrifugation, a suitable cell separation technique (e.g., magnetic beads), and / or the like. See, generally, Temples et al (Journal of Immunology and Regenerative Medicine 202010: 100031) and Fernandez et al, (Cancers 202113: 577), among others, for further details. A cell population manufactured according to this method is also provided. Methods of treatment A method of treatment is also provided. This method may comprise administering an effective amount of a population of the NK or T cells to a patient in need thereof where, in some embodiments, an effective amount may be 100,000-2 Bn cells. In any embodiment, the NK or T cells may be autologous / autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells are obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human. Cells are typically infused into the patient, although routes of administration can be used.SF2024-070-2 In any embodiment, the patient may be a cancer patient where the treatment may result in least an amelioration of one or more symptoms associated with the condition of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. The cancer being treated may be a blood cancer or a solid tumor, e.g., a carcinoma or a sarcoma. If the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma. Cancers that can be treated with a method disclosed herein include any cancer that can be targeted by NK cells or T cells, including, but not limited to carcinomas, sarcomas, melanoma, leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method disclosed herein include solid tumors, and cancers that begin in blood-forming tissue, i.e., hematological cancers such as leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method disclosed herein include metastatic cancers. Carcinomas that can treated by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma,SF2024-070-2 lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be treated by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. As would be apparent, if the recombinant polypeptide comprises a binding domain, then the choice of binding domain may be determined by the type of cancer being treated. For example, the binding domain may target CD19, CD20 or BCMA for B-cell malignancies, including acute myeloid leukemia (AML), multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), lymphoma, etc. Breast cancer antigens include MUC1, HER2, mesothelin, CEA, CAIX, FR-α, CD171, GD2, EGFRvIII, FAP, and vascular endothelial growth factor receptor2 (VEGF-R2). Ovarian cancer antigens include CA125, MUC16, HER2, hepatocyte growth factor receptor (c-Met), mesothelin, folate receptor alpha (FRα), and cancer / testis antigen 1B. Lung cancer antigens include mesothelin (MSLN), EGFR, PSCA, MUC1, CEA, CD80 / CD86, programmed death-ligand 1 (PD-L1), inactive tyrosine-protein kinase transmembrane receptor (ROR1), and HER2. Colorectal cancer antigens include CEA, EGFR, MUC1, NKG2DL, HER2, and CD133. Pancreatic cancer antigens include mesothelin, CD133, PSCA, claudin 6, claudin 18.2, EGFR, CEA, MUC1, and HER2. Glioblastoma antigens include interleukin-13 receptor alpha 2 (IL-13αR2), epidermal growth factor receptor variant III (EGFRvIII), and HER2. Neuroblastoma antigens include GD2 and L1-CAM (CD171). Melanoma antigens include HER2, ganglioside GD2 and c-MET. Hepatocellular carcinoma antigens include CEA, MUC-1, and GPC-3. Gastric cancer antigens include folate receptor 1 (FOLR1) and NKG2D. Prostate cancer antigens include prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), PSCA, T-cell receptor gamma alternate reading frame protein (TARP), transient receptor potential (trp)-p8, and PSMA.SF2024-070-2 Renal cell carcinoma antigens include VEGFR2, CAIX, CCT301-38, CCT301-59, and CD70. Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, intraventricular, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Combination therapy In some cases, the NK or T cells may be administered along with at least one additional therapeutic agent or therapeutic treatment (together or sequentially). Suitable additional therapeutic agents include, but are not limited to, antibodies that stimulate ADCC, NK / T cell engagers, a small molecule cancer chemotherapeutic agent, and an immune checkpoint inhibitor. Suitable additional therapeutic treatments include, e.g., radiation, surgery (e.g., surgical resection of a tumor), and the like. A treatment method of the present disclosure can comprise co-administration of the NK or T cells and at least one additional therapeutic agent. By “co-administration” it is meant that both the NK cells or T cells and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the NK cells or T cells and the at least one additional therapeutic agent. The administration of the NK or T cells and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, the NK or T cells of the present disclosure are administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the NK or T cells can occur at different times and / or at different frequencies. In embodiments in which the recombinant polypeptide does not contain an extracellular binding domain, the subject may additionally receive a monoclonal antibody that stimulates ADCC, or an NK cell engager (NKCE), such as a BiKE (bispecific killer cell engager) or TriKESF2024-070-2 (trispecific killer cell engager). BiKEs and TriKEs are reviewed in Felices et al (Methods Mol Biol. 2016; 1441: 333–346). Such molecules tether NK cells to a tumor cell and induce their activation at that site. BiKEs and TriKEs are molecules that contain a single variable portion of an antibody linked to one (BiKE) or two (TriKE) variable portions from other antibodies of different specificity. See, e.g., Shanshal et al (Cancers (Basel). 202315: 2824). Similar types of antibodies are available for T cells (e.g., BiKEs and TriKEs, etc.) Multispecific antibodies can be in a variety of different formats, including, but not limited to IgG-like antibody formats (including an Fc domain) and non-IgG-like antibody formats (without an Fc domain). Multispecific antibodies with IgG-like antibody formats can be in a variety of different formats, including, but not limited to knob-into-hole (KIH), TrioMab, Duobody, κλ body, CrossMab, common light chain, strand exchange engineered domain bodies (SEEDBodies), Azymetric heterodimeric Fc, dual action Fab (DAF), dual-variable-domain immunoglobulin (DVD-Ig), IgG-scFv, Fab-Fab-Fc, DutaMab, and DutaFab. Non-IgG-like antibody formats may lack an Fc region entirely. For example, Fab, Fv and VHH antibody regions may be genetically engineered and combined in various orientations and pairings. Multispecific antibodies in non-IgG-like formats can be in a variety of different formats, including, but not limited to bivalent dual-affinity re-targeting protein (DART), tetravalent DART, half-life extended bispecific T-cell engager (HLE-BiTE), bispecific T-cell engager (BiTE), immune mobilizing monoclonal T-cell receptor (ImmTAC), tandem diabody (TandAb), bispecific killer cell engager (BiKE), trispecific killer cell engager (TRiKE), multispecific scFV single-chain variable fragment, trispecific T-cell activation construct (TriTAC), bispecific nanobody, and cross-over dual variable region (CODV). In some cases, the subject may be additionally treated with an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint moleculeSF2024-070-2 selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA. Co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine,procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorocil, cytarabine, 6-mercaptopurine or 6-thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy (e.g., by administering prednisone or dexamethasone and (h) radiation treatment, etc. Alternative therapies include targeted therapies and non-targeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF- 06463922), ropotrectinib (TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib which may be administered to patients having an ROS1 fusion, or dabrafenib (Tafinlar) or trametinib (Mekinist) which may be administered to patients having an activating mutation in BRAF. Many other actionable mutations are known. If the patient is going to be switched to a non-targeted chemotherapy, the therapy may be, for example, a platinum-based doublet chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinum-based agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (S1)). In any embodiment, the present NK cell or T cell therapy may be combined with FOLFOX therapy (i.e., a chemotherapy regimen that comprises administering folinic acid, fluorouracil and oxaliplatin (FOLFOX) to the patient), e.g., for the treatment of colorectal and other cancers.SF2024-070-2 Alternative embodiments In any embodiment, the recombinant polypeptide may alternatively comprise the transmembrane domain of DAP10 or DAP12, or a variant of the transmembrane domain that retains the ability to bind to NKG2D, NKp44 and / or NKG2C. The full-length sequence of the wild-type DAP12 is deposited at Genbank as accession number NP_001166986.1 (which deposit is incorporated by reference herein). DAP12 has a single ITAM. The wild type DAP12 transmembrane domain has the following amino acid sequence: CSCSTVSPGVLAGIVMGDLVLTVLIALAV; SEQ ID NO: 4. See Lanier (Immunol Rev. 2009227: 150–160). The full-length sequence of the wild-type DAP10 is deposited at Genbank as accession number AAD47911.1 (which deposit is incorporated by reference herein). DAP10 has a single YINM sequence . The wild type DAP10 transmembrane domain has the following amino acid sequence: LAGLVAADAVASLLIVGAVFLC; SEQ ID NO: 5. See Lanier (Immunol Rev. 2009 227: 150–160). While many embodiments of the alternative recombinant polypeptide may comprise the wild-type transmembrane domain of DAP12 (i.e., SEQ ID NO: 4) or DAP10 (i.e., SEQ ID NO: 5) in certain instances the recombinant polypeptide may comprise a variant of the wild-type transmembrane domain of DAP12 or DAP10 comprising up to 5 amino acid substitutions (e.g., 1, 2, 3, 4, or 5 substitutions) relative to SEQ ID NO: 4 or 5. For example, since this is a transmembrane domain, up to five of the hydrophobic residues can be substituted with another hydrophobic amino acid. The other components of these alternative polypeptides (e.g., the types of co-stimulatory domain, the types and number of ITAMs, whether the recombinant polypeptide contains or lack an extracellular binding domain, etc.) and methods of use are the same for the embodiments that employ the transmembrane domain of FcεRIγ of CD3ζ, or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and / or CD16, as described above. The following examples are offered by way of illustration and not by way of limitation.SF2024-070-2 EXPERIMENTAL Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. The transmembrane adaptor protein FcεR1γ is required for the expression and signaling of the activating receptors NKp30, NKp46, and CD16. These receptors are naturally expressed by human NK cells and a subset of human T cells. The following example shows that adding a co-stimulatory motif to FcεR1γ greatly enhances the effector function of human NK cells against cancer cells. This construct is referred to as FcεR1γ-41BB in this disclosure. Furthermore, addition of an extracellular binding domain (in this example a binding domain that binds to CD19) to FcεR1γ-41BB retains and / or enhances human NK cell activity in the presence of cancer cells that express an antigen that is recognized by the binding domain. In these embodiments, fusion of the FcεR1γ-41BB protein with the anti-human CD19 chimeric antigen receptor (CD19CAR) resulted in a new anti-CD19CAR-FcεR1γTM-41BB-FcεR1γ- ITAM product that can enhance human CAR NK cell activity, which is believed to be mediated by NKp30, NKp46, and / or CD16 expression and downstream signaling. Therefore, these fusion proteins have the potential to provide a better human NK cell cancer immunotherapy treatment. It is noted that the experimental section of this application provides data for NK cells. However, a subset of T cells express NKp30, NKp46, or CD16 and the mechanism for NKp30, NKp46, or CD16 activation is the same (see, e.g., Correia et a, Proc. Natl. Acad. Sci. 115 (26) E5980- E5989). As such, the present findings can be extrapolated to T cells. Materials and methods Cloning and Lentivirus particles preparation:

[0001] Human FcɛR1γ cDNA (FCRG2, Sequence ID: NM_004106.2), FcɛR1γ41BB(FCRG8.2), Myc-tag- CD19ScFvCD8hingeFcεR1γ41BB(FCRG10), human CD16 cDNA (Sequence ID: NM_000569.8), human CD19 cDNA (Sequence ID: NM_001178098.2), human CD20 cDNA (Sequence ID: NM_152866.3), human B7H6 cDNA (generously provided by Dr. A. Cerwenka, University of Heidelberg, Heidelberg, Germany), Myc-tag-CD19ScFv-CD8Hinge-TM-41BB-CD3ζ chimericSF2024-070-2 antigen receptor (conventional CD19CAR, generously provided by Dr. K. T. Roybal, University of California, San Francsico, USA), or HLA-E-β2M-HLA-GSignal Peptide(generously provided by Dr. F. Momburg, German Cancer Research Center (DKFZ), Heidelberg, Germany) constructs were cloned into the lentivirus vectors pHR containing the SFFV promotor using an In-Fusion® HD Cloning Kit (TAKARA). FCRG2 contained nucleic acids 1-273 of human FCER1G (Sequence ID: NM_004106.2). FCRG8 contained nucleic acids 1-273 of human FCER1G and a human 41BB co stimulatory motif (e.g., KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ; SEQ ID NO:6) inserted between exon 3 and exon 4 of FCER1G cDNA sequence. FCRG10 contained nucleic acids 1- 954 of CD19ScFv-CD8Hinge-41BB-CD3ζ chimeric antigen receptor (CAR) ectodomain (e.g., CD8 signal peptide, Myc-tag, CD19ScFv, and CD8hinge) fused to transmembrane and intracellular domains of FCRG8 at amino acid # 17 (e.g., FCER1G exon 2). DNA fragments were obtained from Integrated DNA Technologies (e.g., IDT). Lentivirus preparation was done by using the pMD2.G and pCMV dr8.91 packaging vectors and transfection of the Lenti-X™ 293T cell Line (TAKARA, cat. 632180) cultured in complete DMEM plus 10% FCS. Lentivirus was concentrated using a Lenti-X™ concentrator (TAKARA, cat. 631232) and resuspended in 1 ml RPMI-1640 + 10% fetal calf serum (FCS) with protamine sulfate (1 μg / ml). Aliquots were kept at -20°C. Cell culture and transduction: NK92 cells (CRL-2407) were obtained from “The American Type Culture Collection” (ATCC). NK92 cell culture media: Minimum Essential Medium Eagle (Sigma Aldrich, cat. M0200-500ML) + 20% horse serum (Sigma Aldrich, cat. H1138) + human IL-2 (25 ng / ml, Peprotech, cat 200-02-1MG). mouse pro-B cell line Ba / F3 pre- engineered to express mouse IL-3 or human K562 cells were generously provided by Dr. L. Lanier. Ba / F3 or K562 culture media: complete RPMI-1640 + 10% heat-inactivated FCS. For lentiviral transductions, 50,000 cells were mixed with concentrated lentiviral particles in 96-well round-bottom plate (200 µl of culture media) and were centrifuged at 1000 RCF for 1 hour at room temperature. After centrifugation, cells were incubated at 37°C, 5% CO2) for 24 hours. Cells were then transferred to T25 flask for expansion. Flow cytometry and sorting: The following antibodies or fusion proteins were used for the detection of cell surface receptors, or intracellular proteins, and NK cell receptor binding.SF2024-070-2 Anti-FcεR1γ (Milipore, cat. FCABS440F), anti-CD3ζ (BioLegend, cat. 644103), anti-NKp30 (BioLegend, cat. 325208, cat. 325210), anti-NKp46 (BioLegend, cat. 331908, cat. 331936), anti- NKG2A (Miltenyi Biotech, act. 130-113-563), anti-NKG2D (R&D, cat. FAB139P), anti-NKp44 (BioLegend, cat. 352112), anti-41BB (BioLegend, cat. 309809), anti-TRAIL / CD253 (BioLegend, cat. 308209), anti-B7H6 (R&D, cat. FAB7144P, cat. FAB7144A), anti-HLA-E (BioLegend, cat. 342605. eBioscience, cat, 12-9953-71), anti-CD16 (BioLegend, cat. 302012, cat. 302015, cat. 302038), anti-CD19 (BioLegend, cat. 302208, cat. 302242), anti-Myc-tag (Cell Signaling, cat 3739S), hNKG2D-hFc (R&D, cat. 1299-NK), hNKp30-hFc (R&D, cat. 1849-NK), hNKp44-hFc (R&D, cat. 2249-NK), hNKp46-hFc (R&D, cat. 1850-NK), hFc (R&D, cat. 110- HG), and anti-human IgG (Jackson, cat. 109-117-008, cat. 109-236-170). Staining and wash buffer: PBS + 2% FCS. For surface antigen detection, antibodies were incubated with the cells for 30 minutes at 4oC. For receptor-human Fc chimera surface staining, proteins were incubated with the cells for 60 minutes at 4oC, washed off and then cells were incubated with anti-human IgG for 30 minutes at 4oC. For intracellular antigen detection, cells were incubated for 20 min at 4°C with 100 µl / well Cytofix / Cytoperm buffer (51-2090KZ; Becton Dickinson). Following incubation, cells were washed twice using Perm Wash buffer (cat. 421002; BioLegend) diluted 1:10 in PBS and then the antibodies were incubated with the cells for 60 minutes at room temperature. Protein expression, or co-expression was detected by flow cytometry LSR-II; Becton Dickinson Immunocytometry Systems. Engineered FCRG2, FCRG8, FCRG10, or CD19CAR NK92 cells were sorted by the increased expression or co-expression of NKp30, NKp46, CD16, or Myc-tag. Ba / F3 cells were sorted by the expression or co-expression of B7H6, HLA-E, CD20, CD19 using FACSAria: Becton Dickinson Immunocytometry Systems. Dead cells were excluded by using Zombie NIR™ Fixable Viability Kit (BioLegend, cat. 423106) diluted 1:500 in PBS + 2% FCS. CRISPR-Cas9 knockout of FCER1G or NCR3LG1 (B7H6): FcεR1γ41BBpositive NK92 cells or K562 cells were edited using the non-viral CRISPR-Cas9 system. crRNA was resuspended in IDT duplex buffer at a final concentration of 160 µM. crRNA and tracrRNA, obtained from IDT, were mixed at a 1:1 volume ratio and were incubated at 37 °C for 30 min to form gRNA solution. gRNA solution and Cas9-NLS (Berkeley QB3 MacroLab) were mixed at a 1:1 volume ratio and were incubated at 37 °C for 15–30 min. Cells were then resuspended in 18 µl Lonza P3 buffer + Cas9 / gRNA solution and were then transferred to the Lonza 96-wellSF2024-070-2 electroporation shuttle. Electroporation codes: NK92 cells; CM137, K562 cells; FF120. FCER1G: crRNA-1; GCCCAAGATGATTCCAGCAG (SEQ ID NO:7), crRNA-2: CAGCTCTGCTATATCCTGGA (SEQ ID NO:8). NCR3LG1: crRNA; AAGTAGAGATGATGGCAGGG (SEQ ID NO:9), ATGGTGACATTGTCATTCAG (SEQ ID NO:10). Antibody-conjugated beads and cell stimulation: Antibody-conjugated beads were prepared according to the company’s protocol (Invitrogen Dynabeads Antibody Coupling Kit, cat. 14311D) at 10 µg antibody per 1 mg beads. Following conjugation, beads were resuspended in sterile PBS at an antibody concentration of 0.1 µg / µl. Antibody conjugation was evaluated by flow cytometry with APC-conjugated anti-mouse or rat IgG. BioLegend: anti-CD16 (cat. 302002, IgG1k), anti-NKp30 (cat. 325204, IgG1k), (cat. 325102, IgG1k), anti-NKp46 (cat. 331904, IgG1k), anti-NKG2D (cat. 320802, IgG1k), and mouse IgG1k isotype-matched control (cat. 400102). Antibody-conjugated beads were kept at 4°C until further use. The assays were performed in 96 well round-bottom plates. Antibody-coated beads were diluted at 1:1,000 or as indicated in NK92 cell culture media and used by adding 50 µl / well. NK92 cells (5 × 104cells / well) were added at 150 µl / well in NK92 cell culture media. Alexa Fluor 647 anti- human CD107a antibody (BioLegend, cat. 328620) was added at the beginning of the assay at a final dilution of 0.1 µl / well. NK92 cells were incubated at 37°C with 5% CO2for the duration of the assay. Cells were analyzed for the detection of CD107a by flow cytometry (LSR-II; Becton Dickinson Immunocytometry Systems). Dead cells were excluded by using propidium iodide (1 mg / ml, 1:500). NK92 cells incubated without beads were used for CD107a background control and the increase in CD107a signal was calculated relative to each cell line. NK cell lysis assay and ADCC: NK92 cells were labeled with cell trace violet (CTV) according to the company’s protocol (Invitrogen, cat. C34557;). Ba / F3 or K562 cells (target cells) were labeled with CFSE (Invitrogen, cat. C34554). The assays were done only when the viability of the NK92 cells was above 90% as measured by Trypan Blue and performed in 96 well round-bottom plates. NK92 cells were resuspended in NK92 cell culture at a concentration of 1 × 105cells / 100 µl / well, and were diluted 1:21:4 or as indicated in NK92 cell culture media. Target cells were resuspended in complete RPMI-1640 + 10% heat-inactivated FCS, at a concentration of 5 × 104cells / 100 µl / well. Cell co-cultures were incubated at 37°C with 5%SF2024-070-2 CO2 for the assay duration. For ADCC, anti-human Rituximab (anti-CD20, Selleckchem, cat. A2009) was added to the co-culture at a final concentration of 5 µg / ml. At the end of the assay, cells were stained using propidium iodide (1 mg / ml, 1:500) for the detection of dead cells and percentage of dead cells was calculated as PI+CFSE+relative to PI-CFSE+cells while CTV+cells were excluded. Samples were analyzed by flow cytometry (LSR-II; Becton Dickinson Immunocytometry Systems). Example 1 FcɛR1γ41BBoverexpression leads to NKp30 and NKp46 upregulation FcɛR1γ is a transmembrane adaptor protein with one immunoreceptor tyrosine-based activation motif (ITAM). In human NK cells, FcɛR1γ protein expression is reported to regulate the expression and function of the activating receptors NKp30, NKp46, and CD16. Loss of FcɛR1γ protein expression in human adaptive NK cells, g-NK cells, or following FCER1G CRISPR knockout is associated with loss of NKp30 and NKp46 surface protein expression. Additionally, Loss of FcɛR1γ protein expression leads to a mild decrease in CD16 protein expression but increases CD16 function due to transmembrane interaction with CD3ζ homodimerize. Thus, engineering FcɛR1γ with a co-stimulatory motif, such as 41BB (FIG. 1A), may increase function and protein expression of NKp30, NKp46, and CD16 in human NK cells. To test this, the native form of FcɛR1γ (FCRG) or FcɛR1γ41BB(FCRG41BB) was overexpressed in the FcɛR1γnegativeCD16negativehuman NK cell line, NK92 (FIG. 1B). Overexpression of FcɛR1γ or FcɛR1γ41BBin NK cells led to increased expression of FcɛR1γ without affecting the expression levels of CD3ζ (FIG. 1C). Additionally, FcɛR1γ or FcɛR1γ41BBoverexpression increased NKp30 or NKp46 surface protein expression in NK cells (FIG. 1D), while FCER1G CRISPR knockout led to decreased NKp30 or NKp46 expression (FIG. 1E). FcɛR1γ or FcɛR1γ41BBoverexpression did not lead to changes in surface protein expression of the inhibitory receptor NKG2A, the activating receptors NKG2D or NKp44, and 41BB or TRAIL surface expression (FIG. 1F). Thus, these results indicate that FcεR1γ modification with an intracellular 41BB motif does not prevent the upregulation of NKp30 and NKp46 by FcεR1γ. Moreover, FcɛR1γ or FcɛR1γ41BBoverexpression does not lead to alterations in the expression of main activating or inhibitory surface membrane receptors or to non-specific NK cell activation. Example 2SF2024-070-2 FcɛR1γ41BBincrease NKp30 function against B7H6 positive cells To test if the engineering of FcɛR1γ with 41BB motif would lead to an increase in activating receptor function, the NKp30-B7H6 interaction was investigated. For that purpose, the mouse pro-B cell line, Ba / F3, was engineered to express B7H6. Moreover, B7H6+ Ba / F3 cells were engineered to co-express the NKG2A inhibitory HLA-EHLA-G, as NK cell activation by activating receptors is further regulated by signaling from inhibitory receptors such as NKG2A (FIG. 2A). Challenging the FcɛR1γ or FcɛR1γ41BBpositive NK cells with parental Ba / F3 cells did not show unspecific target cell lysis relative to parental (not transduced) NK cells or no effector control (FIGS. 2B, 2C). FcɛR1γ41BBpositive NK cells displayed an increased target cell lysis of B7H6+or B7H6+HLA-E+Ba / F3 cells relative to FcɛR1γ positive or parental NK cells (FIGS. 2B, 2C). This shows that FcεR1γ41BBexpression leads to a higher NKp30-dependent (B7H6) target cell lysis even in the presence of high levels of an inhibitory ligand such as HLA- E. To further test FcεR1γ41BBfunction against human cancer cells, the chronic myelogenous leukemia (CML) cell line, K562, which expresses low levels of HLA-E, was used. Activating receptor-human Fc fusion protein binding assay confirmed the binding of NKp30-hFc and NKG2D-hFc to K562 cells (FIG. 3A). Relative to B7H6+ Ba / F3 cells, K562 cells express lower levels of surface B7H6 (FIG. 3B). In line with the results with B7H6+ Ba / F3 cells, FcɛR1γ41BBpositive NK cells displayed an increased target cell lysis of K562 cells relative to parental or FcɛR1γ positive NK cells (FIG. 3C), while B7H6 knockout in K562 cells resulted in no target cell lysis (FIG.3D). Thus, further confirming FcεR1γ41BBincreases NKp30-dependent NK cell function against B7H6 positive cells. Example 3 FcɛR1γ41BBincreases CD16 function and ADCC To test the effect of FcɛR1γ41BBexpression on other activating receptors in a ligand- dependent manner, NK92 cells were engineered to express human CD16 (FIG. 4A). A CD107a assay using mouse IgG1 antibody-coated beads against activating receptors confirmed CD16 function in NK92 cells, while CD16 expression did not impact the function of NKp30, NKp46, or NKG2D (FIG. 4B). Similar to the upregulation of NKp30 expression, FcɛR1γ or FcɛR1γ41BBexpression increased CD16 expression relative to parental NK cells (FIG. 4C). Thus, FcεR1γSF2024-070-2 modification with an intracellular 41BB motif does not prevent the upregulation of CD16. To test CD16 function in FcɛR1γ or FcɛR1γ41BBpositive NK cells, a CD107a assay was performed using anti-human CD16 mouse IgG1 coated beads. Following CD16 cross-linking, FcɛR1γ41BBpositive NK cells displayed a significant increase in CD107a surface expression relative to FcεR1γ positive or parental cells (FIG. 4D). To test CD16 function during antibody human Fc recognition, Ba / F3 cells were engineered to express human CD20 and tested for Antibody- dependent cellular cytotoxicity (ADCC) using Rituximab (FIG. 4E). FcɛR1γ41BBpositive NK cells displayed a significant increase in ADCC relative to FcεR1γ positive or parental NK cells. Therefore, FcεR1γ modification with an intracellular 41BB motif increases CD16 function in NK cells. Example 4 CD19ScFv-CD8hingefused to FcεR1γ41BBincreases NKp30 and CD16 expression Chimeric antigen receptors (CARs) are used in cancer immunotherapy. Therefore, it was tested whether an anti-human CD19 CAR with a FcεR1γ41BBtransmembrane and intracellular domains would increase NKp30 and CD16 expression. For that purpose, the CD19ScFv-CD8hingeextracellular domain of a Myc-tag-CD19ScFv-CD8Hinge-TM-41BB-CD3ζ CAR was fused with FcεR1γ41BBtransmembrane and intracellular domains (FCRG10, FIG. 5A). Similar to FcεR1γ41BB, FCRG10 positive NK cells expressed higher levels of NKp30 and CD16 (FIG. 5B). Relative to FCRG10, CD19CAR positive NK cells did not display upregulation of NKp30 or CD16 expression (FIG. 5B, top row). The increase in NKp30 or CD16 expressions in FCRG10 positive NK cells corresponded to the expression of Myc-tag, located in the N-terminal of the CAR (FIG. 5B, bottom row). This shows that FcεR1γ modification with an intracellular 41BB motif and an extracellular CD19ScFv-CD8hingedoes not prevent the upregulation of NKp30 and CD16 by FcεR1γ. Example 5 CD19ScFv-CD8hinge-FcεR1γ41BBCAR improves NK cell function against HLA-E+target cells To test the CD19ScFv-CD8hinge-FcεR1γ41BBCAR (FCRG10) function, Ba / F3, Ba / F3- B7H6+, or Ba / F3-B7H6+HLA-E+ were engineered to express human CD19 (FIGS. 6A, 6B). A lysis assay against parental Ba / F3 cells did not show unspecific target cell lysis by FCRG10 positive NK cells relative to parental NK cells or no effector control (FIG. 6C). Against Ba / F3-SF2024-070-2 CD19+cells, FCRG10 or CD19CAR positive NK cells displayed increase target cell lysis, indicating the CD19ScFv-CD8hinge-FcεR1γ41BBCAR is functional. Still, FCRG10 positive NK cells displayed a higher target cell lysis, which may be due to the transmembrane dimerization of FcεR1γ with CD3ζ (FIG. 6D). Against Ba / F3-CD19+B7H6+cells, FCRG10 positive NK cells displayed increased target cell lysis relative to FcεR1γ41BBpositive NK cells, indicating that the recognition of two activating ligands increases NK cell mediated lysis (FIG. 6E), which can explain the similar outcome between FCRG10 or CD19CAR positive NK cells. However, against Ba / F3-CD19+B7H6+HLA-E+cells, FCRG10 positive NK cells displayed a significant increase in target cell lysis relative to CD19CAR positive NK cells (FIG. 6F). The results indicate that CD19ScFv-CD8hinge-FcεR1γ41BBCAR (X1 ITAM) leads to a similar NK cell mediated lysis as a CD19CAR41BB-CD3ζ(X3 ITAMs) against CD19+cells (single activating ligand). Second, in the presence of an inhibitory ligand (HLA-E), and dual activating ligands recognition (B7H6 and CD19), the CD19ScFv-CD8hinge-FcεR1γ41BBCAR (X1 ITAM) improves NK cell function relative to a CD19CAR41BB-CD3ζ(X3 ITAMs). Example 6 CD19ScFv-CD8hinge-FcεR1γ41BBCAR is functional in primary human NK cells To test the CD19ScFv-CD8hinge-FcεR1γ41BBCAR (FCRG10) function in primary human NK cells, human peripheral blood NK cells were transduced with FCRG10 (FIG. 7A). A lysis assay of FCRG10+ NK cells or non-transduced NK cells against CD19+ Ba / F3 cells was then performed. CD19ScFv-CD8hinge-FcεR1γ41BBCAR positive NK cells mediated a significant cell lysis of CD19+ Ba / F3 cells relative to non-transduced NK cells (FIG. 7B). Thus, the data show that CD19ScFv-CD8hinge-FcεR1γ41BBCAR is functional in primary human NK cells. Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, allSF2024-070-2 statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein

Claims

SF2024-070-2 CLAIMS What is claimed is:

1. A recombinant polypeptide comprising: (i) the transmembrane domain of FcεR1γ of CD3ζ, or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and / or CD16; (ii) a co-stimulatory domain; and (iii) one or more immunoreceptor tyrosine-based activation motifs (ITAMs).

2. The recombinant polypeptide of claim 1, wherein the polypeptide lacks an extracellular binding domain.

3. The recombinant polypeptide of claim 1, wherein the polypeptide comprises an extracellular binding domain.

4. The recombinant polypeptide of claim 3, wherein the extracellular binding domain is an scFv or nanobody.

5. The recombinant polypeptide of any prior claim, wherein the co-stimulatory domain is the co-stimulatory domain of CD28, ICOS, CD27, 4‐1BB, OX40, CD40L, DNAM1 or 2B4.

6. The recombinant polypeptide of any prior claim, wherein the co-stimulatory domain is the 4-1BB co-stimulatory domain.

7. The recombinant polypeptide of any prior claim, wherein the one or more ITAMs are independently selected from the ITAMs of CD3γ, CD3δ, CD3ε, CD3ζ, TYROBP (DAP12), DAP10, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD28, and CD72.

8. The recombinant polypeptide of claim 7, wherein the one or more ITAMs comprises the ITAM of FcεR1γ or CD3ζ.SF2024-070-2 9. The recombinant polypeptide of any prior claim, wherein the polypeptide further comprises one or more other signaling motifs and / or protein binding motifs that are not already present in the adapter protein.

10. The recombinant polypeptide of any prior claim wherein the transmembrane domain has an amino acid sequence of: LCYXLDA / GILFXYGXXXTXLX / YX / CXK (SEQ ID NO: 3) where X is a hydrophobic amino acid independently selected from G, A, V, L, I, P, F, M and W.

11. The recombinant polypeptide of any prior claim wherein the recombinant polypeptide comprises the transmembrane domain of FcεR1γ or CD3ζ, or a variant of the transmembrane domain in which up to 5 hydrophobic amino acid residues are substituted with another hydrophobic amino acid residue.

12. A nucleic acid encoding the recombinant polypeptide of any prior claim.

13. A natural killer (NK) cell or T cell comprising a nucleic acid of claim 12, wherein the NK cell or T cell expresses the recombinant polypeptide.

14. A method of killing a cancer cell, comprising: contacting the cancer cell with the NK cell or T cell of claim 13.

15. The method of claim 14, wherein the polypeptide comprises an extracellular binding domain that recognizes a marker on the cancer cell.

16. The method of claim 14, wherein the polypeptide lacks an extracellular binding domain.

17. The method of claim 16, wherein the contacting is done in the presence of a monoclonal antibody that stimulates antibody-dependent cellular cytotoxicity (ADCC), a bi-specific killer cell engager (BiKE) or a tri-specific killer cell engager (TriKE).SF2024-070-2 18. The method of any of claims 14-17, wherein the contacting is done in vivo, ex vivo or in vitro.

19. A method of treatment, comprising: administering a cell of claim 13 to a subject that has cancer.

20. The method of claim 19, wherein the polypeptide comprises an extracellular binding domain that recognizes a marker on the cancer cell.

21. The method of claim 19, wherein the polypeptide lacks an extracellular binding domain.

22. The method of claim 21, further comprising administering a monoclonal antibody that stimulates antibody-dependent cellular cytotoxicity (ADCC), a bi-specific killer cell engager (BiKE) or tri-specific killer cell engager (TriKE) to the subject.

23. The method of any of claims 19-22, wherein the patient has a solid tumor.

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