Chimeric antigen receptors on natural killer cells and their use in immunotherapy

Abstract

Provided are natural killer cells engineered to express a chimeric antigen receptor (CAR) having a Toll / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain, e.g., a Toll-like receptor (TLR) signaling domain or an IL-1 receptor (IL-1R) subfamily signaling domain. Also provided are compositions and methods for making such natural killer cells and using them in cancer immunotherapy. The CAR-NK cells can be CISH- / - iPSC-NK cells.

Description

[Technical field]

[0001] (cross reference) This application claims the benefit of U.S. Provisional Application No. 63 / 325,441, filed March 30, 2022, and U.S. Provisional Application No. 63 / 440,169, filed January 20, 2023, which are hereby fully incorporated by reference in their entireties.

[0002] (Sequence Listing) This application contains a computer readable sequence listing that has been submitted herewith in XML file format, the entire contents of which are hereby incorporated by reference in their entirety. The Sequence Listing XML file submitted herewith is titled "14735-026-228_SEQ_LISTING.xml", was created on Mar. 28, 2023, and is 347,806 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to chimeric antigen receptors (CARs) for natural killer (NK) cells and their use in immunotherapy. In particular, the present invention relates to CARs having a cytoplasmic portion that includes a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain. [Background technology]

[0004] (background) NK cells are cytotoxic lymphocytes that are a major component of the innate immune system. They are called "natural killers" due to the early idea that they do not require prior activation to kill targets. In humans, natural killer cells normally express the surface markers CD16 (FCyRIII) and CD56. NK cells normally have cytotoxic activity. Endogenous NK cells provide a rapid response to virus-infected cells and respond to transformed cells. Small granules in the cytoplasm contain perforin, granzymes, and other bioactive molecules. Upon contact with target cells, NK cells release these granules. Perforin forms pores in the plasma membrane of the target cell through which granzymes and related molecules can enter and act to induce apoptosis of the target cell.

[0005] NK cells are considered innate lymphoid cells, in contrast to adaptive lymphoid cells (T cells and B cells) and myeloid cells (e.g., macrophages). Unlike T cells, which specifically detect pathogen peptides presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, NK cells recognize stressed cells regardless of whether pathogen-derived peptides are present on the MHC molecules. Instead, receptors on the cell surface of NK cells that recognize other biomolecules control the activation of NK cells.

[0006] Artificial NK cells can be generated from stem cells, such as induced pluripotent stem cells, by known methods, such as those described in Zhu and Kaufman, Methods Mol Biol. 2048:107-119 (2019). Of particular interest are NK cells with inactivating mutations in the gene encoding cytokine-inducible SH2-containing protein (CISH). These mutations include, but are not limited to, substitutions, insertions, and / or deletions. CISH is a key negative regulator of IL-15 signaling. Inactivation of the CISH gene in induced pluripotent stem cells (iPSCs) followed by differentiation of the iPSCs into natural killer (NK) cells results in modified NK cells with increased persistence and potency compared to NK cells in which CISH is not inactivated, as described in US 2021 / 0145883 A1. It is also believed that inactivation of CISH can enhance the cytotoxicity of T cells in cancer therapy.

[0007] By engineering NK cells to express chimeric antigen receptors (CARs), the innate targeting ability of NK cells can be redirected to selected cell populations. Chimeric antigen receptors have an extracellular domain that includes a ligand-binding domain designed to specifically bind to a target antigen; a transmembrane domain; and a cytoplasmic portion that includes one or more signaling domains. Various structures for the cytoplasmic portion of CARs are known. The first generation of CARs contained only the signaling domain for the T cell receptor zeta chain, called the CD3ζ domain. Later generation CARs incorporated domains from costimulatory molecules, in particular the signaling domains of cluster of differentiation 28 (CD28) and 41BB (also known as CD137).

[0008] There remains a need in the art for chimeric antigen receptors for use in modified natural killer (NK) cells and uses thereof. The present invention addresses that need. Summary of the Invention

[0009] (Summary of the invention) Provided are chimeric antigen receptors for use in engineered natural killer (NK) cells and uses thereof. In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) having a cytoplasmic portion that includes a Toll / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

[0010] In one aspect, the present disclosure provides a CAR having a cytoplasmic portion that includes a Toll-like receptor (TLR) signaling domain. As used herein, the term "TLR signaling domain" refers to the TIR domain of a TLR receptor or a functional variant thereof. The TLR signaling domain can be a TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10 signaling domain - preferably, a TLR2 signaling domain.

[0011] Thus, the cytoplasmic portion of the CAR can comprise a signaling domain having a polypeptide sequence selected from SEQ ID NOs: 1-10, or a functional variant thereof. The signaling domain can have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 1-10.

[0012] In a further aspect, the present disclosure provides a CAR having a cytoplasmic portion comprising an interleukin-1 receptor (IL-1R) subfamily signaling domain. As used herein, the term "IL-1R subfamily signaling domain" refers to a TIR domain of a member of the IL-1R receptor subfamily or a functional variant thereof. The IL-1R subfamily signaling domain can be an IL-1R signaling domain or an interleukin-18 receptor (IL-18R) signaling domain.

[0013] Thus, the cytoplasmic portion of the CAR can comprise a signaling domain having a polypeptide sequence selected from SEQ ID NO: 11 or 12, or a functional variant thereof. The signaling domain can have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 21-22.

[0014] In another aspect, the present disclosure provides a CAR having a cytoplasmic portion comprising a TIR signaling domain and a CD3ζ domain (also referred to herein as "CD3-zeta") - preferably a CD3ζ domain at the C-terminus of the CAR. The CARs disclosed herein may further comprise one or more other signaling domains. In a variation, the cytoplasmic portion of the CAR may comprise a 2B4 signaling domain or an OX40 signaling domain. However, in a further variation, the cytoplasmic portion of the CAR lacks the 2B4 signaling domain, lacks the OX40 signaling domain, or lacks both the 2B4 signaling domain and the OX40 signaling domain.

[0015] The CAR disclosed herein further comprises a transmembrane domain. A variety of suitable transmembrane domains are known in the art. In a variant, the transmembrane domain may be a transmembrane domain of the CD8 alpha chain (CD8α), CD28, CD16, NKp44, NKp46, TLR, IL-1R subfamily, or a functional variant thereof. The transmembrane domain may be a polypeptide sequence of [ka] or a variant having one, two, three, four, five or more substitutions.

[0016] In various embodiments, the disclosure provides natural killer (NK) cells engineered to express these CARs, nucleic acids encoding these CARs, vectors for delivering such nucleic acids to cells, stem cells comprising such nucleic acids, therapeutic compositions, kits, methods of manufacture, and methods of treatment.

[0017] In some embodiments, the present disclosure provides a natural killer cell comprising a chimeric antigen receptor (CAR) comprising an extracellular portion, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises a Toll / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain, and the extracellular protein comprises a ligand binding domain. In certain embodiments, the ligand binding domain specifically binds to a CD19 antigen, a CD20 antigen, a Her2 antigen, or a BCMA antigen.

[0018] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising an extracellular portion, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises a Toll / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain. In some embodiments, the TIR signaling domain is a Toll-like receptor (TLR) signaling domain. In some embodiments, the TLR signaling domain is a TLR2 signaling domain or a functional variant thereof. In some embodiments, the TLR signaling domain has a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:2. In some embodiments, the cytoplasmic portion comprises a CD3 zeta signaling domain. In some embodiments, the cytoplasmic portion comprises a 2B4 signaling domain. In some embodiments, the cytoplasmic portion lacks the 2B4 signaling domain. In some embodiments, the cytoplasmic portion consists essentially of, in order from N-terminus to C-terminus, a 2B4 signaling domain, an IL-18R signaling domain, and a CD3 zeta signaling domain. In some embodiments, the cytoplasmic portion consists essentially of, from N-terminus to C-terminus, a 2B4 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain. In some embodiments, the cytoplasmic portion consists essentially of, from N-terminus to C-terminus, a 2B4 signaling domain, a TLR2 signaling domain, and a CD3 zeta signaling domain.

[0019] In some embodiments, the extracellular protein comprises a ligand binding domain. In some embodiments, the ligand binding domain comprises an antibody-like domain. In some embodiments, the ligand binding domain comprises a heavy chain variable (VH) domain and / or a light chain variable (VL) domain. In some embodiments, the ligand binding domain comprises a single chain variable fragment (scFv). In some embodiments, the ligand binding domain specifically binds to a CD19 antigen, a CD20 antigen, a Her2 antigen, or a BCMA antigen.

[0020] In some embodiments, (i) the extracellular domain comprises a polypeptide derived from a CD19 ligand binding domain, a CD20 ligand binding domain, a BCMA binding domain, or a HER2 binding domain; (ii) the transmembrane domain is a transmembrane domain selected from the group consisting of aTM, CD8 alpha chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2Rβ, TLR, and IL-1R subfamily; and (iii) the cytoplasmic domain comprises a signaling domain of TLR2, 41BB, or 2B4.

[0021] In some embodiments, the cytoplasmic domain further comprises a signaling domain of CD3zeta. In some embodiments, the CAR comprises (i) an aTM transmembrane domain, a TLR2 signaling domain, and a CD3zeta signaling domain; or (ii) a CD28 transmembrane domain, a TLR2 signaling domain, and a CD3zeta signaling domain.

[0022] In some embodiments, the C-terminus of the extracellular domain is operably linked to the N-terminus or C-terminus of the transmembrane domain without a linker. In some embodiments, the C-terminus of the extracellular domain is operably linked to the N-terminus or C-terminus of the transmembrane domain with a linker or hinge domain.

[0023] In some embodiments, the hinge domain is a CD8a hinge. In some embodiments, the CD8a hinge comprises the amino acid sequence set forth in SEQ ID NO: 158.

[0024] In some embodiments, the N-terminus or C-terminus of the transmembrane domain is operably linked to the N-terminus of the cytoplasmic domain with or without a linker, in some embodiments, the transmembrane domain is operably linked to the cytoplasmic domain with a Gly-Ser linker.

[0025] In another aspect, provided herein is a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor of the present disclosure.

[0026] In yet another embodiment, provided herein is a population of cells comprising immune cells comprising a chimeric receptor of the present disclosure or a polynucleotide of the present disclosure. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the NK cells are induced pluripotent stem cell-derived natural killer (iPSC-NK) cells. In some embodiments, the immune cells comprise a homozygous inactivating mutation in the cytokine-inducible SH2-containing protein (CISH) gene. In some embodiments, the immune cells comprise a homozygous inactivating mutation in the CISH gene. - / - It is.

[0027] In another aspect, provided herein is a pharmaceutical composition comprising a population of cells of the present disclosure.

[0028] In a further aspect, provided herein is a method of killing a target cell, comprising contacting a population of target cells with a population of cells of the present disclosure or a pharmaceutical composition of the present disclosure, wherein a ligand binding domain of the CAR specifically binds to an antigen on the target cell, and wherein the cell induces specific killing of the target cell.

[0029] In yet another embodiment, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present disclosure.

[0030] Advantages of the present invention can include, but are not limited to, increased activity of the disclosed CARs in NK cells or related cell types compared to CARs known in the art.

[0031] Other aspects and advantages of the present invention will become apparent from the following description and accompanying drawings. [Brief description of the drawings]

[0032] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0033] [Figure 1] Figure 1A shows an illustration of a polynucleotide sequence encoding a chimeric antigen receptor (CAR) with a labeled portion of the CAR. A leader sequence is included to promote integration into the membrane.

[0034] FIG. 1B shows an illustration of the polypeptide sequence of the chimeric antigen receptor (CAR) after cleavage of the N-terminal leader, with the portion of the CAR labeled.

[0035] [Diagram 2] FIG. 2A shows an illustration of an embodiment of a CAR with a general ligand binding domain and specific hinge (CD8α) and signaling domains (TLR2 and CD3ζ).

[0036] FIG. 2B shows an illustration of an embodiment of a CAR with a general ligand binding domain and specific hinge (CD8α) and signaling domains (2B4, TLR2, and CD3ζ).

[0037] [Diagram 3] Figure 3 shows an illustration of CAR within the cell membrane.

[0038] [Figure 4] FIG. 4 shows an illustration of an embodiment of a CAR within the cell membrane with specific hinge (CD8α) and signaling domains (TLR2 and CD3ζ).

[0039] [Diagram 5] FIG. 5A shows a nearest-neighbor-joining phylogenetic tree of TIR signaling domains generated from an alignment of the polypeptide sequences of the TIR signaling domains.

[0040] FIG. 5B shows a nearest-neighbor-joining phylogenetic tree of TLR signaling domains generated from an alignment of the polypeptide sequences of the TLR signaling domains.

[0041] [Figure 6] FIG. 6 shows a multiple sequence alignment of the signaling domains of TLR1 (SEQ ID NO:1), TLR2 (SEQ ID NO:2), TLR4 (SEQ ID NO:4), TLR6 (SEQ ID NO:6), and TLR10 (SEQ ID NO:10).

[0042] [Figure 7] FIG. 7 shows a table of pairwise sequence identities of TLR signaling domains.

[0043] [Figure 8] FIG. 8 shows the sequence of one embodiment of a CAR (SEQ ID NO: 100) separated into its components (SEQ ID NOs: 37, 38, 41, 2, and 63).

[0044] [Figure 9]Figure 9 shows the expression of individual CAR constructs in iPSC-NK cells. Histogram of flow cytometry data showing CAR expression in mature iPSC-derived NK cells. CAR expression was detected using an HA-tag artificially introduced into the extracellular portion of the CAR and an anti-HA antibody (APC).

[0045] [Figure 10] Figure 10 shows a bar graph of CAR expression data. Bar graph showing frequency of CAR expression in iPSC-derived NK cells. The dotted line represents the average CAR expression across all constructs.

[0046] [Figure 11] FIG. 11 shows the long-term killing assay: the line graph shows the frequency of NK-specific killing against CD19+ Raji target cells using the eSight impedance assay. The data is normalized to target cells only (no NK cells), which are represented by the horizontal dotted line. 50% NK cell killing means that there were half as many live cells in the culture at that time point as in the untreated (no NK cells) control. The arrows point to the curves for CARs with TLR2 signaling domain and CD3ζ cytoplasmic domain ("TLR2-zeta").

[0047] [Figure 12] Figure 12 shows a 4 hour caspase 3 / 7 cytotoxicity assay: the bar graph shows NK specific killing of CD19+ SupB15 target cells after 4 hours of co-culture. The dotted line represents the specific killing of the reference CAR ("2B4-Zeta"). The arrows point to the curve for the CAR with the TLR2 signaling domain and the CD3ζ cytoplasmic domain ("TLR2-Zeta").

[0048] [Figure 13] FIG. 13A shows an illustration of an embodiment of a CAR having a TLR2 signaling domain, optionally a 2B4 signaling domain, and a CD3 zeta cytoplasmic domain in three different orders.

[0049] FIG. 13B shows the long-term killing assay for the three constructs shown in FIG. 13A: the line graph shows the CD19 + The frequency of NK-specific killing of Raji target cells is shown.

[0050] FIG. 13C shows a 4 hour caspase 3 / 7 cytotoxicity assay for the three constructs shown in FIG. 13A.

[0051] [Figure 14] FIG. 14A shows a bar graph of CAR expression data showing the frequency of CAR expression following lentiviral transduction of CISH knockout (KO) iPSC-derived NK cells as detected by recombinant HER2.

[0052] FIG. 14B shows a 24-hour killing assay of the CAR constructs referenced in FIG. 14A: the line graph shows HER2 killing using eSight impedance assay. + The frequency of NK-specific killing of target cells is shown.

[0053] FIG. 14C shows HER2 + 14B shows a bar graph depicting the release of cytokines IFN-gamma and TNF-alpha following co-culture of target cells with CISH KO iPSC-derived NK cells expressing the CAR construct referenced in FIG. 14A.

[0054] FIG. 14D shows a bar graph depicting killing of NK cell-resistant solid tumor HER2+ target cells (2:1 E:T) by CISH KO iPSC-derived NK in monolayers 24 hours after exposure to CISH KO iPSC-derived NK cells expressing the CAR construct referenced in FIG. 14A.

[0055] [Figure 15]FIG. 15A shows stress test killing assay of CISH KO iPSC-derived NK cells expressing three different CAR constructs, each containing the same HER2 binding domain, against HER2+ target cells using an eSight impedance assay.

[0056] FIG. 15B shows HER2 expression in CISH KO iPSC-derived NK cells expressing the same CAR construct and three different HER2 binding domains using an eSight impedance assay. + 1 shows a stress test killing assay for target cells.

[0057] [Figure 16] FIG. 16A shows a line graph depicting a caspase 3 / 7 cytotoxicity assay demonstrating NK killing of HER2+ target cells after 4 hours of co-culture with iPSC-derived NK cells expressing the same CAR construct and three different HER2 binding domains.

[0058] FIG. 16B shows representative images of HER2+ target cell killing and caspase 3 / 7 activation after 30 h co-culture of NK cells with NK cell-resistant solid tumor targets (2:1 E:T).

[0059] [Figure 17] FIG. 17 shows representative images of HER2+ 3D tumor spheroids after treatment with non-transduced iPSC-derived NK cells (no CAR expression) and iPSC-derived NK cells expressing the same CAR construct and three different HER2 binding domains for 18 and 36 hours.

[0060] [Figure 18] FIG. 18 shows a sequential killing assay of HER2+ target cells by iPSC-derived NK cells either expressing a CAR with a HER2-binding domain or not expressing a CAR (UT) using an eSight impedance assay.

[0061] [Figure 19] FIG. 19 shows representative images of HER2+ 3D tumor spheroids at days 0 and 3 after treatment with NK cells ("UT") and NK cells expressing a CAR with a HER2-binding domain.

[0062] [Figure 20] FIG. 20 shows an illustration of triple-edited ("TE") iPSC cells according to the present disclosure.

[0063] [Figure 21] FIG. 21 shows representative histograms of flow cytometry data showing CAR and NK marker expression in wild-type (WT) NK cells, CISH KO iPSC-derived NK cells, and triple-edited ("TE") iPSC-derived NK cells.

[0064] [Figure 22] FIG. 22A shows a long-term killing assay in HER2+ 3D tumor spheroids showing the frequency of NK killing against HER2+ target cells by WT NK cells, CISH KO iPSC-derived NK cells, and triple-edited ("TE") iPSC-derived NK cells.

[0065] FIG. 22B shows representative images of HER2+ 3D tumor spheroids from day 0 and day 3 onward of co-culture with either WT NK cells, CISH KO iPSC-derived NK cells, and triple-edited ("TE") iPSC-derived NK cells.

[0066] [Diagram 23] Figures 23A and 23B show the synergistic effect of sIL-15 co-expression with a HER2-CAR construct on NK cell killing against the HER+ breast cancer line BT474. A comparison is made with cells expressing an anti-CD20 CAR. CAR expression with and without sIL-15 is shown in Figure 23A, and NK cell killing against BT474 clone 5 with and without sIL-15 is shown in Figure 23B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Detailed Description of the Invention The present invention relates to chimeric antigen receptors (CARs) for natural killer (NK) cells and their use in immunotherapy. In some variations, the present invention relates to CARs having a cytoplasmic portion that includes a Toll / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

[0068] In various embodiments, the disclosure provides natural killer (NK) cells engineered to express the disclosed CARs, nucleic acids encoding these CARs, vectors for delivering such nucleic acids to cells, stem cells comprising such nucleic acids, therapeutic compositions, kits, methods of manufacture, and methods of treatment. As shown in Figures 1A-1B, the CAR of the disclosure may be a single polypeptide encoded by a single polynucleotide sequence. The CAR may have a conventional arrangement of components as shown in Figure 1A. After expression of the CAR as a polypeptide, the leader peptide is cleaved from the CAR as shown in Figure 1B.

[0069] Exemplary embodiments of CARs are shown in Figures 2A and 2B. The cytoplasmic portion of the CAR may have a single TIR signaling domain, such as the TLR2 signaling domain shown in Figure 2A. In this embodiment, the CAR also has a CD3ζ signaling domain. Such CARs may lack other signaling domains. An embodiment having a 2B4 signaling domain is shown in Figure 2B.

[0070] Figure 3 shows a CAR polypeptide expressed at the cell membrane. The ligand binding domain is shown as a single chain variable domain (scFv), although other ligand binding domains may be used. Figure 4 shows an embodiment of a CAR with a single TIR signaling domain, shown as the TLR2 signaling domain, and a CD3ζ signaling domain, in which the cytoplasmic portion of the CAR.

[0071] In endogenous receptors, TIR signaling domains have shared functional attributes. However, they are diverse at the sequence level. Figure 5A shows a phylogenetic tree illustrating the sequence divergence among TIR signaling domains and their clustering into groups. A similar tree for only TLR signaling domains is shown in Figure 5B. Figure 6 shows a sequence alignment of selected TLR signaling domains: TLR1, TLR2, TLR4, TLR6, and TLR10. Functional variants of TLR signaling domains can be generated using such alignments by selecting residues that can vary and conserved residues that should be retained to facilitate folding and function of the domain.

[0072] Figure 7 shows pairwise sequence comparisons of TLR signaling domains. Sequence identity between TLR signaling domains is usually less than 55%, with very few exceptions. For example, the sequence identity between TLR2 and TLR4 signaling domains is 41%.

[0073] Figure 8 shows the polypeptide sequence of an embodiment of a CAR. In this embodiment, the CAR comprises a ligand binding domain specific for CD19. However, other ligand binding domains may be used, as described in detail below. Furthermore, the present disclosure contemplates each of the various components shown. Many variations on this disclosed embodiment.

[0074] (Natural Killer Cells) In one aspect, the disclosure provides natural killer (NK) cells modified to express the disclosed CAR. The NK cells may be primary NK cells. Primary NK cells can be isolated from umbilical cord blood or peripheral blood using known methods. A polynucleotide encoding a CAR can be introduced into the primary NK cells using a viral vector, a non-viral vector, or electroporation. The NK cells may alternatively be induced pluripotent stem cell (iPSC)-derived NK cells or human embryonic stem cell (hESC)-derived NK cells. Methods for generating iPSC-NK cells can be obtained by differentiating iPSCs using known methods. In one variation, the iPSC-NK cells are generated using embryoid bodies. Exemplary methods are provided in US 2013 / 0287751 A1 and Zhu et al., Methods Mol. Biol. 2048:107-119 (2019). In another variation, hESC-NK cells are generated using single cell differentiation, for example as described in Woll et al., Blood 113:6094-6101 (2009). In a further variation, iPSC-NK cells are generated by differentiating single cells. Exemplary methods are provided in US 2021 / 0024891 A1 and US 2016 / 0097035 A1. hESC- / iPSC-derived NK cells can be phenotyped by measuring surface marker expression, e.g., CD16, NKG2D, NKp44, NKp46, TRAIL, FasL, or combinations thereof. Killing activity of hESC- / iPSC-derived NK cells can be assessed. Functional assays to assess NK cell differentiation include direct cytolytic activity of tumor cells (e.g., killing of K562 cells), caspase-3 / 7 flow cytometry assays, immunological assays of cytotoxic granules or cytokine release, or antitumor activity in in vivo xenograft models.See, e.g., Woll et al., Blood 113:6094-6101 (2009); Hermanson et al., Hematopoietic differentiation of human pluripotent stem cells. 69-19 (2015).

[0075] In some embodiments, the NK cells are CD45+ / CD56+ double positive. In some embodiments, the immune cells are CD45+. In some embodiments, the immune cells are CD56+. In some embodiments, the immune cells are CD45+, CD56+, or CD45+ / CD56+. Further exemplary methods for making and using modified cells are provided in U.S. Patent Application Publications US 2018 / 0298101 A1, US 2021 / 0230548 A1, and US 2021 / 0145883 A1; and International Patent Application Publications WO 2018 / 075664 A1 (corresponding to U.S. Patent Application Serial No. 17 / 481,404) and WO 2020 / 113029 A2 (corresponding to U.S. Patent Application Serial No. 17 / 309,408), the disclosures of which are incorporated herein by reference in their entireties.

[0076] The cell populations of the present disclosure may be purified cell populations. As used herein, a composition containing a "purified cell population" or a "purified cell composition" means that at least 30%, 50%, 60%, usually at least 70%, and more preferably 80%, 90%, 95%, 98%, 99%, or more of the cells in the composition are of the same specified cell phenotype.

[0077] The cell population may be an isolated cell population. As used herein, the term "isolated" refers to a material that is substantially or essentially free from components that normally accompany it in its natural state. In certain embodiments, the terms "obtained" or "derived" are used synonymously with "isolated."

[0078] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in such publications as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (J. Enzymology (Academic Press); Handbook of Experimental Immunology (eds. D. M. Weir and C. C. Blackwell); Gene Transfer Vectors for Mammalian Cells (eds. J. M. Miller and M. P. Calos, 1987); Current Protocols in Molecular Biology (FMAusubel et al., 1987; PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., eds., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press. 2000); Using antibodies: a laboratory manual. These are explained thoroughly in such references as The Antibodies manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0079] The introduction of the polynucleotide encoding the CAR can be achieved by transduction of either NK cells or iPSC or hESC progenitor cells with a viral vector, such as a lentiviral vector. Suitable lentiviral vectors are known in the art and are described, for example, in Zufferey et al. (1997); Dull et al. (1998), US6013516A, and US5994136A. In a variation, the polynucleotide can be introduced into the cell using a non-viral vector, such as lipid nanoparticles, transfection reagents, or electroporation.

[0080] In one aspect, the present disclosure provides a cell comprising the chimeric antigen receptor described herein and / or a polynucleotide encoding the chimeric antigen receptor. The cell may be a stem cell or a progenitor cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is an iPSC-derived NK cell.

[0081] In some embodiments, the cells are cells (e.g., CISH). - / - In some embodiments, the cells comprise a homozygous inactivating mutation in the cytokine-inducible SH2-containing protein (CISH) gene of IL-15. In some embodiments, the cells comprise expression and / or overexpression of a gene encoding soluble IL-15 or membrane-bound IL-15.

[0082] (Chimeric Antigen Receptor) Provided are chimeric antigen receptors for use in engineered natural killer (NK) cells and uses thereof. In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) having a cytoplasmic portion that includes a Toll-like / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

[0083] (Toll-like receptor signaling domain) NK cells are known to express pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), that recognize pathogen-associated molecular patterns (PAMPs) on infected cells. Noh et al. (2020) J. Immunol. Res. 2020:204860.

[0084] The Toll / Interleukin-1 (IL-1) Receptor (TIR) ​​family is defined by having a cytoplasmic portion that contains a structurally homologous signaling domain called the TIR domain. TIR domains are found not only in Toll-like receptors (TLRs) but also in two interleukin receptors, the interleukin-1 receptor (IL1R) and the interleukin-18 receptor (IL18), which are classified as interleukin receptors but participate in a signaling pathway similar to that of TLRs. Thus, the TIR family is considered to have two subfamilies defined by the specificity of their extracellular domains: (i) the Toll-like receptor (TLR) subfamily and (ii) the IL-1 receptor (IL-1R) subfamily.

[0085] In response to extracellular signals, both TLRs can form dimeric receptor complexes that recruit accessory proteins. Martin et al. (2002) Biochimica et Biophysica Acta 1592:265-280. Humans have 10 TLRs, TLR1-TLR10. Signaling by TLRs is variously dependent on heterodimerization or homodimerization. TLR4 is known to signal by homodimerization. TLR2 signaling is induced by heterodimerization with TLR1 or TLR6. Unlike TLR4, TLR2 is known not to signal as a homodimer. Zhang et al. (2002) FEBS Lett. 532:171.

[0086] In one aspect, the present disclosure provides a CAR having a cytoplasmic portion that includes a Toll-like receptor (TLR) signaling domain. As used herein, the term "TLR signaling domain" refers to the TIR domain of a TLR receptor or a functional variant thereof. The TLR signaling domain may be a TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10 signaling domain - preferably, a TLR2 signaling domain.

[0087] Thus, the cytoplasmic portion of the CAR can comprise a signaling domain having a polypeptide sequence selected from SEQ ID NOs: 1-10, or a functional variant thereof. The signaling domain can have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 1-10.

[0088] Upon activation of receptors containing Toll / interleukin-1 (IL-1) receptor (TIR) ​​domains, the TIR domains trigger conserved cell signaling pathways and control cell functions by recruitment of cytosolic signaling molecules, including TIR domain-containing cytosolic adaptor proteins. TIR signaling domains typically contain three conserved regions (e.g., the Box 1 motif) that are responsible for mediating protein-protein interactions between toll-like receptors (TLRs) and signaling components. [ka] , Box 2 motif: [ka] TIR signaling domains have been characterized as containing a Box 3 motif: a conserved W adjacent to a FW or basic residue (Slack et al., 2000). Each TIR signaling domain contains a five-stranded parallel β-sheet (A-E) and six surrounding α-helices (A-E) with their respective connecting loop structures (Bovijen et al., 2013). The conserved residues are located in the hydrophobic core, and large insertions and deletions can occur in various loop regions of different TIR signaling domains. For example, differences in the Box 2 motif, which includes the "BB loop" region of the signaling domain, are thought to play a role in the specificity of TIR-TIR interactions (Xu et al., 2000; Poltorak et al., 1998). Approximately 20 receptors with TIR domains have been identified in humans, including Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). The general structure of TIR signaling domains contains conserved residues within a core sequence ranging from about 125 to about 200 amino acid residues, although sequence conservation between these domains may be only 20-30%. There is a large degree of variability in the external residues between TIR domains, resulting in differences in the distribution of electrostatic potential and the resulting signaling function (Dunne et al., 2003). Sequence variability between TIR signaling domains may include insertions, deletions, and / or mutations in one or more of the α-helices, β-sheets, loops, or box motifs.

[0089] In some embodiments, the TIR signaling domain is a Toll-like receptor (TLR) signaling domain. Human Toll-like receptors (TLR1-10) are single-pass transmembrane proteins that each recognize a unique ligand, which activates intracellular signaling (Armant, M and Fenton, M, 2002) via the TIR signaling domain (Xu et al., 2000; Horng et al., 2002; Brown et al., 2006). TLR1, 2, 4, 5, 6, and 10 reside in the plasma membrane and recognize extracellular pathogen-associated molecular patterns (PAMPs). TLR3, 7, 8, and 9 reside in intracellular endosomes and recognize internalized PAMPs (Akira et al., 2006; Kawai, T. and Akira, S., 2007). TLRs undergo homo- or heterodimerization, which initiates their activation (Gay, N. and Gangloff, M., 2007). The signal cascade continues with TLR signaling through the conserved cytosolic TIR domain present in both the cytoplasmic signaling domain and TLR adaptor proteins such as myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like (MAL / TIRAP), TRIF-related adaptor molecule (TRAM / TICAM2), TIR domain-containing adaptor protein inducing interferon-β (TRIF / TICAM1), and sterile alpha and armadillo motif-containing protein (SARM) (O'Neill, L. and Bowie, A., 2007). The MyD88-dependent pathway signals MyD88 to phosphorylate interleukin-1 receptor-associated kinase (IRAK) 4, which then continues to IRAK1 and IRAK2 (Lin et al., 2010). MyD88 and TRIF interact with the kinases TRAM and MAL (Yamamoto et al., 2003; Yamamoto et al., 2002). The TRAM and TIRAP pathways recruit MyD88 and TRIF to their respective TLRs. All TLRs except TLR3 activate MyD88-dependent pathways (Horng et al., 2002; Kawai and Akira, 2010).The TRIF-dependent pathway signals through the downstream kinases TANK-binding kinase 1 (TBK1) and IKKε, activating IRF3 and subsequently producing type 1 interferons (Yamamoto et al., 2002b; Oshiumi et al., 2003). The SARM pathway functions as a negative regulator of TRIF-dependent Toll-like receptor signaling (Carty et al., 2006).

[0090] The TLR signaling domain may be a TLR2 signaling domain or a functional variant thereof. The TLR2 signaling domain is derived from Toll-like receptor 2 (TLR2), a type of TLR that interacts with TLR1, TLR6, or TLR10 prior to stimulation with a ligand (Jin et al., 2007; Toshchakov, V. and Neuwald, A., 2020). Heterodimerization promotes interaction with the intracellular TIR domain to promote signal initiation. TLR2 acts through MYD88 and TRAF6 to cause activation of the transcription factor NF-κ-B, cytokine secretion, and inflammatory responses; and can also promote apoptosis in response to lipid moieties of lipoproteins (Brightbill et al., 1999; Aliprantis et al., 1999). The TLR2 signaling domain is derived from residues 639-782 (144aa) of human TLR2 (SEQ ID NO:26) or a functional variant thereof. [ka] The TIR signaling domain of TLR2 may comprise the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR2 is encoded by a nucleotide sequence set forth in SEQ ID NO: 180. In some embodiments, the TIR signaling domain of TLR2 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 180.

[0091] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:2. Some or all of the functional variants and / or conserved residues of the TLR2 signaling domain may be retained. Functional variants may include N- to C-terminal truncations, insertions, and / or mutations. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the TLR2 signaling pathway. The conserved residues of the TLR2 TIR signaling domain may include residues 698-707 (Toshchakov, V. and Neuwald, A., 2020). Additional conserved residues in the TLR2 TIR may include P681, K709, K714, KK742-743, K751, K754, S636, R677, Y715, and / or R753 (Xu et al., 2000; Mckelvey et al., 2016; Ben-Ali et al., 2011; Kang et al., 2001; Bochud et al., 2003; Georgel et al., 2009).

[0092] The TLR signaling domain may be a TLR8 signaling domain or a functional variant thereof. The TLR8 signaling domain is derived from Toll-like receptor 8 (TLR8), a type of TLR that functions as an endosomal nucleic acid-sensing TLR that can undergo dimerization upon recruitment of the TIR-containing downstream adaptor MYD88 via homotypic interactions (Tanji et al., 2013; Tanji et al., 2015). The resulting signal propagation through the myddosome is generated by IRAK4, IRAK1, TRAF6, TRAF3, which activates NF-κ-B and IRF7, inducing pro-inflammatory cytokines and interferons (Qin et al., 2006; Doyle et al., 2007; Zhang et al., 2018). The TLR8 signaling domain may comprise residues 878-1022 of human TLR8 (SEQ ID NO: 32). The TIR signaling domain of TLR8 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR8 is encoded by a nucleotide sequence set forth in SEQ ID NO: 188. In some embodiments, the TIR signaling domain of TLR8 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 188.

[0093] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:8. Some or all of the functional variants and / or conserved residues of the TLR8 signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N-terminus to the C-terminus. Functional variants may include sufficient polypeptide sequence to facilitate signal propagation using the TLR8 signaling pathway. Conserved residues of the TLR8 TIR signaling domain may include the conserved pattern of the N-terminal half of the BB loop, -E9E9R9D9W9xP9G9- (where glutamate and tryptophan residues remain present); D9 at the C-terminus of β-sheet A; and / or conserved residues within α-helix C and α-helix D (Toshchakov, V. and Neuwald, A., 2020).

[0094] In some embodiments, the TLR signaling domain is a TLR3 signaling domain or a functional variant thereof. The TLR3 signaling domain is derived from Toll-like receptor 3 (TLR3), an endosomal nucleic acid sensing TLR that undergoes dimerization, where ligand binding results in interaction with the TRIF / TICAM1 adaptor, resulting in activation of NF-κ-B, IRF3 nuclear translocation, cytokine secretion, and inflammatory responses (Oshiumi et al., 2003). The TLR3 signaling domain may comprise residues 754-897 of human TLR3 (SEQ ID NO: 27) or a functional variant thereof. The TIR signaling domain of TLR3 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR3 is encoded by a nucleotide sequence set forth in SEQ ID NO: 183. In some embodiments, the TIR signaling domain of TLR3 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 183.

[0095] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:3. Some or all of the functional variants and / or conserved residues of the TLR3 signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N-terminus to the C-terminus. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the TLR3 signaling pathway. Conserved residues of the TLR3 TIR signaling domain may include N759, Y858, R867, and / or M870 (Sakar et al., 2007; Hu et al., 2015; Zhang et al., 2020).

[0096] In some embodiments, the TLR signaling domain is a TLR7 signaling domain or a functional variant thereof. The TLR7 signaling domain is derived from Toll-like receptor 7 (TLR7), an endosomal receptor that can respond to uridine-containing single-stranded RNA or guanosine analogs (Davenne et al., 2020; Lee et al., 2003). Association with an agonist results in dimerization that allows recruitment of the TIR-containing adaptor MYD88 through homotypic interactions (Zhang et al., 2016). The endosomal signaling complex interacts with IRAK4, IRAK1, TRAF6, and TRAF3, resulting in activation of NF-κ-B and IRF7, inducing cytokines and interferons (Zhang et al., 2016). The TLR7 signaling domain can include residues 889-1033 (145 aa) of human TLR7 (SEQ ID NO: 31). The TIR signaling domain of TLR7 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR7 is encoded by a nucleotide sequence set forth in SEQ ID NO: 187. In some embodiments, the TIR signaling domain of TLR7 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 187.

[0097] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 7. Some or all of the functional variants and / or conserved residues of the TLR7 signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N-terminus to the C-terminus. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the TLR7 signaling pathway. Conserved residues of the TLR7 TIR signaling domain may include >90% of the conserved pattern in the N-terminal half of the BB loop; -E9E9R9D9W9xP9G9- (where glutamate and tryptophan residues remain present, D9 at the C-terminus of β-sheet A); and / or conserved residues within α-helix C and α-helix D (Toshchakov, V. and Neuwald, A., 2020).

[0098] The TLR signaling domain may be a TLR4 signaling domain or a functional variant thereof. The TLR4 signaling domain is derived from Toll-like receptor 4 (TLR4), a type of TLR that functions by interacting with K96 and CD14 via homodimerization to initiate responses to lipopolysaccharide (LPS) (Tatematsu et al., 2016). TLR4 interacts with MYD88, TIRAP, and TRAF6, resulting in activation of NF-κ-B, cytokine secretion, and inflammatory responses (Medzhitov et al., 1997; Arbour et al., 2000; Tatematsu et al., 2016). TLR4 can function through an LPS-independent response whose activation is induced by free fatty acids and Ni(2+), which is non-conserved and species-specific (Marc et al., 2010). The TLR4 signaling domain may comprise residues 672-815 (144 aa) of human TLR4 (SEQ ID NO: 28) or a functional variant thereof. The TIR signaling domain of TLR4 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR4 is encoded by a nucleotide sequence set forth in SEQ ID NO: 184. In some embodiments, the TIR signaling domain of TLR4 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 184.

[0099] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4. Functional variants and / or some or all of the conserved residues of the TLR4 signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N-terminus to the C-terminus. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the TLR4 signaling pathway. Variants of the TLR4 TIR signaling domain may include R763H and / or Q834H (Smirnova et al., 2001). Conserved residues of the TLR4 TIR signaling domain include C747 on α-helix C; WX C747 the presence of an XXE motif; and / or P712 in the BB loop (Shirey et al., 2020; Poltorak et al., 1998). Additional conserved residues in the TLR4 TIR may include residues between the BB loop and α-helix E (Toshchakov et al., 2011).

[0100] The TLR signaling domain may be a TLR1 signaling domain or a functional variant thereof. The TLR1 signaling domain is derived from Toll-like receptor 1 (TLR1), a type of TLR that interacts with TLR2 to regulate immune responses stimulated by the presence of certain lipopeptides (Lancioni et al., 2011). The TLR1 signaling domain may be residues 635-776 (142 aa) of human TLR1 (SEQ ID NO: 25) or a functional variant thereof. [ka] The TIR signaling domain of TLR1 may comprise the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR1 is encoded by a nucleotide sequence set forth in SEQ ID NO: 182. In some embodiments, the TIR signaling domain of TLR1 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 182.

[0101] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:1. Functional variants and / or some or all of the conserved residues of the TLR1 signaling domain may be retained. Functional variants may include N- to C-terminal truncations, insertions, and / or mutations. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the TLR1 signaling pathway.

[0102] The TLR signaling domain may be a TLR5 signaling domain or a functional variant thereof. The TLR5 signaling domain is derived from Toll-like receptor 5 (TLR5), a type of TLR that has been shown to play a role in immune responses through the recognition of unique molecular motifs (Bielaszewska et al., 2018). The TLR5 signaling domain may comprise residues 691-836 (146 aa) of human TLR5 (SEQ ID NO: 29) or a functional variant thereof. The TIR signaling domain of TLR5 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR5 is encoded by a nucleotide sequence set forth in SEQ ID NO: 185. In some embodiments, the TIR signaling domain of TLR5 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 185.

[0103] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5. Functional variants of the TLR5 signaling domain and / or some or all of the conserved residues may be retained. Functional variants may include N- to C-terminal truncations, insertions, and / or mutations. Functional variants may include sufficient polypeptide sequence to facilitate signal propagation using the TLR5 signaling pathway.

[0104] The TLR signaling domain may be a TLR6 signaling domain or a functional variant thereof. The TLR6 signaling domain is derived from Toll-like receptor 6 (TLR6), a type of TLR that has been shown to play a role in the recognition of di- and triacylated lipopeptides via interaction with TLR2 (Stewart et al., 2010; Triantafilou et al., 2006). The TLR6 signaling domain may comprise residues 640-781 (142 aa) of human TLR6 (SEQ ID NO: 30) or a functional variant thereof. The TIR signaling domain of TLR6 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR6 is encoded by a nucleotide sequence set forth in SEQ ID NO: 186. In some embodiments, the TIR signaling domain of TLR6 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 186.

[0105] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:6. Functional variants of the TLR6 signaling domain and / or some or all of the conserved residues may be retained. Functional variants may include N- to C-terminal truncations, insertions, and / or mutations. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the TLR6 signaling pathway.

[0106] The TLR signaling domain may be a TLR9 signaling domain or a functional variant thereof. The TLR9 signaling domain is derived from Toll-like receptor 9 (TLR9), a type of TLR that has been shown to play a role in nucleotide sensing stimulated by the presence of unmethylated cytidine-phosphate-guanosine (CpG) dinucleotides (Takeshita et al., 2001; Doyle et al., 2007). The TLR9 signaling domain may comprise residues 868-1013 (146 aa) of human TLR9 (SEQ ID NO: 33) or a functional variant thereof. The TIR signaling domain of TLR9 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR9 is encoded by a nucleotide sequence set forth in SEQ ID NO: 189. In some embodiments, the TIR signaling domain of TLR9 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 189.

[0107] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:9. Functional variants of the TLR9 signaling domain and / or some or all of the conserved residues may be retained. Functional variants may include N- to C-terminal truncations, insertions, and / or mutations. Functional variants may include sufficient polypeptide sequence to facilitate signal propagation using the TLR9 signaling pathway.

[0108] The TLR signaling domain may be a TLR10 signaling domain or a functional variant thereof. The TLR10 signaling domain is derived from Toll-like receptor 10 (TLR10), a member of the TLR receptor family involved in the detection of unique molecules and the regulation of immune responses. The TLR10 signaling domain may comprise residues 632-775 (144 aa) of human TLR10 (SEQ ID NO: 34) or a functional variant thereof. The TIR signaling domain of TLR10 has the polypeptide sequence: [ka] or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR10 is encoded by a nucleotide sequence set forth in SEQ ID NO: 190. In some embodiments, the TIR signaling domain of TLR10 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 190.

[0109] The TLR signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10. Functional variants and / or some or all of the conserved residues of the TLR10 signaling domain may be retained. Functional variants may include N- to C-terminal truncations, insertions, and / or mutations. Functional variants may include sufficient polypeptide sequence to facilitate signal propagation using the TLR10 signaling pathway.

[0110] (Interleukin-1 receptor (IL-1R) subfamily signaling domain) In one aspect, the present disclosure provides a CAR having a cytoplasmic portion that includes an interleukin-1 receptor (IL-1R) subfamily signaling domain. As used herein, the term "IL-1R subfamily signaling domain" refers to a TIR domain of a member of the IL-1R receptor subfamily or a functional variant thereof. Currently, ten IL-1R subfamily receptors have been identified in humans (Fields et al., 2019), nine of which possess a TIR domain. The IL-1R subfamily signaling domain may be an IL-1R signaling domain or an interleukin-18 receptor (IL-18R) signaling domain. In some embodiments, the IL-1R subfamily signaling domain may be an IL-1R4 (IL-33R, ST2) signaling domain, an IL-1R6 (IL-36R, IL1-Rrp2) signaling domain, an IL-1R3 (IL-1RAcp) signaling domain, an IL-1R7 (IL-18Rbeta, AcPl) signaling domain, an IL-1R8 (SIGGIR, TIR8) signaling domain, an IL-1R9 (IL-1RAPL1, TIGIRR-2) signaling domain, or an IL-1R10 (IL-1RAPL2) signaling domain.

[0111] Thus, the cytoplasmic portion of the CAR can comprise a signaling domain having a polypeptide sequence selected from SEQ ID NO: 21 or 22, or a functional variant thereof. The signaling domain can have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 21-22.

[0112] The cytoplasmic portion of the CAR can comprise a signaling domain having a polypeptide sequence selected from SEQ ID NO: 143, 144, 145, 146, 147, 148, or 149, or a functional variant thereof. The signaling domain can have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NO: 143, 144, 145, 146, 147, 148, or 149.

[0113] The IL-1R subfamily signaling domain is derived from IL-1R, a superfamily characterized by an extracellular immunoglobulin-like (Ig-like) domain and an intracellular Toll / Interleukin 1R (TIR) ​​domain. The TIR domain of the IL-1R subfamily shares some homology with the TIR domain of TLRs and functions similarly with respect to signal transduction. IL-1R family members function as heterodimers that recognize cognate cytokines and activate downstream signaling molecules (Boraschi, D. and Tagliabue, A., 2013). Ligand binding leads to oligomerization of the TIR domain on the receptor and subsequent interaction with auxiliary proteins and adaptor molecules (e.g., MYD88). Heterodimerization with the TIR domain leads to conversion of downstream cytoplasmic kinases (e.g., IRAK) and NF-kB activation, which results in an immune response (e.g., cytokine secretion).

[0114] The IL-1R subfamily signaling domain may be an IL-1R signaling domain or a functional variant thereof. The IL-1R signaling domain is derived from the interleukin-1 receptor (IL-1R), a cytokine receptor that functions by binding to interleukin-1 and associates with an accessory protein (IL-1RAcP) to form a complex that mediates interactions with TOLLIP, MYD88, and IRAK, resulting in MAPK signaling and activation of NF-κ-B (Greenfeder et al., 1995; Slack et al., 2000). The IL-1R signaling domain may comprise residues 357-569 (213aa) of human IL-1R (SEQ ID NO: 35) or a functional variant thereof. The IL-1R signaling domain of IL-1R has the polypeptide sequence: [ka] may include.

[0115] The IL-1R signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:21. Some or all of the functional variants and / or conserved residues of the IL-1R signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N-terminus to the C-terminus. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the IL-1R signaling pathway. The conserved residues of the IL-1R signaling domain may include D369A and / or R428A (Slack et al., 2000).

[0116] The IL-1R subfamily signaling domain may be an interleukin-18 receptor (IL-18R) signaling domain. The IL-18R signaling domain is derived from the interleukin-18 receptor (IL-18R), a cytokine receptor that functions by binding to its cognate cytokine, associating in a complex with the IL-18RAcP accessory protein, and engaging one or more adaptor proteins, resulting in signal transduction (Loiarro et al., 2010). The IL-18R signaling domain may comprise residues 373-520 (148 aa) of human IL-18R (SEQ ID NO: 36) or a functional variant thereof. The IL-18R signaling domain may have the polypeptide sequence: [ka] or a functional variant thereof.

[0117] The IL-18R signaling domain may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:22. Some or all of the functional variants and / or conserved residues of the IL-18R signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N-terminus to the C-terminus. Functional variants may include sufficient polypeptide sequence to promote signal propagation using the IL-18R signaling pathway. The conserved residues of the IL-18R signaling domain may include a conserved sequence in the BB loop (Ota et al., 2012).

[0118] (Other components of the CAR cytoplasmic portion) In another aspect, the present disclosure provides a CAR having a cytoplasmic portion comprising a TIR signaling domain and a CD3ζ domain-preferably a CD3ζ domain at the C-terminus of the CAR. The CAR disclosed herein may further comprise one or more other signaling domains. In a variation, the cytoplasmic portion of the CAR may comprise a 2B4 signaling domain or an OX40 signaling domain. However, in a further variation, the cytoplasmic portion of the CAR lacks the 2B4 signaling domain, lacks the OX40 signaling domain, or lacks both the 2B4 signaling domain and the OX40 signaling domain.

[0119] The cytoplasmic portion has the polypeptide sequence: [ka] or a functional variant thereof. The cytoplasmic portion may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:64.

[0120] The cytoplasmic portion has the polypeptide sequence: [ka] or a functional variant thereof. The cytoplasmic portion may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:65.

[0121] The cytoplasmic portion has the polypeptide sequence: [ka] or a functional variant thereof. The cytoplasmic portion may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:66.

[0122] Additional cytoplasmic domains that can be used in CARs are provided in U.S. Patent Publication US 2017 / 0233454 and U.S. Patent No. 10,415,017.

[0123] (Ligand Binding Domain) In one aspect, the present disclosure provides a CAR having an extracellular portion that includes a ligand binding domain. The ligand binding domain of the present disclosure may include an antibody-based binder that specifically binds to an antigen of interest via the antigen or an antigenic fragment thereof. As used herein, the term "antigen" refers to a peptide, polypeptide, glycoprotein, glycan, lipid, glycolipid, or other biomolecule that identifies a target cell for immunotherapy treatment using NK cells.

[0124] As used herein, the term "antibody" collectively refers to immunoglobulin or immunoglobulin-like molecules, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response. The term "antigen-binding fragment" refers to a portion of an antibody that specifically binds to an antigen. Specific binding is considered to mean any level of specificity that has clinical utility. Specific binding is at least 10 times stronger than the binding constant of other molecules in a tissue. 3 M -1 Greater than or at least 10 4 M -1 Greater than or at least 10 5 M -1 It may mean a large binding constant. The antigen-binding fragment may be an artificial fragment, such as a humanized or chimeric antigen-binding fragment.

[0125] The term "antibody-like domain" refers to recombinant antibody fragments that retain antigen-binding properties (e.g., sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), disulfide stabilized Fv proteins ("dsFv"), diabodies, and triabodies, as known in the art, as well as camelid antibodies (e.g., U.S. Pat. Nos. 6,015,695; 6,00 (See, e.g., International Patent Publication Nos. 5,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808). The antibody-like domain may be expressed as a fusion protein to form a single-chain CAR, or it may be associated with other polypeptides by other known means to form a multi-component CAR. Illustrative examples of multi-component CARs are described, for example, in International Patent Publication No. PCT / US2021 / 05676.

[0126] The antibody-like domain may be a single chain variable fragment (scFv). An scFv protein is a fusion protein in which an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) are expressed as a fusion polypeptide in either a VH-VL or VL-VH orientation, optionally with an intervening linkage such as a Gly-Ser linker. The scFv may be a dsFv, in which the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

[0127] When the ligand binding domain is an antigen-like domain, it may comprise the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the binding domain comprises a VH and / or a VL chain. In some embodiments, the ligand binding domain comprises a single polypeptide chain. The ligand binding domain may be an scFv.

[0128] (Ligand-binding domain that specifically binds to CD19) In one aspect, the disclosure provides a chimeric antigen receptor that specifically binds to CD19. The ligand binding domain may be an anti-CD19 scFv comprising the polypeptide sequence shown in SEQ ID NO: 73 or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 73 or other scFv or VH / VL pair generated from SEQ ID NO: 73. The ligand binding domain may comprise a VH sequence according to SEQ ID NO: 74 and / or a VL sequence according to SEQ ID NO: 75 or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 74 and SEQ ID NO: 75, respectively.

[0129] The ligand binding domain (eg, scFv) may comprise the CDR sequences of Table 1, or a functional variant having one, two, three or more substitutions. Table 1: Anti-CD19 ligand binding domain sequences [Table 1]

[0130] In a variant, the ligand binding domain may comprise CDRs extracted from SEQ ID NO: 109 by known methods (see, e.g., Chothia, Martin, Kabat, AHo, and IMGT numbering, available at abysis.org; Swindells et al., J. Mol. Bio. 429(3):356-365 (2017)).

[0131] In some embodiments, the extracellular domain of the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 152. In some embodiments, the extracellular domain of the chimeric receptor consists of the amino acid sequence set forth in SEQ ID NO: 152. In some embodiments, the extracellular domain of the chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 152.

[0132] In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence set forth in SEQ ID NO: 153. In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:153.

[0133] (Ligand-binding domain that specifically binds to CD20) CD20 is a B cell lineage-specific antigen expressed on the cell surface of most B cell lymphomas, especially during the late stages of B cell lymphopoiesis. In one embodiment, the present disclosure provides a chimeric antigen receptor that specifically binds to CD20. The ligand binding domain may be an anti-CD20 scFv comprising the polypeptide sequence shown in SEQ ID NO: 139 or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 139, or other scFv or VH / VL pairs made from SEQ ID NO: 139. [ka]

[0134] In variations, the ligand binding domain may comprise CDRs extracted from SEQ ID NO: 139 by known methods (see, e.g., Chothia, Martin, Kabat, AHo, and IMGT numbering, available at abysis.org; Swindells et al., J. Mol. Bio. 429(3):356-365 (2017)). In some embodiments, the ligand binding domain may be an anti-CD20 scFv comprising SEQ ID NO: 139.

[0135] In some embodiments, the extracellular domain of the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 156. In some embodiments, the extracellular domain of the chimeric receptor consists of the amino acid sequence set forth in SEQ ID NO: 156. In some embodiments, the extracellular domain of the chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 156. The ligand binding domain may comprise a VH sequence according to SEQ ID NO: 154 and / or a VL sequence according to SEQ ID NO: 155, or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 154 and SEQ ID NO: 155, respectively.

[0136] In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence set forth in SEQ ID NO: 157. In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:157.

[0137] (Ligand-binding domain that specifically binds to HER2) In certain embodiments, the ligand binding domain can bind to a cancer antigen. In a specific embodiment, the ligand binding domain can bind to an antigen associated with solid cancer, e.g., an antigen present on a tumor cell. For example, functional activation of human epidermal growth factor receptor 2 (HER2) has been shown to strongly promote carcinogenesis. In one aspect, the present disclosure provides a chimeric antigen receptor that specifically binds to HER2. The ligand binding domain can be an anti-HER2 scFv comprising a polypeptide sequence as set forth in SEQ ID NO: 140-142, or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 140-142, or other scFv or VH / VL pairs made from SEQ ID NO: 140-142. The ligand binding domain (e.g., scFv) can include a CDR sequence or a functional variant having one, two, three, or more substitutions.

[0138] In some embodiments, the extracellular domain of the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 140, 141, or 142. In some embodiments, the extracellular domain of the chimeric receptor consists of the amino acid sequence set forth in SEQ ID NO: 140, 141, or 142. In some embodiments, the extracellular domain of the chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 140, 141, or 142.

[0139] In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence set forth in SEQ ID NO: 198, 199, or 200. In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 198, 199, or 200. Table 2: Anti-HER2 Ligand Binding Domain Sequences [Table 2]

[0140] In variations, the ligand binding domain may comprise CDRs derived from SEQ ID NOs: 140-142 by known methods (see, e.g., Chothia, Martin, Kabat, AHo, and IMGT numbering, available at abysis.org; Swindells et al., J. Mol. Bio. 429(3):356-365 (2017)). In some embodiments, the ligand binding domain may be an anti-HER2 scFv comprising SEQ ID NO: 140. In some other embodiments, the ligand binding domain may be an anti-HER2 scFv comprising SEQ ID NO: 141. In some embodiments, the ligand binding domain may be an anti-HER2 scFv comprising SEQ ID NO: 142.

[0141] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 202-207. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 202 (HER2-2-CD8-aTM-TLR2-CD3ζ without sIL15). In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 203 (HER2-2-CD8-aTM-TLR2-CD3ζ-sIL15). In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 204 (HER2-3-CD8-aTM-TLR2-CD3ζ without sIL15). In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 205 (HER2-3-CD8-aTM-TLR2-CD3ζ-sIL15). In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 206 (HER2-5-CD8-aTM-TLR2-CD3ζ without sIL15). In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 207 (HER2-5-CD8-aTM-TLR2-CD3ζ-sIL15).

[0142] (Ligand-binding domain that specifically binds to B cell maturation antigen (BCMA)) In some embodiments, the chimeric receptor described herein comprises an extracellular domain that comprises the ligand binding domain or a binding fragment thereof of BCMA, which is preferentially expressed on mature B lymphocytes and is important for B cell development.

[0143] In some embodiments, the chimeric receptor described herein comprises an extracellular domain that comprises the ligand-binding domain of BCMA or a binding fragment thereof.

[0144] In some embodiments, the extracellular domain of the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 210, 212, 214, or 216. In some embodiments, the extracellular domain of the chimeric receptor consists of the amino acid sequence set forth in SEQ ID NO: 210, 212, 214, or 216. In some embodiments, the extracellular domain of the chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 210, 212, 214, or 216.

[0145] In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence set forth in SEQ ID NO: 209, 211, 213, or 215. In some embodiments, the extracellular domain of the chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 209, 211, 213, or 215.

[0146] (signal peptide) The chimeric antigen receptor can be expressed from a polynucleotide sequence encoding an N-terminal signal peptide. The signal peptide can direct its fusion partner to be integrated into the plasma membrane of a cell. For example, the signal peptide can be [ka] , or functional variants having one, two, three or more amino acid substitutions.

[0147] (Transmembrane domain) In one aspect, the present disclosure provides a CAR with a transmembrane portion that includes a transmembrane domain. The CAR can be designed to include a transmembrane domain that is fused to the antigen-binding domain of the CAR. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. Optionally, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains binding to the transmembrane domain of the same or different surface membrane protein and minimize interaction with other members of the receptor complex. In some embodiments, the transmembrane domain is operably linked to the extracellular domain with or without a linker. In some embodiments, the transmembrane domain is operably linked to the extracellular domain via a hinge domain as described herein. In some embodiments, the transmembrane domain is a naturally occurring transmembrane domain derived from a protein. In some embodiments, the transmembrane domain is a synthetic transmembrane domain. In some embodiments, the transmembrane domain comprises the complete transmembrane domain of a polypeptide. In some embodiments, the transmembrane domain comprises a fragment of the transmembrane domain of a polypeptide.

[0148] The transmembrane domain may be derived from either natural or synthetic sources. Transmembrane regions that may be used include the transmembrane domains of CD28, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR alpha, TCR beta, or CD3 zeta, or functional variants thereof. In a variant, the transmembrane domain may be the transmembrane domain of the CD8 alpha chain (CD8 alpha), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2R beta, TLR, IL-1R subfamily, or functional variants thereof.

[0149] Exemplary transmembrane domains are provided in Table 3. Table 3: Transmembrane (TM) domains [Table 3]

[0150] In some embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the transmembrane domain is a fragment of the CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the CD28 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 43.

[0151] In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 160. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 160. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 218. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 218.

[0152] In some embodiments, the transmembrane domain is the transmembrane domain of TLR2. In some embodiments, the transmembrane domain is a fragment of the TLR2 transmembrane domain. In some embodiments, the TLR2 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the TLR2 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46.

[0153] In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 161. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:161.

[0154] In some embodiments, the transmembrane domain is an aTM domain. In some embodiments, the transmembrane domain is a fragment of an aTM domain. In some embodiments, the aTM domain comprises the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 163. In some embodiments, the aTM domain consists of the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the aTM domain consists of the amino acid sequence set forth in SEQ ID NO: 163. In some embodiments, the aTM domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41. In some embodiments, the aTM domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 163.

[0155] In some embodiments, the aTM domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 164 or 165. In some embodiments, the aTM domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 164 or 165.

[0156] In some embodiments, the transmembrane domain is the transmembrane domain of DNAM1. In some embodiments, the transmembrane domain is a fragment of the DNAM1 transmembrane domain. In some embodiments, the DNAM1 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the DNAM1 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the DNAM1 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:42.

[0157] In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 162. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:162.

[0158] (hinge domain) In one aspect, the present disclosure provides a CAR having a portion comprising a hinge domain. The hinge domain of the CAR of the present disclosure can comprise a linker or spacer sequence between the extracellular antigen binding domain and the transmembrane domain. Those skilled in the art will understand that a hinge domain (e.g., sequence) is a polypeptide segment that, at least in some instances, promotes flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.

[0159] The hinge may be derived from or may comprise at least a portion of an immunoglobulin Fc region, such as an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the spacer domain comprises at least a portion of an IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulin Fc region contained within its CH2 and CH3 domains. In some embodiments, the spacer domain may also comprise at least a portion of the corresponding immunoglobulin hinge region.

[0160] In some embodiments, a portion of the immunoglobulin constant region serves as a hinge region between the antigen-binding domain, e.g., scFv, and the transmembrane domain. The spacer can be of a length that results in increased cellular responsiveness following antigen binding compared to the absence of the spacer. Exemplary hinges include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and those including any integer between any of the endpoints of the recited ranges. In some embodiments, the hinge has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. In some embodiments, the hinge is 12 amino acids or about 12 amino acids in length. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Publication WO2014031687, and U.S. Patent Publication US 2014 / 0271635.

[0161] The CAR of the present disclosure may comprise a hinge domain isolated or derived from CD4, CD8α, CD28, IgG1, IgG2, IgG4, or IgD.

[0162] In some embodiments, the hinge domain is isolated or derived from a human CD8α molecule. The hinge domain has the polypeptide sequence: [ka] Or it may include variants having one, two, three, four, five or more substitutions.

[0163] In some embodiments, the hinge domain is isolated or derived from a human CD28 molecule. The hinge domain has the polypeptide sequence: [ka] or a variant having one, two, three, four, five or more substitutions. The hinge domain may comprise the polypeptide sequence: [ka] Or it may include variants having one, two, three, four, five or more substitutions.

[0164] In some embodiments, the hinge domain is isolated or derived from a human IgG4 molecule. The hinge domain has the polypeptide sequence: [ka] Or it may include variants having one, two, three, four, five or more substitutions.

[0165] In some embodiments, the hinge domain is isolated or derived from a human CD8 molecule. The hinge domain has the polypeptide sequence: [ka] Or it may include variants having one, two, three, four, five or more substitutions.

[0166] (Linker) In some embodiments, the chimeric receptor described herein comprises one or more linkers between receptor components.In some embodiments, the chimeric receptor described herein comprises a linker between the extracellular domain and the transmembrane domain.In some embodiments, the chimeric receptor described herein comprises a linker between the transmembrane domain and the cytoplasmic domain.In some embodiments, the chimeric receptor described herein comprises a linker between two or more intracellular signaling domains of the cytoplasmic domain.

[0167] (Gly-Ser linker) Suitable linkers for linking polypeptide sequences are known to those skilled in the art. Exemplary linkers include gly-ser polypeptide linkers, glycine-proline polypeptide linkers, and proline-alanine polypeptide linkers. In some embodiments, the linker is a gly-ser polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0168] As used herein, the term "gly-ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3. In some embodiments, n=4, i.e., Ser(Gly4Ser)4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence (Gly3Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Exemplary multiple gly-ser polypeptide linkers comprise the amino acid sequence Ser(Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3. In some embodiments, n=4, i.e., Ser(Gly4Ser)4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10.Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly3Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6.

[0169] In some embodiments, the chimeric receptor described herein comprises a gly-ser linker as set forth in SEQ ID NO: 166. In some embodiments, the gly-ser linker is encoded by the nucleotide sequence of SEQ ID NO: 167. In some embodiments, the chimeric receptor described herein comprises a gly-ser linker as set forth in SEQ ID NO: 168. In some embodiments, the gly-ser linker is encoded by the nucleotide sequence of SEQ ID NO: 169. In some embodiments, the chimeric receptor described herein comprises a gly-ser linker as set forth in SEQ ID NO: 170. In some embodiments, the gly-ser linker is encoded by the nucleotide sequence of SEQ ID NO: 171. In some embodiments, the chimeric receptor described herein comprises a gly-ser linker as set forth in SEQ ID NO: 172. In some embodiments, the gly-ser linker is encoded by the nucleotide sequence of SEQ ID NO: 173.

[0170] (More Linkers) Further linkers suitable for linking polypeptide sequences that allow bicistronic expression are known to those skilled in the art. Exemplary linkers include Furin GSG-T2A.

[0171] In some embodiments, the chimeric receptor described herein comprises a gly-ser linker as set forth in SEQ ID NO: 174. In some embodiments, the gly-ser linker is encoded by the nucleotide sequence of SEQ ID NO: 175.

[0172] CAR Polypeptide and Polynucleotide Sequences Although the present disclosure contemplates combinations of the disclosed signaling domains in any order and in any combination with transmembrane and extracellular domains, the present disclosure provides at least the constructs set forth in Table 4. Table 4: TLR domain constructs [Table 4] TIFF2025510943000033.tif232170TIFF2025510943000034.tif229170TIFF2025510943000035.tif230170TIFF2025510943000036.tif230170TIFF2025510943000037.tif231170TIFF2025510943000038.tif231170TIFF2025510943000039.tif232170TIFF2025510943000040.tif234170TIFF2025510943000041.tif230170TIFF2025510943000042.tif230170TIFF2025510943000043.tif232170TIFF2025510943000044.tif228170TIFF2025510943000045.tif231170TIFF2025510943000046.tif232170TIFF2025510943000047.tif230170TIFF2025510943000048.tif230170TIFF2025510943000049.tif230170TIFF2025510943000050.tif231170TIFF2025510943000051.tif231170TIFF2025510943000052.tif234170TIFF2025510943000053.tif233170TIFF2025510943000054.tif233170TIFF2025510943000055.tif230170TIFF2025510943000056.tif230170TIFF2025510943000057.tif232170TIFF2025510943000058.tif231170TIFF2025510943000059.tif231170TIFF2025510943000060.tif229170TIFF2025510943000061.tif231170TIFF2025510943000062.tif231170TIFF2025510943000063.tif232170TIFF2025510943000064.tif232170TIFF2025510943000065.tif229170TIFF2025510943000066.tif230170TIFF2025510943000067.tif230170TIFF2025510943000068.tif230170TIFF2025510 943000069.tif232170TIFF2025510943000070.tif229170TIFF2025510943000071.tif230170TIFF2025510943000072.tif229170.

[0173] In each case, the CAR polypeptide may comprise a CD8 hinge domain, an artificial transmembrane domain ("aTM"), and one or more signaling domains and / or other cytoplasmic domains, as shown in the first column of Table 4, in that order. It may have a polypeptide sequence according to the second column, or a functional variant having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In a variation, the CAR polypeptide comprises a ligand binding domain specific for CD19. Examples are provided in the third column of each table. In each case, the CAR polypeptide may comprise a polypeptide sequence according to the third column, or a functional variant having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. The fourth column provides the corresponding exemplary polynucleotide sequence for each. In some other variations, the CAR polypeptide comprises a ligand binding domain specific for CD20. In some other variations, the CAR polypeptide comprises a ligand binding domain specific for HER2.

[0174] (Co-expressed partners) In one embodiment, the NK CAR cells of the present disclosure are modified to co-express additional proteins, either membrane-bound or secreted. Exemplary compositions and methods are provided in US 2017 / 0073638 A1.

[0175] In some variations, the NK cells of the present disclosure are engineered to express interleukin-15 (IL-15) (either membrane-bound or secreted). IL-15 is a cytokine that plays a role in regulating immune responses. Without wishing to be bound by theory, expression of IL-15 may improve NK cell function. IL-15 has the polypeptide sequence: [ka] The IL-15 may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:58. In some embodiments, the IL-15 is encoded by a nucleotide sequence set forth in SEQ ID NO:208. In some embodiments, the IL-15 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:208.

[0176] Alternatively, IL-15 has the polypeptide sequence: [ka] The IL-15 may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:57.

[0177] In some variations, the NK cells of the present disclosure are engineered to express interleukin-18 (IL-18) (either membrane-bound or secreted). IL-18 is a pro-inflammatory cytokine that plays a role in immune cell activation and response. Without being bound by theory, expression of IL-18 may enhance NK cell proliferation, maturation, and cytotoxicity. IL-18 has the polypeptide sequence: [ka] The IL-18 may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 60. Alternatively, the IL-18 may have a polypeptide sequence: [ka] The IL-18 may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:59.

[0178] The co-expressed protein of the NK cell of the present disclosure may be a fusion protein. As used herein, "fusion protein" refers to an expression product resulting from the fusion of at least two genes. A fusion protein may be a chimeric polypeptide comprising two or more unrelated proteins or protein fragments conjugated to each other. The co-expressed polypeptide may be linked to the polypeptide encoding the CAR of the present disclosure via a self-cleaving peptide, for example, a self-cleaving (2A) peptide or a functional variant thereof.

[0179] In some variations, the NK cells of the present disclosure are modified to express an interleukin-15 / interleukin-15R (IL15 / IL15R) fusion protein. The interleukin-15 receptor (IL-15R) has been shown to play a role in stimulating the proliferation of immune cells. Without wishing to be bound by theory, expression of IL-15 / IL15R may improve NK cell function. The IL15 / IL15R fusion protein has the polypeptide sequence: [ka] The IL15 / IL15R fusion protein may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:62.

[0180] In some variations, the NK cells of the present disclosure are engineered to express an interleukin-18 / interleukin-18R (IL18 / IL18R) fusion protein. The interleukin-18 receptor (IL-18R) has been shown to play a role in promoting immune cell activation and cytotoxic activity. Without wishing to be bound by theory, expression of IL18 / IL18R may enhance NK cell proliferation, maturation, and cytotoxicity. The IL18 / IL18R fusion protein has the polypeptide sequence: [ka] The IL18 / IL18R fusion protein may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:63.

[0181] In some variations, the NK cells of the present disclosure are modified to express OX40. OX40 (CD134) is a member of the tumor necrosis factor receptor (TNFR) family that plays a role in immune cell signaling and priming. Without being bound by theory, expression of OX40 may enhance activation of the MAPK / ERK, NFkB, and AKT pathways; and / or induce proliferation and cytokine release. OX40 has the polypeptide sequence: [ka] OX40 may have a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:61.

[0182] Any combination of the extracellular ligand binding domains, hinge domains, transmembrane domains, intracellular / cytoplasmic signaling domains, and co-expressed proteins described herein is anticipated to be within the scope of the CARs of the present disclosure.

[0183] (Polynucleotides and Vectors) The polynucleotides of the present disclosure can be delivered to cells as isolated nucleic acids or in vectors. In one aspect, the present disclosure provides a vector comprising a polynucleotide comprising a CAR of the present disclosure. In some embodiments, the vector comprises a polynucleotide encoding a co-expressed partner of the present disclosure. Additional elements present in the vector of the present disclosure can also include selectable markers, and other components required for protein expression, such as enhancers, promoters, and termination sequences. The polynucleotides of the present disclosure can include flanking left and right arms for homology-directed repair. The polynucleotides can include a promoter operably linked to the polynucleotide sequence encoding the CAR.

[0184] The polynucleotide of the present disclosure can also be used for gene editing.For example, the polynucleotide can be inserted into a desired locus using homology-directed repair.The polynucleotide can include promoters and other genetic elements, or the repair template can be omitted and instead rely on host genome sequences for transcription.

[0185] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Another method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0186] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells.Other viral vectors can be obtained from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0187] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0188] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence within the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot), or other assays.

[0189] Polynucleotides comprising CAR, or portions thereof, are provided. Polynucleotides encoding any combination of the extracellular ligand binding domains, hinge domains, transmembrane domains, intracellular / cytoplasmic signaling domains, and co-expressed proteins described herein are contemplated within the scope of the present disclosure.

[0190] (Modified cells) Methods for generating modified cells (e.g., stem cells, progenitor cells, or immune effector cells) are known to those of skill in the art. Methods for generating cells expressing a polypeptide described herein, e.g., a chimeric receptor or a fusion polypeptide, are known in the art. Exemplary methods are described herein.

[0191] In some embodiments, the present disclosure provides modified cells (e.g., modified stem cells, modified progenitor cells, or modified immune effector cells) that express a chimeric antigen receptor described herein. In some embodiments, the present disclosure provides modified cells (e.g., modified stem cells, modified progenitor cells, or modified immune effector cells) that comprise a polynucleotide sequence encoding a chimeric antigen receptor described herein. In some embodiments, the modified cells (e.g., modified stem cells, modified progenitor cells, or modified immune effector cells) are CISH knockout cells.

[0192] In some embodiments, the cell comprises a chimeric antigen receptor described herein comprising a ligand binding domain, a transmembrane domain, and an intracellular signaling domain comprising a TLR2 signaling domain. In some embodiments, the cell comprises a chimeric antigen receptor described herein comprising a ligand binding domain, a transmembrane domain, and an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 zeta signaling domain. In some embodiments, the cell comprises a chimeric antigen receptor described herein comprising a ligand binding domain, a transmembrane domain selected from the CD8 alpha chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2Rβ, TLR, IL-1R subfamily or a functional variant thereof, and an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 zeta signaling domain.

[0193] (Signaling domain) In some embodiments, the polynucleotide of the present disclosure comprises a sequence encoding a signaling domain of the present disclosure. In some embodiments, the polynucleotide encodes a TLR signaling domain. In some embodiments, the polynucleotide encodes an IL-1R subfamily signaling domain.

[0194] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR2 signaling domain. The polynucleotide sequence encoding the TLR2 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 12. The polynucleotide sequence encoding the TLR2 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 12.

[0195] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR8 signaling domain. The polynucleotide sequence encoding the TLR8 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 18. The polynucleotide sequence encoding the TLR8 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 18.

[0196] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR3 signaling domain. The polynucleotide sequence encoding the TLR3 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 13. The polynucleotide sequence encoding the TLR3 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13.

[0197] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR7 signaling domain. The polynucleotide sequence encoding the TLR7 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 17. The polynucleotide sequence encoding the TLR7 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17.

[0198] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR4 signaling domain. The polynucleotide sequence encoding the TLR4 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 14. The polynucleotide sequence encoding the TLR4 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 14.

[0199] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR1 signaling domain. The polynucleotide sequence encoding the TLR1 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 11. The polynucleotide sequence encoding the TLR1 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 11.

[0200] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR5 signaling domain. The polynucleotide sequence encoding the TLR5 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 15. The polynucleotide sequence encoding the TLR5 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15.

[0201] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR6 signaling domain. The polynucleotide sequence encoding the TLR6 signaling domain may comprise the nucleic acid sequence shown in SEQ ID NO: 16. The polynucleotide sequence encoding the TLR6 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16.

[0202] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR9 signaling domain. The polynucleotide sequence encoding the TLR9 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 19. The polynucleotide sequence encoding the TLR9 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 19.

[0203] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding a TLR10 signaling domain. The polynucleotide sequence encoding the TLR10 signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 20. The polynucleotide sequence encoding the TLR10 signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 20.

[0204] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding an IL-1R signaling domain. The polynucleotide sequence encoding the IL-1R signaling domain may comprise a nucleic acid sequence encoding the polypeptide set forth in SEQ ID NO: 21. The polynucleotide sequence encoding the IL-1R signaling domain may have a polynucleotide sequence encoding a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 21.

[0205] In some embodiments, the polynucleotide sequence encoding the signaling domain of the present disclosure comprises a nucleic acid sequence encoding an IL-18R signaling domain. The polynucleotide sequence encoding the IL-18R signaling domain may comprise the nucleic acid sequence set forth in SEQ ID NO: 24. The polynucleotide sequence encoding the IL-1R signaling domain may have a polynucleotide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 24. Table 5: Signaling domains [Table 5] TIFF2025510943000081.tif224170TIFF2025510943000082.tif224170TIFF2025510943000083.tif105170

[0206] (Other components of the CAR cytoplasmic portion) The cytoplasmic portion of the CAR may further comprise one or more of the domains set forth in Table 6, or a functional variant having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. Table 6: Additional cytoplasmic domains [Table 6]

[0207] In some embodiments, the chimeric receptor described herein comprises a cytoplasmic domain. In some embodiments, the cytoplasmic domain is operably linked to the TIR signaling domain using a linker. In some embodiments, the cytoplasmic domain is operably linked to the TIR signaling domain without a linker. In some embodiments, the N-terminus of the cytoplasmic domain is operably linked to the C-terminus of the TIR signaling domain. In some embodiments, the C-terminus of the cytoplasmic domain is operably linked to the N-terminus of the TIR signaling domain.

[0208] In some embodiments, the cytoplasmic domain is a 2B4 signaling domain. In some embodiments, the 2B4 cytoplasmic domain comprises the amino acid sequence set forth in SEQ ID NO:65. In some embodiments, the 2B4 cytoplasmic domain consists of the amino acid sequence set forth in SEQ ID NO:65. In some embodiments, the 2B4 cytoplasmic domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:65. In some embodiments, the 2B4 cytoplasmic domain is encoded by a nucleotide sequence set forth in SEQ ID NO:179. In some embodiments, the 2B4 cytoplasmic domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:179.

[0209] In some embodiments, the cytoplasmic domain is a CD3 zeta cytoplasmic domain. In some embodiments, the CD3 zeta cytoplasmic domain comprises the amino acid sequence set forth in SEQ ID NO:64. In some embodiments, the CD3 zeta cytoplasmic domain consists of the amino acid sequence set forth in SEQ ID NO:64. In some embodiments, the CD3 zeta cytoplasmic domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:64. In some embodiments, the CD3 zeta cytoplasmic domain is encoded by a nucleotide sequence set forth in SEQ ID NO:181. In some embodiments, the CD3 zeta cytoplasmic domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:181.

[0210] In some embodiments, the cytoplasmic domain is a 41BB cytoplasmic domain. In some embodiments, the 41BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, the 41BB cytoplasmic domain consists of the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, the 41BB cytoplasmic domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 176. In some embodiments, the 41BB cytoplasmic domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 177. In some embodiments, the 41BB cytoplasmic domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 177.

[0211] In some embodiments, the cytoplasmic domain is a CD28 cytoplasmic domain. In some embodiments, the CD28 cytoplasmic domain comprises the amino acid sequence set forth in SEQ ID NO:68. In some embodiments, the cytoplasmic domain is a CD28 cytoplasmic domain. In some embodiments, the CD28 cytoplasmic domain comprises the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, the CD28 cytoplasmic domain consists of the amino acid sequence set forth in SEQ ID NO:68. In some embodiments, the CD28 cytoplasmic domain consists of the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, the CD28 cytoplasmic domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:68. In some embodiments, the CD28 cytoplasmic domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 220. In some embodiments, the CD28 cytoplasmic domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 178. In some embodiments, the CD28 cytoplasmic domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 219. In some embodiments, the CD28 cytoplasmic domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 178. In some embodiments, the CD28 cytoplasmic domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:219.

[0212] (Co-expressed partners) The activity of NK cells can be increased by co-expressing various factors, including but not limited to those provided in Table 7. Table 7: Co-expression partners [Table 7]

[0213] Exemplary Chimeric Antigen Receptors In some embodiments, the chimeric antigen receptor described herein comprises a ligand binding domain, a transmembrane domain, and an intracellular signaling domain comprising a TLR2 signaling domain. In some embodiments, the chimeric antigen receptor described herein comprises a ligand binding domain, a transmembrane domain, and an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 zeta signaling domain. In some embodiments, the chimeric antigen receptor described herein comprises (a) a ligand binding domain, (b) a transmembrane domain selected from the group consisting of CD8 alpha chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2D, DNAM1, IL-2Rβ, TLR, IL-1R subfamily or a functional variant thereof, and (c) an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 zeta signaling domain. Illustrative, non-limiting examples include, for example, chimeric antigen receptors described herein comprising a ligand binding domain, an aTM domain, and an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 zeta signaling domain. Further exemplary chimeric antigen receptors comprise a ligand binding domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 zeta signaling domain.

[0214] (kit) In one aspect, the disclosure provides a kit comprising a CAR NK cell or pharmaceutical composition of the disclosure and instructions for use.

[0215] (How to use) In one aspect, the disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a CAR NK cell of the disclosure or a pharmaceutical composition of the disclosure.

[0216] In one aspect, the present disclosure provides a method of killing target cells comprising contacting a population of target cells with a natural killer cell of the present disclosure, wherein a chimeric antigen receptor of the natural killer cell comprises a ligand binding domain that specifically binds to an antigen on the target cell, and wherein the natural killer cell induces specific killing of the target cells.

[0217] In one aspect, the disclosure provides a method of killing target cells, the method comprising contacting a population of target cells with a population of NK cells.

[0218] Cell killing can be assessed by assays known in the art, including but not limited to those described in the Examples.

[0219] Cancer can result from the proliferation of tumor cells that present an antigen that is specifically bound by a CAR or a NK cell expressing a CAR. The CARs provided herein include CARs with a ligand-binding domain that specifically binds to CD19, CD20, and / or HER2. CD19 and CD20 can be expressed on tumor cells of various indolent and aggressive subtypes of B-cell lymphomas and leukemias, including non-Hodgkin's lymphoma (NHL), B-cell chronic lymphocytic leukemia (CLL), and non-T acute lymphoblastic leukemia (ALL). Thus, the CARs described herein can be used to kill CD19+ and / or CD20+ tumor cells and / or treat cancer in subjects with non-Hodgkin's lymphoma (NHL), B-cell chronic lymphocytic leukemia (CLL), and non-T acute lymphoblastic leukemia (ALL). HER2 expression is found in several tumor types, including, but not limited to, breast cancer, esophageal cancer, lung cancer, cervical cancer, endometrial cancer, and ovarian cancer. Thus, the CARs described herein can be used to kill HER2+ tumor cells and / or treat cancer in subjects with breast cancer, esophageal cancer, lung cancer, cervical cancer, endometrial cancer, and / or ovarian cancer.

[0220] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof comprising administering a population of NK cells, or a pharmaceutical composition comprising the population of NK cells.

[0221] The terms "subject," "patient," and "individual" are used interchangeably herein to refer to a vertebrate, e.g., a mammal, e.g., a human.

[0222] As used herein, the term "treatment" or "treating" includes at least an improvement in symptoms associated with a disease or condition in a patient, where improvement is used broadly to refer to a decrease in at least a parameter, e.g., the magnitude of a symptom, associated with the condition being treated. Thus, "treatment" or "treating" also includes situations in which a disease, disorder, or pathological condition, or at least a symptom associated therewith, is completely inhibited (e.g., prevented from occurring) or halted (e.g., terminated) such that the patient is no longer afflicted by the condition or at least the symptoms that characterize the condition.

[0223] In some embodiments, the method of treating cancer in a subject in need thereof comprises administering to the subject a therapeutically effective amount of the CAR NK cells and / or pharmaceutical compositions of the present disclosure.

[0224] As used herein, "therapeutically effective" refers to an amount of cells or pharmaceutical composition sufficient to treat or ameliorate, or in some way alleviate, the symptoms associated with a disease such as cancer.

[0225] In some embodiments, the CAR NK cells and pharmaceutical compositions comprising same described herein are used as part of a combination therapy, hi some embodiments, the CAR NK cells or pharmaceutical compositions of the present disclosure are co-administered to a subject with a tumor-targeting antibody.

[0226] In some embodiments, the CAR NK cells or pharmaceutical compositions of the present disclosure exhibit increased cytotoxicity compared to cells or pharmaceutical compositions in which the cells have not been modified with a method as disclosed herein. In some embodiments, the cells or pharmaceutical compositions of the present disclosure exhibit increased efficacy compared to comparable cells or pharmaceutical compositions in which the cells have not been modified with a method as disclosed herein.

[0227] The population of NK cells or pharmaceutical composition can be administered in an amount effective to kill target cells and / or in a therapeutically effective amount for the treatment of cancer.

[0228] Pharmaceutical Composition In one aspect, the disclosure provides a pharmaceutical composition comprising a CAR NK cell or cell population of the disclosure and one or more pharma- ceutically acceptable excipients or diluents.

[0229] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, in addition to other formulations that are safe for use in animals, and more particularly, in humans and / or non-human mammals.

[0230] As used herein, the term "pharmaceutical acceptable excipient or diluent" refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which the cells of the present disclosure are administered. The use of such vehicles and agents for pharmaceutical active substances is well known in the art. See, for example, Remington's The Science and Practice of Pharmacy, 20th Edition (Lippincott, Williams & Wilkins 2003).

[0231] The composition of the present invention may further contain other auxiliary components that are conventionally found in pharmaceutical compositions.Thus, for example, the composition may contain additional compatible medicament active materials, such as antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials that are useful for physically compounding various dosage forms of the composition.

[0232] (Manufacturing method) In one aspect, the disclosure provides a method of generating NK cells, comprising providing stem cells or progenitor cells, such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), and differentiating the stem cells or progenitor cells into NK cells. The stem cells or progenitor cells can be genetically modified to include a polynucleotide encoding a CAR before or after stem differentiation. Known vectors can be used to deliver polynucleotides that can be stably maintained by the cells and / or integrated into the genome of the cells. The stem cells or progenitor cells are CISH- / - iPSCs and can be used to generate CISH- / - iPSC-NK cells. The stem cells or progenitor cells are soluble IL-15 or membrane-bound IL-15 knock-in iPSCs and can be used to generate IL-15 knock-in iPSC-NK cells. In some embodiments, the iPSCs can be edited to not express CISH, to express soluble IL-15 or membrane-bound IL-15, and to express any one or more of the CAR constructs disclosed herein.

[0233] (cancer treatment) In some embodiments, the present disclosure provides a method for treating cancer with the chimeric receptor-expressing cells provided herein or a pharmaceutical composition or formulation thereof. In some embodiments, the method includes administering the chimeric receptor-expressing cells provided herein, the pharmaceutical composition or formulation described herein to a subject in need thereof. As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and intramuscular, or a combination thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0234] In some embodiments, cells expressing the chimeric receptors described herein exhibit enhanced anti-tumor efficacy compared to cells lacking the chimeric receptor. Methods for measuring anti-tumor efficacy in vitro and in vivo are known to those skilled in the art and include, for example, in vitro killing assays and tumor implantation in mouse models.

[0235] Cell killing can be assessed by assays known in the art, including but not limited to those described in the Examples.

[0236] In some embodiments, cells expressing a chimeric receptor described herein exhibit about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 1000% enhanced cell killing compared to cells lacking the chimeric receptor and / or fusion polypeptide.

[0237] In some embodiments, cells expressing a chimeric receptor described herein exhibit 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 100%-1000% enhanced cell killing compared to cells lacking the chimeric receptor and / or fusion polypeptide.

[0238] In some embodiments, cells expressing a chimeric receptor described herein exhibit about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 1000% enhanced specific killing compared to cells lacking the chimeric receptor and / or fusion polypeptide.

[0239] In some embodiments, cells expressing a chimeric receptor described herein exhibit 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, or 100%-1000% enhanced specific killing compared to cells lacking the chimeric receptor and / or fusion polypeptide.

[0240] (definition) All publications, patents, and patent applications, including any drawings and appendices contained therein, are incorporated by reference in their entirety for all purposes to the same extent as if each such publication, patent, and patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0241] The reference in this specification to any prior publication (or information derived therefrom) or to any publicly known matter is not intended to be, and should not be construed as, an acknowledgment or admission or any form of suggestion that the prior publication (or information derived therefrom) or publicly known matter forms part of the common general knowledge in the field to which this specification pertains.

[0242] All embodiments of any aspect of the invention can be used in combination unless the context clearly indicates otherwise.

[0243] Unless the context requires otherwise, throughout this description and the claims, the words "comprise", "comprising", and the like are to be interpreted in an inclusive sense; that is, in the sense of "including, but not limited to," as opposed to an exclusive or exhaustive sense.

[0244] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "and" is used interchangeably with "or" unless expressly stated otherwise. Words using the singular or plural number also include the plural and singular number, respectively.

[0245] The section headings are for convenience only, and combinations of elements from different sections are envisioned. Further, the words "herein," "above," "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0246] The term "functional variant" refers to a homology (by sequence or structure) of a domain that retains sufficient signaling activity to activate NK cells.

[0247] The term "sequence identity" refers to the percentage of identity of a polypeptide or polynucleotide sequence of interest to a reference sequence, calculated as the number of perfect matches in an optimal alignment of the sequence of interest to the reference sequence divided by the total length of the reference sequence (including gaps), multiplied by 100. Optimal alignments of sequences can be generated using the Needle program of the European Molecular Biology Open Software Suite (EMBOSS), available at www.ebi.ac.uk, as described in Maderia et al., Nucleic Acids Res. 47(W1): W636-W641 (2019).

[0248] As used herein, the term "inactivating mutation" refers to a mutation in a genomic sequence that disrupts the function of a gene. Inactivating mutations can occur in any sequence region (e.g., coding or non-coding) that contributes to gene expression. Examples include, but are not limited to, cis-acting elements (enhancers) or sequences that are subject to transcription (e.g., mRNA transcription sequences). Inactivating mutations include mutations that render a gene or its encoded protein non-functional or that reduce the function of a gene or its encoded protein. EXAMPLES

[0249] (Example) The present disclosure will now be described with reference to the following examples, which are provided for illustrative purposes only, and the present disclosure should in no way be construed as being limited to these examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0250] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and use the presently disclosed method and practice the claimed method. The following working examples therefore, specifically point out embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0251] The materials and methods utilized in these experiments will now be described.

[0252] Example 1: Screening of chimeric antigen receptor designs In this example, the design and testing of chimeric antigen receptors in induced pluripotent stem cell (iPSC)-derived natural killer (NK cells) is described. We constructed a library of 44 novel CAR constructs containing signaling modules derived from diverse NK cell-specific signaling receptors. We then developed a virus-based CAR expression protocol in mature iPSC-derived NK cells that resulted in high CAR expression (>75% CAR+) while maintaining high viability (>90%). We then screened for CAR activity using two co-culture target cell killing assays (eSight impedance assay and caspase 3 / 7 killing assay) and two NK cell-resistant CD19+ target cell lines (Raji and SupB15). The tested constructs, all with anti-CD19 scFv as the ligand-binding domain, are provided as SEQ ID NOs: 100-117. Our screen identified seven novel CAR signaling modules that outperformed both a second generation T cell CAR (CD28-Zeta) and three previously described NK cell-CARs (2B4-Zeta, OX40-Zeta, and OX40L-Zeta). These results were consistent across both functional assays and both target cell lines tested, lending support to our findings.

[0253] The designed CAR sequence was linked at the C-terminus to membrane-bound IL-15 (mbIL-15) via a self-cleaving (2A) peptide for co-expression in natural killer cells. Lentiviral particles were generated and concentrated ~100-fold to facilitate the high viral titers required to transduce iPSC-derived NK cells.

[0254] The concentrated virus stock was loaded by centrifugation onto a retronectin-coated 96-well plate, and IL-2-activated iPSC-derived NK cells were added, centrifuged in the presence of the virus supernatant, and incubated in the presence of IL-2 for 72 hours. CAR expression was detected by flow cytometry using the HA-tag incorporated into the extracellular portion of the CAR polypeptide (Figure 9). CAR expression was uniform across most CAR constructs, with an average CAR expression of ~78% (Figure 10). We did not observe any loss of cell viability when comparing CAR-transduced cells with untransduced controls.

[0255] We used two co-culture target cell killing assays (eSight impedance assay and caspase 3 / 7 killing assay) and two NK cell-resistant CD19 + The target cell lines (Raji and SupB15) were used to screen for CAR activity. The results of the screening are shown in Figures 11 and 12. We identified six CAR constructs that outperformed the best comparator CAR (2B4-zeta). Twelve outperformed another comparator CAR (OX40L-zeta). All but one of the tested CARs outperformed a third comparator CAR (OX40-zeta).

[0256] Selected data comparing CAR designs with and without the 2B4 domain are shown in Figures 13A-13C. 2B4-based CARs have been shown to be effective in activating NK cells. Surprisingly, our CAR TLR2-Zeta outperforms both the benchmark 2B4-Zeta and our CAR design that combines both the 2B4 and TLR2 domains (2B4-Zeta) in both assays (Figures 13B and 13C).

[0257] Overall, we have successfully developed a CAR screening platform in a therapeutically important allogeneic cell source and identified NK-optimized CARs that enable increased NK cell potency compared to current CAR solutions. Of note, intracellular signaling modules derived from Toll-like receptors and IL-1R subfamily receptors showed enhanced NK CAR activity compared to CARs containing standard NK cell activating receptor domains used in previously developed NK-optimized CAR designs - i.e., 2B4 (shown), FCER1G, DNAM1, DAP10. These findings support the use of diverse immune cell signaling domains in NK-optimized CARs.

[0258] (method) (Construction of lentiviral expression vector and viral packaging:) Polypeptide sequences of all candidate CAR components were obtained from the Ensemble™ database (Howe et al., Nucleic Acids Res. 2021, vol. 49(1):884-891) and assembled into a complete CAR design using SnapGene™ molecular biology software. Nucleic acid sequences were determined using a publicly available codon optimization tool (Integrated DNA Technologies™). All CAR constructs contained a CD19 scFv binder (FMC63; SEQ ID NO: 109) as a representative ligand-binding domain, an mbIL-15 co-expression partner (SEQ ID NO: 48), and an HA-tag for CAR detection. CAR constructs were synthesized and cloned into a lentiviral expression vector (pCDH-EF1α-MCS-T2A-Puro; System Biosciences™) under the EF-1 alpha promoter, maxipreps were generated, and sequences were confirmed. Lentiviral particles were generated using the above constructs, packaging plasmids (Rev, gag-pol, and VSVG; Cell Biolabs™), and the Lenti-X™ 293T cell line (Takara™). Viral supernatants were concentrated 100-fold using a PEG-based concentrating solution (Takara™), and viral titers were determined using Lenti-X GoStix Plus™ (Takara™).

[0259] (Generation of iPSC-NK cells:) iPSCs are harvested and transferred to culture in serum-free medium and formed into embryoid bodies by spin aggregation. On day 6, CD34 +Once cells emerge, EBs are transferred to NK cell differentiation medium containing a 2:1 mixture of Dulbecco's modified Eagle's medium / Ham's F12, 2 mM L-, 1% penicillin / streptomycin, 25 mM b-mercaptoethanol, 20% heat-inactivated human serum AB, 5 ng / mL sodium selenite, 50 mM ethanolamine, 20 mg / mL ascorbic acid, interleukin-3; for the first week only, stem cell factor, interleukin-15, Fms-like tyrosine kinase 3 ligand, and interleukin-7. EBs are left in these conditions for 28 days, receiving weekly medium changes, to differentiate into NK cells (as determined by flow cytometry, CD45 + CD56 + CD33 - CD3 - The NK cells are then harvested for expansion.

[0260] (Proliferation:) After differentiation, the proliferation of NK cells is stimulated using the immortalized leukemia cell line K562, which expresses mbIL21 and 4-1BB ligand (CD137). NK cells are seeded with K562-41BBL-mbIL21 feeder cells and incubated at a 1:2 ratio in stromal cell growth medium (SCGM) containing 100 U / mL IL-2. Non-adherent cells are removed and analyzed by flow cytometry to determine the purity of CD56+ NK cells. These cells are then stimulated 2:1 with aAPC (irradiated at 10,000 Gy) to a concentration of 350,000 NK cells per mL of medium containing RPMI 1640, 2 mM L-glutamine, 1% penicillin / streptomycin, 1% non-essential amino acids, and 10% standard FBS or 10% human serum AB) supplemented with 50-100 U / mL IL-2.

[0261] (Transduction of iPSC-NK cells:) Untreated 96-well plates were coated with RetroNectin (Takara). An equal amount of concentrated lentivirus was added to the plate and centrifuged at 2000×g at 32° C. for 2 h. iPSC-derived NK cells (6e4 cells) were added to each well in medium containing IL-2 (50 U / ml) and centrifuged at 800×g at 32° C. for 1 h. Cells were incubated at 37° C. for 48 h. Fresh medium (with IL-2) was added and cells were incubated for an additional 24 h. CAR expression was determined 72 h after transduction using flow cytometry to detect the HA-tag incorporated into the extracellular domain of each CAR. CAR+ cells were used for functional assays within 24 h after CAR detection.

[0262] Long-Term Target Cell Killing Assay Using eSight™: RTCA E-plates were coated overnight at 4°C with CD40 binding reagent (Agilent™) diluted in PBS. Plates were washed with PBS and target cells (either CD19+ target cells (Raji; ATCC™) or HER2+ target cells (BT474 clone 5) were added to each well and impedance readings were allowed to equilibrate for 2-4 hours. CAR+ iPSC-NK cells were added to each well at the indicated E:T ratio and impedance readings were measured every 15 minutes for 24 hours. NK-specific killing was determined by normalizing impedance readings to "target cell only" control wells and plotted as frequency of target killing.

[0263] (4 hour caspase 3 / 7 cytotoxicity assay:) SupB15 (CD19+ target cells) were pre-stained with CellTrace™ Violet at a final concentration of 5 μM in PBS for 15 min at 37° C. and then washed in complete culture medium. CAR+ iPSC-NK cells were added to each well at an E:T ratio of 2:1 in a final volume of 100 μl. Co-cultures were centrifuged at 100×g for 1 min and incubated at 37° C. for 3.5 h. After incubation, CellEvent® Caspase-3 / 7 Green Detection Reagent and SYTOX™ AADvanced™ Dead Cell Stain Solution were added to the co-cultures and incubated for an additional 30 min at 37° C. After staining, the assay was read on a NovoCyte Advanteon and analyzed by FlowJo software. NK-specific killing was determined by normalizing impedance readings to “target cell only” control wells and plotted as frequency of target killing.

[0264] Example 2: In vivo functional testing of CARs with CD19 binding domains iPSC-derived NK cells expressing functional CAR constructs (as determined by in vitro assay results) are tested for efficacy in vivo using CD19-expressing models of B-cell malignancies. To initiate disease, 10-12 week old NOD-SCID-gamma mice are cultured in vitro. - / - (NSG) mice (Jackson Laboratory) are injected intravenously with luciferase-expressing Raji cells (CD19 positive). One day later, mice are administered iPSC-derived NK cells engineered to express (a) no CAR (negative control), (b) a second generation T cell CAR (benchmark control), or (c) an NK CAR (test group). Subject mice are administered IL-2 every other day for the remainder of the experiment. Disease progression is monitored by bioluminescence imaging of survival, weight loss, and tumor burden (measured weekly). Peripheral blood-derived CD45 NK cells were analyzed to assess the number of infused NK cells present over the course of the experiment. + CD56 + CD3 - Cells are quantified weekly.

[0265] Example 3: Functional testing of CARs with HER2 binding domains Concentrated lentiviral stocks containing different CARs with HER2 scFv binders were loaded onto retronectin-coated 96-well plates by centrifugation. IL-2-activated iPSC-derived NK cells were added, centrifuged in the presence of viral supernatant, and incubated in the presence of IL-2 for 72 hours. CAR expression was detected by flow cytometry using the HA-tag incorporated into the extracellular portion of the CAR polypeptide. CAR expression was uniform across most CAR constructs (Figure 14A).

[0266] CAR activity in NK cell-resistant HER2 + The CARs were screened using a co-culture target cell killing assay (eSight impedance assay) against a target cell line (BT474). The results of the screening are shown in Figure 14B and Figure 14D. In addition, a cytokine secretion assay was performed using ELISA on the co-culture supernatant. The results of the secretion assay are shown in Figure 14C. The results confirmed that CD8-aTM-TLR2-CD3ζ (SEQ ID NO: 82) was the best among the NK-optimized CARs tested, and consistently performed as well / better than the others across multiple CAR targets and assays. Furthermore, similar levels of sIL15 (150-200 pg / ml) were observed between (1) CD8-aTM-TLR2-CD3ζ, (2) CD8-aTM-DNAM1-LFA1-CD3ζ, and (3) 2B4-Zeta (lower at ~100 pg / ml for aTM-IL18R-2B4-CD3ζ) (data not shown).

[0267] Similarly, the CD8-aTM-TLR2-CD3ζ construct carrying the CD19 scFv demonstrated potent cell killing of Raji 2.0 cells at multiple effector-to-target cell ratios (data not shown). The CD8-CD28TM-TLR2-CD3ζ construct also demonstrated similar activity.

[0268] Next, CAR-HER2 activity was screened using a stress test killing assay. Briefly, CISH KO iPSC-derived NK cells were retrovirally transduced with one of three different CAR constructs with the same HER2 scFv binding domain (HER2v3) and different intracellular domains (Figure 15A). In a second experiment, CISH KO iPSC-derived NK cells were retrovirally transduced with one of three different CAR constructs with the same intracellular domain and different HER2 scFv binding domain (HER2v3) (Figure 15B). Effector CISH KO iPSC-derived NK cells were transduced with NK cell-resistant HER2 at an effector cell to target cell ratio of 5:1 without the addition of cytokines or co-expression of sIL-15. + By co-culturing with a target cell line (BT474) and utilizing a target cell killing assay (eSight impedance assay), we identified which CAR constructs exhibited high killing activity.

[0269] Using different CAR constructs with the same HER2 scFv binding domain (HER2v3), CD8-aTM-TLR2-CD3ζ (SEQ ID NO: 82) was found to be the best of the NK-optimized CAR-HER2 constructs tested (Figure 15A). Using the same intracellular domain with different HER2 scFv binding domains (CD8-aTM-TLR2-CD3ζ), HER2 scFv variant 3 (v3) was found to be the best of the NK-optimized CAR-HER2 constructs tested (Figure 15B).

[0270] The CAR activity of CD8-aTM-TLR2-CD3ζ CAR constructs with different HER2 scFv binding domains was also assessed in NK cell-resistant HER2 +Caspase 3 / 7 killing assays using the target cell line (BT474) were assessed (Figures 16A-16B). Briefly, caspase 3 / 7 activation was measured after co-culture (2:1 E:T) of CISH KO iPSC-derived NK cells expressing one of three different CAR constructs with the same intracellular domain and different HER2 scFv binding domains (HER2v2, HER2v3, or HER2v5) with resistant solid tumor targets (Figure 16A). Representative images of target cell killing and caspase 3 / 7 activation after 30 h of co-culture of CISH KO iPSC-derived NK cells + / - CAR with NK cell-resistant solid tumor targets showed robust NK cell killing upon co-culture of CISH KO iPSC-derived NK cells expressing CAR, but not without CAR (Figure 16B). Target cells were labeled with Cell Tracker Red and caspase 3 / 7 activation was visualized in green.

[0271] Next, the killing activity of CISH KO iPSC-derived NK cells expressing the CD8-aTM-TLR2-CD3ζ CAR and one of three different HER2 scFv binding domains (HER2v2, HER2v3, or HER2v5) was tested in 3D single tumor spheroids. Briefly, human HER+ ductal carcinoma cells, BT474 cells, were seeded and cultured using a standard protocol for spheroid formation. After spheroids were formed (approximately 36 hours after seeding), the cells were co-cultured with CISH KO iPSC-derived NK cells that: 1) did not express a CAR; 2) expressed a HER2v2-CD8-aTM-TLR2-CD3ζ CAR; 3) expressed a HER2v3-CD8-aTM-TLR2-CD3ζ CAR; or 4) expressed a HER2v5-CD8-aTM-TLR2-CD3ζ CAR. Figure 17 shows representative images of NK cell-resistant solid tumor targeting spheroids at 0, 18, and 36 hours after treatment. Collectively, these data demonstrated that CAR-HER2 NKs, particularly those expressing HER2v3-CD8-aTM-TLR2-CD3ζ, potently killed HER2+ target cancer cells.

[0272] A serial cell killing assay (eSight impedance assay) was performed in real time using iPSC-derived NK cells transduced with the HER2v3-CD8-aTM-TLR2-CD3ζ construct. Briefly, by repeatedly adding HER2v3-CD8-aTM-TLR2-CD3ζ CISH KO iPSC-derived NK cells to the target cells, a repeated killing and depletion profile of a HER2+ target cell line (SKOV3) was observed. As shown in Figure 18, HER2v3-CD8-aTM-TLR2-CD3ζ NK cells maintained a high percentage of NK cell killing capacity after three rounds, whereas NK cells without a CAR (untransduced, or "UT") no longer killed cells after two rounds.

[0273] The killing activity of CISH KO iPSC-derived NK cells expressing HER2v3-CD8-aTM-TLR2-CD3ζ was tested in 3D single HER+ tumor spheroids. After spheroids were formed, target cells were co-cultured with different ratios of either 1) CISH KO iPSC-derived NK cells not transduced with a CAR construct (untransduced NK or "UT") or 2) CISH KO iPSC-derived NK cells expressing HER2v3-CD8-aTM-TLR2-CD3ζ. The ratio of effector cells (untransduced CISH KO iPSC-derived NK cells or HER2v3-CD8-aTM-TLR2-CD3ζ CISH KO iPSC-derived NK cells) to target cells (BT474 cells) added was either 5:1, 2.5:1, or 1.25:1. Spheroid cell killing was monitored 0 and 3 days after addition of NK cells. FIG. 19 shows that target BT474 cells in HER+ spheroids treated with NK expressing HER2v3-CD8-aTM-TLR2-CD3ζ shrank and underwent apoptosis in a concentration-dependent manner on day 3.

[0274] To investigate the effect of sIL-15 on CAR-HER2 activity, a CAR expression assay (Figure 23A) and a long-term killing assay (Figure 23B) were performed on cell line BT474 clone 5. Expression of sIL-15 did not affect CAR expression, but expression of sIL-15 (HER2-3-CD8-aTM-TLR2-CD3ζ-sIL15 (SEQ ID NO: 205)) dramatically increased CAR NK cell killing of BT474 cells compared to controls (CD20-2b4-zeta-sIL15 and HER2-3-CD8-aTM-TLR2-CD3ζ without sIL15 (SEQ ID NO: 204)). After about 10 hours, about 80% NK cell killing was observed for HER2-3-CD8-aTM-TLR2-CD3ζ-sIL15 (SEQ ID NO: 205), whereas only about 55% and 30% were observed for CD20-2b4-zeta-sIL15 and HER2-3-CD8-aTM-TLR2-CD3ζ without sIL15 (SEQ ID NO: 204), respectively. These results indicate that sIL-15 has a synergistic effect with the HER2-CAR construct in NK cell killing of HER+ breast cancer cell lines.

[0275] Taken together, these results indicate that CISH KO iPSC-derived NK cells expressing HER2-CD8-aTM-TLR2-CD3ζ CAR exhibit potent activity against NK cell-resistant solid tumor target cells.

[0276] Example 4. Functional testing of HER2-CAR CISH KO iPSC-derived NK cells This example describes that testing of CISH KO iPSC-derived NK cells expressing HER2 scFv-CAR and soluble IL-15 demonstrated potent killing of solid tumor target cell lines.

[0277] First, triple edited (TE) iPSCs were generated using a gene editing method by introducing a HER2-CAR sequence linked to soluble IL-15 (sIL-15) at the C-terminus via a self-cleaving (2A) peptide into CISH KO iPSCs. The resulting TE iPSCs 1) did not express the CISH gene (CISH KO), 2) expressed HER2v3-CD8-aTM-TLR2-CD3ζ (HER-CAR), and 3) expressed soluble IL-15 (SEQ ID NO: 58) that allowed IL-15 secretion (Figure 20). The TE iPSCs were then differentiated to form functional NK cells (TE iPSC-derived NK cells) that lacked CISH, expressed HER-CAR, and exhibited IL-15 secretion.

[0278] To determine whether triple editing of iPSCs results in a change in NK phenotype, NK cell marker expression was measured in TE iPSC-derived NK cells by flow cytometry. As shown in Figure 21, expression of 12 NK phenotype markers was not significantly different between wild-type (WT) NK cells, NKs differentiated from iPSCs with only CISH KO, and NKs differentiated from TE iPSCs evaluated.

[0279] The function of TE iPSC-derived NK cells was evaluated in a spheroid killing assay. Briefly, HER2+ (BT474 clone 5) target cell spheroids were prepared as described in the Examples herein. Either wild-type (WT) NK cells, NK cells differentiated from iPSCs with only CISH KO, or NK cells differentiated from TE iPSCs were added to the HER2+ spheroids (E:T of 10:1). NK-mediated killing of HER2+ spheroids, monitored using eSight impedance assay, demonstrated that TE iPSC-derived NK cells were effective in killing NK-resistant HER2+ solid tumor targets (Figure 22A). Figure 22 shows that spheroids treated with TE iPSC-derived NK cells were significantly reduced in size after 3 days compared to spheroids treated with WT NK or CISH KO iPSC-derived NK cells.

[0280] Taken together, these data demonstrate that TE iPSC-derived NK cells potently kill solid tumor target cell lines.

[0281] While the invention has been described with respect to suggested specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and as may be applied to the essential features described above and as set forth in the appended claims.

Claims

1. 1. A natural killer cell comprising a chimeric antigen receptor (CAR) comprising an extracellular portion, a transmembrane domain, and a cytoplasmic portion, The natural killer cell, wherein the cytoplasmic portion comprises a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

2. The cell of claim 1, wherein the TIR signaling domain is a Toll-like receptor (TLR) signaling domain.

3. The cell of claim 2 , wherein the TLR signaling domain is a TLR2 signaling domain or a functional variant thereof.

4. The cell of claim 2 or claim 3, wherein the TLR signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

2.

5. The cell of claim 2 , wherein the TLR signaling domain is a TLR8 signaling domain or a functional variant thereof.

6. The cell of claim 2 or claim 5, wherein the TLR signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

8.

7. The cell of claim 2 , wherein the TLR signaling domain is a TLR3 signaling domain or a functional variant thereof.

8. The cell of claim 2 or claim 7, wherein the TLR signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

3.

9. The cell of claim 2 , wherein the TLR signaling domain is a TLR7 signaling domain or a functional variant thereof.

10. The cell of claim 2 or claim 9, wherein the TLR signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

7.

11. The cell of claim 2 , wherein the TLR signaling domain is a TLR4 signaling domain or a functional variant thereof.

12. The cell of claim 2 or claim 11, wherein the TLR signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

4.

13. The cell of claim 2 , wherein the TLR signaling domain is selected from the group consisting of a TLR1, TLR5, TLR6, TLR9, or TLR10 signaling domain or a functional variant thereof.

14. The cell of claim 2 or claim 13, wherein the TLR signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 1, 5, 6, 9, or 10.

15. The cell of claim 1, wherein the TIR signaling domain is an interleukin-1 receptor (IL-1R) subfamily signaling domain.

16. The cell of claim 15, wherein the IL-1R subfamily signaling domain is an interleukin-1 receptor (IL-1R) signaling domain or a functional variant thereof.

17. The cell of claim 15 or claim 16, wherein the IL-1R subfamily signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

21.

18. The cell of claim 15, wherein the IL-1R subfamily signaling domain is an interleukin-18 receptor (IL-18R) signaling domain or a functional variant thereof.

19. The cell of claim 15 or claim 18, wherein the IL-1R subfamily signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

22.

20. 16. The cell of claim 15, wherein the IL-1R subfamily signaling domain is selected from the group consisting of interleukin-1 receptor 4 (IL-1R4), interleukin-1 receptor 6 (IL-1R6), interleukin-1 receptor 3 (IL-R3), interleukin-1 receptor 7 (IL-1R7), interleukin-1 receptor 8 (IL-R8), interleukin-1 receptor 9 (IL-1R9), or interleukin-1 receptor 10 (IL-1R10) signaling domain, or a functional variant thereof.

21. 21. The cell of claim 15 or claim 20, wherein the IL-1R subfamily signaling domain has a polypeptide sequence that is at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 143, 144, 145, 146, 147, 148, or 149.

22. The cell of any one of claims 1 to 21, wherein the cytoplasmic portion comprises a CD3ζ signaling domain.

23. The cell of any one of claims 1 to 22, wherein the cytoplasmic portion comprises a 2B4 signaling domain.

24. The cell of any one of claims 1 to 22, wherein said cytoplasmic portion lacks the 2B4 signaling domain.

25. The cell of claim 22, wherein the cytoplasmic portion essentially consists of, in N-terminal to C-terminal order, a TLR2 signaling domain and a CD3 zeta signaling domain.

26. The cell of claim 22, wherein the cytoplasmic portion consists essentially of, in N-terminal to C-terminal order, a 2B4 signaling domain, an IL-18R signaling domain, and a CD3 zeta signaling domain.

27. The cell of claim 22, wherein the cytoplasmic portion essentially consists of, in N-terminal to C-terminal order, a 2B4 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain.

28. The cell of claim 22, wherein the cytoplasmic portion essentially consists of, in N-terminal to C-terminal order, a 2B4 signaling domain, a TLR2 signaling domain, and a CD3 zeta signaling domain.

29. The cell of any one of claims 1 to 28, wherein the extracellular protein comprises a ligand binding domain.

30. The cell of claim 29 , wherein the ligand binding domain comprises an antibody-like domain.

31. The cell of claim 30, wherein the ligand binding domain comprises a heavy chain variable (VH) domain and / or a light chain variable (VL) domain.

32. The cell of claim 30, wherein the ligand binding domain comprises a single chain variable fragment (scFv).

33. The cell of any one of claims 29 to 32, wherein the ligand binding domain specifically binds to a CD19 antigen.

34. The cell of claim 33, wherein the ligand binding domain comprises CDR-H1 according to SEQ ID NO: 76; CDR-H2 according to SEQ ID NO: 77; CDR-H3 according to SEQ ID NO: 78; CDR-L1 according to SEQ ID NO: 79; CDR-L2 according to SEQ ID NO: 80; and / or CDR-L3 according to SEQ ID NO:

81.

35. The cell of claim 33, wherein the ligand binding domain comprises a VL according to SEQ ID NO: 74 and / or a VL according to SEQ ID NO: 75, or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical thereto.

36. The cell of claim 33, wherein the ligand binding domain comprises an scFv according to SEQ ID NO: 73, or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical thereto.

37. The cell of any one of claims 29 to 32, wherein the ligand binding domain specifically binds to a CD20 antigen.

38. The cell of claim 37, wherein the ligand binding domain comprises an scFv according to SEQ ID NO: 139, or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical thereto.

39. The cell of any one of claims 29 to 32, wherein the ligand binding domain specifically binds to a HER2 antigen.

40. 40. The cell of claim 39, wherein the ligand binding domain comprises an scFv according to SEQ ID NO: 140-142, or a functional variant thereof having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical thereto.

41. The transmembrane domain has the polypeptide sequence 【Chemistry 1】 or a variant having one, two, three, four, five or more substitutions.

42. 42. The cell of any one of claims 1 to 41, wherein the transmembrane domain is DNAM1, CD28, IL-2Rβ, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, IL-1R, or IL-18R transmembrane domain, or a functional variant thereof.

43. The cell of any one of claims 1 to 42, wherein the CAR further comprises a hinge domain, optionally wherein the hinge domain is a CD8a hinge, a CD28 hinge, or an IgG4 hinge.

44. The cell of any one of claims 1 to 43, wherein the cell comprises a homozygous inactivating mutation in a cytokine-inducible SH2-containing protein (CISH) gene of said cell.

45. The cell of any one of claims 1 to 44, wherein the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell.

46. 46. ​​A pharmaceutical composition comprising the cells of any one of claims 1 to 45 and a pharma- ceutically acceptable solution.

47. 46. ​​A method for killing target cells, comprising contacting a population of target cells with a population of natural killer cells according to any one of claims 1 to 45, wherein the chimeric antigen receptor of the natural killer cell comprises a ligand binding domain that specifically binds to an antigen on the target cell; and wherein the natural killer cells induce specific killing of the target cells. The method.

48. 47. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 46.

49. Use of a natural killer cell or a pharmaceutical composition according to any one of claims 1 to 46 for the treatment of cancer.

50. A chimeric antigen receptor contained in any of the natural killer cells according to any one of claims 1 to 45.

51. A polynucleotide comprising a polynucleotide sequence encoding a chimeric antigen receptor contained in any one of the natural killer cells according to any one of claims 1 to 45.

52. 52. A vector comprising the polynucleotide of claim 51.

53. 52. A stem or progenitor cell comprising the polynucleotide of claim 51.

54. 52. A method for producing a natural killer cell, comprising providing a stem or progenitor cell comprising the polynucleotide of claim 51 and differentiating the stem or progenitor cell into a natural killer cell.

55. 52. A natural killer cell produced by differentiating a stem or progenitor cell comprising the polynucleotide of claim 51 into a natural killer cell.

56. The cell of any one of claims 29 to 32, wherein the ligand binding domain specifically binds to a CD19 antigen, a CD20 antigen, a Her2 antigen, or a BCMA antigen.

57. A chimeric antigen receptor (CAR) comprising an extracellular portion, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises a Toll / Interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

58. The CAR of claim 57, wherein the TIR signaling domain is a Toll-like receptor (TLR) signaling domain.

59. The CAR of claim 58, wherein the TLR signaling domain is a TLR2 signaling domain or a functional variant thereof.

60. The CAR of claim 58 or 59, wherein the TLR signaling domain has a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

2.

61. The CAR of any one of claims 57 to 60, wherein the cytoplasmic portion comprises a CD3 zeta signaling domain.

62. The CAR of any one of claims 57-61, wherein said cytoplasmic portion comprises a 2B4 signaling domain.

63. The CAR of any one of claims 57-61, wherein said cytoplasmic portion lacks the 2B4 signaling domain.

64. The CAR of any one of claims 57 to 61, wherein the cytoplasmic portion consists essentially of, in N-terminal to C-terminal order, a 2B4 signaling domain, an IL-18R signaling domain, and a CD3 zeta signaling domain.

65. The CAR of any one of claims 57 to 61, wherein the cytoplasmic portion consists essentially of, in N-terminal to C-terminal order, a 2B4 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain.

66. The CAR of any one of claims 57 to 61, wherein the cytoplasmic portion consists essentially of, in N-terminal to C-terminal order, a 2B4 signaling domain, a TLR2 signaling domain, and a CD3 zeta signaling domain.

67. The CAR of any one of claims 57 to 66, wherein the extracellular protein comprises a ligand binding domain.

68. The CAR of claim 67, wherein the ligand binding domain comprises an antibody-like domain.

69. The CAR of claim 67, wherein the ligand binding domain comprises a heavy chain variable (VH) domain and / or a light chain variable (VL) domain.

70. The CAR of claim 67, wherein the ligand binding domain comprises a single chain variable fragment (scFv).

71. The CAR of any one of claims 57 to 70, wherein the ligand binding domain specifically binds to a CD19 antigen, a CD20 antigen, a Her2 antigen, or a BCMA antigen.

72. (i) the extracellular domain comprises a polypeptide derived from a CD19 ligand binding domain, a CD20 ligand binding domain, a BCMA binding domain, or a HER2 binding domain; (ii) the transmembrane domain is a transmembrane domain selected from the group consisting of aTM, CD8 alpha chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2Rβ, TLR, and IL-1R subfamily; and (iii) the cytoplasmic domain comprises a signaling domain of TLR2, 41BB, or 2B4; The CAR of claim 57.

73. The CAR of claim 72, wherein the cytoplasmic domain further comprises a signaling domain of CD3zeta.

74. (i) an aTM transmembrane domain, a TLR2 signaling domain, and a CD3 zeta signaling domain; or (ii) the CD28 transmembrane domain, the TLR2 signaling domain, and the CD3 zeta signaling domain 58. The CAR of claim 57, comprising:

75. The CAR of any one of claims 57-74, wherein the C-terminus of the extracellular domain is operably linked to the N-terminus or C-terminus of the transmembrane domain without a linker.

76. The CAR of any one of claims 57-74, wherein the C-terminus of the extracellular domain is operably linked to the N-terminus or C-terminus of the transmembrane domain using a linker or hinge domain.

77. The CAR of claim 76, wherein the hinge domain is a CD8a hinge.

78. The CAR of claim 77, wherein the CD8a hinge comprises the amino acid sequence set forth in SEQ ID NO:

158.

79. The CAR of any one of claims 57-78, wherein the N-terminus or C-terminus of the transmembrane domain is operably linked, with or without a linker, to the N-terminus of the cytoplasmic domain.

80. The CAR of claim 79, wherein the transmembrane domain is operably linked to the cytoplasmic domain with a Gly-Ser linker.

81. A polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 57 to 80.

82. 82. A population of cells comprising an immune cell comprising the chimeric receptor of any one of claims 57 to 80 or the polynucleotide of claim 81.

83. 83. The population of cells of claim 82, wherein said immune cells are natural killer (NK) cells.

84. 84. The population of cells of claim 83, wherein said NK cells are induced pluripotent stem cell-derived natural killer (iPSC-NK) cells.

85. The population of cells of any one of claims 82-84, wherein said immune cells comprise a homozygous inactivating mutation in the cytokine-inducible SH2-containing protein (CISH) gene.

86. The immune cells are subjected to CISH - / - 85. The population of cells of any one of claims 82 to 84,

87. 87. A pharmaceutical composition comprising the population of cells of any one of claims 82 to 86.

88. A method for killing a target cell, comprising the steps of: treating a population of target cells with a population of cells according to any one of claims 82 to 86 or 88. The method of claim 87, wherein the ligand binding domain of the CAR specifically binds to an antigen on the target cell, and wherein the cell induces specific killing of the target cell.

89. 87. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 86.

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