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

Abstract

Natural killer cells engineered to express a chimeric antigen receptor (CAR) having a Toll / interleukin-1 (IL-1) receptor (TIR) signaling domain, such as a Toll-like receptor (TLR) signaling domain or an IL-1 receptor (IL-1 R) subfamily signaling domain, are provided. Compositions and methods for making and using the natural killer cells in immunotherapy for cancer are also provided. And the CAR-NK cell can be a CISH <-> / -iPSC-NK cell.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 325,441, filed on March 30, 2022, and U.S. Provisional Patent Application No. 63 / 440,169, filed on January 20, 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] The present application contains a computer-readable sequence list submitted in XML file format together with the present application, and the entire contents of the sequence list are incorporated herein by reference in their entirety. The title of the sequence list XML file submitted with the present application is "14735-026-228_SEQ_LISTING.xml", created on March 28, 2023 and has a size of 347,806 bytes. Field of the Invention

[0005] The present invention relates to chimeric antigen receptors (CARs) of natural killer (NK) cells and their use in immunotherapy. In particular, the present invention relates to CARs having a cytoplasmic portion comprising a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain. Background Art

[0006] NK cells are cytotoxic lymphocytes that constitute the main component of the innate immune system. They are named "natural killers" because they do not need to be activated in advance to kill the initial concept of the target. In humans, natural killer cells usually express surface markers CD16 (FCyRIII) and CD56. NK cells usually have cytotoxic activity. Endogenous NK cells provide rapid responses to virus-infected cells and respond to transformed cells. The small particles in the cytoplasm contain perforin, granzymes and other bioactive molecules. Once in contact with the target cell, the NK cell releases these particles. Perforin forms holes in the cell membrane of the target cell, and granzymes and related molecules can enter through the hole and act to induce apoptosis of the target cell.

[0007] In contrast to adaptive lymphoid cells (T cells and B cells) and myeloid cells (such as macrophages), NK cells are considered innate lymphoid cells. Unlike T cells, which specifically detect peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, NK cells can recognize stressed cells regardless of the presence or absence of peptides from pathogens on MHC molecules. In contrast, receptors on the cell surface of NK cells that recognize other biomolecules control NK cell activation.

[0008] Artificial NK cells can be prepared 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). NK cells with inactivating mutations in genes encoding cytokine-inducible SH2 proteins (CISH) are of particular concern. These mutations include, but are not limited to, substitutions, insertions, and / or deletions. CISH is an important negative regulator of IL-15 signaling. As described in US 2021 / 0145883 A1, the CISH gene is inactivated in induced pluripotent stem cells (iPSCs), followed by differentiation of iPSCs into natural killer (NK) cells, which results in engineered NK cells having increased persistence and potency relative to NK cells in which CISH is not inactivated. Inactivation of CISH is also believed to be able to enhance the cytotoxicity of T cells in cancer therapy.

[0009] By engineering NK cells to express chimeric antigen receptors (CARs), the innate targeting ability of NK cells can be redirected to a selected cell population. Chimeric antigen receptors have an extracellular portion, which includes a ligand binding domain designed to specifically bind to a target antigen; a transmembrane domain; and a cytoplasmic portion comprising one or more signal transduction domains. The various structures of the cytoplasmic portion of CAR are known. The first generation of CAR contains only the signal transduction domain of the T cell receptor ζ chain, referred to as the CD3ζ domain. Later generations of CAR introduced domains from costimulatory molecules, particularly the signal transduction domains of clusters of differentiation 28 (CD28) and 41BB (also referred to as CD137).

[0010] There remains a need in the art for chimeric antigen receptors for engineering natural killer (NK) cells and uses thereof. The present invention addresses this need. Summary of the invention

[0011] Chimeric antigen receptors for engineering natural killer (NK) cells and uses thereof are provided.In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) having a cytoplasmic portion comprising a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

[0012] In one aspect, the present disclosure provides a CAR with a cytoplasmic portion, the cytoplasmic portion including a Toll-like receptor (TLR) signal transduction domain. As used herein, the term "TLR signal transduction domain" refers to the TIR domain of a TLR receptor or a functional variant thereof. The TLR signal transduction domain can be a TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR10 signal transduction domain, preferably a TLR2 signal transduction domain.

[0013] Thus, the cytoplasmic portion of the CAR may comprise a signal transduction domain having a polypeptide sequence selected from SEQ ID NOs: 1-10, or a functional variant thereof. The signal transduction domain may have a polypeptide sequence having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity to any one of SEQ ID NOs: 1-10.

[0014] In another aspect, the present disclosure provides a CAR having a cytoplasmic portion, the cytoplasmic portion including an interleukin-1 receptor (IL-1R) subfamily signal transduction domain. As used herein, the term "IL-1R subfamily signal transduction domain" refers to a TIR domain or a functional variant thereof of an IL-1R receptor subfamily member. The IL-1R subfamily signal transduction domain can be an IL-1R signal transduction domain or an interleukin 18 receptor (IL-18R) signal transduction domain.

[0015] Thus, the cytoplasmic portion of the CAR may comprise a signal transduction domain having a polypeptide sequence selected from SEQ ID NO: 11 or 12, or a functional variant thereof. The signal transduction domain 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 any one of SEQ ID NO: 21-22.

[0016] In another aspect, the present disclosure provides a CAR having a cytoplasmic portion, the cytoplasmic portion including a TIR signaling domain and a CD3 ζ domain (also referred to herein as "CD3-ζ")-preferably, a CD3 ζ domain located at the C-terminus of the CAR. The CAR disclosed herein may also include one or more other signaling domains. In a variation, the cytoplasmic portion of the CAR may include a 2B4 signaling domain or an OX40 signaling domain. However, in other variations, the cytoplasmic portion of the CAR lacks a 2B4 signaling domain, lacks an OX40 signaling domain, or lacks both 2B4 and OX40 signaling domains.

[0017] CAR disclosed herein also includes a transmembrane domain. A variety of suitable transmembrane domains are known in the art. In a variation, the transmembrane domain can be a transmembrane domain of a CD8 alpha chain (CD8 alpha), CD28, CD16, NKp44, NKp46, TLR, IL-1R subfamily, or a functional variant thereof. The transmembrane domain can be an artificial transmembrane domain (referred to as "aTM") with a polypeptide sequence of NLFVASWIAVMIIFRIGMAVAIFCCFFFP (SEQ ID NO: 41), or a variant with 1, 2, 3, 4, 5 or more replacements.

[0018] In various embodiments, the present disclosure provides natural killer (NK) cells engineered to express these CARs, nucleic acids encoding these CARs, vectors for delivering these nucleic acids to cells, stem cells comprising these nucleic acids, therapeutic compositions, kits, manufacturing methods, and methods of treatment.

[0019] In some embodiments, the present disclosure provides a natural killer cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) comprises an extracellular portion, a transmembrane domain and a cytoplasmic portion, wherein the cytoplasmic portion comprises a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signal transduction 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.

[0020] On the other hand, 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 with at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity to SEQ ID NO: 2. In some embodiments, the cytoplasmic portion comprises a CD3ζ signaling domain. In some embodiments, the cytoplasmic portion comprises a 2B4 signaling domain. In some embodiments, the cytoplasmic portion lacks a 2B4 signaling domain. In some embodiments, the cytoplasmic portion consists essentially of the 2B4 signaling domain, the IL-18R signaling domain, and the CD3ζ signaling domain in the order from N-terminus to C-terminus. In some embodiments, the cytoplasmic portion consists essentially of the 2B4 signaling domain, the CD3ζ signaling domain, and the TLR2 signaling domain in the order from N-terminus to C-terminus. In some embodiments, the cytoplasmic portion consists essentially of the 2B4 signaling domain, the TLR2 signaling domain, and the CD3ζ signaling domain in the order from N-terminus to C-terminus.

[0021] 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 variable heavy chain (VH) domain and / or a variable light chain (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.

[0022] 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 aTM, CD8 α chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2Rβ, TLR, and IL-1R subfamily; and (iii) the cytoplasmic domain comprises a signal transduction domain of TLR2, 41BB, or 2B4.

[0023] In some embodiments, the cytoplasmic domain further comprises a signal transduction domain of CD3ζ. In some embodiments, the CAR comprises (i) an aTM transmembrane domain, a TLR2 signal transduction domain, and a CD3ζ signal transduction domain; or (ii) a CD28 transmembrane domain, a TLR2 signal transduction domain, and a CD3ζ signal transduction domain.

[0024] In some embodiments, the C-terminus of the extracellular domain is operably connected 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 connected to the N-terminus or C-terminus of the transmembrane domain through a linker or hinge domain.

[0025] 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.

[0026] 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 via a Gly-Ser linker.

[0027] In another aspect, provided herein is a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor of the present disclosure.

[0028] In another aspect, provided herein is a cell colony comprising an immune cell, the immune cell comprising a chimeric receptor of the present disclosure or a polynucleotide of the present disclosure. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the NK cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell. In some embodiments, the immune cell comprises a homozygous inactivation mutation in a protein (CISH) gene containing SH2 that is inducible by cytokines. In some embodiments, the immune cell is CISH- / -.

[0029] In another aspect, provided herein is a pharmaceutical composition comprising a cell population of the present disclosure.

[0030] In another aspect, provided herein is a method for killing target cells, comprising contacting a target cell population with a cell population of the present disclosure or a pharmaceutical composition of the present disclosure, 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.

[0031] In another aspect, 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.

[0032] Advantages of the present invention may 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.

[0033] Other aspects and advantages of the present invention will become apparent from the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] This patent or patent application publication contains at least one drawing drawn 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.

[0035] Figure 1A A schematic representation of a polynucleotide sequence encoding a chimeric antigen receptor (CAR) is shown, with the CAR portion labeled. A leader sequence is included to facilitate membrane integration.

[0036] Figure 1B A schematic representation of the polypeptide sequence of the chimeric antigen receptor (CAR) after cleavage of the N-terminal leader sequence is shown, with the CAR portion labeled.

[0037] Figure 2A A schematic representation of an embodiment of a CAR having a general ligand binding domain and specifically illustrated hinge (CD8α) and signaling domains (TLR2 and CD3ζ) is shown.

[0038] Figure 2B A schematic representation of an embodiment of a CAR having a general ligand binding domain and specifically illustrated hinge (CD8α) and signaling domains (2B4, TLR2, and CD3ζ) is shown.

[0039] Figure 3 A schematic representation of CAR in the cell membrane is shown.

[0040] Figure 4 A schematic representation of an embodiment of a CAR in a cell membrane with specified hinge (CD8α) and signaling domains (TLR2 and CD3ζ) is shown.

[0041] Figure 5A Shown is a nearest neighbor joining phylogenetic tree of TIR signaling domains generated from an alignment of their polypeptide sequences.

[0042] Figure 5B Shown is a nearest neighbor joining phylogenetic tree of TLR signaling domains generated from an alignment of their polypeptide sequences.

[0043] Figure 6A 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) is shown.

[0044] Figure 7 A table showing pairwise sequence identities of TLR signaling domains.

[0045] Figure 8 Shown is the sequence of an embodiment of a CAR (SEQ ID NO: 100) divided into CAR components (SEQ ID NOs: 37, 38, 41, 2, and 63).

[0046] Fig. 9 Shown is the expression of a single CAR construct in iPSC-NK cells. Flow cytometry data bar graph shows CAR expression in mature iPSC-derived NK cells. CAR expression is detected using HA-tags and anti-HA antibodies (APC) engineered into the extracellular part of CAR.

[0047] Fig.10 A bar graph of CAR expression data is shown. A bar graph showing the frequency of CAR expression in iPSC-derived NK cells. The dotted line represents the average CAR expression across all constructs.

[0048] Fig.11 Long-term killing assay shown: Line graph shows the use of eSight impedance assay for CD19 + Frequency of NK-specific killing of Raji target cells. Data are normalized to individual target cells (no NK cells), which are represented by horizontal dotted lines. 50% NK cell killing means that at this time point, the number of live cells in the culture is half of the untreated (no NK cells) control. Arrows point to the curve of CAR with TLR2 signal transduction domain and CD3 ζ cytoplasmic domain ("TLR2-ζ").

[0049] Fig.12 4 hr caspase 3 / 7 cytotoxicity assay is shown: The bar graph shows CD19 after 4 hr of co-culture + NK-specific killing of SupB15 target cells. The dotted line indicates specific killing of the reference CAR ("2B4-ξ"). The arrow points to the curve of the CAR with a TLR2 signaling domain and a CD3ζ cytoplasmic domain ("TLR2-ζ").

[0050] Fig.13AShown are schematic representations of embodiments of CARs having a TLR2 signaling domain, optionally a 2B4 signaling domain, and a CD3ζ cytoplasmic domain in three different orders.

[0051] Fig. 13B Shows Fig.13A Long-term killing assay for the three constructs shown in Figure 2: Line graph shows the effect of eSight impedance assay on CD19 + Frequency of NK-specific killing of Raji target cells.

[0052] Fig. 13C Shows Fig.13A 4 hr caspase 3 / 7 cytotoxicity assay of the 3 constructs shown in FIG.

[0053] Fig.14A Shown is 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.

[0054] Fig. 14B Shows Fig.14A 24-hour killing assay of the referenced CAR constructs: Line graph shows the effect of eSight impedance assay on HER2 + Frequency of NK-specific killing of target cells.

[0055] Fig. 14C A bar graph is shown showing HER2 + Target cells and expression Fig.14A Release of cytokines IFN-γ and TNF-α after co-culture with CISH KO iPSC-derived NK cells containing the reference CAR construct.

[0056] Fig.14D A bar graph is shown showing the Fig.14A Figure 3. CISH KO iPSC-derived NK cells with the referenced CAR constructs in Figure 3. CISH KO iPSC-derived NK killing of NK cell-tolerant solid tumor HER2+ target cells (2:1E:T) in monolayer 24 hours later.

[0057] Fig.15A Shown is a stress test killing assay of CISH KO iPSC-derived NK cells expressing 3 different CAR constructs, each containing a CAR against HER2, using the eSight impedance assay. + The same HER2 binding domain is expressed on target cells.

[0058] Fig. 15BFigure 3 shows the use of eSight impedance assay for cells expressing the same CAR construct and targeting HER2 + Stress testing killing assay of CISH KO iPSC-derived NK cells with 3 different HER2 binding domains of target cells.

[0059] Fig.16A Shown is a line graph showing 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 3 different HER2 binding domains.

[0060] Fig. 16B Shown are representative graphs of HER2+ target cell killing and caspase 3 / 7 activation after 30 h of co-culture of NK cells and NK cell-tolerant solid tumor targets (2:1 E:T).

[0061] Fig.17 Shown are representative images of HER2+ 3D tumor spheroids after 18 and 36 hours of treatment with untransduced iPSC-derived NK cells (no CAR expression) and iPSC-derived NK cells expressing the same CAR construct and 3 different HER2 binding domains.

[0062] Fig.18 Figure 2 shows the expression of HER2 by iPSC-derived NK cells expressing either a CAR with a HER2 binding domain or without a CAR (UT) using the eSight impedance assay. + Serial killing assay of target cells.

[0063] Fig.19 Shown are representative images of HER2+ 3D tumor spheroids at day 0 and day 3 treated with NK cells (“UT”) and NK cells expressing a CAR with a HER2 binding domain.

[0064] Fig. 20 Shown is a schematic representation of triple-edited ("TE") iPSC cells according to the present disclosure.

[0065] Fig.21 Shown are representative bar graphs of flow cytometry data showing the expression of CAR and NK markers in wild-type (WT) NK cells, CISHKO iPSC-derived NK cells, and triple-edited (“TE”) iPSC-derived NK cells.

[0066] Fig.22ALong-term killing assay in HER2+ 3D tumor spheroids showing the effects of WT NK cells, CISHKO iPSC-derived NK cells, and triple-edited (“TE”) iPSC-derived NK cells on HER2 + NK killing frequency of target cells.

[0067] Fig. 22B Shown are representative images of HER2+ 3D tumor spheroids after day 0 and day 3 of co-culture with WT NK cells, CISH KO iPSC-derived NK cells, and triple-edited (“TE”) iPSC-derived NK cells.

[0068] Fig.23A and Fig. 23B The synergistic effect of sIL-15 co-expressed with HER2-CAR construct on NK cell killing of HER+ breast cancer cell line BT474 was shown. Comparison was made with cells expressing anti-CD20 CAR. Fig.23A CAR expression with or without sIL-15 is shown in Fig. 23B Killing of BT474 clone 5 by NK cells with or without sIL-15 is shown.

[0069] Detailed description of the invention

[0070] The present invention relates to chimeric antigen receptors (CARs) of natural killer (NK) cells and their use in immunotherapy. In some variations, the present invention relates to CARs having a cytoplasmic portion comprising a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

[0071] In various embodiments, the present disclosure provides natural killer (NK) cells engineered to express the disclosed CARs, nucleic acids encoding these CARs, vectors for delivering these nucleic acids to cells, stem cells containing these nucleic acids, therapeutic compositions, kits, manufacturing methods, and methods of treatment. Figure 1A-1B As shown, the CAR of the present disclosure can be a single polypeptide encoded by a single polynucleotide sequence. The CAR can have a conventional component arrangement, such as Figure 1A After the CAR is expressed as a polypeptide, the leader peptide is cleaved from the CAR, such as Figure 1B shown.

[0072] Figure 2A and Figure 2B An illustrative embodiment of a CAR is shown in FIG. The cytoplasmic portion of the CAR may have a single TIR signaling domain, such as Figure 2AIn this embodiment, the CAR also has a CD3ζ signal transduction domain. This CAR may lack other signal transduction domains. Figure 2B An embodiment with a 2B4 signaling domain is shown.

[0073] Figure 3 Expression of the CAR polypeptide in the cell membrane is shown. The ligand binding domain is shown as a single chain variable domain (scFv), although other ligand binding domains can be used. Figure 4 An embodiment of a CAR is shown in which the cytoplasmic portion of the CAR has a single TIR signaling domain (shown as a TLR2 signaling domain) and a CD3ζ signaling domain.

[0074] Among endogenous receptors, TIR signaling domains share common functional properties, however, they differ at the sequence level. Figure 5A A phylogenetic tree is shown to illustrate the sequence differences between TIR signaling domains and their clustering into groups. Figure 5B A similar phylogenetic tree showing only the TLR signaling domains is shown in . Figure 6 Shown are sequence alignments of selected TLR signaling domains: TLR1, TLR2, TLR4, TLR6 and TLR10. These alignments can be used to generate functional variants of TLR signaling domains to select residues that can be varied and conserved residues that should be retained to promote folding and function of the domain.

[0075] Figure 7 Pairwise sequence comparisons of TLR signaling domains are shown. With a few exceptions, the sequence identity between TLR signaling domains is generally less than 55%. For example, the sequence identity between TLR2 and TLR4 signaling domains is 41%.

[0076] Figure 8 The polypeptide sequence of the embodiment of CAR is shown. In this embodiment, CAR comprises a ligand binding domain specific for CD19. However, as described in detail below, other ligand binding domains can be used. In addition, the present disclosure considers changing each of the components shown. As disclosed, various changes have been made to this embodiment.

[0077] Natural Killer Cells

[0078] In one aspect, the present disclosure provides natural killer (NK) cells engineered to express the disclosed CAR. NK cells may be primary NK cells. Primary NK cells may be isolated from cord blood or peripheral blood using well-known methods. Polynucleotides encoding CARs may be introduced into primary NK cells using viral vectors, non-viral vectors, or electroporation. Alternatively, NK cells may be induced pluripotent stem cells (iPSC)-derived NK cells or human embryonic stem cells (hESC)-derived NK cells. The method for preparing iPSC-NK cells can be obtained by differentiating iPSC using known methods. In one variation, iPSC-NK cells are prepared 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 prepared using single cell differentiation, as described in, for example, Woll et al. Blood 113: 6094-6101 (2009). In other variations, iPSC-NK cells are prepared by differentiating single cells. Exemplary methods are provided in US 2021 / 0024891 A1 and US2016 / 0097035 A1. NK cells derived from hESC- / iPSC- can be phenotyped by measuring surface marker expression, such as CD16, NKG2D, NKp44, NKp46, TRAIL, FasL, or a combination thereof. The killing activity of hESC- / iPSC-derived NK cells can be evaluated. Functional assays to evaluate differentiation into NK cells include direct cytolytic activity of tumor cells (such as killing of K562 cells), caspase-3 / 7 flow cytometry assay, immunological assay of cytotoxic granules or cytokine release, or anti-tumor activity in vivo xenograft models. See, for example, Woll et al. Blood 113: 6094-6101 (2009); Hermanson et al. in Hematopoietic differentiation of human pluripotent stem cells. 69-19 (2015).

[0079] In some embodiments, NK cells are CD45+ / CD56+ double positive. In some embodiments, immune cells are CD45+. In some embodiments, immune cells are CD56+. In some embodiments, immune cells are CD45+, CD56+ or CD45+ / CD56+. Other exemplary methods for preparing and using engineered cells are provided in U.S. Patent Application Publication Nos.US 2018 / 0298101 A1, US 2021 / 0230548 A1, and US 2021 / 0145883 A1; and International Patent Application Publication Nos.WO 2018 / 075664 A1 (corresponding to U.S. Patent Application No. 17 / 481,404) and WO 2020 / 113029 A2 (corresponding to U.S. Patent Application No. 17 / 309,408), the disclosures of the above patents are incorporated herein by reference in their entirety.

[0080] The cell colony of the present disclosure can be a purified cell colony. As used herein, a composition containing a "purified cell colony" or a "purified cell composition" means that at least 30%, 50%, 60%, typically at least 70%, and more preferably 80%, 90%, 95%, 98%, 99% or more of the cells in the composition have a similarly identified cell phenotype.

[0081] The cell population can be an isolated cell population. As used herein, the term "isolated" means substantially or essentially free of the material of the components that normally accompany it in its natural state. In a specific embodiment, the term "obtained" or "derived" is used synonymously with isolated.

[0082] 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. These techniques are fully explained in the literature, such 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 (J E Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J Pather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle. J B Griffiths, and D G Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC. Blackwell (Editor-in-Chief)); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos Editor-in-Chief, 1987); Current Protocols in Molecular Biology (FMAusubel el al. Editor-in-Chief, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al. Chief Editor, 1994); Current Protocols in Immunology (JEColigan) e1al. Editor-in-Chief, 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C A.Janewayand P.Travers.1997); Antibodies (P.Finch.1997); Antibodies: a practical approach (D.Catty. Editor-in-Chief, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C.Dean Editor-in-Chief, Oxford University Press.2000); Using antibodies: alaboratory manual (E.Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by VT DeVita et al., JBLippincott Company, 1993).

[0083] The introduction of polynucleotides encoding CAR can be achieved by transducing NK cells or iPSC or hESC precursors with viral vectors, such as lentiviral vectors. Suitable lentiviral vectors are known in the art and are described in (for example) Zufferey et al., (1997); Dull et al., (1998); US6013516A and US5994136A. In some variations, non-viral vectors, such as lipid nanoparticles, transfection reagents or electroporation can be used to introduce polynucleotides into cells.

[0084] In one aspect, the disclosure provides cells comprising a chimeric antigen receptor as 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.

[0085] In some embodiments, the cell comprises a homozygous inactivating mutation in the cytokine-inducible SH2-containing protein (CISH) gene of the cell (e.g., CISH- / -). In some embodiments, the cell comprises expression and / or overexpression of a gene encoding soluble IL-15 or membrane-bound IL-15.

[0086] Chimeric Antigen Receptor

[0087] Provided are chimeric antigen receptors for engineered natural killer (NK) cells and uses thereof.In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) having a cytoplasmic portion comprising a Toll-like / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain.

[0088] Toll-like receptor signaling domain

[0089] NK cells are known to express pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) on infected cells, including Toll-like receptors (TLRs). Noh et al. (2020) J. Immunol. Res. 2020:204860.

[0090] The Toll / interleukin-1 (IL-1) receptor (TIR) ​​family is defined as having a cytoplasmic portion containing a structurally homologous signal transduction domain, called the TIR domain. TIR domains are present in Toll-like receptors (TLRs) as well as two interleukin receptors, the interleukin-1 receptor (IL1R) and the interleukin-18 receptor (IL18), which, although classified as interleukin receptors, participate in signal transduction pathways similar to those of TLRs. Therefore, the TIR family is considered to be 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.

[0091] 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. Signal transduction by TLRs depends differently on heterodimerization or homodimerization. TLR4 is known to signal by homodimerization. TLR2 signal transduction is triggered by heterodimerization of TLR1 or TLR6. It is known that, unlike TLR4, TLR2 does not signal as a homodimer. Zhang et al. (2002) FEBS Lett. 532: 171.

[0092] In one aspect, the present disclosure provides a CAR with a cytoplasmic portion, the cytoplasmic portion including a Toll-like receptor (TLR) signal transduction domain. As used herein, the term "TLR signal transduction domain" refers to the TIR domain of a TLR receptor or a functional variant thereof. The TLR signal transduction domain can be a TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR10 signal transduction domain, preferably a TLR2 signal transduction domain.

[0093] Thus, the cytoplasmic portion of the CAR may comprise a signal transduction domain having a polypeptide sequence selected from SEQ ID NOs: 1-10, or a functional variant thereof. The signal transduction domain may have a polypeptide sequence having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity to any one of SEQ ID NOs: 1-10.

[0094] When Toll / interleukin-1 (IL-1) receptor (TIR) ​​domain-containing receptors are activated, the TIR domain triggers a conserved cell signaling pathway that controls cellular function by recruiting cytoplasmic signaling molecules, including cytoplasmic adaptor proteins containing the TIR domain. The TIR signaling domain is generally identified as containing three conserved regions (e.g., box 1 motif: (F / Y)DA or FADFISY (SEQ ID NO: 136), box 2 motif: RDXXPG (SEQ ID NO: 137) or GYKLC-RD-PG (SEQ ID NO: 138), and box 3 motif: FW or a conserved W adjacent to a basic residue) responsible for mediating protein-protein interactions between Toll-like receptors (TLRs) and signaling components (Slack et al., 2000). Each TIR signaling domain contains 5 strands of parallel β-sheets (A- to -E) and 6 surrounding α-helices (A- to -E) with respective connecting loop structures (Bovijen et al., 2013). Conserved residues are located in the hydrophobic core, and large insertions and deletions can be present in multiple loop regions of different TIR signaling domains. For example, differences in the box 2 motif of the "BB loop" region comprising the signaling domain are believed to play a role in the specificity of TIR-TIR interactions (Xu et al., 2000; Poltorak et al., 1998). About 20 receptors with TIR domains have been identified in humans, including Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1R). The general structure of the TIR signaling domain contains conserved residues within the core sequence, which range from about 125 to about 200 amino acid residues, however, the sequence conservation between these domains can be as low as only 20-30%. External residues vary widely between TIR domains, which results in differential distributions of electrostatic potential and resulting signaling functions (Dunne et al. 2003). Sequence variation between TIR signaling domains may include insertions, deletions, and / or mutations in one or more α-helices, β-sheets, loops, or box motifs.

[0095] In some embodiments, the TIR signal transduction domain is a Toll-like receptor (TLR) signal transduction domain. Human Toll-like receptors (TLR 1-10) are single-pass transmembrane proteins that recognize unique ligands, respectively, which activate intracellular signal transduction (Armant, M and Fenton, M, 2002) through TIR signal transduction domains (Xu et al., 2000; Horng et al., 2002; Brown et al., 2006). TLR 1, 2, 4, 5, 6 and 10 are present in the plasma membrane and recognize extracellular pathogen-associated molecular patterns (PAMPs). TLR 3, 7, 8 and 9 are located in intracellular endosomes, which recognize internalized PAMPs (Akira et al., 2006; Kawai, T. and Akira, S., 2007). TLRs undergo homo- or hetero-dimerization, which initiates their activation (Gay, N. and Gangloff, M., 2007). The signaling cascade is followed by TLR signaling, which is through the conserved cytoplasmic TIR domain present in both the cytoplasmic signaling domain and TLR adapter proteins, such as myeloid differentiation factor 88 (MyD88), MyD88 adapter protein-like protein (MAL / TIRAP), TRIF-related adapter protein molecule (TRAM / TICAM2), TIR domain-containing adapter protein inducing interferon-β (TRIF / TICAM1), and sterile α- and armadillo motif-containing protein (SARM) (O'Neill, L. and Bowie, A. 2007). The MyD88-dependent pathway transmits the MyD88 signal to the phosphorylation of interleukin-1 receptor-associated kinase (IRAK) 4 and then 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 the MyD88-dependent pathway (Horng et al., 2002; Kawai and Akira, 2010). The TRIF-dependent pathway signals through downstream kinases (TANK-binding kinase 1 (TBK1) and IKKε) to activate IRF3 and subsequently produce type 1 interferons (Yamamoto et al., 2002b; Oshiumi et al., 2003). The SARM pathway acts as a negative regulator of TRIF-dependent Toll-like receptor signaling (Carty et al., 2006).

[0096] The TLR signaling domain can be a TLR2 signaling domain or a functional variant thereof. The TLR2 signaling domain is derived from Toll-like receptor 2 (TLR2), a TLR that interacts with TLR1, TLR6 or TLR10 prior to stimulation with a ligand (Jin et al., 2007; Toshchakov, V. and Neuwald, A., 2020). The formation of heterodimers facilitates interaction with intracellular TIR domains to promote signal initiation. TLR2 acts via MYD88 and TRAF6, leading to activation of transcription factor NF-KB, cytokine secretion, and inflammatory responses; and can also promote apoptosis in response to the lipid portion of lipoproteins (Brightbill et al., 1999; Aliprantis et al., 1999). The TLR2 signaling domain can comprise residues 639-782 (144 aa) of human TLR2 (SEQ ID NO: 26) or a functional variant thereof

[0097] The TIR signaling domain of TLR2 may comprise a polypeptide sequence:

[0098] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR2 is encoded by the nucleotide sequence shown in SEQ ID NO: 180. In some embodiments, the TIR signaling domain of TLR2 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: 180.

[0099] The TLR signaling domain 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: 2. Functional variants and / or some or all of the conserved residues of the TLR2 signaling domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient 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). Other conserved residues of 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).

[0100] 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), which is a class of TLRs that function as endosomal, nucleic acid-sensing TLRs that can undergo dimerization via homotypic interactions in the recruitment of the downstream adaptor protein MYD88 containing TIR (Tanji et al., 2013; Tanji et al., 2015). The signal generated by activating IRAK4, IRAK1, TRAF6, TRAF3 of NF-KB and inducing IRF7 production of proinflammatory cytokines and interferons is propagated through the Mydd body (Myddosome) (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 may comprise a polypeptide sequence:

[0101] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR8 is encoded by the nucleotide sequence shown 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.

[0102] The TLR signaling domain 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: 8. Functional variants and / or some or all of the conserved residues of the TLR8 signaling domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR8 signaling pathway. The conserved residues of the TLR8 TIR signaling domain may include a conserved pattern in the N-terminal half of the BB loop, -E9E9R9D9W9xP9G9-, in which glutamic acid and tryptophan residues are still 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).

[0103] 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), which is an endosomal nucleic acid sensing TLR that undergoes dimerization, wherein ligand binding results in interaction with the TRIF / TICAM1 adaptor protein, leading to activation of NF-κ-B, IRF3 nuclear translocation, cytokine secretion, and inflammatory response (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 may comprise a polypeptide sequence:

[0104] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR3 is encoded by the nucleotide sequence shown in SEQ ID NO: 183. In some embodiments, the TIR signaling domain of TLR3 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: 183.

[0105] The TLR signaling domain 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: 3. Functional variants and / or some or all of the conserved residues of the TLR3 signaling domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR3 signaling pathway. The 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).

[0106] 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), which is an endosomal receptor that can respond to single-stranded RNA containing uridine or guanosine analogs (Davenne et al., 2020; Lee et al., 2003). Binding to an agonist leads to dimerization, which enables the recruitment of the TIR-containing adaptor protein MYD88 via homotypic interactions (Zhang et al., 2016). The Mydd body signaling complex interacts with IRAK4, IRAK1, TRAF6 and TRAF3, resulting in activation of NF-κ-B and IRF7 to induce cytokines and interferons (Zhang et al., 2016). The TLR7 signaling domain may comprise residues 889-1033 (145 aa) of human TLR7 (SEQ ID NO: 31). The TIR signaling domain of TLR7 may comprise a polypeptide sequence:

[0107] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR7 is encoded by the nucleotide sequence shown in SEQ ID NO: 187. In some embodiments, the TIR signaling domain of TLR7 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: 187.

[0108] The TLR signaling domain 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: 7. Functional variants and / or some or all of the conserved residues of the TLR7 signaling domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR7 signaling pathway. The conserved residues of the TLR7TIR signaling domain may include a >90% conserved pattern in the N-terminal half of the BB loop, -E9E9R9D9W9xP9G9-, in which glutamic acid and tryptophan residues are still 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).

[0109] The TLR signal transduction domain can be a TLR4 signal transduction domain or a functional variant thereof. The TLR4 signal transduction domain is derived from Toll-like receptor 4 (TLR4), which is a class of TLRs that initiate responses to lipopolysaccharide (LPS) by homodimerization, through interactions with K96 and CD14 (Tatematsu et al., 2016). TLR4 interacts with MYD88, TIRAP and TRAF6, leading to activation of NF-KB, cytokine secretion and inflammatory responses (Medzhitov et al., 1997; Arbour et al., 2000; Tatematsu et al., 2016). TLR4 can also act via responses independent of LPS, where activation is triggered by free fatty acids and Ni (2+), which is non-conservative and species-specific (Marc et al., 2010). The TLR4 signal transduction domain can comprise residues 672-815 (144 aa) of human TLR4 (SEQ ID NO: 28) or a functional variant thereof. The TIR signaling domain of TLR4 may comprise a polypeptide sequence:

[0110] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR4 is encoded by the nucleotide sequence shown in SEQ ID NO: 184. In some embodiments, the TIR signaling domain of TLR4 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: 184.

[0111] The TLR signaling domain 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: 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 to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR4 signaling pathway. A variation of the TLR4 TIR signaling domain may include R763H and / or Q834H (Smirnova et al., 2001). The conserved residues of the TLR4 TIR signaling domain may include C747 on α-helix C; WX C747 The presence of the XXE motif; and / or P712 of the BB loop (Shirey et al. 2020; Poltorak et al., 1998). Other conserved residues of TLR4 TIR may include residues between the BB loop and α-helix E (Toshchakov et al., 2011).

[0112] 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), which is a type of TLR that interacts with TLR2 to regulate immune responses stimulated by the presence of specific lipopeptides (Lancioni et al., 2011). The TLR1 signaling domain may comprise residues 635-776 (142 aa) of human TLR1 (SEQ ID NO: 25) or a functional variant thereof

[0113] The TIR signaling domain of TLR1 may comprise a polypeptide sequence:

[0114] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR1 is encoded by the nucleotide sequence shown in SEQ ID NO: 182. In some embodiments, the TIR signaling domain of TLR1 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: 182.

[0115] The TLR signal transduction domain 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: 1. Functional variants and / or some or all of the conserved residues of the TLR1 signal transduction domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may comprise a polypeptide sequence that is sufficient to promote signal propagation using the TLR1 signal transduction pathway.

[0116] 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), which is a class of TLRs that have been shown to play a role in immune responses via 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 may comprise a polypeptide sequence:

[0117] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR5 is encoded by the nucleotide sequence shown in SEQ ID NO: 185. In some embodiments, the TIR signaling domain of TLR5 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: 185.

[0118] The TLR signal transduction domain 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: 5. Functional variants and / or some or all of the conserved residues of the TLR5 signal transduction domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR5 signal transduction pathway.

[0119] 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 class of TLRs that have been shown to play a role in the recognition of diacylated 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 may comprise the polypeptide sequence:

[0120] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR6 is encoded by the nucleotide sequence shown in SEQ ID NO: 186. In some embodiments, the TIR signaling domain of TLR6 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: 186.

[0121] The TLR signal transduction domain 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: 6. Functional variants and / or some or all of the conserved residues of the TLR6 signal transduction domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR6 signal transduction pathway.

[0122] 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 class of TLRs that have been shown to play a role in nucleotide sensing stimulated by the presence of unmethylated cytosine-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 may comprise a polypeptide sequence:

[0123] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR9 is encoded by the nucleotide sequence shown in SEQ ID NO: 189. In some embodiments, the TIR signaling domain of TLR9 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: 189.

[0124] The TLR signal transduction domain 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: 9. Functional variants and / or some or all of the conserved residues of the TLR9 signal transduction domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may include polypeptide sequences that are sufficient to promote signal propagation using the TLR9 signal transduction pathway.

[0125] 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), which is a member of the TLR receptor family that is involved in the detection of unique molecules and in regulating 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 may comprise a polypeptide sequence:

[0126] Or a functional variant thereof. In some embodiments, the TIR signaling domain of TLR10 is encoded by the nucleotide sequence shown in SEQ ID NO: 190. In some embodiments, the TIR signaling domain of TLR10 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: 190.

[0127] The TLR signal transduction domain 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: 10. Functional variants and / or some or all of the conserved residues of the TLR10 signal transduction domain may be retained. Functional variants may include truncations, insertions and / or mutations from the N to C termini. Functional variants may comprise a polypeptide sequence that is sufficient to promote signal propagation using the TLR10 signal transduction pathway.

[0128] Interleukin-1 receptor (IL-1R) subfamily signaling domain

[0129] In one aspect, the present disclosure provides a CAR having a cytoplasmic portion, the cytoplasmic portion including an interleukin-1 receptor (IL-1R) subfamily signal transduction domain. As used herein, the term "IL-1R subfamily signal transduction domain" refers to a TIR domain or a functional variant thereof of an IL-1R receptor subfamily member. Currently, in humans, 10 IL-1R subfamily receptors have been identified (Fields et al., 2019), 9 of which have TIR domains. The IL-1R subfamily signal transduction domain can be an IL-1R signal transduction domain or an interleukin 18 receptor (IL-18R) signal transduction domain. In some embodiments, the IL-1R subfamily signaling domain can 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-18Rβ, 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.

[0130] Thus, the cytoplasmic portion of the CAR may comprise a signal transduction domain having a polypeptide sequence selected from SEQ ID NO: 21 or 22, or a functional variant thereof. The signal transduction domain 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 any one of SEQ ID NO: 21-22.

[0131] The cytoplasmic portion of the CAR may comprise a signal transduction domain having a polypeptide sequence selected from SEQ ID NO: 143, 144, 145, 146, 147, 148 or 149, or a functional variant thereof. The signal transduction domain 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 any one of SEQ ID NO: 143, 144, 145, 146, 147, 148 or 149.

[0132] The IL-1R subfamily signal transduction domain is derived from IL-1R, which is 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 a certain degree of homology with the TIR domain of TLR and plays a similar function in signal transduction. IL-1R family members act as heterodimers that recognize homologous cytokines and activate downstream signal transduction molecules (Boraschi, D. and Tagliabue, A., 2013). Ligand binding leads to oligomerization of the TIR domain on the receptor, followed by interaction of auxiliary proteins and adapter protein molecules (e.g., MYD88). Heterodimerization with the TIR domain leads to transduction of downstream cytoplasmic kinases (e.g., IRAK) and activation of NF-kB, which produces an immune response (e.g., cytokine secretion).

[0133] 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), which is a cytokine receptor that functions by binding to interleukin-1 and forming a complex with an accessory protein (IL-1RAcp), the complex formed above mediating interaction with TOLLIP, MYD88 and IRAK, leading to 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 (213 aa) of human IL-1R (SEQ ID NO: 35) or a functional variant thereof. The IL-1R signaling domain of IL-1R may comprise a polypeptide sequence:

[0134]

[0135] The IL-1R signaling domain 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: 21. Functional variants and / or some or all of the conserved residues of the IL-1R signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N to C termini. Functional variants may comprise a polypeptide sequence that is sufficient to promote signal propagation using the IL-1R signaling pathway. The conserved residues of the IL-1R signaling domain may comprise D369A and / or R428A (Slack et al. 2000).

[0136] 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), which is a cytokine receptor that functions by binding to a cognate cytokine, as a complex with the IL-18RAcP accessory protein, and engaging one or more adaptor proteins to cause 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 comprise the following polypeptide sequence:

[0137] or functional variants thereof.

[0138] The IL-18R signaling domain 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: 22. Functional variants and / or some or all of the conserved residues of the IL-18R signaling domain may be retained. Functional variants may include truncations, insertions, and / or mutations from the N to C termini. Functional variants may comprise a polypeptide sequence that is sufficient to promote signal propagation using the IL-18R signaling pathway. The conserved residues of the IL-18R signaling domain may comprise a conserved sequence within the BB loop (Ota et al., 2012).

[0139] Other components of the CAR cytoplasm

[0140] In another aspect, the present disclosure provides a CAR having a cytoplasmic portion including a TIR signaling domain and a CD3 ζ domain, preferably a CD3 ζ domain located at the C-terminus of the CAR. The CAR disclosed herein may also include one or more other signaling domains. In a variation, the cytoplasmic portion of the CAR may include a 2B4 signaling domain or an OX40 signaling domain. However, in other variations, the cytoplasmic portion of the CAR lacks a 2B4 signaling domain, lacks an OX40 signaling domain, or lacks both a 2B4 and an OX40 signaling domain.

[0141] The cytoplasmic portion may comprise the following polypeptide sequence:

[0142] 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.

[0143] The cytoplasmic portion may comprise the following polypeptide sequence:

[0144] 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.

[0145] The cytoplasmic portion may comprise the following polypeptide sequence:

[0146] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 66) 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.

[0147] Other cytoplasmic domains that can be used in CARs are provided in U.S. Patent Publication No. US 2017 / 0233454 and U.S. Patent No. 10,415,017.

[0148] Ligand binding domain

[0149] In one aspect, the present disclosure provides a CAR having an extracellular portion comprising a ligand binding domain. The ligand binding domain of the present disclosure may include an antibody-based binding agent that specifically binds to an antigen of interest via an 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 treatment by immunotherapy using NK cells.

[0150] As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, for example but not limited to, IgA, IgD, IgE, IgG, and IgM, and 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 refer to any level of specificity that has clinical utility. Specific binding can mean a binding constant that is at least 10 greater than that of other molecules in the tissue. 3 M- 1 , at least 10 4 M-1 or at least 10 5 The binding constant is M-1. The antigen-binding fragment may be an artificial fragment, such as a humanized or chimeric antigen-binding fragment.

[0151] The term "antibody-like domain" refers to a recombinant antibody fragment (such as an sFv fragment, a dsFv fragment, a dual-specific sFv fragment, a dual-specific dsFv fragment, a F(ab)'2 fragment, a single-chain Fv protein ("scFv"), a disulfide-stabilized Fv protein ("dsFv"), a diabody and a triabody (as known in the art) and a camelid antibody that retains antigen binding properties (see, e.g., U.S. Patent Nos. 6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808). The antibody-like domain can be expressed as a fusion protein to form a single-chain CAR, or it can be combined with other polypeptides by other known means to form a multipartite CAR. Illustrative examples of multipartite CARs are described in, for example, International Patent Publication No. PCT / US2021 / 05676.

[0152] The antibody-like domain may be a single-chain variable fragment (scFv). A scFv protein is a fusion protein in which the light chain variable region (VL) of an antibody and the heavy chain variable region (VH) of an antibody are expressed as a fusion polypeptide (optionally, in a VH-VL or VL-VH orientation, with an intervening connection, such as a Gly-Ser linker). The scFv may be a dsFv, whose chains have been mutated to introduce disulfide bonds to stabilize the binding of the chains.

[0153] When the ligand binding domain is an antigen-like domain, it may comprise complementary 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 VL chain. In some embodiments, the ligand binding domain comprises a single polypeptide chain. The ligand binding domain may be a scFv.

[0154] Ligand binding domain that specifically binds to CD19

[0155] In one aspect, the present disclosure provides a chimeric antigen receptor that specifically binds to CD 19. The ligand binding domain may be an anti-CD 19 scFv comprising the polypeptide sequence set forth in SEQ ID NO: 73, or a functional variant thereof having 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: 73, or other scFvs or VH / VL pairs 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 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: 74 and SEQ ID NO: 75, respectively.

[0156] The ligand binding domain (eg, scFv) may comprise the CDR sequences in Table 1, or functional variants having 1, 2, 3 or more substitutions.

[0157] Table 1: Anti-CD19 ligand binding domain sequences

[0158]

[0159] In a variation, the ligand binding domain can comprise CDRs extracted from SEQ ID NO: 109 by known methods (e.g., Chothia, Martin, Kabat, AHo, and IMGT numbering, available from, for example, abysis.org; see, Swindells et al. J. Mol. Bio. 429(3): 356-365 (2017)).

[0160] 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 152.

[0161] In some embodiments, the extracellular domain of the chimeric receptor is encoded by the 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 153.

[0162] Ligand binding domain that specifically binds to CD20

[0163] CD20 is a B cell lineage-specific antigen expressed on the cell surface of most B cell lymphomas, especially in the late stages of B cell lymphopoiesis. In one aspect, the present disclosure provides a chimeric antigen receptor that specifically binds to CD20. The ligand binding domain can be an anti-CD20 scFv comprising a polypeptide sequence as set forth in SEQ ID NO: 139, or a functional variant thereof having 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: 139, or other scFv or VH / VL pairs generated from SEQ ID NO: 139.

[0164] CD20-scFv-4 (SEQ ID NO: 139):

[0165]

[0166] In a variation, the ligand binding domain may comprise CDRs extracted from SEQ ID NO: 139 by known methods (e.g., Chothia, Martin, Kabat, AHo, and IMGT numbering, available from, for example, abysis.org; see, 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.

[0167] 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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 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: 154 and SEQ ID NO: 155, respectively.

[0168] In some embodiments, the extracellular domain of the chimeric receptor is encoded by the 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 157.

[0169] Ligand binding domain that specifically binds to HER2

[0170] 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 a solid cancer, such as 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 shown in SEQ ID NO: 140-142, or a functional variant thereof having a polypeptide sequence with at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity to SEQ ID NO: 140-142, or other scFv or VH / VL pairs generated from SEQ ID NO: 140-142. The ligand binding domain (e.g., scFv) can comprise a CDR sequence, or a functional variant having 1, 2, 3 or more substitutions.

[0171] 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140, 141, or 142.

[0172] In some embodiments, the extracellular domain of the chimeric receptor is encoded by the 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 198, 199 or 200.

[0173] Table 2: Anti-HER2 ligand binding domain sequences

[0174]

[0175] In variations, the ligand binding domain may comprise CDRs extracted from SEQ ID NOs: 140-142 by known methods (e.g., Chothia, Martin, Kabat, AHo, and IMGT numbering, available from, for example, abysis.org; see, 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.

[0176] In some embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 202-207. In some embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 202 (HER2-2-CD8-aTM-TLR2-CD3ζ, without sIL15). In some embodiments, 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, 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, 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, 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 207 (HER2-5-CD8-aTM-TLR2-CD3ζ-sIL15).

[0177] Ligand binding domain that specifically binds to B cell maturation antigen (BCMA)

[0178] In some embodiments, the chimeric receptors described herein comprise an extracellular domain comprising a ligand binding domain of BCMA or a binding fragment thereof. BCMA is preferentially expressed in mature B lymphocytes and is important for B cell development.

[0179] In some embodiments, a chimeric receptor as described herein comprises an extracellular domain comprising a ligand binding domain of BCMA or a binding fragment thereof.

[0180] 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 210, 212, 214, or 216.

[0181] In some embodiments, the extracellular domain of the chimeric receptor is encoded by the 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 209, 211, 213, or 215.

[0182] Signal peptide

[0183] Chimeric antigen receptors can be expressed from a polynucleotide sequence encoding an N-terminal signal peptide. The signal peptide can direct their fusion partner to integrate into the plasma membrane of the cell. For example, the signal peptide comprises the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 37), or a functional variant having 1, 2, 3 or more amino acid substitutions.

[0184] Transmembrane domain

[0185] In one aspect, the present disclosure provides a CAR with a transmembrane portion, the transmembrane portion including a transmembrane domain. CAR can be designed to include a transmembrane domain fused to the antigen binding domain of CAR. In some embodiments, a transmembrane domain naturally bound to one of the domains in CAR is used. In some cases, the transmembrane domain can be selected and modified by amino acid replacement to avoid these domains from binding to the transmembrane domain of the same or different surface membrane proteins to minimize the interaction with other members of the receptor complex. In some embodiments, the transmembrane domain is operably connected to the extracellular domain with or without a joint. In some embodiments, the transmembrane domain is operably connected 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 includes a complete transmembrane domain of a polypeptide. In some embodiments, the transmembrane domain includes a fragment of a polypeptide transmembrane domain.

[0186] The transmembrane domain can be derived from a natural source or a synthetic source. The transmembrane region that can be used includes the following transmembrane domains: CD28, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCRα, TCRβ or CD3ζ or its functional variants. In a variation, the transmembrane domain can be the following transmembrane domain: CD8α chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2Rβ, TLR, IL-1R subfamily, or its functional variants.

[0187] Illustrative transmembrane domains are provided in Table 3.

[0188] Table 3: Transmembrane (TM) domains

[0189] abbreviation Peptide sequence SEQ ID NO: aTM NLFVASWIAVMIIFRJGMAVAIFCCFFFP 41 DNAM1TM GGTVLLLLFVISITTIIVIFL 42 CD28TM FW VLVVVGGVLACYSLLVTVAFIIFW V 43 IL-2Rβ IPWLGHLLVGLSGAFGFIILVYLLI 44 TLR1TM LLIVTIVATMLVLAVTVTSLC 45 TLR2TM ALVSGMCCALFLLILLTGVLC 46 TLR3TM FFMINTSILLIFIFIVLLIHF 47 TLR4TM TIIGVSVLSVLVVSVVAVLVY 48 TLR5TM FSLFIVCTVTLTLFLMTILTV 49 TLR6TM LIVTIGATMLVLAVTVTSLCI 50 TLR7TM LILFSLSISVSLFLMVMMTAS 51 TLR8TM AVILFFFTFFITTMVMLAALA 52 TLR9TM FALSLLAVALGLGVPMLHHLC 53 TLR10TM ALLIVTIVVIMLVLGLAVAFC 54 IL1RTM HMIGICVTLTVIIVCSVFIY 55 IL18R TM GMIIAVLILVAVVCLVTVCVI 56

[0190] In some embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the transmembrane domain is a fragment of the transmembrane domain of CD28. In some embodiments, the transmembrane domain of CD28 comprises the amino acid sequence shown in SEQ ID NO: 43. In some embodiments, the transmembrane domain of CD28 consists of the amino acid sequence shown in SEQ ID NO: 43. In some embodiments, the transmembrane domain of CD28 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43.

[0191] In some embodiments, the CD28 transmembrane domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 160. In some embodiments, the CD28 transmembrane 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: 160. In some embodiments, the CD28 transmembrane domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 218. In some embodiments, the CD28 transmembrane 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: 218.

[0192] In some embodiments, the transmembrane domain is a transmembrane domain of TLR2. In some embodiments, the transmembrane domain is a fragment of the transmembrane domain of TLR2. In some embodiments, the transmembrane domain of TLR2 comprises the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the transmembrane domain of TLR2 consists of the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the transmembrane domain of TLR2 comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 46.

[0193] In some embodiments, the TLR2 transmembrane domain is encoded by the nucleotide sequence shown in SEQ ID NO: 161. In some embodiments, the TLR2 transmembrane 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: 161.

[0194] 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. In some embodiments, the aTM domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 163.

[0195] In some embodiments, the aTM domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 164 or 165. In some embodiments, the aTM 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: 164 or 165.

[0196] In some embodiments, the transmembrane domain is a transmembrane domain of DNAM1. In some embodiments, the transmembrane domain is a fragment of the transmembrane domain of DNAM1. In some embodiments, the transmembrane domain of DNAM1 comprises the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the transmembrane domain of DNAM1 consists of the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the transmembrane domain of DNAM1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 42.

[0197] In some embodiments, the DNAM1 transmembrane domain is encoded by the nucleotide sequence shown in SEQ ID NO: 162. In some embodiments, the DNAM1 transmembrane 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: 162.

[0198] Hinge domain

[0199] In one aspect, the present disclosure provides a CAR having a portion including a hinge domain. The hinge domain of the CAR disclosed herein may include a joint or spacer sequence between an extracellular antigen binding domain and a transmembrane domain. It will be understood by those skilled in the art that a hinge domain (e.g., sequence) is a polypeptide segment that contributes to flexibility in at least some examples (see, e.g., Woof et al., Nat. Rev. Immunol., 4 (2): 89-99 (2004)). A hinge sequence may be any suitable sequence derived from or derived from any suitable molecule.

[0200] The hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, 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 inter-arm domain includes at least a portion of an IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulin Fc region belonging to its CH2 and CH3 domains. In some embodiments, the inter-arm domain may also include at least a portion of a corresponding immunoglobulin hinge region.

[0201] In some embodiments, a part of an immunoglobulin constant region is used as a hinge region between an antigen-binding domain (e.g., scFv) and a transmembrane domain. Compared to the absence of an inter-arm, the inter-arm may have a length of cell reactivity that provides antigen binding and improves. Exemplary hinges include at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and include any integer number of amino acids between the endpoints of any listed range. 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 length of the hinge is 12 amino acids or about 12 amino acids. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Application Publication No. WO2014031687, and U.S. Patent Publication No. US 2014 / 0271635.

[0202] The CAR of the present disclosure may comprise a hinge domain isolated or derived from CD4, CD8α, CD28, IgG1, IgG2, IgG4 or IgD.

[0203] In some embodiments, the hinge domain is isolated or derived from a human CD8a molecule.The hinge domain may comprise the polypeptide sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 38) or a variant having 1, 2, 3, 4, 5 or more substitutions.

[0204] In some embodiments, the hinge domain is isolated or derived from a human CD28 molecule. The hinge domain may comprise the polypeptide sequence: CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 39) or a variant having 1, 2, 3, 4, 5 or more substitutions. The hinge domain may comprise the polypeptide sequence: LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 217) or a variant having 1, 2, 3, 4, 5 or more substitutions.

[0205] In some embodiments, the hinge domain is isolated or derived from a human IgG4 molecule.The hinge domain may comprise the polypeptide sequence: ESKYGPPCPPCP (SEQ ID NO: 40) or a variant having 1, 2, 3, 4, 5 or more substitutions.

[0206] In some embodiments, the hinge domain is isolated or derived from a human CD8 molecule.The hinge domain may comprise the polypeptide sequence: TTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 158) or a variant having 1, 2, 3, 4, 5 or more substitutions.

[0207] Connectors

[0208] In some embodiments, the chimeric receptors described herein comprise one or more linkers between receptor components. In some embodiments, the chimeric receptors described herein comprise a linker between an extracellular domain and a transmembrane domain. In some embodiments, the chimeric receptors described herein comprise a linker between a transmembrane domain and a cytoplasmic domain. In some embodiments, the chimeric receptors described herein comprise a linker between two or more intracellular signaling domains of a cytoplasmic domain.

[0209] Gly-Ser linker

[0210] Suitable linkers for connecting peptide sequences are known to those skilled in the art. Exemplary linkers include gly-ser peptide linkers, glycine-proline peptide linkers, and proline-alanine peptide linkers. In certain embodiments, the linker is a gly-ser peptide linker, i.e., a peptide consisting of glycine and serine residues.

[0211] 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. 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 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 certain 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.

[0212] In some embodiments, the chimeric receptors described herein comprise 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 set forth in SEQ ID NO: 167. In some embodiments, the chimeric receptors described herein comprise 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 set forth in SEQ ID NO: 169. In some embodiments, the chimeric receptors described herein comprise 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 set forth in SEQ ID NO: 171. In some embodiments, the chimeric receptors described herein comprise 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 set forth in SEQ ID NO: 173.

[0213] Other connectors

[0214] Other linkers suitable for joining polypeptide sequences to allow bicistronic expression are known to those skilled in the art. Exemplary linkers include Furin GSG-T2A.

[0215] 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 a nucleotide sequence set forth in SEQ ID NO: 175.

[0216] CAR polypeptide and polynucleotide sequences

[0217] While the present disclosure contemplates combinations of the signal transduction domains of the present disclosure in any order and with any transmembrane and extracellular domains, the present disclosure provides at least the constructs listed in Table 4.

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] In each case, the CAR polypeptide may include a CD8 hinge domain, an artificial transmembrane domain ("aTM"), and one or more signal transduction domains and / or other cytoplasmic domains in a continuous order, as shown in the first column of Table 4. 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 include 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 respective corresponding exemplary polynucleotide sequences. 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.

[0259] Co-expression partner

[0260] In one aspect, the NK CAR cells of the present disclosure are engineered to co-express other proteins (membrane-bound or secreted proteins). Exemplary compositions and methods are provided in US 2017 / 0073638 A1.

[0261] In some variations, the NK cells of the present disclosure are engineered to express interleukin-15 (IL-15) (membrane-bound or secreted). IL-15 is a cytokine that plays a role in regulating immune responses. Without being bound by theory, expression of IL-15 can improve NK cell function. IL-15 can comprise the following polypeptide sequence:

[0262] IL-15 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 SEQ ID NO: 58. In some embodiments, IL-15 is encoded by the nucleotide sequence set forth in SEQ ID NO: 208. In some embodiments, IL-15 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: 208.

[0263] Alternatively, IL-15 may comprise the following polypeptide sequence:

[0264] 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.

[0265] In some variations, the NK cells of the present disclosure are engineered to express interleukin-18 (IL-18) (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 can enhance NK cell proliferation, maturation, and cytotoxicity. IL-18 can comprise the following polypeptide sequence:

[0266] 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, IL-18 may comprise the following polypeptide sequence:

[0267] IL-18 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 SEQ ID NO:59.

[0268] The co-expressed proteins of NK cells disclosed herein can be fusion proteins. As used herein, "fusion protein" refers to the expression product produced by the fusion of at least two genes. A fusion protein can be a chimeric polypeptide comprising two or more unrelated proteins or protein fragments conjugated to each other. The co-expressed polypeptide can be connected to the polypeptide encoding the CAR disclosed herein by a self-cleaving peptide, such as a self-cleaving (2A) peptide, or a functional variant thereof.

[0269] In some variations, the NK cells of the present disclosure are engineered to express interleukin-15 / interleukin-15R (IL15 / IL15R) fusion proteins. Interleukin-15 receptor (IL-15R) has been shown to play a role in stimulating immune cell proliferation. Without being bound by theory, expression of IL-15 / IL15R can improve NK cell function. The IL15 / IL15R fusion protein can comprise the following polypeptide sequence:

[0270] The IL15 / IL15R fusion protein 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 SEQ ID NO:62.

[0271] In some variations, the NK cells of the present disclosure are engineered to express interleukin-18 / interleukin-18R (IL18 / IL18R) fusion proteins. Interleukin-18 receptor (IL-18R) has been shown to play a role in immune cell activation and promotion of cytotoxic activity. Without being bound by theory, expression of IL18 / IL18R can enhance NK cell proliferation, maturation, and cytotoxicity. The IL18 / IL18R fusion protein can comprise the following polypeptide sequence:

[0272] The IL18 / IL18R fusion protein 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 SEQ ID NO:63.

[0273] In some variations, the NK cells of the present disclosure are engineered to express OX40. OX40 (CD134) is a member of the tumor necrosis factor receptor (TNFR) family that plays a role in signal transduction and sensitization of immune cells. Without being bound by theory, expression of OX40 may enhance activation of MAPK / ERK, NFkB, and AKT pathways; and / or induce proliferation and cytokine release. OX40 may comprise the following polypeptide sequence:

[0274] 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.

[0275] 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 CARs of the present disclosure.

[0276] Polynucleotides and vectors

[0277] 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-expression partner of the present disclosure. Other elements present in the vector of the present disclosure may also include selectable markers and other components necessary for protein expression, such as enhancers, promoters, and termination sequences. The polynucleotides of the present disclosure may include a flanking left arm and a right arm (arm) for homology-mediated repair. The polynucleotides may include a promoter operably connected to a polynucleotide sequence encoding a CAR.

[0278] The polynucleotides disclosed herein can also be used for gene editing. For example, homology-mediated repair can be used to insert the polynucleotide into a desired genomic locus. The polynucleotide can include a promoter and other genetic elements, or the repair template can omit these and instead rely on the host genomic sequence for transcription.

[0279] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating 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). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0280] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and in particular retroviral vectors, have become the most widely used methods for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adenovirus-associated viruses, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0281] Chemical methods 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 used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0282] Regardless of the method used to introduce the exogenous nucleic acid into the host cell, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. These assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as (fluid) detecting the presence or absence of a specific peptide by immunological means (ELISA and immunoblotting) or other assays.

[0283] Provided are polynucleotides comprising a CAR or a portion thereof. 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.

[0284] Modified cells

[0285] Methods for preparing modified cells (e.g., stem cells, progenitor cells, or immune effector cells) are known to those skilled in the art. Methods for preparing cells expressing polypeptides, such as chimeric receptors or fusion polypeptides described herein, are known in the art. Exemplary methods are described herein.

[0286] In some embodiments, the present disclosure provides modified cells (e.g., modified stem cells, modified progenitor cells, or modified immune effector cells) expressing chimeric antigen receptors described herein. In some embodiments, the present disclosure provides modified cells (e.g., modified stem cells, modified progenitor cells, or modified immune effector cells) comprising polynucleotide sequences encoding chimeric antigen receptors 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.

[0287] In some embodiments, the cell comprises a chimeric antigen receptor described herein, comprising a ligand binding domain, a transmembrane domain, and an intracellular signal transduction domain comprising a TLR2 signal transduction domain. In some embodiments, the cell comprises a chimeric antigen receptor described herein, comprising a ligand binding domain, a transmembrane domain, and an intracellular signal transduction domain comprising a TLR2 signal transduction domain and a CD3ζ signal transduction domain. In some embodiments, the cell comprises a chimeric antigen receptor described herein, comprising a ligand binding domain, a transmembrane domain selected from the following: CD8α chain (CD8α), CD28, CD16, NKp44, NKp46, NKG2, DNAM1, IL-2Rβ, TLR, IL-1R subfamily, or a functional variant thereof and an intracellular signal transduction domain comprising a TLR2 signal transduction domain and a CD3ζ signal transduction domain.

[0288] Signal transduction domain

[0289] In some embodiments, the polynucleotides of the present disclosure comprise a sequence encoding a signal transduction domain of the present disclosure. In some embodiments, the polynucleotides encode a TLR signal transduction domain. In some embodiments, the polynucleotides encode an IL-1R subfamily signal transduction domain.

[0290] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR2 signal transduction domain. The polynucleotide sequence encoding the TLR2 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 12. The polynucleotide sequence encoding the TLR2 signal transduction domain may have a polynucleotide 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: 12.

[0291] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR8 signal transduction domain. The polynucleotide sequence encoding the TLR8 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 18. The polynucleotide sequence encoding the TLR8 signal transduction domain may have a polynucleotide sequence having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity to SEQ ID NO: 18.

[0292] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR3 signal transduction domain. The polynucleotide sequence encoding the TLR3 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 13. The polynucleotide sequence encoding the TLR3 signal transduction domain may have a polynucleotide 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: 13.

[0293] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR7 signal transduction domain. The polynucleotide sequence encoding the TLR7 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 17. The polynucleotide sequence encoding the TLR7 signal transduction domain may have a polynucleotide 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: 17.

[0294] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR4 signal transduction domain. The polynucleotide sequence encoding the TLR4 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 14. The polynucleotide sequence encoding the TLR4 signal transduction domain may have a polynucleotide 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: 14.

[0295] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR1 signal transduction domain. The polynucleotide sequence encoding the TLR1 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 11. The polynucleotide sequence encoding the TLR1 signal transduction domain may have a polynucleotide 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: 11.

[0296] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR5 signal transduction domain. The polynucleotide sequence encoding the TLR5 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 15. The polynucleotide sequence encoding the TLR5 signal transduction domain may have a polynucleotide 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: 15.

[0297] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR6 signal transduction domain. The polynucleotide sequence encoding the TLR6 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 16. The polynucleotide sequence encoding the TLR6 signal transduction domain may have a polynucleotide sequence having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity with SEQ ID NO: 16.

[0298] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR9 signal transduction domain. The polynucleotide sequence encoding the TLR9 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 19. The polynucleotide sequence encoding the TLR9 signal transduction domain may have a polynucleotide 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: 19.

[0299] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding a TLR10 signal transduction domain. The polynucleotide sequence encoding the TLR10 signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 20. The polynucleotide sequence encoding the TLR10 signal transduction domain may have a polynucleotide 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: 20.

[0300] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding an IL-1R signal transduction domain. The polynucleotide sequence encoding the IL-1R signal transduction domain may comprise a nucleic acid sequence encoding the polypeptide shown in SEQ ID NO: 21. The polynucleotide sequence encoding the IL-1R signal transduction domain may have a polynucleotide sequence encoding a polypeptide sequence having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 21.

[0301] In some embodiments, the polynucleotide sequence encoding the signal transduction domain of the present disclosure comprises a nucleic acid sequence encoding an IL-18R signal transduction domain. The polynucleotide sequence encoding the IL-18R signal transduction domain may comprise the nucleic acid sequence shown in SEQ ID NO: 24. The polynucleotide sequence encoding the IL-1R signal transduction domain may have a polynucleotide 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: 24.

[0302] Table 5: Signal transduction domains

[0303]

[0304]

[0305]

[0306] Other components of the CAR cytoplasm

[0307] The cytoplasmic portion of the CAR may further comprise one or more domains listed in Table 6, or a functional variant thereof having at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100% identity.

[0308] Table 6: Other cytoplasmic domains

[0309]

[0310]

[0311]

[0312] In some embodiments, the chimeric receptor described herein comprises a cytoplasmic domain. In some embodiments, the cytoplasmic domain is operably connected to the TIR signaling domain via a joint. In some embodiments, the cytoplasmic domain is operably connected to the TIR signaling domain without a joint. In some embodiments, the N-terminus of the cytoplasmic domain is operably connected to the C-terminus of the TIR signaling domain. In some embodiments, the C-terminus of the cytoplasmic domain is operably connected to the N-terminus of the TIR signaling domain.

[0313] 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 the 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.

[0314] 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 64. In some embodiments, the CD3 zeta cytoplasmic domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 181. In some embodiments, the CD3 zeta cytoplasmic 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: 181.

[0315] In some embodiments, the cytoplasmic domain is the 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 the 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.

[0316] 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 the nucleotide sequence set forth in SEQ ID NO: 178. In some embodiments, the CD28 cytoplasmic domain is encoded by the 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.

[0317] Co-expression partner

[0318] The activity of NK cells can be enhanced by co-expressing a variety of factors, including but not limited to those provided in Table 7.

[0319] Table 7: Co-expression partners

[0320]

[0321] Exemplary Chimeric Antigen Receptors

[0322] 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 CD3ζ signaling domain. In some embodiments, the chimeric antigen receptor described herein comprises (a) a ligand binding domain, (b) a transmembrane domain selected from CD8α 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 signaling domain. Exemplary 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 ζ signaling domain. Other exemplary chimeric antigen receptors include a ligand binding domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising a TLR2 signaling domain and a CD3 ζ signaling domain.

[0323] Reagent test kit

[0324] In one aspect, the present disclosure provides a kit comprising the CARNK cells or pharmaceutical compositions described in the present disclosure and instructions for use.

[0325] How to use

[0326] In one aspect, the present disclosure provides a method for treating or preventing a disease or disorder in a subject in need thereof, comprising administering the CARNK cell of the present disclosure or the pharmaceutical composition of the present disclosure to the subject.

[0327] In one aspect, the present disclosure provides a method of killing a target cell, comprising contacting the target cell population with a natural killer cell population of the present disclosure, 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 cell induces specific killing of the target cell.

[0328] In one aspect, the present disclosure provides a method of killing target cells, comprising contacting a target cell population with a NK cell population.

[0329] Cell killing can be assessed by assays known in the art, including, but not limited to, those described in the Examples.

[0330] The cancer can be caused by the proliferation of tumor cells showing antigens specifically bound by CAR or NK cells expressing CAR. CAR provided herein includes CAR with ligand binding domains specifically binding to CD19, CD20 and / or HER2. CD19 and CD20 can be expressed on tumor cells of various inert and aggressive subtypes of B cell lymphoma and leukemia, non-Hodgkin's lymphoma (NHL), B cell chronic lymphocytic leukemia (CLL) and non-T acute lymphoblastic leukemia (ALL). Therefore, CAR 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 has been found in several tumor types, including but not limited to breast cancer, esophageal cancer, lung cancer, cervical cancer, endometrial cancer and ovarian cancer. Therefore, 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.

[0331] In one aspect, the present 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 a population of NK cells.

[0332] The terms "subject," "patient," and "individual" are used interchangeably herein to refer to a vertebrate, such as a mammal, eg, a human.

[0333] As used herein, the term "treatment" encompasses at least an improvement in symptoms associated with a disease or condition in a patient, wherein improvement is used in a broad sense to mean at least a decrease in the magnitude of a parameter (e.g., a symptom associated with the condition being treated). As such, "treatment" also includes situations in which a disease, disorder or pathological condition, or at least symptoms associated therewith, are completely suppressed (e.g., prevented from occurring) or stopped (e.g., terminated), such that the patient no longer suffers from the condition or at least the symptoms characterizing the condition.

[0334] In some embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a CARNK cell and / or a pharmaceutical composition of the present disclosure.

[0335] 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 (eg, cancer).

[0336] In some embodiments, the CARNK cells described herein and the pharmaceutical compositions comprising them are used as part of a combination therapy. In some embodiments, the CARNK cells or pharmaceutical compositions of the present disclosure are co-administered to a subject together with a tumor-targeting antibody.

[0337] In some embodiments, the CARNK cells or pharmaceutical compositions of the present disclosure show increased cytotoxicity relative to cells or pharmaceutical compositions in which cells are not engineered in the manner of the present disclosure. In some embodiments, the cells or pharmaceutical compositions of the present disclosure show increased effectiveness relative to equivalent cells or pharmaceutical compositions in which cells are not engineered in the manner of the present disclosure.

[0338] The NK cell population or pharmaceutical composition can be administered in an amount effective to kill target cells and / or in an amount therapeutically effective for cancer treatment.

[0339] Pharmaceutical composition

[0340] In one aspect, the present disclosure provides a pharmaceutical composition comprising the CARNK cells or cell populations described in the present disclosure and one or more pharmaceutically acceptable excipients or diluents.

[0341] As used herein, the term "pharmaceutically acceptable" means a formulation approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, other generally recognized pharmacopeia, and otherwise safe for use in animals, and more particularly in humans and / or non-human mammals.

[0342] As used herein, the term "pharmaceutically 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 pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th edition, (Lippincott, Williams & Wilkins 2003).

[0343] The compositions of the present invention may additionally contain other auxiliary components conventionally present in pharmaceutical compositions. Thus, for example, the compositions may contain other, compatible pharmaceutically active materials, such as, for example, antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain other materials useful in physically formulating the composition in various dosage forms.

[0344] Production method

[0345] In one aspect, the present disclosure provides a method for preparing NK cells, which includes providing stem cells or progenitor cells, such as induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC), and differentiating the stem cells or progenitor cells into NK cells. Stem cells or progenitor cells can be genetically modified to include polynucleotides encoding CAR before or after differentiation of stem cells. Known vectors can be used to deliver polynucleotides, which can be stably maintained and / or integrated into the genome of the cell by the cell. Stem cells or progenitor cells can be CISH- / -iPSC and can be used to produce CISH- / -iPSC-NK cells. Stem cells or progenitor cells can be soluble IL-15 or membrane-bound IL-15 knock-in iPSC, and can be used to produce IL-15 knock-in iPSC-NK cells. In some embodiments, iPSC can be edited to not express CISH, express soluble IL-15 or membrane-bound IL-15, and express any one or more CAR constructs disclosed herein.

[0346] Cancer treatment

[0347] In some embodiments, the present disclosure provides a method for treating cancer with a chimeric receptor expressing cell provided herein or a pharmaceutical composition or formulation thereof. In some embodiments, the method comprises administering a chimeric receptor expressing cell provided herein, a pharmaceutical composition or formulation described herein to a subject in need thereof. As used herein, the term "administering" refers to delivering a bioactive composition or formulation by the following administration routes, 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.

[0348] In some embodiments, cells expressing the chimeric receptors described herein exhibit enhanced anti-tumor efficacy relative to cells lacking the chimeric receptor. Methods for measuring in vitro and in vivo anti-tumor efficacy are known to those skilled in the art and include, for example, in vitro killing assays and tumor implantation in mouse models.

[0349] Cell killing can be assessed by assays known in the art, including, but not limited to, those described in the Examples.

[0350] In some embodiments, cells expressing the 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.

[0351] In some embodiments, cells expressing the chimeric receptor described herein exhibit between 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, or 100% to 1000% enhanced cell killing compared to cells lacking the chimeric receptor and / or fusion polypeptide.

[0352] In some embodiments, cells expressing the 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.

[0353] In some embodiments, cells expressing the chimeric receptor described herein exhibit between 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, or 100% to 1000% enhanced specific killing compared to cells lacking the chimeric receptor and / or fusion polypeptide.

[0354] definition

[0355] All patent publications, patents and patent applications, including any figures and appendices therein, are incorporated by reference in their entirety for all purposes to the same extent as if each individual patent publication, patent or patent application, figure or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0356] Reference in this specification to any prior publication (or information derived from it) or to any known matter is not and should not be taken as an acknowledgement or acknowledgment or any form of statement that the prior publication (or information derived from it) or any known matter forms part of the common general knowledge in the field to which this specification relates.

[0357] Unless the context clearly dictates otherwise, all embodiments of any aspect of the invention may be used in combination.

[0358] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise," "comprising," and the like will be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to."

[0359] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "and" and "or" are used interchangeably unless expressly indicated otherwise. Words using the singular or plural number also include the plural and singular number, respectively.

[0360] The section headings are for convenience only, and the combination of elements from different sections is contemplated. In addition, when used in this application, the words "herein," "above," and "below," and words of similar meaning shall refer to this application as a whole and not to any particular portion of this application.

[0361] The term "functional variant" refers to a homology (by sequence or structure) to a domain that retains sufficient signal transduction activity to activate NK cells.

[0362] The term "sequence identity" refers to the percent identity of a polypeptide or polynucleotide sequence of interest to a reference sequence, calculated as the number of exact 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 European Molecular Biology Open Software Suite (EMBOSS) Needle program available at www.ebi.ac.uk, as described in Maderia et al. Nucleic Acids Res. 47(W1): W636-W641 (2019).

[0363] As used herein, the term "inactivating mutation" refers to a mutation in a genomic sequence that disrupts gene function. Inactivating mutations can be in any sequence region (e.g., coding or non-coding region) that contributes to gene expression. Examples include, but are not limited to, cis-acting elements (enhancers) or sequences that undergo transcription (e.g., mRNA transcript sequences). Inactivating mutations include mutations that render a gene or its encoded protein nonfunctional or reduce the function of a gene or its encoded protein. Example

[0364] The present disclosure will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present disclosure should in no way be considered limited to these examples, but rather should be considered to encompass any and all variations that become evident as a result of the teachings provided herein.

[0365] Without further description, it is believed that those skilled in the art can use the above description and the following illustrative examples to prepare and use the methods described in the present disclosure and practice the claimed methods. Therefore, the following working examples specifically point out the embodiments of the present disclosure and should not be considered as limiting the rest of the present disclosure in any way.

[0366] The materials and methods used in these experiments will now be described.

[0367] Example 1: Screening of chimeric antigen receptor designs

[0368] This example describes the design and testing of chimeric antigen receptors in induced pluripotent stem cells (iPSC)-derived natural killer cells (NK cells). We constructed a library of 44 novel CAR constructs containing signal transduction modules from different NK cell-specific signal transduction receptors. We then developed a viral-based CAR expression protocol in mature iPSC-derived NK cells, which produced high CAR expression (>75% CAR+) while maintaining high viability (>90%). We then screened CAR activity using two co-cultured target cell killing assays (eSight impedance assay and caspase 3 / 7 killing assay) and two NK cell-tolerant CD19+ target cell lines (Raji and SupB15). The tested constructs are provided as SEQ ID NOs: 100-117, all of which have anti-CD19scFv as ligand binding domains. Our screen identified seven novel CAR signaling modules that outperformed a second-generation T cell CAR (CD28-ξ) and three previously described NK cell CARs (2B4-ξ, OX40-ξ, and OX40L-ξ). These results were consistent across both functional assays and the two target cell lines tested, supporting our findings.

[0369] The designed CAR sequence was C-terminally linked to membrane-bound IL-15 (mbIL-15) via a self-cleaving (2A) peptide for co-expression in natural killer cells. Lentiviral particles were produced and concentrated ~100-fold to facilitate the high viral titers required for transduction of iPSC-derived NK cells.

[0370] The concentrated virus stock was loaded onto a 96-well plate coated with recombinant human fibronectin by centrifugation, IL-2 activated iPSC-derived NK cells were added, and centrifuged in the presence of viral supernatant and incubated for 72 hr in the presence of IL-2. CAR expression was detected by flow cytometry using an HA-tag engineered into the extracellular portion of the CAR polypeptide ( Fig. 9CAR expression was uniform across most CAR constructs, with an average CAR expression of ~78% ( Fig.10 We did not observe any loss of cell viability when comparing CAR-transduced cells to non-transduced controls.

[0371] We used two co-culture target cell killing assays (eSight impedance assay and caspase 3 / 7 killing assay) and two NK cell tolerance CD19 + Target cell lines (Raji and SupB15) were screened for CAR activity. Fig.11 and Fig.12 The screening results are shown in . We identified 6 CAR constructs that outperformed the best comparison CAR (2B4-ξ). 12 outperformed another comparison CAR (OX40L-ξ). All but one of the tested CARs outperformed the third comparison CAR (OX40-ξ).

[0372] Figures 13A-13C Selected data comparing CAR designs with and without the 2B4 domain are shown in . CARs based on 2B4 have been shown to be effective for activating NK cells. Unexpectedly, our CAR TLR2-ξ outperformed the comparison 2B4-ξ and our CAR design combining the 2B4 and TLR2 domains (2B4-ξ) in both assays ( Fig. 13B and Fig. 13C ).

[0373] Overall, we have successfully developed a CAR screening platform in a therapeutically relevant allogeneic cell source and have identified NK-optimized CARs that can improve the potency of NK cells compared to current CAR solutions. Notably, compared to the CARs containing classical NK cell activation receptor domains used in previously developed NK-optimized CAR designs, namely 2B4 (as shown), FCER1G, DNAM1, and DAP10, the intracellular signaling modules from Toll-like receptors and IL-1R subfamily receptors showed enhanced NK CAR activity. These findings validate the use of different immune cell signaling domains in NK-optimized CARs.

[0374] method

[0375] Construction of lentiviral expression vector and viral packaging: The polypeptide sequences of all candidate CAR components were obtained from Ensemble TM Database (Howe et al. Nucleic Acids Res. 2021, Vol. 49(1): 884-891) and using SnapGene TMMolecular biology software was used to assemble the complete CAR design. Publicly available codon optimization tools (Integrated DNA Technologies TM ) to determine the nucleic acid sequence. All CAR constructs included a CD19 scFv binder (FMC63; SEQ ID NO: 109), an mbIL-15 co-expression partner (SEQ ID NO: 48), and an HA-tag for CAR detection as a representative ligand binding domain. CAR constructs were synthesized and cloned into the EF-1α promoter (pCDH-EF1α-MCS-T2A-Puro; System Biosciences TM )-controlled lentiviral expression vector, and maxi-preps were generated and sequence verified. The constructs described above, packaging plasmids (Rev, gag-pol, and VSVG; Cell Biolabs TM ) and Lenti-X TM 293T cell line (Takara TM ) to produce lentiviral particles. PEG-based concentrated solution (Takara TM ) The viral supernatant was concentrated ~100-fold and lysed using Lenti-x GoStix Plus TM (Takara TM ) Determine the virus titer.

[0376] Generation of iPSC-NK cells: iPSCs were harvested and transferred to serum-free medium and aggregated to form embryoid bodies by rotation. Once CD34 + If EBs were grown, they were 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 D-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 were placed under these conditions for 28 days with weekly medium changes to generate NK cells (CD45 + CD56 + CD33-CD3- cells, as determined by flow cytometry). NK cells were then harvested for expansion.

[0377] Amplification: After differentiation, the immortalized leukemia cell line K562 expressing mbIL21 and 4-1BB ligand (CD137) was used to stimulate NK cell expansion. NK cells were inoculated with K562-41BBL-mbIL21 feeder cells and incubated in a stromal cell growth medium (SCGM) containing 100U / mL IL-2 at a ratio of 1:2. Non-adherent cells were removed and analyzed by flow cytometry to determine the purity of CD56+NK cells. Then, 2:1 aAPC (irradiated with 10000Gy) was used to stimulate these cells with 350000 NK cells / mL culture medium (containing RPMI 1640, 2mM L-glutamine, 1% penicillin / streptomycin, 1% non-essential amino acids and 10% standard FBS or 10% human serum AB, supplemented with 50-100U / mL IL-2).

[0378] Transduction of iPSC-NK cells: Untreated 96-well plates were coated with recombinant human fibronectin (Takara). An equal amount of concentrated lentivirus was added to the plate and centrifuged at 2000 × g for 2 hr at 32 ° C. iPSC-derived NK cells (6e4 cells) were added to each well in a medium containing IL-2 (50U / ml) and centrifuged at 800 × g for 1 hr at 32 ° C. The cells were incubated at 37 ° C for 48 hr. Fresh medium (with IL-2) was added, and the cells were incubated for another 24 hr. CAR expression was determined using flow cytometry 72 hr after transduction to detect the HA-tag engineered into the extracellular domain of each CAR. Within 24 hr of CAR detection, CAR+ cells were used for functional assays.

[0379] Using eSight TM Long-term target cell killing assay: CD40 tethering reagent (Agilent TM ) were coated with RTCA E-plates overnight at 4°C. The plates were washed with PBS and target cells (CD19+ target cells (Raii; ATCC TM ) or HER2+ target cells (BT474 clone 5)) were added to each well, and the impedance readings were allowed to balance for 2-4 hr. CAR+iPSC-NK cells were added to each well at a specified E:T ratio, and impedance readings were measured every 15 min for 24 hr. NK-specific killing was determined by normalizing the impedance readings to the "target cell alone" control wells and plotted as target killing frequency.

[0380] 4hr caspase 3 / 7 cytotoxicity assay: CellTrace TMViolet solution in PBS was pre-stained with SupB15 (CD19+ target cells) at 37°C for 15 min and then washed in complete 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. The co-culture was centrifuged at 100×g for 1 min and incubated at 37°C for 3.5 hr. After incubation, Caspase-3 / 7 Green Detection Reagent and SYTOX TM AADvancedT M Dead cell staining solution was added to the co-culture and incubated for an additional 30 min at 37°C. After staining, the assay was read on the NovoCyte Advanteon and analyzed by FlowJo software. NK-specific killing was determined by normalizing the impedance readings to the "target cells alone" control wells and plotted as target killing frequency.

[0381] [Prophetic] Example 2: In vivo functional testing of CAR with CD19 binding domain

[0382] The in vivo efficacy of iPSC-derived NK cells expressing functional CAR constructs (determined by in vitro assay results) was tested using a CD19 expression model of B cell malignancies. In order to induce disease, 10-12 week-old NOD-SCID-γγ- / -(NSG) mice (Jackson Laboratory) were intravenously injected with Raji cells expressing luciferase (CD19 positive). One day later, iPSC-derived NK cells were administered to mice, and the NK cells were engineered to express (a) no CAR (negative control); (b) second-generation T cell CAR (benchmark control); or (c) NK CAR (test group). During the remainder of the experiment, IL-2 was administered to the test mice every other day. Disease development was monitored by bioluminescent imaging of survival, weight loss, and tumor burden (measured weekly). In order to evaluate the number of infused NK cells present during the experiment, CD45 from peripheral blood was measured weekly. + CD56 + CD3- cells were quantified.

[0383] Example 3: Functional testing of CARs with HER2 binding domains

[0384] Concentrated lentiviral stocks containing different CARs with HER2scFv binders were loaded onto 96-well plates coated with recombinant human fibronectin by centrifugation. IL-2 activated iPSC-derived NK cells were added, centrifuged in the presence of viral supernatant, and incubated for 72 hours in the presence of IL-2. CAR expression was detected by flow cytometry using an HA-tag engineered to the extracellular portion of the CAR polypeptide. CAR expression was uniform in most CAR constructs ( Fig.14A ).

[0385] HER2 in NK cell tolerance + CAR activity was screened on the target cell line (BT474) using a co-culture target cell killing assay (eSight impedance assay). Fig. 14B and Fig.14D The screening results are shown in. In addition, the co-culture supernatants were assayed for cytokine secretion using ELISA. Fig. 14C The secretion assay results are shown in . The results identified CD8-aTM-TLR2-CD3ζ (SEQ ID NO: 82) as the best NK optimized CAR tested and consistently performed the same / better than the others in multiple CAR targets and assays. In addition, similar sIL15 levels (150-200pg / ml) were observed between (1) CD8-aTM-TLR2-CD3ζ, (2) CD8-aTM-DNAM 1-LFA1-CD3ζ, and (3) 2B4-ζ (lower for aTM-IL18R-2B4-CD3ζ, ~100pg / ml) (data not shown).

[0386] Similarly, the CD8-aTM-TLR2-CD3ζ construct with CD19 scFv exhibited robust cell killing of Raji 2.0 cells at multiple effector to target cell ratios (data not shown). The CD8-CD28TM-TLR2-CD3ζ construct also exhibited similar activity.

[0387] Then, CAR-HER2 activity was screened using a stress test killing assay. Briefly, CISH KO iPSC-derived NK cells were transduced with retroviruses containing one of three different CAR constructs, which had the same HER2 scFv binding domain (HER2v3) and different intracellular domains. ( Fig.15A ). In a second set of experiments, CISH KO iPSC-derived NK cells were transduced with retroviruses carrying one of three different CAR constructs, which had the same intracellular domain and a different HER2 scFv binding domain (HER2v3). ( Fig. 15B). In the absence of added cytokines or co-expression of sIL-15, effector CISH KO iPSC-derived NK cells were co-expressed with NK cell-tolerant HER2 + The target cell line (BT474) was co-cultured at an effector cell to target cell ratio of 5:1, and a target cell killing assay (eSight impedance assay) was used to identify which CAR constructs exhibited high killing activity.

[0388] Results using different CAR constructs with the same HER2 scFv binding domain (HER2v3) identified CD8-aTM-TLR2-CD3ζ (SEQ ID NO: 82) as the best NK optimized CAR-HER2 construct tested ( Fig.15A ). The results using the same intramolecular domain (CD8-aTM-TLR2-CD3ζ) with different HER2scFv binding domains identified HER2scFv variant 3 (V3) as the best NK-optimized CAR-HER2 construct tested. ( Fig. 15B ).

[0389] NK cell tolerance to HER2 was also used in the caspase 3 / 7 killing assay + The CAR activity of CD8-aTM-TLR2-CD3ζCAR constructs with different HER2scFv binding domains was evaluated in the target cell line (BT474) Figures 16A-16B Briefly, caspase 3 / 7 activation was measured after CISH KO iPSC-derived NK cells expressing one of three different CAR constructs with identical intracellular domains and different HER2 scFv binding domains (HER2v2, HER2v3, or HER2v5) were co-cultured with resistant solid tumor targets (E:T of 2:1). Fig.16A ). Representative images of target cell killing and caspase 3 / 7 activation after 30 hr co-culture of CISH KO iPSC-derived NK cells + / - CAR with NK cell-tolerant solid tumor targets demonstrate robust NK cell killing after co-culture of CISH KO iPSC-derived NK cells expressing CAR, but not in the absence of CAR. ( Fig. 16B ). Target cells were labeled with Cell TrackerRed and caspase 3 / 7 activation was visualized in green.

[0390] Then, the killing activity of CISH KO iPSC-derived NK cells expressing CD8-aTM-TLR2-CD3ζCAR and one of three different HER2scFv binding domains (HER2v2, HER2v3 or HER2v5) was tested in 3D single tumor spheroids. In brief, BT474 cells were inoculated and cultured using the standard protocol of spheroid formation, and the BT474 cells were human HER+ ductal carcinoma cells. After spheroid formation (about 36 hours after inoculation), cells were co-cultured with NK cells derived from CISH KO iPSC with the following characteristics: 1) CAR is not expressed; 2) HER2v2-CD8-aTM-TLR2-CD3ζCAR is expressed; 3) HER2v3-CD8-aTM-TLR2-CD3ζCAR is expressed; or 4) HER2v5-CD8-aTM-TLR2-CD3ζCAR is expressed. Fig.17 Representative images of NK cell-tolerant solid tumor target spheroids are shown at 0, 18 and 36 hours after treatment. In summary, the data show that CAR-HER2NK, specifically NK expressing HER2v3-CD8-aTM-TLR2-CD3ζ, effectively kills HER2+ target cancer cells.

[0391] A real-time serial cell killing assay (eSight impedance assay) was performed using iPSC-derived NK cells transduced with the HER2v3-CD8-aTM-TLR2-CD3ζζ construct. Briefly, a repetitive killing and depletion profile of the HER2+ target cell line (SKOV3) was observed by repeated addition of HER2v3-CD8-aTM-TLR2-CD3ζCISH KO iPSC-derived NK cells to the target cells. Fig.18 As shown, HER2v3-CD8-aTM-TLR2-CD3ζ NK cells maintained a high percentage of NK cell killing ability after 3 rounds, while NK cells without CAR (untransduced or "UT") no longer killed cells after the second round.

[0392] 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 spheroid formation, target cells were co-cultured with different ratios of 1) CISH KO iPSC-derived NK cells not transduced with CAR constructs (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-CD3ζCISH KO iPSC-derived NK cells) added to target cells (BT474 cells) was 5: 1, 2.5: 1 or 1.25: 1. The killing of spheroid cells was monitored on days 0 and 3 after the addition of NK cells. Fig.19 It was shown that target BT474 cells of HER+ spheroids treated with NK expressing HER2v3-CD8-aTM-TLR2-CD3ζ shrank and underwent apoptosis in a concentration-dependent manner on day 3.

[0393] To examine the effect of sIL-15 on CAR-HER2 activity, a CAR expression assay was performed ( Fig.23A ) and long-term killing assay against cell line BT474 clone 5 ( Fig. 23B ). Although the expression of sIL-15 did not affect CAR expression, sIL-15 expression (HER2-3-CD8-aTM-TLR2-CD3ζ-sIL15 (SEQ ID NO: 205)) significantly increased CARNK cell killing of BT474 cells compared to controls (CD20-2b4-ζ-sIL15 without sIL15 and HER2-3-CD8-aTM-TLR2-CD3ζ (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), while only about 55% and 30% NK cell killing were observed for CD20-2b4-ζ-sIL15 without sIL15 and HER2-3-CD8-aTM-TLR2-CD3ζ (SEQ ID NO: 204), respectively. These results demonstrate that sIL-15 and HER2-CAR constructs synergize in NK cell killing of HER+ breast cancer cell lines.

[0394] Taken together, these results demonstrate that CISH KO iPSC-derived NK cells expressing the HER2-CD8-aTM-TLR2-CD3ζCAR exhibit robust activity against NK cell-resistant solid tumor target cells.

[0395] Example 4. Functional testing of HER2-CAR CISH KO iPSC-derived NK cells

[0396] This example describes that testing of CISH KO iPSC-derived NK cells expressing HER2scFv-CAR and soluble IL-15 demonstrated effective killing of solid tumor target cell lines.

[0397] First, using gene editing methods, triple-edited (TE) iPSCs were generated by introducing the HER2-CAR sequence connected to soluble IL-15 (sIL-15) via the C-terminus of a self-cleaving (2A) peptide into CISH KO iPSCs. The generated TEiPSCs: 1) do not express the CISH gene (CISH KO); 2) express HER2v3-CD8-aTM-TLR2-CD3ζ (HER-CAR) and 3) express soluble IL-15 (SEQ ID NO: 58), thereby allowing IL-15 secretion ( Fig. 20 ). Then, TE iPSCs were differentiated to form functional NK cells (TE iPSC-derived NK cells) that lack CISH, express HER-CAR, and exhibit IL-15 secretion.

[0398] To determine whether triple editing of iPSCs resulted in altered NK phenotype, the expression of NK cell markers in TE iPSC-derived NK cells was measured by flow cytometry. Fig.21 As shown, the expression of 12 NK phenotype markers was not significantly different between wild-type (WT) NK cells, NK differentiated from iPSCs with CISH KO only, and NK differentiated from the evaluated TE iPSCs.

[0399] The function of TE iPSC-derived NK cells was evaluated in a spheroid killing assay. In brief, HER2+ (BT474 clone 5) target cell spheroids were prepared as described in the Examples herein. Any of wild-type (WT) NK cells, NK differentiated from iPSCs with only CISH KO, or NK differentiated from TE iPSCs was added to HER2+ spheroids (E:T of 10:1). NK-mediated killing of HER2+ spheroids monitored using eSight impedance assays showed that TE iPSC-derived NK cells were effective in killing NK-resistant HER2+ solid tumor targets ( Fig.22A22 shows that the spheroids treated with TE iPSC-derived NK cells were significantly reduced in size after 3 days compared with the spheroids treated with WT NK or CISHKO iPSC-derived NK cells.

[0400] In summary, the data demonstrate that TE iPSC-derived NK cells effectively kill solid tumor target cell lines.

[0401] ****

[0402] While the invention has been described in conjunction with particular embodiments thereof proposed, it will be understood that it is capable of further modifications, and the application is intended to cover any variations, uses, or modifications of the invention which generally follow from the principles of the invention and include such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and which may be applied to the essential characteristics herein above described and which come within the scope of the claims appended hereto.

Claims

1. A natural killer cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular portion, a transmembrane domain and a cytoplasmic portion, 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 according to 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 according to 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 according to 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 according to 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 according to 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 according to claim 2, wherein the TLR signaling domain is selected from TLR1, TLR5, TLR6, TLR9 or TLR10 signaling domains or functional variants 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 according to 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. The cell according to claim 15, wherein the IL-1R subfamily signaling domain is selected from 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. 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 CD3 zeta 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 the cytoplasmic portion lacks the 2B4 signaling domain.

25. The cell of claim 22, wherein the cytoplasmic portion consists essentially of a TLR2 signaling domain and a CD3zeta signaling domain in order from N-terminus to C-terminus.

26. The cell of claim 22, wherein the cytoplasmic portion consists essentially of a 2B4 signaling domain, an IL-18R signaling domain, and a CD3ζ signaling domain in order from N-terminus to C-terminus.

27. The cell of claim 22, wherein the cytoplasmic portion consists essentially of a 2B4 signaling domain, a CD3ζ signaling domain, and a TLR2 signaling domain in order from N-terminus to C-terminus.

28. The cell of claim 22, wherein the cytoplasmic portion consists essentially of a 2B4 signaling domain, a TLR2 signaling domain, and a CD3ζ signaling domain in order from N-terminus to C-terminus.

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 variable heavy chain (VH) domain and / or a variable light chain (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 the CD 19 antigen.

34. A cell according to 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. A cell according to 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 having a polypeptide sequence that is at least 55%, at least 70%, at least 80%, at least 90%, at least 95% or 100% identical thereto.

36. A cell according to claim 33, wherein the ligand binding domain comprises a scFv according to SEQ ID NO: 73, or a functional variant having a polypeptide sequence that is 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 the CD20 antigen.

38. The cell of claim 37, wherein the ligand binding domain comprises a scFv according to SEQ ID NO: 139, or a functional variant having a polypeptide sequence that is 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 the HER2 antigen.

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

41. The cell of any one of claims 1 to 40, wherein the transmembrane domain comprises a polypeptide having the polypeptide sequence NLFVASWIAVMIIFRIGMAVAIFCCFFFP (SEQ ID NO: 41), or a variant having 1, 2, 3, 4, 5 or more substitutions.

42. The cell of any one of claims 1 to 41, wherein the transmembrane domain is a DNAM1, CD28, IL-2Rbeta, 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 the cell.

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

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

47. A method of killing a target cell, comprising contacting a target cell population with a natural killer cell population 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 target cells.

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

49. Use of the natural killer cells or pharmaceutical composition according to any one of the preceding claims for treating cancer.

50. A chimeric antigen receptor comprised in any one of the natural killer cells described above.

51. A polynucleotide comprising a polynucleotide sequence encoding a chimeric antigen receptor contained in any one of the natural killer cells described above.

52. A vector comprising the polynucleotide according to claim 51.

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

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

55. Natural killer cells produced by differentiating stem cells or progenitor cells comprising the polynucleotide of claim 51 into natural killer cells.

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 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.

61. The CAR of any one of claims 57-60, wherein the cytoplasmic portion comprises a CD3ζ signaling domain.

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

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

64. The CAR of any one of claims 57-61, wherein the cytoplasmic portion consists essentially of a 2B4 signaling domain, an IL-18R signaling domain, and a CD3ζ signaling domain in order from N-terminus to C-terminus.

65. The CAR of any one of claims 57-61, wherein the cytoplasmic portion consists essentially of a 2B4 signaling domain, a CD3ζ signaling domain, and a TLR2 signaling domain in order from N-terminus to C-terminus.

66. The CAR of any one of claims 57-61, wherein the cytoplasmic portion consists essentially of a 2B4 signaling domain, a TLR2 signaling domain, and a CD3ζ signaling domain in order from N-terminus to C-terminus.

67. A CAR according to any one of claims 57-66, 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 variable heavy chain (VH) domain and / or a variable light chain (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 57-70, wherein the ligand binding domain specifically binds to CD19 antigen, CD20 antigen, Her2 antigen or BCMA antigen.

72. The CAR of claim 57, wherein (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 signal transduction domain of TLR2, 41BB or 2B4.

73. The CAR of claim 72, wherein the cytoplasmic domain further comprises a signal transduction domain of CD3ζ.

74. The CAR according to claim 57, comprising (i) aTM transmembrane domain, TLR2 signaling domain and CD3ζ signaling domain; or (ii) CD28 transmembrane domain, TLR2 signaling domain and CD3ζ signaling domain.

75. The CAR of any one of claims 57-74, wherein the C-terminus of the extracellular domain is operably connected to the N-terminus or C-terminus of the transmembrane domain in the absence of a linker.

76. The CAR of any one of claims 57-74, wherein the C-terminus of the extracellular domain is operably connected to the N-terminus or C-terminus of the transmembrane domain via 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 shown 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 to the N-terminus of the cytoplasmic domain with or without a linker.

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

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

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

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

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

85. A cell population according to any one of claims 82-84, wherein the immune cells contain a homozygous inactivating mutation in the cytokine-inducible SH2-containing protein (CISH) gene.

86. The cell population of any one of claims 82-84, wherein the immune cells are CISH- / -.

87. A pharmaceutical composition comprising a cell population according to any one of claims 82-86.

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

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

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