Modified cells and uses thereof

Abstract

The present invention provides genetically modified mammalian immune cells or populations thereof, and provides pharmaceutical compositions comprising the immune cells or populations thereof and methods of treating diseases, e.g., autoimmune diseases.

Description

Invention Field

[0001] This invention relates to modified cells (e.g., modified immune cells), pharmaceutical compositions thereof, and methods of using these cells and pharmaceutical compositions to prevent and / or treat diseases, cancer, viral infectious diseases, neurodegenerative diseases, autoimmune diseases, etc., such as diseases related to autoreactive lymphocytes (e.g., T cell-mediated autoimmune diseases). Background Technology

[0002] Chimeric natural killer receptors (CNK) can redirect immune cells to specifically recognize and kill target cells expressing natural killer (NK) receptor ligands. CNK is an artificial multi-molecule protein containing an NK receptor domain and an NK signaling connective domain, such as CNK-T and CNK-UT mentioned in "Chimeric Natural Killer Cell Receptor and Methods of Using Therewith" (US Patent Application No. 16 / 827697) and "Universal T Cell and Methods of Using Therewith" (US Patent Application No. 17 / 164834), as well as NKG2D-based CAR-T; Chang et al., A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells. Cancer Res; 73(6) March15, 2013; Han et al., Control of triple-negative breast cancer using ex vivo self-enriched, costimulated NKG2D CAR T cells. Journal of Hematology & Oncology (2018) 11:92; Li et al., Human iPSC-derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-Tumor Activity. Cell Stem Cell. 2018August 02; 23(2): 181–192.e5.; VanSeggelen et al., T Cells Engineered WithChimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice. Molecular Therapy vol. 23 no. 10 oct. 2015.

[0003] However, existing immune cells expressing the aforementioned CNK or NK receptor-based CARs have shown low proliferative capacity or efficacy in treating diseases, especially in treating diseases associated with autoreactive lymphocytes, such as autoimmune diseases.

[0004] Therefore, there is a need to develop novel NK receptor-based adoptive cell therapies with improved efficacy, especially for autoimmune diseases. Invention Summary

[0005] One aspect of this disclosure provides a novel CNK or NKG2D-based CAR that protects modified T cells from fratricide caused by the interaction between NK receptors and NK ligands by introducing an NK protective element (NK-PE) to inhibit the expression or targeted degradation of NK ligands in T cells.

[0006] One aspect of this disclosure provides a genetically modified mammalian immune cell or a population thereof, comprising surface molecules and protective elements, characterized in that... The surface molecules are expressed on the surface of immune cells and can bind to one or more target NK receptor ligands (tNKRL) expressed on target cells, thereby activating immune cells and producing cytotoxic effects on target cells. The immune cells, upon activation, can be induced to express one or more inducible NK receptor ligands (iNKRLs) that can bind to the surface molecules; and The protective element substantially inhibits the expression of one or more induced NK receptor ligands on the surface of the immune cells after the immune cells are activated, thereby preventing the activated immune cells from becoming target cells.

[0007] In some embodiments, at least one target NK receptor ligand is the same as at least one inducible NK receptor ligand, or the surface molecule cross-reacts with at least one target NK receptor ligand and at least one inducible NK receptor ligand.

[0008] In some embodiments, one or more target NK receptor ligands are selected from the group consisting of: NKG2D ligand, NCR ligand, KIR ligand, CD226 ligand, TIGIT ligand, CD96 ligand, or any combination thereof.

[0009] In some implementations, one or more target NK receptor ligands comprise one or more NKG2D ligands.

[0010] In some implementations, the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6.

[0011] In some embodiments, one or more induced NK receptor ligands are selected from the group consisting of: NKG2D ligand, NCR ligand, KIR ligand, CD226 ligand, TIGIT ligand, CD96 ligand, or any combination thereof.

[0012] In some implementations, one or more induced NK receptor ligands comprise one or more NKG2D ligands.

[0013] In some implementations, the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6.

[0014] In some embodiments, at least one target NK receptor ligand is identical to at least one inducible NK receptor ligand and comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6.

[0015] In some embodiments, the expression of one or more inducible NK receptor ligands on the surface of immune cells is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) relative to the expression of one or more inducible NK receptor ligands on the surface of a reference immune cell without a protective element.

[0016] In some embodiments, the expression of one or more induced NK receptor ligands on the surface of immune cells is reduced such that the binding of one or more induced NK receptor ligands to surface molecules is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

[0017] In some embodiments, after the surface molecule binds to one or more target NK receptor ligands, the proportion of cells that induce NK receptor ligand expression in the immune cell population can be detected to be no more than 50% (e.g., no more than 40%, no more than 30%, no more than 20%, or no more than 10%).

[0018] In some embodiments, one or more inducible NK receptor ligands comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6, and the expression levels of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6 on the surface of immune cells are reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

[0019] In some embodiments, one or more inducible NK receptor ligands comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5, and ULBP6, and the expression level of each of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5, and ULBP6 on the surface of immune cells is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

[0020] In some implementations, activation-induced cell death of immune cells is substantially reduced, and optionally, the reduction in activation-induced cell death of immune cells can be determined by detecting the reduction in apoptosis using Annexin V / 7-AAD.

[0021] In some implementations, immune cells are able to proliferate upon activation (e.g., the proliferation rate is increased compared to modified mammalian immune cells without protective elements).

[0022] In some implementations, the protective element is capable of silencing the expression of one or more induced NK receptor ligands, or of inducing intracellular retention and / or degradation of one or more induced NK receptor ligands.

[0023] In some implementations, the protective element comprises a small RNA (e.g., siRNA, miRNA, or shRNA) that targets one or more mRNAs that induce NK receptor ligands.

[0024] In some embodiments, the protective element comprises one or more protein degradation elements capable of inducing intracellular retention and / or degradation of one or more NK receptor ligands.

[0025] In some embodiments, at least one or all of the protein degradation elements comprise an endoplasmic reticulum (ER) retention domain and an iNKRL binding domain capable of binding one or more induced NK receptor ligands.

[0026] In some embodiments, the protein degradation element further includes an ER retention transmembrane domain, characterized in that one end of the ER retention transmembrane domain is operatively connected to an iNKRL binding domain and the other end is operatively connected to an ER retention domain.

[0027] In some implementations, the ER retention domain and / or the ER retention transmembrane domain are derived from ER-retaining viral proteins.

[0028] In some implementations, the ER-retaining viral proteins are selected from the group consisting of: HCMV protein US2, HCMV protein US11, HCMV protein US18, HCMV protein US20, HCMV protein US3, HCMV protein US10, HCMV protein US6, HSVICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV protein UL16, HCMV protein UL141, HCMV protein UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, vaccinia virus protein CPXV203, and Ad E3 / 19K.

[0029] In some implementations, both the ER retention domain and the ER retention transmembrane domain are derived from the same ER-retaining viral protein.

[0030] In some implementations, the iNKRL binding domain includes an antibody domain (e.g., scFv) capable of binding one or more inducible NK receptor ligands.

[0031] In some implementations, the iNKRL binding domain is derived from a viral protein capable of binding one or more induced NK receptor ligands.

[0032] In some implementations, one or more induced NK receptor ligands comprise one or more NKG2D ligands.

[0033] In some embodiments, one or more NKG2D ligands are selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0034] In some embodiments, the viral protein capable of binding one or more NK receptor ligands is derived from a virus selected from the group consisting of adenovirus (Ad) (e.g., human Ad), cytomegalovirus (CMV) (human CMV, i.e., HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV).

[0035] In some embodiments, the viral protein capable of binding one or more NK receptor ligands is derived from a viral protein selected from the group consisting of AdE3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHV K5.

[0036] In some implementations, the ER retention domain and binding domain are derived from the same viral protein.

[0037] In some implementations, the ER retention domain, ER retention transmembrane domain, and binding domain are all derived from the same viral protein.

[0038] In some implementations, the same viral protein is selected from the group consisting of Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHV K5.

[0039] In some embodiments, the protein degradation element comprises a viral protein selected from the group consisting of Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, KSHV K5, or analogues thereof, or derivatives thereof.

[0040] In some implementations, the protective element comprises small RNAs, such as HCMV-encoded microRNAs (miR-20a, miR-UL112), which can regulate the expression of NKG2D ligands.

[0041] In some embodiments, the one or more protein degradation elements comprise a combination of viral proteins, wherein the combination of viral proteins comprises HCMV protein UL16 and another viral protein selected from the group consisting of: Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHV K5.

[0042] In some embodiments, one or more protein degradation elements further include a protein degradation pathway (PDP) binding domain capable of binding to members of a protein degradation pathway.

[0043] In some implementations, the PDP binding domain is operatively connected to the ER retention domain.

[0044] In some implementations, the protein degradation pathway is the ubiquitination-proteasome pathway, the endosome-lysosome pathway, or the autophagy degradation pathway.

[0045] In some implementations, members of the ubiquitination-proteasome pathway include E1 ubiquitin activator, E2 ubiquitin conjugate, or E3 ubiquitin ligase.

[0046] In some implementations, members of the endosome-lysosome pathway include AP-1, AP-2, AP-3, endosome, lysosome, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sortingnexin, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, or C3G / CrkL / Shp2 / Gab2.

[0047] In some implementations, members of the autophagy degradation pathway include molecular chaperone-mediated autophagy (CMA) USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1 / 2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, or NUFIP1.

[0048] In some embodiments, the surface molecule includes a) a tNKRL-binding extracellular domain capable of binding one or more target NK receptor ligands, and b) a surface molecule (SM) intracellular domain capable of activating immune cells after the tNKRL-binding extracellular domain binds to one or more target NK receptor ligands, and the tNKRL-binding extracellular domain is associated with the SM intracellular domain to allow binding signals to be transduced from the tNKRL-binding extracellular domain to the SM intracellular domain.

[0049] In some implementations, the tNKRL binding extracellular domain includes an antibody domain capable of binding one or more target NK receptor ligands.

[0050] In some implementations, the tNKRL binding extracellular domain includes an NK receptor extracellular domain capable of binding one or more target NK receptor ligands.

[0051] In some implementations, the tNKRL binding extracellular domain includes the NKG2D extracellular domain.

[0052] In some embodiments, the receptor further comprises an SM transmembrane domain, wherein one end of the SM transmembrane domain is operatively connected to the tNKRL-binding extracellular domain and the other end is operatively connected to the SM intracellular domain.

[0053] In some implementations, the SM transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD3, CD4, CD8, CD28, B7, ICOS, CD226, 41-BB, OX40, CD27, GITR, HVEM, CD40, BAFFR, BAFF, NKG2D, NKG2C, NKG2A, NKp44, NKp46, and NKp30.

[0054] In some implementations, the surface molecule contains tNKRL-binding chimeric antigen receptor (CAR).

[0055] In some embodiments, the receptor comprises a CNK receptor protein complex comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a tNKRL-binding extracellular domain operatively linked to a first CNK transmembrane domain, and the second polypeptide comprising a CNK intracellular domain operatively linked to a second CNK transmembrane domain, the first CNK transmembrane domain and the second CNK transmembrane domain being associated with each other to form a protein complex.

[0056] In some implementations, the first CNK transmembrane domain includes an NK receptor transmembrane domain, optionally an NKG2D transmembrane domain.

[0057] In some implementations, the first transmembrane domain is capable of forming a first dimer.

[0058] In some implementations, the first polypeptide comprises the NKG2D protein.

[0059] In some embodiments, the NKG2D protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0060] In some embodiments, the second transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a 10 kDa DNAX activating protein (DAP10) and a 12 kDa DNAX activating protein (DAP12).

[0061] In some implementations, the CNK intracellular domain comprises a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR.

[0062] In some implementations, the CNK intracellular domain includes the cytoplasmic domain of CD3 (e.g., CD3 zeta).

[0063] In some implementations, the CNK intracellular domain contains the amino acid sequence of SEQ ID NO: 5.

[0064] In some embodiments, the second polypeptide comprises a chimeric adaptor protein comprising: a transmembrane domain of DAP10 operably linked to an intracellular domain of CD3 (e.g., CD3 zeta); a transmembrane domain of DAP12 operably linked to an intracellular domain of CD3 (e.g., CD3 zeta); or a transmembrane domain of DAP10 operably linked to an intracellular domain of DAP12.

[0065] In some embodiments, the second polypeptide comprises a full-length DAP10 that is operatively linked to an intracellular domain of CD3 zeta.

[0066] In some embodiments, the chimeric adaptor protein comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0067] In some implementations, the second CNK transmembrane domain is capable of forming a second dimer, and the second dimer is associated with the first CNK transmembrane domain to form a complex.

[0068] In some embodiments, the CNK receptor comprises a hexameric protein complex containing one homodimer of the NKG2D protein and two homodimers of the chimeric adaptor protein.

[0069] In some implementations, the immune cells are further modified to be deficient in a) MHC class I proteins, or b) MHC class II proteins or MHC class II transactivators, or c) both a) and b).

[0070] In some implementations, the immune cells are T cells, and these immune cells are further modified to be defective in the endogenous TCR.

[0071] In some implementations, the genetically modified mammalian immune cells provided herein further express target-binding (TB) receptors capable of binding target markers.

[0072] In some implementations, the target marker is expressed on the target cells.

[0073] In some embodiments, the TB receptor includes a target-binding extracellular domain capable of binding a target marker and a TB intracellular domain capable of activating immune cells upon binding of the target marker to the target marker in the target-binding extracellular domain.

[0074] In some implementations, the TB receptor further includes a TB transmembrane domain.

[0075] In some implementations, one end of the TB transmembrane domain is operatively connected to the target marker binding extracellular domain, and the other end is operatively connected to the TB intracellular domain.

[0076] In some implementations, the target marker binding extracellular domain includes the extracellular domain of a natural cytotoxic receptor (e.g., NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1)).

[0077] In some implementations, the TB transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of: CD3, CD4, CD8, CD28, B7, ICOS, CD226, 41-BB, OX40, CD27, GITR, HVEM, CD40, BAFFR, BAFF, NKG2D, NKG2C, NKG2A, NKp44, NKp46, and NKp30.

[0078] In some implementations, the intracellular domain of TB comprises a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR.

[0079] In some implementations, the TB intracellular domain includes a CD28 cytoplasmic domain operatively linked to the CD3 zeta chain.

[0080] In some implementations, the target markers are tumor antigens, autoreactive cell antigens or viral antigens, bacterial antigens, microbially infected cells, or damaged and / or aging cells.

[0081] In some implementations, the target cells are tumor cells, autoreactive cells, senescent cells, or virus-infected cells.

[0082] In some implementations, immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, hematopoietic cells (e.g., bone marrow cells), thymocytes, dendritic cells (e.g., mature dendritic cells), macrophages, tumor-infiltrating lymphocytes, monocytes, and granulocytes.

[0083] In some implementations, the immune cells are T cells, such as CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, or tumor-infiltrating lymphocytes.

[0084] In another respect, this disclosure provides a pharmaceutical composition comprising genetically modified mammalian immune cells or populations thereof as provided herein.

[0085] On the other hand, this disclosure provides a method for treating a disease in a subject in need, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition provided herein.

[0086] In some implementations, the disease is cancer, a disease associated with self-reactive lymphocytes (e.g., an autoimmune disease), an immunodeficiency disease, or an age-related disease.

[0087] In some implementations, autoimmune diseases are organ-specific autoimmune diseases or systemic autoimmune diseases.

[0088] In some implementation schemes, organ-specific autoimmune diseases are selected from the group consisting of chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0089] In some implementation schemes, systemic autoimmune diseases are selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0090] In some implementations, the autoimmune disease is graft-versus-host disease (GvHD).

[0091] In another aspect, this disclosure provides expression constructs encoding surface molecules and protective elements that can be expressed in immune cells, wherein the surface molecules are as defined above and the protective elements are as defined above.

[0092] In another aspect, this disclosure provides a method for generating the genetically modified mammalian immune cells provided herein, comprising introducing the expression construct provided herein into the initiating immune cell under conditions that allow surface molecules to be expressed on the surface of the immune cell and allow protective elements to be expressed in the immune cell. Attached Figure Description

[0093] Figure 1A and 1B The study showed that T cell activation significantly upregulated the expression of NKG2D ligands (e.g., MICA, MICB, ULBP-1, ULPB-2, 5, 6, ULBP-3).

[0094] Figure 2A : A schematic diagram illustrating the structure of the lentiviral vector. (1) The CNK vector contains the EF1a promoter, DAP10-CD3Z and NKG2D separated by the self-cutting sequence T2A; (2) The CNK+PE vector contains the EF1a promoter, DAP10-CD3Z, NKG2D and UL16 separated by the self-cutting sequence T2A; (3) CNK+PE / NCR2-CD28-CD3Z (i.e. CNK+PE / NCR2 ED-CD28TM-CD28ICD-CD3Z ICD) vector 1 contains the EF1a promoter, DAP10-CD3Z, NKG2D and UL16 separated by the self-cutting sequence T2A; vector 2 contains the EF1a promoter and NCR2 ED-CD28-CD3Z.

[0095] Figure 2B Phenotypes of CNK-T cells with or without protective elements.

[0096] Figure 3 The protective element (PE) can inhibit the expression of NKG2D ligand on CNK-T cells.

[0097] Figure 4Display protective elements (PEs) can increase CNK-T cell viability and proliferation.

[0098] Figure 5 The phenotype of NCR2 CNK-UT cells is shown.

[0099] Figure 6 CNK-UT effectively eliminates activated T cells.

[0100] Figure 7 CNK-UT selectively kills activated T cells rather than inactivates them.

[0101] Figure 8 The Jurkat T cells were shown to express NKG2D ligand and NCR ligand.

[0102] Figure 9 shows that NCR2 CNK-UT cells effectively kill Jurkat T cells. Figure 9A Flow cytometry images of cocultures with different E:T ratios (1:2, 1:1, 2:1); Figure 9B Flow cytometry image showing Jurkat T cells (CD3+, CD8-) that are residual tumor cells after 48 hours of co-culture.

[0103] Figure 10 A schematic diagram illustrating the mechanism of CNK-UT therapy for T cell-mediated autoimmune diseases.

[0104] Figure 11 This is a schematic diagram illustrating the experimental protocol for the GVHD mouse model.

[0105] Figure 12A-12D This is a representative FACS chart, showing the results on day 11 ( Figure 12A Day 18 Figure 12B Day 25 Figure 12C ) and the 32nd day ( Figure 12D The percentage of human CD45+ and CD3+ T cells in the PBL of GVHD mice treated with CNK-UT002.

[0106] Figure 13 The percentage of donor T cells is shown in the GVHD mouse model. Untreated GVHD mice were used as a control. The data are representative plots from 8 mice.

[0107] Figure 14 The GVHD scores of mice that received or did not receive treatment after allogeneic PBMC infusion are shown.

[0108] Figure 15 The changes in body weight of GVHD mice over time are shown.

[0109] Figure 16 The percentage of GVHD mice that survived over time is shown. Detailed description

[0110] The following description of the invention is intended only to illustrate various embodiments thereof. Therefore, the specific modifications discussed should not be considered as limiting the scope of the invention. Those skilled in the art will understand that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and it is also understood that such equivalent embodiments should be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0111] Although the various technical features of this disclosure are described in a single embodiment, these features may also be provided individually and / or in any suitable combination. Conversely, although the various technical features of this disclosure are described in a single embodiment for clarity, any one of these features may also be implemented in a single embodiment.

[0112] definition

[0113] As used herein, terms (or terrminology) are used only for the purpose of describing a particular implementation and are not intended to be limiting.

[0114] The singular terms “a / an” and “the” include plural referents unless the context clearly indicates otherwise. For example, the reference to “cell” refers to one or more cells, the reference to “method” includes the equivalent steps and methods disclosed herein and / or known to those skilled in the art, and so on. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and”. Although similar or equivalent methods and materials to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation “eg” is derived from the Latin *exempli gratia* and is used herein to denote a non-limiting example. Therefore, the abbreviation “eg” is synonymous with the term “for example”.

[0115] In all instances of this application, when a series of listed numerical values ​​are presented, it should be understood that any one of the listed numerical values ​​can be an upper or lower limit of the numerical range. It should also be understood that the invention covers all such numerical ranges, i.e., ranges having a combination of an upper and a lower numerical limit, wherein the value of each of the upper and lower limits can be any value mentioned herein. The ranges provided herein should be understood to include all values ​​within that range. For example, 1-10 should be understood to include all values ​​1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as appropriate fractional values. Similarly, ranges defined with “at least” should be understood to include the provided lower limit value and all larger numerical values.

[0116] As used herein, “about” should be understood to include within three standard deviations of the mean or within the standard tolerance range in a particular technique. In some embodiments, “about” should be understood to include a variation of no more than 0.5.

[0117] The term "including" is used herein to mean the phrase "including but not limited to," and may be used interchangeably with the phrase "including but not limited to." Similarly, "for example" is used herein to mean the phrase "for example but not limited to," and may be used interchangeably with the phrase "for example but not limited to."

[0118] As used herein, the terms “comprising” or “comprises” are used to refer to a composition, a method, and the corresponding components necessary for said method or composition, but do not exclude unspecified elements, whether or not they are necessary.

[0119] The term "composed of" refers to the compositions, methods and their respective components described herein, and does not include any elements not described in the description of the embodiments.

[0120] As used herein, the term “substantially inhibited” in relation to protein expression levels means that the expression level is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% after inhibition.

[0121] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms also apply to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0122] As used herein, the terms “nucleotide,” “nucleic acid,” or “polynucleotide” include oligonucleotides (i.e., short polynucleotides). They also refer to synthetic and / or non-naturally occurring nucleic acid molecules (e.g., those containing nucleotide analogs or modified backbone residues or bonds). These terms also refer to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form. These terms cover nucleic acids containing natural nucleotide analogs. These terms also encompass nucleic acid-like structures with synthetic backbones. Unless otherwise specified, a particular polynucleotide sequence also implicitly encompasses its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0123] "Conservative substitution" involving amino acid sequences refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitution can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitution generally does not cause significant changes in protein conformation and structure, thus preserving the protein's biological activity.

[0124] As used herein, the term "functional equivalent" refers to a different form of the parent molecule (e.g., variant, fragment, fusion, derivative, and mimic) that retains the substantial biological activity of the parent molecule despite differences in amino acid sequence or chemical structure. The expression "retains substantial biological activity" as used herein means exhibiting at least some (e.g., not less than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or all of the biological activity of the parent molecule. Functional equivalents of the parent protein can include naturally occurring variant forms and non-naturally occurring forms, such as those obtained through recombinant methods or chemical synthesis. Functional equivalents may contain non-natural amino acid residues.

[0125] As used herein, the term “defect” means insufficient activity or level, and may also include, for example, below normal activity or level, or absence or null of activity or level.

[0126] As used herein, the term "CAR" is used interchangeably with the term "chimeric antigen receptor" and refers to an engineered receptor or artificial receptor or the polynucleotide encoding it. An engineered or synthetic receptor comprises an extracellular domain including an antigen-binding domain, a transmembrane domain, and / or an intracellular signaling domain, which are interconnected or operatively interconnected, conferring specificity of immune effector cells to antigens and bypassing MHC class I and II restrictions.

[0127] The term "chimeric antigen receptor T cell" is used interchangeably with "CAR-T cell" or "CAR-T cell" and refers to T cells or populations thereof that have been engineered (e.g., through genetic engineering) to express CAR on the surface of T cells. CAR-T cells can be T helper CD4+ cells and / or T effector CD8+ cells. CAR-T cells can bind to target cells expressing target antigens and initiate an immune response against those target cells.

[0128] The term "TCR" as used herein is used interchangeably with "T cell receptor" or "TCR complex," referring to either a natural (or endogenous) TCR or an engineered TCR. A TCR is a disulfide-linked membrane-anchored heterodimeric protein complex, typically containing highly variable α and β chains, complexed with CD3γ, CD3δ, two CD3ε chains, and a CD3-zeta chain. The amino acid sequences of the α and β chains differ among different T cells. The transmembrane regions of the α and β chains are surrounded by an open barrel-shaped CD3 transmembrane region. The intracellular tails of the CD3γ, CD3δ, and CD3ε molecules each contain a single conserved motif called the immunoreceptor tyrosine activation motif (ITAM), which is crucial for the signal transduction capabilities of the TCR complex and for initiating signal transduction upon binding to the antigen-MHC complex.

[0129] As used herein, the term "antigen" refers to a molecule that can be specifically recognized and bound by an antibody. Those skilled in the art will understand that an antigen can be any large or small molecule, and almost all proteins or peptides can be used as antigens.

[0130] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A naturally occurring, complete antibody consists of two heavy chains (H) and two light chains (L). Mammalian heavy chains are divided into α, δ, ε, γ, and μ chains, each containing a variable region (V). H) and the first, second and third constant regions (C respectively) H1、 C H2、 C H3 Mammalian light chains are divided into λ or κ, and each light chain contains a variable region (V). LAntibodies are Y-shaped, with the stem of the Y containing the second and third constant regions of two heavy chains linked together by disulfide bonds. Each arm of the Y contains a variable region and a first constant region of a single heavy chain that bind to the variable and constant regions of a single light chain. The variable regions of both the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3; heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibody and antigen binding domains disclosed in this paper can be defined or identified by the Kabat, IMGT, AbM, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); NR Whitelegg et al., Protein Engineering, v13(12), 819-824 (2000); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, 1989; ... of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). Three CDRs are inserted between flanking segments called framework regions (FRs), which are much more conserved than CDRs, forming a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified into several classes based on the amino acid sequence of their heavy chain constant regions.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are subclassed, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some embodiments, the antibodies described herein encompass any of their antigen-binding fragments.

[0131] As used herein, the term "antigen-binding fragment" refers to an antibody fragment comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the complete structure of a native antibody. Examples of antigen-binding fragments include, but are not limited to, diabody, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody, single-chain antibody molecule (scFv), scFv dimer (bivalent dimeric antibody), multispecific antibody, camel-derived single-domain antibody, nanobody, domain antibody, and bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen that binds to the parent antibody. In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a specific parent antibody.

[0132] The term "Fab" in antibody refers to the monovalent antigen-binding fragment of the antibody, which consists of a single light chain (variable and constant regions) bound by disulfide bonds, and a single heavy chain with a variable region and a first constant region. Fab can be obtained by digesting the antibody with papain at residues near the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0133] "Fab" refers to a Fab fragment containing a portion of the hinge region. It can be obtained by digesting antibodies with pepsin at residues near the C-terminus of the disulfide bonds between the heavy chains in the hinge region. Therefore, it differs from Fab in that it has a few residues (including one or more cysteine ​​residues) in the hinge region.

[0134] "F(ab')2" refers to a Fab' dimer containing two light chains and part of two heavy chains.

[0135] In the context of antibodies, "Fv" refers to the smallest antibody fragment with a complete antigen-binding site. An Fv fragment consists of a variable region of a single light chain linked to a variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment, where the connection between the variable regions of the single light chain and the variable regions of the single heavy chain is a disulfide bond.

[0136] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of light chain variable regions and heavy chain variable regions linked together directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad SciUSA, 85:5879 (1988)). An "scFv dimer" refers to a single chain containing two heavy chain variable regions, two light chain variable regions, and a linker. In some embodiments, the "scFv dimer" is a divalent disomeric antibody or divalent scFv (BsFv), which contains a linker with another V... H -V L Partially dimerized V H -V L (Linked via peptide linkers), thus forming a moiety of V H With another part of V L They coordinate to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific disomeric antibody containing V L1 -V H2 (Linked via peptide linkers) associated V H1 -V L2 (Also linked via peptide linkers) so that V H1 and V L1 Paired and V H2 and V L2 Pairing, and each pair has a different antigen specificity.

[00137] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody formed by linking scFv with the Fc region of the antibody.

[0137] "Camel-derived single-domain antibody," "heavy chain antibody," "nanobody," or "HCAb" refers to antibodies containing two V... HThe domain also lacks antibodies against light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001);WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were initially obtained from camelids (camels, dromedaries, and llamas). Despite lacking light chains, camel-derived antibodies possess a true antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8(1993); Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domain (VHH domain) of heavy-chain antibodies represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)). "Dimeric antibodies" comprise small antibody fragments having two antigen-binding sites, wherein the fragments contain linked V molecules within a single polypeptide chain. L Domain and V H Domain (V) H -V L or V L -V H (See, for example, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15;90(14):6444-8 (1993);EP404097; WO93 / 11161). Because the linker is too short, two domains on the same strand cannot pair. Therefore, the domain is forced to pair with a complementary domain on the other strand, resulting in two antigen-binding sites. These antigen-binding sites can target the same or different antigens (or epitopes).

[0138] "Domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. In some embodiments, two or more V... H Domains are covalently linked to peptide linkers to form bivalent or multivalent domain antibodies. Bivalent domain antibodies have two V... H A domain can target the same or different antigens.

[0139] In some implementations, "(dsFv)2" comprises three peptide chains: two V... H Partially linked by peptide linkers and connected to two V via disulfide bonds. L Partial combination.

[0140] In some implementations, the "bispecific ds disodium antibody" comprises via V H1 and V L1 V-shaped disulfide bridges between them H1 -V L2 (linked via peptide linkers) and V L1 -V H2 (Also linked via peptide linkers).

[0141] In some implementations, "bispecific dsFv" or "dsFv-dsFv" comprises three peptide chains: V H1- V H2 In some cases, the heavy chain is bound via peptide linkers (e.g., long flexible linkers) and via disulfide bridges to V. L1 and V L2 Partially paired. Each pair of heavy and light chains, linked by disulfide bonds, has different antigen specificities.

[0142] As used herein, the term "complex" refers to an aggregate or assembly of more than one protein (e.g., two, three, four, or more). Preferably, the complex has a function that the individual proteins in the complex do not possess. In a complex, the proteins forming the complex may or may not be in contact with each other.

[0143] The term “chimerism” as used in this article, when used in conjunction with proteins (e.g., adaptor proteins), refers to a protein in which one part is derived from one species and another part is derived from another species.

[0144] As used herein, the term "functional variant" refers to a polypeptide / protein that shares at least 70% sequence identity with its parent polypeptide / protein while retaining the function of the parent polypeptide / protein. The difference between the variant and the parent polypeptide / protein can be one or more amino acid residues. For example, a functional variant can be a substitution, addition, deletion, insertion, or truncation of one or more amino acid residues in the parent polypeptide / protein.

[0145] The "percentage (%) sequence identity" of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence after alignment and, where necessary, the introduction of vacancies to achieve maximum correspondence. For example, to determine the percentage of amino acid (or nucleic acid) sequence identity, comparisons can be made using publicly available tools such as BLASTN, BLASTp (available from the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403–410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389–3402 (1997)), ClustalW2 (available from the website of the European Institute for Bioinformatics, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tool, or can customize the alignment parameters as needed, for example, by selecting a suitable algorithm. In some embodiments, non-identical residue positions may differ due to conserved amino acid substitutions. A “conserved amino acid substitution” means that an amino acid residue is replaced by another amino acid residue in a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage or degree of similarity can be increased to correct for the conservatism of the substitution. Methods for making such adjustments are generally known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference.

[0146] The term "operably linked" or "operably linked" refers to the juxtaposition of two or more target biological sequences (e.g., polynucleotide or amino acid sequences) in a manner that allows them to function as intended, with or without spacers or linkers. When used for proteins (e.g., protein degradation elements, chimeric adaptors), it is intended to indicate that protein sequences are linked in a manner that allows the ligation product to have the intended biological function. The term can also be used for polynucleotides. For example, when a polynucleotide encoding a protein is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to indicate that the polynucleotide sequence is linked in a manner that allows the protein to be expressed in a regulated manner from said polynucleotide.

[0147] As used in this article, the term "activation" refers to a state in which T cells have been adequately stimulated, thereby inducing detectable cell proliferation. Activation can also be associated with the induction of cytokine production and detectable effector function. The term "activated T cell" specifically refers to a T cell undergoing cell division.

[0148] The term “chimeric natural killer receptor” as used herein is interchangeable with the term “CNK receptor” and refers to a protein complex comprising a chimeric NK-activating receptor element and a chimeric NK signaling linker element that are coupled to allow signal transduction.

[0149] The term "CNKT cell" as used in this article refers to T cells that express CNK receptors.

[0150] The term “chimeric natural killer receptor-universal T cell” used in this article is interchangeable with the term “CNK-UT”, which refers to T cells that express CNK receptors and are genetically modified to knock out TCR and HLA, such modification allowing CNK-T cells to be generated from allogeneic healthy donors.

[0151] As used herein, the term "UT" refers to a modified T cell (e.g., an immune cell) that is deficient in molecules that confer cell GVHD and immunogenicity (e.g., TCR, MHC class I protein, MHC class II protein, or MHC class II transactivator).

[0152] As used herein, the term "therapeuticly effective amount" refers to the amount of cells, compositions, formulations, or any materials described herein that effectively achieve the desired biological outcome. Such outcomes may include, but are not limited to, the elimination of target cells, such as target T cells associated with a disease (e.g., an autoimmune disease).

[0153] As used herein, the terms “subject” or “individual” or “animal” or “patient” refer to human or non-human animals, including mammals or primates, that require diagnosis, prognosis, improvement, prevention, and / or treatment of a disease or condition. Mammal subjects include humans, livestock, farm and zoo animals, sporting animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0154] The term "treatment" or "therapeutic method" as used in this article includes relieving the indication, slowing the onset or progression of the indication, reducing the risk of developing the indication, preventing or delaying the development of symptoms associated with the indication, reducing or terminating symptoms associated with the indication, producing complete or partial remission of the indication, curing the indication, or a combination of the above.

[0155] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted, thereby producing expression of said protein. Vectors can be used to transform, transduce, or transfect host cells, thereby producing expression of the genetic elements they carry within the host cells. Examples of vectors include plasmids, phagemids, granules, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages (such as λ phage or M13 phage), and animal viruses. Animal virus classes used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Vectors may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, optional elements, and reporter genes. Additionally, vectors may contain an origin of replication. Vectors may also include materials that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coatings. Vectors may be expression vectors or cloning vectors. This disclosure provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding a fusion protein provided herein, and at least one promoter operatively linked to said nucleic acid sequence. Vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, multifocal papillomaviruses (e.g., SV40), λ phage and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg- GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, p MONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV -SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0156] The phrase “host cell” as used in this article refers to a cell in which exogenous polynucleotides and / or vectors have been introduced.

[0157] I. Overview

[0158] This disclosure is based, at least in part, on the surprising finding that modified cells (e.g., mammalian immune cells) or populations thereof expressing surface molecules can be induced to express one or more inducible NK receptor ligands (iNKRLs) capable of binding to said surface molecules upon activation. This may lead to self-killing or allotoxic effects of the modified cells (e.g., mammalian immune cells) or populations thereof, thus resulting in poor therapeutic efficacy of the modified cells (e.g., mammalian immune cells) or populations thereof, especially when used to treat diseases in subjects associated with target cells (e.g., autoreactive lymphocytes) expressing target NK receptor ligands (tNKRLs).

[0159] Therefore, to improve the therapeutic efficacy of cell therapy, this disclosure provides modified cells (e.g., mammalian immune cells) or populations thereof designed to substantially eliminate self-killing or allotoxic effects of the modified cells (e.g., mammalian immune cells) or populations thereof. This disclosure also provides methods for substantially eliminating self-killing or allotoxic effects and / or improving the therapeutic efficacy of modified cells (e.g., mammalian immune cells) or populations thereof by substantially inhibiting the expression level of one or more iNKRLs in the modified cells (e.g., mammalian immune cells) or populations thereof, or by directly degrading the modified cells (e.g., mammalian immune cells) or populations thereof. This disclosure also provides a method for treating a disease (e.g., autoimmune disease) in a subject that is associated with target cells (e.g., self-reactive lymphocytes) expressing target NK receptor ligands (tNKRLs), comprising administering to the subject a therapeutically effective amount of the modified cells (e.g., mammalian immune cells) or populations thereof provided herein.

[0160] II. Cell Modification

[0161] In one aspect, this disclosure provides a modified cell (e.g., a mammalian immune cell) or a population thereof comprising a surface molecule and a protective element. In some embodiments, the surface molecule is expressed on the surface of the modified cell (e.g., an immune cell) and is capable of binding to one or more target NK receptor ligands (tNKRLs) expressed on a target cell, thereby activating the modified cell (e.g., the immune cell) to exert cytotoxic effects on the target cell. In some embodiments, the modified cell (e.g., an immune cell) upon activation is induced to express one or more inducible NK receptor ligands (iNKRLs) capable of binding to the surface molecule. In some embodiments, the protective element substantially inhibits the expression or degradation of one or more iNKRLs on the surface of the modified cell (e.g., an immune cell) after activation, thereby preventing the activated modified cell (e.g., an immune cell) from becoming a target cell.

[0162] In some embodiments, at least one tNKRL is identical to at least one iNKRL, or the surface molecule cross-reacts with at least one tNKRL and at least one iNKRL. In some embodiments, one or more tNKRLs are selected from the group consisting of NKG2D ligands, NCR ligands, KIR ligands, CD226 ligands, TIGIT ligands, CD96 ligands, or any combination thereof. In some embodiments, one or more tNKRLs comprise one or more NKG2D ligands. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. In some embodiments, one or more iNKRLs are selected from the group consisting of NKG2D ligands, NCR ligands, KIR ligands, DNAM1 ligands, CD226 ligands, TIGIT ligands, CD96 ligands, or any combination thereof. In some embodiments, one or more inducible NK receptor ligands comprise one or more NKG2D ligands. In some embodiments, the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. In some embodiments, at least one tNKRL is identical to at least one iNKRL and comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6.

[0163] In some embodiments, the expression of one or more iNKRLs on the surface of immune cells is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) relative to the expression of one or more iNKRLs on the surface of a reference immune cell without a protective element.

[0164] In some embodiments, the expression of one or more iNKRLs on the surface of modified cells (e.g., immune cells) is reduced such that the binding of one or more iNKRLs to surface molecules is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

[0165] In some embodiments, after the surface molecule binds to one or more tNKRLs, the expression of one or more iNKRLs can be detected on no more than 50% (e.g., at least 40%, at least 30%, at least 20%, or at least 10%) of an immune cell population.

[0166] In some embodiments, one or more iNKRLs comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6, and the expression levels of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6 on the surface of immune cells are reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

[0167] In some embodiments, one or more iNKRLs comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5, and ULBP6, and the expression levels of each of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5, and ULBP6 on the surface of immune cells are reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

[0168] The expression of iNKRL can be determined using methods known in the art, such as flow cytometry, Western blotting, or qPCR.

[0169] In some implementations, modified cells (e.g., immune cells) exhibit substantially reduced activation-induced cell death. The reduction in activation-induced cell death of immune cells can be determined by the reduction in apoptosis detected by Annexin V / 7-AAD. In short, cell counting using Annexin V and 7-AAD can be used to differentiate between apoptotic and necrotic cells. 7-Aminoactinomycin D (7-AAD) can be excited by a 488 nm argon laser line and separated from the emission of FITC and PE, thereby characterizing necrotic cells (7-AAD positive, annexin V negative, and FITC positive cells), apoptotic cells (7-AAD negative, annexin V negative, and FITC positive cells), and live cells (7-AAD negative, annexin V negative, and FITC negative cells) within a phycoerythrin (PE) positive cell subset. For more details, see Herault, O. et al., British Journal of Haematology, 1999, 104, 530–537.

[0170] In some implementations, immune cells are able to proliferate upon activation (e.g., with an increased proliferation rate compared to modified mammalian immune cells without protective elements).

[0171] A. Protective components

[0172] In some implementations, the protective element is capable of silencing the expression of one or more iNKRLs or inducing intracellular retention and / or degradation of one or more iNKRLs.

[0173] In some embodiments, the protective element comprises a small RNA (e.g., antisense RNA, shRNA, siRNA, miRNA, or guide RNA) that targets one or more iNKRL mRNAs. The small RNA may also be a vector expressing the aforementioned small RNA, such as antisense RNA, siRNA, miRNA, or guide RNA.

[0174] The aforementioned small RNAs are typically 15 to 30 bp in length. RNA interference (RNAi) initiators with larger (>21 bp) RNA duplexes interact with the RNAi pathway enzyme Dicer, cleaving them and delivering them to the RNA-induced silencing complex (RISC), which loads the Argonaute (Ago1-Ago4) protein. Shorter (<21 bp) siRNAs and analogues can bypass Dicer cleavage and enter the RISC via TAR RNA-binding protein (TRBP)-mediated interactions. Mature RISCs can regulate gene expression by inhibiting mRNA translation, inducing mRNA sequestration in cytoplasmic P-body and / or GW-body, promoting mRNA degradation, and directing transcriptional silencing at target genomic sites. For a detailed description, see, for example, Ryan et al., The current state and future directions of RNAi-based therapeutics. Nature Reviews Drug Discovery volume 18, pages 421–446 (2019).

[0175] In some implementations, the protective element comprises small RNAs, such as HCMV-encoding microRNAs (miR-20a, miR-93, miR-UL112) capable of regulating NKG2D ligand expression, as described in, for example, Shen J et al., Silencing NKG2D ligand-targeting miRNAs enhances natural killer cell-mediated cytotoxicity in breast cancer. Cell Death Dis. 2017 Apr 6;8(4):e2740; Codo P et al., MicroRNA-mediated down-regulation of NKG2D ligands contributes to gliomaimmune escape. Oncotarget (2014) 5(17):7651–62. 10.18632 / oncotarget.2287; and Alves E et al., Manipulating the NKG2D Receptor-Ligand Axis Using CRISPR: Novel Technologies for Improved Host Immunity. Front Immunol. 2021 Aug. 12;12:712722. (in Chinese).

[0176] In some embodiments, the protective element comprises a genome editing system, such as a CRISPR / Cas system, to inactivate one or more genes encoding one or more iNKRLs. In some embodiments, the CRISPR / Cas system comprises a Cas9 protein and one or more guide RNAs (gRNAs). In some embodiments, the gRNA comprises a targeting region that hybridizes to a target sequence, and an activation region that hybridizes to the targeting region to form a double-stranded RNA duplex, wherein the activation region and the targeting region are covalently linked together by an intermediate nucleotide. Once the targeting region of the gRNA hybridizes to the target DNA, the target DNA-gRNA forms a complex with the Cas9 protein, causing the Cas9 protein to cleave the target DNA molecule (e.g., a gene encoding iNKRLs).

[0177] By co-expressing a single Cas9 protein with two or more gRNAs, the CRISPR / Cas system can simultaneously target multiple genomic sites, making it suitable for multi-gene inactivation. In some embodiments, the CRISPR / Cas system comprises a single Cas9 protein and one or more gRNAs that specifically target one or more genes encoding one or more iNKRLs. Examples of CRISPR / Cas systems for repressing gene expression are described in U.S. Publication No. 2014 / 0068797 and U.S. Patent Nos. 8,771,945 and 8,697,359. Endonucleases other than Cas9 can also be used, such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas1O, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, T7, Fokl, other nucleases known in the art, their homologs, or their modified versions. Methods for inactivating target genes in immune cells (e.g., T cells) using CRISPR / Cas9 technology are described, for example, in U.S. Patent Publications 2016 / 0272999, 2017 / 0204372, and 2017 / 0119820.

[0178] In some embodiments, the protective element comprises one or more protein degradation elements capable of inducing intracellular retention and / or degradation of one or more iNKRLs.

[0179] As used in this article, the term "protein degradation element" refers to an element that can induce protein degradation through any protein degradation pathway, such as the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, the ubiquitin-proteasome system (UPS)-associated degradation pathway, the ubiquitination-proteasome pathway or the endosome-lysosome pathway, and the autophagy degradation pathway.

[0180] Endoplasmic reticulum-associated protein degradation (ERAD) is a cellular pathway that targets misfolded proteins within the endoplasmic reticulum (ER), ubiquitinates them, and then degrades them via the proteasome. ERAD is a major stress management mechanism for clearing misfolded proteins and normal proteins from the ER for cytoplasmic proteasome degradation. The ERAD process can be divided into three steps: (1) recognition of misfolded or mutant proteins within the ER; (2) reverse transport of the recognized misfolded or mutant proteins from the ER back to the cytoplasm: terminal misfolded proteins must be reverse transported from the ER back to the cytoplasm, where the ubiquitin-proteasome system (UPS) is located; (3) ubiquitin-dependent degradation of the recognized misfolded or mutant proteins by the proteasome (see Annamaria et al., ER-associated degradation: Protein quality control and beyond. J. Cell Biol. Vol. 204 No. 6 869–879).

[0181] In some embodiments, at least one (or all) protein degradation elements comprise an endoplasmic reticulum (ER) retention domain and one or more iNKRL binding domains, the iNKRL binding domains being capable of binding one or more iNKRLs.

[0182] In some embodiments, the iNKRL binding domain includes an antibody domain (e.g., scFv) capable of binding one or more iNKRLs. In some embodiments, one or more iNKRLs include one or more NKG2D ligands. In some embodiments, one or more NKG2D ligands are selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0183] In some embodiments, the iNKRL binding domain is derived from a viral protein capable of binding one or more iNKRLs. In some embodiments, the iNKRL binding domain is derived from an ER-retaining viral protein.

[0184] In some implementations, the iNKRL binding domain is derived from a human receptor capable of binding one or more iNKRLs. In some implementations, the iNKRL binding domain is derived from an NK receptor.

[0185] In some embodiments, the protein degradation element further includes an ER retention transmembrane domain, wherein one end of the ER retention transmembrane domain is operatively connected to an iNKRL binding domain and the other end is operatively connected to an ER retention domain.

[0186] In some implementations, the ER retention domain and / or the ER retention transmembrane domain are derived from human ER retention proteins.

[0187] In some implementations, the ER retention domain and / or the ER retention transmembrane domain are derived from ER-retaining viral proteins.

[0188] In some implementations, the ER retention domain and / or the ER retention transmembrane domain are derived from recombinant ER retention proteins.

[0189] As used in this article, the term "human ER retention protein" refers to proteins involved in the ERAD mechanism or pathway. The term "ER retention domain" refers to a portion or region of a human ER retention protein capable of binding and / or utilizing it, such as the transmembrane domain (or its functional variants) and cytoplasmic domain (or its functional variants) of ER-retaining viral proteins. The cytoplasmic domain (or its functional variants) of human ER retention proteins is also referred to as the "ER retention domain".

[0190] As used in this article, the term "ER-retaining viral protein" refers to a protein involved in the ERAD mechanism or pathway. The term "ER retention domain" as used in this article refers to a portion or region of an ER-retaining viral protein that can bind to and / or utilize it, such as the transmembrane domain (or its functional variants) and cytoplasmic domain (or its functional variants) of the ER-retaining viral protein. The cytoplasmic domain (or its functional variants) of the ER-retaining viral protein is also referred to as the "ER retention domain".

[0191] As used in this article, the term "recombinant ER retention protein" refers to a protein involved in the ERAD mechanism or pathway. The term "ER retention domain" as used in this article refers to a portion or recombinant domain of a human or viral ER retention protein, such as the transmembrane domain (or its functional variant) and the cytoplasmic domain (or its functional variant). The cytoplasmic domain (or its functional variant) of a recombinant ER retention protein is also referred to as the "ER retention domain".

[0192] In some implementations, human ER-retaining viral proteins can be ER-resident proteins of ERAD mechanism components. These proteins work together to recognize and target misfolded or unwanted proteins for degradation, such as Hrd1 and Derlin-1.

[0193] In some implementations, the ER-retaining viral protein can be an ER-resident protein of any virus, which can hijack the ERAD mechanism and promote ubiquitination and proteasome-mediated degradation of target proteins.

[0194] In some implementations, the ER-retaining viral proteins are selected from the group consisting of: HCMV protein US2, HCMV protein US11, HCMV protein US18, HCMV protein US20, HCMV protein US3, HCMV protein US10, HCMV protein US6, HSVICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV protein UL16, HCMV protein UL141, HCMV protein UL142, HIVNef, HIVVpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, vaccinia virus protein CPXV203, and Ad E3 / 19K.

[0195] In some implementations, the viral protein capable of binding to one or more NK receptor ligands is derived from a virus selected from the group consisting of: adenovirus (Ad) (e.g., human Ad), cytomegalovirus (CMV) (human CMV, i.e., HCMV), and Kaposi's sarcoma-associated herpesvirus (KSHV).

[0196] In some implementations, both the ER retention domain and the ER retention transmembrane domain are derived from the same ER-retaining viral protein.

[0197] In some implementations, the ER retention domain and the ER retention transmembrane domain are both derived from different ER-retaining viral proteins.

[0198] In some embodiments, at least one of the ER transmembrane domain, the ER retention transmembrane domain, and the binding domain, and the other of the ER retention domain, the ER retention transmembrane domain, and the binding domain, are derived from different human ERAD proteins or different viral proteins.

[0199] In some implementations, the ER retention domain and binding domain are derived from the same human ERAD protein or the same viral protein or fragments thereof.

[0200] In some implementations, the ER retention domain, the ER retention transmembrane domain, and the binding domain are all derived from the same viral protein or fragments thereof.

[0201] In some implementations, the same viral protein is selected from the group consisting of HCMV protein UL16, HCMV protein UL141, HCMV protein UL142, HCMV protein US18, HCMV protein US20, HHV-7 U21, Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5E3 / 19K), and KSHV K5.

[0202] In some embodiments, the protein degradation element comprises viral proteins selected from the group consisting of: HCMV protein UL16, HCMV protein UL141, HCMV protein UL142, HCMV protein US18, HCMV protein US20, KSHV K5, and AdE3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), analogues thereof, or derivatives thereof.

[0203] CMV UL16 binds to MICBs and ULBPs (David Cosman et al., 2001); UL141 promotes potent downregulation of DNAM1 ligands CD155 and CD112 and exhibits strong NK cell escape capabilities (Tomasec P. et al., 2005; Prod'homme V et al., 2010). Furthermore, UL141 binding promotes intracellular retention of the TRAIL death receptor, thereby protecting virus-infected cells from TRAIL-dependent NK cell-mediated killing (Smith W et al., 2013). UL142 recognizes and arrests full-length MICA in the endoplasmic reticulum (ER) and cis-Golgi apparatus within the organelles (Omodele Ashiru et al., 2009). HCMV targets MICA via US18 and US20 for lysosomal degradation (Ceri A. Fielding et al., 2014). HHV-7 U21 can bind to NKG2D ligands MICA / MICB and ULBP1, redirecting them to lysosomes for degradation (Schneider CL, Hudson AW. PLoS Pathog. 2011). HHV-8 (KSHV) K5 ubiquitin ligase promotes the degradation of MICA and MICB (Thomas M et al., PNAS, 2008). The adenovirus E3 19K protein has been found to directly bind to MHC class I molecules and MICA / MICB in the endoplasmic reticulum (ER), leading to the retention and downregulation of MHC class I molecules (Brian P. McSharry et al., 2008).

[0204] In some implementations, one or more protein degradation elements comprise a combination of viral proteins.

[0205] In some implementations, the combination of viral proteins is selected from the group consisting of: a) HCMV protein UL16 and HCMV protein UL142; b) HCMV protein UL16 and HCMV protein US18; c) HCMV protein UL16 and HCMV protein US20; d) Proteins UL142 and HCMV protein US18; e) Protein UL142 and HCMV protein US20; f) HCMV protein US18 and HCMV protein US20; g)Ad E3 / 19K and HCMV protein UL16; h)Ad E3 / 19K and HCMV protein UL142; i)Ad E3 / 19K and HCMV protein US18; j)Ad E3 / 19K and HCMV protein US20; k) HCMV protein US20, HCMV protein US18 and HCMV protein UL142; l) HCMV protein US20, HCMV protein UL16 and HCMV protein US18; m)HCMV protein US20, HCMV protein UL16 and HCMV protein US20; n) HCMV protein US20, HCMV protein UL16 and HCMV protein UL142; o) HCMV protein US20, HCMV protein US20 and HCMV protein US18; p)Ad E3 / 19K, HCMV protein UL142 and HCMV protein US20; q)Ad E3 / 19K, HCMV protein US18 and HCMV protein UL142; r)Ad E3 / 19K, HCMV protein UL16 and HCMV protein US20; s)Ad E3 / 19K, HCMV protein UL16 and HCMV protein US18; t)Ad E3 / 19K, HCMV protein UL16 and HCMV protein UL142; u)Ad E3 / 19K, HCMV protein US18, HCMV protein UL142 and HCMV protein US20; v)Ad E3 / 19K, HCMV protein UL16, HCMV protein US20 and HCMV protein UL142; w)Ad E3 / 19K, HCMV protein UL16, HCMV protein US18 and HCMV protein UL142; x)Ad E3 / 19K, HCMV protein UL16, HCMV protein US18 and HCMV protein US20; y) HCMV protein UL16, HCMV protein US18, HCMV protein US20, and HCMV protein UL142; and z)Ad E3 / 19K, HCMV protein UL16, HCMV protein US18, HCMV protein US20 and HCMV protein UL142.

[0206] In some embodiments, the combination of viral proteins comprises HCMV protein UL16 and another ER-retaining viral protein. In some embodiments, the other ER-retaining viral protein is selected from the group consisting of HCMV protein UL141, HCMV protein UL142, HCMV protein US18, HCMV protein US20, HHV-7 U21, HHV-8 K5, and Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad3 E3 / 19K, Ad5 E3 / 19K, Ad6 E3 / 19K, Ad11 E3 / 19K, Ad35 E3 / 19K).

[0207] The amino acid sequences of the ER-retained viral proteins are provided in Table 2. These sequences are also available from the published PCT application WO2022161502A1, the full text of which is incorporated herein by reference.

[0208] In some embodiments, one or more protein degradation elements further include a protein degradation pathway (PDP) binding domain capable of binding to members of a protein degradation pathway.

[0209] In some implementations, the PDP binding domain is operatively connected to the ER retention domain.

[0210] In some implementations, the protein degradation pathway is the ubiquitination-proteasome pathway, the endosome-lysosome pathway, or the autophagy degradation pathway.

[0211] In some implementations, members of the ubiquitination-proteasome pathway include E1 ubiquitin activator, E2 ubiquitin conjugate, or E3 ubiquitin ligase.

[0212] Examples of E1 ubiquitin activators include UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, and SAE1.

[0213] Examples of E2 ubiquitin-binding enzymes include hCdc34, Ubc-Uev1A, UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2O, UBE2Q1, UBE2Q2, UBE2R1(CDC34), UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, UBE2Z, ATG3, BIRC6, and UFC1.

[0214] Examples of E3 ubiquitin ligases include von Hippel-Lindau (VHL), Cereblon (CRBN), inhibitor of apoptosis protein (IAP), Kelch-like ECH-associated protein 1 (Keap1), RNF4, RNF114, MDM2, LUBAC, FBW7, Met30, HECT, SKP2, beta TRCP1, HUWEI, TRAF6, SMURF1, and E6AP. In some embodiments, examples of E3 ubiquitin ligases include E3A, MDM2, late-promoting complex (APC), UBR5 (EDD1), SOCS / BC-box / eloBC / CUL5 / RING, LNXp80, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HERC6, and HU. WE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, ST UB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin and MKRN1.

[0215] In some implementations, members of the endosome-lysosome pathway include AP-1, AP-2, AP-3, endosome, lysosome, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sorting linker, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, or C3G / CrkL / Shp2 / Gab2.

[0216] In some implementations, members of the autophagy degradation pathway include molecular chaperone-mediated autophagy (CMA) USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1 / 2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, or NUFIP1.

[0217] B. Surface molecules

[0218] In some embodiments, the surface molecule comprises: a) a tNKRL-binding extracellular domain capable of binding to one or more target NK receptor ligands, and b) a surface molecule (SM) intracellular domain capable of activating immune cells after the tNKRL-binding extracellular domain binds to one or more target NK receptor ligands, and wherein the tNKRL-binding extracellular domain is associated with the SM intracellular domain to allow binding signals to be transduced from the tNKRL-binding extracellular domain to the SM intracellular domain.

[0219] In some implementations, the tNKRL binding extracellular domain includes an antibody domain capable of binding one or more target NK receptor ligands.

[0220] In some implementations, the tNKRL binding extracellular domain includes an NK receptor extracellular domain capable of binding one or more target NK receptor ligands.

[0221] In some implementations, the tNKRL binding extracellular domain includes the NKG2D extracellular domain.

[0222] In some embodiments, the intracellular domain of the SM comprises the cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR.

[0223] In some implementations, the intracellular domain of SM includes the cytoplasmic domain of CD3 (e.g., CD3ζ).

[0224] In some implementations, the intracellular domain of the SM contains the amino acid sequence of SEQ ID NO: 5.

[0225] In some embodiments, the receptor further includes an SM transmembrane domain, wherein one end of the SM transmembrane domain is operatively connected to the tNKRL-binding extracellular domain and the other end is operatively connected to the SM intracellular domain.

[0226] In some implementations, the SM transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of CD3, CD4, CD8, CD28, and CD137.

[0227] In some implementations, the surface molecule contains tNKRL-binding chimeric antigen receptor (CAR).

[0228] In some embodiments, the surface molecule comprises a CNK receptor. In some embodiments, the CNK receptor comprises a protein complex comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a tNKRL-binding extracellular domain operatively linked to a first CNK transmembrane domain, and the second polypeptide comprising a CNK intracellular domain operatively linked to a second CNK transmembrane domain, wherein the first CNK transmembrane domain and the second CNK transmembrane domain are associated with each other to form the protein complex.

[0229] In some implementations, the first CNK transmembrane domain includes an NK receptor transmembrane domain, optionally an NKG2D transmembrane domain.

[0230] In some implementations, the first CNK transmembrane domain is capable of forming a first dimer.

[0231] In some implementations, the first polypeptide comprises the NKG2D protein.

[0232] In some embodiments, the NKG2D protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0233] In some embodiments, the second CNK transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a 10 kDa DNAX activating protein (DAP10) and a 12 kDa DNAX activating protein (DAP12).

[0234] In some implementations, the CNK intracellular domain comprises a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR.

[0235] In some implementations, the CNK intracellular domain includes the cytoplasmic domain of CD3 (e.g., CD3ζ).

[0236] In some embodiments, the second polypeptide comprises a chimeric adaptor protein, said chimeric adaptor protein comprising: a) DAP10 transmembrane domain operatively linked to the intracellular domain of CD3 (e.g., CD3ζ); b) A DAP12 transmembrane domain operatively linked to the CD3 intracellular domain (e.g., CD3ζ); or c) The DAP10 transmembrane domain is operatively linked to the intracellular domain of DAP12.

[0237] In some embodiments, the chimeric adaptor protein comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0238] In some embodiments, the second transmembrane domain is capable of forming a second dimer, wherein the second dimer is associated with the first transmembrane domain to form a complex.

[0239] In some embodiments, the CNK receptor comprises a hexameric protein complex comprising a homodimer of an NKG2D protein and homodimers of two chimeric adaptor proteins.

[0240] In some implementations, the genetically modified immune cells include CNK-UT cells.

[0241] In some implementations, the immune cells are further modified to be deficient in a) MHC class I proteins, or b) MHC class II proteins or MHC class II transactivators, or c) both a) and b).

[0242] In some embodiments, MHC class I proteins comprise HLA-A, HLA-B, HLA-C, B2M, or any combination thereof; optionally, the cells are also deficient in HLA-A and HLA-B. In some embodiments, MHC class II proteins comprise HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, or any combination thereof; optionally, the cells are also deficient in HLA-DR. In some embodiments, the MHC class II transactivator is CIITA; optionally, the cells are deficient in CIITA. In some embodiments, the cells are also deficient in endogenous TCRs; optionally, the cells are also deficient in the T cell receptor α chain constant region (TRAC), T cell receptor β constant region 1 (TRBC1), T cell receptor β constant region 2 (TRBC2), or any combination thereof.

[0243] Cells may be modified to be defective in any suitable manner, such as by gene editing, by interfering with the expression of endogenous a) MHC class I protein, or b) MHC class II protein or MHC class II transactivator, or c) both a) and b), or by promoting the degradation of endogenous a) MHC class I protein, or b) MHC class II protein or MHC class II transactivator, or c) both a) and b), or their encoding mRNA.

[0244] In some embodiments, the modified cells (e.g., mammalian immune cells) or populations thereof contain UT elements. In some embodiments, the UT element contains a small RNA (e.g., siRNA, miRNA, or guide RNA) that targets the following mRNAs: a) MHC class I proteins, or b) MHC class II proteins or MHC class II transactivators, or c) both a) and b). In some embodiments, the UT element contains a genome editing system, such as a CRISPR / Cas system, to inactivate one or more genes encoding the following proteins: a) MHC class I proteins, or b) MHC class II proteins or MHC class II transactivators, or c) both a) and b). In some embodiments, the UT element contains one or more protein degradation elements capable of inducing intracellular retention and / or degradation of the following proteins: a) MHC class I proteins, or b) MHC class II proteins or MHC class II transactivators, or c) both a) and b).

[0245] In some implementations, the immune cells are T cells, and the immune cells are further modified to be in endogenous TCR deficiency.

[0246] C. Other synthetic receptors (e.g., CAR, TCR)

[0247] In some implementations, the genetically modified immune cells provided herein further express target-binding (TB) receptors capable of binding to target markers.

[0248] In some embodiments, the target-binding (TB) receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a target protein binding domain (e.g., an extracellular recognition domain targeting a tumor), a transmembrane domain, and an immune receptor activation signaling domain (ITAM, also known as an "intracellular signaling domain"). In some embodiments, the CAR may also include a co-stimulatory domain. In some embodiments, hinges or connectors may exist between the extracellular recognition domain, transmembrane domain, and / or intracellular signaling domain of the target marker.

[0249] In some implementations, the target-binding (TB) receptor comprises an engineered T-cell receptor (TCR) that is engineered to specifically bind to a target.

[0250] In some implementations, the target marker is expressed on the target cells.

[0251] In some embodiments, the TB receptor comprises a target-binding extracellular domain capable of binding to a target marker and a TB intracellular domain capable of activating immune cells upon binding of the target marker to the extracellular domain. In some embodiments, the extracellular recognition domain of the CAR is selected from antibodies or functional fragments thereof capable of specifically recognizing tumor-associated antigens, T-cell receptors (TCRs), or combinations thereof. Functional fragments of antibodies include Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), single-chain antibodies (scFv), or nanobodies, as well as bispecific, trispecific, and tetravalent antibodies.

[0252] In some embodiments, the TB receptor further includes a TB transmembrane domain. In some embodiments, the transmembrane domain of the CAR can be derived from any membrane-binding or transmembrane protein, including but not limited to BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134, CD137 (4-1BB), CD150 ( IPO-3, SLAMF1, SLAM), CD154, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD244 (2B4, SLAMF4), CD278 (ICOS), CEACAM1, CRTAM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, T cell receptor α, β or δ chain, TNFR2, VLA1 or VLA-6.

[0253] In some implementations, the transmembrane domain of the CAR is derived from the NK cell activation receptor transmembrane domain, the DAP10 transmembrane domain, the DAP12 transmembrane domain, the CD8 transmembrane domain, the CD28 transmembrane domain, the CD4 transmembrane domain, the 4-1BB transmembrane domain, the OX40 transmembrane domain, the ICOS transmembrane domain, the CTLA-4 transmembrane domain, the PD-1 transmembrane domain, the LAG-3 transmembrane domain, the 2B4 transmembrane domain, or the BTLA transmembrane domain, and combinations thereof.

[0254] In some implementations, one end of the TB transmembrane domain is operatively connected to the target marker binding extracellular domain, and the other end is operatively connected to the TB intracellular domain.

[0255] In some embodiments, the immune receptor activation signaling domain (ITAM) of the CAR is derived from the intracellular activation signaling domain of an immune receptor. Preferably, the immune receptor is selected from TCRζ, CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B, or any combination thereof. Optionally, the ITMAM of the immune receptor is fused with an NK cell signaling molecule or a functional variant thereof, wherein the immune receptor is CD3ζ.

[0256] In some preferred embodiments, the ITM of the CAR is derived from the intracellular signaling domain of the immune receptor, including but not limited to CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI CD66d, DAP10, or DAP12.

[0257] In some embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling domain and / or an intracellular signaling domain of the NK cell activation receptor. In some embodiments, the T cell co-stimulatory signaling domain of the CAR is derived from the intracellular signaling domain of the co-stimulatory molecule. Preferably, the co-stimulatory molecule is selected from MHC. Class I molecules, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, lymphocyte activation signaling molecule (SLAM protein), activated NK cell receptor, BTLA, Toll-like receptor ligand, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ. IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA -1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRA, TCRp, TRIM, ZAP70, PTCH2.More preferably, the co-stimulatory signal transduction domain of the CAR can be derived from the intracellular signal domain of NKG2D, DAP10, DAP12, NCR, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, or any combination thereof.

[0258] In some implementations, the target markers are tumor antigens, autoreactive cell antigens, viral antigens, bacterial antigens, microbially infected cells, or damaged and / or aging cells.

[0259] In some implementations, the target cells are tumor cells, autoreactive cells, or virus-infected cells.

[0260] In some implementations, immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, hematopoietic cells (e.g., bone marrow cells), thymocytes, dendritic cells (e.g., mature dendritic cells), macrophages, tumor-infiltrating lymphocytes, monocytes, and granulocytes.

[0261] In some implementations, the immune cells are T cells, such as CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, or tumor-infiltrating lymphocytes.

[0262] II. Expression Builders and Production Methods

[0263] In another aspect, this disclosure provides expression constructs encoding the surface molecules and protective elements described herein, wherein the constructs can be expressed in immune cells.

[0264] In some embodiments, the vector is selected from plasmids, nanoparticles, viral vectors, episomes, RNA vectors, or linear or circular DNA (e.g., transposon DNA) or RNA molecules.

[0265] In some implementations, the viral vector is selected from retroviruses, lentiviral vectors, adenoviruses, adeno-associated virus (AAV) vectors, coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as piconemaviruses and tunica albuginea viruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, smallpox virus, and canarypox virus), norovirus, reovirus, yellow fever virus, calicivirus, hepatitis C virus, piconemaviruses, porcine circoviruses, hepatitis viruses, and virus-like particles (VLPs).

[0266] In some implementations, the viral vector is a retroviral vector.

[0267] In some implementations, the retrovirus is selected from avian leukosis-sarcoma virus, mammalian type C, type B, and type D viruses, HTLV-BLV complexes, lentiviruses, and foam viruses.

[0268] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an oncolytic virus vector. An oncolytic virus vector refers to an oncolytic virus-based viral vector capable of inserting exogenous nucleic acid sequences.

[0269] In some implementations, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Maddie-Visna lentivirus.

[0270] In some implementations, the vector is a transposon-based expression vector. A transposon is a DNA sequence whose location within the genome can be altered. In transposon systems, the nucleic acid molecule encoding the chimeric protein construct provided herein has a terminal repeat sequence recognized by a transposase that mediates transposition. The transposase can be co-delivered as a protein, encoded on the same vector as the chimeric protein construct, or encoded on a separate vector. Non-limiting examples of transposon systems include Sleeping Beauty, PiggyBac, Frog Prince, and Prince Charming.

[0271] In some embodiments, the vector further includes a promoter; preferably, the promoter is an EF1α promoter or a CMV promoter.

[0272] In another aspect, this disclosure provides a method for producing genetically modified mammalian immune cells as described herein, comprising introducing the expression construct described herein into an initiating immune cell under conditions that allow surface molecules to be expressed on the surface of the immune cell and allow protective elements to be expressed in the immune cell.

[0273] Many methods known in the art for generating engineered T cells can also be applied to generate the engineered cells described herein. For example, Zhang et al. described a method for generating CAR-T cells in "Engineered CAR-T Cells" in Biomarker Research (2017) 5:22. The methods provided herein may include one or more of the following steps: obtaining starting cells, culturing (including expansion, optionally activation) the starting cells, and genetically modifying the cells. Starting cells may be stem cells, such as hematopoietic progenitor cells (e.g., T cell progenitor cells, NK cell progenitor cells, macrophage progenitor cells), hematopoietic stem cells (HSCs), CD34+ cells, embryonic stem cells, mesenchymal stem cells, or iPSCs. Starting cells may also be differentiated cells derived from stem cells, such as the aforementioned immune cells.

[0274] Starting cells can be obtained from any source, such as isolated from immune cells (e.g., T cells) of a subject (e.g., a human subject). In some embodiments, immune cells are obtained from a target subject, such as a subject suspected of having a specific disease or indication, a subject susceptible to a specific disease or indication, a subject receiving a specific treatment for a certain disease or indication, a healthy volunteer, or a healthy donor. Immune cells may also be obtained from a blood bank. Immune cells used for the target subject can be autologous or allogeneic. Immune cells can be collected from any location in the subject's body, including but not limited to blood, cord blood, spleen, thymus, lymph nodes, pleural fluid, spleen tissue, tumors, and bone marrow. Isolated immune cells can be used directly or stored for a period of time, such as cryopreservation.

[0275] In some embodiments, the initiating immune cells (e.g., T cells) are derived from a healthy donor (e.g., a mammalian donor) and expanded and activated in vitro. In some embodiments, the initiating mammalian immune cells (e.g., T cells) are modified in vitro to the genetically modified mammalian immune cells or populations thereof provided herein. The initiating immune cells (e.g., T cells) most active in diseases associated with self-reactive T lymphocytes, or the genetically modified mammalian immune cells or populations thereof provided herein, are cultured in large quantities in vitro for approximately 2 weeks. The genetically modified mammalian immune cells or populations thereof cultured in vitro provided herein are infused into a patient.

[0276] In some embodiments, immune cells are activated and / or expanded. In some embodiments, immune cells are activated and expanded simultaneously with, before, and / or after genetic modification. In some embodiments, immune cells are activated and / or expanded in vitro, in vivo, or ex vivo. Methods for activating and expanding immune cells have been described in the art and can be used in the methods described herein. For example, T cells can be activated and expanded by contacting T cells with a reagent that provides a signal via a stimulating CD3 / TCR complex and co-stimulatory molecules on the surface of the T cells. Specifically, T cell populations can be stimulated by contacting a population of T cells with an anti-CD3 antibody or its antigen-binding fragment or an antibody immobilized on the surface, or with a protein kinase C activator (e.g., phorbol ester) and a calcium ionophore. Co-stimulatory molecules on the surface of T cells can be stimulated using ligands that bind to the co-stimulatory molecules. For example, under conditions suitable for stimulating T cell proliferation, a population of T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. Anti-CD3 antibodies and anti-CD28 antibodies can be used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. In some implementations, primary stimulation signals and co-stimulation signals for T cells can be provided in different ways.

[0277] Cellular genetic modification can be achieved by transducing nucleic acid molecules encoding the surface molecules and protective elements provided herein into substantially homogeneous starting cells. In some embodiments, the nucleic acid molecules provided herein can be introduced into starting cells using retroviral vectors (e.g., lentiviral vectors). For example, the nucleic acid molecules provided herein can be cloned into lentiviral vectors and expressed under the control of their endogenous promoters, lentiviral long terminal repeat sequences, or promoters specific to the target cell type. Common delivery methods for viral vectors include, but are not limited to, electroporation, microinjection, gene gun, and magnetic transfection.

[0278] Cellular genetic modification can also be achieved by delivering nucleic acid molecules using lipid nanoparticles (LNPs). Other non-viral methods for nucleic acid delivery include in vitro transfection using calcium phosphate, DEAE-glucan, electroporation, and protoplast fusion. Transposases or targeting nucleases (e.g., zinc finger nucleases, macronucleases, TALE nucleases, CRISPR) can also be used to initiate genetic modification of cells and obtain engineered cells as described in this disclosure.

[0279] In some embodiments, the engineered cells provided herein can be prepared by transfecting starter cells with nucleic acid molecules encoding the constructs provided herein prior to administration. In some embodiments, the engineered cells provided herein can be prepared by transfecting immune cells with nucleic acid molecules encoding chimeric protein constructs prior to administration, for example using a viral vector. The engineered cells provided herein exhibit decreased expression of immunogenic molecules (e.g., TCR, HLA, etc.) and / or immunosuppressive molecules (e.g., PD-1) on the cell surface and / or increased expression of tumor-targeting receptors (e.g., CAR, engineered TCR, CNK receptor) on the cell surface.

[0280] III. Pharmaceutical Compositions

[0281] On the other hand, this disclosure also provides pharmaceutical compositions comprising genetically modified mammalian immune cells or populations thereof as provided herein, and a pharmaceutically acceptable medium. As used herein, the term "pharmaceutical composition" refers to a composition formulated for pharmaceutical use.

[0282] The term “pharmaceutically acceptable” means that the specified carrier, medium, diluent, excipient and / or salt is generally chemically and / or physically compatible with other components contained in the formulation and physiologically compatible with their recipients.

[0283] "Pharmaceutically acceptable medium" refers to a component in a pharmaceutical preparation other than the active ingredient that has acceptable biological activity and is non-toxic to the subject. Pharmaceutically acceptable mediums used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous or non-aqueous carriers, antibacterial agents, buffers, antioxidants, isotonic agents, suspending / dispersing agents, separating or chelating agents, diluents, adjuvants, excipients or non-toxic excipients, or various combinations thereof.

[0284] The pharmaceutical compositions disclosed herein can be prepared using various techniques known in the art, see, for example, Remington, The Science and Practice of Pharmacy (21st ed. 2005). In short, genetically modified mammalian immune cells or populations thereof are mixed with a suitable medium prior to use or storage. A suitable pharmaceutically acceptable medium typically contains an inert substance that facilitates: 1) administering the pharmaceutical composition to a subject, 2) processing the pharmaceutical composition into a deliverable formulation, and / or 3) storing the pharmaceutical composition prior to administration. In some embodiments, the pharmaceutically acceptable medium comprises a formulation that can stabilize, optimize, or alter the form, consistency, viscosity, pH, pharmacokinetics, and / or solubility of the formulation. Such agents include, but are not limited to, buffers, wetting agents, emulsifiers, diluents, encapsulating agents, and skin penetration enhancers, such as saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

[0285] Exemplary pharmaceutically acceptable media include sugars (e.g., lactose, glucose, and sucrose), starches (e.g., corn starch and potato starch), cellulose and their derivatives (e.g., sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate), tragacanth powder, malt, gelatin, lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc), excipients (e.g., cocoa butter and suppository waxes), oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), and glycols. (e.g., propylene glycol), polyols (e.g., glycerol, sorbitol, mannitol, and polyethylene glycol (PEG)), esters (e.g., ethyl oleate and ethyl laurate), agar, buffers (e.g., magnesium hydroxide and aluminum hydroxide), alginate, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, pH buffer solutions, polyesters, polycarbonates, polyanhydrides, swelling agents (e.g., peptides and amino acids), serum alcohols (e.g., ethanol), (sterile) phosphate buffer solutions, Ringer's solution, dextrorotatory glucose solution, and other non-toxic compatible substances used in pharmaceutical preparations.

[0286] The pharmaceutical compositions of the present invention may comprise genetically modified mammalian immune cells or populations thereof as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0287] IV. Reagent Kit

[0288] In another aspect, this disclosure also provides kits comprising the genetically modified mammalian immune cells or populations thereof provided herein. In yet another aspect, this disclosure also provides kits comprising the expression constructs provided herein for generating genetically modified mammalian immune cells or populations thereof.

[0289] In some embodiments, the kits disclosed herein include written instructions for using the kit. In some embodiments, the instructions include at least one of the following: clinical studies, precautions, warnings, and / or references. The instructions may be printed directly on the container (if any), or provided as a label affixed to the container, or as a separate sheet of paper, brochure, card, or folder within or with the container. Suitable containers include, for example, bottles, syringes, ampoules, and test tubes. Containers may be formed from a variety of materials, such as plastic or glass. In some embodiments, the container contains the pharmaceutical composition provided herein and has a sterile interface.

[0290] In some embodiments, the kit further includes a second container containing the pharmaceutically acceptable media described above. In some embodiments, the kit further includes other commercially desirable or user-friendly materials, such as additional diluents, buffers, needles, filters, syringes, and packaging inserts with instructions for use.

[0291] V. Treatment Methods

[0292] In another aspect, this disclosure provides a method of treating a disease in a subject in need, comprising administering to the subject a therapeutically effective amount of the genetically modified mammalian immune cells or populations thereof provided herein, or a pharmaceutical composition provided herein. In some embodiments, the disease is related to target cells expressing tNKRL (e.g., autoreactive lymphocytes). In some embodiments, the disease is cancer. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an immunodeficiency disease, or an age-related disease.

[0293] In some implementations, the disease associated with an increase in self-reactive lymphocytes is an autoimmune disease.

[0294] In some implementations, autoimmune diseases are organ-specific autoimmune diseases or systemic autoimmune diseases.

[0295] In some implementation schemes, organ-specific autoimmune diseases are selected from the group consisting of chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0296] In some implementations, systemic autoimmune diseases are selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0297] In some implementations, the autoimmune disease is graft-versus-host disease (GvHD).

[0298] In some embodiments, the genetically modified mammalian immune cells or populations thereof of this disclosure are administered to a subject or recipient, proliferate in vivo, and can persist in the subject's body for an extended period of time.

[0299] In some implementations, at least 1 × 10⁻⁶ is administered to the subject or recipient. 4 1 cell, at least 5 × 10 4 10 cells, at least 1×10 5 1 cell, at least 5 × 10 5 10 cells, at least 1×10 6 1 cell, at least 5 × 10 6 10 cells, at least 1×10 7 1 cell, at least 5 × 10 7 10 cells, at least 1×10 8 1 cell, at least 5 × 10 8 10 cells, at least 1×10 9 1 cell, at least 2 × 10 9 1 cell, at least 3 × 10 9 1 cell, at least 4 × 10 9 1 cell, at least 5 × 10 9 One cell, or at least 1 × 10 10 Cells. In some embodiments, at least 1 × 10⁻⁶ cells are administered to the subject or recipient. 3Cells / kg body weight, at least 5×10 3 Cells / kg body weight, at least 1×10 4 Cells / kg body weight, at least 5×10 4 Cells / kg body weight, at least 1×10 5 Cells / kg body weight, at least 5×10 5 Cells / kg body weight, at least 1×10 6 Cells / kg body weight, at least 5×10 6 Cells / kg body weight, at least 1×10 7 Cells / kg body weight, at least 5×10 7 Cells / kg body weight, at least 1×10 8 Cells / kg body weight, at least 2×10 8 Cells / kg body weight, at least 3×10 8 Cells / kg body weight, at least 4×10 8 Cells / kg body weight, at least 5×10 8 Cells / kg body weight, or at least 6 × 10 8 Cells per kg of body weight.

[0300] Those skilled in the art will understand that the dosage of the pharmaceutical compositions provided herein can be determined based on various factors of the subject or recipient, such as body type, age, sex, weight, and indications. Those skilled in the art can readily determine the dosage based on this disclosure and knowledge in the art.

[0301] Those skilled in the art can readily determine the amount of the genetically modified mammalian immune cells provided herein, as well as the amounts of optional additives, vehicles, mediators, and / or carriers, in the compositions disclosed herein and in the methods applied herein. Typically, additives (if any) are present in amounts of 0.001 to 50% (wt) of a solution in phosphate-buffered saline, and the active ingredient (e.g., the genetically modified mammalian immune cells provided herein) is present in the order of micrograms to milligrams, for example, from about 0.0001 to about 5 wt%, preferably from about 0.0001 to about 1 wt%, more preferably from about 0.0001 to about 0.05 wt%, or from about 0.001 to about 20 wt%, preferably from about 0.01 to about 10 wt%, more preferably from about 0.05 to about 5 wt%.

[0302] In some embodiments, the composition is formulated in a solution containing Plasma-Lyte A and CS10 in a 50:50 ratio.

[0303] In some embodiments, the main components are formulated in a cell cryopreservation solution comprising human serum albumin (HSA), CS10, and Plasma-Lyte A. The cell cryopreservation solution provided by this invention contains 0.5–5% by weight of HSA, 50–75% by volume of CS10 cryopreservation solution, and 25–50% by volume of Plasma-Lyte A. Preferably, the cell cryopreservation solution provided by this invention contains 2.5% by weight of HSA, 50% by volume of CS10 cryopreservation solution, and 40% by volume of Plasma-Lyte A.

[0304] It is preferable to determine the toxicity of a particular dose, for example by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., mice). It is also preferable to determine the time of administration of the composition that elicits a suitable response. Based on the knowledge of those skilled in the art and this disclosure, such determinations do not require excessive experimentation.

[0305] The pharmaceutical compositions provided herein can be administered using a variety of conventional techniques, including but not limited to infusion, blood transfusion, or parenteral administration. In some embodiments, parenteral administration includes intravascular, intratumoral, intravenous, intradermal, intramuscular, intraarterial, intratracheal, intrathecal, intraperitoneal, subcutaneously, subcutaneously, intra-articular, subcapsular, subarachnoid, and intrasternal infusion or injection.

[0306] In some embodiments, the pharmaceutical compositions provided herein are applied topically to a diseased site (e.g., a tumor site). In some embodiments, the pharmaceutical compositions provided herein are administered to a subject via injection, catheter, suppository, or implant (e.g., a porous, non-porous, or gel-like material, such as a membrane, like a silicone membrane). In some embodiments, the pharmaceutical compositions provided herein are delivered using a controlled-release system.

[0307] Treatment with the methods described herein results in an increase in the survival of recipients (e.g., human subjects) compared to the expected survival of human subjects treated without the pharmaceutical compositions provided herein. In another embodiment, the survival of human subjects is increased by at least 30 days. The increase in survival of human subjects may be at least 3 months, at least 6 months, or at least 1 year. In some embodiments, the recipients (e.g., human subjects) treated with the methods provided herein are children (e.g., 0–18 years old) or adults (e.g., 18+ years old).

[0308] In some embodiments, the therapeutic methods of this disclosure involve using genetically modified mammalian immune cells or populations thereof that express a CAR. The CAR can specifically target antigenic targets on cells present in the host that are not desired, such as cancer cells or self-reactive lymphocytes. In some embodiments, the genetically modified mammalian immune cells exhibit an enhanced cytotoxic response to their targets. In some embodiments, the genetically modified mammalian immune cells or populations thereof induce an enhanced cytotoxic response to their targets compared to reference cells, such as natural T cells.

[0309] In some implementations, genetically modified mammalian immune cells exhibit at least a 1.2-fold, 1.4-fold, 1.6-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, or greater cytotoxic response compared to reference cells (e.g., native T cells).

[0310] In some embodiments, genetically modified mammalian immune cells or populations thereof can kill at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 200%, at least 500%, at least 1000%, or at least 2000% more target cells than reference cells (e.g., natural T cells). In some embodiments, the genetically modified mammalian immune cells or populations thereof are administered to an allogeneic host, wherein the genetically modified mammalian immune cells or populations thereof have reduced or no (or negligible or minimal) host rejection. [Implementation Scheme 1] Genetically modified mammalian immune cells or populations thereof, comprising surface molecules and protective elements, characterized in that, The surface molecules are expressed on the surface of immune cells and can bind to one or more target NK receptor ligands (tNKRL) expressed on target cells, thereby activating immune cells and producing cytotoxic effects on target cells. The immune cells, upon activation, can be induced to express one or more inducible NK receptor ligands (iNKRLs) that can bind to surface molecules, and The protective element substantially inhibits the expression of one or more induced NK receptor ligands on the surface of the immune cells after the immune cells are activated, thereby preventing the activated immune cells from becoming target cells. [Implementation Scheme 2] The genetically modified mammalian immune cells as described in Implementation Scheme 1 are characterized in that at least one of the target NK receptor ligands is the same as at least one of the inducible NK receptor ligands, or the surface molecule is cross-reactive with at least one of the target NK receptor ligands and at least one of the inducible NK receptor ligands. [Implementation Scheme 3] The genetically modified mammalian immune cells as described in Implementation Scheme 1 are characterized in that the one or more target NK receptor ligands are selected from the group consisting of: NKG2D ligand, NCR ligand, KIR ligand, CD226 ligand, TIGIT ligand, CD96 ligand, or any combination thereof. [Implementation Scheme 4] The genetically modified mammalian immune cells as described in Implementation Scheme 1, characterized in that the one or more target NK receptor ligands comprise one or more NKG2D ligands. [Implementation Scheme 5] The genetically modified mammalian immune cells as described in Implementation Scheme 4 are characterized in that the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. [Implementation Scheme 6] A genetically modified mammalian immune cell as described in any of the foregoing embodiments, characterized in that the expression of the one or more induced NK receptor ligands on the surface of the immune cell is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) relative to the expression of the one or more induced NK receptor ligands on the surface of a reference immune cell without the protective element. [Implementation Scheme 7] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that the expression of one or more induced NK receptor ligands on the surface of the immune cell is reduced such that the binding of one or more induced NK receptor ligands to the surface molecules is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). [Implementation Scheme 8] Genetically modified mammalian immune cells as described in any of the foregoing implementation schemes, characterized in that, after the surface molecule binds to the one or more target NK receptor ligands, the expression of one or more induced NK receptor ligands can be detected in an immune cell population of no more than 50% (e.g., no more than 40%, no more than 30%, no more than 20%, or no more than 10%). [Implementation Scheme 9] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that the one or more inducible NK receptor ligands comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and / or ULBP6, and the expression levels of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and / or ULBP6 on the surface of the immune cell are reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). [Implementation Scheme 10] A genetically modified mammalian immune cell as described in any of the foregoing embodiments, characterized in that the one or more inducible NK receptor ligands comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5 and ULBP6, and the expression levels of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5 and ULBP6 on the surface of the immune cell are reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). [Implementation Scheme 11] A genetically modified mammalian immune cell as described in any of the foregoing embodiments, characterized in that the immune cell has substantially reduced activation-induced immune cell death, optionally determined by a reduction in apoptosis detected by Annexin V / 7-AAD. [Example 12] A genetically modified mammalian immune cell as described in any of the foregoing embodiments, characterized in that the immune cell is capable of proliferating upon activation (e.g., having an increased proliferation rate compared to modified mammalian immune cells without protective elements). [Implementation Scheme 13] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that the protective element is capable of silencing the expression of one or more inducible NK receptor ligands, or capable of inducing intracellular retention and / or degradation of one or more inducible NK receptor ligands. [Example 14] Genetically modified mammalian immune cells as described in Example 13, characterized in that the protective element comprises a small RNA (e.g., siRNA, miRNA, or shRNA) that targets one or more NK receptor ligands. [Example 15] Genetically modified mammalian immune cells as described in Embodiment 13, characterized in that the protective element comprises one or more protein degradation elements capable of inducing the retention and / or degradation of the one or more NK receptor ligands within the cell. [Implementation Scheme 16] The genetically modified mammalian immune cell as described in Implementation Scheme 15 is characterized in that the at least one (or all) protein degradation element comprises an endoplasmic reticulum (ER) retention domain and an iNKRL binding domain capable of binding to the one or more inducible NK receptor ligands. [Implementation Scheme 17] The genetically modified mammalian immune cell as described in Implementation Scheme 16 is characterized in that the protein degradation element further comprises an ER retention transmembrane domain, one end of which is operatively connected to an iNKRL binding domain and the other end of which is operatively connected to an ER retention domain. [Implementation Scheme 18] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 16-17, characterized in that the ER retention domain and / or ER retention transmembrane domain are derived from ER retention viral proteins. [Example 19] The genetically modified mammalian immune cells as described in Example 18 are characterized in that the ER-retaining viral proteins are selected from the group consisting of: HCMV protein US2, HCMV protein US11, HCMV protein US18, HCMV protein US20, HCMV protein US3, HCMV protein US10, HCMV protein US6, HSV ICP47, CPXV12, BHVUL49.5, EBV BNFL2a, HCMV protein UL16, HCMV protein UL141, HCMV protein UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, vaccinia virus protein CPXV203, and AdE3 / 19K. [Implementation Scheme 20] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 18-19, characterized in that the ER retention domain and the ER retention transmembrane domain are both derived from the same endoplasmic reticulum-retaining viral protein. [Implementation Scheme 21] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 16-20, characterized in that the iNKRL binding domain comprises an antibody domain (e.g., scFv) capable of binding to one or more inducible NK receptor ligands. [Implementation Scheme 22] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 16 to 20, characterized in that the iNKRL binding domain is derived from a viral protein capable of binding one or more inducible NK receptor ligands. [Implementation Scheme 23] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 21 to 22, characterized in that the one or more inducible NK receptor ligands comprise one or more NKG2D ligands. [Implementation Scheme 24] The genetically modified mammalian immune cells as described in Implementation Scheme 23 are characterized in that the one or more NKG2D ligands are selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. [Implementation Scheme 25] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 18 to 24, characterized in that the viral protein capable of binding one or more NK receptor ligands is derived from a virus selected from the group consisting of: adenovirus (Ad) (e.g., human Ad), cytomegalovirus (CMV) (human CMV, i.e., HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV). [Implementation Scheme 26] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 18-25, characterized in that the viral protein capable of binding one or more NK receptor ligands is derived from a viral protein selected from the group consisting of: Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHV K5. [Implementation Scheme 27] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 22 to 26, characterized in that the ER retention domain and the binding domain are both derived from the same viral protein. [Implementation Scheme 28] The genetically modified mammalian immune cells as described in any one of Implementation Schemes 22 to 26 are characterized in that the ER retention domain, ER retention transmembrane domain and binding domain are all derived from different viral proteins. [Implementation Scheme 29] The genetically modified mammalian immune cells as described in Implementation Scheme 27 are characterized in that the same viral protein is selected from the group consisting of Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHV K5. [Embodiment 30] A genetically modified mammalian immune cell as described in any one of embodiments 18-27 and 29, characterized in that the protein degradation element comprises a viral protein selected from the group consisting of: Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, KSHV K5 or analogues thereof, or derivatives thereof. [Example 31] Genetically modified mammalian immune cells as described in Example 14, characterized in that the protective element comprises small RNAs capable of regulating NKG2D ligand expression, such as HCMV-encoded microRNAs (miR-20a, miR-UL112). [Implementation Scheme 32] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 18-30, characterized in that the one or more protein degradation elements comprise a combination of viral proteins, characterized in that the combination of viral proteins comprises HCMV protein UL16 and another viral protein selected from the group consisting of: Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHVK5. [Implementation Scheme 33] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 15 to 32, characterized in that the one or more protein degradation elements further comprises a protein degradation pathway (PDP) binding domain capable of binding to members of a protein degradation pathway. [Implementation Scheme 34] The genetically modified mammalian immune cell as described in Implementation Scheme 33 is characterized in that the PDP binding domain is operatively linked to the ER retention domain. [Implementation Scheme 35] The genetically modified mammalian immune cells as described in any one of Implementation Schemes 33 to 34 are characterized in that the protein degradation pathway is a ubiquitination-proteasome pathway, an endosome-lysosome pathway, or an autophagy degradation pathway. [Implementation Scheme 36] The genetically modified mammalian immune cells as described in Implementation Scheme 35 are characterized in that the members of the ubiquitination-proteasome pathway include E1 ubiquitin activator, E2 ubiquitin conjugate, or E3 ubiquitin ligase. [Implementation Scheme 37] The genetically modified mammalian immune cells as described in Implementation Scheme 35 are characterized in that the members of the endosome-lysosome pathway include AP-1, AP-2, AP-3, endosome, lysosome, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sorting connexin, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, or C3G / CrkL / Shp2 / Gab2. [Implementation Scheme 38] The genetically modified mammalian immune cells as described in Implementation Scheme 35 are characterized in that the members of the autophagy degradation pathway include molecular chaperone-mediated autophagy (CMA) USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1 / 2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, or NUFIP1. [Implementation Scheme 39] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that the surface molecule comprises a) a tNKRL-binding extracellular domain capable of binding one or more target NK receptor ligands, and b) a surface molecule (SM) intracellular domain capable of activating the immune cell after binding one or more target NK receptor ligands in the tNKRL-binding extracellular domain, and the tNKRL-binding extracellular domain is associated with the SM intracellular domain to allow binding signals to be transduced from the tNKRL-binding extracellular domain to the SM intracellular domain. [Implementation Scheme 40] The genetically modified mammalian immune cell as described in Implementation Scheme 39 is characterized in that the tNKRL binding extracellular domain comprises an antibody domain capable of binding one or more target NK receptor ligands. [Implementation Scheme 41] The genetically modified mammalian immune cell as described in Implementation Scheme 39 is characterized in that the tNKRL binding extracellular domain comprises an NK receptor extracellular domain capable of binding one or more target NK receptor ligands. [Implementation Scheme 42] The genetically modified mammalian immune cell as described in Implementation Scheme 39 is characterized in that the tNKRL binding extracellular domain comprises the NKG2D extracellular domain. [Implementation Scheme 43] The genetically modified mammalian immune cell as described in Implementation Scheme 41, characterized in that the receptor further comprises an SM transmembrane domain, one end of which is operatively connected to a tNKRL-binding extracellular domain and the other end of which is operatively connected to an SM intracellular domain. [Implementation Scheme 44] A genetically modified mammalian immune cell as described in Implementation Scheme 43, characterized in that the SM transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD3, CD4, CD8, CD28, B7, ICOS, CD226, 41-BB, OX40, CD27, GITR, HVEM, CD40, BAFFR, BAFF, NKG2D, NKG2C, NKG2A, NKp44, NKp46, and NKp30. [Implementation Scheme 45] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that the surface molecule comprises a tNKRL-binding chimeric antigen receptor (CAR). [Implementation Scheme 46] The genetically modified mammalian immune cell as described in Implementation Scheme 45, characterized in that the receptor comprises a CNK receptor protein complex, the CNK receptor protein complex comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a tNKRL-binding extracellular domain operably linked to a first CNK transmembrane domain, the second polypeptide comprising a CNK intracellular domain operably linked to a second CNK transmembrane domain, wherein the first CNK transmembrane domain and the second CNK transmembrane domain are associated with each other to form a protein complex. [Implementation Scheme 47] The genetically modified mammalian immune cell as described in Implementation Scheme 46 is characterized in that the first CNK transmembrane domain comprises an NK receptor transmembrane domain, optionally an NKG2D transmembrane domain. [Implementation Scheme 48] The genetically modified mammalian immune cell as described in Implementation Scheme 46 is characterized in that the first transmembrane domain is capable of forming a first dimer. [Implementation Scheme 49] The genetically modified mammalian immune cell as described in Implementation Scheme 46, characterized in that the first polypeptide comprises NKG2D protein. [Implementation Scheme 50] The genetically modified mammalian immune cell as described in Implementation Scheme 49 is characterized in that the NKG2D protein comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. [Implementation Scheme 51] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46 to 50, characterized in that the second transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a 10 kDa DNAX-activating protein (DAP10) and a 12 kDa DNAX-activating protein (DAP12). [Implementation Scheme 52] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46-51, characterized in that the intracellular domain of the CNK comprises a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR. [Implementation Scheme 53] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46 to 51, characterized in that the intracellular domain of the CNK comprises a cytoplasmic domain of CD3 (e.g., CD3zeta). [Implementation Scheme 54] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46 to 53, characterized in that the CNK intracellular domain comprises the amino acid sequence of SEQ ID NO:5. [Implementation Scheme 55] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46-54, characterized in that the second polypeptide comprises a chimeric adaptor protein, said chimeric adaptor protein comprising: a) The transmembrane domain of DAP10, which is operatively linked to the intracellular domain of CD3 (e.g., CD3ζ); b) The transmembrane domain of DAP12, which is operatively linked to the intracellular domain of CD3 (e.g., CD3ζ); or c) The transmembrane domain of DAP10, which is operatively linked to the intracellular domain of DAP12. [Implementation Scheme 56] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46 to 54, characterized in that the second polypeptide comprises a full-length DAP10 operatively linked to an intracellular domain of CD3ζ. [Implementation Scheme 57] The genetically modified mammalian immune cells as described in Implementation Scheme 55 are characterized in that the chimeric adaptor protein comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. [Implementation Scheme 58] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46 to 56, characterized in that the second CNK transmembrane domain is capable of forming a second dimer, and the second dimer is associated with the first CNK transmembrane domain to form a complex. [Implementation Scheme 59] A genetically modified mammalian immune cell as described in any one of Implementation Schemes 46 to 56, characterized in that the CNK receptor comprises a hexameric protein complex comprising a homodimer of an NKG2D protein and homodimers of two chimeric adaptor proteins. [Implementation Scheme 60] A genetically modified mammalian immune cell as described in any of the foregoing embodiments, characterized in that the immune cell is further modified to be defective in a) MHC class I protein, or b) MHC class II protein or MHC class II transactivator, or c) both a) and b). [Implementation Scheme 61] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that the immune cell is a T cell and the immune cell is further modified to be defective in the endogenous TCR. [Implementation Scheme 62] A genetically modified mammalian immune cell as described in any of the foregoing implementation schemes, characterized in that it further expresses a target-binding (TB) receptor capable of binding to a target marker. [Implementation Scheme 63] The genetically modified mammalian immune cells as described in Implementation Scheme 62 are characterized in that the target marker is expressed in the target cells. [Implementation Scheme 64] The genetically modified mammalian immune cell as described in Implementation Scheme 63 is characterized in that the TB receptor comprises a target-binding extracellular domain capable of binding a target marker and a TB intracellular domain capable of activating the immune cell after binding to the target marker in the target-binding extracellular domain. [Implementation Scheme 65] The genetically modified mammalian immune cell as described in Implementation Scheme 64, characterized in that the TB receptor further comprises a TB transmembrane domain. [Implementation Scheme 66] The genetically modified mammalian immune cell as described in Implementation Scheme 65 is characterized in that one end of the TB transmembrane domain is operatively connected to the target marker binding extracellular domain and the other end is operatively connected to the TB intracellular domain. [Implementation Scheme 67] The genetically modified mammalian immune cell as described in Implementation Scheme 66 is characterized in that the target marker binding extracellular domain comprises the extracellular domain of a natural cytotoxic receptor (e.g., NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1)). [Implementation Scheme 68] The genetically modified mammalian immune cell as described in Implementation Scheme 67 is characterized in that the TB transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD3, CD4, CD8, CD28, B7, ICOS, CD226, 41-BB, OX40, CD27, GITR, HVEM, CD40, BAFFR, BAFF, NKG2D, NKG2C, NKG2A, NKp44, NKp46, and NKp30. [Implementation Scheme 69] A genetically modified mammalian immune cell as described in Implementation Scheme 67 or 68, characterized in that the intracellular domain of the TB comprises a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR. [Implementation Scheme 70] The genetically modified mammalian immune cell as described in Implementation Scheme 69 is characterized in that the intracellular domain of the TB includes a cytoplasmic domain of CD28 operatively linked to the CD3ζ chain. [Implementation Scheme 71] The genetically modified mammalian immune cells as described in any one of Implementation Schemes 62 to 70 are characterized in that the target marker is a tumor antigen, an autoreactive cell antigen or a viral antigen, a bacterial antigen, a microbially infected cell, or a damaged and / or senescent cell. [Implementation Scheme 72] The genetically modified mammalian immune cells as described in any one of Implementation Schemes 63 to 70 are characterized in that the target cells are tumor cells, autoreactive cells, senescent cells or virus-infected cells. [Example 73] Genetically modified mammalian immune cells as described in any of the foregoing embodiments, characterized in that the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, hematopoietic cells (e.g., bone marrow cells), thymocytes, dendritic cells (e.g., mature dendritic cells), macrophages, tumor-infiltrating lymphocytes, monocytes, and granulocytes. [Implementation Scheme 74] Genetically modified mammalian immune cells as described in any of the foregoing implementation schemes, wherein the immune cells are T cells, such as CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, or tumor-infiltrating lymphocytes. [Implementation Scheme 75] A pharmaceutical composition characterized in that it comprises genetically modified mammalian immune cells or a population thereof. [Implementation Scheme 76] A method for treating a disease in a subject in need, characterized in that it comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of Implementation Scheme 75. [Implementation Scheme 77] The method as described in Implementation Scheme 76 is characterized in that the disease is cancer, a disease related to self-reactive lymphocytes (e.g., an autoimmune disease), an immunodeficiency disease, or an aging-related disease. [Implementation Scheme 78] The method described in Implementation Scheme 77 is characterized in that the autoimmune disease is an organ-specific autoimmune disease or a systemic autoimmune disease. [Implementation Scheme 79] The method described in Implementation Scheme 78 is characterized in that the organ-specific autoimmune disease is selected from the group consisting of chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis. [Implementation Scheme 80] The method described in Implementation Scheme 78 is characterized in that the systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis. [Implementation Scheme 81] The method as described in Implementation Scheme 77, wherein the autoimmune disease is graft-versus-host disease (GvHD). [Implementation Scheme 82] An expression construct encoding a surface molecule and a protective element, characterized in that the construct can be expressed in immune cells, wherein the surface molecule is defined as in any one of Implementation Schemes 43 to 49, and the protective element is defined as in any one of Implementation Schemes 17 to 42. [Implementation Scheme 83] A method for producing genetically modified mammalian immune cells as described in any one of Implementation Schemes 1 to 74, characterized in that the expression construct as described in Implementation Scheme 82 is introduced into the initiating immune cell under conditions that allow surface molecules to be expressed on the surface of the immune cell and allow protective elements to be expressed in the immune cell. Example

[0311] Although this disclosure has been specifically shown and described with reference to specific embodiments, some of which are preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure as disclosed herein.

[0312] Example 1. Activated T cells significantly increased the expression of NKG2D ligand.

[0313] Two days after T cells were activated using Dynabeads® Human T-Activator CD3 / CD28 (Thermo Scientific) (microbeads:T cells = 1:1), the expression of NKG2D ligands (MICA, MICB, ULBP1-6) was detected by flow cytometry. The results showed that MICA, MICB, and ULBP2, 5, 6, and ULBP3 were all upregulated in activated T cells, but ULBP4 was not upregulated. Figure 1A and Figure 1B ).

[0314] Example 2. Production of lentiviruses encoding chimeric natural killer receptors and CNK-T cells

[0315] To prepare the viral solution, a CNK plasmid expressing a specific chimeric natural killer receptor (NKG2D / NCR2) (constructed by GENEWIZ) (e.g.) was used. Figure 2A (As shown) Along with the packaging plasmids gal-pol, Rev, and VSVG (OBIO), it was mixed with the transfection reagent PEI pro (Polyplus) at a ratio of CNK:gal-pol:rev:vsvg = 12:5:4:4, and then transfected into 293T cells in a 1:1 ratio. Four hours after transfection, OptiPRO SFM medium (Thermofisher) was added to replace the complete medium, and the supernatant was collected after 48 hours of downstream purification.

[0316] The clarified filtrate culture medium containing the virus was filtered through a Diamond Layer 400 filter and then concentrated by ultrafiltration. After ultrafiltration concentration, a 25% sucrose solution equivalent to 10% of the volume of the collected solution was added to obtain the final product (containing 2.5% sucrose). Subsequently, the lentivirus was stored at -70±10°C.

[0317] According to the manufacturer's instructions, human CD8+ and CD4+ cells were isolated from normal donor PBMCs using a CD3+ T cell isolation kit (Miltenyi Biotec). These cells were activated with anti-CD3 / CD28 magnetic beads (Life Technologies) and transduced using lentiviral vectors encoding CNK, CNK+PE (UL16), and CNK+PE / NCR2 ED-CD28 TMD-CD28 ICD-CD3Z ICD, respectively. Transduction was performed on day 3 post-activation by centrifugation at 2,100 rpm for 45 minutes at 32°C with 0.8 μg / mL polybrominated chloride (Millipore, Bedford, Mass.). T cells were expanded in RPMI, 10% human serum, 2 mM L-glutamine, and 1% penicillin-streptomycin (CTL medium), and recombinant human (rh) IL-2 was supplemented every 48 hours until a final concentration of 50 U / mL was reached. Twelve days after amplification, aliquots of each transduced T cell line were stained with CD8-Pacific Blue, NKG2D-PE-Cy7, NKp44-PE, and 7-AAD (Biolegend) and then analyzed by flow cytometry. The results showed that the introduction of CNK elements significantly increased NKG2D expression in both the CD4 and CD8 populations. Furthermore, T cells transduced with a lentiviral vector encoding CNK+PE / NCR2-CD28-CD3Z simultaneously expressed NKG2D and NKp44. Interestingly, FSC / SSC flow cytometry indicated that the introduction of PE (UL16) improved cell viability in the culture medium. Figure 2B ).

[0318] Example 3. Protective elements (PE) can inhibit the expression of NKG2D ligands on CNK-T cells.

[0319] T cells were activated for 2 days using Dynabeads® Human T-Activator CD3 / CD28 (Thermo Scientific) (M:T=3:1), then transduced with a lentiviral vector encoding CNK and a protective element (UL 16), and expanded as described above. T cells were stained with NKG2D ligand antibody (Biolegend) or NKG2D-Fc recombinant protein (SGE Biotech) and then subjected to flow cytometry to detect the expression of NKG2D ligands (MICA, MICB, ULBP1-6) on days 2, 4, and 6 of culture. Untransduced T cells served as a control. The results showed that the protective element significantly inhibited the expression of NKG2D ligands on the surface of T cells during activation. Figure 3 ).

[0320] Example 4. Protective elements (PE) can increase CNK-T cell viability and proliferation.

[0321] T cells were activated for 2 days using Dynabeads® Human T-Activator CD3 / CD28 (Thermo Scientific), and then transduced with a lentiviral vector encoding a CNK element (CNK-T) and a CNK element containing a PE element (UL16) (CNK+PE), with untransduced T cells as a control. Cell viability and number were assessed using an automated cell counter (Countstar) on days 2, 4, 6, 8, 10, and 12. The results showed that the protective element can enhance cell viability and promote cell proliferation in the early stages of cell expansion. Including UL16 significantly improved CNK-T survival and proliferation. Figure 4 ).

[0322] Example 5. Preparation and characterization of NCR2 CNK-UT cells

[0323] NCR2 CNK-UT cells derived from healthy donor T cells were generated using lentivirus and CRISPR / Cas9 genome editing technology. In short, donor CD3+ T cells were activated with TransAct (Miltenyi Biotec) and cultured at 37°C and 5% CO2. The following day, lentiviral vectors encoding CNK+PE and NCR2 ED-CD28 TMD-CD28 ICD-CD3z ICD were thawed at room temperature from a -80°C freezer, added to a cell mixture with an MOI of 1, centrifuged at 1000×g for 60 minutes at 32°C, and then cultured overnight at 37°C and 5% CO2. NCR2 CNK-UT cells derived from healthy donor T cells were then generated using lentivirus and CRISPR / Cas9 genome editing technology. Cells were collected and washed twice with D-PBS (Thermofisher), then resuspended in opti-MEM (Thermofisher) at a density of 5–10 E7 / ml. Ribonuclear protein (RNP) complexes were prepared by incubating Cas9 protein and previously described sgRNA targeting TRAC, B2M, and CIITA (Yuki Kagoya et al., 2020) at room temperature for 15 minutes, followed by electroporation into cells using an X-Porator H1 (Etta Biotech). Immediately after electroporation, cells were cultured at 37°C and 5% CO2 for 7 days, and residual TCR+ cells were depleted using CliniMACS. Cells were analyzed by flow cytometry. The results showed that NKG2D was expressed on both CD8+ and CD4+ T cells, with significantly higher expression levels than control T cells, which only expressed NKG2D on CD8+ T cells. 91.7% NCR2 CNK-UT cells also expressed NCR2 (Nkp44). HLA class II, TCR, and b2M knockout efficiencies were quantified by flow cytometry. Prior to CliniMACS selection, 91.5% of cells were TCRαβ and CD3 negative. After CD3-negative selection, up to 99.3% of cells were TCRαβ and CD3 negative. Furthermore, 91.9% and 88.4% of cells were HLA-DR and HLA-abc negative, respectively. Figure 5 ).

[0324] Example 6. UT effectively eliminates activated T cells and IL2 does not protect activated T cells from NCR2 CNK-UT attack.

[0325] T cells were activated for 2 days using Dynabeads® Human T-Activator CD3 / CD28 (Thermo Scientific), and then co-cultured with NCR2 CNK-UT cells at an effector-to-target ratio of E:T = 3:1, with untransduced T cells as a control. After 72 hours of incubation, cells were collected and submitted for flow cytometry analysis. CRISPR-Cas9 gene editing to knock out TCR resulted in CD3 expression deficiency on NCR2 CNK-UT cells. Therefore, it was easy to distinguish NCR2 CNK-UT cells (TCR-, CD3-), control cells, and activated T cells (TCR+, CD3+). Flow cytometry showed that NCR2 CNK-UT cells effectively eliminated activated T cells, while control T cells did not. Figure 6 ).

[0326] Example 7. UT cells effectively eliminate activated T cells, but do not eliminate the remaining T cells.

[0327] To examine whether NCR2 CNK-UT exhibits cytotoxicity against resting T cells, activated T cells and NCR2 CNK-UT cells were co-cultured at a 3:1 E:T ratio for 48 h. Cells were then collected and subjected to flow cytometry. Results showed that in the NCR2 CNK-UT co-culture group with activated T cells, CD3(+)TCR(+) cells completely disappeared after 48 h of co-culture. Interestingly, in the NCR2 CNK-UT co-culture group with inactivated T cells, CD3(+)TCR(+) cells were still present after 48 h. This suggests that NCR2 CNK-UT can only effectively eliminate activated T cells, but has no effect on resting T cells. Figure 7 ).

[0328] Example 8. Jurkat cells express NKG2D ligand and NCR ligand.

[0329] Flow cytometry was used to detect the expression of NKG2D ligands (MICA, MICB, ULBP1-6) in the T-ALL cell line Jurkat using antibodies (Biolegend), NKG2D-Fc, and different NCR-Fc recombinant proteins (SGE Biotech). IgG isotypes (Biolegend) were used as controls. Results indicated that, except for ULBP-3 and ULBP-4, Jurkat cells highly expressed ULBP-1 and ULBP2, 5, and 6, and mildly expressed MICA and MICB. Both NKG2D-Fc and NKp44L effectively bound to Jurkat cells. Figure 8 ).

[0330] Example 9. NC R2 CNK-UT cells effectively kill Jurkat T cells

[0331] To investigate whether NCR2 CNK-UT cells exhibit cytotoxicity against Jurkat T cells, NCR2 CNK-UT cells were co-cultured with Jurkat T cells at E:T ratios of 1:2, 1:1, and 2:1 for 48 h, with control T cells serving as a control. NCR2 CNK-UT cells had their TCR knocked out, thus exhibiting a lack of CD3 expression compared to Jurkat T cells (CD3+, CD8-). After 48 h of co-culture, the co-cultured cells were collected for flow cytometry. The results showed that in the NCR2 CNK-UT co-culture group, CD3(+)CD8(-) cells almost disappeared after 48 h of co-culture (Figure 9).

[0332] Example 10. Mechanism of CNK-UT in treating T cell-mediated autoimmune diseases

[0333] Figure 10 A. Allogeneic T cells or autoreactive T cells recognize specific antigens presented on tissue cells by MHC molecules (such as HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR). During the T cell response, activated allogeneic or autoreactive T cells upregulate NKG2D ligands (such as MICA / B and ULBP1-6). Then, the allogeneic or autoreactive T cells attack and destroy the tissue cells. Figure 10 B. CNK-UT cells have been infused with CNK elements (NKG2D) and have had their TCRs knocked out, completely preventing TCR-mediated GVHD. During the T cell response, activated allogeneic T cells or autoreactive T cells upregulate NKG2D ligands that can be recognized by CNK elements in CNK-UT. CNK-UT can recognize and directly kill NKG2D-dependent activated allogeneic T cells or autoreactive T cells, protecting tissue cells from T cell-mediated autoimmunity.

[0334] Example 11. Schematic diagram of GVHD experimental scheme

[0335] Twenty mice were intraperitoneally injected with 30 mg / kg busulfan, defined as day 0; on days 1 and 2, 16 mice were each injected with 200 μL of PBMCs, 5E6 cells each time; on day 4, 8 mice were each intravenously injected with 1E7 NCR2 CNK-UT cells. GVHD symptoms were then assessed three times a week, and blood samples were collected weekly for flow cytometry analysis. Figure 11 ).

[0336] Example 12. NCR2 CNK-UT effectively eliminates allogeneic T leukocytes in GVHD mice.

[0337] TCR knockout in NCR2 CNK-UT results in CD3 deficiency in T cells, so UT cells are CD45(+)CD3(-), T cells in allogeneic PBMCs are CD45(+)CD3(+), while mouse peripheral blood cells are CD45(-)CD3(-). Results showed that the percentage of CD45(+)CD3(+) cells in PBLs significantly increased during the development of GVHD in mice. Three weeks after PBMC infusion in GVHD mice, allogeneic T cells expanded to ~50% of total PBLs. Interestingly, in GVHD mice treated with NCR2 CNK-UT, CD45(+)CD3(+) cells were completely eliminated by NCR2 CNK-UT cells. NCR2 CNK-UT reduced activated allogeneic T cells in PBLs to almost undetectable levels after 3 weeks of treatment, similar to control mice without PBMC infusion. In summary, NCR2 CNK-UT infusion can effectively inhibit the development of GVHD, suppress the proliferation of allogeneic human T cells, and ultimately eliminate all allogeneic T cells in GVHD mice. Figure 12A-12D ).

[0338] Example 13. NCR2 CNK-UT treatment can prevent the development of GVHD in mice.

[0339] We observed a characteristic pattern of physical symptoms of GvHD in untreated mice, including weight loss, skin desquamation, kyphosis, reduced activity, and diarrhea. Mice were weighed weekly and monitored for visible signs of GVHD, and scored as shown in Table 1. The activity and posture of NCR2 CNK-UT treated mice remained relatively normal post-transplantation. In untreated mice, the cumulative GVHD score steadily increased during the first 2–3 weeks post-transplantation. Interestingly, as... Figure 14 As shown, the GvHD scores of animals treated with NCR2 CNK-UT were significantly reduced.

[0340] Table 1. GVHD Scoring Criteria

[0341]

[0342] Example 14. NCR2 CNK-UT treatment prevents weight loss in GVHD mice.

[0343] Within 7–10 days after PBMC infusion, GVHD mice also began to lose weight. Compared to control mice, GVHD mice experienced weight loss of up to 20%, while mice treated with NCR2 CNK-UT began to gain weight like normal mice. Figure 15 ).

[0344] Example 15. NCR2 CNK-UT treatment significantly improved the survival rate of mice with GVHD.

[0345] Importantly, the symptoms in untreated mice worsened over time, with only 25% surviving 40 days after PBMC infusion. In contrast, the survival time of NCR2 CNK-UT-treated mice was as long as that of control T cells. Therefore, the results indicate that NCR2 CNK-UT effectively protects mice from GVHD and improves the survival rate of GVHD mice after PBMC infusion. The median survival time of untreated NKG2D mice was significantly shorter than that of NCR2 CNK-UT-treated mice. Figure 16 ).

[0346] Table 2. Sequences mentioned or used in this application Serial Number sequence annotation 1 MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKN NCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV Human NKG2D 2 MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASMFVVRVLAIALAIRFTLNTLMWLAIFKETFQPVLFNQEVQIPLTESYCGPCPKNWICY KNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV Chimeric human NKG2D with mouse NKG2D transmembrane ... 3 MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRG DAP10 4 MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK DAP12 5 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3ζeta ICD 6 EPKSCDKTHTCPPCP IgG1 hinge 7 ESKYGPPCPSCP IgG4 hinge 8 LVAADAVASLLIVGAVF DAP10 transmembrane domain 9 GVLAGIVMGDLVLTVLIALAV DAP12 transmembrane domain 10 GVLAGIVMGDLVLTVLIALAVLVAADAVASLLIVGAVF DAP10 - DAP12 transmembrane domain fusion 11 GSGEGRGSLLTCGDVEENPGP T2A 12 MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASP Human NKG2D intracellular domain 13 GGGGS Linker 14 IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV Human NKG2D extracellular domain 15 <![CDATA[MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRGGGGS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR ]]> DAP10 - CD3ZICD 16 MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRGGGGSYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK DAP10 - DAP12CD 17 MNNLWKAWVGLWTSMGPLIRLPDGITKAGEDALRPWKSTAKHPWFEIEDNRCYIDNGKLFARGSIVGNMSRFVFDPKADYGGVGENLYVHADDVEFVPGESLKWNVRNLDVMPIFETLALRLVLQGDVIWLRCVPELRVDYTSSAYMWNMQYGMVRKSYTHVAWTIVFYSINITLLVLFIVYVTVDCNLSMMWMRFFVC HCMVRUS2 18 MNLVMLILALWAPVAGSMPELSLTLFDEPPPLVETEPLPPLSDVSEYRVEYSEARCVLRSGGRLEALWTLRGNLSVPTPTPRVYYQTLEGYADRVPTPVEDVSESLVAKRYWLRDYRVPQRTKLVLFYFSPCHQCQTYYVECEPRCLVPWVPLWSSLEDIERLLFEDRRLMAYYALTIKSAQYTLMMVAVIQVFWGLYVKGWLHRHFPWMFSDQW HCMV US11 19 MGDTASVSEHHESPTVTIVPLHRSHALVAEQQLFQWLKRFKLLMEVYHGLVWQLACTLTVCLLAWLAFPDVQGQCANGIVPALSSIVPVSTLAMLRGFAEFRPHTTNFAHLTVACLLINTGITVCTGFCGERRVIGLSFALVMVFFVLCSGLTYLAGNNPTRWKVIGIGYGWSVIVFYLLLYFSPVLWVSKIYSGLYVLVVTAASAVLIYETLDLIYQRGTLSKNSVCVSVVLYTIVMSLLNMSVAIFSGHVWVQQYAEKHGGRIDGVSLLSLL HCMV US18 20 MQAQEANALLLSRMEALEWFKKFTVWLRVYAIFIFQLAFSFGLGSVFWLGFPQNRNFCVENYSFFLTVLVPIVCMFITYTLGNEHPSNATVLFIYLLANSLTAAIFQMCSESRVLVGSYVMTLALFISFTGLAFLGGRDRRRWKCISCVYVVMLLSFLTLALLSDADWLQKIVVTLCAFSISFFLGILAYDSLMVIFFCPPNQCIRHAVCLYLDSMAIFLTLLLMLSGPRWISLSDGAPLDNGTLTAASTTGKS HCMV US20 21 MKPVLVLAILAVLFLRLADSVPRPLDVVVSEIRSAHFRVEENQCWFHMGMLYFKGRMSGNFTEKHFVNVGIVSQSYMDRLQVSGEQYHHDERGAYFEWNIGGHPVTHTVDMVDITLSTRWGDPKKYAACVPQVRMDYSSQTINWYLQRSMRDDNWGLLFRTLLVYLFSLVVLVLLTVGVSARLRFI HCMV US3 22 MLRRGSLRNPLAICLLWWLGVVAAATEETREPTYFTCGCVIQNHVLKGAVKLYGQFPSPKTLRASAWLHDGENHERHRQPILVEGTATATEALYILLPTELSSPEGNRPRNYSATLTLASRDCYERFVCPVYDSGTPMGVLMNLTYLWYLGDYGAILKIYFGLFCGACVITRSLLLICGYYPPRE HCMV US10 23 MDLLIRLGFLLMCALPTPGERSSRDPKTLLSLSPRQQACVPRTKSHRPVCYNDTGDCTDADDSWKQLGEDFAHQCLQAAKKRPKTHKSRPNDRNLEGRLTCQRVRRLLPCDLDIHPSHRLLTLMNNCVCDGAVWNAFRLIERHGFFAVTLYLCCGITLLVVILALLCSITYESTGRGIRRCGS HCMV US6 24 MSWALEMADTFLDTMRVGPRTYADVRDEINKRGREDREAARTAVHDPERPLLRSPGLLPEIAPNASLGVAHRRTGGTVTDSPRNPVTR ICP47 25 MFIMRELVYRVVIVILSLSLLSSFLVICFIEHRCFKEETRYKICPKNTISINNICYYFSEDTTNWTASFEYCKNMNGRLACIINDTFPKICRYKGTFDYWVGLYRTNSQSPWLNCDNKPYNNIIPIRGVEDYAYLNNNGISTARIYADRRWICEVPMVDY CPXV12 26 MPRSPLIVAVVAAALFAIVRGRDPLLDAMRREGAMDFWSAGCYARGVPLSEPPQALVVFYVALTAVMVAVALYAYGLCFRLMGASGPNKKESRGRG BHV UL49.5 27 MVHVLERALLEQQSSACGLPGSSTETRPSHPCPEDPDVSRLRLLLAVLCVLFGLLCLLLI EBV BNFL2a 28 MERRRGTVPLGWVFFVLCLSASSSCAVDLGSKSSNSTCRLNVTELASIHPGETWTLHGMCISICYYENVTEDEIIGVAFTWQHNESVVDLWLYQNDTVIRNFSDITTNILQDGLKMRTVPVTKLYTSRMVTNLTVGRYDCLRCENGTTKIIERLYVRLGSLYPRPPGSGLAKHPSVSADEELSATLARDIVLVSAITLFFFLLALRIPQRLCQRLRIRLPHRYQRLRTED HCMV protein UL16 29 MCRRESLRTLPWLFWVLLSCPRLLEYSSSSFPFATADIAEKMWAENYETTSPAPVLVAEGEQVTIPCTVMTHSWPMVSIRARFCRSHDGSDELILDAVKGHRLMNGLQYRLPYATWNFSQLHLGQIFSLTFNVSTDTAGMYECVLRNYSHGLIMQRFVILTQLETLSRPDEPCCTPALGRYSLGDQIWSPTPWRLRNHDCGMYRGFQRNYFYIGRADAEDCWKPACPDEEPDRCWTVIQRYRLPGDCYRSQPHPPKFLPVTPAPPADIDTGMSPWATRGIAAFLGFWSIFTVCFLCYLCYLQCCGRWCPTPGRGRRGGEGYRRLPTYDSYPGVKKMKR HCMV protein UL141 30 MRIEWACWLFGYFVSSVGSERSLSYRYHLESNSSANVVCNGNISVFVNGTLGVRYNITVGISSSLLIGHLTIQTLESWFTPWVQNKSYSKQPLSTTETLYNIDSENIHRVSQYFHTRWIKSLQENHTCDLTNSTPTYTYQANVNNTNYLTLTSSGWQDRLNYTAINSTHFNLTESNITSIHKYLNTTCIERLRNYTLEPVYTTAVPQNVTPEHAITTLYTTPPNAITIKDTTQSHTVQTPSFNDTHNVTEHTLNISYVLSQKTNNTTSPWVYAIPMGATATIGAGLYIGKHFTPVKFVYEVWRGQ HCMV protein UL142 31 MGGKWSKSKTAGWPEVRERIRNAPSAAAPGVGAVSQDLAKHGAITSSNANHPSCVWLEAQEDEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSRRRQEILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEEVEKATEGENNSLLHPICQHGMDDEEGEVLKWQFDPRLALKHRAQELHPEFYKDC HIV Nef 32 QPIPIVAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVEMGVEMGHHAPWDVDDL HIV Vpu 33 MWTILLFCVPVIYGELYPDFCPLAVVDFDVNATVDDLLLFDISLSKQCSDDKIRHSAVAAMTDNAFFFGNSETQIETDFGKYLAFNCYQVFSTLNHFLFKNFKKTKGLMKRYDKLCLDVESYIHIQIICSPFKSFIRLRRMNETGISPRILETTFYLQNKRNSTWVAIKNYLGEDDPFTYRIWHTLTHAKNFLINSCENDFNQLFFWQRKYLSLAKTFEATFKQGFNPMIEQRNEQRYRTNNIDCSFSKFRQNGVKVAVCKYTGWGVSGFGSLEVLQKIKSPFGEEWKRVGFNSTGAFTPLYGSDVLWGLIFLRVEMTTYVCTCTNKNTGTQIQVTLPDVDLDLLDSEKTSSNVFVDMLCYTLIAILFLAFVTAVVLLGVSCLDGVQKVLTWPLQHIQKEPVSEKIINLTNLMFGQEPLPKKESLKQQCL HHV-7 U21 34 MEDEDVPVCWICNEELGNERFRACGCTGELENVHRSCLSTWLTISRNTACQICGVVYNTRVVWRPLREMTLLPRLTYQEGLELIVFIFIMTLGAAGLAAATWVWLYIVGGHDPEIDHVAAAAYYVFFVFYQLFVVFGLGAFFHMMRHVGRAYAAVNTRVEVFPYRPRPTSPECAVEEIELQEILPRGDNQDEEGPAGAAPGDQNGPAGAAPGDQDGPADGAPVHRDSEESVDEAAGYKEAGEPTHNDGRDDNVEPTAVGCDCNNLGAERYRATYCGGYVGAQSGDGAYSVSCHNKAGPSSLVDILPQGLPGGGYGSMGVIRKRSAVSSALMFH HHV-8 KK3 35 MASKDVEEGVEGPICWICREEVGNEGIHPCACTGELDVVHPQCLSTWLTVSRNTACQMCRVIYRTRTQWRSRLNLWPEMERQEIFELFLLMSVVVAGLVGVALCTWTLLVILTAPAGTFSPGAVLGFLCFFGFYQIFIVFAFGGICRVSGTVRALYAANNTRVTVLPYRRPRRPTANEDNIELTVLVGPAGGTDEEPTDESSEGDVASGDKERDGSSGDEPDGGPNDRAGLRGTARTDLCAPTKKPVRKNHPKNNG HHV-8 KK5 36 MDSTGEFCWICHQPEGPLKRFCGCKGSCAVSHQDCLRGWLETSRRQTCALCGTPYSMKWKTKPLREWTWGEEEVLAAMEACLPLVLIPLAVLMIVMGTWLLVNHNGFLSPRMQVVLVVIVLLAMIVFSASASYVMVEGPGCLDTCTAKNSTVTVNSIDEAIATQQPTKTDLGLARETLSTRFRRGKCRSCCRLGCVRLCCV MHV-68 MK3 37 MLFRLLSPLSPLALTALLLFLLPPSDVSGLLLRPPPAPCLLLFLPFQILSGLLFLLFLPLFFSLPLLLSPSLPITMRFPARWRFLPWKAPSQPAAAFLF HTLV-1 p12 38 MKIKLSIIRWLTLLALTIFRMRSLVIVLLFPSIIYSMVIRRCEKMEEETWKLKIGMCIQAKDFYSKRTDCSVHRSDVGGGLITEGNGYRVVVHDQCEEPNPFIIATTKQTHFGVTHSYIEFSNSNTGAPENIPDCSKHILISVYCDQEASGLDFHTLKYVESNYLHITVKYDTSCINHLGVNYSFMNECERKLKSIYETDTLTCGAKDTQTRDKYLKTCTNTKFDRSVYKTHMQKSKILHVKTEL CPXV203 39 MRYMILGLLALAAVCSAAKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGKYAVYAIWQPGDTNDYNVTVFQGENRKTFMYKFPFYEMCDITMYMSKQYKLWPPQKCLENTGTFCSTALLITALALVCTLLYLKYKSRRSFIDEKKMP E3-19K(HAdV-2) 40 MGAILVVLALLSLLGLGSANLNPLDHDPCLDFDPENCTLTFAPDTSRLCGVLIKCGWDCRSVEITHNNKTWNNTLSTTWEPGVPQWYTVSVRGPDGSIRISNNTFIFSEMCDLAMFMSRQYDLWPPSKENIVAFSIAYCLVTCIITAIICVCIHLLIVIRPRQSNEEKEKMP E3-19K(HAdV-3) 41 MIRYIILGLLTLASAHGTTQKVDFKEPACNVTFAAEANECTTLIKCTTEHEKLLIRHKNKIGKYAVYAIWQPGDTTEYNVTVFQGKSHKTFMYTFPFYEMCDITMYMSKQYKLWPPQNCVENTGTFCCTAMLITVLALVCTLLYIKYKSRRSFIEEKKMP E3-19K(HAdV-5) 42 MRYMILGLLALAAVCSAAKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGKYAVYAIWQPGDTNDYNVTVFQGENRKTFMYKFPFYEMCDITMYMSKQYKLWPPQKCLENTGTFCSTALLITALALVCTLLYLKYKSRRSFIDEKKMP E3-19K(HAdV-6) 43 MGPILVLLVLLSLLEPGSANYDPCLDFDPENCTLTFAPDTSRICGVLIKCGWECRSVEITHNNKTWNNTLSTTWEPGVPEWYTVSVRGPDGSIRISNNTFIFSEMCDLAMFMSKQYSLWPPSKDNIVTFSIAYCLCACLLTALLCVCIHLLVTTRIKNANNKEKMP E3-19K(HAdV-11) 44 MGPILVLLVLLSLLEPGSANYDPCLDFDPENCTLTFAPDTSRICGVLIKCGWECRSVEITHNNKTWNNTLSTTWEPGVPEWYTVSVRGPDGSIRISNNTFIFSEMCDLAMFMSKQYSLWPPSKDNIVTFSIAYCLCACLLTALLCVCIHLLVTTRIKNANNKEKMP E3-19K(HAdV-35) 45 MSSTLPALLCVGLCLSQRISAQQQTLPKPFIWAEPHFMVPKEKQVTICCQGNYGAVEYQLHFEGSLFAVDRPKPPERINKVKFYIPDMNSRMAGQYSCIYRVGELWSEPSNLLDLVVTEMYDTPTLSVHPGPEVISGEKVTFYCRLDTATSMFLLLKEGRSSHVQRGYGKVQAEFPLGPVTTAHRGTYRCFGSYNNHAWSFPSEPVKLLVTGDIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQNLLRMGLAFLVLVALVWFLVEDWLSRKRTRERASRASTWEGRRRLNTQTL NCR1(Nkp46) 46 MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIALVPVFCGLLVAKSLVLSALLVWWGDIWWKTMMELRSLDTQKATCHLQQVTDLPWTSVSSPVEREILYHTVARTKISDDDDEHTL NCR2(Nkp44) 47 MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPGG NCR3(NKp30) 48 <![CDATA[MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIAFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR ]]> NCR2 ED-CD28 TM-CD28 ICD-CD3Z ICD

Claims

1. A genetically modified mammalian immune cell or a population thereof, comprising surface molecules and protective elements, characterized in that, The surface molecules are expressed on the surface of immune cells and can bind to one or more target NK receptor ligands (tNKRL) expressed on target cells, thereby activating immune cells and producing cytotoxic effects on target cells. The immune cells, upon activation, can be induced to express one or more inducible NK receptor ligands (iNKRLs) that can bind to the surface molecules, and The protective element substantially inhibits the expression of one or more induced NK receptor ligands on the surface of the immune cells after the immune cells are activated, thereby preventing the activated immune cells from becoming target cells. Preferably, at least one of the target NK receptor ligands is the same as at least one of the inducible NK receptor ligands, or the surface molecule cross-reacts with at least one of the target NK receptor ligands and at least one of the inducible NK receptor ligands; Preferably, one or more of the target NK receptor ligands are selected from the group consisting of: NKG2D ligand, NCR ligand, KIR ligand, CD226 ligand, TIGIT ligand, CD96 ligand, or any combination thereof; More preferably, one or more of the target NK receptor ligands comprise one or more NKG2D ligands; More preferably, the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6.

2. The genetically modified mammalian immune cells as described in claim 1, characterized in that, Compared to the expression of one or more inducible NK receptor ligands on the surface of a reference immune cell without protective elements, the expression of one or more inducible NK receptor ligands on the surface of the immune cell is reduced by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Optionally, the expression of one or more induced NK receptor ligands on the surface of immune cells is reduced such that the binding of one or more induced NK receptor ligands to surface molecules is reduced by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%; Optionally, after the surface molecule binds to one or more target NK receptor ligands, it can be detected that the proportion of cells that induce NK receptor ligand expression in the immune cell population does not exceed 50%, for example, not more than 40%, not more than 30%, not more than 20%, or not more than 10%. Optionally, one or more inducible NK receptor ligands comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6, and the expression levels of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6 on the surface of immune cells are reduced by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%; Optionally, one or more inducible NK receptor ligands comprise MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5, and ULBP6, and the expression level of each of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP5, and ULBP6 on the surface of immune cells is reduced by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

3. The genetically modified mammalian immune cells as described in claim 1 or 2, characterized in that, The activation-induced cell death of the immune cells is substantially reduced, and optionally, the reduction in activation-induced cell death of immune cells can be determined by detecting the reduction in apoptosis using Annexin V / 7-AAD. Optionally, the immune cells are capable of proliferation upon activation, for example, with an increased proliferation rate compared to modified mammalian immune cells without protective elements.

4. The genetically modified mammalian immune cells as described in any one of claims 1 to 3, characterized in that, The protective element can silence the expression of one or more induced NK receptor ligands, or can induce intracellular retention and / or degradation of one or more induced NK receptor ligands; Optionally, the protective element comprises a small RNA, such as siRNA, miRNA, or shRNA, that targets one or more mRNAs that induce NK receptor ligands. Optionally, the protective element comprises a small RNA, such as HCMV-encoded microRNAs (miR-20a, miR-UL112), capable of regulating the expression of the NKG2D ligand.

5. The genetically modified mammalian immune cells as described in claim 4, characterized in that, The protective element includes one or more protein degradation elements capable of inducing intracellular retention and / or degradation of one or more NK receptor ligands; Optionally, at least one or all of the protein degradation elements comprise an endoplasmic reticulum (ER) retention domain and an iNKRL binding domain capable of binding one or more induced NK receptor ligands; Optionally, the protein degradation element further includes an ER retention transmembrane domain, one end of which is operatively connected to the iNKRL binding domain and the other end of which is operatively connected to the ER retention domain. Optionally, the ER retention domain and / or the ER retention transmembrane domain are derived from ER-retaining viral proteins; Optionally, the ER-retaining viral proteins are selected from the group consisting of: HCMV protein US2, HCMV protein US11, HCMV protein US18, HCMV protein US20, HCMV protein US3, HCMV protein US10, HCMV protein US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV protein UL16, HCMV protein UL141, HCMV protein UL142, HIVNef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, vaccinia virus protein CPXV203, and Ad E3 / 19K; Optionally, the ER retention domain and the ER retention transmembrane domain both originate from the same ER-retaining viral protein; Optionally, the iNKRL binding domain includes an antibody domain capable of binding one or more inducible NK receptor ligands, such as scFv; Optionally, the iNKRL binding domain is derived from a viral protein capable of binding one or more induced NK receptor ligands; Optionally, the one or more induced NK receptor ligands comprise one or more NKG2D ligands; Optionally, the one or more NKG2D ligands are selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. Optionally, the viral protein capable of binding one or more NK receptor ligands is derived from a virus selected from the group consisting of adenovirus (Ad), human Ad, cytomegalovirus (CMV), human CMV (i.e., HCMV), and Kaposi's sarcoma-associated herpesvirus (KSHV). Optionally, the viral protein capable of binding one or more NK receptor ligands is derived from a viral protein selected from the group consisting of Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, and KSHV K5. Optionally, the ER retention domain and the binding domain are both derived from the same viral protein; Optionally, the ER retention domain, the ER retention transmembrane domain, and the binding domain are all derived from different viral proteins; Optionally, the same viral protein is selected from the group consisting of Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20 and KSHV K5; Optionally, the protein degradation element comprises viral proteins selected from the group consisting of: for example, Ad2 E3 / 19K, Ad5 E3 / 19K, HCMV protein UL16, HCMV protein UL142, HCMV protein US18, HCMV protein US20, KSHV K5, or analogues thereof, or derivatives thereof. Optionally, the one or more protein degradation elements comprise a combination of viral proteins, the combination of viral proteins comprising HCMV protein UL16 and another viral protein selected from the group consisting of: Ad E3 / 19K (e.g., Ad2 E3 / 19K, Ad5 E3 / 19K), HCMV protein UL142, HCMV protein US18, HCMV protein US20 and KSHV K5.

6. The genetically modified mammalian immune cells as described in claim 5, characterized in that, The one or more protein degradation elements further include a protein degradation pathway (PDP) binding domain capable of binding members of a protein degradation pathway; Optionally, the PDP binding domain is operatively connected to the ER retention domain; Optionally, the protein degradation pathway is a ubiquitination-proteasome pathway, an endosome-lysosome pathway, or an autophagy degradation pathway; Optionally, the members of the ubiquitination-proteasome pathway include E1 ubiquitin activator, E2 ubiquitin conjugate, or E3 ubiquitin ligase. Optionally, the members of the endosome-lysosome pathway include AP-1, AP-2, AP-3, endosome, lysosome, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sorting linker protein, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, or C3G / CrkL / Shp2 / Gab2; Optionally, the members of the autophagy degradation pathway include molecular chaperone-mediated autophagy (CMA) USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1 / 2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, or NUFIP1.

7. The genetically modified mammalian immune cells as described in any one of claims 1 to 6, characterized in that, The surface molecule comprises: a) a tNKRL-binding extracellular domain capable of binding one or more target NK receptor ligands, and b) a surface molecule (SM) intracellular domain capable of activating the immune cell after the tNKRL-binding extracellular domain binds to one or more of the target NK receptor ligands, wherein the tNKRL-binding extracellular domain is associated with the SM intracellular domain to allow binding signals to be transduced from the tNKRL-binding extracellular domain to the SM intracellular domain; Optionally, the tNKRL binding extracellular domain includes an antibody domain capable of binding one or more target NK receptor ligands; Optionally, the tNKRL binding extracellular domain includes an NK receptor extracellular domain capable of binding one or more target NK receptor ligands. Optionally, the tNKRL binding extracellular domain includes the NKG2D extracellular domain; Optionally, the receptor further comprises an SM transmembrane domain, one end of which is operatively connected to the tNKRL-binding extracellular domain and the other end of which is operatively connected to the SM intracellular domain. Optionally, the SM transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: CD3, CD4, CD8, CD28, B7, ICOS, CD226, 41-BB, OX40, CD27, GITR, HVEM, CD40, BAFFR, BAFF, NKG2D, NKG2C, NKG2A, NKp44, NKp46, and NKp30.

8. The genetically modified mammalian immune cells as described in any one of claims 1 to 7, characterized in that, The surface molecules contain tNKRL-binding chimeric antigen receptors (CARs). Optionally, the tNKRL-binding chimeric antigen receptor (CAR) comprises a CNK receptor protein complex comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a tNKRL-binding extracellular domain operatively linked to a first CNK transmembrane domain, and the second polypeptide comprising a CNK intracellular domain operatively linked to a second CNK transmembrane domain, the first CNK transmembrane domain and the second CNK transmembrane domain being associated with each other to form a protein complex. Optionally, the first CNK transmembrane domain includes an NK receptor transmembrane domain, optionally an NKG2D transmembrane domain; Optionally, the first transmembrane domain is capable of forming a first dimer; Optionally, the first polypeptide comprises NKG2D protein; Optionally, the NKG2D protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; Optionally, the second transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a 10 kDa DNAX-activating protein (DAP10) and a 12 kDa DNAX-activating protein (DAP12); Optionally, the CNK intracellular domain comprises a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR; Optionally, the CNK intracellular domain includes a cytoplasmic domain of CD3 (e.g., CD3ζ); Optionally, the CNK intracellular domain comprises the amino acid sequence of SEQ ID NO:

5.

9. The genetically modified mammalian immune cells as described in claim 8, characterized in that, The second polypeptide comprises a chimeric adaptor protein, said chimeric adaptor protein comprising: a) The transmembrane domain of DAP10, which is operatively linked to the intracellular domain of CD3 (e.g., CD3 zeta); b) The transmembrane domain of DAP12, which is operatively linked to the intracellular domain of CD3 (e.g., CD3 zeta); or c) The transmembrane domain of DAP10, which is operatively linked to the intracellular domain of DAP12; Optionally, the second polypeptide comprises a full-length DAP10 operatively linked to an intracellular domain of the CD3zeta. Optionally, the chimeric adaptor protein comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16; Optionally, the second CNK transmembrane domain can form a second dimer, and the second dimer is associated with the first CNK transmembrane domain to form a complex; Optionally, the CNK receptor comprises a hexamethylenetetrameric protein complex comprising a homodimer of the NKG2D protein and two homodimers of the chimeric adaptor protein.

10. Genetically modified mammalian immune cells as described in any one of the preceding claims, characterized in that, The immune cells were further modified to lack a) MHC class I proteins, or b) MHC class II proteins or MHC class II transcription activators, or c) both a) and b); Optionally, the immune cells are T cells and the immune cells are further modified to lack endogenous TCRs.

11. The genetically modified mammalian immune cells as described in any one of claims 1 to 10, characterized in that, Further expression of the target-binding TB receptor capable of binding to the target marker; Optionally, the target marker is expressed in target cells; Optionally, the TB receptor includes a target-binding extracellular domain capable of binding a target marker and a TB intracellular domain capable of activating immune cells after the target-binding extracellular domain binds to the target marker. Optionally, the TB receptor further comprises a TB transmembrane domain; Optionally, one end of the TB transmembrane domain is operatively connected to the target marker binding extracellular domain, and the other end is operatively connected to the TB intracellular domain; Optionally, the target marker binding extracellular domain comprises the extracellular domain of a natural cytotoxic receptor (e.g., NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1)). Optionally, the TB transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: CD3, CD4, CD8, CD28, B7, ICOS, CD226, 41-BB, OX40, CD27, GITR, HVEM, CD40, BAFFR, BAFF, NKG2D, NKG2C, NKG2A, NKp44, NKp46, NKp30; Optionally, the intracellular domain of the TB includes a cytoplasmic domain of proteins selected from the group consisting of: 4-1BB (CD137), CD2, CD3, CD35, CD25, CD27, CD28, CD30, CD40, CD79A, CD79B, CARD11, DAP10, DAP12, Fc receptor, Fyn, LIGHT, LTβR, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRα, vTCRP, TRIM, Zap70, PTCH2, IL7 receptor, IL15 receptor, and GITR; Optionally, the TB intracellular domain includes a CD28 cytoplasmic domain operatively linked to the CD3 zeta chain; Optionally, the target marker is a tumor antigen, an autoreactive cell antigen or a viral antigen, a bacterial antigen, a microbially infected cell, or a damaged and / or aging cell; Optionally, the target cells are tumor cells, autoreactive cells, senescent cells, or virus-infected cells.

12. The genetically modified mammalian immune cells as described in any one of the preceding claims, characterized in that, The immune cells mentioned are T cells, natural killer (NK) cells, NKT cells, B cells, hematopoietic cells (e.g., bone marrow cells), thymocytes, dendritic cells (e.g., mature dendritic cells), macrophages, tumor-infiltrating lymphocytes, monocytes, and granulocytes. Optionally, the immune cells are T cells, such as CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, or tumor-infiltrating lymphocytes.

13. A pharmaceutical composition, characterized in that, Mammalian immune cells or populations thereof comprising the genetically modified forms of any one of claims 1 to 12.

14. A method for treating a disease in a subject in need, characterized in that, The pharmaceutical composition of claim 13 comprising, when administered to the subject, a therapeutically effective amount; Optionally, the disease is cancer, a disease related to self-reactive lymphocytes (such as an autoimmune disease), an immunodeficiency disease, or an age-related disease; Optionally, the autoimmune disease is an organ-specific autoimmune disease or a systemic autoimmune disease; Optionally, the organ-specific autoimmune disease is selected from the group consisting of chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis. Optionally, the systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis. Optionally, the autoimmune disease is graft-versus-host disease (GvHD).

15. An expression construct encoding surface molecules and protective elements, characterized in that, The construct can be expressed in immune cells, the surface molecule is defined as in any one of claims 1 to 12, and the protective element is defined as in any one of claims 1 to 12.

16. A method for producing genetically modified mammalian immune cells according to any one of claims 1 to 12, characterized in that, The expression construct of claim 15 is introduced into the initiating immune cell, provided that surface molecules are allowed to be expressed on the surface of immune cells and protective elements are allowed to be expressed in immune cells.

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