Intracellular signaling and co-stimulatory domains adapted to long-term expression of chimeric antigen receptors

Abstract

Provided herein are proteins containing CD3 zeta intracellular domains having improved properties, such as BCMA-specific chimeric antigen receptors (CARs). The use of CD3 zeta intracellular domain-containing proteins such as CARs in immune cells (e.g., T cells), compositions (e.g., CARs and nucleic acid constructs encoding them), and methods is also intended.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application U.S.SN 63 / 491,038, filed on 17 March 2023 pursuant to 35 U.S.C § 119(e), the entire contents of which are incorporated herein by reference.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (C154070004WO00-SEQ-AZW.xml, size: 137,935 bytes, created: March 15, 2024) are incorporated in their entirety hereby by reference. [Background technology]

[0003] background Chimeric antigen receptors (CARs) are synthetic transmembrane proteins that include an extracellular antigen recognition domain (e.g., antibody single-chain variable fragment), a transmembrane domain, and an intracellular signaling domain (e.g., T cell signaling domain, e.g., CD3 zeta). When a CAR is expressed artificially in or by a first cell (e.g., a T cell), the CAR instructs the first cell to kill a second cell, such as a cancer cell, where the second cell expresses a surface antigen that is intentionally recognized by the CAR's extracellular antigen recognition domain. Cells modified to express CARs, such as T cells (CAR T cells, etc.), can be administered to patients to kill tumor cells or other pathogenic cells. To this end, CARs have been developed that have extracellular antigen recognition domains that specifically bind to surface antigens (markers) such as CD19, BCMA, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, and CD30. CAR-expressing cells, such as CAR T cells, are being developed for the treatment of hematological malignancies, solid tumors, and non-cancerous diseases such as autoimmune diseases. See, for example, Alnefaie et al., “Chimeric Antigen Receptor T-Cells: an overview of concepts, applications, limitations, and proposed solutions” Front. Bioeng. Biotechnol. 2022; 10:797440 (doi 10.3389); and U.S. Patent No. 10,934,337; each of these is incorporated herein by reference. [Disclosure of the Invention]

[0004] overview Nevertheless, efforts to achieve optimal CAR expression in target cells, such as T cells, have been hampered by undesirable downregulation or internalization of CARs after binding to the corresponding antigen. See, for example, Caruso et al. (Cancer Res. 2015;75:3505); Davenport et al. (Immunol. Res. 2015;3:483); Walker et al. (Mol. Ther. 2017;25:2189); Hamieh et al. (Nature 2019;568:7750); and Li (Immunity 2020;53:456). This phenomenon can reduce the durability and benefits of CAR-based therapies. Therefore, there is a need for novel CAR molecules that are resistant to, or less resistant to, downregulation or internalization after binding to the corresponding antigen.

[0005] This disclosure is based on the recognition that certain novel modifications (i.e., a series of amino acid substitutions) to the intracellular domain of CAR, CD3 zeta (the terms “CD3 zeta,” “CD3-Z,” and “CD3-ζ” are used interchangeably herein) confer to CAR: (1) a remarkable degree of CAR expression despite (e.g., after) exposure to its corresponding antigen (e.g., its intended target); (2) superior target cytotoxicity; and / or (3) superior cytokine (e.g., interferon-γ) secretion. The aforementioned superior experimental performance of the CAR proteins of this disclosure was compared to otherwise identical CAR proteins containing the wild-type CD3 zeta intracellular domain (e.g., SEQ ID NO: 18), where exposure of the CAR to its corresponding antigen results in a significant decrease in CAR expression. It has also been observed that some of the amino acid substitutions of this disclosure confer superior CAR expression even before exposure of the CAR to its corresponding antigen. This, too, was determined by experimental comparison with otherwise identical CAR proteins containing the wild-type intracellular CD3 zeta intracellular domain.

[0006] Therefore, in one aspect, what is provided herein is a protein comprising an intracellular domain capable of intracellular signal transduction, wherein the intracellular domain comprises an intracellular signal transduction domain, a co-stimulatory domain, or both, and wherein at least two lysine amino acids of the intracellular domain are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0007] In some embodiments, the intracellular domain includes a CD3 zeta domain. In some embodiments, the intracellular domain includes a domain that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NOs. 18, 61, or 66. In some embodiments, the CD3 zeta intracellular domain is 100% identical to SEQ ID NOs. 18, 61, or 66, except for substituted lysine amino acids.

[0008] In some embodiments, at least three lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0009] In a given environment, at least 4, at least 5, at least 6, at least 7, or at least 8 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0010] In some embodiments, at least nine lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0011] In one embodiment, at least 6, at least 7, at least 8, or at least 9 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0012] In another aspect of this disclosure, at least two lysine amino acids of the CD3 zeta intracellular domain are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0013] In one embodiment, the intracellular domain includes a domain that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NOs. 18, 61, or 66.

[0014] In some embodiments, the CD3 zeta intracellular domain is 100% identical to SEQ ID NOs. 18, 61, or 66, except for the substituted lysine amino acids.

[0015] In one embodiment, at least three lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0016] In some embodiments, at least 4, at least 5, at least 6, at least 7, or at least 8 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0017] In certain embodiments, at least nine lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0018] In some embodiments, at least six, at least seven, at least eight, or at least nine lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0019] In certain embodiments, the CD3 zeta intracellular domain is a domain of a protein.

[0020] In some embodiments, the CD3 zeta intracellular domain is a domain of a transmembrane protein.

[0021] In some embodiments, the CD3 zeta intracellular domain is a domain of an intercellular signaling protein.

[0022] In certain embodiments, the CD3 zeta intracellular domain is a domain of a CAR.

[0023] In one aspect, provided herein is a protein comprising the CD3 zeta intracellular domain of the present disclosure.

[0024] In another aspect of the present disclosure, provided herein is a protein comprising a CD3 zeta intracellular domain, wherein the CD3 zeta intracellular domain is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 18, and wherein at least two lysine amino acids of SEQ ID NO: 18 are either (a) deleted or (b) substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0025] In some embodiments, the CD3 zeta intracellular domain is 100% identical to SEQ ID NO: 18, except for deleted or substituted lysine amino acids.

[0026] In one embodiment, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids in SEQ ID NO: 18 are (a) deleted or (b) substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0027] In some embodiments, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids in SEQ ID NO: 18 are substituted with alanine.

[0028] In one embodiment, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids in SEQ ID NO: 18 are substituted with aspartic acid.

[0029] In some embodiments, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids in SEQ ID NO: 18 are substituted with glutamic acid.

[0030] In another respect, what is provided is a protein containing an amino acid sequence that is at least 80% identical to one of sequence numbers 2-7, 19-66, and 68-86.

[0031] In some embodiments, the protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to one of sequence numbers 2-7, 19-66, and 68-86.

[0032] In one aspect, proteins are chimeric antigen receptors (CARs).

[0033] In some embodiments, the protein further includes a co-stimulatory domain.

[0034] In one embodiment, the co-stimulatory domain is selected from the group consisting of the CD8 alpha domain, 41BB domain, CD28 domain, FcR gamma domain, CD27 domain, OX40 domain, CD30 domain, CD40 domain, PD-1 domain, ICOS domain, LFA-1 domain, CD2 domain, CD7 domain, LIGHT domain, NKG2C domain, and B7 H3 domain, and any variant thereof.

[0035] In some embodiments, the co-stimulatory domain is CD28.

[0036] In some embodiments, the co-stimulatory domain is 41BB.

[0037] In one embodiment, at least one lysine amino acid of the co-stimulatory domain is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0038] In some embodiments, at least two lysine amino acids in the co-stimulatory domain are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0039] In one aspect, the protein further includes an extracellular antigen-binding domain.

[0040] In some embodiments, the extracellular antigen-binding domain binds to CD19, BCMA, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, or CD30.

[0041] In one aspect, the extracellular antigen-binding domain binds to BCMA.

[0042] In some embodiments, the extracellular antigen-binding domain binds to CD19.

[0043] In one embodiment, the extracellular antigen-binding domain is scFv.

[0044] In some embodiments, the protein further includes a transmembrane domain.

[0045] In one embodiment, the transmembrane domain includes: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, 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, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, 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 The transmembrane domain of a receptor that specifically binds to (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, or CD83.

[0046] In one embodiment, the protein further includes a hinge region.

[0047] In some embodiments, the protein of this disclosure further comprises a leader domain.

[0048] In one aspect, the protein further includes one or more spacer sequences between one or more domains.

[0049] In one aspect, the spacer array is a polypeptide linker.

[0050] In another aspect, this disclosure provides nucleic acid constructs encoding the proteins disclosed herein.

[0051] In some embodiments, the nucleic acid construct is RNA.

[0052] In one aspect, the nucleic acid construct is DNA.

[0053] In one aspect, a vector encoding a protein described herein is provided herein.

[0054] In one embodiment, the vector includes a nucleic acid construct as disclosed herein.

[0055] In some forms, the vector is a viral vector.

[0056] In another aspect, compositions comprising proteins disclosed herein, nucleic acid constructs described herein, or vectors described herein are provided herein.

[0057] In another aspect of this disclosure, pharmaceutical compositions comprising a protein composition, a nucleic acid construct, or a vector as described herein are provided herein.

[0058] In one embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0059] In one aspect, cells containing the protein of this disclosure are provided herein.

[0060] In another aspect, this disclosure provides cells comprising nucleic acid constructs or vectors described herein.

[0061] In some embodiments, the cells are human cells.

[0062] In one sense, the cells are immune cells.

[0063] In some embodiments, the cells are T cells, CD3+ cells, CD8+ cells, CD4+ cells, NK cells, stem cells, hematopoietic stem cells, or mesenchymal stem cells.

[0064] In one aspect, what is provided is a method for producing a cell therapy for treating a disease, the method comprising transfecting a plurality of cells with a vector described herein.

[0065] Provided in another aspect of this disclosure is a method for treating a disease in a subject that requires such treatment, the method comprising administering to the subject the cells described herein.

[0066] In some embodiments, the cells are human cells.

[0067] In one sense, the cells are immune cells.

[0068] In some aspects, the cells are T cells.

[0069] In one aspect, the cells are CD3+ cells.

[0070] In some embodiments, the cells are CD8+ cells.

[0071] In one embodiment, the cells are CD4+ cells.

[0072] In one aspect, the cells are NK cells.

[0073] In one aspect, cells are stem cells.

[0074] In some embodiments, stem cells are hematopoietic stem cells.

[0075] In some embodiments, the stem cells are mesenchymal stem cells.

[0076] In one embodiment, the method further includes cytokines.

[0077] In some embodiments, the disease is cancer, an autoimmune disease, or an allergic disease.

[0078] In one aspect, the disease is myeloma.

[0079] In some aspects, the disease is myeloma.

[0080] In one aspect, the disease is myasthenia gravis (MG).

[0081] In some embodiments, the method is characterized by increased intracellular secretion of cytokines.

[0082] In one embodiment, the secreted cytokine is interferon-gamma.

[0083] In some embodiments, the method is characterized by the selective killing of cancer cells.

[0084] In some embodiments, the method is characterized by the selective killing of immune cells.

[0085] In one embodiment, the method is characterized by the selective killing of BCMA+ or CD19+ cells.

[0086] In one aspect, what is provided is the use of the proteins disclosed herein, the nucleic acid constructs disclosed herein, the viral vectors disclosed herein, or the cells disclosed herein, for treating cancer.

[0087] In another aspect, a kit for treating cancer is provided, comprising one or more of the proteins disclosed herein, nucleic acid constructs provided herein, viral vectors described herein, or cells disclosed herein. [Brief explanation of the drawing]

[0088] [Figure 1A] Figure 1A shows the results of measuring CAR expression in CAR T cells generated using wild-type CD3 zeta intracellular domain sequences (SEQ ID NO: 13) and lysine mutant constructs (SEQ ID NOs: 14-17) in the absence of the target ligand (left) and in the presence of the target ligand (right), as measured by median fluorescence intensity (MFI).

[0089] [Figure 1B] Figure 1B shows the expression of interferon-γ in the supernatant of co-cultures of CAR T cells generated using lysine mutant constructs (SEQ ID NOs. 14-17) or wild-type CAR T cells (SEQ ID NOs. 13) with BCMA+ MM1S multiple myeloma, as analyzed by specific ELISA. Prior to co-culture, CAR T cells were either pre-exposed to BCMA+ cells (gray) or not (black).

[0090] [Figure 1C] Figure 1C shows the cytotoxicity of pre-exposed CAR T cells, generated using constructs encoding lysine mutant CARs (SEQ ID NOs. 14-17) and wild-type CARs (SEQ ID NOs. 13), against MM1S-GFP target cells expressing BCMA ligands. Cytotoxicity was evaluated by co-culture at various effector:target ratios.

[0091] [Figure 2A] Figure 2A shows the results of measuring CAR expression in CAR T cells generated using wild-type constructs (SEQ ID NOs: 18 and 61) and mutant constructs (SEQ ID NOs: 19-27 and 43) in the absence of the target ligand (left) and in the presence of the target ligand (right), as measured by median fluorescence intensity (MFI).

[0092] [Figure 2B] Figure 2B shows the expression of interferon-γ in CAR T cells generated using various test constructs (SEQ ID NOs: 19-27 and 43) and wild-type CAR T cells (SEQ ID NOs: 18 and 61) in co-culture with MM1S target cells.

[0093] [Figure 2C] Figure 2C shows the results of measuring CAR expression as median fluorescence intensity (MFI) in CAR T cells generated using wild-type constructs (SEQ ID NOs. 18 and 61) and mutant constructs (SEQ ID NOs. 34-43), both in the absence of the target ligand (left) and in the presence of the target ligand (right).

[0094] [Figure 2D] Figure 2D shows the expression of interferon-γ in CAR T cells generated using various test constructs (SEQ ID NOs: 34-43) and wild-type CAR T cells (SEQ ID NOs: 18 and 61) in co-culture with MM1S target cells.

[0095] [Figure 2E] Figure 2E shows the results of measuring CAR expression as median fluorescence intensity (MFI) in the absence of the target ligand (left) and in the presence of the target ligand (right) for CAR T cells generated using wild-type constructs (SEQ ID NOs. 18 and 61) and mutant constructs (SEQ ID NOs. 28-33 and 43).

[0096] [Figure 2F] Figure 2F shows the expression of interferon-γ in CAR T cells and wild-type CAR T cells (SEQ ID NOs. 18 and 61) generated using various test constructs (SEQ ID NOs. 28-33 and 43) when co-cultured with MM1S target cells.

[0097] [Figure 3A]Figure 3A shows the median fluorescence intensity (MFI) measurements of CAR expression in CAR T cells expressing various test constructs (SEQ ID NOs. 43-56) and CAR T cells expressing wild-type constructs (SEQ ID NOs. 18 and 61), both in the absence of BCMA ligand (left) and in the presence of BCMA ligand (right).

[0098] [Figure 3B] Figure 3B shows the expression of interferon-γ in CAR T cells generated using various test constructs (SEQ ID NOs: 43-56) and wild-type CAR T cells (SEQ ID NOs: 18 and 61) in co-culture with MM1S target cells.

[0099] [Figure 4A] Figure 4A shows the median fluorescence intensity (MFI) measurements of CAR T cells generated using various test constructs (SEQ ID NOs. 43 and 57-60) and CAR T cells generated using the wild-type construct (SEQ ID NO. 18), both in the absence of BCMA ligand (left) and in the presence of the ligand (right).

[0100] [Figure 4B] Figure 4B shows the expression of interferon-γ in CAR T cells generated using various test constructs (SEQ ID NOs. 43 and 57-60) and the wild-type construct (SEQ ID NO: 18) upon exposure to MM1S target cells.

[0101] [Figure 4C] Figure 4C shows the cytotoxicity of CAR T cells generated using separate constructs containing wild-type CD3 zeta (SEQ ID NO: 61), mutant CD3 zeta only, CD28-CD3 zeta, and 41BB-CD3 zeta signaling domains (SEQ ID NOs: 43, 57, and 59, respectively). Cytotoxicity was assessed by pre-exposing cells to MM1S and then performing cytotoxicity assays against BCMA+ MM1S-GFP cells at various effector:target ratios for 72 hours.

[0102] [Figure 5A] Figure 5A shows the median fluorescence intensity (MFI) measurements of CAR expression in CAR T cells expressing variations of anti-BCMA CAR protein (SEQ ID NOs. 43, 56-58, 62-65) and CAR T cells expressing the wild-type construct (SEQ ID NOs. 18), both in the absence of BCMA ligand (left) and in the presence of ligand (right).

[0103] [Figure 5B] Figure 5B shows the cytotoxicity of CAR T cells generated using various constructs (SEQ ID NOs: 18, 43, 57, 58, and 62). Cytotoxicity was assessed by pre-exposing cells to MM1S and then performing cytotoxicity assays against BCMA+ MM1S-GFP cells at various effector:target ratios for 72 hours.

[0104] [Figure 5C] Figure 5C shows the cytotoxicity of CAR T cells generated using various constructs (SEQ ID NOs. 56 and 63-65). Cytotoxicity was assessed by pre-exposing cells to MM1S and then performing cytotoxicity assays against BCMA+ MM1S-GFP cells at various effector:target ratios for 72 hours.

[0105] [Figure 6A] Figures 6A-6B show flow cytometry evaluations of BCMA CAR T (SEQ ID NO: 43) or control CD8+ T cells without CAR, using the activation induction markers CD69 and CD137 (41BB). Activation of BCMA CAR T (SEQ ID NO: 43) and control CD8+ T cells was evaluated as a response to co-culture with plasmablasts (Figure 6A) and plasmacytoid dendritic cells (Figure 6B). [Figure 6B]Figures 6A-6B show flow cytometry evaluations of BCMA CAR T (SEQ ID NO: 43) or control CD8+ T cells without CAR, using the activation induction markers CD69 and CD137 (41BB). Activation of BCMA CAR T (SEQ ID NO: 43) and control CD8+ T cells was evaluated as a response to co-culture with plasmablasts (Figure 6A) and plasmacytoid dendritic cells (Figure 6B).

[0106] [Figure 7A] Figures 7A-7B show the bioluminescence of mice carrying MM1S-fluc myeloma and treated with control CD8+ T cells or CAR T cells expressing CARs with sequence numbers 5 and 68. Figure 7A shows the bioluminescence measurements of mice on day 9 and day 12. [Figure 7B] Figure 7B shows summary statistics for each group of mice at a given time point. Arrows indicate the day of CAR T cell administration. Mean ± standard deviation for n=4.

[0107] [Figure 8A] Figures 8A-8B show the bioluminescence (total flux) of mice harboring MM1S-fluc myeloma, treated with control CD8+ T cells or CAR T cells containing anti-BCMA CARs with KE mutant intracellular CD28-CD3 zeta (SEQ ID NO: 65) or wild-type intracellular CD28-CD3 zeta (SEQ ID NO: 66) signaling domains. Figure 8A shows summary statistics of bioluminescence (photons / second) of mice treated with 12.5 million CAR T cells or control cells. Arrows indicate the day of CAR T cell administration. [Figure 8B] Figure 8B shows bioluminescence data from individual mice at day 21 in groups treated with 2 million or 12.5 million CAR T cells generated using anti-BCMA CARs containing KE mutant intracellular CD28-CD3 zeta (SEQ ID NO: 65) or wild-type intracellular CD28-CD3 zeta (SEQ ID NO: 66) signaling domains. Mean ± standard deviation of n=4.

[0108] [Figure 8C]Figure 8C shows flow cytometry evaluation of whole blood and bone marrow from mice administered with CAR T cells generated using the IVT mRNA construct SEQ ID NO: 68, which encodes the signaling domain of SEQ ID NO: 65. Cells were stained with antibodies against human CD45, CD3, and recombinant BCMA-APC, and CAR expression was evaluated by flow cytometry. CD3 and CAR expression in CD45-gated cells are shown.

[0109] [Figure 9A] Figure 9A shows the cytotoxicity of anti-BCMA CAR T cells from two myasthenia gravis (MG) donors, prepared using the CAR construct of SEQ ID NO: 68 and SEQ ID NO: 94 (anti-PSMA CAR as a negative control), against autologous plasma cells differentiated from MG patients.

[0110] [Figure 9B] Figure 9B shows the differences between donors in interferon-γ cytokine production after cytotoxicity.

[0111] [Figure 9C] Figure 9C shows the functionality of MG donor CAR T cells as evaluated using a malignant MM1S-GFP cell line.

[0112] [Figure 9D] Figure 9D shows the interferon-γ cytokine production of MG donor CAR T cells, as evaluated using a malignant MM1S-GFP cell line.

[0113] [Figure 10A] Figure 10A shows the apparent affinity of anti-BCMA CAR to soluble BCMA.

[0114] [Figure 10B]Figure 10B shows the expression of anti-CD19 CAR by CAR T cells expressing CARs containing the CD28-CD3 zeta intracellular domain, which have wild-type sequences or mutant sequences containing glutamate-mutated lysine, after culture in the absence or presence of BCMA+ MM1S cells.

[0115] definition When used herein and in the claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "drugs" includes a single drug and multiple such drugs.

[0116] The terms “allergy” and “allergic” as used herein refer to a medical condition characterized by an abnormal hypersensitivity reaction to a substance that is normally harmless, i.e., an allergen. Exemplary allergic diseases include anaphylaxis, asthma, food allergies, insect bite allergies, drug allergies, allergic rhinitis, urticaria, angioedema, eczema, atopic dermatitis, contact dermatitis, and eosinophilic esophagitis.

[0117] The amino acids in a polypeptide sequence can be identified by their non-abbreviated form or by three-letter or one-letter abbreviations, which are known in the art and are used interchangeably herein. Naturally occurring amino acids include: alanine (Ala) (A); arginine (Arg) (R); asparagine (Asn) (N); aspartic acid (Asp) (D); cysteine ​​(Cys) (C); glutamine (Gln) (Q); glutamic acid (Glu) (E); glycine (Gly) (G); histidine (His) (H); isoleucine (Ile) (I); leucine (Leu) (L); lysine (Lys) (K); methionine (Met) (M); phenylalanine (Phe) (F); proline (Pro) (P); serine (Ser) (S); threonine (Thr) (T); tryptophan (Trp) (W); tyrosine (Tyr) (Y); and valine (V). The term "aspartate" includes aspartate and aspartic acid. The term "glutamate" includes both glutamate and glutamic acid. Basic amino acids include lysine, arginine, and histidine, which are positively charged at neutral pH.

[0118] An antibody (the plural is also used interchangeably) is an immunoglobulin molecule that can specifically bind to a target such as a carbohydrate, polynucleotide, lipid, or polypeptide via at least one antigen-recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv), their variants, fusion proteins containing the antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, and any other modified forms of the immunoglobulin molecule containing the antigen-recognition site of the desired specificity (including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies). The term "antibody" refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, variant, variant, or derivative thereof of the Ig molecule that retains its essential epitope-binding properties. Formats of such variant, variant, or derivative antibodies are known in the art. Non-limiting embodiments thereof are described below.

[0119] In full-length antibodies, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1, and IgA2), or a subclass.

[0120] The “antigen-binding portion” (or simply “antibody portion”) of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., BCMA). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may be bispecific, dual-specific, or multispecific, specifically binding to two or more different antigens. Multispecific, bispecific, and dual-specific antibody constructs are well known in the art and are described and characterized in Kontermann (ed.), Bispecific Antibodies, Springer, NY (2011) and Spiess et al., Mol. Immunol. 67(2):96-106 (2015).

[0121] Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments containing a single variable domain (Ward et al. (1989) Nature 341:544-546, Winter et al., PCT Publication WO 90 / 05144 A1, these are incorporated herein by reference); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together using a synthetic linker via recombination to create a single protein chain in which the VL and VH regions pair up to form a monovalent molecule (this is known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included. Diabody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain. However, by using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on the other chain, forming two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Such antibody-binding moieties are known in the field (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)).

[0122] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a viral vector. This term should also be interpreted as meaning an antibody produced by the synthesis of an antibody-encoding DNA molecule, which expresses an antibody protein or an amino acid sequence that identifies the antibody, and where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0123] In some embodiments, “antigen” or “Ag” is defined herein as a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens may originate from recombinant DNA or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will encode the term “antigen” as used herein. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by the full-length nucleotide sequence of a gene. It will be readily apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It will be readily apparent that antigens can be produced by synthesis or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0124] As used herein, the term “tumor antigen” refers to molecules (typically proteins, carbohydrates, or lipids) expressed on the surface of cancer cells, either whole or as fragments (e.g., MHC / peptides), that are useful for preferential targeting of drugs against cancer cells. In some embodiments, tumor antigens are markers expressed by both normal and cancer cells, e.g., lineage markers, e.g., CD19 on B cells. In some embodiments, tumor antigens are cell surface molecules that are overexpressed in cancer cells compared to normal cells. In some embodiments, tumor antigens are cell surface molecules that are improperly synthesized within cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed in normal cells. In some embodiments, tumor antigens are expressed only on the cell surface of cancer cells, either whole or as fragments (e.g., MHC / peptides), and are not synthesized or expressed on the surface of normal cells. Examples of tumor antigens include, but are not limited to, BCMA, CD19, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, and CD30.

[0125] As used herein, the term “antitumor effect” refers to a biological effect that may manifest as a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an extension of life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. “Antitumor effect” may also manifest as the ability of the peptides, polynucleotides, cells, and antibodies of this disclosure to prevent tumor development in the first place.

[0126] The term "autoimmunity" refers to a disease or illness in which an individual's immune system or its components attack the individual's normal cells or tissues. Autoimmune diseases can be mediated by autoantibodies, i.e., antibodies produced by the individual, recognizing antigens in the individual's cells or tissues. Examples of autoimmune diseases include myasthenia gravis, lupus erythematosus (SLE), rheumatoid arthritis, vesicular skin diseases such as pemphigus, psoriasis, inflammatory bowel disease, celiac disease, pernicious anemia, idiopathic thrombocytopenic purpura, scleroderma, Graves' disease, Sjögren's syndrome, Goodpasture syndrome, multiple sclerosis, and type 1 diabetes.

[0127] As used herein, the term “self” refers to any material originating from the same individual that is later reintroduced into that individual.

[0128] As used herein, the term "homogeneous" refers to grafts derived from different animals of the same species. "Heterogeneous" refers to grafts derived from animals of different species.

[0129] As used herein, the term “cancer” is defined as a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers, but not limited to, include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In some embodiments, cancer is cancer that expresses BCMA. Exemplary cancers that express BCMA include multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and glioblastoma. In some embodiments, cancer refers to multiple myeloma. Multiple myeloma is a cancer of plasma cells. Multiple myeloma can be diagnosed by blood tests (serum protein electrophoresis, serum free kappa / lambda light chain assay), bone marrow examination, urine protein electrophoresis, and / or commonly involved bone X-ray examinations. In some aspects, cancer refers to Hodgkin lymphoma (HL). HL is a B-cell cancer.

[0130] "Effective dose" means a therapeutic amount sufficient to reduce or improve the severity and / or duration of the disability or one or more of its symptoms, prevent the progression of the disability, cause regression of the disability, prevent recurrence, onset, onset or progression of one or more symptoms associated with the disability, detect the disability, or enhance or improve the preventive or therapeutic effect (or multiple) of another treatment (preventive or therapeutic agent).

[0131] As used herein, the term “exogenous” refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0132] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied from host cells or in an in vitro expression system. Expression vectors include any vectors known in the art, such as cosmids, plasmids (e.g., naked or liposome-encapsulated), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0133] As used herein, “CD3 zeta intracellular domain” refers to the protein domain, which is the intracellular portion of the CD3 zeta protein. For example, in humans, the CD3 zeta intracellular domain corresponds, approximately corresponds to, or is similar to, amino acids 351–463, indicated by the sequential numbers in the full-length CD3 zeta amino acid sequence of SEQ ID NO: 1, and approximately corresponds to or is similar to the partial CD3 zeta amino acid sequence of SEQ ID NO: 18. The definition of “approximately corresponds” is as follows: Since the boundary between one protein domain and another (e.g., between the CD3 zeta transmembrane domain and the intracellular domain) is not always strictly defined, the “CD3 zeta intracellular domain” may consist of more or fewer amino acids than described above (e.g., 1 to 10 more or fewer amino acids on the amino and carboxyl sides of the enumerated, numerically separated, or separately specified sequences), but still approximately corresponds to those described above, namely the amino acid sequence of SEQ ID NO: 18 or amino acids 351–463 of SEQ ID NO: 1. Furthermore, the term “CD3 zeta intracellular domain” is intended to include all allelic variants and naturally occurring or artificial mutations of CD3 zeta, not contrary to the amino acid substitutions described herein.

[0134] As used herein, the terms “immunoglobulin” or “Ig” refer to a class of proteins that function as antibodies, and the terms have the common meaning in the art.

[0135] In this specification, when used in relation to the selection of two or more amino acids from a list, such as a Markush group, "independently selected" means that the selection of one amino acid from the list is independent of, or can be independent of, the selection of each subsequent amino acid from the list, and so on, meaning that the first selection and the subsequent selection may be different or the same.

[0136] "Isolated" means modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0137] Unless otherwise specified, “nucleic acids that encode a nucleotide sequence or an amino acid sequence” includes all nucleotide sequences that encode the same amino acid sequence in degenerate versions of each other. The phrase “nucleotide sequences that encode a protein or RNA” may also include introns, insofar as nucleotide sequences that encode proteins may contain introns in several versions.

[0138] As used herein, the terms “modulate” or “to modulate” mean mediating a detectable increase or decrease in the response level compared to the response level in the absence of the treatment or compound, and / or compared to the response level under otherwise identical circumstances. This term encompasses disturbing and / or influencing a natural signal or response, thereby mediating a beneficial effect.

[0139] As used herein, the term “linker” refers to a bond (e.g., a covalent bond), chemical group, or molecule that connects two molecules or parts (e.g., two domains of a fusion protein, e.g., a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., adenosine deaminase)). Typically, a linker is located between or aside two groups, molecules, or other parts, connected to each other via covalent bonds, thereby connecting the two. In some embodiments, the linker is an amino acid or a group of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical component. In some embodiments, the linker is 5 to 100 amino acid long, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acid long. Longer or shorter linkers are also intended.

[0140] Parenteral administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0141] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal or its cells in vitro or in situ to which the methods herein are applicable. In some embodiments, the patient, subject, or individual is a human. Other examples include dogs, cats, mice, rats, and their transgenic species. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically modified, for example, a genetically modified non-human subject. The subject may be of any sex and at any stage of development. In some embodiments, the subject has cancer (e.g., multiple myeloma). In other embodiments, the subject is a healthy volunteer.

[0142] In this specification, the terms “specifically binding” or “specific to” used with respect to antigen-recognition domains, e.g., antibodies, e.g., scFv, mean a protein or domain that recognizes a particular antigen (or surface marker) but substantially does not recognize or bind to other molecules in the sample (or, in certain circumstances, within the body of an individual). For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species, but such interspecies cross-reactivity itself does not change the classification that the antibody is specific. An antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, such cross-reactivity itself does not change the classification that the antibody is specific. In some cases, the terms “specific binding” or “specifically binding” refer to an interaction between an antibody, protein (or its domain), or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure, rather than a general protein. If the antibody is specific to epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0143] In this specification, "substitution" as used with respect to amino acids or the position of an amino acid in an amino acid sequence means that one type of amino acid is substituted for another type of amino acid at the same (or corresponding) position in the amino acid sequence. The corresponding amino acid positions in two or more similar sequences, for example, a wild-type sequence and a substituted sequence, can be confirmed by aligning those sequences using readily available informatics tools, such as BLAST. If two similar proteins have one or more amino acid deletions or insertions that reference each other, or sequences containing such deletions or insertions, the optimal alignment can be obtained, for example, by including one or more gaps using BLAST, thereby identifying the corresponding amino acid positions between the two proteins. In this specification, "mutation," "mutate," and "mutated" as used with respect to amino acid substitutions in a sequence refer to amino acid substitutions, typically amino acid substitutions that alter the wild-type sequence. In this specification, "mutation," "mutate," and "mutated" as used with respect to modifications of amino acid sequences refer to the substitution of one or more amino acids in a sequence. Mutations can be artificially created. In this specification, as used in reference to modifications of amino acid sequences, “deletion,” “to delete,” and “deleted” mean the removal of one or more amino acids from a sequence, typically a wild-type sequence. In this disclosure, amino acid substitutions or mutations may be described by notations such as “XZ substitution,” “XZ mutation,” etc., where “X” refers to one or more positions in the first sequence occupied by amino acid “X,” and “Z” refers to the second sequence in which such positions are substituted or mutated by amino acid “Z.” Generally, unless otherwise indicated in the context, the first sequence is the reference sequence (e.g., wild-type), and the second sequence is the modified sequence (e.g., the modified sequence of the present invention). In some embodiments, the formula “XZ substitution” refers to only one amino acid position; in some embodiments, to multiple amino acid positions; and in some embodiments, to all amino acid positions occupied by amino acid “X” or “Z” in the sequence (or a particular part thereof); in any case, the number of amino acid positions in which such substitutions or mutations occur is evident from the context.Therefore, for example, a "KA" or "Lys-Ala" substitution at nine amino acid positions means that each of the nine positions occupied by lysine in the first sequence is replaced by alanine in the second sequence.

[0144] As used herein, unless otherwise clearly indicated by the context, the term “surface marker” means an antigen or other molecular portion present on the surface of a cell to which CAR can specifically bind. Examples of useful surface markers include BCMA, CD19, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, and CD30. Tumor antigens, which are specific or relatively specific to cancer cells, can function as surface markers. Many (but not all) surface markers are membrane-bound proteins or their domains, which may include glycosylation and other post-translational modifications.

[0145] As used herein, the terms “target” and derived terms such as “target cell surface marker” refer to a surface marker to which a CAR specifically binds, or to a cell, tissue, or tumor, unless otherwise clearly indicated by the context. Where “target” refers to a type of cell, tissue, or tumor, such cells, tissues, or tumors typically express (i.e., display) a surface marker to which a CAR specifically binds. Thus, as used herein, “target cell” refers to a cell to which a particular CAR or CAR-expressing cell, such as a CAR T cell, specifically binds.

[0146] As used herein, the term “treatment” means procedure and / or prevention. Therapeutic effects are achieved by suppression, remission, or eradication of a disease state.

[0147] As used herein, the term “therapeutic dose” refers to the amount of a compound of interest that elicits a biological or medical response in a tissue, system, or subject as sought by a researcher, veterinarian, medical professional, or other clinician. The term “therapeutic dose” includes the amount of a compound that, when administered, is sufficient to prevent or, to some extent, alleviate the onset of one or more signs or symptoms of the disorder or disease being treated. The therapeutic dose varies depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. The therapeutic dose does not necessarily have to be the amount required for clinical efficacy.

[0148] As used in this specification, the terms “treatment,” “to treat,” and “to treat” refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more of its symptoms. In some embodiments, treatment may be performed after the appearance of one or more symptoms and / or after the disease has been diagnosed. In other embodiments, treatment may be performed even when there are no symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of the disease. For example, treatment may be performed on a susceptible individual before the onset of symptoms (for example, taking into account a history of symptoms and / or taking into account genetic or other susceptibility factors). Treatment may also be continued after the disappearance of symptoms, for example, to prevent or delay relapse.

[0149] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. This includes primary target cells and their progeny cells.

[0150] A "vector" is a composition containing isolated nucleic acids that can be used to deliver isolated nucleic acids into cells. In the art, a large number of vectors are known, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmidal and non-viral compounds that facilitate the movement of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0151] Detailed description of a specific aspect CAR's overall structure In one aspect, this disclosure provides novel CARs each comprising a modified CD3 zeta intracellular domain (i.e., in which specific amino acid residues are selectively modified or mutated from the wild-type sequence), thereby providing CARs that offer better and sustained expression and CAR-mediated cellular functions such as cytotoxicity and cytokine secretion.

[0152] The overall design of CAR proteins is known in the art. See, for example, Alnefaie and U.S. Patent No. 10,934,337; the entire contents of these are incorporated herein by reference. In some embodiments, the CARs of this disclosure include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular (T cell signaling) domain as described herein, which are specific to a particular surface antigen (marker) (e.g., CD19, BCMA, EGFR / HER, CD22, mesoserine, CD123, CD20, PD1, or CD30). Each intracellular domain of this disclosure includes a modified CD3 zeta intracellular domain, as disclosed below. Optionally, the intracellular domain may further include CD8-α protein, CD28 protein, FcR gamma protein, CD27 protein, OX40 protein, 4-1BB protein, CD30 protein, CD40 protein, PD-1 protein, ICOS protein, LFA-1 protein, CD2 protein, CD7 protein, LIGHT protein, NKG2C protein, B7 H3 protein, or modified versions or parts of any of these, or other costimulatory domains known to be used in CAR, and combinations thereof.

[0153] Generally, when a suitable class of extracellular (antigen-binding) domains is known for the purpose of CAR construction, and another suitable class of intracellular domains is also known, a functional CAR can be obtained by combining any particular species of such extracellular domains with any particular species of such intracellular domains (via transmembrane domains). Thus, when a functional CAR is provided, a different species of extracellular domain can be substituted in that CAR to confer, for example, CAR-binding specificity to a specific antigen, i.e., a target cell surface marker. Similarly, when a functional CAR is provided, a different type of intracellular domain can be substituted in that CAR to affect other CAR properties, for example, as described herein. This phenomenon is beneficial to this disclosure because the novel CD3 zeta intracellular domains of this disclosure described herein are typically suitable for use in any CAR containing CD3 zeta intracellular domains, including, but not limited to, many CARs described herein. Furthermore, the novel CD3 zeta intracellular domains of this disclosure are suitable for any system containing CD3 zeta intracellular domains. Therefore, even if, for example, novel CD3 zeta intracellular domains are described in this disclosure in relation to BCMA-specific CARs, those same novel CD3 zeta intracellular domains are suitable for use in CARs that include different extracellular antigen recognition domains that bind to different surface antigens (e.g., CD19, EGFR / HER, CD22, mesoserine, CD123, CD20, PD1, or CD30, etc., but not limited to these).

[0154] In some embodiments, spacer and / or hinge domains may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the transmembrane domain and the intracellular domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligo or polypeptide that has the function of linking a transmembrane domain to either an extracellular or intracellular domain in a polypeptide chain. Spacer domains for use in CARs are known in the art. See, for example, U.S. Patent No. 10,934,337.

[0155] In some embodiments, the CAR of this disclosure comprises the structure NH2-[extracellular antigen-binding domain]-[transmembrane domain]-[intracellular domain of this disclosure]-COOH. In some embodiments, the CAR comprises the structure NH2-[extracellular antigen-binding domain]-[hinge region]-[transmembrane domain]-[intracellular domain of this disclosure]-COOH. In some embodiments, the CAR comprises one or more spacer sequences. In some embodiments, each example of ``]-['' indicates the optional presence of a spacer sequence.

[0156] In some embodiments, the intracellular domain comprises the CD3 zeta protein of the present disclosure and optionally comprises CD8-α protein, CD28 protein, FcR gamma protein, CD27 protein, OX40 protein, 41BB protein, CD30 protein, CD40 protein, PD-1 protein, ICPS protein, LFA-1 protein, CD2 protein, CD7 protein, LIGHT protein, NKG2C protein, B7 H3 protein, or any part thereof, other intracellular costimulatory molecules known to be used in CARs, and any combination thereof. In some embodiments, the CAR of the present invention of the present disclosure comprises at least one of the following structures: NH2-[extracellular antigen-binding domain]-[transmembrane domain]-[intracellular domain]-COOH; NH2-[extracellular antigen-binding domain]-[hinge region]-[transmembrane domain]-[intracellular domain]-COOH; NH2-[signal peptide]-[extracellular antigen-binding domain]-[transmembrane domain]-[intracellular domain]-COOH; or NH2-[signal peptide]-[extracellular antigen-binding domain]-[hinge region]-[transmembrane domain]-[intracellular domain]-COOH.

[0157] In some embodiments, the CAR includes an intracellular domain having a configuration selected from the following exemplary and non-limiting configurations: NH2-[the intracellular domain of the CD3 zeta in this disclosure]-COOH; NH2-[CD28]-[CD3 zeta intracellular domain of this disclosure]-COOH; NH2-[41BB]-[CD3 zeta intracellular domain of this disclosure]-COOH; NH2-[CD27]-[CD3 zeta intracellular domain of this disclosure]-COOH; NH2-[CD40]-[CD3 zeta intracellular domain of this disclosure]-COOH; NH2-[ICOS]-[the intracellular domain of CD3 zeta in this disclosure]-COOH; NH2-[CD40L]-[CD3 zeta intracellular domain of this disclosure]-COOH; NH2-[OX40]-[the intracellular domain of CD3 zeta in this disclosure]-COOH; or NH2-[41BB]-[OX40]-[the intracellular domain of the CD3 zeta in this disclosure]-COOH.

[0158] In some embodiments, the above exemplary and non-limiting arrangements are from left to right, N-terminus to C-terminus of the CAR. In some embodiments, each example of "[]-[" indicates the arbitrary presence of a spacer sequence.

[0159] In some embodiments, CARs are designed to have a leader domain (also referred to as a “signal peptide”) for guiding the translated chimeric protein to the membrane. In some embodiments, CARs contain a leader sequence at the amino terminus of the CAR protein. For example, a CAR may contain a leader sequence at the N terminus of an extracellular antigen-binding domain, where the leader sequence is optionally selected based on its tendency or ability to be cleaved from the antigen-binding domain during cell processing and localization of the CAR to the cell membrane. Leader domains are further described in U.S. Patent No. 10,934,337.

[0160] Transmembrane domains used in CARs are also described in U.S. Patent No. 10,934,337. Transmembrane domains may be derived from naturally occurring sequences or may be synthetic. In the case of naturally occurring sequences, the domain may be derived from any membrane-bound or transmembrane protein, as long as it enables signaling to the intracellular domain(s) each time the CAR binds to a target. Transmembrane domains particularly used in the present invention may include, for example, at least the transmembrane regions of the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.In some embodiments, the transmembrane domain is associated with co-stimulatory molecules, such as MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, and NKp80. (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 It may contain at least one transmembrane domain (or more) of ligands that specifically bind to CD83, 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, and CD83. In some embodiments, the transmembrane domain may be synthetic and in which case it mainly comprises hydrophobic residues such as leucine and valine. In some embodiments, one or both ends of the synthetic transmembrane domain contain a triplet of phenylalanine, tryptophan, and valine.Optionally, short oligo or polypeptide linkers, for example, 2 to 10 amino acids in length, may form a link between the transmembrane domain and the intracellular signaling domain of the CAR. Glycine-serine doublets provide exemplary suitable linkers.

[0161] In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD8 transmembrane domain or a CD28 transmembrane domain. The sequence of CD8 for this purpose is known in the art and is described in PCT publication number WO 2014 / 055771, which is incorporated herein by reference.

[0162] intracellular domain The CAR protein of the present invention, or any other protein containing a CD3 zeta intracellular domain, comprises a novel intracellular domain which is a substitution, modification, or mutation of the CD3 zeta intracellular domain.

[0163] In some embodiments, the novel intracellular domain contains a sequence similar to the wild-type CD3 zeta intracellular domain sequence (SEQ ID NO: 18), except for substitutions of specific lysine amino acids naturally present in the wild-type sequence. For example, the novel intracellular domain contains a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or 100% identical to SEQ ID NO: 18, except for substitutions of specific lysine amino acids naturally present in the wild-type sequence.

[0164] Therefore, in some embodiments, any other protein containing the CAR or CD3 zeta intracellular domain contains the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that each of the at least six lysine amino acids of SEQ ID NO: 18 is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0165] Therefore, in some embodiments, any other protein containing the CAR or CD3 zeta intracellular domain contains the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that each of the at least seven lysine amino acids of SEQ ID NO: 18 is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0166] Therefore, in some embodiments, any other protein containing the CAR or CD3 zeta intracellular domain contains the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that each of the at least eight lysine amino acids of SEQ ID NO: 18 is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0167] Therefore, in some embodiments, any other protein containing the CAR or CD3 zeta intracellular domain contains the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that each of the at least nine lysine amino acids of SEQ ID NO: 18 is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0168] Therefore, in some embodiments, any other protein containing the CAR, or CD3 zeta intracellular domain, contains the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that each of the nine lysine amino acids of SEQ ID NO: 18 is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0169] In some embodiments of SEQ ID NO: 18 in which at least 6, 7, 8, or 9 lysine amino acids are substituted, the substitutions can be selected from the group consisting of alanine, aspartic acid, and glutamic acid. In some embodiments of SEQ ID NO: 18 in which at least 6, 7, 8, or 9 lysine amino acids are substituted, all substitutions may be due to alanine. In some embodiments of SEQ ID NO: 18 in which at least 6, 7, 8, or 9 lysine amino acids are substituted, all substitutions may be due to aspartic acid. In some embodiments of SEQ ID NO: 18 in which at least 6, 7, 8, or 9 lysine amino acids are substituted, all substitutions may be due to glutamic acid.

[0170] The above amino acid substitutions can be further understood by referring to sequences of specific embodiments of other proteins containing the CAR or CD3 zeta intracellular domain of the Disclosure. Thus, in some embodiments, any other protein containing the CAR or CD3 zeta intracellular domain of the Disclosure contains a sequence selected from the group consisting of SEQ ID NOs: 2-5 (corresponding to a modified CD3 zeta domain). In some embodiments, any other protein containing the CAR or CD3 zeta intracellular domain of the Disclosure contains a sequence selected from the group consisting of SEQ ID NOs: 19-66 and 68-86 (corresponding to a modified CD3 zeta intracellular domain).

[0171] In some embodiments, the CAR of the Disclosure, or any other protein containing the CD3 zeta intracellular domain, contains the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that at least six lysine amino acids of SEQ ID NO: 18 are deleted. In some such embodiments, at least seven such lysine amino acids are deleted. In some such embodiments, at least eight such lysine amino acids are deleted. In some such embodiments, at least nine such lysine amino acids are deleted. In some such embodiments, nine such lysine amino acids are deleted.

[0172] In some embodiments, the CAR of this disclosure, or any other protein comprising the CD3 zeta intracellular domain, comprises the human CD3 zeta intracellular domain of SEQ ID NO: 18, except that at least six lysine amino acids of SEQ ID NO: 18 are substituted or deleted in any of the manner described herein. In some such embodiments, at least seven such lysine amino acids are substituted or deleted. In some such embodiments, at least eight such lysine amino acids are substituted or deleted. In some such embodiments, at least nine such lysine amino acids are substituted or deleted. In some such embodiments, nine such lysine amino acids are substituted or deleted.

[0173] Some of the intracellular domains used in this disclosure include, in addition to the CD3 zeta sequence of this disclosure or any other protein containing a CD3 zeta intracellular domain, a co-stimulatory domain corresponding to a wild-type co-stimulatory domain, or a co-stimulatory domain in which one or more lysine amino acids of the co-stimulatory domain are substituted or mutated with, for example, alanine amino acids. See, for example, Examples 4 and 5, and the co-stimulatory domain amino acid sequences of SEQ ID NOs. 57-60 and 62-66. Thus, in some embodiments, the CAR of this disclosure, or any other protein containing a CD3 zeta intracellular domain, includes a novel co-stimulatory domain in which one, two or more, or all of the naturally occurring lysine amino acids in the co-stimulatory domain (or a portion thereof incorporated into the CAR) are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine. In some embodiments, the substitutions can be selected from the group consisting of alanine, aspartic acid, and glutamic acid. In some embodiments, all substitutions are by alanine. In some embodiments, all substitutions are by aspartic acid. In some embodiments, all substitutions are by glutamic acid.

[0174] Examples of proteins whose entirety can be incorporated into CAR as a co-stimulatory domain in each example include: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIR2DS1, KIR2DS2, KIR3DS1, SLAMF7, and NKp80. (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 Ligands that specifically bind to 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, and CD83, etc.

[0175] nucleic acids and vectors In some embodiments, the present invention encompasses a nucleic acid molecule (e.g., DNA or RNA) encoding any other protein including a CAR of the present disclosure (e.g., any of the CARs provided herein) or a CD3 zeta intracellular domain. In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the nucleic acid molecule comprises a sequence of any other protein including a CAR or a CD3 zeta intracellular domain, wherein the sequence comprises a nucleic acid sequence encoding any other protein including a CAR of the present disclosure or a CD3 zeta intracellular domain.

[0176] None of the nucleic acid molecules provided herein may have other features, such as a 5′ untranslated region (5′ UTR), a 3′ untranslated region (3′ UTR), a polyadenine tail (polyA), or a 7-methylguanosine cap (m 7 G) may include other features such as an internal ribosome entry site (IRES) and / or an open reading frame. In some embodiments, the present disclosure provides RNA, or DNA encoding it, having the following arrangement of features: 5′-[CAR]-3′ 5'-[5' UTR]-[CAR]-3' 5′-[m7G cap]-[5′ UTR]-[CAR]-3′ 5′-[m7G cap]-[5′ UTR]-[CAR]-[Poly A]-3′ 5′-[CAR]-[Poly A]-3′ 5'-[CAR]-[3' UTR]-[Poly A]-3' 5′-[5′ UTR]-[CAR]-[3′ UTR]-3′ 5′-[5′ UTR]-[CAR]-[3′ UTR]-[Poly A]-3′ 5′-[m7G cap]-[5′ UTR]-[CAR]-[3′ UTR]-[Poly A]-3′

[0177] The construction of the nucleic acids encoding the CARs of this disclosure can be further understood by referring to U.S. Patent No. 10,934,337, which is incorporated herein by reference.

[0178] The present invention also provides vectors into which the DNA or RNA of the present invention is inserted. The construction of such vectors for CARs of the present disclosure can be further understood by reference to U.S. Patent No. 10,934,337, which is incorporated herein by reference.

[0179] Cells modified to express CAR Any of the CARs described herein, or any other proteins described herein that include the CD3 zeta intracellular domain, can be expressed in suitable cells. Examples of suitable cells include T cells, which become CAR T cells when modified to express the CAR. In some embodiments, the cells are CD3+ cells. In some embodiments, the cells are CD8+ cells. In some embodiments, the cells are CD4+ cells. Other suitable cells for the expression of the CAR, or any other protein including the CD3 zeta intracellular domain, include NK cells and stem cells, such as hematopoietic stem cells.

[0180] Methods for introducing and expressing genes in cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. In some embodiments, the host cell is a T cell. Physical methods for introducing polynucleotides into host cells include electroporation, mechanical membrane disruption (e.g., cell squeezing or nanoparticle-based delivery), calcium phosphate precipitation, lipofection, particle guns, and microinjection. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is electroporation.

[0181] Biological methods for introducing target polynucleotides into host cells include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for gene insertion into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0182] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). When nonviral delivery systems are utilized, the exemplary delivery vehicle is liposomes. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, and in vivo).

[0183] Regardless of the method used to introduce exogenous nucleic acids into host cells or to expose cells to the inhibitors of the present invention, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; and “biochemical” assays, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blotting) or by assays described herein for identifying agents included within the scope of this disclosure.

[0184] RNA transfection In some embodiments, CAR T cells of the present disclosure, or other cells containing a protein comprising the modified CD3 zeta intracellular domain of the present disclosure, are obtained by introduction of RNA (e.g., mRNA containing a sequence encoding the CAR described herein). In some embodiments, in vitro transcribed RNA CARs can be introduced into cells as a form of transient transfection. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Desired DNA from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable source of DNA. The desired template for in vitro transcription can be the CAR of the present invention.

[0185] RNA can be introduced into target cells using a variety of methods, including, but are not limited to, commercially available methods such as: electroporation (Amaxa® Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), Gene Pulser II® (BioRad, Denver, Colo.), Multiporator® (Eppendorf, Hamburg, Germany), mechanical membrane disruption (e.g., cell compression, see U.S. Patent Publication 2014 / 287509A1), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or bioristic particle delivery systems such as "gene guns" (e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0186] Disclosed herein are methods for generating in vitro transcribed RNA CARs, or in vitro transcribed RNA of any other protein containing a CD3 zeta intracellular domain. The present invention also includes RNA constructs encoding CARs, or RNA constructs of any other protein containing a CD3 zeta intracellular domain, that are directly transfectable into cells. Methods for generating mRNA for use in transfection include in vitro transcription (IVT) of a template using specially designed primers, followed by poly-A addition, thereby enabling the generation of constructs typically 50–400, 50–2000, 150–400, or 150–2000 nucleotides long, containing 5' and 3' untranslated sequences ("UTR"), a 5' cap, the nucleic acid to be expressed, and a poly-A tail. RNA thus generated can be efficiently transfected into different types of cells. In one aspect, the template comprises the sequence of a CAR of the present disclosure. In another aspect, the template comprises the sequence of any other protein of the present disclosure containing a CD3 zeta intracellular domain.

[0187] For example, the generation of mRNA by in vitro transcription (IVT) from a DNA template is known in the art. For example, one method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template using specially designed primers. Optionally, the mRNA may be 3' polyadenylated by methods known in the art and may contain a 3' polyadenine tail consisting of, for example, about 25, 50, 100, 150, 250, 500, or 1000 adenine nucleotides.

[0188] In some embodiments, the nucleic acid is self-amplified RNA (saRNA) prepared according to methods known in the art. In some embodiments, the RNA, e.g., mRNA, contains pseudouridine. In some embodiments, the RNA is artificially enriched with pseudouridine. In some embodiments, substantially all of the uridine nucleotides in the RNA (e.g., more than 90%, 95%, 97%, 99%, or 99.9%) are replaced with pseudouridine. Methods for incorporating pseudouridine into RNA are known in the art.

[0189] In some embodiments, the nucleic acid is a circular RNA prepared according to methods known in the art.

[0190] therapeutic use In some embodiments, the present invention encompasses cells (e.g., T cells) modified to express the CARs of the present disclosure or other proteins of the present disclosure, including the CD3 zeta intracellular domain. Thus, in some cases, transduced immune cells (e.g., T cells) can induce CAR-mediated immune (e.g., T cell) responses, cytotoxic responses, or antitumor responses. In some embodiments, the present disclosure provides the use of the CARs of the present disclosure to redirect the specificity of primary T cells to surface markers or tumor antigens. Thus, in some embodiments, the present invention also provides a method for stimulating a T cell-mediated cytotoxic or immune response against a target cell population or tissue in a mammal, the method comprising the step of administering T cells expressing the CARs of the present disclosure to a mammal, wherein the CARs include an antigen-binding domain that specifically binds to a given target or surface marker (e.g., BCMA). In some embodiments, the present invention includes a type of cell therapy in which T cells are genetically modified to express the CARs of the present disclosure and the CAR T cells are injected into a recipient that requires them. The injected cells can kill the target cells of the recipient. Unlike antibody therapy, some CAR T cells can replicate in vivo, so these cells persist for a long period of time.

[0191] CAR-modified T cells of the present disclosure, or cells modified with the proteins of the present disclosure, including the CD3 zeta intracellular domain, may also function as a type of vaccine for ex vivo immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0192] With respect to ex vivo cell production, at least one of the following is performed in vitro before administering the cells to a mammal: i) cell proliferation, ii) introduction of nucleic acid encoding the CAR into the cells, and / or iii) cell cryopreservation. The ex vivo procedure is well known in the art and will be discussed in more detail below. Briefly, cells are isolated from a mammal (e.g., human) and genetically modified (e.g., transduced or transfected in vitro) as disclosed herein using nucleic acid or vector expressing the CAR of this disclosure. The CAR-modified cells can be administered to a mammalian recipient to obtain a therapeutic effect. The mammalian recipient may be human, and the CAR-modified cells may be autologous to the recipient. Alternatively, the cells may be homogeneous, syngeneic, or heterologous to the recipient.

[0193] The CAR-modified immune cells (e.g., CAR T cells) of the present invention, or compositions comprising such cells, can be used or administered in effective amounts to subjects in need for conferring antitumor immunity; treating or preventing cancer; treating or preventing autoimmune diseases; or treating or preventing allergic diseases. In some embodiments, cancer is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, or glioblastoma. In some embodiments, autoimmune diseases are myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pemphigus, psoriasis, inflammatory bowel disease, celiac disease, pernicious anemia, idiopathic thrombocytopenic purpura, scleroderma, Graves' disease, Sjögren's syndrome, Goodpasture syndrome, or type 1 diabetes. In some aspects, allergic diseases include anaphylaxis, asthma, food allergies, insect bite allergies, drug allergies, allergic rhinitis, urticaria, angioedema, eczema, atopic dermatitis, contact dermatitis, and eosinophilic esophagitis.

[0194] The CAR-modified immune cells of the present invention (e.g., CAR T cells) may be administered alone or as a composition (e.g., a pharmaceutical composition) in combination with diluents and / or other components such as IL-2 or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may contain the target cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0195] The compositions of the present invention are preferably formulated for intravenous administration, but can also be formulated for other parenteral administration routes.

[0196] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0197] Where “immunologically effective dose,” “antitumor effective dose,” “tumor inhibitory effective dose,” or “therapeutic dose” is indicated, the exact amount of the composition of the present invention administered may be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition. The pharmaceutical compositions comprising CAR-modified immune cells (e.g., CAR T cells) as described herein are generally administered in 10 doses. 4 ~10 9 Cells / kg body weight, preferably 10 5 ~10 9 It can be said that the T cell composition can be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). Multiple doses of the T cell composition can also be administered at these doses. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment plan for a particular patient can be easily determined by a person skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0198] Without further explanation, those skilled in the art will be able to make the most of this disclosure based on the above description. Therefore, the following specific embodiments should be construed as merely illustrative and not to limit the remainder of this disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter described herein. [Examples]

[0199] To allow for a deeper understanding of the disclosures described herein, the following examples are provided. The synthesis examples described herein are provided to illustrate the compounds and methods provided herein and should not be construed as limiting their scope.

[0200] Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art relating to this disclosure. Any methods and materials similar to or equivalent to those described herein may be used in carrying out the tests of the present invention, but preferred materials and methods are those described herein. The following terms are used in the description and claims of the present invention:

[0201] Example 1. CAR T cells were generated using the mRNA construct of this disclosure encoding the CAR protein of this disclosure, which contains a mutant CD3 zeta intracellular domain to prevent protein downregulation. A series of experiments were then performed on these cells. The CAR proteins investigated contained one of the following: wild-type CD3 zeta intracellular domain sequence (SEQ ID NO: 1), mutant sequence in which all lysines in the CD3 zeta intracellular domain were mutated to arginine (SEQ ID NO: 2), mutant sequence in which all lysines in the CD3 zeta intracellular domain were mutated to alanine (SEQ ID NO: 3), mutant sequence in which all lysines in the CD3 zeta intracellular domain were mutated to aspartate (SEQ ID NO: 4), or mutant sequence in which all lysines in the CD3 zeta intracellular domain were mutated to glutamate (SEQ ID NO: 5). The CAR T cells were observed to express high levels of CAR protein, bind to BCMA, and kill BCMA-positive tumor cells. Exposure of CAR T cells to target cells expressing BCMA ligands resulted in low levels of downregulation of cell surface CAR protein.

[0202] The mRNA constructs of this disclosure each contain the nucleotide sequences of SEQ ID NOs: 13–17 and were generated by in vitro transcription from a PCR-amplified DNA template. In vitro transcription was performed using T7 RNA polymerase and a PCR product template containing a 180-nucleotide poly(A) tail. A 7-methylguanosine cap (CleanCap) was incorporated into the 5' end of the mRNA during co-transcription mRNA synthesis.

[0203] The mRNA construct of this disclosure, including SEQ ID NO: 13, contained, from 5' to 3', a 5' cap, a 5' UTR described as SEQ ID NO: 11, an open reading frame (ORF) described as SEQ ID NO: 6, a 3' UTR described as SEQ ID NO: 12, and a 3' polyadenine tail of 150 adenine units or more. The ORF encoded the CAR protein of this disclosure having the amino acid sequence of SEQ ID NO: 1.

[0204] Another mRNA construct of the present disclosure, SEQ ID NO: 14, contained a 5' cap, a 5' UTR described as SEQ ID NO: 11, an open reading frame (ORF) described as SEQ ID NO: 7, a 3' UTR described as SEQ ID NO: 12, and a 3' polyadenine tail of 150 adenine units or more, from 5' to 3'. The ORF encoded the CAR protein of the present disclosure having the amino acid sequence of SEQ ID NO: 2.

[0205] Another mRNA construct of the present disclosure, including SEQ ID NO: 15, contained, from 5' to 3', a 5' cap, a 5' UTR described as SEQ ID NO: 11, an open reading frame (ORF) described as SEQ ID NO: 8, a 3' UTR described as SEQ ID NO: 12, and a 3' polyadenine tail of 150 adenine units or more. The ORF encoded the CAR protein of the present disclosure having the amino acid sequence of SEQ ID NO: 3.

[0206] Another mRNA construct of the present disclosure, including SEQ ID NO: 16, contained, from 5' to 3', a 5' cap, a 5' UTR described as SEQ ID NO: 11, an open reading frame (ORF) described as SEQ ID NO: 9, a 3' UTR described as SEQ ID NO: 12, and a 3' polyadenine tail of 150 adenine units or more. The ORF encoded the CAR protein of the present disclosure having the amino acid sequence of SEQ ID NO: 4.

[0207] Another mRNA construct of the present disclosure, including SEQ ID NO: 17, contained, from 5' to 3', a 5' cap, a 5' UTR described as SEQ ID NO: 11, an open reading frame (ORF) described as SEQ ID NO: 10, a 3' UTR described as SEQ ID NO: 12, and a 3' polyadenine tail of 150 adenine units or more. The ORF encoded the CAR protein of the present disclosure having the amino acid sequence of SEQ ID NO: 5.

[0208] Lymphocytes were obtained from whole blood of healthy human donors to prepare CAR T cells from mRNA constructs. CD8+ T cells were positively selected from these lymphocytes using paramagnetic microbeads conjugated with anti-CD8 antibody. This resulted in cells with 95% CD8+ T cell and 95% viable cells. These enriched CD8+ T cells were incubated at 37°C and 5% CO2 for up to 14 days in the presence of anti-CD3 antibody (clone OKT3), IL-7, and IL-15. The cells were transfected with a 0.1 μg / μl mRNA construct by electroporation (4D Nucleofector, Lonza) according to the manufacturer's instructions. The cells were then cultured overnight in complete medium containing IL-7 and IL-15.

[0209] CAR T cells obtained through the above process were tested for viability, CAR protein expression, BCMA binding, cytotoxicity (i.e., ability to kill BCMA-positive myeloma (tumor) cells), and cytokine production. CAR protein resistance to downregulation was tested by incubating CAR T cells with or without BCMA-positive myeloma cells, followed by analysis of CAR expression. Viability, CAR expression, and BCMA binding were determined by flow cytometry on a Guava® EasyCyte® 12HT flow cytometer (Luminex). To test viability, CAR T cell samples were mixed with propidium iodide and acridine orange and analyzed by fluorescence microscopy using a Nexcelom Auto 2000 cytometer. To test CAR protein expression and BCMA binding, CAR T cell samples were incubated with 0.4 μg / mL allophycocyanin (APC)-conjugated BCMA (recombinant human TNFRSF17 protein, Fc-tagged, APC-labeled; Creative Biomart, Shirley, NY). CAR expression was assessed using a flow cytometer that electron-gated the fluorescence of the red channel to detect the presence or absence of emission from BCMA-APC on CAR-positive and CAR-negative cells. BCMA binding was determined by measuring the fluorescence intensity of the red channel to determine the relative amount of BCMA-APC bound to labeled CAR-positive cells. Control (non-CAR) CD8+ T cells generated by electroporation without IVT mRNA were tested as a parallel control in viability, expression, cytotoxicity, cytokine production, and BCMA binding assays.

[0210] To test the ability of the wild-type and mutant CAR proteins (SEQ ID NOs. 1-5) of this disclosure to resist antigen-mediated downregulation, 50,000 CAR T cells generated using the mRNA sequences (SEQ ID NOs. 13-17) of this disclosure were co-cultured with 100,000 MM1S tumor cells in wells of a 96-well V-bottom plate. The MM1S tumor cell line was derived from patients with IgA-lambda type multiple myeloma. See, for example, Greenstein et al., Exp. Hematol (2003) 31(4): 271-82 (DOI=10.1016 / S0301-472X(03)00023-7). Cultures of CAR T cells alone (without target cells) were also prepared as a control. After incubation at 37°C for 24 hours, tissue culture supernatant and cells were collected. CAR expression was analyzed by staining cells with CD8-BV421 antibody, propidium iodide, and BCMA-APC. Viable CD8+ T cells were identified by excluding dead cells with propidium iodide staining (near-infrared fluorescence) and selecting CD8+ cells that emitted blue fluorescence using a violet laser. CAR expression in all viable CD8+ T cells was quantified by the red fluorescence intensity from a red laser. CAR signaling was evaluated by analyzing interferon-γ production in tissue culture supernatant using a specific ELISA.

[0211] The signaling capacity of residual expressed CAR proteins was determined by secondary culture. Secondary culture was set up by co-culturing pre-exposed CAR T cells with MM1S-GFP tumor cells. Aliquots of 50,000 MM1S-GFP tumor cells were dispensed into the wells of a 96-well plate. CAR T cells obtained from the primary cell culture were washed to remove residual components of the culture medium. Approximately 1,500 to 50,000 washed CAR T cells were added to each well to obtain various effector:target ratios (i.e., the ratio of CAR T cells to BCMA+ myeloma cells) between 1:1 and 1:32. After incubation for 24 to 72 hours, dead cells were stained with propidium iodide. Surviving target cells were identified by GFP (green fluorescence from a blue laser) expression and propidium iodide elimination, and cell density was measured by flow cytometry. The degree of myeloma cell death by CAR T cells was calculated by comparing the number of myeloma cells in a simultaneous control well without CAR T cells. Signal transduction was evaluated by analyzing interferon-γ production in the supernatant using specific ELISA.

[0212] The CAR T cells described herein showed similar viability after electroporation using all of the constructs described herein (including sequences SEQ ID NOs. 13–17). The percentage of CAR T cells expressing anti-BCMA CAR was similar across all constructs (91.3–94.0%). Table 1 CAR T cell survival rate and CAR expression after electroporation with IVT mRNA [Table 1]

[0213] When the CAR T cells described herein were exposed to BCMA, a ligand (target) on MM1S myeloma target cells, for 24 hours, downregulation of CAR expression occurred. CAR T cells generated using a construct (SEQ ID NO: 13) encoding a protein containing the wild-type CD3 zeta intracellular domain sequence showed downregulation of CAR expression from a median fluorescence intensity (MFI) of 8,633 in the absence of BCMA ligand to an MFI of 950 in the presence of BCMA ligand expressed by MM1S myeloma target cells (i.e., an 89% decrease in CAR expression) (Figure 1A). Construct SEQ ID NO: 14 was designed to encode a protein in which all lysine residues in the CD3 zeta intracellular domain were mutated to arginine. Generation of CAR T cells with this construct (SEQ ID NO: 14) resulted in cells with lower anti-BCMA CAR expression than those of the wild-type construct SEQ ID NO: 13 (2,188 MFI compared to 8,633 MFI). When these CAR T cells were exposed to the target ligand BCMA on MM1S target cells, surface CAR expression was further reduced (405 MFIs of SEQ ID NO: 14 compared to 950 MFIs of the wild-type construct SEQ ID NO: 13) (Figure 1A).

[0214] Other amino acid substitutions were introduced in place of lysine in the intracellular domain of CAR CD3 zeta. IVT mRNA SEQ ID NO: 15 encodes a protein in which all intracellular lysines of CD3 zeta are mutated to alanine. IVT mRNA SEQ ID NO: 16 encodes a protein in which all intracellular lysines of CD3 zeta are mutated to aspartate. IVT mRNA SEQ ID NO: 17 encodes a protein in which all intracellular lysines of CD3 zeta are mutated to glutamate. Generation of CAR T cells with these constructs (SEQ ID NOs: 15, 16, 17) resulted in CAR T cells with significantly improved CAR expression and enhanced resistance to ligand-mediated CAR downregulation. CAR T cells generated using these constructs showed higher CAR expression than wild-type CAR (10,493–11,678 MFI compared to 8,633 for the wild-type construct). For sequence numbers 15, 16, and 17, respectively, CAR expression on MM1S myeloma cells after exposure to BCMA ligands decreased dramatically to 44.2%, 73.0%, and 85.3%, respectively. In contrast, CAR expression was maintained at 11.0% for the wild-type construct sequence number 13 (Figure 1A). Table 2 Anti-BCMA CAR expression by CAR T cells after culture with and without MM1S target cells. [Table 2]

[0215] CAR T cells (SEQ ID NOs: 14-17) in which the lysine in the intracellular domain of the CD3 zeta molecule was mutated to arginine, alanine, aspartic acid, and glutamic acid, all of which maintained signal transduction capacity. Interferon-gamma expression was analyzed for the CAR T cells of this disclosure and the supernatant of co-cultured BCMA+ MM1S multiple myeloma cells using specific ELISA. Prior to co-culture, the CAR T cells were either pre-exposed to BCMA+ cells or not. All constructs of this disclosure showed high levels of interferon-gamma production. Interferon-gamma expression was similar among CAR T cells generated using the lysine mutant constructs SEQ ID NOs: 14-17 (10,326-12,767 pg / mL). CAR T cells generated using the lysine mutant construct showed higher interferon-gamma expression than wild-type CAR T cells (SEQ ID NOs: 13; 6,881 pg / mL). Therefore, by mutating the lysine in the intracellular domain of CAR CD3 zeta to other amino acids such as arginine, alanine, aspartic acid, and glutamic acid, CAR T cell activation was possible and was not inhibited (Figure 1B). Table 3 Cytokine (interferon-γ) production by anti-BCMA CAR T cells cultured in the presence of MM1S target cells. [Table 3]

[0216] CAR T cells generated using SEQ ID NOs. 15–17 maintained higher CAR expression than wild-type cells (SEQ ID NOs. 13) even after exposure to BCMA ligands. The superiority of these constructs was tested in secondary cultures. Cytotoxicity and cytokine production against BCMA ligand-expressing MM1S-GFP target cells were evaluated using pre-exposed CAR T cells generated with the constructs of this disclosure encoding lysine mutant CARs and wild-type CARs. Cytotoxicity was evaluated by co-culture at various effector:target ratios. At a 1:2 effector:target ratio, control T cells showed no cytotoxicity against MM1S-GFP target cells (<10%), while pre-exposed CAR T cells generated using SEQ ID NOs. 13–17 completely or nearly completely eliminated target cells (98–100% cytotoxicity) (Figure 1C). Effectors: At low target cell ratios, wild-type pre-exposed CAR-T cells (SEQ ID NO: 13) showed a dramatic reduction in cytotoxicity (49.3% at 1:8 and 4.7% at 1:32) (Figure 1C). CAR T cells generated using IVT mRNA of SEQ ID NO: 14, which encodes a protein with a lysine-to-arginine variant, showed a similar cytotoxicity profile (Figure 1C). In contrast, pre-exposed CAR T cells generated using IVT mRNA for SEQ ID NOs: 15, 16, and 17, respectively, showed high levels of cytotoxicity up to a 1:32 ratio (59.0%, 68.7%, and 72.3%, respectively) (Figure 1C). Very low effectors: Maintenance of cytolytic activity at target ratios was consistent with high levels of interferon-γ production (1:2 ratio) by pre-exposed CAR T cells generated with these constructs (Table 4 and Figure 1B, right). Pre-exposed CAR T cells generated using a construct in which the lysine in the intracellular domain was mutated with amino acids bearing the opposite negative charge (aspartic acid (SEQ ID NO: 16) and glutamic acid (SEQ ID NO: 17)) showed the highest cytotoxic activity and cytokine production. Table 4 Cytotoxicity of CAR T cells at 72 hours and cytokine production activity at 24 hours after exposure to BCMA ligand (MM1S). [Table 4]

[0217] Therefore, CAR T cells generated using IVT mRNA encoding the CARs of this disclosure (SEQ ID NOs: 14, 15, 16, and 17) provide superior cytotoxicity and cytokine production compared to wild-type CAR T constructs.

[0218] Example 2. A series of experiments were conducted to test how changes in the number of alanine residues in the lysine residues of the CAR CD3 zeta intracellular domain affect CAR's resistance to downregulation by target binding.

[0219] Numerous IVT mRNA constructs encoding variations of the anti-BCMA CAR protein were prepared, each identical except for the intracellular polypeptide sequence encoding the signaling domain. The constructs encoded CARs containing one of the following: wild-type CD3 zeta intracellular domain sequence (SEQ ID NO: 18), CD3 zeta intracellular domain sequences with one lysine residue mutated to alanine (SEQ ID NOs: 19-27), CD3 zeta intracellular domain sequences with 2-7 lysine residues mutated to alanine (SEQ ID NOs: 28-33), CD3 zeta intracellular domain sequences with all but one (i.e., 8) lysine residues mutated to alanine (SEQ ID NOs: 34-42), or CD3 zeta intracellular domain sequences with all nine lysine residues mutated to alanine (SEQ ID NO: 43). These constructs were otherwise identical with respect to the cap, 5'UTR, open reading frame encoding the signal peptide, scFv and transmembrane sequence, 3'UTR, and poly(A) tail. The contribution of individual lysines or lysine amino acid aggregates to the downregulation of CAR after exposure to the CAR ligand (BCMA) was investigated.

[0220] For each mRNA construct, CAR T cells were prepared substantially as described in Example 1. Twenty-four hours after transfection, CAR T cells generated from each mRNA construct were either exposed to MM1S or left unexposed to target cells, as described in Example 1. After 24 hours, the cells were evaluated for cell number, viability, maintenance of CAR expression, and cytokine production, as described in Example 1. The constructs and their effects on downregulating CAR expression are shown below.

[0221] CAR T cells generated using all test constructs showed good viability after electroporation (viability >70%). All CAR T cells showed high anti-BCMA CAR expression rates after electroporation (>85%). 24-hour exposure of CAR T cells to the ligand BCMA on MM1S myeloma target cells resulted in downregulation of CAR expression. CAR T cells generated using IVT mRNA containing a wild-type intracellular polypeptide sequence (SEQ ID NO: 18) showed downregulation of CAR expression from 6,358 MFIs to 705 MFIs, an 89% decrease in CAR expression. In contrast, CAR T cells generated using IVT mRNA in which all nine intracellular lysines were mutated to alanine (SEQ ID NO: 43) showed downregulation of CAR expression from 8,264 MFIs to 3,165 MFIs, and maintained 38% CAR expression after exposure to the BCMA ligand. CAR T cells generated using constructs containing individual CD3 zeta-intracellular lysine-alanine mutations (SEQ ID NOs: 19-27) showed downregulation of CAR expression from 5,707 ± 609 MFI in the absence of ligand to 617 ± 119 MFI in the presence of BCMA+ MM1S myeloma cells (mean ± SD of all constructs) (Figure 2A). This decrease in anti-BCMA CAR expression represented an 89% ± 3% reduction in CAR expression, and was therefore comparable to the 89% reduction in CAR expression observed with the wild-type construct (SEQ ID NO: 18) (Figure 2A). CAR T cells generated using constructs containing CD3 zeta intracellular lysine-alanine mutations in 8 of the 9 lysines (SEQ ID NOs: 34-42) showed downregulation of CAR expression from 6,736 ± 299 MFI in the absence of ligand to 2,064 ± 234 MFI in the presence of BCMA+ MM1S myeloma cells (mean ± SD of all constructs) (Figure 2C). After exposure to the BCMA ligand, anti-BCMA CAR expression remained at 31% ± 3% of the CAR expression observed in the absence of ligand, showing a similar expression pattern to CAR cells generated using IVT mRNA in which all lysines in the CD3 zeta intracellular domain were mutated to alanine (SEQ ID NO: 43) (Figure 2A).CAR T cells generated using IVT mRNA constructs (SEQ ID NOs. 28-33) containing 2-7 lysine-alanine intracellular lysine-alanine mutations in the CD3 zeta molecule showed intermediate effects, with 25% ± 6% of the MFI of anti-BCMA CARs remaining after exposure to BCMA ligands compared to culture in the absence of ligands (Figure 2E). A similar trend was observed in interferon-γ production from CAR T cells generated using constructs containing CD3 zeta molecule intracellular domains with varying numbers of lysine-alanine mutations. CAR T cells generated using the wild-type IVT mRNA construct (SEQ ID NOs. 18) produced 2,291 pg / mL of interferon-γ after co-culture with MM1S (Figure 2B). CAR T cells generated using the 9-lysine-alanine mutant construct (SEQ ID NOs. 43) produced 7,853 ± 285 pg / mL of interferon-γ in co-culture (Figure 2B). CAR T cells generated using constructs containing a CD3 zeta intracellular domain with one lysine-alanine mutation (sequence numbers 19-27), similar to wild-type IVT mRNA (sequence number 18), produced 2,498 ± 583 pg / mL of interferon-γ during co-culture with MM1S (Figure 2B). CAR T cells generated using constructs containing a CD3 zeta intracellular domain with eight lysine-alanine mutations (sequence numbers 34-42) produced 4,871 ± 739 pg / mL of interferon-γ during co-culture with MM1S. This level was higher than that of the wild-type construct but lower compared to the fully mutant construct (sequence number 43) (Figure 2D). Finally, constructs with intermediate lysine-alanine mutations of number 2–7 (SEQ ID NOs. 28–33) generated CAR T cells producing an average of 4,241 ± 1,279 pg / mL of interferon-γ during co-culture with MM1S, which showed intermediate expression levels compared to the wild-type (SEQ ID NOs. 18) and fully mutant (SEQ ID NOs. 43) constructs (Figure 2F). Table 5 Expression of anti-BCMA CARs by CAR T cells expressing CARs containing the intracellular domain of CD3 zeta with lysine-alanine mutations, after culturing with or without MM1S target cells. [Table 5] Table 6 Interferon-γ production by anti-BCMA CAR T cells expressing CARs containing a CD3 zeta intracellular domain lysine-alanine mutation in the presence of MM1S target cells. [Table 6]

[0222] In summary, the improvement in resistance to downregulation brought about by mutations in the lysines of the CD3 zeta intracellular domain of anti-BCMA CARs was optimal when all nine lysines were mutated. Mutation of any one lysine alone did not result in resistance to downregulation of the CAR in the presence of BCMA ligands. Mutation of any eight of the nine lysines resulted in resistance to downregulation similar to that of the completely mutated construct. Intermediate numbers of mutations showed intermediate effects. Mutation (substitution) of 6, 7, 8, or 9 lysines as a whole resulted in substantial improvement proportional to the number of such mutations (substitutions).

[0223] Example 3. A series of experiments were conducted to test how substituting lysine residues in the CD3 zeta intracellular domain of CAR with different amino acids affects CAR's resistance to downregulation by target ligands.

[0224] Numerous IVT mRNA constructs encoding variations of the anti-BCMA CAR protein were prepared. These constructs were identical except for the intracellular polypeptide sequence encoding the signaling domain. The constructs encoded CARs containing either the wild-type CD3 zeta intracellular polypeptide sequence (SEQ ID NO: 18) or the CD3 zeta intracellular domain sequence (SEQ ID NOs: 43-56) in which all lysine residues were mutated to one of several different amino acids. A total of 14 different amino acid variants (Ala, Val, Ile, Leu, Met, Phe, Tyr, Ser, Thr, Asn, Gln, His, Asp, Glu) were tested. These IVT mRNA constructs were otherwise identical with respect to the cap, 5'UTR, open reading frame encoding the signal peptide, scFv and transmembrane sequence, 3'UTR, and poly(A) tail. The objective was to compare the effects of different amino acid substitutions (mutations) on the preservation of CAR expression, maintenance of CAR signaling, and resistance of the protein to ligand-mediated downregulation after exposure to the CAR ligand (BCMA).

[0225] For each IVT mRNA construct, CAR T cells were prepared substantially as described in Example 1. Twenty-four hours after transfection, CAR T cells generated from each mRNA construct were either exposed to MM1S or left unexposed to target cells using the method described in Example 1. After 24 hours, the cells were evaluated for cell number, viability, maintenance of CAR expression, and cytokine production using the method described in Example 1.

[0226] CAR T cells generated using all test constructs showed good viability after electroporation (>70% viability). All tested constructs were able to drive anti-BCMA CAR expression. In the absence of BCMA ligands, CAR expression by CAR T cells expressing constructs with lysine-phenylalanine mutations (SEQ ID NO: 48;340 MFI), lysine-tyrosine mutations (SEQ ID NO: 49;177 MFI), and lysine-isoleucine mutations (SEQ ID NO: 45;1,634 MFI) was lower than that of the wild-type construct (SEQ ID NO: 18;4,084 MFI) (Figure 3A). Conversely, CAR expression on CAR T cells expressing constructs with lysine-aspartate mutations (SEQ ID NO: 55; MFI of 7,562), lysine-glutamate mutations (SEQ ID NO: 56; MFI of 8,346), and lysine-alanine mutations (SEQ ID NO: 43; MFI of 5,656) was higher than that of the wild-type construct (SEQ ID NO: 18) (Figure 3A). Other constructs tested showed CAR expression comparable to the wild-type construct (Figure 3A). All mutant CAR constructs showed improved maintenance of anti-BCMA CAR expression after ligand exposure compared to the wild-type construct (11%) (32%–53%). Of these constructs, IVT mRNA containing SEQ ID NO: 55 (lysine-aspartate intracellular domain mutation) or SEQ ID NO: 56 (lysine-glutamate intracellular domain mutation) showed the highest maintenance rate of CAR expression after BCMA ligand exposure (52%) (Figure 3A). CAR T cells generated using all tested constructs were capable of ligand-dependent signaling and produced interferon-γ at levels of 1,729–7,853 pg / mL during co-culture with MM1S target cells (Figure 3B). The highest levels of interferon-γ production were observed when using IVT mRNA constructs containing SEQ ID NO: 55 (lysine-aspartate), SEQ ID NO: 56 (lysine-glutamate), or SEQ ID NO: 43 (lysine-alanine) (Figure 3B). Table 7 Expression of anti-BCMA CARs by CAR T cells expressing CARs containing an intracellular domain of CD3 zeta with lysine mutated to another amino acid, after culturing with or without MM1S target cells. [Table 7] Table 8 Cytokine (interferon-γ) production by anti-BCMA CAR T cells that express CARs containing lysine residues in the intracellular domain of CD3 zeta cells mutated to other amino acids after being cultured in the presence of MM1S target cells. [Table 8]

[0227] In summary, the improved resistance to downregulation resulting from lysine mutations in the intracellular domain of the CD3 zeta of anti-BCMA CARs was compatible with mutations to various different amino acids, including alanine, valine, isoleucine, leucine, methionine, serine, threonine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid. CAR constructs in which the lysine amino acid was mutated to aspartic acid, glutamic acid, or alanine were found to be BCMA + The study showed the highest conservation of CAR expression and the highest level of signal transduction upon exposure to target cells.

[0228] Example 4. A series of experiments were conducted to test how incorporating an additional costimulatory domain into the intracellular portion of the lysine-alanine mutant CAR receptor affects CAR's resistance to downregulation by the target ligand BCMA.

[0229] A series of IVT mRNA constructs encoding variations of anti-BCMA CAR proteins were prepared. These constructs were identical except for the intracellular polypeptide sequence encoding the intracellular domain. These constructs encoded proteins containing one of the following: a wild-type intracellular CD3 zetapolypeptide sequence (SEQ ID NO: 18), or an intracellular CD3 zetapolypeptide sequence in which all lysine residues have been mutated to alanine (SEQ ID NO: 43), or an intracellular CD3 zetapolypeptide sequence in which all lysine residues have been mutated to alanine, plus a preceding co-stimulatory sequence, namely: a wild-type CD28 co-stimulatory domain (SEQ ID NO: 57), a CD28 co-stimulatory domain in which lysine has been mutated to alanine (SEQ ID NO: 58), a wild-type 41BB co-stimulatory domain (SEQ ID NO: 59), or a 41BB co-stimulatory domain in which all lysine has been mutated to alanine (SEQ ID NO: 60). The constructs were otherwise identical with respect to the cap, 5'UTR, open reading frame encoding the signal peptide, scFv and transmembrane sequence, 3'UTR, and poly(A) tail. Next, we tested the compatibility of wild-type or lysine-alanine mutant CD28 and 41BB costimulatory domains to preserve CAR expression, maintain CAR signaling, and provide resistance of the CAR protein to ligand-mediated downregulation after exposure to CAR ligand (BCMA).

[0230] For each IVT mRNA construct, CAR T cells were prepared substantially as described in Example 1. Twenty-four hours after transfection, CAR T cells generated from each mRNA construct were either exposed to MM1S or left unexposed to target cells, as described in Example 1. After 24 hours, the cells were evaluated for cell number, viability, maintenance of CAR expression, cytotoxicity, and cytokine production, as described in Example 1. The constructs and their effects on downregulating CAR expression are described below.

[0231] CAR T cells generated using all test constructs showed high viability after electroporation. CAR expression in CAR T cells generated using constructs encoding only the intracellular CD3 zeta signaling domain, SEQ ID NO: 18 (wild-type), and SEQ ID NO: 43 (KA) showed maximum anti-BCMA CAR expression in the absence of the BCMA ligand (Figure 4A). Addition of the wild-type CD28 (SEQ ID NO: 57) or 41BB (SEQ ID NO: 59) signaling domain reduced CAR expression in the absence of the ligand (7,389 MFIs for SEQ ID NO: 43 compared to 896 and 2,886 MFIs) (Figure 4A). Co-stimulatory domain mutations due to lysine-to-alanine mutations (SEQ ID NOs: 58 and 60) further reduced anti-BCMA CAR expression in the absence of the ligand (625 and 114 MFIs, respectively) (Figure 4A). In the presence of ligands, CAR T cells generated using all constructs encoding proteins containing mutant lysine-alanine CD3 zeta signaling domains showed a higher retention rate of CAR expression on the cell surface after exposure to BCMA+ MM1S target cells (35%–75%) compared to construct SEQ ID NO: 18 (11%) containing wild-type CD3 zeta (Figure 4A). Similarly, CAR T cells generated using all constructs were capable of signal transduction and interferon-γ production upon exposure to MM1S target cells (Figure 4B). Interferon-γ production was highest with construct SEQ ID NO: 59, containing both the wild-type 41BB signaling domain and the KA mutant CD3 zeta signaling domain (9,101 pg / mL), followed by construct SEQ ID NO: 43, containing only the KA mutant CD3 zeta signaling domain (8,054 pg / mL) (Figure 4B). Interferon-γ production driven by constructs SEQ ID NOs. 57 and 58, which contain wild-type and KA mutant CD28 signaling domains (2,998 and 3,886 pg / mL, respectively), was higher than that of the wild-type CAR construct SEQ ID NO: 18 (2,291 pg / mL) (Figure 4B).The cytotoxicity of CAR T cells generated using separate constructs containing only CD3 zeta, CD28-CD3 zeta, and 41BB-CD3 zeta signaling domains was assessed by a 72-hour cytotoxicity assay against BCMA+ MM1S-GFP cells after pre-exposure of cells to MM1S. All tested constructs (SEQ ID NOs. 61, 43, 57, and 59) showed higher cytotoxicity against MM1S GFP target cells with an effector:target ratio of 1:2 compared to control T cells (Figure 4C).

[0232] At lower effector-to-target ratios, CAR T cells generated using SEQ ID NO: 43 (containing the KA CD3 zeta signaling domain) and SEQ ID NO: 59 (containing WT 41BB and the KA CD3 zeta signaling domain) showed high specific cytotoxicity against MM1S-GFP (67.4% and 51.4%, respectively, at an effector-to-target ratio of 1:32), which was 3–4 times higher than the values ​​observed with the wild-type construct (SEQ ID NO: 61, containing only the wild-type CD3 zeta signaling domain) (Figure 4C). Table 9 Anti-BCMA CAR expression by CAR T cells expressing a CD3 zeta intracellular domain in which lysine has been mutated to alanine, and a CAR containing a wild-type or mutant costimulatory domain in the intracellular domain. [Table 9] Table 10 Cytokine (interferon-γ) production by anti-BCMA CAR T cells expressing a CD3 zeta intracellular domain in which lysine has been mutated to alanine, and a CAR containing a wild-type or mutant costimulatory domain in the intracellular domain. [Table 10] Table 11 72-hour cytotoxicity of CAR T cells against MM1S-GFP cells after exposure to BCMA ligand (MM1S) [Table 11]

[0233] To summarize, we successfully combined a CAR construct encoding a protein containing a CD3 zeta intracellular signaling domain conferring resistance to ligand-dependent downregulation by lysine amino acid mutations with additional signaling domains from CD28 and 41BB. The addition of these domains did not prevent downregulation, even in the context of wild-type CD28 and 41BB signaling domain sequences. CAR constructs containing mutated CD28-CD3 zeta or 41BB-CD3 zeta intracellular signaling domains were able to efficiently drive CAR-dependent cytokine production and cytotoxicity.

[0234] Example 5. Experiments were conducted to test how the incorporation of a CD28 co-stimulatory domain into the intracellular portion of a lysine-alanine or lysine-glutamic acid mutant CAR receptor affects the resistance of the CAR to downregulation by the target BCMA.

[0235] A series of IVT mRNA constructs encoding variations of anti-BCMA CAR proteins were prepared. These constructs were identical except for the intracellular polypeptide sequence encoding the signaling domain. The control construct encoded a protein containing the wild-type intracellular CD3 zeta polypeptide sequence (SEQ ID NO: 18). Two series of test constructs encoded proteins containing either an intracellular CD3 zeta polypeptide sequence in which all lysine residues were mutated to alanine (SEQ ID NOs: 43, 57, 58, 62) or an intracellular CD3 zeta polypeptide sequence in which all lysine residues were mutated to glutamate (SEQ ID NOs: 56, 63, 64, 65). The constructs differed in the presence and sequence composition of the CD28 co-stimulatory domain in the encoded protein: two control constructs encoded proteins lacking the CD28 co-stimulatory domain (SEQ ID NOs. 43, 56); two constructs encoded proteins containing the wild-type CD28 co-stimulatory domain (SEQ ID NOs. 57, 63); two constructs encoded proteins containing the CD28 co-stimulatory domain with a lysine residue mutated to alanine (SEQ ID NOs. 58, 64); and two constructs encoded proteins containing the CD28 co-stimulatory domain with a lysine residue mutated to glutamate (SEQ ID NOs. 62, 65). The constructs were otherwise identical with respect to the cap, 5'UTR, open reading frame encoding the signal peptide, scFv and transmembrane sequence, 3'UTR, and poly(A) tail.

[0236] This experiment evaluated the ability of second-generation CARs, including lysine-alanine or lysine-glutamate mutant CD28-CD3 zeta intracellular polypeptides, to maintain CAR expression, preserve CAR signaling, and provide resistance of CAR proteins to ligand-mediated downregulation after exposure to CAR ligands (BCMAs).

[0237] For each IVT mRNA construct, CAR T cells were prepared substantially as described in Example 1. Twenty-four hours after transfection, CAR T cells generated from each mRNA construct were either exposed to MM1S or left unexposed to target cells, as described in Example 1. After 24 hours, the cells were evaluated for cell number, viability, maintenance of CAR expression, cytotoxicity, and cytokine production, as described in Example 1. The constructs and their effects on downregulating CAR expression are described below.

[0238] CAR T cells generated using all test constructs showed high viability after electroporation. CAR expression in CAR T cells generated using constructs encoding proteins including the intracellular CD3 zeta signaling domain, SEQ ID NO: 18 (wild-type), SEQ ID NO: 43 (KA), and SEQ ID NO: 56 (KE) showed high anti-BCMA CAR expression in the absence of BCMA ligands. Addition of the CD28 costimulatory domain resulted in decreased CAR expression compared to the CD3 zeta-only construct (16,848 MFIs for SEQ ID NO: 43 compared to 2,507-3,306 MFIs for SEQ ID NO: 57, 58, and 62 in the context of lysine-alanine mutations, and 24,850 MFIs for SEQ ID NO: 56 compared to 12,784-13,598 MFIs for SEQ ID NO: 63, 64, and 65 in the context of lysine-glutamate mutations) (Figure 5A). In the presence of BCMA, CAR T cells generated using constructs encoding proteins containing mutant lysine-alanine CD3 zeta signaling domains (SEQ ID NOs. 43, 57, 58, 62) or lysine-glutamate CD3 zeta signaling domains (SEQ ID NOs. 56, 63, 64, 65) showed higher retention rates of CAR expression on the cell surface after exposure to BCMA+ MM1S target cells (6–26% and 12–47%, respectively) compared to construct SEQ ID NO. 18 containing wild-type CD3 zeta (2%) (Figure 5A). Maintenance of CAR expression after exposure to BCMA ligands was highest in CAR T cells generated using constructs encoding proteins containing lysine-glutamate mutations in the intracellular domain of CD3 zeta (including those without a costimulatory domain (SEQ ID NO: 56), or those with a CD28 costimulatory domain where the lysine residue is mutated to alanine (SEQ ID NO: 63), or a costimulatory domain where the lysine residue is mutated to glutamate (SEQ ID NO: 64)), showing surface CAR expression maintenance rates of 47%, 30%, and 31%, respectively (Figure 5A). Cytotoxicity of CAR T cells generated using constructs containing only CD3 zeta and those containing the CD28-CD3 zeta signaling domain was assessed by a 72-hour cytotoxicity assay against BCMA+ MM1S-GFP cells after pre-exposure of cells to MM1S.All constructs tested (SEQ ID NOs. 43, 56, 57, 58, 62, 63, 64, and 65) showed high cytotoxicity against MM1S GFP-targeted cells with an effector:target ratio of 1:2 compared to control T cells. At lower effector:target ratios, CAR T cells generated using SEQ ID NO: 43 (containing only the KA CD3 zeta signaling domain), SEQ ID NO: 56 (containing only the KE CD3 zeta signaling domain), SEQ ID NO: 63 (containing both the KA CD28 signaling domain and the KE CD3 zeta signaling domain), and SEQ ID NO: 64 (containing both the KE CD28 signaling domain and the KE CD3 zeta signaling domain) conferred high levels of specific cytotoxicity against MM1S-GFP (89.4%, 98.4%, 72.7%, and 70.2%, respectively, at 1,8 effector:target ratios), which were 9–13 times higher than the values ​​observed with the wild-type construct (SEQ ID NO: 18, containing only the wild-type CD3 zeta signaling domain) (7.5%) (Figures 5B and 5C). Table 12 Expression of anti-BCMA CARs by CAR T cells expressing a CD3 zeta intracellular domain in which lysine is mutated to alanine or glutamate, and a CAR containing a wild-type or mutant costimulatory domain in the intracellular domain. [Table 12] Table 13 72-hour cytotoxicity of CAR T cells against MM1S-GFP cells after exposure to BCMA ligand (MM1S) [Table 13]

[0239] In summary, a CAR construct encoding a CD3 zeta intracellular signaling domain conferred resistance to ligand-dependent downregulation through lysine amino acid mutations was combined with a CD28 signaling domain. Addition of the CD28 signaling domain to the lysine-glutamate mutant CD3 zeta intracellular signaling domain was consistent with preventing BCMA ligand-mediated downregulation of the CAR. This effect was most pronounced when CD28 lysine was mutated to alanine or glutamate. In contrast, addition of the CD28 signaling domain to the lysine-alanine mutant CD3 zeta intracellular signaling domain dramatically reduced the prevention of BCMA ligand-mediated downregulation of the CAR. Therefore, mutations such as lysine to glutamate throughout the entire CD28-CD3 zeta intracellular sequence result in a CAR with high resistance to BCMA ligand-dependent downregulation.

[0240] Example 6. A series of experiments were conducted to investigate how downregulation-resistant anti-BCMA CARs, containing lysine-alanine mutations in their intracellular signaling domains, activate CAR T cells and drive effector function in response to normal plasmablasts and plasmacytoid dendritic cells (pDCs).

[0241] In autoimmune diseases such as SLE, plasmablasts and further differentiated plasma cells produce autoantibodies that contribute to the pathogenesis, while pDCs are activated by inflammatory triggers such as immune complexes and drive the pathogenic immune response through cytokine production and T cell stimulation. We determined the ability of downregulatory resistant anti-BCMA CAR to drive CAR-specific T cell activation in autologous plasmablasts and pDCs and to modulate the immune response.

[0242] IVT mRNA constructs encoding an anti-BCMA CAR protein with an intracellular CD3 zetapolypeptide sequence (SEQ ID NO: 43) in which all lysine residues were mutated to alanine were prepared. For this construct, CAR T cells were prepared substantially as described in Example 1. Plasmablasts and pDCs were isolated from whole blood of the same (autologous) donor by isolation using antibody magnetic beads (Plasma Cell Isolation Kit II and Diamond Plasmacytoid Dendritic Cell Isolation Kit II, human, Miltenyi). The isolated plasmablasts and pDCs were stained with characteristic cell surface markers CD38 / CD138 and CD123, respectively. Co-cultures were established using BCMA CAR T (SEQ ID NO: 43) or CAR-free control CD8+ T cells, and each of the autologous plasmablast and pDC populations (7.5K T cells and 1.5K plasmablasts for plasmablasts, and 10K T cells and 10K pDCs for pDCs). The cultures were incubated overnight at 37°C, and the following day, the activation of CAR T cells or control cells using the activation-inducing markers CD69 and CD137 (41BB) by flow cytometry, and the production of interferon-γ in the supernatant by ELISA were analyzed.

[0243] BCMA CAR T cells generated using IVT mRNA encoding a protein containing the sequence of SEQ ID NO: 43 showed upregulation of CD69 and 41BB in response to exposure to blasts and pDCs (in blast co-culture, CD69: 0.93 - 12.74%, 41BB: 3.92 - 23.93%; in pDC co-culture, CD69: 2.65 - 34.05%, 41BB: 1.92 - 28.99%) (Figure 6A and Figure 6B). Similarly, interferon γ was also produced in the supernatant of the co-cultures (187 pg / mL in blast co-culture, 5 pg / mL in pDCs co-culture). In contrast, in control cells lacking IVT mRNA, upregulation of activation markers in response to blasts and pDCs was not observed (CD69+ 2.45% and 41BB+ 2.28% in blast co-culture, CD69+ 12.98% and 41BB+ 2.36% in pDC co-culture), and interferon γ remained at a concentration 5.25-fold lower compared to BCMA CAR T (7.3 pg / mL in blast co-culture, <LOD in pDC co-culture) (Figure 6A and Figure 6B). Thus, CAR T cells expressing downregulation-resistant CARs are activated in a CAR-dependent manner in the context of blasts and pDCs and produce immunomodulatory cytokines of the types known to control autoimmune diseases.

[0244] Example 7. In this example, for each of multiple CAR proteins expressed in CAR T cells (and thus each targeting CD19, PSMA, and CCR4 individually), experiments and processes for conferring resistance to downregulation by mutating intracellular lysine amino acid residues to alanine or glutamic acid are described.

[0245] Prepare a series of mRNA constructs, each containing an scFv specific to one of the following cell surface targets: BCMA, CD19, PSMA, or CCR4, and encoding a variation of the target CAR protein. For each of these cell surface targets, construct an mRNA construct encoding the CAR containing one of the following: wild-type intracellular CD3 zetapolypeptide sequence (SEQ ID NO: 18), wild-type intracellular CD28-CD3 zetapolypeptide sequence (SEQ ID NO: 66), intracellular CD3 zetapolypeptide sequence with all lysine residues mutated to alanine (SEQ ID NO: 43), intracellular CD3 zetapolypeptide sequence with all lysine residues mutated to glutamate (SEQ ID NO: 56), or intracellular CD28-CD3 zetapolypeptide sequence with all lysine residues mutated to glutamate (SEQ ID NO: 65). These constructs are otherwise identical with respect to the cap, 5'UTR', 3'UTR, and polyA tail.

[0246] For each mRNA construct, CAR T cells are prepared substantially as described in Example 1. Approximately 24 hours after transfection, CAR T cells produced from each mRNA construct are either exposed to different target cell lines (MM1S for BCMA CAR; Raji for CD19 CAR; LNCaP for PSMA CAR; and CEM for CCR4 CAR) or left unexposed to target cells, as described in Example 1. After 24 hours, the cells are evaluated for cell number, viability, and CAR expression as described in Example 1 (using BCMA-APC for BCMA CAR, FMC63 antibody for CD19 CAR, PSMA-PE for PSMA CAR, and anti-idiotype antibody for CCR4). The pre-exposed CAR T cells are then evaluated for their ability to perform effector functions (cytotoxicity and interferon-γ production) in response to additional target cell lines, as described substantially in Example 1.

[0247] CAR T cells generated using IVT mRNA that exhibits specificity for BCMA, CD19, PSMA, and CCR4, and contains lysine-alanine or lysine-glutamate mutations in the intracellular signaling domains of CD3 zeta or CD28-CD3 zeta, are expected to exhibit less downregulation, higher cytotoxicity, and greater cytokine production compared to their wild-type counterparts.

[0248] Example 8. This example describes experiments and processes for controlling tumor loading in mice using CAR T cells generated with IVT mRNA encoding a BCMA CAR resistant to ligand-induced downregulation. Different mRNA CAR constructs contain sequences of SEQ ID NOs. 17, 67, 98, or 99, respectively. These mRNA constructs encode CAR proteins containing sequences of SEQ ID NOs. 5, 68, 101, or 102, respectively.

[0249] CAR T cells are prepared by transfecting human CD8+ cells with an mRNA CAR construct as described in Example 1, except that the endogenous T cell receptor is genetically removed from the cells by CRISPR / Cas engineering. Negative controls used in this example include control CD8+ cells without IVT mRNA. NOD-scid-gamma (NSG) mice are inoculated with 2 million MM1S-fluc human multiple myeloma cells. Tumor burden is monitored by serial bioluminescence imaging. On day 5, mice are randomly assigned to receive intravenous administration of either control CD8+ T cells or CAR T cells transfected to express CAR SEQ ID NO: 5 or 68. Tumor burden is measured daily, and a reduction in tumor burden over time is observed in mice treated with vehicle or control CD8+ cells compared to CAR T cells transfected to express CAR SEQ ID NO: 5 or 68. CAR T cells generated using (SEQ ID NO: 5 or 68) are expected to significantly control tumor burden and suppress tumor growth.

[0250] In conclusion, CAR T cells prepared from three examples of the mRNA construct encoding the anti-BCMA CAR of the present invention (and the corresponding CAR protein) are expected to suppress tumor growth of human myeloma in a predictive animal model.

[0251] Example 9. In this example, experiments and processes for expressing one or more downregulation-resistant CAR proteins in T cells using carriers and nucleic acids other than IVT mRNA will be described.

[0252] CAR T cells are prepared and modified using the Sleeping Beauty transposon system to express the CAR proteins of this disclosure. A “wild-type CAR” plasmid is constructed containing the EFla promoter, IgG 5'UTR, an open reading frame encoding the amino acid sequence of SEQ ID NO: 1, and a polyadenylated sequence, where the above elements are collectively flanked by reverse-terminal repeats of the Sleeping Beauty transposon. A “KE CAR” plasmid (where KE refers to an amino acid substitution) is constructed containing the EFla promoter, IgG 5'UTR, an open reading frame encoding the amino acid sequence of SEQ ID NO: 5, and a polyadenylated sequence, where the above elements are collectively flanked by reverse-terminal repeats of the Sleeping Beauty transposon. The “SB11” transposase plasmid is constructed containing the EFI a promoter, IgG 5'UTR, a Kozak consensus sequence, an open reading frame encoding SB II, and a polyadenylated sequence. To generate wild-type CAR T cells, peripheral blood mononuclear cells from a normal human donor are washed and resuspended in P3 buffer (Lonza) in the presence of both the wild-type CAR transposon plasmid and the SB11 plasmid. Using the same strategy, lysine-mutated CAR T cells are generated using the KE CAR plasmid and the SB11 plasmid. Cells are electroporated (4D Nucleofector, Lonza) and plasmid(s) are introduced into the cells. Cells are then transferred to culture and stimulated with CD3 / CD28 Dynabeads® (ThermoFisher). Proliferating CD4+ and CD8+ T cells are analyzed for CAR expression by staining with BCMA-APC reagent and flow cytometry. T cells are expected to proliferate for at least 14 days and express anti-BCMA CAR protein.

[0253] CAR expression and functional CAR T cell activity will be evaluated using the cytotoxicity assay with the multiple myeloma target cell line MMIS-GFP, as described in Example 1. CAR T cells generated using a transposon encoding the lysine-glutamate mutant intracellular signaling domain (SEQ ID NO: 5) are expected to show higher expression levels and greater toxicity in the cytotoxicity assay than CAR T cells generated using a transposon encoding wild-type CAR.

[0254] Example 10. This example describes a strategy for eradicating myeloma cells and treating the disease in patients with multiple myeloma (MM) using anti-BCMA CAR T cells expressing one or more of the CAR proteins of this disclosure.

[0255] Anti-BCMA CAR T cells are prepared substantially according to the method of Example 1 using the CAR protein of this disclosure containing sequence numbers 3, 4, 5, 68, 101, or 102. 0.2–100 × 10⁶ anti-BCMA CAR T cells of this disclosure are administered to MM patients (optionally prepared using lymphodermectomy). 9 The CAR is injected in small quantities. Serum M protein levels, free light chains of MM-associated immunoglobulins, soluble serum BCMA levels, peripheral blood CAR+ T cell count, serum cytokine levels (e.g., interferon-γ, IL-2, IL-10), and bone marrow biopsy are analyzed before treatment and at weeks 2, 4, 8, 12, and 24 after treatment. CAR T cells generated using the CAR described herein are expected to effectively reduce and / or eradicate MM, as measured by the reduction in serum M protein levels, free light chains of MM-associated immunoglobulins, soluble serum BCMA levels, and MM cells in bone marrow biopsy.

[0256] Example 11. This example describes experiments and processes for controlling autoimmune diseases using anti-BCMA CAR T cells expressing one or more of the CAR proteins of this disclosure. Anti-BCMA CAR T cells are prepared substantially according to the method of Example 1 using CAR receptors containing sequence numbers 3, 4, 5, 68, 101, or 102. 0.2–100 × 10⁶ anti-BCMA CAR T cells of this disclosure are administered to MG patients (optionally prepared using lymphodermectomy). 9 The cells are injected individually. Clinical assessments of the disease, such as anti-autoantigen antibodies (e.g., anti-AChR or anti-MUSK), soluble serum BCMA levels, peripheral blood CAR+ T cell count, serum cytokine levels (e.g., TNF, IL-6, IL-2, interferon-γ, IL-10), and myasthenia gravis daily living scale (MG-ADL), are assessed in patients at weeks 2, 4, 8, 12, 24, and 52 post-treatment. CAR T cells generated using the CARs of this disclosure are expected to effectively control autoimmune diseases, which are measured by the reduction of clinical symptoms of the disease, as determined by a decrease in autoantibody levels, a decrease in circulating cytokine concentrations, and a decline in MG-ADL or other clinical scores.

[0257] Example 12. This example describes an experiment testing how substituting glutamate residues in the CD3 zeta and CD28 intracellular domains of anti-CD19 CARs affects resistance to downregulation by the target ligand CD19.

[0258] A series of IVT mRNA constructs encoding variations of a CAR protein having an scFv targeting CD19 were prepared. The constructs contained either wild-type intracellular CD3 zeta and wild-type intracellular CD28-CD3 zeta polypeptide sequences (SEQ ID: 18 and 66), or an intracellular CD28-CD3 zeta polypeptide sequence in which all lysine residues were mutated to glutamic acid (SEQ ID: 56 and 65). The anti-CD19 scFv was derived from clone FMC63 (scFv1: SEQ ID: 87), or an anti-CD19 scFv having a tyrosine to alanine mutation at position 261, as described in He et al. (doi.org / 10.1126 / sciimmunol.adf1426) (scFv2: SEQ ID: 88). These constructs were identical otherwise with respect to the cap, 5’UTR, 3’UTR, and polyA tail. Incorporating mutations of these lysine residues into glutamic acid residues of the intracellular tail is a strategy to protect CARs with different target specificities from downregulation by their specific ligands.

[0259] For each mRNA construct, CAR T cells were prepared as described in Example 1. Approximately 24 hours after transfection, the CAR T cells generated from each mRNA construct were exposed to CD19 + Raji target cells or not exposed to the target cells, by the method described in Example 1. After 24 hours, the cells were evaluated for CAR expression (using an anti-Myc-Alexa 647 antibody, an extracellular tag on the CAR) by the method described in Example 1. The CAR T cells were also evaluated substantially by the method described in Example 1 for their ability to exert cytotoxicity and effector function (IFN-γ production) in response to the CD19+ Raji cell line.

[0260] CAR T cells generated using IVT mRNA containing KE mutant signaling domains SEQ ID NOs. 89 and 90 (KE CD3 zeta and CD28) showed the highest anti-CD19 CAR expression in the absence of the CD19 ligand. In the presence of the ligand, CAR T cells generated using all constructs containing mutant lysine-glutamine CD3 zeta and CD28 signaling domains (SEQ ID NOs. 89 and 90) showed a higher retention rate of CAR expression on the cell surface after exposure to Raji target cells (30-50%) compared to those observed with comparator constructs containing wild-type CD3 zeta (SEQ ID NOs. 91 and 92) (4-5%). CAR T cells generated using constructs containing mutant intracellular CD3 and CD28 domains had higher cytolytic activity (Table 14). CAR T cells generated using all constructs were able to signal and produce interferon-γ when exposed to Raji target cells at a 1:2 E:T ratio (Table 15). Interferon-γ production was highest with construct SEQ ID NO: 90, an anti-CD19 CAR of scFv2 containing KE mutant CD28 and CD3 zeta signaling domain (614 pg / mL), followed by construct SEQ ID NO: 89, an anti-CD19 CAR of scFv1 containing mutant CD28 and CD3 zeta signaling domain (393 pg / mL) (Table 15). Table 14 CD19 + Expression of anti-CD19 CAR by CAR T cells expressing a CAR containing a CD28-CD3 zeta intracellular domain, having a wild-type sequence or a mutant sequence containing glutamate-mutated lysine, after culture in the absence or presence of Raji target cells. [Table 14] Table 15 Cytotoxicity (effector vs. target: 1:2) on CD19+ Raji cells by anti-CD19 CAR T cells expressing CARs containing wild-type or mutant CD28-CD3 zeta intracellular domains. [Table 15] Table 16 CD19 induced by anti-CD19 CAR T cells expressing CARs containing wild-type or mutant CD28-CD3 zeta intracellular domains. + Cytokine (interferon-γ) production after exposure to Raji cells [Table 16]

[0261] In summary, we successfully generated a CAR construct containing a CD28-CD3 zeta intracellular signaling domain that exhibits resistance to ligand-dependent downregulation due to lysine amino acid mutations, using a CD19-targeting scFv. The mutated anti-CD19 CAR T cells showed CAR-dependent cytokine production and cytolytic activity. CAR T cells generated using a CAR containing a KE mutation in the intracellular domain showed resistance to CD19 + In the presence of Raji cells, they exhibited resistance to ligand-mediated downregulation and superior effector function (cytokines) compared to their wild-type counterparts.

[0262] Example 13. This example describes an experiment to test how tumor loading is regulated in vivo using CAR T cells generated with IVT mRNA encoding a BCMA CAR in which the lysine residue in the intracellular signaling domain has been mutated to glutamate, thereby conferring resistance to ligand-induced downregulation. These mRNA CAR constructs contain the sequences (SEQ ID NOs. 17 and 67). These mRNA structures encode CAR proteins containing the sequences (SEQ ID NOs. 5 and 68), respectively.

[0263] CAR T cells were prepared by transfecting human CD8+ T cells with mRNA CAR constructs as described in Example 1, except that the endogenous T cell receptor was genetically removed from the cells by CRISPR / Cas engineering. Negative control cells without IVT mRNA were prepared using the same batch of TCR knockout CD8+ cells. + Prepared from T cells. Immunodeficient Nod-Scid IL2R-γ knockout (NSG) mice were inoculated with 2 million MM1S-fluc human multiple myeloma cells on day 0. Tumor burden was monitored by continuous bioluminescence imaging. On day 5, mice were randomly assigned to treatment groups. On day 6, mice were inoculated with 2 million control CD8 cells. + Two million CAR T cells, or two million CAR T cells engineered to express CAR SEQ ID NO: 5, or two million CAR T cells engineered to express CAR SEQ ID NO: 68, were administered intravenously. Tumor burden was measured on days 9 and 12. Tumor burden measured on day 9 by BLI (photons / second) was compared to the control CD8 + Compared to mice treated with T cells, mice treated with CAR cells expressing SEQ ID NO: 5 showed a 4.7-fold lower BLI score, and mice treated with CAR cells expressing SEQ ID NO: 68 showed a 7.9-fold lower BLI score. On day 12, BLI was observed to be reduced 10.8-fold (SEQ ID NO: 5) and 16.0-fold (SEQ ID NO: 68) compared to control mice. All comparisons showed high significance (p<0.001; two-way ANOVA on log-transformed data). Figures 7A-7B and Table 17 show individual mouse BLI data (Figure 7A) and summary statistics (Figure 7B). Table 17 Bioluminescence imaging of MM1S-fluc tumor loading in NSG mice treated with control CD8+ T cells or cells expressing anti-BCMA CARs containing lysine mutations in the intracellular signaling domain. [Table 17]

[0264] In summary, CAR T constructs containing either CD3 zeta alone or a CD28-CD3 zeta signaling domain with a lysine residue mutated to glutamate, as well as the sequences of SEQ ID NOs. 5 and 68, generated CART cells that potently suppressed BCMA+ tumor burden in vivo.

[0265] Example 14. This example describes an experiment to evaluate the activity and duration of CAR T cells generated using the IVT mRNA construct of this disclosure in vivo in a mouse model of multiple myeloma.

[0266] CAR T cells, as described in Example 1, use IVT mRNA CAR constructs with human CD8 + T cells were prepared by transfecting them. CAR T cells were generated using IVT mRNA encoding sequence number 68, which is a sequence encoded by IVT mRNA containing a CD28-CD3 zeta intracellular signaling domain (SEQ ID NO: 65) in which the lysine residue has been mutated to glutamate, and control CARs encoding sequence number 66, which contains the wild-type CD28-CD3 zeta intracellular signaling domain. Negative control cells without IVT mRNA were prepared using CD8 from the same batch. + Prepared from T cells. Immunodeficient Nod-Scid IL2R-γ knockout (NSG) mice were inoculated with 2 million MM1S-fluc human multiple myeloma cells on day 0. Tumor burden was monitored by continuous bioluminescence imaging. On day 8, mice were randomly assigned to treatment groups. On day 9, mice were intravenously administered either 2 million or 12.5 million of the following cells: control CD8 + T cells, CAR T cells engineered to express KE mutant CAR sequence number 65, and CAR T cells engineered to express wild-type CAR sequence number 66. Tumor burden was measured on days 11, 16, and 21. Pre-selected individual mice were harvested on days 10, 11, and 14, and CAR expression by transplanted cells in blood, spleen, and bone marrow was analyzed.

[0267] A single dose of 12.5 million anti-BCMA CAR T cells generated using IVT mRNA constructs containing the KE mutation in SEQ ID NO: 65 or the wild-type SEQ ID NO: 66 on day 9 suppressed tumor burden compared to control CD8+ T cells (tumor burden was reduced 21.4-fold and 12.4-fold, respectively, on day 21) (Figure 8A). The suppression of tumor burden was dose-dependent (Figure 8B). CAR T cells generated using the SEQ ID NO: 65 CAR construct containing a CD28-CD3 zeta signaling domain with a lysine residue mutated to glutamate showed better control of tumor burden than SEQ ID NO: 66 CAR T cells containing the wild-type CD28-CD3 zeta signaling domain (p<0.05 at 2 million cells; Figure 8B).

[0268] Analysis of whole blood and bone marrow from mice administered anti-BCMA CAR T cells containing KE mutation sequence number 65 identified human CAR T cells that expressed anti-BCMA CAR up to day 14 (Figure 8C).

[0269] In summary, the CAR T construct of sequence SEQ ID NO: 68, which contains a CD28-CD3 zeta signaling domain in which the lysine residue has been mutated to glutamate, is BCMA in vivo. + We generated CAR T cells that strongly suppressed tumor burden and demonstrated durable CAR expression in vivo.

[0270] Example 15. This example describes an experiment to test the ability of target T cells of an autoimmune disease to generate anti-BCMA CAR T cells expressing the downregulated resistant CAR protein of this disclosure.

[0271] Anti-BCMA CAR T cells were prepared from two myasthenia gravis donors using CAR receptors generated with SEQ ID NOs. 68 and 94 (anti-PSMA CAR as the negative control CAR) according to the method of Example 1. Vehicle EP was set as the negative EP control. Autologous plasma cells were isolated from the same two myasthenia gravis donors. 24 hours after transfection, CAR-T cells were co-cultured with either autologous plasma cells or malignant MM1S-GFP cells. CAR-T cells generated from MG donors were evaluated for their CAR expression (MFI) and cytolytic activity (i.e., cytotoxicity percentage, interferon-γ).

[0272] CAR T cells generated using the CARs of this disclosure expressed anti-BCMA CARs. Differences in CAR expression 24 hours post-EP were observed between donors (MFI = 1887 vs 3766). CAR T cells were able to effectively kill autologous plasma cells differentiated from MG patients in vitro, as measured by cytotoxicity (E:T = 1:2, 67.5% cytotoxicity for donor 1, 62% cytotoxicity for donor 2), whereas vehicle-transfected T cells or anti-PSMA control CARs resulted in lower killing rates (32% and 18% for donor 1, and 18% and -5.5% for donor 2). Differences between donors were also observed in interferon-γ cytokine production after cytotoxicity (9.82 pg / mL for donor 1, and 81.68 pg / mL for donor 2). The functionality of CAR T cells from MG donors was also evaluated using malignant MM1S-GFP cell lines. Effective killing of MM1S-GFP cells was observed in both donors (73% in donor 1 and 89.5% in donor 2), and cytokine production was also observed (217.71 pg / mL in donor 1 and 138.38 pg / mL in donor 2). No significant killing or cytokine production was observed in the control CAR T cell group. Table 18 Expression of an anti-BCMA CAR containing a CD28-figCD3 zeta intracellular domain with a mutant sequence containing glutamic acid-mutated lysine, and an anti-PSMA CAR as a control CAR. [Table 18] Table 19 Functionality of MG donor CAR T cells generated using a downregulated resistant CAR containing a mutant CD28-CD3 zeta intracellular signaling domain: relative to autologous plasma cells (n=2) [Table 19] Table 20 Functionality of MG donor CAR T cells generated using a downregulated resistant CAR containing a mutant CD28-CD3 zeta intracellular signaling domain: against malignant MM1S-GFP cells (n=2) [Table 20]

[0273] In summary, CAR T cells containing a downregulation-resistant signaling domain can be generated using CD8 T cells from patients with autoimmune diseases. These CAR-T cells potently express anti-BCMA CARs and can exert cytolytic activity against either autologous plasma cells or malignant MM1S cells with cytokine production.

[0274] Example 16. This example describes an experiment to further enhance CAR expression and CAR T cell functionality by incorporating a low-affinity scFv into the downregulation resistance signaling domain. Mutation of lysine residues in the intracellular domain with glutamate residues is a strategy to protect CARs with different target specificities from downregulation by their specific ligands. Incorporating a low-affinity scFv enhances this strategy, further improving CAR retention after target exposure.

[0275] A series of IVT mRNA constructs encoding variations of the CAR protein with two scFvs targeting BCMA were prepared. scFv1 had an apparent affinity KD of 3.2 nM, and scFv2 had an apparent affinity KD of 13.3 nM (Figure 10A). The constructs contained either wild-type intracellular CD3 zeta and wild-type intracellular CD28-CD3 zeta polypeptide sequences, or intracellular CD28-CD3 zeta polypeptide sequences in which all lysine residues were mutated to glutamate. These constructs were otherwise identical with respect to the cap, 5'UTR', 3'UTR, and polyA tail. For each mRNA construct, CAR T cells were prepared as described in Example 1. Approximately 24 hours after transfection, CAR T cells prepared from each mRNA construct were exposed to BCMA+ target cells (MM1S) or not, as described in Example 1. After 24 hours, CAR expression was evaluated as described in Example 1. CAR T cells also expressed BCMA + The ability to exert cytotoxicity in response to cell lines was also evaluated using essentially the method described in Example 1.

[0276] All generated CAR T cells showed the highest anti-BCMA expression in the absence of BCMA ligand, with CARs containing scFv1 and the mutant intracellular signaling domain CD3 zeta KE (including SEQ ID NO: 101, encoded by RNA SEQ ID NO: 98) showing the highest expression (MFI=5336). In the presence of BCMA ligand, CAR T cells generated using all constructs containing the mutant lysine-glutamine CD3 zeta signaling domain showed higher CAR retention on the cell surface after exposure to BCMA target cells compared to constructs containing wild-type CD3 zeta (10% and 13%) (26%, 46%, 24%, and 50%). Among these intracellular signaling domain mutant CAR T cells, cells containing low-affinity scFv2 showed higher retention of CAR expression after MM1S exposure compared to their high-affinity scFv1 counterparts (24% and 26%) (46% and 50%). CAR T cells generated using SEQ ID NO: 98 showed higher BCMA +Following exposure to target cells, CAR expression was maintained at its highest level. CAR T cells generated using constructs containing low-affinity scFv2 (sequence number 98 encoding the CAR of sequence number 101, and sequence number 99 encoding the CAR of sequence number 102) also exhibited higher cytolytic capacity (94% and 91%) compared to high-affinity scFv1 CAR T cells (40-58%). Table 21 Expression of anti-CD19 CARs by CAR T cells expressing CARs containing the intracellular domain of CD28-CD3 zeta, which has either a wild-type sequence or a mutant sequence in which lysine is mutated to glutamate, after culture in the absence or presence of BCMA+ MM1S cells. [Table 21] Table 22 48-hour cytotoxicity (effector vs. target: 1:4) on MM1S GFP cells by anti-CD19 CAR T cells expressing wild-type or mutant CD28-CD3 zeta intracellular domains and CARs containing scFvs of different affinity. [Table 22]

[0277] In summary, mutations in the CD28 and CD3 zeta signaling domains, along with modulation of scFv affinity to targets, demonstrate improved maintenance of CAR function using RNA CARs.

[0278] Example 16. The various substitutions and their respective lysine positions in this disclosure are shown below: [Table 23-1] [Table 23-2] [Table 23-3]

[0279] In this Specified, all publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) described in, for example, the sections of Background Art, Overview, Detailed Description, Examples, and / or References are incorporated herein by reference in whole, as if each individual publication, patent, patent application, publication, and database entry were incorporated herein by reference specifically and individually. In the event of any conflict, the present application, including the definitions herein, shall prevail.

[0280] Equivalents and range Those skilled in the art will recognize, or can verify by ordinary experimentation alone, many equivalents of the embodiments described herein. The scope of this disclosure is not intended to be limited to the foregoing description, but rather to the claims set forth in the appended claims.

[0281] Articles such as "a," "an," and "the" can mean one or more unless otherwise indicated or the context makes it clear. A claim or statement containing "or" between two or more members of a group is deemed satisfied if one, more, or all of the members of the group are present, unless otherwise indicated or the context makes it clear. A disclosure of a group containing "or" between two or more members of a group provides embodiments in which exactly one member of the group is present, embodiments in which two or more members of the group are present, and embodiments in which all members of the group are present. For the sake of brevity, these embodiments are not described separately herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or waived.

[0282] It should be understood that this disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more claims or relevant parts of the specification are introduced into another claim. For example, a claim dependent on another claim may be modified to include one or more limitations found in other claims dependent on the same basic claim. Furthermore, if a claim describes a composition, it should be understood that, unless otherwise specified or unless it is obvious to a person skilled in the art that this would result in a contradiction or inconsistency, it also includes a method of producing or using the composition according to either the manufacturing method or method of use disclosed herein, or any method known in the art.

[0283] When elements are presented as a list, for example in Markush group format, it should be understood that all possible subgroups of the elements are also disclosed, and any element or subgroup of an element may be removed from the group. It should also be noted that the term “comprising” is intended to be open, allowing for the inclusion of additional elements or steps. Generally, when an aspect, product, or method is referred to as including a particular element, feature, or step, it should be understood that an aspect, product, or method consisting of, or essentially consisting of, such element, feature, or step is also provided. For brevity, these aspects are not described individually herein, but it should be understood that each of these aspects is provided herein and may be specifically claimed or waived.

[0284] Where a range is indicated, it includes the endpoints. Furthermore, unless otherwise specified, or as is evident from the context and / or from the understanding of those skilled in the art, values ​​expressed as a range may, in some embodiments, take any specific value within the stated range to the same precision as one-tenth of the lower limit of the range, unless otherwise explicitly indicated by the context. For brevity, the values ​​of each range are not individually specified herein, but it will be understood that these values ​​are provided herein and may be specifically claimed or waived. Also, unless otherwise specified, or as is evident from the context and / or from the understanding of those skilled in the art, values ​​expressed as a range may take any subrange within a given range, where the endpoints of the subrange are expressed with the same precision as one-tenth of the lower limit of the range.

[0285] Furthermore, it should be understood that any particular aspect of the present invention may be expressly excluded from one or more claims. Where a range is indicated, any value within that range may be expressly excluded from one or more claims. Any aspect, element, feature, use, or aspect of the compositions and / or methods of this disclosure may be excluded from any one or more claims. For the sake of brevity, not all aspects from which one or more elements, features, uses, or aspects are excluded are expressly described herein.

[0286] manner The following embodiments are within the scope of this disclosure. 1. A protein capable of intracellular signal transduction, comprising a CD3 zeta intracellular domain, wherein the CD3 zeta intracellular domain is 80% identical to that of SEQ ID NO: 18, and wherein at least six lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine. 2. The protein according to embodiment 1, wherein the CD3 zeta intracellular domain is 100% identical to that of SEQ ID NO: 18, except for the substituted lysine amino acid. 3. The protein according to embodiment 1 or 2, wherein at least seven lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine. 4. The protein according to any one of embodiments 1 to 3, wherein at least eight lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine. 5. The protein according to any one of embodiments 1 to 4, wherein the nine lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine. 6. The protein according to any one of embodiments 1 to 5, wherein at least six lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 7. The protein according to any one of embodiments 1 to 6, wherein at least seven lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 8. The protein according to any one of embodiments 1 to 7, wherein at least eight lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 9. The protein according to any one of embodiments 1 to 8, wherein the nine lysine amino acids of SEQ ID NO: 18 are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 10. A protein comprising a CD3 zeta intracellular domain, wherein the CD3 zeta intracellular domain is 80% identical to SEQ ID NO: 18, and wherein at least six lysine amino acids of SEQ ID NO: 18 are (a) deleted or (b) substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 11. The protein according to embodiment 10, wherein the CD3 zeta intracellular domain is 100% identical to SEQ ID NO: 18, except for deleted or substituted lysine amino acids. 12. The protein according to embodiment 10 or 11, wherein at least seven lysine amino acids of SEQ ID NO: 18 are (a) deleted or (b) substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 13. The protein according to any one of embodiments 10 to 12, wherein at least eight lysine amino acids of SEQ ID NO: 18 are (a) deleted or (b) substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 14. The protein according to any one of embodiments 10 to 13, wherein the nine lysine amino acids of SEQ ID NO: 18 are (a) deleted or (b) substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, and glutamic acid. 15. The protein according to any one of embodiments 1 to 14, wherein at least six lysine amino acids of SEQ ID NO: 18 are substituted with alanine. 16. The protein according to any one of embodiments 1 to 15, wherein at least seven lysine amino acids of SEQ ID NO: 18 are substituted with alanine. 17. The protein according to any one of embodiments 1 to 16, wherein at least eight lysine amino acids of SEQ ID NO: 18 are substituted with alanine. 18. The protein according to any one of embodiments 1 to 17, wherein nine lysine amino acids of sequence number 18 are substituted with alanine. 19. The protein according to any one of embodiments 1 to 14, wherein at least six lysine amino acids of SEQ ID NO: 18 are substituted with aspartic acid. 20. The protein according to any one of embodiments 1 to 14 or 19, wherein at least seven lysine amino acids of SEQ ID NO: 18 are substituted with aspartic acid. 21. The protein according to any one of embodiments 1 to 14, 19, or 20, wherein at least eight lysine amino acids of SEQ ID NO: 18 are substituted with aspartic acid. 22. The protein according to any one of embodiments 1 to 14 or 19 to 21, wherein nine lysine amino acids of sequence number 18 are substituted with aspartic acid. 23. The protein according to any one of embodiments 1 to 14, wherein at least six lysine amino acids of SEQ ID NO: 18 are substituted with glutamic acid. 24. The protein according to any one of embodiments 1 to 14 or 23, wherein at least seven lysine amino acids of SEQ ID NO: 18 are substituted with glutamic acid. 25. The protein according to any one of embodiments 1 to 14, 23, or 24, wherein at least eight lysine amino acids of SEQ ID NO: 18 are substituted with glutamic acid. 26. The protein according to any one of embodiments 1 to 14 or 23 to 25, wherein nine lysine amino acids of sequence number 18 are substituted with glutamic acid. 27. A protein according to any one of embodiments 1 to 26, comprising an amino acid sequence that is 80% identical to any one of sequence numbers 2-7, 19-66, and 68-86. 28. The protein according to embodiment 1, comprising an amino acid sequence that is 80% identical to any one of sequence numbers 2-7, 19-66, and 68-86. 29. The protein according to any one of embodiments 1 to 28, wherein the protein is a chimeric antigen receptor (CAR). 30. A nucleic acid encoding a protein as described in any one of embodiments 1 to 29. 31. The nucleic acid according to embodiment 30, wherein the nucleic acid includes ribonucleic acid. 32. The nucleic acid according to embodiment 30 or 31, wherein the nucleic acid is ribonucleic acid. 33. A cell containing the nucleic acid described in any one of embodiments 30 to 32. 34. The cell according to embodiment 33, wherein the cell is a human cell. 35. A viral vector adapted to express the protein described in any one of embodiments 1 to 29. 36. A cell modified to express the protein described in any one of embodiments 1 to 29. 37. The cell according to embodiment 36, wherein the cell is a human cell. 38. A method for preparing cell therapy, comprising combining cells with a nucleic acid encoding a protein as described in any one of embodiments 1 to 29. 39. The method according to embodiment 38, wherein the cells are human cells. 40. The method according to embodiment 39, wherein the human cell is a T cell. 41. The method according to embodiment 39, wherein the human cells are CD3+ cells. 42. The method according to embodiment 39, wherein the human cells are CD8+ cells. 43. The method according to embodiment 39, wherein the human cells are CD4+ cells. 44. The method according to embodiment 39, wherein the human cells are NK cells. 45. The method according to embodiment 39, wherein the human cells are stem cells. 46. ​​The method according to embodiment 45, wherein the stem cells are hematopoietic stem cells. 47. The method according to embodiment 45, wherein the stem cells are mesenchymal stem cells. 48. A kit comprising one or more of the following: a protein according to any one of embodiments 1 to 29, a nucleic acid according to any one of embodiments 30 to 32, a cell according to embodiment 33, 34, 36, or 37, or a vector according to embodiment 35. array

[0287] In this specification, the following amino acid (AA) sequences or nucleotide (nt) sequences are referenced. For the following amino acid sequences: SEQ ID NOs: 1-5, and nucleic acid sequences: SEQ ID NOs: 6-10, signal peptide-scFv-CD8 sequences or nucleic acids encoding signal peptide-scFv-CD8 sequences are shown in bold, and signal transduction domains or nucleic acids encoding signal transduction domains are shown in underline. Double underlined letters indicate mutated amino acids. Unless otherwise specified, in the nucleic acid sequences described below and in this application, representative DNA sequences may contain "T," but when the sequence represents RNA, "T" is replaced with "U." Therefore, any DNA disclosed herein and identified by a particular sequence also discloses the corresponding RNA sequence in which each "T" in the DNA sequence is replaced with "U": Sequence ID 1 (a polypeptide containing only AA, WT CD3z, and anti-BCMA CAR) [ka] Sequence ID No. 2 (Potentiometer anti-BCMA CAR containing only AA, KR CD3z) [ka] Sequence ID 3 (Polypeptide anti-BCMA CAR containing only AA, KA CD3z) [ka] Sequence ID No. 4 (Polypeptide anti-BCMA CAR containing only AA, KD CD3z) [ka] Sequence ID No. 5 (a polypeptide containing only AA, KE CD3z, as an anti-BCMA CAR) [ka] Sequence ID 6 (ORF+STOP anti-BCMA CAR containing only nt, WT CD3z) [ka] Sequence ID 7 (ORF+STOP anti-BCMA CAR containing only nt, KR CD3z) [ka] Sequence ID 8 (ORF+STOP anti-BCMA CAR containing only nt, KA CD3z) [ka] Sequence ID 9 (ORF+STOP anti-BCMA CAR containing only nt, KD CD3z) [ka] Sequence ID 10 (ORF+STOP anti-BCMA CAR containing only nt, KE CD3z) [ka] Sequence ID 11 (nt, 5'UTR) aggactcttctggtccccacagactcagagagaacccaccgccacc Sequence ID 12 (nt, 3'UTR) tgcccgtcctcaccaagactgactgcctgctgctttgctactgcccgggcccatgagactgacttcccactgctctgcctgcctctccccactgcactggcacagccccgccttgccgctgctgatccattgccggtgtgacc caagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacacca Sequence ID 13 (nt, IVT mRNA) Sequence ID 14 (nt, IVT mRNA) Sequence ID 15 (nt, IVT mRNA) Sequence ID 16 (nt, IVT mRNA) Sequence ID 17 (nt, IVT mRNA) Sequence ID No. 18 (AA, polypeptide WT CD3z intracellular domain) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 19 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 20 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 21 (AA, polypeptide single KA CD3z intracellular domain) [ka] Sequence ID No. 22 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 23 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 24 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 25 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 26 (AA, polypeptide single KA CD3z intracellular domain) [ka] Sequence ID No. 27 (AA, single polypeptide KA CD3z intracellular domain) [ka] Sequence ID No. 28 (AA, polypeptide 2-7 KA CD3z intracellular domain) [ka] Sequence ID No. 29 (AA, polypeptide 2-7 KA CD3z intracellular domain) [ka] Sequence ID 30 (AA, polypeptide 2-7 KA CD3z intracellular domain) [ka] Sequence ID 31 (AA, polypeptide 2-7 KA CD3z intracellular domain) [ka] Sequence ID 32 (AA, polypeptide 2-7 KA CD3z intracellular domain) [ka] Sequence ID 33 (AA, polypeptide 2-7 KA CD3z intracellular domain) [ka] Sequence ID 34 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID 35 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID 36 (AA, polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID No. 37 (AA, Polypeptide 8 K-ACD3z intracellular domain) [ka] Sequence ID 38 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID 39 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID No. 40 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID No. 41 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID No. 42 (AA, Polypeptide 8 KA CD3z intracellular domain) [ka] Sequence ID 43 (AA, polypeptide 9 KA CD3z intracellular domain) [ka] Sequence ID No. 44 (AA, Polypeptide 9 KV CD3z intracellular domain) [ka] Sequence ID No. 45 (AA, Polypeptide 9 KI CD3z intracellular domain) [ka] Sequence ID No. 46 (AA, Polypeptide 9 KL CD3z intracellular domain) [ka] Sequence ID No. 47 (AA, Polypeptide 9 KM CD3z intracellular domain) [ka] Sequence ID No. 48 (AA, Polypeptide 9 KF CD3z intracellular domain) [ka] Sequence ID 49 (AA, Polypeptide 9 KY CD3z intracellular domain) [ka] Sequence ID 50 (AA, Polypeptide 9 KS CD3z intracellular domain) [ka] Sequence ID 51 (AA, Polypeptide 9 KT CD3z intracellular domain) [ka] Sequence ID 52 (AA, Polypeptide 9 KN CD3z intracellular domain) [ka] Sequence ID 53 (AA, Polypeptide 9 KQ CD3z intracellular domain) [ka] Sequence ID 54 (AA, Polypeptide 9 KH CD3z intracellular domain) [ka] Sequence ID 55 (AA, Polypeptide 9 KD CD3z intracellular domain) RVDFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDDRRGRDPEMGGDPQRRDNPQEGLYNELQDDDMAEAYSEIGMDGERRRGDGHDGLYQGLSTATDDTYDALHMQALPPR Sequence ID 56 (AA, Polypeptide 9 KE CD3z intracellular domain) [ka] Sequence ID 57 (AA, polypeptide WT CD28 KA CD3z intracellular domain) [ka] Sequence ID 58 (AA, polypeptide KA CD28 KA CD3z intracellular domain) [ka] Sequence ID 59 (AA, polypeptide WT 41BB KA CD3z intracellular domain) [ka] Sequence ID 60 (AA, polypeptide KA 41BB KA CD3z intracellular domain) [ka] Sequence ID 61 (AA, polypeptide WT CD3z intracellular domain (polymorphic variant)) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 62 (AA, polypeptide KE CD28 KA CD3z intracellular domain) [ka] Sequence ID 63 (AA, polypeptide WT CD28 KE CD3z intracellular domain) [ka] Sequence ID 64 (AA, polypeptide KA CD28 KE CD3z intracellular domain) [ka] Sequence ID 65 (AA, polypeptide KE CD28 KE CD3z intracellular domain) [ka] Sequence ID 66 (AA, polypeptide WT CD28-CD3z intracellular domain (polymorphic variant)) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 67 (nt, IVT mRNA) Sequence ID No. 68 (AA, KE CD28-CD3z-containing polypeptide anti-BCMA CAR) [ka] Sequence ID 69 (AA, polypeptide CD3z intracellular domain KE and one KA (position 1)) [ka] Sequence ID 70 (AA, polypeptide CD3z intracellular domain KE and one KA (position 2)) [ka] Sequence ID 71 (AA, polypeptide CD3z intracellular domain KE and one KA (position 3)) [ka] Sequence ID 72 (AA, polypeptide CD3z intracellular domain KE and one KA (position 4)) [ka] Sequence ID 73 (AA, polypeptide CD3z intracellular domain KE and one KA (position 5)) [ka] Sequence ID 74 (AA, polypeptide CD3z intracellular domain KE and one KA (position 6)) [ka] Sequence ID 75 (AA, polypeptide CD3z intracellular domain KE and one KA (position 7)) [ka] Sequence ID 76 (AA, polypeptide CD3z intracellular domain KE and one KA (position 8)) [ka] Sequence ID 77 (AA, polypeptide CD3z intracellular domain KE and one KA (position 9)) [ka] Sequence ID 78 (AA, polypeptide CD3z intracellular domain KA and one KE (position 1)) [ka] Sequence ID 79 (AA, polypeptide CD3z intracellular domain KA and one KE (position 2)) [ka] Sequence ID 80 (AA, polypeptide CD3z intracellular domain KA and one KE (position 3)) [ka] Sequence ID 81 (AA, polypeptide CD3z intracellular domain KA and one KE (position 4)) [ka] Sequence ID 82 (AA, polypeptide CD3z intracellular domain KA and one KE (position 5)) [ka] Sequence ID 83 (AA, polypeptide CD3z intracellular domain KA and one KE (position 6)) [ka] Sequence ID 84 (AA, polypeptide CD3z intracellular domain KA and one KE (position 7)) [ka] Sequence ID 85 (AA, polypeptide CD3z intracellular domain KA and one KE (position 8)) [ka] Sequence ID 86 (AA, polypeptide CD3z intracellular domain KA and one KE (position 9)) [ka] Sequence ID 87 (AA, scFv) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGS TKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS Sequence ID 88 (AA, scFv) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGS TKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYAGGSYAMDYWGQGTSVTVSS Sequence ID 89 (nt, anti-BCMA CAR full length ntd) Sequence ID 90 (nt, anti-BCMA CAR full length ntd) Sequence ID 91 (nt, anti-BCMA CAR full length ntd) Sequence ID 92 (nt, anti-BCMA CAR full length ntd) Sequence ID 93 (AA, anti-BCMA CAR full length aa) MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGG GTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYS YAMDYWGQGTSVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 94 (AA, anti-BCMA CAR full length aa) MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKTGKVPKFLIYEASTLQSGVPSRFSGGGSGTDFTLTISSLQPEDVATYYCQNYNSAPFTFGPGTKVDIKGSTSGSGKPGSGEGSTKGQVQLVESGGGVVQPGRSLRLSCAASGFAFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTQYLQMNSLRAEDTAVYYCARGGDFLYYYYYGMDVWGQGTTVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence number 95 (nt, full-length ntd of anti-BCMA CAR) Sequence ID 96 (nt, anti-BCMA CAR full length ntd) Sequence ID 97 (nt, anti-BCMA CAR full length ntd) Sequence ID 98 (nt, anti-BCMA CAR full length ntd) Sequence ID 99 (nt, anti-BCMA CAR full length ntd) Sequence ID 100 (nt, anti-BCMA CAR full length ntd) Sequence ID 101 (AA, anti-BCMA CAR full length) MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPGQPPKLLIYSASNLQSGVPARFSGSGSGTDFTLTISSVEPEDTANYYCLQ SRTLPRTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGGSVKISCKASGYTFTSYSINWVRQAPGKGLEWVGWINTETREPAYAQGFTGRFTFSADTSKSMAYL QINSLRAEDTAVYYCALDYLYSLDFWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRVEFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDERRGRDPEMGGEPQRRENPQEGLYNELQEDEMAEAYSEIGMEGERRRGEGHDGLYQGLSTATEDTYDALHMQALPPR Sequence ID 102 (AA, anti-BCMA CAR full length) MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPGQPPKLLIYSASNLQSGVPARFSGSGSGTDFTLTISSVEPEDTANYYCLQSRTLPRTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGGSVKISCKASGYTFTSYSINWVRQAPGKGLEWVGWINTETREPAYAQGFTGRFTFSADTSKSMAYLQINSLRAEDTAVYYCALDYLYSLDFWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSERSRLLHSDYMNMTPRRPGPTREHYQPYAPPRDFAAYRSRVEFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDERRGRDPEMGGEPQRRENPQEGLYNELQEDEMAEAYSEIGMEGERRRGEGHDGLYQGLSTATEDTYDALHMQALPPR

Claims

1. A protein comprising an intracellular domain capable of intracellular signal transduction, wherein the intracellular domain comprises an intracellular signal transduction domain, a co-stimulatory domain, or both, and wherein at least two lysine amino acids of the intracellular domain are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

2. The protein according to claim 1, wherein the intracellular domain includes a CD3 zeta domain.

3. The protein according to claim 1 or 2, wherein the intracellular domain comprises a domain that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 18, 61, or 66.

4. The protein according to any one of claims 1 to 3, wherein the CD3 zeta intracellular domain is 100% identical to that of SEQ ID NO: 18, 61, or 66, except for the substituted lysine amino acid.

5. The protein according to any one of claims 1 to 4, wherein at least three lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

6. The protein according to any one of claims 1 to 5, wherein at least 4, at least 5, at least 6, at least 7, or at least 8 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

7. The protein according to any one of claims 1 to 6, wherein at least nine lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

8. The protein according to any one of claims 1 to 7, wherein at least 6, at least 7, at least 8, or at least 9 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

9. The CD3 zeta intracellular domain wherein at least two lysine amino acids of the CD3 zeta intracellular domain are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

10. The CD3 zeta intracellular domain according to claim 9, wherein the intracellular domain comprises a domain that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 18 or 61.

11. The CD3 zeta intracellular domain according to claim 9 or 10, wherein the CD3 zeta intracellular domain is 100% identical to that of SEQ ID NO: 18 or 61, except for the substituted lysine amino acid.

12. The CD3 zeta intracellular domain according to any one of claims 9 to 11, wherein the substituted lysine amino acid is selected from the group consisting of positions 3, 37, 48, 53, 65, 67, 78, 85, and 99 of the amino acid sequence of SEQ ID NO:

18.

13. The CD3 zeta intracellular domain according to any one of claims 9 to 12, wherein at least three lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

14. The CD3 zeta intracellular domain according to any one of claims 9 to 13, wherein at least 4, at least 5, at least 6, at least 7, or at least 8 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

15. The CD3 zeta intracellular domain according to any one of claims 9 to 14, wherein at least nine lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

16. The CD3 zeta intracellular domain according to any one of claims 9 to 15, wherein at least 6, at least 7, at least 8, or at least 9 lysine amino acids are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

17. The CD3 zeta intracellular domain according to any one of claims 9 to 16, wherein the CD3 zeta intracellular domain is a protein domain.

18. The CD3 zeta intracellular domain according to any one of claims 9 to 17, wherein the CD3 zeta intracellular domain is a domain of a transmembrane protein.

19. The CD3 zeta intracellular domain according to any one of claims 9 to 18, wherein the CD3 zeta intracellular domain is a domain of an intercellular signaling protein.

20. The CD3 zeta intracellular domain according to any one of claims 9 to 19, wherein the CD3 zeta intracellular domain is a CAR domain.

21. A protein comprising the CD3 zeta intracellular domain described in any one of claims 9 to 20.

22. A protein comprising a CD3 zeta intracellular domain, wherein the CD3 zeta intracellular domain is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 18, wherein at least two lysine amino acids of SEQ ID NO: 18 are (a) deleted or (b) substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

23. The protein according to claim 22, wherein the CD3 zeta intracellular domain is 100% identical to that of SEQ ID NO: 18, except for deleted or substituted lysine amino acids.

24. The protein according to claim 22 or 23, wherein at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids of SEQ ID NO: 18 are (a) deleted or (b) substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

25. The protein according to any one of claims 22 to 24, wherein at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids of SEQ ID NO: 18 are substituted with alanine.

26. The protein according to any one of claims 22 to 25, wherein at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids of SEQ ID NO: 18 are substituted with aspartic acid.

27. The protein according to any one of claims 22 to 26, wherein at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 lysine amino acids of SEQ ID NO: 18 are substituted with glutamic acid.

28. The protein according to claim 22, comprising an amino acid sequence that is at least 80% identical to one of sequence numbers 2-7, 19-66, and 68-86.

29. The protein according to claim 22, comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of sequence numbers 2-7, 19-66, and 68-86.

30. The protein according to any one of claims 1 to 8 or 22 to 29, wherein the protein is a chimeric antigen receptor (CAR).

31. The protein according to any one of claims 1 to 8 or 22 to 29, further comprising a co-stimulatory domain.

32. The protein according to claim 31, wherein the co-stimulatory domain is selected from the group consisting of the CD8 alpha domain, 41BB domain, CD28 domain, FcR gamma domain, CD27 domain, OX40 domain, CD30 domain, CD40 domain, PD-1 domain, ICOS domain, LFA-1 domain, CD2 domain, CD7 domain, LIGHT domain, NKG2C domain, and B7 H3 domain, and any variant thereof.

33. The protein according to claim 31, wherein the co-stimulatory domain is CD28.

34. The protein according to claim 31, wherein the co-stimulatory domain is 41BB.

35. The protein according to any one of claims 31 to 34, wherein at least one lysine amino acid of the co-stimulatory domain is substituted with an amino acid independently selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

36. The protein according to any one of claims 31 to 35, wherein at least two lysine amino acids of the co-stimulatory domain are substituted with amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

37. The protein according to any one of claims 1 to 8 or 22 to 36, further comprising an extracellular antigen-binding domain.

38. The protein according to claim 37, wherein the extracellular antigen-binding domain binds to CD19, BCMA, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, or CD30.

39. The protein according to claim 37 or 38, wherein the extracellular antigen-binding domain binds to BCMA.

40. The protein according to claim 37 or 38, wherein the extracellular antigen-binding domain binds to CD19.

41. The protein according to any one of claims 37 to 40, wherein the extracellular antigen-binding domain is scFv.

42. The protein according to any one of claims 1 to 8 or 22 to 41, further comprising a transmembrane domain.

43. The protein according to claim 42, wherein the transmembrane domain is one of the following: MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activator molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, 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, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 The protein comprising a transmembrane domain of a receptor that specifically binds to (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, or CD83.

44. A protein according to any one of claims 1 to 8 or 22 to 43, further comprising a hinge region.

45. A protein according to any one of claims 1 to 8 or 22 to 44, further comprising a leader domain.

46. The protein according to any one of claims 31 to 45, further comprising one or more spacer sequences between one or more domains.

47. The protein according to claim 46, wherein the spacer sequence is a polypeptide linker.

48. The CD3 zeta intracellular domain according to claim 9, comprising one of the sequences of sequence numbers 28-56 or 69-86.

49. The protein according to claim 22, comprising any one of the sequences of sequence numbers 57-60 and 62-65.

50. A protein containing the sequence of sequence numbers 5, 68, 101, or 102.

51. A nucleic acid construct encoding a protein according to any one of claims 1 to 8 or 22 to 50, or a CD3 zeta intracellular domain according to any one of claims 9 to 21.

52. The nucleic acid construct according to claim 51, wherein the nucleic acid construct is RNA.

53. The nucleic acid construct according to claim 51, wherein the nucleic acid construct is DNA.

54. Nucleic acid constructs containing sequence numbers 13-17 or 67.

55. A vector encoding the protein according to any one of claims 1 to 8 or 22 to 50, or the CD3 zeta intracellular domain according to any one of claims 9 to 21.

56. A vector comprising a nucleic acid construct according to any one of claims 51 to 54.

57. The vector according to claim 55 or 56, wherein the vector is a viral vector.

58. A composition comprising a protein according to any one of claims 1 to 8 or 22 to 50, a CD3 zeta intracellular domain according to any one of claims 9 to 21, a nucleic acid construct according to any one of claims 51 to 54, or a vector according to any one of claims 55 to 57.

59. A pharmaceutical composition comprising the composition described in claim 58.

60. The pharmaceutical composition according to claim 59, further comprising a pharmaceutically acceptable excipient.

61. A cell comprising the protein according to any one of claims 1 to 8 or 22 to 50.

62. A cell comprising a nucleic acid construct according to any one of claims 51 to 54 or a vector according to any one of claims 55 to 57.

63. The cell according to claim 61 or 62, wherein the cell is a human cell.

64. The cell according to any one of claims 61 to 63, wherein the cell is an immune cell.

65. The cell according to any one of claims 61 to 63, wherein the cell is a T cell, a CD3+ cell, a CD8+ cell, a CD4+ cell, an NK cell, a stem cell, a hematopoietic stem cell, or a mesenchymal stem cell.

66. A method for preparing a cell therapy for treating a disease, comprising transfecting a plurality of cells with the vector described in claim 55 or 56.

67. A method for transfecting a cell having multiple cells with a vector according to any one of claims 55 to 57.

68. The method according to claim 67, wherein the method is in vitro.

69. The method according to claim 67, wherein the method is ex vivo.

70. The method according to claim 67, wherein the method is in vivo.

71. A method for treating a disease in a subject requiring such treatment, comprising administering to the subject the cells described in any one of claims 61 to 65.

72. A method for transfecting cells with the vector described in any one of claims 55 to 57.

73. The method according to claim 72, wherein the cells are human cells.

74. The method according to claim 72 or 73, wherein the cells are immune cells.

75. The method according to any one of claims 72 to 74, wherein the cell is a T cell.

76. The method according to any one of claims 72 to 74, wherein the cells are CD3+ cells.

77. The method according to any one of claims 72 to 74, wherein the cells are CD8+ cells.

78. The method according to any one of claims 72 to 74, wherein the cells are CD4+ cells.

79. The method according to any one of claims 72 to 74, wherein the cells are NK cells.

80. The method according to any one of claims 72 to 74, wherein the cells are stem cells.

81. The method according to claim 80, wherein the stem cells are hematopoietic stem cells.

82. The method according to claim 80, wherein the stem cells are mesenchymal stem cells.

83. The method according to any one of claims 71 or 73 to 82, further comprising administering cytokines.

84. The method according to any one of claims 71 or 73 to 83, wherein the disease is cancer, an autoimmune disease, or an allergic disease.

85. The method according to any one of claims 71 or 73 to 84, wherein the disease is cancer.

86. The method according to any one of claims 71 or 73 to 84, wherein the disease is myeloma.

87. The method according to any one of claims 71 or 73 to 84, wherein the disease is myasthenia gravis (MG).

88. The method according to any one of claims 71 or 73 to 87, wherein the method is characterized by an increase in intracellular secretion of cytokines.

89. The method according to claim 88, wherein the secreted cytokine is interferon-γ.

90. The method according to any one of claims 71 or 73 to 89, wherein the method is characterized by selective killing of cancer cells.

91. The method according to any one of claims 71 or 73 to 89, wherein the method is characterized by the selective killing of immune cells.

92. The method according to any one of claims 71 or 73-89, wherein the method is characterized by selective killing of BCMA+ or CD19+ cells.

93. Use of the protein according to any one of claims 1 to 8 or 22 to 50, the CD3 zeta intracellular domain according to any one of claims 9 to 21, the nucleic acid construct according to any one of claims 51 to 54, the vector according to any one of claims 55 to 57, the composition according to claim 58, the pharmaceutical composition according to claim 59 or 60, or the cells according to any one of claims 61 to 65, for treating cancer.

94. A kit for treating cancer, comprising one or more of the following: a protein according to any one of claims 1 to 8 or 22 to 50; a CD3 zeta intracellular domain according to any one of claims 9 to 21; a nucleic acid construct according to any one of claims 51 to 54; a vector according to any one of claims 55 to 57; a composition according to claim 58; a pharmaceutical composition according to claim 59 or 60; or a cell according to any one of claims 61 to 65.

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