Intracellular signaling and costimulatory domains suitable for prolonged expression of chimeric antigen receptors

Abstract

Provided herein are proteins, such as chimeric antigen receptors (CARs), such as those specific for BCMA, comprising a CD3-zeta intracellular domain with improved properties. Also contemplated are uses of proteins, such as CARs, comprising a CD3-zeta intracellular domain in immune cells (e.g., T cells), compositions (e.g., CARs and nucleic acid constructs encoding the same), and methods.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application USSN 63 / 491,038, filed March 17, 2023, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic serial number (C154070004WO00-SEQ-AZW.xml; size: 137,935 bytes; and creation date: March 15, 2024) are incorporated herein by reference in their entirety. Background Technology

[0005] Chimeric antigen receptors (CARs) are synthetic transmembrane proteins comprising an extracellular antigen recognition domain (e.g., a variable single-chain fragment of an antibody), a transmembrane domain, and an intracellular signaling domain (e.g., a T-cell signaling domain, such as CD3-ζ). When a CAR is expressed artificially in or by a first cell (e.g., a T cell), the CAR directs the first cell to kill a second cell (such as a cancer cell) that expresses a surface antigen that is designed to be recognized by the CAR's extracellular antigen recognition domain. Cells modified to express CARs, such as T cells (e.g., CAR T cells), can be administered to patients to kill tumor cells or other pathogenic cells. For this purpose, CARs with an extracellular antigen recognition domain that specifically binds to surface antigens (markers), such as CD19, BCMA, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, and CD30, have been developed. Cells expressing CARs, such as CAR T cells, have been developed to treat hematologic malignancies, solid tumors, and non-cancerous conditions 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 10,934,337, each of which is incorporated herein by reference. Summary of the Invention

[0006] However, efforts to achieve optimal CAR expression in cells of interest (e.g., T cells) encounter an obstacle: undesirable downregulation or internalization of CARs upon binding to their corresponding antigens. 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 may reduce the durability and benefit of CAR-based therapies. Therefore, there is a need for novel CAR molecules that resist or are less prone to downregulation or internalization upon binding to their corresponding antigens.

[0007] This disclosure is based on the recognition that certain novel modifications (i.e., a series of amino acid substitutions) to the intracellular domain CD3-ζ of the CAR (the terms “CD3-zeta”, “CD3-Z”, and “CD3-ζ” are used interchangeably herein) confer upon the CAR: (1) a significant degree of CAR expression maintenance, even after the CAR has been exposed to its corresponding antigen (e.g., its intended target) (e.g., after such exposure); (2) superior target cell cytotoxicity; and / or (3) superior cytokine (e.g., interferon-γ) secretion. The aforementioned superior experimental performance of the CAR protein of this disclosure is compared to other CAR proteins containing the same aspects of the wild-type CD3-ζ intracellular domain (e.g., SEQ ID NO: 18), where exposure to the corresponding antigen of the CAR results in a substantial 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 the CAR has been exposed to its corresponding antigen. Again, this was determined through experimental comparisons with other CAR proteins containing the same aspects of the wild-type intracellular CD3-ζ intracellular domain.

[0008] Therefore, in one aspect, this article provides a protein capable of intracellular signal transduction comprising an intracellular domain, 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 independently substituted with amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0009] In some embodiments, the intracellular domain comprises a CD3-ζ domain. In some embodiments, the intracellular domain comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the same domain as SEQ ID NO: 18, 61, or 66. In some embodiments, the CD3-ζ intracellular domain is 100% identical to SEQ ID NO: 18, 61, or 66, except for the substituted lysine amino acid.

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

[0011] Under certain conditions, at least four, at least five, at least six, at least seven, or at least eight lysine amino acids are replaced by amino acids independently selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0012] In some embodiments, at least nine lysine amino acids are replaced independently by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0013] In some embodiments, at least six, at least seven, at least eight, or at least nine lysine amino acids are replaced independently by amino acids selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0014] In another aspect of this disclosure, a CD3-ζ intracellular domain is provided, wherein at least two lysine amino acids of the CD3-ζ intracellular domain are independently replaced by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0015] In some embodiments, the intracellular domain comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the same domain as SEQ ID NO: 18, 61, or 66.

[0016] In some embodiments, the CD3-ζ intracellular domain is 100% identical to SEQ ID NO: 18, 61 or 66, except for the substituted lysine amino acid.

[0017] In some embodiments, at least three lysine amino acids are replaced independently by amino acids 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 four, at least five, at least six, at least seven, or at least eight lysine amino acids are replaced independently by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0019] In some embodiments, at least nine lysine amino acids are replaced independently by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0020] In some embodiments, at least six, at least seven, at least eight, or at least nine lysine amino acids are replaced independently by amino acids selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0021] In some implementations, the CD3-ζ intracellular domain is a protein domain.

[0022] In some implementations, the CD3-ζ intracellular domain is a transmembrane protein domain.

[0023] In some implementations, the CD3-ζ intracellular domain is a domain of an intercellular signaling protein.

[0024] In some implementations, the CD3-ζ intracellular domain is a CAR domain.

[0025] In one aspect, this document provides a protein comprising the CD3-ζ intracellular domain of the present disclosure.

[0026] In another aspect of this disclosure, a protein comprising a CD3-ζ intracellular domain is provided, wherein the CD3-ζ 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) missing, or (b) substituted by amino acids independently selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0027] In some embodiments, the CD3-ζ intracellular domain is 100% identical to SEQ ID NO: 18, except for the absence or substitution of lysine amino acids.

[0028] In some embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are (a) missing or (b) substituted by amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0029] In some embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids in SEQ ID NO: 18 are replaced by alanine.

[0030] In some embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are replaced by aspartic acid.

[0031] In some embodiments, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids in SEQ ID NO: 18 are replaced with glutamic acid.

[0032] On the other hand, a protein is provided that contains at least 80% of the same amino acid sequence as any one of SEQ ID NO: 2-7, 19-66 and 68-86.

[0033] In some embodiments, the protein comprises at least 85%, at least 90%, at least 95%, or at least 99% of the same amino acid sequence as any one of SEQ ID NO: 2-7, 19-66, and 68-86.

[0034] In some implementations, the protein is a chimeric antigen receptor (CAR).

[0035] In some embodiments, the protein further includes a co-stimulatory domain.

[0036] In some embodiments, the co-stimulatory domain is selected from the group consisting of: CD8-α domain, 41BB domain, CD28 domain, FcR γ 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 B7H3 domain, and any variant thereof.

[0037] In some implementations, the co-stimulatory domain is CD28.

[0038] In some implementations, the co-stimulatory domain is 41BB.

[0039] In some embodiments, at least one lysine amino acid of the co-stimulatory domain is independently replaced by an amino acid selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0040] In some embodiments, at least two lysine amino acids of the co-stimulatory domain are replaced by amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0041] In some embodiments, the protein further includes an extracellular antigen-binding domain.

[0042] In some embodiments, the extracellular antigen-binding domain binds to CD19, BCMA, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, or CD30.

[0043] In some implementations, the extracellular antigen-binding domain binds to BCMA.

[0044] In some implementations, the extracellular antigen-binding domain binds to CD19.

[0045] In some implementations, the extracellular antigen-binding domain is scFv.

[0046] In some embodiments, the protein further includes a transmembrane domain.

[0047] In some embodiments, the transmembrane domain comprises transmembrane regions of the following: class I MHC molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activating 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, CD8alpha, CD8beta, 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 (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a or receptors that specifically bind to CD83.

[0048] In some embodiments, the protein further includes a hinge region.

[0049] In some embodiments, the proteins disclosed herein further include a leader domain.

[0050] In some embodiments, the protein further comprises one or more spacer sequences located between one or more of the domains.

[0051] In some embodiments, the spacer sequence is a peptide linker.

[0052] On the other hand, this disclosure provides nucleic acid constructs encoding the proteins disclosed herein.

[0053] In some implementations, the nucleic acid construct is RNA.

[0054] In some implementations, the nucleic acid construct is DNA.

[0055] In one respect, this article provides vectors that encode the proteins described herein.

[0056] In some implementations, the vector comprises the nucleic acid constructs disclosed herein.

[0057] In some implementations, the vector is a viral vector.

[0058] On the other hand, this document provides a composition comprising the proteins disclosed herein, the nucleic acid constructs described herein, or the vectors described herein.

[0059] In another aspect of this disclosure, a pharmaceutical composition comprising the protein, nucleic acid construct or carrier described herein is provided.

[0060] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0061] In one aspect, this document provides a cell comprising the proteins disclosed herein.

[0062] On the other hand, this disclosure provides a cell comprising the nucleic acid construct or vector described herein.

[0063] In some implementations, the cells are human cells.

[0064] In some implementations, the cells are immune cells.

[0065] In some implementations, the cells are T cells, CD3+ cells, CD8+ cells, CD4+ cells, NK cells, stem cells, hematopoietic stem cells, or mesenchymal stem cells.

[0066] In one aspect, a method for producing a cell therapy for treating a disease is provided, the method comprising transfecting multiple cells with the vector described herein.

[0067] In another aspect of this disclosure, a method for treating a disease in a subject in need is provided, the method comprising administering the cells described herein to the subject.

[0068] In some implementations, the cells are human cells.

[0069] In some implementations, the cells are immune cells.

[0070] In some implementations, the cell is a T cell.

[0071] In some implementations, the cells are CD3+ cells.

[0072] In some implementations, the cells are CD8+ cells.

[0073] In some implementations, the cells are CD4+ cells.

[0074] In some implementations, the cell is an NK cell.

[0075] In some implementations, the cell is a stem cell.

[0076] In some implementations, the stem cells are hematopoietic stem cells.

[0077] In some implementations, the stem cells are mesenchymal stem cells.

[0078] In some embodiments, the method further includes cytokines.

[0079] In some implementations, the disease is cancer, an autoimmune disease, or an allergic disease.

[0080] In some implementations, the disease is myeloma.

[0081] In some implementations, the disease is myeloma.

[0082] In some implementations, the disease is myasthenia gravis (MG).

[0083] In some implementations, the method is characterized by increased cellular secretion of cytokines.

[0084] In some embodiments, the secreted cytokine is interferon-γ.

[0085] In some implementations, the method is characterized by selectively killing cancer cells.

[0086] In some implementations, the method is characterized by selectively killing immune cells.

[0087] In some implementations, the method is characterized by selectively killing BCMA+ or CD19+ cells.

[0088] In one aspect, the uses of the proteins disclosed herein, the nucleic acid constructs described herein, the viral vectors disclosed herein, or the cells described herein for the treatment of cancer are provided.

[0089] On the other hand, a kit is provided comprising one or more of the following: the proteins disclosed herein, the nucleic acid constructs provided herein, the viral vectors described herein, or the cells disclosed herein. Attached Figure Description

[0090] Figure 1A The image shows CAR expression in CART cells generated with wild-type CD3-ζ intracellular domain sequence (SEQ ID NO: 13) and with lysine mutant constructs (SEQ ID NO: 14-17) in the absence of target ligand (left) and in the presence of target ligand (right), measured by median fluorescence intensity (MFI).

[0091] Figure 1B The expression of interferon-γ in the supernatant of co-cultures between CAR T cells generated with lysine mutant constructs (SEQ ID NO: 14-17) or wild-type CAR T cells (SEQ ID NO: 13) and BCMA+ MM1S multiple myeloma is shown by specific ELISA analysis. CAR T cells were pre-exposed (gray) or not pre-exposed (black) to BCMA+ cells prior to co-culture.

[0092] Figure 1C The cytotoxicity of pre-exposed CAR T cells against MM1S-GFP target cells expressing the BCMA ligand was demonstrated. These CAR T cells were generated using constructs encoding lysine-mutant CARs (SEQ ID NO: 14-17) and wild-type CARs (SEQ ID NO: 13). Cytotoxicity was assessed by co-culturing at various effector:target ratios.

[0093] Figure 2A The image shows CAR expression in CAR T cells generated with wild-type constructs (SEQ ID NO: 18 and 61) and mutant constructs (SEQ ID NO: 19-27 and 43) in the absence of target ligands (left) and in the presence of target ligands (right), measured by median fluorescence intensity (MFI).

[0094] Figure 2B The expression of interferon-γ in CAR T cells generated with various test constructs (SEQ ID NO: 19-27 and 43) and wild-type CAR T cells (SEQ ID NO: 18 and 61) during co-culture with MM1S target cells is shown.

[0095] Figure 2C The image shows CAR expression in CAR T cells generated with wild-type constructs (SEQ ID NO: 18 and 61) and mutant constructs (SEQ ID NO: 34-43) in the absence of target ligands (left) and in the presence of target ligands (right), measured by median fluorescence intensity (MFI).

[0096] Figure 2D The expression of interferon-γ in CAR T cells generated with various test constructs (SEQ ID NO: 34-43) and wild-type CAR T cells (SEQ ID NO: 18 and 61) during co-culture with MM1S target cells is shown.

[0097] Figure 2E The image shows CAR expression in CAR T cells generated with wild-type constructs (SEQ ID NO: 18 and 61) and mutant constructs (SEQ ID NO: 28-33 and 43) in the absence of target ligands (left) and in the presence of target ligands (right), measured by median fluorescence intensity (MFI).

[0098] Figure 2F The expression of interferon-γ in CAR T cells generated with various test constructs (SEQ ID NO: 28-33 and 43) and wild-type CAR T cells (SEQ ID NO: 18 and 61) during co-culture with MM1S target cells is shown.

[0099] Figure 3A The image shows CAR expression in CAR T cells expressing various test constructs (SEQ ID NO: 43-56) and CAR cells expressing wild-type constructs (SEQ ID NO: 18 and 61) in the absence of BCMA ligand (left) and in the presence of BCMA ligand (right), measured by median fluorescence intensity (MFI).

[0100] Figure 3B The expression of interferon-γ in CAR T cells generated with various test constructs (SEQ ID NO: 43-56) and wild-type CAR T cells (SEQ ID NO: 18 and 61) during co-culture with MM1S target cells is shown.

[0101] Figure 4A The images show CAR expression in CAR T cells generated with various test constructs (SEQ ID NO: 43 and 57-60) and CAR cells generated with the wild-type construct (SEQ ID NO: 18) in the absence of BCMA ligand (left) and in the presence of ligand (right), measured by median fluorescence intensity (MFI).

[0102] Figure 4B The expression of interferon-γ in CAR T cells generated using various test constructs (SEQ ID NO: 43 and 57-60) and wild-type construct (SEQ ID NO: 18) was shown when exposed to MM1S target cells.

[0103] Figure 4C The cytotoxicity of CAR T cells generated using individual constructs containing wild-type CD3-ζ (SEQ ID NO: 61) or mutant CD3-ζ, CD28-CD3-ζ, and 41BB-CD3-ζ signaling domains (SEQ ID NO: 43, 57, and 59, respectively) was demonstrated. Cytotoxicity was assessed by pre-exposing cells to MM1S followed by a 72-hour cytotoxicity assay of BCMA+MM1S-GFP cells at various effector:target ratios.

[0104] Figure 5A The images show CAR expression in CAR T cells expressing variants of anti-BCMACAR protein (SEQ ID NO: 43, 56-58, and 62-65) and CAR T cells expressing the wild-type construct (SEQ ID NO: 18) in the absence of BCMA ligand (left) and in the presence of BCMA ligand (right), measured by median fluorescence intensity (MFI).

[0105] Figure 5B The cytotoxicity of CAR T cells generated using various constructs (SEQ ID NO: 18, 43, 57, 58, and 62) was demonstrated. Cytotoxicity was assessed by pre-exposing cells to MM1S followed by a 72-hour cytotoxicity assay of BCMA+MM1S-GFP cells at various effector:target ratios.

[0106] Figure 5C The cytotoxicity of CAR T cells generated using various constructs (SEQ ID NO: 56 and 63-65) was demonstrated. Cytotoxicity was assessed by pre-exposing cells to MM1S followed by a 72-hour cytotoxicity assay of BCMA+ MM1S-GFP cells at various effector:target ratios.

[0107] Figures 6A-6B Flow cytometry assessment of activation of BCMA CAR T (SEQ ID NO: 43) or control CD8+ T cells without CAR using the activation-induced markers CD69 and CD137 (41BB) is shown. Response to plasmablasts ( Figure 6A ) and plasmacytoid dendritic cells ( Figure 6B) co-cultured to evaluate the activation of BCMA CAR T (SEQ ID NO: 43) and control CD8+ T cells.

[0108] Figures 7A-7B Bioluminescence was shown in mice carrying MM1S-fluc myeloma and treated with control CD8+ T cells or CAR T cells expressing CARs of SEQ ID NO: 5 and 68. Figure 7A Bioluminescence measurements of individual mice on days 9 and 12 are shown. Figure 7B Summary statistics for mice in each group at a given time point are shown. Arrows indicate the date of CAR T cell administration. Mean ± SD, n=4.

[0109] Figures 8A-8B Bioluminescence (total flux) was shown for mice carrying MM1S-fluc myeloma and treated with the following cells: control CD8+ T cells or CAR T cells containing anti-BCMA CAR with intracellular CD28-CD3-ζ (SEQ ID NO: 65) signaling domains of KE-mutant or wild-type intracellular CD28-CD3-ζ (SEQ ID NO: 66). Figure 8A Summary statistics of bioluminescence (photons / second) in mice treated with 12.5 million CAR T cells or control cells are shown. Arrows indicate the date of CAR T cell administration. Figure 8B Bioluminescence data from individual mice on day 21 in groups treated with 2 million or 12.5 million CAR T cells generated using anti-BCMA CARs containing the intracellular CD28-CD3-ζ (SEQ ID NO: 65) signaling domain with a KE mutation or wild-type intracellular CD28-CD3-ζ (SEQ ID NO: 66) are shown. Mean ± SD, n=4.

[0110] Figure 8C Flow cytometry evaluation of whole blood and bone marrow from mice administered CAR T cells generated using the IVT mRNA construct SEQ ID NO: 68 encoding the signal transduction domain of SEQ ID NO: 65 is shown. Cells were stained with antibodies against human CD45, CD3, and recombinant BCMA-APC, and CAR expression was assessed by flow cytometry. Data showed CD3 and CAR expression on CD45-gated cells.

[0111] Figure 9AThe cytotoxicity of anti-BCMA CAR T cells derived from two myasthenia gravis (MG) disease donors to autologous plasma cells differentiated from MG patients was demonstrated. The anti-BCMA CAR T cells were prepared using SEQ ID NO: 68 CAR construct and SEQ ID NO: 94 (anti-PSMA CAR as a negative control).

[0112] Figure 9B This study demonstrated donor differences in interferon-γ cytokine production following cytotoxicity.

[0113] Figure 9C The function of MG donor CAR T cells, as evaluated using the malignant MM1S-GFP cell line, is shown.

[0114] Figure 9D Interferon-γ cytokine production in MG donor CAR T cells, as assessed using the malignant MM1S-GFP cell line, is shown.

[0115] Figure 10A The apparent affinity of the anti-BCMA CAR for soluble BCMA was demonstrated.

[0116] Figure 10B The image shows an anti-CD19 CAR expressed by CAR T cells containing a CD28-CD3-ζ intracellular domain after culture in the absence or presence of BCMA+ MM1S cells. The CD28-CD3-ζ intracellular domain has a wild-type sequence or a mutant sequence containing a lysine-to-glutamic acid mutation.

[0117] definition

[0118] Unless the context clearly indicates otherwise, as used herein and in the claims, the singular forms “a,” “an,” and “the” include both single and multiple references. Thus, for example, reference to “reagent” includes both a single reagent and multiple such reagents.

[0119] As used herein, the terms “allergy” and “hypersensitivity” refer to medical conditions involving an abnormally hypersensitive reaction to normally harmless substances (i.e., allergens). Exemplary allergic conditions include allergic reactions, asthma, food allergies, insect sting allergies, drug allergies, allergic rhinitis, urticaria, angioedema, eczema, atopic dermatitis, contact dermatitis, and eosinophilic esophagitis.

[0120] Amino acids in a polypeptide sequence can be identified by their unabbreviated names or by three-letter or single-letter abbreviations known in the art, and such identifiers 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 (S) (S); threonine (Thr) (T); tryptophan (Trp) (W); tyrosine (Tyr) (Y); and valine (V) (V). "Aspartic acid" includes aspartate and aspartic acid. "Glutamic acid" includes glutamate and glutamic acid. Basic amino acids include lysine, arginine, and histidine, and are positively charged at neutral pH.

[0121] An antibody (used interchangeably with the plural form) is an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, biantibodies, nanobodies, linear antibodies, single-chain antibodies, and any other modified structures of immunoglobulin molecules containing antigen recognition sites of 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 consisting of four polypeptide chains, namely two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope binding characteristics of an Ig molecule. Such mutant, variant, or derived antibody forms are known in the art. The following discussion covers its non-restrictive implementation scheme.

[0122] In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated as HCVR or VH in this article) 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 as LCVR or VL in this article) 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 hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0123] As used herein, the term “antigen-binding portion” (or simply “antibody portion”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., BCMA). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Such antibody implementations can also be bispecific, dual-specific, or multispecific; specifically binding two or more different antigens. Multispecific, dual-specific, and bispecific antibody constructs are well known in the art and have been characterized in Kontermann (ed.), Bispecific Antibodies, Springer, NY (2011), and Spiess et al., Mol. Immunol. 67(2):96-106 (2015).

[0124] Examples of binding fragments covered within the term "antigen-binding moiety" of antibodies include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (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, which is incorporated herein by reference); and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked using recombination methods via synthetic adapters that allow them to be made into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a 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 covered within the term "antigen-binding moiety" of antibodies. Other forms of single-chain antibodies, such as biantibodies, are also included. Biantibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain. This forces the domains to pair with complementary domains on another chain, creating 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 art (Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

[0125] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed, for example, by a viral vector. The term should also be interpreted to mean an antibody produced by synthesizing a DNA molecule encoding the antibody, and that the DNA molecule expresses an antibody protein or a specified amino acid sequence of the antibody, wherein said DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0126] In some embodiments, as used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune-competent cells, or both. Those skilled in the art will understand that any macromolecule can act as an antigen, including virtually all proteins or peptides. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding an immune response therefore encodes an "antigen," as used herein. Furthermore, those skilled in the art will understand that an antigen does not need to be encoded solely 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 more than one gene, and the arrangement of these nucleotide sequences in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen does not need to be encoded by a "gene" at all. It will be readily apparent that antigens can be generated, synthetic, or can be derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0127] As used herein, the term "tumor antigen" refers to a molecule (typically a protein, carbohydrate, or lipid) that is expressed, either entirely or as a fragment (e.g., MHC / peptide), on the surface of cancer cells and can be used to preferentially target drugs to cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal cells and cancer cells, such as a lineage marker, like CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule overexpressed in cancer cells compared to normal cells. In some embodiments, the tumor antigen is a cell surface molecule inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, the tumor antigen will be expressed, either entirely or as a fragment (e.g., MHC / peptide), only on the cell surface of cancer cells and will not be 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.

[0128] As used herein, the term "antitumor effect" refers to biological effects that can manifest as a reduction in tumor volume, a decrease in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer. "Antitumor effect" can also manifest as the ability of the peptides, polynucleotides, cells, and antibodies disclosed herein to prevent tumorigenesis.

[0129] The term "autoimmune" refers to a disease or disorder 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, which are antibodies produced by an individual that recognize antigens in their own cells or tissues. Exemplary autoimmune diseases include myasthenia gravis, systemic 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, Goodpassuia syndrome, multiple sclerosis, and type 1 diabetes.

[0130] As used in this article, the term "self" means any material derived from the same individual and subsequently reintroduced into that individual.

[0131] As used in this article, the term "allogeneic" refers to a graft derived from a different animal of the same species. "Xenograft" refers to a graft derived from an animal of a different species.

[0132] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth 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 include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In some embodiments, cancer refers to cancer expressing BCMA. Exemplary cancers expressing 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 κ / λ light chain assay), bone marrow examination, urine protein electrophoresis, and / or X-rays of the commonly affected bones. In some embodiments, cancer refers to Hodgkin lymphoma (HL). HL is a cancer of B cells.

[0133] "Effective amount" means the amount of a therapy sufficient to reduce or improve the severity and / or duration of a disease or one or more of its symptoms, prevent the progression of the disease, cause the remission of the disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, detect the disease, or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent).

[0134] As used herein, the term “exogenous” means any material introduced from or generated outside an organism, cell, tissue, or system.

[0135] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as viscera, plasmids (e.g., naked or contained in liposomes), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0136] As used herein, “CD3-ζ intracellular domain” refers to a protein domain that is the intracellular portion of the CD3-ζ protein. For example, in humans, the CD3-ζ intracellular domain corresponds to, substantially corresponds to, or resembles amino acids 351 to 463 sequentially numbered in the full-length CD3-ζ amino acid sequence of SEQ ID NO: 1, and substantially corresponds to or resembles a portion of the CD3-ζ amino acid sequence of SEQ ID NO: 18. “Substantially corresponds” is defined as follows: Since the boundary between one protein domain and another protein domain (e.g., between the CD3-ζ transmembrane domain and the intracellular domain) is not necessarily strictly defined, the “CD3-ζ intracellular domain” may consist of more or fewer amino acids than those described above (e.g., 1 to 10 more or fewer amino acids on each side of the amino and carboxyl sides of the sequence as listed, described numerically, or otherwise specified), but still substantially 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-ζ intracellular domain" is intended to include all allelic variants and naturally occurring or artificially mutated CD3-ζ that do not have the same amino acid substitutions as described herein.

[0137] As used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that act as antibodies, and the term has its usual meaning in the art.

[0138] When used in this document with regard to the selection of two or more amino acids from a list (e.g., the Markush group), “independent selection” means that the selection of an amino acid from the list is independent of or may be independent of the selection of each consecutive amino acid from the list, and so on, and the first and consecutive selections may be different or the same.

[0139] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their natural counterparts are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-natural environments, such as, for example, host cells.

[0140] Unless otherwise stated, "nucleotide sequence or nucleic acid encoding an amino acid sequence" includes all nucleotide sequences that are degenerate in form and encode the same amino acid sequence. If a nucleotide sequence encoding a protein may contain one or more introns in some forms, then a nucleotide sequence encoding a protein or RNA phrase may also include introns.

[0141] As used herein, the term "modulate" or "modulating" refers to a detectable increase or decrease in response level compared to the response level in the absence of treatment or compound, and / or compared to the response level under otherwise identical conditions. The term encompasses perturbations and / or influences on natural signals or responses that mediate beneficial effects.

[0142] As used herein, the term "connector" 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, such as the inactive Cas9 domain of a nuclease and a nucleic acid editing domain (e.g., adenosine deaminase)). Typically, a connector is located between or flanking two groups, molecules, or other parts and is covalently linked to each group, molecule, or other part, thereby connecting them. In some embodiments, the connector is one or more amino acids (e.g., a peptide or protein). In some embodiments, the connector is an organic molecule, group, polymer, or chemical part. In some implementations, the linker length is 5-100 amino acids, 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 acids. Longer or shorter linkers have also been considered.

[0143] "Parenteral" administration of immunogenic compositions includes techniques such as subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion.

[0144] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, suitable for the methods described herein. 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 engineered, for example, a genetically engineered non-human subject. The subject can be of any sex and at any developmental stage. In some embodiments, the subject has cancer (e.g., multiple myeloma). In other embodiments, the subject is a healthy volunteer.

[0145] As used herein with respect to antigen recognition domains (e.g., antibodies, such as scFv), the term "specific binding" or "specific to..." refers to a protein or its domain that recognizes a particular antigen (or surface marker) but substantially does not recognize or bind to other molecules in the sample (or, in some cases, within 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 this cross-species reactivity itself does not change the antibody's classification as specific. An antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, this cross-reactivity itself does not change the antibody's classification as specific. In some cases, the term "specific binding" refers to the interaction of an antibody, protein (or its domain), or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than the protein in general. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and an 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.

[0146] When used herein with respect to amino acids or their positions in an amino acid sequence, “substituted” means that an amino acid of one type is substituted for another type at the same (or corresponding) position in the amino acid sequence. The corresponding amino acid positions in two or more similar sequences (e.g., wild-type and substituted sequences) can be determined by aligning those sequences using readily available informatics tools (e.g., BLAST). When two similar proteins have sequences that have or contain one or more amino acid deletions or insertions relative to each other, optimal alignment can be obtained, for example, by using BLAST with one or more vacancies, and the corresponding amino acid positions between the two proteins can be identified. When used herein with respect to amino acid substitutions in a sequence, “mutation” and “mutated” refer to amino acid substitutions, typically altering amino acid substitutions in the wild-type sequence. When used herein with respect to modifications of an amino acid sequence, “mutation” and “mutated” refer to the substitution of one or more amino acids in the sequence. Mutations can be artificially generated. When used herein with respect to modifications of an amino acid sequence, “deletion” and “deleted” mean the removal of one or more amino acids from the sequence, typically from the wild-type sequence. In this disclosure, amino acid substitutions or mutations may be described using texts such as “XZ substitution” or “XZ mutation”, where “X” refers to one or more positions in a first sequence occupied by amino acid “X”, and “Z” refers to a second sequence in which this position is substituted or mutated to amino acid “Z”. Typically, but not necessarily when otherwise indicated by the context, the first sequence is a reference (e.g., wild-type) sequence, and the second sequence is a modified sequence (e.g., a modified sequence of the present invention). In some embodiments, the formula “XZ substitution” refers to only one amino acid position; in some embodiments, it refers to more than one amino acid position; and in some embodiments, depending on the context, it refers to all amino acid positions in the sequence (or a designated portion thereof) occupied by amino acid “X” or “Z”; in all such cases, the number of amino acid positions in which such substitution or mutation occurs is clear from the context. Thus, for example, a “KA” or “Lys-Ala” substitution at nine amino acid positions means that each of the nine lysine positions in the first sequence is replaced by an alanine in the second sequence.

[0147] As used herein, unless the context otherwise clarifies, the term "surface marker" means an antigen or other molecular motif present on the cell surface that a CAR can specifically bind to. Examples of available surface markers are BCMA, CD19, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, and CD30. Tumor antigens are antigens that are specific or relatively specific to cancer cells and can act 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.

[0148] As used herein, unless the context otherwise clarifies, the term “target” and derivatives such as “target cell surface marker” refer to a surface marker or cell, tissue, or tumor that is specifically bound by a CAR. In such cases where the target refers to a cell, tissue, or tumor type, such cells, tissue, or tumor typically express (i.e., exhibit) a surface marker that is specifically bound by a CAR. Therefore, as used herein, “target cell” refers to a cell that is specifically bound by a particular CAR or a cell expressing a CAR (e.g., CAR T cell).

[0149] As used in this article, the term "therapeutic" refers to treatment and / or prevention. Therapeutic effects are achieved by suppressing, alleviating, or eradicating a disease state.

[0150] As used herein, the term "therapeuticly effective amount" refers to the amount of a subject compound that will elicit a biological or medical response in the tissue, system, or subject being sought by researchers, veterinarians, physicians, or other clinicians. The term "therapeuticly effective amount" includes, when administered, an amount sufficient to prevent the development of one or more signs or symptoms of the treated disorder or disease, or to alleviate to some extent one or more signs or symptoms of the treated disorder or disease. Therapeuticly effective amounts will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. A therapeutically effective amount does not need to be the amount required for clinical efficacy.

[0151] As used herein, the term "treatment" refers to a clinical intervention aimed at reversing a disease or disorder or one or more symptoms thereof, alleviating a disease or disorder or one or more symptoms thereof, delaying the onset of a disease or disorder or one or more symptoms thereof, or inhibiting the progression of a disease or disorder or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after the disease has been diagnosed. In other embodiments, for example, treatment may be administered in the absence of symptoms to prevent or delay the onset of symptoms or inhibit the onset or progression of the disease. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0152] As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary subject cells and their progeny.

[0153] A “vector” is a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into the cell. Many vectors are known in the art, 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-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc. Detailed Implementation

[0154] CAR overall structure

[0155] In one aspect, this disclosure provides novel CARs, each comprising a modified CD3-ζ intracellular domain, wherein certain amino acid residues are selectively modified or mutated relative to the wild-type sequence to obtain CARs that provide better and more durable expression and CAR-mediated cellular functions such as cytotoxicity and cytokine secretion.

[0156] The general design of CAR proteins is known in the art. See, for example, Alnefaie, ibid., and U.S. Patent 10,934,337, the entire contents of which are incorporated herein by reference. In some embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular (T cell signaling) domain of this disclosure, as described herein, that are specific to a particular surface antigen (marker) (e.g., CD19, BCMA, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, or CD30). Each intracellular domain of this disclosure comprises a modified CD3-ζ intracellular domain, as disclosed below. Optionally, the intracellular domain may further comprise CD8-α protein, CD28 protein, FcR γ 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 a modified form or portion thereof—or other co-stimulatory domains known for use in CAR, and combinations thereof.

[0157] Generally, for the purpose of constructing a CAR, one suitable class of extracellular (antigen-binding) domains is known and another suitable class of intracellular domains is known. Any particular type of such extracellular domain can be combined (via a transmembrane domain) with any particular type of such intracellular domain to obtain an operable CAR. Therefore, in the case of providing a functional CAR, different types of extracellular domains can be substituted in the CAR, for example, to confer binding specificity to a specific antigen (i.e., a target cell surface marker). Similarly, in the case of providing a functional CAR, different types of intracellular domains can be substituted in the CAR, for example, to affect other CAR properties, as described herein. This phenomenon is beneficial to the present disclosure because the novel CD3-ζ intracellular domain of the present disclosure, as described herein, is generally suitable (non-limitingly) for use in any CAR containing a CD3-ζ intracellular domain, including many CARs described to date. Furthermore, the novel CD3-ζ intracellular domain of the present disclosure is applicable to any system containing a CD3-ζ intracellular domain. Therefore, for example, wherever the novel CD3-ζ intracellular domains described in this disclosure with respect to BCMA-specific CARs are, those same novel CD3-ζ intracellular domains are suitable for use in CARs that include different extracellular antigen recognition domains that bind different surface antigens (such as, but not necessarily limited to, CD19, EGFR / HER, CD22, mesothelin, CD123, CD20, PD1, or CD30).

[0158] In some embodiments, a spacer domain and / or hinge domain may be incorporated between the extracellular and transmembrane domains of the CAR or between the transmembrane and intracellular domains of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide used to link a transmembrane domain to an extracellular or intracellular domain in a polypeptide chain. Spacer domains for CARs are known in the art. See, for example, U.S. Patent 10,934,337.

[0159] 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 instance of "]-[" indicates the optional presence of a spacer sequence.

[0160] In some embodiments, the intracellular domain comprises the CD3-ζ protein of this disclosure, and optionally comprises CD8-α protein, CD28 protein, FcR γ 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 a portion thereof—other intracellular co-stimulatory molecules known for use in CARs, and any combinations thereof. In some embodiments, the CAR of this disclosure comprises at least one of the following structures:

[0161] NH2-[extracellular antigen-binding domain]-[transmembrane domain]-[intracellular domain]-COOH;

[0162] NH2-[extracellular antigen-binding domain]-[hinge region]-[transmembrane domain]-[intracellular domain]-COOH;

[0163] NH2-[signal peptide]-[extracellular antigen-binding domain]-[transmembrane domain]-[intracellular domain]-COOH; or

[0164] NH2-[signal peptide]-[extracellular antigen-binding domain]-[hinge region]-[transmembrane domain]-[intracellular domain]-COOH.

[0165] In some implementations, the CAR includes intracellular domains having an arrangement selected from one of the following exemplary non-limiting arrangements:

[0166] NH2-[CD3-ζ intracellular domain disclosed herein]-COOH;

[0167] NH2-[CD28]-[the CD3-ζ intracellular domain disclosed herein]-COOH;

[0168] NH2-[41BB]-[the CD3-ζ intracellular domain disclosed herein]-COOH;

[0169] NH2-[CD27]-[the CD3-ζ intracellular domain disclosed herein]-COOH;

[0170] NH2-[CD40]-[the CD3-ζ intracellular domain disclosed herein]-COOH;

[0171] NH2-[ICOS]-[the CD3-ζ intracellular domain disclosed herein]-COOH;

[0172] NH2-[CD40L]-[the CD3-ζ intracellular domain disclosed herein]-COOH;

[0173] NH2-[OX40]-[the CD3-ζ intracellular domain disclosed herein]-COOH; or

[0174] NH2-[41BB]-[OX40]-[the CD3-ζ intracellular domain disclosed herein]-COOH.

[0175] In some embodiments, the exemplary non-limiting arrangement described above is the CAR from left to right, from the N-terminus to the C-terminus. In some embodiments, each instance of “]-[” indicates the optional presence of a spacer sequence.

[0176] In some embodiments, the CAR is designed to have a leader domain (also referred to as a “signal peptide”) for guiding the translated chimeric protein toward the membrane. In some embodiments, the CAR includes a leader sequence at the N-terminus of the CAR protein. For example, the CAR may include a leader sequence located at the N-terminus of an extracellular antigen-binding domain, wherein, optionally, the leader sequence is selected because of its tendency or ability to cleave from the antigen-binding domain during cell processing and CAR localization to the cell membrane. The leader domain is further described in U.S. Patent 10,934,337.

[0177] Transmembrane domains for CARs are also described in U.S. Patent 10,934,337. Transmembrane domains can be derived from naturally occurring sequences or can be synthetic. When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein, provided that the transmembrane domain allows signal transduction to the intracellular domain whenever the CAR has bound to the target. Transmembrane domains specifically used in this invention may include at least, for example, the transmembrane region of the α, β, or ζ chain of a T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8 α, CD8 β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.In some implementations, the transmembrane domain may at least include the transmembrane region of a co-stimulatory molecule, such as class I MHC molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activating 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, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7R α, 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 (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83. In some embodiments, the transmembrane domain may be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is synthesized at one or both ends of the transmembrane domain. Optionally, short oligopeptides or polypeptide linkers (e.g., between 2 and 10 amino acids in length) may form a link between the transmembrane domain of the CAR and the intracellular signaling domain. Glycine-serine duplexes provide an exemplary suitable linker.

[0178] 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 used for this purpose is known in the art and is set forth in PCT Publication No. WO 2014 / 055771, which is incorporated herein by reference.

[0179] Intracellular domains

[0180] The CAR protein of the present invention, or any other protein containing the CD3-ζ intracellular domain, contains a novel intracellular domain that is a substituted, modified, or mutated form of the CD3-ζ intracellular domain.

[0181] In some embodiments, the novel intracellular domain comprises a sequence similar to the wild-type CD3-ζ intracellular domain sequence (SEQ ID NO: 18), except for substitutions of certain lysine amino acids naturally present in the wild-type sequence. For example, the novel intracellular domain comprises 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 certain lysine amino acids naturally present in the wild-type sequence.

[0182] Therefore, in some embodiments, the CAR or any other protein containing the CD3-ζ intracellular domain comprises the human CD3-ζ intracellular domain of SEQ ID NO: 18, except that each of the at least six lysine amino acids of SEQ ID NO: 18 is independently replaced by an amino acid selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0183] In some embodiments, the CAR or any other protein containing the CD3-ζ intracellular domain comprises the human CD3-ζ intracellular domain of SEQ ID NO: 18, except that at least seven lysine amino acids of SEQ ID NO: 18 are replaced by amino acids independently selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0184] In some embodiments, the CAR or any other protein containing the CD3-ζ intracellular domain comprises the human CD3-ζ intracellular domain of SEQ ID NO: 18, except that at least eight lysine amino acids of SEQ ID NO: 18 are replaced by amino acids independently selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0185] In some embodiments, the CAR or any other protein containing the CD3-ζ intracellular domain comprises the human CD3-ζ intracellular domain of SEQ ID NO: 18, except that at least nine lysine amino acids of SEQ ID NO: 18 are substituted independently with amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0186] In some embodiments, the CAR or any other protein containing the CD3-ζ intracellular domain comprises the human CD3-ζ intracellular domain of SEQ ID NO: 18, except that the nine lysine amino acids of SEQ ID NO: 18 are replaced by amino acids independently selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0187] In some embodiments in which at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are substituted, the substitution may be selected from the group consisting of alanine, aspartic acid, and glutamic acid. In some embodiments in which at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are substituted, all substitutions may be with alanine. In some embodiments in which at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are substituted, all substitutions may be with aspartic acid. In some embodiments in which at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are substituted, all substitutions may be with glutamic acid.

[0188] The above-described amino acid substitutions can be further understood by referring to the sequences of certain embodiments of the CAR of this disclosure or any other protein containing a CD3-ζ intracellular domain. Therefore, in some embodiments, the CAR of this disclosure or any other protein containing a CD3-ζ intracellular domain comprises a sequence selected from SEQ ID NO: 2-5 (which corresponds to the modified CD3-ζ domain). In some embodiments, the CAR of this disclosure or any other protein containing a CD3-ζ intracellular domain comprises a sequence selected from SEQ ID NO: 19-66 and 68-86 (which corresponds to the modified CD3-ζ intracellular domain).

[0189] In some embodiments, the CAR of this disclosure or any other protein containing a CD3-ζ intracellular domain comprises the human CD3-ζ intracellular domain of SEQ ID NO: 18, except that at least six lysine amino acids of SEQ ID NO: 18 are missing. In some such embodiments, at least seven such lysine amino acids are missing. In some such embodiments, at least eight such lysine amino acids are missing. In some such embodiments, at least nine such lysine amino acids are missing. In some such embodiments, nine such lysine amino acids are missing.

[0190] In some embodiments, the CAR of this disclosure or any other protein containing a CD3-ζ intracellular domain comprises the human CD3-ζ 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 ways described above. 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.

[0191] In addition to the CD3-ζ sequence of this disclosure or any other protein containing the CD3-ζ intracellular domain, some intracellular domains used in this disclosure also include a co-stimulatory domain corresponding to the wild-type co-stimulatory domain, or wherein one or more lysine amino acids of the co-stimulatory domain have been substituted or mutated to, for example, alanine amino acids. See, for example, Examples 4 and 5 and the amino acid sequences of the co-stimulatory domains in SEQ ID NO: 57-60 and 62-66. Therefore, in some embodiments, the CAR of this disclosure or any other protein containing the CD3-ζ intracellular domain includes a novel co-stimulatory domain in which one, more than one, or all of the lysine amino acids naturally present in the co-stimulatory domain (or a portion thereof, as incorporated into the CAR) are independently substituted with amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine. In some embodiments, the substitution may be selected from the group consisting of: alanine, aspartic acid, and glutamic acid. In some embodiments, all substitutions are with alanine. In some embodiments, all substitutions are made with aspartic acid. In some embodiments, all substitutions are made with glutamic acid.

[0192] Examples of proteins that can be at least partially incorporated into a CAR (as a co-stimulatory domain in each case) include: class I MHC molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activating 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, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA M, 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 (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83, etc.

[0193] Nucleic acid and vector

[0194] In some embodiments, the invention encompasses nucleic acid molecules (e.g., DNA or RNA) encoding a CAR of the present disclosure (e.g., any CAR provided herein) or any other protein comprising a CD3-ζ 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 the sequence of a CAR or any other protein comprising a CD3-ζ intracellular domain, wherein the sequence comprises a nucleic acid sequence encoding a CAR of the present disclosure, or any other protein comprising a CD3-ζ intracellular domain.

[0195] Any nucleic acid molecule provided herein may include other features such as a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), a polyadenylation tail (polyA), a 7-methylguanosine cap (m 7 G), an internal ribosome entry site (IRES), and / or an open reading frame. In some embodiments, the present disclosure provides an RNA or DNA encoding the RNA having the following arrangement of features:

[0196] 5′-[CAR]-3’

[0197] 5’-[5′ UTR]-[CAR]-3′

[0198] 5′-[m 7 G cap]-[5′ UTR]-[CAR]-3′

[0199] 5′-[m 7 G cap]-[5′ UTR]-[CAR]-[polyA]-3′

[0200] 5′-[CAR]-[polyA]-3’

[0201] 5’-[CAR]-[3′ UTR]-[polyA]-3′

[0202] 5′-[5′ UTR]-[CAR]-[3′ UTR]-3′

[0203] 5′-[5′ UTR]-[CAR]-[3′ UTR]-[polyA]-3′

[0204] 5′-[m 7 G cap]-[5′ UTR]-[CAR]-[3′ UTR]-[polyA]-3′

[0205] The construction of the nucleic acid encoding a CAR of the present disclosure can be further understood by reference to U.S. Patent 10,934,337, which is incorporated herein by reference.

[0206] The present invention also provides vectors in which the DNA or RNA of the present invention is inserted. The construction of such vectors for the CAR of the present disclosure can be further understood by reference to U.S. Patent 10,934,337, which is incorporated herein by reference.

[0207] Cells modified to express CAR

[0208] Any CAR or any other protein of this disclosure containing a CD3-ζ intracellular domain as presented herein can be expressed in suitable cells. Examples of suitable cells are T cells, which, once modified to express a CAR, become CAR T cells. 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 cells suitable for expressing a CAR or any other protein containing a CD3-ζ intracellular domain include NK cells and stem cells, such as hematopoietic stem cells.

[0209] Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector 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 extrusion or nanoparticle-based delivery), calcium phosphate precipitation, lipofection, particle bombardment, microinjection, etc. 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). Electroporation is a preferred method for introducing polynucleotides into host cells.

[0210] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., 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. Patent Nos. 5,350,674 and 5,585,362.

[0211] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In cases using non-viral delivery systems, an exemplary delivery medium is a liposome. This covers the use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).

[0212] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of the present invention, a variety of assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a specific peptide, for example by immunological means (ELISA and Western blotting) or by assays described herein to identify reagents falling within the scope of this disclosure.

[0213] RNA transfection

[0214] In some embodiments, the CAR T cells of this disclosure or other cells containing proteins, such as the modified CD3-ζ intracellular domain of this disclosure, are obtained by introducing RNA (e.g., mRNA containing a sequence encoding a CAR as described herein). In some embodiments, the in vitro transcribed RNA CAR can be introduced into cells as a transient transfection. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted into a template by PCR, using suitable primers and RNA polymerase for in vitro mRNA synthesis. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. The desired template for in vitro transcription can be the CAR of this invention.

[0215] RNA can be introduced into target cells using any of a number of different methods, such as commercially available methods including, but not limited to, 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 squeezing, see U.S. Patent Publication No. 2014 / 287509 A1), transfection mediated by cationic liposomes using lipid transfection, polymer encapsulation, peptide-mediated transfection, or biological projectile particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0216] This document discloses methods for generating in vitro transcribed RNA CARs or generating in vitro transcribed RNA containing any other protein with a CD3-ζ intracellular domain. The invention also includes RNA constructs encoding CARs, or RNA constructs containing any other protein with a CD3-ζ intracellular domain, that can be directly transfected into cells. Methods for generating mRNA for transfection may involve in vitro transcription (IVT) of a template using specially designed primers, followed by the addition of a polyA tail to generate a construct containing 5' and 3' untranslated sequences (“UTR”), a 5' cap, the nucleic acid to be expressed, and a polyA tail, typically 50-400, 50-2000 bases, 150-400 bases, or 150-2000 bases in length. The resulting RNA can be efficiently transfected into various cell types. In one aspect, the template contains the sequence of a CAR disclosed herein. In another aspect, the template contains the sequence of another protein disclosed herein, said other protein containing a CD3-ζ intracellular domain.

[0217] For example, the production of mRNA by in vitro transcription (IVT) from a DNA template is known in the art. One method for producing mRNA for transfection, for example, involves in vitro transcription (IVT) of a template using specially designed primers. Optionally, the mRNA is 3' polyadenylated by methods known in the art and may contain a 3' polyadenylated tail, for example, about 25, 50, 100, 150, 250, 500, or 1000 adenine nucleotides.

[0218] 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, pseudouridine is artificially enriched in the RNA. In some embodiments, substantially all uridine nucleotides of the RNA (e.g., greater than 90%, 95%, 97%, 99%, or 99.9%) are replaced by pseudouridine. Methods for incorporating pseudouridine into RNA are known in the art.

[0219] In some implementations, the nucleic acid is a circular RNA prepared according to methods known in the art.

[0220] Therapeutic applications

[0221] In some embodiments, the present invention covers cells (e.g., T cells) modified to express the CAR of the present disclosure or other proteins of the present disclosure containing a CD3-ζ intracellular domain. Therefore, in some cases, the transduced immune cells (e.g., T cells) can elicit a CAR-mediated immune (e.g., T cell) response, a cytotoxic response, or an antitumor response. In some embodiments, the present disclosure provides the use of the CAR of the present disclosure for the specific redirection of primary T cells to a surface marker or tumor antigen. Therefore, 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 mammals, comprising the step of administering T cells expressing the CAR of the present disclosure to the mammal, wherein the CAR contains an antigen-binding domain that specifically binds to a predetermined target marker or surface marker (e.g., BCMA). In some embodiments, the present invention includes a class of cell therapies in which T cells are genetically modified to express the CAR of the present disclosure and the CAR T cells are infused into a recipient in need. The infused cells are capable of killing target cells in the recipient. Unlike antibody therapy, some CAR T cells are able to replicate in the body, resulting in the long-term persistence of these cells.

[0222] The CAR-modified T cells of this disclosure, or cells modified with proteins of this disclosure containing CD3-ζ intracellular domains, can also serve as a class of vaccines for ex vivo immunization and / or in vivo therapy in mammals. Preferably, the mammal is a human.

[0223] Regarding the generation of ex vivo cells, at least one of the following occurs in vitro prior to administration of the cells to a mammal: i) cell expansion, ii) introduction of nucleic acid encoding a CAR into the cells, and / or iii) cryopreservation of the cells. Ex vivo procedures are well known in the art and are discussed more fully below. In short, cells are isolated from a mammal (e.g., a human) and genetically modified (e.g., in vitro transduction or transfection) with a nucleic acid or vector expressing the CAR of this disclosure, as disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0224] The CAR-modified immune cells (e.g., CAR T cells) or compositions containing such cells of the present invention can be used or administered in an effective amount to a subject in need to provide anti-tumor immunity; treat or prevent cancer; treat or prevent autoimmune diseases; or treat or prevent allergic conditions. In some embodiments, the cancer is multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or glioblastoma. In some embodiments, the autoimmune disease is 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 implementations, the allergic condition is anaphylaxis, asthma, food allergy, insect sting allergy, drug allergy, allergic rhinitis, urticaria, angioedema, eczema, atopic dermatitis, contact dermatitis, and eosinophilic esophagitis.

[0225] The CAR-modified immune cells (e.g., CAR T cells) of the present invention can be administered alone or as a composition (e.g., a pharmaceutical composition) in combination with a diluent and / or other components (such as IL-2 or other cytokines or cell populations). In short, the pharmaceutical compositions of the present invention may comprise a combination of a target cell population as described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0226] The compositions of the present invention are preferably formulated for intravenous administration, but can also be formulated for other routes of parenteral administration.

[0227] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined based on factors such as the patient's condition and the type and severity of the patient's disease, but an appropriate dosage can be determined through clinical trials.

[0228] When referring to "immunely effective dose," "antitumor effective dose," "tumor-suppressive effective dose," or "therapeutic dose," the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in the condition. Generally, it can be stated that a pharmaceutical composition comprising CAR-modified immune cells (e.g., CAR T cells) as described herein can be administered at the following dosage: 10 4 Up to 10 9 Cells / kg body weight, preferably 10 5 Up to 10 9 Cells per kg body weight, including all integer values ​​within those ranges. T-cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Medical professionals can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's signs of disease and adjusting treatment accordingly.

[0229] Without further elaboration, it is believed that those skilled in the art can make full use of this disclosure based on the foregoing description. Therefore, the following specific embodiments should be construed as illustrative only and not as limiting the remainder of this disclosure in any way. All publications cited herein for the purposes or subject matter are incorporated herein by reference.

[0230] Example

[0231] To provide a fuller understanding of this disclosure, the following embodiments are illustrated. The synthetic examples described herein are provided to illustrate the compounds and methods provided herein and should not be construed as limiting their scope in any way.

[0232] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the testing practices of this invention, preferred materials and methods are described herein. The following terms will be used in describing and claiming protection for this invention.

[0233] Example 1.

[0234] CAR T cells were generated using an mRNA construct encoding the CAR protein of this disclosure, the CAR protein containing a mutated CD3-ζ intracellular domain to prevent downregulation of the protein. A series of experiments were then performed on them. The CAR proteins studied contained wild-type CD3-ζ intracellular domain sequences (SEQ ID NO: 1), mutant sequences of the CD3-ζ intracellular domain with all lysine mutated to arginine (SEQ ID NO: 2), mutant sequences of the CD3-ζ intracellular domain with all lysine mutated to alanine (SEQ ID NO: 3), mutant sequences of the CD3-ζ intracellular domain with all lysine mutated to aspartic acid (SEQ ID NO: 4), or mutant sequences of the CD3-ζ intracellular domain with all lysine mutated to glutamate (SEQ ID NO: 5). CAR T cells were observed to express high levels of the CAR protein, bind BCMA, and kill BCMA+ tumor cells. Exposure of CAR T cells to target cells expressing BCMA ligands resulted in low levels of downregulation of the cell surface CAR protein.

[0235] The mRNA constructs of this disclosure, comprising the nucleotide sequences of SEQ ID NO: 13-17, 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 poly-A tail of 180 nucleotides. During co-transcriptional mRNA synthesis, a 7-methylguanosine cap (CleanCap) was incorporated into the 5' end of the mRNA.

[0236] The mRNA construct disclosed herein comprises SEQ ID NO: 13 and includes, from 5' to 3': a 5' cap, a 5' UTR as described in SEQ ID NO: 11, an open reading frame (ORF) as described in SEQ ID NO: 6, a 3' UTR as described in SEQ ID NO: 12, and a 3' polyadenine tail of 150 or more adenine units. The ORF encodes the CAR protein of this disclosure having the amino acid sequence of SEQ ID NO: 1.

[0237] Another mRNA construct of this disclosure comprises SEQ ID NO: 14 and includes, from 5' to 3': a 5' cap, a 5' UTR as described in SEQ ID NO: 11, an open reading frame (ORF) as described in SEQ ID NO: 7, a 3' UTR as described in SEQ ID NO: 12, and a 3' polyadenine tail of 150 or more adenine units. The ORF encodes the CAR protein of this disclosure having the amino acid sequence of SEQ ID NO: 2.

[0238] Another mRNA construct of this disclosure comprises SEQ ID NO: 15 and includes, from 5' to 3': a 5' cap, a 5' UTR as described in SEQ ID NO: 11, an open reading frame (ORF) as described in SEQ ID NO: 8, a 3' UTR as described in SEQ ID NO: 12, and a 3' polyadenine tail of 150 or more adenine units. The ORF encodes the CAR protein of this disclosure having the amino acid sequence of SEQ ID NO: 3.

[0239] Another mRNA construct of this disclosure comprises SEQ ID NO: 16 and includes, from 5' to 3': a 5' cap, a 5' UTR as described in SEQ ID NO: 11, an open reading frame (ORF) as described in SEQ ID NO: 9, a 3' UTR as described in SEQ ID NO: 12, and a 3' polyadenine tail of 150 or more adenine units. The ORF encodes the CAR protein of this disclosure having the amino acid sequence of SEQ ID NO: 4.

[0240] Another mRNA construct of this disclosure comprises SEQ ID NO: 17 and includes, from 5' to 3': a 5' cap, a 5' UTR as described in SEQ ID NO: 11, an open reading frame (ORF) as described in SEQ ID NO: 10, a 3' UTR as described in SEQ ID NO: 12, and a 3' polyadenine tail of 150 or more adenine units. The ORF encodes the CAR protein of this disclosure having the amino acid sequence of SEQ ID NO: 5.

[0241] To prepare CAR T cells from the mRNA construct, lymphocytes were obtained from whole blood of healthy human donors. CD8+ T cells were positively selected from these lymphocytes using paramagnetic microbeads conjugated with an anti-CD8 antibody. This yielded 95% CD8+ T cells and 95% viable cells. These enriched CD8+ T cells were expanded by incubation at 37 °C and 5% CO2 for up to 14 days in the presence of an anti-CD3 antibody (clone OKT3), IL-7, and IL-15. Cells were transfected with the mRNA construct at 0.1 μg / μL using electroporation (4DNucleofector, Lonza) according to the manufacturer's instructions. The cells were then returned to complete medium containing IL-7 and IL-15 and cultured overnight.

[0242] CAR T cells obtained from the above methods were tested for viability, CAR protein expression, BCMA binding, cytotoxicity (i.e., the ability to kill BCMA+ myeloma (tumor) cells), and cytokine production. Resistance to CAR protein downregulation was tested by incubating CAR T cells with or without BCMA+ myeloma cells and then analyzing CAR expression. Viability, CAR expression, and BCMA binding were determined by flow cytometry on a Guava® EasyCyte® 12HT 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; CreativeBiomart, Shirley, NY). CAR expression was assessed by electrically gating fluorescence in the red channel on a flow cytometer to detect the presence or absence of BCMA-APC emission on CAR-positive and CAR-negative cells. BCMA binding was determined by measuring fluorescence intensity in the red channel to ascertain the relative amount of BCMA-APC bound to labeled CAR-positive cells. For viability, expression, cytotoxicity, cytokine production, and BCMA binding assays, control (non-CAR) CD8+ T cells generated by electroporation in the absence of IVT mRNA served as parallel controls.

[0243] To test the ability of the wild-type and mutant CAR proteins (SEQ ID NO: 1-5) of this disclosure to resist antigen-mediated downregulation, 50,000 CAR T cells generated with the mRNA sequences of this disclosure (SEQ ID NO: 13-17) were co-incubated with 100,000 MM1S tumor cells in the wells of a 96-well V plate. The MM1S tumor cell line was derived from patients with IgA λ multiple myeloma. See, for example, Greenstein et al., Exp. Hematol (2003) 31(4): 271-82 (DOI=10.1016 / S0301-472X(03)00023-7). Additional cultures of CAR T cells (without target cells) were prepared as controls. After incubation at 37°C for 24 hours, the tissue culture supernatant and cells were harvested. CAR expression was analyzed using CD8-BV421 antibody, propidium iodide, and BCMA-APC staining. Live CD8+ T cells were identified by exclusion of dead cells using propidium iodide (near-infrared fluorescence) staining and CD8+ cell selection using blue fluorescence with a violet laser. CAR expression on total live CD8+ T cells was quantified by the intensity of red fluorescence using a red laser. CAR signaling was assessed by analyzing interferon-γ production in tissue culture supernatant using a specific ELISA.

[0244] The signal transduction capacity of residual expressed CAR proteins was determined by secondary culture. Secondary cultures were established by co-culturing pre-exposed CAR T cells with MM1S-GFP tumor cells. Aliquots of 50,000 MM1S-GFP tumor cells were placed in the wells of a 96-well plate. CAR T cells 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 24–72 hours, dead cells were stained with propidium iodide. Live target cells were identified by the expression of GFP (green fluorescence of a blue laser) and the exclusion of propidium iodide, and cell density was determined by flow cytometry. The killing effect of CAR T cells on myeloma cells was calculated by comparing the number of myeloma cells in each well with the number of parallel control wells containing no CAR T cells. Signal transduction was assessed by analyzing interferon-γ production in the supernatant using a specific ELISA.

[0245] Following electroporation with all constructs of this disclosure (containing sequences of SEQ ID NO: 13-17), the CAR T cells of this disclosure exhibited similar viability. The percentage of CAR T cells expressing anti-BCMA CAR was similar across all constructs (91.3% to 94.0%).

[0246] Table 1

[0247] CAR T cell viability and CAR expression after electroporation using IVT mRNA

[0248]

[0249] Mean ± SD (n=3)

[0250] Exposure of the CAR T cells disclosed herein to the ligand (target) BCMA on MM1S myeloma target cells for 24 hours resulted in downregulation of CAR expression. CAR T cells generated using a construct (SEQ ID NO: 13) encoding a protein containing a wild-type CD3-ζ intracellular domain sequence showed a downregulation of CAR expression from a median fluorescence intensity (MFI) of 8,633 in the absence of BCMA ligand to 950 MFI 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 with all CD3-ζ intracellular domain lysine residues mutated to arginine. CAR T cells generated using this construct (SEQ ID NO: 14) produced cells with lower anti-BCMA CAR expression compared to the wild-type construct SEQ ID NO: 13 (2,188 MFI compared to 8,633 MFI). Exposure of these CAR T cells to the target ligand BCMA on MM1S target cells resulted in a further reduction in surface CAR expression (405 MFI for SEQ ID NO: 14 compared to 950 MFI for the wild-type construct SEQ ID NO: 13). Figure 1A ).

[0251] Other amino acids were introduced to replace lysine residues in the CAR CD3-ζ intracellular domain. The IVT mRNA of SEQ ID NO: 15 encodes a protein in which all lysine residues in the CD3-ζ intracellular domain are mutated to alanine. The IVT mRNA of SEQ ID NO: 16 encodes a protein in which all lysine residues in the CD3-ζ intracellular domain are mutated to aspartic acid. The IVT mRNA of SEQ ID NO: 17 encodes a protein in which all lysine residues in the CD3-ζ intracellular domain are mutated to glutamate. CAR T cells generated using these constructs (SEQ ID NO: 15, 16, 17) resulted in significantly higher 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 to 11,678 MFI, compared to 8,633 MFI for the wild-type construct). Compared to the 11.0% CAR expression maintained by the wild-type construct SEQ ID NO: 13, downregulation was significantly reduced in each of SEQ ID NO: 15, 16, and 17, with CAR expression at 44.2%, 73.0%, and 85.3% respectively after exposure to BCMA ligand on MM1S myeloma cells. Figure 1A ).

[0252] Table 2

[0253] Anti-BCMA CAR expressed by CAR T cells after culture in the absence or presence of MM1S target cells.

[0254]

[0255] Mean ± SD (n=3)

[0256] The CAR T cells of this disclosure, generated by mutations in the lysine domain of CD3-ζ cells to arginine, alanine, aspartic acid, and glutamate (SEQ ID NO: 14-17), all maintained signal transduction capabilities. Specific ELISA analysis of the CAR T cells of this disclosure and BCMA... +Interferon-γ expression in the supernatant of co-cultures between MM1S multiple myeloma patients. CAR T cells were pre-exposed or not pre-exposed to BCMA+ cells prior to co-culture. All constructs of this disclosure showed high levels of interferon-γ production. Interferon-γ expression was comparable between CARs generated using lysine mutant constructs SEQ ID NO: 14-17 (10,326 to 12,767 pg / mL). CAR T cells generated using lysine mutant constructs showed higher interferon-γ expression than wild-type CAR T cells (SEQ ID NO: 13; 6,881 pg / mL). Therefore, the mutation of the lysine domain in the CAR CD3-ζ cell domain to other amino acids (including arginine, alanine, aspartic acid, and glutamate) allows and does not interfere with CAR T cell activation. Figure 1B ).

[0257] Table 3

[0258] Cytokines (interferon-γ) produced by anti-BCMA CAR T cells cultured in the presence of MM1S target cells.

[0259]

[0260] Mean ± SD (n=3); N / A = Not applicable

[0261] CAR T cells generated using SEQ ID NO: 15-17 maintained higher CAR expression than wild-type (SEQ ID NO: 13) after exposure to BCMA ligand. 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 from constructs of this disclosure encoding lysine-mutant and wild-type CARs. Cytotoxicity was assessed by co-culturing at various effector:target ratios. At an effector:target ratio of 1:2, control T cells showed no cytotoxicity against MM1S-GFP target cells (<10%), while pre-exposed CAR T cells generated using each of SEQ ID NO: 13-17 showed complete or near-complete elimination of target cells (98-100% cytotoxicity). Figure 1C At lower effector:target cell ratios, wild-type pre-exposed CAR-T cells (SEQ ID NO: 13) showed a sharp decrease in cytotoxicity (49.3% at 1:8 and 4.7% at 1:32). Figure 1C CAR T cells generated using the IVT mRNA of SEQ ID NO: 14 (encoding a protein with a lysine-to-arginine mutation) showed a similar cytotoxic profile. Figure 1C In contrast, pre-exposed CAR T cells generated with IVT mRNA from each of SEQ ID NO: 15, 16, and 17 showed high levels of cytotoxicity, down to 1:32 ratios (59.0%, 68.7%, and 72.3%, respectively). Figure 1C This maintenance of cytolytic activity at extremely low effector:target ratio was achieved by matching the higher levels of interferon-γ produced (at a 1:2 ratio) in pre-exposed CAR T cells generated by these constructs (Table 4 and ). Figure 1B (Right). Pre-exposed CAR T cells generated from constructs in which the intracellular domain lysine is mutated to amino acids with opposite negative charges (aspartic acid (SEQ ID NO: 16) and glutamic acid (SEQ ID NO: 17)) showed the highest cytotoxic activity and cytokine production.

[0262] Table 4

[0263] 72-hour cytotoxicity and 24-hour cytokine production activity of CAR T cells after exposure to BCMA ligand (MM1S)

[0264]

[0265] Mean ± SD (n=3)

[0266] Therefore, compared with wild-type CAR T constructs, CAR T cells generated using IVT mRNA encoding the CAR of this disclosure (SEQ ID NO: 14, 15, 16 and 17) provide superior cytotoxicity and cytokine production.

[0267] Example 2.

[0268] A series of experiments were conducted to test how changes in the number of lysine residues in the intracellular domain of CAR CD3-ζ cells mutated to alanine residues would affect the CAR's resistance to downregulation after target binding.

[0269] Numerous IVT mRNA constructs were prepared encoding variants of the anti-BCMA CAR protein, which were identical except for the intracellular polypeptide sequence encoding the signal transduction domain. The constructs encoded CARs containing: wild-type CD3-ζ intracellular domain sequence (SEQ ID NO: 18), CD3-ζ intracellular domain sequence with a single lysine mutated to alanine (SEQ ID NO: 19-27), CD3-ζ intracellular domain sequence with 2 to 7 lysine residues mutated to alanine (SEQ ID NO: 28-33), CD3-ζ intracellular domain sequence with all but one (i.e., 8) lysine residues mutated to alanine (SEQ ID NO: 34-42), or CD3-ζ intracellular domain sequence 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 study investigated the contribution of lysine alone or the lysine amino acid group to CAR downregulation after exposure to CAR ligand (BCMA).

[0270] For each mRNA construct, CAR T cells were prepared essentially as described in Example 1. Twenty-four hours post-transfection, CAR T cells prepared from each mRNA construct were exposed to MM1S or not exposed to target cells using the method described in Example 1. Twenty-four hours later, cell count, viability, CAR expression maintenance, and cytokine production were assessed using the method described in Example 1. The constructs and their effects on CAR expression downregulation are shown below.

[0271] CAR T cells generated using all test constructs showed good viability (viability >70%) after electroporation. All CAR T cells showed high percentage anti-BCMA CAR expression (>85%) after electroporation. Exposure of CAR T cells to the ligand BCMA on MM1S myeloma target cells for up to 24 hours resulted in downregulation of CAR expression. CAR T cells generated using IVT mRNA containing the wild-type intracellular polypeptide sequence (SEQ ID NO: 18) showed a downregulation of CAR expression from 6,358 MFI to 705 MFI, representing an 89% reduction in CAR expression. In contrast, CAR T cells generated using IVT mRNA containing all nine intracellular lysine-to-alanine mutations (SEQ ID NO: 43) showed a downregulation of CAR expression from 8,264 MFI to 3,165 MFI, with 38% of CAR expression maintained after exposure to the BCMA ligand. CAR T cells generated using a construct containing a single intracellular lysine-alanine mutation in CD3-ζ cells (SEQ ID NO: 19-27) showed that CAR expression was downregulated from 5,707 ± 609 MFI in the absence of a ligand to 617 ± 119 MFI in the presence of BCMA+ MM1S myeloma cells (all constructs are mean ± SD). Figure 2A This reduction in anti-BCMA CAR expression represents an 89% ± 3% decrease in CAR expression, and is therefore comparable to the 89% decrease in CAR expression observed with the wild-type construct (SEQ ID NO: 18). Figure 2A CAR T cells generated using a construct containing an intracellular lysine-alanine mutation at 8 of the 9 lysine residues (SEQ ID NO: 34-42) showed CAR expression downregulated from 6,736 ± 299 MFI in the absence of a ligand to 2,064 ± 234 MFI in the presence of BCMA+ MM1S myeloma cells (all constructs are mean ± SD). Figure 2C Following exposure to BCMA ligand, anti-BCMA CAR expression remained at 31% ± 3% of the CAR expression observed in the absence of the ligand, showing an expression pattern similar to that of CAR cells generated using IVT mRNA (SEQ ID NO: 43) with all CD3-ζ intracellular domain lysine mutated to alanine. Figure 2A CAR T cells generated using an IVT mRNA construct containing a CD3-ζ intracellular lysine-alanine mutation at 2-7 lysines (SEQ ID NO: 28-33) showed moderate efficacy, retaining 25% ± 6% anti-BCMA CAR MFI after exposure to BCMA ligand compared to culture without ligand. Figure 2EA similar trend was observed in interferon-γ production in CAR T cells generated from constructs containing intracellular domains of CD3-ζ with varying numbers of lysine-alanine mutations. CAR T cells generated from a wild-type IVT mRNA construct (SEQ ID NO: 18) produced 2,291 pg / mL of interferon-γ after co-culturing with MM1S. Figure 2B CAR-T cells generated using the 9-lysine-alanine mutant construct (SEQ ID NO: 43) produced 7,853 ± 285 pg / mL of interferon-γ (γ-γ) during co-culture. Figure 2B Similar to wild-type IVT mRNA (SEQ ID NO: 18), CAR T cells generated using a construct (SEQ ID NO: 19-27) containing a CD3-ζ intracellular domain with a lysine-alanine mutation produced 2,498 ± 583 pg / mL of interferon-γ during co-culture with MM1S. Figure 2B CAR T cells generated using a construct (SEQ ID NO: 34-42) containing an intracellular domain of CD3-ζ with eight lysine-alanine mutations 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 than that of the fully mutant construct (SEQ ID NO: 43). Figure 2D Finally, CAR T cells generated from constructs with an intermediate number of 2-7 lysine-alanine mutations (SEQ ID NO: 28-33) produced an average of 4,241 ± 1,279 pg / mL of interferon-γ when co-cultured with MM1S, showing an intermediate expression level compared to wild-type (SEQ ID NO: 18) and fully mutant (SEQ ID NO: 43) constructs. Figure 2F ).

[0272] Table 5

[0273] After culturing in the absence or presence of MM1S target cells, CAR T cells expressing CAR express anti-BCMACAR, wherein the CAR contains a CD3-ζ intracellular domain with a lysine-alanine mutation.

[0274]

[0275] Table 6

[0276] In the presence of MM1S target cells, anti-BCMA CAR T cells expressing CAR produce interferon-γ, wherein the CAR contains a CD3-ζ intracellular domain lysine-alanine mutation.

[0277]

[0278] In summary, lysine mutations in the CD3-ζ intracellular domain of the anti-BCMA CAR provide the best improved resistance to downregulation when all nine lysines are mutated. Mutation of any single lysine does not provide resistance to CAR downregulation in the presence of BCMA ligand. Mutations of any eight of the nine lysines provide resistance to downregulation similar to the fully mutated construct. Intermediate numbers of mutations show moderate effects. Overall mutations (substitutions) of six, seven, eight, or nine lysines provide substantial improvements proportional to the number of such mutations (substitutions).

[0279] Example 3.

[0280] A series of experiments were conducted to test how replacing lysine residues in the CD3-ζ intracellular domain of CAR with different amino acids affects the CAR's resistance to downregulation caused by target ligands.

[0281] Numerous IVT mRNA constructs encoding variants of the anti-BCMA CAR protein were prepared. These constructs are identical except for the intracellular polypeptide sequence encoding the signal transduction domain. The constructs encode the CAR and contain either the wild-type CD3-ζ intracellular polypeptide sequence (SEQ ID NO: 18) or the CD3-ζ intracellular domain sequence with all lysine residues mutated to one of several different amino acids (SEQ ID NO: 43-56). 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 are otherwise identical in terms of the cap, 5'UTR, open reading frame encoding the signal peptide, scFv, and transmembrane sequence, 3'UTR, and poly-A tail. The aim was to compare the effects of different amino acid substitutions (mutations) on the preservation of CAR expression, maintenance of CAR signaling, and resistance of proteins to ligand-mediated downregulation after exposure to CAR ligands (BCMA).

[0282] For each IVT mRNA construct, CAR T cells were prepared essentially as described in Example 1. Twenty-four hours post-transfection, the CAR T cells prepared from each mRNA construct were exposed to MM1S or not exposed to target cells using the method described in Example 1. Twenty-four hours later, cell count, viability, CAR expression maintenance, and cytokine production were assessed using the method described in Example 1.

[0283] CAR T cells generated using all test constructs showed good viability (>70% viability) after electroporation. All test constructs were able to drive anti-BCMA CAR expression. In the absence of BCMA ligand, CAR T cells expressed by 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) showed lower CAR expression than wild-type constructs (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; 7,562 MFI), lysine-glutamate mutations (SEQ ID NO: 56; 8,346 MFI), and lysine-alanine mutations (SEQ ID NO: 43; 5,656 MFI) was higher than that of the wild-type construct (SEQ ID NO: 18). Figure 3A Other constructs tested showed comparable CAR expression to the wild-type construct. Figure 3A Compared to the wild-type construct (11%), all mutant CAR constructs showed improved maintenance of anti-BCMA CAR expression (32% to 53%) after ligand exposure. Among these constructs, the 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 of CAR expression (52%) after BCMA ligand exposure. Figure 3A CAR T cells generated using all tested constructs were capable of ligand-dependent signaling, producing interferon-γ levels ranging from 1,729 to 7,853 pg / mL during co-culture with MM1S target cells. Figure 3B The highest levels of interferon-γ production were observed using IVT mRNA constructs containing SEQ ID NO: 55 (lysine-aspartic acid), SEQ ID NO: 56 (lysine-glutamic acid), or SEQ ID NO: 43 (lysine-alanine). Figure 3B ).

[0284] Table 7

[0285] After culturing in the absence or presence of MM1S target cells, CAR T cells expressing CAR express anti-BCMACAR, wherein the CAR contains a CD3-ζ intracellular domain with lysine mutated to other amino acids.

[0286]

[0287] Table 8

[0288] After being cultured in the presence of MM1S target cells, anti-BCMA CAR T cells expressing CAR produced interferon-γ cytokines. The CAR contained a CD3-ζ intracellular domain with lysine residues mutated to other amino acids.

[0289]

[0290] nd-Undetermined

[0291] In summary, the improved resistance to downregulation provided by the lysine mutation in the CD3-ζ intracellular domain of the anti-BCMA CAR is compatible with its mutation to a variety of different amino acids, including alanine, valine, isoleucine, leucine, methionine, serine, threonine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid. CAR constructs with lysine amino acid mutations to aspartic acid, glutamic acid, or alanine are compatible with BCMA after exposure. + The target cells showed the highest retention of CAR expression and the highest level of signal transduction.

[0292] Example 4.

[0293] A series of experiments were conducted to test how the inclusion of an additional co-stimulatory domain in the intracellular portion of the lysine-alanine mutant CAR receptor affects the CAR's resistance to downregulation induced by the target ligand BCMA.

[0294] A series of IVT mRNA constructs encoding anti-BCMA CAR protein variants were prepared. These constructs are identical except for the intracellular polypeptide sequence encoding the intracellular domain. The protein encoded by the constructs comprises a wild-type intracellular CD3-ζ polypeptide sequence (SEQ ID NO: 18), or an intracellular CD3-ζ polypeptide sequence with all lysine residues mutated to alanine (SEQ ID NO: 43), or an intracellular CD3-ζ polypeptide sequence with all lysine residues mutated to alanine except for the preceding costimulatory sequence, namely: wild-type CD28 costimulatory domain (SEQ ID NO: 57), CD28 costimulatory domain with lysine mutated to alanine (SEQ ID NO: 58), wild-type 41BB costimulatory domain (SEQ ID NO: 59), or 41BB costimulatory domain with all lysine mutated to alanine (SEQ ID NO: 60). The constructs are otherwise identical in terms of the cap, 5'UTR, encoding signal peptide, scFv, open reading frame of the transmembrane sequence, 3'UTR, and poly-A tail. The experiment then tested the compatibility of wild-type or lysine-alanine mutant CD28 and 41BB co-stimulatory domains to preserve CAR expression, maintain CAR signaling, and provide resistance to ligand-mediated downregulation of the CAR protein upon exposure to the CAR ligand (BCMA).

[0295] For each IVT mRNA construct, CAR T cells were prepared essentially as described in Example 1. Twenty-four hours post-transfection, CAR T cells prepared from each mRNA construct were exposed to MM1S or not exposed to target cells using the method described in Example 1. Twenty-four hours later, cell counts, maintenance of CAR expression activity, cytotoxicity, and cytokine production were assessed using the method described in Example 1. The constructs and their effects on CAR expression downregulation are shown below.

[0296] 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-ζ signaling domains SEQ ID NO: 18 (wild-type) and SEQ ID NO: 43 (KA) showed the highest anti-BCMA CAR expression in the absence of BCMA ligand. Figure 4A The addition of the wild-type CD28 (SEQ ID NO: 57) or 41BB (SEQ ID NO: 59) signaling domains resulted in reduced CAR expression in the absence of ligands (896 MFI and 2,886 MFI, compared to 7,389 MFI in SEQ ID NO: 43). Figure 4AMutations in the co-stimulatory domain, via lysine to alanine (SEQ ID NO: 58 and 60), resulted in further loss of anti-BCMA CAR expression in the absence of ligands (625 MFI and 114 MFI, respectively). Figure 4A In the presence of a ligand, CAR T cells generated from all constructs encoding a protein containing a lysine-alanine mutant CD3-ζ signaling domain showed higher CAR expression maintenance (35%–75%) on the cell surface after exposure to BCMA+ MM1S target cells compared to the construct containing wild-type CD3-ζ (11%). Figure 4A Similarly, CAR T cells generated using all constructs can signal and produce interferon-γ upon exposure to MM1S target cells. Figure 4B The construct containing both the wild-type 41BB signaling domain and the KA-mutated CD3-ζ signaling domain, SEQ ID NO: 59, produced the highest interferon-γ yield (9,101 pg / mL), followed by SEQ ID NO: 43, which contains only the KA-mutated CD3-ζ signaling domain (8,054 pg / mL). Figure 4B Interferon-γ production driven by constructs SEQ ID NO: 57 and 58, which respectively contain wild-type and KA mutant CD28 signaling domains, was 2,998 and 3,886 pg / mL, respectively, 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 from individual constructs containing separate CD3-ζ, CD28-CD3-ζ, and 41BB-CD3-ζ signaling domains was evaluated by pre-exposing cells to MM1S followed by a 72-hour cytotoxicity assay against BCMA+ MM1S-GFP cells. All constructed constructs tested (SEQ ID NO: 61, 43, 57, and 59) showed high cytotoxicity against MM1S.

[0297] Compared to control T cells, GFP target cells with an effector:target ratio of 1:2 ( Figure 4CAt lower effector:target ratios, CAR T cells generated with SEQ ID NO: 43 (containing the KA CD3-ζ signaling domain) and SEQ ID NO: 59 (containing both WT41BB and the KA CD3-ζ signaling domain) exhibited high levels of specific cytotoxicity against MM1S-GFP (67.4% and 51.4%, respectively, at an effector:target ratio of 1:32), which was 3 to 4 times higher than the cytotoxicity observed with the wild-type construct (SEQ ID NO: 61, containing only the wild-type CD3-ζ signaling domain). Figure 4C ).

[0298] Table 9

[0299] An anti-BCMA CAR expressed by CAR T cells, wherein the CAR comprises a CD3-ζ intracellular domain with a lysine-to-alanine mutation and a wild-type or mutant co-stimulatory domain within the intracellular domain.

[0300]

[0301] Table 10

[0302] Cytokines (interferon-γ) produced by anti-BCMA CAR T cells expressing CAR, wherein the CAR comprises a CD3-ζ intracellular domain with lysine mutated to alanine and a wild-type or mutant co-stimulatory domain within the intracellular domain.

[0303]

[0304] Table 11

[0305] 72-hour cytotoxicity of CAR T cells to MM1S-GFP cells after exposure to BCMA ligand (MM1S)

[0306]

[0307] Mean ± SD (n=3)

[0308] In summary, the CAR construct encoding the protein was successfully combined with additional signaling domains from CD28 and 41BB, the protein containing the CD3-ζ intracellular signaling domain, which is resistant to ligand-dependent downregulation via lysine amino acid mutation. The addition of these domains did not interfere with the prevention of downregulation, even in the presence of wild-type CD28 and 41BB signaling domain sequences. CAR constructs containing mutated CD28-CD3-ζ or 41BB-CD3-ζ intracellular signaling domains effectively drove CAR-dependent cytokine production and cytotoxicity.

[0309] Example 5.

[0310] Experiments were conducted to test how the inclusion of a CD28 co-stimulatory domain in the intracellular portion of the CAR receptor with lysine-alanine or lysine-glutamate mutations affects the CAR's resistance to downregulation induced by the target BCMA.

[0311] A series of IVT mRNA constructs encoding variants against BCMA CAR protein were prepared. These constructs were identical except for the intracellular polypeptide sequence encoding the signal transduction domain. The control construct encoded a protein containing the wild-type intracellular CD3-ζ polypeptide sequence (SEQ ID NO: 18). The two test constructs encoded proteins containing either an intracellular CD3-ζ polypeptide sequence with all lysine residues mutated to alanine (SEQ ID NO: 43, 57, 58, 62) or an intracellular CD3-ζ polypeptide sequence with all lysine residues mutated to glutamate (SEQ ID NO: 56, 63, 64, 65). The constructs differ in the presence and sequence composition of the CD28 costimulatory domain encoding the protein: two control constructs (SEQ ID NO: 43, 56) encode a protein lacking the CD28 costimulatory domain; two constructs encode a protein containing the wild-type CD28 costimulatory domain (SEQ ID NO: 57, 63); two constructs encode a protein containing a CD28 costimulatory domain with a lysine residue mutated to alanine (SEQ ID NO: 58; 64); and two constructs encode a protein containing a CD28 costimulatory domain with a lysine residue mutated to glutamic acid (SEQ ID NO: 62, 65). The constructs are otherwise identical in terms of the cap, 5' UTR, open reading frame encoding the signal peptide, scFv and transmembrane sequence, 3' UTR, and poly-A tail.

[0312] This experiment evaluated the ability of second-generation CARs containing lysine-alanine or lysine-glutamate mutant CD28-CD3-ζ intracellular peptides to retain CAR expression, maintain CAR signaling, and provide resistance to ligand-mediated downregulation of CAR proteins after exposure to CAR ligands (BCMA).

[0313] For each IVT mRNA construct, CAR T cells were prepared essentially as described in Example 1. Twenty-four hours post-transfection, CAR T cells prepared from each mRNA construct were exposed to MM1S or not exposed to target cells using the method described in Example 1. Twenty-four hours later, cell counts, maintenance of CAR expression activity, cytotoxicity, and cytokine production were assessed using the method described in Example 1. The constructs and their effects on CAR expression downregulation are shown below.

[0314] CAR T cells generated using all test constructs exhibited high viability after electroporation. CAR-expressing CAR T cells showed high anti-BCMA CAR expression in the absence of BCMA ligand, and these CAR-expressing CAR T cells were generated using constructs encoding proteins containing intracellular CD3-ζ signaling domains SEQ ID NO: 18 (wild-type), SEQ ID NO: 43 (KA), and SEQ ID NO: 56 (KE). Compared with the CD3-ζ construct alone, the addition of the CD28 co-stimulatory domain resulted in decreased CAR expression (MFI of 2,507-3,306 for SEQ ID NO: 57, 58, and 62 with the lysine-alanine mutation, compared to 16,848 for SEQ ID NO: 43; and MFI of 12,784-13,598 for SEQ ID NO: 63, 64, and 65 with the lysine-glutamate mutation, compared to 24,850 for SEQ ID NO: 56). Figure 5A In the presence of BCMA, CAR T cells generated from constructs encoding proteins containing mutant lysine-alanine CD3-ζ signaling domains (SEQ ID NO: 43, 57, 58, 62) or lysine-glutamate CD3-ζ signaling domains (SEQ ID NO: 56, 63, 64, 65) showed higher CAR expression maintenance on the cell surface after exposure to BCMA+ MM1S target cells (6-26% and 12-47%, respectively), compared to those on the construct containing wild-type CD3-ζ (SEQ ID NO: 18, 2%). Figure 5A In CAR T cells generated using constructs encoding proteins containing a lysine-glutamate mutation in the CD3-ζ intracellular domain, CAR expression was maintained at its highest level after exposure to BCMA ligands, including those lacking a co-stimulatory domain (SEQ ID NO: 56) or a CD28 co-stimulatory domain with a lysine residue mutated to alanine (SEQ ID NO: 63) or a co-stimulatory domain with a lysine residue mutated to glutamate (SEQ ID NO: 64), showing surface CAR expression maintenance of 47%, 30%, and 31%, respectively. Figure 5AThe cytotoxicity of CAR T cells generated using constructs containing individual CD3-ζ and CD28-CD3-ζ signaling domains was evaluated by pre-exposing cells to MM1S followed by a 72-hour cytotoxicity assay against BCMA+ MM1S-GFP cells. All tested constructs (SEQ ID NO: 43, 56, 57, 58, 62, 63, 64, and 65) conferred high cytotoxicity against MM1S-GFP target cells at an effector:target ratio of 1:2 compared to control T cells. At lower effector:target ratios, CAR T cells generated with SEQ ID NO: 43 (containing a single KA CD3-ζ signaling domain), SEQ ID NO: 56 (containing a single KE CD3-ζ signaling domain), SEQ ID NO: 63 (containing both KA CD28 and KE CD3-ζ signaling domains), and SEQ ID NO: 64 (containing both KE CD28 and KE CD3-ζ signaling domains) conferred high levels of specific cytotoxicity against MM1S-GFP (89.4%, 98.4%, 72.7%, and 70.2%, respectively, at a 1:8 effector:target ratio), which was 9 to 13 times higher than the cytotoxicity (7.5%) observed with the wild-type construct (SEQ ID NO: 18, containing only a wild-type CD3-ζ signaling domain). Figure 5B and 5C ).

[0315] Table 12

[0316] Anti-BCMA CAR expressed by CAR T cells expressing CAR, wherein the CAR comprises a CD3-ζ intracellular domain with lysine mutated to alanine or glutamate and a wild-type or mutant co-stimulatory domain within the intracellular domain.

[0317]

[0318] Table 13

[0319] 72-hour cytotoxicity of CAR T cells to MM1S-GFP cells after exposure to BCMA ligand (MM1S)

[0320]

[0321] Mean ± SD (n=3)

[0322] In summary, combining a CAR construct encoding a protein with a CD28 signaling domain, wherein the protein contains a CD3-ζ intracellular signaling domain resistant to ligand-dependent downregulation via lysine amino acid mutations, yields a CAR. Adding the CD28 signaling domain to a lysine-glutamate-mutated CD3-ζ intracellular signaling domain is compatible with prevention of BCMA ligand-induced CAR downregulation. This effect is most pronounced when CD28 lysine is mutated to alanine or glutamate. In contrast, adding the CD28 signaling domain to a lysine-alanine-mutated CD3-ζ intracellular signaling domain shows significantly lower prevention of BCMA ligand-induced CAR downregulation. Therefore, lysine mutations (e.g., to glutamate) throughout the CD28-CD3-ζ intracellular sequence produce a CAR highly resistant to BCMA ligand-dependent downregulation.

[0323] Example 6.

[0324] A series of experiments were conducted to test how downregulated anti-BCMA CARs containing lysine-alanine mutations in intracellular signal transduction domains activate CAR T cells and drive effector functions in response to normal plasmablasts and plasmacytoid dendritic cells (pDCs).

[0325] In autoimmune diseases such as SLE, plasmablasts and further differentiated plasma cells produce autoantibodies that contribute to pathogenesis, while pDCs are activated by inflammatory triggers such as immune complexes and drive pathogenic immune responses by producing cytokines and stimulating T cells. The ability of downregulating anti-BCMA CARs to drive CAR-specific T cell activation and modulate immune responses in the context of autologous plasmablasts and pDCs was determined.

[0326] An IVT mRNA construct encoding an anti-BCMA CAR protein of an intracellular CD3-ζ polypeptide sequence (SEQ ID NO: 43) with all lysine residues mutated to alanine was prepared. For this construct, CAR T cells were prepared essentially as described in Example 1. Plasma cells and pDCs were isolated from whole blood of the same (autologous) donor using antibody-magnetic bead-based separation (Plasma Cell Separation Kit II and Diamond Plasma-like Dendritic Cell Separation Kit II, human, Miltenyi). The isolated plasma cells and pDCs were stained with the 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, as well as each of the autologous plasma cell and pDC populations (7.5K T cells and 1.5K plasma cells for plasma cells; 10K T cells and 10K pDCs for pDCs). The cultures were incubated overnight at 37 °C, and the activation of CAR T cells or control cells was analyzed by flow cytometry using activation-induced markers CD69 and CD137 (41BB) the next day. The production of interferon-γ in the supernatant was analyzed by ELISA.

[0327] BCMA CAR T cells generated from IVT mRNA encoding a protein containing the sequence SEQ ID NO: 43 showed upregulation of CD69 and 41BB in response to exposure to plasmablasts and pDCs (for plasmablast cocultures, CD69: from 0.93% to 12.74%, 41BB: from 3.92% to 23.93%; for pDC cocultures, CD69: from 2.65% to 34.05%, 41BB: from 1.92% to 28.99%). Figure 6A and 6B Similarly, interferon-γ was produced in the supernatant of the co-cultures (187 pg / mL in plasmablast co-cultures and 5 pg / mL in pDC co-cultures). In contrast, control cells lacking IVT mRNA did not show upregulation of activation markers in response to plasmablasts and pDCs (2.45% for CD69+ and 2.28% for 41BB+ in plasmablast co-cultures, and 12.98% for CD69+ and 2.36% for 41BB+ in pDC co-cultures), and interferon-γ remained at concentrations 5–25 times lower than in the BCMA CAR T case (7.3 pg / mL in plasmablast co-cultures and 5 pg / mL in pDC co-cultures). <LOD)( Figure 6A and 6BTherefore, CAR T cells expressing downregulated resistance CARs are activated in a CAR-dependent manner in the context of plasmablasts and pDCs, and produce a class of immunomodulatory cytokines known to regulate autoimmune diseases.

[0328] Example 7.

[0329] This embodiment describes experiments and methods for conferring resistance to downregulation to each of a variety of CAR proteins, each of which is expressed in CAR T cells (and thus each targets CD19, PSMA, and CCR4 respectively), obtained by mutating intracellular lysine amino acid residues to alanine or glutamic acid.

[0330] A series of mRNA constructs encoding variants of the CAR protein of interest were prepared, each containing an scFv specific to one of the following cell surface targets: BCMA, CD19, PSMA, or CCR4. For each such cell surface target, an mRNA construct encoding the CAR was prepared, the CAR comprising a wild-type intracellular CD3-ζ polypeptide sequence (SEQ ID NO: 18), a wild-type intracellular CD28-CD3-ζ polypeptide sequence (SEQ ID NO: 66), an intracellular CD3-ζ polypeptide sequence with all lysine residues mutated to alanine (SEQ ID NO: 43), an intracellular CD3-ζ polypeptide sequence with all lysine residues mutated to glutamate (SEQ ID NO: 56), or an intracellular CD28-CD3-ζ polypeptide sequence with all lysine residues mutated to glutamate (SEQ ID NO: 65). These constructs were otherwise identical in terms of the cap, 5'UTR', 3'UTR, and poly-A tail.

[0331] For each mRNA construct, CAR T cells were prepared essentially as described in Example 1. Approximately 24 hours post-transfection, CAR T cells prepared from each mRNA construct were exposed to different target cell lines (MM1S for BCMA CAR; Raji for CD19 CAR; LNCaP for PSMA CAR; and CEM for CCR4 CAR) or not exposed to target cells, according to the methods described in Example 1. After 24 hours, cell counts, viability, and CAR expression were assessed using the methods described in Example 1 (BCMA-APC for BCMA CAR, FMC63 antibody for CD19 CAR, PSMA-PE for PSMA CAR, and an anti-idiotype antibody for CCR4). The ability of the pre-exposed CAR T cells to perform effector functions (cytotoxicity and interferon-γ production) in response to additional target cell lines was then assessed using methods essentially described in Example 1.

[0332] CAR T cells generated using IVT mRNAs specific to BCMA, CD19, PSMA, and CCR4 are expected to show less downregulation and higher cytotoxicity and cytokine production than wild-type counterparts, wherein the IVT mRNAs contain lysine-alanine or lysine-glutamate mutations in their intracellular signaling domains of CD3-ζ or CD28-CD3-ζ cells.

[0333] Example 8.

[0334] This embodiment describes an experimental method for controlling tumor burden in mice using CAR T cells generated from IVT mRNA encoding ligand-induced downregulated BCMA CAR. Different mRNA CAR constructs contain sequences of SEQ ID NO: 17, 67, 98, or 99, respectively. These mRNA constructs encode CAR proteins containing sequences of SEQ ID NO: 5, 68, 101, or 102, respectively.

[0335] As described in Example 1, CAR T cells were prepared by transfecting an mRNA CAR construct into human CD8+ cells, except that the endogenous T cell receptor was genetically removed from the cells through CRISPR / Cas engineering. The negative controls used in this example included control CD8+ cells without IVT mRNA. NOD-scid-γ (NSG) mice were inoculated with 2 million MM1S-fluc human multiple myeloma tumor cells. Tumor burden was monitored by continuous bioluminescence imaging. On day 5, mice were randomized and then intravenously injected with either control CD8+ T cells or CAR T cells transfected to express SEQ ID NO: 5 or 68. Tumor burden was measured daily to confirm the reduction in burden over time in mice treated with the vector or control CD8+ cells compared to CAR T cells transfected to express SEQ ID NO: 5 or 68. It is expected that CAR T cells generated with (SEQ ID NO: 5 or 68) will significantly control tumor burden and inhibit tumor growth.

[0336] In summary, CAR T cells prepared from three examples of the present invention encoding anti-BCMA CAR mRNA constructs (and their corresponding CAR proteins) are expected to suppress the growth of human myeloma tumors in predictive animal models.

[0337] Example 9.

[0338] This embodiment describes the experiments and methods for using vectors and nucleic acids other than IVT mRNA to induce T cells to express one or more downregulated resistance CAR proteins.

[0339] CAR T cells were prepared and modified using the Sleeping Beauty transposon system to express the CAR protein of this disclosure. A "wild-type CAR" plasmid was constructed comprising an EF1a promoter, an IgG 5'UTR, an open reading frame encoding the amino acid sequence of SEQ ID NO: 1, and a polyadenylated sequence element, wherein the aforementioned elements are collectively flanked by the inverted terminal repeat sequence of the Sleeping Beauty transposon. A "KE CAR" plasmid (where KE refers to an amino acid substitution) was constructed comprising an EF1a promoter, an IgG 5'UTR, an open reading frame encoding the amino acid sequence of SEQ ID NO: 5, and a polyadenylated sequence element, wherein the aforementioned elements are collectively flanked by the inverted terminal repeat sequence of the Sleeping Beauty transposon. An "SB11" transposase plasmid was constructed comprising an EFI a promoter, an IgG 5'UTR, a Kozak concordant sequence, an open reading frame encoding SB II, and a polyadenylated sequence. To generate wild-type CAR T cells, peripheral blood mononuclear cells from healthy human donors were washed and resuspended in P3 buffer (Lonza) in the presence of both the wild-type CAR transposon plasmid and the SB11 plasmid. Lysine-mutant CAR T cells were generated using the same strategy with the KE CAR plasmid and the SB11 plasmid. Cells were electroporated (4D Nucleofector, Lonza) to introduce the plasmids into the cells. Cells were then transferred to cultures and stimulated with CD3 / CD28 Dynabeads® (ThermoFisher). During expansion, CAR expression in CD4+ and CD8+ T cells was analyzed by staining with BCMA-APC reagent and flow cytometry. T cells were expected to expand and express anti-BCMA CAR protein for at least 14 days.

[0340] As described in Example 1, CAR expression and functional CAR T cell activity were assessed using a cytotoxicity assay with the multiple myeloma target cell line MMIS-GFP. CAR T cells generated with a transposon encoding a lysine-glutamate mutant intracellular signal transduction domain (SEQ ID NO: 5) were expected to show higher BCMA CAR expression levels and greater potency in the cytotoxicity assay compared with CAR T cells generated with a transposon encoding wild-type CAR.

[0341] Example 10.

[0342] This embodiment 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 CAR proteins of this disclosure.

[0343] Essentially according to the method described in Example 1, anti-BCMA CAR T cells were prepared using the CAR protein of this disclosure containing the sequence of SEQ ID NO: 3, 4, 5, 68, 101, or 102. 0.2 to 100 x 10⁻⁶ cells were infused into patients with MM (optionally prepared with lympholytic chemotherapy). 9 This disclosure describes anti-BCMA CAR T cells. Serum M protein levels, free light chains of MM-associated immunoglobulins, soluble serum BCMA levels, peripheral blood CAR+ T cell counts, serum cytokine levels (e.g., interferon-γ, IL-2, IL-10), and bone marrow biopsy were analyzed before treatment and at 2, 4, 8, 12, and 24 weeks after treatment. Based on measurements of serum M protein levels, free light chains of MM-associated immunoglobulins, soluble serum BCMA levels, and reductions in MM cells in bone marrow biopsy, it is anticipated that CAR T cells generated using the CAR described in this disclosure will effectively reduce and / or eradicate MM.

[0344] Example 11.

[0345] This embodiment describes experiments and methods for controlling autoimmune diseases using anti-BCMA CAR T cells expressing one or more CAR proteins of this disclosure.

[0346] Essentially following the method described in Example 1, anti-BCMA CAR T cells were prepared using a CAR receptor containing the sequence of SEQ ID NO: 3, 4, 5, 68, 101, or 102. 0.2 to 100 x 10⁻⁶ cells were infused into patients with MG (optionally prepared with lympholytic chemotherapy). 9 This disclosure describes anti-BCMA CAR T cells. Patients were assessed at 2, 4, 8, 12, 24, and 52 weeks post-treatment for anti-autoantigen antibodies (e.g., anti-AChR or anti-MUSK), soluble serum BCMA levels, peripheral blood CAR+ T cell counts, serum cytokine levels (e.g., TNF, IL-6, IL-2, interferon-γ, IL-10), and clinical assessments of the disease such as the Myasthenia Gravis Activities of Daily Living Scale (MG-ADL). Based on reductions in autoantibody levels, circulating cytokine concentrations, and clinical manifestations of the disease as measured by reductions in MG-ADL or other clinical scores, CAR T cells generated from the CAR of this disclosure are expected to effectively control autoimmune diseases.

[0347] Example 12.

[0348] This embodiment describes an experiment testing how replacing intracellular lysine residues in the CD3-ζ and CD28 intracellular domains of anti-CD19 CARs with glutamate affects resistance to downregulation caused by the target ligand CD19.

[0349] A series of IVT mRNA constructs encoding variants of CAR proteins targeting CD19 in scFv were prepared. The constructs contained wild-type intracellular CD3-ζ and wild-type intracellular CD28-CD3-ζ polypeptide sequences (SEQ ID: 18 and 66), or intracellular CD28-CD3-ζ polypeptide sequences with all lysine residues mutated to glutamate (SEQ ID: 56 and 65). The anti-CD19 scFv was derived from clone FMC63 (scFv1: SEQ ID NO: 87) or, as described by He et al. (doi.org / 10.1126 / sciimmunol.adf1426), the anti-CD19 scFv with a tyrosine mutation to alanine at position 261 (scFv2: SEQ ID NO: 88). These constructs were otherwise identical in terms of the cap, 5'UTR', 3'UTR, and poly-A tail. Incorporating these mutations from lysine residues to glutamate residues into the intracellular tail is a strategy to protect CARs with different target specificities from downregulation caused by their specific ligands.

[0350] For each mRNA construct, CAR T cells were prepared as described in Example 1. Approximately 24 hours post-transfection, the CAR T cells prepared from each mRNA construct were exposed to CD19 using the method described in Example 1. + Raji target cells or no exposure to target cells. 24 hours later, CAR expression in the cells was assessed using the method described in Example 1 (using anti-Myc-Alexa 647 antibody, an extracellular tag on the CAR). The ability of CAR T cells to perform cytotoxic and effector functions (IFN-γ production) in response to CD19+ Raji cell lines was also assessed using a method substantially as described in Example 1.

[0351] CAR T cells generated using IVT mRNA containing KE mutant signaling domains SEQ ID NO: 89 and 90 (KE CD3-ζ and CD28) showed the highest anti-CD19 CAR expression in the absence of CD19 ligand. In the presence of ligand, CAR T cells generated using all constructs containing lysine-glutamine mutant CD3-ζ and CD28 signaling domains (SEQ ID NO: 89 and 90) showed higher CAR expression maintenance (30%-50%) on the cell surface after exposure to Raji target cells compared to the CAR expression observed on the comparative construct containing wild-type CD3-ζ (4-5%). CAR T cells generated using constructs containing mutant intracellular CD3 and CD28 domains had higher cell lysis capacity (Table 14). CAR T cells generated using all constructs were able to signal and produce interferon-γ upon exposure to Raji target cells at an E:T ratio of 1:2 (Table 15). The construct SEQ ID NO: 90 (anti-CD19 CAR containing the KE-mutated CD28 and CD3-ζ signaling domains of scFv2) produced the highest amount of interferon-γ (614 pg / mL), followed by SEQ ID NO: 89, anti-CD19 CAR containing scFv1 and the mutated CD28 and CD3-ζ signaling domains (393 pg / mL) (Table 15).

[0352] Table 14

[0353] In the absence or presence of CD19 + After being cultured in Raji target cells, an anti-CD19 CAR was expressed by CAR T cells expressing a CAR containing a CD28-CD3-ζ intracellular domain, wherein the CD28-CD3-ζ intracellular domain has a wild-type sequence or a mutant sequence containing a lysine-to-glutamic acid mutation.

[0354]

[0355] Table 15

[0356] Cytotoxicity of anti-CD19 CAR T cells against CD19+ Raji cells (effectant to target ratio: 1:2), wherein the anti-CD19 CAR T cells express CAR containing wild-type or mutant CD28-CD3-ζ intracellular domains.

[0357]

[0358] Table 16

[0359] Exposure to CD19 +Raji cells followed by anti-CD19 CAR T cells produce cytokines (interferon-γ), and these anti-CD19 CAR T cells express CARs containing wild-type or mutant CD28-CD3-ζ intracellular domains.

[0360]

[0361] In summary, CAR constructs containing the CD28-CD3-ζ intracellular signaling domain were successfully generated using CD19-targeting scFv, exhibiting resistance to ligand-dependent downregulation via lysine amino acid mutation. The mutant anti-CD19 CAR T cells exhibited CAR-dependent cytokine production and cytolytic activity. CAR T cells generated using the CAR containing the KE mutation in the intracellular domain, compared to their wild-type counterparts, showed superior resistance in the presence of CD19. + In the case of Raji cells, resistance to ligand-mediated downregulation and superior effector function (cytokines) were observed.

[0362] Example 13.

[0363] This embodiment describes an experiment testing the use of CAR T cells generated from IVT mRNA encoding BCMA CAR to control tumor burden in vivo. The BCMA CAR is resistant to ligand-induced downregulation by mutating lysine residues in its intracellular signaling domain to glutamate. These mRNA CAR constructs contain sequences (SEQ ID NO: 17 and 67). These mRNA constructs encode CAR proteins containing sequences (SEQ ID NO: 5 and 68), respectively.

[0364] As described in Example 1, CAR T cells were prepared by transfecting the mRNA CAR construct into human CD8+ T cells, the difference being that the endogenous T cell receptor was genetically removed from the cells through CRISPR / Cas engineering. CD8+ T cells were generated from the same batch of TCR-knockout T cells. + T cell preparation was used to generate negative control cells that did not contain IVT mRNA. On day 0, immunocompromised Nod-Scid IL2R-γ knockout (NSG) mice were inoculated with 2 million MM1S-fluc human multiple myeloma tumor cells. Tumor burden was monitored by continuous bioluminescence imaging (BLI). On day 5, mice were randomly assigned to treatment groups. On day 6, mice were administered 2 million control CD8+ cells via IV. +T cells, or 2 million CAR T cells engineered to express SEQ ID NO: 5, or 2 million CAR T cells engineered to express SEQ ID NO: 68. Tumor burden was measured on days 9 and 12. On day 9, tumor burden was compared with control CD8 administration based on results measured by BLI (photons / second). + In mice receiving T-cell therapy, tumor burden was 4.7-fold lower in mice given CAR cells expressing SEQ ID NO: 5 and 7.9-fold lower in mice given CAR cells expressing SEQ ID NO: 68. On day 12, a 10.8-fold (SEQ ID NO: 5) and 16.0-fold (SEQ ID NO: 68) reduction in BLI was observed compared to control mice. All comparisons were highly significant (p < 0.001; two-way ANOVA for log-transformed data). Figures 7A-7B Table 17 shows BLI data from individual mice. Figure 7A ) and summary statistics ( Figure 7B ).

[0365] Table 17

[0366] Bioluminescent imaging of MM1S-fluc tumor burden in NSG mice treated with control CD8+ T cells or cells expressing anti-BCMA CAR, wherein the anti-BCMA CAR contains a lysine mutation in its intracellular signal transduction domain.

[0367]

[0368] In summary, the CAR T constructs of sequences SEQ ID NO: 5 and SEQ ID NO: 68 generate robust control of BCMA in vivo. + CART cells with tumor burden, wherein the construct contains a single CD3-ζ or a CD28-CD3-ζ signaling domain with a lysine residue mutated to glutamate.

[0369] Example 14.

[0370] This embodiment describes an experiment evaluating the activity and duration of CAR T cells generated using the IVT mRNA construct of this disclosure in a mouse model of multiple myeloma.

[0371] As described in Example 1, the IVT mRNA CAR construct was transfected into human CD8. +CAR T cells were prepared from T cells. CAR T cells were generated using IVT mRNA encoding the sequence SEQ ID NO: 68 and IVT mRNA encoding a control CAR, wherein the sequence SEQ ID NO: 68 contains a CD28-CD3-ζ intracellular signaling domain (SEQ ID NO: 65) with a lysine residue mutated to glutamate, and the control CAR contains a wild-type CD28-CD3-ζ intracellular signaling domain SEQ ID NO: 66. From the same batch of CD8... + T cell preparation was used to generate negative control cells that did not contain IVT mRNA. On day 0, immunocompromised Nod-Scid IL2R-γ knockout (NSG) mice were inoculated with 2 million MM1S-fluc human multiple myeloma tumor cells. Tumor burden was monitored by continuous bioluminescence imaging (BLI). On day 8, mice were randomly assigned to treatment groups. On day 9, mice were administered either 2 million or less of the following cells via IV: control CD8 + T cells, CAR T cells engineered to express the KE mutant CAR of SEQ ID NO: 65, and CAR T cells engineered to express the wild-type CAR of SEQ ID NO: 66. Tumor burden was measured at days 11, 16, and 21. Pre-selected individual mice were harvested at days 10, 11, and 14 for analysis of CAR expression in metastatic cells in blood, spleen, and bone marrow.

[0372] Compared with control CD8 + Compared to T cells, a single administration of 12.5 million anti-BCMA CAR T cells generated from an IVT mRNA construct containing the KE mutation SEQ ID NO:65 or wild-type SEQ ID NO:66 on day 9 controlled tumor burden (reducing tumor burden by 21.4-fold and 12.4-fold, respectively, by day 21). Figure 8A Tumor burden control is dose-dependent. Figure 8B CAR T cells generated from the SEQ IDNO: 65 CAR construct containing a CD28-CD3-ζ signaling domain with lysine residues mutated to glutamate showed improved tumor burden control compared to SEQ IDNO: 66 CAR T cells containing a wild-type CD28-CD3-ζ signaling domain (p<0.05 for 2 million cells). Figure 8B ).

[0373] Analysis of whole blood and bone marrow from mice still identified human CAR T cells expressing anti-BCMA CAR up to day 14. Figure 8CThe mice were administered anti-BCMA CAR T cells containing a CAR containing the KE mutation SEQ ID NO: 65.

[0374] In summary, the CAR T construct of sequence SEQ ID NO: 68 produced robust control of BCMA in vivo. + The tumor burden and demonstrated persistent expression of CAR in CART cells in vivo, the construct containing a CD28-CD3-ζ signaling domain with a lysine residue mutated to glutamate.

[0375] Example 15.

[0376] This embodiment describes an experiment testing the ability to generate anti-BCMA CAR T cells expressing the downregulated resistance CAR protein of this disclosure in T cells of subjects with autoimmune diseases.

[0377] According to the method of Example 1, anti-BCMA CAR T cells were prepared from two myasthenia gravis donors using CAR receptors generated with SEQ ID NO: 68 and SEQ ID NO: 94 (anti-PSMA CAR as a negative control CAR). The mediator EP was used as a negative EP control. Autologous plasma cells were isolated from the same two myasthenia gravis donors. Twenty-four hours after transfection, CAR-T cells were co-cultured with autologous plasma cells or malignant MM1S-GFP cells. CAR expression (MFI) and cytolytic activity (i.e., percentage of cytotoxicity, interferon-γ) of CAR-T cells generated from MG donors were evaluated.

[0378] CAR T cells generated using the CAR disclosed herein expressed anti-BCMA CAR. Donor changes in CAR expression were observed 24 hours after EP (MFI=1887 vs 3766). CAR T cells were able to effectively kill autologous plasma cells in vitro, as measured by cytotoxicity of autologous plasma cells differentiated from MG patients (E:T=1:2, cytotoxicity of donor 1 was 67.5%, and that of donor 2 was 62%), while neither vector-transfected T cells nor anti-PSMA control CAR produced a lower percentage of killing (donor 1: 32%, 18%, donor 2: 18%, -5.5%). Donor changes were also observed in interferon-γ cytokine production after cytotoxicity (donor 1: 9.82 pg / mL, donor 2: 81.68 pg / mL). The function of MG donor CAR T cells was also evaluated using the malignant MM1S-GFP cell line. Effective killing of MM1S-GFP cells was observed in both donors (73% for donor 1 and 89.5% for donor 2), accompanied by cytokine production (217.71 pg / mL for donor 1 and 138.38 pg / mL for donor 2). No significant killing or cytokine production was observed in the control CAR T group.

[0379] Table 18

[0380] Expression of an anti-BCMA CAR containing the CD28-figCD3-ζ intracellular domain and an anti-PSMA CAR as a control CAR, wherein the CD28-figCD3-ζ intracellular domain has a mutant sequence containing a lysine-to-glutamic acid mutation.

[0381]

[0382] Table 19

[0383] Functional use of MG donor CAR T cells generated from resistant CARs containing a mutant CD28-CD3-ζ intracellular signal transduction domain: targeting autologous plasma cells (n=2).

[0384]

[0385] Table 20

[0386] Functional use of MG donor CAR T cells generated from resistant CARs containing a mutant CD28-CD3-ζ intracellular signal transduction domain: targeting malignant MM1S-GFP cells (n=2).

[0387]

[0388] In summary, CD8 T cells from patients with autoimmune diseases can be used to generate CAR T cells containing downregulated resistance signaling domains. These CAR-T cells robustly express anti-BCMA CAR, carry cytolytic activity against autologous plasma cells or malignant MM1S cells, and produce cytokines.

[0389] Example 16.

[0390] This embodiment describes experiments to further enhance CAR expression and CAR T cell function by incorporating a lower affinity scFv into the downregulated resistance signaling domain. Mutating lysine residues in the intracellular domain to glutamate residues is a strategy to protect CARs with different target specificities from downregulation caused by their specific ligands. Incorporation of a lower affinity scFv enhances this strategy to further increase CAR retention after target exposure.

[0391] A series of IVT mRNA constructs were prepared, encoding variants of CAR proteins with two scFv targets targeting BCMA. scFv1 has an epigenetic affinity KD of 3.2 nM, and scFv2 has an epigenetic affinity KD of 13.3 nM. Figure 10A The constructs contain wild-type intracellular CD3-ζ and wild-type intracellular CD28-CD3-ζ polypeptide sequences or intracellular CD28-CD3-ζ polypeptide sequences with all lysine residues mutated to glutamate. These constructs are otherwise identical in terms of the cap, 5'UTR', 3'UTR, and poly-A tail. CAR T cells were prepared for each mRNA construct as described in Example 1. Approximately 24 hours post-transfection, the CAR T cells prepared from each mRNA construct were exposed to BCMA+ target cells (MM1S) or not exposed to target cells, as described in Example 1. CAR expression in the cells was assessed 24 hours later, as described in Example 1. The ability of CAR T cells to exhibit cytotoxicity in response to BCMA+ cell lines was also assessed, substantially as described in Example 1.

[0392] In the absence of BCMA ligand, all generated CAR T cells showed the highest anti-BCMA expression, with the CAR containing scFv1 and the mutant intracellular signaling domain CD3-ζ KE (encoded by SEQ ID NO: 101 and RNA SEQ ID: 98) showing the highest expression (MFI=5336). In the presence of BCMA ligand, CAR T cells generated from all constructs containing the lysine-glutamine mutant CD3-ζ signaling domain showed higher CAR retention on the cell surface after exposure to BCMA target cells (26%, 46%, 24%, 50%) compared to constructs containing wild-type CD3-ζ (10% and 13%). Among these intracellular signaling domain mutant CAR T cells, cells containing the lower affinity scFv2 showed higher CAR expression maintenance after MM1S exposure (46% and 50%) compared to their higher affinity scFv1 counterparts (24% and 26%). CAR T cells generated with SEQ ID NO: 98 showed higher CAR retention after exposure to BCMA. + The highest maintenance of CAR expression was observed after targeting cells. CAR T cells generated using constructs containing lower affinity scFv2 (SEQ ID: 98, encoding SEQ ID: 101 CAR and SEQ ID: 99, encoding SEQ ID: 102 CAR) also showed higher cell lysis (94% and 91%, respectively) compared to higher affinity scFv1 CAR T cells (40%–58%).

[0393] Table 21

[0394] After culturing in the absence or presence of BCMA+ MM1S cells, an anti-CD19 CAR was expressed by CAR T cells expressing a CAR containing a CD28-CD3-ζ intracellular domain, wherein the CD28-CD3-ζ intracellular domain has a wild-type sequence or a mutant sequence containing a lysine-to-glutamic acid mutation.

[0395]

[0396] Table 22

[0397] 48-hour cytotoxicity of anti-CD19 CAR T cells expressing CAR against MM1S GFP cells (effectant to target ratio: 1:4), wherein the CAR contains wild-type or mutant CD28-CD3-ζ intracellular domains and scFvs with different affinities.

[0398]

[0399] In summary, apart from mutations in the CD28 and CD3-ζ signaling domains, the modulation of scFv affinity for the target demonstrates an improvement in maintaining CAR function using RNA CAR.

[0400] Example 16.

[0401] The various substitutions and corresponding lysine positions of this disclosure are provided below:

[0402]

[0403]

[0404]

[0405] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned herein (e.g., in the background, summary, detailed description, embodiments, and / or references sections) are incorporated herein by reference in their entirety, as if each individual publication, patent, patent application, publication, and database entry were specifically and separately incorporated herein by reference. In case of conflict, this application (including any definitions herein) shall prevail.

[0406] Equivalents and scope

[0407] Those skilled in the art will recognize or be able to determine multiple equivalents of the embodiments described herein using only conventional experiments. The scope of this disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0408] Articles such as “a,” “an,” and “the” may indicate one or more members unless indicated to the contrary or obvious from the context. A claim or description including “or” between two or more members of a group is considered satisfied if one, more than one, or all members of that group are present, unless indicated to the contrary or obvious from the context. The disclosure of a group including “or” between two or more members 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 set forth separately herein, but it should be understood that each of these embodiments is provided herein and may be specifically claimed or waived.

[0409] It should be understood that this disclosure covers all variations, combinations, and arrangements of one or more limitations, elements, clauses, or descriptive terms derived from one or more claims or from one or more related portions of the specification that are incorporated into another claim. For example, a claim dependent on another claim may be modified to include one or more limitations present in any other claim dependent on the same basic claim. Furthermore, in the case of claims reciting compositions, it should be understood that, unless otherwise stated or unless a contradiction or inconsistency would be apparent to a person skilled in the art, this disclosure includes methods of preparing or using the composition according to any method disclosed herein or according to methods known in the art (if any).

[0410] When elements are presented in a list format, such as in Markush group format, it should be understood that every possible subgroup of the elements is also disclosed, and any element or subgroup of elements can be removed from that group. It should also be noted that the term "comprises" is intended to be open-ended and allows for the inclusion of additional elements or steps. It should be understood that, generally, when an embodiment, product, or method is referred to as comprising a particular element, feature, or step, embodiments, products, or methods consisting of or substantially consisting of those elements, features, or steps are also provided. For the sake of brevity, these embodiments are not described separately herein, but it should be understood that each of these embodiments is provided herein and can be specifically claimed or waived.

[0411] Where a range is given, endpoints are included. Furthermore, it should be understood that, unless otherwise indicated or otherwise apparent from the context and / or understanding of one of ordinary skill in the art, in some embodiments, values ​​represented as ranges may assume any particular value within the range, up to one-tenth of the unit of the lower limit of the range, unless the context expressly specifies otherwise. For the sake of brevity, values ​​in each range are not individually stated herein, but it should be understood that each of these values ​​is provided herein and may be specifically claimed or waived. It should also be understood that, unless otherwise indicated or otherwise apparent from the context and / or understanding of one of ordinary skill in the art, values ​​represented as ranges may assume any subrange within a given range, wherein the endpoints of the subranges are represented with the same accuracy as one-tenth of the unit of the lower limit of the range.

[0412] Furthermore, it should be understood that any particular embodiment of the invention may be expressly excluded from any one or more claims. Where a scope is given, any value within that scope may be expressly excluded from any one or more claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods disclosed herein may be excluded from any one or more claims. For the sake of brevity, not all embodiments in which one or more elements, features, objects, or aspects are explicitly excluded herein.

[0413] Implementation Plan

[0414] The following implementation schemes are within the scope of this disclosure.

[0415] 1. A protein capable of intracellular signal transduction comprising a CD3-ζ intracellular domain, wherein the CD3-ζ 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 by amino acids independently selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0416] 2. The protein according to embodiment 1, wherein the CD3-ζ intracellular domain is 100% identical to SEQ ID NO: 18 except for the substituted lysine amino acid.

[0417] 3. The protein according to embodiment 1 or 2, wherein at least seven lysine amino acids of SEQ ID NO: 18 are independently replaced by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

[0418] 4. The protein according to any one of embodiments 1-3, wherein at least eight lysine amino acids of SEQ ID NO: 18 are independently substituted with amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine and valine.

[0419] 5. The protein according to any one of embodiments 1-4, wherein the nine lysine amino acids of SEQ ID NO: 18 are independently replaced by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine and valine.

[0420] 6. The protein according to any one of embodiments 1-5, wherein at least six lysine amino acids of SEQ ID NO: 18 are independently replaced by amino acids selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0421] 7. The protein according to any one of embodiments 1-6, wherein at least seven lysine amino acids of SEQ ID NO: 18 are independently replaced by amino acids selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0422] 8. The protein according to any one of embodiments 1-7, wherein at least eight lysine amino acids of SEQ ID NO: 18 are independently replaced by amino acids selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0423] 9. The protein according to any one of embodiments 1-8, wherein the nine lysine amino acids of SEQ ID NO: 18 are independently replaced by amino acids selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0424] 10. A protein comprising a CD3-ζ intracellular domain, wherein the CD3-ζ intracellular domain is 80% identical to SEQ ID NO: 18, wherein at least six lysine amino acids of SEQ ID NO: 18 are (a) missing, or (b) substituted by amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

[0425] 11. The protein according to embodiment 10, wherein the CD3-ζ intracellular domain is 100% identical to SEQ ID NO: 18, except for the missing or substituted lysine amino acids.

[0426] 12. The protein according to embodiment 10 or 11, wherein at least seven lysine amino acids of SEQ ID NO: 18 are (a) missing, or (b) substituted by amino acids independently selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0427] 13. The protein according to any one of embodiments 10-12, wherein at least eight lysine amino acids of SEQ ID NO: 18 are (a) missing or (b) substituted by amino acids independently selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0428] 14. The protein according to any one of embodiments 10-13, wherein nine lysine amino acids of SEQ ID NO: 18 are (a) missing or (b) substituted by amino acids independently selected from the group consisting of alanine, aspartic acid and glutamic acid.

[0429] 15. The protein according to any one of embodiments 1-14, wherein at least six lysine amino acids of SEQ ID NO: 18 are replaced by alanine.

[0430] 16. The protein according to any one of embodiments 1-15, wherein at least seven lysine amino acids of SEQ ID NO: 18 are replaced by alanine.

[0431] 17. The protein according to any one of embodiments 1-16, wherein at least eight lysine amino acids of SEQ ID NO: 18 are replaced by alanine.

[0432] 18. The protein according to any one of embodiments 1-17, wherein nine lysine amino acids of SEQ ID NO: 18 are replaced by alanine.

[0433] 19. The protein according to any one of embodiments 1-14, wherein at least six lysine amino acids of SEQ ID NO: 18 are replaced by aspartic acid.

[0434] 20. The protein according to any one of embodiments 1-14 or 19, wherein at least seven lysine amino acids of SEQ ID NO: 18 are replaced by aspartic acid.

[0435] 21. The protein according to any one of embodiments 1-14, 19 or 20, wherein at least eight lysine amino acids of SEQ ID NO: 18 are replaced by aspartic acid.

[0436] 22. The protein according to any one of embodiments 1-14 or 19-21, wherein nine lysine amino acids of SEQ ID NO: 18 are replaced by aspartic acid.

[0437] 23. The protein according to any one of embodiments 1-14, wherein at least six lysine amino acids of SEQ ID NO: 18 are replaced by glutamic acid.

[0438] 24. The protein according to any one of embodiments 1-14 or 23, wherein at least seven lysine amino acids of SEQ ID NO: 18 are replaced by glutamic acid.

[0439] 25. The protein according to any one of embodiments 1-14, 23 or 24, wherein at least eight lysine amino acids of SEQ ID NO: 18 are replaced by glutamic acid.

[0440] 26. The protein according to any one of embodiments 1-14 or 23-25, wherein nine lysine amino acids of SEQ ID NO: 18 are replaced by glutamic acid.

[0441] 27. The protein according to any one of embodiments 1-26, comprising an amino acid sequence that is 80% identical to any one of SEQ ID NO: 2-7, 19-66 and 68-86.

[0442] 28. The protein according to embodiment 1, comprising an amino acid sequence that is 80% identical to any one of SEQ ID NO: 2-7, 19-66 and 68-86.

[0443] 29. The protein according to any one of embodiments 1-28, wherein the protein is a chimeric antigen receptor (CAR).

[0444] 30. A nucleic acid that encodes a protein according to any one of embodiments 1-29.

[0445] 31. The nucleic acid according to embodiment 30, wherein the nucleic acid comprises ribonucleic acid.

[0446] 32. The nucleic acid according to embodiment 30 or 31, wherein the nucleic acid is ribonucleic acid.

[0447] 33. A cell comprising the nucleic acid described in any one of embodiments 30-32.

[0448] 34. The cell according to embodiment 33, wherein the cell is a human cell.

[0449] 35. A viral vector suitable for expressing the protein described in any one of embodiments 1-29.

[0450] 36. A cell modified to express a protein according to any one of embodiments 1-29.

[0451] 37. The cell according to embodiment 36, wherein the cell is a human cell.

[0452] 38. A method for producing a cell therapy, the method comprising combining cells with nucleic acids encoding proteins according to any one of embodiments 1-29.

[0453] 39. As described in embodiment 38, wherein the cell is a human cell.

[0454] 40. The method according to embodiment 39, wherein the human cell is a T cell.

[0455] 41. The method according to embodiment 39, wherein the human cell is a CD3+ cell.

[0456] 42. The method according to embodiment 39, wherein the human cell is a CD8+ cell.

[0457] 43. The method according to embodiment 39, wherein the human cell is a CD4+ cell.

[0458] 44. The method according to embodiment 39, wherein the human cell is an NK cell.

[0459] 45. The method according to embodiment 39, wherein the human cell is a stem cell.

[0460] 46. ​​The method according to embodiment 45, wherein the stem cell is a hematopoietic stem cell.

[0461] 47. The method according to embodiment 45, wherein the stem cell is a mesenchymal stem cell.

[0462] 48. A kit comprising one or more of the following: a protein according to any one of embodiments 1-29, a nucleic acid according to any one of embodiments 30-32, a cell according to embodiments 33, 34, 36 or 37, or a vector according to embodiment 35.

[0463] sequence

[0464] This document refers to the following amino acid (AA) or nucleotide (nt) sequences. For the amino acid sequences SEQ ID NO: 1-5 and nucleic acid sequences SEQ ID NO: 6-10 provided below, the signal peptide-scFv-CD8 sequence or nucleic acid encoding the signal peptide-scFv-CD8 sequence is indicated in bold, and the signal transduction domain or nucleic acid encoding the signal transduction domain is indicated in underscore. Double underscores indicate mutated amino acids. Unless otherwise stated, nucleic acid sequences listed below and in this application may be described as “T” in a representative DNA sequence, but in the case where the sequence represents RNA, the “T” will be replaced with “U”. Therefore, any DNA disclosed herein and identified by a specific sequence also discloses a corresponding RNA sequence, wherein each “T” in the DNA sequence is replaced with a “U”:

[0465] SEQ ID NO: 1 (AA, a peptide anti-BCMA CAR containing only WT CD3z)

[0466]

[0467] SEQ ID NO: 2 (AA, a peptide anti-BCMA CAR containing only KR CD3z)

[0468]

[0469] SEQ ID NO: 3 (AA, a peptide anti-BCMA CAR containing only KA CD3z)

[0470]

[0471] SEQ ID NO: 4 (AA, a peptide anti-BCMA CAR containing only KD CD3z)

[0472]

[0473] SEQ ID NO: 5 (AA, a peptide anti-BCMA CAR containing only KE CD3z)

[0474]

[0475] SEQ ID NO: 6 (nt, ORF+STOP anti-BCMA CAR containing only WT CD3z)

[0476]

[0477] SEQ ID NO: 7 (nt, ORF+STOP anti-BCMA CAR containing only KR CD3z)

[0478]

[0479] SEQ ID NO: 8 (nt, ORF+STOP anti-BCMA CAR containing only KA CD3z)

[0480]

[0481] SEQ ID NO: 9 (nt, ORF+STOP anti-BCMA CAR containing only KD CD3z)

[0482]

[0483] SEQ ID NO: 10 (nt, ORF+STOP anti-BCMA CAR containing only KE CD3z)

[0484]

[0485] SEQ ID NO: 11 (nt, 5'UTR)

[0486] aggactcttctggtccccacagactcagagagaacccaccgccacc

[0487] SEQ ID NO: 12 (nt, 3'UTR)

[0488] tgcccgtcctcaccaagactgactgcctgctgctttgctactgcccgggcccatgagactgacttcccactgctctgcctgcctctccccactgcactggcacagccccgccttgccgctgctgatccattgccggtgtgacccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacacca

[0489] SEQ ID NO: 13 (nt, IVT mRNA)

[0490]

[0491] SEQ ID NO: 14 (nt, IVT mRNA)

[0492]

[0493] SEQ ID NO: 15 (nt, IVT mRNA)

[0494]

[0495] SEQ ID NO: 16 (nt, IVT mRNA)

[0496]

[0497] SEQ ID NO: 17 (nt, IVT mRNA)

[0498]

[0499] SEQ ID NO: 18 (AA, intracellular domain of peptide WT CD3z)

[0500]

[0501] SEQ ID NO: 19 (AA, single KA CD3z intracellular domain of polypeptide)

[0502]

[0503] SEQ ID NO: 20 (AA, single KA CD3z intracellular domain of polypeptide)

[0504]

[0505] SEQ ID NO: 21 (AA, single KA CD3z intracellular domain of peptide)

[0506]

[0507] SEQ ID NO: 22 (AA, single KA CD3z intracellular domain of polypeptide)

[0508]

[0509] SEQ ID NO: 23 (AA, single KA CD3z intracellular domain of polypeptide)

[0510]

[0511] SEQ ID NO: 24 (AA, single KA CD3z intracellular domain of polypeptide)

[0512]

[0513] SEQ ID NO: 25 (AA, single KA CD3z intracellular domain of peptide)

[0514]

[0515] SEQ ID NO: 26 (AA, single KA CD3z intracellular domain of polypeptide)

[0516]

[0517] SEQ ID NO: 27 (AA, single KA CD3z intracellular domain of polypeptide)

[0518]

[0519] SEQ ID NO: 28 (AA, polypeptide 2-7 KA CD3z intracellular domain)

[0520]

[0521] SEQ ID NO: 29 (AA, polypeptide 2-7 KA CD3z intracellular domain)

[0522]

[0523] SEQ ID NO: 30 (AA, polypeptide 2-7 KA CD3z intracellular domain)

[0524]

[0525] SEQ ID NO: 31 (AA, polypeptide 2-7 KA CD3z intracellular domain)

[0526]

[0527] SEQ ID NO: 32(AA, polypeptide 2-7 KA CD3z intracellular domain)

[0528]

[0529] SEQ ID NO: 33 (AA, polypeptide 2-7 KA CD3z intracellular domain)

[0530]

[0531] SEQ ID NO: 34 (AA, peptide 8 KA CD3z intracellular domain)

[0532]

[0533] SEQ ID NO: 35 (AA, peptide 8 KA CD3z intracellular domain)

[0534]

[0535] SEQ ID NO: 36 (AA, peptide 8 KA CD3z intracellular domain)

[0536]

[0537] SEQ ID NO: 37 (AA, peptide 8 KA CD3z intracellular domain)

[0538]

[0539] SEQ ID NO: 38 (AA, peptide 8 KA CD3z intracellular domain)

[0540]

[0541] SEQ ID NO: 39 (AA, peptide 8 KA CD3z intracellular domain)

[0542]

[0543] SEQ ID NO: 40 (AA, peptide 8 KA CD3z intracellular domain)

[0544]

[0545] SEQ ID NO: 41 (AA, peptide 8 KA CD3z intracellular domain)

[0546]

[0547] SEQ ID NO: 42 (AA, peptide 8 KA CD3z intracellular domain)

[0548]

[0549] SEQ ID NO: 43 (AA, peptide 9 KA CD3z intracellular domain)

[0550]

[0551] SEQ ID NO: 44 (AA, peptide 9 KV CD3z intracellular domain)

[0552]

[0553] SEQ ID NO: 45 (AA, peptide 9 KI CD3z intracellular domain)

[0554]

[0555] SEQ ID NO: 46 (AA, peptide 9 KL CD3z intracellular domain)

[0556]

[0557] SEQ ID NO: 47 (AA, peptide 9 KM CD3z intracellular domain)

[0558]

[0559] SEQ ID NO: 48 (AA, peptide 9 KF CD3z intracellular domain)

[0560]

[0561] SEQ ID NO: 49 (AA, peptide 9 KY CD3z intracellular domain)

[0562]

[0563] SEQ ID NO: 50 (AA, peptide 9 KS CD3z intracellular domain)

[0564]

[0565] SEQ ID NO: 51 (AA, intracellular domain of peptide 9 KT CD3z)

[0566]

[0567] SEQ ID NO: 52 (AA, peptide 9 KN CD3z intracellular domain)

[0568]

[0569] SEQ ID NO: 53 (AA, peptide 9 KQ CD3z intracellular domain)

[0570]

[0571] SEQ ID NO: 54 (AA, peptide 9 KH CD3z intracellular domain)

[0572]

[0573] SEQ ID NO: 55 (AA, peptide 9 KD CD3z intracellular domain)

[0574]

[0575] SEQ ID NO: 56 (AA, peptide 9 KE CD3z intracellular domain)

[0576]

[0577] SEQ ID NO: 57 (AA, peptide WT CD28 KA CD3z intracellular domain)

[0578]

[0579] SEQ ID NO: 58 (AA, peptide KA CD28 KA CD3z intracellular domain)

[0580]

[0581] SEQ ID NO: 59 (AA, peptide WT 41BB KA CD3z intracellular domain)

[0582]

[0583] SEQ ID NO: 60 (AA, polypeptide KA 41BB KA CD3z intracellular domain)

[0584]

[0585] SEQ ID NO: 61(AA, peptide WT CD3z intracellular domain (polymorphic variant))

[0586]

[0587] SEQ ID NO: 62 (AA, peptide KE CD28 KA CD3z intracellular domain)

[0588]

[0589] SEQ ID NO: 63 (AA, peptide WT CD28 KE CD3z intracellular domain)

[0590]

[0591] SEQ ID NO: 64 (AA, peptide KA CD28 KE CD3z intracellular domain)

[0592]

[0593] SEQ ID NO: 65 (AA, intracellular domain of peptide KE CD28 KE CD3z)

[0594]

[0595] SEQ ID NO: 66(AA, peptide WT CD28-CD3z intracellular domain (polymorphic variant))

[0596]

[0597] SEQ ID NO: 67 (nt, IVT mRNA)

[0598]

[0599] SEQ ID NO: 68 (AA, a peptide containing KE CD28-CD3z to fight BCMA CAR)

[0600]

[0601] SEQ ID NO: 69 (AA, intracellular domain KE of peptide CD3z with a KA (position 1))

[0602]

[0603] SEQ ID NO: 70 (AA, intracellular domain KE of peptide CD3z with a KA (position 2))

[0604]

[0605] SEQ ID NO: 71 (AA, intracellular domain KE of peptide CD3z with a KA (position 3))

[0606]

[0607] SEQ ID NO: 72 (AA, intracellular domain KE of peptide CD3z with a KA (position 4))

[0608]

[0609] SEQ ID NO: 73 (AA, intracellular domain KE of peptide CD3z with a KA (position 5))

[0610]

[0611] SEQ ID NO: 74 (AA, intracellular domain KE of peptide CD3z with a KA (position 6))

[0612]

[0613] SEQ ID NO: 75 (AA, intracellular domain KE of peptide CD3z with a KA (position 7))

[0614]

[0615] SEQ ID NO: 76 (AA, intracellular domain KE of peptide CD3z with a KA (position 8))

[0616]

[0617] SEQ ID NO: 77 (AA, intracellular domain KE of peptide CD3z with a KA (position 9))

[0618]

[0619] SEQ ID NO: 78 (AA, intracellular domain KA of peptide CD3z with a KE (position 1))

[0620]

[0621] SEQ ID NO: 79 (AA, intracellular domain KA of peptide CD3z with a KE (position 2))

[0622]

[0623] SEQ ID NO: 80 (AA, intracellular domain KA of peptide CD3z with a KE (position 3))

[0624]

[0625] SEQ ID NO: 81 (AA, intracellular domain KA of peptide CD3z with a KE (position 4))

[0626]

[0627] SEQ ID NO: 82 (AA, intracellular domain KA of peptide CD3z with a KE (position 5))

[0628]

[0629] SEQ ID NO: 83 (AA, intracellular domain KA of peptide CD3z with a KE (position 6))

[0630]

[0631] SEQ ID NO: 84 (AA, intracellular domain KA of peptide CD3z with a KE (position 7))

[0632]

[0633] SEQ ID NO: 85 (AA, intracellular domain KA of peptide CD3z with a KE (position 8))

[0634]

[0635] SEQ ID NO: 86 (AA, Polypeptide CD3z intracellular domain K - A, having a K - E (position 9))

[0636]

[0637] SEQ ID NO: 87 (AA, scFv)

[0638] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0639] SEQ ID NO: 88 (AA, scFv)

[0640] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYAGGSYAMDYWGQGTSVTVSS

[0641] SEQ ID NO: 89 (nt, anti - BCMA CAR full - length ntd)

[0642]

[0643] SEQ ID NO: 90 (nt, full-length anti-BCMA CAR ntd)

[0644]

[0645] SEQ ID NO: 91 (nt, full-length anti-BCMA CAR ntd)

[0646]

[0647] SEQ ID NO: 92 (nt, full-length anti-BCMA CAR ntd)

[0648]

[0649] SEQ ID NO: 93 (AA, full-length anti-BCMA CAR aa)

[0650] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0651] SEQ ID NO: 94 (AA, full-length anti-BCMA CAR aa)

[0652] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKTGKVPKFLIYEASTLQSGVPSRFSGGGSGTDFTLTISSLQPEDVATYYCQNYNSAPFTFGPGTKVDIKGSTSGSGKPGSGEGSTKGQVQLVESGGGVVQPGRSLRLSCAASGFAFSRYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTQYLQMNSLRAEDTAVYYCARGGDFLYYYYYGMDVWGQGTTVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0653] SEQ ID NO: 95 (nt, full-length ntd of anti-BCMA CAR)

[0654]

[0655] SEQ ID NO: 96 (nt, full-length anti-BCMA CAR ntd)

[0656]

[0657] SEQ ID NO: 97 (nt, full-length anti-BCMA CAR ntd)

[0658]

[0659] SEQ ID NO: 98 (nt, full-length anti-BCMA CAR ntd)

[0660]

[0661] SEQ ID NO: 99 (nt, full-length anti-BCMA CAR ntd)

[0662]

[0663] SEQ ID NO: 100 (nt, full-length anti-BCMA CAR ntd)

[0664]

[0665] SEQ ID NO: 101 (AA, full-length anti-BCMA CAR)

[0666] MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPGQPPKLLIYSASNLQSGVPARFSGSGSGTDFTLTISSVEPEDTANYYCLQSRTLPRTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGGSVKISCKASGYTFTSYSINWVRQAPGKGLEWVGWINTETREPAYAQGFTGRFTFSADTSKSMAYLQINSLRAEDTAVYYCALDYLYSLDFWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRVEFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDERRGRDPEMGGEPQRRENPQEGLYNELQEDEMAEAYSEIGMEGERRRGEGHDGLYQGLSTATEDTYDALHMQALPPR

[0667] SEQ ID NO: 102 (AA, full-length anti-BCMA CAR)

[0668] MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPGQPPKLLIYSASNLQSGVPARFSGSGSGTDFTLTISSVEPEDTANYYCLQSRTLPRTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGGSVKISCKASGYTFTSYSINWVRQAPGKGLEWVGWINTETREPAYAQGFTGRFTFSADTSKSMAYLQINSLRAEDTAVYYCALDYLYSLDFWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSERSRLLHSDYMNMTPRRPGPTREHYQPYAPPRDFAAYRSRVEFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDERRGRDPEMGGEPQRRENPQEGLYNELQEDEMAEAYSEIGMEGERRRGEGHDGLYQGLSTATEDTYDALHMQALPPR

Claims

1. A protein capable of intracellular signal transduction, comprising an intracellular domain, 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 independently substituted with amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

2. The protein of claim 1, wherein the intracellular domain comprises a CD3-ζ domain.

3. The protein according to claims 1 and 2, wherein the intracellular domain comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the same domain as SEQ ID NO: 18, 61, or 66.

4. The protein according to any one of claims 1-3, wherein the CD3-ζ 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 1-4, wherein at least three lysine amino acids are independently replaced by amino acids 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 1-5, wherein at least four, at least five, at least six, at least seven, or at least eight lysine amino acids are independently substituted by amino acids 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 1-6, wherein at least nine lysine amino acids are substituted by 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 1-7, wherein at least six, at least seven, at least eight, or at least nine lysine amino acids are independently substituted with amino acids selected from the group consisting of alanine, aspartic acid, and glutamic acid.

9. A CD3-ζ intracellular domain, wherein at least two lysine amino acids of the CD3-ζ intracellular domain are independently replaced by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

10. The CD3-ζ intracellular domain of claim 9, wherein the intracellular domain comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the same domain as SEQ ID NO: 18 or 61.

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

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

18.

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

14. The CD3-ζ intracellular domain according to any one of claims 9-13, wherein at least four, at least five, at least six, at least seven, or at least eight lysine amino acids are independently substituted by amino acids selected from the group consisting of: alanine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, leucine, methionine, serine, threonine, and valine.

15. The CD3-ζ intracellular domain according to any one of claims 9-14, wherein at least nine lysine amino acids are substituted by 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-ζ intracellular domain according to any one of claims 9-15, wherein at least six, at least seven, at least eight, or at least nine lysine amino acids are independently substituted with amino acids selected from the group consisting of alanine, aspartic acid, and glutamic acid.

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

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

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

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

21. A protein comprising the CD3-ζ intracellular domain of any one of claims 9-20.

22. A protein comprising a CD3-ζ intracellular domain, wherein the CD3-ζ 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) missing or (b) substituted by amino acids 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-ζ intracellular domain is 100% identical to that of SEQ ID NO: 18, except for missing or substituted lysine amino acids.

24. The protein according to claim 22 or 23, wherein at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are (a) missing, or (b) substituted by amino acids independently selected from the group consisting of alanine, aspartic acid, and glutamic acid.

25. The protein according to any one of claims 22-24, wherein at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are replaced by alanine.

26. The protein according to any one of claims 22-25, wherein at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are replaced by aspartic acid.

27. The protein according to any one of claims 22-26, wherein at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine lysine amino acids of SEQ ID NO: 18 are replaced by glutamic acid.

28. The protein of claim 22, comprising at least 80% identical amino acid sequence to any one of SEQ ID NO: 2-7, 19-66 and 68-86.

29. The protein of claim 22, comprising at least 85%, at least 90%, at least 95%, or at least 99% of the same amino acid sequence as any one of SEQ ID NO: 2-7, 19-66, and 68-86.

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

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

32. The protein of claim 31, wherein the co-stimulatory domain is selected from the group consisting of: CD8-α domain, 41BB domain, CD28 domain, FcRγ 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 B7H3 domain, and any variant thereof.

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

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

35. The protein according to any one of claims 31-34, wherein at least one lysine amino acid of the co-stimulatory domain is independently replaced by an amino acid 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 31-35, wherein at least two lysine amino acids of the co-stimulatory domain are independently replaced by amino acids selected from the group consisting of alanine, aspartic acid, and glutamic acid.

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

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

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

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

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

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

43. The protein of claim 42, wherein the transmembrane domain comprises a transmembrane region of the following: class I MHC molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activating 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, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7R α, 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 (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a or receptors that specifically bind to CD83.

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

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

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

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

48. The CD3-ζ intracellular domain according to claim 9, comprising any one of the sequences SEQ ID NO: 28-56 or 69-86.

49. The protein of claim 22, comprising any one of sequences SEQ ID NO: 57-60 and 62-65.

50. A protein comprising the sequence of SEQ ID NO: 5, 68, 101 or 102.

51. A nucleic acid construct encoding the protein of any one of claims 1-8 or 22-50 or the CD3-ζ intracellular domain of any one of claims 9-21.

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. A nucleic acid construct comprising SEQ ID NO: 13-17 or 67.

55. A vector encoding the protein of any one of claims 1-8 or 22-50 or the CD3-ζ intracellular domain of any one of claims 9-21.

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

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

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

59. A pharmaceutical composition comprising the composition of claim 58.

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

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

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

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 61-63, wherein the cell is an immune cell.

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

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

67. A method for transfecting cells in a plurality of cells using the vector according to any one of claims 55-57.

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

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

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

71. A method for treating a disease in a subject in need, the method comprising administering to the subject the cells of any one of claims 61-65.

72. A method for transfecting cells using the vector according to any one of claims 55-57.

73. The method of claim 72, wherein the cell is a human cell.

74. The method according to claim 72 or 73, wherein the cell is an immune cell.

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

76. The method according to any one of claims 72-74, wherein the cell is a CD3+ cell.

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

78. The method according to any one of claims 72-74, wherein the cell is a CD4+ cell.

79. The method according to any one of claims 72-74, wherein the cell is an NK cell.

80. The method according to any one of claims 72-74, wherein the cell is a stem cell.

81. The method of claim 80, wherein the stem cell is a hematopoietic stem cell.

82. The method of claim 80, wherein the stem cell is a mesenchymal stem cell.

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

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

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

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

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

88. The method according to any one of claims 71 or 73-87, wherein the method is characterized by increased cellular secretion of cytokines.

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

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

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

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

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

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

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