Compositions and methods of cell immunotherapy

Abstract

This paper relates to chimeric receptors with anti-transplant rejection function, manipulated cells expressing chimeric receptors, and methods for antitumor and anti-transplant immune rejection, particularly methods for anti-NK cell immune rejection. It also relates to cell therapy.

Description

[Technical Field]

[0001] This application relates to a chimeric receptor, engineered cells expressing the chimeric receptor, and further to a method for using the chimeric receptor and engineered cells in immunotherapy.

[0002] cross reference This application claims the rights of Chinese patent applications CN2023108420804 filed on 10 July 2023, CN2023109052320 filed on 21 July 2023, and CN2023110354213 filed on 16 August 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Simultaneous submission of sequence listing file The contents of the following XML file are incorporated herein by reference in their entirety: Computer-Readable Format (CRF) Sequence Listing FH01069PCT-Sequence listing (created July 10, 2024, file size 159KB). [Background technology]

[0004] Due to immunogenetic differences between donors and recipients, in exogenous donor transplantation, the donor may be recognized and attacked by the recipient's immune cells as an exogenous graft, potentially leading to suppression or rejection, and causing a host-versus-graft reaction (HVGR). Knocking out MHC molecules in transplanted cells can effectively suppress graft rejection by host T cells, but it may trigger rejection by other immune cells in the host. For example, in allogeneic cell transplantation, the absence of MHC class I molecules in allogeneic cells leads to rejection by host NK cells, promoting the elimination of allogeneic cells. Therefore, effectively suppressing immune rejection by host NK cells is crucial in the development of allogeneic cell transplantation therapy.

[0005] CD38 is a membrane-localized glycoprotein widely expressed in various hematopoietic malignancies (e.g., multiple myeloma (MM), acute myeloid leukemia (AML)) and is a potential therapeutic target. CD38 is also strongly expressed in immunosuppressive cells and may be a potential target for immunosuppressive cells. Because CD38 is also expressed in T cells, cannibalism (fratricide) occurs between cells during the culture and proliferation of CD38-CAR-T cells, leading to culture failure and consequently reducing the effectiveness of CD38-CAR-T therapy for hematopoietic malignancies.

[0006] Because activated T cells express the NKG2D ligand on their surface, NKG2D-CAR-T cells exhibit cannibalism (flatorization) of CAR-T cells in in vitro and in vivo culture. This phenomenon not only makes in vitro culture and proliferation of NKG2D-CAR-T cells difficult, but also reduces the survival rate of CAR-T cells in vivo, affecting the production and clinical application of NKG2D-CAR-T cells.

[0007] Therefore, it is extremely important to effectively prevent cannibalization of CD38-CAR-T cells or NKG2D-CAR-T cells during culture and to enhance the role of CAR-T cells in antitumor and anti-immune transplantation. [Disclosure of the Invention]

[0008] Within the scope of this application, it should be understood that new or preferred technical configurations can be formed by combining the above-mentioned technical features of this application with the technical features specifically described below (e.g., embodiments). Due to space limitations, these will not be explained in detail here.

[0009] (i) One aspect of this application includes the following technical configurations and combinations thereof: The first part of this application provides a chimeric receptor comprising a CD38-binding antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain is linked to the transmembrane domain via an IgG4 hinge region or a fragment thereof.

[0010] Preferably, the antigen-binding domain further binds to a tumor antigen and / or a pathogen antigen.

[0011] The second part of the present application provides a polynucleotide encoding the chimeric receptor described in the first part.

[0012] The third part of the present application provides a polynucleotide encoding a chimeric receptor that binds to CD38 and a tumor antigen, or encoding a first chimeric receptor that binds to CD38 and a second chimeric receptor that binds to a tumor antigen. Here, the polynucleotide component encoding a chimeric receptor that binds to CD38 and a tumor antigen is represented by any of the following formulas: L-VL1-VH2-I-VL2-VH1-H-TM-C-CD3ζ;L-VL1-VL2-I-VH2-VH1-H-TM-C-CD3ζ; L-VH1-VH2-I-VL2-VL1-H-TM-C-CD3ζ;L-VH1-VL2-I-VH2-VL1-H-TM-C-CD3ζ; L-VL2-VH1-I-VL1-VH2-H-TM-C-CD3ζ;L-VH2-VH1-I-VL1-VL2-H-TM-C-CD3ζ; L-VL2-VL1-I-VH1-VH2-H-TM-C-CD3ζ;L-VH2-VL1-I-VH1-VL2-H-TM-C-CD3ζ; Here, the polynucleotide component encoding a first chimeric receptor that binds to CD38 and a second chimeric receptor that binds to a tumor antigen is represented by any of the following formulas: (1)L-VL1-VH1-H-TM-C-CD3ζ-2A-L-VH2-VL2-H-TM-C-CD3ζ (2)L-VL1-VH1-H-TM-C-CD3ζ-2A-L-VL2-VH2-H-TM-C-CD3ζ (3)L-VH1-VL1-H-TM-C-CD3ζ-2A-L-VH2-VL2-H-TM-C-CD3ζ (4) L-VH1-VL1-H-TM-C-CD3ζ-2A-L-VL2-VH2-H-TM-C-CD3ζ (5) L-VL2-VH2-H-TM-C-CD3ζ-2A-L-VH1-VL1-H-TM-C-CD3ζ (6) L-VL2-VH2-H-TM-C-CD3ζ-2A-L-VL1-VH1-H-TM-C-CD3ζ (7) L-VH2-VL2-H-TM-C-CD3ζ-2A-L-VH1-VL1-H-TM-C-CD3ζ (8) L-VH2-VL2-H-TM-C-CD3ζ-2A-L-VL1-VH1-H-TM-C-CD3ζ In the formula, each "-" independently represents a linker peptide or a peptide bond component. L is any nucleic acid component encoding a signal peptide. I is a nucleic acid component encoding a flexible linker. H is any hinge region nucleic acid component. TM is a transmembrane domain nucleic acid component. C is a costimulatory signaling molecule nucleic acid component. CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ. 2A is any 2A self-cleaving peptide nucleic acid component. VH1 is the variable region of the heavy chain of the CD38 antibody. VL1 is the variable region of the light chain of the CD38 antibody. VL2 is the variable region of the light chain of an antibody that binds to a tumor antigen. VH2 is the variable region of the heavy chain of an antibody that binds to a tumor antigen. "-" is a linker peptide or a peptide bond.

[0013] Preferably, the polynucleotide is codon-optimized for expression in human cells.

[0014] Preferably, the nucleotide sequences encoding the same components have different codons.

[0015] Preferably, the 2A peptide is selected from T2A, P2A, E2A, or F2A.

[0016] Preferably, the hinge region contains an IgG4 hinge region or a fragment thereof.

[0017] The fourth part of this application provides a vector comprising the polynucleotides described in the second and third parts.

[0018] The fifth part of this application provides engineered cells that contain the polynucleotides described in the second and third parts, or that express the chimeric receptor described in the first part.

[0019] Preferably, the manipulated cells can enhance the survival and proliferation of second T cells and / or second CAR-T cells carrying tumor target antigens that are introduced into the subject prior to, simultaneously with, or later, as well as the killing effect of the second T cells and / or second CAR-T cells on tumor cells.

[0020] Preferably, the manipulated cells can be used to eliminate alloimmune cells.

[0021] Preferably, the manipulated cells are immune cells, and preferably, the immune cells are selected from T cells, NK cells, cytotoxic T cells, NKT cells, dendritic cells, macrophages, CIK cells, and stem cell-derived immune cells, or a combination thereof. Preferably, the manipulated cells are autologous cells or allogeneic cells. Preferably, the manipulated cells express endogenous TCR, B2M, HLA-II, and / or NKG2A at low levels or not at all.

[0022] (ii) Another aspect of this application includes the following technical configurations and combinations thereof: The first part of this application provides a chimeric receptor, which is a chimeric receptor, (i) A target molecule binding domain; (ii) A membrane-bound receptor related to a degradation pathway or a fragment thereof, Here, (i) and (ii) are linked directly or via a linker fragment, the target molecule binding domain includes an antibody or a fragment thereof, a receptor or a fragment thereof, an antigen or a fragment thereof, and the degradation pathway-related membrane-bound receptor includes a lysosomal shuttle receptor.

[0023] Preferably, the chimeric receptor is (i) the extracellular domain, transmembrane domain, and / or intracellular domain of a degradation pathway-related membrane-bound receptor, or a combination thereof; or (ii) the transmembrane domain and intracellular domain of a degradation pathway-related membrane-bound receptor.

[0024] Preferably, the membrane-bound receptor includes mannose-6-phosphate receptor (M6PR), cation-independent mannose-6-phosphate receptor (CI-M6PR), ASGPR, insulin-like growth factor 2 receptor (IGF2R), or a combination thereof.

[0025] Preferably, the membrane-bound receptor is ASGPR1 and / or ASGPR2.

[0026] Preferably, the target molecule includes a cell marker or its ligand, a tissue marker or its ligand, an immune checkpoint protein or its ligand, a pathogenic autoantibody or its fragment, a pathogenic target, a non-protein substance, or a combination thereof.

[0027] Preferably, the target molecule is an immune cell antigen, a tumor antigen, a pathogen antigen, or a combination thereof.

[0028] Preferably, the target molecule is a T cell antigen and / or an NK cell antigen.

[0029] Preferably, the target molecule is selected from CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, NKG2A, NKG2C, NKG2E, CD279, NKG2D, CD305, NKP46, CD337, CS1, TCRα, TCRβ, SLAMF7, or their ligands.

[0030] Preferably, the target molecule is an NKG2D ligand. Preferably, the target molecule binding domain is full-length NKG2D, the extracellular domain or fragment thereof of NKG2D, an antibody or fragment thereof that recognizes NKG2D, or an antibody or fragment thereof that recognizes an NKG2D ligand, and preferably, the full-length NKG2D or fragment thereof is located at the C-terminus of the chimeric receptor.

[0031] Preferably, the target molecule binding domain includes Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), single-domain antibody, or single-chain variable fragment (scFv).

[0032] Preferably, the linker fragment is a (G4S)n linker, where n is an integer between 1 and 20.

[0033] Preferably, the chimeric receptor comprises, in order from the N-terminus to the C-terminus, SEQ ID NOs: 80 and 76, or SEQ ID NOs: 81 and 76.

[0034] The second part provides a chimeric receptor that recognizes NKG2D, and the chimeric receptor is (i) an antibody or fragment thereof that recognizes NKG2D; (ii) a degradation pathway-related membrane-bound receptor or fragment thereof.

[0035] The third part provides a chimeric receptor that recognizes the NKG2D ligand, (i) an antibody or fragment thereof that recognizes an NKG2D ligand; (ii) a degradation pathway-related membrane-bound receptor or fragment thereof.

[0036] The fourth part provides a chimeric receptor that recognizes the NKG2D ligand, and the chimeric receptor, in turn, (i) Degradation pathway-related membrane-bound receptors or fragments thereof; (ii) Full-length NKG2D, extracellular NKG2D, or fragments thereof.

[0037] Preferably, the membrane-bound receptor includes M6PR, CI-M6PR, ASGPR, IGF2R, or a combination thereof.

[0038] Preferably, the membrane-bound receptor is ASGPR1 and / or ASGPR2.

[0039] Preferably, (i) and (ii) are linked by a linker fragment, preferably the linker fragment is a (G4S)n linker, where n is an integer from 1 to 20.

[0040] The fifth part provides engineered cells containing the chimeric receptor described in the first, second, third, or fourth part.

[0041] Preferably, the cells include immune cells, neurons, epithelial cells, endothelial cells, stem cells, or a combination thereof. Preferably, the cells are selected from B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, peripheral blood monocytes (PBMCs), stem cell-derived immune cells, or a combination thereof.

[0042] Preferably, the cells are autologous cells or allogeneic cells.

[0043] Preferably, the cells further express chimeric receptor 2. Preferably, the chimeric receptor 2 recognizes the same target molecule as the chimeric receptor. Preferably, the chimeric receptor 2 is a CAR and / or recombinant TCR.

[0044] Preferably, the chimeric receptor 2 recognizes tumor antigens and / or pathogen antigens.

[0045] Preferably, the tumor antigen is selected from CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesoserine, NKG2D, NKG2A, or CD94, or a combination thereof.

[0046] Preferably, the manipulated cells are a) Low or absent expression of endogenous MHC-I molecules; b) Low or absent expression of endogenous TCR molecules; c) Low or absent expression of endogenous MHC-II molecules; d) Low or absent expression of endogenous NKG2A molecules; e) Low or absent expression of endogenous FAS and / or CD58 molecules; and / or f) comprising being genetically engineered to increase the expression, activity, and / or signaling of GRM polypeptides in cells.

[0047] Preferably, the manipulated cells further comprise NKG2D-CAR.

[0048] Preferably, the extracellular domain of the NKG2D-CAR contains an antibody or fragment thereof that recognizes NKG2D.

[0049] Preferably, the extracellular domain of the NKG2D-CAR comprises full-length NKG2D, a fragment of the NKG2D extracellular domain or the antibody or a fragment of the antibody that recognizes an NKG2D ligand.

[0050] Preferably, the intracellular domain of the NKG2D-CAR includes a CD28 intracellular domain, a CD137 intracellular domain, a CD3Z intracellular domain, or a combination thereof. Preferably, the intracellular domain of the NKG2D-CAR includes a CD3Z intracellular domain.

[0051] Preferably, the manipulated cells further include a second CAR that does not recognize NKG2D or its ligand.

[0052] Preferably, the second CAR recognizes tumor antigens and / or pathogen antigens.

[0053] Preferably, the tumor antigen is selected from CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesoserine, NKG2D, NKG2A, or CD94, or a combination thereof.

[0054] Preferably, the manipulated cells are a) Low or absent expression of endogenous MHC-I molecules; b) Low or absent expression of endogenous TCR molecules; c) Low or absent expression of endogenous MHC-II molecules; d) Low or absent expression of endogenous NKG2A molecules; e) Low or absent expression of endogenous FAS and / or CD58 molecules; and / or f) comprising being genetically engineered to increase the expression, activity, and / or signaling of GRM polypeptides in cells.

[0055] Preferably, the manipulated cells are T cells, NK cells, NKT cells, or a combination thereof.

[0056] Preferably, the manipulated cells are autologous cells or allogeneic cells.

[0057] The sixth part provides a composition comprising the manipulated cells described in the fifth part, and further comprising another manipulated cell, preferably the other manipulated cell comprising the chimeric receptor described in the first, second, third, or fourth part.

[0058] Preferably, the other manipulated cells express a chimeric receptor 3 that recognizes tumor antigens and / or pathogen antigens, and the chimeric receptor 3 does not recognize NKG2D or its ligand. Preferably, the chimeric receptor 3 is a CAR or recombinant TCR.

[0059] Preferably, the tumor antigen is selected from CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesoserine, NKG2D, NKG2A, or CD94, or a combination thereof.

[0060] Preferably, the other manipulated cells are a) Low or absent expression of endogenous MHC-I molecules; b) Low or absent expression of endogenous TCR molecules; c) Low or absent expression of endogenous MHC-II molecules; d) Low or absent expression of endogenous NKG2A molecules; e) Low or absent expression of endogenous FAS and / or CD58 molecules; and / or f) comprising being genetically engineered to increase the expression, activity, and / or signaling of GRM polypeptides in cells.

[0061] The seventh part provides a method for treating a subject's disease, comprising administering to the subject the manipulated cells described in the fifth part or the composition described in the sixth part.

[0062] Preferably, the method promotes the survival and / or proliferation of the manipulated cells and / or other manipulated cells.

[0063] Preferably, the method reduces the fructides between the manipulated cells.

[0064] Preferably, the method further includes reducing the killing of manipulated cells and / or other manipulated cells by the subject's immune cells, and optionally, consuming the subject's immune cells.

[0065] The eighth part provides a method for improving cell survival and / or proliferation in vivo and / or in vitro culture, comprising genetically engineering cells to express the chimeric receptors described in the first to fourth parts.

[0066] The ninth part provides a method for improving the survival and / or proliferation of CAR-T cells in vivo, comprising administering the engineered cells described in the fifth part, wherein the engineered cells recognize immune cell antigens, and the CAR-T cells recognize tumor antigens and / or pathogen antigens.

[0067] The tenth part provides a method for reducing the levels and / or activity of membrane proteins in engineered cells, comprising causing the engineered cells to express the chimeric receptors described in parts one through four, wherein the chimeric receptors recognize the membrane proteins.

[0068] Preferably, the level and / or activity of the membrane protein is reduced by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more compared to the reference.

[0069] Preferably, this method is carried out in vivo, in vitro, or ex vivo.

[0070] Preferably, the manipulated cells are derived from a subject or a healthy individual that requires them.

[0071] The eleventh part provides a method for reducing the levels and / or activity of NKG2D or its ligand in engineered cells, comprising causing the engineered cells to express the chimeric receptor described in the second, third, and fourth parts.

[0072] Preferably, the level and / or activity of NKG2D or its ligand is reduced by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more compared to a reference.

[0073] Preferably, this method is carried out in vivo, in vitro, or ex vivo.

[0074] Preferably, the manipulated cells are derived from a subject or a healthy individual that requires them.

[0075] The twelfth part provides a method for improving the viability of immune cells, including an NKG2D-CAR, which comprises genetically engineering immune cells to express the chimeric receptor described in the second, third, and fourth parts, wherein the NKG2D-CAR recognizes NKG2D or its ligand.

[0076] Preferably, the method reduces the level and / or activity of NKG2D or its ligand in the immune cells.

[0077] Preferably, the levels and / or activity of NKG2D or its ligand in the immune cells are reduced by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more compared to a reference.

[0078] Preferably, the immune cells are derived from a subject or a healthy individual who needs them.

[0079] The chimeric receptors described in parts 1, 2, 3, and 4, the manipulated cells described in part 5, the compositions described in part 6, and the methods described in parts 7 to 12 are used for the treatment of tumors.

[0080] The chimeric receptors described in parts 1, 2, 3, and 4, the manipulated cells described in part 5, the compositions described in part 6, and the methods described in parts 7 to 12 are used for the treatment, prevention, or improvement of autoimmune or inflammatory diseases.

[0081] The thirteenth part provides a method for increasing the survival and / or proliferative capacity of engineered cells targeting tumors and / or pathogens in the presence of host immune cells, comprising administering the engineered cells described in the fifth part, the composition described in the sixth part, and the method described in the seventh to twelfth parts to a subject in need thereof. Preferably, the subject is human, and preferably, the manipulated cells are autologous T cells, NK cells, NKT cells, or allogeneic T cells, NK cells, NKT cells.

[0082] The fourteenth part provides a pharmaceutical composition comprising the manipulated cells described in the fifth part and / or the cell composition described in the sixth part, as well as a pharmaceutically acceptable excipient.

[0083] (iii) Further aspects of this application include the following technical configurations and combinations thereof: (1) The present application provides a chimeric receptor comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds to GPRC5D and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the included HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 104, 105, 106, 107, 108, and 109, respectively, and the second antigen-binding domain binds to a multiple myeloma-associated antigen or immune cell marker different from GPRC5D.

[0084] (2) The VH and VL contained in the first antigen-binding domain each contain an amino acid sequence having at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NOs: 99 and 102, respectively. Preferably, the scFv contained in the first antigen-binding domain includes an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 103. Preferably, the second antigen is selected from BCMA, CD3, CD19, CD20, CD22, CD123, CD38, CD138, CS-1, BAFF-R, TACI, FcRH5, NKG2A, TIGIT, FasL, CD94, CD300A, or a combination thereof, the chimeric receptor as described in (1).

[0085] (3) The structure of the chimeric receptor from the N-terminus to the C-terminus is shown by the following formulas (1) to (4), where one of the following is selected: (1) VL1-VH2-VL2-VH1-H-TM-C; (2) VL2-VH1-VL1-VH2-H-TM-C; (3) VH1-VL2-VH2-VL1-H-TM-C; (4) VH2-VL1-VH1-VL2-H-TM-C, where each "-" is independent. A chimeric receptor as described in (1) or (2), wherein the linker peptide or peptide bond is represented, H is an arbitrary hinge region, TM is a transmembrane domain, C is an intracellular signaling region, VH1 is a GPRC5D antibody heavy chain variable region, VL1 is a GPRC5D antibody light chain variable region, VH2 is an antibody heavy chain variable region of a second antigen, and VL2 is an antibody light chain variable region of a second antigen.

[0086] (4) The VH2 comprises HCDR1, HCDR2, and HCDR3, and the LCDR1, LCDR2, and LCDR3 contained in the VL2 comprises the amino acid sequences shown in SEQ ID NOs. 110, 111, 112, 113, 114, and 115, respectively. Preferably, the chimeric receptor according to (3), wherein VH2 and VL2 each contain an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NOs. 100 and 101, or contains an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NOs. 116 and 117.

[0087] (5) The chimeric receptor according to (3) or (4), wherein the transmembrane domain is selected from the transmembrane domains of CD3ε, CD3δ, CD3ζ, CD8, CD28, CD134, CD137, CD150, CD152, DAP10, FcRα, FcRβ, FcRγ, or Zap70, and preferably the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 7, 8, or 9.

[0088] (6) The chimeric receptor according to any one of (1) to (5), wherein the intracellular signaling domain is selected from the intracellular signaling domains of CD3, CD28, CD137, OX40, DAP10, or ICOS or a combination thereof, and preferably the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NOs: 10, 11, 12, 13 or a combination thereof.

[0089] (7) The chimeric receptor according to any one of (1) to (6), wherein the hinge region comprises a CD8 hinge, a CD28 hinge, or an IgG4 hinge.

[0090] (8) The chimeric receptor according to any one of (1) to (7), wherein the chimeric receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence encoded by the nucleic acid shown in SEQ ID NO: 37, or comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NOs: 84, 96, 97.

[0091] (9) The present application provides a polynucleotide encoding a chimeric receptor as described in any one of (1) to (8).

[0092] (10) The polynucleotide according to (9), wherein the polynucleotide comprises a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NOs: 37, 90, 91, 92, 93, 94, 95, and preferably further comprises a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NOs: 4, 18, 23, 25, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 78, 79.

[0093] (11) The present application provides a vector comprising the polynucleotide described in (9) or (10).

[0094] (12). The present application provides engineered cells comprising a chimeric receptor as described in any one of (1) to (8), a polynucleotide as described in (9) or (10), or a vector as described in (11).

[0095] (13) The manipulated cells described in (12) are selected from T cells, natural killer cells, natural killer T cells, NK92 cells, cytotoxic T cells, dendritic cells, macrophages, CIK cells, stem cell-derived immune cells, or a combination thereof, wherein the immune cells preferably include native T cells and / or T cells derived from pluripotent stem cells, wherein the T cells preferably include autologous T cells and / or allogeneic T cells, wherein the T cells preferably include primary T cells, and preferably the T cells are derived from human autologous T cells.

[0096] (14) The manipulated cells according to (12) or (13), wherein the manipulated cells further include low or no expression of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS, and / or CD58, preferably the manipulated cells further include no expression of endogenous TCR / B2M, no expression of endogenous TCR / B2M / FAS, no expression of endogenous TCR / B2M / NKG2A, or no expression of endogenous TCR / B2M / NKG2A / FAS.

[0097] (15) The manipulated cells according to any one of (12) to (14), wherein the immune cells are prepared by a rapid preparation method, preferably the total time including the activation of the immune cells and introduction by the virus does not exceed 24 hours, and preferably the immune cells do not undergo significant in vitro proliferation after introduction.

[0098] (16) The present application provides a pharmaceutical composition comprising an effective amount of a chimeric receptor according to any one of (1) to (8), a polynucleotide according to (9) or (10), a vector according to (11), or an engineered cell according to any one of (12) to (15), and a pharmaceutically acceptable carrier, diluent, or excipient.

[0099] (17) The present application provides agents for the prevention or treatment of disease, comprising a chimeric receptor as described in any one of (1) to (8), a polynucleotide as described in (9) or (10), a vector as described in (11), or an engineered cell as described in any one of (12) to (15).

[0100] (18) The present application provides a chimeric receptor according to any one of (1) to (8) or engineered cells according to any one of (12) to (15) for killing or inhibiting GPRC5D-positive pathological cells, preferably they are used in the preparation of agents for treating autoimmune or inflammatory diseases, preferably for treating, preventing or improving autoimmune or inflammatory diseases in subjects who require them, preferably the autoimmune or inflammatory diseases are selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis, or dermatomyositis, preferably they are used in the preparation of agents for treating tumors, preferably for treating, preventing or improving tumors in subjects who require them, preferably the tumors include leukemia, lymphoma, and myeloma.

[0101] (19) The present application provides a method for treating, preventing or improving an autoimmune disease, inflammatory disease or tumor in a subject in need of treatment, prevention or improvement, comprising administering to the subject a chimeric receptor described in any one of (1) to (8), a polynucleotide described in (9) or (10), a vector described in (11), or an engineered cell described in any one of (12) to (15), or a pharmaceutical composition described in (16).

[0102] Preferably, the autoimmune or inflammatory disease is selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis, or dermatomyositis. The tumors include leukemia, lymphoma, and myeloma.

[0103] (iv) Further aspects of this application include the following technical configurations and combinations thereof: (1) This application provides a chimeric receptor comprising (i) a target molecule binding domain and (ii) a transmembrane protein or a fragment thereof, wherein (i) and (ii) are linked directly or via a linker fragment, and the target molecule binding domain comprises an antibody or a fragment thereof, a receptor or a fragment thereof, or an antigen or a fragment thereof.

[0104] (2) The chimeric receptor according to (1), wherein the target molecule binding domain is a type II membrane protein.

[0105] (3) The chimeric receptor according to (1) or (2), wherein the transmembrane protein or fragment thereof is a type II membrane protein or fragment thereof.

[0106] (4) The chimeric receptor according to any one of (1) to (3), wherein the transmembrane protein is selected from ASGPR1, ASGPR2, CD137L, or CD40L.

[0107] (5) A chimeric receptor according to any one of (1) to (4), wherein the target molecule comprises a cell marker or its ligand, a tissue marker or its ligand, an immune checkpoint protein or its ligand, a pathogenic autoantibody or its fragment, a pathogenic target, a nonprotein substance, or a combination thereof.

[0108] (6) The chimeric receptor according to any one of (1) to (5), wherein the target molecule is an immune cell antigen, a tumor antigen, a pathogen antigen, or a combination thereof.

[0109] (7) The chimeric receptor according to any one of (1) to (6), wherein the target molecule is a T cell antigen and / or an NK cell antigen.

[0110] (8).The target molecule is CD1b, CD1c, CD1d, CD3d, CD3e, CD4, CD7, CD8, CD11, CD13, CD16b, CD20, CD22, CD25, C D26, CD27, CD28, CD30, CD33, CD34, CD38, CD39, CD40, CD44, CD45, CD45RA, CD45RO, CD48, CD52, CD5 6, CD58, CD59, CD62L, CD66a, CD69, CD70, CD80, CD83, CD85, CD86, CD93, CD94, CD95, CD95L, CD96, C D99, CD100, CD102, CD103, CD107a, CD107b, CD111, CD112, CD117, CD119, CD122, CD123, CD126, CD1 27, CD132, CD134, CD137, CD150, CD152, CD153, CD154, CD155, CD158, CD159a, CD160, CD161, CD178 , CD183, CD185, CD191, CD192, CD196, CD212, CD215, CD218, CD223, CD226, CD244, CD252, CD253, CD Chimeric receptors as described in any one of (1) to (7), selected from 275, CD278, CD279, CD281, CD282, CD283, CD284, CD286, CD300, CD305, CD314, CD319, CD336, CD337, CD355, CD360, CD366, CD371, B2MG, NKG2DL, or any combination thereof.

[0111] Preferably, the target molecule is selected from CD1d, CD7, CD13, CD20, CD22, CD25, CD26, CD30, CD33, CD38, CD45, CD52, CD56, CD70, CD80, CD83, CD86, CD93, CD94, CD95L, CD99, CD112, CD117, CD123, CD155, CD159a, CD319, CD366, CD371, NKG2DL, or a combination thereof.

[0112] Preferably, the target molecule is an NKG2D ligand (NKG2DL).

[0113] (9) The chimeric receptor according to any one of (1) to (8), wherein the target molecule binding domain is full-length NKG2D, the extracellular domain of NKG2D or a fragment thereof, an antibody that recognizes NKG2D or a fragment thereof, or an antibody that recognizes an NKG2D ligand or a fragment thereof.

[0114] (10) The chimeric receptor according to (9), wherein the full-length NKG2D, the extracellular domain or fragment thereof of NKG2D, an antibody or fragment thereof that recognizes NKG2D, or an antibody or fragment thereof that recognizes an NKG2D ligand is located at the C-terminus of the chimeric receptor.

[0115] (11) The chimeric receptor according to any one of (1) to (10), wherein the target molecule binding domain comprises Fab, Fab', F(ab')2, F(ab)2, a variable fragment (Fv), a domain antibody (dAb), a single-domain antibody, or a single-chain variable fragment (scFv).

[0116] (12) The chimeric receptor according to any one of (1) to (11), wherein the linker fragment is (G4S)n, where n is an integer from 1 to 20.

[0117] (13) The chimeric receptor according to any one of (1) to (12), wherein the chimeric receptor comprises, in order from the N-terminus to the C-terminus, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 80 and an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 76, or an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 81 and an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 76, preferably the chimeric receptor comprises, in order from the N-terminus to the C-terminus, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 80, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 16, and an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 76, or an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 81, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 16, and an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 76.

[0118] (14) The present application provides a chimeric receptor composition comprising first and second chimeric receptors that recognize the same target molecule, wherein the first chimeric receptor comprises a target molecule binding domain 1, a transmembrane domain, and an intracellular domain, and the first chimeric receptor mediates the killing of target cells expressing the target molecule, and the second chimeric receptor comprises a target molecule binding domain 2, a transmembrane protein or a fragment thereof, and the second chimeric receptor does not mediate the killing of target cells expressing the target molecule.

[0119] (15) The chimeric receptor composition according to (14), wherein the target molecule binding domain 2 is linked directly to the transmembrane protein or a fragment thereof, or via a linker fragment.

[0120] (16) The chimeric receptor composition according to (14) or (15), wherein the second chimeric receptor comprises a type II membrane protein or a fragment thereof.

[0121] (17) The chimeric receptor composition according to any one of (14) to (16), wherein the second chimeric receptor comprises ASGPR1 or a fragment thereof, ASGPR2 or a fragment thereof, CD137L or a fragment thereof, and CD40L or a fragment thereof.

[0122] (18) The chimeric receptor composition according to any one of (14) to (17), wherein the target molecule comprises a cell marker or its ligand, a tissue marker or its ligand, an immune checkpoint protein or its ligand, a pathogenic autoantibody or its fragment, a pathogenic target, a nonprotein substance, or a combination thereof.

[0123] (19) The chimeric receptor composition according to any one of (14) to (18), wherein the target molecule is an immune cell antigen, a tumor antigen, a pathogen antigen, a T cell antigen, an NK cell antigen, or a combination thereof.

[0124] (20) The chimeric receptor composition according to any one of (14) to (19), wherein the target molecule is expressed on tumor cells or inflammatory cells and on T cells or NK cells.

[0125] (21) The chimeric receptor composition according to any one of (14) to (20), wherein the target molecule binding domain 1 or the target molecule binding domain 2 comprises Fab, Fab', F(ab')2, F(ab)2, a variable fragment (Fv), a domain antibody (dAb), a single-domain antibody, or a single-chain variable fragment (scFv).

[0126] (22) The chimeric receptor composition according to any one of (14) to (21), wherein the target molecule is selected from CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, NKG2A, NKG2C, NKG2E, CD279, NKG2D, CD305, NKP46, CD337, CS1, TCRα, TCRβ, SLAMF7, or ligands thereof.

[0127] Preferably, the target molecule is an NKG2D ligand.

[0128] (23) The chimeric receptor composition according to any one of (14) to (22), wherein the target molecule binding domain 1 or 2 is selected from full-length NKG2D, the extracellular domain or fragment thereof of NKG2D, an antibody or fragment thereof that recognizes NKG2D, or an antibody or fragment thereof that recognizes an NKG2D ligand, and preferably both the target molecule binding domains 1 and 2 contain full-length NKG2D.

[0129] (24) The chimeric receptor composition according to any one of (14) to (23), wherein the intracellular domain of the first chimeric receptor comprises a functional signaling domain of a stimulating molecule and / or a co-stimulating molecule.

[0130] (25) The chimeric receptor composition according to any one of (14) to (24), wherein the linker fragment is (G4S)n (where n is an integer from 1 to 20), or the linker fragment comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 14 or 15.

[0131] (26) A chimeric receptor composition according to any one of (14) to (25), wherein the first chimeric receptor is induced to be expressed.

[0132] (27) The chimeric receptor composition according to any one of (14) to (26), wherein the composition further comprises a third chimeric receptor, the third chimeric receptor recognizes another target molecule different from the target molecule, the third chimeric receptor, after recognizing the other target molecule, induces the expression of the first chimeric receptor, preferably, after binding to the other target molecule, induces cleavage of the third chimeric receptor to release a transcription factor, the transcription factor regulates the expression of the first chimeric receptor, preferably, the second chimeric receptor and the third chimeric receptor are linked by a cleavable peptide, preferably, the second chimeric receptor and the third chimeric receptor are linked by a 2A peptide, preferably, the 2A peptide comprises P2A, T2A, E2A, or F2A.

[0133] (28) The chimeric receptor composition according to (27), wherein the third chimeric receptor is located on the same expression vector as the first chimeric receptor, and preferably the first, second, and third chimeric receptors are located on the same expression vector.

[0134] (29) The chimeric receptor composition according to (27) or (28), wherein the other target molecule is selected from tumor antigens, tissue-specific markers, and / or pathogen antigens.

[0135] Preferably, the tissue-specific antigen includes the brain tissue-specific marker MOG, the liver tissue-specific marker ASGR1, the prostate tissue marker PSA, or a combination thereof.

[0136] Preferably, the tumor antigens are ALPPL2, ALPI, Axl, B7H3, BCMA, CD117, CD123, CD171, CD179a, CD19, CD213A2, CD20, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD7, CD71, CD94, CD97, CEA, Claudin18.2, CLDN6, CLECL1, CLL1, cMet, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FAP, FCRH5, FLT3, GD2, GD3, GM3, GPC3, GPRC5D, HER2 (ERBB2), IGLL1, IL Select from llRa, IL13Ra2, Mesothelin, MUC1, NCAM, NKG2D-Ligand, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, or WT1.

[0137] (30) The first chimeric receptor contains an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NOs. 78, 79, or The second chimeric receptor comprises, for example, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 80, and an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 76, in order from the N-terminus to the C-terminus, and preferably, the second chimeric receptor comprises, in order from the N-terminus to the C-terminus, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 80, an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 16, and an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 76, or The third chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence encoded by the nucleic acid sequence shown in Sequence ID No. 82. A chimeric receptor composition according to any one of items (27) to (29).

[0138] (31). The present application provides engineered cells comprising a chimeric receptor as described in any one of paragraphs (1) to (13).

[0139] (32). The present application provides engineered cells comprising a chimeric receptor composition as described in any one of paragraphs (14) to (30).

[0140] (33) The manipulated cells according to (31) or (32), wherein the manipulated cells have low or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / NKG2A, or TCR / B2M / FAS / NKG2A.

[0141] Preferably, the manipulated cells are autologous cells or allogeneic cells selected from immune cells, neurons, epithelial cells, endothelial cells, stem cells, or combinations thereof. Preferably, the manipulated cells are selected from B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof. Preferably, the manipulated cells are allogeneic T cells.

[0142] Preferably, the manipulated cells significantly block the target antigen on the autologous cells.

[0143] Preferably, the manipulated cells have an extended survival time or enhanced proliferative capacity in the presence of host immune cells. Preferably, the host immune cells are NK cells.

[0144] Preferably, the manipulated cells can enhance the survival, proliferation, and pathological cell-killing effects of other manipulated cells introduced into the subject prior to, simultaneously with, or afterward.

[0145] (34). The present application provides a chimeric receptor according to any one of (1) to (13), and a polynucleotide encoding a first, second, or third chimeric receptor in a chimeric receptor composition according to any one of (14) to (30).

[0146] Preferably, the polynucleotide encoding the first chimeric receptor is linked to the polynucleotide encoding the second chimeric receptor via a 2A fragment, or the polynucleotide encoding the second chimeric receptor is linked to the polynucleotide encoding the third chimeric receptor via a 2A fragment.

[0147] (35) The present application provides a vector comprising the polynucleotide described in (34).

[0148] (36) The present application provides a virus comprising the vector described in (35).

[0149] (37). The present application provides a pharmaceutical composition comprising an effective amount of a chimeric receptor according to any one of (1) to (13), a chimeric receptor composition according to any one of (14) to (30), an engineered cell according to any one of (31) to (33), a polynucleotide according to (34), a vector according to (35), or a virus according to (36), and a pharmaceutically acceptable carrier.

[0150] (38) The use of a chimeric receptor according to any one of items (1) to (13), a chimeric receptor composition according to any one of items (14) to (30), or an engineered cell according to any one of items (31) to (33) in the preparation of a drug to prevent or resist transplant immune rejection, or to prepare a drug to prevent or resist transplant immune rejection, or to treat an autoimmune or inflammatory disease, or to treat, prevent or improve an autoimmune or inflammatory disease in a subject who requires treatment, prevention or improvement of a disease, or to treat a tumor, or to treat, prevent or improve a tumor in a subject who requires treatment, prevention or improvement of a disease.

[0151] Preferably, the autoimmune or inflammatory disease is selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis or dermatomyositis, Waldenström macroglobulinemia, primary systemic amyloidosis, membranous glomerulonephritis, idiopathic thrombocytopenic purpura, or myasthenia gravis.

[0152] Preferably, the tumor is a solid tumor or a hematological tumor. Preferably, the tumor is selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, triple-negative breast cancer, and sarcoma, and the hematological malignancy is selected from leukemia, lymphoma, myeloma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), NK cell leukemia, NK / T cell lymphoma (NKTCL), or myelodysplastic syndrome.

[0153] (39) The present application provides a subject in need of a method for treating, preventing or improving an autoimmune disease, inflammatory disease or tumor, or a method for preventing or resisting transplant immune rejection, comprising administering to the subject a chimeric receptor according to any one of (1) to (13), a chimeric receptor composition according to any one of (14) to (30), an engineered cell according to any one of (31) to (33), a polynucleotide according to (34), a vector according to (35), a virus according to (36), or a pharmaceutical composition according to (37).

[0154] Preferably, the autoimmune or inflammatory disease is selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis or dermatomyositis, Waldenström macroglobulinemia, primary systemic amyloidosis, membranous glomerulonephritis, idiopathic thrombocytopenic purpura, or myasthenia gravis. Preferably, the tumor is a solid tumor or a hematological tumor, and preferably, the tumor is selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, triple-negative breast cancer, or sarcoma, and the hematological tumor is selected from leukemia, lymphoma, myeloma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), NK cell leukemia, NK / T cell lymphoma (NKTCL), or myelodysplastic syndrome. [Brief explanation of the drawing]

[0155] [Figure 1] Figures 1A-1E show the CAR positivity rates of CD38-CAR-T cells constructed using different CD38 antibodies (top of Figures 1A, 1B, and 1C; left side of Figures 1D and 1E). In the figures, CD38-sBBZ and CD38-s28Z, constructed using different antibodies, significantly blocked the CD38 antigen (bottom of Figures 1A, 1B, and 1C; right side of Figures 1D and 1E). [Figure 2] Figure 2 shows that the in vitro proliferation rate of CD38-sBBZ cells constructed using CD38 antibody 1 was significantly increased (Figure 2 left), the in vitro proliferation rate of CD38-s28Z cells constructed using CD38 antibody 1 was significantly increased (Figure 2 right), and the in vitro proliferation rate of CD38-sBBZ cells constructed using CD38 antibodies 2 and 3 were significantly increased, respectively (Figure 2 bottom). [Figure 3]Figures 3A and 3B show that CD38-CAR-T1, 2, 3, 4, 6, 9, and 10 killed tumor cells or NK cells (Figure 3A), and secreted high levels of IL-2, TNF-α, and IFN-γ after co-incubation with tumor cells (Figure 3B). [Figure 4] Figures 4A-4B show that CD38-CAR-T6 and 10 significantly inhibited THP-1 orthotopic tumors in NPG mice. [Figure 5] Figures 5A and 5B show that CD38-UCAR-T6 and 10 significantly killed rNK cells and aNK cells (Figure 5A), and that they secreted high levels of IL-2, TNF-α, and IFN-γ after co-incubation with NK cells (Figure 5B). [Figure 6] Figure 6 shows that CD38-CAR-T6 significantly inhibited THP-1 orthotopic tumors in NPG mice. [Figure 7] Figures 7A and 7B show that CD38-UCAR-T6 cells expressing HLA-E fragments 1, 2, 3, or 4 significantly killed rNK cells and aNK cells (Figure 7A), and secreted high levels of IL-2, TNF-α, and IFN-γ after co-incubation with NK cells (Figure 7B). [Figure 8] Figures 8A and 8B show that bispecific UCAR-T, which targets NKG2A and CD38, killed tumor cells in vitro (Figure 8A), and that it secreted high levels of IL-2, TNF-α, and IFN-γ after co-incubation with tumor cells (Figure 8B). [Figure 9] Figures 9A and 9B show that bispecific UCAR-T, which targets NKG2A and CD38, killed NK cells in vitro (Figure 9A), and that it secreted high levels of IL-2, TNF-α, and IFN-γ after co-incubation with tumor cells (Figure 9B). [Figure 10] Figure 10 shows that bispecific UCAR-T cells targeting NKG2A and CD38 significantly inhibited THP-1 orthotopic tumors in NPG mice. [Figure 11]Figure 11 shows that bispecific UCAR-T cells targeting NKG2A and CD38 significantly inhibited THP-1 orthotopic tumors in NPG mice in the presence of NK cells. [Figure 12] Figures 12A and 12B show that trispecific UCAR-T cells targeting NKG2A, CD38, and the tumor antigen GPRC5D significantly killed tumor cells (Figure 12A left) and NK cells (Figure 12A right), and that trispecific UCAR-T cells targeting NKG2A, CD38, and the tumor antigen BCMA, as well as bispecific UCAR-T cells targeting CD94 and CD38, significantly killed NK cells (Figure 12B). [Figure 13] Figures 13A and 13B show that the proliferation and viability of NKG2Dz cells expressing the full-length ASGPR1-NKG2D extracellular domain polypeptide or the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide were significantly improved compared to NKG2Dz cells (Figure 13A), and that the proliferative capacity of NKG2Dz cells expressing the full-length ASGPR1-NKG2D extracellular domain polypeptide or the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide was significantly improved after co-incubation with target cells THP1 (Figure 13B). [Figure 14] Figure 14 shows that NKG2Dz cells expressing the full-length ASGPR1-NKG2D extracellular domain polypeptide or the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide significantly killed tumor cells BxPC3 and U251. [Figure 15]Figure 15 shows that NKG2Dz cells containing the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide or the full-length CD137L-NKG2D extracellular domain polypeptide significantly killed THP-1 orthotopic tumors in NPG mice (Figure 15 left), and that the number of in vivo viable NKG2Dz cells containing the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide or the full-length CD137L-NKG2D extracellular domain polypeptide significantly increased 7 days after CAR-T cell injection (Figure 15 right). [Figure 16] Figure 16 shows that induced NKG2Dz cells or NKG2D-BBZ cells structurally expressing the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide showed a significantly increased proliferation ratio upon stimulation with target cell THP-1, compared to induced NKG2Dz cells or NKG2D-BBZ cells. [Figure 17] Figures 17A and 17B show that induced NKG2Dz cells or NKG2D-BBZ cells structurally expressing the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide significantly killed tumor cells U251 and PLC / PRF5 in vitro (Figure 17A), and that they secreted high levels of IFN-γ and TNF cytokines after co-incubation with tumor cells THP-1 (Figure 17B). [Figure 18] Figures 18A and 18B show that induced NKG2D-BBZ cells structurally expressing the full-length ASGPR1-linker peptide-NKG2D extracellular domain polypeptide significantly inhibited THP1 orthotopic tumors in NPG mice (Figure 18A), and that the number of in vivo viable cells increased on days 8 and 14 after CAR-T cell injection (Figure 18B). [Figure 19]Figures 19A, 19B, and 19C show that BG-BBZ, BGp-BBZ, GB-BBZ, BLG-BBZ, BLGp-BBZ, and GLBp-BBZ cells significantly killed BCMA and GPRC5D bipositive tumor cells, and BCMA or GPRC5D monopositive tumor cells (Figure 19A), significantly killed mixed BCMA monopositive and GPRC5D monopositive tumor cells (Figure 19B), and secreted high levels of IL-2, TNF-α, and IFN-γ after co-incubation with tumor cells (Figure 19C). [Figure 20] Figure 20 shows that the BG-BBZ, GB-BBZ, BGp-BBZ, BLG-BBZ, BLGp-BBZ, and GPRC5D-CART groups significantly inhibited tumor growth. [Figure 21] Figures 21A, 21B, and 21C show that rapidly prepared R-BGp-BBZ cells and R-BLGp-BBZ cells significantly killed BCMA-positive, GPRC5D-positive, and BCMA and GPRC5D-double-positive tumor cells (Figure 21A), significantly killed MM.1S-BCMA KO cells and MM.1S-GPRC5D KO cells in mixed cells containing BCMA-positive and GPRC5D-positive cells (Figure 21B), and secreted high levels of IL-2, TNF-α, and IFN-γ after culture with tumor cells (Figure 21C). [Figure 22] Figure 22 shows that the rapidly prepared R-BGp-BBZ group and R-BLGp-BBZ group exhibit a significant advantage in in vivo CAR-T amplification. Details of the invention

[0156] This application also provides a CD38-targeting chimeric receptor, which was unexpectedly obtained through the design and screening of various structures. The chimeric receptor can significantly block the CD38 antigen on immune cells expressing the chimeric receptor. This application also discloses engineered cells containing the chimeric receptor. The engineered cells show a significant increase in proliferation in in vitro and in vivo culture. The engineered cells show a significant killing effect against NK cells and CD38-positive tumor cells in vitro and in vivo.

[0157] This application provides a second chimeric receptor that can reduce cannibalism in engineered cells expressing a first chimeric receptor during in vitro and in vivo culture. The viability and / or proliferative capacity of engineered cells expressing the first and second chimeric receptors is significantly improved during in vitro and in vivo culture. Engineered cells expressing the first and second chimeric receptors significantly kill target cells recognized by the first chimeric receptor.

[0158] Unless otherwise specified, all technical and scientific terms used herein have the meaning generally understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics, and molecular biology. All methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this application, where the methods and materials described herein are appropriate. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification, including definitions, shall prevail. Furthermore, unless otherwise specified, the materials, methods, and examples of this application are for illustrative purposes only and are not intended to limit the scope. Based on the content of this application, those skilled in the art will understand that many variations or modifications can be made to the specific embodiments disclosed, and that identical or similar results can be obtained without departing from the spirit and scope of this application. This application is not limited to the specific embodiments described herein (which are intended only as examples of various aspects of this application), but functionally equivalent methods and components are also within the scope of this application. This application includes variations and modifications to the subject matter to suit various uses and conditions.

[0159] Unless otherwise specified, the practice of this application utilizes prior arts in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which fall within the scope of this art. These techniques are adequately described in the literature.

[0160] In this application, the description of scope is for convenience and conciseness only and should not be considered an immutable limitation on the scope of this application. Therefore, the description of scope should be considered to specifically disclose all possible subscopes and individual figures within that scope.

[0161] definition The term "approximately" refers to the normal error range of each value, which is readily known to those skilled in the art. Values ​​or parameters modified with "approximately" in this specification include embodiments that refer to the value or parameter itself. For example, the statement "approximately X" includes a description of "X". In this specification, "approximately" may refer to a range of tolerance in the art. For example, it may refer to a value or parameter within ±10% of the value or parameter modified with "approximately". For example, approximately 5 μM may include any number from 4.5 μM to 5.5 μM.

[0162] Term "NKG2A": NKG2A (gene ID: 3821). NKG2A and CD94 form a dimer on NK cells and act as an inhibitory receptor for NK cells. Its ligand is HLA-E.

[0163] Term "CD38": CD38 (gene ID: 952). Expressed on the surface of T lymphocytes, B lymphocytes, and NK cells.

[0164] The term "NKG2D ligand (NKG2DL)" refers to ligands that bind to NKG2D, including MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, and ULBP-6.

[0165] The term "NKG2D" refers to a type C lectin family receptor (gene ID: 22914) present on the surface of NK cells. The NKG2D described in this application includes the sequence shown in SEQ ID NO: 77, or the nucleic acid sequence shown in SEQ ID NO: 76 or the amino acid sequence encoded thereby.

[0166] The term "ASGPR" refers to the asialoglycoprotein receptor of gene ID:432, which includes two subunits: ASGPR1 (Uniprot:P07306) and ASGPR2 (Uniprot:P07307). The ASGPR1 described in this application has the nucleic acid sequence shown in SEQ ID NO: 80 or the amino acid sequence encoded thereby.

[0167] The term "CD137L" refers to the ligand for the CD137 protein (gene ID: 8744), which mediates the activation of T cell costimulatory signals. The CD137L described in this application has the nucleic acid sequence shown in SEQ ID NO: 81 or the amino acid sequence encoded thereby.

[0168] The term "CD40L" refers to the ligand for the CD40 protein (gene ID: 959) expressed on the surface of T cells.

[0169] The term "GPRC5D" refers to G protein-coupled receptor class C group 5 member D expressed in malignant myeloid plasma cells. "GPRC5D" also refers to any variant, derivative, or isotype of the GPRC5D gene or the protein it encodes. A GPRC5D polypeptide has an amino acid sequence or fragment having at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the amino acid sequence encoded by the transcript expressed by the gene represented by NCBI GenBank Gene ID: 55507, and / or optionally up to one, up to two, or up to three conserved amino acid substitutions.

[0170] The term "BCMA" generally refers to BCMA polypeptides that are specifically highly expressed in plasma cells and multiple myeloma cells. "BCMA" refers to any variant, derivative, or isotype of the BCMA gene or the protein it encodes. BCMA polypeptides have an amino acid sequence or fragment that has at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the amino acid sequence encoded by the transcript expressed by the gene represented by NCBI GenBank Gene ID:608, and / or optionally contain up to one, up to two, or up to three conserved amino acid substitutions.

[0171] The term "host-versus-graft reaction (HVGR)" generally refers to a situation in exogenous donor transplantation where, due to immunogenetic differences between the donor and recipient (or host), the donor is recognized and attacked as an exogenous graft by the host's immune cells (e.g., NK cells), and subsequently suppressed or eliminated by the host's immune cells.

[0172] The term "graft-versus-host disease (GVHD)" generally refers to a situation in which, due to the diversity of the TCR of exogenous transplanted donor T lymphocytes and incompatibility with host HLA molecules, donor T lymphocytes recognize and amplify antigens on normal host tissue, release a series of cytokines, and attack host cells.

[0173] The term "antibody" is used in its broadest sense herein and includes, but is not limited to, various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), domain antibodies, and antibody fragments thereof that can specifically bind to an antigen or antigenic determinant, provided that they exhibit desired antigen-binding activity. The term "antibody fragment" is a molecule distinct from the complete antibody and includes the portion of the complete antibody that binds to the antigen to which the complete antibody is bound. Examples of antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL, and CH1 domains (including Fab' and Fab'-SH), (ii) Fd fragments consisting of a VH domain and a CH1 domain, (iii) Fv fragments consisting of the VL domain and VH domain of a single antibody, (iv) dAb fragments consisting of a single variable region, (v) F(ab')2 fragments which are bivalent fragments formed by linking two Fab fragments, (vi) antigen-binding sites of single-chain Fv molecules, (vii) bispecific single-chain Fv dimers, (viii) polyvalent or multispecific fragments of "dimers" or "trimers" constructed by gene fusion, and (ix) scFv formed by gene fusion with the same or different antibodies. For example, the antibody may be selected from complete antibodies, scFv, single-domain antibodies, Fab fragments, Fab' fragments, Fv fragments, F(ab')2 fragments, Fd fragments, sdAbs, multifunctional antibodies, scFv-Fc antibodies, or IgG4 antibodies. For example, the antibodies of this application include typical antibodies, scFvs, and combinations thereof, where, for instance, DDpp is covalently bonded (e.g., via peptide bonds or chemical linkers) to the N-terminus of the heavy and / or light chains of a typical full-length antibody, or inserted into the H and / or L chains of a full-length antibody. DDpp antibodies that recognize CD38 and NKG2DL are prepared according to the method of CN111727250A.

[0174] The terms "recognize," "bind," and "target" are used interchangeably to refer to the selective binding of a target antigen. For example, binding to a target cell means binding to a target antigen (e.g., the target molecule) on the target cell.

[0175] The term “scFv” refers to a chimeric receptor comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are adjacent (e.g., linked by a synthetic linker such as a short, flexible polypeptide linker), and the scFv is expressible as a single-chain polypeptide, while retaining the complete specificity of the antibody from which it is derived. Unless otherwise specified, as used herein, an scFv may have VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and an scFv may contain VL-linker-VH or VH-linker-VL. The antigen-binding function of an antibody may be performed by naturally occurring antibody fragments. These fragments are collectively referred to as “antigen-binding units.” The term “antigen-binding unit” also includes any molecular structure containing a polypeptide chain having a specific shape suitable for recognizing an epitope, and the complex between the molecular structure and the epitope is stabilized by one or more non-covalent interactions.

[0176] The term "DDpp" refers to target-binding D-domain (DD) polypeptides based on a non-traditional antibody structural skeleton. D-domain polypeptides (DDpp) are characterized by high target-binding affinity and a non-antibody structural skeleton. DDpp can be monovalent or polyvalent. For example, DDpp can be single-specific or multi-specific. For example, DDpp can be single-specific and polyvalent. For example, DDpp can be multi-specific and polyvalent. For further details, see CN111727250A.

[0177] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The heavy chain variable domain (VH) and light chain variable domain (VL) of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). A single VH or VL domain can confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains using the VH or VL domain of the antibody that binds to that antigen. The variable regions of the heavy and light chains each consist of four frame regions (FRs) and three complementarity-determining regions (CDRs) (also called "hypervariable regions"). The CDRs of each chain are tightly bound by the frame regions and, together with the CDRs of the other chain, promote the formation of the antibody's antigen-binding site.

[0178] The terms "hypervariable region," "complementarity-determining region," or "CDR" refer to regions within the variable domain of an antibody that exhibit hypervariability in their sequence, and / or regions that form structurally defined loops ("hypervariable loops"), and / or regions that contain residues that come into contact with the antigen ("antigen contact sites"). Typically, an antibody has six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).

[0179] The term "Fc region" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. This term includes both the native sequence Fc region and the variant Fc region.

[0180] A "frame (FR)" refers to a variable domain residue distinct from the hypervariable region (CDR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. In VH (or VL), the CDR and FR sequences usually appear in the order FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0181] Unless otherwise specified, CDR residues and other residues within the variable domain (e.g., FR residues) are numbered in accordance with Kabat et al. above.

[0182] The term "natural antibody" refers to naturally occurring immunoglobulin molecules that have multiple structures. For example, a natural IgG antibody is a heterotetraglycoprotein weighing approximately 150,000 daltons and consists of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) from the N-terminus to the C-terminus, followed by a light chain constant (CL) domain. Based on the amino acid sequence of the constant domains, antibody light chains are classified into two types called κ (κ) and λ (λ).

[0183] The terms "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably to refer to full-length antibodies having a structure substantially similar to that of a natural antibody, or full-length antibodies having a heavy chain containing an Fc region as defined herein, or complete full-length antibodies having an antigen-binding region.

[0184] The term "single-domain antibody (sdAb)" is also known as "VHH" or "VHH polypeptide" and contains a variable VHH domain responsible for antigen recognition. Antigen binding to the VHH domain is mediated by three CDRs, flanked by four relatively constant frame regions (FRs). Nanobodies are a type of antibody that lacks an antibody light chain and contains only the heavy chain variable region; they are also called nanobodies due to their small molecular weight. For example, VHH can be cleaved at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of these frame regions, as long as antigen-binding and specificity are substantially maintained.

[0185] The term "single-domain antibody" refers to an antibody that contains all or part of the heavy chain variable domain, or all or part of the light chain variable domain. A single-domain antibody may be a human single-domain antibody.

[0186] The terms "monoclonal antibody" and "mAb" refer to antibodies derived from a substantially homologous antibody population. This means that the antibody molecules constituting this population are identical and / or bind to the same epitope. However, this excludes possible mutant antibodies that are normally present in small amounts, such as those containing spontaneous mutations or those generated during the preparation of monoclonal antibody preparations. In contrast to polyclonal antibody preparations (which typically contain different antibodies against different antigenic determinants (epitopes)), each monoclonal antibody in a monoclonal antibody preparation targets a single antigenic determinant on an antigen. Therefore, the name "monoclonal" indicates that the antibody originates from a substantially homologous antibody population and does not require a specific preparation method. For example, it can be prepared using various techniques, such as hybridoma, recombinant DNA, phage display, or methods using transgenic animals containing all or part of the human immunoglobulin locus.

[0187] The term "fully human antibody" (or "full-length human antibody") refers to an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody that has an amino acid sequence of an antibody derived from a non-human antibody using a human antibody library or other human antibody coding sequence. Humanized antibodies containing non-human antigen-binding residues are explicitly excluded from the definition of fully human antibodies. Fully human antibodies can be produced using phage display technology. Fully human antibodies can also be produced by engineered strains and / or engineered cells.

[0188] The term "mutant" refers to a different protein or polypeptide that has one or more amino acid substitutions, deletions, and / or additions (e.g., about 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5) compared to a protein or polypeptide with specific sequence characteristics ("control protein" or "reference polypeptide"). Amino acid sequence changes may be amino acid substitutions. Amino acid sequence changes may be substitutions of conserved amino acids. Functional fragments or functional mutants of a protein or polypeptide maintain the basic structural and functional characteristics of the control protein or polypeptide.

[0189] The term "cell marker" is also called a cell surface molecule or cell surface protein, and is preferably a molecule present on the surface of an immune cell membrane. For example, "T cell and / or NK cell markers" refers to markers present on T cells, NK cells, or both T cells and NK cells, such as CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD This list includes, but is not limited to, CD100, CD102, CD122, CD127, CD132, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, SLAMF7, TIGIT, and FasL. For example, NK cell markers are selected from the NKG2 receptor family, the cytotoxic immunoglobulin-like receptor (KIR) family, native cytotoxic receptors (NCRs), and / or other NK cell-specific antigens. The NKG2 receptor family includes NKG2A, NKG2D, and NKG2C. The KIR family includes KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, and KIR3DS1. NCRs include NKP30, NKP44, NKP46, and NKp80. Other NK cell-specific antigens include CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1, IL-15R, and FasL.

[0190] The term "NK inhibitory receptor (NKIR)" refers to a type of receptor on NK cells that inhibits the cytotoxic function of NK cells by transmitting a killing inhibitory signal. These include HLA-specific and non-HLA-specific inhibitory receptors. NKIRs include, but are not limited to, immune receptor tyrosine-based inhibitory motifs (ITIMs). For example, NKIRs include NKG2 / CD94 components, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, immune checkpoint inhibitors, SIGLEC family members, Ly49 family members, or combinations thereof. For example, NKG2 / CD94 components are selected from NKG2A, NKG2C, and CD94. For example, KIR family members are selected from KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3. For example, LIR family members are selected from LIR1, LIR2, LIR3, LIR5, and LIR8. For example, NKR-P1 family members are selected from NKR-P1B and NKR-P1D. For example, immune checkpoint inhibitors include (a) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor α), T (b) one or more antagonists of checkpoint molecules including LR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (b) one or more atezolizumab, avelumab, duvalmab, ipilimumab, IPH4102, IPH43, IPH33, lirelimumab, monolizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or one or more of venetoclax, azacitidine, and pomalidomide.For example, immune checkpoint receptors are selected from PD-1, TIGIT, CD96, TIM3, and LAG3. For example, SIGLEC family members are selected from SIGLEC7 and SIGLEC9. For example, Ly49 family members are selected from Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4.

[0191] The term "pathological cells" generally refers to any type of cell thought to contribute to the deterioration of a patient's health, or to malignant or infected cells that need to be reduced or removed to achieve remission in the patient. This application relates to a novel adoptive immunotherapy strategy for treating diseases associated with the development of pathological cells, such as cancer (tumors), infectious diseases, and autoimmune diseases.

[0192] The term "tumor antigen" refers to an antigen that newly appears or is overexpressed during the onset and progression of a hyperproliferative disease. In some respects, hyperproliferative disease in this application refers to cancer. Hyperproliferative diseases are called cancer or tumors. Tumor antigens include solid tumor antigens and hematological tumor antigens. The tumors described in this application are solid tumors, but may also be hematological tumors. Non-limiting examples of tumors include hematological malignancies (e.g., leukemia, lymphoma, myeloma), ovarian cancer, breast cancer, bladder cancer, brain tumors, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various cancers (including prostate cancer and small cell lung cancer), astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumors (PNETs), chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell lung adenocarcinoma and large cell lung adenocarcinoma, chordoma, angiosarcoma, endosarcoma, and squamous cell carcinoma. These include bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, cholangiocarcinoma, gallbladder cancer, synovial tumor, mesothelioma, Ewing's tumor, rhabdomyosarcoma, basal cell carcinoma, bronchial cancer, renal cell carcinoma, choriocarcinoma, seminomas, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, multiple myeloma, Waldenström macroglobulinemia and heavy chain disease, breast cancer such as ductal carcinoma and lobular adenocarcinoma, cervical cancer, ovarian cancer, bladder cancer, plasmacytoma, acute leukemia, and chronic leukemia.

[0193] The term "chimeric receptor" refers to a fusion molecule created by combining corresponding cDNAs of DNA fragments or proteins of different origins using genetic engineering techniques, and includes an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs) and recombinant TCR receptors.

[0194] The term “chimeric antigen receptor” (CAR) includes at least one extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes functional signaling domains for stimulating and / or co-stimulating molecules. For example, the stimulating molecule is a ζ chain (e.g., CD3Z) that binds to the T cell receptor complex. For example, the intracellular signaling domain may further include functional signaling domains for one or more co-stimulating molecules such as 4-1BB (i.e., CD137), CD27, and / or CD28. For example, polypeptide groups are linked to each other. For example, the intracellular signaling domain (or structural region) may be selected from the intracellular co-stimulating domains of one or more polypeptides including CD27, CD28, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, and CD83.

[0195] The term "recombinant T cell receptor (recombinant TCR)" includes a chimeric receptor derived from one or more TCR subunits. For example, a recombinant TCR includes at least a portion of the extracellular domain, transmembrane domain, and intracellular domain of a TCR subunit, the TCR subunit effectively bound to an antigen-binding domain. For example, TCR subunits in a recombinant TCR include CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRγ, and / or TCRδ subunits. For example, a recombinant TCR may be incorporated into a TCR / CD3 complex expressed on T cells. For example, a recombinant TCR includes the constant regions and intracellular domains of the TCRα and TCRβ subunits, the constant regions of these subunits effectively bound to an antigen-binding domain. For example, a recombinant TCR includes the constant regions and intracellular domains of the TCRγ and TCRδ subunits, the constant regions of these subunits effectively bound to an antigen-binding domain. For example, recombinant TCRs contain CD3ζ, CD3ε, CD3γ, or CD3δ subunits, and the extracellular domains of these subunits effectively bind to the antigen-binding domain.

[0196] The "primary signaling domain" or "primary signaling structure domain" stimulates the initial activation of the TCR complex. Conversely, the primary signaling domain is activated, for example, by the binding of the TCR / CD3 complex to peptide-supported MHC molecules, and mediates T cell responses (including, but not limited to, proliferation, activation, and differentiation). Stimulating primary signaling domains may contain activation motifs based on immune receptor tyrosine or ITAM signaling motifs. For example, fragments containing the primary signaling domain of ITAM include, but are not limited to, intracellular signaling domains derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD79a, CD79b, CD278, and CD66d.

[0197] The term "signaling domain" refers to the functional portion of a protein that transmits information and functions within a cell, regulating cellular activity through specific signaling pathways by producing second messengers or acting as effectors in response to such messengers. Intracellular signaling domains include the entire intracellular portion of a molecule, all native intracellular signaling domains, or their functional fragments or derivatives.

[0198] The terms “costimulatory signaling domain” or “costimulatory molecule” generally refer to the intracellular domain of a costimulatory molecule that binds to cell-stimulatory signaling molecules such as TCR / CD3 and induces signals that lead to T cell proliferation and / or upregulation or downregulation of major molecules. Costimulatory molecules are typically associated-binding ligands on T cells that specifically bind to costimulatory ligands and mediate T cell costimulatory responses, including but not limited to proliferation. Costimulatory molecules are non-antigen receptor cell surface molecules or their ligands necessary for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0199] The terms "immunosuppressive signaling domain" or "immunosuppressive receptor molecule" generally refer to receptors that negatively regulate and suppress activation signals of immune cells, such as classical receptors containing ITIM domains. Non-classical ITIM domains, such as the CD300A receptor, are also included. Furthermore, ITIM-independent inhibitory receptors or enzyme molecules, such as CTLA-4, IDO1, and IDO2, are also included.

[0200] The term "CD3ζ (also known as CD3 zeta)" includes the protein described in GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, and apes. In this application, "CD3ζ" is used interchangeably with "CD3z" and "CD3Z".

[0201] The term "T cell receptor (TCR)" refers to the T cell receptor that mediates the recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens by T cells, including classical TCR receptors and optimized TCR receptors. Classical TCR receptors consist of two polypeptide chains, an α chain and a β chain. Each chain is further divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. Its antigen specificity resides in the V region, and each of the V regions (Vα, Vβ) has three hypervariable regions (CDR1, CDR2, and CDR3). In one embodiment, T cells expressing classical TCRs can be induced, such as by antigen stimulation, to have their TCRs respond specifically to target antigens.

[0202] The term "signal peptide (SP)" refers to a short polypeptide chain (approximately 5-30 amino acids long) that transfers newly synthesized proteins or polypeptides into the secretory pathway. SPs are short peptides located at the N-terminus of a protein.

[0203] The term "cell" refers to cells derived from humans, non-human organisms, or animals.

[0204] The terms "host" or "subject" refer to an individual that can receive a transplant, such as a human, or any other individual capable of receiving a transplant of foreign cells. Examples of subjects include clinical patients, volunteers in clinical trials, and laboratory animals. Subjects may have, have, or have been diagnosed with a disease characterized by cell proliferation. For example, subjects may have or be at risk of having an autoimmune disease or other immune disorder, a tumor, or an inflammatory disease.

[0205] The terms "engineered" or "genetically modified" refer to the application of principles and methods of cell biology and molecular biology to alter the genetic material within a cell or organelle, or to obtain cell products according to human intent, through some engineering means. For example, "engineered" refers to one or more changes to nucleic acids (e.g., nucleic acids in the genome of an organism). "Engineered" may refer to the modification, addition, and / or deletion of genes. "Engineered cell" may refer to a cell in which genes have been added, deleted, and / or modified.

[0206] In one example, the manipulated cells are immune cells, neurons, epithelial cells, endothelial cells, or stem cells. Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSCs) and human embryonic stem cells). For example, the manipulated cells are immune cells. For example, the manipulated cells are primary cells. For example, the manipulated cells are B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, or a combination thereof. The manipulated cells are either autologous cells or allogeneic cells. For example, the manipulated cells are obtained by genetically modifying T cells isolated from human peripheral blood mononuclear cells (PBMCs).

[0207] The term "immune cells" refers to cells that are involved in the immune response and exert an immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, and mast cells. For example, the immune cells mentioned above are T cells, NK cells, and NKT cells. For example, the T cells mentioned above may be autologous T cells, heterologous T cells, or allologous T cells. For example, the NK cells mentioned above may be autologous NK cells or allologous NK cells. For example, immune cells can be obtained by selecting peripheral blood mononuclear cells (PBMCs) from a donor.

[0208] The term "T cell" may refer to native T cells obtained from PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, and infection sites, ascites, pleural fluid, spleen tissue, and tumor tissue. It may also refer to a population of cells with specific phenotypic characteristics obtained through selection or other means, or a mixed population of cells with different phenotypic characteristics. For example, "T cell" may refer to a cell containing at least one T cell subset, namely stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (Treg), and / or effector memory T cells (Tem). For example, "T cell" may refer to a specific T cell subtype such as αβT cells or γδT cells. In some cases, T cells can be obtained from blood collected from an individual using various techniques known to those skilled in the art, such as the Ficol® isolation method and / or apheresis method. For example, T cells can be obtained by differentiation induction of pluripotent stem cells. For example, cells can be obtained from an individual's circulating blood by apheresis. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. For example, cells obtained by apheresis can be washed to remove plasma molecules and placed in an appropriate buffer or culture medium for subsequent processing steps. The T cells may be derived from a healthy donor or from an individual diagnosed with cancer. The T cells may be autologous T cells or allogeneic T cells. The T cells may be primary cultured T cells. For example, "T cells" may be T cells that have a chimeric receptor that binds to CD38. Examples include CAR-T cells and recombinant TCR-T cells.

[0209] The term “cell composition” generally refers to a combination comprising at least two types of cells, the first type of cell being capable of binding to at least CD38, and the second type of cell being capable of binding to target antigens on NK cell markers and / or pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases). For example, each type of cell may be present in different containers and may be formulated simultaneously or separately with appropriate adjuvants as needed to form the desired formulation. Each type of cell may originate from different sources (e.g., prepared, manufactured, or sold by different manufacturers; e.g., native T cells isolated from a donor and T cells derived from stem cells, respectively). Each type of cell may be prepared into an independent formulation (solid, liquid, gel, etc.). Alternatively, each type of cell may exist in a mixed form. The cell composition may contain effective amounts of antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells, and may also contain pharmaceutically acceptable carriers.

[0210] The term "MHC" is an abbreviation for histocompatibility complex. In human cells, MHC is called HLA antigen and plays an important role in transplantation reactions, with rejection being mediated through T cells that react to histocompatibility antigens on the surface of transplanted tissue.

[0211] The term "human leukocyte antigen" (HLA) refers to genes that encode the major human histocompatibility complex, which is closely related to the function of the human immune system. HLA includes gene segments of classes I, II, and III. HLA class I is a heterodimer consisting of a heavy chain (α chain) and a light chain β2-microglobulin (B2M). The term "B2M" refers to β2-microglobulin (also called B2M), which is the light chain of the MHC class I molecule. HLA class II genes include the HLA-D family, mainly HLA-DP, HLA-DQ, and HLA-DR, which are distributed mainly on the surface of antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells.

[0212] The term "exogenous" refers to nucleic acid molecules, polypeptides, cells, or tissues that are not endogenously expressed within the body, or whose expression level is insufficient to achieve the desired function when overexpressed.

[0213] The term "endogenous" refers to nucleic acid molecules or polypeptides that are native to the organism itself.

[0214] The terms “activate” and “activate” are used interchangeably and refer to the process by which a cell transitions from a quiescent state to an active state. This process includes phenotypic or genetic changes in response to antigens, migration, and / or functional activity. For example, the term “activation” refers to the stepwise activation of T cells. This activation process is jointly controlled by primary and co-stimulatory signals. T cell activation is a dynamic process, and its duration and activation intensity are influenced by external stimuli. “T cell activation” or “to activate T cells” refers to a state in which T cells are stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function. The use of CD3 / CD28 magnetic beads, in vitro antigen stimulation, or in vivo antigen stimulation can all affect the degree and duration of T cell activation. For example, the aforementioned engineered T cells are activated after co-incubation with tumor cells containing a specific target antigen or after viral infection.

[0215] The term "gene editing" refers to a genetic engineering technique that alters the DNA sequence by inserting, knocking out, modifying, or replacing DNA at specific locations within the genome of an organism using site-specific nucleases. Gene editing can be used to achieve precise and efficient gene knockout or knock-in. Nuclease-based gene knockout techniques include CRISPR / Cas, ZFN, TALEN, TALEN-CRISPR / Cas techniques, base editing techniques, prime editing techniques, and meganuclease homing techniques. A guide sequence (gRNA) is a polynucleotide sequence that has sufficient complementarity to hybridize with a target polynucleotide sequence, and the gRNA induces a CRISPR complex to specifically bind to the target sequence. In this application, the gRNA sequence may be a target DNA sequence, or a complete Cas9 guide sequence formed by the corresponding ribonucleotides of the DNA and crRNA or TracrRNA. The gRNA is used to induce, bind to, or recognize the Cas enzyme. For example, when using an appropriate alignment algorithm to obtain optimal alignment, the complementarity between the guide sequence and the corresponding target sequence is approximately 80%, 85%, 90%, 95%, 97.5%, 99%, or higher. For example, using CRISPR technology, engineered cells can be constructed with endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / CD38, TCR / B2M / CD38 / NKG2A, TCR / B2M / NKG2A, TCR / B2M / FAS / CD38, or TCR / B2M / FAS / NKG2A knocked out. The gRNA sequences targeting TCR, B2M, NKG2A, and CD38 are shown in SEQ ID NOs: 39, 40, 41, and 42, respectively.

[0216] In this application, the term "low expression" refers to a lower level of protein and / or RNA expressed in target genes in manipulated cells compared to the expression level before cell manipulation. For example, low expression of B2M, TCR, CD38, FAS, and NKG2A means that the expression of B2M, TCR, CD38, FAS, and NKG2A in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The expression or content of specific proteins in cells can be measured using specific antibodies by any suitable method known in the art, such as ELISA, immunohistochemistry, Western blotting, or flow cytometry. CRISPR-based library screening technologies are widely used to screen for a variety of biological problems, including screening for drug resistance control genes in tumors and screening for genes that enhance the function of cell therapy products.

[0217] The term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and bioristics (gene guns).

[0218] The terms "encoded by a nucleic acid molecule," "coding DNA sequence," and "coding DNA" refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid strand. For example, a nucleic acid sequence codes for an amino acid sequence. When referring to a nucleotide sequence, "sequence" can include DNA or RNA and may be single-stranded or double-stranded.

[0219] The term "individual" refers to any animal, including mammals and marsupials. In this application, "individual" includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and all kinds of poultry.

[0220] The term "peripheral blood mononuclear cells" (PBMCs) refers to cells in peripheral blood that have a single nucleus, including lymphocytes and monocytes. PBMCs can be obtained using density-based cell separation methods, such as lysing or delysing red blood cells and separating peripheral blood, apheresis samples, or leukocyte-removed samples by Percoll-Ficoll density gradient centrifugation.

[0221] The term "effective dose" or "therapeutic dose" refers to an amount sufficient to prevent or treat an individual's disease (cancer). The effective dose for treatment or prevention varies depending on the stage and severity of the disease of the subject being treated, the subject's age, weight, and overall health, as well as the prescribing physician's judgment. The dose also depends on the selected active ingredient, method of administration, timing and frequency of administration, the presence, nature and extent of any potential side effects associated with the administration of a particular active ingredient, and the expected physiological effects. Depending on the judgment of the prescribing physician or a person skilled in the art, one or more rounds or multiple doses of the manipulated cells of this application may be required.

[0222] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence effectively ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, with other expression elements potentially provided by host cells or an in vitro expression system. Expression vectors include plasmids and viruses (e.g., lentiviruses, retroviruses, adenoviruses, adeno-associated viruses).

[0223] The term "vector" refers to a composition containing isolated nucleic acids that can be used to deliver isolated nucleic acids into cells. This includes, but is not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Examples include autonomously replicating plasmids or viruses. It also includes non-plasmid and non-viral compounds that facilitate the transport of nucleic acids into cells, such as polylysine compounds and liposomes.

[0224] The term "adjustment" refers to a positive or negative change. Examples of adjustments include changes of 1%, 2%, 10%, 25%, 50%, 75%, or 100%. In certain embodiments, it refers to a negative change.

[0225] The terms “homology” or “identity” refer to the identity of subunit sequences between two polymer molecules, such as two DNA molecules or two RNA molecules, or two polypeptide molecules. The terms “substantial identity” or “substantial homology” refer to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity with a reference amino acid sequence or nucleic acid sequence. For example, such a sequence may have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence or nucleic acid sequence used for comparison. Polypeptides having an amino acid sequence that is modified based on the amino acid sequence provided in this application, and / or substituted with one or more amino acids, and / or deleted and / or added with one or more amino acids, and that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity with the amino acid sequence provided in this application, and that has the same function, are also included in the scope of protection of this application.

[0226] The term "treatment" refers to any intervention, whether preventive or interventional in the clinicopathological process, that attempts to alter the course of a disease. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of the direct or indirect pathological effects of the disease, prevention of metastasis, delay of disease progression, improvement or relief of symptoms, and improvement or relief of prognosis.

[0227] The term "prevention" refers to interventions attempted before the onset of a disease (such as rejection reactions resulting from cell transplantation).

[0228] The term "transplant immune rejection" refers to the phenomenon where, after a host transplants an allogeneic tissue, organ, or cell, the host's immune system recognizes the foreign source graft as a "foreign component" and initiates an immunological response against the graft, such as attack, destruction, and removal. This application provides cells and methods that resist transplant immune rejection.

[0229] The term “graft” refers to a biological material or preparation used to isolate from an individual other than a host and to transplant into a host. Grafts may be from any animal source, preferably from humans, such as mammalian sources. The graft may be isolated from a host; for example, cells isolated from a host may be cultured in vitro or modified and transplanted back into the host. The graft may be isolated from another individual of the same species or lineage; for example, cells from another human may be cultured in vitro or modified and transplanted into the host. The graft may originate from an individual of a different species, such as transplanting organs from another species (such as mice, pigs, and monkeys) into a human. Xenografts include, but are not limited to, angiogenic xenografts, partial angiogenic xenografts, non-angiogenic xenografts, xenodressings, xenobandages, and xenostructures.

[0230] The term "self" refers to originating from the same organism. For example, a sample (e.g., cells) can be collected from a subject at a later date, processed, and then returned to that subject (e.g., a patient). Self-processes are distinguished from allogeneic processes, where the donor and recipient are different subjects.

[0231] The term "autotransplantation" encompasses any procedure involving the transplantation, transfer, or injection of cells, tissues, or organs into a recipient, where the subject and donor are the same individual. The transplantation of cells, organs, and / or tissues described herein can be used for human autotransplantation. Autotransplantation includes, but is not limited to, neovascular autotransplantation, partial neovascular autotransplantation, non-angiogenic autotransplantation, autodressing, autobandaging, and autostructures.

[0232] The term "allogeneic transplantation" encompasses any procedure involving the transplantation, transfer, or injection of cells, tissues, or organs into a recipient, wherein the subject and donor are different individuals of the same species. The transplantation of cells, organs, and / or tissues described herein can be used for allogeneic transplantation into humans. Allogeneic transplantation includes, but is not limited to, neovascular allogeneic transplantation, partial neovascular allogeneic transplantation, non-angiogenic allogeneic transplantation, allogeneic dressings, allogeneic bandages, and allogeneic structures.

[0233] "Blocking" refers to inhibiting the recognition of a target molecule on the cell surface, thereby inhibiting the biological response triggered by the recognition of that target molecule.

[0234] The statistical analysis used in this application: The t-test (also known as the Student's t-test) is a tool for evaluating the mean of one or two populations using hypothesis testing. The p-value indicates the probability that an outcome more extreme than the observed sample outcome occurs, given that the null hypothesis is true. A small p-value means that the probability of the observed outcome occurring under the null hypothesis is small. In this application, *: p-value ≤ 0.05, **: p-value ≤ 0.01, and ***: p-value ≤ 0.001.

[0235] Chimeric receptor This application also provides a chimeric receptor according to the present invention, comprising an extracellular domain, a transmembrane domain (or transmembrane structural domain, transmembrane region), and an intracellular signaling domain (or intracellular structural domain, intracellular domain) from the N-terminus to the C-terminus. If the intracellular signaling domain includes one or more signaling domains or motifs such as an immunoreceptor tyrosine activation motif (ITAM), a kinase domain, or a costimulatory domain, it can directly promote a cellular response. The intracellular signaling domain indirectly promotes a cellular response by associating with one or more other proteins, and the other proteins directly promote a cellular response. The intracellular signaling domain or its functional fragment may be derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD278(ICOS), CD357(GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. The chimeric receptor provided by the present invention includes an intracellular signaling domain. In one example, the intracellular signaling domain includes an immune receptor tyrosine activation motif or an ITAM signaling motif.

[0236] In one example, the intracellular signaling domain includes an intracellular signaling domain selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof. In another example, the intracellular signaling domain includes the CD3ζ intracellular signaling domain (SEQ ID NO: 12 or 13).

[0237] For example, the intracellular signaling domain contained in the chimeric receptor provided by the present invention further includes a co-stimulatory domain. For example, the co-stimulatory domain is selected from the intracellular signaling domains of CD137, CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, or CD83, or a combination thereof. For example, the co-stimulatory structure is the intracellular signaling domain of CD137 (SEQ ID NO: 11). For example, the co-stimulatory structure is the intracellular signaling domain of CD28 (SEQ ID NO: 10).

[0238] In one example, the chimeric receptor further includes a spacer region (or hinge, hinge region) located between the antigen-binding domain and the transmembrane domain. For example, the spacer region includes a portion of immunoglobulin. For example, the spacer region includes sequences of the hinge region, the CH2 region, and the CH3 region. For example, the hinge region is selected from the CD28 hinge region, the CD8 hinge region, the IgG4 hinge region, or fragments thereof, or combinations thereof. For example, the hinge region is the CD8 hinge region (SEQ ID NO: 3) or the IgG4 spacer region (SEQ ID NO: 5 or 6).

[0239] The transmembrane domain can fix (anchoreograph) the CAR to the cell membrane. The transmembrane domain of the chimeric receptor of the present invention is the α, β, or ζ transmembrane domain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD1 9, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, I TGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, IT GB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD16 It includes a transmembrane domain selected from 0(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0240] In one example, the transmembrane domains are CD2, CD3ε, CD3δ, CD3ζ, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95(Fas), CD134(OX40), CD137, CD150(SLAMF1), CD152(CTLA4), CD200R, CD223(LAG3), CD270(HVEM), CD272(BTLA), CD273(PD-L2), CD274(PD-L1), C The transmembrane domain is selected from D278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, or Zap70. For example, the transmembrane domain is the CD28 transmembrane domain (SEQ ID NO: 8 or 9). For example, the transmembrane domain is a variant of the CD28 transmembrane domain (SEQ ID NO: 8 or 9) having three or fewer amino acid mutations. For example, the transmembrane domain is a variant of the CD8 transmembrane domain (SEQ ID NO: 7) having three or fewer amino acid mutations.

[0241] In one example, the chimeric receptor includes a signal peptide. For example, the signal peptide is the CD8 signal peptide (SEQ ID NO: 1) or the GMCSFRα signal peptide (SEQ ID NO: 2).

[0242] For example, the chimeric receptor of the present invention targets a target antigen on pathological cells. For example, pathological cells include malignant cells or infected cells. For example, pathological cells are tumor cells or pathological cells of autoimmune diseases. For example, pathological cells include any of the diseases disclosed in this application. For example, tumors include solid tumors and hematological malignancies. For example, solid tumors are selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma. Hematological malignancies are selected from leukemia, lymphoma, and myeloma, and autoimmune diseases are selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), and polymyositis / dermatomyositis.

[0243] For example, the target antigens include: thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; type C lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neuronal cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; Ephrin A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-ase Chill GD2 ganglioside (OAcGD2); folate receptor; tumor vascular endothelial marker 1 (TEM1 / CD248); tumor vascular endothelial marker 7-related (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variant of tumor necrotic zone; G protein-coupled receptor class C group 5 member D (GPRC5D);X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific protein 1 (PLAC1); hexose portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation mutation gene 6 (ETV6-AML); sperm protein 17 (S PA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma carcinoma testicular antigen-1 (MAD-CT-1); melanoma carcinoma testicular antigen 2 (MAD-CT-2); Fos-related antigen 1; p53 variant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; apoptosis-induced melanoma inhibitor (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocyte virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2);The target antigens are selected from CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin λ-like polypeptide 1 (IGLL1). For example, the target antigen is selected from CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesoserine, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its variants, or combinations thereof.

[0244] For example, the target antigen is a pathogen. For example, the target antigen is selected from antigens of viruses, bacteria, fungi, protozoa, or parasites. For example, the target antigen is a viral antigen. The viral antigen is selected from cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.

[0245] Manipulated cells For example, the manipulated cells are immune cells, neurons, epithelial cells, endothelial cells, stem cells, or a combination thereof. For example, the manipulated cells are immune cells. For example, the immune cells are autologous cells. For example, the immune cells are allogeneic cells. For example, the immune cells are selected from B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof. For example, the manipulated cells are T cells. For example, the manipulated cells are allogeneic T cells. For example, the manipulated cells are stem cell-derived T cells. T cells may be cytotoxic T cells, helper T cells, or αβT, γδT, CD4+ / CD8+ double-positive T cells, CD4+ T cells, CD8+ T cells, CD4 / CD8 double-negative T cells, CD3+ T cells, naive T cells, effector T cells, cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like central memory TCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, or γδT cells. For example, the T cell is a cytotoxic T cell. In some embodiments, the genetically modified T cells provided in this application are isolated. For example, the genetically modified T cells provided in this application are substantially purified. For example, the modified cells provided in this application are obtained by genetically modifying cells isolated from a subject. When used in this application, the manipulated cells of this application are obtained by genetically modifying cells isolated from a subject. Alternatively, the manipulated cells of this application can be obtained by subjecting cells isolated from a subject, subcultured in vitro, or otherwise genetically modified to the genetic modification described in this application.

[0246] For example, the genetically modified cells provided in this application are obtained from cells isolated from the human body after genetic modification. Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, infection site tissue, ascites, pleural fluid, splenic tissue, and tumors. For example, T cell lines available in the art can be used. For example, the genetically modified cells provided in this application are obtained from cells isolated from peripheral blood after genetic modification. For example, the genetically modified cells provided in this application are obtained from cells isolated from bone marrow after genetic modification. For example, the genetically modified cells provided in this application are obtained from cells isolated from peripheral blood mononuclear cells (PBMCs) after genetic modification.

[0247] For example, the manipulated cells provided in this application are obtained from cells differentiated in vitro from stem cells or progenitor cells after genetic manipulation. For example, the stem cells or progenitor cells are selected from the group consisting of T cell progenitor cells, hematopoietic stem cells / progenitor cells, hematopoietic pluripotent progenitor cells, embryonic stem cells, and induced pluripotent cells. For example, the manipulated cells provided in this application are obtained by genetically manipulating cells differentiated in vitro from T cell progenitor cells. For example, the manipulated cells provided in this application are obtained by genetically manipulating cells differentiated from hematopoietic stem cells / progenitor cells in vitro. For example, the manipulated cells provided in this application are obtained by genetically manipulating cells differentiated from embryonic stem cells in vitro. For example, the manipulated cells provided in this application are obtained by genetically manipulating cells differentiated from induced pluripotent cells in vitro.

[0248] Immunogenetic differences between donor and recipient (or host) can lead to host-versus-graft (HVGR) in exogenous donor transplantation, where the donor is recognized and attacked by host immune cells (e.g., NK cells) as an exogenous graft, potentially resulting in the donor being inhibited or eliminated. In allogeneic cell transplantation, the absence of HLA-I class I molecules in allogeneic cells may reduce host CD8+-mediated cellular immune rejection. For example, the absence of HLA-I class I molecules in allogeneic cells may enhance host NK cell-mediated cellular immune rejection. For example, this application provides engineered cells with low or no expression of endogenous TCR and / or B2M.

[0249] Graft-versus-host disease (GVHD) occurs when the diversity of the TCR of exogenous graft donor T lymphocytes and their incompatibility with host HLA molecules cause donor T lymphocytes to recognize and amplify antigens on normal host tissue, releasing a series of cytokines to enhance the graft's immune response to host antigens and attack host cells. Graft-versus-host disease can be prevented by knocking out the TCR of an exogenous graft donor (e.g., T cells). For example, we provide engineered cells with low or no expression of endogenous HLA-I / TCR. For example, this application creates cells with low or no expression of endogenous TCR by knocking out the TRAC gene of the α chain of the endogenous TCR using the CRISPR system. For example, we create endogenous HLA-I deletion cells by knocking out endogenous B2M using the CRISPR system. For example, we provide cells with low or no expression of HLA-A and HLA-B and / or TCR. For example, we provide engineered cells in which endogenous HLA-I / TCR and / or HLA-II expression is low or absent. For example, we provide engineered cells in which endogenous HLA-I / TCR expression is low or absent. For example, we provide engineered cells in which endogenous TCR / B2M, TCR / B2M / CD38, TCR / B2M / FAS, TCR / B2M / NKG2A, TCR / B2M / CD38 / NKG2A, or TCR / B2M / FAS / NKG2A expression is low or absent at all.

[0250] In one example, the present application provides a method for genetically engineering cells by introducing the polynucleotides provided in the present application into cells using gene editing. If necessary, techniques such as nuclease, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN), regularly clustered short palindromic repeats (CRISPR), homologous recombination, non-homologous end joining, microhomology-mediated end joining, and homologous end joining can be used to achieve site-specific integration.

[0251] In one example, the CRISPR / Cas9 technique is used to knockout endogenous TCR / B2M, TCR / B2M / CD38, TCR / B2M / NKG2A, or TCR / B2M / CD38 / NKG2A. For example, the sgRNA sequences targeting TRAC, B2M, NKG2A, and CD38 are shown in SEQ ID NO: 39, 40, 41, and 42, respectively.

[0252] In one embodiment, first, T cells expressing a specific CAR are constructed, and then the endogenous TCR / B2M, TCR / B2M / CD38, TCR / B2M / NKG2A, or TCR / B2M / CD38 / NKG2A of the CAR-T cells are knocked out by the CRISPR / Cas9 technique to construct the corresponding UCAR-T cells. For example, first, the endogenous TCR / B2M, TCR / B2M / CD38, TCR / B2M / NKG2A, or TCR / B2M / CD38 / NKG2A of T cells are knocked out using the CRISPER / Cas9 technique to construct universal T cells, and then a specific CAR is expressed to construct UCAR-T cells. For example, UCAR-T cells are constructed by knocking out endogenous TCR / B2M, TCR / B2M / CD38, TCR / B2M / NKG2A, or TCR / B2M / CD38 / NKG2A using the CRISPR / Cas9 technique and simultaneously expressing a specific CAR.

[0253] For example, the present application provides engineered T cells that express a chimeric receptor, and these engineered T cells also include non-expression of endogenous TCR / B2M. This chimeric receptor includes, from the N-terminus to the C-terminus, an extracellular domain, a transmembrane domain, and an optional intracellular signaling domain. The extracellular domain includes the anti-CD38 antibodies disclosed herein, the transmembrane domain includes, for example, the transmembrane domain of CD28 or CD8, and the intracellular signaling domain includes, for example, the intracellular signaling domain of CD3ζ, the intracellular signaling domain of CD28, and / or the intracellular signaling domain of CD137. The extracellular domain and the transmembrane domain may be linked by, for example, a CD8 hinge region, a CD28 hinge region, or an IgG spacer region, or fragments thereof. This chimeric receptor may also include a domain that binds to an immune cell marker different from CD38 and / or a domain that binds to a target antigen on a pathological cell.

[0254] In one example, the engineered cells expressing the chimeric receptor of the present application can bind to target cells that express CD38. For example, the engineered cells of the present application can bind to NK cells. For example, the engineered cells of the present application can bind to target cells that express CD38 and an immune cell marker. For example, the engineered cells of the present application can bind to target cells that express CD38 and an NK cell marker. For example, the engineered cells of the present application can bind to target cells that express CD38 and an NK inhibitory receptor.

[0255] For example, engineered cells expressing the chimeric receptor of this application do not induce host rejection of grafts. For example, in the presence of host immune cells (e.g., NK cells), engineered cells expressing the chimeric receptor of this application have a longer survival time and / or proliferative capacity. For example, engineered cells can kill host immune cells. For example, engineered cells can kill host NK cells. For example, engineered cells can kill allogeneic NK cells. For example, engineered cells can be resistant to killing by host NK cells. For example, engineered cells can be resistant to killing by allogeneic NK cells. For example, engineered cells expressing the chimeric receptor of this application have a significantly improved ability to kill pathological cells compared to cells that do not express the chimeric receptor of this application.

[0256] For example, the manipulated cells exhibit extended survival time or improved proliferative capacity in the presence of host immune cells. For instance, the manipulated cells have inhibitory or cytotoxic functions against host immune cells. For instance, the manipulated cells have inhibitory or cytotoxic functions against host T cells or NK cells. For instance, the manipulated cells have inhibitory or cytotoxic functions against host NK cells. For instance, the manipulated cells can enhance the survival, proliferation, and pathological cell-killing effects of other manipulated cells introduced into a subject prior to, simultaneously with, or afterward.

[0257] For example, in the presence of host immune cells (e.g., NK cells), engineered cells expressing CD38 of this application have a longer survival time and / or proliferative capacity. For example, engineered cells have a stronger ability to kill host NK cells. For example, engineered cells have a stronger ability to kill allogeneic NK cells. For example, engineered cells have a stronger resistance to the killing effect of host NK cells. For example, engineered cells have a stronger resistance to the killing effect of allogeneic NK cells.

[0258] For example, in the presence of host immune cells (e.g., NK cells), dual-target engineered cells expressing CD38 according to this application and binding to target antigens on pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases) have a longer survival time and / or proliferative capacity. For instance, the engineered cells exhibit a stronger cytotoxic effect in vivo and in vitro against pathological cells possessing the target antigen.

[0259] In one example, engineered cells expressing the chimeric receptor also express immune checkpoint inhibitors. Immune checkpoint inhibitors include any agents that block, inhibit, or reduce the activity or function of suppressive pathways in the immune system. Such inhibitors may include small molecule inhibitors or antibodies or their antigen-binding fragments that bind to and block or inhibit immune checkpoint receptors, ligands, and / or receptor-ligand interactions. For example, modulation, enhancement, and / or stimulation of specific receptors may disable components of the immune checkpoint pathway. Exemplary immune checkpoint molecules that can be targeted for inhibition, suppression, modulation, enhancement, and / or stimulation include PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor-associated antigen (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (a molecule belonging to the CD2 family), Examples of immune checkpoint inhibitors include, but are not limited to, antibodies, antigen-binding fragments, or other binding proteins that bind to one or more of these molecules and block or inhibit their activity, and / or enhance or stimulate it.

[0260] This application discloses engineered cells containing a chimeric receptor targeting CD38. This application discloses engineered cells containing a chimeric receptor targeting NKG2A and CD38. This application discloses engineered cells containing a chimeric receptor targeting CD94 and CD38. This application discloses engineered cells containing a chimeric receptor targeting BCMA, NKG2A, and CD38. This application discloses engineered cells containing a chimeric receptor targeting GPRC5D, NKG2A, and CD38.

[0261] This specification provides autologous or allogeneic immune cells (e.g., T cells) genetically engineered to express NKG2D-CAR to resist NK cell killing, thereby providing a method for improving the persistence and / or transplant survival rate of autologous or allogeneic first immune cells in the presence of second immune cells of the host. For clarity, the “host” refers to the recipient of the “first immune cells,” such as a subject or patient. After the “first immune cells” have been genetically engineered and transplanted into the host, any other immune cells in the host body besides the “first immune cells” are referred to as “second immune cells.” The “first immune cells” and “second immune cells” may be cells derived from the same individual or allogeneic cells.

[0262] For example, the first immune cell is genetically engineered to express NKG2D-CAR. For instance, the first immune cell is genetically engineered to express NKG2D-CAR, and endogenous NKG2D ligand is knocked out using gene editing technology. For instance, the first immune cell is genetically engineered to express NKG2D-CAR, and endogenous B2M and TCR are knocked out using gene editing technology. For instance, the first immune cell is genetically engineered to express NKG2D-CAR, and endogenous NKG2D ligand, B2M, and TCR are knocked out using gene editing technology. For instance, the first immune cell is genetically engineered to express NKG2D-CAR, and endogenous B2M and TCR are knocked out using gene editing technology, and it expresses the ASGPR chimeric receptor. For example, the first immune cell is genetically engineered to express a CAR or TCR that recognizes CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesothelin, NKG2D, NKG2A, or CD94 or a combination thereof, and also expresses an ASGPR1 chimeric receptor, an ASGPR2 chimeric receptor, an M6PR chimeric receptor, or an IGF2R chimeric receptor. For example, a first immune cell is genetically engineered to express a CAR or TCR that recognizes CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesothelin, NKG2D, NKG2A, or CD94, or a combination thereof, and uses gene editing technology to knock out endogenous B2M and TCR, and further expresses an ASGPR1 chimeric receptor, an ASGPR2 chimeric receptor, an M6PR chimeric receptor, or an IGF2R chimeric receptor. For example, the NKG2D-CAR described in this application includes the nucleic acid sequence shown in SEQ ID NOs. 78, 79, or the amino acid sequence encoded therein.

[0263] composition This application also provides pharmaceutical compositions comprising the chimeric receptor disclosed herein. This application also provides cell compositions comprising the engineered cells disclosed herein. This application also provides pharmaceutical compositions comprising the engineered cells disclosed herein. For example, a pharmaceutical composition comprises an effective amount of the chimeric receptor disclosed herein and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition may comprise an effective amount of the engineered cells disclosed herein and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition may be used to inhibit host rejection of a graft. For example, a pharmaceutical composition may be used in immunotherapy. For example, a pharmaceutical composition may be used in the treatment of oncology. For example, a pharmaceutical composition may be used to inhibit tumor growth in a subject (e.g., a human patient). For example, a pharmaceutical composition may be used in the treatment of viral infections. For example, a pharmaceutical composition may be used in the treatment of autoimmune diseases.

[0264] For example, the cell composition provided in this application comprises a first engineered cell and a second engineered cell. For instance, the first engineered cell in the cell composition comprises a chimeric receptor that binds to the CD38 domain disclosed in this application, and the second engineered cell binds to a target antigen (e.g., CD19, BCMA, CD20, GPRC5D, CLD18.2, GPC3, CLDN6, B7H3, FAP, mesoserine, FcRH5, EGFR and its variants, ASGPR1, IL13RA2, WT1, CLL1, or a combination thereof) on pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases). The second engineered cell is an engineered immune cell used in the prior art for tumor treatment. For example, the second engineered cell is a CAR-T cell in the prior art.

[0265] For example, the types of cells in the cell composition provided in this application may be the same or different. For instance, the first or second manipulated cell may be any suitable known immune cell, and the type of the first manipulated cell does not affect the type of the second manipulated cell. For example, the first manipulated cell may be a T cell, NK cell, or NKT cell, and the second manipulated cell may be a T cell, NK cell, or NKT cell, or any other suitable immune cell.

[0266] For example, this application provides a pharmaceutical composition comprising a chimeric receptor or cells provided herein. For example, the composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). For example, the composition is suitable for topical administration. For example, topical administration includes intratumoral injection, peritumoral injection, near-tumor injection, intralesional injection, and / or injection into tumor inflow area lymph nodes, or substantially any tumor-targeted injection, where the antitumor agent is expected to leak into primary lymph nodes adjacent to the target solid tumor.

[0267] Depending on the route of administration, the active ingredient (i.e., chimeric receptor or engineered cell) may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate the active ingredient. Pharmaceutically acceptable carriers usable in the compositions or formulations provided in this application include any solvent, dispersion medium, coating layer, antimicrobial and antifungal agents, isotonic and absorption retardants, and other physiologically compatible substances. For example, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., genetically engineered chimeric receptor or cell) may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate the active ingredient.

[0268] This application provides a kit for preparing a pharmaceutical composition comprising the chimeric receptor disclosed herein. For example, the kit comprises the chimeric receptor disclosed herein and pharmaceutically acceptable excipients in one or more containers. For example, the kit may contain the chimeric receptor disclosed herein for administration to a subject. For example, the kit may include instructions (protocol) for the preparation and / or administration of the chimeric receptor.

[0269] This application provides a kit for preparing the cells disclosed herein. For example, the kit includes one or more vectors for generating engineered cells (e.g., T cells) expressing the chimeric receptor disclosed herein. The kit can be used to generate genetically modified cells from autologous or non-autologous cells. For example, the kit may include the cells disclosed herein for administration to a subject. For example, the kit includes the cells disclosed herein in one or more containers. For example, the kit includes instructions relating to the preparation and / or administration of the genetically modified cells.

[0270] This application provides a kit for inducing and / or enhancing an immune response in a subject, and / or for the treatment and / or prevention of tumor or pathogen infections, autoimmune diseases, inflammatory diseases. For example, the kit comprises an effective amount of the chimeric receptor of this application and / or a composition of the chimeric receptor that binds to pathological cells, as well as a pharmaceutical composition. For example, the kit comprises a sterile container. Such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such a container may be made from plastic, glass, laminated paper, metal foil, or other material suitable for containing the drug. For example, the kit comprises molecules encoding the chimeric receptor of this application, recombinant TCR, exogenous cytokine, and / or therapeutic monoclonal antibody, which may optionally be contained in one or more carriers.

[0271] Methods and Uses For example, this application provides the use of chimeric receptors or engineered cells disclosed herein. For example, the chimeric receptors or engineered cells are used in the preparation of agents for preventing or suppressing transplant rejection. For example, the chimeric receptors or engineered cells are used for preventing or suppressing transplant rejection.

[0272] In one example, the present application provides a method for preventing, alleviating, and / or treating tumors, autoimmune diseases, and inflammatory diseases, comprising administering a chimeric receptor, engineered cells, or pharmaceutical composition to a subject in need thereof. The pharmaceutical compositions provided in the present application are as described above, and the methods for preventing, alleviating, and / or treating tumors, autoimmune diseases, and inflammatory diseases provided in the present application encompass all of their technical configurations.

[0273] In one example, the chimeric receptor or engineered cells disclosed herein can induce and / or enhance an immune response in a subject. This application provides a method for inhibiting the activity of pathological cells in a subject, the method comprising administering to the subject a therapeutically effective amount of engineered cells expressing the chimeric receptor of this application, the engineered cells inhibiting the activity of pathological cells in the subject and / or killing the pathological cells. For example, the pathological cells are tumor cells. For example, the pathological cells include, but are not limited to, acute myeloma leukemia cells, undifferentiated lymphoma cells, astrocytoma cells, B-cell cancer cells, breast cancer cells, colorectal cancer cells, ependymoma cells, esophageal cancer cells, glioblastoma cells, glioma cells, leiomyosarcoma cells, liposarcoma cells, liver cancer cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T-cell lymphoma cells, renal cancer cells, sarcoma cells, gastric cancer cells, liver cancer cells, mesothelioma cells, or sarcoma cells. For example, the target cells are immune cells.

[0274] In one example, the chimeric receptor or engineered cell disclosed in the present application can be used for the treatment and / or prevention of tumors in a subject. For example, the chimeric receptor or engineered cell is used to extend the survival period of a subject having a tumor. For example, the chimeric receptor or engineered cell is used for the treatment and / or prevention of pathogen infections or other infectious diseases in immunocompromised human subjects. This method includes administering an effective amount of the composition of the present application to achieve a desired effect, such as alleviating existing symptoms or preventing recurrence. In treatment, the dosage is an amount that effectively brings about the desired effect. The effective amount can be provided by a single administration or multiple administrations. The effective amount can be provided by a high dose or continuous infusion. The tumor can be any of those disclosed in the present application.

[0275] In one example, the chimeric receptor or engineered cell disclosed in the present application is used to prepare a medicament for the treatment of an autoimmune disease (AID) or an inflammatory disease. For example, the chimeric receptor or engineered cell is used for the treatment, prevention, or improvement of an autoimmune disease or an inflammatory disease in a subject who needs it. The autoimmune disease or inflammatory disease can be any of those disclosed in the present application.

[0276] For example, inflammatory diseases associated with autoimmune diseases are selected from arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis, and osteoarthritis) and rheumatic diseases (including inflammatory and rheumatic diseases with bone loss and inflammatory pain), spinal joint diseases (including ankylosing spondylitis), Reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteroarthritis, enthesitis, hypersensitivity reactions (including airway hypersensitivity and skin hypersensitivity), and allergies. The manipulated T cells provided in this application are used in autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura, etc.), systemic lupus erythematosus (SLE), lupus nephritis, inflammatory myopathy (dermatomyositis), periodontitis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, and Steven Johnson. This includes conditions such as idiopathic stomatitis and diarrhea, autoimmune inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, irritable bowel syndrome), endocrine eye diseases, Graves' disease, sarcoidosis, multiple sclerosis, systemic sclerosis, fibrous diseases, primary biliary cirrhosis, juvenile diabetes (type 1 diabetes), uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, osteolysis around artificial joints, and glomerulonephritis. The following conditions are selected from: (including idiopathic nephrotic syndrome or minimal change disease, with or without the presence of nephrotic syndrome), multiple myeloma, inflammatory diseases of the skin and cornea, myositis, loosening of bone implants, metabolic disorders (including obesity, arteriosclerosis, and other cardiovascular diseases such as dilated cardiomyopathy, myocarditis, type 2 diabetes, and dyslipidemia), autoimmune thyroid diseases (including Hashimoto's disease), primary vasculitis of small and medium vessels, vasculitis of large vessels including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjögren's syndrome, Behçet's disease, atopic dermatitis and contact dermatitis, bronchiolitis, inflammatory muscle diseases, autoimmune peripheral neuropathy, immune kidney, liver and thyroid diseases, inflammatory and atherosclerotic arteriosclerosis, autoinflammatory febrile syndromes, immunohemorrhagic diseases, and vesicular diseases of the skin and mucous membranes. For example, AID refers to a group of diseases that affect multiple tissues, organs, or organ systems, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis, and dermatomyositis.

[0277] For example, the chimeric receptors or manipulated cells disclosed in this application may be used to treat, prevent, or improve asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema, and other obstructive or inflammatory airway diseases.

[0278] In another example, the present application provides a method for treating a disease in a subject suffering from a tumor, an immune disease (e.g., an autoimmune disease), or an inflammatory disease, the method comprising: i) genetically modifying autologous or allogeneic T cells, NK cells, and / or NKT cells using a vector comprising encoding the chimeric receptor of the present application, wherein the chimeric receptor is capable of specifically binding to immune cells (e.g., NK cells) and pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases, pathological cells of inflammatory diseases) in the subject; and ii) introducing the genetically modified T cells, NK cells, and / or NKT cells into the subject, wherein the genetically modified T cells, NK cells, and / or NKT cells recognize and kill tumor cells, and the genetically modified cells are resistant to attack by the subject's immune cells.

[0279] For example, the methods described in this application may also be interpreted as therapeutic uses. That is, all methods described in this application may be considered therapeutic uses of the compositions, nucleic acid molecules, or engineered cells of this application, or pharmaceutical uses for corresponding therapeutic uses. For example, this application relates to the use of the aforementioned engineered cells or nucleic acid molecules to modulate the activity of engineered cells, or the use of the aforementioned engineered cells in the preparation of agents for modulating the activity of engineered cells, the use of the aforementioned engineered cells or nucleic acid molecules to activate engineered cells, or the use of the aforementioned engineered cells in the preparation of agents for activating engineered cells. This application relates to the use of the aforementioned engineered cells or nucleic acid molecules to inhibit the activity of target cells in a subject, or to their use in the preparation of agents for inhibiting the activity of target cells in a subject. This application relates to the use of the aforementioned engineered cells or nucleic acid molecules for improving or treating the health of a subject who needs improvement or treatment of their health, or for preparing agents for improving or treating the health of a subject who needs improvement or treatment of their health. All of the above descriptions in this application are also applicable to therapeutic or pharmaceutical uses.

[0280] The manipulated cells of this application can be administered, for example, as a single active ingredient or in combination with other agents such as immunosuppressants, immunomodulators, other anti-inflammatory agents, other cytotoxic agents, and anticancer agents (e.g., as an adjuvant or in combination with them) to treat or prevent immunodeficiency-related diseases. For example, the antibodies of this application include gold salts, sulfasalazine, antimalarial drugs, methotrexate, D-penicillamine, azathioprine, mycophenolate mofetil, tacrolimus, sirolimus, dimethylaminotetracycline, leflunomide, glucocorticoids, and other DMARDs; calcineurin inhibitors such as cyclosporine A or FK506; lymphocyte recirculation regulators such as FTY720 and FTY720 analogs; mTOR inhibitors such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573, or TAFA-93; ABT-281, ASM98 It can be used in combination with other immunomodulatory compounds such as ascomycin, corticosteroids, cyclophosphamide, azathioprine, leflunomide, mizoribine, mycophenolate mofetil, 15-deoxyspergualin or its immunosuppressive congeners, analogs, or derivatives; immunosuppressive monoclonal antibodies (monoclonal antibodies against leukocyte receptors such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands); and other immunomodulatory compounds.

[0281] CD38 expression was detected in T cells, B cells, and NK cells derived from peripheral blood collected from various healthy human donors. For example, the CD38-targeting chimeric receptor and engineered cells expressing the chimeric receptor disclosed herein can be used to treat chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma (MCL), acute myeloid leukemia, acute lymphoblastic leukemia, NK cell leukemia, NK / T cell lymphoma (NKTCL), multiple myeloma, melanoma, glioma, esophageal cancer, cervical cancer, lung cancer, myelodysplastic syndrome, lymphoma, membranous glomerulonephritis, non-small cell lung cancer, prostate cancer, triple-negative breast cancer, head and neck cancer, idiopathic thrombocytopenic purpura, myasthenia gravis, and systemic lupus erythematosus.

[0282] Trait introduction This application also provides a method suitable for constructing engineered cells containing any chimeric receptor disclosed herein. For example, a method for transducing a viral vector into cells (e.g., immune effector cells) is provided, which involves activation and transduction of the cells to be transduced, and which may be performed simultaneously, i.e., co-incubating a transduction composition containing the cells to be transduced, a cell stimulant to be transduced, and viral vector particles having recombinant nucleic acid encoding the chimeric receptor of the present invention. Alternatively, transduction may be performed after activation, for example, by co-incubating the transduction composition containing the cells to be transduced with a cell stimulant to activate it, and then adding and incubating viral vector particles having recombinant nucleic acid encoding the chimeric receptor of the present invention. The total time for activation and transduction of the recombinant nucleic acid is controlled to be completed within 72 hours, preferably within 48 hours, or within 36 hours, or within 24 hours. For example, the provided method includes incubating and / or contacting retroviral vector particles (e.g., lentiviral vectors) with a cell population (e.g., immune cells such as T cells), and activating and / or making the T cells active using an extracorporeal cell activator (e.g., anti-CD3 / anti-CD28 reagent) before and / or simultaneously with and / or after contacting or incubating the cells with the viral particles. Preferably, the cells are activated first, followed by viral transduction. For example, when co-incubating a transduction composition containing cells to be transduced, a cell stimulant to be transduced, and viral vector particles having recombinant nucleic acid encoding the chimeric receptor of the present invention, the incubation time until harvesting to obtain the transduction composition containing cells transduced with recombinant nucleic acid is 72 hours or less, preferably 1 to 72 hours, more preferably 2 to 48 hours, even more preferably 2 to 36 hours, even more preferably 12 to 36 hours, even more preferably 12 to 24 hours, and even more preferably 15 to 24 hours.For example, after purification processes such as washing and centrifugation, the introduced composition can be used to prepare a pharmaceutical formulation without further in vitro amplification culture. That is, the drug prepared using the introduced composition does not require in vitro amplification before use in a subject (or patient).

[0283] CD38-targeting chimeric receptor and engineered cells expressing the chimeric receptor This application provides a CD38-targeting chimeric receptor having an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes a CD38-binding domain, and the extracellular domain and the transmembrane domain are linked by a spacer region. For example, the spacer region includes an IgG4 hinge region (or hinge) and a CD8 hinge region (or hinge). The chimeric receptor has anti-immune rejection and antitumor effects. For example, engineered cells expressing the chimeric receptor have a low CD38 positivity rate or almost no detectable CD38. For example, engineered cells expressing the chimeric receptor have low fructicide levels. For example, engineered cells expressing the chimeric receptor exhibit strong cell proliferation in in vivo and in vitro culture. For example, engineered cells expressing the chimeric receptor exhibit strong cell viability in in vivo and in vitro culture. For example, engineered cells expressing the chimeric receptor can significantly kill CD38-positive tumor cells in vivo and in vitro. For example, engineered cells expressing the chimeric receptor can significantly kill NK cells in vivo and in vitro. For example, engineered cells expressing the chimeric receptor can resist killing by NK cells in vitro and in vivo.

[0284] The CD38-targeting chimeric receptor disclosed in this application comprises, in order from the N-terminus to the C-terminus, an antigen-binding unit that binds to CD38, an IgG4 hinge region or a fragment thereof, a transmembrane domain, and an intracellular domain (intracellular signaling domain).

[0285] For example, the antigen-binding unit that binds to CD38 includes a natural CD38 ligand extracellular segment or a fragment or variant thereof capable of binding to CD38, an anti-CD38 antibody or a fragment thereof, or a binding domain that binds to synthetic CD38. Any antibody with high affinity for CD38 described in this application or known in the art can be the CD38-binding domain in the CD38-targeting chimeric receptor of this application. For example, the anti-CD38 antibody or a fragment thereof is selected from a complete antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody. For example, the anti-CD38 antibody is scFv. For example, the anti-CD38 antibody is a single-domain antibody. For example, the anti-CD38 antibody is a DDPP antibody.

[0286] For example, an anti-CD38 antibody or fragment thereof contains an scFv. For instance, the scFv has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NOs. For example, an anti-CD38 antibody or fragment thereof contains a heavy chain variable region (VH) and a light chain variable region (VL). For example, the VH and VL have an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 49 and 50, or SEQ ID NOs. 57 and 58, or SEQ ID NOs. 65 and 66, or SEQ ID NOs. 73 and 74, respectively. For example, VH includes HCDR1, HCDR2, and HCDR3, and VL includes LCDR1, LCDR2, and LCDR3, and each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 has an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NOs. 43, 44, 45, 46, 47, and 48, or the amino acid sequence shown in SEQ ID NOs. 51, 52, 53, 54, 55, and 56, or the amino acid sequence shown in SEQ ID NOs. 59, 60, 61, 62, 63, and 64, or the amino acid sequence shown in SEQ ID NOs. 67, 68, 69, 70, 71, and 72.

[0287] It is known in the art that the VH and VL regions can undergo several amino acid substitutions without altering the CDR sequence, while maintaining affinity to the target. Similarly, the CDR sequence can undergo several amino acid substitutions without altering the amino acids in contact with the target, while maintaining affinity to the target.

[0288] For example, an anti-CD38 antibody or a fragment thereof is an scFv. The VH (heavy chain variable region) and VL (light chain variable region) in the scFv can be linked by a linker peptide (or linker fragment), and their positions can be interchanged. For example, an anti-CD38 antibody contains VH, a linker peptide, and VL from the N-terminus to the C-terminus. For example, an anti-CD38 antibody contains VL, a linker peptide chain, and VH from the N-terminus to the C-terminus. Any linker peptide chain is suitable for linking VH and VL to form a fully functional single-chain antibody. For example, the linker peptide is a GS linker peptide such as GGGGS, (GGGGS)3, or (GGGGS)4. For example, an scFv in a chimeric receptor contains VH and VL in order from the N-terminus to the C-terminus. For example, an scFv in a chimeric receptor contains VL and VH in order from the N-terminus to the C-terminus.

[0289] For example, the binding domain in the extracellular domain of a CD38-targeting chimeric receptor may include the extracellular segment of a CD38 ligand or a variant thereof capable of binding to CD38.

[0290] For example, the spacer region of a CD38-targeting chimeric receptor is derived in whole or in part from an IgG4 hinge, preferably a human IgG4 hinge, the IgG4 hinge comprising a cleaved IgG4 sequence, or the IgG4 hinge comprising a modified IgG4 hinge having at least one amino acid substitution, the IgG4 hinge being the amino acid sequence shown in SEQ ID NO: 5 or 6, or comprising the amino acid sequence shown in SEQ ID NO: 5 or 6, or comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 5 or 6.

[0291] For example, the transmembrane domain of a chimeric receptor targeting CD38 is CD2, CD3ε, CD3δ, CD3ζ, CD8, CD9, CD16, CD22, CD25, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154, CD200R, CD223(LAG3), CD270(HVEM), CD272(BTLA), The transmembrane domain is selected from CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, PDGFR, or Zap70. For example, the transmembrane domain is the transmembrane domain of CD8 (SEQ ID NO: 7) or the transmembrane domain of CD28 (SEQ ID NOs: 8, 9). For example, the transmembrane domain is a mutant of the CD8 transmembrane domain (SEQ ID NO: 7) or the CD28 transmembrane domain (SEQ ID NOs: 8, 9) having three or fewer amino acid mutations.

[0292] For example, an intracellular signaling domain can directly promote a cellular response if it contains one or more signaling domains or motifs, such as an immune receptor tyrosine-based activation motif (ITAM), a kinase domain, or a costimulatory domain. The intracellular signaling domain can indirectly promote a cellular response by associating with one or more other proteins, while the other proteins may also directly promote a cellular response. For example, the intracellular signaling domain or its functional fragment may include the intracellular signaling domains of CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD278(ICOS), CD357(GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. For example, the CD38-targeting chimeric receptor provided by the present invention includes an intracellular signaling domain. For example, the CD38-targeting chimeric receptor provided by the present invention does not include an intracellular signaling domain. For example, the intracellular signaling domain includes an immune receptor tyrosine-based activation motif or an ITAM signaling motif.

[0293] For example, an intracellular signaling domain in a CD38-targeting chimeric receptor includes an intracellular signaling domain selected from TCRα, TCRβ, TCRγ, TCRδ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof. For example, the intracellular signaling domain includes the CD3ζ intracellular signaling domain (SEQ ID NOs: 12, 13).

[0294] For example, the intracellular signaling domain contained in the CD38-targeting chimeric receptor provided by this application further includes a co-stimulatory domain. For instance, the co-stimulatory domain is selected from the intracellular signaling domains of CD137(4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CARD11, CD30, CD54, OX40, CD150, CD152, CD223, CD270, PD-L2, PD-L1, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, ICAM-1, LFA-1(CD11a / CD18), 41BBL, or CD83, or a combination thereof. For example, the aforementioned co-stimulatory structure is the intracellular signaling domain of CD137(4-1BB) (SEQ ID NO: 11). For example, the aforementioned co-stimulatory structure is the intracellular signaling domain of CD28 (SEQ ID NO: 10).

[0295] For example, a chimeric receptor targeting CD38 includes a signal peptide. For instance, the signal peptide has the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0296] For example, a CD38-targeting chimeric receptor provided in this application comprises, from the N-terminus to the C-terminus, (1) a domain containing an anti-CD38 antibody (scFv selected from SEQ ID NOs: 19, 20, 21, 22), (2) an IgG4 spacer fragment (SEQ ID NOs: 5, 6), (3) a CD8 transmembrane domain (SEQ ID NOs: 7) or a CD28 transmembrane domain (SEQ ID NOs: 8, 9), as well as (4) an intracellular signaling domain of CD28 (SEQ ID NOs: 10) and / or an intracellular signaling domain of CD137 (SEQ ID NOs: 11), and (5) an intracellular signaling domain of CD3ζ (SEQ ID NOs: 12, 13). Its extracellular region may include any binding domain that binds to CD38 as referred to in this application, such as an extracellular segment of a CD38 ligand or an antibody that binds to CD38. For example, the chimeric receptors are, in order, SEQ ID NOs: 19, 6, 7, 10 and 12, or SEQ ID NOs: 19, 6, 7, 10 and 13, or SEQ ID NOs: 19, 6, 7 and 12, or SEQ ID NOs: 19, 6, 7 and 13, or SEQ ID NOs: 19, 6, 7 and 10, or SEQ ID NOs: 19, 6, 8, 10 and 12, or SEQ ID NOs: 19, 6, 8 and 12, or SEQ ID NOs: 19, 6, 8 and 13, or SEQ ID NOs: 19, 6, 8 and 10, or SEQ ID NOs: 19, 6, 9, 10 and 12, or SEQ ID NOs: 19, 6, 9 and 10 and 13, or SEQ ID NOs: 19, 6, 9 and 10, or SEQ ID NOs: 19, 5, 7, 10 and 12, or SEQ ID NOs 19, 5, 7, 10 and 13, or SEQ ID NOs 19, 5, 7 and 12, or SEQ ID NOs 19, 5, 7 and 13, or SEQ ID NOs 19, 5, 7 and 10, or SEQ ID NOs 19, 5, 8, 10 and 12, or SEQ ID NOs 19, 5, 8 and 12, or SEQ ID NOs 19, 5, 8 and 13, or SEQ ID NOs 19, 5, 8 and 10, or SEQ ID NOs 19, 5, 9, 10 and 12, or SEQ ID NOs 19, 5, 9, 10 and 13, or SEQ ID NOs 19, 5, 9 and 12, or SEQ ID NOs 19, 5, 9 and 13, or SEQ ID NOs 19, 6, 7, 11 and 12, or SEQ ID NOs 19, 6, 7, 11 and 13, or SEQ ID NOs 19, 6, 7 and 11,or SEQ ID NOs: 19, 6, 8, 11 and 12, or SEQ ID NOs: 19, 6, 8, 11 and 13, or SEQ ID NOs: 19, 6, 8 and 11, or SEQ ID NOs: 19, 6, 9, 11 and 12, or SEQ ID NOs: 19, 6, 9, 11 and 13, or SEQ ID NOs: 19, 6, 9 and 11, or SEQ ID NOs: 19, 5, 7, 11 and 12, or SEQ ID NOs: 19, 5, 7, 11 and 13, or SEQ ID NOs: 19, 5, 7 and 11, or SEQ ID NOs: 19, 5, 8, 11 and 12, or SEQ ID NOs: 19, 5, 8, 11 and 13, or SEQ ID NOs: 19, 5, 8 and 11, or SEQ ID NOs: 19 , 5, 9, 11 and 12, or sequence number 19, 5, 9, 11 and 13, or sequence number 19, 5, 9 and 11, or sequence number 20, 6, 7, 10 and 12, or sequence number 20, 6, 7, 10 and 13, or sequence number 20, 6, 7 and 12, or sequence number 20, 6, 7 and 13, or sequence number 20, 6, 7 and 10, or sequence number 20, 6, 8, 10 and 12, or sequence number 20, 6, 8 and 12, or sequence number 20, 6, 8 and 13, or sequence number 20, 6, 8 and 10, or sequence number Sequence numbers 20, 6, 9, 10 and 12, or sequence numbers 20, 6, 9, 10 and 13, or sequence numbers 20, 6, 9 and 12, or sequence numbers 20, 6, 9 and 13, or sequence numbers 20, 6, 9 and 10, or sequence numbers 20, 5, 7, 10 and 12, or sequence numbers 20, 5, 7, 10 and 13, or sequence numbers 20, 5, 7 and 12, or sequence numbers 20, 5, 7 and 13, or sequence numbers 20, 5, 7 and 10, or sequence numbers 20, 5, 8, 10 and 12, or sequence numbers 20, 5, 8, 10 and 13, or sequence numbers 20, 5, 8 and 12, or Sequence numbers 20, 5, 8 and 13, or sequence numbers 20, 5, 8 and 10, or sequence numbers 20, 5, 9, 10 and 12, or sequence numbers 20, 5, 9, 10 and 13, or sequence numbers 20, 5, 9 and 12, or sequence numbers 20, 5, 9 and 13, or sequence numbers 20, 5, 9 and 10, or sequence numbers 20, 6, 7, 11 and 12, or sequence numbers 20, 6, 7, 11 and 13, or sequence numbers 20, 6, 7 and 11, or sequence numbers 20, 6, 8, 11 and 12, or sequence numbers 20, 6, 8, 11 and 13, or sequence numbers 20, 6, 8 and 11,or SEQ ID NOs. 20, 6, 9, 11 and 12, or SEQ ID NOs. 20, 6, 9, 11 and 13, or SEQ ID NOs. 20, 6, 9 and 11, or SEQ ID NOs. 20, 5, 7, 11 and 12, or SEQ ID NOs. 20, 5, 7, 11 and 13, or SEQ ID NOs. 20, 5, 7 and 11, or SEQ ID NOs. 20, 5, 8, 11 and 12, or SEQ ID NOs. 20, 5, 8, 11 and 13, or SEQ ID NOs. 20, 5, 8 and 11, or SEQ ID NOs. 20, 5, 9, 11 and 12, or SEQ ID NOs. 20, 5, 9, 11 and 13, or SEQ ID NOs. 20, 5, 9 and 11, or SEQ ID NOs. 21, 6, 7, 10 and 12, or SEQ ID NO: 21, 6, 7, 10 and 13, or SEQ ID NO: 21, 6, 7 and 12, or SEQ ID NO: 21, 6, 7 and 13, or SEQ ID NO: 21, 6, 7 and 10, or SEQ ID NO: 21, 6, 8, 10 and 12, or SEQ ID NO: 21, 6, 8, and 12, or SEQ ID NO: 21, 6, 8, and 13, or SEQ ID NO: 21, 6, 8, and 10, or SEQ ID NO: 21, 6, 9, 10 and 12, or SEQ ID NO: 21, 6, 9, 10 and 13, or SEQ ID NO: 21, 6, 9 and 12, or Sequence numbers 21, 6, 9 and 13, or sequence numbers 21, 6, 9 and 10, or sequence numbers 21, 5, 7, 10 and 12, or sequence numbers 21, 5, 7, 10 and 13, or sequence numbers 21, 5, 7 and 12, or sequence numbers: 21, 5, 7 and 13, or sequence numbers: 21, 5, 7 and 10, or sequence numbers: 21, 5, 8, 10 and 12, or sequence numbers: 21, 5, 8, 10 and 13, or sequence numbers 21, 5, 8 and 12, or sequence numbers 21, 5, 8 and 13, or sequence numbers 21, 5, 8 and 10, or sequence numbers 21, 5, 9, 10 and 12, or SEQ ID NOs. 21, 5, 9, 10 and 13, or SEQ ID NOs. 21, 5, 9 and 12, or SEQ ID NOs. 21, 5, 9 and 13, or SEQ ID NOs. 21, 5, 9 and 10, or SEQ ID NOs. 21, 6, 7, 11 and 12, or SEQ ID NOs. 21, 6, 7 and 11, or SEQ ID NOs. 21, 6, 8, 11 and 12, or SEQ ID NOs. 21, 6, 8, 11 and 13, or SEQ ID NOs. 21, 6, 8 and 11, or SEQ ID NOs. 21, 6, 9, 11 and 12, or SEQ ID NOs. 21, 6, 9, 11 and 13, or SEQ ID NOs. 21, 6,9, and 11, or SEQ ID NOs. 21, 5, 7, 11, and 12, or SEQ ID NOs. 21, 5, 7, 11, and 13, or SEQ ID NOs. 21, 5, 7, and 11, or SEQ ID NOs. 21, 5, 8, 11, and 12, or SEQ ID NOs. 21, 5, 8, 11, and 13, or SEQ ID NOs. 21, 5, 8, and 11, or SEQ ID NOs. 21, 5, 9, 11, and 12, or SEQ ID NOs. 21, 5, 9, 11, and 13, or SEQ ID NOs. 22, 6, 7, 10, and 12, or SEQ ID NOs. 22, 6, 7, 10, and 13, or SEQ ID NOs. 22, 6, 7 and 12, or sequence numbers 22, 6, 7 and 13, or sequence numbers 22, 6, 7 and 10, or sequence numbers 22, 6, 8, 10 and 12, or sequence numbers 22, 6, 8, 10 and 13, or sequence numbers 22, 6, 8 and 12, or sequence numbers 22, 6, 8 and 13, or sequence numbers 22, 6, 8 and 10, or sequence numbers 22, 6, 9, 10 and 12, or sequence numbers 22, 6, 9, 10 and 13, or sequence numbers 22, 6, 9 and 12, or sequence numbers 22, 6, 9 and 13, or sequence numbers 22, 6, 9 and 10, or sequence numbers 22, 5, 7, 10 and 12, or SEQ ID NOs: 22, 5, 7, 10 and 13, or SEQ ID NOs: 22, 5, 7 and 12, or SEQ ID NOs: 22, 5, 7 and 13, or SEQ ID NOs: 22, 5, 7 and 10, or SEQ ID NOs: 22, 5, 8, 10 and 12, or SEQ ID NOs: 22, 5, 8 and 12, or SEQ ID NOs: 22, 5, 8 and 13, or SEQ ID NOs: 22, 5, 8 and 10, or SEQ ID NOs: 22, 5, 9, 10 and 12, or SEQ ID NOs: 22, 5, 9, 10 and 13, or SEQ ID NOs: 22, 5, 9 and 12, or SEQ ID NOs: 22, 5, 9 and 13, or SEQ ID NO: 22, 5, 9 and 10, or SEQ ID NO: 22, 6, 7, 11 and 12, or SEQ ID NO: 22, 6, 7, 11 and 13, or SEQ ID NO: 22, 6, 7 and 11, or SEQ ID NO: 22, 6, 8, 11 and 12, or SEQ ID NO: 22, 6, 8, 11 and 13, or SEQ ID NO: 22, 6, 8 and 11, or SEQ ID NO: 22, 6, 9, 11 and 12, or SEQ ID NO: 22, 6, 9, 11 and 13, or SEQ ID NO: 22, 6, 9 and 11, or SEQ ID NO: 22, 5, 7, 11 and 12, or SEQ ID NO: 22, 5, 7, 11 and 13,Or it contains an amino acid sequence that has at least approximately 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NOs. 22, 5, 5, 7, and 11, or SEQ ID NOs. 22, 5, 8, 11, and 12, or SEQ ID NOs. 22, 5, 9, 11, and 13, or SEQ ID NOs. 22, 5, 9, and 11.

[0297] For example, a CD38-targeting chimeric receptor includes an amino acid sequence having approximately 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any of SEQ ID NOs. 24, 26, 27, or 28. For example, the chimeric receptor includes an amino acid sequence encoded by a nucleotide sequence having approximately 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in any of SEQ ID NOs. 18, 23, 25, or 29.

[0298] Examples of CD38-targeting chimeric receptor variants in these cases include, but are not limited to, chimeric receptors obtained by swapping the positions of VH and VL in the scFv structure of an anti-CD38 antibody, chimeric receptors obtained by substituting one linker peptide with another, chimeric receptors obtained by substituting a portion of an anti-CD38 antibody with another antibody structure (e.g., Fab, single-domain antibody), and chimeric receptors in which the sequences of the VH and VL regions of an anti-CD38 antibody can be modified to some extent without affecting the binding of the anti-CD38 antibody to CD38. Those skilled in the art can obtain the above-mentioned possible variants through common knowledge and conventional experiments in the art.

[0299] For example, the CD38-targeting chimeric receptor may include a domain that binds to an immune cell marker different from CD38, and this domain, together with the CD38-binding domain, may form the extracellular domain of the chimeric receptor. For instance, the extracellular domain of the chimeric receptor may also include a domain that binds to an immune cell marker different from CD38.

[0300] In one example, the binding domain that binds to the immune cell marker includes a ligand that binds to the immune cell marker, a synthetic binding domain, an antibody, or a fragment thereof. For example, the extracellular domain includes an antibody or fragment thereof that binds to the immune cell marker. For example, the antibody or fragment that binds to the immune cell marker is selected from a complete antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody. For example, the antibody or fragment that binds to the immune cell marker is scFv. For example, the antibody or fragment that binds to the immune cell marker is a single-domain antibody. For example, the antibody or fragment that binds to the immune cell marker is a Fab fragment.

[0301] In one example, the antibody or fragment that binds to the immune cell marker includes a heavy chain variable region (VH2) and a light chain variable region (VL2). For example, the extracellular domain of the chimeric receptor includes an antibody or fragment against CD38 and an antibody or fragment that binds to the immune cell marker, where the antibody or fragment against CD38 includes a heavy chain variable region (VH1) and a light chain variable region (VL1), and the antibody or fragment against the immune cell marker includes a heavy chain variable region (VH2) and a light chain variable region (VL2). For example, VH1, VL1, VH2, and VL2 are linked in a tandem structure. The tandem structure is formed such that VH1 and VL1 are linked as scFv and VH2 and VL2 are linked to both ends of the scFv, or VH2 and VL2 are linked as scFv and VH1 and VL1 are linked to both ends of the scFv, for example, VL2-VH1-VL1-VH2, VH2-VL1-VH1-VL2, VL1-VH2-VL2-VH1, VH1-VL2-VH2-VL1, VH2-VH1-VL1-VL2, VL2-VL1-VH1-VH2, VH1-VH2-VL2-VL1, or VL1-VL2-VH2-VH1. For example, the anti-CD38 antibody or a fragment thereof in the chimeric receptor is linked in tandem with the antibody or a fragment thereof that binds to the immune cell marker. Tandem linkages are denoted as VH1-VL1-VH2-VL2, VH2-VL2-VH1-VL1, VL1-VH1-VL2-VH2, VL2-VH2-VL1-VH1, VH1-VL1-VL2-VH2, VH2-VL2-VL1-VH1, VL1-VH1-VH2-VL2, or VL2-VH2-VH1-VL1. "-" indicates any linker peptide or peptide bond. Different variable regions are linked by linker peptide chains. For example, the linker peptide chain is a GS linker peptide chain, e.g., (GGGGS)n, where n can be any number from 1 to 20. For example, the linker peptides include GGGGS, (GGGGS)3, or (GGGGS)4. For example, the linker peptide chain contains the amino acid sequence shown in SEQ ID NO: 14 or 15.

[0302] In one example, the CD38-targeting chimeric receptor includes a domain that binds to a target antigen on a pathological cell. The domain that binds to the target antigen on the pathological cell, together with the CD38-binding domain, can form the extracellular domain of the chimeric receptor.

[0303] For example, the extracellular domain of the CD38-targeting chimeric receptor further includes a domain that binds to a target antigen on a pathological cell. For example, the binding domain that binds to the target antigen includes a ligand that binds to the target antigen, a synthetic binding domain, an antibody, or a fragment thereof. For example, the extracellular domain includes an antibody or a fragment thereof that binds to the target antigen. For example, the antibody or fragment is selected from a complete antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody.

[0304] In one example, the antibody or fragment that binds to the target antigen on the pathological cell includes a heavy chain variable region (VH3) and a light chain variable region (VL3). For example, the extracellular domain of the CD38-targeting chimeric receptor includes an antibody or fragment against CD38 and an antibody or fragment that binds to the target antigen on the pathological cell, where the antibody or fragment against CD38 includes a heavy chain variable region (VH1) and a light chain variable region (VL1), and the antibody or fragment against the target antigen on the pathological cell includes a heavy chain variable region (VH3) and a light chain variable region (VL3). For example, VH1, VL1, VH3, and VL3 are linked in a tandem structure. The tandem structure is formed such that VH1 and VL1 are linked as an scFv and VH3 and VL3 are linked to both ends of the scFv, or VH3 and VL3 are linked as an scFv and VH1 and VL1 are linked to both ends of the scFv, for example, VL3-VH1-VL1-VH3, VH3-VL1-VH1-VL3, VL1-VH3-VL3-VH1, VH1-VL3-VH3-VL1, VH3-VH1-VL1-VL3, VL3-VL1-VH1-VH3, VH1-VH3-VL3-VL1, or VL1-VL3-VH3-VH1. For example, the anti-CD38 antibody or a fragment thereof in the chimeric receptor is linked in tandem with an antibody or a fragment thereof that binds to a target antigen on the pathological cell. The aforementioned tandem linkages are denoted as VH1-VL1-VH3-VL3, VH3-VL3-VH1-VL1, VL1-VH1-VL3-VH3, VL3-VH3-VL1-VH1, VH1-VL1-VL3-VH3, VH3-VL3-VL1-VH1, VL1-VH1-VH3-VL3, or VL3-VH3-VH1-VL1. "-" indicates any linker peptide or peptide bond. Different variable regions are linked by linker peptide chains. For example, the linker peptide chain is a GS linker peptide chain, e.g., (GGGGS)n, where n can be any number from 1 to 20. For example, the linker peptides include GGGGS, (GGGGS)3, or (GGGGS)4. For example, the linker peptide chain contains the amino acid sequence shown in SEQ ID NO: 14 or 15.

[0305] This application discloses a chimeric receptor targeting CD38 as shown in Table 1, a dual-target chimeric receptor targeting NKG2A and CD38 as shown in Table 2, and a chimeric receptor targeting NK cell markers (e.g., NKG2A, CD94, FasL, CD300A, TIGIT) as shown in Table 3. TIFF2026525409000001.tif40170TIFF2026525409000002.tif39170TIFF2026525409000003.tif58170In Tables 1, 2, and 3: αCD38(scFv): The scFv of anti-CD38 antibodies 1, 2, 3, and 4 are SEQ ID NOs: 19, 20, 21, and 22, respectively; αCD38-VH: The heavy chain variable regions of anti-CD38 antibodies 1, 2, 3, and 4 are SEQ ID NOs: 49, 57, 65, and 73, respectively; αCD38-VL: The light chain variable region of anti-CD38 antibodies 1, 2, 3, and 4 The chain-variable regions are sequence numbers 50, 58, 66, and 74, respectively; CD8(H): CD8 hinge region (sequence number 3); IgG4(H): IgG4 hinge region (sequence number 5); CD8(TM): CD8 transmembrane domain (sequence number 7); CD28(TM): CD28 transmembrane domain (sequence number 9); CD137(C): ​​CD137 intracellular domain (sequence number 11); CD28(C): CD28 intracellular domain (sequence number 10); CD3ζ(C): CD3ζ intracellular domain (sequence number 12); (L): linker peptide (sequence numbers 14, 15, 16).

[0306] CD38-CAR1, 2, 3, and 4 in the examples disclosed in the present invention contain CD38-BBZ shown in Table 1, constructed from CD38 antibody 1 (SEQ ID NO: 19), CD38 antibody 2 (SEQ ID NO: 20), CD38 antibody 3 (SEQ ID NO: 21), and CD38 antibody 4 (SEQ ID NO: 22), respectively. CD38-CAR2s contain CD38-sBBZ shown in Table 1, constructed from CD38 antibody 2 (SEQ ID NO: 20). CD38-CAR3s contain CD38-sBBZ shown in Table 1, constructed from CD38 antibody 3 (SEQ ID NO: 21). CD38-CAR6 contains CD38-sBBZ shown in Table 1, constructed from CD38 antibody 1 (SEQ ID NO: 19). CD38-CAR9 contains CD38-28Z shown in Table 1, constructed from CD38 antibody 1 (SEQ ID NO: 19). CD38-CAR10 contains CD38-s28Z, which is constructed from CD38 antibody 1 (SEQ ID NO: 19), as shown in Table 1.

[0307] In the examples, the CD38 / NKG2A-DCAR1 fragment contains CD38-sBBZ, 2A peptide, and NKG2A-28Z from Table 1, from the 5' end to the 3' end. The CD38 / NKG2A-DCAR2 fragment contains CD38-s28Z, 2A peptide, and NKG2A-28Z from Table 1, in that order. The CD38 / NKG2A-DCAR3 fragment contains NKG2A-28Z, 2A peptide, and CD38-s28Z from Table 1, in that order.

[0308] This application provides engineered cells containing a CD38-targeting chimeric receptor disclosed herein.

[0309] In one example, the manipulated cells further include a chimeric receptor 2 that binds to at least one immune cell marker different from CD38. For example, the immune cell marker is selected from T cell markers and / or NK cell markers. For example, the immune cell marker is an NK inhibitory receptor (NKIR). For example, the immune cell marker is selected from NKG2 / CD94, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, SIGLEC family members, Ly49 family members, or a combination thereof. For example, the NKG2 / CD94 component is selected from NKG2A, NKG2C, and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5, and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3, and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; and the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4.For example, the aforementioned immune cell markers are CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD244, and CD159a (NKG2A). Selected from CD159c (NKG2C), NKG2E, CD279, CD300A, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectins, native cytotoxic receptors NCR1, NCR2, NCR3, or combinations thereof.

[0310] In one example, the manipulated cells include a chimeric receptor 1 that binds to CD38 as disclosed in the present invention, and a chimeric receptor 2 that binds to at least one immune cell marker different from CD38. For example, the structure of chimeric receptor 2 is shown in Table 3. For example, chimeric receptors that bind to CD38 are shown in Table 1. For example, chimeric receptor 1 and chimeric receptor 2 are constructed on a single vector. For example, a polypeptide fragment contains, from the N-terminus to the C-terminus, chimeric receptor 2, 2A peptide, and chimeric receptor 1 in that order. For example, the 2A peptide is P2A, T2A, E2A, or F2A. For example, chimeric receptor 1 and chimeric receptor 2 are linked by a cleavable peptide. For example, a polypeptide fragment contains, from the N-terminus to the C-terminus, chimeric receptor 1, 2A peptide, and chimeric receptor 2 in that order. For example, chimeric receptor 1 and chimeric receptor 2 are constructed on different vectors. For example, chimeric receptor 2 contains a ligand that binds to the immune cell marker, a synthetic binding domain, an antibody, or a fragment thereof. For example, chimeric receptor 2 contains an antibody or fragment thereof that binds to an immune cell marker. For example, the antibody or fragment that binds to the immune cell marker is selected from a complete antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody. For example, the antibody or fragment that binds to the immune cell marker is scFv. For example, the antibody or fragment that binds to the immune cell marker is a single-domain antibody. For example, the antibody or fragment that binds to the immune cell marker is a Fab fragment. For example, chimeric receptor 2 also binds to a target antigen on pathological cells. Chimeric receptor 1 and chimeric receptor 2 can each be independently selected to have the same or different hinge regions, transmembrane domains, and intracellular domains.

[0311] In one example, the manipulated cells include a CD38-binding chimeric receptor 1 disclosed in the present invention and a chimeric receptor 2 that binds to a target antigen on at least one pathological cell. For example, CD38-binding chimeric receptors are shown in Table 1. For example, the chimeric receptor 1 and chimeric receptor 2 are constructed on a single vector. For example, a polypeptide fragment contains, from the N-terminus to the C-terminus, chimeric receptor 2, 2A peptide, and chimeric receptor 1 in that order. For example, the 2A peptide is P2A, T2A, E2A, or F2A. For example, the chimeric receptor 1 and chimeric receptor 2 are linked by the 2A peptide. For example, a polypeptide fragment contains, from the N-terminus to the C-terminus, chimeric receptor 1, 2A peptide, and chimeric receptor 2 in that order. For example, the chimeric receptor 1 and chimeric receptor 2 are constructed on different vectors. For example, chimeric receptor 2 contains a ligand that binds to the target antigen on the pathological cell, a synthetic binding domain, an antibody, or a fragment thereof. For example, chimeric receptor 2 includes an antibody or fragment thereof that binds to a target antigen on the pathological cell. For example, the antibody or fragment that binds to the target antigen on the pathological cell is selected from a complete antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody. For example, the antibody or fragment that binds to the target antigen on the pathological cell is scFv. For example, the antibody or fragment that binds to the target antigen on the pathological cell is a single-domain antibody. For example, the antibody or fragment that binds to the target antigen on the pathological cell is a Fab fragment. Chimeric receptor 1 and chimeric receptor 2 can each be independently selected to have the same or different hinge regions, transmembrane domains, and intracellular domains.

[0312] In one example, the manipulated cells include a CD38-binding chimeric receptor 1 and a chimeric receptor 2 disclosed in the present invention, wherein the chimeric receptor 2 binds to at least one immune cell marker different from CD38 and at least one target antigen on pathological cells. For example, CD38-binding chimeric receptors are shown in Table 1. For example, the chimeric receptor 1 and chimeric receptor 2 are constructed on a single vector. For example, the chimeric receptor 1 and chimeric receptor 2 are linked by a 2A peptide. For example, the 2A peptide is P2A, T2A, E2A, or F2A. For example, the chimeric receptor 1 and chimeric receptor 2 are linked by a 2A peptide. For example, a polypeptide fragment contains, from the N-terminus to the C-terminus, the chimeric receptor 1, the 2A peptide, and the chimeric receptor 2 in that order. For example, the chimeric receptor 1 and chimeric receptor 2 are constructed on different vectors. For example, the chimeric receptor 2 includes a ligand that binds to the immune cell marker and the target antigen on pathological cells, a synthetic binding domain, an antibody, or a fragment thereof. For example, the chimeric receptor 2 includes an antibody or fragment thereof that binds to the immune cell marker and the target antigen on pathological cells. For example, the antibody or fragment that binds to the immune cell marker and the target antigen on pathological cells is selected from a complete antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody. For example, the antibody or fragment that binds to the immune cell marker and the target antigen on pathological cells is scFv. For example, the antibody or fragment that binds to the immune cell marker and the target antigen on pathological cells is a single-domain antibody. For example, the antibody or fragment that binds to the immune cell marker and the target antigen on pathological cells is a Fab fragment. For example, the antibody or fragment of the chimeric receptor 2 that binds to the immune cell marker includes a heavy chain variable region (VH2) and a light chain variable region (VL2), and the antibody or fragment that binds to the target antigen on pathological cells includes a heavy chain variable region (VH3) and a light chain variable region (VL3). For example, VH2, VL2, VH3, and VL3 are connected in a tandem structure.This tandem structure is formed in which VH2 and VL2 are linked as an scFv, with VH3 and VL3 linked to each end of the scFv, or VH3 and VL3 are linked as an scFv, with VH2 and VL2 linked to each end of the scFv, for example, VL3-VH2-VL2-VH3, VH3-VL2-VH2-VL3, VL2-VH3-VL3-VH2, VH2-VL3-VH3-VL2, VH3-VH2-VL2-VL3, VL3-VL2-VH2-VH3, VH2-VH3-VL3-VL2, or VL2-VL3-VH3-VH2. For example, an antibody or fragment that binds to the immune cell marker in chimeric receptor 2 is linked in tandem with an antibody or fragment that binds to a target antigen on pathological cells. The aforementioned tandem linkages are denoted as VH2-VL2-VH3-VL3, VH3-VL3-VH2-VL2, VL2-VH2-VL3-VH3, VL3-VH3-VL2-VH2, VH2-VL2-VL3-VH3, VL3-VH3-VL2-VH2, VL2-VH2-VH3-VL3, or VL3-VH3-VH2-VL2. "-" indicates any linker peptide or peptide bond. Different variable regions are linked by linker peptide chains. For example, the linker peptide chain is a GS linker peptide chain, e.g., (GGGGS)n, where n can be any number from 1 to 20. For example, the linker peptides include GGGGS, (GGGGS)3, or (GGGGS)4. For example, the linker peptide chain contains the amino acid sequence shown in SEQ ID NO: 14 or 15. Chimeric receptor 1 and chimeric receptor 2 can each be independently selected to have the same or different hinge region, transmembrane domain, and intracellular domain.

[0313] In one example, the manipulated cells include chimeric receptor 1, chimeric receptor 2, and chimeric receptor 3 that bind to CD38, where chimeric receptor 2 binds to the immune cell marker and chimeric receptor 3 binds to a target antigen on pathological cells. For example, chimeric receptor 1, chimeric receptor 2, and chimeric receptor 3 are constructed on a single vector. For example, chimeric receptor 1 and chimeric receptor 3 are constructed on a single vector, while chimeric receptor 2 is constructed on a different vector. For example, chimeric receptor 2 and chimeric receptor 3 are constructed on a single vector, while chimeric receptor 1 is constructed on a different vector. Chimeric receptors constructed on the same vector are linked by a coding sequence of 2A peptides. For example, the 2A peptide is P2A, T2A, E2A, or F2A. For example, chimeric receptor 1, chimeric receptor 2, and chimeric receptor 3 are each constructed on different vectors. The chimeric receptors 1, 2, and 3 can each independently select the same or different hinge regions, transmembrane domains, and intracellular domains.

[0314] The CD38-targeting engineered cells provided in this application include the chimeric receptors shown in Table 1. For example, the engineered cells include CD38-sBBZ or CD38-s28Z. In one example, the engineered cells include CD38-sBBZ or CD38-s28Z and further include another polypeptide, which includes a second antigen-binding domain or second chimeric receptor that binds to another NK cell marker, a target antigen on pathological cells, or a combination thereof, or an HLA-E polypeptide or a fragment thereof, or CD300A-PDGFR. For example, the engineered cells include CD38-sBBZ or CD38-s28Z and further include NKG2A-s28Z or NKG2A-28Z. For example, the engineered cells include CD38-sBBZ or CD38-s28Z and further include TIGIT-s28Z or TIGIT-28Z. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z, and also FasL-s28Z or FasL-28Z. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z, and also CD300A-PDGFR. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z, and also HLA-E. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z, and also BCMA-CAR. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z, and also CD94-CAR. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z, and also BCMA / NKG2A-CAR. For example, the manipulated cells include CD38-sBBZ or CD38-s28Z, and also include GPRC5D / NKG2A-CAR. For example, the manipulated cells include CD38-sBBZ or CD38-s28Z, and also include CD19-CAR. For example, the manipulated cells include CD38-sBBZ or CD38-s28Z, and also include CD19 / CD20-CAR. For example, the manipulated cells include CD38-sBBZ or CD38-s28Z, and also include CD19 / CD20 / NKG2A-CAR.For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z and also contain antigen-binding domains that recognize NKG2A, TIGIT, FasL, or CD94. For example, the manipulated cells contain CD38-sBBZ or CD38-s28Z and also contain antigen-binding domains that recognize BCMA, GPRC5D, or CD19.

[0315] For example, the engineered cells disclosed in this application include an amino acid sequence having approximately 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in any of SEQ ID NOs: 24, 26, 27, or 28. For example, the engineered cells include an amino acid sequence encoded by a nucleotide sequence having approximately 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in any of SEQ ID NOs: 18, 23, 25, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38. For example, the engineered cells include an amino acid sequence encoded by the nucleic acid sequences shown in SEQ ID NOs: 18 and 30. The manipulated cells contain amino acid sequences that have at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences encoded by the nucleic acid sequences shown in SEQ ID NOs: 23 and 30, or SEQ ID NOs: 25 and 30, or SEQ ID NOs: 18 and 31, or SEQ ID NOs: 23 and 31, or SEQ ID NOs: 25 and 31, or SEQ ID NOs: 29 and 31, or SEQ ID NOs: 18 and 32, or SEQ ID NOs: 23 and 32, or SEQ ID NOs: 25 and 32, or SEQ ID NOs: 29 and 32, or SEQ ID NOs: 18 and 33, or SEQ ID NOs: 23 and 33, or SEQ ID NOs: 25 and 33, or SEQ ID NOs: 29 and 33, or SEQ ID NOs: 18 and 34, or SEQ ID NOs: 23 and 34, or SEQ ID NOs: 25 and 34, or SEQ ID NOs: 29 and 34, or SEQ ID NOs: 18 and 35, or SEQ ID NOs: 23 and 35, or SEQ ID NOs: 25 and 35, or SEQ ID NOs: 29 and 35.

[0316] For example, the manipulated cells contain an amino acid sequence that has at least approximately 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 24 and 84, or SEQ ID NOs: 26 and 84, or SEQ ID NOs: 24 and 85, or SEQ ID NOs: 26 and 85, or SEQ ID NOs: 27 and 85, or SEQ ID NOs: 28 and 85, or SEQ ID NOs: 24 and 86, or SEQ ID NOs: 26 and 86, or SEQ ID NOs: 27 and 86, or SEQ ID NOs: 28 and 86.

[0317] In one example, the target antigen is selected from CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its variants, ASGPR1, IL13RA2, WT1, CLL1, or a combination thereof.

[0318] In one example, pathological cells are selected from malignant or infected cells. For example, pathological cells are selected from solid tumors, hematological malignancies, or autoimmune diseases. For example, the solid tumors are selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the hematological malignancies are selected from leukemia, lymphoma, and myeloma; and the autoimmune diseases are selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis, or dermatomyositis.

[0319] For example, CD38-CARs with an IgG4 hinge region significantly reduce the expression of CD38 antigen on engineered cells. For instance, CD38 antigen on engineered cells expressing CD38-CARs with an IgG4 hinge region is blocked, and its expression is almost undetectable. For instance, engineered cells expressing CD38-CARs with an IgG4 hinge region exhibit high viability and robust proliferative capacity in in vitro and in vivo cultures. For instance, expressed CD38-CARs with an IgG4 hinge region promote the survival and / or proliferation of immune cells that recognize tumor antigens in the host. For instance, expressed CD38-CARs with an IgG4 hinge region enhance the antitumor activity of immune cells that recognize tumor antigens. For instance, expressed CD38-CARs with an IgG4 hinge region promote the survival and / or proliferation of autoimmune cells that recognize solid tumor antigens in the host. For instance, expressed CD38-CARs with an IgG4 hinge region enhance the antitumor activity of autoimmune cells that recognize solid tumor antigens in the host. For example, endogenous TCR, B2M, TCR / B2M, TCR / B2M / NKG2A, TCR / B2M / CD38, TCR / B2M / FAS, TCR / B2M / NKG2A / CD38, TCR / B2M / NKG2A / FAS, or TCR / B2M / NKG2A / CIITA are either low-expressed or not expressed at all in autoimmune cells containing the CD38-CAR of the present invention.

[0320] For example, the antitumor activity of immune cells containing CD38-CARs with an IgG4 hinge region is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, 2, 3, 4, 5 times, or more, in killing or inhibiting target antigen-positive pathological cells compared to immune cells containing chimeric receptors that recognize only target antigens on pathological cells. For instance, immune cells containing CD38-CARs with an IgG4 hinge region have approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, 2, 3, 4, 5 times, or more, in host viability and / or proliferation compared to immune cells containing chimeric receptors that recognize only target antigens on pathological cells.

[0321] This application provides polynucleotides encoding a CD38-targeting chimeric receptor disclosed herein, and / or a second antigen-binding domain or second chimeric receptor that binds to another NK cell marker, a target antigen on pathological cells, or a combination thereof, or an HLA-E peptide or a fragment thereof, or CD300A-PDGFR. This application provides vectors comprising the polynucleotides disclosed herein. This application provides viruses comprising the vectors disclosed herein.

[0322] Chimeric receptor composition and cells manipulated therein This application discloses engineered cells (e.g., NK cells, T cells, NKT cells) expressing a first chimeric receptor and a second chimeric receptor, wherein the extracellular domains of both the first and second chimeric receptors recognize a target antigen, and pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases), cells that mediate transplant rejection (e.g., NK cells, T cells, NKT cells), and engineered cells all express the target antigen, the first chimeric receptor mediates the killing of pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases) expressing the target antigen and / or cells that mediate transplant rejection (e.g., NK cells, T cells, NKT cells), and the second chimeric receptor can block the target antigen expressed on the engineered cells (e.g., NK cells, T cells, NKT cells).

[0323] This application discloses engineered cells (e.g., NK cells, T cells, NKT cells) expressing a first chimeric receptor and a second chimeric receptor, wherein the extracellular domains of both the first and second chimeric receptors recognize a target antigen, and pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases), cells that mediate transplant rejection (e.g., NK cells, T cells, NKT cells), and the engineered cells all express the target antigen, the first chimeric receptor mediates the killing of pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases) expressing the target antigen and / or cells that mediate transplant rejection (e.g., NK cells, T cells, NKT cells), and the first chimeric receptor does not mediate the killing of the engineered cells (e.g., NK cells, T cells, NKT cells) or has a significantly reduced ability to mediate it.

[0324] In one example, the extracellular domain of the second chimeric receptor is capable of binding to a target antigen to which the first chimeric receptor binds, the first chimeric receptor mediates the killing of target cells expressing the target antigen, and the second chimeric receptor further comprises ASGPR, full-length CD137L or a fragment thereof, or further comprises full-length ASGPR1 or a fragment thereof, or further comprises full-length ASGPR2 or a fragment thereof, or further comprises full-length CD137L or a fragment thereof, or further comprises the transmembrane domain and intracellular domain of ASGPR1, or further comprises the transmembrane domain and intracellular domain of ASGPR2, or further comprises the transmembrane domain and intracellular domain of CD137L.

[0325] For example, a chimeric receptor composition is 1. A first chimeric receptor selected from (1) a full-length native ligand (or receptor) and an intracellular domain, (2) an extracellular region, transmembrane region and intracellular domain of a native ligand (or receptor), and (3) an antibody or a fragment thereof, transmembrane region and intracellular domain: and 2. A second chimeric receptor selected from (1) comprising the extracellular domain of the same innate ligand (or receptor) as the first chimeric receptor, and further comprising full-length ASGPR1 or a fragment thereof, full-length ASGPR2 or a fragment thereof, the transmembrane domain and intracellular domain of ASGPR1, the transmembrane domain and intracellular domain of ASGPR2, full-length CD137L or a fragment thereof, or the transmembrane domain and intracellular domain of CD137L; or (2) comprising an antibody or a fragment thereof that recognizes a target antigen recognized by the first chimeric receptor, and further comprising full-length ASGPR1 or a fragment thereof, full-length ASGPR2 or a fragment thereof, the transmembrane domain and intracellular domain of ASGPR1, the transmembrane domain and intracellular domain of ASGPR2, full-length CD137L or a fragment thereof, or the transmembrane domain and intracellular domain of CD137L. Includes.

[0326] Preferably, the first chimeric receptor in the chimeric receptor composition mediates the killing of target cells expressing the target antigen.

[0327] For example, antigens recognized by the first and second chimeric receptors are CD1b, CD1c, CD1d, CD3d, CD3e, CD4, CD7, CD8, CD11, CD13, CD16b, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD34, CD38, CD39, CD40, CD44, CD45, C D45RA, CD45RO, CD48, CD52, CD56, CD58, CD59, CD62L, CD66a, CD69, CD70, CD80, CD83, CD85, CD86, C D93, CD94, CD95, CD95L, CD96, CD99, CD100, CD102, CD103, CD107a, CD107b, CD111, CD112, CD117, CD 119, CD122, CD123, CD126, CD127, CD132, CD134, CD137, CD150, CD152, CD153, CD154, CD155, CD158 , CD159a, CD160, CD161, CD178, CD183, CD185, CD191, CD192, CD196, CD212, CD215, CD218, CD223, C Select from D226, CD244, CD252, CD253, CD275, CD278, CD279, CD281, CD282, CD283, CD284, CD286, CD300, CD305, CD314, CD319, CD336, CD337, CD355, CD360, CD366, CD371, B2MG, NKG2DL, or a combination thereof. For example, the antigens recognized by the first and second chimeric receptors are selected from CD1d, CD7, CD13, CD20, CD22, CD25, CD26, CD30, CD33, CD38, CD45, CD52, CD56, CD70, CD80, CD83, CD86, CD93, CD94, CD95L, CD99, CD112, CD117, CD123, CD155, CD159a, CD319, CD366, CD371, NKG2DL, or combinations thereof.

[0328] This application discloses engineered cells expressing a first chimeric receptor and a second chimeric receptor. For example, the engineered cells are immune cells, neurons, epithelial cells, endothelial cells, stem cells, or a combination thereof. For example, the engineered cells are immune cells. For example, the immune cells are autologous cells. For example, the immune cells are allogeneic cells. For example, the immune cells are selected from B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof. For example, the engineered cells are T cells. In one example, the engineered cells are allogeneic T cells. For example, the engineered cells are stem cell-derived T cells. T cells can be cytotoxic T cells, helper T cells, or αβT cells, γδT cells, CD4+ / CD8+ double-positive T cells, CD4+ T cells, CD8+ T cells, CD4 / CD8 double-negative T cells, CD3+ T cells, naive T cells, effector T cells, cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like central memory TCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, or γδT cells. For example, a T cell is a cytotoxic T cell.

[0329] This application discloses engineered cells (e.g., NK cells, T cells, NKT cells) expressing a first chimeric receptor and a second chimeric receptor, wherein the extracellular domains of the first and second chimeric receptors recognize NKG2DL. The first chimeric receptor mediates the killing of target cells expressing NKG2DL (e.g., tumor cells, pathological cells of autoimmune diseases, NK cells, T cells, NKT cells), and the second chimeric receptor can block the NKG2DL polypeptide on the engineered cells (e.g., NK cells, T cells, NKT cells).

[0330] In one example, manipulated cells (e.g., NK cells, T cells, NKT cells) with a first chimeric receptor and a second chimeric receptor have extracellular domains that recognize NKG2DL, the first chimeric receptor mediates the killing of target cells expressing NKG2DL (e.g., tumor cells, pathological cells of autoimmune diseases, NK cells, T cells, NKT cells), and the first chimeric receptor does not mediate the killing of the manipulated cells (e.g., NK cells, T cells, NKT cells), or has a significantly reduced ability to mediate it.

[0331] For example, a chimeric receptor composition is 1. A first chimeric receptor selected from the following, extending from the N-terminus to the C-terminus: (1) containing a CD3 intracellular domain and full-length NKG2D; (2) containing a CD3Z intracellular domain, a CD137 intracellular domain, and full-length NKG2D; (3) containing a CD3Z intracellular domain, a CD28 intracellular domain, and full-length NKG2D; (4) containing an NKG2D extracellular domain, a CD8 or CD28 or IgG1 or IgG4 hinge region, a CD8 or CD28 intracellular domain, and a CD3Z intracellular domain; (5) containing an NKG2D extracellular domain, a CD8 or CD28 or IgG1 or IgG4 hinge region, a CD8 or CD28 transmembrane domain, a CD137 intracellular domain, and a CD3Z intracellular domain; and 2. From the N-terminus to the C-terminus, (1) containing the extracellular domains of full-length ASGPR1 and NKG2D, (2) containing the extracellular domains of full-length ASGPR2 and NKG2D, (3) containing the extracellular domains of full-length CD137L and NKG2D, (4) containing an antibody or fragment thereof that binds to full-length ASGPR1 and NKG2DL, (5) containing an antibody or fragment thereof that binds to full-length ASGPR2 and NKG2DL, (6) containing full-length CD137L and NK (7) Contains an antibody or fragment thereof that binds to G2DL, (8) Contains the ASGPR1 intracellular domain and full-length NKG2D, (9) Contains the CD137L intracellular domain and full-length NKG2D, (10) Contains the ASGPR1 intracellular domain, the NKG2D transmembrane domain and the extracellular domain of NKG2D, (11) Contains the ASGPR1 intracellular domain, the NKG2D transmembrane domain and the extracellular domain of NKG2D A second chimeric receptor selected from the following: (12) containing the main domain, (13) containing the CD137L intracellular domain, the NKG2D transmembrane domain, and the NKG2D extracellular domain, (14) containing the ASGPR1 intracellular domain, the ASGPR1 transmembrane domain, and the NKG2D extracellular domain, (15) containing the CD137L intracellular domain, the CD137L transmembrane domain, and the NKG2D extracellular domain, (16) containing an antibody or fragment that binds to NKG2DL, the CD8 or CD28 transmembrane domain, and the ASGPR1 intracellular domain, (17) containing an antibody or fragment that binds to NKG2DL, the CD8 or CD28 transmembrane domain, and the ASGPR2 intracellular domain, and (18) containing an antibody or fragment that binds to NKG2DL, the CD8 or CD28 transmembrane domain, and the CD137L intracellular domain. Includes.

[0332] For example, the chimeric receptor composition includes, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain and a full-length NKG2D, and a second chimeric receptor comprising a full-length ASGPR1 and an NKG2D extracellular domain. For example, the chimeric receptor composition includes, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain and a full-length NKG2D, and a second chimeric receptor comprising a full-length CD137L and an NKG2D extracellular domain. For example, the chimeric receptor composition includes, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD137 intracellular domain, and a full-length NKG2D, and a second chimeric receptor comprising a full-length ASGPR1 and an NKG2D extracellular domain. For example, a chimeric receptor composition may include, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD137 intracellular domain, and full-length NKG2D, and a second chimeric receptor comprising full-length CD137L and NKG2D extracellular domains. For example, a chimeric receptor composition may include, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD28 intracellular domain, and full-length NKG2D, and a second chimeric receptor comprising full-length ASGPR1 and NKG2D extracellular domains. For example, a chimeric receptor composition may include, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD28 intracellular domain, and full-length NKG2D, and a second chimeric receptor comprising full-length CD137L and NKG2D extracellular domains.

[0333] For example, a chimeric receptor composition comprises, from the N-terminus to the C-terminus, a first chimeric receptor containing a CD3Z intracellular domain and full-length NKG2D, and a second chimeric receptor containing full-length ASGPR1, a linker peptide, and an NKG2D extracellular domain. For example, a chimeric receptor composition comprises, from the N-terminus to the C-terminus, a first chimeric receptor containing a CD3Z intracellular domain and full-length NKG2D, and a second chimeric receptor containing full-length CD137L, a linker peptide, and an NKG2D extracellular domain. For example, a chimeric receptor composition comprises, from the N-terminus to the C-terminus, a first chimeric receptor containing a CD3Z intracellular domain, a CD137 intracellular domain, and full-length NKG2D, and a second chimeric receptor containing full-length ASGPR1, a linker peptide, and an NKG2D extracellular domain. For example, a chimeric receptor composition may include, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD137 intracellular domain, and full-length NKG2D, and a second chimeric receptor comprising full-length CD137L, a linker peptide, and an NKG2D extracellular domain. For example, a chimeric receptor composition may include, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD28 intracellular domain, and full-length NKG2D, and a second chimeric receptor comprising full-length ASGPR1, a linker peptide, and an NKG2D extracellular domain. For example, a chimeric receptor composition may include, from the N-terminus to the C-terminus, a first chimeric receptor comprising a CD3Z intracellular domain, a CD28 intracellular domain, and full-length NKG2D, and a second chimeric receptor comprising full-length CD137L, a linker peptide, and an NKG2D extracellular domain. In one example, the linker peptide chain is a GS linker peptide chain, e.g., (GGGGS)n, where n can be any number from 1 to 20. For example, the linker peptide may contain GGGGS, (GGGGS)3, or (GGGGS)4. In one example, the linker peptide chain may contain the amino acid sequence shown in SEQ ID NO: 14 or 15.

[0334] For example, a chimeric receptor composition comprises: 1. A first chimeric receptor comprising an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence encoded by the nucleic acid shown in SEQ ID NOs. 78 or 79; and 2. A second chimeric receptor comprising an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence encoded by the nucleic acid shown in SEQ ID NOs. 80 and 76, or 80, 16 and 76, or 81 and 76, or 81, 16 and 76.

[0335] This application discloses engineered cells that inductively express a first chimeric receptor and constitutively express a second chimeric receptor, the extracellular domains of both the first and second chimeric receptors capable of recognizing a target antigen, the first chimeric receptor mediating the killing of target cells expressing the target antigen (e.g., tumor cells, pathological cells of autoimmune diseases, NK cells, T cells, NKT cells, etc., which mediate transplant immune rejection), and the first chimeric receptor not mediating the killing of the engineered cells (e.g., NK cells, T cells, NKT cells), or having a significantly reduced ability to mediate it. For example, the second chimeric receptor is constitutively expressed, and the first chimeric receptor is regulated by the synNOTCH receptor. For example, see PCT / CN2022 / 102395 for information on the synNOTCH receptor.

[0336] Manipulated cells comprising the first and second chimeric receptors provided in this application can not only resist killing by autologous or allogeneic NK cells but can also significantly kill pathological cells. For example, this application provides T cells expressing CARs that recognize NKG2D ligand and CARs that recognize tumor antigens. This application provides universal T cells that express and contain CARs that recognize NKG2D ligand and CARs that recognize tumor antigens, have knocked out endogenous TCRs and B2Ms, and express ASGPR1 chimeric receptors or ASGPR2 chimeric receptors.

[0337] This application provides the use of a combination of T cells resistant to self- or allogeneic NK cell killing and T cells expressing a second protein (e.g., CAR) that recognizes tumor antigens. The self- or allogeneic NK cell killing resistant T cells provided in this application can promote the survival of T cells expressing a second protein (e.g., CAR) that recognizes tumor antigens in the presence of self- or allogeneic immune cells.

[0338] The chimeric receptor compositions disclosed in this application include any of the first chimeric receptors targeting NKG2DL shown in Table 4 and any of the second chimeric receptors targeting NKG2DL shown in Table 5. TIFF2026525409000004.tif66170TIFF2026525409000005.tif79170

[0339] In Tables 4 and 5 above: NKG2D: full-length NKG2D (SEQ ID NO: 77); NKG2Dex: NKG2D extracellular domain (SEQ ID NO: 76); CD8(H): CD8 hinge region (SEQ ID NO: 3); IgG4(H): IgG4 hinge region (SEQ ID NO: 5); CD8(TM): CD8 transmembrane domain (SEQ ID NO: 7); CD28(TM): CD28 transmembrane domain (SEQ ID NO: 8, 9); CD137(C): ​​CD137 intracellular domain (SEQ ID NO: 11); CD28(C): CD28 intracellular domain (SEQ ID NO: 10); CD3ζ(C): CD3ζ intracellular domain (SEQ ID NO: 12, 13); ASGPR1: full-length ASGPR1 (SEQ ID NO: 80); (L): linker peptide (SEQ ID NO: 76), numbers 14, 15, 16); CD137L: full-length CD137L (SEQ ID NO: 81).

[0340] The A-N1 / NKG2Dz fragment disclosed in this application comprises, in order from 5' to 3', the NKG2Dz fragment, the 2A peptide, and the A-N1 fragment. The A-N2 / NKG2Dz fragment comprises, in order from 5' to 3', the NKG2Dz fragment, the 2A peptide, and the A-N2 fragment. The CD137L-N / NKG2Dz fragment comprises, in order from 5' to 3', the NKG2Dz fragment, the 2A peptide, and the CD137L-N fragment. The CD40L-N / NKG2Dz fragment comprises, in order from 5' to 3', the NKG2Dz fragment, the 2A peptide, and the CD40L-N fragment. The A-N1 / NKG2D-BBZ fragment comprises, in order from 5' to 3', the NKG2D-BBZ fragment, the 2A peptide, and the A-N1 fragment. A-N2 / NKG2D-BBZ fragment: From 5' to 3', it sequentially contains the NKG2D-BBZ fragment, the 2A peptide, and the A-N2 fragment. CD137L-N / NKG2D-BBZ fragment: From 5' to 3', it sequentially contains the NKG2D-BBZ fragment, the 2A peptide, and the CD137L-N fragment. CD40L-N / NKG2D-BBZ fragment: From 5' to 3', it sequentially contains the NKG2D-BBZ fragment, the 2A peptide, and the CD40L-N fragment. For example, the 2A peptide is P2A, T2A, E2A, or F2A.

[0341] This application discloses engineered cells modified with the aforementioned A-N1 / NKG2Dz fragment, A-N2 / NKG2Dz fragment, CD137L-N / NKG2Dz fragment, CD40L-N / NKG2Dz fragment, A-N1 / NKG2D-BBZ fragment, A-N2 / NKG2D-BBZ fragment, CD137L-N / NKG2D-BBZ fragment, or CD40L-N / NKG2D-BBZ fragment.

[0342] To enhance the protective effect of the second chimeric receptor on engineered cells, the first chimeric receptor is inductively expressed, and the second chimeric receptor is constitutively expressed. Engineered cells with controlled expression of the first chimeric receptor were generated according to PCT / CN2022 / 102395. For example, regulatory NKG2D-CAR-T cells are T cells containing a chimeric polypeptide (see PCT / CN2022 / 102395) and NKG2D-CAR (see Table 4) that bind to tumor antigens or tissue-specific antigens. The chimeric polypeptide synE or its variants, truncated or modified forms, synJagged2EC, synE-APLP2®, synNOTCH, and syn-CD8 can induce transcriptional regulation of NKG2D-CAR expression.

[0343] Regulatory NKG2D-CAR-T cells possess one or more of the following characteristics: 1) When incubated with tumor antigen or tissue-specific antigen-positive cells, they effectively kill NKG2D ligand-positive tumor cells (MV-4-11, THP-1, etc.) or immune cells (NK cells, T cells, NKT cells, etc.), but show weak killing effects against cells with low NKG2D ligand expression (KG-1, Molm13, etc.). 2) When incubated with cells that have low or no expression of tumor antigen or tissue-specific antigen, their killing effect against NKG2D ligand-positive cells is weakened or completely eliminated (SK-Hep1, K562, etc.). The cytotoxicity of synE or synE-del3-regulated NKG2D-CAR-T cells is NKG2D ligand-dependent, but cells with high expression of tumor antigen or tissue-specific antigen alone do not induce NKG2D ligand-independent cytotoxicity. By placing NKG2D-CAR under the control of synE or synE-del3, the in vivo viability and cytotoxicity of regulatory NKG2D-CAR-T cells against target cells (pathological cells or immune-rejecting cells) can be improved. Regulatory NKG2D-CAR-T cells can recognize target cells (tumor antigen or tissue-specific antigen-positive cells) via synE or synE-del3, the cytotoxic effect of NKG2D-CAR-T cells requires induction of NKG2D ligands on the target cell surface, and downstream signaling of synE or synE-del3 does not cross-influence with downstream signaling of NKG2D-CAR.

[0344] This application discloses a CD123-synE fragment that recognizes the CD123 antigen, comprising, from 5' to 3', a CD8 signal peptide, CD123-scFv, ephrin B2 extracellular domain, Notch transmembrane domain, and GAL4-VP64 in that order. The CD123-synE-NKG2Dz fragment comprises, from 5' to 3', a UAS-CMV, an NKG2Dz fragment, an EF1a promoter, and a CD123-synE fragment in that order. The CD123-synE-NKG2D-BBZ fragment comprises, from 5' to 3', a UAS-CMV, an NKG2D-BBZ fragment, an EF1a promoter, and a CD123-synE fragment in that order.

[0345] The A-N1 / CD123-synE-NKG2Dz fragment contains, from 5' to 3', UAS-CMV, NKG2Dz fragment, EF1a promoter, CD123-synE fragment, 2A peptide, and A-N1 fragment in that order. The A-N1 / CD123-synE-NKG2D-BBZ fragment contains, from 5' to 3', UAS-CMV, NKG2D-BBZ fragment, EF1a promoter, CD123-synE fragment, 2A peptide, and A-N1 fragment in that order. The A-N2 / CD123-synE-NKG2Dz fragment contains, from 5' to 3', UAS-CMV, NKG2D-BBZ fragment, EF1a promoter, CD123-synE fragment, 2A peptide, and A-N2 fragment in that order. The A-N2 / CD123-synE-NKG2D-BBZ fragment contains, from 5' to 3', UAS-CMV, the NKG2D-BBZ fragment, the EF1a promoter, the CD123-synE fragment, the 2A peptide, and the A-N2 fragment in that order. For example, the 2A peptide is P2A, T2A, E2A, or F2A.

[0346] The regulatory NKG2D-CAR-T cells disclosed in this application further comprise a constitutively expressed second chimeric receptor. For example, engineered cells comprising the CD123-synE-NKG2Dz fragment and one of the second chimeric receptors listed in Table 5. For example, engineered cells comprising the CD123-synE-NKG2Dz fragment and A-N1 fragment, the CD123-synE-NKG2Dz fragment and A-N2 fragment, the CD123-synE-NKG2Dz fragment and A-N3 fragment, the CD123-synE-NKG2Dz fragment and A-N4 fragment, the CD123-synE-NKG2Dz fragment and CD137L-N fragment, CD123 The CD123-synE-NKG2Dz fragment and CD40L-N fragment, the CD123-synE-NKG2Dz fragment and CD137L-N1 fragment, the CD123-synE-NKG2Dz fragment and CD40L-N1 fragment, the CD123-synE-NKG2Dz fragment and CD137L-N2 fragment, or the CD123-synE-NKG2Dz fragment and CD40L-N2 fragment. For example, the CD123-synE-NKG2Dz fragment and the second chimeric receptor are constructed on a single vector. For example, the CD123-synE-NKG2Dz fragment and the second chimeric receptor are linked by the coding sequence of the 2A peptide. For example, from the 5' end to the 3' end, the CD123-synE-NKG2Dz fragment, the coding sequence of the 2A peptide, and the second chimeric receptor are included in that order. For example, 2A peptides are P2A, T2A, E2A, or F2A.

[0347] For example, the manipulated cells contain the CD123-synE-NKG2D-BBZ fragment and A-N1 fragment, the CD123-synE-NKG2D-BBZ fragment and A-N2 fragment, the CD123-synE-NKG2D-BBZ fragment and A-N3 fragment, the CD123-synE-NKG2D-BBZ fragment and A-N4 fragment, the CD123-synE-NKG2D-BBZ fragment and CD137L-N fragment, and CD123 The CD123-synE-NKG2D-BBZ fragment and CD40L-N fragment, the CD123-synE-NKG2D-BBZ fragment and CD137L-N1 fragment, the CD123-synE-NKG2D-BBZ fragment and CD40L-N1 fragment, the CD123-synE-NKG2D-BBZ fragment and CD137L-N2 fragment, or the CD123-synE-NKG2D-BBZ fragment and CD40L-N2 fragment. For example, the CD123-synE-NKG2D-BBZ fragment and the second chimeric receptor are linked by the coding sequence of the 2A peptide. For example, from the 5' end to the 3' end, the CD123-synE-NKG2D-BBZ fragment, the coding sequence of the 2A peptide, and the second chimeric receptor are included in this order. For example, the 2A peptide is P2A, T2A, E2A, or F2A.

[0348] Bispecific CARs that bind to GPRC5D and BCMA, and bispecific CARs that bind to GPRC5D and NKG2A This application discloses a bispecific CAR targeting GPRC5D and another antigen, as well as engineered cells containing this bispecific CAR. For example, the other antigen is either an antigen that specifically binds to an antigen expressed in multiple myeloma or a second antigen associated with multiple myeloma. For example, the other antigen includes BCMA, CD3, CD19, CD20, CD22, CD123, CD38, CD138, CS-1, BAFF-R, TACI, and FcRH5. For example, the other antigen is an immune cell marker. For example, the other antigen is either a T cell or NK cell marker disclosed in this application. For example, the other antigen is NKG2A, CD38, TIGIT, FasL, CD94, CD300A, or a combination thereof.

[0349] This application provides immune cells capable of recognizing GPRC5D and a second antigen, thereby enhancing the killing effect or immune rejection effect of immune cells against tumor cells such as multiple myeloma cells.

[0350] This application discloses bispecific CARs targeting GPRC5D and another antigen, as shown in Table 6. CARs targeting GPRC5D or BCMA are shown in Table 7. TIFF2026525409000006.tif85170TIFF2026525409000007.tif27170

[0351] Tables 6 and 7: αGPRC5D_VH: GPRC5D antibody VH (SEQ ID NO: 99); αGPRC5D_VL: GPRC5D antibody VL (SEQ ID NO: 102); αBCMA_VH: BCMA antibody VH (SEQ ID NO: 100); αBCMA_VL: BCMA antibody VL (SEQ ID NO: 101); αNKG2A_VH: NKG2A antibody VH (SEQ ID NO: 116); αNKG2A_VL: NKG2A antibody VL (SEQ ID NO: 117); (L): Linker peptide chain ( SEQ ID NOs: 14, 15, 16); CD8(H): CD8 hinge (SEQ ID NO: 3); CD8(TM): CD8 transmembrane domain (SEQ ID NO: 7); CD28(TM): CD28 transmembrane domain (SEQ ID NO: 9); CD28(C): CD28 intracellular domain (SEQ ID NO: 10); CD137(C): ​​CD137 intracellular domain (SEQ ID NO: 11); CD3ζ: CD3ζ intracellular domain (SEQ ID NO: 12); IgG4(H): IgG4 spacer region (SEQ ID NO: 5).

[0352] This application provides a bispecific chimeric receptor of BCMA-CAR and GPRC5D-CAR as shown in Table 7, or BLG-sBBZ, BLG-BBZ, GLB-sBBZ, GLB-BBZ, GLN-BBZ as shown in Table 6, or GPRC5D-CAR as shown in Table 7 and NKG2A-s28z or NKG2A-28z as shown in Table 3.

[0353] The GPRC5D-targeting chimeric receptor provided by the present invention comprises a GPRC5D-binding domain. For example, the GPRC5D-binding domain comprises a heavy-chain variable region VH and a light-chain variable region VL. For example, VH and VL of GPRC5D comprise the amino acid sequences shown in SEQ ID NOs. 99 and 102, respectively, or comprise amino acid sequences having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs. 99 and 102. For example, VH comprises CDR1, CDR2, and CDR3, which comprise the amino acid sequences shown in SEQ ID NOs. 104, 105, and 106, respectively, and VL comprises CDR1, CDR2, and CDR3, which comprise the amino acid sequences shown in SEQ ID NOs. 107, 108, and 109, respectively. For example, GPRC5D scFv contains the amino acid sequence shown in SEQ ID NO: 103, or contains an amino acid sequence that is at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 103.

[0354] For example, a bispecific CAR targeting GPRC5D further includes a BCMA-binding domain. For instance, the BCMA-binding domain comprises VH and VL, which each contain the amino acid sequences shown in SEQ ID NOs. 100 and 101, or amino acid sequences having at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs. 100 and 101. For example, CDR1, CDR2, and CDR3 contained in VH each contain the amino acid sequences shown in SEQ ID NOs. 110, 111, and 112, and CDR1, CDR2, and CDR3 contained in VL each contain the amino acid sequences shown in SEQ ID NOs. 113, 114, and 115.

[0355] For example, a bispecific CAR targeting GPRC5D further includes an NKG2A binding domain. For instance, the NKG2A binding domain includes VH and VL, which contain amino acid sequences that are at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 116 and 117, respectively.

[0356] This application discloses a bispecific chimeric receptor targeting GPRC5D and BCMA, and an immune cell thereof, which comprises (1) an anti-GPRC5D antibody or a fragment thereof (including a heavy chain variable region (VH1) and a light chain variable region (VL1), where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences shown in SEQ ID NOs. 104, 105, 106, 107, 108, and 109); and (2) an anti-BCMA antibody or a fragment thereof (including a heavy chain variable region (VH2) and a light chain variable region (VL2), where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences shown in SEQ ID NOs. 110, 111, 112, 113, 114, and 115). For example, the immune cell comprises a chimeric receptor targeting GPRC5D and a chimeric receptor targeting BCMA. For example, the immune cells contain a chimeric receptor that simultaneously targets GPRC5D and BCMA. For example, the immune cells contain an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the nucleic acid sequence shown in SEQ ID NOs. 90, 91, 92, 93, 94, 95, or an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NOs. 90, 91, 92, 93, 94, 95.

[0357] This application discloses a bispecific chimeric receptor targeting GPRC5D and NKG2A, and immune cells thereof, comprising: (1) an anti-GPRC5D antibody or a fragment thereof (comprising a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein CDR1, CDR2, and CDR3 contained in VH1 comprise the amino acid sequences shown in SEQ ID NOs. 104, 105, and 106, respectively, and CDR1, CDR2, and CDR3 contained in VL1 comprise the amino acid sequences shown in SEQ ID NOs. 107, 108, and 109, respectively); and (2) an anti-NKG2A antibody or a fragment thereof (comprising a heavy chain variable region (VH3) and a light chain variable region (VL3), comprising amino acid sequences having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs. 116 and 117, respectively). For example, the immune cell includes a chimeric receptor targeting GPRC5D and a chimeric receptor targeting NKG2A. For example, the immune cell includes a chimeric receptor that simultaneously targets GPRC5D and NKG2A. For example, the immune cell includes an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in Sequence ID No. 84.

[0358] The transmembrane domain of a bispecific chimeric receptor targeting GPRC5D and BCMA, or GPRC5D and NKG2A, may be any transmembrane domain disclosed in this application. For example, a CD8 or CD28 transmembrane domain. The hinge of a bispecific chimeric receptor targeting GPRC5D and BCMA, or GPRC5D and NKG2A, may be any hinge disclosed in this application. For example, a CD8 hinge, a CD28 hinge, or an IgG4 hinge. The intracellular signaling domain of the chimeric receptor comprises one or more co-stimulatory signaling domains and / or primary signaling domains, the primary signaling domain being selected from TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS, CD66d, and CD3ζ, and / or the co-stimulatory signaling molecule being selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88 intracellular domains, or combinations thereof. For example, the intracellular domain may include the co-stimulatory signaling 4-1BB intracellular signaling domain and the primary signaling CD3ζ intracellular activation domain.

[0359] Compared to CARs targeting a single antigen, affinity is improved, T cell activity is increased, and these targets exhibit additive or synergistic effects. CAR-T cells that simultaneously target BCMA and GPRC5D on the tumor cell surface can reduce the possibility of antigen escape caused by the downregulation or absence of a single surface antigen. CAR-T cells that simultaneously target NKG2A and GPRC5D can provide CAR-T cells with anti-immune rejection capabilities. Antibodies against GPRC5D and BCMA are linked in tandem. Antibodies against GPRC5D and NKG2A are linked in tandem. For example, GPRC5D antibody VH1 and GPRC5D antibody VL1 are linked as antibody scFv1, and BCMA antibody VH2 and BCMA antibody VL2 are linked to both ends of the antibody scFv1, respectively. For example, GPRC5D antibody VH1 and VL1 are linked as antibody scFv1, and NKG2A antibody VH3 and VL3 are linked to both ends of the antibody scFv1, respectively. For example, VH2 and VL2 of the BCMA antibody are linked as antibody scFv2, and VH1 and VL1 of the GPRC5D antibody are linked to both ends of the antibody scFv2, respectively. For example, VH3 and VL3 of the NKG2A antibody are linked as antibody scFv3, and VH1 and VL1 of the GPRC5D antibody are linked to both ends of the antibody scFv3, respectively. The antigen-binding domain of the chimeric receptor can have different three-dimensional structures.

[0360] For example, the antigen-binding domain includes VH1 and VL1 of the GPRC5D antibody, and VH2 and VL2 of the BCMA antibody, and from the N-terminus to the C-terminus, it is VH1-VL1-VH2-VL2, VL1-VH1-VL2-VH2, VH1-VL1-VL2-VH2, VH1-VH2-VL2-VL1, VL1-VH2-VL2-VH1, VH1-VL2-VH2-VL1, VL1-VL2-VH2-VH1, VH2-VH1-VL1-VL2, VL2-VH1-VL1-VH2, VH2-VL1-VH1-VL2, VL2-VL1-VH1-VH2.

[0361] For example, the antigen-binding domain includes VH1 and VL1 of the GPRC5D antibody and VH3 and VL3 of the NKG2A antibody, and from the N-terminus to the C-terminus, it is VH1-VL1-VH3-VL3, VL1-VH1-VL3-VH3, VH1-VL1-VL3-VH3, VL1-VH3-VL3-VL1, VL1-VH3-VL3-VH1, VH1-VL3-VH3-VL1, VL1-VL3-VH3-VH1, VH3-VH1-VL1-VL3, VL3-VH1-VH1-VL3, VL3-VL1-VH1-VH3, VL3-VL1-VH1-VH3.

[0362] It is known in the art that the VH and VL regions can undergo several amino acid substitutions without altering the CDR sequence, while maintaining affinity to the target. Similarly, the CDR sequence can undergo several amino acid substitutions without altering the amino acids in contact with the target, while maintaining affinity to the target.

[0363] For example, an anti-GPRC5D, BCMA, or NKG2A antibody or a fragment thereof is an scFv. The VH (heavy chain variable region) and VL (light chain variable region) in the scFv can be linked by a linker peptide chain (linker), and their positions can be swapped. For example, an anti-GPRC5D, BCMA, or NKG2A antibody contains VH, a linker peptide chain, and VL from the N-terminus to the C-terminus. For example, an anti-GPRC5D, BCMA, or NKG2A antibody contains VL, a linker peptide chain, and VH from the N-terminus to the C-terminus. Any linker peptide chain is suitable for linking VH and VL to form a fully functional single-chain antibody. For example, the linker peptide chain is a GS linker peptide chain such as GGGGS, (GGGGS)3, or (GGGGS)4.

[0364] In one example, the chimeric receptor provided by the present invention comprises (1) a domain of an anti-GPRC5D antibody (VH / VL selected from SEQ ID NOs. 99 and 102), a domain of an anti-BCMA antibody (VH / VL selected from SEQ ID NOs. 100 and 101), and / or a domain of an NKG2A antibody (VH / VL selected from SEQ ID NOs. 116 and 117), (2) a CD8 hinge region (SEQ ID NOs. 3), or an IgG4 spacer fragment (SEQ ID NOs. 5 or 6), (3) a CD28 transmembrane domain (SEQ ID NOs. 8 or 9) or a CD8 transmembrane domain (SEQ ID NOs. 7), (4) a CD28 intracellular signaling domain (SEQ ID NOs. 10) or a CD137 intracellular signaling domain (SEQ ID NOs. 11), and optionally further comprising (5) a CD3ζ intracellular signaling domain (SEQ ID NOs. 12 or 13).

[0365] The chimeric receptors provided by the present invention include nucleic acid sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the nucleic acid sequences shown in SEQ ID NOs. 90, 91, 92, 93, 94, and 95, or include amino acid sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequences encoded by nucleic acid sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the nucleic acid sequences shown in SEQ ID NOs. 90, 91, 92, 93, 94, and 95. The chimeric receptors provided by the present invention include amino acid sequences that have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequences shown in SEQ ID NOs. 84 and 96.

[0366] The manipulated cells provided by the present invention include a chimeric receptor 1 that binds to GPRC5D, comprising an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in SEQ ID NOs. 84 and 96, and optionally further comprising a chimeric receptor 2 comprising an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in SEQ ID NOs. 24, 26, 27, 28, 83, 85, 86, 96, or 98. For example, the manipulated cells include a chimeric receptor 1 that binds to GPRC5D, comprising an amino acid sequence having at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in SEQ ID NOs. 84 and 96, and further comprising a chimeric receptor 2 that comprises an amino acid sequence having at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NOs. 4, 18, 23, 25, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 78, or 79. For example, the manipulated cells include a chimeric receptor 1 that binds to GPRC5D, comprising an amino acid sequence having at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in SEQ ID NO: 97, and further comprising a chimeric receptor 2 that comprises an amino acid sequence having at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in SEQ ID NO: 24, 26, 27, 28, 83, 85, 86, 96, or 98.For example, the manipulated cell contains a chimeric receptor 1 that binds to GPRC5D, comprising an amino acid sequence having at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence shown in SEQ ID NO: 97, and further comprises a chimeric receptor 2 that comprises an amino acid sequence having at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NOs: 4, 18, 23, 25, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 78, or 79. In one example, the chimeric receptors 1 and 2 are constructed on a single vector. For example, the polypeptide fragment contains, in order from the N-terminus to the C-terminus, chimeric receptor 2, 2A peptide, and chimeric receptor 1. For example, the 2A peptide is P2A, T2A, E2A, or F2A. For example, chimeric receptor 1 and chimeric receptor 2 are linked by a cleavable peptide. For example, the polypeptide fragment contains, in order from the N-terminus to the C-terminus, chimeric receptor 1, 2A peptide, and chimeric receptor 2. For example, chimeric receptor 1 and chimeric receptor 2 are constructed on different vectors.

[0367] The advantages of this invention are as follows: Through the design and screening of various structures, the present invention was obtained by screening for a structure that can effectively block CD38 antigen expression in engineered cells. This reduces cannibalism in in vivo and in vitro culture of engineered cells containing CD38-targeting chimeric receptors, extends the in vivo and in vitro survival period of said engineered cells, and improves their proliferative capacity. This can significantly enhance the role of CD38-CAR-T cells in antitumor and anti-immune transplantation.

[0368] This invention was obtained by screening various structures through design and screening to identify a structure that can effectively block NKG2DL expression in manipulated cells. This reduces cannibalism in in vitro and in vivo culture of manipulated cells containing chimeric receptors that target NKG2DL, extends the survival period of the manipulated cells, and improves their proliferative capacity. This can significantly enhance the role of NKG2D-CAR-T cells that recognize NKG2DL in antitumor and anti-immune transplantation.

[0369] This invention discloses CARs that simultaneously target GPRC5D and BCMA, and CARs that simultaneously target GPRC5D and NKG2A. Compared to CARs that target a single antigen, these CARs exhibit improved affinity and increased T cell activity, and these targets show additive or synergistic effects. CAR-T cells that simultaneously target BCMA and GPRC5D on the surface of tumor cells can reduce the possibility of antigen escape caused by the downregulation or absence of a single surface antigen. CAR-T cells that simultaneously target the NK cell marker NKG2A and GPRC5D can enhance the immune rejection capacity of GPRC5D-CAR-T cells.

[0370] The following specific examples further illustrate this application. It should be understood that these examples are for illustrative purposes only and do not limit the scope of this application. The experimental methods in the following examples are carried out according to the conventional conditions described in J. Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or the manufacturer's recommended conditions, unless otherwise specified. All publications, patents, and patent applications described herein are incorporated herein by reference to the extent that each individual publication, patent, or patent application is incorporated herein by reference individually. [Examples]

[0371] Example 1. Biomaterials MM.1S cells (Chinese Academy of Sciences Cell Bank, SCSP-5017), THP-1 cells (Chinese Academy of Sciences Cell Bank, SCSP-567), and MV-4-11 cells (Chinese Academy of Sciences Cell Bank, SCSP-5031) were cultured and grown in vitro. Using conventional cell biology techniques, lentiviruses overexpressing Luciferase-GFP were transfected to prepare MM.1S-luci-gfp, THP-1-luci-gfp, and MV-4-11-luci-gfp cells. U251 cells were obtained from TCHU58 of the Chinese Academy of Sciences Cell Bank. BxPC3 cells were obtained from CRL-1687 of the ATCC Cell Bank. MM.1S cells were infected with lentiviruses overexpressing Luciferase-GFP and mCherry to produce MM.1S-GFP / mCherry cells. Using CRISPR editing technology, we constructed endogenous GPRC5D knockout cells (MM.1S-GFP-GPRC5D KO cells, also known as MM.1S-GPRC5D KO cells) and endogenous BCMA knockout cells (MM.1S-mCherry-BCMA KO cells, also known as MM.1S-BCMA KO cells).

[0372] Flow cytometry analysis of multiple myeloma cell lines MM.1S, NCI-H929 (ATCC, CRL-9068), RPMI-8226 (Cell Bank of the Chinese Academy of Sciences (Shanghai), TCHU234), and the monoclonal cell line MM1S-GFP, which was stably transfected with GFP, revealed that BCMA expression in MM.1S, NCI-H929, RPMI-8226, and MM.1S-GFP cells was approximately 80%, 78%, 90%, and 83%, respectively, while GPRC5D expression was approximately 78%, 54%, 38%, and 84%, respectively.

[0373] rNK (resting NK cells): Peripheral blood was collected from healthy human donors and isolated to obtain PBMCs. rNK cells were obtained from PBMCs using a negative NK cell selection method (Miltenyi, 130-092-657). rNK cells were activated and proliferated in vitro using cytokines (500 IU / mL IL-2, 150 IU / mL IL-15) to obtain aNK cells (activated NK cells). NPG mice were obtained from Beijing Vitalstar Biotechnology Co., Ltd., and their genotype was NOD.Cg-Prkdcscid Il2rgtm1Vst / Vst.

[0374] Example 2. Vector preparation The vectors expressing the chimeric receptors disclosed in this application were constructed using conventional molecular biology techniques. Full-length DNA was synthesized to construct CD38-targeting CARs, which were then inserted between the MluI and SalI restriction endonuclease sites of the pRRLSIN lentiviral vector (Addgene) for lentiviral packaging. CD38-CAR1, 2, 3, and 4 contain CD38-BBZ, as shown in Table 1, constructed from CD38 antibody 1, CD38 antibody 2, CD38 antibody 3, and CD38 antibody 4, respectively. CD38-CAR9 contains CD38-28Z, as shown in Table 1, constructed from CD38 antibody 1. CD38-CAR6, CD38-CAR10, CD38-CAR2, and CD38-CAR3 contain the sequences shown in SEQ ID NOs. 23, 25, 18, and 29, respectively, and were incorporated into the pRRLSIN vector (Addgene). Lentiviruses corresponding to these expression vectors were prepared using conventional molecular biology techniques.

[0375] Example 3. Preparation of CAR-T cells CAR-T cells were prepared using conventional methods in the art. Donor PBMCs were collected and activated using magnetic beads containing anti-CD3 and CD28 antibodies (Life Technologies, 40203D). Next, T cells were infected with lentiviruses carrying the aforementioned vectors CD38-CAR1, 2, 3, 4, 6, 9, 10, 2s, and 3s to prepare CD38-CAR-T1, CD38-CAR-T2, CD38-CAR-T3, CD38-CAR-T4, CD38-CAR-T6, CD38-CAR-T9, CD38-CAR-T10, CD38-CAR-T2s, and CD38-CAR-T3s cells expressing the corresponding chimeric receptors. Untransfected T cells were used as the UTD. The CAR positivity rate was detected using FACS.

[0376] Example 4. Detection of CD38 expression in T cells, B cells, and NK cells in PBMCs derived from different human donors. PBMC cells isolated from the peripheral blood of different healthy human donors were collected and stained in two separate groups. The control group was labeled with CD3-percp, CD19 APC-cy7, and CD56 APC, while the experimental group (anti-CD38) was labeled and stained with CD3-percp, CD19 APC-cy7, CD56 APC, and CD38 BV421. After incubation at 4°C for 30 minutes, the cells were classified into CD3+, CD19+, and CD56+ populations using flow cytometry, and CD38 expression was detected. The results showed that CD38 was expressed in T cells, B cells, and NK cells.

[0377] Example 5. CD38-CAR-T positivity rate and detection of CD38 expression in CAR-T cells CD38-CAR-T cells were cultured in vitro for 7 days, washed twice with PBS + 1% FBS, and then divided into two equal parts. One part was analyzed for positivity using biotin-anti-huF(ab)2 and streptavidin-PE, while the other part was co-incubated with anti-CD38 BV421 to detect CD38 expression. Negative controls (NC) were T cells without antibody labeling.

[0378] Figure 1A (top) shows that the CAR positivity rate in CD38-CAR-T1, 2, 3, 4, 6, and 10 cells was generally above 70%. Figure 1A (bottom) shows that the CD38 positivity rate of CD38-CAR-T cells was significantly lower than that of UTD cells, with the most significant decrease observed in CD38-CAR-T6 and 10 cells. Figure 1B (bottom) shows that CD38-sBBZ constructed with CD38 antibody 2 or 3, as well as CD38-CAR-T6 constructed with CD38 antibody 1, significantly blocked the CD38 antigen on autologous T cells on day 8 or 13. Figure 1D (right) shows that CD38-sBBZ constructed with CD38 antibodies 1 and 3 almost completely blocked the CD38 antigen from day 5, and CD38-sBBZ constructed with CD38 antibody 2 also almost completely blocked the CD38 antigen from day 13.

[0379] The above experiment was repeated using PBMCs from another healthy human donor. Figures 1C (bottom) and 1E (right) show that CD38-sBBZ constructed with CD38 antibodies 1 and 3 almost completely blocked the CD38 antigen from day 5, and CD38-sBBZ constructed with CD38 antibody 2 also almost completely blocked the CD38 antigen from day 13.

[0380] Example 6. Detection of in vitro proliferation of CD38-CAR-T cells Human peripheral blood PBMCs were activated with CD3 / CD28 magnetic beads, and 48 hours later (recorded as day 2), the same number of T cells were infected with a virus containing CD38-CAR to prepare CD38-CAR-T cells. These cells were cultured in vitro and the cell count was measured. Cells were harvested and counted every 2-3 days, and after counting, 2e5 cells were centrifuged, resuspended in 2e5 / mL, and seeded on culture plates. Culture and counting were continued. Figure 2 (left) shows that the proliferation rate of CD38-CAR-T6 cells significantly increased from day 8, maintaining high proliferative capacity. Figure 2 (right) shows that when cultured by day 10, the proliferative capacity of CD38-CAR-T6 and T10 cells was significantly higher than that of CD38-CAR-T9 cells. Figure 2 (bottom) shows that the proliferation rate of CD38-sBBZ cells constructed using CD38 antibodies 1, 2, or 3 began to significantly increase from day 13.

[0381] Example 7. Flow cytometry detection of CD38-CAR-T cell killing and cytokine secretion against tumor cells or NK cells. CAR-T cells and target cells (THP-1, MV-4-11, MM.1S, and aNK cells) were co-incubated for 20 hours at effector cell-to-target cell ratios of 1:1 or 1:3. T cells were labeled with anti-CD3-PE, dead cells were labeled with 7-AAD staining, and finally count beads (Invitrogen) were added. Data were read using FACS, and the number of target cells and T cells was statistically analyzed. Cytotoxic lysis was calculated using the following formula: cytotoxicity % = (1 - experimental group / control group) × 100%. Figure 3A shows that CD38-CAR-T1, 2, 3, 4, 6, 9, and 10 cells all significantly killed THP-1, MV-4-11, MM.1S, and aNK cells.

[0382] CD38-CAR-T6, 9, and 10 cells were co-incubated with MM.1S, THP-1, and MV-4-11, respectively, for 18 hours, and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected using the supernatant by the CBA method (BD, 558264). As shown in Figure 3B, compared to UTD, all of the above CAR-T cells secreted high levels of cytokines after co-incubation with tumor cells.

[0383] Example 8: Treatment of orthotopic tumors in NPG mice with CD38-CAR-T 1x10 NPG mouse 7 THP-1-Luci cells were injected into the tail vein (recorded as day D0), and the mice were divided into three groups of 5 mice each. The photon intensity of the tumors was observed at different time points using fluorescence imaging (IVIS LUMINA III SYSTEM). Day 14 (average photon intensity was approximately 1.5 × 10⁶) 4 p / s / cm 2 At the point when / sr is reached, 5 × 10 per mouse 6 Individual CAR-T cells were injected via the tail vein. As shown in Figure 4A, one mouse in the UTD group died on day 42, while significant tumor suppression was observed in the CD38-CAR-T6 group, with all mice surviving and in good condition.

[0384] 1x10 NPG mouse 7 THP-1-Luci cells were injected into the tail vein (recorded as day 0), and the mice were divided into three groups of 5. The photon intensity of the tumors was observed at different time points using fluorescence imaging (IVIS LUMINA III SYSTEM). On day 15, the photon intensity was approximately 1.5 × 10⁶. 4 p / s / cm 2 At the point when / sr is reached, 4 × 10 per mouse 6 Individual CAR-T cells were injected via the tail vein. As shown in Figure 4B, on day 36, both the CD38-CAR-T6 group and the CAR-T10 group showed a significant tumor-suppressing effect compared to the control group.

[0385] Example 9. Construction of CD38-UCAR-T cells sgRNA sequences targeting TRAC and B2M (shown in SEQ ID NOs. 39 and 40, respectively) were synthesized in vitro (CARSGEN diagnosis), and UCAR-T (or dko) cells with TRAC and B2M knocked out were prepared using CRISPR / Cas9 technology (Cas9 protein, Kactus Biosystems (Shanghai), catalog number CAS-EE109). As a control, UTD cells (UUTD) with TCR / B2M knocked out without gene transduction were used. CAR expression positivity was measured using biotin-anti-huF(ab)2 and streptavidin-PE. The results showed that T cells in each group successfully expressed CAR molecules.

[0386] Example 10. Detection of NK cell killing and cytokine secretion by CD38-UCAR-T cells CD38-UCAR-T cells and NK cells were seeded in a 1:1 or 1:2 ratio. 20,000 CD38-UCAR-T cells were seeded per well. After 24 hours of co-incubation, staining was performed using anti-HLA-ABC-APC (Thermo, 17-9983-42). HLA-ABC-negative cells were CD38-UCAR-T cells, and HLA-ABC-positive cells were NK cells. Dead cells were labeled with 7-AAD (BD, 559925). Finally, counting beads (Invitrogen, C36995) were added, and FACS analysis was performed. Cytotoxic lysis was calculated using the following formula: relative cytolysis (%) = (1 - experimental group / control group) × 100%. As shown in Figure 5A, after 24 hours of co-incubation with NK cells, CD38-UCAR-T6 and T10 cells significantly killed both quiescent and activated NK cells.

[0387] CD38 UCAR-T cells and NK cells were co-incubated in a 1:1 ratio for 24 hours. The culture supernatant was used to detect the secretion of IL-2, TNF-α, and IFN-γ cytokines using the CBA method (BD, 558264). As shown in Figure 5B, CD38 UCAR-T cells and NK cells secreted higher levels of cytokines after co-incubation compared to UUTD cells.

[0388] Example 11. Treatment of syngeneic tumors in NPG mice with CD38-UCAR-T 1×10 7 THP-1-Luci cells per NPG mouse were injected via the tail vein, and the mice were divided into 3 groups with 5 mice in each group. The photon intensity of the tumor was observed using fluorescence imaging (IVIS LUMINA III SYSTEM) at different time points. In the NK cell injection group, on day 13 (when the photon intensity reached an average of about 1.5×10 4 p / s / cm 2 / sr), 2×10 6 NK cells per mouse were injected via the tail vein continuously for 3 days. On day 14, 5×10 6 UCAR-T cells were injected via the tail vein of the mice. Figure 6 shows that the CD38-UCAR-T6 group still showed a significant tumor suppression effect even in the presence of NK cells. As a result of observation until day 48, all the mice survived and were in good condition. All the mice in the control group died on day 48.

[0389] Example 12. Preparation of CD38-HLA-E UCAR-T cells According to Example 9, CD38-HLA-E UCAR-T1, 2, 3, and 4 cells with endogenous TRAC and B2M knocked out were constructed. From the 5'-end to the 3'-end, the CD38-HLA-E1 fragment sequentially contained the sequences shown in SEQ ID NO: 23, 75, and 30, the CD38-HLA-E2 fragment sequentially contained the sequences shown in SEQ ID NO: 23, 75, and 31, the CD38-HLA-E3 fragment sequentially contained the sequences shown in SEQ ID NO: 23, 75, and 32, and the CD38-HLA-E4 fragment sequentially contained the sequences shown in SEQ ID NO: 23, 75, and 33.

[0390] Example 13. Detection of NK cell killing and cytokine secretion by CD38-HLA-E UCAR-T cells See Example 7. UCAR-T cells were co-incubated with NK cells for 24 or 72 hours. Compared to UTD, CD38-HLA-E-UCAR-T1, 2, 3, and 4 cells all significantly killed aNK and rNK cells (Figure 7A). After co-incubating UCAR-T cells with NK cells for 24 hours, the supernatant was collected and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected using the CBA method (BD, 558264). As shown in Figure 7B, compared to UUTD, all of the above UCAR-T cells co-incubated with NK cells secreted high levels of cytokines.

[0391] Example 14. Preparation of NKG2A / CD38-CAR-T cells and CD94 / CD38-CAR-T cells Following Examples 2 and 3, NKG2A / CD38-CAR-T cells were constructed: CD38 / N-DCAR1, CD38 / N-DCAR2, CD38 / N-DCAR3, CD38 / N-s28z, N / CD38-s28Z, CD38 / N-DCAR4, and CD38 / N-DCAR5. CD94-CD38-CAR-T cells were constructed. CD300A / CD38-CAR-T cells were constructed. GPRC5D / N / CD38-CAR-T cells targeting the tumor antigen GPRC5D were constructed. BCMA / N / CD38-CAR-T cells targeting the tumor antigen BMCA were constructed.

[0392] Table 2 shows the structures of CD38 / N-s28z and N / CD38-s28Z. CD38 / N-DCAR2 contains the sequences shown in SEQ ID NOs. 26, 87, and 83 in order from the N-terminus to the C-terminus. CD38 / N-DCAR4 contains the sequences shown in SEQ ID NOs. 27, 87, and 83 in order from the N-terminus to the C-terminus. CD38 / N-DCAR5 contains the sequences shown in SEQ ID NOs. 28, 87, and 83 in order from the N-terminus to the C-terminus, respectively. CD38 / N-DCAR1, CD38 / N-DCAR3, GPRC5D / N / CD38-CAR, and BCMA / N / CD38-CAR contain the sequences shown in SEQ ID NOs. 34, 4, 37, and 38 in order.

[0393] Referring to Example 9, tko cells with endogenous TRAC, B2M, and NKG2A knocked out, and qko cells with endogenous TRAC, B2M, NKG2A, and CD38 knocked out were prepared. The sgRNA sequences targeting TRAC, B2M, NKG2A, and CD38 are shown in SEQ ID NOs. 39, 40, 41, and 42, respectively. CAR expression positivity was detected using biotin-anti-huF(ab)2 and streptavidin-PE. The results indicate that T cells in each group successfully expressed CAR molecules.

[0394] Example 15. In vitro tumor cell killing and cytokine secretion detection using NKG2A / CD38-UCAR-T UCAR-T cells and MV-4-11 cells were co-incubated in a 1:3 ratio for 16 hours. Staining was performed using anti-HLA-ABC APC (Thermo, 17-9983-42). UCAR-T cells were HLA-ABC negative, while MV-4-11 cells were HLA-ABC positive. Dead cells were labeled with 7-AAD (BD, 559925). Finally, counting beads (Invitrogen, C36995) were added, and FACS analysis was performed. Figure 8A shows that CD38 / N-DCAR1-tko, CD38 / N-DCAR1-qko, CD38 / N-DCAR2-tko, CD38 / N-DCAR3-tko, CD38 / N-s28z-tko, and N / CD38-s28Z-tko significantly killed MV-4-11 cells.

[0395] The above UCAR-T cells were co-incubated with MV-4-11 cells for 16 hours, and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected in the supernatant using the CBA method (BD, 558264). Figure 8B shows that, compared to UUTD, all of the above UCAR-T cells secreted high levels of cytokines after co-incubation with tumor cells.

[0396] Example 16. Detection of aNK cell toxicity and cytokine secretion by NKG2A / CD38-UCAR-T cells See Example 7. UCAR-T cells were co-incubated with aNK cells for 24 or 72 hours. Compared to the control group, CD38 / N-DCAR1-tko, CD38 / N-DCAR1-qko, CD38 / N-DCAR2-tko, CD38 / N-DCAR3-tko, CD38 / N-s28Z-tko, and N / CD38-s28Z-tko significantly killed NK cells (Figure 9A). Secretion of IL-2, TNF-α, and IFN-γ cytokines was detected after 24 hours of co-incubation with NK cells. As shown in Figure 9B, compared to UUTD, all of the above UCAR-T cells secreted high levels of cytokines after co-incubation with NK cells.

[0397] Example 17. Treatment of orthotopic tumors in NPG mice with NKG2A / CD38-UCAR-T 1 x 10 per NPG mouse 7 THP-1-Luci cells were injected into the tail vein, and the mice were divided into four groups of six. The photon intensity of the tumors was observed at different time points using fluorescence imaging (IVIS LUMINA III SYSTEM). On day 10, the photon intensity was 2.5 × 10⁶ per mouse. 6 Individual UCAR-T cells were injected via the tail vein. As shown in Figure 10, after observation up to day 37, all mice in the control group died, while the CD38-UCAR-T10 group, CD38 / N-DCAR1-tko group, and CD38 / N-DCAR3-tko group all showed significant tumor suppression effects.

[0398] Example 18. Treatment of orthotopic tumors in NPG mice with NKG2A / CD38-UCAR-T cells in the presence of NK cells 1 x 10 per NPG mouse 7 Each mouse was injected with THP-1-Luci cells via the tail vein and divided into three groups of six mice. The photon intensity of the tumors was observed at different time points using fluorescence imaging (IVIS LUMINA III SYSTEM). In the NK cell injection group, on day 9, the average photon intensity was approximately 5.3 × 10⁶. 3 p / s / cm 2At the point when / sr is reached, 1.5 × 10 per mouse 6 NK cells were injected into the tail vein for three consecutive days. On day 10, 2.5 × 10⁶ NK cells were injected into the tail vein of the mouse. 6 Each UCAR-T cell was injected. Figure 11 shows that even in the presence of NK cells, the CD38 / N-DCAR1-tko group and the CD38 / N-DCAR3-tko group still showed significant tumor suppression effects.

[0399] Example 19. Killing of tumor cells or NK cells and detection of cytokine secretion by CAR-T cells CAR-T cells were co-incubated with MM.1S cells in a 1:5 ratio for 24 hours. Staining was performed using anti-CD3-APC (Biolegend, 300412). CD3+ cells were identified as T cells, and CD3- cells as MM.1S cells. Dead cells were labeled with 7-AAD (BD, 559925). Finally, counting beads (Invitrogen, C36995) were added, and FACS analysis was performed. Figure 12A (left) shows that GPRC5D / N / CD38-CAR-T cells significantly killed tumor cells.

[0400] See Example 7. Figures 12A (right) and 12B show that after co-incubating with NK cells for 24 hours or 72 hours, GPRC5D / N / CD38-CAR-T, BCMA / N / CD38-CAR-T-tko, and CD94 / CD38-CAR-T-dko cells all significantly killed NK cells, and after co-incubating with NK cells for 24 hours, BCMA / N / CD38-CAR-T-tko cells and CD94 / CD38-CAR-T-dko cells significantly secreted IL-2, TNF-α, and IFN-γ.

[0401] Example 20. Combination use of CD38-CAR-T cells and CAR-T cells targeting tumor antigens Claudin 18.2-CAR-T cells, GPC3-CAR-T cells, and mesothelin-CAR-T cells were constructed according to Examples 2 and 3. Claudin 18.2 / N-CAR-T cells that bind to Claudin 18.2 and NKG2A were also constructed.

[0402] CD38-CAR-T cells and Claudin 18.2 / N-CAR-T cells were mixed in ratios of 1:1, 1:2, and 2:1 and administered to subjects for the treatment of Claudin 18.2-positive tumors.

[0403] Example 21. Preparation and proliferation of CAR-T cells targeting NKG2DL. The vectors expressing the chimeric receptors disclosed in this application were constructed using conventional molecular biology techniques. Full-length DNA was synthesized, chimeric receptors targeting NKG2DL were constructed, and each was incorporated into a pRRLSIN lentiviral vector (Addgene).

[0404] The NKG2Dz fragment and the NKG2D-BBZ fragment contain the sequences shown in sequence numbers 78 and 79, respectively.

[0405] The A-N1 fragment contains the full-length ASGPR1 (SEQ ID NO: 80) and the NKG2D extracellular region (SEQ ID NO: 76) in that order. The A-N2 fragment contains the full-length ASGPR1 (SEQ ID NO: 80), the G4S linker (SEQ ID NO: 16), and the NKG2D extracellular region (SEQ ID NO: 76) in that order. The CD137L-N fragment contains the full-length CD137L (SEQ ID NO: 81) and the NKG2D extracellular region (SEQ ID NO: 76) in that order.

[0406] From 5' to 3', the A-N1 / NKG2Dz fragment sequentially contained NKG2Dz (SEQ ID NO: 78), P2A (SEQ ID NO: 17), full-length ASGPR1 (SEQ ID NO: 80), and the NKG2D extracellular region (SEQ ID NO: 76); the A-N2 / NKG2Dz fragment sequentially contained NKG2Dz (SEQ ID NO: 78), P2A (SEQ ID NO: 17), full-length ASGPR1 (SEQ ID NO: 80), G4S linker (SEQ ID NO: 16), and the NKG2D extracellular region (SEQ ID NO: 76); and the CD137L-N / NKG2Dz fragment sequentially contained NKG2Dz (SEQ ID NO: 78), P2A (SEQ ID NO: 17), full-length CD137L (SEQ ID NO: 81), and the NKG2D extracellular region (SEQ ID NO: 76).

[0407] From 5' to 3', the A-N1 / NKG2D-BBZ fragment sequentially contained NKG2D-BBZ (SEQ ID NO: 79), P2A (SEQ ID NO: 17), full-length ASGPR1 (SEQ ID NO: 80), and the NKG2D extracellular region (SEQ ID NO: 76); the A-N2 / NKG2D-BBZ fragment sequentially contained NKG2D-BBZ (SEQ ID NO: 79), P2A (SEQ ID NO: 17), full-length ASGPR1 (SEQ ID NO: 80), G4S linker (SEQ ID NO: 16), and the NKG2D extracellular region (SEQ ID NO: 76); and the CD137L-N / NKG2D-BBZ fragment sequentially contained NKG2D-BBZ (SEQ ID NO: 79), P2A (SEQ ID NO: 17), full-length CD137L (SEQ ID NO: 81), and the NKG2D extracellular region (SEQ ID NO: 76).

[0408] The above sequence was inserted between the MluI and SalI restriction endonuclease sites of the pRRLSIN lentiviral vector (Addgene) for lentiviral packaging.

[0409] CAR-T cells were prepared using conventional methods in this art. Donor PBMCs were collected, and anti-CD3 and CD28 antibodies were activated with magnetic beads (Life Technologies, 40203D). Then, lentiviruses containing the above vector were used to infect T cells, and A-N1 / NKG2Dz, A-N2 / NKG2Dz, CD137L-N / NKG2Dz, CD40L-N / NKG2Dz, A-N1 / NKG2D-BBZ, A-N2 / NKG2D-BBZ, and CD137L-N / NKG2D-BBZ cells expressing the corresponding chimeric receptors were prepared. T cells that had not been transduced by the virus were used as UTDs.

[0410] Expression vectors were introduced into T cells via lentivirus, and the cells were cultured in vitro for two weeks to promote growth. Cell counting and cell viability analysis (PI staining) were performed every 2-3 days. Measurement of NKG2D expression on the surface of CD4 T cells (endogenous NKG2D negative) showed a positive rate of at least approximately 93%. As shown in Figure 13A, the proliferation rate and viability of A-N1 / NKG2Dz cells and A-N2 / NKG2Dz cells were significantly increased compared to the control group.

[0411] Example 22. Detection of NKG2D-CAR-T cell proliferation under target cell stimulation. CAR-T cells and THP1 cells were co-incubated in an effector cell:target cell ratio of 1:1 (THP1 cells were pre-treated with mitomycin C to halt proliferation). After 48 hours, cell counting was performed, and THP1 cells were added again in an effector cell:target cell ratio of 1:1 for stimulation, for a total of three rounds. As shown in Figure 13B, co-expression of A-N1 or A-N2 peptide increased the proliferation multiplier of NKG2D-CAR-T cells under target cell stimulation compared to the control group.

[0412] Example 23. Detection of in vitro euthanasia of tumor cells by NKG2D-CAR-T cells Using the xCELLigence RTCA real-time monitoring method, CAR-T cells and target cells (U251, BxPC3) were co-incubated in an effector-to-target cell ratio of 1:3 (target cells were seeded on electrode plates 24 hours prior). As shown in Figure 14, A-N1 / NKG2Dz cells and A-N2 / NKG2Dz cells significantly killed tumor cells U251 and BxPC3.

[0413] Example 24. Treatment of orthotopic tumors in NPG mice with NKG2D-CAR-T 1 × 10 per NPG mouse 7 THP1-luci cells were inoculated via the tail vein (recorded as day 0). On day 15, 5 × 10⁶ cells were inoculated. 6 Individual CAR-T cells were injected via the tail vein. Luciferase substrate was administered weekly, and tumor load was observed by imaging. Figure 15 (left) shows that CD137L-N / NKG2Dz cells and A-N2 / NKG2Dz cells showed a significant in vivo antitumor effect compared to UTD cells.

[0414] Peripheral blood was collected from mice 7 days after CAR-T injection, and the number of CD3 / CD4 / CD8 T cells in the peripheral blood was quantitatively analyzed by flow cytometry (BD Trucount Tubes). Figure 15 (right) shows that the number of viable CD137L-N / NKG2Dz cells and A-N2 / NKG2Dz cells in vivo increased compared to UTD.

[0415] Example 25. Preparation of regulatory NKG2D-CAR-T cells To further enhance the protective effect of the AN fragment, we prepared T cells constitutively expressing the AN fragment and T cells regulatoryly expressing NKG2D-CAR-CAR. Following PCT / CN2022 / 102395, we prepared a chimeric polypeptide that recognizes the CD123-synE fragment of CD123.

[0416] The vectors expressing the chimeric receptors disclosed in this application were constructed using conventional molecular biology techniques. Full-length DNA was synthesized, chimeric receptors targeting NKG2DL were constructed, and each was incorporated into a pRRLSIN lentiviral vector (Addgene).

[0417] Full-length DNA was synthesized, and from 5' to 3', the A-N2 / CD123-synE fragment contained, in order, the CD123-synE fragment (SEQ ID NO: 82), P2A (SEQ ID NO: 17), full-length ASGPR1 (SEQ ID NO: 80), G4S linker (SEQ ID NO: 16), and NKG2D extracellular region (SEQ ID NO: 76).

[0418] In short, the UAS-CMV promoter (SEQ ID NO: 88), NKG2Dz (SEQ ID NO: 78) or NKG2D-BBZ (SEQ ID NO: 79), EF1a promoter (SEQ ID NO: 89), and CD123-synE fragment (SEQ ID NO: 82) were sequentially ligated from 5' to 3'. Finally, the resulting fragment was inserted between the ClaI and SalI restriction endonuclease sites of the pRRLSIN lentiviral vector (Addgene) to prepare the lentiviral vectors CD123-synE-NKG2Dz and CD123-synE-NKG2D-BBZ.

[0419] The UAS-CMV promoter (SEQ ID NO: 88), NKG2Dz (SEQ ID NO: 78) or NKG2D-BBZ (SEQ ID NO: 79), EF1a promoter (SEQ ID NO: 89), and A-N2 / CD123-synE fragment were sequentially ligated from the 5' end to the 3' end. Finally, the resulting fragment was inserted between the ClaI and SalI restriction endonuclease sites of the pRRLSIN lentiviral vector (Addgene) to prepare the lentiviral vectors A-N2 / CD123-synE-NKG2Dz and A-N2 / CD123-synE-NKG2D-BBZ.

[0420] CAR-T cells were prepared using conventional methods in this art. Donor PBMCs were collected, and anti-CD3 and CD28 antibodies were activated with magnetic beads (Life Technologies, 40203D). Then, lentiviruses containing the above vector were used to infect T cells, and T cells expressing the corresponding chimeric receptors (CD123-synE-NKG2Dz cells, CD123-synE-NKG2D-BBZ cells, A-N2 / CD123-synE-NKG2Dz cells, and A-N2 / CD123-synE-NKG2D-BBZ cells) were prepared. T cells that had not been transduced by the virus were designated as UTDs.

[0421] The delivery efficiency was measured by detecting the expression of CD123 single-chain antibodies on the surface of T cells. The results showed that all expression vectors were effectively delivered to T cells.

[0422] Example 26. Cell proliferation of regulatory NKG2D-CAR-T cells under target cell stimulation. CAR-T cells and THP1 cells were co-incubated in an effector cell:target cell ratio of 1:1 (THP1 cells were pre-treated with mitomycin C to stop proliferation). After 48 hours, cell counting was performed, and THP1 cells were added again in an effector cell:target cell ratio of 1:1 for stimulation, for a total of two rounds. As shown in Figure 16, the cell proliferation rate of A-N2 / CD123-synE-NKG2Dz cells and A-N2 / CD123-synE-NKG2D-BBZ cells under target cell stimulation was significantly increased compared to CD123-synE-NKG2Dz cells and CD123-synE-NKG2D-BBZ cells, respectively.

[0423] Example 27. Killing function and cytokine secretion of target cells by regulatory NKG2D-CAR-T cells. The xCELLigence RTCA real-time monitoring method was used. Target cells were seeded on electrode plates 24 hours prior to the study. CAR-T cells and target cells U251 and PLC / PFR / 5 were co-incubated in a 1:1 ratio of effector cells to target cells. Figure 17A shows that A-N2 / CD123-synE-NKG2Dz cells and A-N2 / CD123-synE-NKG2D-BBZ cells significantly killed tumor cells. Figure 17B shows that significant cytokine secretion was detected in the supernatant after co-incubating A-N2 / CD123-synE-NKG2Dz cells and A-N2 / CD123-synE-NKG2D-BBZ cells with THP-1 for 24 hours in a 1:1 ratio of effector cells to target cells.

[0424] Example 28. Treatment of orthotopic tumors in NPG mice with regulatory NKG2D-CAR-T cells NPG mouse, 1x10 7 THP1-luci cells were inoculated via the tail vein (recorded as day 0). On day 15, 5 × 10⁶ cells were administered to each mouse. 6 Nine CAR-T cells were injected via the tail vein. Luciferase substrates were administered weekly, and tumor load was observed by imaging. As shown in Figure 18A, the A-N2 / CD123-synE-NKG2D-BBZ group, which co-expresses the A-N2 peptide, showed superior results in in vivo antitumor effects, with a median survival time of 77 days for mice, significantly exceeding the 61 days for the CD123-synE-NKG2D-BBZ group. Peripheral blood was collected from mice on days 8 and 14 after CAR-T cell injection, and the number of CD3 / CD4 / CD8 T cells in the peripheral blood was quantitatively analyzed using flow cytometry (BD Trucount Tubes). As shown in Figure 18B, the number of surviving CAR-T cells in vivo increased in the A-N2 / CD123-synE-NKG2D-BBZ group.

[0425] Example 29. Preparation of BCMA / GPRC5D-CAR-T cells Following the procedures in Examples 2 and 3, full-length DNA was synthesized, and CARs targeting BCMA and GPRC5D were constructed. These were then inserted between the MluI and SalI restriction endonuclease sites of the pRRLSIN lentiviral vector (Addgene) for lentiviral packaging. The BG-BBZ, BGp-BBZ, GB-BBZ, BLG-BBZ, BLGp-BBZ, and GLBp-BBZ fragments contained the sequences shown in sequence numbers 90, 91, 92, 94, 93, and 95, respectively. The CAR positivity rate in each BCMA / GPRC5D-CAR-T cell population was detected using FACS, and each T cell population successfully expressed the CAR molecule.

[0426] Example 30. In vitro tumor cell killing and cytokine secretion detection using BCMA / GPRC5D-CAR-T cells Approximately 3×10 4 Tumor cells were seeded in 96-well plates. CAR-T cells were added in a 1:1 ratio of effector cells to target cells. After 24 hours of culture, the number of GPF and mCherry-positive cells was detected by flow cytometry, and the lysis values ​​of CAR-T cells against tumor cells MM.1S-GFP, MM.1S-BCMA KO, and MM.1S-GPRC5D KO were calculated.

[0427] MM.1S-BCMA KO cells and MM.1S-GPRC5D KO cells were mixed in a 1:1 ratio. The mixed target cells were 1 × 10⁶ 4Cells were seeded into 96-well plates. CAR-T cells were added in a 1:1 ratio of effector cells to target cells. After 24 hours of incubation, the number of GPF and mCherry-positive cells was detected using flow cytometry, and tumor cell lysis values ​​were calculated. Figures 19A and 19B show that BG-BBZ, BGp-BBZ, GB-BBZ, BLG-BBZ, BLGp-BBZ, and GLBp-BBZ cells effectively inhibited the proliferation of mixed tumor cells, including BCMA-positive, GPRC5D-positive, BCMA and GPRC5D-double-positive, or BCMA-positive and GPRC5D-positive cells. CBA analysis showed that the above CAR-T cells secreted high levels of cytokines after 24 hours of co-incubation with tumor cells (Figure 19C).

[0428] Example 31. Detection of in vivo antitumor effects of BCMA / GPRC5D-CAR-T cells 3 x 10 6 Individual MM.1S-luciferase / GFP cells were subcutaneously inoculated into NPG mice (recorded as day 0). The tumor load on day 11 was approximately 300 mm². 3 The mice were divided into 7 groups, with 5 mice in each group. On the 12th day, each mouse was 0.8 × 10 6 Numerous CAR-T cells were injected via the tail vein, and tumor growth was monitored. As shown in Figure 20, compared to the UTD group, the BG-BBZ, GB-BBZ, BGp-BBZ, BLG-BBZ, BLGp-BBZ, and GPRC5D-CAR-T groups significantly suppressed tumor growth. On day 28, peripheral blood was collected from mice and analyzed by flow cytometry, revealing significant proliferation of CAR-T cells in the BCMA / GPRC5D-CAR-T group.

[0429] Example 32. Detection of the antitumor effect of BCMA / GPRC5D-CAR-T cells BLG-sBBZ and GLB-sBBZ were constructed by substituting IgG4(H) and CD28(TM) for the hinge and transmembrane domain of BLG-BBZ (SEQ ID NO: 94, 96) and GLBp-BBZ (SEQ ID NO: 95) fragments, respectively. BLG-sBBZ and GLB-sBBZ cells were constructed according to Examples 2 and 3.

[0430] CAR-T cells and tumor cells were seeded in ratios of 3:1, 1:1, and 1:3 and cultured for 24 hours. Flow cytometry analysis showed that BLG-sBBZ cells and GLB-sBBZ cells effectively inhibited BCMA-positive, GPRC5D-positive, and BCMA- and GPRC5D-double-positive tumor cells and secreted high levels of cytokines IFNγ, IL-2, and TNFα. In vivo antitumor experiments showed that BLG-sBBZ cells and GLB-sBBZ cells effectively inhibited the growth of MM.1S subcutaneous tumors in NPG mice.

[0431] Example 33. Rapid preparation of R-CAR-T cells PBMC cells isolated from human donor peripheral blood were revived for approximately 24 hours and activated with anti-CD3 / CD28 TransAct magnetic beads. 22 hours after activation, lentiviral transfection with BG-BBZ, GB-BBZ, BLG-BBZ, BGp-BBZ, or BLGp-BBZ was performed. 2 hours after infection, R-BG-BBZ, R-GB-BBZ, R-BLG-BBZ, R-BGp-BBZ, and R-BLGp-BBZ cells were collected. The collected R-CAR-T cells were grown in vitro for 4 days, and the positive rate was measured. R-UTD, R-BCMA-CART, and R-GPRC5D-CART cells were prepared using the same method.

[0432] Example 34. In vitro tumor cell killing and cytokine secretion detection by R-CAR-T cells MM.1S-GPF, MM.1S-BCMA KO, MM.1S-GPRC5D KO, and MM.1S-BCMA KO + MM.1S-GPRC5D KO (mixed in a 1:1 ratio) were used as target cells and seeded in 96-well plates at a ratio of 3 × 10^4 cells. R-CAR-T cells proliferated in vitro by day 5 were used as effector cells, and the effector cells were co-incubated for 24 hours at ratios of 1:1, 1:5, and 1:15, respectively. The number of tumor cells was detected and quantified by flow cytometry. Figure 21A shows that R-BGp-BBZ cells and R-BLGp-BBZ cells significantly killed BCMA-positive, GPRC5D-positive, and BCMA and GPRC5D-double-positive tumor cells. Figure 21B shows that R-BGp-BBZ cells and R-BLGp-BBZ cells significantly killed MM.1S-BCMA KO cells and MM.1S-GPRC5D KO cells in a mixed cell population including BCMA-positive and GPRC5D-positive cells.

[0433] R-CAR-T cells and tumor cells were seeded in a 1:1 ratio and cultured for 24 hours. The supernatant was collected, and the concentrations of INFγ, IL-2, and TNFα were measured using the CBA method. Figure 21C shows that R-BGp-BBZ cells and R-BLGp-BBZ cells significantly secreted cytokines after tumor cell stimulation.

[0434] Example 35. In vivo antitumor effect of R-CAR-T cells 2 × 10 per NPG mouse 6 Individual MM.1S-luciferase / GFP cells were intravenously inoculated (recorded as day 0). On day 10, the tumor burden was approximately 3-5 × 10⁶. 4 (Photon intensity)(p / s / cm 2 The result was / sr). The mice were divided into four groups, with 5 mice in each group. On day 11, each mouse had 0.1 × 10 6 Nine CAR-T cells were injected via the tail vein, and tumor growth was monitored. On day 44, the mean fluorescence values ​​of tumors in the UTD group, R-BGp-BBZ group, and R-BLGp-BBZ group were 3.47 × 10⁶, respectively. 6 , 3.36 × 10 4 , and 2.27 × 103 p / s / cm 2 The sr was / sr. The tumor suppression rates for the R-BGp-BBZ group and the R-BLGp-BBZ group compared to the UTD group were 99.90% and 99.93%, respectively. The number of T cells in the peripheral blood of mice was measured by flow cytometry on days 8, 14, and 21 after CAR-T cell injection. Figure 22 shows that the R-BGp-BBZ group and the R-BLGp-BBZ group showed a significant advantage in the in vivo proliferation of CAR-T cells.

[0435] The embodiments described in this application include any single embodiment, or combinations with other embodiments or parts thereof. Furthermore, after reading the foregoing teachings of this application, it should be understood that various changes or modifications can be made to this application, and that these equivalents also fall within the scope defined by the appended claims. TIFF2026525409000008.tif234170TIFF2026525409000009.tif232170TIFF2026525409000010.tif229170TIFF2026525409000011.tif235170TIFF202 6525409000012.tif234170TIFF2026525409000013.tif227170TIFF2026525 409000014.tif227170TIFF2026525409000015.tif246170TIFF20265254090 00016.tif242170TIFF2026525409000017.tif234170TIFF2026525409000018.tif228170TIFF2026525409000019.tif226170TIFF2026525409000020.t if227170TIFF2026525409000021.tif233170TIFF2026525409000022.tif228170TIFF2026525409000023.tif227170TIFF2026525409000024.tif175170

Claims

1. A chimeric receptor comprising an antigen-binding domain that binds to CD38, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain is linked to the transmembrane domain via an IgG4 hinge region or a fragment thereof.

2. The IgG4 hinge region or its fragment is a cut IgG4 hinge or a modified IgG4 hinge. Preferably, the IgG4 hinge region or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 5 or 6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 or 6, according to claim 1, the chimeric receptor.

3. The chimeric receptor according to claim 1 or 2, wherein the antigen-binding domain comprises a natural CD38 ligand extracellular segment or a fragment or variant thereof capable of binding to CD38, an anti-CD38 antibody or a fragment thereof, or a synthetic CD38-binding domain, and preferably the anti-CD38 antibody or fragment thereof is selected from a whole antibody, scFv, a single-domain antibody, a Fab fragment, a Fab' fragment, an Fv fragment, an F(ab')2 fragment, an Fd fragment, an sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody, or an IgG4 antibody.

4. The antigen-binding domain is one of the following (1) to (3): (1) an anti-CD38 antibody comprising scFv, wherein the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 19, 20, 21, or 22; (2) An anti-CD38 antibody comprising VH and VL, wherein VH and VL each comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NOs: 49, 50, 57, 58, 65, 66, 73, 74, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence; (3) An anti-CD38 antibody comprising VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3, VL comprises LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each have the amino acid sequence shown in SEQ ID NOs: 43, 44, 45, 46, 47, 48, or the amino acid sequence shown in SEQ ID NOs: 51, 52, 53, 54, 55, 56, or the amino acid sequence shown in SEQ ID NOs: 59, 60, 61, 62, 63, 64, or the amino acid sequence shown in SEQ ID NOs: 67, 68, 69, 70, 71, 72 A chimeric receptor according to any one of claims 1 to 3, which is an anti-CD38 antibody selected from the following.

5. The chimeric receptor according to any one of claims 1 to 4, wherein the transmembrane domain is selected from the transmembrane domains of CD3ε, CD3δ, CD3ζ, CD8, CD28, CD134, CD137, CD150, CD152, DAP10, FcRα, FcRβ, FcRγ, or Zap70, and preferably the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 7, 8, or 9.

6. The chimeric receptor according to any one of claims 1 to 5, wherein the intracellular domain is selected from the intracellular signaling domains of CD3, CD28, CD137, OX40, DAP10, or ICOS, or a combination thereof, and preferably the intracellular domain comprises the amino acid sequence shown in SEQ ID NOs. 10, 11, 12, or 13, or a combination thereof.

7. The chimeric receptor according to any one of claims 1 to 6, wherein the chimeric receptor is expressed on a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NOs: 4, 18, 23, 25, 29, 34, 36, 37, 38, or comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NOs: 24, 26, 27, 28.

8. The chimeric receptor according to any one of claims 1 to 7, wherein the antigen-binding domain further binds to another antigen, preferably the other antigen is a pathological cell antigen or an immune cell antigen, preferably the pathological cell is selected from solid tumor cells, hematological tumor cells, and pathological cells of autoimmune diseases, and the immune cell antigen is selected from NK cell antigen or T cell antigen.

9. A manipulated cell comprising the chimeric receptor according to any one of claims 1 to 8.

10. The manipulated cells according to claim 9, wherein the manipulated cells further comprise another polypeptide, the other polypeptide comprising another NK cell marker, a second chimeric receptor that binds to a target antigen on a pathological cell or a combination thereof, or an HLA-E polypeptide or a fragment thereof, or CD300A-PDGFR.

11. The pathological cells are selected from malignant cells or infected cells, preferably from solid tumor cells, hematological tumor cells, and pathological cells of autoimmune diseases. Preferably, the manipulated cells according to claim 10, wherein the solid tumor is selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the hematological malignancy is selected from leukemia, lymphoma, and myeloma; and the autoimmune disease is selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes mellitus, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis, and dermatomyositis.

12. The target antigen is CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin 18.2, FAP, mesoserine, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its variants, ASGPR1, IL13RA2, WT1, CLL1, or combinations thereof, and the other NK cell marker is selected from CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD1 The manipulated cells according to claim 10 or 11, selected from 78, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), FasL, NKG2E, CD279, CD300A, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, native cytotoxic receptors NCR1, NCR2, NCR3, or combinations thereof.

13. The manipulated cell according to any one of claims 10 to 12, wherein the other polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NOs: 83, 84, 85, or 86, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 30, 31, 32, 33, or 35.

14. The manipulated cells exhibit low or no expression of endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / CD38, TCR / B2M / NKG2A, TCR / B2M / CD38 / NKG2A, or TCR / B2M / FAS / NKG2A, preferably endogenous TCR / B2M, TCR / B2M / FAS, TCR / B2M / CD38, TCR / B2M / NKG2A, TCR / B2M / CD38 The manipulated cell according to any one of claims 9 to 13, wherein / NKG2A or TCR / B2M / FAS / NKG2A is knocked out or knocked down by CRISPR technology, and more preferably the gRNA sequences used to target TCR, B2M, NKG2A, and CD38 during knockout or knockdown using CRISPR technology are shown in SEQ ID NOs. 39, 40, 41, and 42, respectively.

15. The manipulated cells are autologous cells or allogeneic cells selected from immune cells, neurons, epithelial cells, endothelial cells, stem cells, or combinations thereof. Preferably, the manipulated cells are selected from B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof, and preferably, the manipulated cells are allogeneic T cells, according to any one of claims 9 to 14.

16. The manipulated cells according to any one of claims 9 to 15, wherein the manipulated cells significantly block the CD38 antigen on the autologous cells.

17. The manipulated cells have an extended survival period or enhanced proliferative capacity in the presence of host immune cells, or the manipulated cells can enhance the survival, proliferation, and pathological cell-killing effect of other manipulated cells introduced into the subject prior to, simultaneously with, or afterward. Preferably, the host immune cells are NK cells, according to any one of claims 9 to 16.

18. A polynucleotide which is a nucleic acid molecule encoding a chimeric receptor according to any one of claims 1 to 8.

19. The polynucleotide according to claim 18, wherein the polynucleotide is linked via a 2A fragment to a polynucleotide encoding another polypeptide contained in the manipulated cell according to any one of claims 10 to 17, the polynucleotide encoding the other polypeptide comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence shown in any one of SEQ ID NOs. 30, 31, 32, 33, or 35, or comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence encoding the amino acid sequence shown in any one of SEQ ID NOs. 83, 84, 85, or 86.

20. The polynucleotide according to claim 19, wherein the polynucleotide includes a nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence shown in any one of SEQ ID NOs: 34, 36, 37, or 38.

21. A vector comprising a polynucleotide according to any one of claims 18 to 20.

22. A virus comprising the vector described in claim 21.

23. A pharmaceutical composition comprising an effective amount of a chimeric receptor according to any one of claims 1 to 8, an engineered cell according to any one of claims 9 to 17, a polynucleotide according to any one of claims 18 to 20, a vector according to claim 21, or a virus according to claim 22, and a pharmaceutically acceptable carrier.

24. The use of a chimeric receptor according to any one of claims 1 to 8, or a manipulated cell according to any one of claims 9 to 17, in the preparation of a drug to prevent or resist transplant immune rejection, or to prepare a drug to prevent or resist transplant immune rejection, or to treat an autoimmune or inflammatory disease, or to prepare a drug to treat, prevent or improve an autoimmune or inflammatory disease in a subject who requires treatment, prevention or improvement of the disease, or to treat a tumor, or to treat, prevent or improve a tumor in a subject who requires treatment, prevention or improvement.

25. The autoimmune disease or inflammatory disease is selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis or dermatomyositis, Waldenström macroglobulinemia, primary systemic amyloidosis, membranous glomerulonephritis, idiopathic thrombocytopenic purpura, or myasthenia gravis, and the tumor is selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer The use according to claim 24, selected from cancer, colon cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, triple-negative breast cancer, sarcoma, leukemia, lymphoma, myeloma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), NK cell leukemia, NK / T cell lymphoma (NKTCL), or myelodysplastic syndrome.

26. A method for preventing or resisting transplant immune rejection, or a method for treating an autoimmune disease or inflammatory disease, or a method for treating a tumor, comprising administering to a subject a chimeric receptor according to any one of claims 1 to 8, an engineered cell according to any one of claims 9 to 17, a polynucleotide according to any one of claims 18 to 20, a vector according to claim 21, or a virus according to claim 22, or a pharmaceutical composition according to claim 23, preferably the autoimmune disease or inflammatory disease being multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome ( A method in which a tumor is selected from SS), polymyositis or dermatomyositis, Waldenström macroglobulinemia, primary systemic amyloidosis, membranous glomerulonephritis, idiopathic thrombocytopenic purpura, or myasthenia gravis, and the tumor is selected from esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, triple-negative breast cancer, sarcoma, leukemia, lymphoma, myeloma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), NK cell leukemia, NK / T cell lymphoma (NKTCL), or myelodysplastic syndrome.

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