CN115521917A - Engineered immune cells and uses thereof - Google Patents

Engineered immune cells and uses thereof Download PDF

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CN115521917A
CN115521917A CN202110710299.XA CN202110710299A CN115521917A CN 115521917 A CN115521917 A CN 115521917A CN 202110710299 A CN202110710299 A CN 202110710299A CN 115521917 A CN115521917 A CN 115521917A
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熊瑛
邢芸
任江涛
贺小宏
王延宾
韩露
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Nanjing Bioheng Biotech Co Ltd
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Nanjing Bioheng Biotech Co Ltd
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Priority to PCT/CN2022/099374 priority patent/WO2022267983A1/en
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Abstract

The present invention relates to an engineered immune cell expressing (i) a cell surface molecule that specifically recognizes an antigen, and (ii) one or more LTBR ligands. The invention also provides the use of the engineered immune cells in the treatment of cancer, infection or autoimmune disease. Compared with the traditional engineered immune cell, the engineered immune cell of the invention has obviously improved tumor killing activity.

Description

Engineered immune cells and uses thereof
Technical Field
The present invention is in the field of immunotherapy. More specifically, the invention relates to an engineered immune cell that expresses (i) a cell surface molecule that specifically recognizes an antigen, and (ii) one or more LTBR ligands. More preferably, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor or a recombinant T cell receptor.
Background
Currently, CAR-T cell therapy has shown an attractive prospect in the field of tumor immunotherapy. The basic structure of the CAR comprises a tumor associated antigen binding region, a transmembrane region, and an intracellular signaling region. Once CAR binds to tumor associated antigens, T cells can be activated by intracellular domains, which in turn manifest as CAR-dependent cell killing, proliferation, and cytokine release. However, CAR-T cell therapy still has some problems in clinical applications, such as a large number of tumor recurrence phenomena in hematologic tumor treatment, a low response rate in solid tumor treatment, etc., which may be caused by factors such as complex tumor microenvironment, CAR-T cell depletion, etc.
Thus, there remains a need for improvements in existing CART T cell therapies to promote proliferation of CAR-T cells in vivo, to counteract the immunosuppressive effects of the tumor microenvironment, and to improve the overall therapeutic effect of CAR-T cell therapy on tumors.
Disclosure of Invention
In a first aspect, the present invention provides a novel engineered immune cell expressing (i) a cell surface molecule that specifically recognizes an antigen, and (ii) one or more LTBR ligands.
In one embodiment, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor or a recombinant T cell receptor, preferably a chimeric antigen receptor.
In one embodiment, the LTBR ligand is selected from LTA, LTB, LIGHT and anti-LTBR antibody.
In one embodiment, the immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell. Preferably, the T cell is a CD4+/CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γ δ -T cell, or an α β -T cell.
In one embodiment, the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor comprising an antigen binding region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. Wherein the antigen-antigen binding region may be selected from IgG, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin and DARPIN. Preferably, the antigen-antigen binding region is selected from the group consisting of scFv, fab, single domain antibody and nanobody.
In one embodiment, the cell surface molecule specifically recognizing an antigen binds to a target selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1 DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/neu), erbB3, erbB4 MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, C-Met, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, ephA2, fucosyl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVR 1, ADRB3, PANX3, pNX 3 GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-L/Galectin8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, LAP, LAM, TMPRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF β, APRIL, NKG2D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecule expressed by HIV, HCV, HBV and/or other pathogen; and/or a neoepitope or neoantigen. Preferably, the target is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, folate receptor alpha, CEA, PSCA, PSMA, her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin, and any combination thereof.
In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ∈ subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28 and CD 278.
In one embodiment, the intracellular signaling domain is selected from the intracellular regions of the following proteins: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling domain comprises a CD3 ζ intracellular region.
In one embodiment, the co-stimulatory domain is one or more intracellular regions of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof. Preferably, the co-stimulatory domain is selected from the intracellular region of CD27, CD28, CD134, CD137, DAP10, DAP12, or CD278 or a combination thereof.
In a second aspect, the invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a cell surface molecule which specifically recognizes an antigen, and (ii) a nucleic acid sequence encoding an LTBR ligand. Preferably, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor or a recombinant T cell receptor, more preferably a chimeric antigen receptor. Preferably, the nucleic acid is DNA or RNA.
The invention also provides a vector comprising the nucleic acid molecule described above. In particular, the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous Sarcoma Viruses (RSV), polyoma viruses, and adeno-associated viruses (AAV). In some embodiments, the vector further comprises elements such as an origin of autonomous replication in an immune cell, a selectable marker, a restriction enzyme cleavage site, a promoter, a poly A tail (polyA), a 3'UTR, a 5' UTR, an enhancer, a terminator, an insulator, an operator, a selectable marker, a reporter gene, a targeting sequence, and/or a protein purification tag. In a specific embodiment, the vector is an in vitro transcription vector.
In one embodiment, the invention also provides a kit comprising an engineered immune cell, nucleic acid molecule or vector of the invention.
In one embodiment, the invention also provides a pharmaceutical composition comprising an engineered immune cell, nucleic acid molecule or vector of the invention, and one or more pharmaceutically acceptable excipients.
Antigen in a third aspect, the present invention also provides a method of treating a subject suffering from cancer, an infection or an autoimmune disease, comprising administering to the subject an effective amount of an immune cell, a nucleic acid molecule, a vector or a pharmaceutical composition according to the invention.
In one embodiment, the cancer is a solid tumor or a hematological tumor. More specifically, the cancer is selected from: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, cancer of the biliary tract, cancer of the bladder, bone, brain and CNS cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, cancer of connective tissue, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, kidney cancer, larynx cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland carcinoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, ovarian cancer, pancreatic cancer, cancer of the bladder, cancer of the bone, cancer of the brain and CNS, cancer of the breast, peritoneum, cervical cancer, choriocarcinoma, hepatoma, intraepithelial tumors, lymphoma, melanoma, myeloma, neuroblastoma, and neuroblastoma malignant tumors of the urinary system, vulvar and other carcinomas and sarcomas, as well as B-cell lymphomas, B-lymphoblastic lymphomas (B-LBL), mantle cell lymphomas, AIDS-related lymphomas, and Waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumors, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmacytic lymphoma, and marginal zone lymphoma, pre-leukemic, plasmacytoid dendritic cell tumors, and post-transplant lymphoproliferative disorders (PTLD).
In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
In one embodiment, the autoimmune disease includes, but is not limited to, type I diabetes, celiac disease, graves 'disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, addison's disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus, among others.
Drawings
FIG. 1: schematic structure of CAR plasmid.
FIG. 2: CAR expression levels of CAR-T cells determined by flow cytometry.
FIG. 3: killing activity of CAR-T cells against target cells.
FIG. 4: (iii) IFN- γ release levels after co-culture of CAR-T cells with target and non-target cells, respectively.
FIG. 5: survival curves of mice after treatment of mouse B-lymphomas with CAR-T cells.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Cell surface molecules that specifically recognize antigens
As used herein, the term "cell surface molecule that specifically recognizes an antigen" refers to a molecule expressed on the surface of a cell that is capable of specifically binding to a target molecule (e.g., an antigen). Such surface molecules typically comprise an antigen-binding region capable of specifically binding to an antigen, a transmembrane domain that anchors the surface molecule to the cell surface, and an intracellular domain responsible for signaling. Examples of common such surface molecules include, for example, recombinant T cell receptors or chimeric antigen receptors.
As used herein, the term "recombinant T cell receptor" or "TCR" refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. Stimulation of TCRs is triggered by major histocompatibility complex Molecules (MHC) on antigen presenting cells that present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling. TCRs are composed of six peptide chains that form heterodimers, which are generally classified into α β type and γ δ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to specific antigens and MHC molecules. The variable region of the TCR can comprise or be operably linked to an antigen-binding region, wherein the antigen-binding region is defined as follows.
As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen binding region (e.g., an antigen-binding portion of an antibody), a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, each of which is linked by a linker. CARs are able to redirect the specificity and reactivity of T cells and other immune cells to a selected target in a non-MHC-restricted manner using the antigen binding properties of monoclonal antibodies. non-MHC-restricted antigen recognition gives CAR cells the ability to recognize antigens independent of antigen processing, thus bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with the alpha and beta chains of an endogenous recombinant T Cell Receptor (TCR).
As used herein, "antigen binding region" refers to any structure or functional variant thereof that can bind to an antigen. The antigen binding region can be an antibody structure including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies and functional fragments thereof. For example, antigen binding regions include, but are not limited to, igG, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin, DARPIN, and the like, preferably selected from Fab, scFv, sdAb, and nanobody. In the present invention, the antigen binding region may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody. In another embodiment, the antigen binding region may also be a specific binding polypeptide or receptor structure for a particular protein, such as PD1, PDL2, TGF β, APRIL and NKG2D.
"Fab" refers to either of the two identical fragments produced by papain cleavage of an immunoglobulin molecule, consisting of the entire light and heavy chain N-terminal portions linked by disulfide bonds, wherein the heavy chain N-terminal portion includes the heavy chain variable region and CH1. Compared to intact IgG, fab has no Fc fragment, higher mobility and tissue penetration, and binds antigen monovalent without mediating antibody effects.
"Single-chain antibody" or "scFv" is an antibody comprising an antibody variable region (VH) and an antibody variable region (VL) linked via a linker. The optimal length and/or amino acid composition of the linker may be selected. The length of the linker can significantly affect the variable region folding and interaction profile of the scFv. In fact, if shorter linkers are used (e.g., between 5-10 amino acids), intra-strand folding may be prevented. For selection of the size and composition of the linker, see, e.g., hollinger et al, 1993proc Natl acad.sci.u.s.a.90; U.S. patent application publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT publication Nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference in their entirety. The scFv may comprise a VH and a VL connected in any order, for example a VH-linker-VL or a VL-linker-VH.
A "single domain antibody" or "sdAb" refers to an antibody that naturally lacks a light chain and comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also referred to as a "heavy chain antibody".
"Nanobody" or "Nb" refers to a VHH structure that is cloned and expressed individually, has structural stability comparable to that of an original heavy chain antibody and binding activity to an antigen, and is the smallest unit currently known to bind to a target antigen.
The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of a parent, but contains at least one amino acid modification (i.e., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.
As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Conservative modifications may be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
Thus, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a parent amino acid sequence and retains the biological activity, e.g., binding activity, of the parent amino acid.
As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity. One skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al (1997) Nucleic Acids Res.25: 3389-3402), blast2 (Altschul et al (1990) J.mol.biol.215: 403-410), smith-Waterman (Smith et al (1981) J.mol.biol.147: 195-197), and ClustalW.
The choice of antigen binding region depends on the cell surface marker on the target cell to be identified as being associated with a particular disease state, e.g., a tumor specific antigen or a tumor associated antigen. Thus, in one embodiment, the antigen binding region of the invention binds to one or more targets selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1 DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/neu), erbB3, erbB4 MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, C-Met, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, ephA2, fucosyl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globh, NY-BR-1, UPK2, HAVC 1, ADRB3, PANX3, NY-BR-1, and pK2 GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-L/Galectin8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, LAP, and, TMPRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LIA 2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF β, APRIL, NKG2D ligand, and/or a pathogen-specific antigen, biotinylated molecule, a molecule expressed by HIV, HCV, HBV, and/or other pathogens; and/or a neoepitope or neoantigen. Depending on the antigen to be targeted, the CAR of the invention may be designed to include an antigen binding region specific for that antigen. For example, if CD19 is the antigen to be targeted, CD19 antibodies may be used as the antigen binding region of the invention.
As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and to direct the cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to a target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 epsilon subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8a having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 4 or 16, or the coding sequence of the transmembrane domain of CD8a has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID No. 3 or 15.
In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antigen binding region and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an antigen binding region. In particular, the hinge region serves to provide greater flexibility and accessibility to the antigen binding region. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from a native molecule, such as from the extracellular region of CD8, CD4 or CD28, or from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a portion of the hinge region of a CD8 α chain, fc γ RIII α receptor, igG4 or IgG1, more preferably a CD8 α hinge, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 12, 22, 33 or 35, or the coding sequence of a CD8 α hinge has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID No. 11, 21, 34 or 36.
As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specified function. The intracellular signaling domain is responsible for intracellular primary signaling after binding of the antigen at the antigen-binding region, resulting in activation of the immune cell and immune response. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.
In one embodiment, the intracellular signaling domain comprised by the chimeric antigen receptor of the present invention may be the cytoplasmic sequences of the recombinant T cell receptor and co-receptor that work together to trigger primary signaling upon antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence with the same or similar function. The intracellular signaling domain may contain a number of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the invention include, but are not limited to, those derived from FcR γ, fcR β, CD3 γ, CD3 δ, CD3 e, CD3 ζ, CD22, CD79a, CD79b, and CD66d. In preferred embodiments, the signalling domain of a CAR of the invention may comprise a CD3 ζ intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence as set forth in SEQ ID No. 8 or 20, or a coding sequence thereof having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID No. 7 or 19.
In one embodiment, the chimeric antigen receptor of the present invention comprises one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule, comprising the entire intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of co-stimulatory domains of the invention include, but are not limited to, intracellular regions derived from: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70. Preferably, the co-stimulatory domain of a CAR of the invention is from 4-1BB, CD28, CD27, OX40, ICOS, DAP10, DAP12 or a combination thereof, more preferably 4-1BB and/or CD28. In one embodiment, the CAR of the invention comprises a co-stimulatory domain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence depicted in SEQ ID No. 6, 18 or 31, or the coding sequence of the co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence depicted in SEQ ID No. 5, 17 or 32.
In one embodiment, the CAR of the invention may further comprise a signal peptide such that when it is expressed in a cell, for example a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long hydrophobic amino acid segment that has a tendency to form a single alpha-helix. At the end of the signal peptide there is usually a stretch of amino acids which is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during translocation or after completion to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, such as those derived from CD8 α, igG1, GM-CSFR α, and the like. In one embodiment, the signal peptide useful in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 10, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO. 9.
In one embodiment, the CAR of the invention may further comprise a switch structure to regulate the time of expression of the CAR. For example, the switch structure may be in the form of a dimerization domain that causes a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain to allow binding to the targeted antigen, thereby activating the signaling pathway. Alternatively, a switch domain may be used to connect the binding domain and the signaling domain, respectively, such that the binding domain and the signaling domain are connected together via a dimer only when the switch domains are bound to each other (e.g., in the presence of an inducing compound), thereby activating the signaling pathway. The switch structure may also be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain, preventing its binding to the antigen to be targeted, and when the masking peptide is cleaved, for example by a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure. Various switch configurations known to those skilled in the art may be used with the present invention.
In one embodiment, the CAR of the invention may also comprise a suicide gene, i.e. such that it expresses a cell death signal that can be induced by foreign substances, to eliminate CAR cells when needed (e.g. when severe toxic side effects are produced). For example, the suicide gene may be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when desired, the CAR cells can be eliminated by adding antibodies or agents that target these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir therapy. The suicide gene can also be iCaspase-9, and iCaspase-9 can be induced to dimerize through chemical induction drugs such as AP1903, AP20187 and the like, so that downstream Caspase3 molecules are activated, and apoptosis is caused. Various suicide genes known to those skilled in the art can be used in the present invention.
LTBR ligands
LTBR refers to the lymphotoxin beta receptor, a type I single transmembrane protein with a molecular weight of 61 kDa. The extracellular region of LTBR is responsible for ligand binding and contains four cysteine-rich repeats characteristic of the TNF motif. The intracellular region of LTBR consists of 175 amino acid residues, which do not themselves have kinase or other enzymatic activity. LTBR is constitutively expressed on most cells, such as stromal cells of lymphoid tissues, myeloid lineage cells, blood monocytes, alveolar macrophages, mast cells, dendritic cells, and the like. However, LTBR is not expressed in T cells, B cells and NK cells, which is also its most prominent feature. In contrast, ligands for LTBR are often expressed in T cells and B cells. This expression pattern suggests that for LT, signaling may be unidirectional, requiring direct contact between lymphocytes and LTBR-bearing cells to deliver the signal.
The natural LTBR ligands known to date mainly include two: lymphotoxin (LT) and LIGHT. Lymphotoxin (LT) is a cytokine secreted by lymphocytes after being activated by an antigen or mitogen or the like and in the case of some tumors and autoimmune diseases. Lymphotoxin, also known as tumor necrosis factor β, is two closely related cytokines with tumor necrosis factor α (TNF- α), belonging to the TNF family. LT is expressed predominantly in B cells, T cells and NK cells. LT comprises two subunits: LT α (also known as LTA) and LT β (also known as LTB). Wherein LT α can be secreted extracellularly and then anchored to the cell surface by non-covalent association with LT β to form a heterotrimer. LT plays an important role in various biological activities such as killing tumor cells, regulating immune response, regulating inflammatory response, inducing antigen expression of various differentiated cells and the like. LIGHT is a type II transmembrane protein, also belonging to the TNF family, produced by activated T cells, monocytes, granulocytes, and immature dendritic cells. Binding between LTBR and its ligands activates NK κ B and c-Jun N-terminal kinase (JNK) signaling pathways, leading to expression of chemokines, such as CXCL12, CXCL13, CCL19, CCL21, etc., which in turn induces apoptosis in certain cells (e.g., tumor cells). Furthermore, signaling between LTBR receptor-ligand may also be initiated by anti-LTBR antibodies or by overexpression of the receptor.
In one embodiment, the LTBR ligand is LTA, which is homologous to SEQ ID NO: 26. 40, 46 or 48, or a sequence encoding the same as or identical to the amino acid sequence of SEQ ID No.: 25. 39, 45 or 47, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity. The amino acid sequence of SEQ ID NO:46 is represented by SEQ ID NO:26 (amino acids 1 to 33 of SEQ ID NO:26 is a signal peptide), and the amino acid sequence of SEQ ID NO:48 is represented by SEQ ID NO:40 (amino acids 1 to 34 of SEQ ID NO:40 is a signal peptide).
In one embodiment, the LTBR ligand is LTB, which is identical to SEQ ID NO:24 or 42, or a coding sequence thereof, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:23 or 41, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
In one embodiment, the LTBR ligand is LIGHT, which is identical to SEQ ID NO:28 or 44, or a sequence whose coding sequence shares at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:27 or 43 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
In one embodiment, the LTBR ligand is an anti-LTBR antibody, e.g. an antibody selected from the group consisting of IgG, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody. Antibodies known in the art can be used as LTBR ligands of the present invention, including, but not limited to, CBE11 (ATCC accession number 11793), BKA11 (ATCC accession number 11799), CDH10 (ATCC accession number 11797), BCG6 (ATCC accession number 11794), BHA10 (ATCC accession number 11795), BDA8 (ATCC accession number HB 11798) (see U.S. Pat. nos. 5,925,351 and 631,269,1, the entire contents of which are incorporated herein by reference).
Nucleic acids
The invention also provides a nucleic acid molecule comprising (i) a nucleic acid sequence encoding a cell surface molecule which specifically recognizes an antigen, and (ii) a nucleic acid sequence encoding an LTBR ligand.
In one embodiment, the cell surface molecule specifically recognizing an antigen is a recombinant T cell receptor or a chimeric antigen receptor, preferably a chimeric antigen receptor. The chimeric antigen receptor is defined as above.
As used herein, the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in linear or circular form, in single-and/or double-stranded form, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.
Nucleic acids may comprise conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as found in Peptide Nucleic Acids (PNAs)). The nucleic acids of the invention may also contain one or more modified bases such as, for example, tritylated bases and unusual bases such as inosine. Other modifications, including chemical, enzymatic, or metabolic modifications are also contemplated, so long as the multi-chain CAR of the invention can be expressed from a polynucleotide. The nucleic acid may be provided in isolated form. In one embodiment, the nucleic acid may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.
The nucleic acid sequences of the invention may be codon optimized for optimal expression in a desired host cell (e.g., an immune cell); or for expression in bacterial, yeast or insect cells. Codon optimization refers to the replacement of codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, with the codons before and after the replacement encoding the same amino acid. Thus, the choice of optimal codons depends on the codon usage bias of the host genome.
Carrier
The invention also provides a vector comprising a nucleic acid according to the invention. Wherein the nucleic acid sequence encoding a cell surface molecule specifically recognizing the antigen, and the nucleic acid encoding the LTBR ligand may be located in one or more vectors.
For example, two chimeric antigen receptor structures can be expressed independently without interaction, although in the same vector, by inserting a nucleic acid encoding a 2A peptide between the two nucleic acid sequences. As used herein, the term "2A peptide" is a cis-hydrolase acting element, originally found in foot-and-mouth disease virus (FMDV). The 2A peptides have an average length of 18 to 22 amino acids. During protein translation, the 2A peptide can be cleaved from its last2 amino acids C-terminus by ribosome skipping. Specifically, the peptide chain binding group between glycine and proline is impaired at the 2A site, and initiates ribosome skipping to start translation from the 2 nd codon, thereby allowing independent expression of 2 proteins in1 transcription unit. This 2A peptide-mediated cleavage is widely present in eukaryotic animal cells. The expression efficiency of heterologous polyproteins (such as cell surface receptors, cytokines, immunoglobulins, etc.) can be improved by utilizing the higher cleavage efficiency of the 2A peptide and the ability to promote balanced expression of upstream and downstream genes. The conventional 2A peptide comprises: P2A, T2A, E2A, F2A, etc.
As used herein, the term "vector" is a vector nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into a host cell where it can, for example, be replicated and/or expressed.
Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering an isolated nucleic acid to the interior of a cell, for example, by homologous recombination or by using a hybrid recombinase that targets sequences at a site specifically. An "expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding the chimeric antigen receptor polypeptides of the invention) in a suitable host cell, as well as the translation of their mRNA. Suitable carriers for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the invention include, but are not limited to, plasmids, viruses (e.g., retrovirus, lentivirus, adenovirus, vaccinia virus, rous sarcoma virus (RSV, polyoma virus and adeno-associated virus (AAV), etc.), bacteriophage, phagemid, cosmid, and artificial chromosomes (including BAC and YAC). The vectors themselves are typically nucleotide sequences, typically DNA sequences comprising an insert (transgene) and a larger sequence as the "backbone" of the vector.
Engineered immune cells
The invention also provides an engineered immune cell comprising a nucleic acid or vector of the invention. In other words, the engineered immune cells of the invention express a cell surface molecule that specifically recognizes an antigen, and an LTBR ligand.
As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cells may be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells obtained from stem cell sources such as cellular umbilical cord blood. Preferably, the immune cell is a T cell. The T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, e.g., jurkat, supT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. The T cells may be at any developmental stage, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (e.g., th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γ δ -T cells, α β -T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll isolation. In the present invention, immune cells are engineered to express a chimeric antigen receptor and an exogenous LTBR ligand.
In yet another embodiment, the immune cell of the invention further comprises at least one inactivated gene selected from the group consisting of: CD52, GR, TCR alpha, TCR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD247 zeta, HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More particularly, at least the TCR component (including TCR α, TCR β genes) or the CD3 component (including CD3 γ, CD3 δ, CD3 ε, CD247 ζ) in the immune cell is inactivated. This inactivation renders the TCR-CD3 complex non-functional in the cell. This strategy is particularly useful for avoiding graft versus host disease (GvHD). Methods of inactivating a gene are known in the art, for example, by mediating DNA cleavage by meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems, thereby inactivating the gene.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising the engineered immune cell, nucleic acid molecule or vector of the invention as an active agent, and one or more pharmaceutically acceptable excipients. Thus, the invention also encompasses the use of said nucleic acid molecule, vector or engineered immune cell for the preparation of a pharmaceutical composition or medicament.
As used herein, the term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesirable local or systemic effects) and are well known in the art (see, e.g., remington's Pharmaceutical sciences. Edded by Gennaro AR,19th ed. Pennsylvania. Mack Publishing company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients to prepare the desired pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
The pharmaceutical composition according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or extracts of fluid extracts, or in a form which is particularly suitable for the desired method of administration. Processes known in the art for the manufacture of medicaments may comprise, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.
The pharmaceutical compositions according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the diseases to be treated. Preferred examples of the pharmaceutical agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rebeccin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium II (sorfimer sodium phosphate II), temozolomide, topotecan, glucuronic acid trimetrexate (trimetrenate glucoside), oritavastin E (auristatin E), vincristine and adriamycin; peptide cytotoxins such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin a, dnase and rnase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225, and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants such as platelet factor 4, melanoma growth stimulating protein, and the like; antibodies or fragments thereof, such as anti-CD 3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains, and viral/bacterial peptides. In addition, the pharmaceutical compositions of the present invention may also be used in combination with one or more other therapeutic methods, such as chemotherapy, radiation therapy.
Therapeutic applications
The invention also provides a method of treating a subject having cancer, an infection or an autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the invention. Thus, the invention also encompasses the use of said engineered immune cells in the preparation of a medicament for the treatment of cancer, infection or autoimmune disease.
In one embodiment, the method of treatment comprises administering to a subject an effective amount of an immune cell and/or pharmaceutical composition of the invention.
In one embodiment, the immune cell is an autologous or allogeneic cell, preferably a T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell, more preferably a T cell, NK cell or NKT cell.
As used herein, the term "autologous" means that any material derived from an individual will be reintroduced into the same individual at a later time.
As used herein, the term "allogeneic" refers to any material derived from a different animal or patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered allogeneic to each other. In some cases, genetic differences in allogenic material from individuals of the same species may be sufficient for antigen interactions to occur.
As used herein, the term "subject" is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.
In one embodiment, the cancer is a cancer associated with expression of a target bound by an antigen binding region. For example, the cancer includes, but is not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, kidney cancer, larynx cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer and squamous lung cancer), lymphoma (including hodgkin lymphoma and non-hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, and prostate cancer retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant neoplasms of the urinary system, vulval cancer as well as other carcinomas and sarcomas, and B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cracked cellular NHL, large lump disease NHL), B lymphoblastic lymphoma (B-LBL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablatic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); and other diseases associated with target expression. Preferably, the diseases that can be treated with the engineered immune cells or the pharmaceutical compositions of the invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, and the like.
In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
In one embodiment, the autoimmune disease includes, but is not limited to, type I diabetes, celiac disease, graves 'disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, addison's disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus, among others.
In one embodiment, the method further comprises administering to the subject one or more additional chemotherapeutic agents, biologies, drugs, or treatments. In this embodiment, the chemotherapeutic agent, biological agent, drug or treatment is selected from the group consisting of radiation therapy, surgery, antibody agents and/or small molecules and any combination thereof.
The invention will be described in detail below with reference to the drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.
Detailed Description
Example 1 preparation of CAR-T cells
1.1. Construction of retroviral plasmids
A fragment of the coding sequence of the CD8a signal peptide (SEQ ID NO: 10), CD19-scFv (SEQ ID NO: 14), CD8a hinge region (SEQ ID NO: 22), CD8a transmembrane region (SEQ ID NO: 16), 4-1BB endodomain (SEQ ID NO: 18) and CD3 zeta endodomain (SEQ ID NO: 20) connected in sequence was synthesized and cloned into an MSCV vector to obtain a bbz plasmid.
A fragment of the coding sequence of T2A (SEQ ID NO: 37) and LTB (SEQ ID NO: 24) ligated in sequence was synthesized artificially and cloned into the bbz vector to obtain the bbz-T2A-Ltb plasmid.
A coding sequence fragment of T2A (SEQ ID NO: 37), LTB (SEQ ID NO: 24) and LTA (SEQ ID NO: 26) which are connected in sequence is artificially synthesized and cloned into a bbz vector to obtain a bbz-T2A-Ltba plasmid.
A sequence fragment of the coding sequence of T2A (SEQ ID NO: 37) and LIGHT (SEQ ID NO: 28) which are connected in sequence is artificially synthesized and cloned into a bbz vector to obtain a bbz-T2A-LIGHT plasmid.
The structure of each plasmid is shown in FIG. 1.
1.2. Preparation of retrovirus
In T175 flasks at 30X 10 6 Density of Individual cells/flask 293T cells were seeded in 30ml DMEM medium containing 10% foetal calf serum at 37 ℃ 5% 2 Cultured overnight in an incubator for virus packaging.
To a sterile tube, 3ml of Opti-MEM (Gibco, cat # 31985-070), 45. Mu.g of the prepared retroviral plasmid and 15. Mu.g of the packaging vector pCL-Eco (Shanghai cereal noon Biotech Co., ltd., cat # P3029) were added. Then 120. Mu.l of X-trememe GENE HP DNA transfection reagent (Roche, cat # 06366236001) was added, mixed immediately and incubated at room temperature for 15min. Then the plasmid/vector/transfection reagent mixture was added dropwise to a 293T cell culture flask prepared in advance, at 37 ℃ 5% 2 Incubated under conditions overnight. The culture was collected 72 hours after transfection, and centrifuged (2000 g,4 ℃,10 minutes) to obtain a retrovirus supernatant.
1.3 preparation of CAR-T cells
T lymphocytes were isolated from mouse spleen and CTS with DynaBeads CD3/CD28 TM (Gibco, cat # 40203D) activates T cells, then CO at 37 ℃ and 5% 2 The culture was performed for 1 day.
At a rate of 3X 10 per hole 6 Density of individual cells/mL activated T cells were seeded into 24-well plates previously coated overnight with RetroNectin, and then 500 μ L complete medium (NT, control) or retroviral supernatant prepared in step 2 was added, respectively, and supplemented to 2mL.
The 24-well plate was placed in a centrifuge for centrifugal infection and centrifuged at 2000g for 2h at 32 ℃. Then, the 24-well plate was immediately placed in a CO2 incubator at 37 ℃ for static culture. The next day, the fresh medium was replaced and the cell density was adjusted to 1X10 6 Individual cells/mL. Three days after infection, cells were harvested for subsequent analysis. The collected cells are NT cells, bbz CAR-T cells, bbz-T2A-Ltb CAR-T cells, bbz-T2A-Ltba CAR-T cells, and bbz-T2A-LIGHT CAR-T cells.
1.4 detection of CAR expression levels
Taking 2X 10 5 The expression level of CAR on each CAR-T cell was examined by flow cytometry using Biotin-SP-AffiniPure Goat Anti-Rat IgG, F (ab') 2Fragment specificity (Jackson ImmunoResearch, cat # 112-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, cat # 554067) as the secondary antibody, as shown in FIG. 2.
It can be seen that the CAR can be expressed efficiently in bbz CAR-T cells, bbz-T2A-Ltb CAR-T cells, bbz-T2A-Ltba CAR-T cells and bbz-T2A-LIGHT CAR-T cells.
Example 2 killing effect and cytokine release of CAR-T cells on target cells
2.1Killing effect on target cells
At 1x10 4 A20-CD19 target cells (screened CD19 positive AD20 cells) carrying a fluorescein gene were plated in 96-well plates, then the CAR-T cells and NT cells prepared in example 1 were plated in 96-well plates for co-culture at a potency-to-target ratio (i.e., effector T cell to target cell ratio) of 5. According to the calculation formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and the killing efficiency was calculated, and the results are shown in FIG. 3.
It can be seen that CAR-T cells additionally expressing LTB, LTB + LTA or LIGHT have slightly improved killing ability against target cells compared to traditional bbz-CAR-T cells at various target ratios.
2.2Cytokine release levels
(1) Collecting cell co-culture supernatant
At 1x10 5 Concentration per well target cells P02-CD19 or non-target cells P02 were plated in 96-well plates, and then CAR-T cells and NT cells prepared in example 1 were co-cultured with the target cells at a ratio of 10.
(2) ELISA detection of IFN-gamma secretion in supernatant
Use the trapObtained Antibody Purified anti-human IFN-. Gamma.antibody (Biolegend, cat. No. 506502) was coated on 96-well plates and incubated overnight at 4 ℃ followed by removal of the Antibody solution and addition of 250. Mu.L of PBST (1 XPBS with 0.1% Tween) solution containing 2% BSA (sigma, cat. No. V900933-1 kg) and incubation at 37 ℃ for 2 hours. The plates were then washed 3 times with 250 μ L of PBST (1 XPBS with 0.1% Tween). mu.L of cell co-culture supernatant or standard was added to each well and incubated at 37 ℃ for 1 hour, after which the plates were washed 3 times with 250. Mu.L of PBST (1 XPBS with 0.1% Tween). Then 50. Mu.L of an Anti-Interferon gamma antibody [ MD-1 ] was added to each well](Biotin) (abcam, cat # ab 25017), after 1 hour incubation at 37 ℃ the plates were washed 3 times with 250. Mu.L PBST (1 XPBS with 0.1% Tween). HRP Streptavidin (Biolegend, cat # 405210) was added, and after incubation at 37 ℃ for 30 minutes, the supernatant was discarded, 250. Mu.L of PBST (1 XPBS containing 0.1% Tween) was added, and washed 5 times. To each well 50 μ L of TMB substrate solution was added. The reaction was allowed to occur at room temperature in the dark for 30 minutes, after which 50. Mu.L of 1mol/L H was added to each well 2 SO 4 To stop the reaction. Within 30 minutes of stopping the reaction, absorbance at 450nm was measured using a microplate reader, and the content of cytokine was calculated from a standard curve (plotted according to the reading and concentration of the standard), and the result is shown in FIG. 4.
It can be seen that the secretion level of IFN γ is significantly increased after coculture of CAR-T cells with target cells compared to non-target cells, indicating that CAR-T cells are specific for killing of target cells.
Example 3 demonstration of tumor-inhibiting Effect of CAR-T cells
24 Balb/c immunized healthy mice 6 weeks old were randomly divided into 4 groups of 6 mice each, and 1X10 mice were injected via tail vein 6 An A20 cell (a B lymphoma cell). The day of inoculation of A20 cells was defined as D0. Each group of mice was injected intraperitoneally at D10 with 3mg/20g of cyclophosphamide. Mice in each group were injected via tail vein with 5X 10 at D13 6 Individual bbz CAR-T cells, bbz-T2A-Ltb CAR-T cells, bbz-T2A-Ltba CAR-T cells, or bbz-T2A-LIGHT CAR-T cells. Mice survival was monitored until the end of the experiment. The results are shown in FIG. 5.
It can be seen that CAR-T cells additionally expressing LTB, LTB + LTA or LIGHT significantly enhance the tumor suppressive effect and thus increase survival rate, compared to conventional bbz CAR-T cells.
It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Sequence listing
<110> Nanjing Beijing Heng Biotechnology Ltd
<120> engineered immune cells and uses thereof
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115 120 125
Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys
130 135 140
Lys Val Ser Gly Asp Thr Ile Thr Phe Tyr Tyr Met His Phe Val Lys
145 150 155 160
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Glu
165 170 175
Asp Glu Ser Thr Lys Tyr Ser Glu Lys Phe Lys Asn Lys Ala Thr Leu
180 185 190
Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Lys Leu Ser Ser Leu
195 200 205
Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ile Tyr Gly Gly Tyr Tyr
210 215 220
Phe Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser
225 230 235
<210> 15
<211> 63
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mCD8 alpha transmembrane domain
<400> 15
atctgggcac ccttggccgg aatctgcgtg gcccttctgc tgtccttgat catcactctc 60
atc 63
<210> 16
<211> 21
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mCD 8a transmembrane domain
<400> 16
Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu
1 5 10 15
Ile Ile Thr Leu Ile
20
<210> 17
<211> 126
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> m4-1BB co-stimulatory domain
<400> 17
aggaaaaaat tcccccacat attcaagcaa ccatttaaga agaccactgg agcagctcaa 60
gaggaagatg cttgtagctg ccgatgtcca caggaagaag aaggaggagg aggaggctat 120
gagctg 126
<210> 18
<211> 42
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> m4-1BB co-stimulatory domain
<400> 18
Arg Lys Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys Lys Thr Thr
1 5 10 15
Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys Pro Gln Glu
20 25 30
Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
35 40
<210> 19
<211> 327
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mCD3 zeta signaling domain
<400> 19
agcaggagtg cagagactgc tgccaacctg caggacccca accagctcta caatgagctc 60
aatctagggc gaagagagga atatgacgtc ttggagaaga agcgggctcg ggatccagag 120
atgggaggca aacagcagag gaggaggaac ccccaggaag gcgtatacaa tgcactgcag 180
aaagacaaga tggcagaagc ctacagtgag atcggcacaa aaggcgagag gcggagaggc 240
aaggggcacg atggccttta ccagggtctc agcactgcca ccaaggacac ctatgatgcc 300
ctgcatatgc agaccctggc ccctcgc 327
<210> 20
<211> 109
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mCD3 zeta signaling domain
<400> 20
Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp Pro Asn Gln Leu
1 5 10 15
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Glu
20 25 30
Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys Gln Gln Arg Arg
35 40 45
Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln Lys Asp Lys Met
50 55 60
Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu Arg Arg Arg Gly
65 70 75 80
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
85 90 95
Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro Arg
100 105
<210> 21
<211> 135
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mCD8 α hinge region
<400> 21
actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60
ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120
gcctgtgata tttac 135
<210> 22
<211> 45
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mCD8 α hinge region
<400> 22
Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr
1 5 10 15
Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser
20 25 30
Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr
35 40 45
<210> 23
<211> 921
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mLTB
<400> 23
atggggacac ggggactgca gggcctgggt gggagacccc aggggagggg ctgcctcttg 60
ctggctgtgg caggagctac ttccctggtg accctgttgt tggcagtgcc tatcactgtc 120
ctggctgtgc tggccttggt gccgcaggat cagggacgtc gggttgagaa gatcattggc 180
tcaggagcac aggctcagaa aagactggat gacagcaaac cgtcgtgcat cttgccctca 240
ccctctagcc tctcagagac tcctgacccc cgtctgcatc ctcagagatc caatgcttcc 300
aggaatctag cctccacatc ccagggccct gttgcgcagt cctctcggga ggcatctgca 360
tggatgacca tcctgtctcc agctgcggat tctacaccag atccaggggt tcaacagctg 420
ccaaaggggg aaccagaaac tgacctcaac cctgagctcc ctgctgccca cctcataggc 480
gcttggatga gtgggcaagg gctcagctgg gaggcgagcc aagaagaagc gtttctgagg 540
agcggcgcgc agttctcccc cacccacggg ctggcgctgc cacaggacgg cgtctattac 600
ctctactgcc acgtcgggta caggggcagg acgccccctg ccggccgaag ccgtgctcgc 660
tcgctcacgc tgcgcagcgc cctgtaccgc gcggggggcg cctacgggcg aggttccccc 720
gagttgctgc tggagggcgc ggagacagtc acacctgttg tggaccccat cgggtacggg 780
tcgttatggt acacgagcgt ggggttcggc ggcctggcgc agctccggag cggcgagagg 840
gtctacgtta acatcagtca ccccgacatg gtggactaca ggagagggaa gaccttcttc 900
ggggcggtga tggtggggtg a 921
<210> 24
<211> 306
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mLTB
<400> 24
Met Gly Thr Arg Gly Leu Gln Gly Leu Gly Gly Arg Pro Gln Gly Arg
1 5 10 15
Gly Cys Leu Leu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu
20 25 30
Leu Leu Ala Val Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro
35 40 45
Gln Asp Gln Gly Arg Arg Val Glu Lys Ile Ile Gly Ser Gly Ala Gln
50 55 60
Ala Gln Lys Arg Leu Asp Asp Ser Lys Pro Ser Cys Ile Leu Pro Ser
65 70 75 80
Pro Ser Ser Leu Ser Glu Thr Pro Asp Pro Arg Leu His Pro Gln Arg
85 90 95
Ser Asn Ala Ser Arg Asn Leu Ala Ser Thr Ser Gln Gly Pro Val Ala
100 105 110
Gln Ser Ser Arg Glu Ala Ser Ala Trp Met Thr Ile Leu Ser Pro Ala
115 120 125
Ala Asp Ser Thr Pro Asp Pro Gly Val Gln Gln Leu Pro Lys Gly Glu
130 135 140
Pro Glu Thr Asp Leu Asn Pro Glu Leu Pro Ala Ala His Leu Ile Gly
145 150 155 160
Ala Trp Met Ser Gly Gln Gly Leu Ser Trp Glu Ala Ser Gln Glu Glu
165 170 175
Ala Phe Leu Arg Ser Gly Ala Gln Phe Ser Pro Thr His Gly Leu Ala
180 185 190
Leu Pro Gln Asp Gly Val Tyr Tyr Leu Tyr Cys His Val Gly Tyr Arg
195 200 205
Gly Arg Thr Pro Pro Ala Gly Arg Ser Arg Ala Arg Ser Leu Thr Leu
210 215 220
Arg Ser Ala Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Arg Gly Ser Pro
225 230 235 240
Glu Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Val Asp Pro
245 250 255
Ile Gly Tyr Gly Ser Leu Trp Tyr Thr Ser Val Gly Phe Gly Gly Leu
260 265 270
Ala Gln Leu Arg Ser Gly Glu Arg Val Tyr Val Asn Ile Ser His Pro
275 280 285
Asp Met Val Asp Tyr Arg Arg Gly Lys Thr Phe Phe Gly Ala Val Met
290 295 300
Val Gly
305
<210> 25
<211> 609
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mLTA
<400> 25
atgacactgc tcggccgtct ccacctcttg agggtgcttg gcacccctcc tgtcttcctc 60
ctggggctgc tgctggccct gcctctaggg gcccagggac tctctggtgt ccgcttctcc 120
gctgccagga cagcccatcc actccctcag aagcacttga cccatggcat cctgaaacct 180
gctgctcacc ttgttgggta ccccagcaag cagaactcac tgctctggag agcaagcacg 240
gatcgtgcct ttctccgaca tggcttctct ttgagcaaca actccctcct gatccccacc 300
agtggcctct actttgtcta ctcccaggtg gttttctctg gagaaagctg ctcccccagg 360
gccattccca ctcccatcta cctggcacac gaggtccagc tcttttcctc ccaatacccc 420
ttccatgtgc ctctcctcag tgcgcagaag tctgtgtatc cgggacttca aggaccgtgg 480
gtgcgctcaa tgtaccaggg ggctgtgttc ctgctcagta agggagacca gctgtccacc 540
cacaccgacg gcatctccca tctacacttc agccccagca gtgtattctt tggagccttt 600
gcactgtag 609
<210> 26
<211> 202
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mLTA
<400> 26
Met Thr Leu Leu Gly Arg Leu His Leu Leu Arg Val Leu Gly Thr Pro
1 5 10 15
Pro Val Phe Leu Leu Gly Leu Leu Leu Ala Leu Pro Leu Gly Ala Gln
20 25 30
Gly Leu Ser Gly Val Arg Phe Ser Ala Ala Arg Thr Ala His Pro Leu
35 40 45
Pro Gln Lys His Leu Thr His Gly Ile Leu Lys Pro Ala Ala His Leu
50 55 60
Val Gly Tyr Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Ser Thr
65 70 75 80
Asp Arg Ala Phe Leu Arg His Gly Phe Ser Leu Ser Asn Asn Ser Leu
85 90 95
Leu Ile Pro Thr Ser Gly Leu Tyr Phe Val Tyr Ser Gln Val Val Phe
100 105 110
Ser Gly Glu Ser Cys Ser Pro Arg Ala Ile Pro Thr Pro Ile Tyr Leu
115 120 125
Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val Pro
130 135 140
Leu Leu Ser Ala Gln Lys Ser Val Tyr Pro Gly Leu Gln Gly Pro Trp
145 150 155 160
Val Arg Ser Met Tyr Gln Gly Ala Val Phe Leu Leu Ser Lys Gly Asp
165 170 175
Gln Leu Ser Thr His Thr Asp Gly Ile Ser His Leu His Phe Ser Pro
180 185 190
Ser Ser Val Phe Phe Gly Ala Phe Ala Leu
195 200
<210> 27
<211> 720
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mLIGHT
<400> 27
atggagagtg tggtacagcc ttcagtgttt gtggtggatg gacagacgga catcccattc 60
aggcggctgg aacagaacca ccggagacgg cgctgtggca ctgtccaggt cagcctggcc 120
ctggtgctgc tgctaggtgc tgggctggcc actcagggct ggtttctcct gagactgcat 180
caacgtcttg gagacatagt agctcatctg ccagatggag gcaaaggctc ctgggagaag 240
ctgatacaag atcaacgatc tcaccaggcc aacccagcag cacatcttac aggagccaac 300
gccagcttga taggtattgg tggacctctg ttatgggaga cacgacttgg cctggccttc 360
ttgaggggct tgacgtatca tgatggggcc ctggtgacca tggagcccgg ttactactat 420
gtgtactcca aagtgcagct gagcggcgtg ggctgccccc aggggctggc caatggcctc 480
cccatcaccc atggactata caagcgcaca tcccgctacc cgaaggagtt agaactgctg 540
gtcagtcggc ggtcaccctg tggccgggcc aacagctccc gagtctggtg ggacagcagc 600
ttcctgggcg gcgtggtaca tctggaggct ggggaagagg tggtggtccg cgtgcctgga 660
aaccgcctgg tcagaccacg tgacggcacc aggtcctatt tcggagcttt catggtctga 720
<210> 28
<211> 239
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> mLIGHT
<400> 28
Met Glu Ser Val Val Gln Pro Ser Val Phe Val Val Asp Gly Gln Thr
1 5 10 15
Asp Ile Pro Phe Arg Arg Leu Glu Gln Asn His Arg Arg Arg Arg Cys
20 25 30
Gly Thr Val Gln Val Ser Leu Ala Leu Val Leu Leu Leu Gly Ala Gly
35 40 45
Leu Ala Thr Gln Gly Trp Phe Leu Leu Arg Leu His Gln Arg Leu Gly
50 55 60
Asp Ile Val Ala His Leu Pro Asp Gly Gly Lys Gly Ser Trp Glu Lys
65 70 75 80
Leu Ile Gln Asp Gln Arg Ser His Gln Ala Asn Pro Ala Ala His Leu
85 90 95
Thr Gly Ala Asn Ala Ser Leu Ile Gly Ile Gly Gly Pro Leu Leu Trp
100 105 110
Glu Thr Arg Leu Gly Leu Ala Phe Leu Arg Gly Leu Thr Tyr His Asp
115 120 125
Gly Ala Leu Val Thr Met Glu Pro Gly Tyr Tyr Tyr Val Tyr Ser Lys
130 135 140
Val Gln Leu Ser Gly Val Gly Cys Pro Gln Gly Leu Ala Asn Gly Leu
145 150 155 160
Pro Ile Thr His Gly Leu Tyr Lys Arg Thr Ser Arg Tyr Pro Lys Glu
165 170 175
Leu Glu Leu Leu Val Ser Arg Arg Ser Pro Cys Gly Arg Ala Asn Ser
180 185 190
Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu
195 200 205
Glu Ala Gly Glu Glu Val Val Val Arg Val Pro Gly Asn Arg Leu Val
210 215 220
Arg Pro Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val
225 230 235
<210> 29
<211> 27
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> CD28 transmembrane Domain
<400> 29
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 30
<211> 81
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> CD28 transmembrane domain
<400> 30
ttttgggtcc tcgtcgtagt tggaggggta cttgcctgtt atagcctcct ggttaccgta 60
gcatttatta tattctgggt g 81
<210> 31
<211> 41
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> CD28 Co-stimulatory Domain
<400> 31
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 32
<211> 123
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> CD28 Co-stimulatory Domain
<400> 32
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 33
<211> 39
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> CD28 hinge region
<400> 33
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro
35
<210> 34
<211> 117
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> CD28 hinge region
<400> 34
attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60
catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117
<210> 35
<211> 12
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> IgG4 hinge region
<400> 35
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 36
<211> 36
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> IgG4 hinge region
<400> 36
gaaagcaaat acgggccgcc gtgtccaccc tgtccg 36
<210> 37
<211> 18
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> T2A
<400> 37
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 38
<211> 22
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> F2A
<400> 38
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
1 5 10 15
Glu Ser Asn Pro Gly Pro
20
<210> 39
<211> 618
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLTA
<400> 39
atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc 60
cttctggggc tgctgctggt tctgctgcct ggggcccagg ggctccctgg tgttggcctc 120
acaccttcag ctgcccagac tgcccgtcag caccccaaga tgcatcttgc ccacagcacc 180
ctcaaacctg ctgctcacct cattggagac cccagcaagc agaactcact gctctggaga 240
gcaaacacgg accgtgcctt cctccaggat ggtttctcct tgagcaacaa ttctctcctg 300
gtccccacca gtggcatcta cttcgtctac tcccaggtgg tcttctctgg gaaagcctac 360
tctcccaagg ccacctcctc cccactctac ctggcccatg aggtccagct cttctcctcc 420
cagtacccct tccatgtgcc tctcctcagc tcccagaaga tggtgtatcc agggctgcag 480
gaaccctggc tgcactcgat gtaccacggg gctgcgttcc agctcaccca gggagaccag 540
ctatccaccc acacagatgg catcccccac ctagtcctca gccctagtac tgtcttcttt 600
ggagccttcg ctctgtag 618
<210> 40
<211> 205
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLTA
<400> 40
Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr
1 5 10 15
Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala
20 25 30
Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala
35 40 45
Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala
50 55 60
Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg
65 70 75 80
Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn
85 90 95
Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln
100 105 110
Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro
115 120 125
Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe
130 135 140
His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln
145 150 155 160
Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr
165 170 175
Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val
180 185 190
Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu
195 200 205
<210> 41
<211> 735
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLTB
<400> 41
atgggggcac tggggctgga gggcaggggt gggaggctcc aggggagggg ttccctcctg 60
ctagctgtgg caggagccac ttctctggtg accttgttgc tggcggtgcc tatcactgtc 120
ctggctgtgc tggccttagt gccccaggat cagggaggac tggtaacgga gacggccgac 180
cccggggcac aggcccagca aggactgggg tttcagaagc tgccagagga ggagccagaa 240
acagatctca gccccgggct cccagctgcc cacctcatag gcgctccgct gaaggggcag 300
gggctaggct gggagacgac gaaggaacag gcgtttctga cgagcgggac gcagttctcg 360
gacgccgagg ggctggcgct cccgcaggac ggcctctatt acctctactg tctcgtcggc 420
taccggggcc gggcgccccc tggcggcggg gacccccagg gccgctcggt cacgctgcgc 480
agctctctgt accgggcggg gggcgcctac gggccgggca ctcccgagct gctgctcgag 540
ggcgccgaga cggtgactcc agtgctggac ccggccagga gacaagggta cgggcctctc 600
tggtacacga gcgtggggtt cggcggcctg gtgcagctcc ggaggggcga gagggtgtac 660
gtcaacatca gtcaccccga tatggtggac ttcgcgagag ggaagacctt ctttggggcc 720
gtgatggtgg ggtga 735
<210> 42
<211> 244
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLTB
<400> 42
Met Gly Ala Leu Gly Leu Glu Gly Arg Gly Gly Arg Leu Gln Gly Arg
1 5 10 15
Gly Ser Leu Leu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu
20 25 30
Leu Leu Ala Val Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro
35 40 45
Gln Asp Gln Gly Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln
50 55 60
Ala Gln Gln Gly Leu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu
65 70 75 80
Thr Asp Leu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro
85 90 95
Leu Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe
100 105 110
Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro
115 120 125
Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg
130 135 140
Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg
145 150 155 160
Ser Ser Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu
165 170 175
Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala
180 185 190
Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly
195 200 205
Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser
210 215 220
His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala
225 230 235 240
Val Met Val Gly
<210> 43
<211> 723
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLIGHT
<400> 43
atggaggaga gtgtcgtacg gccctcagtg tttgtggtgg atggacagac cgacatccca 60
ttcacgaggc tgggacgaag ccaccggaga cagtcgtgca gtgtggcccg ggtgggtctg 120
ggtctcttgc tgttgctgat gggggccggg ctggccgtcc aaggctggtt cctcctgcag 180
ctgcactggc gtctaggaga gatggtcacc cgcctgcctg acggacctgc aggctcctgg 240
gagcagctga tacaagagcg aaggtctcac gaggtcaacc cagcagcgca tctcacaggg 300
gccaactcca gcttgaccgg cagcgggggg ccgctgttat gggagactca gctgggcctg 360
gccttcctga ggggcctcag ctaccacgat ggggcccttg tggtcaccaa agctggctac 420
tactacatct actccaaggt gcagctgggc ggtgtgggct gcccgctggg cctggccagc 480
accatcaccc acggcctcta caagcgcaca ccccgctacc ccgaggagct ggagctgttg 540
gtcagccagc agtcaccctg cggacgggcc accagcagct cccgggtctg gtgggacagc 600
agcttcctgg gtggtgtggt acacctggag gctggggaga aggtggtcgt ccgtgtgctg 660
gatgaacgcc tggttcgact gcgtgatggt acccggtctt acttcggggc tttcatggtg 720
tga 723
<210> 44
<211> 240
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLIGHT
<400> 44
Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Gln
1 5 10 15
Thr Asp Ile Pro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser
20 25 30
Cys Ser Val Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly
35 40 45
Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg
50 55 60
Leu Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp
65 70 75 80
Glu Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala
85 90 95
His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu
100 105 110
Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr
115 120 125
His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr
130 135 140
Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser
145 150 155 160
Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu
165 170 175
Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser
180 185 190
Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His
195 200 205
Leu Glu Ala Gly Glu Lys Val Val Val Arg Val Leu Asp Glu Arg Leu
210 215 220
Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val
225 230 235 240
<210> 45
<211> 510
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> signal peptide-free mLTA
<400> 45
ctctctggtg tccgcttctc cgctgccagg acagcccatc cactccctca gaagcacttg 60
acccatggca tcctgaaacc tgctgctcac cttgttgggt accccagcaa gcagaactca 120
ctgctctgga gagcaagcac ggatcgtgcc tttctccgac atggcttctc tttgagcaac 180
aactccctcc tgatccccac cagtggcctc tactttgtct actcccaggt ggttttctct 240
ggagaaagct gctcccccag ggccattccc actcccatct acctggcaca cgaggtccag 300
ctcttttcct cccaataccc cttccatgtg cctctcctca gtgcgcagaa gtctgtgtat 360
ccgggacttc aaggaccgtg ggtgcgctca atgtaccagg gggctgtgtt cctgctcagt 420
aagggagacc agctgtccac ccacaccgac ggcatctccc atctacactt cagccccagc 480
agtgtattct ttggagcctt tgcactgtag 510
<210> 46
<211> 169
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> signal peptide-free mlTA
<400> 46
Leu Ser Gly Val Arg Phe Ser Ala Ala Arg Thr Ala His Pro Leu Pro
1 5 10 15
Gln Lys His Leu Thr His Gly Ile Leu Lys Pro Ala Ala His Leu Val
20 25 30
Gly Tyr Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Ser Thr Asp
35 40 45
Arg Ala Phe Leu Arg His Gly Phe Ser Leu Ser Asn Asn Ser Leu Leu
50 55 60
Ile Pro Thr Ser Gly Leu Tyr Phe Val Tyr Ser Gln Val Val Phe Ser
65 70 75 80
Gly Glu Ser Cys Ser Pro Arg Ala Ile Pro Thr Pro Ile Tyr Leu Ala
85 90 95
His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val Pro Leu
100 105 110
Leu Ser Ala Gln Lys Ser Val Tyr Pro Gly Leu Gln Gly Pro Trp Val
115 120 125
Arg Ser Met Tyr Gln Gly Ala Val Phe Leu Leu Ser Lys Gly Asp Gln
130 135 140
Leu Ser Thr His Thr Asp Gly Ile Ser His Leu His Phe Ser Pro Ser
145 150 155 160
Ser Val Phe Phe Gly Ala Phe Ala Leu
165
<210> 47
<211> 516
<212> DNA
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLTA without Signal peptide
<400> 47
ctccctggtg ttggcctcac accttcagct gcccagactg cccgtcagca ccccaagatg 60
catcttgccc acagcaccct caaacctgct gctcacctca ttggagaccc cagcaagcag 120
aactcactgc tctggagagc aaacacggac cgtgccttcc tccaggatgg tttctccttg 180
agcaacaatt ctctcctggt ccccaccagt ggcatctact tcgtctactc ccaggtggtc 240
ttctctggga aagcctactc tcccaaggcc acctcctccc cactctacct ggcccatgag 300
gtccagctct tctcctccca gtaccccttc catgtgcctc tcctcagctc ccagaagatg 360
gtgtatccag ggctgcagga accctggctg cactcgatgt accacggggc tgcgttccag 420
ctcacccagg gagaccagct atccacccac acagatggca tcccccacct agtcctcagc 480
cctagtactg tcttctttgg agccttcgct ctgtag 516
<210> 48
<211> 171
<212> PRT
<213> Artificial sequence(Artificial Sequence)
<220>
<223> hLTA without Signal peptide
<400> 48
Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln
1 5 10 15
His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala Ala His
20 25 30
Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Asn
35 40 45
Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn Asn Ser
50 55 60
Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln Val Val
65 70 75 80
Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro Leu Tyr
85 90 95
Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val
100 105 110
Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln Glu Pro
115 120 125
Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr Gln Gly
130 135 140
Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val Leu Ser
145 150 155 160
Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu
165 170

Claims (18)

1. An engineered immune cell that expresses (i) a cell surface molecule that specifically recognizes an antigen, and (ii) one or more LTBR ligands.
2. The engineered immune cell of claim 1, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a recombinant T cell receptor.
3. The engineered immune cell of claim 2, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor comprising: an antigen binding region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.
4. The engineered immune cell of any one of claims 1-3, wherein the LTBR ligand is selected from the group consisting of LTA, LTB, LIGHT and an anti-LTBR antibody.
5. The engineered immune cell of any one of claims 1-3, wherein LTA is identical to SEQ ID NO: 26. 40, 46 or 48, and LTB has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:24 or 42, LIGHT is at least 90% identical to SEQ ID NO:28 or 44 has at least 90% identity.
6. The engineered immune cell of any one of claims 1-4, wherein the immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.
7. The engineered immune cell of claim 5, wherein the T cell is a CD4+/CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γ δ -T cell, or an α β -T cell.
8. The engineered immune cell of any one of claims 3-6, wherein the antigen binding region is selected from the group consisting of IgG, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin, and DARPIN antigen.
9. The engineered immune cell of claim 3, wherein the antigen-antigen binding region binds to a target selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1 DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/neu), erbB3, erbB4 MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, C-Met, prostase, PAP, ELF2M, ephrinB2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, ephA2, fucosyl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVR 1, ADRB3, PANX3, and GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-L/Galectin8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, LAP, LAM, TMPRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF β, APRIL, NKG2D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecule expressed by HIV, HCV, HBV and/or other pathogen; and/or a neoepitope or neoantigen. The engineered immune cell of any one of claims 3-8, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
10. The engineered immune cell of any one of claims 3-9, wherein the intracellular signaling domain is selected from the signaling domains of the following proteins: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 epsilon, CD3 ζ, CD22, CD79a, CD79b, and CD66d.
11. The engineered immune cell of any one of claims 3-10, wherein the costimulatory domain is one or more costimulatory signaling domains of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.
12. The engineered immune cell of any one of claims 1-11, wherein the immune cell further comprises at least one inactivated gene selected from the group consisting of: CD52, GR, TCR α, TCR β, CD3 γ, CD3 δ, CD3 ε, CD247 ζ, HLA-I, HLA-II, B2M, PD1, CTLA-4, LAG3, and TIM3.
13. A nucleic acid molecule comprising: (i) A nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, and (ii) a nucleic acid sequence encoding an LTBR ligand.
14. The nucleic acid molecule of claim 13, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor.
15. A vector comprising the nucleic acid molecule of any one of claims 13-14.
16. The vector of claim 18, wherein the vector is selected from the group consisting of a plasmid, a retrovirus, a lentivirus, an adenovirus, a vaccinia virus, a Rous Sarcoma Virus (RSV), a polyoma virus, and an adeno-associated virus (AAV).
17. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 1-12, a nucleic acid molecule according to any one of claims 13-14 or a vector according to any one of claims 15-16, and one or more pharmaceutically acceptable excipients.
18. A method of treating a subject having cancer, an infection, or an autoimmune disease, comprising administering to the subject the engineered immune cell of any one of claims 1-12 or the pharmaceutical composition of claim 17.
CN202110710299.XA 2021-06-25 2021-06-25 Engineered immune cells and uses thereof Pending CN115521917A (en)

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WO2024114767A1 (en) * 2022-12-01 2024-06-06 南京北恒生物科技有限公司 Immune rejection-resistant engineered cell

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US20220025001A1 (en) * 2016-04-28 2022-01-27 The Trustees Of Dartmouth College Nucleic acid constructs for co-expression of chimeric antigen receptor and transcription factor, cells containing and therapeutic use thereof
CN110343667A (en) * 2019-07-17 2019-10-18 贝赛尔特(北京)生物技术有限公司 Immunocyte of engineering and its preparation method and application
CN112063588A (en) * 2020-08-13 2020-12-11 南京北恒生物科技有限公司 Engineered immune cells and uses thereof
CN112778427B (en) * 2021-01-29 2022-03-15 武汉思安医疗技术有限公司 Bispecific CS1-BCMA CAR-T cells and uses thereof

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* Cited by examiner, † Cited by third party
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WO2024114767A1 (en) * 2022-12-01 2024-06-06 南京北恒生物科技有限公司 Immune rejection-resistant engineered cell

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