WO2022223049A1 - Cellule immunitaire modifiée et son utilisation - Google Patents

Cellule immunitaire modifiée et son utilisation Download PDF

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WO2022223049A1
WO2022223049A1 PCT/CN2022/089109 CN2022089109W WO2022223049A1 WO 2022223049 A1 WO2022223049 A1 WO 2022223049A1 CN 2022089109 W CN2022089109 W CN 2022089109W WO 2022223049 A1 WO2022223049 A1 WO 2022223049A1
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cells
cell
engineered immune
immune cell
domain
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PCT/CN2022/089109
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Chinese (zh)
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邢芸
李国坤
任江涛
贺小宏
王延宾
韩露
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南京北恒生物科技有限公司
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Publication of WO2022223049A1 publication Critical patent/WO2022223049A1/fr

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Definitions

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell that expresses (i) cell surface molecules that specifically recognize ligands, (ii) exogenous cytokines selected from IL12, IL18, IL-21, IL23 and IL33, and (iii) an exogenous chemokine selected from XCL2XCL1. More preferably, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor.
  • Tumor immunotherapy mainly relies on autoimmunity to eliminate tumor cells by regulating the human immune system and tumor microenvironment.
  • the immune system is a unified whole, and innate immunity also plays a very important role in tumor immunity.
  • Some antigen-presenting cells such as dendritic cells and macrophages, are the bridge between innate and adaptive immunity. Antigen-presenting cells can recognize and present tumor antigens to the adaptive immune system, activate tumor-specific T cells, and then eliminate tumors. Therefore, enhancing the tumor-killing effect of the immune system by enhancing the antigen presentation process is an important research direction of tumor immunity.
  • CAR cell therapy is an important tumor cell immunotherapy.
  • the successful tumor control of CAR cells generally requires the following processes: activation of the immune system, activation and expansion of CAR cells, infiltration of tumor tissue by activated CAR cells and killing of tumor cells.
  • the tumor microenvironment has an inhibitory effect on CAR cells, so that CAR cells cannot infiltrate tumor tissue. Therefore, how to reduce the inhibitory effect of the tumor microenvironment on CAR cells, improve the survival time of CAR cells, or recruit other immune cells to synergize with CAR cells is very important to improve the therapeutic effect of CAR cells.
  • an improved immunotherapy method which can improve the efficiency of tumor antigen presentation, induce the adoptive immune response of the body, solve the problem of tumor heterogeneity and the inhibitory effect of the tumor microenvironment, so as to improve the efficacy of CAR cell therapy.
  • the present invention provides a novel engineered immune cell that expresses (i) a cell surface molecule that specifically recognizes a ligand, (ii) an exogenous cytokine selected from the group consisting of IL12, IL18, IL-21, IL23 and IL33, and (iii) an exogenous chemokine selected from XCL2 and XCL1.
  • the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler or a T cell receptor, preferably a chimeric antigen receptor.
  • the cytokine or chemokine protein is a fusion protein or mutant that is resistant to proteolysis.
  • the immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells.
  • the T cells are CD4+/CD8+ T cells, CD4+ helper T cells, CD8+ T cells, CD4-CD8- T cells, tumor infiltrating cells, memory T cells, naive T cells, regulatory T cells, ⁇ -T cells or ⁇ -T cells.
  • the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain and an intracellular domain comprising a common Stimulatory domains and/or primary signaling domains.
  • the ligand binding domain can be selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin and Darpin.
  • the ligand binding domain is selected from the group consisting of scFv, Fab, single domain antibodies and Nanobodies.
  • the cell surface molecule that specifically recognizes the ligand binds to a target selected from CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23 , CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD179a, 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
  • the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain is selected from the transmembrane domains of CD8 ⁇ , CD4, CD28 and CD278.
  • the primary signaling domain is selected from the signaling domains of the following proteins: FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is a CD3 ⁇ -comprising signaling domain.
  • the costimulatory domain is one or more intracellular domains selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 ( B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.
  • the costimulatory domain is the intracellular region of CD27, CD28, CD134, CD137 or CD278 or a combination thereof.
  • the expression or activity of exogenous cytokines eg, IL12, IL18, IL21, IL23, and IL33
  • chemokines eg, XCL1, XCL2
  • the expression or activity of the exogenous cytokine and/or chemokine is conditional expression.
  • Conditional expression is achieved, for example, by operably linking the exogenous gene to an inducible, repressible, or tissue-specific promoter.
  • cytokines and/or chemokines can be operably linked to a localization domain that can localize the exogenous gene of the invention for expression at a specific cellular location, eg, cell membrane, cytoplasm Specific organelles such as endoplasmic reticulum, Golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, guide peptides, transmembrane domains, and the like.
  • the exogenous genetic cytokines and/or chemokines of the invention are operably linked to the transmembrane domain so as to be anchored for expression on the surface of engineered immune cells.
  • the present invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes a ligand, (ii) a nucleic acid sequence encoding a cytokine selected from the group consisting of IL21, IL12 and IL23, and (iii) a nucleic acid sequence encoding a chemokine selected from the group consisting of XCL1 and XCL2.
  • the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler or a T cell receptor, more preferably a chimeric antigen receptor.
  • the nucleic acid is DNA or RNA.
  • the present invention also provides a vector comprising the above-described nucleic acid molecule.
  • the vector is selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV).
  • the vector further comprises an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a polyadenylation tail (polyA), 3'UTR, 5'UTR, enhancer elements such as promoters, terminators, insulators, operons, selectable markers, reporter genes, targeting sequences and/or protein purification tags.
  • the vector is an in vitro transcribed vector.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered immune cells, nucleic acid molecules or vectors of the present invention, and one or more pharmaceutically acceptable excipients.
  • the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell according to the present invention, Nucleic acid molecules, vectors or pharmaceutical compositions.
  • the cancer is a solid tumor or a hematological tumor. More specifically, the cancer is selected from the group consisting of: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, 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, glioblastoma (GBM), liver cancer, hepatocellular tumor, Intraepithelial tumor, kidney cancer, throat cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectum cancer, cancer of the respiratory system
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.
  • the advantage of the engineered immune cells of the present invention is that the co-expressed cytokines and/or chemokines can effectively promote the differentiation or recruitment of DC cells at the tumor site, increase the number of DC cells, increase the proliferation of engineered immune cells and
  • the inhibitory effect of the tumor microenvironment on engineered immune cells can be reduced, and the tumor killing ability of engineered immune cells can be improved.
  • the increased DC cells can activate the adoptive immune recognition of the body's own T cells.
  • the immune cells form a synergistic effect, which ultimately enhances tumor suppression.
  • Figure 1 CAR expression levels of CAR-T cells determined by flow cytometry.
  • Figure 2 XCL1 expression levels of CAR-T cells determined by ELISA.
  • Figure 3 IL-21 expression levels of CAR-T cells determined by ELISA.
  • FIG. 4 IFN- ⁇ release levels after CAR-T cells were co-cultured with target cells and non-target cells, respectively.
  • Figure 5 The killing effect of CAR-T cells on target cells under different effector-target ratios.
  • Figure 6 Changes in body weight of mice after treating pancreatic cancer with CAR-T cells.
  • Figure 7 Tumor growth curve in mice after treatment of mouse pancreatic cancer with CAR-T cells.
  • Figure 8 IFN- ⁇ release levels after CAR-T cells expressing IL12, IL23 or their combination with XCL1 were co-cultured with target cells and non-target cells, respectively.
  • Figure 9 IFN- ⁇ release levels after CAR-T cells expressing IL18, IL33 or their combination with XCL1 were co-cultured with target cells and non-target cells, respectively.
  • Figure 10 Tumor inhibitory effect of CAR-T cells expressing IL12, IL23 or their combination with XCL1 in vivo.
  • Figure 11 Tumor inhibitory effect of CAR-T cells expressing IL18, IL33 or their combination with XCL1 in vivo.
  • cell surface molecule that specifically recognizes a ligand refers to a molecule expressed on the surface of a cell capable of specifically binding to a target molecule (eg, a ligand).
  • a target molecule eg, a ligand
  • Such surface molecules generally comprise a ligand binding domain capable of specifically binding the ligand, 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, T cell receptors (TCRs), chimeric antigen receptors (CARs), T cell fusion proteins (TFPs), or T cell antigen couplers (TACs).
  • T cell receptor refers to a membrane protein complex that is involved in T cell activation in response to antigen presentation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHCs) on antigen-presenting cells that present antigenic peptides to T cells and bind to the TCR complex to induce a cascade of intracellular signaling.
  • MHCs major histocompatibility complex molecules
  • TCR consists of six peptide chains that form heterodimers respectively, which are generally classified into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specificity to a specific antigen and MHC molecule.
  • the variable region of the TCR may comprise or be operably linked to a ligand binding domain, wherein the ligand binding domain is defined below.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes a ligand-binding domain (eg, an antigen-binding portion of an antibody), a transmembrane domain, and An intracellular domain comprising a costimulatory domain and/or a primary signaling domain, each of which is connected by a linker.
  • CARs can exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in an MHC-non-restricted manner.
  • Non-MHC-restricted antigen recognition gives CAR cells the ability to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape.
  • CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).
  • T cell fusion protein refers to a recombinant polypeptide derived from components of the TCR, typically consisting of a TCR subunit and an antigen-binding region linked thereto, and expressed on the cell surface.
  • the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.
  • T cell antigen coupler includes three functional domains: 1 Tumor targeting domains, including single chain antibodies, designed ankyrin repeat proteins (DARPins) or Other targeting groups; 2 extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor close to the TCR receptor; 3 transmembrane region and intracellular region of the CD4 co-receptor, where the intracellular Domain-linked protein kinase LCK, catalyzes the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) of the TCR complex as an initial step in T cell activation.
  • TAC tumor targeting domains
  • DARPins ankyrin repeat proteins
  • Other targeting groups 2 extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor close to the TCR receptor
  • 3 transmembrane region and intracellular region of the CD4 co-receptor where the intracellular Domain-linked protein kinase LCK, cata
  • ligand binding domain refers to any structure or functional variant thereof that can bind a ligand (eg, an antigen).
  • the ligand binding domain can be an antibody structure including, but not limited to, monoclonal, polyclonal, recombinant, human, humanized, murine, chimeric, and functional fragments thereof.
  • ligand binding domains include, but are not limited to, IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies, Anticalin, DARPIN, etc., are preferably selected from Fab, scFv, sdAb and Nanobodies.
  • the ligand binding domain may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody.
  • the ligand binding domain may also be a native specific binding polypeptide or native receptor structure of a particular protein, such as PDl, PDLl, PDL2, TGF[beta], APRIL and NKG2D.
  • Fab refers to either of two identical fragments produced upon cleavage of an immunoglobulin molecule by papain, consisting of an intact light chain and an N-terminal portion of a heavy chain linked by disulfide bonds, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared to intact IgG, Fab has no Fc fragment, has higher mobility and tissue penetration, and can bind antigen monovalently without mediating antibody effects.
  • a “single chain antibody” or “scFv” is an antibody composed of an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker significantly affects the variable region folding and interaction of scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids).
  • linker size and composition see, eg, Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448; US Patent Application Publication Nos.
  • the scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.
  • Single-domain antibody refers to an antibody that is naturally deficient in its light chain, and which contains only one variable heavy chain region (VHH) and two conventional CH2 and CH3 regions, also referred to as a “heavy chain” Antibody”.
  • VHH variable heavy chain region
  • CH2 and CH3 regions also referred to as a “heavy chain” Antibody
  • Nemobody or “Nb” refers to a single cloned and expressed VHH structure, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind to the 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 (ie, substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variants retain the biological activity of the parent amino acid sequence.
  • the amino acid modification is preferably a conservative modification.
  • conservative modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These 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 substitutions in which amino acid residues are replaced by amino acid residues having similar side chains.
  • Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • threonine valine, isoleucine
  • aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • a “functional variant” or “functional fragment” is at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.
  • sequence identity refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical.
  • sequence identity can be determined 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 ligand binding domains of the invention bind 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, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD179a , 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, EG
  • the CAR of the present invention can be designed to include a ligand binding domain specific for that antigen.
  • a ligand binding domain specific for that antigen for example, if CD19 is the antigen to be targeted, an antibody to CD19 can be used as the ligand binding domain of the invention.
  • the CAR of the present invention comprises a CD19 scFv comprising at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 2, positions 1-107, or SEQ ID NO: 14, positions 1-107 A light chain variable region sequence of % or 99% or 100% sequence identity and having at least 90%, Heavy chain variable region sequences of 95%, 97% or 99% or 100% sequence identity.
  • the cell surface molecule of the invention that specifically recognizes a ligand is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain and an intracellular domain, wherein the intracellular structure Domains comprise costimulatory domains and/or primary signaling domains.
  • transmembrane domain refers to a polypeptide that enables expression of a chimeric antigen receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells structure.
  • immune cells eg, lymphocytes, NK cells, or NKT cells
  • the transmembrane domain can be natural or synthetic, and can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling when the chimeric antigen receptor binds to the target antigen.
  • Transmembrane domains particularly useful in the present invention may be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments.
  • the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from the CD8 alpha chain, which is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence of the CD8 ⁇ transmembrane domain and the nucleotide sequence shown in SEQ ID NO: 3 or 15 have at least 70%, preferably at least 80%, more preferably at least 90% %, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the present invention may further comprise a hinge region between the ligand binding domain and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand binding domain. Specifically, the hinge region serves to provide greater flexibility and accessibility to the ligand binding domain.
  • 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 can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises a hinge region portion of a CD8 ⁇ chain, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably a CD8 ⁇ hinge, which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 12 or 22 , preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence of the CD8 ⁇ hinge and the nucleotide sequence shown in SEQ ID NO: 11 or 21 have At least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • intracellular domain refers to the intracellular portion of a cell surface molecule that specifically recognizes a ligand, including costimulatory domains and/or primary signaling domains.
  • primary signaling domain refers to the portion of a protein that transduces effector function signals and directs cells to perform specified functions.
  • the primary signaling domain is responsible for the intracellular primary signaling following binding of the ligand binding domain to the antigen, resulting in the activation of immune cells and immune responses.
  • the primary signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed.
  • the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.
  • the chimeric antigen receptors of the invention comprise primary signaling domains that may be cytoplasmic sequences of T cell receptors and co-receptors that act together upon antigen receptor binding to initiate primary signaling , as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar function.
  • the primary signaling domain can contain a number of immunoreceptor tyrosine-based activation motifs (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • Non-limiting examples of primary signaling domains of the invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the signaling domain of the CAR of the present invention may comprise a CD3 ⁇ signaling domain having at least 70%, preferably at least 80% of the amino acid sequence shown in SEQ ID NO: 8 or 20. , more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, with the nucleotide sequence shown in SEQ ID NO: 7 or 19 %, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the invention comprise one or more costimulatory domains.
  • a costimulatory domain can be an intracellular functional signaling domain from a costimulatory molecule that comprises the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.
  • a "costimulatory molecule” refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, 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 costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA) , CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.
  • costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 ,
  • the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40 or a combination thereof.
  • the CAR of the present invention comprises a costimulatory domain that is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 6 or 18 or 99% or 100% sequence identity, or the coding sequence of the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleotide sequence shown in SEQ ID NO: 5 or 17 , 95%, 97% or 99% or 100% sequence identity.
  • the CAR of the present invention may further comprise a signal peptide such that when it is expressed in a cell such as 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 segment of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptidases At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases.
  • the signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • Signal peptides useful in the present invention are well known to those skilled in the art, eg, signal peptides derived from CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the signal peptide useful in the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence set forth in SEQ ID NO: 10 or 30 or 100% sequence identity, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or the amino acid sequence shown in SEQ ID NO: 9 or 29 99% or 100% sequence identity.
  • the CAR of the present invention may further comprise a switch structure to regulate the expression time of the CAR.
  • the switch structure can be in the form of a dimerization domain that induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain for binding to the targeted antigen, thereby activating a signaling pathway.
  • switch domains can be used to connect the binding and signaling domains, respectively, which can dimerize only when the switch domains bind to each other (eg, in the presence of an inducing compound) linked together, thereby activating signaling pathways.
  • the switch structure can also be in the form of a masked peptide.
  • the masking peptide can mask the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure.
  • Various switch structures known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may further comprise a suicide gene, i.e., causing it to express a cell death signal that can be induced by a foreign substance, to clear the CAR cells when needed (eg, when severe toxic side effects occur).
  • suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when desired, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes.
  • the suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir treatment.
  • HSV-TK herpes simplex virus thymidine kinase
  • the suicide gene can also be iCaspase-9, which can be induced to dimerize by chemical inducing drugs such as AP1903, AP20187, etc., thereby activating the downstream Caspase3 molecule, leading to apoptosis.
  • chemical inducing drugs such as AP1903, AP20187, etc.
  • Various suicide genes known to those skilled in the art can be used in the present invention.
  • the chimeric antigen receptor comprises: (a) a 4-1BB costimulatory domain and a CD3 ⁇ intracellular signaling domain, (b) a CD27 costimulatory domain and a CD3 ⁇ intracellular signaling domain Transduction domain, (c) CD28 costimulatory domain and CD3 ⁇ intracellular signaling domain, (d) OX40 costimulatory domain and CD3 ⁇ intracellular signaling domain, (e) CD28 costimulatory domain, 4-1BB Costimulatory domain and CD3 ⁇ intracellular signaling domain, (f) OX40 costimulatory domain, 4-1BB costimulatory domain and CD3 ⁇ intracellular signaling domain, or (g) CD28 costimulatory domain, OX40 costimulatory domain Stimulatory domain and CD3 ⁇ intracellular signaling domain.
  • the engineered immune cells of the present invention also express one or more exogenous cytokines and one or more exogenous chemokines selected from the group consisting of IL12, IL18, IL21, IL23 and IL33, the chemokine is selected from XCL1 and XCL2.
  • the cytokines used in the present invention are mainly interleukins, which are selected from IL-21, IL12, IL18, IL33 and IL23.
  • Interleukins are produced by leukocytes and function among leukocytes, such as transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and playing an important role in inflammatory responses.
  • the biological effects of interleukins are achieved through their binding to the corresponding receptors, for example, the biological properties of IL-21 are achieved through the binding of IL-21 to its receptor IL-21R.
  • At least 38 interleukins have been found, named IL1 to IL38 respectively, and their functions are complex.
  • interleukins can be divided into several protein families, such as IL1 family (including IL1 ⁇ , IL1 ⁇ , IL18, IL37, IL38, IL33, etc.), IL2 family (including IL2, IL4, IL13, IL15, IL21, etc.) ), IL6 family (including IL6, IL12, IL23, IL27, IL35, etc.), IL10 family (including IL10, IL19, IL20, IL22, IL26, etc.), IL17 family (including IL17 and IL25) and other interleukins (including IL5, IL7 , IL9, IL11, IL14, IL16, IL31, IL32, etc.).
  • the cytokine used in the present invention is a wild-type or a variant thereof that has the same or similar, or even better, function than the wild-type.
  • amino acid sequence of the propeptide of wild-type hIL-21 is shown in SEQ ID NO: 32, and the amino acid sequence of the mature peptide is shown in SEQ ID NO: 34 (composed of amino acids 30-162 of SEQ ID NO: 32).
  • IL-21 is produced by activated CD4+ T cells, NKT cells, Tfh cells and Th17 cells, and has high homology with IL-2 and IL-15.
  • IL-21 has a wide range of immunoregulatory functions, and activation of it can enhance the proliferation of activated CD8+ T cells, enhance the cytotoxic activity of NK cells, and promote the proliferation and differentiation of B cells.
  • the IL-21 used in the present invention is wild-type IL-21 or an IL-21 variant, eg, an IL-21 variant that retains at least all or most of the IL-21 activity, even with improved activity.
  • the IL-21 variant used in the present invention may be, for example, a variant sequence obtained by deleting and/or substituting one or more amino acids in amino acids 65-96 of SEQ ID NO: 34, such as : (1) deletion and/or substitution of amino acids 83-86, amino acids 83-88, amino acids 83-90, amino acids 82-88, amino acids 77-92 of SEQ ID NO: 34, Variant sequences obtained from amino acids 71-92, amino acids 65-92, and amino acids 77-96; (2) Variant sequences containing additional N-terminal Met on the basis of (1); (3) Variant sequences obtained by conservatively modifying up to 10 amino acids in the sequences of (1) and (2). See, eg, WO2006111524, the entire contents of which are incorporated herein by reference.
  • the polypeptide encoded by the IL-21 gene used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 32, 34 or 36 , 97% or 99% or 100% sequence identity, or the IL-21 gene has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleic acid sequence shown in SEQ ID NO: 31, 33 or 35, 95%, 97% or 99% or 100% sequence identity.
  • IL12 is a heterodimeric cytokine encoded by two independent genes IL-12A (p35) and IL-12B (p40). IL12 is produced by dendritic cells, macrophages, neutrophils, and other antigen-presenting cells, and can promote the proliferation of T helper 1 (Th1) cells; induce NK cells and T cells to produce gamma interferon, and improve NK cells Toxicity; promote the formation of cytotoxic T cells, etc.
  • Th1 T helper 1
  • the IL12 used in the present invention is wild-type IL12 or an IL12 variant, eg, an IL12 variant that retains at least all or most of the IL12 activity, even with improved activity.
  • the IL12 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 40 or 42 % sequence identity, or the gene encoding IL-12 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% with the nucleic acid sequence shown in SEQ ID NO: 39 or 41 or 100% sequence identity.
  • IL23 is also a heterodimeric cytokine composed of two subunits, p40 and p19. IL23 is mainly produced by activated dendritic cells, macrophages and monocytes. IL23 has been reported to play an important role in experimental autoimmune encephalomyelitis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, tumor development, and infection.
  • the IL-23 used in the present invention is wild-type IL-23 or an IL-23 variant, eg, an IL-23 variant that retains at least all or most of the IL-23 activity, even with improved activity.
  • the IL-23 used in the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence shown in SEQ ID NO: 48 or 50 or 100% sequence identity, or the gene encoding IL-23 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or the nucleic acid sequence shown in SEQ ID NO: 47 or 49 99% or 100% sequence identity.
  • IL18 is mainly produced by macrophages, but can also be produced by various immune and non-immune cells such as monocytes, dendritic cells, epithelial cells, and fibroblasts.
  • IL18 mediates the MyD88-NF ⁇ B signaling pathway by binding to the heterodimeric receptor IL18R ⁇ /R ⁇ and can strongly induce the secretion of IFN ⁇ . Since IL18 can induce the proliferation and enhance the activity of immune cells, its role in anti-tumor, anti-infection and immune regulation has been widely studied in recent years.
  • the IL18 used in the present invention is wild-type IL18 or an IL18 variant, eg, an IL18 variant that retains at least all or most of the IL18 activity, even with improved activity.
  • the IL18 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 44 or 46 % sequence identity, or the gene encoding IL18 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleic acid sequence shown in SEQ ID NO: 43 or 45 % sequence identity.
  • IL33 is a bifunctional protein that can act as a transcription factor as a molecule located in the nucleus, and can be secreted to the outside of the cell to act as a cytokine. It is associated with many diseases such as allergies, autoimmune diseases, cardiovascular Infections and tumors are closely related. IL33 is expressed in a variety of cells, including epithelial cells, fibroblasts, macrophages, endothelial cells, mast cells, dendritic cells, and osteoblasts. These cells release IL33 when they are injured, and IL33 can produce different inflammatory responses depending on the cell type.
  • the IL33 used in the present invention is wild-type IL33 or an IL33 variant, eg, an IL33 variant that retains at least all or most of the IL33 activity, even with improved activity.
  • full-length IL33 can be cleaved into mature forms IL33 95-270 (consisting of amino acids 95-270 of full-length IL33), IL33 99-270 (consisting of amino acids 99-270 of full-length IL33) composition) and IL33 109-270 (consisting of amino acids 109-270 of full-length IL33), these fragments are both released extracellularly from damaged or necrotic cells and are 10-fold more biologically active than full-length IL33 (see Lefrancais, E.
  • the IL33 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 52, 54, 55, 56 or 57 % or 99% or 100% sequence identity, or the gene encoding IL33 has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleic acid sequence shown in SEQ ID NO: 51, 53, 58, 59 or 60 %, 95%, 97% or 99% or 100% sequence identity.
  • the C-type chemokine family also known as lymphoid chemokines, includes two members, XCL1 and XCL2, which are mainly produced by CD8+ T cells and natural killer cells.
  • XCL1 has unique sequence features and two interchangeable protein spatial conformations, which make XCL1 different from other chemokines and play unique functions.
  • the XCL1-specific receptor XCR1 is a member of the G protein-coupled receptor family. The interaction between the two not only plays an important role in the negative selection of the thymus and the establishment of autoimmune tolerance, but also can initiate cross-antigen presentation and mediate cytotoxic immune responses. .
  • XCL1 not only regulates immune system balance and maintains intestinal immune homeostasis, but is also associated with various diseases, such as autoimmune diseases, nephritis, tuberculosis and human immunodeficiency virus infection.
  • the nucleic acid sequences of XCL2 and XCL1 are 97% identical, with two amino acid residues different at positions 7 and 8: Asp and Lys in XCL1, and His and Arg in XCL2.
  • Studies have found that XCL2 and XCL1 are very similar in expression profile, structure and function. For example, like XCL1, XCL2 also has two interconvertible protein spatial conformations, haplotype and dimer, in which the monomeric conformation binds and interacts with each other.
  • XCL1 Activates XCR1, and the dimerized conformation has a higher affinity for the hairpin structure in Glycosaminoglycans (GAGs).
  • GAGs Glycosaminoglycans
  • XCL1 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 24 or 26 % sequence identity, or the coding sequence of XCL1 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleic acid sequence shown in SEQ ID NO: 23 or 25 % sequence identity.
  • XCL2 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 38 Sequence identity, or the coding sequence of XCL2 is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 37 sex.
  • exogenous genes eg cytokines and/or chemokines in the present invention may be constitutive or conditional.
  • the expression of exogenous cytokines and/or chemokines is conditional expression.
  • the exogenous gene of the present invention can be operably linked to an inducible or tissue-specific promoter to regulate the expression level of the introduced exogenous gene at a specific time or in a specific tissue or cell type.
  • the promoter is an inducible promoter, ie, a promoter that initiates transcription only in the presence of specific environmental conditions, developmental conditions, or inducers.
  • environmental conditions include, for example, a tumor acidic microenvironment, a tumor hypoxic microenvironment, and the like.
  • Such inducers include, for example, ciccycline, tetracycline, or analogs thereof, and analogs of tetracycline include, for example, chlortetracycline, oxytetracycline, desmethylchlorotetracycline, methycycline, doxycycline, and minocycline white.
  • Inducible promoters include, for example, Lac operon sequences, tetracycline operon sequences, galactose operon sequences, or doxycycline operon sequences, and the like.
  • the expression of cytokines and/or chemokines is secreted or anchored, eg, can be operably linked to a localization domain that can localize an exogenous gene of the invention Expressed at specific cellular locations, such as cell membranes, specific organelles in the cytoplasm such as endoplasmic reticulum, Golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, guide peptides, transmembrane domains, and the like.
  • the exogenous gene of the present invention is operably linked to the transmembrane domain, thereby anchored for expression on the surface of engineered immune cells.
  • the exogenous gene in the present invention eg, IL12, IL18, IL-21, IL23, IL33, XCL2 or XCL1 protein may be wild type or a fusion protein with specific properties (eg resistance to proteolysis) or mutant.
  • the present invention also provides a nucleic acid molecule comprising (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes a ligand, (ii) a nucleic acid sequence encoding a cytokine (selected from IL12, IL18, IL-21, IL23 and IL33). a nucleic acid sequence, and (iii) a nucleic acid sequence encoding a chemokine (selected from XCL2 and XCL1).
  • the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler, or a T cell receptor, preferably a chimeric antigen receptor.
  • Chimeric antigen receptors are defined as above.
  • 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 form, or their mixtures (including hybrid molecules).
  • nucleic acids according to the present invention include DNA (eg dsDNA, ssDNA, cDNA), RNA (eg dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (eg PNA).
  • the nucleic acid is DNA or RNA, more preferably mRNA.
  • Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNA)). Nucleic acids of the invention may also contain one or more modified bases, such as, for example, tritylated bases and uncommon bases such as inosine. Other modifications are also contemplated, including chemical, enzymatic, or metabolic modifications, so long as the multi-chain CARs of the invention can be expressed from polynucleotides. Nucleic acids can be provided in isolated form. In one embodiment, nucleic acids 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.
  • 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 can be codon optimized for optimal expression in desired host cells (eg, immune cells); 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, and the codons before and after the replacement code for the same amino acid. Therefore, the choice of optimal codons depends on the codon usage preferences of the host genome.
  • the present invention also provides a vector comprising the nucleic acid according to the present invention.
  • the nucleic acid sequences encoding cell surface molecules that specifically recognize ligands the nucleic acid sequences encoding cytokines (selected from IL12, IL18, IL-21, IL23, and IL33), the nucleic acid sequences encoding chemokines (selected from XCL1 and XCL2) Nucleic acid sequences can be located in one or more vectors. When located in the same vector, these nucleic acid sequences can be linked by, for example, the 2A peptide.
  • vector is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell, in which the nucleic acid molecule can eg be replicated and/or expressed.
  • Targeting vector is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a specific targeting sequence at the site.
  • An “expression vector” is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric antigen receptor polypeptides of the invention, in suitable host cells and the translation of their mRNAs. Suitable carriers for use in the present invention are known in the art and many are commercially available.
  • the vectors of the present invention include, but are not limited to, plasmids, viruses (eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)), and the like ), phages, phagemids, cosmids, and artificial chromosomes (including BAC and YAC).
  • viruses eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)
  • RSV Rous sarcoma virus
  • AAV adeno-associated virus
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector .
  • Engineered vectors typically also contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (eg, a multiple cloning site, MCS).
  • the vector may additionally contain a promoter , polyadenylation tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements.
  • the vector is an in vitro transcribed vector.
  • the present invention also provides an engineered immune cell comprising the nucleic acid or vector of the present invention.
  • the engineered immune cells of the present invention express a cell surface molecule that specifically recognizes a ligand, one or more exogenous cytokines (selected from IL12, IL18, IL-21, IL23, IL33), and a or a plurality of exogenous chemokines selected from the XCL1 and XCL2 genes.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells obtained from stem cell sources such as cord blood.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, eg, primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject.
  • 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 sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4-CD8- T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells) ), tumor infiltrating cells, memory T cells, naive T cells, regulatory T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.
  • immune cells are engineered to express chimeric antigen receptors as well as exogenous cytokines, and chemokines.
  • Nucleic acid sequences encoding chimeric antigen receptor polypeptides, as well as cytokines and chemokines, can be introduced into immune cells using conventional methods known in the art (eg, by transduction, transfection, transformation, etc.).
  • Transfection is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell.
  • An example is RNA transfection, the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell.
  • the term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes and deposit their cargo inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells).
  • transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means.
  • the cells or bacteria must be in a competent state.
  • techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation.
  • the immune cells of the present invention further comprise at least one inactivated gene selected from the group consisting of CD52, GR, TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ , HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3.
  • at least TCR components (including TCR ⁇ , TCR ⁇ genes) or CD3 components (including CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ ) in immune cells are 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, eg, by mediated DNA fragmentation by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, thereby inactivating the gene.
  • the present invention also provides a pharmaceutical composition comprising the engineered immune cell, nucleic acid molecule or carrier of the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also encompasses the use of the nucleic acid molecule, vector or engineered immune cell in the preparation of a pharmaceutical composition or medicament.
  • the term "pharmaceutically acceptable excipient” means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited 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, antiadherents, 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 modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention.
  • excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends, among other things, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • compositions according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration.
  • Processes known in the present invention for the manufacture of pharmaceuticals may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.
  • compositions according to the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetate glucuronate, auristatin E, vincristine and doxorubicin; 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, act
  • the present invention also provides a combination therapy comprising (1) an engineered immune cell and a chemokine expressing an exogenous cytokine; (2) an engineered immune cell and a cytokine expressing an exogenous chemokine; or (3) engineered immune cells, cytokines and chemokines, wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, and the cytokines are selected from the group consisting of IL12, IL18, IL-21, IL23 and IL33, The chemokine is selected from XCL1 and XCL2.
  • the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the present invention. Accordingly, the present invention also encompasses the use of the engineered immune cells in the manufacture of a medicament for the treatment of cancer, infection or autoimmune disease.
  • an effective amount of an immune cell and/or pharmaceutical composition of the invention is administered directly to a subject.
  • the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, more preferably T cells, NK cells cells or NKT cells.
  • autologous refers to any material derived from an individual to be later reintroduced into that same individual.
  • allogeneic refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.
  • the term "subject" is a mammal.
  • the mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples.
  • Mammals other than humans can advantageously be used as subjects representing animal models of cancer.
  • the subject is a human.
  • the cancer is a cancer associated with expression of the target bound by the ligand binding domain, eg, a hematological tumor or a solid tumor.
  • cancers include, but are not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, 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, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous lung cancer), lymphoma (including Hod
  • the diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, and the like.
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.
  • the method further comprises administering to the subject one or more additional chemotherapeutic agents, biological agents, drugs or treatments.
  • the chemotherapeutic agent, biologic, drug or treatment is selected from radiation therapy, surgery, antibody agents and/or small molecules and any combination thereof.
  • the coding sequence fragments of T2A and murine IL-21 (SEQ ID NO: 35) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-IL-21 plasmid.
  • the coding sequence fragments of T2A and murine XCL1 (SEQ ID NO: 25) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-XCL1 plasmid.
  • 293T cells were seeded in 30 ml of DMEM medium containing 10 % fetal bovine serum at a density of 30 x 106 cells/flask, and cultured overnight in a 37°C, 5% CO2 incubator for Virus packaging.
  • the plasmid/vector/transfection reagent mixture was then added dropwise to a pre-prepared culture flask of 293T cells and incubated overnight at 37°C under 5% CO2 conditions. Cultures were harvested 72 hours after transfection and centrifuged (2000 g, 4[deg.]C, 10 minutes) to obtain retroviral supernatant.
  • T lymphocytes were isolated from mouse spleen and T cells were activated with DynaBeads CD3/CD28CTS TM (Gibco, Cat. No. 40203D) and then cultured at 37°C and 5% CO 2 for 1 day.
  • Activated T cells were seeded into 24-well plates pre-coated with RetroNectin overnight at a density of 3 ⁇ 10 6 cells/mL per well, and then 500 ⁇ L of complete medium (NT, control), MSCV-mCD19-CAR, and 500 ⁇ L of complete medium (NT, control) were added, respectively.
  • the 24-well plate was placed in a centrifuge for centrifugation, and centrifuged at 32°C and 2000g for 2h. Then, the 24-well plate was immediately placed in a 37°C, CO2 incubator for static culture. Change fresh medium the next day and adjust the cell density to 1 x 10 6 cells/mL. Three days after infection, cells were collected for subsequent analysis. The collected cells are NT cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells, and mCD19-CAR-IL-21-XCL1 cells.
  • CAR T cells containing mCD19-CAR-XCL1 can effectively secrete XCL1.
  • both CAR T cells containing mCD19-CAR-IL-21 can efficiently express IL-21.
  • NT cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells, and mCD19-CAR-IL-21-XCL1 cells were added at a concentration of 2 x 105 cells/100 ⁇ l in a 96-well round bottom plate, respectively.
  • Target cells Panc02-mCD19 cells or non-target cells Panc02 cells were then added to each well at a concentration of 1 ⁇ 10 4 cells/100 ⁇ l, respectively. After 24 h incubation at 37°C, the culture supernatant was collected. The expression levels of IFN- ⁇ in culture supernatants were detected using the Mouse IFN-gamma DuoSet ELISA kit (R&D, Cat. No. DY485) according to the manufacturer's recommendations.
  • IL-21 slightly increased the killing ability of CAR-T cells against target cells compared with mCD19-CAR cells alone.
  • the inventors found that compared with CAR-T cells expressing only IL-21, the combination of expressing XCL1+IL-21 can further significantly improve the killing ability of CAR-T cells to target cells, especially in low-efficiency targets. than (eg 1.25:1) case.
  • Panc02-mCD19 pancreatic cancer cells overexpressing CD19 were subcutaneously inoculated into the axilla of the left forelimb of healthy C57BL/6 mice.
  • mice inoculated with pancreatic cancer cells were randomly divided into 5 groups of 7 mice each. When the tumor volume grew to 100 mm, each mouse was injected with PBS, 5 x 10 NT cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells or mCD19-CAR-IL-21- XCL1 cells.
  • mice were monitored for changes in body weight and tumor volume until the end of the experiment.
  • CAR-T cells co-expressing IL15(SEQ ID NO:61)+XCL1 and IL17(SEQ ID NO:62)+XCL1 were also prepared as controls.
  • the IFN- ⁇ secretion level of the above-mentioned CAR-T cells was detected.
  • the IFN- ⁇ secretion level of CAR-T cells expressing IL12+XCL1 and IL23+XCL1 was comparable to that of CAR-T cells expressing only IL12 or IL23 (see Figure 8), while the The IFN- ⁇ secretion level of IL18+XCL1 and IL33+XCL1 CAR-T cells was significantly higher than that of CAR-T cells expressing only IL18 or IL33 (see Figure 9).
  • Example 5 the tumor suppressive effect of the above CAR-T cells in vivo was detected.
  • CAR-T cells expressing IL12 or IL23 alone showed very excellent tumor suppressive effect
  • CAR-T cells expressing IL12+XCL1 or IL23+XCL1 combination still had significantly better tumor suppressive effect than CAR expressing IL12 or IL23 alone -T cells (see Figure 10).
  • CAR-T cells expressing IL18+XCL1 and IL33+XCL1 also showed significant tumor suppressive effects
  • CAR-T cells expressing IL15+XCL1 and IL17+XCL1 had a similar tumor suppressive effect to traditional mCD19- CAR T cells were comparable or slightly worse (see Figure 11).
  • This also shows that not any combination of interleukin and XCL1 can achieve a significant inhibitory effect on tumors, which may be related to the complex function of interleukin.

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Abstract

La présente invention concerne une cellule immunitaire modifiée, exprimant les éléments suivants : (i) une molécule de surface cellulaire reconnaissant particulièrement un ligand ; (ii) une ou plusieurs cytokines exogènes, choisies parmi IL12, IL18, IL-21, IL23 et IL33 ; et (iii) un ou plusieurs facteurs chimiotactiques exogènes choisis parmi XCL2 et XCL1. La présente invention concerne également l'utilisation de la cellule immunitaire modifiée dans le traitement du cancer, d'une infection ou d'une maladie auto-immune. La cellule immunitaire modifiée présente une activité de destruction tumorale significativement améliorée par comparaison avec des cellules immunitaires génétiquement modifiées classiques.
PCT/CN2022/089109 2021-04-23 2022-04-25 Cellule immunitaire modifiée et son utilisation WO2022223049A1 (fr)

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