WO2022267983A1 - Engineered immune cell and use thereof - Google Patents

Engineered immune cell and use thereof Download PDF

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WO2022267983A1
WO2022267983A1 PCT/CN2022/099374 CN2022099374W WO2022267983A1 WO 2022267983 A1 WO2022267983 A1 WO 2022267983A1 CN 2022099374 W CN2022099374 W CN 2022099374W WO 2022267983 A1 WO2022267983 A1 WO 2022267983A1
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cells
antigen
immune cell
engineered immune
cell
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Chinese (zh)
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熊瑛
邢芸
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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Definitions

  • the invention belongs to the field of immunotherapy. More specifically, the present 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 that specifically recognizes an antigen is a chimeric antigen receptor or a recombinant T cell receptor.
  • CAR-T cell therapy has shown attractive prospects in the field of tumor immunotherapy.
  • the basic structure of CAR includes tumor-associated antigen binding region, transmembrane region and intracellular signal region. Once CAR binds to tumor-associated antigens, it can activate T cells through the intracellular region, and then manifest CAR-dependent cell killing, proliferation and cytokine release.
  • CAR-T cell therapy for example, there are a large number of tumor recurrences in the treatment of hematological tumors, and the response rate is not high in the treatment of solid tumors, etc. These may be caused by the complex tumor microenvironment, CAR - Caused by factors such as T cell exhaustion.
  • 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.
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a recombinant T cell receptor, preferably a chimeric antigen receptor.
  • the LTBR ligand is selected from LTA, LTB, LIGHT and anti-LTBR antibodies.
  • 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, tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells or ⁇ -T cells.
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor comprising an antigen binding region, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
  • the antigen binding region 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 antigen binding region is selected from scFv, Fab, single domain antibodies and nanobodies.
  • the cell surface molecule that specifically recognizes the 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, CD40, 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,
  • the target is selected from 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.
  • 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 intracellular signaling domain is selected from the intracellular regions of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b and CD66d.
  • said intracellular signaling domain comprises a CD3 ⁇ intracellular region.
  • 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(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 co-stimulatory domain is selected from the intracellular region of CD27, CD28, CD134, CD137, DAP10, DAP12 or CD278 or a combination thereof.
  • the present invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, and (ii) a nucleic acid sequence encoding a LTBR ligand.
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a recombinant 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-mentioned nucleic acid molecule.
  • the vector is selected from plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV).
  • RSV Rous sarcoma virus
  • AAV adeno-associated virus
  • the vector further comprises an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a polyA tail (polyA), a 3'UTR, a 5'UTR, an enhancer Elements such as promoters, terminators, insulators, operators, selectable markers, reporter genes, targeting sequences and/or protein purification tags.
  • said vector is an in vitro transcribed vector.
  • the present invention also provides a kit comprising the engineered immune cells, nucleic acid molecules or vectors described in the present invention.
  • the present invention also provides a pharmaceutical composition, which comprises the engineered immune cells, nucleic acid molecules or vectors described in the present invention, and a variety of pharmaceutically acceptable excipients.
  • the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to said subject an effective amount of the Immune cells, 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, gastric cancer, glioblastoma (GBM), liver cancer, hepatoma, Intraepithelial neoplasms, kidney cancer, laryngeal cancer, liver tumors, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma,
  • the infections include, but are 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.
  • Figure 1 Schematic diagram of the structure of the CAR plasmid.
  • Figure 2 CAR expression levels of CAR-T cells determined by flow cytometry.
  • Figure 3 The killing activity of CAR-T cells on target cells.
  • FIG. 4 IFN- ⁇ release levels after CAR-T cells were co-cultured with target cells and non-target cells.
  • Figure 5 Survival curves of mice treated with CAR-T cells for B lymphocytic tumors.
  • 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 (eg, an antigen).
  • a target molecule eg, an antigen
  • Such surface molecules generally 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 signal transmission.
  • Common examples of such surface molecules include, for example, recombinant T cell receptors or chimeric antigen receptors.
  • TCR refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling.
  • MHC major histocompatibility complex molecules
  • TCR is composed of six peptide chains that form heterodimers, which are generally divided into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specific antigens and MHC molecules.
  • the variable region of the TCR may comprise or be operably linked to an antigen binding region, wherein the definition of the antigen binding region is as follows.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding region (such as the antigen-binding portion of an antibody), a transmembrane domain, a co-stimulatory Domain and intracellular signaling domain, each domain is connected by a linker.
  • CARs are able to exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to a target of choice in a non-MHC-restricted manner.
  • Non-MHC-restricted antigen recognition confers on CAR cells the ability to recognize antigens independently of antigen processing, thus bypassing major mechanisms of tumor escape.
  • CAR advantageously does not dimerize with the alpha and beta chains of the endogenous recombinant T cell receptor (TCR) when expressed in T cells.
  • 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 antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, murine antibody, chimeric antibody and functional fragments thereof.
  • antigen 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, anticalins, DARPIN etc., preferably selected from Fab, scFv, sdAb and Nanobodies.
  • the antigen binding domain may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody.
  • the antigen binding region may also be a specific binding polypeptide or receptor structure of a specific protein, such as PD1, PDL1, PDL2, TGF ⁇ , APRIL and NKG2D.
  • Fab refers to either of two identical fragments of an immunoglobulin molecule produced after cleavage by papain, consisting of the complete light chain and the N-terminal portion of the heavy chain linked by a disulfide bond, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared with intact IgG, Fab has no Fc fragment, has higher fluidity and tissue penetration ability, and can monovalently bind antigen without mediating antibody effect.
  • a “single-chain antibody” or “scFv” is an antibody in which the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker can significantly affect the variable domain folding and interaction of scFv. In fact, if shorter linkers (eg, between 5-10 amino acids) are used, intrachain folding can be prevented.
  • linker size and composition see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448; U.S. Patent Application Publication Nos.
  • a scFv may comprise VH and VL linked in any order, eg VH-linker-VL or VL-linker-VH.
  • Single domain antibody refers to an antibody naturally devoid of light chains, which contains only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as “heavy chain Antibody”.
  • VHH heavy chain variable region
  • CH2 and CH3 regions also known as “heavy chain Antibody”.
  • Nemobody or “Nb” refers to the VHH structure cloned and expressed separately, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is currently the smallest unit known to bind the target antigen .
  • the term "functional variant” or “functional fragment” refers to a variant comprising essentially the amino acid sequence of a parent but containing at least one amino acid modification (i.e. substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the Such variants retain the biological activity of the parent amino acid sequence.
  • the amino acid modification is preferably a conservative modification.
  • conservative modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment comprising the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain.
  • Families of amino acid residues with similar side chains have been defined in the art and include 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), non-polar side chains (e.g. alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.
  • 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, tyros
  • threonine valine, isoleucine
  • aromatic side chains eg, tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • a “functional variant” or “functional fragment” has 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 means the degree to which two (nucleotide or amino acid) sequences in an alignment have the same residue at the same position, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies of the exact same sequence have 100% identity.
  • 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 Clustal W.
  • 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, CD40, 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, EGFR
  • the CAR of the invention can be designed to include an antigen-binding region specific for that antigen.
  • an antigen-binding region specific for that antigen for example, if CD19 is the antigen to be targeted, a CD19 antibody can be used as the antigen binding region of the present invention.
  • transmembrane domain refers to a polypeptide capable of expressing a chimeric antigen receptor on the surface of an immune cell (such as a lymphocyte, NK cell or NKT cell) and directing a cellular response of the immune cell against a target cell structure.
  • Transmembrane domains 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 suitable for use 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 functional fragments thereof.
  • the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from CD8 ⁇ , which has 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: 4 or 16. % or 100% sequence identity, or the coding sequence of the CD8 ⁇ transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90% of the nucleotide sequence shown in SEQ ID NO:3 or 15 , 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antigen binding region and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an antigen binding region. Specifically, the hinge region is used 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 natural 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 an entirely synthetic hinge sequence.
  • the hinge region comprises the hinge region part of CD8 ⁇ chain, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably CD8 ⁇ hinge, which has the same amino acid sequence as shown in SEQ ID NO: 12, 22, 33 or 35
  • the coding sequence of the CD8 ⁇ hinge is identical to SEQ ID NO: 11, 21, 34 or 36
  • the nucleotide sequences shown have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a given function.
  • the intracellular signaling domain is responsible for primary intracellular signal transmission following antigen binding at the antigen binding region, resulting in activation of immune cells and immune responses.
  • 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.
  • the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.
  • the intracellular signaling domains comprised by the chimeric antigen receptors of the invention may be cytoplasmic sequences of recombinant T cell receptors and co-receptors, which act together to elicit primary Signal transduction, and any derivatives or variants of these sequences and any synthetic sequences having the same or similar function.
  • the intracellular signaling domain can contain many immunoreceptor tyrosine-based activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM).
  • Non-limiting examples of intracellular 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 ⁇ intracellular region, and the signaling domain has 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.
  • a chimeric antigen receptor of the invention comprises one or more co-stimulatory domains.
  • a co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule comprising the entire intracellular portion of said co-stimulatory 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 co-stimulatory domains of the invention include, but are not limited to, intracellular regions derived from the following proteins: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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.
  • CD94 intracellular regions derived from the following proteins: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7
  • the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40, ICOS, DAP10, DAP12 or a combination thereof, more preferably 4-1BB and/or CD28.
  • the costimulatory domain contained in the CAR of the present invention has 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 costimulatory domain and the nucleotide sequence shown in SEQ ID NO: 5, 17 or 32 have at least 70%, preferably at least 80%, more A sequence identity of at least 90%, 95%, 97% or 99% or 100% is preferred.
  • the CAR of the invention may also 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 stretch of hydrophobic amino acids with a propensity to form a single ⁇ -helix.
  • At the end of the signal peptide there is usually a stretch of amino acids that is recognized and cleaved by the signal peptidase.
  • the signal peptidase can cleave during translocation or after completion to generate a free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • Signal peptides that can be used in the present invention are well known to those skilled in the art, for example, signal peptides derived from CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the signal peptide that can be 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: 10 % 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 99% of the nucleotide sequence shown in SEQ ID NO:9 or 100% sequence identity.
  • the CAR of the present invention may also include a switch structure to regulate the expression time of the CAR.
  • the switch structure can be in the form of a dimerization domain, which induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain to allow it to bind to the targeted antigen, thereby activating the signaling pathway.
  • a switch domain can be used to link the binding and signaling domains separately, and the binding and signaling domains can pass through the dimer only when the switch domains are associated with each other (e.g. in the presence of an inducing compound). Link together to activate the signaling pathway.
  • the switch structure can also be in the form of a masking peptide.
  • the masking peptide can mask the extracellular binding domain and prevent it from binding to the targeted antigen.
  • the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it an "ordinary" CAR structure.
  • Various switch configurations known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may also contain a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance, so as to eliminate CAR cells when necessary (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 necessary, 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, and the dimerization of iCaspase-9 can be induced by chemically inducing drugs such as AP1903 and AP20187, thereby activating the downstream Caspase3 molecule and leading to cell apoptosis.
  • chemically inducing drugs such as AP1903 and AP20187
  • LTBR refers to lymphotoxin beta receptor, which is a type I single transmembrane protein with a molecular weight of 61 kDa.
  • the extracellular domain of LTBR is responsible for ligand binding and contains four cysteine-rich repeats characteristic of TNF motifs.
  • the intracellular region of LTBR consists of 175 amino acid residues, which itself does not have kinase or other enzymatic activities.
  • LTBR is constitutively expressed on most cells, such as stromal cells of lymphoid tissue, myeloid cells, blood monocytes, alveolar macrophages, mast cells, dendritic cells, etc.
  • LTBR is not expressed in T cells, B cells and NK cells, which is also its most prominent feature.
  • LTBR ligands are often expressed on T cells and B cells. This expression pattern suggests that signaling may be unidirectional for LT, requiring direct contact between lymphocytes and LTBR-bearing cells to transmit the signal.
  • LTBR ligands mainly include two: lymphotoxin (LT) and LIGHT.
  • Lymphotoxin (LT) is a cytokine that is secreted by lymphocytes after being stimulated by antigens or mitogens and in certain tumors and autoimmune diseases.
  • Lymphotoxin also known as tumor necrosis factor ⁇ , and tumor necrosis factor ⁇ (TNF- ⁇ ) are two closely related cytokines that belong to the TNF family.
  • LT is mainly expressed in B cells, T cells and NK cells.
  • LT includes two subunits: LT ⁇ (also known as LTA) and LT ⁇ (also known as LTB).
  • LT ⁇ can be secreted out of the cell, and then anchored on the cell surface by non-covalent combination with LT ⁇ to form a heterotrimer.
  • LT plays an important role in various biological activities such as killing tumor cells, regulating immune response, regulating inflammatory response, and inducing antigen expression of various differentiated cells.
  • LIGHT is a type II transmembrane protein that also belongs to the TNF family and is produced by activated T cells, monocytes, granulocytes, and immature dendritic cells.
  • LTBR receptor-ligand signaling can also be initiated by anti-LTBR antibodies or by overexpression of the receptor.
  • the LTBR ligand is LTA, which 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: 26, 40, 46 or 48. % or 99% or 100% sequence identity (the 1-33 amino acids of SEQ ID NO: 26 are signal peptides), or its coding sequence and the nucleic acid sequence shown in SEQ ID NO: 25, 39, 45 or 47 Having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • SEQ ID NO: 46 consists of amino acids 34-202 of SEQ ID NO: 26 (amino acids 1-33 of SEQ ID NO: 26 are signal peptides), and SEQ ID NO: 48 consists of amino acids of SEQ ID NO: 40 Amino acid composition at positions 35-205 (amino acids 1-34 of SEQ ID NO: 40 are signal peptides).
  • the LTBR ligand is LTB, which has 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: 24 or 42 Or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the nucleic acid sequence shown in SEQ ID NO: 23 or 41 or 100% sequence identity.
  • the LTBR ligand is LIGHT, which has 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: 28 or 44 Or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the nucleic acid sequence shown in SEQ ID NO: 27 or 43 or 100% sequence identity.
  • the LTBR ligand is an anti-LTBR antibody, e.g. selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, single domain antibody, Nanobodies, diabodies.
  • Antibodies known in the art can be used as LTBR ligands of the invention, including but not limited to CBE11 (ATCC Accession No. 11793), BKA11 (ATCC Accession No. 11799), CDH10 (ATCC Accession No. 11797), BCG6 (ATCC Accession No. 11794), BHA10 (ATCC Accession No. 11795), BDA8 (ATCC Accession No. HB11798) (see US Patent Nos. 5,925,351 and 631,269,1, the entire contents of which are incorporated herein by reference).
  • the present invention also provides 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.
  • the cell surface molecule that specifically recognizes an antigen is a recombinant T cell receptor or a chimeric antigen receptor, preferably a chimeric antigen receptor.
  • Chimeric antigen receptors are defined above.
  • nucleic acid molecule includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single- and/or double-stranded form, linear or circular shape, or their mixtures (including hybrid molecules).
  • 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).
  • the nucleic acid is DNA or RNA, more preferably mRNA.
  • Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds such as amide bonds such as those found in peptide nucleic acids (PNAs). 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 are also contemplated, including chemical, enzymatic or metabolic modifications, so long as the multi-chain CAR of the invention can be expressed from the polynucleotide. Nucleic acids can be provided in isolated form. In one embodiment, a nucleic acid may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcriptional termination signals), ribosome binding sites, introns, and the like.
  • transcriptional control elements including promoters, enhancers, operators, repressors, and transcriptional termination signals
  • ribosome binding sites introns, and the like.
  • nucleic acid sequences of the present invention can be codon-optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacteria, yeast or insect cells.
  • Codon optimization refers to the replacement of codons 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 this species, while the codons before and after the replacement code for the same amino acid. Therefore, the choice of optimal codons depends on the codon usage bias of the host genome.
  • the present invention also provides a vector comprising the nucleic acid according to the present invention.
  • the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen, and the nucleic acid encoding the LTBR ligand may be located in one or more vectors.
  • the two chimeric antigen receptor structures can be independently expressed in the same vector without affecting each other.
  • the term "2A peptide” is a cis-hydrolase action element originally discovered in foot-and-mouth disease virus (FMDV).
  • FMDV foot-and-mouth disease virus
  • the average length of 2A peptides is 18-22 amino acids.
  • the 2A peptide can be cleaved from the C-terminus of the last 2 amino acids of itself by ribosome jumping.
  • the peptide chain binding group between glycine and proline is damaged at the 2A site, which can trigger ribosome jumping and start translation from the second codon, so that two proteins in one transcription unit independent expression.
  • This 2A peptide-mediated cleavage is ubiquitous in eukaryotic cells.
  • the expression efficiency of heterologous polyproteins (such as cell surface receptors, cytokines, immunoglobulins, etc.) can be improved by utilizing the higher shearing efficiency of 2A peptides and the ability to promote the balanced expression of upstream and downstream genes.
  • Conventional 2A peptides include: P2A, T2A, E2A, F2A, etc.
  • vector is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell where it 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 sequence at a specific targeting site.
  • An “expression vector” is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding chimeric antigen receptor polypeptides of the invention, and the translation of their mRNA in a suitable host cell. Suitable vectors for use in the present invention are known in the art and many are commercially available.
  • vectors of the present invention include, but are not limited to, plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc. ), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs).
  • viruses such as retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc.
  • phages phagemids
  • cosmids include BACs and YACs.
  • the vector itself is usually a sequence of nucleotides, usually a DNA sequence containing an insert (transgene) and a larger sequence that acts as the "backbone" of the vector
  • the engineered vector usually also contains 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 (such as a multiple cloning site, MCS).
  • the vector may additionally contain a promoter , polyadenylation tail (polyA), 3' UTR, enhancer, terminator, insulator, operator, 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 cell surface molecules that specifically recognize antigens, as well as LTBR ligands.
  • 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 may be any T cells, such as T cells cultured in vitro, such as 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 enriched or purified.
  • T cells can 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, ⁇ -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 LTBR ligands.
  • 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. More specifically, 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 nonfunctional in the cell. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD). Methods for gene inactivation are known in the art, such as DNA fragmentation mediated by meganuclease, zinc finger nuclease, TALE nuclease or Cas enzyme in CRISPR system, thereby inactivating the gene.
  • GvHD graft-versus-host
  • the present invention also provides a pharmaceutical composition, which comprises the engineered immune cell, nucleic acid molecule or carrier described in the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the nucleic acid molecules, vectors or engineered immune cells in the preparation of pharmaceutical compositions or medicaments.
  • the term "pharmaceutically acceptable excipient” means pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse desired local or systemic effect), which are well known in the art (see, for example, 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, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators .
  • suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical compositions of the present invention.
  • excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends inter alia 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 are suitable for various routes of administration. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • the pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, especially 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 especially adapted to the desired method of administration.
  • Processes known in the present invention for the production of medicaments 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.
  • the pharmaceutical composition according to the invention can also be administered in combination with one or more other agents suitable for the treatment and/or prophylaxis 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, trimetreate 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
  • the present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the immune cell or the pharmaceutical composition according to the present invention. Therefore, the present invention also covers the use of the engineered immune cells in the preparation of medicaments for treating cancer, infection or autoimmune diseases.
  • the method of treatment comprises administering to a subject an effective amount of an immune cell and/or a pharmaceutical composition of the present invention.
  • 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 that will later be reintroduced into that same individual.
  • allogeneic refers to any material derived from a different animal of the same species or a different patient as the individual into whom the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci differ. 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. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cows, but are 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.
  • the cancer is a cancer associated with expression of a target bound by the antigen binding region.
  • the 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 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, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatoma, intraepithelial neoplasia, renal cancer, laryngeal cancer, liver tumors, lung cancer (such as small cell lung cancer, non-small cell lung cancer, adenoid lung cancer, and squamous lung cancer), lymphoma (including Hodgkin lymphoma and non-
  • ALL B-cell acute lymphoblastic leukemia
  • T-ALL T-cell acute lymphoblastic leukemia
  • B-cell prolymphocytic leukemia blastic plasmacytoid dendritic cell tumor, Burkitt Lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, Myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and post-transplantation lymphoproliferative disorder (PTLD); and other disorders associated with target expression.
  • CML chronic myelogenous leukemia
  • MALT lymphoma hairy cell leukemia
  • marginal zone lymphoma multiple myeloma
  • Myelodysplasia plasmablastic lymphoma
  • the diseases that can be treated with the engineered immune cells or the pharmaceutical composition of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer and the like.
  • the infections include, but are 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, biologics, 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.
  • T2A SEQ ID NO: 37
  • LTB SEQ ID NO: 24
  • T2A SEQ ID NO: 37
  • LTB SEQ ID NO: 24
  • LTA SEQ ID NO: 26
  • T2A SEQ ID NO: 37
  • LIGHT SEQ ID NO: 28
  • each plasmid is shown in FIG. 1 .
  • Opti-MEM Gibco, Cat. No. 31985-070
  • 45 ⁇ g of prepared retroviral plasmid 15 ⁇ g of packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029)
  • packaging vector pCL-Eco Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029
  • 120 ⁇ l of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001)
  • the plasmid/vector/transfection reagent mixture was added dropwise into the pre-prepared 293T cell culture flask, and cultured overnight at 37°C and 5% CO 2 . Cultures were harvested 72 hours after transfection and centrifuged (2000g, 4°C, 10 minutes) to obtain retroviral supernatants.
  • T lymphocytes were isolated from mouse spleen, and T cells were activated with DynaBeads CD3/CD28 CTS TM (Gibco, Cat. No. 40203D), and then cultured at 37°C and 5% CO 2 for 1 day.
  • the 24-well plate Place the 24-well plate in a centrifuge for centrifugal infection, and centrifuge at 2000g for 2h at 32°C. Then, immediately place the 24-well plate in a 37°C, CO2 incubator for static culture. Replace the fresh medium the next day, and adjust the cell density to 1 ⁇ 106 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.
  • Example 2 The killing effect and cytokine release of CAR-T cells on target cells
  • A20-CD19 target cells (screened CD19 positive AD20 cells) carrying the fluorescein gene into 96-well plates at 1x10 4 /well, and then use the effect-to-target ratio of 5:1, 10:1 or 20:1 respectively (i.e. the ratio of effector T cells to target cells)
  • the CAR-T cells and NT cells prepared in Example 1 were placed in a 96-well plate for co-culture, and the fluorescence value was measured with a microplate reader after 16-18 hours. According to the calculation formula: (average fluorescence value of target cells-average fluorescence value of samples)/average fluorescence value of target cells ⁇ 100%, the killing efficiency was calculated, and the results are shown in FIG. 3 .
  • CAR-T cells additionally expressing LTB, LTB+LTA or LIGHT have slightly improved killing ability on target cells.
  • the target cells P02-CD19 or non-target cells P02 in a 96-well plate at a concentration of 1 ⁇ 10 5 /well, and then co-combine the CAR-T cells and NT cells prepared in Example 1 with the target cells at a ratio of 10:1. After culturing, the cell co-culture supernatant was collected after 18-24 hours.
  • the reaction was allowed to take place in the dark at room temperature for 30 minutes, after which 50 ⁇ L of 1 mol/L H2SO4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, use a microplate reader to detect the absorbance at 450nm, and calculate the cytokine content according to the standard curve (drawn according to the reading value and concentration of the standard), and the results are shown in Figure 4.
  • mice Twenty-four 6-week-old Balb/c immunocompetent mice were randomly divided into 4 groups, 6 mice in each group, and 1 ⁇ 10 6 A20 cells (a type of B lymphocytoma cells) were injected through the tail vein. The day of inoculation of A20 cells was defined as D0. On D10, 3 mg/20 g body weight of cyclophosphamide was intraperitoneally injected into the mice of each group. On D13, 5 ⁇ 10 6 bbz CAR-T cells, bbz-T2A-Ltb CAR-T cells, bbz-T2A-Ltba CAR-T cells or bbz-T2A-LIGHT CAR-T cells were injected into mice in each group via tail vein cell. Mouse survival was monitored until the end of the experiment. The result is shown in Figure 5.
  • CAR-T cells additionally expressing LTB, LTB+LTA or LIGHT can significantly enhance the inhibitory effect on tumors, thereby improving the survival rate.

Abstract

Provided are an engineered immune cell and a use thereof in the treatment of cancer, infections or autoimmune diseases. The engineered immune cell expresses (i) a cell surface molecule that specifically recognizes an antigen, and (ii) one or more LTBR ligands. The engineered immune cell has significantly improved tumor-killing activity.

Description

工程化免疫细胞及其用途Engineered immune cells and uses thereof 技术领域technical field
本发明属于免疫治疗领域。更具体地，本发明涉及一种工程化免疫细胞，其表达(i)特异性识别抗原的细胞表面分子，和(ii)一种或多种LTBR配体。更优选地，所述特异性识别抗原的细胞表面分子是嵌合抗原受体或重组T细胞受体。The invention belongs to the field of immunotherapy. More specifically, the present 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 that specifically recognizes an antigen is a chimeric antigen receptor or a recombinant T cell receptor.
背景技术Background technique
目前，CAR-T细胞治疗已经在肿瘤免疫治疗领域中展现出诱人的前景。CAR的基础结构包含肿瘤相关抗原结合区、跨膜区和胞内信号区。CAR一旦与肿瘤相关抗原结合，可通过胞内胞内区使T细胞活化，进而表现为CAR依赖的细胞杀伤、增殖及细胞因子释放。然而，CAR-T细胞疗法在临床应用中仍然存在一些问题，例如在血液瘤治疗中存在大量肿瘤复发现象，在实体瘤治疗中应答率不高等等，这些可能是由复杂的肿瘤微环境、CAR-T细胞耗竭等因素造成。At present, CAR-T cell therapy has shown attractive prospects in the field of tumor immunotherapy. The basic structure of CAR includes tumor-associated antigen binding region, transmembrane region and intracellular signal region. Once CAR binds to tumor-associated antigens, it can activate T cells through the intracellular region, and then manifest CAR-dependent cell killing, proliferation and cytokine release. However, there are still some problems in the clinical application of CAR-T cell therapy, for example, there are a large number of tumor recurrences in the treatment of hematological tumors, and the response rate is not high in the treatment of solid tumors, etc. These may be caused by the complex tumor microenvironment, CAR - Caused by factors such as T cell exhaustion.
因此，仍然需要对现有的CART细胞疗法进行改进，以促进CAR-T细胞在体内的增殖，抵抗肿瘤微环境的免疫抑制作用，进而提高CAR-T细胞疗法对肿瘤的整体治疗效果。Therefore, there is still a need to improve the existing CAR T cell therapy to promote the proliferation of CAR T cells in vivo, resist the immunosuppressive effect of the tumor microenvironment, and improve the overall therapeutic effect of CAR T cell therapy on tumors.
发明内容Contents of the invention
在第一个方面，本发明提供一种新的工程化免疫细胞，其表达(i)特异性识别抗原的细胞表面分子，和(ii))一种或多种LTBR配体。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.
在一个实施方案重，所述特异性识别抗原的细胞表面分子是嵌合抗原受体或重组T细胞受体，优选是嵌合抗原受体。In one embodiment, the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a recombinant T cell receptor, preferably a chimeric antigen receptor.
在一个实施方案中，LTBR配体选自LTA、LTB、LIGHT和抗LTBR抗体。In one embodiment, the LTBR ligand is selected from LTA, LTB, LIGHT and anti-LTBR antibodies.
在一个实施方案中，所述免疫细胞是选自T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。优选地，所述T细胞是CD4+/CD8+T细胞、CD4+辅助T细胞、CD8+T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。In one embodiment, the immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells. Preferably, the T cells are CD4+/CD8+ T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells or αβ-T cells.
在一个实施方案中，所述特异性识别抗原的细胞表面分子是嵌合抗原受体，其包含 抗原结合区、跨膜结构域、共刺激结构域和胞内信号传导结构域。其中，抗原抗原结合区可以选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双体、anticalin和DARPIN。优选地，所述抗原抗原结合区选自scFv、Fab、单结构域抗体和纳米抗体。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 co-stimulatory domain and an intracellular signaling domain. Wherein, the antigen binding region 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. Preferably, the antigen binding region is selected from scFv, Fab, single domain antibodies and nanobodies.
在一个实施方案中，所述特异性识别抗原的细胞表面分子与选自以下的靶标结合：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、CD 179a、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CD133、IFNAR1、DLL3、kappa轻链、TIM3、TSHR、CD19、BAFF-R、CLL-1、EGFRvIII、tEGFR、GD2、GD3、BCMA、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、IL-llRa、IL-22Ra、IL-2、间皮素、PSCA、PRSS21、VEGFR2、LewisY、PDGFR-β、SSEA-4、AFP、Folate受体α、ErbB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、NCAM、Claudin18.2、c-Met、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gpl00、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM7R、CLDN6、GPRC5D、CXORF61、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、MAGE-A3、MAGE-A6、豆荚蛋白、HPV E6、E7、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PSA、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、TMPRSS2ETS融合基因、NA17、PAX3、雄激素受体、孕酮受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2D配体，和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。优选地，所述靶标选自CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、AFP、Folate受体α、CEA、PSCA、PSMA、Her2、EGFR、IL13Ra2、GD2、NKG2D、EGFRvIII、 CS1、BCMA、间皮素和它们的任意组合。In one embodiment, the cell surface molecule that specifically recognizes the 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, CD40, 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, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY- ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT -2, Fos-related antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRS S2ETS 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, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and/or other pathogens and/or neo-epitopes or neoantigens. Preferably, the target is selected from 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.
在一个实施方案中，所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。优选地，跨膜结构域选自CD8α、CD4、CD28和CD278的跨膜结构域。In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8α, CD4, CD28 and CD278.
在一个实施方案中，所述胞内信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。优选地，所述胞内信号传导结构域包含CD3ζ胞内区。In one embodiment, the intracellular signaling domain is selected from the intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. Preferably, said intracellular signaling domain comprises a CD3ζ intracellular region.
在一个实施方案中，所述共刺激结构域是一个或多个选自以下蛋白质的胞内区：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、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以及它们的组合。优选地，所述共刺激结构域选自CD27、CD28、CD134、CD137、DAP10、DAP12或CD278的胞内区或它们的组合。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(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. Preferably, the co-stimulatory domain is selected from the intracellular region of CD27, CD28, CD134, CD137, DAP10, DAP12 or CD278 or a combination thereof.
在第二个方面，本发明提供一种核酸分子，(i)编码特异性识别抗原的细胞表面分子的核酸序列，和(ii)编码LTBR配体的核酸序列。优选地，所述特异性识别抗原的细胞表面分子是嵌合抗原受体或重组T细胞受体，更优选嵌合抗原受体。优选地，所述核酸是DNA或RNA。In a second aspect, the present invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, and (ii) a nucleic acid sequence encoding a LTBR ligand. Preferably, the cell surface molecule that specifically recognizes 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.
本发明还提供包含上述核酸分子的载体。具体地，所述载体选自质粒、逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV)、多瘤病毒和腺相关病毒(AAV)。在一些实施方案中，该载体还包含在免疫细胞中自主复制的起点、选择标记、限制酶切割位点、启动子、多聚腺苷酸尾(polyA)、3’UTR、5’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。The present invention also provides a vector comprising the above-mentioned nucleic acid molecule. Specifically, the vector is selected from plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV). In some embodiments, the vector further comprises an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a polyA tail (polyA), a 3'UTR, a 5'UTR, an enhancer Elements such as promoters, terminators, insulators, operators, selectable markers, reporter genes, targeting sequences and/or protein purification tags. In a specific embodiment, said vector is an in vitro transcribed vector.
在一个实施方案中，本发明还提供一种试剂盒，其包含本发明所述的工程化免疫细胞、核酸分子或载体。In one embodiment, the present invention also provides a kit comprising the engineered immune cells, nucleic acid molecules or vectors described in the present invention.
在一个实施方案中，本发明还提供一种药物组合物，其包含本发明所述的工程化免疫细胞、核酸分子或载体，和一种多种药学上可接受的赋型剂。In one embodiment, the present invention also provides a pharmaceutical composition, which comprises the engineered immune cells, nucleic acid molecules or vectors described in the present invention, and a variety of pharmaceutically acceptable excipients.
抗原抗原抗原在第三个方面，本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的免疫细胞、核酸分子、载体或药物组合物。Antigen Antigen In a third aspect, the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to said subject an effective amount of the Immune cells, nucleic acid molecules, vectors or pharmaceutical compositions.
在一个实施方案中，所述癌症是实体瘤或血液肿瘤。更具体地，所述癌症选自：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌、淋巴瘤、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤、B淋巴母细胞淋巴瘤(B-LBL)、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)。In one embodiment, 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, gastric cancer, glioblastoma (GBM), liver cancer, hepatoma, Intraepithelial neoplasms, kidney cancer, laryngeal cancer, liver tumors, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer Carcinoma, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system malignancies, vulvar and other carcinomas and sarcomas, and B cell Lymphoma, B-lymphoblastic lymphoma (B-LBL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse Acute large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasma Blastic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and posttransplantation lymphoproliferative disorder (PTLD).
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。In one embodiment, the infections include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites.
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。In one embodiment, 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.
附图说明Description of drawings
图1：CAR质粒的结构示意图。Figure 1: Schematic diagram of the structure of the CAR plasmid.
图2：通过流式细胞术测定的CAR-T细胞的CAR表达水平。Figure 2: CAR expression levels of CAR-T cells determined by flow cytometry.
图3：CAR-T细胞对靶细胞的杀伤活性。Figure 3: The killing activity of CAR-T cells on target cells.
图4：CAR-T细胞分别与靶细胞和非靶细胞共培养后的IFN-γ释放水平。Figure 4: IFN-γ release levels after CAR-T cells were co-cultured with target cells and non-target cells.
图5：用CAR-T细胞治疗小鼠B淋巴细胞瘤后，小鼠的存活曲线。Figure 5: Survival curves of mice treated with CAR-T cells for B lymphocytic tumors.
发明详述Detailed description of the invention
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
特异性识别抗原的细胞表面分子Cell surface molecules that specifically recognize antigens
如本文所用，术语“特异性识别抗原的细胞表面分子”是指在细胞表面表达的能够与靶分子(例如抗原)特异性结合的分子。此类表面分子一般包含能够与抗原特异性结合的抗原结合区、将表面分子锚定在细胞表面的跨膜结构域，以及负责信号传递的胞内结构域。常见的此类表面分子的实例包括例如重组T细胞受体或嵌合抗原受体。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 (eg, an antigen). Such surface molecules generally 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 signal transmission. Common examples of such surface molecules include, for example, recombinant T cell receptors or chimeric antigen receptors.
如本文所用，术语“重组T细胞受体”或“TCR”是指响应于抗原呈递并参与T细胞活化的膜蛋白复合体。TCR的刺激由抗原呈递细胞上的主要组织相容性复合体分子(MHC)触发，所述抗原呈递细胞将抗原肽呈递至T细胞并且结合至TCR复合体以诱发一系列胞内信号传导。TCR由分别形成异二聚体的六条肽链组成，其一般分为αβ型和γδ型。每条肽链包括恒定区和可变区，其中可变区负责结合特异性的特定的抗原和MHC分子。TCR的可变区可以包含抗原结合区或与抗原结合区可操作连接，其中抗原结合区的定义如下所述。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 the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling. TCR is composed of six peptide chains that form heterodimers, which are generally divided into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specific antigens and MHC molecules. The variable region of the TCR may comprise or be operably linked to an antigen binding region, wherein the definition of the antigen binding region is as follows.
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括抗原结合区(例如抗体的抗原结合部分)、跨膜结构域、共刺激结构域和细胞内信号传导结构域，各个结构域之间通过接头连接。CAR能够利用单克隆抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。非MHC限制性的抗原识别给予CAR细胞与抗原处理无关的识别抗原的能力，因此绕过了肿瘤逃逸的主要机制。此外，当在T细胞内表达时，CAR有利地不与内源性重组T细胞受体(TCR)的α链和β链二聚化。As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding region (such as the antigen-binding portion of an antibody), a transmembrane domain, a co-stimulatory Domain and intracellular signaling domain, each domain is connected by a linker. CARs are able to exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to a target of choice in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR cells the ability to recognize antigens independently of antigen processing, thus bypassing major mechanisms of tumor escape. Furthermore, CAR advantageously does not dimerize with the alpha and beta chains of the endogenous recombinant T cell receptor (TCR) when expressed in T cells.
如本文所用，“抗原结合区”是指可以与抗原结合的任何结构或其功能性变体。抗原结合区可以是抗体结构，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其功能性片段。例如，抗原结合区包括但不限于IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双体、anticalin、DARPIN等，优选选自Fab、scFv、sdAb和纳米抗体。在本发明中，抗原结合区可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。在另一个实施方案中，抗原结合区也可以是特定蛋白的特异性结合多肽或受体结构，所述特定 蛋白是例如PD1、PDL1、PDL2、TGFβ、APRIL和NKG2D。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 antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, murine antibody, chimeric antibody and functional fragments thereof. For example, antigen 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, anticalins, DARPIN etc., preferably selected from Fab, scFv, sdAb and Nanobodies. In the present invention, the antigen binding domain 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 of a specific protein, such as PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D.
“Fab”是指免疫球蛋白分子被木瓜蛋白酶裂解后产生的两个相同片段中的任一个，由通过二硫键连接的完整轻链和重链N端部分组成，其中重链N端部分包括重链可变区和CH1。与完整的IgG相比，Fab没有Fc片段，流动性和组织穿透能力较高，并且无需介导抗体效应即可单价结合抗原。"Fab" refers to either of two identical fragments of an immunoglobulin molecule produced after cleavage by papain, consisting of the complete light chain and the N-terminal portion of the heavy chain linked by a disulfide bond, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared with intact IgG, Fab has no Fc fragment, has higher fluidity and tissue penetration ability, and can monovalently bind antigen without mediating antibody effect.
“单链抗体”或“scFv”是由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。可以选择接头的最佳长度和/或氨基酸组成。接头的长度会明显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人,1993Proc Natl Acad.Sci.U.S.A.90:6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。A "single-chain antibody" or "scFv" is an antibody in which the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linker. The optimal length and/or amino acid composition of the linker can be selected. The length of the linker can significantly affect the variable domain folding and interaction of scFv. In fact, if shorter linkers (eg, between 5-10 amino acids) are used, intrachain folding can be prevented. For selection of linker size and composition, see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448; U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication Nos. WO2006/020258 and WO2007/024715 are hereby incorporated by reference in their entirety. A scFv may comprise VH and VL linked in any order, eg VH-linker-VL or VL-linker-VH.
“单结构域抗体”或“sdAb”是指一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，也称为“重链抗体”。"Single domain antibody" or "sdAb" refers to an antibody naturally devoid of light chains, which contains only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as "heavy chain Antibody".
“纳米抗体”或“Nb”是指单独克隆并表达出来的VHH结构，其具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是目前已知的可结合目标抗原的最小单位。"Nanobody" or "Nb" refers to the VHH structure cloned and expressed separately, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is currently the smallest unit known to bind the target antigen .
术语“功能性变体”或“功能性片段”是指基本上包含亲本的氨基酸序列但与该亲本氨基酸序列相比含有至少一个氨基酸修饰(即取代、缺失或插入)的变体，条件是所述变体保留亲本氨基酸序列的生物活性。在一个实施方案中，所述氨基酸修饰优选是保守型修饰。The term "functional variant" or "functional fragment" refers to a variant comprising essentially the amino acid sequence of a parent but containing at least one amino acid modification (i.e. substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the Such variants retain the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守修饰包括氨基酸取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。 保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。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 comprising the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include 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), non-polar side chains (e.g. alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
因此，“功能性变体”或“功能性片段”与亲本氨基酸序列具有至少75％，优选至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性，并且保留亲本氨基酸的生物活性，例如结合活性。Thus, a "functional variant" or "functional fragment" has 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.
如本文所用，术语“序列同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。As used herein, the term "sequence identity" means the degree to which two (nucleotide or amino acid) sequences in an alignment have the same residue at the same position, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies of the exact same sequence have 100% identity. Those 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 Clustal W.
抗原结合区的选择取决于待识别的与具体疾病状态相关的靶细胞上的细胞表面标记，例如肿瘤特异性抗原或肿瘤相关抗原。因此，在一个实施方案中，本发明的抗原结合区与选自以下的一个或多个靶标结合：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、CD 179a、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CD133、IFNAR1、DLL3、kappa轻链、TIM3、TSHR、CD19、BAFF-R、CLL-1、EGFRvIII、tEGFR、GD2、GD3、BCMA、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、IL-llRa、IL-22Ra、IL-2、间皮素、PSCA、PRSS21、VEGFR2、LewisY、PDGFR-β、SSEA-4、AFP、Folate受体α、ErbB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、NCAM、Claudin18.2、c-Met、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gpl00、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM7R、CLDN6、GPRC5D、CXORF61、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、MAGE-A3、MAGE-A6、豆荚蛋白、HPV E6、E7、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、 MAD-CT-2、Fos相关抗原1、p53、p53突变体、PSA、存活蛋白和端粒酶、PCTA-l/Galectin8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、TMPRSS2ETS融合基因、NA17、PAX3、雄激素受体、孕酮受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2D配体，和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。根据待靶向的抗原，本发明的CAR可以被设计为包括对该抗原具有特异性的抗原结合区。例如，如果CD19是待靶向的抗原，则CD19抗体可用作本发明的抗原结合区。The choice of the antigen binding region depends on the cell surface markers to be recognized on the target cells associated with the particular disease state, such as tumor-specific or tumor-associated antigens. 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, CD40, 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, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl- GD2, Folate receptor β, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD -CT-2, Fos-related antigen 1, p53, p53 mutants, PSA, survivin and telomerase, PCTA-1/Galectin8, MelanA/MART1, Ras mutants, hTERT, sarcoma translocation breakpoint, ML-IAP 、TMPR SS2ETS 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, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and/or other pathogens and/or neo-epitopes or neoantigens. Depending on the antigen to be targeted, the CAR of the invention can be designed to include an antigen-binding region specific for that antigen. For example, if CD19 is the antigen to be targeted, a CD19 antibody can be used as the antigen binding region of the present invention.
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154及其功能性片段。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD8α，其与SEQ ID NO:4或16所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或所述CD8α跨膜结构域的编码序列与SEQ ID NO:3或15所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。As used herein, the term "transmembrane domain" refers to a polypeptide capable of expressing a chimeric antigen receptor on the surface of an immune cell (such as a lymphocyte, NK cell or NKT cell) and directing a cellular response of the immune cell against a target cell structure. Transmembrane domains 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 suitable for use in the present invention may be derived from, for example, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α , 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 comprise predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8α, which has 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: 4 or 16. % or 100% sequence identity, or the coding sequence of the CD8α transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90% of the nucleotide sequence shown in SEQ ID NO:3 or 15 , 95%, 97% or 99% or 100% sequence identity.
在一个实施方案中，本发明的嵌合抗原受体还可以包含位于抗原结合区和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包 含CD8α链、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选CD8α铰链，其与SEQ ID NO:12、22、33或35所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或者CD8α铰链的编码序列与SEQ ID NO:11、21、34或36所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。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 link a transmembrane domain to an antigen binding region. Specifically, the hinge region is used 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 natural 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. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In a preferred embodiment, the hinge region comprises the hinge region part of CD8α chain, FcγRIIIα receptor, IgG4 or IgG1, more preferably CD8α hinge, which has the same amino acid sequence as shown in SEQ ID NO: 12, 22, 33 or 35 Have 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 CD8α hinge is identical to SEQ ID NO: 11, 21, 34 or 36 The nucleotide sequences shown have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在抗原结合区结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应子功能的至少一种。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。As used herein, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a given function. The intracellular signaling domain is responsible for primary intracellular signal transmission following antigen binding at the antigen binding region, resulting in activation of immune cells and immune responses. 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 can be cytolytic activity or helper activity, including secretion of cytokines.
在一个实施方案中，本发明的嵌合抗原受体包含的胞内信号传导结构域可以是重组T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发初级信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。胞内信号传导结构域可以包含许多免疫受体酪氨酸激活基序(Immunoreceptor Tyrosine-based Activation Motifs,ITAM)。本发明的胞内信号传导结构域的非限制性施例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在优选的实施方式中，本发明CAR的信号传导结构域可以包含CD3ζ胞内区，该信号传导结构域与SEQ ID NO:8或20所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:7或19所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In one embodiment, the intracellular signaling domains comprised by the chimeric antigen receptors of the invention may be cytoplasmic sequences of recombinant T cell receptors and co-receptors, which act together to elicit primary Signal transduction, and any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signaling domain can contain many immunoreceptor tyrosine-based activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM). Non-limiting examples of intracellular signaling domains of the invention include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR of the present invention may comprise a CD3ζ intracellular region, and the signaling domain has 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.
在一个实施方案中，本发明的嵌合抗原受体包含一个或多个共刺激结构域。共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的胞内区：CD94、LTB、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、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以及ZAP70。优选地，本发明CAR的共刺激结构域来自4-1BB、CD28、CD27、OX40、ICOS、DAP10、DAP12或其组合，更优选4-1BB和/或CD28。在一个实施方案中，本发明的CAR包含的共刺激结构域与SEQ ID NO:6、18或31所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或该共刺激结构域的编码序列与SEQ ID NO:5、17或32所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In one embodiment, a chimeric antigen receptor of the invention comprises one or more co-stimulatory domains. A co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule comprising the entire intracellular portion of said co-stimulatory 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 co-stimulatory domains of the invention include, but are not limited to, intracellular regions derived from the following proteins: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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. Preferably, the costimulatory domain of the CAR of the present 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 costimulatory domain contained in the CAR of the present invention has 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 costimulatory domain and the nucleotide sequence shown in SEQ ID NO: 5, 17 or 32 have at least 70%, preferably at least 80%, more A sequence identity of at least 90%, 95%, 97% or 99% or 100% is preferred.
在一个实施方案中，本发明的CAR还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自CD8α、IgG1、GM-CSFRα等的信号肽。在一个实施方案中，可用于本发明的信号肽与SEQ ID NO:10所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或该信号肽的编码序列与SEQ ID NO:9所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In one embodiment, the CAR of the invention may also 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 stretch of hydrophobic amino acids with a propensity to form a single α-helix. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by the signal peptidase. The signal peptidase can cleave during translocation or after completion to generate a free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases. Signal peptides that can be used in the present invention are well known to those skilled in the art, for example, signal peptides derived from CD8α, IgG1, GM-CSFRα, and the like. In one embodiment, the signal peptide that can be 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: 10 % 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 99% of the nucleotide sequence shown in SEQ ID NO:9 or 100% sequence identity.
在一个实施方案中，本发明的CAR还可以包含开关结构，以调控CAR的表达时间。例如，开关结构可以是二聚化结构域的形式，通过与其相应配体的结合引起构象变化，暴露胞外结合结构域，使其与被靶向抗原结合，从而激活信号传导通路。或者，也可以使用开关结构域分别连接结合结构域和信号传导结构域，仅当开关结构域互相结合(例如在诱导化合物的存在下)时，结合结构域和信号传导结构域才能通过二聚体连接在一起，从而激活信号通路。开关结构还可以是掩蔽肽的形式。掩蔽肽可以遮蔽胞外结合结构域，阻止其与被靶向抗原的结合，当通过例如蛋白酶切割掩蔽肽后，就可以暴露胞外结合结构域，使其成为一个“普通”的CAR结构。本领域技术人员知晓的各种开关结构均可用于本发明。In one embodiment, the CAR of the present invention may also include a switch structure to regulate the expression time of the CAR. For example, the switch structure can be in the form of a dimerization domain, which induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain to allow it to bind to the targeted antigen, thereby activating the signaling pathway. Alternatively, a switch domain can be used to link the binding and signaling domains separately, and the binding and signaling domains can pass through the dimer only when the switch domains are associated with each other (e.g. in the presence of an inducing compound). Link together to activate the signaling pathway. The switch structure can also be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain and prevent it from binding to the targeted antigen. When the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it an "ordinary" CAR structure. Various switch configurations known to those skilled in the art can be used in the present invention.
在一个实施方案中，本发明的CAR还可以包含自杀基因，即，使其表达一个可通过外源物质诱导的细胞死亡信号，以在需要时(例如产生严重的毒副作用时)清除CAR细胞。例如，自杀基因可以是插入的表位的形式，例如CD20表位、RQR8等，当需要时，可以通过加入靶向这些表位的抗体或试剂来消除CAR细胞。自杀基因也可以是单 纯疱疹病毒胸苷激酶(HSV-TK)，该基因可使细胞在接受更昔洛韦治疗诱导下死亡。自杀基因还可以是iCaspase-9，可以通过化学诱导药物如AP1903、AP20187等诱导iCaspase-9发生二聚化，从而激活下游的Caspase3分子，导致细胞凋亡。本领域技术人员知晓的各种自杀基因均可用于本发明。In one embodiment, the CAR of the present invention may also contain a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance, so as to eliminate CAR cells when necessary (eg, when severe toxic side effects occur). For example, suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when necessary, 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. The suicide gene can also be iCaspase-9, and the dimerization of iCaspase-9 can be induced by chemically inducing drugs such as AP1903 and AP20187, thereby activating the downstream Caspase3 molecule and leading to cell apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.
LTBR配体LTBR ligand
LTBR是指淋巴毒素β受体，其是分子量为61kDa的I型单跨膜蛋白。LTBR的胞外区负责与配体结合，含有TNF基序特有的四个富含半胱氨酸的重复序列。LTBR的胞内区由175个氨基酸残基组成，其本身并不具有激酶货其他酶活性。LTBR在大多数细胞上组成型表达，例如淋巴组织的基质细胞、髓系细胞、血液单核细胞、肺泡巨噬细胞、肥大细胞、树突细胞等。然而，LTBR却不在T细胞、B细胞和NK细胞中表达，这也是其最突出的特点。与此相反，LTBR的配体则常在T细胞和B细胞中表达。这种表达模式表明对于LT而言，信号传导可能是单向的，需要淋巴细胞和携带LTBR的细胞之间的直接接触以传递信号。LTBR refers to lymphotoxin beta receptor, which is a type I single transmembrane protein with a molecular weight of 61 kDa. The extracellular domain of LTBR is responsible for ligand binding and contains four cysteine-rich repeats characteristic of TNF motifs. The intracellular region of LTBR consists of 175 amino acid residues, which itself does not have kinase or other enzymatic activities. LTBR is constitutively expressed on most cells, such as stromal cells of lymphoid tissue, myeloid cells, blood monocytes, alveolar macrophages, mast cells, dendritic cells, etc. However, LTBR is not expressed in T cells, B cells and NK cells, which is also its most prominent feature. In contrast, LTBR ligands are often expressed on T cells and B cells. This expression pattern suggests that signaling may be unidirectional for LT, requiring direct contact between lymphocytes and LTBR-bearing cells to transmit the signal.
目前已知的天然LTBR配体主要包括两个：淋巴毒素(LT)和LIGHT。淋巴毒素(LT)是淋巴细胞受抗原或有丝分裂原等刺激活化后及在某些肿瘤、自身免疫病的情况下产生分泌的一种细胞因子。淋巴毒素又称肿瘤坏死因子β，与肿瘤坏死因子α(TNF-α)是两个密切相关的细胞因子，同属TNF家族。LT主要在B细胞、T细胞和NK细胞中表达。LT包括两种亚单位：LTα(也称为LTA)和LTβ(也称为LTB)。其中，LTα可以分泌到细胞外，然后通过与LTβ非共价结合形成异源三聚体而锚定于细胞表面。LT在杀伤肿瘤细胞、调节免疫反应、调节炎症反应、诱导多种分化细胞的抗原表达等多种生物学活动中发挥重要作用。LIGHT是一种II型跨膜蛋白，也属于TNF家族，由激活的T细胞、单核细胞、粒细胞以及未成熟的树突细胞产生。LTBR与其配体之间的结合激活NKκB和c-Jun N末端激酶(JNK)信号通路，导致趋化因子，例如CXCL12、CXCL13、CCL19、CCL21等的表达，进而在某些细胞(例如肿瘤细胞)中诱导细胞凋亡。此外，LTBR受体-配体之间的信号传导也可以通过抗LTBR抗体或通过受体的过表达来启动。Currently known natural LTBR ligands mainly include two: lymphotoxin (LT) and LIGHT. Lymphotoxin (LT) is a cytokine that is secreted by lymphocytes after being stimulated by antigens or mitogens and in certain tumors and autoimmune diseases. Lymphotoxin, also known as tumor necrosis factor β, and tumor necrosis factor α (TNF-α) are two closely related cytokines that belong to the TNF family. LT is mainly expressed in B cells, T cells and NK cells. LT includes two subunits: LTα (also known as LTA) and LTβ (also known as LTB). Among them, LTα can be secreted out of the cell, and then anchored on the cell surface by non-covalent combination with LTβ to form a heterotrimer. LT plays an important role in various biological activities such as killing tumor cells, regulating immune response, regulating inflammatory response, and inducing antigen expression of various differentiated cells. LIGHT is a type II transmembrane protein that also belongs to the TNF family and is produced by activated T cells, monocytes, granulocytes, and immature dendritic cells. The binding between LTBR and its ligands activates NKκB and c-Jun N-terminal kinase (JNK) signaling pathways, resulting in the expression of chemokines, such as CXCL12, CXCL13, CCL19, CCL21, etc., and in some cells (such as tumor cells) inducing apoptosis. In addition, LTBR receptor-ligand signaling can also be initiated by anti-LTBR antibodies or by overexpression of the receptor.
在一个实施方案中，LTBR配体是LTA，其与SEQ ID NO：26、40、46或48所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性(SEQ ID NO：26的第1-33位氨基酸为信号肽)，或其编码序列与SEQ ID NO：25、39、45或47所示的核酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。SEQ ID NO：46由SEQ ID NO： 26的第34-202位氨基酸组成(SEQ ID NO：26的第1-33位氨基酸为信号肽)，SEQ ID NO：48由SEQ ID NO：40的第35-205位氨基酸组成(SEQ ID NO：40的第1-34位氨基酸为信号肽)。In one embodiment, the LTBR ligand is LTA, which 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: 26, 40, 46 or 48. % or 99% or 100% sequence identity (the 1-33 amino acids of SEQ ID NO: 26 are signal peptides), or its coding sequence and the nucleic acid sequence shown in SEQ ID NO: 25, 39, 45 or 47 Having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity. SEQ ID NO: 46 consists of amino acids 34-202 of SEQ ID NO: 26 (amino acids 1-33 of SEQ ID NO: 26 are signal peptides), and SEQ ID NO: 48 consists of amino acids of SEQ ID NO: 40 Amino acid composition at positions 35-205 (amino acids 1-34 of SEQ ID NO: 40 are signal peptides).
在一个实施方案中，LTBR配体是LTB，其与SEQ ID NO：24或42所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：23或41所示的核酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In one embodiment, the LTBR ligand is LTB, which has 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: 24 or 42 Or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the nucleic acid sequence shown in SEQ ID NO: 23 or 41 or 100% sequence identity.
在一个实施方案中，LTBR配体是LIGHT，其与SEQ ID NO：28或44所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：27或43所示的核酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。In one embodiment, the LTBR ligand is LIGHT, which has 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: 28 or 44 Or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the nucleic acid sequence shown in SEQ ID NO: 27 or 43 or 100% sequence identity.
在一个实施方案中，LTBR配体是抗LTBR抗体，例如选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双体的抗体。本领域已知的抗体均可用作本发明的LTBR配体，包括但不限于CBE11(ATCC登录号11793)、BKA11(ATCC登录号11799)、CDH10(ATCC登录号11797)、BCG6(ATCC登录号11794)、BHA10(ATCC登录号11795)、BDA8(ATCC登录号HB11798)(参见美国专利号5,925,351和631,269,1，其全部内容通过引用并入本文)。In one embodiment, the LTBR ligand is an anti-LTBR antibody, e.g. selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, single domain antibody, Nanobodies, diabodies. Antibodies known in the art can be used as LTBR ligands of the invention, including but not limited to CBE11 (ATCC Accession No. 11793), BKA11 (ATCC Accession No. 11799), CDH10 (ATCC Accession No. 11797), BCG6 (ATCC Accession No. 11794), BHA10 (ATCC Accession No. 11795), BDA8 (ATCC Accession No. HB11798) (see US Patent Nos. 5,925,351 and 631,269,1, the entire contents of which are incorporated herein by reference).
核酸nucleic acid
本发明还提供一种核酸分子，其包含(i)编码特异性识别抗原的细胞表面分子的核酸序列，和(ii)编码LTBR配体的核酸序列。The present invention also provides 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.
在一个实施方案重，所述特异性识别抗原的细胞表面分子是重组T细胞受体或嵌合抗原受体，优选嵌合抗原受体。嵌合抗原受体的定义如上所述。In one embodiment, the cell surface molecule that specifically recognizes an antigen is a recombinant T cell receptor or a chimeric antigen receptor, preferably a chimeric antigen receptor. Chimeric antigen receptors are defined above.
如本文所用，术语“核酸分子”包括核糖核苷酸和脱氧核糖核苷酸的序列，如经修饰的或未经修饰的RNA或DNA，各自为单链和/或双链形式的线性或环状，或它们的混合物(包括杂合分子)。因此，根据本发明的核酸包括DNA(比如dsDNA、ssDNA、cDNA)、RNA(比如dsRNA、ssRNA、mRNA、ivtRNA)，它们的组合或衍生物(比如PNA)。优选地，所述核酸是DNA或RNA，更优选mRNA。As used herein, the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single- and/or double-stranded form, linear or circular shape, or their mixtures (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.
核酸可以包含常规的磷酸二酯键或非常规的键(如酰胺键，比如在肽核酸(PNA)中发现的)。本发明的核酸还可含有一种或多种经修饰的碱基，比如，例如三苯甲基化的碱基和不常见的碱基(比如肌苷)。也可以想到其它修饰，包括化学、酶促或代谢修饰，只要 本发明的多链CAR可以从多核苷酸表达即可。核酸可以以分离的形式提供。在一个实施方案中，核酸也可以包括调节序列，比如转录控制元件(包括启动子、增强子、操纵子、抑制子和转录终止信号)、核糖体结合位点、内含子等。Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds such as amide bonds such as those found in peptide nucleic acids (PNAs). 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 are also contemplated, including chemical, enzymatic or metabolic modifications, so long as the multi-chain CAR of the invention can be expressed from the polynucleotide. Nucleic acids can be provided in isolated form. In one embodiment, a nucleic acid may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcriptional termination signals), ribosome binding sites, introns, and the like.
可以对本发明的核酸序列进行密码子优化以在所需的宿主细胞(如，免疫细胞)中进行最佳表达；或者用于在细菌、酵母菌或昆虫细胞中表达。密码子优化是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。The nucleic acid sequences of the present invention can be codon-optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacteria, yeast or insect cells. Codon optimization refers to the replacement of codons 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 this species, while the codons before and after the replacement code for the same amino acid. Therefore, the choice of optimal codons depends on the codon usage bias of the host genome.
载体carrier
本发明还提供一种载体，包含如本发明所述的核酸。其中，编码特异性识别抗原的细胞表面分子的核酸序列、和编码LTBR配体的核酸可以位于一个或多个载体中。The present invention also provides a vector comprising the nucleic acid according to the present invention. Among them, the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen, and the nucleic acid encoding the LTBR ligand may be located in one or more vectors.
例如，可以通过在两个核酸序列之间插入编码2A肽的核酸，使得两个嵌合抗原受体结构虽然位于同一载体但却可以独立表达而互不影响。如本文所用，术语“2A肽”是一种cis-水解酶作用元件，最初在口蹄疫病毒(FMDV)中发现。2A肽的平均长度为18～22氨基酸。在蛋白翻译时，2A肽可以通过核糖体跳跃从自身最后2个氨基酸C末端断裂。具体地，甘氨酸和脯氨酸之间的肽链结合群在2A位点是受损的，能引发核糖体跳跃而从第2个密码子开始翻译，从而使1个转录单元里的2个蛋白独立表达。这种2A肽介导的剪切广泛存在于真核动物细胞中。利用2A肽较高的剪切效率及促使上下游基因平衡表达的能力，可以改进异源多聚蛋白(如细胞表面受体、细胞因子、免疫球蛋白等)的表达效率。常规2A肽包含：P2A、T2A、E2A、F2A等。For example, by inserting a nucleic acid encoding 2A peptide between the two nucleic acid sequences, the two chimeric antigen receptor structures can be independently expressed in the same vector without affecting each other. As used herein, the term "2A peptide" is a cis-hydrolase action element originally discovered in foot-and-mouth disease virus (FMDV). The average length of 2A peptides is 18-22 amino acids. During protein translation, the 2A peptide can be cleaved from the C-terminus of the last 2 amino acids of itself by ribosome jumping. Specifically, the peptide chain binding group between glycine and proline is damaged at the 2A site, which can trigger ribosome jumping and start translation from the second codon, so that two proteins in one transcription unit independent expression. This 2A peptide-mediated cleavage is ubiquitous in eukaryotic cells. The expression efficiency of heterologous polyproteins (such as cell surface receptors, cytokines, immunoglobulins, etc.) can be improved by utilizing the higher shearing efficiency of 2A peptides and the ability to promote the balanced expression of upstream and downstream genes. Conventional 2A peptides include: P2A, T2A, E2A, F2A, etc.
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。As used herein, the term "vector" is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell where it can eg be replicated and/or expressed.
载体一般包括靶向载体和表达载体。“靶向载体”是通过例如同源重组或使用特异性靶向位点处序列的杂合重组酶将分离的核酸递送至细胞内部的介质。“表达载体”是用于异源核酸序列(例如编码本发明的嵌合抗原受体多肽的那些序列)在合适的宿主细胞中的转录以及它们的mRNA的翻译的载体。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一个实施方案中，本发明的载体包括但不限于质粒、病毒(例如逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV、多瘤病毒和腺相关病毒(AAV)等)、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核苷酸序列，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。 工程化载体通常还包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。Vectors generally include targeting vectors and expression vectors. A "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 sequence at a specific targeting site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding chimeric antigen receptor polypeptides of the invention, and the translation of their mRNA in a suitable host cell. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, vectors of the present invention include, but are not limited to, plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc. ), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is usually a sequence of nucleotides, usually a DNA sequence containing an insert (transgene) and a larger sequence that acts as the "backbone" of the vector The engineered vector usually also contains 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 (such as a multiple cloning site, MCS). The vector may additionally contain a promoter , polyadenylation tail (polyA), 3' UTR, enhancer, terminator, insulator, operator, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements. In a specific embodiment In, the vector is an in vitro transcribed vector.
工程化免疫细胞engineered immune cells
本发明还提供一种工程化免疫细胞，其包含本发明的核酸或载体。换言之，本发明的工程化免疫细胞表达特异性识别抗原的细胞表面分子、以及LTBR配体。The present invention also provides an engineered immune cell comprising the nucleic acid or vector of the present invention. In other words, the engineered immune cells of the present invention express cell surface molecules that specifically recognize antigens, as well as LTBR ligands.
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，或者是从细胞脐带血等干细胞来源获得的免疫细胞。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+/CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。在本发明中，免疫细胞被工程化以表达嵌合抗原受体以及外源性的LTBR配体。As used herein, 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). For example, 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. Preferably, the immune cells are T cells. The T cells may be any T cells, such as T cells cultured in vitro, such as 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. 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 sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be enriched or purified. T cells can 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, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, 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. In the present invention, immune cells are engineered to express chimeric antigen receptors as well as exogenous LTBR ligands.
还在一个实施方案中，本发明的免疫细胞还包含至少一种失活基因，其选自以下：CD52、GR、TCRα、TCRβ、CD3γ、CD3δ、CD3ε、CD247ζ、HLA-I、HLA-II、B2M、免疫检查点基因如PD1、CTLA-4、LAG3和TIM3。更特别地，免疫细胞中的至少TCR组分(包括TCRα、TCRβ基因)或CD3组分(包括CD3γ、CD3δ、CD3ε、CD247ζ)被失活。这种失活使得TCR-CD3复合物在细胞中没有功能。该策略对于避免移植物抗宿主病(GvHD)特别有用。使基因失活的方法是本领域已知的，例如通过大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂，从而使该基因失活。In yet another embodiment, the immune cells of the present invention further comprise at least one inactivated gene selected from the group consisting of: CD52, GR, TCRα, TCRβ, CD3γ, CD3δ, CD3ε, CD247ζ, HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More specifically, at least TCR components (including TCRα, TCRβ genes) or CD3 components (including CD3γ, CD3δ, CD3ε, CD247ζ) in immune cells are inactivated. This inactivation renders the TCR-CD3 complex nonfunctional in the cell. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD). Methods for gene inactivation are known in the art, such as DNA fragmentation mediated by meganuclease, zinc finger nuclease, TALE nuclease or Cas enzyme in CRISPR system, thereby inactivating the gene.
药物组合物pharmaceutical composition
本发明还提供一种药物组合物，其包含本发明所述的工程化免疫细胞、核酸分子或载体作为活性剂，和一种或多种药学上可接受的赋型剂。因此，本发明还涵盖所述核酸分子、载体或工程化免疫细胞在制备药物组合物或药物中的用途。The present invention also provides a pharmaceutical composition, which comprises the engineered immune cell, nucleic acid molecule or carrier described in the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the nucleic acid molecules, vectors or engineered immune cells in the preparation of pharmaceutical compositions or medicaments.
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。As used herein, the term "pharmaceutically acceptable excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse desired local or systemic effect), which are well known in the art (see, for example, 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, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . The selection of suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water. In general, the selection 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.
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。The pharmaceutical compositions according to the present invention are suitable for various routes of administration. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。The pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, especially 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 especially adapted to the desired method of administration. Processes known in the present invention for the production of medicaments 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.
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、 假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。The pharmaceutical composition according to the invention can also be administered in combination with one or more other agents suitable for the treatment and/or prophylaxis of the disease to be treated. Preferred examples of 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, trimetreate 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, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains and viral/bacterial peptides. In addition, the pharmaceutical composition of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.
治疗应用therapeutic application
本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的免疫细胞或药物组合物。因此，本发明还涵盖所述工程化免疫细胞在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。The present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the immune cell or the pharmaceutical composition according to the present invention. Therefore, the present invention also covers the use of the engineered immune cells in the preparation of medicaments for treating cancer, infection or autoimmune diseases.
在一个实施方案中，所述治疗方法包括向受试者施用有效量的本发明的免疫细胞和/或药物组合物。In one embodiment, the method of treatment comprises administering to a subject an effective amount of an immune cell and/or a pharmaceutical composition of the present invention.
在一个实施方案中，所述免疫细胞是自体或同种异体的细胞，优选T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，更优选T细胞、NK细胞或NKT细胞。In one embodiment, 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.
如本文所用，术语“自体”是指来源于个体的任何材料稍后将被再引入该相同个体中。As used herein, the term "autologous" refers to any material derived from an individual that will later be reintroduced into that same individual.
如本文所用，术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些情况下，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species or a different patient as the individual into whom the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci differ. In some cases, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.
如本文所用，术语“受试者”是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。As used herein, the term "subject" is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cows, but are 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.
在一个实施方案中，所述癌症是与抗原结合区结合的靶标表达有关的癌症。例如，所述癌症包括但不限于：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌(包括胃肠癌)、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌(例如小细胞肺癌、非小细胞肺癌、腺状肺癌和鳞状肺癌)、淋巴瘤(包括霍奇金淋巴 瘤和非霍奇金淋巴瘤)、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中间级/滤泡性NHL、中间级扩散性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小型非裂化细胞性NHL、大肿块病NHL)、B淋巴母细胞淋巴瘤(B-LBL)、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病In one embodiment, the cancer is a cancer associated with expression of a target bound by the antigen binding region. For example, the 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 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, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatoma, intraepithelial neoplasia, renal cancer, laryngeal cancer, liver tumors, lung cancer (such as small cell lung cancer, non-small cell lung cancer, adenoid lung cancer, and squamous lung cancer), lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (eg, lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer Carcinoma, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system malignancies, vulvar and other carcinomas and sarcomas, and B cell Lymphoma (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-cleaved cellular NHL, large-bulk disease NHL), B-lymphoblastic lymphoma (B-LBL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia
(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)；以及其他与靶标表达有关的疾病。优选地，可以用本发明的工程化免疫细胞或药物组合物治疗的疾病选自：白血病、淋巴瘤、多发性骨髓瘤、脑神经胶质瘤、胰腺癌、胃癌等。(ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt Lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, Myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and post-transplantation lymphoproliferative disorder (PTLD); and other disorders associated with target expression. Preferably, the diseases that can be treated with the engineered immune cells or the pharmaceutical composition of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer and the like.
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。In one embodiment, the infections include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites.
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。In one embodiment, 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.
在一个实施方案中，所述方法还进一步包括向所述受试者施用一种或多种额外的化疗剂、生物制剂、药物或治疗。在该实施方案中，化疗剂、生物制剂、药物或治疗选自放射疗法、手术、抗体试剂和/或小分子和它们的任意组合。In one embodiment, the method further comprises administering to the subject one or more additional chemotherapeutic agents, biologics, drugs or treatments. In this embodiment, the chemotherapeutic agent, biologic, drug or treatment is selected from radiation therapy, surgery, antibody agents and/or small molecules and any combination thereof.
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。The present invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the drawings and the embodiments of the present invention are only for the purpose of illustration, and shall not constitute any limitation to the present invention. In the case of no contradiction, the embodiments in the present application and the features in the embodiments can be combined with each other.
具体实施方式detailed description
实施例1.制备CAR-T细胞Example 1. Preparation of CAR-T cells
1.1.构建逆转录病毒质粒1.1. Construction of retroviral plasmids
人工合成依次连接的CD8a信号肽(SEQ ID NO：10)、CD19-scFv(SEQ ID NO：14)、CD8a铰链区(SEQ ID NO：22)、CD8a跨膜区(SEQ ID NO：16)、4-1BB胞内域(SEQ ID NO：18)和CD3ζ胞内域(SEQ ID NO：20)的编码序列片段，并将其克隆入MSCV载体，获得bbz质粒。Artificially synthesized sequentially connected 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), The coding sequence fragments of 4-1BB intracellular domain (SEQ ID NO: 18) and CD3ζ intracellular domain (SEQ ID NO: 20) were cloned into MSCV vector to obtain bbz plasmid.
人工合成依次连接的T2A(SEQ ID NO：37)和LTB(SEQ ID NO：24)的编码序列片段，并将其克隆入bbz载体，获得bbz-T2A-Ltb质粒。The coding sequence fragments of T2A (SEQ ID NO: 37) and LTB (SEQ ID NO: 24) connected sequentially were artificially synthesized and cloned into the bbz vector to obtain the bbz-T2A-Ltb plasmid.
人工合成依次连接的T2A(SEQ ID NO：37)、LTB(SEQ ID NO：24)和LTA(SEQ ID NO：26)的编码序列片段，并将其克隆入bbz载体，获得bbz-T2A-Ltba质粒。The coding sequence fragments of T2A (SEQ ID NO: 37), LTB (SEQ ID NO: 24) and LTA (SEQ ID NO: 26) were artificially synthesized and cloned into the bbz vector to obtain bbz-T2A-Ltba plasmid.
人工合成依次连接的T2A(SEQ ID NO：37)和LIGHT(SEQ ID NO：28)的编码序列片段，并将其克隆入bbz载体，获得bbz-T2A-LIGHT质粒。The coding sequence fragments of T2A (SEQ ID NO: 37) and LIGHT (SEQ ID NO: 28) connected sequentially were artificially synthesized and cloned into the bbz vector to obtain the bbz-T2A-LIGHT plasmid.
各质粒的结构如图1所示。The structure of each plasmid is shown in FIG. 1 .
1.2.制备逆转录病毒1.2. Preparation of retrovirus
在T175培养瓶中，以30×10 6个细胞/瓶的密度将293T细胞接种于30ml含有10％胎牛血清的DMEM培养基中，于37℃、5％CO 2培养箱中培养过夜，用于病毒包装。 In a T175 culture flask, inoculate 293T cells in 30ml of DMEM medium containing 10 % fetal bovine serum at a density of 30×106 cells/flask, and cultivate overnight in a 37°C, 5% CO2 incubator, and use in virus packaging.
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)、45μg制备的逆转录病毒质粒和15μg包装载体pCL-Eco(上海禾午生物科技有限公司，货号P3029)。然后加入120μl X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，室温孵育15min。然后将该质粒/载体/转染试剂混合物逐滴加入到预先准备好的293T细胞的培养瓶中，于37℃，5％CO 2条件下培养过夜。在转染后72小时收集培养物，离心(2000g，4℃，10分钟)，获得逆转录病毒上清液。 Add 3ml of Opti-MEM (Gibco, Cat. No. 31985-070), 45 μg of prepared retroviral plasmid and 15 μg of packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029) into a sterile tube. Then add 120 μl of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, and incubate at room temperature for 15 minutes. Then the plasmid/vector/transfection reagent mixture was added dropwise into the pre-prepared 293T cell culture flask, and cultured overnight at 37°C and 5% CO 2 . Cultures were harvested 72 hours after transfection and centrifuged (2000g, 4°C, 10 minutes) to obtain retroviral supernatants.
1.3制备CAR-T细胞1.3 Preparation of CAR-T cells
从小鼠脾脏分离T淋巴细胞，并用DynaBeads CD3/CD28 CTS TM(Gibco,货号40203D)激活T细胞，然后在37℃和5％CO 2下培养1天。 T lymphocytes were isolated from mouse spleen, and T cells were activated with DynaBeads CD3/CD28 CTS TM (Gibco, Cat. No. 40203D), and then cultured at 37°C and 5% CO 2 for 1 day.
以每孔3×10 6个细胞/mL的密度将激活的T细胞接种至预先用RetroNectin包被过夜的24孔板中，然后分别加入500μL完全培养基(NT，对照)或步骤2制备的逆转录病毒上清液，并补充完全培养基至2mL。 Inoculate activated T cells at a density of 3 ×106 cells/mL per well into a 24-well plate pre-coated overnight with RetroNectin, and then add 500 μL of complete medium (NT, control) or reverse prepared in step 2, respectively. Record the virus supernatant, and supplement the complete medium to 2mL.
将24孔板放于离心机进行离心感染，于32℃，2000g离心2h。然后，立刻将24孔板放置于37℃、CO2培养箱静置培养。第二天更换新鲜培养基，并调整细胞密度为1×10 6个细胞/mL。感染三天后，收集细胞用于后续分析。收集的细胞即为NT细胞、bbz CAR-T细胞、bbz-T2A-Ltb CAR-T细胞、bbz-T2A-Ltba CAR-T细胞和bbz-T2A-LIGHT  CAR-T细胞。 Place the 24-well plate in a centrifuge for centrifugal infection, and centrifuge at 2000g for 2h at 32°C. Then, immediately place the 24-well plate in a 37°C, CO2 incubator for static culture. Replace the fresh medium the next day, and adjust the cell density to 1× 106 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检测CAR表达水平1.4 Detection of CAR expression level
取2×10 5个CAR-T细胞，用Biotin-SP-AffiniPure Goat Anti-Rat IgG,F(ab’)2 Fragment Specific(Jackson ImmunoResearch，货号112-065-072)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)作为二抗，通过流式细胞术检测各种CAR-T细胞上的CAR的表达水平，结果如图2所示。 Take 2×10 5 CAR-T cells, use Biotin-SP-AffiniPure Goat Anti-Rat IgG, F(ab')2 Fragment Specific (Jackson ImmunoResearch, Cat. No. 112-065-072) as primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) was used as a secondary antibody to detect the expression levels of CAR on various CAR-T cells by flow cytometry, and the results are shown in Figure 2.
可以看出，bbz CAR-T细胞、bbz-T2A-Ltb CAR-T细胞、bbz-T2A-Ltba CAR-T细胞和bbz-T2A-LIGHT CAR-T细胞中的CAR均可有效表达。It can be seen that the CARs in bbz CAR-T cells, bbz-T2A-Ltb CAR-T cells, bbz-T2A-Ltba CAR-T cells and bbz-T2A-LIGHT CAR-T cells can all be effectively expressed.
实施例2.CAR-T细胞对靶细胞的杀伤效果和细胞因子释放Example 2. The killing effect and cytokine release of CAR-T cells on target cells
2.1 对靶细胞的杀伤效果 2.1 Killing effect on target cells
以1x10 4/孔将携带荧光素基因的A20-CD19靶细胞(经筛选的CD19阳性AD20细胞)铺入96孔板中，然后分别以5:1、10:1或20:1的效靶比(即效应T细胞与靶细胞之比)将实施例1制备的CAR-T细胞和NT细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图3所示。 Spread the A20-CD19 target cells (screened CD19 positive AD20 cells) carrying the fluorescein gene into 96-well plates at 1x10 4 /well, and then use the effect-to-target ratio of 5:1, 10:1 or 20:1 respectively (i.e. the ratio of effector T cells to target cells) The CAR-T cells and NT cells prepared in Example 1 were placed in a 96-well plate for co-culture, and the fluorescence value was measured with a microplate reader after 16-18 hours. According to the calculation formula: (average fluorescence value of target cells-average fluorescence value of samples)/average fluorescence value of target cells×100%, the killing efficiency was calculated, and the results are shown in FIG. 3 .
可以看出，在各种效靶比下，与传统的bbz-CAR-T细胞相比，额外表达LTB、LTB+LTA或LIGHT的CAR-T细胞对靶细胞的杀伤能力略有提高。It can be seen that under various effect-to-target ratios, compared with traditional bbz-CAR-T cells, CAR-T cells additionally expressing LTB, LTB+LTA or LIGHT have slightly improved killing ability on target cells.
2.2 细胞因子释放水平 2.2 Cytokine release level
(1)收集细胞共培养上清液(1) Collect the cell co-culture supernatant
以1x10 5/孔的浓度将靶细胞P02-CD19或非靶细胞P02铺于96孔板中，然后以10:1的比例将实施例1制备的CAR-T细胞和NT细胞分别与靶细胞共培养，18-24小时后收集细胞共培养上清液。 Spread the target cells P02-CD19 or non-target cells P02 in a 96-well plate at a concentration of 1×10 5 /well, and then co-combine the CAR-T cells and NT cells prepared in Example 1 with the target cells at a ratio of 10:1. After culturing, the cell co-culture supernatant was collected after 18-24 hours.
(2)ELISA检测上清中IFN-γ的分泌量(2) ELISA detection of IFN-γ secretion in the supernatant
使用捕获抗体Purified anti-human IFN-γAntibody(Biolegend，货号506502)包被96孔板4℃孵育过夜，然后移除抗体溶液，加入250μL含有2％BSA(sigma，货号V900933-1kg)的PBST(含0.1％吐温的1XPBS)溶液，37℃孵育2小时。然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。每孔加入50μL细胞共培养上清液或标准品，并在37℃孵育1小时，然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。然后向各孔分别加入50μL检测抗体Anti-Interferon gamma抗体[MD-1] (Biotin)(abcam，货号ab25017)，在37℃孵育1小时后，用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，加入250μL PBST(含0.1％吐温的1XPBS)，清洗5次。向各孔加入50μL TMB底物溶液。使反应在室温下于暗处发生30分钟，之后向各孔中加入50μL 1mol/L H 2SO 4以停止反应。在停止反应的30分钟内，使用酶标仪检测450nm处吸光度，并根据标准曲线(根据标准品的读值和浓度绘制)计算细胞因子的含量，结果如图4所示。 Use the capture antibody Purified anti-human IFN-γAntibody (Biolegend, product number 506502) to coat the 96-well plate and incubate overnight at 4°C, then remove the antibody solution, add 250 μL PBST (containing 2% BSA (sigma, product number V900933-1kg) 0.1% Tween in 1XPBS) solution, incubated at 37°C for 2 hours. Plates were then washed 3 times with 250 [mu]L PBST (1XPBS with 0.1% Tween). Add 50 μL of cell co-culture supernatant or standard to each well and incubate at 37° C. for 1 hour, then wash the plate 3 times with 250 μL PBST (1XPBS containing 0.1% Tween). Then add 50 μL detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, product number ab25017) to each well, incubate at 37°C for 1 hour, wash with 250 μL PBST (1XPBS containing 0.1% Tween) Plate 3 times. Then add HRP Streptavidin (Biolegend, Cat. No. 405210), incubate at 37°C for 30 minutes, discard the supernatant, add 250 μL PBST (1XPBS containing 0.1% Tween), and wash 5 times. Add 50 μL of TMB substrate solution to each well. The reaction was allowed to take place in the dark at room temperature for 30 minutes, after which 50 μL of 1 mol/L H2SO4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, use a microplate reader to detect the absorbance at 450nm, and calculate the cytokine content according to the standard curve (drawn according to the reading value and concentration of the standard), and the results are shown in Figure 4.
可以看出，与非靶细胞相比，CAR-T细胞与靶细胞共培养后IFNγ的分泌水平显著升高，表明CAR-T细胞对靶细胞的杀伤是特异性的。It can be seen that compared with non-target cells, the secretion level of IFNγ was significantly increased after CAR-T cells were co-cultured with target cells, indicating that the killing of target cells by CAR-T cells was specific.
实施例3.CAR-T细胞的肿瘤抑制效果验证Example 3. Verification of the tumor suppressive effect of CAR-T cells
将24只6周龄的Balb/c免疫健全小鼠随机分为4组，每组6只，经尾静脉注射1×10 6个A20细胞(一种B淋巴细胞瘤细胞)。接种A20细胞的当天定义为D0。于D10向各组小鼠经腹腔注射3mg/20g体重的环磷酰胺。于D13向各组小鼠经尾静脉注射5×10 6个bbz CAR-T细胞、bbz-T2A-Ltb CAR-T细胞、bbz-T2A-Ltba CAR-T细胞或bbz-T2A-LIGHT CAR-T细胞。监测小鼠存活情况，直至实验结束。结果如图5所示。 Twenty-four 6-week-old Balb/c immunocompetent mice were randomly divided into 4 groups, 6 mice in each group, and 1×10 6 A20 cells (a type of B lymphocytoma cells) were injected through the tail vein. The day of inoculation of A20 cells was defined as D0. On D10, 3 mg/20 g body weight of cyclophosphamide was intraperitoneally injected into the mice of each group. On D13, 5×10 6 bbz CAR-T cells, bbz-T2A-Ltb CAR-T cells, bbz-T2A-Ltba CAR-T cells or bbz-T2A-LIGHT CAR-T cells were injected into mice in each group via tail vein cell. Mouse survival was monitored until the end of the experiment. The result is shown in Figure 5.
可以看出，与传统的bbz CAR-T细胞相比，额外表达LTB、LTB+LTA或LIGHT的CAR-T细胞能显著增强对肿瘤的抑制效果，进而提高存活率。It can be seen that compared with traditional bbz CAR-T cells, CAR-T cells additionally expressing LTB, LTB+LTA or LIGHT can significantly enhance the inhibitory effect on tumors, thereby improving the survival rate.
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。It should be noted that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (18)

  1. 一种工程化免疫细胞，其表达(i)特异性识别抗原的细胞表面分子，和(ii)一种或多种LTBR配体。An engineered immune cell that expresses (i) a cell surface molecule that specifically recognizes an antigen, and (ii) one or more LTBR ligands.
  2. 权利要求1所述的工程化免疫细胞，其中所述特异性识别抗原的细胞表面分子是嵌合抗原受体或重组T细胞受体。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. 权利要求2所述的工程化免疫细胞，其中所述特异性识别抗原的细胞表面分子是包含以下的嵌合抗原受体：抗原结合区、跨膜结构域、共刺激结构域和胞内信号传导结构域。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 co-stimulatory domain, and intracellular signaling domain.
  4. 权利要求1-3任一项所述的工程化免疫细胞，其中所述LTBR配体选自LTA、LTB、LIGHT和抗LTBR抗体。The engineered immune cell according to any one of claims 1-3, wherein the LTBR ligand is selected from LTA, LTB, LIGHT and anti-LTBR antibodies.
  5. 权利要求1-3任一项所述的工程化免疫细胞，其中LTA与SEQ ID NO：26、40、46或48所示的氨基酸序列具有至少90％同一性，LTB与SEQ ID NO：24或42所示的氨基酸序列具有至少90％同一性，LIGHT与SEQ ID NO：28或44所示的氨基酸序列具有至少90％同一性。The engineered immune cell according to any one of claims 1-3, wherein LTA has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 26, 40, 46 or 48, and LTB has at least 90% identity with SEQ ID NO: 24 or The amino acid sequence set forth in 42 has at least 90% identity, and LIGHT has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28 or 44.
  6. 权利要求1-4任一项所述的工程化免疫细胞，其中所述免疫细胞选自T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞、或NKT细胞。The engineered immune cell according to any one of claims 1-4, wherein the immune cell is selected from T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.
  7. 权利要求5所述的工程化免疫细胞，其中所述T细胞是CD4+/CD8+T细胞、CD4+辅助T细胞、CD8+T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。The engineered immune cell of claim 5, wherein the T cells are CD4+/CD8+T cells, CD4+ helper T cells, CD8+T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells or αβ-T cells.
  8. 权利要求3-6任一项所述的工程化免疫细胞，其中所述抗原结合区选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双体、anticalin和DARPIN抗原。The engineered immune cell according to any one of claims 3-6, wherein the antigen binding region is selected from the group consisting of IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear Antibodies, single domain antibodies, nanobodies, diabodies, anticalins and DARPIN antigens.
  9. 权利要求3所述的工程化免疫细胞，其中所述抗原抗原结合区与选自以下的靶标结合：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、CD 179a、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CD133、IFNAR1、DLL3、kappa轻链、TIM3、TSHR、CD19、BAFF-R、CLL-1、EGFRvIII、tEGFR、GD2、GD3、BCMA、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、 IL-llRa、IL-22Ra、IL-2、间皮素、PSCA、PRSS21、VEGFR2、LewisY、PDGFR-β、SSEA-4、AFP、Folate受体α、ErbB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、NCAM、Claudin18.2、c-Met、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gpl00、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM7R、CLDN6、GPRC5D、CXORF61、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、MAGE-A3、MAGE-A6、豆荚蛋白、HPV E6、E7、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PSA、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、TMPRSS2 ETS融合基因、NA17、PAX3、雄激素受体、孕酮受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2D配体，和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。权利要求3-8任一项所述的工程化免疫细胞，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。The engineered immune cell of claim 3, wherein the 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, CD40, 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-11Ra, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl- GD2, Folate receptor β, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD -CT-2, Fos-related antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML- IAP, TM PRSS2 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, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, expressed by HIV, HCV, HBV, and/or other pathogens molecules; and/or neo-epitopes or neoantigens. The engineered immune cell according to any one of claims 3-8, wherein the transmembrane domain is selected from the transmembrane domain of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  10. 权利要求3-9任一项所述的工程化免疫细胞，其中所述胞内信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。The engineered immune cell according to any one of claims 3-9, wherein the intracellular signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a , CD79b and CD66d.
  11. 权利要求3-10任一项所述的工程化免疫细胞，其中所述共刺激结构域是一个或多个选自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、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以及它们的组合。The engineered immune cell according to any one of claims 3-10, wherein the co-stimulatory domain is one or more co-stimulatory signaling domains selected from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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.
  12. 权利要求1-11任一项所述的工程化免疫细胞，其中所述免疫细胞还包含至少一种失活基因，其选自以下：CD52、GR、TCRα、TCRβ、CD3γ、CD3δ、CD3ε、CD247ζ、HLA-I、HLA-II、B2M、PD1、CTLA-4、LAG3和TIM3。The engineered immune cell according to 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, TCRα, TCRβ, CD3γ, CD3δ, CD3ε, CD247ζ , HLA-I, HLA-II, B2M, PD1, CTLA-4, LAG3, and TIM3.
  13. 一种核酸分子，其包含：(i)编码特异性识别抗原的细胞表面分子的核酸序列，和(ii)编码LTBR配体的核酸序列。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. 权利要求13所述的核酸分子，其中所述特异性识别抗原的细胞表面分子是嵌合抗原受体。The nucleic acid molecule of claim 13, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor.
  15. 一种载体，其包含权利要求13-14任一项所述的核酸分子。A vector comprising the nucleic acid molecule of any one of claims 13-14.
  16. 权利要求18所述的载体，其中所述载体选自质粒、逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV)、多瘤病毒和腺相关病毒(AAV)。The vector of claim 18, wherein the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyoma virus, and adeno-associated virus (AAV).
  17. 一种药物组合物，其包含权利要求1-12任一项所述的工程化免疫细胞、权利要求13-14任一项所述的核酸分子或权利要求15-16任一项所述的载体，和一种或多种药学上可接受的赋型剂。A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1-12, the nucleic acid molecule according to any one of claims 13-14 or the carrier according to any one of claims 15-16 , and one or more pharmaceutically acceptable excipients.
  18. 一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用权利要求1-12任一项所述的工程化免疫细胞或权利要求17所述的药物组合物。A method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject the engineered immune cell of any one of claims 1-12 or the subject of claim 17 pharmaceutical composition.
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