Chimeric antigen receptor comprising novel co-stimulatory domain and use thereof

Abstract

The present invention relates to a chimeric antigen receptor, comprising a ligand binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, wherein the co-stimulatory domain comprises a mutated CD27 intracellular region. The present invention also relates to an engineered immune cell comprising the chimeric antigen receptor, and a use thereof in treatment of diseases, such as cancer, autoimmune diseases, infections.

Description

Chimeric antigen receptors containing novel costimulatory domains and uses thereof Technical Field

[0001] The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to chimeric antigen receptors comprising novel costimulatory domains, as well as engineered immune cells comprising such chimeric antigen receptors and uses thereof. Background Art

[0002] Adoptive cell therapy refers to the in vitro activation and expansion of autologous immune cells, which are then reinfused back into the patient's body to directly kill tumors or stimulate the body's immune response to kill tumor cells. Common adoptive cell therapies include non-specific immunotherapies such as LAK, CIK and NK, as well as specific immunotherapies such as TIL, CAR-T and TCR-T. Among them, CAR-T cell therapy has attracted widespread attention due to its outstanding performance in the field of hematological tumors. In view of the rapid development of CAR-T cell therapy, a series of new cell therapies with CAR as the core technology have gradually emerged, such as CAR-NK, CAR-NKT, CAR-Treg, CAR-γδT, etc., which have jointly led CAR cell therapy to become a major trend in tumor cell immunotherapy.

[0003] Chimeric antigen receptor (CAR) is a cell surface receptor protein that gives immune cells the ability to target specific antigen proteins. Structurally, CAR consists of at least three functional domains: antigen binding domain, transmembrane domain, and intracellular signaling domain. The intracellular signaling domain is composed of co-stimulatory domain and / or primary signaling domain. The design of the CAR structure has gone through the following process: the first-generation CAR has only one intracellular signaling component, CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause short-term immune cell proliferation and less cytokine secretion, but cannot provide long-term immune cell proliferation signals and sustained in vivo anti-tumor effects, so it has not achieved good clinical efficacy; the second-generation CAR introduces a co-stimulatory molecule, such as CD28, 4-1BB, OX40, ICOS, based on the original structure. Compared with the first-generation CAR, the function has been greatly improved, further strengthening the CAR -T cell persistence and tumor cell killing ability; the third-generation CAR contains two co-stimulatory molecules, with CD28 and 4-1BB, and CD28 and OX40 combinations as co-stimulatory molecules being the most common in the third-generation CAR; the fourth-generation CAR is mainly designed to overcome the inhibition of the tumor immune microenvironment, and pro-inflammatory cytokines (IL-12, IL-15, IL-18, etc.) and co-stimulatory ligands are introduced during the design; the fifth-generation CAR-T adds co-stimulatory domains that activate other signaling pathways based on the second generation, such as the IL2-2Rβ intracellular binding SAAT3 / 5 domain. Currently, most companies' CAR products are still explored and developed based on the second-generation CAR structure.

[0004] Studies have shown that different functional domains have a significant impact on the function of CAR. For example, in the international application WO2016014789A, without changing the antigen binding domain and intracellular signaling domain, only by changing the transmembrane domain, the performance of CAR can be greatly improved, and the non-specific killing effect of CAR-T on non-target cells can be reduced. For example, it is generally believed in the field of CAR cell therapy that CAR cells containing the CD28 costimulatory domain show better cell proliferation activity, and CAR cells containing the 4-1BB costimulatory domain show better persistence.

[0005] Currently, there are still some problems in the clinical application of CAR cell therapy, such as poor efficacy and poor sustainability in treating solid tumors. Therefore, it is necessary to develop new CAR structures to further enhance the therapeutic effect of CAR cell therapy.

[0006] Summary of the Invention

[0007] Unless otherwise defined, 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.

[0008] Chimeric antigen receptor

[0009] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes a ligand binding domain (e.g., an antibody or its antigen binding portion), a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, each of which is connected by a linker. CAR is able to redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner using the antigen binding properties of antibodies. Non-MHC restricted antigen recognition gives CAR-expressing immune cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape.

[0010] In a first aspect, the present invention provides a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain, wherein the costimulatory domain comprises a mutated CD27 intracellular region.

[0011] Costimulatory domain is the intracellular functional signal transduction domain from costimulatory molecules, which includes the entire intracellular part of the costimulatory molecules, or its functional fragment." costimulatory molecules" refers to specifically binding to costimulatory ligands on T cells, thereby mediating the cognate binding partner of the costimulatory response (such as proliferation) of T cells. Traditional chimeric antigen receptors generally use CD28 and / or 4-1BB as costimulatory domains. The chimeric antigen receptor of the present invention includes novel costimulatory domains, i.e., mutated CD27 intracellular regions.

[0012] CD27 is a dimeric transmembrane glycoprotein composed of disulfide-linked monomers with a relative molecular weight of 55,000 Da. It is a key member of the tumor necrosis factor (TNF) superfamily. CD27 is widely expressed on the surface of both naive and memory T cells. CD27 primarily activates the downstream TRAF2 and TRAF5 signaling pathways by binding to its ligand CD70, further inducing activation of the NF-κB and JNK signaling pathways. It plays a crucial regulatory role in the activation, proliferation, and apoptosis of immune cells, particularly T cells.

[0013] Compared to the wild-type CD27 intracellular region (SEQ ID NO: 1), the mutated CD27 intracellular region of the present invention comprises at least one lysine mutation (i.e., amino acid deletion, substitution, or insertion), for example, one lysine mutation, two lysine mutations, or three lysine mutations. Preferably, the mutated CD27 intracellular region of the present invention comprises at least one lysine substitution. More preferably, the substitution is to replace lysine with other basic amino acids, such as arginine or histidine.

[0014] Alternatively, compared to the wild-type CD27 intracellular region (SEQ ID NO: 1), the mutated CD27 intracellular region of the present invention comprises at least one (e.g., one, two, three, four, five, or six) amino acid deletion at the 1st to 6th amino acid positions near the C-terminus. Preferably, the mutated CD27 intracellular region of the present invention comprises at least one (e.g., one, two, three, four, five, or six) consecutive deletion of amino acids at the 1st to 6th amino acid positions near the C-terminus. More preferably, the mutated CD27 intracellular region of the present invention comprises a consecutive deletion of four amino acids at the 1st to 4th amino acid positions near the C-terminus.

[0015] The CD27 intracellular region in the present invention has undergone the above-mentioned mutation, which can significantly increase the proliferation level of CAR cells compared to the wild-type CD27 intracellular region, thereby improving the inhibitory effect on tumors.

[0016] In some embodiments, the mutated CD27 intracellular region of the present invention has the amino acid sequence shown in SEQ ID NO: 2 or 3, or has at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0017] In addition to the mutated CD27 intracellular region provided by the present invention, the chimeric antigen receptor of the present invention may further comprise one or more additional costimulatory domains selected from the costimulatory signaling domains of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM or ZAP70, preferably selected from 4-1BB, CD28, CD27, OX40, CD278 and any combination thereof, more preferably selected from 4-1BB and CD28.

[0018] In some embodiments, the CAR of the present invention includes a mutated CD27 intracellular region as a costimulatory domain. In some embodiments, the CAR of the present invention includes a mutated CD27 intracellular region and a 4-1BB intracellular region as a costimulatory domain. In some embodiments, the CAR of the present invention includes a mutated CD27 intracellular region and a CD28 intracellular region as a costimulatory domain.

[0019] 4-1BB and CD28 costimulatory domains known to those skilled in the art can be used in the present invention. For example, the 4-1BB costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or 18; the CD28 costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0020] As used herein, "ligand binding domain" refers to any structure or functional variant thereof that can bind to a ligand (e.g., an antigen). The ligand binding domain can be an antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, and antigen-binding fragments thereof. For example, the ligand binding domain includes but is not limited to complete antibodies, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, linear antibodies, sdAb (e.g., VH or VL), nanobodies (Nanobody, Nb), recombinant fibronectin domains, anticalins, and DARPINs, etc., preferably selected from Fab, scFv, sdAb, and nanobodies. In the present invention, the ligand binding domain can be monovalent or divalent, and can be a monospecific, bispecific, or multispecific antibody.

[0021] "Single-chain antibody" or "scFv" is an antibody formed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody via a linker. The optimal length and / or amino acid composition of the linker can be selected. The length of the linker significantly affects the folding and interaction of the variable regions of the scFv. In fact, if a shorter linker (e.g., between 5-10 amino acids) is used, intrachain folding can be prevented. Regarding the selection of the size and composition of the linker, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90: 6444-6448; U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794; and PCT Publication Nos. WO2006 / 020258 and WO2007 / 024715, the entireties of which are incorporated herein by reference. scFv can comprise VH and VL connected in any order, such as VH-linker-VL or VL-linker-VH. Preferably, the linker has the amino acid sequence shown in SEQ ID NO: 30 or 31.

[0022] The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of the parent but contains at least one amino acid modification (i.e., substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. For example, for an antibody, a functional fragment thereof is an antigen-binding portion thereof. Preferably, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the parent amino acid sequence and retains the biological activity of the parent amino acid, such as binding activity.

[0023] As used herein, the term "sequence identity" refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity. One skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.

[0024] The choice of ligand binding domain depends on the cell surface marker on the target cell associated with the specific disease state to be identified, such as a tumor-specific antigen or tumor-associated antigen. Thus, in some embodiments, the ligand binding domain of the present invention binds to one or more targets selected from the group consisting of ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, , CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin 6. Claudin 18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2,EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2 VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor β, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variants of tumor necrosis, and any combination thereof. Preferably,The ligand binding domain binds to a target selected from the group consisting of: CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor α, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, and GPRC5D, more preferably CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, CD7, CD70, and BCMA. Depending on the antigen to be targeted, the CAR of the present invention can be designed to include a ligand binding domain that is specific for the antigen. For example, if CD19 is the antigen to be targeted, a CD19 antibody can be used as the ligand binding domain of the present invention.

[0025] In some embodiments, the chimeric antigen receptor of the present invention targets CD19. Preferably, the chimeric antigen receptor of the present invention comprises an anti-CD19 antibody. Antibodies targeting CD19 known in the art can be used in the present invention. In some embodiments, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 10; wherein the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 11. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:4, CDR2-H as shown in SEQ ID NO:5, and CDR3-H as shown in SEQ ID NO:6; the light chain variable region comprises CDR1-L as shown in SEQ ID NO:7, CDR2-L as shown in SEQ ID NO:8, and CDR3-L as shown in SEQ ID NO:9.

[0026] In some embodiments, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11. Preferably, the antibody targeting CD19 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 10 and a light chain variable region as set forth in SEQ ID NO: 11.

[0027] In some embodiments, the antibody targeting CD19 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12. Preferably, the antibody targeting CD19 is as shown in SEQ ID NO: 12.

[0028] As used herein, the term "membrane spaning domain" refers to a polypeptide structure that enables chimeric antigen receptors to be expressed on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells), and guides immune cells to respond to target cells. The membrane spaning domain can be natural or synthetic, or it can be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor is bound to the target antigen, the membrane spaning domain can carry out signal transduction. Particularly suitable for the membrane spaning domain in the present invention can be derived from, for example, TCR alpha chains, TCR beta chains, TCR gamma chains, TCR delta chains, CD3 zeta subunits, CD3 epsilon subunits, CD3 gamma subunits, CD3 delta subunits, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, the membrane spaning domain can be synthetic and can primarily comprise hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8α chain or CD28, and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:13-15.

[0029] As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs cells to perform designated functions. The intracellular signaling domain is responsible for intracellular signaling after the ligand binding domain binds to the antigen, 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 the secretion of cytokines.

[0030] In some embodiments, the intracellular signaling domain included in the chimeric antigen receptor of the present invention can be a cytoplasmic sequence of a T cell receptor and a co-receptor, which works together to initiate signal transduction after the antigen receptor binds, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions. The intracellular signaling domain can include many immunoreceptor tyrosine activation motifs (ITAMs). Non-limiting examples of the intracellular signaling domain of the present 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 can include a CD3ζ signaling domain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 19-22.

[0031] In some embodiments, the costimulatory domain and the intracellular signaling domain can be operably linked in any order. For example, the costimulatory domain can be located proximal to the membrane, while the intracellular signaling domain is located distal to the membrane, or the stimulatory domain can be located distal to the membrane, while the intracellular signaling domain is located proximal to the membrane. When two or more costimulatory domains are present, the costimulatory domain can be located on one side or both sides of the intracellular signaling domain.

[0032] In some embodiments, the chimeric antigen receptor of the present invention may also include a hinge region between the ligand binding domain and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the ligand binding domain. Specifically, the hinge region is used to provide greater flexibility and accessibility for the ligand binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from natural molecules, 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 a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a portion of the hinge region of the CD8α chain, FcγRIIIα receptor, CD28, IgG4 or IgG1, more preferably a hinge from CD8α, CD28 or IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 23-26.

[0033] In some embodiments, the CAR of the present invention may further comprise a signal peptide so 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 hydrophobic amino acid segment that has a tendency to form a single α-helix. At the end of the signal peptide, there is usually an amino acid segment that is recognized and cut by a signal peptidase. The signal peptidase can cut during or after the translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from CD8α, IgG1, GM-CSFRα, B2M, etc. In some embodiments, the signal peptide that can be used in the present invention has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 27-29.

[0034] In some embodiments, the CAR of the present invention may further comprise a switch structure to regulate the expression time of CAR. For example, the switch structure may be in the form of a dimerization domain, which causes a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain so that it binds to the targeted antigen, thereby activating the signal transduction pathway. Alternatively, the switch domain may be used to connect the binding domain and the signal transduction domain respectively, and only when the switch domains bind to each other (for example, in the presence of an inducing compound) can the binding domain and the signal transduction domain be connected together through a dimer, thereby activating the signal pathway. The switch structure may also be in the form of a masking peptide. The masking peptide may shield the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, for example, a protease, the extracellular binding domain may be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art may be used in the present invention.

[0035] In some embodiments, the CAR of the present invention may also include a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance, so as to remove the CAR cells when needed (e.g., when serious toxic side effects are produced). For example, the suicide gene can be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when needed, the 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 can cause cells to die under the induction of ganciclovir treatment. The suicide gene can also be iCaspase-9, which can be induced by chemical induction drugs such as AP1903, AP20187, etc. to dimerize iCaspase-9, thereby activating downstream Caspase3 molecules, leading to cell apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

[0036] Nucleic Acids

[0037] The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor of the present invention. Preferably, the nucleic acid is DNA or RNA, more preferably DNA.

[0038] 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-stranded and / or double-stranded form, linear or circular, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the present invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations thereof, or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

[0039] Nucleic acids may comprise conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as those found in peptide nucleic acids (PNA)). The nucleic acids of the present invention may also contain one or more modified bases, such as, for example, tritylated bases and uncommon bases (such as inosine). Other modifications are also contemplated, including chemical, enzymatic or metabolic modifications, as long as the multi-chain CAR of the present invention can be expressed from polynucleotides. Nucleic acids may be provided in an isolated form. In some embodiments, nucleic acids may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.

[0040] The nucleic acid sequences of the present invention can be codon-optimized for optimal expression in a desired host cell (e.g., immune cell); or for expression in bacteria, yeast, or insect cells. Codon optimization refers to replacing codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, with the codons before and after the replacement encoding the same amino acid. Therefore, the selection of the optimal codon depends on the codon usage preference of the host genome.

[0041] carrier

[0042] The present invention also provides a vector comprising the nucleic acid molecule of the present invention.

[0043] As used herein, the term "vector" is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell where the nucleic acid molecule can, for example, be replicated and / or expressed.

[0044] Vector generally includes targeting vector and expression vector. "Targeting vector" is a medium that separates nucleic acid by, for example, homologous recombination or using a hybrid recombinase at a specific targeting site sequence to deliver it to the cell interior. "Expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding chimeric antigen receptor polypeptides of the present invention) in suitable host cells and the translation of their mRNA. Suitable vectors that can be used for the present invention are known in the art, and many are commercially available. In some embodiments, vectors of the present invention include but are not limited to plasmids, viruses such as retroviruses, slow viruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyomaviruses and adeno-associated viruses (AAV), phages, phagemids, cosmids and artificial chromosomes (including BAC and YAC). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton". Engineered vectors are also typically included in the starting point (if stable expression of polynucleotides is desired), selective markers and restriction enzyme cleavage sites (such as multiple cloning sites MCS) for autonomous replication in host cells. The vector may further comprise elements such as a promoter, a polyadenylic acid tail (polyA), a 3'UTR, an enhancer, a terminator, an insulator, an operator, a selection marker, a reporter gene, a targeting sequence and / or a protein purification tag. In a specific embodiment, the vector is an in vitro transcription vector.

[0045] Engineered immune cells

[0046] The present invention provides engineered immune cells comprising a chimeric antigen receptor or a nucleic acid encoding the chimeric antigen receptor.

[0047] As used herein, the term "immune cell" refers to any cell with one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC) of the immune system. Immune cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. Immune cells can also be derived from stem cells such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. For example, immune cells can be T cells, macrophages, dendritic cells, neutrophils, monocytes, NK cells and / or NKT cells. Preferably, immune cells are T cells. T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines such as Jurkat, SupT1, etc. cultured in vitro, or T cells obtained from experimenter. Examples of experimenters include people, dogs, cats, mice, rats and transgenic species thereof. T cell can also be concentrated or purified.T cell can be any type of T cell and can be in any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as, cytotoxic T cells), tumor infiltrating cells, memory T cells, immature T cells, γδ-T cells, αβ-T cells etc. Preferably, immune cell is human T cell, NK cell or NKT cell. Can use multiple technology well known to those skilled in the art, such as Ficoll separation and obtain T cell from experimenter's blood. In the present invention, immune cell is engineered to express chimeric antigen receptor polypeptide.

[0048] Nucleic acid sequences encoding chimeric antigen receptor polypeptides can be introduced into immune cells using conventional methods known in the art (e.g., by transduction, transfection, transformation, etc.) to cause them to express the chimeric antigen receptor polypeptides of the present invention. "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, which is the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells generally involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, also into non-animal eukaryotic cells (including plant cells). Therefore, transformation is the genetic alteration of bacteria or non-animal eukaryotic cells, which is produced by direct uptake from their surroundings by the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be achieved by artificial means. In order for transformation to occur, the cell or bacterium must be in a competent state. For prokaryotic transformation, techniques may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation.

[0049] In some embodiments, the expression of endogenous HLA-I class genes and / or HLA-II class genes in the engineered immune cells of the present invention is not modified. That is, the expression level of any endogenous HLA-I class gene and / or HLA-II class gene is not altered by any artificial intervention method (gene editing or non-gene editing).

[0050] In some embodiments, the expression of at least one endogenous HLA-I class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene and at least one endogenous HLA-I class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-I class and HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene, at least one endogenous HLA-I class gene, and at least one endogenous HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. Preferably, the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C, and B2M. Preferably, the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA. Preferably, the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε and CD3ζ.

[0051] In some embodiments, the expression of one or more endogenous genes selected from the group consisting of CD52, GR, dCK, and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP1 is suppressed or silenced in the engineered immune cells of the present invention. 0. CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, I L10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0052] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to, for example, DNA or RNA breakage mediated by large-range nucleases, zinc finger nucleases, TALENs, CRISPR / Cas systems, base editors, or gene inactivation by antisense oligonucleotides, RNAi, shRNA, transposons, mutations, and the like.

[0053] Pharmaceutical composition

[0054] In a third aspect, the present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor, nucleic acid, vector, or engineered immune cell described herein as an active agent and one or more pharmaceutically acceptable excipients. Therefore, the present invention also encompasses the use of the chimeric antigen receptor, nucleic acid, vector, or engineered immune cell in the preparation of a pharmaceutical composition or medicament.

[0055] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which is 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, coatings, adsorbents, antiadherents, glidants, antioxidants, flavorings, colorants, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coatings, isotonic agents, absorption delaying agents, stabilizers, and tension modifiers. It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. In general, the choice of suitable excipients depends on, inter alia, the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

[0056] The pharmaceutical composition according to the present invention can be applied in a variety of ways. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0057] The pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. The processes known in the present invention for producing drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions comprising, for example, immune cells as described herein are typically provided in solution form and preferably comprise a pharmaceutically acceptable buffer.

[0058] The pharmaceutical composition according to the present invention can also be used in combination with one or more other medicaments that are applicable to the treatment and / or prevention of the disease to be treated. Preferred examples of the medicament that is applicable to 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, dapoxetine ... E), vincristine, and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes, and RNA enzymes; 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 IL-2, chemokines such as IL-8, 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 compositions of the present invention can be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0059] Methods for preparing engineered immune cells

[0060] In a fourth aspect, the present invention also provides a method for preparing engineered immune cells, comprising introducing the chimeric antigen receptor of the present invention or a nucleic acid sequence encoding the chimeric antigen receptor into immune cells, so that the immune cells express the chimeric antigen receptor of the present invention.

[0061] In some embodiments, the immune cells are human immune cells, more preferably human T cells, macrophages, dendritic cells, neutrophils, monocytes, NK cells and / or NKT cells.

[0062] Methods for introducing nucleic acid or vector into immune cells and expressing them are known in the art. For example, nucleic acid or vector can be introduced into immune cells by physical methods, such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Alternatively, chemical methods can be used, such as by colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In addition, biological methods can also be used to introduce nucleic acid or vector. For example, viral vectors, especially retroviral vectors, have become the most commonly used methods for inserting genes into mammals, such as human cells. Other viral vectors can be derived from slow viruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.

[0063] After the nucleic acid or vector is introduced into the immune cells, those skilled in the art can amplify and activate the obtained immune cells using conventional techniques.

[0064] Pharmaceutical uses / therapeutic applications

[0065] 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 a chimeric antigen receptor, nucleic acid molecule, vector, engineered immune cell, or pharmaceutical composition according to the present invention. Therefore, the present invention also encompasses the use of the chimeric antigen receptor, nucleic acid molecule, vector, or engineered immune cell in the preparation of a medicament for treating cancer, infection, or autoimmune disease.

[0066] In some embodiments, an effective amount of the immune cells and / or pharmaceutical compositions of the invention are administered directly to a subject.

[0067] In another embodiment, the treatment method of the present invention is ex vivo treatment. Specifically, the method comprises the following steps: (a) providing a sample of a subject, the sample comprising immune cells; (b) introducing the chimeric antigen receptor of the present invention into the immune cells in vitro to obtain modified immune cells, and (c) administering the modified immune cells to a subject in need thereof. Preferably, the immune cells provided in step (a) are selected from T cells, NK cells and / or NKT cells; and the immune cells can be obtained from a sample (particularly a blood sample) of the subject by conventional methods known in the art. However, other immune cells capable of expressing the chimeric antigen receptor of the present invention and exerting the desired biological effector function as described herein can also be used. In addition, the immune cells generally selected are compatible with the subject's immune system, i.e., preferably, the immune cells do not trigger an immunogenic response. For example, "universal acceptor cells" can be used, i.e., lymphocytes that can grow and expand in vitro and are generally compatible with the desired biological effector function. The use of such cells will not require the acquisition and / or provision of the subject's own lymphocytes. The ex vivo introduction of step (c) can be carried out by introducing the nucleic acid or vector described herein into immune cells via electroporation or by infecting immune cells with a viral vector, wherein the viral vector is a lentiviral vector, adenoviral vector, adeno-associated viral vector or retroviral vector as described above. Other conceivable methods include the use of a transfection agent (such as liposomes) or transient RNA transfection.

[0068] In some embodiments, the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, neutrophils, monocytes, NK cells and / or NKT cells, more preferably T cells, NK cells or NKT cells.

[0069] As used herein, the term "autologous" refers to any material derived from an individual that will later be reintroduced into that same individual.

[0070] As used herein, the term "allogeneic" refers to any material derived from a different animal or patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to one another when they differ genetically at one or more loci. In some cases, allogeneic material from individuals of the same species may differ genetically enough to allow antigenic interaction.

[0071] As used herein, the term "subject" is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.

[0072] In some embodiments, the cancer is a cancer associated with expression of a target to which the ligand binding domain binds. For example, the cancer includes, but is not limited to, brain gliomas, blastomas, sarcomas, basal cell carcinomas, 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, cancers 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, kidney cancer, laryngeal cancer, liver tumors, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous cell lung cancer), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue cancer), ovarian cancer (e.g., ovarian ... , mouth and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, mesothelioma, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancers of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignancies of the urinary system, vulvar cancer, Waldenstrom macroglobulinemia, lymphomas (including Hodgkin lymphoma and non-Hodgkin lymphoma, such as B-cell lymphomas (including low-grade / follicular non-Hodgkin 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 cell NHL, large mass disease NHL), mantle cell lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, MALT lymphoma, marginal zone lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, etc.), leukemia (including acute leukemia, such as acute lymphoblastic leukemia, acute myeloid leukemia, acute nonlymphocytic leukemia such as acute myeloid leukemia (including undifferentiated and partially differentiated types), acute promyelocytic leukemia, acute myelomonocytic leukemia, acute The invention also includes the following diseases: acute monocytic leukemia, erythroleukemia, acute megakaryocytic leukemia; chronic leukemia, such as chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia; and other special types of leukemia, such as hairy cell leukemia, prolymphocytic leukemia, plasma cell leukemia, adult T-cell leukemia, eosinophilic leukemia, basophilic leukemia, etc.), blastic plasmacytoid dendritic cell neoplasm, malignant lymphoproliferative disease, myelodysplasia, multiple myeloma, myelodysplasia, and post-transplantation lymphoproliferative disorder (PTLD); and other diseases related to target expression. Preferably, the diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from the group consisting of leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, ovarian cancer, mesothelioma, breast cancer, lung cancer, prostate cancer, melanoma, myeloma, sarcoma, gastric cancer, etc.

[0073] In some embodiments, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0074] In some embodiments, the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus.

[0075] In some embodiments, 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 reagents and / or small molecules and any combination thereof.

[0076] The present invention will be further described below with reference to specific examples and accompanying drawings. It should be understood that these examples and accompanying drawings are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, where specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1: scFv expression levels of CAR-T cells determined by flow cytometry.

[0078] Figure 2: Killing effect of CAR-T cells on target cells.

[0079] Figure 3: IL-2 release levels after co-culture of CAR-T cells with target cells.

[0080] Figure 4: Long-term killing effect of CAR-T cells on target cells in vitro.

[0081] Figure 5: Expansion curve of CAR-T cells after multiple co-culture with tumor cells. DETAILED DESCRIPTION

[0082] Example 1: Construction of CAR T cells

[0083] The following coding sequences were synthesized and cloned into the pGEM-T Easy vector in sequence: CD8α signal peptide (SEQ ID NO: 28), anti-CD19 scFv (SEQ ID NO: 12), CD8α hinge region (SEQ ID NO: 24), CD8α transmembrane region (SEQ ID NO: 14), CD27 co-stimulatory domain (SEQ ID NO: 1), CD3ζ intracellular signaling domain (SEQ ID NO: 20), and the CAR19-27 plasmid was obtained, and the correct insertion of the target sequence was confirmed by sequencing. The same method was used to obtain CAR19-27 (-4) and CAR19-27K4R plasmids, which differ from the CAR19-27 plasmid in that 4 amino acids were removed from the intracellular segment (SEQ ID NO: 3) or the fourth amino acid K in the intracellular segment was mutated to R (SEQ ID NO: 2).

[0084] After diluting the plasmid in 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) in a sterile tube, add the packaging vector psPAX2 (Addgene, Catalog No. 12260) and the envelope vector pMD2.G (Addgene, Catalog No. 12259) at a ratio of 4:2:1. Next, add 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche, Catalog No. 06366236001), mix immediately, and incubate at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture is then added dropwise to the 293T cell culture flask. Viruses are harvested at 24 and 48 hours, pooled, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours).

[0085] T cells were activated with DynaBeads CD3 / CD28 CTS™ (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Concentrated lentivirus was then added, and after 3 days of culture, CD19-targeted CAR19-27 T cells, CAR19-27(-4) T cells, and CAR19-27K4R T cells were obtained, respectively.

[0086] After culturing for 12 days at 37°C and 5% CO2, the expression level of scFv on CAR T cells was detected by flow cytometry using Fluorescein (FITC) AffiniPure Rabbit Anti-Mouse IgG, F(ab') fragment specific (Jackson Immunoresearch, Catalog No. 315-095-006) as the primary antibody. The results are shown in Figure 1 (NT is unmodified wild-type T cells).

[0087] It can be seen that CAR T cells containing different mutations in the CD27 intracellular region can effectively express scFv.

[0088] Example 2: Killing effect of CAR T cells on target cells and cytokine release

[0089] 2.1 Killing effect of CAR T cells on target cells

[0090] When T cells kill target cells, the number of target cells decreases. When T cells are co-cultured with target cells expressing luciferase, the number of target cells decreases, and the secretion of luciferase also decreases. Luciferase catalyzes the conversion of luciferin to oxidized luciferin. During this oxidation process, bioluminescence is generated, and the intensity of this luminescence depends on the level of luciferase expressed by the target cells. Therefore, the detected fluorescence intensity can reflect the ability of T cells to kill target cells.

[0091] In order to detect the killing ability of CAR T cells on target cells, 1×10 4 / well Raji target cells carrying the fluorescent gene were plated into a 96-well plate, and then CAR19-27 T cells, CAR19-27(-4) T cells, CAR19-27K4R T cells and NT cells were plated into a 96-well plate for co-culture at different effector-target ratios (i.e., the ratio of effector T cells to target cells). After 16-18 hours, the fluorescence value of the Raji target cells was measured using a microplate reader. The killing efficiency was calculated according to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, and the results are shown in Figure 2.

[0092] It can be seen that compared with the NT group, CAR19-27 T cells, CAR19-27(-4) T cells, and CAR19-27K4R T cells all have the ability to specifically kill target cells, and there is no significant difference.

[0093] 2.2 Cytokine Release by CAR T Cells

[0094] When T cells kill target cells, the number of target cells decreases and cytokines (such as IL-2 and IFN-γ) are released. Follow the steps below to use an enzyme-linked immunosorbent assay (ELISA) to measure the level of IL-2 released when CAR T cells kill target cells.

[0095] (1) Collecting cell co-culture supernatant

[0096] 1×10 5 Target cells (Raji) were plated in a 96-well plate at 1:1 ratio. CAR19-27 T cells, CAR19-27(-4) T cells, CAR19-27K4R T cells, and NT cells were co-cultured with Raji cells at a ratio of 1:1. The cell co-culture supernatant was collected after 18-24 hours.

[0097] (2) ELISA detection of IL-2 secretion in the supernatant

[0098] A 96-well plate was coated with the capture antibody Purified anti-human IL-2 Antibody (R&D, Cat. No. DY202) and incubated overnight at 4°C. The antibody solution was then removed and 250 μL of PBST (1X PBS containing 0.1% Tween) containing 2% BSA (Sigma, Cat. No. V900933-1kg) was added, followed by incubation at 37°C for 2 hours. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or standard was added to each well and incubated at 37°C for 1 hour. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). 50 μL of detection antibody, Anti-IL-2 (R&D, Catalog No. DY202), was then added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). HRP Streptavidin (Biolegend, Catalog No. 405210) was then added. After incubation at 37°C for 30 minutes, the supernatant was discarded, and 250 μL of PBST (1X PBS containing 0.1% Tween) was added and washed five times. 50 μL of TMB substrate solution was added to each well. The reaction was allowed to proceed in the dark at room temperature for 30 minutes, after which 50 μL of 1 mol / L HSO was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, absorbance at 450 nm was measured using a microplate reader, and cytokine levels were calculated using a standard curve (plotted based on the readings and concentrations of the standard). The results are shown in Figure 3.

[0099] It can be seen that compared with the NT group, CAR19-27 T cells, CAR19-27(-4) T cells and CAR19-27K4R T cells can specifically secrete IL-2, and the IL-2 release levels of CAR19-27(-4) T cells and CAR19-27K4R T cells are significantly higher than those of CAR19-27 T cells. It is worth noting that the IL-2 release level of CAR19-27(-4) T cells is significantly higher than that of CAR19-27 T cells and CAR19-27K4R T cells.

[0100] Example 3: Long-term ability of CAR T to kill tumor cells

[0101] 5×10 5 Target cells (Raji) were plated in 48-well plates at 48-well plates per well. CAR19-27 T cells, CAR19-27(-4) T cells, and CAR19-27K4R T cells were then co-cultured with Raji cells at an E:T ratio of 1:2. Raji cells were added on D3, D6, and D10 after culture (E:T = 1:1). Before each addition of Raji cells, the residual tumor cells were detected by flow cytometry to evaluate the tumor-killing ability of each group of CAR T cells, and the amplification curves of each group of CAR T cells were recorded. The results are shown in Figures 4 and 5.

[0102] It can be seen that the long-term tumor killing ability and expansion curve of CAR19-27(-4) T cells and CAR19-27K4R T cells are significantly better than those of the CAR19-27 T group, and CAR19-27K4R T cells are better than CAR19-27K4R T cells.

[0103] The above results show that compared with the wild-type CD27 intracellular region, the CAR-T cells containing the mutated CD27 intracellular region of the present invention can promote the secretion of cytokines and the expansion of immune cells, and enhance the ability of immune cells to kill tumors in the long term.

[0104] 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 be modified and varied in various ways. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the co-stimulatory domain comprises a mutated CD27 intracellular region; compared with the wild-type CD27 intracellular region, the mutated CD27 intracellular region comprises at least one lysine mutation, or the 1st to 6th amino acid positions near the C-terminus comprise at least one amino acid deletion.

2. The chimeric antigen receptor of claim 1, wherein the mutated CD27 intracellular region comprises a deletion or substitution of the 4th lysine residue at the C-terminus, or comprises a continuous deletion of at least one amino acid at the 1st to 6th amino acid positions near the C-terminus.

3. The chimeric antigen receptor of claim 1 or 2, wherein the mutated CD27 intracellular region has the amino acid sequence shown in SEQ ID NO: 2 or 3 or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 3.

4. The chimeric antigen receptor of any one of claims 1-3, wherein the co-stimulatory domain further comprises a signaling domain selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.

5. The chimeric antigen receptor of claim 4, wherein the co-stimulatory domain further comprises a signaling domain of CD28, CD134, CD137 or CD278, or a combination thereof.

6. The chimeric antigen receptor of any one of claims 1-5, 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.

7. The chimeric antigen receptor of any one of claims 1-6, wherein 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.

8. The chimeric antigen receptor of any one of claims 1-7, wherein the ligand binding domain is an antibody or an antigen binding portion thereof.

9. The chimeric antigen receptor of claim 8, wherein the antigen binding portion is selected from the group consisting of intact antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, nanobodies, diabodies, and sdAbs.

10. The chimeric antigen receptor of any one of claims 1-9, wherein the ligand binding domain binds to a target selected from the group consisting of ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, C D30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD8 0. CD81, CD86, CD97, CD123, CD133, CD137, CD138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin 6. Claudin 18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2, EGP40, FA P, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor β, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variant of tumor necrosis zone, and any combination thereof. , 11. A nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 10. A vector comprising the nucleic acid molecule according to claim 11 .

13. An engineered immune cell comprising the chimeric antigen receptor according to any one of claims 1 to 10 or the nucleic acid molecule according to claim 11.

14. The engineered immune cell of claim 13, wherein the immune cell is selected from a T cell, a macrophage, a dendritic cell, a neutrophil, a monocyte, a NK cell, or a NKT cell.

15. The engineered immune cell of claim 14, wherein the T cell is a CD4+ / CD8+ double positive T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell, or an αβ-T cell.

16. The engineered immune cell of any one of claims 13-15, wherein the immune cell is derived from an adult stem cell, an embryonic stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell.

17. The engineered cell according to any one of claims 13-16, wherein in the engineered cell, the expression of one or more genes among endogenous HLA-I class genes, HLA-II class genes, and TCR / CD3 genes is inhibited or silenced.

18. The engineered cell according to claim 17, wherein the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C, B2M and any combination thereof; the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and any combination thereof; the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and any combination thereof.

19. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 13 to 18, and one or more pharmaceutically acceptable excipients.

20. Use of the chimeric antigen receptor of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the vector of claim 12, the engineered immune cell of any one of claims 13 to 18, or the pharmaceutical composition of claim 19 in the preparation of a medicament for treating cancer, infection or autoimmune disease.

Images