WO2023083003A1 - Cellule immunitaire modifiée et son utilisation - Google Patents

Cellule immunitaire modifiée et son utilisation Download PDF

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WO2023083003A1
WO2023083003A1 PCT/CN2022/127718 CN2022127718W WO2023083003A1 WO 2023083003 A1 WO2023083003 A1 WO 2023083003A1 CN 2022127718 W CN2022127718 W CN 2022127718W WO 2023083003 A1 WO2023083003 A1 WO 2023083003A1
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cells
engineered immune
cell
immune cell
antigen
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PCT/CN2022/127718
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邢芸
任江涛
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北恒医疗有限公司
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Definitions

  • the invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance the expression of IL9.
  • the present invention provides a novel engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance the expression of IL9.
  • the exogenous IL9 is wild-type IL9 or a variant thereof, and the variant has the same or similar function as wild-type IL9. More preferably, said IL9 has at least 90% identity to the amino acid sequence shown in SEQ ID NO: 21 or 23.
  • the modified promoter is selected from constitutive and inducible promoters. For example, by replacing the endogenous promoter with a constitutive promoter or an inducible promoter, or inserting it into the promoter region of the endogenous IL9 gene, so that the modified promoter is operably linked to the endogenous IL9 gene, And then enhance the expression of IL9.
  • the constitutive promoter includes but not limited to CMV promoter, EF1a promoter, SV40 promoter, PGK1 promoter, Ubc promoter, ⁇ -actin promoter, CAG promoter;
  • the inducible promoter Promoters include, but are not limited to, the tetracycline response element (TRE) promoter, the estrogen response element (ERE) promoter.
  • the engineered immune cells further express exogenous chemokines selected from XCL1 and XCL2.
  • said XCL1 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 25 or 27;
  • XCL2 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 29.
  • the engineered immune cell further expresses a cell surface molecule that specifically recognizes an antigen comprising an antigen binding region, which is selected from a chimeric antigen receptor, a T cell receptor, a T cell fusion protein, or a T cell antigen
  • a cell surface molecule that specifically recognizes an antigen comprising an antigen binding region, which is selected from a chimeric antigen receptor, a T cell receptor, a T cell fusion protein, or a T cell antigen
  • the coupler is preferably a chimeric antigen receptor.
  • the antigen binding region may be selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies , 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 one or more targets selected from: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD179a , DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII , tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA,
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor comprising an antigen binding region, a transmembrane domain and an intracellular domain comprising a co-stimulatory domain and/or primary signaling domain.
  • the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain is selected from the transmembrane domains of CD8 ⁇ , CD4, CD28 and CD278.
  • the primary signaling domain is an intracellular region of a protein selected from the group consisting of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • said primary signaling domain comprises a CD3 ⁇ intracellular region.
  • the co-stimulatory domain comprises one or more intracellular regions selected from the group consisting of 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, 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 immune cells are selected from T cells, B cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells.
  • the T cells are CD4+CD8+T cells, CD4+T cells, CD8+T cells, CD4-CD8-T cells, tumor infiltrating cells, memory T cells, regulatory T cells, naive T cells, ⁇ - T cells or ⁇ -T cells.
  • the expression of IL9 and/or chemokine is secreted expression or anchored expression.
  • IL9 and/or chemokines can be operably linked to a localization domain (such as a transmembrane domain) that can localize the expression of the exogenous gene of the invention to a specific cellular location, such as a cell membrane .
  • the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding exogenous IL9 or a nucleic acid sequence encoding an endogenous IL9 operably linked to a modified promoter, preferably further comprising an exogenous IL9 encoding A nucleic acid sequence of a chemokine selected from XCL1 and XCL2.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, and the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor, a T cell receptor, a T cell fusion A protein or T cell antigen coupler, more preferably a chimeric antigen receptor.
  • 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 pharmaceutical composition, which comprises the engineered immune cells, nucleic acid molecules or vectors described in the present invention, and one or more pharmaceutically acceptable excipients.
  • 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 cells according to the present invention, Nucleic acid molecule, vector or pharmaceutical composition.
  • the present invention also provides a combination therapy comprising engineered immune cells expressing cell surface molecules that specifically recognize antigens and exogenous IL9.
  • the combination therapy comprises: (1) engineered immune cells expressing exogenous IL9 and exogenous chemokines; (2) engineered immune cells expressing exogenous IL9 and exogenous chemokines; or (3) engineered immune cells and exogenous IL9 and chemokines; wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, and the chemokines are selected from XCL1 and XCL2 .
  • Figure 1 CAR expression levels of CAR-T cells determined by flow cytometry.
  • Figure 2 The expression level of IL9 in CAR-T cells determined by ELISA.
  • FIG. 3 IFN- ⁇ release levels after CAR-T cells were co-cultured with target cells and non-target cells.
  • Figure 4 The body weight change curve of mice after pancreatic cancer was treated with CAR-T cells.
  • Figure 5 Tumor growth curves of mice treated with CAR-T cells for pancreatic cancer.
  • the present invention provides a novel engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance the expression of IL9.
  • IL9 is an interleukin that activates the JAK-STAT signaling pathway by binding to the receptor IL9R, and then plays an important role in the activation and regulation of immune cells, proliferation and differentiation, and in inflammatory responses.
  • IL9 is mainly produced by mast cells, Th2 cells, Th9 cells, Th17 cells, Treg cells, NKT cells, innate lymphoid cells (ILCs) and so on.
  • the relationship between IL9 and other interleukins is not yet clear, but the obvious difference from IL2 is that it cannot induce cytotoxic activities such as CTL and LAK, but can maintain the long-term growth of non-antigen-dependent Th cells.
  • the expression of IL9 is enhanced by introducing exogenous IL9 into immune cells or operably linking endogenous IL9 with a modified promoter.
  • any genome editing approach can be used to modify the promoter/enhancer region of the IL9 locus, thereby enhancing endogenous expression of IL9 in immune responsive cells, including replacing the endogenous expression of IL9 with a constitutive or inducible promoter.
  • the original promoter, or a constitutive or inducible promoter was inserted into the promoter region of the IL9 locus.
  • a constitutive promoter is located at the IL9 locus to drive gene expression of the endogenous IL9 gene.
  • Suitable constitutive promoters include, but are not limited to, the CMV promoter, the EF1a promoter, the SV40 promoter, the PGK1 promoter, the Ubc promoter, the ⁇ -actin promoter, and the CAG promoter.
  • an inducible promoter is located at the IL9 locus to drive gene expression of the endogenous IL9 gene.
  • inducible promoters include, but are not limited to, tetracycline response element (TRE) promoters and estrogen response element (ERE) promoters.
  • TRE tetracycline response element
  • ERE estrogen response element
  • enhancer elements can also be placed in regions other than the promoter region.
  • the exogenous IL9 is wild-type (eg, from human or murine) or a variant thereof that has the same or similar function as wild-type IL9. More preferably, the IL9 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 21 or 23 % or 100% identity, or its coding sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the C-type chemokine family also known as lymphatic chemokines, includes two members, XCL1 and XCL2, which are mainly produced by CD8+ T cells and natural killer cells.
  • XCL1 has unique sequence characteristics and two interconvertible protein spatial conformations, which make XCL1 different from other chemokines and exert unique functions.
  • the XCL1-specific receptor XCR1 is a member of the G protein-coupled receptor family, and the interaction between the two not only plays an important role in the negative selection of the thymus and the establishment of autoimmune tolerance, but also initiates cross-antigen presentation and mediates cytotoxic immune responses .
  • XCL1 can not only regulate the balance of the immune system and maintain intestinal immune homeostasis, but also is associated with various diseases, such as autoimmune diseases, nephritis, tuberculosis and human immunodeficiency virus infection.
  • the nucleic acid sequences of XCL2 and XCL1 have 97% identity, and the amino acid sequences differ only by two residues.
  • GAG glycosaminoglycans
  • XCR1 the receptor of XCL1 and XCL2
  • DC DC
  • XCL1 and/or XCL2 used in the present invention is wild-type (eg, from human or mouse) or a variant thereof, which has the same or similar function as the wild-type.
  • XCL1 has at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 25 or 27 , 97%, 98%, 99% or 100% sequence identity
  • the coding sequence of XCL1 has at least 70%, preferably at least 80%, more preferably at least 90% of the nucleotide sequence shown in SEQ ID NO: 24 or 26 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.
  • XCL2 has at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 29 %, 99% or 100% sequence identity, or the coding sequence of XCL2 has at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the expression of exogenous genes (eg, IL9 and/or chemokines) of the present invention is secreted expression.
  • the exogenous gene is anchored expression, for example, it is operably linked with a localization domain, and the localization domain can localize the expression of the exogenous gene of the present invention on a specific cell location, For example cell membrane etc. Localization domains include, but are not limited to, nuclear localization signals, leader peptides, transmembrane domains, and the like.
  • the exogenous gene of the present invention is operably linked to a transmembrane domain, thereby anchoring expression on the surface of engineered immune cells.
  • the engineered immune cells of the present invention further express cell surface molecules that specifically recognize antigens.
  • 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 eg T cell receptor (TCR), chimeric antigen receptor (CAR), T cell fusion protein (TFP) or T cell antigen coupler (TAC).
  • T cell receptor or "TCR” is a characteristic marker on the surface of T cells that binds to CD3 in a non-covalent bond to form a complex.
  • Antigen presenting cells present antigenic peptides to T cells through major histocompatibility complex molecules (MHC) and bind to TCR complexes 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 antigen and MHC molecules.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding region (eg, the antigen-binding portion of an antibody), a transmembrane domain, and an intracellular Domains (comprising co-stimulatory domains and/or primary signaling domains), 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 T cell receptor (TCR) when expressed in T cells.
  • TCR T cell receptor
  • T cell fusion protein refers to a recombinant polypeptide derived from each component of TCR, usually composed of a TCR subunit and an antigen-binding region linked thereto, and expressed on the cell surface.
  • TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.
  • T cell antigen coupler includes three functional domains: 1 tumor targeting domain, including single chain antibody, designed ankyrin repeat protein (DARPin) or Other targeting groups; 2 extracellular region domain, single-chain antibody binding to CD3, thus bringing TAC receptor and TCR receptor closer; 3 transmembrane region and intracellular region of CD4 co-receptor, wherein, intracellular
  • the domain-linked protein kinase, LCK catalyzes the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) of the TCR complex as an initial step in T cell activation.
  • ITAM immunoreceptor tyrosine activation motif
  • 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.
  • 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.
  • the target is selected from CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2 , NKG2D, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, mesothelin, and any combination thereof.
  • 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.
  • 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 CD28, 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:3 or 100% sequence identity; or derived from CD8 ⁇ , it has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% with the amino acid sequence shown in SEQ ID NO: 4 or 5 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, FcyRIIIa receptor, IgG4, IgGl, 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 a CD28 hinge, 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:15.
  • CD8 ⁇ hinge which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity; or comprising an IgG4 hinge having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% to the amino acid sequence shown in SEQ ID NO: 18 % or 100% sequence identity.
  • intracellular domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a given function, which includes costimulatory domains and/or primary signaling domains.
  • the intracellular domain is responsible for intracellular signaling following antigen binding by the antigen binding region, resulting in activation of immune cells and immune responses.
  • the chimeric antigen receptors of the invention comprise a primary signaling domain, which may be the cytoplasmic sequence of the T cell receptor and co-receptor that function together to elicit primary signaling following antigen receptor binding , and any derivatives or variants of these sequences and any synthetic sequences having the same or similar function.
  • Primary signaling domains can contain many immunoreceptor tyrosine activation motifs.
  • Non-limiting examples of primary signaling domains of the invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain of the CAR of the present invention may comprise a CD3 ⁇ intracellular region, and the signaling domain has at least 70% of the amino acid sequence shown in SEQ ID NO: 9, 10 or 11, preferably at least 80%, 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 co-stimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40, ICOS, DAP10, DAP12 or a combination thereof.
  • the CAR of the present invention comprises a CD28 co-stimulatory domain, 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:6 Or 99% or 100% sequence identity; and/or comprising a 4-1BB costimulatory domain, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • 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.
  • 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 B2M, CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the signal peptide that can be used in the present invention is a B2M signal peptide, 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:12.
  • CD8 ⁇ signal peptide it has at least 70%, preferably at least 80%, more preferably at least 90%, 95% with the amino acid sequence shown in SEQ ID NO: 13 or 14 , 97% or 99% or 100% sequence identity.
  • the present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding exogenous IL9 or a nucleic acid sequence encoding endogenous IL9 operably linked to a modified promoter.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding one or more chemokines selected from XCL1 and XCL2.
  • the nucleic acid molecule further comprises cell surface molecules that specifically recognize antigens, such as chimeric antigen receptors, T cell receptors, T cell fusion proteins and T cell antigen couplers as described 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.
  • the present invention also provides a vector comprising the nucleic acid molecule according to the present invention.
  • the nucleic acid sequence encoding the exogenous IL9 or the nucleic acid sequence encoding the endogenous IL9 operably linked to the modified promoter, the nucleic acid sequence encoding the chemokine, and the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen can be in one or more vectors.
  • the nucleic acid sequences can be operably linked by a 2A peptide.
  • 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 invention include, but are not limited to, plasmids, viruses (such as retroviruses, oncolytic viruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus, polyoma virus, and adeno-associated virus (AAV ), etc.), phage, phagemid, cosmid and artificial chromosome (including BAC and YAC).
  • viruses such as retroviruses, oncolytic viruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus, polyoma virus, and adeno-associated virus (AAV ), etc.
  • phage phagemid
  • cosmid and artificial chromosome including BAC and YAC.
  • the vector itself is usually a sequence of nucleotides, usually a DNA sequence containing the 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 comprise elements such as a promoter, a polyA tail (polyA), a 3' UTR, an enhancer, a terminator, an insulator, an operator, a selectable marker, a reporter gene, a targeting sequence and/or a protein purification tag.
  • said vector is an in vitro transcribed vector.
  • the present invention also provides an engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance the expression of IL9.
  • the engineered immune cells further comprise exogenous chemokines selected from XCL1 and XCL2.
  • the engineered immune cells further comprise cell surface molecules that specifically recognize antigens, such as chimeric antigen receptors, T cell receptors, T cell fusion proteins and T cell antigen couplers as described above.
  • 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).
  • 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 iPSCs, ESCs, hematopoietic stem cells, etc.
  • 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 a site 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 (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), CD4-CD8-T cells, tumor infiltrating cells, memory T cells, regulatory 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.
  • the immune cells of the present invention further comprise suppressed or silenced expression of at least one endogenous 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, the expression of at least TCR components (including TCR ⁇ , TCR ⁇ genes) or CD3 components (including CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ ) in immune cells is inhibited or silenced. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD).
  • GvHD graft-versus-host disease
  • DNA fragmentation is mediated by meganucleases, zinc finger nucleases, TALEN nucleases or Cas enzymes in the CRISPR system, thereby knocking out the gene; or by shRNA, RNAi and other ways to inhibit gene expression.
  • 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.
  • 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.
  • 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.
  • the present invention also provides a combination therapy comprising engineered immune cells expressing cell surface molecules that specifically recognize antigens and exogenous IL9.
  • the combination therapy comprises: (1) engineered immune cells expressing exogenous IL9 and exogenous chemokines; (2) engineered immune cells expressing exogenous chemokines and Exogenous IL9; or (3) engineered immune cells and exogenous IL9 and chemokines; wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, and the chemokines are selected from XCL1 and XCL2.
  • 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 nucleic acid molecule, vector, engineered immune cell according to the present invention or pharmaceutical compositions. Therefore, the present invention also covers the use of the nucleic acid molecules, vectors, and engineered immune cells in the preparation of drugs 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, such as a hematological tumor or a solid tumor.
  • 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), lympho
  • lung cancer
  • 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, liver cancer, breast cancer, esophageal cancer , thyroid cancer, prostate cancer, bone cancer, lung cancer, etc.
  • 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.
  • MSCV-mCD19-CAR plasmid which contains CD19-scFv (SEQ ID NO: 2), CD8 ⁇ hinge region (SEQ ID NO: 17), CD8 ⁇ transmembrane region (SEQ ID NO: 5), 41BB co-stimulatory domain ( SEQ ID NO: 8) and the coding sequence of CD3 ⁇ intracellular region (SEQ ID NO: 11).
  • the MSCV-mCD19-CAR-IL19 plasmid was constructed, which further included the coding sequences of T2A (SEQ ID NO: 19) and IL19 (SEQ ID NO: 23) on the basis of the MSCV-mCD19-CAR plasmid.
  • the MSCV-mCD19-CAR-TSLP plasmid was constructed, which further included the coding sequences of T2A (SEQ ID NO: 19) and XCL1 (SEQ ID NO: 25) on the basis of the MSCV-mCD19-CAR plasmid.
  • the above plasmids were packaged into retroviruses and further transfected into activated T cells to obtain mCD19-CAR cells with traditional structure and CAR-T cells expressing IL9 or IL9+XCL1, that is, mCD19-CAR+IL9 cells, mCD19 -CAR+IL9+XCL1 cells (obtained by co-transfection with retroviruses packaged with MSCV-mCD19-CAR-IL9 plasmid and retroviruses packaged with MSCV-mCD19-CAR-XCL1 plasmid).
  • CAR-T cells Take out 2 ⁇ 10 5 CAR-T cells prepared in Example 1, use Goat Anti-Rat IgG (H&L) Biotin (BioVision, Cat. No. 6910-250) as the primary antibody, and APC Streptavidin (BD Pharmingen, Cat. No. 554067) as the secondary antibody , the expression level of CAR on CAR T cells was detected by flow cytometry, and the results are shown in Figure 1. It can be seen that CAR can be efficiently expressed in all CAR-T cells compared with untreated NT cells.
  • H&L Goat Anti-Rat IgG
  • BioVision BioVision, Cat. No. 6910-250
  • APC Streptavidin BD Pharmingen, Cat. No. 554067
  • the inventors also unexpectedly found that the IFN- ⁇ level of CAR-T cells expressing the combination of IL9+XCL1 was significantly higher than that of CAR-T cells expressing only IL9, indicating that IL9 and XCL1 can produce a synergistic effect to further enhance CAR - Killing activity of T cells.
  • Panc02-mCD19 pancreatic cancer cells were inoculated subcutaneously in the axilla of the left forelimb of healthy C57BL/6 mice.
  • the mice inoculated with pancreatic cancer cells were randomly divided into 4 groups, 6 mice in each group.
  • mice in each group were injected with 1 ⁇ 106 NT cells, mCD19-CAR cells, mCD19-CAR+IL9 cells or mCD19-CAR+IL9+XCL1 cells through the tail vein. Monitor the mice for body weight and tumor volume changes until the end of the experiment.
  • mice The body weight changes of the mice are shown in Figure 4. It can be seen that after administration of CAR-T cells, the body weight of the mice in each group has no significant difference compared with the control group, indicating that the administration of CAR-T cells will not have obvious toxic side effects on the mice.
  • the tumor volume changes in the mice are shown in Figure 5. It can be seen that, compared with traditional ACR-T cells, the additional expression of IL9 can significantly enhance the tumor-suppressive effect of CAR-T cells.
  • the inventors also unexpectedly found that the in vivo tumor suppressive effect of CAR-T cells expressing IL9+XCL1 combination also has a significant advantage compared with CAR-T cells expressing only IL9, indicating that IL9 can interact with chemokines to produce synergy.

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Abstract

L'invention concerne une cellule immunitaire modifiée comprenant de l'IL 9 exogène ou un promoteur modifié fonctionnellement lié à l'IL 9 endogène pour améliorer l'expression d'IL 9, et éventuellement une chimiokine et/ou une molécule de surface cellulaire qui reconnaît spécifiquement un antigène. L'invention concerne en outre une utilisation de la cellule immunitaire modifiée dans le traitement du cancer, d'infections ou de maladies auto-immunes. Par comparaison avec les cellules immunitaires modifiées classiques, la cellule immunitaire modifiée de la présente invention présente une activité de destruction tumorale significativement améliorée.
PCT/CN2022/127718 2021-11-11 2022-10-26 Cellule immunitaire modifiée et son utilisation WO2023083003A1 (fr)

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