WO2024114767A1 - 耐免疫排斥的工程化细胞 - Google Patents

耐免疫排斥的工程化细胞 Download PDF

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WO2024114767A1
WO2024114767A1 PCT/CN2023/135659 CN2023135659W WO2024114767A1 WO 2024114767 A1 WO2024114767 A1 WO 2024114767A1 CN 2023135659 W CN2023135659 W CN 2023135659W WO 2024114767 A1 WO2024114767 A1 WO 2024114767A1
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seq
cells
cell
immunosuppressive
variable region
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PCT/CN2023/135659
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French (fr)
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周亚丽
陈功
黄洁
汤颖秀
任江涛
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南京北恒生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an immunosuppressive molecule, which comprises an immunosuppressive protein binding domain, a transmembrane domain and a co-stimulatory domain and does not comprise a primary signaling domain, wherein the immunosuppressive protein binding domain is selected from an antibody targeting FasL, an antibody targeting IRP60, a ligand of IRP60 or a functional fragment thereof, an antibody targeting SIRP ⁇ , a ligand of SIRPa or a functional fragment thereof, wherein the immunosuppressive molecule can reduce the immune rejection of subject cells.
  • the present invention also relates to engineered cells expressing the immunosuppressive molecule, and a method for inhibiting the immune rejection of subject cells.
  • exogenous transplanted cells or transplanted organs will be recognized and attacked by immune cells in the subject, resulting in immune rejection.
  • allogeneic cell therapy such as CAR-T cells
  • organ transplantation the normal immune system in the patient will reject exogenous cells, thereby producing host-versus-graft disease (HvGD).
  • HvGD host-versus-graft disease
  • CD52 CD52 antibody
  • knocking out CD52 can make CAR-T cells resistant to alemtuzumab (CD52 antibody), thereby avoiding killing the introduced CAR-T cells when alemtuzumab is used to clear T cells in the patient's body.
  • alemtuzumab will increase the production cost of CAR-T products.
  • knocking out HLA-I molecules can ensure that CAR-T cells are not cleared by T cells in the patient's body without using antibodies or other treatments, cells that knock out HLA-I molecules will still be recognized by other immune cells of the patient, such as NK cells, and rejection occurs. Therefore, there is still a need to improve existing cell therapies, especially to reduce the immune rejection of patients.
  • immunosuppressive molecule refers to a molecule that can bind to immunosuppressive proteins (e.g., FasL, IRP60, SIRP ⁇ ) and thereby inhibit the subject's immune rejection of foreign cells, such as reducing the killing function of immune cells (e.g., T cells, NK cells) in the subject's body or inhibiting the excessive proliferation of immune cells.
  • immunosuppressive proteins e.g., FasL, IRP60, SIRP ⁇
  • antibody has the broadest meaning understood by those skilled in the art, and includes monoclonal antibodies (including complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit desired biological activity.
  • the antibodies of the present invention may be of any kind (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).
  • antigen-binding fragment or "antibody fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by a fragment of a full-length antibody.
  • antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, single-chain antibody (scFv), disulfide bond-linked Fv (sdFv), antibody heavy chain variable region (VH) or light chain variable region (VL), linear antibodies, "diabodies” with two antigen-binding sites, single domain antibodies (sdAb), nanobodies, etc. Therefore, unless the context clearly indicates, the present invention is not limited to a fragment of a full-length antibody.
  • the "antibody” herein encompasses antibody fragments or antigen binding fragments as defined above.
  • the antibody of the present invention is selected from IgG, Fab, Fab', F(ab') 2 , Fd, Fd', Fv, scFv, sdFv, linear antibody, diabody, sdAb or nanobody, preferably scFv, sdAb or nanobody.
  • a complete antibody typically includes two heavy chains and two light chains linked together by disulfide bonds, and each light chain is connected to its own heavy chain by a disulfide bond, forming a "Y" shaped structure.
  • Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region, wherein the heavy chain variable region contains three complementary determining regions (CDRs): CDR1-H, CDR2-H and CDR3-H, and the heavy chain constant region contains three constant domains: CH1, CH2 and CH3.
  • VH heavy chain variable region
  • CDRs complementary determining regions
  • Each light chain contains a light chain variable region (VL) and a light chain constant region, wherein the light chain variable region contains three CDRs: CDR1-L, CDR2-L and CDR3-L, and the light chain constant region contains a constant domain CL.
  • CDRs are separated by more conserved framework regions (FRs).
  • the variable region of the heavy chain/light chain is responsible for the recognition and binding to the antigen, while the constant region can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system.
  • the boundaries of a given CDR or FR may differ depending on the scheme used for the definition.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information.
  • Both the Kabat and Chothia schemes are based on numbering for the most common antibody region sequence lengths, with insertions provided by intervening letters (e.g., "30a") and deletions occurring in some antibodies. The two schemes place certain insertions and deletions (indels) in different positions, resulting in different numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used by Oxford Molecular's AbM antibody modeling software.
  • CDR of a given antibody or its region (such as its variable region) covers CDRs defined by any of the above schemes or other known schemes.
  • a specific CDR such as CDR3
  • CDR can also have the sequence of the corresponding CDR (such as CDR3) defined by any of the above schemes or other known schemes.
  • the FR of a given antibody or its region covers FRs defined by any of the above schemes or other known schemes.
  • the numbering scheme used to define the boundaries of CDR and FR herein adopts the Chothia scheme.
  • the immunosuppressive protein binding domain included in the immunosuppressive molecule of the present invention is an antibody targeting FasL.
  • FasL is a type II cell membrane surface glycoprotein and a member of the tumor necrosis factor receptor superfamily (TNFRSF). FasL is distributed on the surface of activated T lymphocytes, NK cells, mononuclear macrophages, etc., and its ligand is Fas. It has been found that the Fas/FasL system is one of the important pathways of cell apoptosis.
  • FasL rheumatoid arthritis, systemic lupus erythematosus, etc.
  • acute immune hyperactivity caused by organ transplantation, multiple trauma, etc.
  • chronic immune hyperactivity Kawasaki disease, idiopathic pulmonary fibrosis, etc.
  • chronic infection EBV infection, viral inflammation, etc.
  • lymphoma etc.
  • T-ALL T cell lymphoma, etc.
  • the antibody targeting FasL comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1-L, CDR2-L and CDR3-L with SEQ ID NO: 8; wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 7 or 13.
  • the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 4, CDR2-L as shown in SEQ ID NO: 5, and CDR3-L as shown in SEQ ID NO: 6;
  • the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 1, CDR2-H as shown in SEQ ID NO: 2, and CDR3-H as shown in SEQ ID NO: 3; or CDR1-H as shown in SEQ ID NO: 10, CDR2-H as shown in SEQ ID NO: 11, and CDR3-H as shown in SEQ ID NO: 12.
  • the antibody targeting FasL comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has 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% identity with SEQ ID NO: 8, or has one or more (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acids compared to the amino acid sequence of SEQ ID NO: 8.
  • the heavy chain variable region has 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% identity with SEQ ID NO: 7 or 13, or has one or several (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 7 or 13.
  • the modification is a conservative modification, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 7 or 13 and a light chain variable region as shown in SEQ ID NO: 8.
  • the antibody targeting FasL has 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% identity with SEQ ID NO: 9 or 14, or has one or several (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 9 or 14.
  • the antibody targeting FasL is as shown in SEQ ID NO: 9 or 14.
  • the immunosuppressive protein binding domain contained in the immunosuppressive molecule of the present invention is an antibody targeting SIRP ⁇ , a ligand of SIRP ⁇ , or a functional fragment thereof.
  • SIRP ⁇ also known as signal regulatory protein a or CD172a, is a transmembrane protein expressed on the surface of macrophages, dendritic cells, NK cells, and neurons, and its intracellular region contains an immunoreceptor tyrosine-based inhibitory motif (ITIM).
  • the ligand of SIRP ⁇ is CD47, a cell surface protein that transmits a "don't eat me" signal on healthy cells.
  • CD47 is also widely expressed on various tumor cells as a mechanism to evade immune detection.
  • the antibody targeting SIRP ⁇ comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 57; wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 56.
  • the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 53, CDR2-L as shown in SEQ ID NO: 54, and CDR3-L as shown in SEQ ID NO: 55;
  • the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 50, CDR2-H as shown in SEQ ID NO: 51, and CDR3-H as shown in SEQ ID NO: 52.
  • the antibody targeting SIRP ⁇ comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has 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% identity with SEQ ID NO: 57, or has one or more (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91
  • the modification is a conservative modification, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 56 and a light chain variable region as shown in SEQ ID NO: 57.
  • the antibody targeting SIRP ⁇ has at least 90%, at least 91%, at least 92%, or at least 93% similarity to SEQ ID NO: 58. 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, or has one or several (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 58.
  • the antibody targeting SIRP ⁇ is as shown in SEQ ID NO: 58.
  • the SIRP ⁇ binding domain contained in the immunosuppressive molecule of the present invention is a ligand of SIRP ⁇ , such as CD47 or a functional fragment thereof (i.e., a fragment having SIRP ⁇ binding function), such as the extracellular region of CD47.
  • the transmembrane region of the immunosuppressive molecule is preferably a CD47 transmembrane region.
  • the immunosuppressive molecule may also include a portion of the CD47 intracellular region.
  • the ligand of SIRP ⁇ 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: 69, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 69.
  • the ligand of SIRP ⁇ is as shown in SEQ ID NO: 69.
  • the immunosuppressive protein binding domain contained in the immunosuppressive molecule of the present invention is an antibody targeting IRP60, a ligand of IRP60 or a functional fragment thereof.
  • IRP60 also known as CD300a, is a type I transmembrane inhibitory receptor. IRP60 is widely expressed on the surface of myeloid cells (e.g., dendritic cells, mast cells, granulocytes, monocytes) and lymphocytes (T cells, B cells, etc.), and its expression level is different based on cell type and degree of differentiation. IRP60 contains 4 ITIMs, and the initiation of its inhibitory signal depends on the phosphorylation of tyrosine residues in ITIM.
  • IRP60 By interacting with ligand aminophospholipids (e.g., PtdEth and/or PtdSer), IRP60 participates in regulating a variety of cell activities, such as cell growth, proliferation, apoptosis, differentiation, immune response, etc. It is reported that IRP60 is closely related to the development of diseases such as hematological malignancies (e.g., leukemia, lymphoma, etc.), infectious diseases, allergic reactions, chronic inflammatory reactions, etc.
  • diseases such as hematological malignancies (e.g., leukemia, lymphoma, etc.), infectious diseases, allergic reactions, chronic inflammatory reactions, etc.
  • Anti-IRP60 antibodies known in the art can be used in the present invention, such as TX41, TX49, etc.
  • the antibody targeting IRP60 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 66; wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 65.
  • the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 62, CDR2-L as shown in SEQ ID NO: 63, and CDR3-L as shown in SEQ ID NO: 64;
  • the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 59, CDR2-H as shown in SEQ ID NO: 60, and CDR3-H as shown in SEQ ID NO: 61.
  • the antibody targeting IRP60 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has 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% identity with SEQ ID NO: 66, or has one or more (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91
  • the modification is a conservative modification, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 65 and a light chain variable region as shown in SEQ ID NO: 66.
  • the antibody targeting IRP60 has 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% identity with SEQ ID NO: 67, or has one or several (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 67.
  • the antibody targeting IRP60 is as shown in SEQ ID NO: 67.
  • conservative modification refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. These conservative modifications include conservative substitutions, additions and deletions of amino acids. Modifications can be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which 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 ...
  • the amino acids in the residues of interest are preferably amino acids, such as amino acids, ...
  • 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 usually expressed as a percentage. Preferably, the identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity.
  • sequence identity 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.
  • transmembrane domain refers to a polypeptide structure that enables the expression of immunosuppressive molecules on the cell surface and anchors the immunosuppressive protein binding domain to the cell membrane.
  • the transmembrane domain can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. When the target binding domain binds to the target, the transmembrane domain is able to conduct signal transduction.
  • Transmembrane domains particularly suitable for use in the present invention can be derived from, for example, TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD28, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, CD18, ICOS, 4-1BB, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, BCMA, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, C 1, 2, 3, 4, 5-6-nitropropene-1 (P ⁇ 0.05), D49a, ITGA4, IA4, CD49D, ITGA6,
  • the transmembrane domain is derived from the following molecules: CD8a, CD4, CD28, 4-1BB, CD47, CD80, CD86, CD152 and PD1.
  • the transmembrane domain may be synthetic and may comprise mainly 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% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 15.
  • the transmembrane domain is derived from CD8a, 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 SEQ ID NO: 16 or 17.
  • the immunosuppressive molecule further comprises a hinge region between the immunosuppressive protein binding domain and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antibody.
  • 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 antibody constant regions.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises a hinge region portion of CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably CD8 ⁇ , CD28 or IgG4 hinge.
  • the hinge region is from CD28, which 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: 27.
  • the hinge region is from CD8a, which 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: 28 or 29.
  • the hinge region is from IgG4, which 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: 30.
  • costimulatory domain refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function, which is an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular region of the costimulatory molecule, or a functional fragment thereof.
  • Cosmetic molecule refers to a cognate binding partner that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (e.g., proliferation and survival).
  • the costimulatory signaling domain of any costimulatory molecule is suitable for use in the immunosuppressive molecules described herein.
  • Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors.
  • the non-limiting examples of the costimulatory domain of the invention include, but are not limited to, the intracellular region derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, 4-1BB, CD270, CD272, B7-H3, ICOS, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70 and combinations thereof.
  • the costimulatory domain of CAR of the present invention is 4-1BB, CD28 or 4-1BB+CD28.
  • the costimulatory domain is from 4-1BB, 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 SEQ ID NO: 19 or 20.
  • the costimulatory domain is from CD28, 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 SEQ ID NO: 18.
  • the immunosuppressive molecules of the present invention do not include a primary signaling domain.
  • the term "primary signaling domain” refers to a protein structure that works together to initiate primary signaling after antigen-receptor binding, which is generally an intracellular sequence of a T cell receptor and a co-receptor.
  • the primary signaling domain generally includes one or more immunoreceptor tyrosine-based activation motifs (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • Non-limiting examples of the primary signaling domain of the present invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2, and CD66d.
  • the immunosuppressive molecules of the present invention do not include a CD3 ⁇ intracellular region.
  • the CD3 ⁇ intracellular region 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 SEQ ID NO:21, 22 or 23.
  • the immunosuppressive molecule of the present invention further comprises 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 ⁇ -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 B2M, CD8 ⁇ , IgG1, GM-CSFR ⁇ , etc.
  • the signal peptide that can be used in the present invention is from B2M, which 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 SEQ ID NO: 24.
  • the signal peptide useful in the present invention is derived from CD8a, which 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 SEQ ID NO: 25 or 26.
  • the present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the immunosuppressive molecule of the present invention.
  • the nucleic acid molecule may also comprise a nucleic acid sequence encoding a functional exogenous receptor (eg, chimeric antigen receptor, chimeric T cell receptor, etc.).
  • 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). Therefore, nucleic acids according to the present invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA.
  • 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, trityl bases and uncommon bases (such as inosine). Other modifications are also conceivable, including chemical, enzymatic or metabolic modifications, as long as the multi-chain CAR of the present invention can be expressed from a polynucleotide.
  • Nucleic acids may be provided in an isolated form.
  • 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.
  • the nucleic acid sequences of the present invention can be codon optimized for optimal expression in desired cells (e.g., immune cells); or for expression in bacteria, yeast, or insect cells. Codon optimization refers to replacing the codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement encode the same amino acid. Therefore, the selection of the optimal codon depends on the codon usage preference of the host genome.
  • the present invention also provides a vector comprising the nucleic acid molecule of the present invention.
  • the nucleic acid encoding the immunosuppressive molecule can be located on the same vector or on different vectors.
  • a functional exogenous receptor eg, chimeric antigen receptor, chimeric T cell receptor, etc.
  • vector is used as a mediating nucleic acid molecule for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule can, for example, be replicated and/or expressed.
  • Suitable vectors that can be used for the present invention are known in the art, and many are commercially available.
  • the vector of the present invention includes, but is not limited to, plasmids, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, vesicular stomatitis viruses, measles viruses, mumps viruses, polioviruses, orthomyxoviruses, small viruses, Maraba viruses, coxsackie viruses, herpes viruses, and slow viruses, etc.), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs).
  • viruses e.g., retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, vesicular stomatitis viruses, measles viruses, mumps viruses, polioviruses, orthomyxoviruses, small viruses, Maraba viruses, coxsackie viruses, herpes viruses, and slow viruses, etc.
  • the vector is generally also included in the starting point of autonomous replication in the cell (if stable expression of polynucleotides is required), a selection marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS).
  • the vector may additionally include 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.
  • the vector is a vector for in vitro transcription.
  • the present invention also provides an engineered cell expressing the immunosuppressive molecule of the present invention, a nucleic acid encoding the immunosuppressive molecule, or a vector comprising the nucleic acid.
  • the engineered cell of the present invention also expresses a functional exogenous receptor (as defined below).
  • the engineered cells of the invention are engineered immune cells.
  • 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.
  • effector functions e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC
  • immune cells can be B cells, T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells.
  • Immune cells can be obtained from a variety of sources, such as from a subject (e.g., from a subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumor, etc.), or from a cell line (e.g., Jurkat, SupT1, NK92, etc.) cultured in vitro, or differentiated from stem cells (e.g., derived from cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSC, etc.).
  • a subject e.g., from a subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumor,
  • immune cells are T cells or NK cells, more preferably T cells.
  • T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including but not limited to, CD4+CD8+T cells, CD4+T cells (e.g., Th1 and Th2 cells), CD8+T cells (e.g., cytotoxic T cells), CD4-CD8-T cells, tumor infiltrating cells, memory T cells, immature T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • immune cells are human T cells.
  • Various techniques known to those skilled in the art can be used, such as Ficoll separation to obtain T cells from the blood of the subject.
  • the expression of at least one endogenous HLA-I class gene of the engineered cells of the present invention is suppressed or silenced. In one embodiment, the expression of at least one endogenous HLA-II class gene of the engineered cells of the present invention is suppressed or silenced. In one embodiment, the expression of at least one endogenous TCR/CD3 gene of the engineered cells of the present invention is suppressed or silenced. In one embodiment, the expression of at least one endogenous TCR/CD3 gene and at least one endogenous HLA-I class gene of the engineered cells of the present invention is suppressed or silenced.
  • 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 cells of the present invention is suppressed or silenced.
  • the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C and B2M.
  • 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.
  • the TCR/CD3 gene is selected from TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • 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, CASP10 is inhibited or silenced in the engineered cells of the present invention.
  • CASP3, CASP6, CASP7 FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.
  • Methods for inhibiting gene expression or silencing genes include, but are not limited to, for example, Nucleases, zinc finger nucleases, TALEN, CRISPR/Cas systems, base editors mediate DNA or RNA breakage, or gene inactivation through antisense oligonucleotides, RNAi, shRNA, transposons, mutagenesis and other technologies.
  • the engineered cells of the present invention are allogeneic cells.
  • allogeneic refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered to be allogeneic to each other when the genes at one or more loci are different. In some cases, the genetic differences of allogeneic material from each individual of the same species may be sufficient to cause antigenic interactions.
  • the engineered cells expressing immunosuppressive molecules of the present invention may also express functional exogenous receptors.
  • the functional exogenous receptors are selected from recombinant T cell receptors, chimeric antigen receptors, T cell fusion proteins or T cell antigen couplers, more preferably chimeric antigen receptors.
  • T cell fusion protein or “TFP” refers to a recombinant polypeptide derived from various components of TCR, usually composed of TCR subunits and antibodies 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. a tumor targeting domain, including a single-chain antibody (such as the anti-FasL antibody, anti-IRP60 antibody or anti-SIRP ⁇ antibody of the present invention), a designed ankyrin repeat protein (DARPin) or other targeting groups; 2. an extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3.
  • a tumor targeting domain including a single-chain antibody (such as the anti-FasL antibody, anti-IRP60 antibody or anti-SIRP ⁇ antibody of the present invention), a designed ankyrin repeat protein (DARPin) or other targeting groups; 2. an extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3.
  • a single-chain antibody such as the anti-FasL antibody, anti-IRP60 antibody or anti-SIRP ⁇ antibody of the present invention
  • transmembrane region and an intracellular region of a CD4 co-receptor wherein the intracellular region is connected to the protein kinase LCK, catalyzing the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) of the TCR complex as the initial step of T cell activation.
  • ITAM immunoreceptor tyrosine activation motif
  • T cell receptor As used herein, the term “T cell receptor” or “TCR” is a characteristic marker on the surface of T cells, which binds to CD3 with non-covalent bonds to form a complex.
  • Antigen presenting cells present antigenic peptides to T cells through major histocompatibility complex molecules (MHC) and bind to the TCR complex to induce a series of intracellular signal transductions.
  • MHC major histocompatibility complex molecules
  • TCR consists 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, wherein the variable region is responsible for binding to specific antigens and MHC molecules.
  • the term "recombinant TCR receptor” refers to an artificially constructed T cell receptor that further comprises an antigen (e.g., tumor antigen) binding domain.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide, which generally includes an antigen (e.g., tumor antigen) binding domain (e.g., a ligand of an antibody or antigen), a transmembrane domain, an optional co-stimulatory domain, and a primary signaling domain, each domain being connected by a linker.
  • an antigen e.g., tumor antigen
  • binding domain e.g., a ligand of an antibody or antigen
  • transmembrane domain e.g., a ligand of an antibody or antigen
  • a primary signaling domain e.g., a primary signaling domain
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises a tumor antigen binding domain, a transmembrane domain, one or more co-stimulatory domains, and a primary signaling domain.
  • the chimeric antigen receptor also includes one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc.
  • the functional exogenous receptor comprises an extracellular domain that specifically recognizes an antigen (e.g., a tumor antigen).
  • the extracellular domain comprises an antibody that specifically binds to an antigen or a ligand of the antigen.
  • the antigen is 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, CD 138, CD151, CD171, CD179a, CD300LF, CLEC12A, CDH16, CSPG4, CS1, CLL-1, Claudin 6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, c-Met, CAIX, CXORF61, CA125
  • the antigen is selected from CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor ⁇ , CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA and GPRC5D, more preferably selected from CD19, Claudin18.2, MSLN, GPRC5D, CD7 and BCMA.
  • the functional exogenous receptor comprises an extracellular domain that specifically recognizes CD19, such as an antibody targeting CD19.
  • the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 35, CDR2-L as shown in SEQ ID NO: 36, and CDR3-L as shown in SEQ ID NO: 37; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 32, CDR2-H as shown in SEQ ID NO: 33, and CDR3-H as shown in SEQ ID NO: 34.
  • the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has 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% identity with SEQ ID NO: 39, 41 or 43, or has one or more (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 ...
  • the heavy chain variable region has 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% identity with SEQ ID NO: 38, 40 or 42, or has one or several (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 38, 40 or 42.
  • the modification is a conservative modification, such as conservative substitution, addition and deletion of amino acids.
  • the antibody of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 38, 40 or 42 and a light chain variable region as shown in SEQ ID NO: 39, 41 or 43.
  • the antibody targeting CD19 has 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% identity with SEQ ID NO: 44, 45, 46, 47 or 48, or has one or several (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 44, 45, 46, 47 or 48.
  • the antibody targeting FasL is as shown in SEQ ID NO: 44, 45, 46, 47 or 48.
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain, one or more co-stimulatory domains and a primary signaling domain.
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, and the chimeric antigen receptor targets CD19, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor ⁇ , CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA or GPRC5D, more preferably targeting CD19, Claudin18.2, MSLN, ROR1, GPRC5D, CD7 and BCMA.
  • the chimeric antigen receptor also comprises a signal peptide, a hinge region, or both.
  • a signal peptide a signal peptide, a hinge region, or both.
  • the definitions of the transmembrane domain, co-stimulatory domain, primary signaling domain, and optional hinge region, signal peptide and other structures that can be used in the CAR of the present invention are shown in the above “immunosuppressive molecules" section.
  • the CAR of the present invention may further comprise a switch structure to regulate the expression time of the CAR.
  • the switch structure may be in the form of a dimerization domain that causes a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain. Domain, so that it binds to the targeted antigen, thereby activating the signal transduction pathway.
  • the switch domain can also be used to connect the binding domain and the signal transduction domain respectively. Only when the switch domains bind to each other (for example, in the presence of an inducing compound), the binding domain and the signal transduction domain can be connected together through a dimer, thereby activating the signal pathway.
  • the switch structure can also be in the form of a masked peptide.
  • the masking peptide can shield the extracellular binding domain to prevent it from binding to the targeted antigen.
  • the masking peptide is cut by, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure.
  • Various switch structures known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may also include a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance to remove CAR cells when needed (e.g., when serious toxic side effects occur).
  • the suicide gene can be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when necessary, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes.
  • the suicide gene may also be herpes simplex virus thymidine kinase (HSV-TK), which can cause cells to die under induced treatment with ganciclovir.
  • HSV-TK herpes simplex virus thymidine kinase
  • the suicide gene may 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 apoptosis.
  • chemical induction drugs such as AP1903, AP20187, etc.
  • Various suicide genes known to those skilled in the art can be used in the present invention.
  • the present invention also provides a pharmaceutical composition, which comprises the immunosuppressive molecule, engineered cell, nucleic acid molecule or vector of the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the immunosuppressive molecule, nucleic acid molecule, vector or engineered cell in the preparation of a pharmaceutical composition or a drug.
  • 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, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, antiadhesives, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension regulators. It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical composition of the present invention.
  • Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • composition according to the present invention can be applied to a variety of routes. 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.
  • 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 the form particularly suitable for the desired method of administration.
  • the process known to 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 generally provided in solution form, and preferably include a pharmaceutically acceptable buffer.
  • the pharmaceutical composition according to the present invention can also be used in combination with one or more other medicaments (biological agents such as antibody reagents, and/or small molecules) or therapeutic methods (such as surgery, chemotherapy or radiotherapy) suitable for treating and/or preventing the disease to be treated.
  • medicaments biological agents such as antibody reagents, and/or small molecules
  • therapeutic methods such as surgery, chemotherapy or radiotherapy
  • Preferred examples of the medicament suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, tadalafil ...
  • 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, tadalafil
  • composition of the invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.
  • peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial 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 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.
  • the pharmaceutical composition of the invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.
  • compositions of the invention are used to treat a subject suffering from cancer, infection or autoimmune disease.
  • the cancer is a cancer associated with expression of a target to which a functional exogenous receptor binds.
  • the cancer includes, but is not limited to, brain gliomas, blastomas, sarcomas, leukemias, 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, 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, kidney cancer, laryngeal cancer, liver tumors, lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous cell lung cancer), lymphoma (including Hodg
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • 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.
  • the present invention also provides a method for reducing immune rejection of exogenous cells by cells of a subject, characterized in that the immunosuppressive molecule of the present invention, a nucleic acid molecule encoding the immunosuppressive molecule or a vector containing the nucleic acid molecule is expressed in the exogenous cells.
  • the present invention also provides a method for reducing immune rejection of exogenous cells by cells of a subject, characterized in that the immunosuppressive molecule of the present invention or the engineered cell expressing the immunosuppressive molecule is administered to the subject.
  • the immunosuppressive molecule or engineered cell of the present invention is administered simultaneously or sequentially with the exogenous cell.
  • the immunosuppressive molecule or engineered cell of the present invention can be administered after the exogenous cell.
  • the term "subject" is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, pig, 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.
  • FIG. 1 shows the CAR expression level of CAR-T cells expressing an immunosuppressive molecule targeting FasL.
  • FIG. 2 shows the killing effect of CAR-T cells expressing immunosuppressive molecules targeting FasL on target cells.
  • FIG. 3 shows the cytokine release level after CAR-T cells expressing immunosuppressive molecules targeting FasL were co-incubated with target cells Nalm6.
  • FIG. 4 shows the cytokine release level after CAR-T cells expressing immunosuppressive molecules targeting FasL were co-incubated with target cells Raji.
  • FIG. 5 shows the inhibitory effect of immunosuppressive molecules targeting FasL on the proliferation of allogeneic PBMCs.
  • FIG. 6 shows the in vivo tumor-suppressive effect of CAR-T cells expressing immunosuppressive molecules targeting FasL.
  • FIG. 7 shows the CAR expression level of CAR-T cells expressing immunosuppressive molecules targeting SIRP ⁇ .
  • FIG. 8 shows the expression level of anti-SIRP ⁇ antibodies in CAR-T cells expressing immunosuppressive molecules targeting SIRP ⁇ .
  • FIG. 9 shows the cytokine release level after CAR-T cells expressing immunosuppressive molecules targeting SIRP ⁇ were co-incubated with target cells Nalm6.
  • FIG. 10 shows the cytokine release level after CAR-T cells expressing immunosuppressive molecules targeting SIRP ⁇ were co-incubated with target cells Raji.
  • FIG. 11 shows the inhibitory effect of immunosuppressive molecules targeting SIRP ⁇ on NK cell killing.
  • FIG. 12 shows the CAR expression level of CAR-T cells expressing immunosuppressive molecules targeting IRP60.
  • FIG. 13 shows the expression level of anti-IRP60 antibodies in CAR-T cells expressing immunosuppressive molecules targeting IRP60.
  • FIG. 14 shows the cytokine release levels after co-incubation of CAR-T cells expressing immunosuppressive molecules targeting IRP60 with target cells Nalm6.
  • FIG. 15 shows the cytokine release levels after CAR-T cells expressing immunosuppressive molecules targeting IRP60 were co-incubated with target cells Raji.
  • FIG. 16 shows the inhibitory effect of immunosuppressive molecules targeting IRP60 on NK cell killing.
  • FIG. 17 shows the CAR expression levels of CAR-T cells expressing immunosuppressive molecules including CD47.
  • FIG. 18 shows the expression level of CD47 in CAR-T cells expressing immunosuppressive molecules including CD47.
  • FIG. 19 shows the killing effect of CAR-T cells expressing immunosuppressive molecules including CD47 on target cells Raji.
  • FIG. 20 shows the cytokine release level after CAR-T cells expressing immunosuppressive molecules including CD47 were co-incubated with target cells Raji.
  • FIG. 21 shows the inhibitory effect of immunosuppressive molecules including CD47 on NK cell killing.
  • the following coding sequences were synthesized and cloned into the pGEM-T Easy vector (Promega) in sequence: anti-CD19 scFv (SEQ ID NO: 44), CD8 ⁇ hinge region (SEQ ID NO: 29), CD8 ⁇ transmembrane region (SEQ ID NO: 17), 4-1BB co-stimulatory domain (SEQ ID NO: 20), and CD3 ⁇ intracellular region (SEQ ID NO: 23) to obtain the CAR19 plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.
  • anti-CD19 scFv SEQ ID NO: 44
  • CD8 ⁇ hinge region SEQ ID NO: 29
  • CD8 ⁇ transmembrane region SEQ ID NO: 17
  • 4-1BB co-stimulatory domain SEQ ID NO: 20
  • CD3 ⁇ intracellular region SEQ ID NO: 23
  • the above-mentioned CAR19 plasmid further expressed an immunosuppressive molecule connected by T2A (SEQ ID NO: 31), whose structure was as follows: B2m signal peptide (SEQ ID NO: 24), anti-FasL scFv-1 (SEQ ID NO: 9) or anti-FasL scFv-2 (SEQ ID NO: 14), IgG4 hinge region (SEQ ID NO: 30), CD28 transmembrane region (SEQ ID NO: 15), CD28 co-stimulatory domain (SEQ ID NO: 18), and CAR19-aFL-1 plasmid and CAR19-aFL-2 plasmid were obtained, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.
  • B2m signal peptide SEQ ID NO: 24
  • anti-FasL scFv-1 SEQ ID NO: 9
  • anti-FasL scFv-2 SEQ ID NO: 14
  • T2A The following structures connected by T2A (SEQ ID NO: 31) were further expressed in the above-mentioned CAR19 plasmid: B2m signal peptide (SEQ ID NO: 24), Fas extracellular region and transmembrane region (SEQ ID NO: 49), IgG4 hinge region (SEQ ID NO: 30), CD28 transmembrane region (SEQ ID NO: 15), and CD28 co-stimulatory domain (SEQ ID NO: 18) to obtain CAR19-Fas plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.
  • B2m signal peptide SEQ ID NO: 24
  • Fas extracellular region and transmembrane region SEQ ID NO: 49
  • IgG4 hinge region SEQ ID NO: 30
  • CD28 transmembrane region SEQ ID NO: 15
  • CD28 co-stimulatory domain SEQ ID NO: 18
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco) and cultured at 37°C and 5% CO2 for 1 day. On the second day, concentrated lentivirus was added, and after 3 days of continuous culture, T cells expressing CD19 CAR (CAR19-T) and T cells expressing CD19 CAR + a combination of immunosuppressive molecules targeting FasL (CAR19-Fas T, CAR19-aFL-1 T, and CAR19-aFL-2 T) were obtained.
  • the target cell Raji cells were plated into a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and then NT cells, CAR19 T cells, CAR19-Fas T cells, CAR19-aFL-1 T cells and CAR19-aFL-2 T cells were plated into a 96-well plate for co-culture at an effector-target ratio of 4:1, 2:1, 1:1 or 0.5:1.
  • the fluorescence value was measured by an ELISA reader after 18 hours.
  • 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.
  • CAR19-Fas T cells, CAR19-aFL-1 T cells and CAR19-aFL-2 T cells showed effective killing of target cells, and at a low efficient target ratio (for example, 0.5:1), their killing effect was slightly better than that of CAR19 T cells that do not express immunosuppressive molecules.
  • target cells (Nalm6 cells and Raji cells) were plated in a 96-well plate at a concentration of 1 ⁇ 10 5 cells/well, and NT cells, CAR19 T cells, CAR19-Fas T cells, CAR19-aFL-1 T cells, and CAR19-aFL-2 T cells were added at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours of co-culture.
  • the inhibitory effect of the immunosuppressive molecule of the present invention on HvGD in vitro was evaluated by one-way mixed lymphocyte reaction (MLR) assay. Specifically, NT cells, CAR19 T cells, CAR19-Fas T cells, CAR19-aFL-1 T cells and CAR19-aFL-2 T cells were co-incubated with allogeneic PBMCs at 37°C and 5% CO2 for 7 days, and then the proliferation of PBMCs was detected by flow cytometry, and the results are shown in Figure 5.
  • MLR mixed lymphocyte reaction
  • CAR19 T cells increased the PBMC count by about 3.5 times (0.4x10 6 vs 1.4x10 6 ), indicating that heterologous CAR-T cells greatly promoted the proliferation of PBMC.
  • additional expression of immunosuppressive molecules can significantly reduce this proliferation of PBMC.
  • the immunosuppressive molecule containing the Fas extracellular region (CAR19-Fas T) reduced PBMC proliferation by about 35% (about 0.9x10 6 ), while the immunosuppressive molecules containing anti-FasL scFv (CAR19-aFL-1 T and CAR19-aFL-2 T) further reduced PBMC proliferation by about 3 times (about 0.3x10 6 ) on the basis of CAR19-Fas T cells, which is basically equivalent to the effect of NT cells that do not express CAR.
  • the additional expression of immunosuppressive molecules targeting FasL can significantly inhibit the proliferation of allogeneic PBMCs in vitro, thereby significantly reducing HvGD.
  • the inhibitory effect of immunosuppressive molecules containing anti-FasL scFv is significantly better than that of immunosuppressive molecules containing the extracellular region of Fas.
  • mice Twenty-five healthy female NPI mice aged 6-8 weeks were divided into 5 groups, with 5 mice in each group: NT group, CAR19 group, CAR19-Fas group, CAR19-aFL-1 group, and CAR19-aFL-2 group.
  • 5 ⁇ 10 5 Raji cells were injected into the tail vein of each mouse on day 0.
  • 2x10 6 NT cells or corresponding CAR-T cells were injected into the tail vein of each mouse according to the grouping. The status of the mice was evaluated weekly. The mouse survival curve is shown in Figure 6.
  • the survival rate of the CAR19-Fas group is comparable to that of the CAR19 group, indicating that the immunosuppressive molecules containing the Fas extracellular domain may not have a good immune rejection inhibitory effect in vivo.
  • the survival rates of the CAR19-aFL-1 group and the CAR19-aFL-2 group were significantly higher than those of the CAR19-Fas group, indicating that the immunosuppressive molecules containing anti-FasL scFv can also effectively inhibit immune rejection in vivo, thereby enhancing the tumor inhibition effect of CAR-T cells and improving the survival rate.
  • Example 5 Construction of CAR-T cells expressing immunosuppressive molecules targeting SIRP ⁇ and verification of their functions
  • CAR-T cells expressing immunosuppressive molecules targeting SIRP ⁇ were constructed, wherein the B2M gene in T cells was knocked out using the CRISPR/Cas9 system before the lentiviral transfection step.
  • the immunosuppressive molecules targeting SIRP ⁇ include: B2m signal peptide (SEQ ID NO: 24), anti-SIRP ⁇ scFv (SEQ ID NO: 58), IgG4 hinge region (SEQ ID NO: 30), CD28 transmembrane region (SEQ ID NO: 15), CD28 co-stimulatory domain (SEQ ID NO: 18).
  • the obtained CAR-T cells were named CAR19- ⁇ T cells.
  • Target cells (Nalm6 cells and Raji cells) were plated in a 96-well plate at a concentration of 1 ⁇ 10 5 cells/well, and CAR19-T cells, CAR19- ⁇ T cells, and NT cells (negative control) were added at a ratio of 1:1. After 18-24 hours of co-culture, the cell co-culture supernatant was collected.
  • IL2 and IFN- ⁇ in the co-culture supernatant were detected using Human IL-2 DuoSet ELISA Kit (R&D systems) and Human IFN-gamma DuoSet ELISA Kit (R&D systems), respectively.
  • the results are shown in Figures 9 and 10. It can be seen that compared with NT cells, the release levels of cytokines IL2 and IFN- ⁇ were significantly increased after CAR19-T and CAR19- ⁇ T were co-cultured with each target cell, indicating that this cytokine release is specific. In addition, the cytokine release levels of CAR19-T and CAR19- ⁇ T cells are comparable, indicating that the additionally expressed immunosuppressive molecules do not impair the cytokine release characteristics of CAR-T cells.
  • the CAR-T cells prepared in this example were labeled with Far-Red (invitrogen).
  • the labeled CAR-T cells were then plated into a 96-well plate at a concentration of 1x10 4 cells/well, and allogeneic NK cells (pNK-1 and pNK-2) derived from peripheral blood were added at a 2:1 effector-target ratio (NK cells: CAR-T cells) for co-culture.
  • NK-1 and pNK-2 allogeneic NK cells derived from peripheral blood were added at a 2:1 effector-target ratio (NK cells: CAR-T cells) for co-culture.
  • NK cells derived from peripheral blood were added at a 2:1 effector-target ratio
  • Example 6 Construction of CAR-T cells expressing immunosuppressive molecules targeting IRP60 and verification of their function
  • CAR-T cells expressing immunosuppressive molecules targeting IRP60 were constructed, wherein the B2M gene in T cells was knocked out using the CRISPR/Cas9 system before the lentiviral transfection step.
  • the immunosuppressive molecules targeting IRP60 include: B2m signal peptide (SEQ ID NO: 24), anti-IRP60 scFv (SEQ ID NO: 67), IgG4 hinge region (SEQ ID NO: 30), CD28 transmembrane region (SEQ ID NO: 15), CD28 co-stimulatory domain (SEQ ID NO: 18).
  • the obtained CAR-T cells were named CAR19-60T cells.
  • the release levels of cytokines IL2 and IFN- ⁇ of the CAR-T cells prepared in this example after co-incubation with target cells Nalm6 cells and Raji cells were detected, and the results are shown in Figures 14 and 15. It can be seen that compared with NT cells, the release levels of cytokines IL2 and IFN- ⁇ were significantly increased after CAR19-T and CAR19-60 T were co-cultured with each target cell, indicating that this cytokine release is specific. In addition, the cytokine release levels of CAR19-T and CAR19-60 T cells are comparable, indicating that the additionally expressed immunosuppressive molecules do not impair the cytokine release of CAR-T cells.
  • Example 7 Construction of CAR-T immune cells expressing immunosuppressive molecules including CD47 and verification of their function
  • the following coding sequences were synthesized and cloned into the pGEM-T Easy vector (Promega, catalog number A1360) in sequence: anti-CD19 scFv (SEQ ID NO: 44), CD8 ⁇ hinge region (SEQ ID NO: 29), CD8 ⁇ transmembrane region (SEQ ID NO: 17), 4-1BB co-stimulatory domain (SEQ ID NO: 20), CD3 ⁇ intracellular region (SEQ ID NO: 23), to obtain the CAR19 plasmid, and confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • anti-CD19 scFv SEQ ID NO: 44
  • CD8 ⁇ hinge region SEQ ID NO: 29
  • CD8 ⁇ transmembrane region SEQ ID NO: 17
  • 4-1BB co-stimulatory domain SEQ ID NO: 20
  • CD3 ⁇ intracellular region SEQ ID NO: 23
  • the above-mentioned CAR19 plasmid was further expressed with an immunosuppressive molecule connected by T2A (SEQ ID NO: 31), whose structure was as follows: CD47 extracellular transmembrane region (SEQ ID NO: 68), CD28 co-stimulatory domain (SEQ ID NO: 18), to obtain the CAR19-47 plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Catalog No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, concentrated lentivirus was added and cultured for 3 days to obtain T cells expressing CD19CAR (CAR19-T) and T cells expressing CD19CAR and immunosuppressive molecules targeting SIRP ⁇ (CAR19-47-T).
  • the target cell Raji cells were plated into a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and then NT cells, CAR19-T cells and CAR19-47-T cells were plated into a 96-well plate for co-culture at an effector-target ratio of 4:1, 2:1, 1:1 or 0.5:1.
  • the fluorescence value was measured using an ELISA reader after 18 hours.
  • 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 19.
  • CAR19-T cells and CAR19-47-T cells showed effective specific killing of target cells, and the killing activity of CAR19-47-T cells was significantly improved compared with CAR19-T.
  • Target cells (Raji cells) were plated in a 96-well plate at a concentration of 1 ⁇ 10 5 cells/well, and CAR19-T cells, CAR19-CD47-T cells, and NT cells (negative control) were added at a ratio of 1:1. After 18-24 hours of co-culture, the cell co-culture supernatant was collected.
  • the levels of IL2 and IFN- ⁇ in the co-culture supernatant were detected using Human IL-2 DuoSet ELISA Kit (R&D systems, catalog number DY202) and Human IFN-gamma DuoSet ELISA Kit (R&D systems, catalog number DY285), respectively. The results are shown in Figure 20.
  • the CAR-T cells prepared by the present invention were labeled with Far-Red (invitrogen, catalog number C34564).
  • the labeled CAR-T cells were then plated into a 96-well plate at a concentration of 1x10 4 cells/well, and NK cells derived from peripheral blood were added at a 2:1 effector-target ratio for co-culture. After 16-18 hours, the survival rate of T cells in the culture was detected by flow cytometry, and the results are shown in Figure 21.
  • the immunosuppressive molecules targeting FasL, IRP60 or SIRP ⁇ of the present invention will not adversely affect the killing activity or cytokine release characteristics of CAR-T cells on the one hand, and on the other hand can significantly inhibit the killing of allogeneic NK cells or inhibit the excessive proliferation of allogeneic PBMCs after activation, thereby effectively reducing the immune rejection of foreign cells by immune cells in the host body.
  • the present invention relates to an immunosuppressive molecule, which comprises an immunosuppressive protein binding domain, a transmembrane domain and a co-stimulatory domain and does not comprise a primary signal transduction domain, wherein the immunosuppressive protein binding domain is selected from an antibody targeting FasL, an antibody targeting IRP60, a ligand of IRP60 or a functional fragment thereof, an antibody targeting SIRP ⁇ , a ligand of SIRPa or a functional fragment thereof, wherein the immunosuppressive molecule can reduce the immune rejection of cells in a subject.
  • the immunosuppressive molecule of the present invention effectively reduces the immune rejection of foreign cells by immune cells in a host body and has excellent industrial applicability.

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Abstract

本发明涉及一种免疫抑制分子，其包含免疫抑制蛋白结合结构域、跨膜结构域和共刺激结构域并且不包含初级信号传导结构域，其中所述免疫抑制蛋白结合结构域选自靶向FasL的抗体、靶向IRP60的抗体、IRP60的配体或其功能性片段、靶向SIRPα的抗体、SIRPa的配体或其功能性片段，其中所述免疫抑制分子能够降低受试者细胞的免疫排斥。本发明还涉及表达所述免疫抑制性分子的工程化细胞以及包含该细胞的组合物。本发明还提供了用所述免疫抑制分子来降低免疫排斥的方法。

Description

耐免疫排斥的工程化细胞
相关申请的交叉引用
本申请要求于2022年12月01日提交中国国家知识产权局的申请号为202211543531.6、名称为“耐免疫排斥的工程化细胞”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及一种免疫抑制分子，其包含免疫抑制蛋白结合结构域、跨膜结构域和共刺激结构域并且不包含初级信号传导结构域，其中所述免疫抑制蛋白结合结构域选自靶向FasL的抗体、靶向IRP60的抗体、IRP60的配体或其功能性片段、靶向SIRPα的抗体、SIRPa的配体或其功能性片段，其中所述免疫抑制分子能够降低受试者细胞的免疫排斥。本发明还涉及表达所述免疫抑制分子的工程化细胞，以及抑制受试者细胞的免疫排斥的方法。
背景技术
在异体移植的情况下，外源的移植细胞或移植器官会受到受试者体内免疫细胞的识别和攻击，发生免疫排斥反应。例如，当患者接受同种异体的细胞疗法(如CAR-T细胞)或器官移植时，患者体内的正常免疫系统会排斥外源的细胞，进而产生宿主抗移植物病(HvGD)。目前，对于HvGD，主要通过敲除CD52或HLA-I类分子的方式来降低或避免。具体地，敲除CD52可以使CAR-T细胞对阿仑单抗(CD52抗体)产生抗性，从而避免在用阿伦单抗清除患者体内T细胞时对引入的CAR-T细胞产生杀伤。然而，使用阿仑单抗会增加CAR-T产品的生产成本。另一方面，敲除HLA-I类分子虽然可以保证在不使用抗体或者其他处理的情况下避免CAR-T细胞被患者体内的T细胞清除，但敲除HLA-I分子的细胞仍然会被患者的其他免疫细胞，例如NK细胞识别而发生排斥反应。因此，仍然需要对现有的细胞疗法进行改进，尤其是降低患者的免疫排斥反应。
发明内容
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
免疫抑制分子
在第一个方面，本发明提供一种免疫抑制分子，其包含免疫抑制蛋白结合结构域、跨膜结构域和共刺激结构域并且不包含初级信号传导结构域，其中所述免疫抑制蛋白结合结构域选自靶向FasL的抗体、靶向IRP60的抗体、IRP60的配体或其功能性片段、靶向SIRPα的抗体、SIRPa的配体或其功能性片段，其中所述免疫抑制分子能够降低受试者细胞的免疫排斥。
如本文所用，术语“免疫抑制分子”是指能够与免疫抑制蛋白(例如FasL、IRP60、SIRPα)结合进而抑制受试者对外源细胞的免疫排斥，例如降低受试者体内的免疫细胞(如T细胞、NK细胞)的杀伤功能或者抑制免疫细胞的过度增殖的分子。
如本文所用，术语“抗体”具有本领域技术人员所理解的最广泛的含义，并且包括单克隆抗体(包含完整抗体)、多克隆抗体、多价抗体、多特异性抗体(例如双特异性抗体)、和能够表现期望的生物活性的携带一个或多个CDR序列的抗体片段或合成多肽。本发明所述抗体可为任何种类(例如IgG、IgE、IgM、IgD、IgA等)或亚类(例如IgG1、IgG2、IgG2a、IgG3、IgG4、IgA1、IgA2等)。
如本文所用，术语“抗原结合片段”或“抗体片段”指抗体中保留特异性结合抗原的能力的一个或更多个片段。已经显示，抗体的抗原结合功能可通过全长抗体的片段来实现。本发明中的抗体片段的实例包括但不限于：Fab、Fab'、F(ab')2、Fd片段、Fd′、Fv片段、单链抗体(scFv)、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、线性抗体、具有两个抗原结合位点的“双体”、单结构域抗体(sdAb)、纳米抗体等。因此，除非上下文明确指出，否则本发 明的“抗体”涵盖如上定义的抗体片段或抗原结合片段。因此，在一个优选的实施方案中，本发明的抗体选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、双体、sdAb或纳米抗体，优选scFv、sdAb或纳米抗体。
通常，完整抗体包括通过二硫键连接在一起的两条重链和两条轻链，每条轻链通过二硫键被连至各自的重链，呈“Y”形结构。每条重链包含重链可变区(VH)和重链恒定区，其中重链可变区包含三个互补决定区(CDR)：CDR1-H、CDR2-H和CDR3-H，重链恒定区包含三个恒定结构域：CH1、CH2和CH3。每条轻链包含轻链可变区(VL)和轻链恒定区，其中轻链可变区包含三个CDR：CDR1-L、CDR2-L和CDR3-L，轻链恒定区包含一个恒定结构域CL。在重链/轻链可变区中，CDR被更保守的框架区(FR)隔开。重链/轻链的可变区负责与抗原的识别和结合，恒定区则可以介导抗体与宿主组织或因子的结合，包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一组分。
可以使用许多本领域熟知的编号方案容易地确定给定CDR或FR的精确氨基酸序列边界，这些方案包括：Kabat等人(1991),“Sequences ofProteins of Immunological Interest,”第5版Public Health Service,NationalInstitutes of Health,贝塞斯达,马里兰州(“Kabat”编号方案)；Al-Lazikani等人,(1997)JMB 273,927-948(“Chothia”编号方案)；MacCallum等人,J.Mol.Biol.262:732-745(1996),“Antibody-antigen interactions:Contact analysis and binding sitetopography,”J.Mol.Biol.262,732-745”(“Contact”编号方案)；Lefranc MP等人,“IMGTunique numbering for immunoglobulin and T cell receptor variable domains andIg superfamily V-like domains,”Dev Comp Immunol,2003年1月；27(1):55-77(“IMGT”编号方案)；Honegger A和Plückthun A,“Yet another numbering scheme forimmunoglobulin variable domains:an automatic modeling and analysis tool,”JMol Biol,2001年6月8日；309(3):657-70(“Aho”编号方案)；和Martin等人,“Modeling antibody hypervariable loops:a combined algorithm,”PNAS,1989,86(23):9268-9272(“AbM”编号方案)。
给定CDR或FR的边界可能取决于用于定义的方案而不同。例如，Kabat方案是基于结构比对，而Chothia方案是基于结构信息。Kabat和Chothia方案的编号都是基于最常见的抗体区域序列长度，其中通过插入字母提供插入(例如“30a”)并且在一些抗体中出现缺失。这两种方案将某些插入和缺失(indel)放置在不同的位置，从而产生不同的编号。Contact方案是基于对复杂晶体结构的分析，并且在许多方面与Chothia编号方案相似。AbM方案是介于Kabat与Chothia定义之间的折衷，其基于Oxford Molecular的AbM抗体建模软件所使用的方案。
因此，除非另有规定，否则应当理解，给定抗体或其区域(如其可变区)的“CDR”涵盖由任何上述方案或其他已知方案所定义的CDR。例如，在指定特定的CDR(例如CDR3)含有给定氨基酸序列的情况下，应理解，这样的CDR还可以具有由任何上述方案或其他已知方案所定义的相应CDR(例如CDR3)的序列。同样，除非另有规定，否则应当理解给定抗体或其区域(如其可变区)的FR涵盖由任何上述方案或其他已知方案所定义的FR。除非特别指出，否则在本文中用于界定CDR和FR的边界的编号方案采用Chothia方案。
在一个实施方案中，本发明的抗体是鼠源抗体、嵌合抗体、驼源抗体、人源化抗体或人抗体。
在一个实施方案中，本发明的免疫抑制分子包含的免疫抑制蛋白结合结构域是靶向FasL的抗体。FasL是一种II型细胞膜表面糖蛋白，属于肿瘤坏死因子受体超家族(TNFRSF)的成员。FasL分布于活化的T淋巴细胞、NK细胞、单核巨噬细胞等表面，其配体是Fas。已经发现Fas/FasL系统是细胞凋亡的重要途径之一。具体地，FasL与Fas的结合会形成诱导死亡的复合体，进而激活caspase信号通路，通过其胞内酪氨酸和丝氨酸的磷酸化最终导致细胞凋亡。据报道，Fas/FasL的功能异常与各种临床疾病密切相关，例如自身免疫性疾病(类风湿性关节炎、系统性红斑狼疮等)、急性免疫过度活跃(器官移植、多创伤等引起)、慢性免疫过度活跃(川崎病、特发性肺纤维化等)、慢性感染(EBV感染、病毒引起的炎症等)、淋巴肿瘤等(T-ALL、T细胞淋巴瘤等)。
本领域已知的抗FasL抗体均可用于本发明。在一个实施方案中，靶向FasL的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO： 8所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO：7或13所包含的CDR1-H、CDR2-H和CDR3-H相同。在一个实施方案中，轻链可变区包含的CDR1-L如SEQ ID NO：4所示，CDR2-L如SEQ ID NO：5所示，CDR3-L如SEQ ID NO：6所示；重链可变区包含的CDR1-H如SEQ ID NO：1所示，CDR2-H如SEQ ID NO：2所示，CDR3-H如SEQ ID NO：3所示；或CDR1-H如SEQ ID NO：10所示，CDR2-H如SEQ ID NO：11所示，CDR3-H如SEQ ID NO：12所示。
在一个实施方案中，所述靶向FasL的抗体包含轻链可变区和重链可变区，其中所述轻链可变区与SEQ ID NO:8具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：8的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰；其中所述重链可变区与SEQ ID NO:7或13具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：7或13的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体包含如SEQ ID NO：7或13所示的重链可变区和如SEQ ID NO：8所示的轻链可变区。
在一个实施方案中，所述靶向FasL的抗体与SEQ ID NO:9或14具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：9或14的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述靶向FasL的抗体如SEQ ID NO：9或14所示。
在一个实施方案中，本发明的免疫抑制分子包含的免疫抑制蛋白结合结构域是靶向SIRPα的抗体、SIRPα的配体或其功能性片段。SIRPα也称为信号调节蛋白a或CD172a，是一种在巨噬细胞、树突状细胞、NK细胞和神经细胞表面均有表达的跨膜蛋白，其胞内区含有免疫受体基于酪氨酸的抑制性基序(ITIM)。SIRPα的配体是CD47，一种在健康细胞上传递“不要吃我”信号的细胞表面蛋白。此外，CD47也广泛地在各种肿瘤细胞上表达，作为逃避免疫检测的一种机制。许多研究发现，抑制癌细胞中CD47-SIRPα信号通路(例如，通过抗CD47抗体或抗SIRPα抗体)可以促进巨噬细胞对肿瘤细胞的吞噬，最终限制肿瘤生长。
本领域已知的抗SIRPα抗体均可用于本发明。在一个实施方案中，靶向SIRPα的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO：57所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO：56所包含的CDR1-H、CDR2-H和CDR3-H相同。在一个实施方案中，轻链可变区包含的CDR1-L如SEQ ID NO：53所示，CDR2-L如SEQ ID NO：54所示，CDR3-L如SEQ ID NO：55所示；重链可变区包含的CDR1-H如SEQ ID NO：50所示，CDR2-H如SEQ ID NO：51所示，CDR3-H如SEQ ID NO：52所示。
在一个实施方案中，所述靶向SIRPα的抗体包含轻链可变区和重链可变区，其中所述轻链可变区与SEQ ID NO:57具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：57的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰；其中所述重链可变区与SEQ ID NO:56具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：56的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体包含如SEQ ID NO：56所示的重链可变区和如SEQ ID NO：57所示的轻链可变区。
在一个实施方案中，所述靶向SIRPα的抗体与SEQ ID NO:58具有至少90％、至少91％、至 少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：58的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述靶向SIRPα的抗体如SEQ ID NO：58所示。
在一个实施方案中，本发明的免疫抑制分子包含的SIRPα结合结构域是SIRPα的配体，例如CD47或其功能性片段(即，具有SIRPα结合功能的片段)，例如CD47的胞外区。在该实施方案中，所述免疫抑制分子的跨膜区优选是CD47跨膜区。优选地，所述免疫抑制分子还可以包含部分CD47胞内区。
在一个实施方案中，所述SIRPα的配体与SEQ ID NO:69具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：69的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述SIRPα的配体如SEQ ID NO：69所示。
在一个实施方案中，本发明的免疫抑制分子包含的免疫抑制蛋白结合结构域是靶向IRP60的抗体、IRP60的配体或其功能性片段。IRP60也称为CD300a，是一种I型跨膜抑制性受体。IRP60在髓系细胞(例如树突状细胞、肥大细胞、粒细胞、单核细胞)以及淋巴细胞(T细胞、B细胞等)表面广泛表达，并且其表达水平基于细胞类型和分化程度不同而具有差异性。IRP60包含4个ITIM，其抑制信号的启动依赖于ITIM中的酪氨酸残基磷酸化。通过与配体氨基磷脂(例如PtdEth和/或PtdSer)相互作用，IRP60参与调控多种细胞活动，例如细胞生长、增殖、凋亡、分化、免疫应答等。据报道，IRP60与血液系统恶性肿瘤(例如白血病、淋巴瘤等)、感染性疾病、过敏反应、慢性炎症反应等疾病的发展密切相关。
本领域已知的抗IRP60抗体均可用于本发明，例如TX41，TX49等。在一个实施方案中，靶向IRP60的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO：66所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO：65所包含的CDR1-H、CDR2-H和CDR3-H相同。在一个实施方案中，轻链可变区包含的CDR1-L如SEQ ID NO：62所示，CDR2-L如SEQ ID NO：63所示，CDR3-L如SEQ ID NO：64所示；重链可变区包含的CDR1-H如SEQ ID NO：59所示，CDR2-H如SEQ ID NO：60所示，CDR3-H如SEQ ID NO：61所示。
在一个实施方案中，所述靶向IRP60的抗体包含轻链可变区和重链可变区，其中所述轻链可变区与SEQ ID NO:66具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：66的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰；其中所述重链可变区与SEQ ID NO:65具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：65的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体包含如SEQ ID NO：65所示的重链可变区和如SEQ ID NO：66所示的轻链可变区。
在一个实施方案中，所述靶向IRP60的抗体与SEQ ID NO:67具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：67的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述靶向IRP60的抗体如SEQ ID NO：67所示。
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守性修饰包括氨基酸的保守性取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守性氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪 氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
如本文所用，术语序列“同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员知晓，可以使用一些算法来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
如本文所用，术语“跨膜结构域”是指能够使免疫抑制分子在细胞表面上表达，并将免疫抑制蛋白结合结构域锚定在细胞膜上的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当靶标结合结构域与靶标结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD28、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD47、CD64、CD80、CD86、CD94、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、CD18、ICOS、4-1BB、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、BCMA、IL-2Rβ、IL-2Rγ、IL-7R a、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CDl ld、ITGAE、CD103、ITGAL、CDl la、LFA-1、ITGAM、CDl lb、ITGAX、CDl lc、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRT AM、Ly9、CD160、PSGL1、CDIOO、SLAMF6、SLAMF1、SLAMF8、CD162、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D或NKG2C。在一些实施方案中，跨膜结构域源自以下分子：CD8a、CD4、CD28、4-1BB、CD47、CD80、CD86、CD152和PD1。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD28，其与SEQ ID NO:15所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。优选地，所述跨膜结构域源自CD8a，其与SEQ ID NO:16或17所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，免疫抑制分子还包含位于免疫抑制蛋白结合结构域和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗体的任何寡肽或多肽。具体地，铰链区用来为抗体提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD8α、CD28、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选CD8α、CD28或IgG4铰链。在一个实施方案中，铰链区来自CD28，其与SEQ ID NO:27所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个实施方案中，铰链区来自CD8a，其与SEQ ID NO:28或29所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个实施方案中，铰链区来自IgG4，其与SEQ ID NO:30所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，“共刺激结构域”是指介导细胞内的信号转导以诱导如效应功能的免疫反应的蛋白质的至少一部分，其是来自共刺激分子的细胞内功能性信号传导结构域，包含所述共刺激分子的整个胞内区，或其功能片段。“共刺激分子”是指在与共刺激配体特异性结合，由此介导共刺激反应(例如增殖和生存)的同源结合配偶体。任何共刺激分子的共刺激信号传导结构域适用于本文所述的免疫抑制分子中。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本 发明的共刺激结构域的非限制性实例包括但不限于源自以下蛋白质的胞内区：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134、4-1BB、CD270、CD272、B7-H3、ICOS、CD357、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70以及它们的组合。优选地，本发明CAR的共刺激结构域是4-1BB、CD28或4-1BB+CD28。在一个实施方案中，共刺激结构域来自4-1BB，其与SEQ ID NO:19或20所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个实施方案中，共刺激结构域来自CD28，其与SEQ ID NO:18所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
本发明的免疫抑制分子不包含初级信号传导结构域。如本文所用，术语“初级信号传导结构域”是指在抗原-受体结合以后一同起作用以引发初级信号传导的蛋白结构，其一般是T细胞受体和共受体的胞内序列。初级信号传导结构域一般包含一个或多个基于免疫受体酪氨酸的激活基序(Immunoreceptor Tyrosine-based Activation Motifs,ITAM)。本发明的初级信号传导结构域的非限制性实例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、CD79a、CD79b、NFAM1、STAM1、STAM2和CD66d的那些。在一个实施方式中，本发明的免疫抑制分子不包含CD3ζ胞内区。例如，CD3ζ胞内区与SEQ ID NO:21、22或23所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的免疫抑制分子还包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自B2M、CD8α、IgG1、GM-CSFRα等的信号肽。在一个实施方案中，可用于本发明的信号肽来自B2M，其与SEQ ID NO：24所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个实施方案中，可用于本发明的信号肽来自CD8a，其与SEQ ID NO：25或26所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
核酸和载体
本发明还提供一种核酸分子，其包含编码本发明的免疫抑制分子的核酸序列。任选地，所述核酸分子还可以包含编码功能性外源受体(例如嵌合抗原受体、嵌合T细胞受体等)的核酸序列。
如本文所用，术语“核酸分子”包括核糖核苷酸和脱氧核糖核苷酸的序列，如经修饰的或未经修饰的RNA或DNA，各自为单链和/或双链形式的线性或环状，或它们的混合物(包括杂合分子)。因此，根据本发明的核酸包括DNA(比如dsDNA、ssDNA、cDNA)、RNA(比如dsRNA、ssRNA、mRNA、ivtRNA)，它们的组合或衍生物(比如PNA)。优选地，所述核酸是DNA或RNA。
核酸可以包含常规的磷酸二酯键或非常规的键(如酰胺键，比如在肽核酸(PNA)中发现的)。本发明的核酸还可含有一种或多种经修饰的碱基，比如，例如三苯甲基化的碱基和不常见的碱基(比如肌苷)。也可以想到其它修饰，包括化学、酶促或代谢修饰，只要本发明的多链CAR可以从多核苷酸表达即可。核酸可以以分离的形式提供。在一个实施方案中，核酸也可以包括调节序列，比如转录控制元件(包括启动子、增强子、操纵子、抑制子和转录终止信号)、核糖体结合位点、内含子等。
可以对本发明的核酸序列进行密码子优化以在所需的细胞(如，免疫细胞)中进行最佳表达；或者用于在细菌、酵母菌或昆虫细胞中表达。密码子优化是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。
本发明还提供一种载体，其包含本发明所述的核酸分子。任选地，编码免疫抑制分子的核酸 序列和编码功能性外源受体(例如嵌合抗原受体、嵌合T细胞受体等)的核酸序列可以位于相同载体或不同载体。
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一个实施方案中，本发明的载体包括但不限于质粒、病毒(例如逆转录病毒、腺病毒、腺伴随病毒、牛痘病毒、水泡性口炎病毒、麻疹病毒、腮腺炎病毒、脊髓灰质炎病毒、正粘液病毒、小病毒、马拉巴病毒、柯萨奇病毒、疱疹病毒和慢病毒等)、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体一般还包含在细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
工程化细胞
本发明还提供一种工程化细胞，其表达本发明的免疫抑制分子、编码所述免疫抑制分子的核酸，或包含所述核酸的载体。在一个实施方案中，本发明的工程化细胞还表达功能性外源受体(如下文所定义)。
在一个实施方案中，本发明的工程化细胞是工程化的免疫细胞。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是B细胞、T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。免疫细胞可以从多种来源获得，例如可以来自受试者(例如，从受试者的外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织、肿瘤等分离而获得)，或来自体外培养的细胞系(例如Jurkat、SupT1、NK92等)，或从干细胞分化而来(例如，源自脐带血干细胞、祖细胞、骨髓干细胞、造血干细胞、成体干细胞、胚胎干细胞、多能干细胞、iPSC等)。优选地，免疫细胞是T细胞或NK细胞，更优选T细胞。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+CD8+T细胞、CD4+T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。
在一个实施方案中，本发明的工程化细胞的至少一种内源性HLA-I类基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化细胞的至少一种内源性HLA-II类基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化细胞的至少一种内源性TCR/CD3基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化细胞的至少一种内源性TCR/CD3基因和至少一种内源性HLA-I类基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化细胞的至少一种内源性TCR/CD3基因，至少一种内源性HLA-I类基因和至少一种内源性HLA-II类基因的表达被抑制或沉默。优选地，所述HLA-I类基因选自HLA-A、HLA-B、HLA-C和B2M。优选地，所述HLA-II类基因选自HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK和CIITA，优选选自RFX5、RFXAP、RFXANK和CIITA。优选地，所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε和CD3ζ。
在一个实施方案中，本发明的工程化细胞的一个或多个选自以下内源性基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的，包括但不限于例如通过大范围 核酸酶、锌指核酸酶、TALEN、CRISPR/Cas系统、碱基编辑器介导DNA或RNA断裂，或通过反义寡核苷酸、RNAi、shRNA、转座子、突变等技术使基因失活。
在一个实施方案中，本发明的工程化细胞是同种异体的细胞。如本文所用，术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些情况下，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。
功能性外源受体
在一个实施方案中，本发明的表达免疫抑制分子的工程化细胞还可以表达功能性外源受体。优选地，所述功能性外源受体选自重组T细胞受体、嵌合抗原受体、T细胞融合蛋白或T细胞抗原耦合器，更优选嵌合抗原受体。
如本文所用，术语“T细胞融合蛋白”或“TFP”是指由TCR各组分衍生的重组多肽，通常由TCR亚基和与其连接的抗体组成并在细胞表面表达。其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内信号结构域。
如本文所用，术语“T细胞抗原耦合器”或“TAC”包括三个功能结构域：1肿瘤靶向结构域，包括单链抗体(例如本发明的抗FasL抗体、抗IRP60抗体或抗SIRPα抗体)、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAM)磷酸化作为T细胞活化的初始步骤。
如本文所用，术语“T细胞受体”或“TCR”是T细胞表面的特征性标志，以非共价键与CD3结合形成复合物。抗原呈递细胞通过主要组织相容性复合体分子(MHC)将抗原肽呈递至T细胞并且结合至TCR复合物以诱发一系列胞内信号传导。TCR由分别形成异二聚体的六条肽链组成，其一般分为αβ型和γδ型。每条肽链包括恒定区和可变区，其中可变区负责结合特异性的抗原和MHC分子。术语“重组TCR受体”是指人工构建的进一步包含抗原(例如肿瘤抗原)结合结构域的T细胞受体。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括抗原(例如肿瘤抗原)结合结构域(例如抗体或抗原的配体)、跨膜结构域、任选的共刺激结构域和初级信号传导结构域，各个结构域之间通过接头连接。CAR能够以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。在一个实施方案中，本发明的功能性外源受体是嵌合抗原受体，其包含肿瘤抗原结合结构域、跨膜结构域、一个或多个共刺激结构域和初级信号传导结构域。在一个实施方案中，所述嵌合抗原受体还包含以下结构中的一个或多个：信号肽、铰链区、自杀基因、开关结构等。
在一个实施方案中，功能性外源受体包含特异性识别抗原(例如肿瘤抗原)的胞外域。在一个实施方案中，所述胞外域包含特异性结合抗原的抗体或所述抗原的配体。在一个实施方案中，所述抗原选自：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、CD 138、CD151、CD171、CD179a、CD300LF、CLEC12A、CDH16、CSPG4、CS1、CLL-1、Claudin 6、Claudin18.1、Claudin 18.2、CEA、CEACAM6、c-Met、CAIX、CXORF61、CA125、CYP1B1、CS1、ELF2M、EGFR、EPCAM、EGFRvIII、EphA2、ERG/TMPRSS2ETS融合基因、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受体、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配体、OY-TES1、OR51E2、OX40、PRSS21、PSCA、PD1、PD-L1、PD-L2、PSMA、Prostase、PAP、PDGFR-β、PCTA-1/半乳凝集素8、p53、p53突变体、prostein、PLAC1、PANX3、PAX3、PAX5、PTCH1、RANK、RAGE-1、ROR1、Ras突变体、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整合素、CA9、叶酸受体α、肝配蛋白B2、酪氨酸酶、岩藻糖基GM1、邻-乙酰-GD2、叶酸受体β、多聚唾液酸、精子蛋白17、存活蛋白和端粒酶、肉瘤易位断点、人端粒末端逆转录酶/hTERT、雄激素受体、肠羧基酯酶、细胞周期蛋白B1、纤连蛋白、腱生蛋白、肿瘤坏死区的癌胚变体或其任意组合。优选地，所述抗原选自CD7、CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、MSLN、AFP、叶酸受体α、CEA、PSCA、PSMA、Her2、EGFR、IL-13Ra、GD2、NKG2D、Claudin18.2、ROR1、EGFRvIII、CS1、BCMA和GPRC5D，更优选选自CD19、Claudin18.2、MSLN、GPRC5D、CD7和BCMA。
在一个实施方案中，功能性外源受体包含特异性识别CD19的胞外域，例如靶向CD19的抗体。在一个实施方案中，所述靶向CD19的抗体包含轻链可变区和重链可变区，其中轻链可变区包含的CDR1-L如SEQ ID NO：35所示，CDR2-L如SEQ ID NO：36所示，CDR3-L如SEQ ID NO：37所示；重链可变区包含的CDR1-H如SEQ ID NO：32所示，CDR2-H如SEQ ID NO：33所示，CDR3-H如SEQ ID NO：34所示。
在一个实施方案中，所述靶向CD19的抗体包含轻链可变区和重链可变区，其中所述轻链可变区与SEQ ID NO:39、41或43具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：39、41或43的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰；其中所述重链可变区与SEQ ID NO:38、40或42具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：38、40或42的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体包含如SEQ ID NO：38、40或42所示的重链可变区和如SEQ ID NO：39、41或43所示的轻链可变区。
在一个实施方案中，所述靶向CD19的抗体与SEQ ID NO:44、45、46、47或48具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与SEQ ID NO：44、45、46、47或48的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰。优选地，所述靶向FasL的抗体如SEQ ID NO：44、45、46、47或48所示。
在一个实施方案中，本发明的功能性外源受体是嵌合抗原受体，其包含抗原结合结构域、跨膜结构域、一个或多个共刺激结构域和初级信号传导结构域。在一个实施方案中，本发明的功能性外源受体是嵌合抗原受体，所述嵌合抗原受体靶向CD19、CD7、CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、MSLN、AFP、叶酸受体α、CEA、PSCA、PSMA、Her2、EGFR、IL-13Ra、GD2、NKG2D、Claudin18.2、ROR1、EGFRvIII、CS1、BCMA或GPRC5D，更优选靶向CD19、Claudin18.2、MSLN、ROR1、GPRC5D、CD7和BCMA。在一个实施方案中，所述嵌合抗原受体还包含信号肽、铰链区，或两者。可用于本发明的CAR的跨膜结构域、共刺激结构域、初级信号传导结构域以及任选的铰链区、信号肽等结构的定义参见上文“免疫抑制分子”章节。
在一个实施方案中，本发明的CAR还可以包含开关结构，以调控CAR的表达时间。例如，开关结构可以是二聚化结构域的形式，通过与其相应配体的结合引起构象变化，暴露胞外结合结 构域，使其与被靶向抗原结合，从而激活信号传导通路。或者，也可以使用开关结构域分别连接结合结构域和信号传导结构域，仅当开关结构域互相结合(例如在诱导化合物的存在下)时，结合结构域和信号传导结构域才能通过二聚体连接在一起，从而激活信号通路。开关结构还可以是掩蔽肽的形式。掩蔽肽可以遮蔽胞外结合结构域，阻止其与被靶向抗原的结合，当通过例如蛋白酶切割掩蔽肽后，就可以暴露胞外结合结构域，使其成为一个“普通”的CAR结构。本领域技术人员知晓的各种开关结构均可用于本发明。
在一个实施方案中，本发明的CAR还可以包含自杀基因，即，使其表达一个可通过外源物质诱导的细胞死亡信号，以在需要时(例如产生严重的毒副作用时)清除CAR细胞。例如，自杀基因可以是插入的表位的形式，例如CD20表位、RQR8等，当需要时，可以通过加入靶向这些表位的抗体或试剂来消除CAR细胞。自杀基因也可以是单纯疱疹病毒胸苷激酶(HSV-TK)，该基因可使细胞在接受更昔洛韦治疗诱导下死亡。自杀基因还可以是iCaspase-9，可以通过化学诱导药物如AP1903、AP20187等诱导iCaspase-9发生二聚化，从而激活下游的Caspase3分子，导致细胞凋亡。本领域技术人员知晓的各种自杀基因均可用于本发明。
药物组合物
本发明还提供一种药物组合物，其包含本发明所述的免疫抑制分子、工程化细胞、核酸分子或载体作为活性剂，和一种多种药学上可接受的赋型剂。因此，本发明还涵盖所述免疫抑制分子、核酸分子、载体或工程化细胞在制备药物组合物或药物中的用途。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂(生物制剂如抗体试剂，和/或小分子)或治疗方法(例如手术、化疗或放疗)组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本 发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
在一个实施方案中，本发明的药物组合物用于治疗患有癌症、感染或自身免疫性疾病的受试者。
在一个实施方案中，所述癌症是与功能性外源受体结合的靶标表达有关的癌症。例如，所述癌症包括但不限于：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌(包括胃肠癌)、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌(例如小细胞肺癌、非小细胞肺癌、腺状肺癌和鳞状肺癌)、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤)、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中间级/滤泡性NHL、中间级扩散性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小型非裂化细胞性NHL、大肿块病NHL)、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)；以及其他与靶标表达有关的疾病。优选地，可以用本发明的工程化免疫细胞或药物组合物治疗的疾病选自：白血病、淋巴瘤、多发性骨髓瘤、脑神经胶质瘤、胰腺癌、胃癌等。
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。
抑制外源细胞被受试者的免疫细胞免疫排斥的方法
在一个方面，本发明还提供一种降低受试者细胞对外源细胞的免疫排斥的方法，其特征在于，在所述外源细胞中表达本发明的免疫抑制分子，编码所述免疫抑制分子的核酸分子或包含所述核酸分子的载体。
在另一个方面，本发明还提供一种降低受试者细胞对外源细胞的免疫排斥的方法，其特征在于，向所述受试者施用本发明所述的免疫抑制分子或表达所述免疫抑制分子的工程化细胞。在一个实施方案中，本发明所述的免疫抑制分子或工程化细胞与所述外源细胞同时或顺次施用。例如，本发明所述的免疫抑制分子或工程化细胞可以在所述外源细胞之后施用。
如本文所用，术语“受试者”是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、猪、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1：示出了表达靶向FasL的免疫抑制分子的CAR-T细胞的CAR表达水平。
图2：示出了表达靶向FasL的免疫抑制分子的CAR-T细胞对靶细胞的杀伤效果。
图3：示出了表达靶向FasL的免疫抑制分子的CAR-T细胞与靶细胞Nalm6共孵育后的细胞因子释放水平。
图4：示出了表达靶向FasL的免疫抑制分子的CAR-T细胞与靶细胞Raji共孵育后的细胞因子释放水平。
图5：示出了靶向FasL的免疫抑制分子对同种异体PBMC增殖的抑制效果。
图6：示出了表达靶向FasL的免疫抑制分子的CAR-T细胞在体内的抑瘤效果。
图7：示出了表达靶向SIRPα的免疫抑制分子的CAR-T细胞的CAR表达水平。
图8：示出了表达靶向SIRPα的免疫抑制分子的CAR-T细胞中抗SIRPα抗体的表达水平。
图9：示出了表达靶向SIRPα的免疫抑制分子的CAR-T细胞与靶细胞Nalm6共孵育后的细胞因子释放水平。
图10：示出了表达靶向SIRPα的免疫抑制分子的CAR-T细胞与靶细胞Raji共孵育后的细胞因子释放水平。
图11：示出了靶向SIRPα的免疫抑制分子对NK细胞杀伤作用的抑制效果。
图12：示出了表达靶向IRP60的免疫抑制分子的CAR-T细胞的CAR表达水平。
图13：示出了表达靶向IRP60的免疫抑制分子的CAR-T细胞中抗IRP60抗体的表达水平。
图14：示出了表达靶向IRP60的免疫抑制分子CAR-T细胞与靶细胞Nalm6共孵育后的细胞因子释放水平。
图15：示出了表达靶向IRP60的免疫抑制分子的CAR-T细胞与靶细胞Raji共孵育后的细胞因子释放水平。
图16：示出了靶向IRP60的免疫抑制分子对NK细胞杀伤作用的抑制效果。
图17：示出了表达包含CD47的免疫抑制分子的CAR-T细胞的CAR表达水平。
图18：示出了表达包含CD47的免疫抑制分子的CAR-T细胞中CD47的表达水平。
图19：示出了表达包含CD47的免疫抑制分子的CAR-T细胞对靶细胞Raji的杀伤效果。
图20：示出了表达包含CD47的免疫抑制分子的CAR-T细胞与靶细胞Raji共孵育后的细胞因子释放水平。
图21：示出了包含CD47的免疫抑制分子对NK细胞杀伤作用的抑制效果。
具体实施方式
实施例1.构建表达免疫抑制分子的CAR-T免疫细胞
合成以下的编码序列，并将其依次克隆至pGEM-T Easy载体(Promega)：抗CD19 scFv(SEQ ID NO：44)、CD8α铰链区(SEQ ID NO：29)、CD8α跨膜区(SEQ ID NO：17)、4-1BB共刺激结构域(SEQ ID NO：20)、CD3ζ胞内区(SEQ ID NO：23)，获得CAR19质粒，并通过测序确认目标序列在质粒中的正确插入。
在上述CAR19质粒中进一步表达通过T2A(SEQ ID NO：31)连接的免疫抑制分子，其结构如下：B2m信号肽(SEQ ID NO：24)、抗FasL scFv-1(SEQ ID NO：9)或抗FasL scFv-2(SEQ ID NO：14)、IgG4铰链区(SEQ ID NO：30)、CD28跨膜区(SEQ ID NO：15)、CD28共刺激结构域(SEQ ID NO：18)，获得CAR19-aFL-1质粒和CAR19-aFL-2质粒，并通过测序确认目标序列在质粒中的正确插入。
在上述CAR19质粒中进一步表达通过T2A(SEQ ID NO：31)连接的以下结构：B2m信号肽(SEQ ID NO：24)、Fas胞外区和跨膜区(SEQ ID NO：49)、IgG4铰链区(SEQ ID NO：30)、CD28跨膜区(SEQ ID NO：15)、CD28共刺激结构域(SEQ ID NO：18)，获得CAR19-Fas质粒，并通过测序确认目标序列在质粒中的正确插入。
在无菌管中加入3ml Opti-MEM(Gibco)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene)和包膜载体pMD2.G(Addgene)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28 CTSTM(Gibco)激活T细胞，并在37℃和5％CO2下培养1天。第 二天，加入浓缩的慢病毒，持续培养3天后，获得表达CD19 CAR的T细胞(CAR19-T)和表达CD19 CAR+靶向FasL的免疫抑制分子组合的T细胞(CAR19-Fas T、CAR19-aFL-1 T和CAR19-aFL-2 T)。
此外，使用流式细胞仪，用FITC goat anti-human Fab(Thermo Fisher)检测CAR-T细胞中的anti-CD19 scFv表达。可以看出，本实施例制备的CAR-T细胞中的CAR均能够有效表达(图1)。
实施例2.CAR-T细胞对靶细胞的杀伤效果和细胞因子释放水平
以1×104个细胞/孔的浓度将靶细胞Raji细胞铺入96孔板中，然后以4:1、2:1、1：1或0.5:1的效靶比将NT细胞、CAR19 T细胞、CAR19-Fas T细胞、CAR19-aFL-1 T细胞和CAR19-aFL-2 T细胞铺入到96孔板进行共培养，18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图2所示。
可以看出，CAR19-Fas T细胞、CAR19-aFL-1 T细胞和CAR19-aFL-2 T细胞对靶细胞呈现有效的杀伤，且在低效靶比下(例如0.5:1)，其杀伤效果略优于不表达免疫抑制分子的CAR19 T细胞。
另外，以1×105个细胞/孔的浓度将靶细胞(Nalm6细胞和Raji细胞)铺于96孔板中，按1:1的比例分别加NT细胞、CAR19 T细胞、CAR19-Fas T细胞、CAR19-aFL-1 T细胞和CAR19-aFL-2 T细胞，共培养18-24小时后收集细胞共培养上清液。
分别使用Human IL-2 DuoSet ELISA Kit(R&D systems)、Human IFN-gamma DuoSet ELISA Kit(R&D systems)检测共培养上清液中IL2和IFN-γ的含量，结果如图3和图4所示。可以看出，与NT细胞相比，各种CAR-T细胞与靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，表明这种细胞因子释放是特异性的。此外，各种CAR-T细胞的细胞因子释放水平基本相当。
这些数据表明，额外表达靶向FasL的免疫抑制分子不会改变CAR-T细胞的细胞因子释放水平，并且在低效靶比下会略微提高CAR-T细胞的体外杀伤活性。
实施例3.体外HvGD
通过单向混合淋巴细胞反应(MLR)测定评估本发明的免疫抑制分子在体外对HvGD的抑制作用。具体地，在37℃和5％CO2下，将NT细胞、CAR19 T细胞、CAR19-Fas T细胞、CAR19-aFL-1 T细胞和CAR19-aFL-2 T细胞与同种异体的PBMC共孵育7天，然后通过流式细胞术检测PBMC的增殖情况，结果如图5所示。
可以看出，与NT细胞相比，CAR19 T细胞将PBMC的计数提高了约3.5倍左右(0.4x106vs1.4x106)，这表明异源的CAR-T细胞极大促进了PBMC的增殖。此外，还注意到，额外表达免疫抑制分子可以显著降低PBMC的这种增殖。尤其值得注意的是，包含Fas胞外区的免疫抑制分子(CAR19-Fas T)将PBMC增殖降低了约35％(约0.9x106)，而包含抗FasL scFv的免疫抑制分子(CAR19-aFL-1 T和CAR19-aFL-2 T)则进一步在CAR19-Fas T细胞的基础上将PBMC增殖降低了约3倍左右(约0.3x106)，这与不表达CAR的NT细胞的效果基本相当。
综上，额外表达靶向FasL的免疫抑制分子能够显著抑制同种异体PBMC的体外增殖，进而显著降低HvGD。并且，包含抗FasL scFv的免疫抑制分子的这种抑制效果显著优于包含Fas胞外区的免疫抑制分子。
实施例4.CAR T细胞对肿瘤的体内抑制效果
将25只6-8周龄的健康雌性NPI小鼠分成5组，每组5只小鼠：NT组、CAR19组、CAR19-Fas组、CAR19-aFL-1组和CAR19-aFL-2组。在第0天向每只小鼠尾静脉注射5×105个Raji细胞。6天后，根据分组情况向每只小鼠尾静脉注射2x106个NT细胞或相应的CAR-T细胞。每周评估小鼠状态。小鼠存活曲线如图6所示。
可以看出，CAR19-Fas组的存活率与CAR19组相当，表明包含Fas胞外区的免疫抑制分子可能在体内没有发挥很好的免疫排斥抑制效果。相反，CAR19-aFL-1组和CAR19-aFL-2组的存活率显著高于CAR19-Fas组，表明包含抗FasL scFv的免疫抑制分子在体内也能非常有效的抑制免疫排斥，进而增强CAR-T细胞的肿瘤抑制效果，提高存活率。
实施例5.构建表达靶向SIRPα的免疫抑制分子的CAR-T细胞并验证其功能
5.1构建CAR-T细胞
根据实施例1的方法构建表达靶向SIRPα的免疫抑制分子的CAR-T细胞，其中在慢病毒转染步骤前，用CRISPR/Cas9系统敲除T细胞中的B2M基因。靶向SIRPα的免疫抑制分子包含：B2m信号肽(SEQ ID NO：24)、抗SIRPα scFv(SEQ ID NO：58)、IgG4铰链区(SEQ ID NO：30)、CD28跨膜区(SEQ ID NO：15)、CD28共刺激结构域(SEQ ID NO：18)。将获得的CAR-T细胞命名为CAR19-αT细胞。
使用流式细胞仪，用FITC goat anti-human Fab(Thermo Fisher)检测CAR-T细胞中的anti-CD19 scFv表达，用Human SIRPα protein(Acro)检测anti-SIRPα scFv表达。可以看出，本实施例制备的CAR-T细胞中的CAR均能够有效表达(图7)，且CAR19-α T细胞中的免疫抑制分子也能有效表达(图8)。
5.2检测与靶细胞共孵育后的细胞因子释放水平
以1×105个细胞/孔的浓度将靶细胞(Nalm6细胞和Raji细胞)铺于96孔板中，按1:1的比例分别加入CAR19-T、CAR19-αT细胞和NT细胞(阴性对照)，共培养18-24小时后收集细胞共培养上清液。
分别使用Human IL-2 DuoSet ELISA Kit(R&D systems)、Human IFN-gamma DuoSet ELISA Kit(R&D systems)检测共培养上清液中IL2和IFN-γ的含量，结果如图9和图10所示。可以看出，与NT细胞相比，CAR19-T和CAR19-αT与各靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，表明这种细胞因子释放是特异性的。此外，CAR19-T和CAR19-αT细胞的细胞因子释放水平相当，表明额外表达的免疫抑制分子没有损害CAR-T细胞的细胞因子释放特性。
5.3检测对NK细胞杀伤作用的抑制效果
用Far-Red(invitrogen)标记本实施例制备的CAR-T细胞。然后按照1x104个细胞/孔的浓度将标记好的CAR-T细胞铺入96孔板，并以2:1的效靶比(NK细胞：CAR-T细胞)加入外周血来源的同种异体NK细胞(pNK-1和pNK-2)进行共培养。16-18小时后，用流式细胞仪检测培养物中T细胞的存活比例，结果如图11所示。
可以看出，与常规CAR19-T细胞相比，表达免疫抑制分子的CAR19-αT细胞与NK细胞共培养后的存活率显著提高。这表明本发明的免疫抑制分子可以有效抑制NK细胞对表达其的CAR-T细胞的杀伤作用。这对于降低免疫排斥反应尤其有效。
实施例6.构建表达靶向IRP60的免疫抑制分子的CAR-T细胞并验证其功能
6.1构建CAR-T细胞
根据实施例1的方法构建表达靶向IRP60的免疫抑制分子的CAR-T细胞，其中在慢病毒转染步骤前，用CRISPR/Cas9系统敲除T细胞中的B2M基因。靶向IRP60的免疫抑制分子包含：B2m信号肽(SEQ ID NO：24)、抗IRP60 scFv(SEQ ID NO：67)、IgG4铰链区(SEQ ID NO：30)、CD28跨膜区(SEQ ID NO：15)、CD28共刺激结构域(SEQ ID NO：18)。将获得的CAR-T细胞命名为CAR19-60T细胞。
使用流式细胞仪，用FITC goat anti-human Fab(Thermo Fisher)检测CAR-T细胞中的anti-CD19 scFv表达，用Human IRP60 protein(Kactus)检测anti-IRP60 scFv表达。可以看出，本实施例制备的CAR-T细胞中的CAR均能够有效表达(图12)，且CAR19-60 T细胞中的免疫抑制分子也能有效表达(图13)。
6.2检测与靶细胞共孵育后的细胞因子释放水平
根据实施例5.2所述的方法检测本实施例制备的CAR-T细胞根本与靶细胞Nalm6细胞和Raji细胞共孵育后的细胞因子IL2和IFN-γ的释放水平，结果如图14和图15所示。可以看出，与NT细胞相比，CAR19-T和CAR19-60 T与各靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，表明这种细胞因子释放是特异性的。此外，CAR19-T和CAR19-60 T细胞的细胞因子释放水平相当，表明额外表达的免疫抑制分子没有损害CAR-T细胞的细胞因子释放。
6.3检测对NK细胞杀伤作用的抑制效果
根据实施例5.3所述的方法检测本实施例制备的CAR-T细胞对同种异体的NK细胞杀伤作用 的抑制效果，结果如图16所示。
可以看出，与常规CAR19-T细胞相比，表达免疫抑制分子的CAR19-60T细胞与NK细胞共培养后的存活率显著提高。这表明本发明的免疫抑制分子可以有效抑制NK细胞对表达其的CAR-T细胞的杀伤作用。
实施例7.构建表达包含CD47的免疫抑制分子的CAR-T免疫细胞并验证其功能
7.1构建CAR-T细胞
合成以下的编码序列，并将其依次克隆至pGEM-T Easy载体(Promega，货号A1360)：抗CD19 scFv(SEQ ID NO：44)、CD8α铰链区(SEQ ID NO：29)、CD8α跨膜区(SEQ ID NO：17)、4-1BB共刺激结构域(SEQ ID NO：20)、CD3ζ胞内区(SEQ ID NO：23)，获得CAR19质粒，并通过测序确认目标序列在质粒中的正确插入。
在上述CAR19质粒中进一步表达通过T2A(SEQ ID NO：31)连接的免疫抑制分子，其结构如下：CD47胞外跨膜区(SEQ ID NO：68)、CD28共刺激结构域(SEQ ID NO：18)，获得CAR19-47质粒，并通过测序确认目标序列在质粒中的正确插入。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28 CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2下培养1天。然后，加入浓缩的慢病毒，持续培养3天后，获得表达CD19CAR的T细胞(CAR19-T)和表达CD19CAR和靶向SIRPα的免疫抑制分子的T细胞(CAR19-47-T)。
此外，使用流式细胞仪，用FITC goat anti-human Fab(Thermo Fisher,货号31628)(检测CAR-T细胞中的anti-CD19 scFv表达，用mouse anti-human CD47 Ab(Biolegend,货号323108)检测CD47表达。可以看出，本发明制备的CAR-T细胞中的CAR均能够有效表达(图17)，且CAR19-CD47-T细胞中的免疫抑制分子也能有效表达(图18)。
7.2检测CAR-T细胞对靶细胞的杀伤效果
以1×104个细胞/孔的浓度将靶细胞Raji细胞铺入96孔板中，然后以4:1、2:1、1：1或0.5:1的效靶比将NT细胞、CAR19-T细胞和CAR19-47-T细胞铺入到96孔板进行共培养，18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图19所示。
可以看出，CAR19-T细胞和CAR19-47-T细胞对靶细胞均呈现有效的特异性杀伤，并且与CAR19-T相比，CAR19-47-T细胞的杀伤活性显著提高。
7.3.检测CAR-T细胞与靶细胞共孵育后的细胞因子释放水平
以1×105个细胞/孔的浓度将靶细胞(Raji细胞)铺于96孔板中，按1:1的比例分别加入CAR19-T、CAR19-CD47-T细胞和NT细胞(阴性对照)，共培养18-24小时后收集细胞共培养上清液。
分别使用Human IL-2 DuoSet ELISA Kit(R&D systems,货号DY202)、Human IFN-gamma DuoSet ELISA Kit(R&D systems,货号DY285)检测共培养上清液中IL2和IFN-γ的含量，结果如图20所示。
可以看出，与NT细胞相比，CAR19-T和CAR19-47-T与靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，表明这种细胞因子释放是特异性的。此外，CAR19-CD47-T IFN-γ的分泌能力显著高于CAR19-T细胞。
7.4.检测免疫抑制分子对NK细胞杀伤作用的抑制效果
用Far-Red(invitrogen，货号C34564)标记本发明制备的CAR-T细胞。然后按照1x104个细胞/孔的浓度将标记好的CAR-T细胞铺入96孔板，并以2:1的效靶比加入外周血来源的NK细胞进行共培养。16-18小时后，用流式细胞仪检测培养物中T细胞的存活比例，结果如图21所示。
可以看出，与常规CAR19-T细胞相比，表达免疫抑制分子的CAR19-47细胞与NK细胞共培养后的存活率显著提高。这表明本发明的免疫抑制分子可以有效抑制NK细胞对CAR-T细胞的杀伤作用。
综上，本发明的靶向FasL、IRP60或SIRPα的免疫抑制分子一方面不会对CAR-T细胞的杀伤活性或细胞因子释放特性产生不利影响，另一方面能够显著抑制同种异体NK细胞的杀伤或抑制同种异体PBMC在被激活后的过度增殖，从而有效降低宿主体内免疫细胞对外源细胞的免疫排斥。
要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
工业实用性
本发明涉及一种免疫抑制分子，其包含免疫抑制蛋白结合结构域、跨膜结构域和共刺激结构域并且不包含初级信号传导结构域，其中所述免疫抑制蛋白结合结构域选自靶向FasL的抗体、靶向IRP60的抗体、IRP60的配体或其功能性片段、靶向SIRPα的抗体、SIRPa的配体或其功能性片段，其中所述免疫抑制分子能够降低受试者细胞的免疫排斥。本发明的免疫抑制分子有效降低宿主体内免疫细胞对外源细胞的免疫排斥，具备优异的工业实用性。

Claims (38)

  1. 一种免疫抑制分子，其包含免疫抑制蛋白结合结构域、跨膜结构域和共刺激结构域并且不包含初级信号传导结构域，其中所述免疫抑制蛋白结合结构域选自靶向FasL的抗体、靶向IRP60的抗体、IRP60的配体或其功能性片段、靶向SIRPα的抗体、SIRPa的配体或其功能性片段，其中所述免疫抑制分子能够降低受试者细胞的免疫排斥。
  2. 权利要求2所述的免疫抑制分子，其中所述抗体选自完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv、线性抗体、sdAb或纳米抗体。
  3. 权利要求1-2任一项所述的免疫抑制分子，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD28、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD47、CD64、CD80、CD86、CD94、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、CD18、ICOS、4-1BB、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、BCMA、IL-2Rβ、IL-2Rγ、IL-7R a、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CDl ld、ITGAE、CD103、ITGAL、CDl la、LFA-1、ITGAM、CDl lb、ITGAX、CDl lc、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRT AM、Ly9、CD160、PSGL1、CDIOO、SLAMF6、SLAMF1、SLAMF8、CD162、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D或NKG2C。
  4. 权利要求1-3任一项所述的免疫抑制分子，其中所述共刺激结构域选自以下蛋白质的胞内区：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134、4-1BB、CD270、CD272、B7-H3、ICOS、CD357、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70以及它们的组合。
  5. 权利要求1-4任一项所述的免疫抑制分子，其中所述初级信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、CD79a、CD79b、NFAM1、STAM1、STAM2和CD66d。
  6. 权利要求1-5任一项所述的免疫抑制分子，其还包含信号肽。
  7. 权利要求1-6任一项所述的免疫抑制分子，其还包含源自以下的铰链区：CD8α、CD28、FcγRIIIα受体、IgG4或IgG1。
  8. 权利要求1-7任一项所述的免疫抑制分子，其中所述免疫抑制蛋白结合结构域是靶向FasL的抗体。
  9. 权利要求8所述的免疫抑制分子，其中所述靶向FasL的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO：8所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO：7或13所包含的CDR1-H、CDR2-H和CDR3-H相同；
    优选地，所述CDR1-L如SEQ ID NO：4所示，CDR2-L如SEQ ID NO：5所示，CDR3-L如SEQ ID NO：6所示，CDR1-H如SEQ ID NO：1或10所示，CDR2-H如SEQ ID NO：2或11所示，CDR3-H如SEQ ID NO：3或12所示；
    优选地，其中所述轻链可变区如SEQ ID NO:8所示，其中所述重链可变区如SEQ ID NO:7或13所示；
    优选地，其中所述靶向FasL的抗体如SEQ ID NO:9或14所示。
  10. 权利要求1所述的免疫抑制分子，其中所述免疫抑制蛋白结合结构域是靶向SIRPα的抗体、SIRPα的配体或其功能性片段。
  11. 权利要求10所述的免疫抑制分子，其中所述靶向SIRPα的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO：57所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO：56所包含的CDR1-H、CDR2-H和CDR3-H相同；
    优选地，所述CDR1-L如SEQ ID NO：53所示，CDR2-L如SEQ ID NO：54所示，CDR3-L如SEQ ID NO：55所示，CDR1-H如SEQ ID NO：50所示，CDR2-H如SEQ ID NO：51所示，CDR3-H如SEQ ID NO：52所示；
    优选地，其中所述轻链可变区如SEQ ID NO:57所示，其中所述重链可变区如SEQ ID NO:56所示；
    优选地，其中所述靶向SIRPα的抗体如SEQ ID NO:58所示。
  12. 权利要求10所述的免疫抑制分子，其中所述SIRPα的配体是CD47或其胞外区；
    优选地，其中所述SIRPα的配体如SEQ ID NO:69所示。
  13. 权利要求1所述的免疫抑制分子，其中所述免疫抑制蛋白结合结构域是靶向IRP60的抗体、IRP60的配体或其功能性片段。
  14. 权利要求13所述的免疫抑制分子，其中所述靶向IRP60的抗体包含轻链可变区和重链可变区，其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO：66所包含的CDR1-L、CDR2-L和CDR3-L相同；其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO：65所包含的CDR1-H、CDR2-H和CDR3-H相同，
    优选地，所述CDR1-L如SEQ ID NO：64所示，CDR2-L如SEQ ID NO：63所示，CDR3-L如SEQ ID NO：62所示，CDR1-H如SEQ ID NO：59所示，CDR2-H如SEQ ID NO：60所示，CDR3-H如SEQ ID NO：61所示；
    选地，其中所述轻链可变区如SEQ ID NO:66所示，其中所述重链可变区如SEQ ID NO:65所示；
    优选地，其中所述靶向IRP60的抗体如SEQ ID NO:67所示。
  15. 一种核酸分子，其编码权利要求1-14任一项所述的免疫抑制分子。
  16. 一种载体，其包含权利要求15所述的核酸分子。
  17. 权利要求15所述的载体，其中所述载体选自逆转录病毒、腺病毒、腺伴随病毒、牛痘病毒、水泡性口炎病毒、麻疹病毒、腮腺炎病毒、脊髓灰质炎病毒、正粘液病毒、小病毒、马拉巴病毒、柯萨奇病毒、疱疹病毒和慢病毒。
  18. 一种工程化细胞，其表达权利要求1-14任一项所述的免疫抑制分子，权利要求15所述的核酸分子，或权利要求16-17任一项所述的载体。
  19. 权利要求18所述的工程化细胞，其特征还在于，所述工程化细胞还表达功能性外源受体。
  20. 权利要求19所述的工程化细胞，其中所述功能性外源受体选自嵌合抗原受体、嵌合T细胞受体、T细胞抗原耦合器和T细胞融合蛋白，优选嵌合抗原受体。
  21. 权利要求19所述的工程化细胞，其中所述功能性外源受体包含特异性识别抗原的胞外域。
  22. 权利要求21所述的工程化细胞，其中所述胞外域包含特异性识别所述抗原的抗体或所述抗原的配体。
  23. 权利要求22所述的工程化细胞，其中所述抗原选自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、CD 138、CD151、CD171、CD179a、CD300LF、CLEC12A、CDH16、CSPG4、CS1、CLL-1、Claudin 6、Claudin18.1、Claudin 18.2、CEA、CEACAM6、c-Met、CAIX、CXORF61、CA125、CYP1B1、CS1、ELF2M、EGFR、EPCAM、EGFRvIII、EphA2、ERG/TMPRSS2ETS融合基因、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受体、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配体、OY-TES1、OR51E2、OX40、PRSS21、PSCA、PD1、PD-L1、PD-L2、PSMA、Prostase、PAP、PDGFR-β、PCTA-1/半乳凝集素8、p53、p53突变体、prostein、PLAC1、PANX3、PAX3、PAX5、PTCH1、RANK、RAGE-1、ROR1、Ras突变体、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整合素、CA9、叶酸受体α、肝配蛋白B2、酪氨酸酶、岩藻糖基GM1、邻-乙酰-GD2、叶酸受体β、多聚唾液酸、精子蛋白17、存活蛋白和端粒酶、肉瘤易位断点、人端粒末端逆转录酶/hTERT、雄激素受体、肠羧基酯酶、细胞周期蛋白B1、纤连蛋白、腱生蛋白、肿瘤坏死区的癌胚变体或其任意组合。
  24. 权利要求23所述的工程化细胞，其中所述功能性外源受体是嵌合抗原受体，所述嵌合抗 原受体靶向CD19、CD7、CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、MSLN、AFP、叶酸受体α、CEA、PSCA、PSMA、Her2、EGFR、IL-13Ra、GD2、NKG2D、Claudin18.2、ROR1、EGFRvIII、CS1、BCMA或GPRC5D。
  25. 权利要求18-24任一项所述的工程化细胞，其中所述所述工程化细胞的至少一种内源性HLA-I类基因的表达被抑制或沉默。
  26. 权利要求25所述的工程化细胞，其中所述内源性HLA-I类基因选自HLA-A、HLA-B、HLA-C和B2M。
  27. 权利要求18-26任一项所述的工程化细胞，其中所述所述工程化细胞的至少一种内源性HLA-II类基因的表达被抑制或沉默。
  28. 权利要求27所述的工程化细胞，其中所述内源性HLA-II类基因选自HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK和CIITA。
  29. 权利要求18-28任一项所述的工程化细胞，其中所述工程化细胞的至少一种内源性TCR/CD3基因的表达被抑制或沉默。
  30. 权利要求29所述的工程化细胞，其中所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合。
  31. 权利要求18-30任一项所述的工程化细胞，其特征还在于，其中选自以下的一个或多个内源性基因的表达被抑制或沉默：CD52、GR、dCK、PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
  32. 权利要求18-31任一项所述的工程化细胞，其中所述工程化细胞是B细胞、T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。
  33. 权利要32所述的工程化免疫细胞，其中所述工程化细胞是CD4+CD8+T细胞、CD4+T细胞、CD8+T细胞、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。
  34. 权利要求18-33任一项所述的工程化细胞，其中所述工程化细胞来源于脐带血干细胞、祖细胞、骨髓干细胞、造血干细胞、成体干细胞、胚胎干细胞、多能干细胞、iPSC。
  35. 一种降低受试者细胞对外源细胞的免疫排斥的方法，其特征在于，在所述外源细胞中表达权利要求1-14任一项所述的免疫抑制分子、权利要求15所述的核酸分子或权利要求16-17任一项所述的载体。
  36. 一种降低受试者细胞对外源细胞的免疫排斥的方法，其特征在于，向所述受试者施用1-14任一项所述的免疫抑制分子或权利要求18-34任一项所述的工程化细胞。
  37. 一种药物组合物，其包含权利要求1-14任一项所述的免疫抑制分子、权利要求15所述的核酸分子、权利要求16-17任一项所述的载体或权利要求18-34任一项所述的工程化免疫细胞，和一种多种药学上可接受的赋型剂。
  38. 权利要求37所述的药物组合物，其中所述药物组合物用于治疗癌症、感染或自身免疫性疾病。
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