WO2024011335A1 - 经修饰的免疫细胞 - Google Patents

经修饰的免疫细胞 Download PDF

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WO2024011335A1
WO2024011335A1 PCT/CN2022/104785 CN2022104785W WO2024011335A1 WO 2024011335 A1 WO2024011335 A1 WO 2024011335A1 CN 2022104785 W CN2022104785 W CN 2022104785W WO 2024011335 A1 WO2024011335 A1 WO 2024011335A1
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cells
constant region
immune cell
antigen
modified immune
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PCT/CN2022/104785
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English (en)
French (fr)
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林欣
郁翰扬
刘玥
赵学强
芮魏
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清华大学
华夏英泰（北京）生物技术有限公司
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Priority to PCT/CN2022/104785 priority Critical patent/WO2024011335A1/zh
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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
    • 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
    • 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
    • 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 relates to the field of biomedicine, and in particular to a modified immune cell.
  • the present invention discloses a modified immune cell that overexpresses nuclear factor- ⁇ B (NF- ⁇ B) family transcription factors and the use of the modified immune cell.
  • NF- ⁇ B nuclear factor- ⁇ B
  • the three most important transcription factor families are AP-1, NFAT and NF- ⁇ B, which are the three transcription factor families mainly expressed by T cells after TCR activation.
  • the NF- ⁇ B family consists of five members: P50 (precursor P105), P52 (precursor P100), P65, c-Rel and RelB. These five members all have an N-terminal Rel homology domain RHD, which is responsible for its binding to DNA and dimerization.
  • the classical NF- ⁇ B signal originates from the heterodimer of RelA and p50 and the heterodimer of c-Rel and p50; the non-classical NF- ⁇ B signal originates from the heterodimer of RelB and p52.
  • RelA, c-Rel and RelB there is a transcription activation region that positively regulates gene expression.
  • p50 and p52 proteins do not have transcription activation regions, so their homodimers will inhibit transcription.
  • NF- ⁇ B signaling is regulated by a variety of membrane receptors, including B lymphoid receptors.
  • B lymphoid receptors In T cells, NF- ⁇ B transcription factors mainly receive signals from the T lymphoid receptor and tumor necrosis factor receptor TNFR family, among which non-classical NF- ⁇ B is mainly regulated by TNFR signals (Philipson B I, O'Connor R S, May M J, et al. 4-1BB costimulation promotes CAR T cell survival through noncanonical NF- ⁇ B signaling. Science signaling, 2020).
  • P52 is a transcription factor of the non-canonical NF- ⁇ B family, and its precursor protein is p100 encoded by the Nfkb2 gene.
  • p100 When non-canonical NF- ⁇ B signaling is not activated, p100 will form a strong dimer with RelB, another transcription factor of the NF- ⁇ B family, and cannot enter the nucleus; when non-canonical NF- ⁇ B is activated, p100 will be After phosphorylation, it is ubiquitinated and cleaved to p52, and combines with RelB to form a dimer and enters the nucleus, thereby regulating downstream gene expression (Shao-cong Sun.
  • NF- ⁇ B-inducing kinase forms a complex with tumor necrosis factor receptor-associated factor (TRAF) and cIAP and is ubiquitinated, making it unable to phosphorylate oxidizes downstream IKK ⁇ , leading to the formation of a complex between RelB and p100 to prevent nuclear entry.
  • TRAF3 After the tumor necrosis factor receptor binds to its ligand, TRAF3 will be recruited and ubiquitinated, causing NIK to be phosphorylated and phosphorylated downstream IKK ⁇ , ultimately causing p100 to be phosphorylated and cleaved into p52 to bind to RelB Incorporated into the core.
  • Chimeric antigen receptor T cell (CAR-T) therapy is an anti-cancer immunotherapy that has achieved good results in recent years. Unlike the way natural T cells recognize tumor cells, the recognition of tumor cells by CAR-T cells does not rely on MHC molecules.
  • the CAR molecule consists of three parts: the extracellular region is the antigen recognition domain derived from the antibody, responsible for recognizing the target antigen; the transmembrane region; and the intracellular region is the signaling molecule and co-stimulatory signaling molecule derived from the T cell receptor, responsible for Transmits T cell activation signals after receiving stimulation.
  • the T cell receptor (TCR) complex molecule contains multiple chains.
  • the TCR ⁇ chain and TCR ⁇ chain are responsible for recognizing MHC-peptide molecules.
  • the other six CD3 subunits combine with the TCR ⁇ / ⁇ chain to perform signal transduction.
  • the natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically conduct stronger signals than CAR. Previous studies have shown that although TCR signals are conducted more slowly than CAR signals, TCR signals are more persistent. Therefore, by utilizing the signaling function of natural TCR, a new type of receptor can be constructed to alleviate T cell incompetence and enable it to better exert its anti-solid tumor effect.
  • the extracellular region of the TCR is very similar to the Fab domain of the antibody, so the TCR variable region sequence can be replaced with the antibody variable region sequence to obtain the synthetic T cell receptor antigen receptor (Snythetic TCR and Antigen Receptor, STAR). It has both the specificity of antibodies and the superior signaling function of natural TCR, which can mediate complete T cell activation.
  • Embodiment 1 A modified immune cell modified to overexpress a nuclear factor- ⁇ B (NF- ⁇ B) family transcription factor.
  • NF- ⁇ B nuclear factor- ⁇ B
  • Embodiment 2 The modified immune cell of embodiment 1, wherein the immune cell is selected from T cells, B cells or NK cells, preferably T cells.
  • Embodiment 3 The modified immune cell of embodiment 1 or 2, wherein the NF- ⁇ B family transcription factor is selected from the group consisting of P50, P105, P52, P100, P65, c-Rel and RelB, or any combination thereof, preferably Preferably, the NF- ⁇ B family transcription factor is p52.
  • Embodiment 4 The modified immune cell of any one of embodiments 1-3, wherein the modified immune cell comprises an introduced nucleotide sequence encoding the NF- ⁇ B family transcription factor, thereby overexpressing The NF- ⁇ B family transcription factors.
  • Embodiment 5 The modified immune cell of embodiment 4, wherein a nucleotide sequence encoding the NF- ⁇ B family transcription factor is introduced into the modified immune cell via an expression vector, optionally encoding the NF-
  • the nucleotide sequence of the ⁇ B family transcription factor is operably linked to regulatory sequences in the expression vector.
  • Embodiment 6 The modified immune cell of any one of embodiments 1-5, wherein the modified immune cell further comprises a target-specific receptor.
  • Embodiment 7 The modified immune cell of Embodiment 6, wherein the modified immune cell comprises an introduced nucleotide sequence encoding the target-specific receptor, thereby expressing the target-specific receptor.
  • Embodiment 8 The modified immune cell of embodiment 7, wherein a nucleotide sequence encoding said target-specific receptor is introduced into said modified immune cell via an expression vector, optionally encoding said target-specific receptor
  • the nucleotide sequence of the receptor is operably linked to regulatory sequences in the expression vector.
  • Embodiment 9 The modified immune cell of any one of embodiments 6-8, wherein the target-specific receptor is selected from the group consisting of chimeric antigen receptors (CARs) and T cell receptors (TCRs).
  • CARs chimeric antigen receptors
  • TCRs T cell receptors
  • Embodiment 10 The modified immune cell of embodiment 9, wherein the target-specific receptor is a modified T-cell receptor (TCR), such as a Synthetic T-Cell Receptor and Antibody Receptor,STAR).
  • TCR modified T-cell receptor
  • Embodiment 11 The modified immune cell of embodiment 10, wherein the modified TCR, such as STAR, is derived from an ⁇ TCR, and wherein the ⁇ chain comprises a first constant region and the ⁇ chain comprises a second constant region.
  • the modified TCR such as STAR
  • Embodiment 12 The modified immune cell of embodiment 11, wherein the first constant region is a native TCR ⁇ chain constant region, eg, a native human TCR ⁇ chain constant region or a native mouse TCR ⁇ chain constant region.
  • the first constant region is a native TCR ⁇ chain constant region, eg, a native human TCR ⁇ chain constant region or a native mouse TCR ⁇ chain constant region.
  • Embodiment 13 The modified immune cell of embodiment 11, wherein the first constant region is a modified TCR ⁇ chain constant region.
  • Embodiment 14 The modified immune cell of embodiment 13, wherein the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region at position 48 relative to the wild-type mouse TCR ⁇ chain constant region. Amino acids such as threonine T are mutated to cysteine C.
  • Embodiment 15 The modified immune cell of embodiment 13 or 14, wherein the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which is at step 112 relative to a wild-type mouse TCR ⁇ chain constant region.
  • the amino acid at position 114 such as serine S, is changed to leucine L
  • the amino acid at position 114 such as methionine M
  • the amino acid at position 115 such as glycine G
  • Embodiment 16 The modified immune cell of any one of embodiments 13-15, wherein the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region relative to a wild-type mouse TCR ⁇ chain constant region.
  • the amino acid at position 48 such as threonine T is mutated to cysteine C
  • the amino acid at position 112 such as serine S is mutated into leucine L
  • the amino acid at position 114 such as methionine M is mutated
  • the amino acid at position 115 such as glycine G is changed to lycine V.
  • Embodiment 17 The modified immune cell of any one of embodiments 11-16, wherein the first constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 1, 3, 5, 7 and 28.
  • Embodiment 18 The modified immune cell of any one of embodiments 11-17, wherein the second constant region is a native TCR beta chain constant region, e.g., a native human TCR beta chain constant region or a native mouse TCR beta chain constant region .
  • the second constant region is a native TCR beta chain constant region, e.g., a native human TCR beta chain constant region or a native mouse TCR beta chain constant region .
  • Embodiment 19 The modified immune cell of any one of embodiments 11-17, wherein the second constant region is a modified TCR beta chain constant region.
  • Embodiment 20 The modified immune cell of embodiment 19, wherein the modified TCR beta chain constant region is derived from a mouse TCR beta chain constant region at position 56 relative to the wild-type mouse TCR beta chain constant region. Amino acids such as serine S are mutated to cysteine C.
  • Embodiment 21 The modified immune cell of any one of embodiments 11-20, wherein the second constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 2, 4 and 6.
  • Embodiment 22 The modified immune cell of any one of embodiments 11-21, wherein the first constant region is derived from a mouse TCR ⁇ chain constant region that is at The amino acid at position 48, such as threonine T, is mutated into cysteine C, the amino acid at position 112, such as serine S, is mutated into leucine L, and the amino acid at position 114, such as methionine M, is mutated into iso.
  • the first constant region is derived from a mouse TCR ⁇ chain constant region that is at The amino acid at position 48, such as threonine T, is mutated into cysteine C, the amino acid at position 112, such as serine S, is mutated into leucine L, and the amino acid at position 114, such as methionine M, is mutated into iso.
  • Leucine I the amino acid at position 115 such as glycine G is changed to leucine V; and the second constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, The amino acid at position 56, such as serine S, is mutated to cysteine C.
  • Embodiment 23 The modified immune cell of embodiment 22, wherein the first constant region includes the amino acid sequence shown in SEQ ID NO: 28; the second constant region includes the amino acid sequence shown in SEQ ID NO: 6.
  • Embodiment 24 The modified immune cell of any one of embodiments 11-23, wherein the modified TCR, such as STAR, is derived from an ⁇ TCR, and wherein the ⁇ chain comprises a first antigen-binding region and the ⁇ chain comprises a second antigen. bonding zone.
  • the modified TCR such as STAR
  • Embodiment 25 The modified immune cell of embodiment 24, wherein the first antigen-binding region and the second antigen-binding region each specifically bind a target antigen, independently or in combination.
  • Embodiment 26 The modified immune cell of embodiment 25, wherein the target antigen is a disease-associated antigen, preferably a cancer-associated antigen, such as a cancer-associated antigen selected from the group consisting of: GPC3, CD16, CD64, CD78, CD96, CLL1 , CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variants III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, ⁇ -human chorionic membrane Gonadotropin, alpha fetoglobulin (AFP), lectin-responsive AFP, thyroglobulin
  • Embodiment 27 The modified immune cell of any one of embodiments 24-26, wherein the first antigen-binding region comprises a heavy chain variable region of an antibody that specifically binds a target antigen, and the second antigen binds The region includes the light chain variable region of the antibody; alternatively, the first antigen-binding region includes the light chain variable region of an antibody that specifically binds the target antigen, and the second antigen-binding region includes the heavy chain variable region of the antibody. chain variable region.
  • Embodiment 28 The modified immune cell of any one of embodiments 24-26, wherein said first antigen-binding region comprises a single chain antibody or single domain antibody that specifically binds a target antigen; and/or said second The antigen-binding region contains single-chain antibodies or single-domain antibodies that specifically bind to the target antigen,
  • the single-chain antibody includes a heavy chain variable region and a light chain variable region connected by a linker, such as a (G4S)n linker, where n represents an integer from 1 to 10, preferably, n is 1 or 3.
  • a linker such as a (G4S)n linker, where n represents an integer from 1 to 10, preferably, n is 1 or 3.
  • Embodiment 29 The modified immune cell of embodiment 28, wherein the first antigen binding region and the second antigen binding region bind the same target antigen.
  • Embodiment 30 The modified immune cell of embodiment 28, wherein the first antigen-binding region and the second antigen-binding region bind to different regions of the same target antigen (eg, different epitopes) or bind to different target antigens .
  • Embodiment 31 A pharmaceutical composition comprising the modified immune cell of any one of embodiments 1-30, and a pharmaceutically acceptable carrier.
  • Embodiment 32 Use of the modified immune cell of any one of embodiments 1-30 or the pharmaceutical composition of embodiment 31 for the preparation of a medicament for treating a disease, such as cancer, in a subject.
  • Embodiment 33 A method of treating a disease, such as cancer, in a subject, comprising administering to the subject a therapeutically effective amount of the modified immune cell of any one of embodiments 1-30 or the pharmaceutical composition of embodiment 31.
  • Embodiment 34 A method of significantly shrinking a subject in a subject having cancer, comprising administering to the subject a therapeutically effective amount of the modified immune cell of any one of embodiments 1-30 or of embodiment 31 Pharmaceutical compositions.
  • Embodiment 35 A method of prolonging the survival time of a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of the modified immune cell of any one of embodiments 1-30 or the pharmaceutical composition of embodiment 31 .
  • Embodiment 36 An isolated nucleic acid molecule comprising a nucleotide sequence encoding an NF- ⁇ B family transcription factor as defined in any one of embodiments 1-30 and/or encoding any one of embodiments 1-30 The nucleotide sequence of the defined target-specific receptor.
  • Embodiment 37 An expression vector comprising i) a nucleotide sequence encoding a target-specific receptor as defined in any one of embodiments 1-30 and/or ii) encoding an implementation operably linked to a regulatory sequence. Nucleotide sequence of the NF- ⁇ B family transcription factor as defined in any of Schemes 1-30.
  • Embodiment 38 A method of preparing modified immune cells, comprising
  • Embodiment 39 The method of embodiment 38, wherein said immune cells are selected from T cells, B cells or NK cells, preferably T cells.
  • Embodiment 40 The method of embodiment 38 or 39, wherein the NF- ⁇ B family transcription factor is selected from the group consisting of P50, P105, P52, P100, P65, c-Rel and RelB, or any combination thereof, preferably, the The NF- ⁇ B family transcription factor is p52.
  • Embodiment 41 The method of any one of embodiments 38-40, wherein said method is performed by combining a nucleic acid molecule comprising a nucleotide sequence encoding said NF- ⁇ B family transcription factor or a coding sequence operably linked to a regulatory sequence.
  • the expression vector of the nucleotide sequence of the NF- ⁇ B family transcription factor is introduced into the immune cell to overexpress the NF- ⁇ B family transcription factor.
  • Embodiment 42 The method of embodiment 41, wherein the NF- ⁇ B family transcription factor is p52, and the nucleotide sequence encoding the p52 is shown in SEQ ID NO: 8.
  • Embodiment 43 The method of any one of embodiments 38-42, wherein the method further comprises c) converting a nucleic acid molecule comprising a nucleotide sequence encoding a target-specific receptor or encoding a nucleic acid molecule operably linked to a regulatory sequence.
  • An expression vector for the nucleotide sequence of a target-specific receptor is introduced into the immune cell.
  • Embodiment 44 The method of Embodiment 43, wherein the target-specific receptor is selected from the group consisting of chimeric antigen receptor (CAR) and T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • Embodiment 45 The method of embodiment 44, wherein the target-specific receptor is a modified T-cell receptor (TCR), such as a Synthetic T-Cell Receptor and Antibody Receptor, STAR).
  • TCR modified T-cell receptor
  • Embodiment 46 The method of any one of embodiments 38-45, wherein said method further comprises the step
  • Embodiment 47 A method for improving T cell proliferation ability, improving T cell effector survival time, increasing the number of T cells in vivo, inducing T cells to differentiate into effector T cells, reducing T cell inhibitory receptor expression, and increasing CD8+ in the T cell population T cell ratio, or a method for improving the target killing ability of T cells, the method includes:
  • Embodiment 48 The method of embodiment 47, wherein the NF- ⁇ B family transcription factor is selected from the group consisting of P50, P105, P52, P100, P65, c-Rel and RelB, or any combination thereof, preferably, the NF- The ⁇ B family transcription factor is p52.
  • Embodiment 49 The method of embodiment 48, wherein the method is accomplished by combining a nucleic acid molecule comprising a nucleotide sequence encoding the NF- ⁇ B family transcription factor or a nucleic acid molecule encoding the NF- ⁇ B family operably linked to a regulatory sequence.
  • An expression vector of the nucleotide sequence of the transcription factor is introduced into the T cells to overexpress the NF- ⁇ B family transcription factor.
  • Embodiment 50 The method of embodiment 49, wherein the NF- ⁇ B family transcription factor is p52, and the nucleotide sequence encoding the p52 is shown in SEQ ID NO: 8.
  • Embodiment 51 The method of any one of embodiments 47-50, wherein the method further comprises the step
  • Embodiment 51 A kit for preparing the modified immune cell of any one of embodiments 1-30, said kit comprising the nucleic acid molecule of embodiment 36 and/or the expression vector of embodiment 37, Optionally, the kit further contains reagents for isolating, culturing and/or expanding immune cells such as T cells, and/or preparations for introducing the nucleic acid molecules or expression vectors into cells.
  • Figure 1 Structure of STAR showing constant region modification.
  • Figure 2 Shows the vector structure for co-expression of p52/RFP and STAR.
  • Figure 3 Shows that overexpression of p52 increases the killing ability of STAR-T cells against target cell A431.
  • Figure 4 Shows that overexpression of p52 increases the proportion of CD8+ T cells.
  • Figure 5 Shows that overexpression of p52 improves the proliferation ability of CD8+T cells.
  • Figure 6 Shows that after 5 days of antigen protein stimulation, overexpression of p52 makes mut-STAR T cells more differentiated into effector T cells.
  • FIG. 9 ELISA results show that overexpression of p52 can increase the secretion of IFN- ⁇ and IL2 by mut-STAR T cells and enhance the effector function of T cells.
  • Figure 10 Shows the experimental design of the low-dose Raji hematoma model.
  • Figure 11 Shows the results of tumor size monitoring in the low-dose Raji hematoma model experiment.
  • Figure 12 Shows the tumor size imaging in the low-dose Raji hematoma model experiment.
  • Figure 13 Shows the count of reinfused human T cells in the peripheral blood of mice during experiments in the low-dose Raji hematoma model.
  • Figure 14 Shows experimental design of excess Raji hematoma model.
  • Figure 15 Shows the count of reinfused human T cells in the peripheral blood of mice during experiments in the excess Raji hematoma model.
  • Figure 16 Shows the count of human CD8+ T cells reinfused in the peripheral blood of mice during the excess Raji hematoma model experiment.
  • Figure 17 Shows the survival curve of mice during the experiment of excess Raji hematoma model.
  • FIG. 18 A431 subcutaneous tumor model experiments show that overexpression of p52 significantly increases the inhibition of tumors by mut-STAR T cells.
  • FIG. 19 A549 subcutaneous tumor model experiments show that overexpression of p52 significantly increases the inhibition of tumors by mut-STAR T cells.
  • FIG. 20 Subcutaneous A431-NYESO-1 tumor model to detect the effect of overexpression of p52 on the tumor suppressive effect of 1G4-TCR T cells.
  • Figure 21 Shows that in the subcutaneous A431-NYESO-1 tumor model, 24 days after T cell infusion, overexpression of p52 significantly increased the tumor suppressive effect of 1G4-TCR T cells.
  • FIG. 22 Shows the tumor size imaging in the subcutaneous A431-NYESO-1 tumor model experiment.
  • Figure 23 shows that overexpression of p52 on first-generation CAR (zCAR) T cells can improve its target cell killing ability, and is stronger than BBzCAR.
  • Figure 24 Shows that overexpression of p52 on first-generation CAR (zCAR) T cells can improve their proliferation ability.
  • the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleic acids at one or both ends of the protein or nucleic acid. glycosides, but still have the activity described in the present invention.
  • those skilled in the art know that the methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain practical circumstances (such as when expressed in a specific expression system), but will not substantially affect the function of the polypeptide.
  • amino acid numbering refers to SEQ ID NO:x
  • SEQ ID NO:x is a specific sequence listed herein
  • amino acid correspondence can be determined according to sequence comparison methods known in the art. For example, amino acid correspondence can be determined through the EMBL-EBI online alignment tool (https://www.ebi.ac.uk/Tools/psa/), where two sequences can be determined using the Needleman-Wunsch algorithm using default parameters. Alignment.
  • the amino acid in the polypeptide can also be described herein. It is "alanine at position 48 of the polypeptide, and the amino acid position refers to SEQ ID NO:x".
  • the amino acid position related to the ⁇ chain constant region refers to SEQ ID NO: 3.
  • the amino acid position related to the ⁇ -chain constant region refers to SEQ ID NO: 4.
  • the invention provides a modified immune cell modified to overexpress a nuclear factor- ⁇ B (NF- ⁇ B) family transcription factor.
  • NF- ⁇ B nuclear factor- ⁇ B
  • the modified immune cells are isolated.
  • the modified immune cells are therapeutic immune cells.
  • the modified immune cells are used for administration to a subject in need thereof to treat a disease, such as a tumor or an infectious disease in the subject.
  • the immune cells described herein can be a variety of different immune cells, including but not limited to T cells, B cells, or NK cells.
  • Immune cells described herein can be obtained by various non-limiting methods from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites fluid , pleural effusion, spleen tissue and tumors.
  • cells can be derived from healthy donors or from patients diagnosed with cancer.
  • cells may be part of a mixed population of cells exhibiting different phenotypic characteristics.
  • T cells can be obtained by isolating peripheral blood mononuclear cells (PBMC) and then activating and amplifying them with specific antibodies.
  • PBMC peripheral blood mononuclear cells
  • the immune cells are derived from the subject's autologous cells.
  • autologous means that a cell, cell line or cell population used to treat a subject is derived from the subject.
  • the immune cells, such as T cells are derived from allogeneic cells, such as from a donor that is human leukocyte antigen (HLA) compatible with the subject. Cells from the donor can be converted into non-alloreactive cells using standard protocols and replicated as needed, producing cells that can be administered to one or more patients.
  • HLA human leukocyte antigen
  • the NF- ⁇ B family transcription factor is selected from P50, P105, P52, P100, P65, c-Rel and RelB, or any combination thereof.
  • the NF- ⁇ B family transcription factor is a transcription factor of the non-canonical NF- ⁇ B family.
  • the non-canonical NF- ⁇ B family transcription factor is selected from RelB and p52. In some preferred embodiments, the non-canonical NF- ⁇ B family transcription factor is p52.
  • Exemplary p52 includes the amino acid sequence set forth in SEQ ID NO:9 or is encoded by the nucleotide sequence set forth in SEQ ID NO:8.
  • the p52 also encompasses functional variants thereof, such as naturally occurring functional variants, for example, the functional variants of p52 comprise at least 75%, at least 80% of the same as SEQ ID NO: 9 , at least 85%, 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%, at least 99.5% sequence identical
  • the unique amino acid sequence but retains the activity of SEQ ID NO:9, such as the activity of binding to RelB and initiating downstream gene expression.
  • overexpression of NF- ⁇ B family transcription factors in immune cells can be achieved by incorporating a nucleic acid molecule encoding a nucleotide sequence encoding the NF- ⁇ B family transcription factor or comprising the nucleic acid molecule encoding the NF- ⁇ B family transcription factor. This is achieved by introducing the expression vector of the nucleotide sequence of the transcription factor into the immune cells.
  • it can also be achieved by targeted modification of the endogenous NF- ⁇ B family transcription factor gene in immune cells.
  • overexpression of the transcription factor can be achieved by targeted modification of the expression regulatory region of the gene.
  • the targeted modification can be achieved, for example, by gene editing technologies known in the art, such as CRISPR technology, TALEN, ZFN and other gene editing technologies.
  • the modified immune cells comprise introduced nucleotide sequences encoding the NF- ⁇ B family transcription factors, thereby overexpressing the NF- ⁇ B family transcription factors.
  • the nucleotide sequence encoding the NF- ⁇ B family transcription factor is introduced into the modified immune cell via an expression vector. In some embodiments, the nucleotide sequence encoding the NF- ⁇ B family transcription factor is operably linked to regulatory sequences in the expression vector.
  • the "expression vector" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or an RNA capable of translation (such as mRNA).
  • the expression vector is a viral vector, such as a lentiviral vector.
  • regulatory sequence and “regulatory element” are used interchangeably and refer to a coding sequence that is located upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) and affects the transcription, RNA processing or Stability or translated nucleotide sequence.
  • Expression regulatory elements refer to nucleotide sequences capable of controlling the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leaders, introns, enhancers, and polyadenylation recognition sequences.
  • operably linked means that a regulatory element (eg, but not limited to, a promoter sequence, a transcription termination sequence, etc.) is linked to a nucleic acid sequence (eg, a coding sequence or an open reading frame) such that the nucleotide Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements.
  • a regulatory element eg, but not limited to, a promoter sequence, a transcription termination sequence, etc.
  • nucleic acid sequence eg, a coding sequence or an open reading frame
  • the modified immune cells further comprise (eg express) a "target-specific receptor.”
  • the target-specific receptor is capable of mediating activation of the immune cell upon specific binding to a target.
  • the modified immune cell comprises an introduced nucleotide sequence encoding the target-specific receptor, thereby expressing the target-specific receptor.
  • the nucleotide sequence encoding the target-specific receptor is introduced into the modified immune cell via an expression vector. In some embodiments, the nucleotide sequence encoding the target-specific receptor is operably linked to regulatory sequences in the expression vector.
  • a nucleotide sequence encoding the target-specific receptor and a nucleotide sequence encoding the NF- ⁇ B family transcription factor are introduced into the modified immune cell in the same expression vector.
  • the target-specific receptor is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • CAR Chimeric antigen receptor
  • the CAR may comprise an extracellular target (antigen) binding region directed to a specific target (eg, an antigen).
  • a specific target eg, an antigen
  • the target (antigen) binding region is a single chain antibody or single domain antibody that specifically binds a target antigen.
  • the CAR may also include a transmembrane domain and an intracellular signaling domain.
  • the intracellular signal transduction domain of the CAR according to the present invention is responsible for intracellular signal transduction after the extracellular target binding region binds to the target, resulting in activation of immune cells and immune response.
  • the intracellular signaling domain has the ability to activate at least one normal effector function of the CAR-expressing immune cell.
  • the intracellular signaling domain for a CAR may be a cytoplasmic sequence such as, but not limited to, that of a T cell receptor and a coreceptor that act in concert to initiate signal transduction upon antigen receptor engagement, as well as derivatives or variants of any of these sequences and any synthetic sequences having the same functional capabilities.
  • Intracellular signaling domains include two distinct types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide secondary or costimulatory signals.
  • the primary cytoplasmic signaling sequence may include a signaling motif known as the immunoreceptor tyrosine activation motif of ITAM.
  • Non-limiting examples of ITAMs used in CARs of the invention may include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain of a CAR of the invention includes a CD3 ⁇ signaling domain.
  • the intracellular signaling domain of the CAR of the invention also includes a costimulatory domain (second generation or higher CAR), such as a 41BB costimulatory domain or a CD28 costimulatory domain.
  • a CAR is expressed on the surface of cells. Therefore, a CAR may also include a transmembrane domain. Suitable transmembrane domains of the CAR of the invention have the ability to: (a) be expressed on the surface of cells, preferably immune cells, such as but not limited to lymphocytes or natural killer (NK) cells, and (b) bind to the ligand binding domain Interacts with intracellular signal transduction domains to guide immune cells to respond to predetermined target cells.
  • Transmembrane domains can be derived from natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain can be derived from subunits of T cell receptors such as alpha subunits, beta subunits, gamma or delta subunits, polypeptides constituting the CD3 complex, p55 ( ⁇ chain), p75 ( ⁇ chain) or ⁇ , subunit chain of an Fc receptor, in particular Fc ⁇ receptor III or CD protein.
  • the transmembrane domain may be synthetic and may consist primarily of hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from human CD8 alpha chain.
  • the transmembrane domain may further comprise a hinge region located between the extracellular ligand binding domain and said transmembrane domain. The hinge region is, for example, derived from the extracellular region of CD8, CD4 or CD28.
  • the CAR is a first generation CAR that does not contain a costimulatory domain.
  • the first-generation CAR may be composed of an extracellular ligand-binding domain, a hinge region, a transmembrane region, and a CD3 ⁇ signaling domain.
  • the target-specific receptor is a T cell receptor (TCR). In some preferred embodiments, the target-specific receptor is a modified T cell receptor (TCR). In some embodiments, the TCR is an ⁇ TCR. In some embodiments, the TCR is a ⁇ TCR.
  • the target-specific receptor is a Synthetic T-Cell Receptor and Antibody Receptor (STAR).
  • STARs are derived from native TCRs and can be generated by replacing the variable region of the native TCR with a different given target binding region, such as a given antibody variable region.
  • Synthetic T cell receptor antibody receptors (STAR) of the present invention include, but are not limited to, those disclosed in WO2021135178A1 or WO2021223707A1.
  • the modified TCR such as STAR
  • the ⁇ chain includes a first constant region and the ⁇ chain includes a second constant region.
  • the alpha chain further comprises a first target binding region, and/or the beta chain further comprises a second target binding region.
  • the modified TCR such as the alpha chain and/or beta chain of STAR, has at least one functional domain attached to its C-terminus.
  • the alpha chain of the modified TCR such as STAR
  • the beta chain of the modified TCR such as STAR, has at least one functional domain attached to its C-terminus.
  • the alpha and beta chains of the STAR have at least one functional domain linked to their C-terminus.
  • the native intracellular region of the alpha chain and/or beta chain of the modified TCR is deleted. In some embodiments, the native intracellular region of the alpha chain of the modified TCR, such as STAR, is deleted. In some embodiments, the native intracellular region of the beta chain of the modified TCR, such as STAR, is deleted. In some embodiments, the native intracellular regions of the alpha and beta chains of the modified TCR, such as STAR, are deleted.
  • the at least one functional domain is linked to the C-terminus of the alpha chain and/or beta chain from which the native intracellular region is deleted, either directly or through a linker. In some embodiments, the at least one functional domain is linked to the C-terminus of the alpha chain from which the native intracellular region is deleted, either directly or through a linker. In some embodiments, the at least one functional domain is linked to the C-terminus of the beta chain from which the native intracellular region is deleted, either directly or through a linker. In some embodiments, the at least one functional domain is linked to the C-terminus of the alpha chain and beta chain from which the native intracellular region is deleted, either directly or through a linker.
  • the alpha chain and/or beta chain in the modified TCR can be linked to the same or different functional domains.
  • the functional domain is a foreign functional domain.
  • the functional domain is an exogenous intracellular domain, eg, a domain that functions in signaling within the cell.
  • exogenous means a protein or nucleic acid sequence from a foreign species or, if from the same species, a protein that has been significantly altered in composition and/or position from its native form by deliberate human intervention or Nucleic acid sequence.
  • a "functional domain” may be an intracellular domain of a co-stimulatory molecule such as CD40, OX40, ICOS, CD28, 4-1BB, CD27, CD137; it may also be an intracellular domain of a co-inhibitory molecule.
  • a co-stimulatory molecule such as CD40, OX40, ICOS, CD28, 4-1BB, CD27, CD137; it may also be an intracellular domain of a co-inhibitory molecule.
  • Structural domain such as the intracellular domain of TIM3, PD1, CTLA4, LAG3; it can also be a cytokine receptor such as an interleukin receptor (such as IL-2 ⁇ receptor, IL-7 ⁇ receptor or IL-21 receptor) , interferon receptor, tumor necrosis factor superfamily receptor, colony-stimulating factor receptor, chemokine receptor, growth factor receptor or the intracellular domain of other membrane proteins; or the domain of an intracellular protein such as NIK.
  • the functional domain can also be a fusion of the intracellular domain of the cytokine receptor and the human STAT5 activation module (SEQ ID NO: 25) directly or through a linker.
  • the functional domain is an intracellular domain of a costimulatory molecule, preferably an intracellular domain of OX40 or ICOS, more preferably an intracellular domain of OX40.
  • An exemplary CD40 intracellular domain includes the amino acid sequence shown in SEQ ID NO:16.
  • An exemplary OX40 intracellular domain includes the amino acid sequence shown in SEQ ID NO:17.
  • An exemplary ICOS intracellular domain includes the amino acid sequence shown in SEQ ID NO: 18.
  • An exemplary CD28 intracellular domain includes the amino acid sequence shown in SEQ ID NO: 19.
  • An exemplary 4-1BB intracellular domain includes the amino acid sequence shown in SEQ ID NO:20.
  • An exemplary CD27 intracellular domain includes the amino acid sequence shown in SEQ ID NO: 21.
  • An exemplary IL-2 ⁇ receptor intracellular domain includes the amino acid sequence shown in SEQ ID NO: 22.
  • An exemplary IL-17 ⁇ receptor intracellular domain includes the amino acid sequence shown in SEQ ID NO: 23.
  • An exemplary IL-21 receptor intracellular domain includes the amino acid sequence shown in SEQ ID NO: 24.
  • An exemplary fusion amino acid sequence of the IL-2 ⁇ receptor intracellular domain and the human STAT5 activation module is shown in SEQ ID NO: 26.
  • An exemplary fusion amino acid sequence of the IL-17 ⁇ receptor intracellular domain and the human STAT5 activation module is shown in SEQ ID NO: 27.
  • the first constant region is a native TCR ⁇ chain constant region, e.g., a native human TCR ⁇ chain constant region (an exemplary human TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 1) or natural mouse TCR ⁇ Chain constant region (an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 3).
  • a native human TCR ⁇ chain constant region an exemplary human TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 1
  • natural mouse TCR ⁇ Chain constant region an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 3
  • the first constant region is a modified TCR ⁇ chain constant region.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, e.g., threonine T, is mutated to Cysteine C.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 112, such as serine S, is changed to leucine relative to the wild-type mouse TCR ⁇ chain constant region.
  • the amino acid at position 112 such as serine S
  • the amino acid at position 114 such as methionine M
  • the amino acid at position 115 such as glycine G
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, in which, relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6, such as E, is replaced by D, and the amino acid at position 13 is replaced by D.
  • the K at position 1 was replaced by R, and amino acids 15-18 were deleted.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, e.g., threonine T, is mutated to Cysteine C, the amino acid at position 112 such as serine S is changed to leucine L, the amino acid at position 114 such as methionine M is changed to isoleucine I, the amino acid at position 115 such as Glycine G is changed to glycine V.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, in which, relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6, such as E, is replaced by D, and the amino acid at position 13 is replaced by D.
  • the K at position 1 is replaced by R, the amino acids 15-18 are deleted, the amino acid at position 48, such as threonine T, is mutated to cysteine C, and the amino acid at position 112, such as serine S, is changed to leucine
  • the amino acid at position 114, such as methionine M is changed to isoleucine I
  • the amino acid at position 115 such as glycine G, is changed to lycine V.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region that lacks the native intracellular region of the constant region relative to the wild-type mouse TCR ⁇ chain constant region, e.g., deletes 136- Amino acid at position 137.
  • the first constant region comprises the amino acid sequence set forth in one of SEQ ID Nos: 1, 3, 5, 7, 28, 29, 31 and 33.
  • the second constant region is a native TCR beta chain constant region, e.g., a native human TCR beta chain constant region (an exemplary human TCR beta chain constant region amino acid sequence is shown in SEQ ID NO: 2) or natural mouse TCR beta Chain constant region (an exemplary mouse TCR beta chain constant region amino acid sequence is shown in SEQ ID NO: 4).
  • a native human TCR beta chain constant region an exemplary human TCR beta chain constant region amino acid sequence is shown in SEQ ID NO: 2
  • natural mouse TCR beta Chain constant region an exemplary mouse TCR beta chain constant region amino acid sequence is shown in SEQ ID NO: 4
  • the second constant region is a modified TCR beta chain constant region.
  • the modified TCR beta chain constant region is derived from a mouse TCR beta chain constant region in which the amino acid at position 56, such as serine S, is mutated to cysteine relative to the wild-type mouse TCR beta chain constant region. Amino acid C.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, in which, relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 3, such as R, is replaced by K, and position 6
  • T is replaced by F
  • K at position 9 is replaced by E
  • S at position 11 is replaced by A
  • L at position 12 is replaced by V
  • amino acids at positions 17 and 21-25 are deleted.
  • the modified TCR beta chain constant region is derived from a mouse TCR beta chain constant region in which the amino acid at position 56, such as serine S, is mutated to cysteine relative to the wild-type mouse TCR beta chain constant region.
  • Amino acid C the amino acid at position 3 such as R is replaced by K
  • the amino acid at position 6 such as T is replaced by F
  • the K at position 9 is replaced by E
  • the S at position 11 is replaced by A
  • the L at position 12 is replaced by V substitution
  • amino acids 17 and 21-25 were deleted.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region that lacks the native intracellular region of the constant region relative to the wild-type mouse TCR ⁇ chain constant region, e.g., deletes 167- Amino acid 172.
  • the second constant region comprises the amino acid sequence set forth in one of SEQ ID Nos: 2, 4, 6, 30, 32, and 34.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, such as threonine T, is mutated relative to the wild-type mouse TCR ⁇ chain constant region.
  • the amino acid at position 48 such as threonine T
  • the amino acid at position 112 such as serine S
  • the amino acid at position 114 such as methionine M
  • isoleucine I
  • amino acid at position 115 For example, glycine G is changed to salinine V
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which has an amino acid at position 56, such as serine, relative to the wild-type mouse TCR ⁇ chain constant region. S is mutated to cysteine C.
  • the modified TCR ⁇ chain constant region includes the amino acid sequence shown in SEQ ID NO:28; the modified TCR ⁇ chain constant region includes the amino acid sequence shown in SEQ ID NO:6.
  • target binding region refers to a domain capable of binding, preferably specifically binding, to a target molecule.
  • the target is an antigen.
  • the target binding region is an "antigen binding region.”
  • the target binding region (preferably an antigen binding region) alone or in combination with another target binding region (preferably an antigen binding region) can specifically bind a target molecule (preferably a target antigen).
  • the first target binding region (first antigen binding region) and the second target binding region (second antigen binding region) each, independently or in combination, specifically bind a target antigen.
  • the antigen-binding region is derived from an antibody that specifically binds a target antigen.
  • the antigen-binding region can also be derived from a specific receptor, in which case the ligand of the receptor can serve as the antigen to be targeted.
  • the specific receptor may be a natural T cell receptor.
  • the antigen binding region comprises a variable region from a native T cell receptor.
  • the antigen-binding region may also be derived from a ligand, particularly where the antigen intended to be targeted is a receptor.
  • the target antigen is a disease-associated antigen, preferably a cancer-associated antigen, such as a cancer-associated antigen selected from: GPC3 (glypican 3), CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR mutation Body III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal mucin, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, ⁇ -human villi Membrane gonadotropin, alpha fetoglobulin (AFP), lectin-responsive AFP, thyroglobulin, RAGE-1, MN-CA IX, human
  • the target antigen is an antigen derived from a pathogen or a surface antigen of a cell infected by a pathogen, such as RSV F (prevention of respiratory syncytial virus), PA (inhalational anthrax), CD4 (HIV infection), etc.
  • RSV F prevention of respiratory syncytial virus
  • PA inhalational anthrax
  • CD4 HIV infection
  • the target antigen is some disease-causing cells or secreted molecules produced by cells, such as CD3 (involved in transplant rejection), CD25 (involved in acute renal transplant rejection), C5 (involved in paroxysmal nocturnal hemoglobin urea), IL-1 ⁇ (cryopyrin-related periodic syndrome), RANKL (involved in cancer-related bone damage), von Willebrand factor (involved in adult-acquired thrombotic platelet purpura), plasma kallikrein (involved in vascular edema), calcitonin gene-related peptide receptor (involved in migraine in adults), FGF23 (involved in X-linked hypophosphatemia), etc.
  • CD3 in transplant rejection
  • CD25 involved in acute renal transplant rejection
  • C5 in paroxysmal nocturnal hemoglobin urea
  • IL-1 ⁇ cryopyrin-related periodic syndrome
  • RANKL involved in cancer-
  • the antigen-binding region may be derived from one or more known antibodies, including any commercially available antibody, such as FMC63, rituximab, alemtuzumab, epratuzumab (epratuzumab), trastuzumab (trastuzumab), bivacizumab (bivatuzumab), cetuximab (cetuximab), labetuzumab (labetuzumab), palivizumab (palivizumab), Sevirumab, tuvirumab, basiliximab, daclizumab, infliximab, omalizumab, according to law efalizumab, Keliximab, siplizumab, natalizumab, clenoliximab, pemtumomab, Edrecolomab, Cantuzumab, etc.
  • FMC63 FMC63
  • the antigen-binding region comprises the heavy chain variable region set forth in SEQ ID NO: 10 and/or the light chain variable region set forth in SEQ ID NO: 11. In some specific embodiments, the antigen-binding region comprises the heavy chain variable region set forth in SEQ ID NO: 12 and/or the light chain variable region set forth in SEQ ID NO: 13.
  • the first antigen-binding region comprises a heavy chain variable region of an antibody that specifically binds a target antigen
  • the second antigen-binding region comprises a light chain variable region of the antibody
  • the first antigen-binding region includes the light chain variable region of an antibody that specifically binds a target antigen
  • the second antigen-binding region includes the heavy chain variable region of the antibody.
  • the first antigen binding region includes a single chain antibody (eg, scFv) or single domain antibody that specifically binds a target antigen; and/or the second antigen binding region includes a single chain antibody that specifically binds a target antigen.
  • a single chain antibody eg, scFv
  • the second antigen binding region includes a single chain antibody that specifically binds a target antigen.
  • the single chain antibody (e.g., scFv) comprises a heavy chain variable region and a light chain variable region connected by a linker, such as a (G4S)n linker, where n represents 1-10 an integer, preferably, n is 1 or 3.
  • a linker such as a (G4S)n linker, where n represents 1-10 an integer, preferably, n is 1 or 3.
  • the first antigen binding region and the second antigen binding region bind the same target antigen.
  • the first antigen binding region and the second antigen binding region bind to different regions of the same target antigen (eg, different epitopes).
  • the first antigen binding region and the second antigen binding region bind different target antigens.
  • the present invention provides a pharmaceutical composition comprising the modified immune cells of the present invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion).
  • the invention provides the use of a modified immune cell of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for treating a disease, such as cancer, in a subject.
  • Subject refers to an organism that suffers from or is susceptible to a disease (eg, cancer) treatable by the cells, methods, or pharmaceutical compositions of the invention.
  • a disease eg, cancer
  • Non-limiting examples include humans, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.
  • the subject is a human.
  • the invention provides a method of treating a disease, such as cancer, in a subject, comprising administering to the subject a therapeutically effective amount of a modified immune cell of the invention or a pharmaceutical composition of the invention.
  • a “therapeutically effective amount” or “therapeutically effective dose” or “effective amount” refers to an amount of a substance, compound, material or cell that is at least sufficient to produce a therapeutic effect when administered to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or condition.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or the prevention of damage or disability resulting from the distress of the disease.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 20%, relative to an untreated subject. Preferably it is at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit tumor cell growth, which inhibition can be determined in vitro by assays well known to those skilled in the art.
  • the dosage levels of the cells in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention to which it is administered, the route of administration, the time of administration, the rate of excretion of the particular compound to which it is administered, the duration of treatment, and the specific conditions for which it is administered.
  • other drugs, compounds and/or materials in combination with the composition the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical field.
  • modified immune cells or pharmaceutical compositions or drugs according to the invention may be performed in any convenient manner, including by injection, infusion, implantation or transplantation.
  • Administration of cells or compositions described herein can be by intravenous, intralymphatic, intradermal, intratumoral, intramedullary, intramuscular, or intraperitoneal administration.
  • the cells or compositions of the invention are preferably administered by intravenous injection.
  • the disease is, for example, cancer, examples of which include, but are not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer , lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anus Area cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, Adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukin.
  • the treatment results in significant tumor shrinkage in a subject with cancer.
  • the tumor shrinks by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more Preferably at least about 80%.
  • the treatment prolongs (eg, significantly prolongs) the survival time of a subject with cancer.
  • survival time is prolonged by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, More preferably at least about 80%, more preferably at least about 90%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or longer.
  • the disease is, for example, infection by a pathogen, examples of which include, but are not limited to, respiratory syncytial virus, Bacillus anthracis, human immunodeficiency virus, and the like.
  • the disease is, for example, cardiovascular, diabetic, neurological, anti-post-transplant rejection, or some other disease.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an NF- ⁇ B family transcription factor as described herein and/or a nucleotide sequence encoding a target-specific receptor as described herein.
  • the invention provides an expression vector comprising i) a nucleotide sequence encoding a target-specific receptor as described herein and/or ii) encoding NF- ⁇ B as described herein operably linked to a regulatory sequence. Nucleotide sequences of family transcription factors.
  • the expression vector comprises i) a nucleotide sequence encoding the target-specific receptor and ii) a nucleotide sequence encoding the NF- ⁇ B family transcription factor operably linked to a regulatory sequence , and i) the nucleotide sequence encoding the target-specific receptor and ii) the nucleotide sequence encoding the NF- ⁇ B family transcription factor are connected through an internal ribosome entry site (IRES), thereby achieving the Co-expression of target-specific receptors and the NF- ⁇ B family of transcription factors.
  • IRS internal ribosome entry site
  • the target-specific receptor is a TCR, such as a modified TCR, preferably a STAR, and the nucleotide sequence encoding the NF- ⁇ B family transcription factor is in the same reading frame as i) encoding The nucleotide sequence of the alpha chain, ii) the nucleotide sequence encoding the beta chain and iii) the nucleotide sequence encoding a self-cleaving peptide located between i) and ii).
  • the nucleotide sequence encoding the alpha chain may be located at the 5' end or the 3' end of the nucleotide sequence encoding the beta chain.
  • Self-cleaving peptide as used herein means a peptide that can achieve self-cleavage within a cell.
  • the self-cleaving peptide may contain a protease recognition site, thereby being recognized and specifically cleaved by intracellular proteases.
  • the self-cleaving peptide may be a 2A polypeptide.
  • 2A polypeptides are short peptides from viruses whose self-cleavage occurs during translation. When 2A polypeptide is used to connect two different target proteins and expressed in the same reading frame, the two target proteins are generated at an almost 1:1 ratio.
  • 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asigna virus, and E2A from equine rhinitis A virus. and F2A from foot-and-mouth disease virus. Among them, P2A has the highest cutting efficiency and is therefore preferred.
  • a variety of functional variants of these 2A polypeptides are also known in the art and may be used in the present invention. Coexpression of alpha and beta chains can be achieved by using self-cleaving peptides.
  • the invention provides the use of the nucleic acid molecule or expression vector of the invention in preparing the modified immune cells of the invention.
  • the invention provides a method for preparing modified immune cells (such as the modified immune cells described above in the invention), comprising
  • the immune cells are selected from T cells, B cells or NK cells, preferably T cells.
  • the NF- ⁇ B family transcription factor is selected from P50, P105, P52, P100, P65, c-Rel and RelB, or any combination thereof.
  • the NF- ⁇ B family transcription factor is p52.
  • the method is performed by combining a nucleic acid molecule comprising a nucleotide sequence encoding the NF- ⁇ B family transcription factor or a nucleoside encoding the NF- ⁇ B family transcription factor operably linked to a regulatory sequence.
  • An expression vector of acidic sequences is introduced into the immune cells to overexpress the NF- ⁇ B family transcription factors.
  • the NF- ⁇ B family transcription factor is p52.
  • the nucleotide sequence encoding p52 is set forth in SEQ ID NO: 8.
  • the method further comprises c) converting a nucleic acid molecule comprising a nucleotide sequence encoding a target-specific receptor herein or comprising a nucleotide sequence encoding a target-specific receptor operably linked to a regulatory sequence
  • the expression vector is introduced into the immune cells.
  • the target-specific receptor is selected from the group consisting of chimeric antigen receptors (CARs) and T cell receptors (TCRs).
  • the target-specific receptor is a modified T-cell receptor (TCR), such as a Synthetic T-Cell Receptor and Antibody Receptor (STAR).
  • TCR T cell receptor
  • STAR Synthetic T-Cell Receptor and Antibody Receptor
  • the target-specific receptor is a target-specific receptor as defined above.
  • step b) is performed before step c). In some embodiments, step c) is performed after step b).
  • steps b) and c) are performed simultaneously.
  • expression of a nucleic acid molecule comprising i) a nucleotide sequence encoding the NF- ⁇ B family transcription factor, and ii) a nucleotide sequence encoding the target-specific receptor operably linked to a regulatory sequence can be The vector is introduced into the immune cells.
  • Nucleic acid molecules or expression vectors may be introduced into cells by any appropriate method, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofectamine transfection ; and infection (e.g., the expression construct is a virus).
  • the expression vector is a viral vector. In some more preferred embodiments, the expression vector is a lentiviral vector.
  • the step of providing isolated immune cells can be performed by methods known in the art for isolating immune cells.
  • immune cells such as T cells can be isolated from the peripheral blood of a subject using commercial kits. Suitable kits include, but are not limited to, EasySep human T cell enrichment kit (Stemcell Technologies).
  • kits include, but are not limited to, EasySep human T cell enrichment kit (Stemcell Technologies).
  • isolated immune cells, such as T cells are not necessarily homogeneous, but may be a mixed population of different cells, preferably in which the desired immune cells, such as T cells, are enriched.
  • Immune cells such as T cells, of the invention can be activated and expanded before or after any modification steps. Immune cells can be expanded in vitro or in vivo.
  • the method further includes the step
  • step d) is performed before and/or after step b). In some embodiments, step d) is performed before and/or after step c).
  • the present invention provides a method to improve the proliferation ability of immune cells such as T cells, improve the survival time of immune cells such as T cells, increase the number of immune cells such as T cells in the body, and induce immune cells such as T cells to effector immune cells such as effector cells.
  • the method includes:
  • the NF- ⁇ B family transcription factor is selected from P50, P105, P52, P100, P65, c-Rel and RelB, or any combination thereof.
  • the NF- ⁇ B family transcription factor is p52.
  • the method is performed by combining a nucleic acid molecule comprising a nucleotide sequence encoding the NF- ⁇ B family transcription factor or a nucleoside encoding the NF- ⁇ B family transcription factor operably linked to a regulatory sequence.
  • the expression vector of the acid sequence is introduced into the immune cells such as T cells to overexpress the NF- ⁇ B family transcription factors.
  • the NF- ⁇ B family transcription factor is p52.
  • the nucleotide sequence encoding said p52 is set forth in SEQ ID NO: 8.
  • the method further includes the step
  • T cells can be expanded, for example, by contact with an agent that stimulates the CD3TCR complex and costimulatory molecules on the surface of the T cell to generate a T cell activation signal.
  • an agent that stimulates the CD3TCR complex and costimulatory molecules on the surface of the T cell to generate a T cell activation signal.
  • chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitotic lectins such as phytohemagglutinin (PHA) can be used to generate activation signals for T cells .
  • T cells can be activated by contact with, for example, an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface in vitro, or by contact with a protein kinase C activator (e.g., bryostatin ) is activated together with contact with calcium ionophores.
  • a protein kinase C activator e.g., bryostatin
  • T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable to stimulate T cell proliferation.
  • Conditions suitable for T cell culture include suitable media (e.g., Minimal Essential Media or RPMI Media 1640, or X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF ⁇ and TNF, or additives for cell growth known to those skilled in the art.
  • Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder, and reducing agents such as N-acetyl-cysteine and 2-mercaptoacetic acid.
  • Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, amino acids, sodium pyruvate and vitamins, serum-free or appropriately supplemented serum (or plasma) or a defined set of hormones, and/or an amount of cytokines sufficient to enable T cell growth and expansion.
  • T cells can be maintained under conditions necessary to support growth, such as appropriate temperature (eg, 37°C) and environment (eg, air plus 5% CO2 ).
  • the proliferation ability of the immune cells of the present invention is increased by at least about 10%, preferably at least about 20%, and more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 100 %, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or more.
  • the survival time of the immune cells, such as T cells, of the present invention after reinfusion into the body is extended by at least about 10%, preferably at least about 20 %, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90% , more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or more.
  • the number of immune cells, such as T cells, of the present invention is increased by at least about 10%, preferably at least about 20%, after reinfusion into the body. , more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, More preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or more.
  • the proportion of effector cells, such as effector T cells, in the immune cells, such as T cells, of the invention is increased by at least about 10%, preferably by at least about 20%. %, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90% , more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or more.
  • inhibitory receptors in the immune cells of the invention is reduced by at least about 10%, preferably at least about 20%, compared to corresponding control immune cells, such as control T cells, which do not express the NF- ⁇ B family transcription factor. , more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%.
  • the proportion of CD8 + T cells in the immune cells of the present invention is increased by at least about 10%, preferably at least about 20%. , more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, More preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or more.
  • the target killing ability of the immune cells of the present invention is increased by at least about 10%, preferably at least about 20%, and more preferably at least About 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, more preferably at least about 300% or more.
  • the present invention also provides a kit for preparing the modified immune cells of the present invention.
  • the kit of the present invention includes the nucleic acid molecule of the present invention and/or the expression vector of the present invention.
  • the kit may also include reagents for isolating, culturing and/or amplifying immune cells such as T cells, preparations for introducing nucleic acid molecules or expression vectors into cells, and the like.
  • the secreted antibody (Antibody, Ab) or B cell receptor (BCR) produced by B cells has great similarities with the T cell receptor (TCR) in gene structure, protein structure and spatial conformation.
  • Both antibodies and TCRs are composed of variable regions and constant regions.
  • the variable regions play a role in antigen recognition and binding, while the constant region domains play a role in structural interaction and signal transduction.
  • a synthetic chimeric molecule can be constructed by replacing the variable regions of TCR ⁇ and ⁇ chains (or TCR ⁇ and ⁇ chains) with the heavy chain variable region (VH) and light chain variable region (VL) of the antibody. , called Synthetic T-Cell Receptor and Antibody Receptor, STAR/WT-STAR), its structure is shown in WT-STAR on the left side of Figure 1.
  • the STAR molecule has two chains.
  • the first chain is the fusion of the antigen recognition sequence (such as the variable region VH of the heavy chain of the antibody) and the constant region (C ⁇ ) of the T cell receptor ⁇ chain (TCR ⁇ ).
  • the second chain is The chain is the fusion of the antigen recognition sequence (such as the variable region VL of the light chain of the antibody) and the constant region (C ⁇ ) of the T cell receptor ⁇ chain (TCR ⁇ ).
  • the antigen recognition domain such as VH, VL or scFv, etc.
  • the constant region domain the constant regions of TCR ⁇ , ⁇ , ⁇ and ⁇
  • the first and second chains of the STAR molecule After the first and second chains of the STAR molecule are expressed in T cells, they will combine with the endogenous CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chains in the endoplasmic reticulum to form an 8-subunit complex, which will be displayed in the form of a complex. on the cell membrane surface.
  • Immunoreceptor Tyrosine-based Activation Motif is a motif that plays a signal transduction role in TCR molecules, and its conserved sequence is YxxL/V.
  • the intracellular region of the CD3 ⁇ , ⁇ , ⁇ , and ⁇ chains contains 1 ITAM sequence, and the intracellular region of the CD3 ⁇ chain contains 3 ITAM sequences, so a complete STAR complex contains a total of 10 ITAM sequences.
  • the intracellular ITAM sequence When the antigen recognition sequence of the STAR receptor binds to its specific antigen, the intracellular ITAM sequence will be phosphorylated one after another, thereby activating downstream signaling pathways, activating transcription factors such as NF- ⁇ , NFAT and AP-1, and triggering T cell activation. , produce effect function.
  • the inventor's previous research showed that compared with conventional chimeric antigen receptor CAR, STAR can better activate T cells, and the background activation in the absence of antigen stimulation is significantly reduced, thus having significant advantages (see Chinese Invention Patent application number: 201810898720.2). However, further improvements to STAR are still expected.
  • the STAR prototype design uses the constant region sequence of the TCR ⁇ / ⁇ chain (or TCR ⁇ and ⁇ chain) of human origin (wild-type human TCR ⁇ constant region, SEQ ID NO:1; wild-type human TCR ⁇ constant region, SEQ ID NO:2 ). Due to the functional conservation of the constant region sequences of human, primate and mouse TCR ⁇ / ⁇ chains (wild-type mouse TCR ⁇ constant region, SEQ ID NO:3; wild-type mouse TCR ⁇ constant region, SEQ ID NO:4) They are highly homogeneous and have the same key amino acid sequences, so they can be replaced with each other.
  • the inventors replaced the constant region of the STAR molecule with a mouse sequence to enhance the function of the STAR molecule after being transferred into human T cells.
  • cysteine point mutations on the STAR molecule to introduce intermolecular disulfide bonds, enhance the mutual pairing between the two chains of the STAR molecule, and reduce the mismatch with the endogenous TCR.
  • the threonine T at position 48 is mutated to cysteine C (mouse TCR ⁇ C-Cys, SEQ ID NO: 5) in the constant region of the TCR ⁇ chain
  • the serine S at position 56 is mutated to cysteine in the constant region of the TCR ⁇ chain.
  • C (mouse TCR ⁇ C-Cys, SEQ ID NO: 6).
  • the inventors substituted hydrophobic amino acids in the transmembrane region of the STAR molecule to increase the stability of the STAR molecule and help it function more permanently. Specifically, three amino acid sites were mutated in the transmembrane region of the TCR ⁇ chain constant region from 111 to 119 amino acids: serine S at position 112 was changed to leucine L, and methionine M at position 114 was changed to hetero.
  • Leucine I and glycine G at position 115 become leucine V.
  • the overall amino acid sequence of this region changed from LSVMGLRIL to LLVIVLRIL.
  • This modification is called mouse TCR ⁇ C-TM9, and the resulting ⁇ chain constant region sequence is SEQ ID NO:7.
  • This design increases the hydrophobicity of the transmembrane region and offsets the instability caused by the positive charge carried by the TCR transmembrane region, allowing the STAR molecule to exist more stably on the cell membrane, thereby obtaining better functions. Its structure See Mut-STAR on the right side of Figure 1.
  • STARs that contain constant region murine sequences, constant region cysteine substitutions, and constant region hydrophobic amino acid substitutions are called mut-STARs.
  • Example 2 In vitro effects of STAR-T cells overexpressing the non-canonical NF- ⁇ B transcription factor p52
  • costimulatory factors 41BB, OX40, etc. from TNFR family receptors
  • overexpressing costimulatory factors can improve the proliferation ability of T cells.
  • the regulation within T cells is very complex, and the in vitro signals after overexpression of co-stimulatory factors may not be effectively transmitted to the nucleus after complex regulation in vivo, resulting in the weakening and reduction of the signals.
  • the present invention directly overexpresses the most important downstream transcription factors of the TNFR family to enhance the signals and functions provided by costimulatory factors. This can bypass the complex regulatory pathways in cells and directly act on the cell nucleus to enhance the proliferation and effector capabilities of T cells. .
  • STAR-T or CAR-T cells (1) improve T cell proliferation ability; (2) improve T cell effector survival time; (3) increase the number of T cells in the body; (4) induce T cells to Effector T cell differentiation; (5) Reduce the expression of T cell inhibitory receptors; (6) Increase the proportion of CD8+ T cells; (7) Significantly shrink the patient's tumor; (8) Prolong the survival time of tumor patients; (9) Improve The inventors further modified STAR-T or CAR-T cells for their tumor-killing ability and ability to infiltrate into the tumor microenvironment and kill target cells efficiently.
  • the non-classical NF- ⁇ B transcription factor refers to the non-full-length protein p52 encoded by the gene Nfkb2 (its coding sequence is SEQ ID NO: 8, and its amino acid sequence is SEQ ID NO: 9).
  • Nfkb2 its coding sequence is SEQ ID NO: 8
  • its amino acid sequence is SEQ ID NO: 9.
  • TNF receptor family members When specific TNF receptor family members are stimulated by ligands (including: LT ⁇ R, CD40, CD27, CD30, BAFF-R, etc.), these members inhibit the action of TFAF3 on NF- ⁇ B-inducing kinase (NIK) by recruiting TRAF2 and TRAF3. Phosphorylation, in turn, causes NIK to phosphorylate IKK ⁇ . The phosphorylated IKK ⁇ phosphorylates p100 and is further processed by the proteasome into p52, which forms a heterodimer with RelB and enters the nucleus, and induces the expression of downstream genes.
  • ligands including: LT ⁇ R, CD40, CD27, CD30, BAFF-R, etc.
  • the vectors used in this example including viral vectors, plasmid vectors, etc., were purchased from or synthesized by commercial companies, and the full-length sequences of these vectors were obtained, and clear enzyme cleavage sites were known.
  • the gene fragments used in this example such as the STAR variable region, TCR constant region, p52 coding sequence, tag sequence, and linker, were all synthesized from commercial companies and connected by PCR.
  • a mut-STAR containing the VH shown in SEQ ID NO:10 and the VL shown in SEQ ID NO:11 (derived from the EGFR-targeting cetuximab Cetuximab) and the corresponding CAR (BBz or 28z CAR ), or a mut-STAR containing the VH shown in SEQ ID NO:12 and the VL shown in SEQ ID NO:13 (derived from the CD19-targeting antibody FMC63) and the corresponding CAR (BBz or 28z CAR), verifying the overexpression of SEQ Optimization of STAR-T or CAR-T cells by p52 shown in ID NO:9.
  • Red fluorescent protein with the amino acid sequence shown in SEQ ID NO:14 was used to replace p52 as a control group.
  • the STAR vector structure is shown in Figure 2, in which the overexpression of p52 is initiated by using the ribosome entry site IRES to achieve co-expression of the two fragments mut-STAR and p52.
  • add a myc-tag (SEQ ID NO:15: GAGCAGAAACTCATCTCTGAAGAGGATCTG) to the N-terminus of mut-STAR, and use a 6 bp sequence (GGATCT) to connect the mut-STAR and myc-tag fragments.
  • the lentiviral vector used in the examples is pHAGE-EF1 ⁇ -IRES-RFP.
  • the linear vector is obtained through restriction endonuclease Not I/Nhe I.
  • the target gene fragment is obtained through synthesis and PCR methods, and the complete vector is obtained through homologous recombination. .
  • the lentiviral packaging, culture and transduction of T cells used to transduce T cells refer to the international patent publication WO 2021/135178A1.
  • the target cells A431 are adherent cells, and the primary T cells are suspension cells. When co-incubating, take the corresponding number of cells, mix them with the target cell culture medium, and then centrifuge them for culture. The specific steps are: use the packaged and purified mut-STAR and mut-STAR-p52 viruses to infect primary T cells, and use flow cytometry to sort out the infected cells for in vitro detection experiments before co-culture. Target cells and primary T cells were co-incubated at 1:2 or 2:1, and the killing of target cells by T cells was detected after 1 day of co-incubation.
  • lactate dehydrogenase LDH kit to detect cell killing ability. After the target cells die, they will release LDH in the body into the cell suspension. Take the co-incubated cell suspension and centrifuge it at 2500rpm/min to remove the precipitate. Take 50uL of the supernatant from each well and add 50uL of LDH detection reagent. After reacting for 15 minutes, add 50uL of stop reagent and use a microplate reader at a wavelength of 490. detection.
  • Target antigen stimulates T cells
  • the target antigen EGFR is a cell surface protein that can be directly used to activate T cells to detect the function of T cells. Usually, 1 ⁇ 10 5 /well of positive T cells is added, centrifuged, and the cell suspension or culture supernatant is collected after 24 hours of activation to detect T cell function, or the value-added ability and memory of T cells are detected after 2.5 days, 5 days, or 7.5 days of activation. T cell swarming and killing of target cells.
  • the results in Figure 4 and Table 2 show that overexpression of p52 can promote an increase in the proportion of CD8+ T cells.
  • the results in Figure 5 show that overexpression of p52 in STAR-T cells can improve the better proliferation ability of CD8+T cells.
  • the results in Figure 6 and Table 3 show that after 5 days of antigen protein stimulation, overexpression of p52 can make mut-STAR T cells differentiate more into effector T cells.
  • the results in Figure 7 show that after long-term antigen stimulation (7.5 days), overexpression of p52 can cause mut-STAR cells to proliferate in greater numbers.
  • the results in Figure 8 and Table 4 show that after long-term antigen stimulation of T cells (7.5 days), the killing effect of T cells on target cells was detected. It was found that STAR-p52 has significantly improved killing ability than STAR.
  • T cells During the activation process of T cells, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves. Common ones include IFN- ⁇ and IL-2. After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken. The amounts of TNF- ⁇ , IFN- ⁇ , and IL-2 were determined by ELISA using kits according to the manufacturer's instructions. TNF- ⁇ , IFN- ⁇ , IL-2 ELISA kits use Human IL-2 Uncoated ELISA, Human TNF- ⁇ Uncoated ELISA, and Human IFN- ⁇ Uncoated ELISA (product numbers are 88-7025, 88-7346, 88-7316 respectively ).
  • the ELISA results in Figure 9 show that overexpression of p52 can increase the secretion of IFN- ⁇ and IL2 by mut-STAR T cells and enhance the effector function of T cells.
  • Example 3 In vivo effects of STAR-T cells overexpressing the non-canonical NF- ⁇ B transcription factor p52
  • Raji cells human Burkitt’s lymphoma cell line Raji cells were used to xenograft NSG immunodeficient mice.
  • Raji cells are cell lines that express luciferase genes through lentiviral vectors.
  • the development and changes of Raji tumors can be monitored in real time in mice through luciferin chemiluminescence and in vivo imaging.
  • the specific experimental design is shown in Figure 10.

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Abstract

本发明涉及生物医药领域，尤其涉及一种经修饰的免疫细胞。具体而言，本发明公开了一种过表达核因子-κB(NF-κB)家族转录因子的经修饰的免疫细胞以及所述经修饰的免疫细胞的用途。

Description

经修饰的免疫细胞 技术领域
本发明涉及生物医药领域，尤其涉及一种经修饰的免疫细胞。具体而言，本发明公开了一种过表达核因子-κB(NF-κB)家族转录因子的经修饰的免疫细胞以及所述经修饰的免疫细胞的用途。
发明背景
在T细胞中，最重要的三个转录因子家族是AP-1、NFAT和NF-κB，其是TCR激活以后T细胞主要表达的三个转录因子家族。
NF-κB家族由P50(前体为P105)、P52(前体为P100)、P65、c-Rel和RelB五个成员组成。这五个成员都具有一个N端Rel同源结构域RHD，负责其与DNA的结合及二聚化。经典NF-κB信号来源于RelA和p50异源二聚体及c-Rel和p50的异源二聚体；非经典的NF-κB信号来源于RelB和p52的异源二聚体。在RelA、c-Rel和RelB中，存在着转录激活区域，对基因表达起正向调节作用。而p50和p52两个蛋白不存在转录激活区域，所以他们的同型二聚体将会抑制转录。
NF-κB信号受多种膜受体调控，包括B淋巴受体等。在T细胞中，NF-κB转录因子主要接受T淋巴受体和肿瘤坏死因子受体TNFR家族信号，其中非经典NF-κB主要受TNFR信号调控(Philipson B I,O'Connor R S,May M J,et al.4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.Science signaling,2020)。
P52是非经典核因子-κB(non-canonical NF-κB)家族的转录因子，其前体蛋白是Nfkb2基因编码的p100。在非经典NF-κB信号未被激活时，p100会与NF-κB家族的另一个转录因子RelB形成牢固的二聚体，且无法入核；而当非经典NF-κB激活后，p100会被磷酸化后进而泛素化切割为p52，并与RelB结合为二聚体的形式入核，进而调控下游基因表达(Shao-cong Sun.The non-canonical NF-κB pathway in immunity and inflammation.NRI.2017)。具体而言，在肿瘤坏死因子受体未与配体结合时，NF-κB诱导激酶(NIK)会与肿瘤坏死因子受体相关因子(TRAF)和cIAP形成复合体并被泛素化，无法磷酸化下游IKKα，进而导致RelB与p100形成复合物阻止入核。而在肿瘤坏死因子受体与配体结合后，TRAF3会被招募并被泛素化，从而导致NIK被磷酸化并磷酸化下游的IKKα，最终导致p100被磷酸化并被切割为p52与RelB结合并入核。
嵌合抗原受体T细胞(CAR-T)治疗是近年来收获良好疗效的一种抗癌免疫疗法。不同于天然的T细胞识别肿瘤细胞的方式，CAR-T细胞对肿瘤细胞的识别不依赖于MHC分子。CAR分子包含三大部分：胞外区是来源于抗体的抗原识别结构域，负责识别靶 标抗原；跨膜区；胞内区是来源于T细胞受体的信号分子和共刺激信号分子，负责在接受刺激后传导T细胞激活信号。其工作原理如下：当CAR分子与其对应抗原结合的时候，CAR分子会发生聚集，从而局部磷酸化水平升高、激活下游信号，最终启动T细胞的效应功能、杀伤靶标肿瘤细胞。
T细胞受体(TCR)复合物分子含有多条链，TCRα链和TCRβ链负责识别MHC-多肽分子，其它6个CD3亚基与TCRα/β链结合，起信号转导的功能。天然TCR复合体中共含有10个ITAM信号序列，理论上能传导比CAR更强的信号。之前研究表明，TCR的信号虽然比CAR的信号传导得缓慢一些，但是TCR的信号更为持久。因此，利用天然TCR的信号传导功能，可构建出一种新型受体来缓解T细胞失能，使其能够更好地发挥抗实体肿瘤作用。TCR的胞外区和抗体的Fab结构域非常相似，因此可以把TCR可变区序列替换成抗体可变区序列，从而得到合成T细胞受体抗原受体(Snythetic TCR and Antigen Receptor,STAR)，其既具有抗体的特异性，也具有天然TCR的优越的信号传导功能，可以介导完全的T细胞激活。
然而，已有的包括CAR-T、TCR-T和STAR-T在内的治疗性T细胞在一些肿瘤特别是实体瘤的治疗上仍没有获得很好的效果，重要的原因包括在肿瘤微环境中功能受到抑制、细胞容易发生耗竭而凋亡等，这可能与下游信号通路性质有关。本领域仍然需要能克服这些缺陷的改进的治疗性免疫细胞，特别是治疗性T细胞。
发明简述
本发明至少提供以下实施方案：
实施方案1.一种经修饰的免疫细胞，其被修饰而过表达核因子-κB(NF-κB)家族转录因子。
实施方案2.实施方案1的经修饰的免疫细胞，其中所述免疫细胞选自T细胞、B细胞或NK细胞，优选是T细胞。
实施方案3.实施方案1或2的经修饰的免疫细胞，其中所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
实施方案4.实施方案1-3中任一项的经修饰的免疫细胞，其中所述经修饰的免疫细胞包含导入的编码所述NF-κB家族转录因子的核苷酸序列，由此过表达所述NF-κB家族转录因子。
实施方案5.实施方案4的经修饰的免疫细胞，其中编码所述NF-κB家族转录因子的核苷酸序列通过表达载体导入所述经修饰的免疫细胞，任选地，编码所述NF-κB家族转录因子的核苷酸序列在所述表达载体中与调控序列可操作地连接。
实施方案6.实施方案1-5中任一项的经修饰的免疫细胞，其中所述经修饰的免疫细胞还包含靶特异性受体。
实施方案7.实施方案6的经修饰的免疫细胞，其中所述经修饰的免疫细胞包含导 入的编码所述靶特异性受体的核苷酸序列，由此表达所述靶特异性受体。
实施方案8.实施方案7的经修饰的免疫细胞，其中编码所述靶特异性受体的核苷酸序列通过表达载体导入所述经修饰的免疫细胞，任选地，编码所述靶特异性受体的核苷酸序列在所述表达载体中与调控序列可操作地连接。
实施方案9.实施方案6-8中任一项的经修饰的免疫细胞，其中所述靶特异性受体选自嵌合抗原受体(CAR)和T细胞受体(TCR)。
实施方案10.实施方案9的经修饰的免疫细胞，其中所述靶特异性受体是经修饰的T细胞受体(TCR)，例如是合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)。
实施方案11.实施方案10的经修饰的免疫细胞，其中所述经修饰的TCR例如STAR衍生自αβTCR，且其中α链包含第一恒定区，β链包含第二恒定区。
实施方案12.实施方案11的经修饰的免疫细胞，其中所述第一恒定区是天然TCRα链恒定区，例如，天然人TCRα链恒定区或天然小鼠TCRα链恒定区。
实施方案13.实施方案11的经修饰的免疫细胞，其中所述第一恒定区是经修饰的TCRα链恒定区。
实施方案14.实施方案13的经修饰的免疫细胞，其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C。
实施方案15.实施方案13或14的经修饰的免疫细胞，其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
实施方案16.实施方案13-15中任一项的经修饰的免疫细胞，其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
实施方案17.实施方案11-16中任一项的经修饰的免疫细胞，其中所述第一恒定区包含SEQ ID NO:1、3、5、7和28之一所示氨基酸序列。
实施方案18.实施方案11-17中任一项的经修饰的免疫细胞，其中所述第二恒定区是天然TCRβ链恒定区，例如，天然人TCRβ链恒定区或天然小鼠TCRβ链恒定区。
实施方案19.实施方案11-17中任一项的经修饰的免疫细胞，其中所述第二恒定区是经修饰的TCRβ链恒定区。
实施方案20.实施方案19的经修饰的免疫细胞，其中所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
实施方案21.实施方案11-20中任一项的经修饰的免疫细胞，其中所述第二恒定区包含SEQ ID NO:2、4和6之一所示氨基酸序列。
实施方案22.实施方案11-21中任一项的经修饰的免疫细胞，其中所述第一恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V；且所述第二恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
实施方案23.实施方案22的经修饰的免疫细胞，其中所述第一恒定区包含SEQ ID NO:28所示氨基酸序列；所述第二恒定区包含SEQ ID NO:6所示氨基酸序列。
实施方案24.实施方案11-23中任一项的经修饰的免疫细胞，其中所述经修饰的TCR例如STAR衍生自αβTCR，且其中α链包含第一抗原结合区，β链包含第二抗原结合区。
实施方案25.实施方案24的经修饰的免疫细胞，其中所述第一抗原结合区和所述第二抗原结合区各自独立地或组合地特异性结合靶抗原。
实施方案26.实施方案25的经修饰的免疫细胞，其中所述靶抗原是疾病相关抗原，优选是癌症相关抗原，例如选自以下的癌症相关抗原：GPC3、CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜***、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、***酶(prostase)、***酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、***癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
实施方案27.实施方案24-26中任一项的经修饰的免疫细胞，其中所述第一抗原结合区包含特异性结合靶抗原的抗体的重链可变区，且所述第二抗原结合区包含所述抗体 的轻链可变区；或者，所述第一抗原结合区包含特异性结合靶抗原的抗体的轻链可变区，且所述第二抗原结合区包含所述抗体的重链可变区。
实施方案28.实施方案24-26中任一项的经修饰的免疫细胞，其中所述第一抗原结合区包含特异性结合靶抗原的单链抗体或单域抗体；和/或所述第二抗原结合区包含特异性结合靶抗原的单链抗体或单域抗体，
例如，所述单链抗体包含通过接头相连接的重链可变区和轻链可变区，所述接头例如是(G4S)n接头，其中n代表1-10的整数，优选地，n是1或3。
实施方案29.实施方案28的经修饰的免疫细胞，其中所述第一抗原结合区和所述第二抗原结合区结合相同的靶抗原。
实施方案30.实施方案28的经修饰的免疫细胞，其中所述第一抗原结合区和所述第二抗原结合区结合相同靶抗原的不同区域(如不同的表位)或结合不同的靶抗原。
实施方案31.一种药物组合物，其包含实施方案1-30中任一项的经修饰的免疫细胞，和药物可接受的载体。
实施方案32.实施方案1-30中任一项的经修饰的免疫细胞或实施方案31的药物组合物在制备用于在对象中治疗疾病例如癌症的药物中的用途。
实施方案33.一种在对象中治疗疾病例如癌症的方法，包括给所述对象施用治疗有效量的实施方案1-30中任一项的经修饰的免疫细胞或实施方案31的药物组合物。
实施方案34.一种在患有癌症的对象中使得患者显著缩小的方法，包括给所述对象施用治疗有效量的实施方案1-30中任一项的经修饰的免疫细胞或实施方案31的药物组合物。
实施方案35.一种延长患有癌症的对象生存时间的方法，包括给所述对象施用治疗有效量的实施方案1-30中任一项的经修饰的免疫细胞或实施方案31的药物组合物。
实施方案36.一种分离的核酸分子，其包含编码实施方案1-30中任一项所定义的NF-κB家族转录因子的核苷酸序列和/或编码实施方案1-30中任一项所定义的靶特异性受体的核苷酸序列。
实施方案37.一种表达载体，其包含与调控序列可操作连接的i)编码实施方案1-30中任一项所定义的靶特异性受体的核苷酸序列和/或ii)编码实施方案1-30中任一项所定义的NF-κB家族转录因子的核苷酸序列。
实施方案38.一种制备经修饰的免疫细胞的方法，包括
a)提供分离的免疫细胞；和
b)在所述免疫细胞中过表达NF-κB家族转录因子。
实施方案39.实施方案38的方法，其中所述免疫细胞选自T细胞、B细胞或NK细胞，优选是T细胞。
实施方案40.实施方案38或39的方法，其中所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
实施方案41.实施方案38-40中任一项的方法，其中所述方法通过将包含编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述免疫细胞来过表达所述NF-κB家族转录因子。
实施方案42.实施方案41的方法，其中所述NF-κB家族转录因子是p52，编码所述p52的核苷酸序列如SEQ ID NO:8所示。
实施方案43.实施方案38-42中任一项的方法，其中所述方法还包括c)将包含编码靶特异性受体的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码靶特异性受体的核苷酸序列的表达载体导入所述免疫细胞。
实施方案44.实施方案43的方法，其中所述靶特异性受体选自嵌合抗原受体(CAR)和T细胞受体(TCR)。
实施方案45.实施方案44的方法，其中所述靶特异性受体是经修饰的T细胞受体(TCR)，例如是合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)。
实施方案46.实施方案38-45中任一项的方法，其中所述方法还包括步骤
d)扩增所述免疫细胞。
实施方案47.一种提高T细胞增殖能力、提高T细胞效应存活时间、提高T细胞体内数量、诱导T细胞向效应T细胞分化、降低T细胞抑制性受体表达、提高T细胞群体中CD8+T细胞比例、或提高T细胞的靶杀伤能力的方法，所述方法包括：
a)提供分离的T细胞；和
b)在所述T细胞中过表达NF-κB家族转录因子。
实施方案48.实施方案47的方法，其中所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
实施方案49.实施方案48的方法，其中所述方法通过将包含编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述T细胞来过表达所述NF-κB家族转录因子。
实施方案50.实施方案49的方法，其中所述NF-κB家族转录因子是p52，编码所述p52的核苷酸序列如SEQ ID NO:8所示。
实施方案51.实施方案47-50中任一项的方法，其中所述方法还包括步骤
c)扩增所述T细胞。
实施方案51.一种试剂盒，其用于制备实施方案1-30中任一项的经修饰的免疫细胞，所述试剂盒包括实施方案36的核酸分子和/或实施方案37的表达载体，任选地，所述试剂盒还包含用于分离、培养和/或扩增免疫细胞如T细胞的试剂，和/或用于将所述核酸分子或表达载体导入细胞的制剂。
附图简述
图1.示出恒定区改造的STAR结构。
图2.示出共表达p52/RFP和STAR的载体结构。
图3.示出过表达p52增加STAR-T细胞对靶细胞A431的杀伤能力。
图4.示出过表达p52提高CD8+T细胞比例。
图5.示出过表达p52提高CD8+T的增值能力。
图6.示出抗原蛋白刺激5天后，过表达p52使得mut-STAR T细胞更多地向效应T细胞分化。
图7.在长期抗原刺激(7.5天)后，过表达p52促进mut-STAR T细胞增殖。
图8.长期抗原刺激(7.5天)后，过表达p52显著增加mut-STAR-T细胞对靶细胞的杀伤能力。
图9.ELISA结果显示过表达p52可以提高mut-STAR T细胞的IFN-γ和IL2的分泌，增强T细胞的效应功能。
图10.示出小剂量Raji血液瘤模型实验设计。
图11.示出小剂量Raji血液瘤模型实验中肿瘤大小监测结果。
图12.示出小剂量Raji血液瘤模型实验中肿瘤大小成像图。
图13.示出小剂量Raji血液瘤模型实验期间小鼠外周血中回输人T细胞计数。
图14.示出过量Raji血液瘤模型实验设计。
图15.示出过量Raji血液瘤模型实验期间小鼠外周血中回输人T细胞计数。
图16.示出过量Raji血液瘤模型实验期间小鼠外周血中回输人CD8+T细胞计数。
图17.示出过量Raji血液瘤模型实验期间小鼠生存曲线。
图18.A431皮下肿瘤模型实验示出过表达p52显著增加mut-STAR T细胞对肿瘤的抑制。
图19.A549皮下肿瘤模型实验示出过表达p52显著增加mut-STAR T细胞对肿瘤的抑制。
图20.皮下A431-NYESO-1肿瘤模型检测过表达p52对1G4-TCR T细胞的肿瘤抑制效果的影响。
图21.示出皮下A431-NYESO-1肿瘤模型中，T细胞输注24天后，过表达p52显著增加1G4-TCR T细胞的肿瘤抑制效果。
图22.示出皮下A431-NYESO-1肿瘤模型实验中肿瘤大小成像图。
图23.示出在一代CAR(zCAR)T细胞上过表达p52可以提高其靶细胞杀伤能力，且比BBzCAR强。
图24.示出在一代CAR(zCAR)T细胞上过表达p52可以提高其增殖能力。
发明详述
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为 本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达***表达时)会被保留，但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的多肽氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸的序列，相应地，其编码核苷酸序列也可以包含起始密码子；反之亦然。
如本文所用，“氨基酸编号参考SEQ ID NO:x”(SEQ ID NO:x为本文所列的某一具体序列)指的是所描述的具体氨基酸的位置编号是该氨基酸在SEQ ID NO:x上对应的氨基酸的位置编号。不同序列中的氨基酸的对应性可以根据本领域公知的序列比对方法确定。例如氨基酸对应性可以通过EMBL-EBI的在线比对工具来确定(https://www.ebi.ac.uk/Tools/psa/)，其中两个序列可以使用Needleman-Wunsch算法，使用默认参数来对齐。例如，一多肽从其N末端起第46位的丙氨酸与SEQ ID NO:x的第48位的氨基酸在序列比对中对齐，则该多肽中的该氨基酸在本文中也可以被描述为“在该多肽的第48位的丙氨酸，所述氨基酸位置参考SEQ ID NO:x”。在本发明中，涉及α链恒定区的氨基酸位置参考SEQ ID NO:3。在本发明中，涉及β链恒定区的氨基酸位置参考SEQ ID NO:4。
在一方面，本发明提供一种经修饰的免疫细胞，其被修饰而过表达核因子-κB(NF-κB)家族转录因子。
在本发明各方面的在一些实施方案中，所述经修饰的免疫细胞是分离的。
在本发明各方面的在一些实施方案，所述经修饰的免疫细胞是治疗性免疫细胞。例如经修饰的所述免疫细胞用于给有需要的对象施用而在所述对象中治疗疾病，例如肿瘤或感染性疾病。
本文所述的免疫细胞可以是各种不同的免疫细胞，包括但不限于T细胞、B细胞或NK细胞。
本文所述的免疫细胞例如T细胞或B细胞或NK细胞可以通过各种非限制性方法从许多非限制性来源获得，包括外周血单核细胞、骨髓、***组织、脐带血、胸腺组织、腹水、胸腔积液、脾组织和肿瘤。在本发明各方面的一些实施方案中，细胞可以衍生自 健康供体或来自诊断为癌症的患者。在本发明各方面的一些实施方案中，细胞可以是呈现不同表型特征的细胞的混合群体的一部分。例如，T细胞可以通过分离外周血单个核细胞(PBMC)，然后用特异性抗体活化、扩增获得。
在本发明各方面的一些实施方案中，所述免疫细胞例如T细胞衍生自对象的自体细胞。如本文所用，“自体”是指用于治疗对象的细胞、细胞系或细胞群源自所述对象。在一些实施方案中，所述免疫细胞例如T细胞衍生自异体细胞，例如源自与所述对象人类白细胞抗原(HLA)相容的供体。可以使用标准方案将来自供体的细胞转化为非同种异体反应性细胞，并根据需要进行复制，从而产生可以施用至一个或多个患者的细胞。
在本发明各方面的一些实施方案中，所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合。
在本发明各方面的一些实施方案中，所述NF-κB家族转录因子是非经典核因子-κB(non-canonical NF-κB)家族的转录因子。在一些实施方案中，所述非经典NF-κB家族转录因子选自RelB和p52。在一些优选实施方案中，所述非经典NF-κB家族转录因子是p52。
示例性的p52包含SEQ ID NO:9所示氨基酸序列或由SEQ ID NO:8所示核苷酸序列编码。在一些实施方案中，所述p52还涵盖其功能性变体，例如天然存在的功能性变体，例如所述p52的功能性变体包含与SEQ ID NO:9具有至少75％、至少80％、至少85％、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％、至少99.5％序列相同性的氨基酸序列，但保留SEQ ID NO:9的活性，例如与RelB结合并启动下游基因表达的活性。
在本发明的各个方面，在免疫细胞中过表达NF-κB家族转录因子可以通过将编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含所述编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述免疫细胞来实现。然而，也可以通过靶向修饰免疫细胞中内源NF-κB家族转录因子基因来实现，例如，可以通过靶向修饰所述基因的表达调控区实现所述转录因子的过表达。所述靶向修饰例如可以通过本领域已知的基因编辑技术，例如CRISPR技术、TALEN、ZFN等基因编辑技术实现。
在一些实施方案中，所述经修饰的免疫细胞包含导入的编码所述NF-κB家族转录因子的核苷酸序列，由此过表达所述NF-κB家族转录因子。
在一些实施方案中，编码所述NF-κB家族转录因子的核苷酸序列通过表达载体导入所述经修饰的免疫细胞。在一些实施方案中，编码所述NF-κB家族转录因子的核苷酸序列在所述表达载体中与调控序列可操作地连接。
本发明的“表达载体”可以是线性的核酸片段、环状质粒、病毒载体，或者可以是能够翻译的RNA(如mRNA)。在一些优选实施方案中，所述表达载体是病毒载体，例如慢病毒载体。
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的 核苷酸序列。表达调控元件指的是能够控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子、增强子和多腺苷酸化识别序列。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
在一些实施方案中，所述经修饰的免疫细胞还包含(如表达)“靶特异性受体”。在一些实施方案中，所述靶特异性受体在与靶特异性结合之后，能够介导所述免疫细胞的活化。
在一些实施方案中，所述经修饰的免疫细胞包含导入的编码所述靶特异性受体的核苷酸序列，由此表达所述靶特异性受体。
在一些实施方案中，编码所述靶特异性受体的核苷酸序列通过表达载体导入所述经修饰的免疫细胞。在一些实施方案中，编码所述靶特异性受体的核苷酸序列在所述表达载体中与调控序列可操作地连接。
在一些实施方案中，编码所述靶特异性受体的核苷酸序列和编码所述NF-κB家族转录因子的核苷酸序列在同一表达载体中被导入所述经修饰的免疫细胞。
在一些实施方案中，所述靶特异性受体是嵌合抗原受体(CAR)。
“嵌合型抗原受体(CAR)”又称人工T细胞受体、嵌合型T细胞受体、嵌合型免疫受体，是一种人工设计的单链受体，可以赋予免疫效应细胞某一种特异性。
所述CAR可以包含针对特定靶(如抗原)的细胞外靶(抗原)结合区。在一些实施方案中，所述靶(抗原)结合区是特异性结合靶抗原的单链抗体或单域抗体。
所述CAR还可以包括跨膜结构域和细胞内信号转导结构域。根据本发明的CAR的细胞内信号转导结构域负责细胞外靶结合区与靶结合之后的细胞内信号转导，导致激活免疫细胞和免疫应答。细胞内信号转导结构域具有激活表达CAR的免疫细胞的至少一种正常效应子功能的能力。
用于CAR的细胞内信号转导结构域可以是细胞质序列，例如但不限于T细胞受体和共同受体(它们一致地起作用从而在抗原受体接合之后启动信号转导)的细胞质序列，以及任何这些序列的衍生物或变体以及具有相同功能能力的任何合成序列。细胞内信号转导结构域包括两种不同类型的细胞质信号转导序列：那些启动抗原依赖性初级激活的序列，以及那些以抗原非依赖性方式作用以提供次级或共刺激信号的序列。初级细胞质信号转导序列可以包括称为ITAM的免疫受体酪氨酸激活基序的信号转导基序。本发明的CAR中使用的ITAM的非限制性实例可以包括衍生自TCRζ、FcRγ、FcRβ、FcRε、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。在一些实施方案中，本发明CAR的细胞内信号转导结构域包括CD3ζ信号转导结构域。在一些实施方案中，本发明的CAR的细胞内信号转导结构域还包括共刺激结构域(第二代或以上的 CAR)，例如41BB共刺激结构域或CD28共刺激结构域。
CAR在细胞的表面上表达。因此，CAR还可以包括跨膜结构域。本发明的CAR的合适跨膜结构域具有以下能力：(a)在细胞表面表达，优选免疫细胞，例如但不限于淋巴细胞或自然杀伤(NK)细胞，和(b)与配体结合结构域和细胞内信号转导结构域相互作用，用于指导免疫细胞对预定靶细胞的细胞应答。跨膜结构域可以衍生自天然来源或合成来源。跨膜结构域可以衍生自任何膜结合蛋白或跨膜蛋白。作为非限制性实例，跨膜结构域可以衍生自T细胞受体的亚基如α亚基、β亚基、γ或δ亚基，组成CD3复合物的多肽，IL-2受体的p55(α链)、p75(β链)或γ，Fc受体的亚基链，特别是Fcγ受体III或CD蛋白。或者，跨膜结构域可以是合成的，并且可以主要包括疏水性残基，如亮氨酸和缬氨酸。在一些实施方案中，所述跨膜结构域衍生自人CD8α链。跨膜结构域可以进一步包括位于细胞外配体结合结构域和所述跨膜结构域之间的铰链区域。所述铰链区域例如来自CD8、CD4或CD28的胞外区。
在一些优选实施方案中，所述CAR是不包含共刺激结构域的第一代CAR。例如，所述第一代CAR可以由细胞外配体结合结构域、铰链区、跨膜区和CD3ζ信号转导结构域组成。
在一些实施方案中，所述靶特异性受体是T细胞受体(TCR)。在一些优选实施方案中，所述靶特异性受体是经修饰的T细胞受体(TCR)。在一些实施方案中，所述TCR是αβTCR。在一些实施方案中，所述TCR是γδTCR。
在一些优选实施方案中，所述靶特异性受体是合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)。STAR衍生自天然TCR，可以通过将天然TCR的可变区替换为不同的给定靶结合区例如给定的抗体可变区来生成STAR。本发明所述的合成T细胞受体抗体受体(STAR)包括但不限于WO2021135178A1或WO2021223707A1中公开的那些。
在一些实施方案中，所述经修饰的TCR例如STAR衍生自αβTCR，且其中α链包含第一恒定区，β链包含第二恒定区。在一些实施方案中，所述α链还包含第一靶结合区，和/或所述β链还包含第二靶结合区。
在一些实施方案中，所述经修饰的TCR例如STAR的α链和/或β链在其C末端连接有至少一个功能结构域。在一些实施方案中，所述经修饰的TCR例如STAR的α链在其C末端连接有至少一个功能结构域。在一些实施方案中，所述经修饰的TCR例如STAR的β链在其C末端连接有至少一个功能结构域。在一些实施方案中，所述STAR的α链和β链在其C末端连接有至少一个功能结构域。
在一些实施方案中，所述经修饰的TCR例如STAR的α链和/或β链的天然胞内区被缺失。在一些实施方案中，所述经修饰的TCR例如STAR的α链的天然胞内区被缺失。在一些实施方案中，所述经修饰的TCR例如STAR的β链的天然胞内区被缺失。在一些实施方案中，所述经修饰的TCR例如STAR的α链和β链的天然胞内区被缺失。
在一些实施方案中，所述至少一个功能结构域通过直接或通过接头连接至所述天然 胞内区被缺失的α链和/或β链的C端。在一些实施方案中，所述至少一个功能结构域通过直接或通过接头连接至所述天然胞内区被缺失的α链的C端。在一些实施方案中，所述至少一个功能结构域通过直接或通过接头连接至所述天然胞内区被缺失的β链的C端。在一些实施方案中，所述至少一个功能结构域通过直接或通过接头连接至所述天然胞内区被缺失的α链和β链的C端。
在一些实施方案中，所述接头例如是(G 4S)n接头，其中n代表1-10的整数，优选地，n是选自1-6的整数，更优选n是2-5的整数，最优选n＝3。
在一些实施方案中，所述经修饰的TCR例如STAR中α链和/或β链可以连接至相同的或不同的功能结构域。
在一些实施方案中，所述功能结构域是外源功能结构域。在一些实施方案中，所述功能结构域是外源胞内结构域，例如在胞内起信号传导作用的结构域。
如本文所用，“外源”意指来自外来物种的蛋白或核酸序列，或者如果来自相同物种，则指通过蓄意的人为干预而从其天然形式发生了组成和/或位置的显著改变的蛋白或核酸序列。
如本文所用，“功能结构域”可以是共刺激分子的胞内结构域诸如CD40、OX40、ICOS、CD28、4-1BB、CD27、CD137的胞内结构域；也可以是共抑制分子的胞内结构域，例如TIM3、PD1、CTLA4、LAG3的胞内结构域；也可以是细胞因子受体如白细胞介素受体(如IL-2β受体、IL-7α受体或IL-21受体)、干扰素受体、肿瘤坏死因子超家族受体、集落刺激因子受体、趋化因子受体、生长因子受体或其他膜蛋白的细胞内结构域；或者胞内蛋白如NIK的结构域。功能结构域还可以是细胞因子受体胞内结构域直接或通过接头与人STAT5激活模块(SEQ ID NO:25)的融合物。在一些优选实施方案中，所述功能结构域是共刺激分子的胞内结构域，优选OX40或ICOS的胞内结构域，更优选OX40的胞内结构域。
示例性的CD40胞内结构域包含SEQ ID NO:16所示氨基酸序列。示例性的OX40胞内结构域包含SEQ ID NO:17所示氨基酸序列。示例性的ICOS胞内结构域包含SEQ ID NO:18所示氨基酸序列。示例性的CD28胞内结构域包含SEQ ID NO:19所示氨基酸序列。示例性的4-1BB胞内结构域包含SEQ ID NO:20所示氨基酸序列。示例性的CD27胞内结构域包含SEQ ID NO:21所示氨基酸序列。示例性的IL-2β受体胞内结构域包含SEQ ID NO:22所示氨基酸序列。示例性的IL-17α受体胞内结构域包含SEQ ID NO:23所示氨基酸序列。示例性的IL-21受体胞内结构域包含SEQ ID NO:24所示氨基酸序列。示例性的IL-2β受体胞内结构域与人STAT5激活模块的融合氨基酸序列示于SEQ ID NO:26。示例性的IL-17α受体胞内结构域与人STAT5激活模块的融合氨基酸序列示于SEQ ID NO:27。
在一些实施方案中，所述第一恒定区是天然TCRα链恒定区，例如，天然人TCRα链恒定区(示例性人TCRα链恒定区氨基酸序列示于SEQ ID NO:1)或天然小鼠TCRα链恒定区(示例性小鼠TCRα链恒定区氨基酸序列示于SEQ ID NO:3)。
在一些实施方案中，所述第一恒定区是经修饰的TCRα链恒定区。
在一些实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C。
在一些实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
在一些实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，其第6位的氨基酸如E被D取代，第13位的K被R取代，且第15-18位氨基酸被缺失。
在一些实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
在一些实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，其第6位的氨基酸如E被D取代，第13位的K被R取代，第15-18位氨基酸被缺失，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
在一些实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，缺失恒定区的天然胞内区，例如缺失第136-137位氨基酸。
在一些实施方案中，所述第一恒定区包含SEQ ID NO:1、3、5、7、28、29、31和33之一所示氨基酸序列。
在一些实施方案中，所述第二恒定区是天然TCRβ链恒定区，例如，天然人TCRβ链恒定区(示例性人TCRβ链恒定区氨基酸序列示于SEQ ID NO:2)或天然小鼠TCRβ链恒定区(示例性小鼠TCRβ链恒定区氨基酸序列示于SEQ ID NO:4)。
在一些实施方案中，所述第二恒定区是经修饰的TCRβ链恒定区。
在一些实施方案中，所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
在一些实施方案中，所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，其第3位的氨基酸如R被K取代，第6位的氨基酸如T被F取代，第9位的K被E取代，第11位的S被A取代，第12位的L被V取代，且第17、21-25位氨基酸被缺失。
在一些实施方案中，所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C，第3位的氨基酸如R被K取代，第6位的氨基酸如T被F取代，第9位的K被E取代，第11位的S被A取代，第12位的L被V取代，且第17、21-25位氨基酸被缺失。
在一些实施方案中，所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，缺失恒定区的天然胞内区，例如缺失第167-172位氨基酸。
在一些实施方案中，所述第二恒定区包含SEQ ID NO:2、4、6、30、32和34之一所示氨基酸序列。
在一些具体实施方案中，所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V；且所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
在一些具体实施方案中，所述经修饰的TCRα链恒定区包含SEQ ID NO:28所示氨基酸序列；所述经修饰的TCRβ链恒定区包含SEQ ID NO:6所示氨基酸序列。
如本文所用，“靶结合区”指的是能够结合(优选特异性结合)靶分子的结构域。在一些优选实施方案中，所述靶是抗原。因此，在一些实施方案中，所述靶结合区是“抗原结合区”。
在一些实施方案中，所述靶结合区(优选抗原结合区)独自或者和另一靶结合区(优选抗原结合区)组合可以特异性结合靶分子(优选靶抗原)。
在一些实施方案中，所述第一靶结合区(第一抗原结合区)和所述第二靶结合区(第二抗原结合区)各自独立地或组合地特异性结合靶抗原。
在一些实施方案中，所述抗原结合区衍生自特异性结合靶抗原的抗体。在一些实施方案中，抗原结合区还可以衍生自特异性受体，此时该受体的配体可以作为待靶向的抗原。例如所述特异性受体可以是天然T细胞受体。在一些实施方案中，所述抗原结合区包含来自天然T细胞受体的可变区。在一些实施方案中，抗原结合区还可以衍生自配体，特别是在旨在靶向的抗原是受体的情况下。
在一些实施方案中，所述靶抗原是疾病相关抗原，优选是癌症相关抗原，例如选自以下的癌症相关抗原：GPC3(磷脂酰肌醇蛋白聚糖3)、CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜***、α胎 儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、***酶(prostase)、***酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、***癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。在一些具体实施方案中，所述靶抗原是EGFR。在一些具体实施方案中，所述靶抗原是CD19。
在一些实施方案中，所述靶抗原是衍生自病原体的抗原或者被病原体侵染细胞的表面抗原，例如RSVF(预防呼吸道合胞病毒)、PA(吸入性炭疽)、CD4(HIV感染)等。
在一些实施方案中，所述靶抗原是一些引起疾病的细胞或者细胞所产生分泌的分子，例如CD3(涉及移植排斥)、CD25(涉及肾移植急性排斥)、C5(涉及阵发性睡眠性血红蛋白尿症)、IL-1β(冷吡啉相关的周期性综合征)、RANKL(涉及癌症相关骨损伤)、von Willebrand factor(涉及成人获得性血栓性血小板紫癜)、血浆激肽释放酶(涉及血管性水肿)、降钙素基因相关肽受体(涉及成年中偏头痛)、FGF23(涉及X连锁低磷血症)等。
所述抗原结合区可以衍生自一或多种已知抗体，包括任何商业可获得的抗体，如FMC63、利妥昔单抗(rituximab)、阿仑珠单抗(alemtuzumab)、依帕珠单抗(epratuzumab)、曲妥珠单抗(trastuzumab)、比伐珠单抗(bivatuzumab)、西妥昔单抗(cetuximab)、拉贝珠单抗(labetuzumab)、帕利珠单抗(palivizumab)、司韦单抗(sevirumab)、妥韦单抗(tuvirumab)、巴利昔单抗(basiliximab)、达克珠单抗(daclizumab)、英利昔单抗(infliximab)、奥马珠单抗(omalizumab)、依法珠单抗(efalizumab)、凯利昔单抗(Keliximab)、西利珠单抗(siplizumab)、那他珠单抗(natalizumab)、克立昔单抗(clenoliximab)、培马单抗(pemtumomab)、依屈洛单抗(Edrecolomab)、坎妥珠单抗(Cantuzumab)等。
在一些具体实施方案中，所述抗原结合区包含SEQ ID NO:10所示的重链可变区和/或SEQ ID NO:11所示的轻链可变区。在一些具体实施方案中，所述抗原结合区包含SEQ ID NO:12所示的重链可变区和/或SEQ ID NO:13所示的轻链可变区。
在一些实施方案中，所述第一抗原结合区包含特异性结合靶抗原的抗体的重链可变区，且所述第二抗原结合区包含所述抗体的轻链可变区；或者，所述第一抗原结合区包含特异性结合靶抗原的抗体的轻链可变区，且所述第二抗原结合区包含所述抗体的重链可变区。
在一些实施方案中，所述第一抗原结合区包含特异性结合靶抗原的单链抗体(例如scFv)或单域抗体；和/或所述第二抗原结合区包含特异性结合靶抗原的单链抗体或单域抗体。
在一些实施方案中，所述单链抗体(例如scFv)包含通过接头相连接的重链可变区和轻链可变区，所述接头例如是(G4S)n接头，其中n代表1-10的整数，优选地，n是1或3。
在一些实施方案中，所述第一抗原结合区和所述第二抗原结合区结合相同的靶抗原。
在一些实施方案中，所述第一抗原结合区和所述第二抗原结合区结合相同靶抗原的不同区域(如不同的表位)。
在一些实施方案中，所述第一抗原结合区和所述第二抗原结合区结合不同的靶抗原。
在另一方面，本发明提供一种药物组合物，其包含本发明的经修饰的免疫细胞，和药物可接受的载体。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。
在另一方面，本发明提供本发明的经修饰的免疫细胞或本发明的药物组合物在制备用于在对象中治疗疾病例如癌症的药物中的用途。
如本文所用“对象”是指患有或者易于患有可以通过本发明的细胞、方法、或药物组合物治疗的疾病(如癌症)的生物体。非限制性例子包括人、牛、大鼠、小鼠、狗、猴、山羊、绵羊、母牛、鹿，及其它非哺乳动物。在优选实施方案中，对象是人。
在另一方面，本发明提供一种在对象中治疗疾病例如癌症的方法，包括给所述对象施用治疗有效量的本发明的经修饰的免疫细胞或本发明的药物组合物。
如本文所用，“治疗有效量”或“治疗有效剂量”或“有效量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或细胞的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。例如，“有效量”的本发明的细胞或药物组合物优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的细胞或药物组合物优选地将肿瘤细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型***中评价。或者，也可以通过检查抑制肿瘤细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。
实际应用中，本发明药物组合物中细胞的剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择 的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物的活性，给药途径，给药时间，应用的特定化合物的***速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
根据本发明的经修饰的免疫细胞或药物组合物或药物的施用可以以任何方便的方式进行，包括通过注射、输注、植入或移植。本文所述的细胞或组合物施用可以通过静脉内、淋巴内、皮内、肿瘤内、髓内、肌内或腹膜内施用。在一个实施方案中，本发明的细胞或组合物优选通过静脉内注射施用。
在本发明各个方面的实施方案中，所述疾病例如是癌症，所述癌症的实例包括但不限于肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、***、子宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、***癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、***癌、***癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌***癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、***癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经***(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
在一些实施方案中，所述治疗使得患有癌症的对象中的肿瘤显著缩小。例如肿瘤缩小至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。
在一些实施方案中，所述治疗延长(如显著延长)患有癌症的对象的生存时间。例如生存时间延长至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更长。在本发明各个方面的实施方案中，所述疾病例如是病原体感染，所述病原体的实例包括但不限于呼吸道合胞病毒、炭疽杆菌、人类免疫缺陷病毒等。
在本发明各个方面的实施方案中，所述疾病例如是心血管、糖尿病、神经性疾病、抗移植后排斥、或一些其他疾病。
在另一方面，本发明提供一种分离的核酸分子，其包含编码本文所述NF-κB家族转录因子的核苷酸序列和/或编码本文所述靶特异性受体的核苷酸序列。
在另一方面，本发明提供一种表达载体，其包含与调控序列可操作连接的i)编码本文所述靶特异性受体的核苷酸序列和/或ii)编码本文所述NF-κB家族转录因子的核苷酸序列。
在一些实施方案中，所述表达载体包含与调控序列可操作连接的i)编码所述靶特异 性受体的核苷酸序列和ii)编码所述NF-κB家族转录因子的核苷酸序列，且i)编码所述靶特异性受体的核苷酸序列和ii)编码所述NF-κB家族转录因子的核苷酸序列通过内部核糖体进入位点(IRES)连接，从而实现所述靶特异性受体和所述NF-κB家族转录因子的共表达。
在一些实施方案中，所述靶特异性受体是TCR，如经修饰的TCR，优选STAR，且编码所述NF-κB家族转录因子的核苷酸序列在同一读码框内的i)编码所述α链的核苷酸序列、ii)编码所述β链的核苷酸序列和iii)位于i)和ii)之间的编码自裂解肽的核苷酸序列。编码所述α链的核苷酸序列可以位于编码所述β链的核苷酸序列的5’端或3’端。
如本文所用“自裂解肽”意指可以在细胞内实现自剪切的肽。例如，所述自裂解肽可以包含蛋白酶识别位点，从而被细胞内的蛋白酶识别并特异性切割。或者，所述自裂解肽可以是2A多肽。2A多肽是一类来自病毒的短肽，其自切割发生在翻译期间。当用2A多肽连接两种不同目的蛋白在同一读码框表达时，几乎以1:1的比例生成两种目的蛋白。常用的2A多肽可以是来自猪捷申病毒(porcine techovirus-1)的P2A、来自明脉扁刺蛾β四体病毒(Thosea asigna virus)的T2A、马甲型鼻病毒(equine rhinitis A virus)的E2A和来自***病毒(foot-and-mouth disease virus)的F2A。其中P2A的切割效率最高，因此是优选的。本领域也已知多种这些2A多肽的功能性变体，这些变体也可以用于本发明。通过使用自裂解肽可以实现α链和β链的共表达。
在另一方面，本发明提供本发明所述核酸分子或表达载体在制备本发明所述的经修饰的免疫细胞中的用途。
在另一方面，本发明提供一种制备经修饰的免疫细胞(例如本发明上文所述的经修饰的免疫细胞)的方法，包括
a)提供分离的免疫细胞；和
b)在所述免疫细胞中过表达NF-κB家族转录因子。
在一些实施方案中，所述免疫细胞选自T细胞、B细胞或NK细胞，优选是T细胞。
在一些实施方案中，所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
在一些实施方案中，所述方法通过将包含编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述免疫细胞来过表达所述NF-κB家族转录因子。
在一些实施方案中，所述NF-κB家族转录因子是p52。在一些实施方案中，编码所述p52的核苷酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述方法还包括c)将包含编码本文靶特异性受体的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码靶特异性受体的核苷酸序列的表达载体导入所述免疫细胞。
在一些实施方案中，所述靶特异性受体选自嵌合抗原受体(CAR)和T细胞受体(TCR)。在一些实施方案中，所述靶特异性受体是经修饰的T细胞受体(TCR)，例如是 合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)。在一些实施方案中，所述靶特异性受体是上文所定义的靶特异性受体。
在一些实施方案中，所述步骤b)在步骤c)之前进行。在一些实施方案中，所述步骤c)在步骤b)之后进行。
在一些优选实施方案中，所述步骤b)和步骤c)同时进行。例如，可以将包含与调控序列可操作连接的i)编码所述NF-κB家族转录因子的核苷酸序列的核酸分子，和ii)编码所述靶特异性受体的核苷酸序列的表达载体导入所述免疫细胞。
核酸分子或表达载体可以通过任何适当的方法引入细胞，包括电穿孔；使用氯化钙、氯化铷、磷酸钙、DEAE-葡聚糖或其他物质的转染；微粒轰击；脂质体转染；和感染(例如，表达构建体是病毒)。
在一些优选实施方案中，所述表达载体是病毒载体。在一些更优选实施方案中，所述表达载体是慢病毒载体。
提供分离的免疫细胞例如T细胞的步骤可以通过本领域已知的分离免疫细胞的方法来进行。例如，可以利用商品化试剂盒从对象的外周血中分离免疫细胞例如T细胞。合适的试剂盒包括但不限于EasySep human T cell enrichment kit(Stemcell Technologies)。如上所述，分离的免疫细胞例如T细胞并不一定是均质的，其可以是不同细胞的混合群体，优选地，在所述群体中期望的免疫细胞例如T细胞被富集。
本发明的免疫细胞例如T细胞可以在任何修饰步骤之前或之后被活化和扩增。免疫细胞可以在体外或体内扩增。
因此，在一些实施方案中，所述方法还包括步骤
d)扩增所述免疫细胞。
在一些实施方案中，所述步骤d)在在步骤b)之前和/或之后进行。在一些实施方案中，所述步骤d)在在步骤c)之前和/或之后进行。
在另一方面，本发明提供一种提高免疫细胞如T细胞增殖能力、提高免疫细胞如T细胞效应存活时间、提高免疫细胞如T细胞体内数量、诱导免疫细胞如T细胞向效应免疫细胞如效应T细胞分化、降低免疫细胞如T细胞抑制性受体表达、提高免疫细胞如T细胞群体中CD8+T细胞比例、或提高免疫细胞如T细胞的靶杀伤能力的方法，所述方法包括：
a)提供分离的免疫细胞如T细胞；和
b)在所述免疫细胞如T细胞中过表达NF-κB家族转录因子。
在一些实施方案中，所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
在一些实施方案中，所述方法通过将包含编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述免疫细胞如T细胞来过表达所述NF-κB家族转录因子。
在一些实施方案中，所述NF-κB家族转录因子是p52。在一些实施方案中，编码所 述p52的核苷酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述方法还包括步骤
c)扩增所述免疫细胞如T细胞。
通常，T细胞可以例如通过与刺激CD3TCR复合物和T细胞表面上的共刺激分子以产生T细胞活化信号的试剂接触来扩增。例如，可以使用诸如钙离子载体A23187、佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)、或有丝***凝集素如植物血凝素(PHA)的化学品来产生T细胞的活化信号。在一些实施方案中，T细胞可以通过在体外与例如抗CD3抗体或其抗原结合片段、或固定在表面上的抗CD2抗体接触被活化，或通过与蛋白激酶C激活剂(例如，苔藓抑素)连同钙离子载体的接触来活化。例如，在适合于刺激T细胞增殖的条件下，T细胞可与抗CD3抗体和抗CD28抗体接触。适用于T细胞培养的条件包括可能含有增殖和活力所必需的因子的合适培养基(例如Minimal Essential Media或RPMI Media 1640、或X-vivo 5、(Lonza))，其中必需的因子包括血清(例如胎牛或人类血清)、白介素-2(IL-2)、胰岛素、IFN-γ、IL-4、IL-7、GM-CSF、IL-10、IL-2、IL-15、TGFβ和TNF，或本领域技术人员已知的用于细胞生长的添加剂。其它用于细胞生长的添加剂包括但不限于表面活性剂、人血浆蛋白粉、和还原剂如N-乙酰-半胱氨酸和2-巯基乙酸。培养基可以包括RPMI 1640、A1M-V、DMEM、MEM、a-MEM、F-12、X-Vivo 1和X-Vivo 20、Optimizer、氨基酸、丙酮酸钠和维生素、无血清或适量补充的血清(或血浆)或一组明确的激素、和/或一定量的足以使T细胞生长和扩增的细胞因子。T细胞可以保持在支持生长所必需的条件下，例如适当的温度(例如37℃)和环境(例如，空气加5％CO 2)。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞的增殖能力提高至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更多。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞在回输至体内后的存活时间延长至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更多。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞在回输至体内后的数量增加至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更多。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞中效应细胞如效应T细胞的比例增加至少约10％，优选至少 约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更多。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞中抑制性受体的表达被降低至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞中CD8+T细胞比例的比例增加至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更多。
与不过表达所述NF-κB家族转录因子的相应对照免疫细胞如对照T细胞相比，本发明的免疫细胞如T细胞的靶杀伤能力增加至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，更优选至少约100％，更优选至少约150％，更优选至少约200％，更优选至少约300％或更多。
在另一方面，本发明还提供一种试剂盒，其用于制备本发明的经修饰的免疫细胞。本发明的试剂盒包括本发明的核酸分子和/或本发明的表达载体。所述试剂盒还可以包含用于分离、培养和/或扩增免疫细胞如T细胞的试剂、用于将核酸分子或表达载体导入细胞的制剂等。
实施例
实施例1、STAR的改进
1.1 STAR的原型设计
B细胞产生的分泌抗体(Antibody，Ab)或B细胞受体(BCR)与T细胞受体(TCR)在基因结构、蛋白结构和空间构象上有很大的相似性。抗体和TCR都是由可变区和恒定区组成，其中可变区起到抗原识别和结合的作用，而恒定区结构域起到结构互作和信号转导的作用。通过把TCRα和β链(或TCRγ和δ链)的可变区替换为抗体的重链可变区(VH)和轻链可变区(VL)，可以构建出一种人工合成的嵌合分子，称为合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR/WT-STAR)，其结构见图1左侧的WT-STAR。
STAR分子有两条链，第一条链是抗原识别序列(如抗体的重链可变区VH)与T细胞受体α链(TCRα)的恒定区(Cα)相融合而得，第二条链是抗原识别序列(如抗体的轻链可变区VL)与T细胞受体β链(TCRβ)的恒定区(Cβ)相融合而得。该构建体中抗原识别结构域(如VH、VL或scFv等)与恒定区结构域(TCRα、β、γ和δ的恒定区)可以进行排列组 合，组成多种构型不同但功能类似的构建体。
STAR分子的第一、二条链在T细胞内表达后，会在内质网中与细胞内源的CD3εδ、CD3γε、CD3ζζ链相结合形成8个亚基的复合物，并以复合物的形式展示在细胞膜表面。免疫受体酪氨酸活化基序(Immunoreceptor Tyrosine-based Activation Motif，ITAM)是TCR分子中起到信号转导作用的基序，其保守序列为YxxL/V。CD3ε、δ、γ、ε链的胞内区含有1个ITAM序列，CD3ζ链的胞内区含有3个ITAM序列，所以一个完整STAR复合体共含有10个ITAM序列。当STAR受体的抗原识别序列与其特异性抗原结合后，胞内的ITAM序列会逐次被磷酸化，进而激活下游信号通路，激活NF-κΒ、NFAT和AP-1等转录因子，引发T细胞激活，产生效应功能。本发明人之前的研究表明，与常规的嵌合抗原受体CAR相比，STAR能够更好地激活T细胞，并且在没有抗原刺激时的背景激活显著降低，因而具有显著的优势(参见中国发明专利申请号:201810898720.2)。然而，仍希望对STAR进行进一步的改进。
1.2.突变体STAR(mut-STAR)的设计
STAR原型设计使用的是人类来源的TCRα/β链(或TCRγ和δ链)的恒定区序列(野生型人TCRα恒定区，SEQ ID NO:1；野生型人TCRβ恒定区，SEQ ID NO:2)。由于人源、灵长类动物来源和鼠源TCRα/β链的恒定区序列(野生型鼠TCRα恒定区，SEQ ID NO:3；野生型鼠TCRβ恒定区，SEQ ID NO:4)的功能保守性较高，且关键氨基酸序列相同，所以可以相互替换。
STAR分子被转导入T细胞中后，会通过恒定区与T细胞内源的TCR发生错配。这一错配问题一方面减少了STAR分子正确配对的效率、削弱其功能，另一方面增加了错配产生未知特异性的可能、增加了安全性风险。为解决这一问题，本发明人将STAR分子的恒定区替换为鼠源序列，以增强STAR分子转入人类T细胞后的功能。为进一步优化STAR分子的设计，本发明人对STAR分子进行半胱氨酸点突变，以引入分子间二硫键，增强STAR分子两条链间的相互配对，减少与内源TCR的错配。具体为于TCRα链恒定区将48位苏氨酸T突变为半胱氨酸C(mouse TCRαC-Cys，SEQ ID NO:5)，于TCRβ链恒定区将56位丝氨酸S突变为半胱氨酸C(mouse TCRβC-Cys，SEQ ID NO:6)。这两个新增的半胱氨酸会在STAR的两条链间形成二硫键，减少STAR两条链与内源性TCR链的错配，帮助STAR分子形成更加稳定的复合体，进而获得更好的功能。此外，为进一步优化STAR分子的设计，本发明人对STAR分子的跨膜区进行疏水氨基酸替换，以增加STAR分子的稳定性，帮助其发挥更持久功能。具体为在TCRα链恒定区跨膜区的111位至119位氨基酸区域内进行3个氨基酸位点的突变：将112位丝氨酸S变成亮氨酸L、114位甲硫氨酸M变成异亮氨酸I、115位甘氨酸G变成颉氨酸V。该区域整体氨基酸序列由LSVMGLRIL变为LLVIVLRIL，该修饰称作mouse TCRαC-TM9，产生的α链恒定区序列为SEQ ID NO:7。这一设计增加了跨膜区的疏水性，抵消了TCR跨膜区携带正电荷所导致的不稳定性，使得STAR分子能在细胞膜上更为稳定地存在，进 而获得更好的功能，其结构见图1右侧的Mut-STAR。包含恒定区鼠源序列、恒定区半胱氨酸取代和恒定区疏水氨基酸替换的STAR称作mut-STAR。
实施例2、过表达非经典NF-κB转录因子p52的STAR-T细胞的体外效果
根据之前的研究，将CAR-T或TCR-T与一些共刺激因子(来自TNFR家族受体的41BB、OX40等)相连或过表达共刺激因子可以提高T细胞的增殖能力。然而，T细胞内的调控非常复杂，过表达共刺激因子后的体外信号经过体内的复杂调控后可能无法有效地传递到细胞核内，导致其信号被削弱和减少。本发明则直接过表达TNFR家族下游最主要的转录因子来加强由共刺激因子提供的信号和功能，这样可以绕过细胞内的复杂调控路径，直接作用于细胞核来增强T细胞的增殖和效应能力。
为进一步提升STAR-T或CAR-T细胞的如下功能：(1)提高T细胞增殖能力；(2)提高T细胞效应存活时间；(3)提高T细胞体内数量；(4)诱导T细胞向效应T细胞分化；(5)降低T细胞抑制性受体表达；(6)提高CD8+T细胞占比；(7)使得患者肿瘤显著缩小；(8)延长肿瘤患者生存时间；(9)提高T细胞肿瘤杀伤能力以及浸润到肿瘤微环境并进行高效杀伤靶细胞的能力，本发明人对STAR-T或CAR-T细胞进行了进一步的改造。通过在转导STAR复合物进入T细胞的同时过表达非经典NF-κB转录因子p52，启动mut-STAR细胞的促增殖、存活相关基因的转录，提高STAR-T的临床响应率，从而实现持久的疗效。
所述的非经典NF-κB转录因子指的是由基因Nfkb2编码的非全长蛋白p52(其编码序列为SEQ ID NO:8，氨基酸序列为SEQ ID NO:9)。在非经典NF-κB通路未被激活时，Nfkb2基因全长表达蛋白p100会与RelB蛋白结合，抑制RelB蛋白入核发挥功能。而当特定TNF受体家族成员受到配体的刺激(包括：LTβR、CD40、CD27、CD30、BAFF-R等)，这些成员通过募集TRAF2和TRAF3，抑制TFAF3对于NF-κB诱导激酶(NIK)的磷酸化，进而使得NIK将IKKα磷酸化，磷酸化的IKKα对p100进行磷酸化并进一步经蛋白酶体加工成p52，与RelB形成异二聚体入核，并诱导下游基因表达。
2.1载体构建
本实施例中所用的载体，包括病毒载体、质粒载体等均购买自商业公司或由商业公司合成，并获得这些载体的全长序列，已知明确的酶切位点。本实施例中使用的例如STAR的可变区、TCR恒定区、p52编码序列、标签序列以及接头(linker)等的基因片段均来源于商业化公司合成，并通过PCR手段连接。
本实施例中使用包含SEQ ID NO:10所示VH和SEQ ID NO:11所示VL(衍生自靶向EGFR的西妥昔单抗Cetuximab)的mut-STAR以及相应的CAR(BBz或28z CAR)，或包含SEQ ID NO:12所示VH和SEQ ID NO:13所示VL(衍生自靶向CD19的抗体FMC63)的mut-STAR以及相应的CAR(BBz或28z CAR)，验证过表达SEQ ID NO:9所示的p52对STAR-T或CAR-T细胞的优化。使用氨基酸序列如SEQ ID NO:14所示的红色荧光蛋 白(RFP)取代p52作为对照组。STAR载体结构如图2所示，其中通过使用核糖体进入位点IRES启动p52的过表达，实现mut-STAR和p52这两个片段的共表达。同时，在mut-STAR在N端加入一段myc-tag(SEQ ID NO:15：GAGCAGAAACTCATCTCTGAAGAGGATCTG)，并用一段6个bp的序列(GGATCT)连接mut-STAR和myc-tag片段。
实施例中使用的慢病毒载体为pHAGE-EF1α-IRES-RFP，通过限制性内切酶Not I/Nhe I获得线性载体，目的基因片段通过合成和PCR方法获得，通过同源重组方法获得完整载体。
用于转导T细胞的慢病毒包装、T细胞的培养和转导参照国际专利公开WO 2021/135178A1。
2.2过表达p52对STAR-T细胞功能的影响
T细胞与靶细胞体外共培养
靶细胞A431为贴壁细胞，原代T细胞为悬浮细胞，共孵育时取相应细胞数量使用靶细胞培养基混匀后离心培养。具体步骤为:利用包装并纯化好的mut-STAR和mut-STAR-p52病毒感染原代T细胞，在共培养前使用流式分选出感染阳性的细胞用于体外检测的实验。靶细胞与原代T细胞以1:2或2:1进行共孵育，在共孵育1天后检测T细胞对靶细胞的杀伤情况。
用乳酸脱氢酶LDH试剂盒检测细胞杀伤能力，靶细胞在死亡后会释放体内的LDH进入细胞悬液中。取共孵育后的细胞悬液以2500rpm/min离心去除沉淀，取50uL每孔的上清液加入LDH检测试剂50uL，反应15min后，加入50uL终止试剂，并使用酶标仪在波长为490处进行检测。
结果如图3和表1所示。发现在STAR-T细胞中过表达p52提高T细胞的杀伤能力，无长时间刺激即可表现出良好杀伤效果。
表1.不同T细胞的杀伤能力
E:T ratio Mock STAR STAR-p52
2:1 4.925485 29.50344 40.29148
1:2 0.70408 21.62349 27.06441
靶抗原刺激T细胞
靶抗原EGFR为细胞表面蛋白，可直接用于T细胞的激活，以检测T细胞的功能。通常加入1×10 5/孔的阳性T细胞，离心，激活24h后收集细胞悬液或培养液上清检测T细胞功能，或激活2.5天、5天或7.5天后检测T细胞的增值能力、记忆T细胞分群以及对靶细胞的杀伤。
图4和表2的结果显示过表达p52可以促进CD8+T细胞比例变多。图5的结果显示在STAR-T细胞中过表达p52可以提高CD8+T的更好的增殖能力。图6和表3的结果显示抗原蛋白刺激5天后，过表达p52可以使得mut-STAR T细胞更多地向效应T细 胞分化。图7的结果显示在长时抗原刺激(7.5天)后，过表达p52可以使mut-STAR细胞增殖数量更多。图8和表4的结果显示长期受抗原刺激的T细胞(7.5天)后，检测T细胞对靶细胞的杀伤效果。发现STAR-p52比STAR有显著提高的杀伤能力。
表2.CD4+T细胞及CD8+T细胞数量比较
  CD4+ CD8+
BB 29.8 68
STAR-p52 35.2 61.2
STAR 44.9 51.8
mock 46.1 52.8
表3.CD62L+T细胞数量比较
  Mock STAR STAR-p52
Day0 65.1 70.6 71.5
Day5 31.8 68.5 54.3
表4.T细胞杀伤能力比较
E:T ratio Mock STAR STAR-p52
1:1 -0.278 8.4475 35.0395
1:2 4.985 8.4475 15.6495
T细胞分泌细胞因子分析
T细胞激活过程中会释放大量细胞因子，以帮助T细胞杀伤靶细胞或者促进T细胞自身的扩增，常见的有IFN-γ、IL-2。T细胞经过与靶细胞或者抗原刺激后，收集T细胞，离心，取上清。使用试剂盒按制造商说明通过ELISA测定TNF-α、IFN-γ、IL-2的量。TNF-α、IFN-γ、IL-2 ELISA试剂盒使用的为Human IL-2 Uncoated ELISA、Human TNF-αUncoated ELISA、Human IFN-γUncoated ELISA(货号分别为88-7025、88-7346、88-7316)。
图9的ELISA结果显示过表达p52可以提高mut-STAR T细胞的IFN-γ和IL2的分泌，增强T细胞的效应功能。
实施例3、过表达非经典NF-κB转录因子p52的STAR-T细胞的体内效果
1)Raji血液瘤模型
a.小剂量Raji血液瘤模型
本实验采用人类Burkitt’s淋巴瘤细胞系Raji细胞对NSG免疫缺陷型小鼠进行异种移植。Raji细胞为通过慢病毒载体表达了荧光素酶(luciferase)基因的细胞株，在小鼠体内通过荧光素化学发光、活体成像的方式实时监测Raji肿瘤的发展变化。具体实验设计见图10。
实验结果见图11-13。结果显示过表达p52显著增强STAR-T细胞的肿瘤抑制能力，使得肿瘤显著缩小(图11和12)，增加STAR-T细胞在小鼠体内的持续时间(图13)。
b.过量Raji血液瘤模型
具体实验设计见图14。结果如图15-17。结果显示，p52过表达显著增加小鼠外周血中的人T细胞计数(图15)，特别是CD8+T细胞计数(图16)，并且显著增加荷瘤小鼠的存活时间(图17)。
2)A431和A549皮下肿瘤模型
此外，还在利用A431细胞系和A549细胞系的皮下肿瘤模型中评估过表达p52对STAR-T细胞的影响。
结果如图18和图19所示。过表达p52显著增加STAR-T对A431肿瘤细胞和A549肿瘤细胞的抑制。
实施例4、过表达非经典NF-κB转录因子p52的对TCR-T和CAR-T的影响
首先在皮下A431-NYESO-1肿瘤模型中测试过表达p52对本领域已知的针对NY-ESO-1的1G4-TCR的影响。
结果如图20-22所示。结果显示，过表达p52能够显著增加1G4-TCR-T细胞在体内对表达NY-ESO-1的A431肿瘤细胞的抑制作用。
然后，还在小鼠模型上测试了过表达p52对zCAR(一代CAR)的影响。结果如图23和24所示。过表达p52可以提高zCAR的靶细胞杀伤能力，甚至强于BBzCAR(图23)。p52可以提高zCAR的增殖能力(图24)。
Figure PCTCN2022104785-appb-000001
Figure PCTCN2022104785-appb-000002
Figure PCTCN2022104785-appb-000003
Figure PCTCN2022104785-appb-000004

Claims (52)

  1. 一种经修饰的免疫细胞，其被修饰而过表达核因子-κB(NF-κB)家族转录因子。
  2. 权利要求1的经修饰的免疫细胞，其中所述免疫细胞选自T细胞、B细胞或NK细胞，优选是T细胞。
  3. 权利要求1或2的经修饰的免疫细胞，其中所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
  4. 权利要求1-3中任一项的经修饰的免疫细胞，其中所述经修饰的免疫细胞包含导入的编码所述NF-κB家族转录因子的核苷酸序列，由此过表达所述NF-κB家族转录因子。
  5. 权利要求4的经修饰的免疫细胞，其中编码所述NF-κB家族转录因子的核苷酸序列通过表达载体导入所述经修饰的免疫细胞，任选地，编码所述NF-κB家族转录因子的核苷酸序列在所述表达载体中与调控序列可操作地连接。
  6. 权利要求1-5中任一项的经修饰的免疫细胞，其中所述经修饰的免疫细胞还包含靶特异性受体。
  7. 权利要求6的经修饰的免疫细胞，其中所述经修饰的免疫细胞包含导入的编码所述靶特异性受体的核苷酸序列，由此表达所述靶特异性受体。
  8. 权利要求7的经修饰的免疫细胞，其中编码所述靶特异性受体的核苷酸序列通过表达载体导入所述经修饰的免疫细胞，任选地，编码所述靶特异性受体的核苷酸序列在所述表达载体中与调控序列可操作地连接。
  9. 权利要求6-8中任一项的经修饰的免疫细胞，其中所述靶特异性受体选自嵌合抗原受体(CAR)和T细胞受体(TCR)。
  10. 权利要求9的经修饰的免疫细胞，其中所述靶特异性受体是经修饰的T细胞受体(TCR)，例如是合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)。
  11. 权利要求10的经修饰的免疫细胞，其中所述经修饰的TCR例如STAR衍生自αβTCR，且其中α链包含第一恒定区，β链包含第二恒定区。
  12. 权利要求11的经修饰的免疫细胞，其中所述第一恒定区是天然TCRα链恒定区，例如，天然人TCRα链恒定区或天然小鼠TCRα链恒定区。
  13. 权利要求11的经修饰的免疫细胞，其中所述第一恒定区是经修饰的TCRα链恒定区。
  14. 权利要求13的经修饰的免疫细胞，其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C。
  15. 权利要求13或14的经修饰的免疫细胞，其中所述经修饰的TCRα链恒定区衍 生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
  16. 权利要求13-15中任一项的经修饰的免疫细胞，其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
  17. 权利要求11-16中任一项的经修饰的免疫细胞，其中所述第一恒定区包含SEQ ID NO:1、3、5、7和28之一所示氨基酸序列。
  18. 权利要求11-17中任一项的经修饰的免疫细胞，其中所述第二恒定区是天然TCRβ链恒定区，例如，天然人TCRβ链恒定区或天然小鼠TCRβ链恒定区。
  19. 权利要求11-17中任一项的经修饰的免疫细胞，其中所述第二恒定区是经修饰的TCRβ链恒定区。
  20. 权利要求19的经修饰的免疫细胞，其中所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
  21. 权利要求11-20中任一项的经修饰的免疫细胞，其中所述第二恒定区包含SEQ ID NO:2、4和6之一所示氨基酸序列。
  22. 权利要求11-21中任一项的经修饰的免疫细胞，其中所述第一恒定区衍生自小鼠TCRα链恒定区，其相对于野生型小鼠TCRα链恒定区，在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C，在第112位的氨基酸例如丝氨酸S被变成亮氨酸L，在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I，在第115位的氨基酸例如甘氨酸G被变成颉氨酸V；且所述第二恒定区衍生自小鼠TCRβ链恒定区，其相对于野生型小鼠TCRβ链恒定区，在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
  23. 权利要求22的经修饰的免疫细胞，其中所述第一恒定区包含SEQ ID NO:28所示氨基酸序列；所述第二恒定区包含SEQ ID NO:6所示氨基酸序列。
  24. 权利要求11-23中任一项的经修饰的免疫细胞，其中所述经修饰的TCR例如STAR衍生自αβTCR，且其中α链包含第一抗原结合区，β链包含第二抗原结合区。
  25. 权利要求24的经修饰的免疫细胞，其中所述第一抗原结合区和所述第二抗原结合区各自独立地或组合地特异性结合靶抗原。
  26. 权利要求25的经修饰的免疫细胞，其中所述靶抗原是疾病相关抗原，优选是癌症相关抗原，例如选自以下的癌症相关抗原：GPC3、CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋 白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜***、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、***酶(prostase)、***酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、***癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
  27. 权利要求24-26中任一项的经修饰的免疫细胞，其中所述第一抗原结合区包含特异性结合靶抗原的抗体的重链可变区，且所述第二抗原结合区包含所述抗体的轻链可变区；或者，所述第一抗原结合区包含特异性结合靶抗原的抗体的轻链可变区，且所述第二抗原结合区包含所述抗体的重链可变区。
  28. 权利要求24-26中任一项的经修饰的免疫细胞，其中所述第一抗原结合区包含特异性结合靶抗原的单链抗体或单域抗体；和/或所述第二抗原结合区包含特异性结合靶抗原的单链抗体或单域抗体，
    例如，所述单链抗体包含通过接头相连接的重链可变区和轻链可变区，所述接头例如是(G4S)n接头，其中n代表1-10的整数，优选地，n是1或3。
  29. 权利要求28的经修饰的免疫细胞，其中所述第一抗原结合区和所述第二抗原结合区结合相同的靶抗原。
  30. 权利要求28的经修饰的免疫细胞，其中所述第一抗原结合区和所述第二抗原结合区结合相同靶抗原的不同区域(如不同的表位)或结合不同的靶抗原。
  31. 一种药物组合物，其包含权利要求1-30中任一项的经修饰的免疫细胞，和药物可接受的载体。
  32. 权利要求1-30中任一项的经修饰的免疫细胞或权利要求31的药物组合物在制备用于在对象中治疗疾病例如癌症的药物中的用途。
  33. 一种在对象中治疗疾病例如癌症的方法，包括给所述对象施用治疗有效量的权利要求1-30中任一项的经修饰的免疫细胞或权利要求31的药物组合物。
  34. 一种在患有癌症的对象中使得患者显著缩小的方法，包括给所述对象施用治疗有效量的权利要求1-30中任一项的经修饰的免疫细胞或权利要求31的药物组合物。
  35. 一种延长患有癌症的对象生存时间的方法，包括给所述对象施用治疗有效量的 权利要求1-30中任一项的经修饰的免疫细胞或权利要求31的药物组合物。
  36. 一种分离的核酸分子，其包含编码权利要求1-30中任一项所定义的NF-κB家族转录因子的核苷酸序列和/或编码权利要求1-30中任一项所定义的靶特异性受体的核苷酸序列。
  37. 一种表达载体，其包含与调控序列可操作连接的i)编码权利要求1-30中任一项所定义的靶特异性受体的核苷酸序列和/或ii)编码权利要求1-30中任一项所定义的NF-κB家族转录因子的核苷酸序列。
  38. 一种制备经修饰的免疫细胞的方法，包括
    a)提供分离的免疫细胞；和
    b)在所述免疫细胞中过表达NF-κB家族转录因子。
  39. 权利要求38的方法，其中所述免疫细胞选自T细胞、B细胞或NK细胞，优选是T细胞。
  40. 权利要求38或39的方法，其中所述NF-κB家族转录因子选自P50、P105、P52、P100、P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
  41. 权利要求38-40中任一项的方法，其中所述方法通过将包含编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述免疫细胞来过表达所述NF-κB家族转录因子。
  42. 权利要求41的方法，其中所述NF-κB家族转录因子是p52，编码所述p52的核苷酸序列如SEQ ID NO:8所示。
  43. 权利要求38-42中任一项的方法，其中所述方法还包括c)将包含编码靶特异性受体的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码靶特异性受体的核苷酸序列的表达载体导入所述免疫细胞。
  44. 权利要求43的方法，其中所述靶特异性受体选自嵌合抗原受体(CAR)和T细胞受体(TCR)。
  45. 权利要求44的方法，其中所述靶特异性受体是经修饰的T细胞受体(TCR)，例如是合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)。
  46. 权利要求38-45中任一项的方法，其中所述方法还包括步骤
    d)扩增所述免疫细胞。
  47. 一种提高T细胞增殖能力、提高T细胞效应存活时间、提高T细胞体内数量、诱导T细胞向效应T细胞分化、降低T细胞抑制性受体表达、提高T细胞群体中CD8+T细胞比例、或提高T细胞的靶杀伤能力的方法，所述方法包括：
    a)提供分离的T细胞；和
    b)在所述T细胞中过表达NF-κB家族转录因子。
  48. 权利要求47的方法，其中所述NF-κB家族转录因子选自P50、P105、P52、P100、 P65、c-Rel和RelB，或它们的任意组合，优选地，所述NF-κB家族转录因子是p52。
  49. 权利要求48的方法，其中所述方法通过将包含编码所述NF-κB家族转录因子的核苷酸序列的核酸分子或包含与调控序列可操作连接的编码所述NF-κB家族转录因子的核苷酸序列的表达载体导入所述T细胞来过表达所述NF-κB家族转录因子。
  50. 权利要求49的方法，其中所述NF-κB家族转录因子是p52，编码所述p52的核苷酸序列如SEQ ID NO:8所示。
  51. 权利要求47-50中任一项的方法，其中所述方法还包括步骤
    c)扩增所述T细胞。
  52. [根据细则91更正 26.08.2022]
    一种试剂盒，其用于制备权利要求1-30中任一项的经修饰的免疫细胞，所述试剂盒包括权利要求36的核酸分子和/或权利要求37的表达载体，任选地，所述试剂盒还包含用于分离、培养和/或扩增免疫细胞如T细胞的试剂，和/或用于将所述核酸分子或表达载体导入细胞的制剂。
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