WO2022218375A1 - Chimeric t cell receptor and use thereof - Google Patents

Chimeric t cell receptor and use thereof Download PDF

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Publication number
WO2022218375A1
WO2022218375A1 PCT/CN2022/086816 CN2022086816W WO2022218375A1 WO 2022218375 A1 WO2022218375 A1 WO 2022218375A1 CN 2022086816 W CN2022086816 W CN 2022086816W WO 2022218375 A1 WO2022218375 A1 WO 2022218375A1
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atc
cells
engineered cell
antigen
peptide
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PCT/CN2022/086816
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French (fr)
Chinese (zh)
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王鹏
李宗海
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克莱格医学有限公司
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Priority to CN202280025488.6A priority Critical patent/CN117480247A/en
Publication of WO2022218375A1 publication Critical patent/WO2022218375A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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
    • C12N15/63—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 belongs to the field of immunotherapy. More specifically, the present invention relates to engineered cells comprising antibody/T cell receptor chimeras and uses thereof.
  • CAR-T Chimeric Antibody Receptor Engineered T Cell
  • CAR-T cell therapy fails to produce good efficacy in solid tumors, and at the same time, severe toxicity often occurs during the treatment process.
  • CRS cytokine release syndrome
  • CRS is the most frequent and symptomatic acute adverse event The reaction can be life-threatening in severe cases. Therefore, the correct and effective management and intervention of CRS to reduce the incidence of adverse events related to CAR-T therapy is an urgent clinical problem to be solved.
  • CRS may be related to the unnatural activation signal provided by CAR, which can lead to uncontrolled activation of T cells, thus releasing a large number of cytokines, increasing the risk of developing CRS, and at the same time Hyperactivated T cells rapidly deplete in the malignancy microenvironment of solid tumors, reducing the therapeutic efficacy of solid tumors.
  • TCR-T cell therapy is less prone to toxic side effects due to its dependence on the natural signal of TCR.
  • TCR-T cells have limited ability to recognize tumor cells because TCR relies on binding to the major histocompatibility complex (MHC), which is often absent or downregulated in cancer cells
  • MHC major histocompatibility complex
  • cancer cells often escape the attack of the immune system by low expression of MHC, which greatly reduces the efficiency of TCR-T cell therapy. Therefore, reducing the direct toxic side effects caused by CAR-T cells and improving the targeting ability of TCR-T cells have become the focus of current immune cell therapy.
  • a first aspect of the present invention provides an engineered cell expressing a T cell receptor (TCR) chimera (ATC), the ATC comprising:
  • the expression, activity and/or signaling of the endogenous TCR subunit of the engineered cell is reduced or inhibited, while the expression, activity and/or signaling of the ATC is not reduced or inhibited.
  • the nucleic acid molecule in the constant region of the TCR subunit of the ATC has a synonymous mutation relative to the wild-type TRAC nucleic acid molecule, TRBC nucleic acid molecule, TRGC nucleic acid molecule and/or TRDC nucleic acid molecule.
  • the TCR subunits in the ATC include natural and/or modified TCR ⁇ chain constant regions (TRAC) and ⁇ chain constant regions (TRBC); or include natural and/or modified TCR ⁇ chain constant regions ( TRGC) and delta chain constant region (TRDC).
  • TCR TCR ⁇ chain constant regions
  • TRBC ⁇ chain constant regions
  • TRGC delta chain constant region
  • TRDC delta chain constant region
  • the antigen recognition unit of the ATC includes one or two antibodies; or the antigen recognition unit of the ATC includes an antibody heavy chain variable region (VH) and/or light chain variable region (VL) .
  • VH antibody heavy chain variable region
  • VL light chain variable region
  • the TRAC peptide in the ATC is directly connected to VH or connected through a hinge region, the TRBC peptide is directly connected to VL or connected through a hinge region; or the TRAC peptide in the ATC is directly connected to VL or through a hinge. or the TRDC peptide in the ATC is directly linked or linked to the VH, the TRGC peptide is directly linked to the VL or linked through the hinge region; or the The TRDC peptide in ATC is directly linked to the VL or linked through the hinge region, the TRGC peptide is linked directly to the VH or linked through the hinge region.
  • the ATC can activate the CD3 molecule associated with the ATC after binding to the antigen.
  • the expression of endogenous TCR is reduced or inhibited by using gene knockout technology and/or gene silencing technology including: TALE nuclease, meganuclease, zinc finger nuclease, CRISPR/Cas9, Argonaute , guided editing technology, homing endonuclease technology, or a combination thereof.
  • the ATC does not contain nucleic acid sequences targeted by gene knockout technology and/or gene silencing technology.
  • the nucleic acid molecule of the ATC comprises a nucleic acid molecule that is no longer the target sequence targeted by the gene knockout technology and/or gene silencing technology after the base synonymous mutation.
  • the ATC does not include a gRNA target sequence.
  • the engineered cells comprise gRNA, the sequences of which are respectively shown in SEQ ID NOs: 25, 28, 33, 34, 35, 36, 37, 38, 39, 40, 41, 44 or a combination thereof; Or comprise gRNA, the sequence is shown as SEQ ID NO:25 and SEQ ID NO:28 respectively; Or comprise gRNA, the sequence is shown as SEQ ID NO:41 and 44 respectively.
  • the ATC comprises the nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12, and the nucleotide sequence shown in SEQ ID NO: 16 or 17; or the ATC comprises The amino acid sequence shown in SEQ ID NO: 5, 9, 11 or 13, and the amino acid sequence shown in SEQ ID NO: 14 or 18; or the ATC comprises the amino acid sequence shown in SEQ ID NO: 21 and SEQ ID NO: 24 Nucleotide sequence; or the ATC comprises the amino acid sequence shown in SEQ ID NO:20 and SEQ ID NO:23.
  • the engineered cells are selected from T cells, cytotoxic T lymphocytes (CTL), regulatory T cells, NK cells, NKT cells, human embryonic stem cells, and multiple cells from which lymphoid cells can be differentiated. able stem cells.
  • the engineered cells are autologous or allogeneic cells.
  • the antigen is a tumor antigen and/or a pathogen antigen
  • the antigen is a tumor antigen
  • the antigen is a solid tumor antigen
  • the antigen is GPC3, EGFR, Claudin18.2, BCMA, mesothelin, CD19.
  • the VL of the antigen recognition unit comprising the amino acid sequence of the antigen recognition unit recognizing GPC3 is selected from SEQ ID NO: 3, 49, 51, 53, 55, 57, 59, 61, 63 or 65 or with The VH having 70-100% sequence identity, and/or recognizing the amino acid sequence of the antigen recognition unit of GPC3 is selected from SEQ ID NO: 1, 48, 50, 52, 54, 56, 58, 60, 62 or 64 or have 70-100% sequence identity to it, or,
  • the VL recognizing the amino acid sequence of the antigen recognition unit of Claudin18.2 is selected from or has 70-100% sequence identity with SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85 , and/or the VH that recognizes the amino acid sequence of the antigen recognition unit of Claudin18.2 is selected from or has 70-100% sequence identity.
  • the engineered cells have one of the following characteristics or a combination thereof:
  • CD4+ and CD8+ are close to those of cells that have not been transduced with polynucleotide fragments encoding ATC;
  • the engineered cells have one of the following or a combination thereof:
  • the ATC positive rate of the engineered cells is increased or increased by about 5% compared to cells that express the same ATC but the expression, activity and/or signaling of endogenous TCR subunits are not reduced or inhibited , 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100 %.
  • a second aspect of the present invention provides ATC molecules in engineered cells according to the present invention.
  • a third aspect of the present invention provides a polynucleotide encoding the ATC or gRNA molecule in the engineered cells of the present invention.
  • a fourth aspect of the present invention provides a vector comprising the polynucleotide according to the present invention.
  • a fifth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the engineered cells of the present invention, the ATC molecules of the present invention, the polynucleotides of the present invention, the The carrier and pharmaceutically acceptable excipients.
  • the pharmaceutical composition according to the present invention is used for the treatment of tumors.
  • the sixth aspect of the present invention provides a kit comprising the engineered cells of the present invention, the ATC molecules of the present invention, the polynucleotides of the present invention, and the vector of the present invention Or the pharmaceutical composition according to the present invention.
  • the kit according to the present invention further includes written instructions for treating and/or preventing tumors, pathogen infections, autoimmune diseases or allotransplantation.
  • a seventh aspect of the present invention provides a method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of the engineered cells of the present invention and the pharmaceutical composition of the present invention.
  • the eighth aspect of the present invention provides a method for treating or preventing tumors in a subject, comprising administering to the subject an effective amount of the engineered cells of the present invention and the pharmaceutical composition of the present invention.
  • the tumor is selected from liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, pancreatic cancer, multiple myeloma, and hematological tumors.
  • the tumor is a GPC3-positive tumor or a Claudin18.2-positive tumor.
  • a ninth aspect of the present invention provides a method for generating antigen-specific immune effector cells, comprising adding a polynucleotide encoding the ATC molecule of the present invention, or the polynucleotide of the present invention, or the The vector of the present invention is introduced into immune effector cells.
  • a tenth aspect of the present invention provides a method for prolonging the survival of a subject suffering from a tumor, comprising administering to the subject an effective amount of the engineered cells of the present invention, the pharmaceutical combination of the present invention thing.
  • the eleventh aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition in therapy.
  • the twelfth aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition for reducing tumor burden in a subject.
  • the thirteenth aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition for treating or preventing tumors in a subject.
  • a fourteenth aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition for prolonging the survival of a subject suffering from a tumor.
  • a fifteenth aspect of the present invention provides an ATC targeting GPC3, the ATC comprising a polypeptide formed by an antigen recognition unit that recognizes GPC3 and TRAC, and a polypeptide formed by an antigen recognition unit that recognizes GPC3 and TRBC; or the ATC comprises a polypeptide that recognizes GPC3 A polypeptide formed by the antigen recognition unit and TRDC, and a polypeptide formed by the antigen recognition unit and TRGC of GPC3.
  • the antigen recognition unit that recognizes GPC3 includes the antibody heavy chain variable region (VH) and/or light chain variable region (VL) of an anti-GPC3 antibody:
  • the TRAC peptide is directly linked to the VH or linked through the hinge region, the TRBC peptide is directly linked to the VL or linked through the hinge region; or the TRAC peptide is directly linked to the VL or linked through the hinge region, the TRBC peptide is directly linked to the VH or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VH or through the hinge region, the TRGC peptide is directly attached to the VL or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VL or through the hinge Region linkage, the TRGC peptide is linked directly to the VH or via a hinge region.
  • the sixteenth aspect of the present invention provides an ATC targeting Claudin18.2, the ATC comprising a polypeptide formed by an antigen recognition unit recognizing Claudin18.2 and TRAC, and a polypeptide formed by an antigen recognition unit recognizing Claudin18.2 and TRBC; or
  • the ATC comprises a polypeptide formed by recognizing the antigen recognition unit of Claudin18.2 and TRDC, and a polypeptide formed by recognizing the antigen recognition unit of Claudin18.2 and TRGC.
  • the antigen recognition unit recognizing Claudin18.2 comprises the antibody heavy chain variable region (VH) and/or light chain variable region (VL) of an anti-Claudin18.2 antibody:
  • the TRAC peptide is directly linked to the VH or linked through the hinge region, the TRBC peptide is directly linked to the VL or linked through the hinge region; or the TRAC peptide is directly linked to the VL or linked through the hinge region, the TRBC peptide is directly linked to the VH or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VH or through the hinge region, the TRGC peptide is directly attached to the VL or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VL or through the hinge Region linkage, the TRGC peptide is linked directly to the VH or via a hinge region.
  • FIG. 1 ATC virus titer determination results.
  • FIG. 1 The detection results of ATCT positive rate and knockout efficiency.
  • Figure 3 In vitro killing results of ATCT cells on target cells.
  • Figure 6 The results of the differentiation test of ATCT cells.
  • the present invention relates to the construction and application of a novel cellular immune technology platform.
  • Exemplarily targeting GPC3, the anti-GPC3 antibody and the mutated T cell receptor are tandemly transferred into T cells with low or no expression of endogenous TCR to construct antibody-T cell receptor chimeric T cells (Antibody).
  • “about” may mean, depending on the circumstances and known or known to those skilled in the art, or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%.
  • the term may mean within an order of magnitude of a numerical value, eg, within about 5-fold or within about 2-fold of a value.
  • activation of immune effector cells refers to changes in intracellular protein expression caused by signal transduction pathways that lead to the initiation of an immune response.
  • a signal transduction cascade occurs when CD3 molecules aggregate in response to ligand binding and immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the immune synapse formed when endogenous TCR or exogenous ATC binds to an antigen includes many near the binding receptor (eg, CD4 or CD8, CD3 ⁇ /CD ⁇ /CD ⁇ /CD ⁇ , etc.). aggregation of molecules. This aggregation of membrane-bound signaling molecules phosphorylates the ITAM motif contained in the CD3 molecule.
  • T cell activation or T cell activation refers to the state of T cells that upon stimulation induces detectable cell proliferation, cytokine production, and/or detectable effector function.
  • CD3/CD28 magnetic beads antigen stimulation in vitro or in vivo will affect the degree and duration of T cell activation.
  • the engineered cells are activated after co-incubation with tumor cells containing a specific target antigen, or the engineered cells are activated after infection with a virus.
  • stimulation of immune effector cells refers to a signal transduction pathway that results in a strong, sustained immune response by immune effector cells. In one embodiment, this occurs following activation of immune effector cells (eg, T cells) or mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS.
  • immune effector cells eg, T cells
  • receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS.
  • antigen recognition unit refers to a molecule that specifically binds an antigenic determinant, including immunoglobulin molecules and immunologically active portions of immunological molecules, i.e., molecules containing an antigen-binding site that specifically binds ("immunoreactive") an antigen .
  • antibody includes not only intact antibody molecules, but also fragments of antibody molecules that retain antigen-binding capacity.
  • antibody is used interchangeably with the terms “immunoglobulin” and "antigen recognition unit” in the present invention.
  • Antibodies including but not limited to monoclonal antibodies, polyclonal antibodies, native antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single chain Antibody molecules (eg scFv), single domain antibodies.
  • the antibody comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains, CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain.
  • VH and VL can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, immune effector cells) and the first component (Clq) of the classical complement system.
  • An antigen recognition unit "specifically binds" to an antigen or is "immune” to an antigen if it binds the antigen with greater affinity (or avidity) than other reference anti
  • chimeric antigen receptor refers to a molecule comprising an extracellular antigen binding domain and a transmembrane domain fused to an intracellular signaling domain capable of activating or stimulating immune effector cells.
  • the extracellular antigen binding domain of the CAR comprises a scFV.
  • scFVs include antibody heavy chain variable regions and light chain variable regions.
  • the CAR comprises a polypeptide in which a scFV, a transmembrane domain and an intracellular signaling domain are linked in sequence.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, including any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof.
  • the nucleic acid molecule only needs to maintain substantial identity with the endogenous nucleic acid sequence, and does not need to be 100% homologous or identical to the endogenous nucleic acid sequence.
  • a polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule.
  • Hybridization refers to the pairing of double-stranded molecules between complementary polynucleotide sequences or portions thereof under various stringent conditions.
  • the term “homology” or “identity” refers to a subunit between two polymer molecules, eg, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules sequence identity.
  • the term “substantially identical” or “substantially homologous” refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence or nucleic acid sequence.
  • such a sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the amino acid or nucleic acid sequence used for comparison Homology or identity.
  • Sequence identity can be measured using sequence analysis software (eg, BLAST, BESTFIT, GAP or the PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine acid lysine, arginine; and phenylalanine, tyrosine.
  • the BLAST program can be used, where a probability score between e-3 and e-100 indicates closely related sequences.
  • disease refers to any condition that impairs or interferes with the normal function of cells, tissues or organs, such as tumors (cancer) or pathogen infections.
  • Refractory cancers include, but are not limited to, radiotherapy-insensitive, relapsed after radiotherapy, chemotherapy-insensitive, relapsed after chemotherapy, insensitive to CAR-T therapy, or cancers that have relapsed after treatment.
  • terapéuticaally effective amount refers to a compound effective to achieve a specified biological result, as described herein, The amount of formulation, substance or composition, pharmaceutical composition, such as, but not limited to, an amount or dose sufficient to promote a T cell response.
  • An effective amount of immune effector cells refers to, but is not limited to: the number of immune effector cells that can increase, enhance or prolong anti-tumor activity; increase the number of anti-tumor immune effector cells or the number of activated immune effector cells; promote the secretion of IFN- ⁇ , tumor regression, tumor shrinkage, and tumor necrosis in the number of immune effector cells.
  • endogenous means that a nucleic acid molecule or polypeptide, etc., is derived from the organism itself.
  • exogenous means that a nucleic acid molecule or polypeptide is not endogenously present in a cell, or is expressed at an insufficient level to achieve the function when overexpressed; any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous , heterologous and overexpressed nucleic acid molecules and polypeptides.
  • the term “recognition” refers to selective binding to a target antigen.
  • Engineered cells that recognize tumors can express receptors (eg, ATCs or CARs) that bind to tumor antigens.
  • the term “specifically binds” refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample.
  • a binding partner eg, tumor antigen
  • signal peptide is a short peptide chain (about 5-30 amino acids in length) that directs the transfer of newly synthesized proteins or polypeptides to the secretory pathway.
  • the coding region of the polynucleotide of the present invention may be linked to a coding region encoding a signal peptide that directs secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of a fusion protein is desired, a polynucleotide encoding a signal peptide can be placed upstream of a polynucleotide encoding a fusion protein or polypeptide of the invention.
  • proteins or polypeptides secreted by vertebrate cells often have a signal peptide fused to the N-terminus of the protein or polypeptide that is cleaved from the translated protein or polypeptide to produce the secreted or "mature" form protein or polypeptide.
  • native signal peptides are used, such as the signal peptide of TCR (TRAV signal peptide sequence is shown in SEQ ID NO: 93, TRBV signal peptide is shown in SEQ ID NO: 94), IL-2 signal peptide sequence As shown in SEQ ID NO: 96 (human), SEQ ID NO: 97 (mouse), the kappa signal peptide sequence is shown in SEQ ID NO: 98 (human), SEQ ID NO: 99 (mouse); CD8 signal The peptide sequence is shown in SEQ ID NO: 95 (human); the truncated human CD8 signal peptide is shown in SEQ ID NO: 100 (human); the albumin signal peptide sequence is shown in SEQ ID NO: 101 (human); The prolactin signal peptide sequence is shown in SEQ ID NO: 102 (human).
  • TCR TCR
  • TCR signal peptide sequence is shown in SEQ ID NO: 93
  • TRBV signal peptide is shown in SEQ ID NO: 94
  • mice rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs , goat, cow, horse, ape, monkey.
  • T cell receptor also known as “TCR subunit”, or “TCR unit”
  • TCR is a characteristic marker on the surface of all T cells that binds non-covalently to CD3 bind to form the TCR-CD3 complex.
  • the TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCR is a heterodimer composed of two different peptide chains, consisting of ⁇ and ⁇ peptide chains, or ⁇ and ⁇ peptide chains; each peptide chain includes a variable region and a constant region (including cellular outer constant region, transmembrane region and cytoplasmic region); it is characterized by a very short cytoplasmic region.
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (V ⁇ , V ⁇ ) has three hypervariable regions, CDR1, CDR2, and CDR3. Among them, CDR3 has the largest variation, which directly determines the antigen of TCR. binding specificity.
  • CDR1 and CDR2 recognize and bind to the side wall of the antigen-binding groove of the MHC molecule, while CDR3 directly binds to the antigen peptide.
  • TCRs are divided into two categories: TCR1 and TCR2; where TCR1 is composed of two chains, ⁇ and ⁇ , while TCR2 is composed of two chains, ⁇ and ⁇ .
  • T cells In peripheral blood, about 90%-95% of T cells express TCR2; and any T cell expresses only TCR2 or TCR1.
  • the recognition capabilities of these natural TCR receptors are often weak and thus cannot form an effective attack on target cells.
  • the "affinity" of the native TCR for the corresponding target antigen can be improved by means of partial genetic modification, that is, high-affinity TCR, such as the antibody-T cell receptor chimera (Antibody-TCR- Chimeric, ATC).
  • isolated means altered or removed from the natural state.
  • a nucleic acid or peptide that occurs naturally in a living animal is not “isolated”, but the same nucleic acid or peptide that is partially or completely separated from the material with which it is found in its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as a host cell.
  • peptide refers to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • the term "synonymous mutation” means that a mutation of a base pair in a DNA fragment does not change the encoded amino acid because the codon at that position is a synonymous codon before and after the mutation.
  • three consecutive nucleotide residues constitute a codon.
  • the remaining 61 codons represent 20 amino acids (Table 1).
  • methionine and tryptophan which each have one codon
  • the other 18 amino acids have two or more codons.
  • Different codons corresponding to the same amino acid are called synonymous codons.
  • the synonymous codons CTA and CTG both encode leucine, and if the A in CTA is mutated to G, the mutation is a synonymous mutation.
  • wild-type gene refers to the most common allele in nature and is often used as a standard control gene in biological experiments. The corresponding concept is mutant gene.
  • engineing refers to applying the principles and methods of cell biology and molecular biology to change the genetic material in cells according to people's wishes at the overall level of cells, organelles, and molecules through some engineering means. Or a comprehensive science and technology to obtain cell products.
  • Antibody-T cell receptor chimeric ATC Antibody-TCR-Chimeric
  • the present invention provides ATC receptors modified by TCR.
  • the ATC comprises a TCR subunit portion modified with an antigen recognition unit.
  • the TCR subunit portion of ATC includes native or modified TCR alpha, beta, gamma and/or delta chains.
  • the ATC includes the constant regions of native or modified TCR alpha and beta chains; or includes the constant regions of native or modified TCR gamma and delta chains.
  • the TCR subunit constant region optionally, further includes a hinge/spacer region.
  • TCR subunit constant regions include TCR ⁇ constant regions (TRAC), TCR ⁇ constant regions (TRBC, eg, TRBC1 or TRBC2), TCR ⁇ constant regions (TRGC, eg, TRGC1 or TRGC2), TCR ⁇ constant regions (TRDC), or any variant or function thereof Fragment.
  • TCR ⁇ constant regions TRBC
  • TRBC TCR ⁇ constant regions
  • TRGC TCR ⁇ constant regions
  • TRDC TCR ⁇ constant regions
  • Wild-type TRAC nucleic acid molecule refers to encoding TRAC polypeptide, has the nucleotide sequence shown in NCBI GenBank Gene ID: 28755, NG_001332.3, 925603 to 930229 (TRAC, SEQ ID NO: 6).
  • Wild-type TRBC nucleic acid molecule refers to encoding a TRBC polypeptide, with NCBI GenBank Gene ID: 28639, NC_000007.14, 142791694 to 142793141 (TRBC1, SEQ ID NO: 15), or NCBI GenBank Gene ID: 28638, NG_001333.2, 655095 to The nucleotide sequence shown in 656583 (TRBC2).
  • Wild-type TRGC nucleic acid molecule which encodes a TRGC polypeptide, has NCBI GenBank Gene ID: 6966, NG_001336.2, 108270 to 113860 (TRGC1, SEQ ID NO: 19), or NCBI GenBank Gene ID: 6967, NG_001336.2, 124376 to The nucleotide sequence shown in 133924 (TRGC2).
  • Wild-type TRDC nucleic acid molecule refers to encoding TRDC polypeptide, has the nucleotide sequence shown in NCBI GenBank Gene ID: 28526, NG_001332.3, 841011 to 844674 (TRDC, SEQ ID NO: 22).
  • the ATC nucleic acid molecule provided by the present invention comprises a nucleic acid molecule that is no longer the target sequence targeted by gene knockout technology and/or gene silencing technology after base mutation.
  • the nucleic acid fragment of the constant region of the TCR subunit comprised by the ATC is mutated.
  • the nucleic acid fragments of TRAC and/or TRBC contained in the ATC are mutated.
  • the nucleic acid fragments of TRGC and/or TRDC contained in the ATC are mutated.
  • the nucleic acid fragments of wild-type TRAC and/or TRBC contained in the ATC are mutated.
  • the nucleic acid fragments of wild-type TRGC and/or TRDC contained in the ATC are mutated.
  • the ATC nucleic acid molecule provided by the present invention comprises a nucleic acid molecule that is no longer the target sequence targeted by gene knockout technology and/or gene silencing technology after synonymous mutation of bases.
  • a synonymous mutation is made to the nucleic acid fragment of the extracellular constant region of the TCR subunit comprised by the ATC.
  • a synonymous mutation is made to the nucleic acid fragment of the extracellular constant region of TRAC and/or TRBC comprised by the ATC.
  • a synonymous mutation is made to the nucleic acid fragment of the extracellular constant region of TRGC and/or TRDC comprised by the ATC.
  • the ATC comprises a synonymous mutation of the nucleic acid fragment of the extracellular constant region of wild-type TRAC and/or TRBC.
  • the ATC comprises a synonymous mutation of the nucleic acid fragment of the extracellular constant region of wild-type TRGC and/or TRDC.
  • the TRAC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions.
  • amino acid 47 of the TRAC polypeptide in the ATC is mutated to cysteine, amino acid 115 to leucine, amino acid 118 to valine, and amino acid 119 to leucine acid or a combination thereof.
  • the TRAC polypeptides included in the ATC polypeptides provided by the present invention have at least about 80% of the amino acid sequence encoded by the transcripts expressed by the genes of NCBI GenBank Gene ID: 28755, NG_001332.3, 925603 to 930229 , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to amino acid sequences or Fragments thereof, and/or may optionally contain up to one or up to two or up to three conservative amino acid substitutions.
  • amino acid 47 of the TRAC polypeptide in the ATC is mutated to cysteine, amino acid 115 to leucine, amino acid 118 to valine, and amino acid 119 to leucine acid or a combination thereof.
  • the TRBC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions.
  • amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
  • the TRBC polypeptides contained in the ATC polypeptides provided by the present invention have the same properties as those provided by NCBI GenBank Gene ID: 28639, NC_000007.14, 142791694 to 142793141 (TRBC1, SEQ ID NO: 15), NCBI GenBank Gene ID: 28638 , NG_001333.2, 655095 to 656583 (TRBC2) genes expressed transcripts encoding amino acid sequences having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% %, at least about 98%, at least about 99%, or at least about 100% homology or identity to amino acid sequences or fragments thereof, and/or may optionally contain at most one or at most two or at most three conservative amino acid substitutions .
  • amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
  • the TRGC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions.
  • amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
  • the TRGC polypeptides contained in the ATC polypeptides provided by the present invention have the same properties as those provided by NCBI GenBank Gene ID: 6966, NG_001336.2, 108270 to 113860 (TRGC1), NCBI GenBank Gene ID: 6967, NG_001336.2, 124376
  • the amino acid sequence encoded by the transcript of the gene expression to 133924 has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homology or identity
  • the amino acid sequence or fragment thereof, and/or may optionally contain up to one or up to two or up to three conservative amino acid substitutions.
  • the TRDC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions.
  • amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
  • the TRDC polypeptides contained in the ATC polypeptides provided by the present invention have at least about 85% of the amino acid sequences encoded by the transcripts expressed by NCBI GenBank Gene ID: 28526, NG_001332.3, 841011 to 844674, amino acid sequences or fragments thereof of about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homology or identity, and/or may optionally comprise at most one or Up to two or up to three conservative amino acid substitutions.
  • the ATCs of the invention comprise an antigen recognition unit (also referred to as an extracellular antigen binding domain) directly linked to the constant region of the TCR subunit.
  • the ATCs of the invention comprise a hinge/spacer region linking an antigen recognition unit (also referred to as an extracellular antigen binding domain) to the constant region of the TCR subunit.
  • the hinge/spacer region can be a hinge region from IgG1, or a CH2CH3 region of an immunoglobulin and a portion of CD3, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, having at least about 80%, at least about 85% of any of the foregoing , a variant of at least about 90% or at least about 95% homology or identity, or a synthetic spacer sequence.
  • the ATC comprises a TCR ⁇ constant region (amino acid sequence shown in SEQ ID NO: 5 or 9, 11, 13), a TCR ⁇ chain constant region (amino acid sequence shown in SEQ ID NO: 14 or 18), a TCR ⁇ chain constant region region (amino acid sequence shown in SEQ ID NO: 20), and/or with the TCR ⁇ chain constant region (amino acid sequence shown in SEQ ID NO: 23).
  • the ATC does not comprise the nucleotide sequence set forth in SEQ ID NO:25 and/or SEQ ID NO:28.
  • the ATC does not comprise the sequence set forth in SEQ ID NO:41 and/or SEQ ID NO:44.
  • the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12), a TCR ⁇ mutated constant region (nucleotide sequence shown in SEQ ID NO: 16 or 17) ), TCR ⁇ mutant constant region (nucleotide sequence shown in SEQ ID NO: 21), and/or TCR ⁇ mutant constant region (nucleotide sequence shown in SEQ ID NO: 24).
  • the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12) and a TCR ⁇ mutated constant region (nucleotide sequence shown in SEQ ID NO: 16 or 17) ).
  • the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 7) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 7) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17).
  • the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 8) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 8) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17).
  • the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 10) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 10) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17).
  • the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 12) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 12) and a TCR ⁇ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17).
  • the extracellular domains of the ATCs of the present invention may be derived from natural or recombinant sources.
  • the domain may be derived from any protein, but in particular membrane-bound or transmembrane proteins.
  • the extracellular domain is capable of associating with the transmembrane domain.
  • Extracellular domains that are particularly useful in the present invention may include at least the following extracellular domains: for example, the alpha, beta or gamma, delta chains of T cell receptors, or CD3 ⁇ , CD3 ⁇ or CD3 ⁇ , or in alternative embodiments, CD28 , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domains of the ATCs of the present invention can be derived from natural or recombinant sources.
  • the domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling to the intracellular domain whenever an ATC binds to a target antigen.
  • Transmembrane domains that are particularly useful in the present invention may include at least the following transmembrane regions: for example, the alpha, beta or gamma, delta chains of T cell receptors, or CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD28, CD45, CD4, CD5, CD8 , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can be linked to the extracellular region of the ATC (eg, the antigen-binding domain of the ATC) via a hinge (eg, from a human protein).
  • the hinge may be the hinge of the alpha, beta chain of the T cell receptor.
  • the ATC provided by the present invention comprises an extracellular antigen binding domain (also referred to as an antigen recognition unit) and a TCR subunit constant region.
  • the antigen binding domain specifically binds an antigen, such as a tumor antigen or a pathogen antigen.
  • the antigen binding domain comprises an antibody or fragment thereof.
  • the antigen binding domain comprises an antibody heavy chain variable region and/or light chain variable region; or comprises a cross-linked Fab; or comprises F(ab) 2 .
  • the antigen binding domain comprises an antibody heavy chain variable region (VH) and light chain variable region (VL), forming a variable fragment (Fv).
  • the antibody heavy chain variable region in the ATC is directly linked to the alpha chain constant region (TRAC) of the TCR, and/or the antibody light chain variable region is directly linked to the TCR beta chain constant region (TRBC); Or the variable region of the antibody light chain is directly linked to the constant region of the alpha chain of the TCR, and/or the variable region of the heavy chain of the antibody is directly linked to the constant region of the beta chain of the TCR.
  • TCR alpha chain constant region
  • TRBC TCR beta chain constant region
  • variable region of the antibody heavy chain in the ATC is directly connected with the constant region of the ⁇ chain of the TCR to form fragment 1
  • variable region of the antibody light chain is directly connected with the constant region of the ⁇ chain of the TCR to form the fragment 2
  • fragment Fragment 1 and Fragment 2 are joined by a linker, which can be swapped in order relative to the linker.
  • variable region of the antibody light chain in the ATC is directly connected to the constant region of the ⁇ chain of the TCR to form fragment 3
  • variable region of the antibody heavy chain is directly connected to the constant region of the ⁇ chain of the TCR to form the fragment 4
  • fragment Fragment 3 and Fragment 4 are joined by a linker, which can be swapped in order relative to the linker.
  • variable region of the antibody heavy chain in the ATC is directly connected with the constant region of the ⁇ chain of the TCR to form fragment 5
  • variable region of the antibody light chain is directly connected with the constant region of the ⁇ chain of the TCR to form the fragment 6
  • fragment Fragment 5 and Fragment 6 are linked by a linker, which can be swapped in the order of the front and rear relative to the linker.
  • variable region of the antibody heavy chain in the ATC is directly linked to the constant region of the delta chain of the TCR to form fragment 7
  • variable region of the antibody light chain is directly linked to the constant region of the gamma chain of the TCR to form fragment 8
  • fragment Fragment 7 and Fragment 8 are linked by a linker, which can be swapped in the order of the front and rear relative to the linker.
  • the "linker” includes a sequence encoding a self-cleaving peptide (eg, 2A sequence) or a protease recognition site (eg, furin).
  • a "self-cleaving peptide” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate post-translationally into component proteins.
  • Various self-cleaving peptides are known to those of skill in the art, including but not limited to those viruses found in members of the Picornaviridae family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), Viruses (TaV) and porcine Texaco virus-1 (PTV-1), and cardioviruses such as Theilovirus and encephalomyocarditis virus.
  • FMDV foot-and-mouth disease virus
  • ERAVO equine rhinitis A virus
  • Viruses TaV
  • PTV-1 porcine Texaco virus-1
  • cardioviruses such as Theilovirus and encephalomyocarditis virus.
  • F2A comprises the sequence shown in SEQ ID NO: 88 or 89
  • P2A comprises the sequence shown in SEQ ID NO: 90 or 91
  • connecting peptide 1 comprises the sequence shown in SEQ ID NO: 92.
  • the present invention includes recombinant DNA molecules (or constructs) encoding ATC, exemplarily, ATC comprising an antibody fragment that specifically binds to GPC3 or Claudin18.2, wherein the antibody fragment sequence is associated with a nucleic acid encoding a TCR subunit or portion thereof The sequences are linked and in the same Open Reading Frame.
  • an antibody recognizing GPC3 the antibody comprising a heavy chain variable region comprising SEQ ID NOs: 1, 48, 50, 52, 54, 56, 58, 60 , 62 or 64; and/or the antibody comprises a light chain variable region comprising SEQ ID NO: 3, 49, 51, 53, 55, 57, 59, 61, The amino acid sequence shown at 63 or 65.
  • an antibody recognizing CLDN18A2 comprising a heavy chain variable region comprising SEQ ID NOs: 66, 68, 70, 72, 74, 76, 78, 80 , 82 or 84; and/or the antibody comprises a light chain variable region comprising SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, The amino acid sequence shown at 83 or 85.
  • the invention contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, VH or VL) that produces a functionally equivalent molecule.
  • an anti-GPC3 or Claudin18.2 binding domain, eg, VH or VL, contained in an ATC can be modified to retain at least about 70%, 71%, 72%, 73% of the anti-GPC3 or Claudin18.2 binding domain, eg, VH or VL , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the present invention contemplates modification of the entire ATC construct, eg, modification of one or more amino acid sequences of individual domains of the ATC construct, in order to generate functionally equivalent molecules.
  • the ATC construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the starting ATC construct , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity.
  • antibodies or antibody fragments of the invention can be further modified such that they vary in amino acid sequence (eg, relative to wild type), but not in the desired activity.
  • additional nucleotide substitutions can be made in the protein, resulting in amino acid substitutions at "non-essential" amino acid residues.
  • a non-essential amino acid residue in a molecule can be substituted with another amino acid residue from the same side chain family.
  • amino acid fragments may be replaced by amino acid fragments that are structurally similar but differ in sequence and/or composition from side chain family members, eg, conservative substitutions may be made in which amino acid residues are replaced by amino acids with similar side chains residues replaced.
  • the ATC binds to a tumor antigen.
  • Any tumor antigen may be used in the tumor-related embodiments of the present invention.
  • Antigens are expressed as polypeptides or intact proteins or parts thereof.
  • the tumor antigens of the present invention include, but are not limited to: Thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Mesothelin; EpCAM; Protease
  • ATC identifies GPC3. In one embodiment, ATC recognizes Claudin18.2. In one embodiment, the human GPC3 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:86. In one embodiment, the ATC binds to the extracellular domain of a GPC3 polypeptide. In one embodiment, the human Claudin18.2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:87. In one embodiment, ATC binds to the extracellular domain of a Claudin18.2 polypeptide.
  • the ATC recognizes pathogen antigens, eg, for the treatment and/or prevention of pathogen infections or other infectious diseases, eg, in immunocompromised subjects.
  • Pathogen antigens include but are not limited to: antigens of viruses, bacteria, fungi, protozoa, or parasites; viral antigens include but are not limited to: cytomegalovirus (CMV) antigens, Epstein-Barr virus (EBV) antigens, human immune Defective virus (HIV) antigen or influenza virus antigen.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • HAV human immune Defective virus
  • the ATC receptor provided by the present invention can associate with CD3 ⁇ polypeptide.
  • the ATC comprises the constant region of the TCR subunit associated with a CD3 ⁇ polypeptide.
  • CD3 ⁇ polypeptides can be endogenous or exogenous.
  • the binding of the extracellular antigen-binding domain of ATC to the antigen is capable of activating the CD3 ⁇ polypeptide associated with the constant region of the TCA subunit.
  • Activated CD3 ⁇ polypeptides can activate and/or stimulate immune effector cells (eg, cells of the lymphoid lineage, eg, T cells).
  • CD3 ⁇ contains three immunoreceptor tyrosine activation motifs (ITAM1, ITAM2, and ITAM3), three basic-rich stretch regions (BRS) (BRS1, BRS2, and BRS3), and binds to extracellular antigens of ATC at the Domain binding transmits an activation signal to cells (eg, cells of the lymphoid lineage, eg, T cells).
  • the intracellular signaling domain of the CD3 ⁇ chain is the primary transmitter of TCR signaling.
  • the ATC receptors provided herein are capable of associating with the CD3 complex (also referred to as "T cell co-receptor").
  • the ATC and CD3 complex form an antigen-recognition receptor complex similar to the native TCR/CD3 complex.
  • the ATC can activate the CD3 molecule associated with the ATC upon binding to the antigen.
  • the CD3 molecules of the present invention include CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • the CD3 complex can be endogenous as well as exogenous.
  • ATC receptors replace native and/or endogenous TCRs in the CD3/TCR complex.
  • the CD3 complex contains two CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and two CD3 ⁇ chains.
  • the ATC receptor provided by the present invention exhibits higher antigen sensitivity than CAR targeting the same antigen.
  • ATCs are capable of inducing an immune response when bound to antigens with low densities on the surface of tumor cells.
  • immune effector cells comprising ATCs of the invention can be used to treat subjects with tumor cells that express low levels of surface antigens, such as due to relapse of the disease, where the subject has received a tumor that resulted in residual Cell therapy.
  • the engineered cells provided by the present invention comprise immune effector cells expressing ATC.
  • ATC can activate the immune effector cells.
  • the engineered cells of the present invention exhibit cytolytic effects on antigen-bearing cells after binding to the antigen.
  • the present invention provides a technical platform for constructing engineered cells expressing ATC.
  • the engineered cells are T cells, also known as ATCT cells, which combine the high affinity and high specificity of the antibody/antigen recognition unit to recognize antigens, Combined with the natural TCR signaling ability of T cells, it can reduce cytokine secretion and improve clinical safety without reducing the killing effect of the engineered cells of the present invention, and ATCT cells have a lower degree of differentiation and exhaustion. , suggesting that the cell survival ability of the present invention is stronger, and has certain advantages in the treatment of solid tumors.
  • the engineered cells of the invention exhibit comparable or better therapeutic efficacy compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention exhibit comparable or better cytolysis compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention secrete anti-tumor cytokines. Cytokines secreted by engineered cells include, but are not limited to, TNF ⁇ , IFN ⁇ , and IL2. In one embodiment, the engineered cells of the invention exhibit comparable or better levels of activation of the engineered cells upon antigen binding compared to cells comprising a CAR targeting the same antigen.
  • the engineered cells of the invention exhibit a comparable or lower degree of differentiation compared to cells comprising a CAR targeting the same antigen, eg, ATCT cells express a high ratio of CCR7 and CD45RA.
  • the engineered cells of the invention exhibit a CD4/CD8 phenotype comparable to or close to the native state compared to cells comprising a CAR targeting the same antigen.
  • the engineered cells of the invention exhibit a comparable or lower degree of depletion compared to cells comprising a CAR targeting the same antigen.
  • the engineered cells of the invention exhibit a proliferative capacity comparable to or closer to the native state compared to cells comprising a CAR targeting the same antigen.
  • the engineered cells have one of the following characteristics or a combination thereof: 1) the ability to kill target cells, and/or after incubation with target cells There was no significant difference in the secretion of IFN- ⁇ and cell proliferation; the expression levels of CD25 and CD69 were high after incubation with target cells; 2) the proportion of primitive T cells was large; 3) the ratio of CD4+/CD8+ was high; 4) PD-1/TIM-3/LAG -3 The positive rate is low.
  • the platform of the present invention combines the high affinity and high specificity of antibody recognition with the natural TCR signaling ability of T cells, can reduce cytokine secretion and improve clinical safety without weakening the killing effect, and ATCT cells have relatively The low degree of differentiation and depletion indicates that the cells of the present invention have stronger viability and have significant advantages in the treatment of solid tumors.
  • the ATCT cells described herein can further express another factor, such as a cytokine, transcription factor, chemokine, and/or a combination thereof, to increase T cell proliferation, cell survival, anti-apoptotic effects , tumor infiltration and other effects to enhance anti-tumor activity.
  • another factor such as a cytokine, transcription factor, chemokine, and/or a combination thereof, to increase T cell proliferation, cell survival, anti-apoptotic effects , tumor infiltration and other effects to enhance anti-tumor activity.
  • the engineered cells provided by the present invention have low expression or no expression of endogenous TCR molecules, and the contained ATC can form a complex with endogenous CD3.
  • the ATC nucleic acid molecule is no longer the nucleic acid molecule of the target sequence targeted by the gene knockout technology and/or the gene silencing technology after including the base mutation.
  • the ATC nucleic acid molecule is no longer the nucleic acid molecule of the target sequence targeted by the gene knockout technology and/or gene silencing technology after comprising the base synonymous mutation, and the ATC polypeptide can interact with endogenous CD3 form a complex.
  • the ATC nucleic acid molecule is no longer the nucleic acid molecule of the target sequence targeted by the gene knockout technology and/or gene silencing technology after comprising the base synonymous mutation, and the ATC amino acid sequence is the same as that of the wild-type TCR subtype.
  • the base constant region has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology amino acid sequences or fragments thereof, and ATC polypeptides can form complexes with endogenous CD3.
  • the endogenous ⁇ TCR molecules in the engineered cells provided by the present invention are underexpressed or not expressed, and the expressed ATC nucleic acid molecules contain synonymous mutations relative to wild-type TRAC nucleic acid molecules and TRBC nucleic acid molecules.
  • the endogenous ⁇ TCR molecule is underexpressed or not expressed in the engineered cell provided by the present invention, and the expressed ATC nucleic acid molecule contains a synonymous mutation relative to the wild-type TRGC nucleic acid molecule and TRDC nucleic acid molecule.
  • the expression, activity and/or signaling of an endogenous TCR in the engineered cell is reduced compared to the expression, activity and/or signaling of the endogenous TCR in the non-engineered cell Greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100%.
  • the exons of the genes encoding the constant regions of the TCR ⁇ and ⁇ chains of the engineered cells are knocked out using CRISPR/Cas technology.
  • the target sequences targeted by the CRISPR/Cas technology are located in the TCR alpha chain and beta chain constant regions.
  • both endogenous TRAC and endogenous TRBC are simultaneously knocked out in the engineered cells.
  • the engineered cell comprises a gRNA, the sequence of which is set forth in SEQ ID NO: 25, 28, 33, 34, 35, 36, 37, 38, 39, 40, or a combination thereof. In one embodiment, the engineered cell comprises a gRNA, the sequence of which is set forth in SEQ ID NO: 41 and/or 44. In one embodiment, the engineered cell comprises a gRNA having a sequence as set forth in SEQ ID NO: 25 and/or 28.
  • the engineered cells have ATC positivity rates and/or ATC and endogenous TCR positivity rates compared to cells expressing the same ATC but without reduced or inhibited expression, activity and/or signaling of endogenous TCR subunits.
  • the ratio of exogenous CD3 complex formation is increased, or about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95% or 100%.
  • the present invention also provides ATCT cells transduced with a nucleic acid encoding the ATC and targeting an inhibitory nucleic acid molecule or gRNA encoding an endogenous TCR gene, or transduced with a recombinant plasmid comprising the nucleic acid, or The virus containing the plasmid is transduced.
  • ATCT cells are modified with two sets of nucleotide fragments
  • the first set of nucleotide fragments includes inhibitory nucleic acid molecules and/or gRNAs, the complementary sequences of the inhibitory nucleic acid molecules or gRNA targets
  • the sequence is located in the constant region of the wild-type TCR subunit;
  • the second group of nucleotide fragments includes an antigen recognition unit encoding an antigen and a nucleotide fragment comprising a TCR subunit constant region with a synonymous mutation of the nucleotide sequence, the second The set of nucleotide fragments does not include the complementary sequence and/or the gRNA target sequence of the inhibitory nucleic acid molecule in the first set of nucleotide fragments.
  • the immune effector cells of the present invention may be cells of the lymphoid lineage.
  • the lymphatic lineage including B, T, and natural killer (NK) cells, provides antibody production, regulation of the cellular immune system, detection of foreign agents in the blood, detection of foreign cells in the host, and the like.
  • Non-limiting examples of immune effector cells of the lymphoid lineage include T cells, natural killer T (NKT) cells, and their precursors, including embryonic stem cells and pluripotent stem cells (eg, stem cells that differentiate into lymphoid cells or pluripotent stem cells).
  • T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system.
  • T cells can be any type of T cell including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-like memory T cells (or stem-like memory T cells), and both effector T cells Memory T cells: eg TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, ⁇ T cells or ⁇ T cells.
  • Cytotoxic T cells are T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • a subject's own T cells can be engineered to express ATCs targeting specific antigens.
  • the immune effector cells are T cells.
  • the T cells may be CD4+ T cells and/or CD8+ T cells.
  • the immune effector cells are CD3+ T cells.
  • the engineered cells comprise a population of cells collected from PBMC cells stimulated with CD3 magnetic beads.
  • Immune effector cells can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells. It can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMC peripheral blood mononuclear cells
  • T cells can be obtained from a blood sample collected from a subject using any number of techniques known to those of skill in the art, such as the Ficoll TM separation technique.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. Multiple rounds of selection can also be used in the context of the present invention. In some aspects, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "Unselected" cells can also undergo other rounds of selection.
  • the engineered cells of the present invention are capable of modulating the tumor microenvironment.
  • the source of unpurified CTL can be any source known in the art, such as bone marrow, fetal, neonatal or adult or other source of hematopoietic cells, such as fetal liver, peripheral blood, or umbilical cord blood.
  • Various techniques can be used to isolate cells. For example, negative selection can initially remove non-CTLs.
  • mAbs are particularly useful for identifying markers associated with specific cell lineages and/or positively and negatively selected differentiation stages.
  • Most terminally differentiated cells can initially be removed by relatively crude dissociation.
  • magnetic bead separation can initially be used to remove large numbers of irrelevant cells.
  • at least about 80%, usually at least about 70%, of the total hematopoietic cells will be removed prior to isolating the cells.
  • Separation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; agents, including but not limited to complement and cytotoxins; and panning with antibodies attached to solid substrates (eg, plates, chips, panning, or any other convenient technique).
  • Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low and obtuse angle light scatter detection channels, impedance channels.
  • Cells can be selected for dead cells by using dyes associated with dead cells, such as propidium iodide (PI).
  • PI propidium iodide
  • cells are collected in medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable eg sterile isotonic medium.
  • FCS fetal calf serum
  • BSA bovine serum albumin
  • engineered cells can be accomplished by transduction of a substantially homogeneous population of cells with recombinant DNA molecules.
  • retroviral vectors gamma-retrovirus or lentivirus
  • ATC-encoding polynucleotides can be cloned into retroviral vectors.
  • Non-viral vectors can also be used.
  • Transduction can use any suitable viral vector or non-viral delivery system.
  • ATCs can be constructed with helper molecules (eg, cytokines) in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors.
  • elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosomal entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-III IRES, NF- ⁇ B IRES, RUNX1 IRES, p53IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, baculovirus IRES, picornavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and cleavable linkers (eg 2A peptides such as P2A, T2A, E2A and F2A peptides).
  • IRES viral and non-viral internal ribosomal entry sites
  • cleavable linkers eg 2A peptides such as P2A, T2A, E2A and F2A peptides.
  • viral vectors that can be used include, for example, adenoviruses, lentiviruses and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus or herpesviruses such as Epstein-Barr virus.
  • Non-viral methods can also be used for genetic modification of engineered cells.
  • nucleic acid molecules can be introduced into immune effector cells by administering the nucleic acid in the context of lipofection, asialosomucoid-polylysine conjugation, or microinjection under surgical conditions.
  • Other non-viral gene transfer methods include in vitro transfection using liposomes, calcium phosphate, DEAE dextran, electroporation and protoplast fusion.
  • Transplantation of the nucleic acid molecule into a subject can also be accomplished by transferring the nucleic acid molecule into a cell type that can be cultured ex vivo (eg, autologous or allogeneic primary cells or progeny thereof), after which the nucleic acid molecule is transferred to the subject.
  • the nucleic acid molecule-modified cells (or progeny thereof) are injected into the target tissue of the subject or injected systemically.
  • Gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR molecules.
  • Gene knockout technologies include Argonaute, CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guide editing technology and/or homing endonuclease technology.
  • Gene silencing techniques include, but are not limited to, antisense RNA, RNA interference, microRNA-mediated translational inhibition, and the like.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • the system includes Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, the RNA that contains the Cas9 uses to guide it to the correct segment of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin) loop form), forms an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA, forms an active complex with Cas9), and an optional fragment of a DNA repair template (which directs cellular repair processes to allow insertion of specific DNA sequence of DNA).
  • Cas9 a protein capable of modifying DNA using crRNA as its guide
  • CRISPR RNA CRISPR RNA
  • tracrRNA the RNA that contains the Cas9 uses to guide it to the correct segment of host DNA
  • tracrRNA transactivating crRNA
  • tracrRNA binds to crRNA, forms an active complex with Cas9
  • CRISPR/Cas9 usually uses plasmids or electroporation to deliver nucleic acid fragments to target cells.
  • crRNA needs to be designed for each application, as this is the sequence Cas9 uses to recognize and bind directly to target DNA in cells.
  • Multiple crRNAs and tracrRNAs can be packaged together to form guide RNAs (gRNAs).
  • the gRNA can be linked to the Cas9 gene and made into a plasmid for transfection into cells.
  • the present invention relates to the sequence of gRNA, it can be a targeted DNA sequence, or a complete Cas9 guide sequence formed by the ribonucleotide corresponding to the DNA, crRNA and TracrRNA.
  • the administered gRNA, tracr pairing sequence and tracr sequence can be administered alone, or a complete RNA sequence can be administered.
  • CRISPR/Cas9 transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
  • Zinc finger nucleases are artificial restriction enzymes produced by binding a zinc finger DNA binding domain to a DNA cleavage domain. Zinc finger domains can be engineered to target specific DNA sequences, which allow zinc finger nucleases to target target sequences within the genome.
  • Transcription activator-like effector nucleases are restriction enzymes that can be engineered to cleave specific sequences of DNA.
  • the TALEN system works almost the same as the ZFN. They are generated by binding transcription activator-like effector DNA binding domains to DNA cleavage domains.
  • the invention also provides nucleic acid molecules encoding one or more of the ATCs described herein, nucleic acid inhibitory molecules targeting endogenous TCRs, or gRNA constructs.
  • immune effector cells such as T cells or NKT cells
  • CRISPR/Cas9 technology to knock out endogenous TCR subunits to obtain the engineering of the present invention. cells.
  • the endogenous TCR subunits of immune effector cells are first knocked out using CRISPR/Cas9 technology, and then a virus comprising a polynucleotide encoding ATC is used to infect.
  • infection of immune effector cells with a virus comprising a polynucleotide encoding ATC and knockout of endogenous TCR subunits in immune effector cells using CRISPR/Cas9 technology are performed simultaneously.
  • the ATC-encoding polynucleotide fragment does not include the target sequence of CRISPR/Cas9.
  • Viruses containing ATC-encoding polynucleotides were added to infect the T cells on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th day after activation of the T cells, and the T cells were infected on the 1st, 2nd, 3rd, On days 4, 5, 6, 7, 8, 9, 10, 15, and 20, the endogenous TCR subunits of the T cells were knocked out by electroporation using the CRISPR/Cas9 technology to prepare the ATCT cells of the present invention.
  • the T cell endogenous TCR subunit was knocked out by electroporation using CRISPR/Cas9 technology.
  • a virus comprising a polynucleotide encoding ATC is added to infect the T cells to prepare the ATCT cells of the present invention.
  • a virus comprising a polynucleotide encoding ATC is added within 1-5 days of T cell activation, and CRISPR/Cas9 technology is used within 1-7 days after infection to knock out the T cell endogenous TCR by electroporation subunit.
  • the T cell endogenous TCR subunit is knocked out by electroporation using CRISPR/Cas9 technology within 1-5 days of T cell activation, and a polynucleoside encoding ATC is added within 1-7 days after the knockout. Acidic virus infects the T cells.
  • a virus comprising a polynucleotide encoding ATC is added on the second day of T cell activation, and the T cell endogenous TCR subunit is knocked out by electroporation using CRISPR/Cas9 technology on the second day after infection .
  • a virus comprising a polynucleotide encoding ATC is added on the second day of T cell activation, and the T cell endogenous TCR subunit is knocked out by electroporation using CRISPR/Cas9 technology on the second day after infection .
  • the endogenous TCR subunit of the T cell is knocked out by electroporation using CRISPR/Cas9 technology on the 2nd day of T cell activation, and on the 2nd day after the knockout, a polynucleotide comprising a polynucleotide encoding ATC is added.
  • the virus infects the T cells.
  • the present invention knocks out the endogenous TCR in the T cell or genetically modifies the T cell to make the endogenous TCR subunit.
  • TCR molecules are low or not expressed, and the extracellular constant regions of the TCR subunits in ATC are genetically modified, so that when the endogenous TCR in T cells is knocked out or genes that cause low or no expression of endogenous TCR molecules are performed Modification does not affect ATC expression in T cells and/or does not affect ATC complexing with endogenous CD3 in T cells.
  • a nucleic acid molecule encoding an ATC targeting a target antigen eg, GPC3 or Claudin 18.2 tumor antigen
  • a nucleic acid inhibitory molecule targeting an endogenous TCR or a gRNA
  • in vitro transcribed ATC nucleic acid molecules, nucleic acid inhibitory molecules targeting endogenous TCRs, or gRNAs can be introduced into cells as a form of transient transfection.
  • An exemplary artificial DNA sequence is a sequence comprising portions of a gene linked together to form an open reading frame encoding a fusion protein. The DNA portions that are linked together can be from a single organism or from more than one organism.
  • compositions comprising the engineered cells of the present invention can be provided to a subject systemically or directly to induce and/or enhance immune responses to antigens and/or to treat and/or prevent tumors, pathogen infections or infectious diseases.
  • the engineered cells of the invention or compositions comprising the same are injected directly into an organ of interest (eg, an organ affected by a tumor).
  • the engineered cells of the invention, or compositions comprising the same are provided indirectly to an organ of interest, eg, by administration to the circulatory system (eg, intravenous, tumor vasculature).
  • Expansion and differentiation agents can be provided before, concurrently with, or after administration of the cells or compositions to increase the production of T cells, NKT cells, or CTL cells in vitro or in vivo.
  • the engineered cells of the present invention may comprise purified cell populations. Those skilled in the art can readily determine the percentage of engineered cells of the invention in a population using a variety of well-known methods, such as fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • suitable ranges of purity are from about 50% to about 55%, from about 5% to about 60%, and from about 65% to about 70%.
  • the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%.
  • the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Dosage can be easily adjusted by one skilled in the art (eg, a decrease in purity may require an increase in dose). Cells can be introduced by injection, catheter, and the like.
  • the composition of the present invention may be a pharmaceutical composition comprising the immune effector cell of the present invention or its progenitor cell and a pharmaceutically acceptable carrier.
  • Administration can be autologous or allogeneic.
  • immune effector or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject.
  • Peripheral blood-derived immune effector cells or progeny thereof eg, in vivo, ex vivo, or in vitro sources
  • the therapeutic compositions of the present subject matter eg, pharmaceutical compositions comprising immune effector cells of the present invention
  • they may be formulated in unit dose injectable forms (solutions, suspensions, emulsions, etc.).
  • compositions comprising the engineered cells of the invention can be conveniently provided in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to a selected pH.
  • sterile liquid preparations such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to a selected pH.
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection.
  • the viscous composition can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues.
  • Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable suitable mixture.
  • a carrier which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable suitable mixture.
  • Sterile injectable solutions can be prepared by incorporating the genetically modified engineered cells in the required amount of the appropriate solvent with other ingredients in varying amounts as required.
  • Such compositions may be admixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose, dextrose, and the like.
  • suitable carriers diluents or excipients
  • Compositions can also be lyophilized.
  • the compositions may contain auxiliary substances such as wetting agents, dispersing agents or emulsifying agents (eg, methyl cellulose), pH buffering agents, gelling or tackifying agents, preservatives, flavoring agents, pigments, and the like, This will depend on the route of administration and the desired formulation.
  • additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption such as aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immune effector cells or their progenitor cells.
  • compositions may be isotonic, ie they may have the same osmotic pressure as blood and/or tears.
  • the desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • Sodium chloride can be particularly useful in buffers containing sodium ions.
  • a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level.
  • methylcellulose is readily and economically available and easy to use.
  • suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like.
  • concentration of thickening agent can depend on the agent chosen. It is important to use an amount that will achieve the chosen viscosity.
  • suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, eg, liquid dosage form (eg, whether the composition is to be formulated as a solution, suspension, gel, or other liquid form, eg time-release form or liquid-filled form).
  • the number of cells to be administered will vary for the subject being treated.
  • the immune effector cells of the invention are administered to a human subject between about 10 4 to about 10 10 , between about 10 5 to about 10 9 , or between about 10 6 to about 10 9 . More potent cells can be administered in smaller numbers.
  • the precise determination of the effective dose can be determined according to individual factors of each subject, including its size, age, sex, weight, and the condition of the subject. Dosages can be readily determined by those skilled in the art from the present invention and knowledge in the art.
  • any additives are present in phosphate buffered saline in an amount ranging from 0.001% to 50% by weight solution, and the active ingredient is in the range of micrograms to milligrams are present in the order of, for example, about 0.0001 wt% to about 5 wt%, about 0.0001 wt% to about 1 wt%, about 0.0001 wt% to about 0.05 wt%, or about 0.001 wt% to about 20 wt%, about 0.01 wt% to about 10 wt% % or about 0.05 wt % to about 5 wt %.
  • toxicity eg, by determining the lethal dose (LD) and LD50 in a suitable animal model, eg, rodents such as mice; the dosage of the composition, wherein Concentrations of components and timing of application of the composition, elicit an appropriate response.
  • the present invention provides methods for inducing and/or increasing an immune response in a subject in need of such engineered cells.
  • the engineered cells of the present invention and compositions comprising the same can be used to treat and/or prevent tumors in a subject.
  • the engineered cells of the present invention and compositions comprising the same can be used to prolong the survival of subjects with tumors.
  • the engineered cells of the present invention and compositions comprising the same can also be used to treat and/or prevent pathogenic infections or other infectious diseases, such as in immunocompromised human subjects.
  • Such methods include administering an effective amount of an engineered cell of the invention or a composition comprising the same (eg, a pharmaceutical composition) to achieve the desired effect, whether reducing an existing condition or preventing relapse.
  • the amount administered is that amount effective to produce the desired effect.
  • the effective amount can be provided in one or more administrations. Effective amounts can be provided in boluses or by continuous infusion.
  • immune effector cells comprising ATCs of the invention can be used to treat subjects with tumor cells that express low levels of surface antigens, for example due to relapse of the disease, where the subject has received a treatment that results in residual tumor cells. treat.
  • tumor cells have a low density of target molecules on the tumor cell surface.
  • an immune effector cell comprising an ATC of the invention can be used to treat a subject suffering from disease relapse, wherein the subject has received an immune effector cell (eg, T cell) comprising a CAR, the CAR
  • An intracellular signaling domain is included, which includes a co-stimulatory signaling domain (eg, a 4-1BBz CAR).
  • tumor cells have a low density of tumor-specific antigens on the tumor cell surface.
  • the disease is a GPC3 positive tumor, a Claudin18.2 positive tumor.
  • the tumor cells have a low density of GPC3 on tumor cells.
  • the tumor cells have a low density of Claudin 18.2 on the tumor cells.
  • Such methods include administering an effective amount of an immune effector cell of the invention or a composition (eg, a pharmaceutical composition) comprising the same to achieve the desired effect, alleviation of an existing condition or prevention of relapse.
  • an “effective amount” is an amount sufficient to produce a beneficial or desired clinical result following treatment.
  • An effective amount can be administered to a subject in one or more doses.
  • an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow disease progression or otherwise reduce the pathological consequences of the disease.
  • Effective amounts are generally determined by the physician on a case-by-case basis and are within the capabilities of those skilled in the art. Several factors are generally considered when determining an appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of immune effector cells administered.
  • T cells For adoptive immunotherapy using antigen-specific T cells, cell doses in the range of about 106-1010 are typically infused. Following administration of the engineered cells of the invention to a host and subsequent differentiation, T cells specific for a particular antigen are induced. Engineered cells can be administered by any method known in the art, including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly to the thymus.
  • the present invention provides methods for treating and/or preventing tumors in a subject.
  • the method can include administering to a subject having a tumor an effective amount of an engineered of the invention or a composition comprising the same.
  • Non-limiting examples of tumors include blood cancers (eg, leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer , gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma and various carcinomas (including prostate cancer and small cell lung cancer).
  • blood cancers eg, leukemia, lymphoma, and myeloma
  • ovarian cancer breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer , gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sar
  • Non-limiting examples of tumors include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), Chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphatic vessels Sarcoma, Lymphoendothelioma, Liver Cancer, Cholangiocarcinoma, Synovialoma, Mesothelioma, Ewing's Tumor, Rhabdomyosarcoma, Colon Cancer, Basal
  • the tumor is selected from hematological cancers (eg, leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer , prostate cancer, skin cancer, stomach cancer, glioblastoma and throat cancer.
  • the engineered cells of the present invention and compositions comprising the same can be used to treat and/or prevent solid tumors that are unsuitable or relapsed or refractory to conventional therapeutic measures, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, etc. cancer, thyroid cancer, stomach cancer, colorectal cancer.
  • the tumor is a hematological tumor.
  • the therapeutic goals of engineered cells of the present invention may include alleviating or reversing disease progression and/or reducing side effects, or therapeutic goals including reducing or delaying the risk of relapse.
  • the present invention provides methods for treating and/or preventing pathogenic infections (eg, viral, bacterial, fungal, parasitic, or protozoan infections) in, eg, immunocompromised subjects.
  • the method may comprise administering to a subject suffering from a pathogen infection an effective amount of an engineered cell of the invention or a composition comprising the same.
  • Exemplary viral infections that are amenable to treatment include, but are not limited to, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus infections.
  • enhancing refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein.
  • an enhanced response can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the responsiveness %, 75%, 80%, 85%, 90%, 95% or 98% or more increase.
  • enhancing can also refer to increasing the number of subjects that respond to treatment, eg, immune effector cell therapy.
  • an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentages are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
  • immune effector cells target GPC3-positive tumors.
  • immune effector cells target tumors that express positive Claudin18.2.
  • the tumor includes, but is not limited to, liver cancer, gastric cancer, lung cancer, esophageal cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, Adrenal cancer, schwannoma, malignant fibrous histiocytoma, esophageal cancer.
  • GPC3 expression-positive tumors or GPC-positive tumors described herein include, but are not limited to, liver cancer, gastric cancer, lung cancer, and esophageal cancer.
  • Claudin18.2-positive tumors or Claudin18.2-positive tumors described herein include, but are not limited to, liver cancer, gastric cancer, lung cancer, esophageal cancer, pancreatic cancer, gallbladder tumor, bile duct cancer.
  • kits for inducing and/or enhancing an immune response and/or treating and/or preventing tumor or pathogen infection in a subject comprises an effective amount of an engineered cell of the invention or a pharmaceutical composition comprising the same.
  • the kit includes a sterile container; such a container may be a box, ampule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art.
  • Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.
  • the kit includes a nucleic acid molecule encoding an ATC of the invention targeting an antigen of interest in an expressible form, optionally contained in one or more vectors.
  • the engineered cells and/or nucleic acid molecules of the invention are administered to a subject having or developing a tumor or pathogen or immune disease. supplied with the user's manual.
  • the instructions generally include information regarding the use of the composition for treating and/or preventing infection by a tumor or pathogen.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for the treatment or prevention of tumors, pathogenic infections, or immune diseases or symptoms thereof; precautions; warnings; indications; indications Symptoms; Medication Information; Adverse Reactions; Animal Pharmacology; Clinical Studies; and/or References.
  • These instructions can be printed directly on the container, or as labels affixed to the container, or provided in or with the container as separate sheets, brochures, cards or folders.
  • the ATCT cells of the present invention can reduce the secretion of cytokines without reducing the killing ability in vitro, and at the same time have longer lasting survival ability and higher antigen sensitivity.
  • the present invention relates to the design and construction method of ATCT cells (including but not limited to GPC3, Claudin18.2 target).
  • the present invention also relates to the research and application of in vitro functional experiments of ATCT cells (including but not limited to GPC3 and Claudin18.2 targets).
  • this example uses CRISPR technology. Endogenous TCR was knocked out.
  • Exemplary, target sequences for TCR ⁇ chain constant region (TRAC) and ⁇ chain constant region (TRBC) and their corresponding primer sequences are shown in Table 2, for TCR ⁇ chain constant region (TRGC) and ⁇ chain constant region (TRDC) The target sequence and its corresponding primer sequence are shown in Table 3; GeneArt TM Precision gRNA Synthesis Kit (Invitrogen) was used for the synthesis of gRNA, and the specific steps were referred to its instructions.
  • the Cas 9 enzyme, TRAC-targeting gRNA and TRBC-targeting gRNA were co-electrotransferred to T cells by electroporation, and finally TCR ⁇ chain and TCR ⁇ chain knockout T cells were successfully obtained;
  • the Cas 9 enzyme, TRGC-targeting gRNA and TRDC-targeting gRNA were co-electroporated into T cells, and finally TCR ⁇ chain and TCR ⁇ chain knockout T cells were successfully obtained.
  • the constant region of the TCR in the provided ATC is mutated in this example, keeping the corresponding amino acid unchanged but the base sequence Make mutations.
  • mutation design is performed for the sequences of the TCR ⁇ chain constant region and ⁇ chain constant region targeted by the gRNAs exemplified in Table 2 in Example 1, and the sequences after mutation are shown in Table 4.
  • mutation design is performed for the sequences of the TCR ⁇ chain constant region and ⁇ chain constant region targeted by the gRNAs exemplified in Table 3 in Example 1, and the sequences after mutation are shown in Table 5.
  • This example takes the construction of an ATC including the mutated sequences of the constant regions of the TCR ⁇ chain and ⁇ chain in Table 4, or the construction of an ATC including the mutated sequences of the constant regions of the TCR ⁇ chain and ⁇ chain in Table 5 as an example.
  • ATC contains two chains, chain 1 includes antibody variable region VH or VL that recognizes target antigen, and TCR ⁇ mutated constant region (SEQ ID NO: 7, 8, 10, 12); chain 2 includes antibody that recognizes target antigen The variable region VL or VH of the antibody, and the TCR ⁇ mutated constant region (SEQ ID NO: 16, 17); the two chain gene sequences are linked by peptides (exemplary, F2A (SEQ ID NO: 88), P2A (SEQ ID NO: 88), P2A (SEQ ID NO: 88) NO: 90) or linker peptide 1 (SEQ ID NO: 92) and constructed in a vector.
  • ATC contains two chains, chain 1 includes the antibody variable region VH or VL that recognizes the target antigen, in tandem with the TCR ⁇ mutated constant region (SEQ ID NO: 21); chain 2 includes the antibody variable region that recognizes the target antigen VL or VH, in tandem with the TCR ⁇ mutant constant region (SEQ ID NO:24); the two chain gene sequences are linked by a linker peptide (exemplarily, F2A (SEQ ID NO:88), P2A (SEQ ID NO:90) or Peptide 1 (SEQ ID NO: 92) was ligated and constructed in the vector.
  • linker peptide exemplarily, F2A (SEQ ID NO:88), P2A (SEQ ID NO:90) or Peptide 1 (SEQ ID NO: 92
  • variable region of ATC targets and binds to the tumor antigen GPC3.
  • the variable region includes a light chain variable region and a heavy chain variable region of an antibody targeting human GPC3.
  • ATC contains two chains, chain 1 includes the anti-GPC3 antibody variable region VH (SEQ ID NO: 1, 2) in tandem with the TCR ⁇ mutated constant region (SEQ ID NO: 7); chain 2 includes the anti-GPC3 antibody variable region VL (SEQ ID NO: 3, 4), in series with the TCR ⁇ mutant constant region (SEQ ID NO: 16); the gene sequences of the two chains were constructed in the pWPT lentiviral vector by linking the linker peptide F2A (SEQ ID NO: 88), called for PWPT-ATC.
  • variable region of ATC targets and binds to the tumor antigen Claudin18.2.
  • the variable region includes a light chain variable region and a heavy chain variable region of an antibody targeting human Claudin18.2.
  • ATC contains two chains, chain 1 includes the anti-Claudin18.2 antibody variable region VH (SEQ ID NO: 84) in tandem with the TCR ⁇ mutated constant region (SEQ ID NO: 7); chain 2 includes the anti-Claudin18.2 antibody variable region Region VL (SEQ ID NO: 85), tandem with TCR ⁇ mutant constant region (SEQ ID NO: 16); the two chain gene sequences are constructed in pWPT lentiviral vector by connecting peptide F2A (SEQ ID NO: 88).
  • the lentivirus was packaged by calcium phosphate method, and the virus supernatant was purified with PEG8000/NaCl. After purification, the titer of the virus was detected by flow cytometry. , 1:2700, 1:8100) virus infected J.RT3-T3.5 cells (ATCC), after 65 hours, the cells were taken and incubated with 5ug/ml antigen (biotinylated human GPC3 protein) at 4 degrees for 45 minutes, two Antibody was used at 1:300 dilution of SA-PE fluorescent antibody (eBioscience), and was detected by flow cytometer after incubation at 4 degrees for 45 minutes.
  • 5ug/ml antigen biotinylated human GPC3 protein
  • the described CRISPR/Cas9 technology uses electroporation to knock out TCR ⁇ chain and ⁇ chain, the target sequence of gRNA on TRAC is shown in SEQ ID NO: 25, and the target sequence of gRNA on TRBC is shown in SEQ ID NO: 28.
  • the ATC positivity rate and TCR knockout efficiency were then detected on days 5-8 after T cell activation.
  • the ATC positive rate detection reagent is 5ug/ml biotinylated human GPC3 protein, and then added with SA-PE fluorescent antibody (eBioscience) at a 1:300 dilution for labeling; the knockout efficiency is reflected by the expression of CD3 on the cell surface, using Anti-CD3- APC (Invitrogen) was used for labeling; finally detected by flow cytometry.
  • UT group was uninfected virus T cells.
  • UT ko was a T cell that was not transduced with ATC vector but knocked out endogenous TCR ⁇ and ⁇ chains.
  • the results are shown in Figure 2.
  • the TCR knockout efficiency in the UT ko group reached 94.6%; the ATC positive rate in the ATCT group reached 68.5%; the ATCT did not knock out the endogenous TCR (ATCT w/o ko) group, due to the presence of endogenous TCR mismatch, the ATCT positive rate was 15.0%.
  • the CAR-T group used as a control was anti-GPC3-CAR T cells (prepared according to the conventional CAR-T cell preparation method, the sequence of CAR is shown in SEQ ID NO: 47), and the endogenous TCR was not knocked out, and its CAR positive rate was 91.8%.
  • ATC structure can integrate with the endogenous CD3 subunit and express on the surface of T cells in ATCT cells to form a complete ATC/CD3 complex.
  • the ATCT cells infected with lentivirus were collected, and the cells were lysed with a protein lysis solution.
  • the ATC and its binding proteins were co-immunoprecipitated with biotin-labeled GPC3 antigen and streptavidin-labeled magnetic beads, and then the western blotting experiment was performed. , and the corresponding antibodies were used to detect the expression of different CD3 subunits.
  • the results showed that ATC could form complexes with CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • Target cells GPC3-expressing liver cancer cell Huh7 (Cell Bank of Chinese Academy of Sciences), liver cancer cell SK-hep-1 (ATCC) not expressing GPC3
  • Effector cells ATCT cells, CAR-T cells, UT cells prepared with reference to Example 3
  • the target cells were adjusted to a density of 0.2x10 6 /mL with AIM-V medium (AIM-V+2%ABS), and 10,000 target cells were added to each well of a 96-well cell culture plate.
  • AIM-V+2%ABS AIM-V medium
  • T 3:1, 1:1 and 1:3 were added to effector cells respectively, and after co-incubating at 37 degrees for 16 hours, the supernatant was taken for color development with LDH kit, and finally OD490 reading and data analysis were carried out with a microplate reader.
  • the killing effect of the ATCT group was more consistent with that of the CAR-T group: it specifically killed Huh7 cells expressing GPC3, with the highest killing efficiency of 86.7%, and had a concentration-dependent characteristic, while the killing effect of SK-hep without GPC3 expression was the same as that of the CAR-T group. -1 cells have no killing effect.
  • cytokine IFN- ⁇ was detected in the supernatant after co-incubating effector cells with target cells in Example 4 for 16 hours. Experiments were carried out using Biolegend's detection kit according to the instructions of the kit.
  • effector cells (ATCT, CAR-T cells, and UT cells prepared in Example 3) were co-incubated with target cells (Huh7, SK-hep-1) at an effector-target ratio of 3:1 for 16 hours. Cells were then taken for flow cytometry. In the experiment, the cells were divided into two groups and respectively added to CD69-PE/CD3-APC (Invitrogen) and CD25-APC/CD3-FITC (Invitrogen) for incubation, and were detected by flow cytometry after incubation at 4 degrees for 45 minutes.
  • the differentiation indicators of ATCT cells and CAR-T cells prepared in Example 3 were detected.
  • the cells were divided into 4 groups and incubated with GranB-488, CCR7-FITC, CD45RA-FITC, and CD45RO-APC antibodies (BD Biosciences), respectively. After 45 minutes of incubation, the cells were detected by flow cytometry.
  • the results are shown in Figure 6.
  • the CAR-T cells have low expression of CCR7 and CD45RA and high expression of CD45RO, indicating that the CAR-T group has a higher degree of differentiation than the ATCT and UT groups, while the ATCT cells have a relatively lower degree of differentiation and more primitive T cells. .
  • the ATCT cells and CAR-T cells prepared in Example 3 were subjected to CD4/CD8 typing detection, and the antibody CD4-PE/CD8-APC (BD Biosciences) was incubated with cells at 4 degrees for 45 minutes and detected by flow cytometry .
  • the results are shown in Figure 7.
  • the CAR-T group maintained a high proportion of CD8+ T cells (greater than 85%) after viral transduction; while the changes of CD8+ T cells and CD4+ T cells in the ATCT group were consistent with those in the UT group. . This indicates that the ATCT cell phenotype is more similar to untransfected T cells and closer to the natural state.
  • Example 3 For GPC3, the cells prepared in Example 3 were tested for depletion. The cells were divided into three groups, and they were first incubated with human GPC3 protein whose antigen was biotinylated at 5ug/ml, and then the secondary antibody was added with SA-APC-Cy7 (BD Biosciences) to detect positive cells.
  • CD4-BV510/CD8-APC (BD Biosciences) antibodies were added for co-incubation, and at the same time, PD-1-BV421 (BD Biosciences), TIM-3-PE (BD Biosciences) and LAG-3 were added to the three groups of cells respectively.
  • -BV421 (BD Biosciences) antibody was co-incubated at 4 degrees and detected by flow cytometry after 45 minutes.
  • Example 3 For GPC3, 1 ⁇ 10 6 CAR-T, ATCT, and UT cells prepared in Example 3 were cultured under the same conditions (AIM-V+2%ABS+300U/ml IL-2), and then every two days. One count to detect cell expansion.

Abstract

Provided are an engineered cell containing an antibody/T cell receptor chimera and the use thereof.

Description

嵌合T细胞受体及其应用Chimeric T cell receptors and their applications 技术领域technical field
本发明属于免疫治疗领域。更具体地，本发明涉及包含抗体/T细胞受体嵌合物的工程化细胞及其用途。The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to engineered cells comprising antibody/T cell receptor chimeras and uses thereof.
背景技术Background technique
表达嵌合抗原受体的T细胞(Chimeric Antibody Receptor Engineered T Cell，CAR-T)疗法在靶向CD19和BCMA的恶性肿瘤的治疗方面已显示出高效的治疗效果。但CAR-T细胞疗法在实体瘤中未能产生良好的疗效，同时在治疗过程中经常会出现严重的毒性作用，其中细胞因子释放综合征(CRS)是发生最频繁、症状最突出的急性不良反应，严重时可危及生命。因此，正确有效地对CRS进行管理和干预，降低CAR-T治疗相关不良事件发生率是亟待解决的临床问题。Chimeric Antibody Receptor Engineered T Cell (CAR-T) therapy has shown high efficacy in the treatment of malignant tumors targeting CD19 and BCMA. However, CAR-T cell therapy fails to produce good efficacy in solid tumors, and at the same time, severe toxicity often occurs during the treatment process. Among them, cytokine release syndrome (CRS) is the most frequent and symptomatic acute adverse event The reaction can be life-threatening in severe cases. Therefore, the correct and effective management and intervention of CRS to reduce the incidence of adverse events related to CAR-T therapy is an urgent clinical problem to be solved.
目前研究表明，CRS的产生可能与CAR提供的非自然的激活信号有关，这种非自然的信号会导致T细胞不受控制的激活，因而释放了大量细胞因子，增加了产生CRS的风险，同时过度激活的T细胞在实体瘤的恶性肿瘤微环境中会迅速衰竭，降低了对实体瘤的治疗效果。相较于此，TCR-T细胞疗法由于依赖TCR的自然信号，因此发生毒副作用事件的概率更低。然而，由于TCR依赖于与主要组织相容性复合体(major histocompatibility complex，MHC)结合，所以TCR-T细胞对肿瘤细胞的识别能力有限，另外这种结合在癌细胞中通常是不存在或下调的，癌细胞常常通过低表达MHC而逃逸免疫系统的攻击，极大降低TCR-T细胞治疗的效率。因此，降低CAR-T细胞带来的直接毒副作用和提高TCR-T细胞的靶向能力成为了目前免疫细胞治疗领域关注的重点。Current research shows that the generation of CRS may be related to the unnatural activation signal provided by CAR, which can lead to uncontrolled activation of T cells, thus releasing a large number of cytokines, increasing the risk of developing CRS, and at the same time Hyperactivated T cells rapidly deplete in the malignancy microenvironment of solid tumors, reducing the therapeutic efficacy of solid tumors. In contrast, TCR-T cell therapy is less prone to toxic side effects due to its dependence on the natural signal of TCR. However, TCR-T cells have limited ability to recognize tumor cells because TCR relies on binding to the major histocompatibility complex (MHC), which is often absent or downregulated in cancer cells However, cancer cells often escape the attack of the immune system by low expression of MHC, which greatly reduces the efficiency of TCR-T cell therapy. Therefore, reducing the direct toxic side effects caused by CAR-T cells and improving the targeting ability of TCR-T cells have become the focus of current immune cell therapy.
发明内容SUMMARY OF THE INVENTION
本发明第一方面提供一种工程化细胞，所述工程化细胞表达T细胞受体(TCR)嵌合物(ATC)，所述ATC包含：A first aspect of the present invention provides an engineered cell expressing a T cell receptor (TCR) chimera (ATC), the ATC comprising:
(a)识别抗原的抗原识别单元，和(a) an antigen recognition unit that recognizes the antigen, and
(b)核苷酸序列同义突变的TCR亚基恒定区；(b) a TCR subunit constant region with a synonymous mutation in the nucleotide sequence;
所述工程化细胞的内源性TCR亚基的表达、活性和/或信号传导被降低或抑制，而所述ATC的表达、活性和/或信号传导不被降低或抑制。The expression, activity and/or signaling of the endogenous TCR subunit of the engineered cell is reduced or inhibited, while the expression, activity and/or signaling of the ATC is not reduced or inhibited.
在一优选例中，相对于野生型TRAC核酸分子、TRBC核酸分子、TRGC核酸分子和/或TRDC核酸分子，所述ATC的TCR亚基恒定区的核酸分子有同义突变。In a preferred embodiment, the nucleic acid molecule in the constant region of the TCR subunit of the ATC has a synonymous mutation relative to the wild-type TRAC nucleic acid molecule, TRBC nucleic acid molecule, TRGC nucleic acid molecule and/or TRDC nucleic acid molecule.
在一优选例中，所述ATC中的TCR亚基包括天然和/或修饰的TCRα链恒定区(TRAC)和β链恒定区(TRBC)；或包括天然和/或修饰的TCRγ链恒定区(TRGC)和δ链恒定区(TRDC)。In a preferred embodiment, the TCR subunits in the ATC include natural and/or modified TCR α chain constant regions (TRAC) and β chain constant regions (TRBC); or include natural and/or modified TCR γ chain constant regions ( TRGC) and delta chain constant region (TRDC).
在一优选例中，所述ATC的抗原识别单元包括一种或两种抗体；或所述ATC的抗原识别单元包括抗体重链可变区(VH)和/或轻链可变区(VL)。In a preferred embodiment, the antigen recognition unit of the ATC includes one or two antibodies; or the antigen recognition unit of the ATC includes an antibody heavy chain variable region (VH) and/or light chain variable region (VL) .
在一优选例中，所述ATC中的TRAC肽与VH直接连接或通过铰链区连接、TRBC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRAC肽与VL直接连接或通过铰链区连接、所述TRBC肽与VH直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VH直接连接或通过铰链区连接、TRGC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VL直接连接或通过铰链区连接、所述TRGC肽与VH直接连接或通过铰链区连接。In a preferred embodiment, the TRAC peptide in the ATC is directly connected to VH or connected through a hinge region, the TRBC peptide is directly connected to VL or connected through a hinge region; or the TRAC peptide in the ATC is directly connected to VL or through a hinge. or the TRDC peptide in the ATC is directly linked or linked to the VH, the TRGC peptide is directly linked to the VL or linked through the hinge region; or the The TRDC peptide in ATC is directly linked to the VL or linked through the hinge region, the TRGC peptide is linked directly to the VH or linked through the hinge region.
在一优选例中，所述ATC与抗原结合后能激活与所述ATC缔合的CD3分子。In a preferred embodiment, the ATC can activate the CD3 molecule associated with the ATC after binding to the antigen.
在一优选例中，所述内源性TCR表达被降低或抑制是通过使用基因敲除技术和/或基因沉默技术包括：TALE核酸酶、巨核酸酶、锌指核酸酶、CRISPR/Cas9、Argonaute、引导编辑技术、归巢核酸内切酶技术或其组合。In a preferred embodiment, the expression of endogenous TCR is reduced or inhibited by using gene knockout technology and/or gene silencing technology including: TALE nuclease, meganuclease, zinc finger nuclease, CRISPR/Cas9, Argonaute , guided editing technology, homing endonuclease technology, or a combination thereof.
在一优选例中，所述ATC不包含基因敲除技术和/或基因沉默技术所靶向的核酸序列。In a preferred embodiment, the ATC does not contain nucleic acid sequences targeted by gene knockout technology and/or gene silencing technology.
在一优选例中，所述ATC的核酸分子包含碱基同义突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子。In a preferred embodiment, the nucleic acid molecule of the ATC comprises a nucleic acid molecule that is no longer the target sequence targeted by the gene knockout technology and/or gene silencing technology after the base synonymous mutation.
在一优选例中，所述ATC不包括gRNA靶序列。In a preferred embodiment, the ATC does not include a gRNA target sequence.
在一优选例中，所述工程化细胞包含gRNA,序列分别如SEQ ID NO：25、28、33、34、35、36、37、38、39、40、41、44或其组合所示；或包含gRNA，序列分别如SEQ ID NO：25和SEQ ID NO：28所示；或包含gRNA，序列分别如SEQ ID NO：41和44所示。In a preferred embodiment, the engineered cells comprise gRNA, the sequences of which are respectively shown in SEQ ID NOs: 25, 28, 33, 34, 35, 36, 37, 38, 39, 40, 41, 44 or a combination thereof; Or comprise gRNA, the sequence is shown as SEQ ID NO:25 and SEQ ID NO:28 respectively; Or comprise gRNA, the sequence is shown as SEQ ID NO:41 and 44 respectively.
在一优选例中，所述ATC包含SEQ ID NO：7、8、10或12所示的核苷酸序列，和SEQ ID NO：16或17所示的核苷酸序列；或所述ATC包含SEQ ID NO：5、9、11或13所示的氨基酸序列，和SEQ ID NO：14或18所示的氨基酸序列；或所述ATC包含SEQ ID NO：21和SEQ ID NO：24所示的核苷酸序列；或所述ATC包含SEQ ID NO：20和SEQ ID NO：23所示的氨基酸序列。In a preferred embodiment, the ATC comprises the nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12, and the nucleotide sequence shown in SEQ ID NO: 16 or 17; or the ATC comprises The amino acid sequence shown in SEQ ID NO: 5, 9, 11 or 13, and the amino acid sequence shown in SEQ ID NO: 14 or 18; or the ATC comprises the amino acid sequence shown in SEQ ID NO: 21 and SEQ ID NO: 24 Nucleotide sequence; or the ATC comprises the amino acid sequence shown in SEQ ID NO:20 and SEQ ID NO:23.
在一优选例中，所述工程化细胞选自T细胞、细胞毒性T淋巴细胞(CTL)、调节性T细胞、NK细胞、NKT细胞、人胚胎干细胞、和可从中分化出淋巴样细胞的多能干细胞。In a preferred embodiment, the engineered cells are selected from T cells, cytotoxic T lymphocytes (CTL), regulatory T cells, NK cells, NKT cells, human embryonic stem cells, and multiple cells from which lymphoid cells can be differentiated. able stem cells.
在一优选例中，所述工程化细胞是自体或同种异体细胞。In a preferred embodiment, the engineered cells are autologous or allogeneic cells.
在一优选例中，所述抗原为肿瘤抗原和/或病原体抗原；In a preferred embodiment, the antigen is a tumor antigen and/or a pathogen antigen;
优选地，所述抗原为肿瘤抗原；Preferably, the antigen is a tumor antigen;
优选地，所述抗原为实体瘤抗原；Preferably, the antigen is a solid tumor antigen;
优选地，所述抗原为GPC3、EGFR、Claudin18.2、BCMA、间皮素、CD19。Preferably, the antigen is GPC3, EGFR, Claudin18.2, BCMA, mesothelin, CD19.
在一优选例中，所述抗原识别单元包含识别GPC3的抗原识别单元的氨基酸序列的VL选自SEQ ID NO：3、49、51、53、55、57、59、61、63或65或与之具有70-100％的序列同一性，和/或识别GPC3的抗原识别单元的氨基酸序列的VH选自SEQ ID NO：1、48、50、52、54、56、58、60、62或64或与之具有70-100％的序列同一性，或者，In a preferred embodiment, the VL of the antigen recognition unit comprising the amino acid sequence of the antigen recognition unit recognizing GPC3 is selected from SEQ ID NO: 3, 49, 51, 53, 55, 57, 59, 61, 63 or 65 or with The VH having 70-100% sequence identity, and/or recognizing the amino acid sequence of the antigen recognition unit of GPC3 is selected from SEQ ID NO: 1, 48, 50, 52, 54, 56, 58, 60, 62 or 64 or have 70-100% sequence identity to it, or,
识别Claudin18.2的抗原识别单元的氨基酸序列的VL选自SEQ ID NO：67、69、71、73、75、77、79、81、83或85或与之具有70-100％的序列同一性，和/或识别Claudin18.2的抗原识别单元的氨基酸序列的VH选自SEQ ID NO：66、68、70、72、74、76、78、80、82或84或与之具有70-100％的序列同一性。The VL recognizing the amino acid sequence of the antigen recognition unit of Claudin18.2 is selected from or has 70-100% sequence identity with SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85 , and/or the VH that recognizes the amino acid sequence of the antigen recognition unit of Claudin18.2 is selected from or has 70-100% sequence identity.
在一优选例中，所述工程化细胞具有以下特点之一或其组合：In a preferred embodiment, the engineered cells have one of the following characteristics or a combination thereof:
1)能杀伤携带所述抗原的靶细胞；1) can kill target cells carrying the antigen;
2)与所述靶细胞孵育后分泌IFN；2) secrete IFN after incubation with the target cells;
3)与所述靶细胞孵育后CD25、CD69阳性率提高；3) The positive rates of CD25 and CD69 are increased after incubation with the target cells;
4)CD4+、CD8+阳性率与未转导编码ATC的多核苷酸片段的细胞接近；4) The positive rates of CD4+ and CD8+ are close to those of cells that have not been transduced with polynucleotide fragments encoding ATC;
5)原始T细胞比例大；和/或5) A large proportion of naive T cells; and/or
6)PD-1/TIM-3/LAG-3阳性率与未转导编码ATC的多核苷酸片段的细胞接近。6) The positive rate of PD-1/TIM-3/LAG-3 was close to that of cells not transduced with polynucleotide fragments encoding ATC.
在一优选例中，与表达相同抗原识别单元的嵌合抗原受体的CAR的细胞相比，所述工程化细胞具有以下之一或其组合：In a preferred embodiment, compared with cells expressing CARs of chimeric antigen receptors of the same antigen recognition unit, the engineered cells have one of the following or a combination thereof:
1)对靶细胞的杀伤能力、和/或与靶细胞孵育后分泌IFN-γ、细胞增殖没有显著差异；与靶细胞孵育后CD25、CD69表达水平高；1) There was no significant difference in the killing ability of target cells, and/or the secretion of IFN-γ and cell proliferation after incubation with target cells; the expression levels of CD25 and CD69 were high after incubation with target cells;
2)原始T细胞比例大；2) The proportion of primitive T cells is large;
3)CD4+/CD8+比例高；3) The ratio of CD4+/CD8+ is high;
4)PD-1/TIM-3/LAG-3阳性率低。4) The positive rate of PD-1/TIM-3/LAG-3 is low.
在一优选例中，与表达相同ATC但内源性TCR亚基的表达、活性和/或信号传导没有被降低或抑制的细胞相比，所述工程化细胞ATC阳性率提高或提高约5％、10％、20％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％。In a preferred embodiment, the ATC positive rate of the engineered cells is increased or increased by about 5% compared to cells that express the same ATC but the expression, activity and/or signaling of endogenous TCR subunits are not reduced or inhibited , 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100 %.
本发明第二方面提供了如本发明所述的工程化细胞中的ATC分子。A second aspect of the present invention provides ATC molecules in engineered cells according to the present invention.
本发明第三方面提供了一种多核苷酸，其编码如本发明所述的工程化细胞中的ATC或gRNA分子。A third aspect of the present invention provides a polynucleotide encoding the ATC or gRNA molecule in the engineered cells of the present invention.
本发明第四方面提供了一种载体，其包含如本发明所述的多核苷酸。A fourth aspect of the present invention provides a vector comprising the polynucleotide according to the present invention.
本发明第五方面提供了一种药物组合物，其包含有效量的如本发明所述的工程化细 胞、如本发明所述的ATC分子、如本发明所述的多核苷酸、如本发明所述的载体和药学上可接受的赋形剂。A fifth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the engineered cells of the present invention, the ATC molecules of the present invention, the polynucleotides of the present invention, the The carrier and pharmaceutically acceptable excipients.
在一优选例中，如本发明所述的药物组合物，其用于治疗肿瘤。In a preferred embodiment, the pharmaceutical composition according to the present invention is used for the treatment of tumors.
本发明第六方面提供了一种试剂盒，其包含如本发明所述的工程化细胞、如本发明所述的ATC分子、如本发明所述的多核苷酸、如本发明所述的载体或者如本发明所述的药物组合物。The sixth aspect of the present invention provides a kit comprising the engineered cells of the present invention, the ATC molecules of the present invention, the polynucleotides of the present invention, and the vector of the present invention Or the pharmaceutical composition according to the present invention.
在一优选例中，如本发明所述的试剂盒还包括用于治疗和/或预防肿瘤、病原体感染、自身免疫性疾病或同种异体移植的书面说明书。In a preferred embodiment, the kit according to the present invention further includes written instructions for treating and/or preventing tumors, pathogen infections, autoimmune diseases or allotransplantation.
本发明第七方面提供了一种降低受试者肿瘤负荷的方法，包括向所述受试者施用有效量的如本发明所述的工程化细胞、如本发明所述的药物组合物。A seventh aspect of the present invention provides a method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of the engineered cells of the present invention and the pharmaceutical composition of the present invention.
本发明第八方面提供了一种治疗或预防受试者肿瘤的方法，包括向所述受试者施用有效量的如本发明所述的工程化细胞、如本发明所述的药物组合物。The eighth aspect of the present invention provides a method for treating or preventing tumors in a subject, comprising administering to the subject an effective amount of the engineered cells of the present invention and the pharmaceutical composition of the present invention.
在一优选例中，所述肿瘤选自肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、胰腺癌、多发性骨髓瘤、血液肿瘤。In a preferred embodiment, the tumor is selected from liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, pancreatic cancer, multiple myeloma, and hematological tumors.
在一优选例中，所述肿瘤是GPC3阳性肿瘤、或Claudin18.2阳性肿瘤。In a preferred embodiment, the tumor is a GPC3-positive tumor or a Claudin18.2-positive tumor.
本发明第九方面提供了一种产生抗原特异性免疫效应细胞的方法，包括将编码如本发明所述的ATC分子的多核苷酸、或将如本发明所述的多核苷酸、或将如本发明所述的载体导入免疫效应细胞。A ninth aspect of the present invention provides a method for generating antigen-specific immune effector cells, comprising adding a polynucleotide encoding the ATC molecule of the present invention, or the polynucleotide of the present invention, or the The vector of the present invention is introduced into immune effector cells.
本发明第十方面提供了一种延长患有肿瘤的受试者存活的方法，包括向所述受试者施用有效量的如本发明所述的工程化细胞、如本发明所述的药物组合物。A tenth aspect of the present invention provides a method for prolonging the survival of a subject suffering from a tumor, comprising administering to the subject an effective amount of the engineered cells of the present invention, the pharmaceutical combination of the present invention thing.
本发明第十一方面提供了如本发明所述的工程化细胞、如本发明所述的ATC分子、如本发明所述的多核苷酸、如本发明所述的载体、如本发明所述的药物组合物在治疗中的用途。The eleventh aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition in therapy.
本发明第十二方面提供了如本发明所述的工程化细胞、如本发明所述的ATC分子、如本发明所述的多核苷酸、如本发明所述的载体、如本发明所述的药物组合物用于减轻受试者的肿瘤负荷的用途。The twelfth aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition for reducing tumor burden in a subject.
本发明第十三方面提供了如本发明所述的工程化细胞、如本发明所述的ATC分子、如本发明所述的多核苷酸、如本发明所述的载体、如本发明所述的药物组合物用于治疗或预防受试者肿瘤的用途。The thirteenth aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition for treating or preventing tumors in a subject.
本发明第十四方面提供了如本发明所述的工程化细胞、如本发明所述的ATC分子、如本发明所述的多核苷酸、如本发明所述的载体、如本发明所述的药物组合物用于延长患有肿瘤的受试者存活的用途。A fourteenth aspect of the present invention provides an engineered cell according to the present invention, an ATC molecule according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and a vector according to the present invention Use of the pharmaceutical composition for prolonging the survival of a subject suffering from a tumor.
本发明第十五方面提供了靶向GPC3的ATC，所述ATC包含识别GPC3的抗原识别单元和TRAC形成的多肽，和识别GPC3的抗原识别单元和TRBC形成的多肽；或所述ATC包含识别GPC3的抗原识别单元和TRDC形成的多肽，和识别GPC3的抗原识别单元和TRGC形成的多肽。A fifteenth aspect of the present invention provides an ATC targeting GPC3, the ATC comprising a polypeptide formed by an antigen recognition unit that recognizes GPC3 and TRAC, and a polypeptide formed by an antigen recognition unit that recognizes GPC3 and TRBC; or the ATC comprises a polypeptide that recognizes GPC3 A polypeptide formed by the antigen recognition unit and TRDC, and a polypeptide formed by the antigen recognition unit and TRGC of GPC3.
在一优选例中，所述识别GPC3的抗原识别单元包括抗GPC3抗体的抗体重链可变区(VH)和/或轻链可变区(VL)：In a preferred embodiment, the antigen recognition unit that recognizes GPC3 includes the antibody heavy chain variable region (VH) and/or light chain variable region (VL) of an anti-GPC3 antibody:
所述TRAC肽与VH直接连接或通过铰链区连接、TRBC肽与VL直接连接或通过铰链区连接；或所述TRAC肽与VL直接连接或通过铰链区连接、所述TRBC肽与VH直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VH直接连接或通过铰链区连接、TRGC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VL直接连接或通过铰链区连接、所述TRGC肽与VH直接连接或通过铰链区连接。The TRAC peptide is directly linked to the VH or linked through the hinge region, the TRBC peptide is directly linked to the VL or linked through the hinge region; or the TRAC peptide is directly linked to the VL or linked through the hinge region, the TRBC peptide is directly linked to the VH or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VH or through the hinge region, the TRGC peptide is directly attached to the VL or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VL or through the hinge Region linkage, the TRGC peptide is linked directly to the VH or via a hinge region.
本发明第十六方面提供了靶向Claudin18.2的ATC，所述ATC包含识别Claudin18.2的抗原识别单元和TRAC形成的多肽，和识别Claudin18.2的抗原识别单元和TRBC形成的多肽；或所述ATC包含识别Claudin18.2的抗原识别单元和TRDC形成的多肽，和识别Claudin18.2的抗原识别单元和TRGC形成的多肽。The sixteenth aspect of the present invention provides an ATC targeting Claudin18.2, the ATC comprising a polypeptide formed by an antigen recognition unit recognizing Claudin18.2 and TRAC, and a polypeptide formed by an antigen recognition unit recognizing Claudin18.2 and TRBC; or The ATC comprises a polypeptide formed by recognizing the antigen recognition unit of Claudin18.2 and TRDC, and a polypeptide formed by recognizing the antigen recognition unit of Claudin18.2 and TRGC.
在一优选例中，所述识别Claudin18.2的抗原识别单元包括抗Claudin18.2抗体的抗体重链可变区(VH)和/或轻链可变区(VL)：In a preferred embodiment, the antigen recognition unit recognizing Claudin18.2 comprises the antibody heavy chain variable region (VH) and/or light chain variable region (VL) of an anti-Claudin18.2 antibody:
所述TRAC肽与VH直接连接或通过铰链区连接、TRBC肽与VL直接连接或通过铰链区连接；或所述TRAC肽与VL直接连接或通过铰链区连接、所述TRBC肽与VH直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VH直接连接或通过铰链区连接、TRGC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VL直接连接或通过铰链区连接、所述TRGC肽与VH直接连接或通过铰链区连接。The TRAC peptide is directly linked to the VH or linked through the hinge region, the TRBC peptide is directly linked to the VL or linked through the hinge region; or the TRAC peptide is directly linked to the VL or linked through the hinge region, the TRBC peptide is directly linked to the VH or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VH or through the hinge region, the TRGC peptide is directly attached to the VL or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VL or through the hinge Region linkage, the TRGC peptide is linked directly to the VH or via a hinge region.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
附图说明Description of drawings
图1 ATC病毒滴度测定结果。Figure 1 ATC virus titer determination results.
图2 ATCT阳性率和敲除效率检测结果。Figure 2 The detection results of ATCT positive rate and knockout efficiency.
图3 ATCT细胞对靶细胞的体外杀伤结果。Figure 3 In vitro killing results of ATCT cells on target cells.
图4 ATCT细胞与靶细胞共孵育后IFN-γ分泌结果。Figure 4 IFN-γ secretion results after co-incubation of ATCT cells with target cells.
图5 ATCT细胞CD25和CD69表达水平检测结果。Figure 5 Detection results of CD25 and CD69 expression levels in ATCT cells.
图6 ATCT细胞分化情况检测结果。Figure 6 The results of the differentiation test of ATCT cells.
图7 ATCT细胞CD4/CD8分型检测结果。Figure 7 Results of CD4/CD8 typing of ATCT cells.
图8 ATCT细胞耗竭情况检测结果。Figure 8 Results of ATCT cell depletion detection.
图9 ATCT细胞增殖情况检测结果。Figure 9 Results of ATCT cell proliferation detection.
具体实施方式Detailed ways
本发明是关于一种新型细胞免疫技术平台的构建和应用。示例性以GPC3为靶点，将抗GPC3抗体与突变后的T细胞受体进行串联转入内源性TCR低表达或不表达的T细胞，构建抗体-T细胞受体嵌合T细胞(Antibody-TCR-Chimeric T cell，ATCT)。The present invention relates to the construction and application of a novel cellular immune technology platform. Exemplarily targeting GPC3, the anti-GPC3 antibody and the mutated T cell receptor are tandemly transferred into T cells with low or no expression of endogenous TCR to construct antibody-T cell receptor chimeric T cells (Antibody). -TCR-Chimeric T cell, ATCT).
除非专门定义，否则本文所用的所有技术和科学术语具有在基因治疗、生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的那些所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容通过引用并入本文。在发生冲突的情况下，以本说明书为准。此外，除非另有规定，否则本发明的材料、方法和实施例仅是说明性的，而并非旨在进行限制。根据本发明内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等价的方法和组分在本发明的范围内。本发明包括对本发明的主题进行变型和修改来用于各种用途和条件。Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, where suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification will control. Furthermore, unless otherwise specified, the materials, methods, and examples of the present invention are illustrative only and not intended to be limiting. Based on this disclosure, those of skill in the art should appreciate that many changes or changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The present invention is not limited in scope to the specific embodiments described herein, which are intended only as illustrations of various aspects of the invention, and functionally equivalent methods and components are within the scope of this invention. This invention includes variations and modifications of the inventive subject matter for various usages and conditions.
除非另有说明，否则本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(Frederick M.AUSUBEL，2000，Wiley and son Inc.，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrook et al.，2001，Cold Spring Harbor，New York:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gait ed.，1984)；Mullis et al.美国专利号4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higgins eds.1984)；Transcription And Translation(B.D.Hames&S.J.Higgins eds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wu et al.，eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Calos eds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和 Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. These techniques are fully explained in the literature. See, eg, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J.Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B.D.Harries & S.J.Higgins eds.1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. , eds.) and Vol.185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand boo k Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986) and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
1.定义：1. Definition:
如本文所用，“约”可表示取决于具体情况并且由本领域技术人员已知或可知的，或表示给定值的至多约1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、25％、30％。可替代的，特别是关于生物系统或方法，该术语可指在数值的一个数量级内，例如在一个值的约5倍之内或在约2倍之内。As used herein, "about" may mean, depending on the circumstances and known or known to those skilled in the art, or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%. Alternatively, particularly with respect to biological systems or methods, the term may mean within an order of magnitude of a numerical value, eg, within about 5-fold or within about 2-fold of a value.
范围：范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本发明的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。Scope: The description in range format is merely for convenience and brevity and should not be construed as an inexorable limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range.
术语“激活免疫效应细胞”，是指信号转导通路引起的细胞内蛋白质表达的变化，导致免疫应答的启动。例如，当CD3分子响应于配体结合和基于免疫受体酪氨酸的活化基序(ITAM)聚集，从而产生信号转导级联反应。在一实施例中，当内源性TCR或外源性ATC与抗原结合后形成的免疫突触，包括在结合受体(例如，CD4或CD8，CD3γ/CDδ/CDε/CDζ等)附近的许多分子的聚集。膜结合信号分子的这种聚集使CD3分子中包含的ITAM基序磷酸化。该磷酸化进而启动T细胞激活通路，最终激活转录因子，例如NF-κB和AP-1。这些转录因子诱导T细胞的整体基因表达，包括上调IL-2生成，促进T细胞增殖，进而启动T细胞介导的免疫应答。“T细胞活化”或“T细胞激活”指被刺激后诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。使用CD3/CD28磁珠，体外抗原刺激或者体内抗原刺激都会对T细胞的活化程度和持续时间造成影响。在一个实施例中，所述工程化细胞与含特定靶抗原肿瘤细胞共孵育后活化、或所述工程化细胞被病毒感染后活化。The term "activation of immune effector cells" refers to changes in intracellular protein expression caused by signal transduction pathways that lead to the initiation of an immune response. For example, a signal transduction cascade occurs when CD3 molecules aggregate in response to ligand binding and immunoreceptor tyrosine-based activation motifs (ITAMs). In one embodiment, the immune synapse formed when endogenous TCR or exogenous ATC binds to an antigen includes many near the binding receptor (eg, CD4 or CD8, CD3γ/CDδ/CDε/CDζ, etc.). aggregation of molecules. This aggregation of membrane-bound signaling molecules phosphorylates the ITAM motif contained in the CD3 molecule. This phosphorylation in turn initiates a T cell activation pathway that ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors induce overall gene expression in T cells, including upregulation of IL-2 production, which promotes T cell proliferation, which in turn initiates T cell-mediated immune responses. "T cell activation" or "T cell activation" refers to the state of T cells that upon stimulation induces detectable cell proliferation, cytokine production, and/or detectable effector function. Using CD3/CD28 magnetic beads, antigen stimulation in vitro or in vivo will affect the degree and duration of T cell activation. In one embodiment, the engineered cells are activated after co-incubation with tumor cells containing a specific target antigen, or the engineered cells are activated after infection with a virus.
术语“刺激免疫效应细胞”，是指信号转导通路导致免疫效应细胞发生强烈、持续的免疫应答。在一实施例中，这发生在免疫效应细胞(例如，T细胞)激活后或通过包括但不限于CD28、CD137(4-1BB)、OX40、CD40和ICOS的受体同时介导。The term "stimulation of immune effector cells" refers to a signal transduction pathway that results in a strong, sustained immune response by immune effector cells. In one embodiment, this occurs following activation of immune effector cells (eg, T cells) or mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS.
术语“抗原识别单元”是指特异性结合抗原决定簇的分子，包括免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗体”不仅包括完整的抗体分子，也包括保留抗原结合能力的抗体分子的片段。本发明中术语“抗体”与术语“免疫球蛋白”“抗原识别单元”可互换使用。抗体，包括 但不限于单克隆抗体、多克隆抗体、天然抗体、双特异性抗体、嵌合抗体、Fv、Fab、Fab’、Fab’-SH、F(ab’)2、线性抗体、单链抗体分子(例如scFv)、单域抗体。在一实施例中，抗体包含通过二硫键连接的至少两个重(H)链和两个轻(L)链。每条重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区有三个结构域CH1、CH2、CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区(CL)。轻链恒定区由一个结构域组成。VH和VL可进一步细分为高变区，称为互补决定区(CDR)，其间散布有更保守的区域，称为框架区(FR)。每个VH和VL均由三个CDR和四个FR组成，从氨基端到羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区包含与抗原相互作用的结合结构域。抗体的恒定区介导免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的各种细胞(例如，免疫效应细胞)与经典补体系统的第一组分(C1q)。如果抗原识别单元以与其它参考抗原(包括多肽或其他物质)结合相比更大亲和力(或称亲合力)结合抗原，则所述抗原识别单元与抗原“特异性结合”或与抗原是“免疫反应性的”。The term "antigen recognition unit" refers to a molecule that specifically binds an antigenic determinant, including immunoglobulin molecules and immunologically active portions of immunological molecules, i.e., molecules containing an antigen-binding site that specifically binds ("immunoreactive") an antigen . The term "antibody" includes not only intact antibody molecules, but also fragments of antibody molecules that retain antigen-binding capacity. The term "antibody" is used interchangeably with the terms "immunoglobulin" and "antigen recognition unit" in the present invention. Antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, native antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single chain Antibody molecules (eg scFv), single domain antibodies. In one embodiment, the antibody comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain. VH and VL can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, immune effector cells) and the first component (Clq) of the classical complement system. An antigen recognition unit "specifically binds" to an antigen or is "immune" to an antigen if it binds the antigen with greater affinity (or avidity) than other reference antigens (including polypeptides or other substances) reactive".
术语“嵌合抗原受体(CAR)”,是指包括与能够激活或刺激免疫效应细胞的细胞内信号传导结构域融合的胞外抗原结合结构域和跨膜结构域的分子。在一实施例中，CAR的胞外抗原结合结构域包含scFV。scFV包括抗体重链可变区和轻链可变区。在一实施例中，CAR包括scFV、跨膜结构域和胞内信号传导结构域顺序连接而成的多肽。The term "chimeric antigen receptor (CAR)" refers to a molecule comprising an extracellular antigen binding domain and a transmembrane domain fused to an intracellular signaling domain capable of activating or stimulating immune effector cells. In one embodiment, the extracellular antigen binding domain of the CAR comprises a scFV. scFVs include antibody heavy chain variable regions and light chain variable regions. In one embodiment, the CAR comprises a polypeptide in which a scFV, a transmembrane domain and an intracellular signaling domain are linked in sequence.
术语“核酸”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物，包括编码目的多肽或其片段的任何核酸分子。所述核酸分子只需要与内源性核酸序列保持基本同一性即可，不需要与内源性核酸序列100％同源性或同一性。与内源性序列具有“基本同一性”的多核苷酸通常能与双链核酸分子的至少一条链杂交。“杂交”是指在各种严格条件下在互补多核苷酸序列或其部分之间形成双链分子的配对。术语“同源性”或“同一性”是指两个聚合物分子之间，例如，两个核酸分子如两个DNA分子或两个RNA分子之间，或两个多肽分子之间的亚单位序列同一性。术语“基本同一性”或“基本同源性”，是指与参考氨基酸序列或核酸序列表现出至少约50％同源性或同一性的多肽或核酸分子。在一实施例中，这样的序列与用于比较的氨基酸或核酸序列为至少约60％、65％、70％、75％、80％、85％、90％、95％、99％或100％同源性或同一性。序列同一性可以通过使用序列分析软件(例如，BLAST、BESTFIT、GAP或PILEUP/PRETTYBOX程序)进行测量。这样的软件通过将同源性程度分配给各种取代、缺失和/或其它修饰来匹配相同或相似的序列。保守取代通常包括以下组内的取代：甘氨酸、丙氨酸；缬氨酸、异亮氨酸、亮氨酸；天冬氨酸、谷氨酸、天冬酰胺、谷氨酰胺；丝氨酸、苏氨酸赖氨酸、精氨酸；和苯丙氨酸、酪氨酸。在确定同一性程度的示例性方法中，可以使用BLAST程序，其中e-3和e-100之间的概率得分指示密切相关的序列。The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, including any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof. The nucleic acid molecule only needs to maintain substantial identity with the endogenous nucleic acid sequence, and does not need to be 100% homologous or identical to the endogenous nucleic acid sequence. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of double-stranded molecules between complementary polynucleotide sequences or portions thereof under various stringent conditions. The term "homology" or "identity" refers to a subunit between two polymer molecules, eg, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules sequence identity. The term "substantially identical" or "substantially homologous" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence or nucleic acid sequence. In one embodiment, such a sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the amino acid or nucleic acid sequence used for comparison Homology or identity. Sequence identity can be measured using sequence analysis software (eg, BLAST, BESTFIT, GAP or the PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine acid lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where a probability score between e-3 and e-100 indicates closely related sequences.
术语“疾病”是指损害或干扰细胞、组织或器官的正常功能的任何病症，例如肿瘤(癌症)或病原体感染。难治性癌症包括但不限于放疗不敏感、放疗后复发、化疗不敏感、化疗后复发、对CAR-T治疗不敏感或治疗后复发的癌症。The term "disease" refers to any condition that impairs or interferes with the normal function of cells, tissues or organs, such as tumors (cancer) or pathogen infections. Refractory cancers include, but are not limited to, radiotherapy-insensitive, relapsed after radiotherapy, chemotherapy-insensitive, relapsed after chemotherapy, insensitive to CAR-T therapy, or cancers that have relapsed after treatment.
术语“治疗有效量”、“治疗有效的”、“有效量”或“以有效的量”在本文中可互换地使用，是指如本文中所述有效地实现特定生物学结果的化合物、制剂、物质或组合物、药物组合物的量，例如但不限于足以促进T细胞应答的量或剂量。有效量的免疫效应细胞，是指但不限于：能使抗肿瘤活性增加、增强或延长的免疫效应细胞的数量；抗肿瘤免疫效应细胞数目或活化免疫效应细胞数目的增加；促进IFN-γ分泌、肿瘤消退、肿瘤缩小、肿瘤坏死的免疫效应细胞的数量。The terms "therapeutically effective amount", "therapeutically effective", "effective amount" or "in an effective amount" are used interchangeably herein to refer to a compound effective to achieve a specified biological result, as described herein, The amount of formulation, substance or composition, pharmaceutical composition, such as, but not limited to, an amount or dose sufficient to promote a T cell response. An effective amount of immune effector cells refers to, but is not limited to: the number of immune effector cells that can increase, enhance or prolong anti-tumor activity; increase the number of anti-tumor immune effector cells or the number of activated immune effector cells; promote the secretion of IFN-γ , tumor regression, tumor shrinkage, and tumor necrosis in the number of immune effector cells.
术语“内源”，是指核酸分子或多肽等来自生物体自身。The term "endogenous" means that a nucleic acid molecule or polypeptide, etc., is derived from the organism itself.
术语“外源”，是指核酸分子或多肽不是内源性存在细胞中的，或表达水平不足以实现过表达时具有的功能；涵盖在细胞中表达的任何重组核酸分子或多肽，例如外源、异源和过表达的核酸分子和多肽。The term "exogenous" means that a nucleic acid molecule or polypeptide is not endogenously present in a cell, or is expressed at an insufficient level to achieve the function when overexpressed; any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous , heterologous and overexpressed nucleic acid molecules and polypeptides.
术语“识别”，是指选择性结合靶抗原。识别肿瘤的工程化细胞可以表达与肿瘤抗原结合的受体(例如ATC或CAR)。The term "recognition" refers to selective binding to a target antigen. Engineered cells that recognize tumors can express receptors (eg, ATCs or CARs) that bind to tumor antigens.
术语“特异性结合”是指识别并且结合存在于样品中的结合配偶体(例如肿瘤抗原)蛋白质的抗体或配体，但是该抗体或配体基本上不会识别或结合样品中的其它分子。The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample.
术语“信号肽(SP)”，是引导新合成的蛋白或多肽向分泌通路转移的短肽链(长度约5-30个氨基酸)。本发明的多核苷酸编码区可以与编码信号肽的编码区连接，所述信号肽指导本发明多核苷酸编码的多肽分泌。例如，如果需要分泌融合蛋白，则可以将编码信号肽的多核苷酸置于编码本发明融合蛋白或多肽的多核苷酸的上游。本领域普通技术人员知晓由脊椎动物细胞分泌的蛋白或多肽通常具有与蛋白或多肽N末端融合的信号肽，所述信号肽从翻译的蛋白或多肽被切下以产生分泌的或“成熟”形式的蛋白或多肽。在某些实施方案中，使用天然信号肽，例如TCR的信号肽(TRAV信号肽序列如SEQ ID NO：93所示、TRBV信号肽如SEQ ID NO：94所示)、IL-2信号肽序列如SEQ ID NO：96(人)、SEQ ID NO：97(小鼠)所示，κ信号肽序列如SEQ ID NO：98(人)、SEQ ID NO：99(小鼠)所示；CD8信号肽序列如SEQ ID NO：95(人)所示；截短的人CD8信号肽如SEQ ID NO：100(人)所示；白蛋白信号肽序列如SEQ ID NO：101(人)所示；催乳素信号肽序列如SEQ ID NO：102(人)所示。The term "signal peptide (SP)" is a short peptide chain (about 5-30 amino acids in length) that directs the transfer of newly synthesized proteins or polypeptides to the secretory pathway. The coding region of the polynucleotide of the present invention may be linked to a coding region encoding a signal peptide that directs secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of a fusion protein is desired, a polynucleotide encoding a signal peptide can be placed upstream of a polynucleotide encoding a fusion protein or polypeptide of the invention. Those of ordinary skill in the art know that proteins or polypeptides secreted by vertebrate cells often have a signal peptide fused to the N-terminus of the protein or polypeptide that is cleaved from the translated protein or polypeptide to produce the secreted or "mature" form protein or polypeptide. In certain embodiments, native signal peptides are used, such as the signal peptide of TCR (TRAV signal peptide sequence is shown in SEQ ID NO: 93, TRBV signal peptide is shown in SEQ ID NO: 94), IL-2 signal peptide sequence As shown in SEQ ID NO: 96 (human), SEQ ID NO: 97 (mouse), the kappa signal peptide sequence is shown in SEQ ID NO: 98 (human), SEQ ID NO: 99 (mouse); CD8 signal The peptide sequence is shown in SEQ ID NO: 95 (human); the truncated human CD8 signal peptide is shown in SEQ ID NO: 100 (human); the albumin signal peptide sequence is shown in SEQ ID NO: 101 (human); The prolactin signal peptide sequence is shown in SEQ ID NO: 102 (human).
术语“个体”和“受试者”可互换，包括人或来自其他种属的动物，其包括但不限于人、小鼠、大鼠、仓鼠和豚鼠、兔子、狗、猫、绵羊、猪、山羊、牛、马、猿、猴子。The terms "individual" and "subject" are interchangeable and include humans or animals from other species including, but not limited to, humans, mice, rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs , goat, cow, horse, ape, monkey.
术语“T细胞(抗原)受体(T cell receptor,TCR)”，也称为“TCR亚基”，或“TCR单 元”，为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。TCR负责识别与主要组织相容性复合体分子结合的抗原。TCR是由两条不同肽链构成的异二聚体，由α、β两条肽链组成，或由γ、δ两条肽链组成；每条肽链包括可变区和恒定区(包括胞外恒定区、跨膜区和胞质区)；其特点是胞质区很短。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时，CDR1、CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。TCR分为两类：TCR1和TCR2；其中TCR1由γ和δ两条链组成，而TCR2由α和β两条链组成。外周血中，约90％-95％的T细胞表达TCR2；而且任一T细胞只表达TCR2或TCR1。这些天然TCR受体的识别能力常常比较弱，因此不能形成对靶细胞的有效攻击。在这种情况下，可以通过部分基因修改的方法来提高天然TCR对相应靶抗原的“亲和力”，即高亲和力TCR，例如本发明提供的抗体-T细胞受体嵌合物(Antibody-TCR-Chimeric，ATC)。The term "T cell receptor (TCR)", also known as "TCR subunit", or "TCR unit", is a characteristic marker on the surface of all T cells that binds non-covalently to CD3 bind to form the TCR-CD3 complex. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is a heterodimer composed of two different peptide chains, consisting of α and β peptide chains, or γ and δ peptide chains; each peptide chain includes a variable region and a constant region (including cellular outer constant region, transmembrane region and cytoplasmic region); it is characterized by a very short cytoplasmic region. The TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (Vα, Vβ) has three hypervariable regions, CDR1, CDR2, and CDR3. Among them, CDR3 has the largest variation, which directly determines the antigen of TCR. binding specificity. When TCR recognizes the MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the side wall of the antigen-binding groove of the MHC molecule, while CDR3 directly binds to the antigen peptide. TCRs are divided into two categories: TCR1 and TCR2; where TCR1 is composed of two chains, γ and δ, while TCR2 is composed of two chains, α and β. In peripheral blood, about 90%-95% of T cells express TCR2; and any T cell expresses only TCR2 or TCR1. The recognition capabilities of these natural TCR receptors are often weak and thus cannot form an effective attack on target cells. In this case, the "affinity" of the native TCR for the corresponding target antigen can be improved by means of partial genetic modification, that is, high-affinity TCR, such as the antibody-T cell receptor chimera (Antibody-TCR- Chimeric, ATC).
术语“分离的”意指从天然状态改变或移出的。例如，天然存在于活动物中的核酸或肽不是“分离的”，但与其天然状态下共同存在的物质部分或完全分离的相同核酸或肽则是“分离的”。分离的核酸或蛋白质可以以基本上纯化的形式存在，或者可存在于非天然环境如宿主细胞中。The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the material with which it is found in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as a host cell.
术语“肽”、“多肽”和“蛋白质”可互换使用，是指由通过肽键共价连接的氨基酸残基组成的化合物。The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds.
术语“同义突变”，是指DNA片段中某个碱基对的突变并不改变所编码的氨基酸，原因在于该位置的密码子在突变前后为同义密码子。核酸序列中，连续的3个核苷酸残基为一个密码子。在64个密码子中，除了3个终止密码子(TAA、TAG、TGA)以外，其余61个密码子代表20种氨基酸(表1)。除了甲硫氨酸、色氨酸各有1个密码子外，其它18种氨基酸均有2个或多个密码子。对应于同一种氨基酸的不同密码子称为同义密码子。比如同义密码子CTA与CTG均编码亮氨酸，若CTA中的A突变为G则该变异为同义突变。The term "synonymous mutation" means that a mutation of a base pair in a DNA fragment does not change the encoded amino acid because the codon at that position is a synonymous codon before and after the mutation. In a nucleic acid sequence, three consecutive nucleotide residues constitute a codon. Among the 64 codons, except for 3 stop codons (TAA, TAG, TGA), the remaining 61 codons represent 20 amino acids (Table 1). Except for methionine and tryptophan, which each have one codon, the other 18 amino acids have two or more codons. Different codons corresponding to the same amino acid are called synonymous codons. For example, the synonymous codons CTA and CTG both encode leucine, and if the A in CTA is mutated to G, the mutation is a synonymous mutation.
表1.密码子表Table 1. Codon Table
Figure PCTCN2022086816-appb-000001
Figure PCTCN2022086816-appb-000001
术语“野生型基因”，指自然界中占多数的等位基因，在生物学实验中常作为标准对照基因。与之相对应的概念为突变型基因。The term "wild-type gene" refers to the most common allele in nature and is often used as a standard control gene in biological experiments. The corresponding concept is mutant gene.
术语“工程化”是指应用细胞生物学和分子生物学的原理和方法，通过某种工程学手段，在细胞整体水平、细胞器水平、分子水平上，按照人们的意愿来改变细胞内的遗传物质或获得细胞产品的一门综合科学技术。The term "engineering" refers to applying the principles and methods of cell biology and molecular biology to change the genetic material in cells according to people's wishes at the overall level of cells, organelles, and molecules through some engineering means. Or a comprehensive science and technology to obtain cell products.
2.抗体-T细胞受体嵌合物ATC(Antibody-TCR-Chimeric)2. Antibody-T cell receptor chimeric ATC (Antibody-TCR-Chimeric)
本发明提供了对TCR进行改造后的ATC受体。所述ATC包含用抗原识别单元修饰的TCR亚基部分。The present invention provides ATC receptors modified by TCR. The ATC comprises a TCR subunit portion modified with an antigen recognition unit.
2.1 TCR亚基部分2.1 TCR subunit part
ATC的TCR亚基部分包括天然或修饰的TCRα链、β链、γ链和/或δ链。在一实施例中，ATC包括天然或修饰的TCRα链和β链的恒定区；或包括天然或修饰的TCRγ链和δ链的恒定区。The TCR subunit portion of ATC includes native or modified TCR alpha, beta, gamma and/or delta chains. In one embodiment, the ATC includes the constant regions of native or modified TCR alpha and beta chains; or includes the constant regions of native or modified TCR gamma and delta chains.
在一实施例中，TCR亚基恒定区，任选地，还包括铰链/间隔区。In one embodiment, the TCR subunit constant region, optionally, further includes a hinge/spacer region.
TCR亚基恒定区包含TCRα恒定区(TRAC)、TCRβ恒定区(TRBC，例如TRBC1或TRBC2)、TCRγ恒定区(TRGC，例如TRGC1或TRGC2)、TCRδ恒定区(TRDC)或其任何变体或功能片段。TCR subunit constant regions include TCRα constant regions (TRAC), TCRβ constant regions (TRBC, eg, TRBC1 or TRBC2), TCRγ constant regions (TRGC, eg, TRGC1 or TRGC2), TCRδ constant regions (TRDC), or any variant or function thereof Fragment.
野生型TRAC核酸分子，指编码TRAC多肽，具有NCBI GenBank Gene ID：28755， NG_001332.3，925603至930229(TRAC，SEQ ID NO：6)所示核苷酸序列。Wild-type TRAC nucleic acid molecule, refers to encoding TRAC polypeptide, has the nucleotide sequence shown in NCBI GenBank Gene ID: 28755, NG_001332.3, 925603 to 930229 (TRAC, SEQ ID NO: 6).
野生型TRBC核酸分子，指编码TRBC多肽，具有NCBI GenBank Gene ID：28639，NC_000007.14，142791694至142793141(TRBC1，SEQ ID NO：15)，或NCBI GenBank Gene ID：28638，NG_001333.2，655095至656583(TRBC2)所示核苷酸序列。Wild-type TRBC nucleic acid molecule, refers to encoding a TRBC polypeptide, with NCBI GenBank Gene ID: 28639, NC_000007.14, 142791694 to 142793141 (TRBC1, SEQ ID NO: 15), or NCBI GenBank Gene ID: 28638, NG_001333.2, 655095 to The nucleotide sequence shown in 656583 (TRBC2).
野生型TRGC核酸分子,指编码TRGC多肽，具有NCBI GenBank Gene ID：6966，NG_001336.2，108270至113860(TRGC1，SEQ ID NO：19)，或NCBI GenBank Gene ID：6967，NG_001336.2，124376至133924(TRGC2)所示核苷酸序列。Wild-type TRGC nucleic acid molecule, which encodes a TRGC polypeptide, has NCBI GenBank Gene ID: 6966, NG_001336.2, 108270 to 113860 (TRGC1, SEQ ID NO: 19), or NCBI GenBank Gene ID: 6967, NG_001336.2, 124376 to The nucleotide sequence shown in 133924 (TRGC2).
野生型TRDC核酸分子,指编码TRDC多肽，具有与NCBI GenBank Gene ID：28526，NG_001332.3，841011至844674(TRDC,SEQ ID NO：22)所示核苷酸序列。Wild-type TRDC nucleic acid molecule, refers to encoding TRDC polypeptide, has the nucleotide sequence shown in NCBI GenBank Gene ID: 28526, NG_001332.3, 841011 to 844674 (TRDC, SEQ ID NO: 22).
本发明提供的ATC核酸分子包含碱基突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子。在一实施例中，对ATC包含的TCR亚基的恒定区的核酸片段进行突变。在一实施例中，对ATC包含的TRAC和/或TRBC的核酸片段进行突变。在一实施例中，对ATC包含的TRGC和/或TRDC的核酸片段进行突变。优选地，对ATC包含的野生型TRAC和/或TRBC的核酸片段进行突变。优选地，对ATC包含的野生型TRGC和/或TRDC的核酸片段进行突变。The ATC nucleic acid molecule provided by the present invention comprises a nucleic acid molecule that is no longer the target sequence targeted by gene knockout technology and/or gene silencing technology after base mutation. In one embodiment, the nucleic acid fragment of the constant region of the TCR subunit comprised by the ATC is mutated. In one embodiment, the nucleic acid fragments of TRAC and/or TRBC contained in the ATC are mutated. In one embodiment, the nucleic acid fragments of TRGC and/or TRDC contained in the ATC are mutated. Preferably, the nucleic acid fragments of wild-type TRAC and/or TRBC contained in the ATC are mutated. Preferably, the nucleic acid fragments of wild-type TRGC and/or TRDC contained in the ATC are mutated.
本发明提供的ATC核酸分子包含碱基同义突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子。在一实施例中，对ATC包含的TCR亚基的胞外恒定区的核酸片段进行同义突变。在一实施例中，对ATC包含的TRAC和/或TRBC的胞外恒定区的核酸片段进行同义突变。在一实施例中，对ATC包含的TRGC和/或TRDC的胞外恒定区的核酸片段进行同义突变。优选地，对ATC包含的野生型TRAC和/或TRBC的胞外恒定区的核酸片段进行同义突变。优选地，对ATC包含的野生型TRGC和/或TRDC的胞外恒定区的核酸片段进行同义突变。The ATC nucleic acid molecule provided by the present invention comprises a nucleic acid molecule that is no longer the target sequence targeted by gene knockout technology and/or gene silencing technology after synonymous mutation of bases. In one embodiment, a synonymous mutation is made to the nucleic acid fragment of the extracellular constant region of the TCR subunit comprised by the ATC. In one embodiment, a synonymous mutation is made to the nucleic acid fragment of the extracellular constant region of TRAC and/or TRBC comprised by the ATC. In one embodiment, a synonymous mutation is made to the nucleic acid fragment of the extracellular constant region of TRGC and/or TRDC comprised by the ATC. Preferably, the ATC comprises a synonymous mutation of the nucleic acid fragment of the extracellular constant region of wild-type TRAC and/or TRBC. Preferably, the ATC comprises a synonymous mutation of the nucleic acid fragment of the extracellular constant region of wild-type TRGC and/or TRDC.
在一实施例中，本发明提供的ATC多肽所包含的TRAC多肽包含与SEQ ID NO：5所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在一个实施例中，ATC中的TRAC多肽第47位氨基酸突变为半胱氨酸、第115位氨基酸突变为亮氨酸、第118位氨基酸突变为缬氨酸、第119位氨基酸突变为亮氨酸或其组合。In one embodiment, the TRAC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions. In one embodiment, amino acid 47 of the TRAC polypeptide in the ATC is mutated to cysteine, amino acid 115 to leucine, amino acid 118 to valine, and amino acid 119 to leucine acid or a combination thereof.
在某些实施方式中，本发明提供的ATC多肽所包含的TRAC多肽具有与由NCBI GenBank Gene ID：28755，NG_001332.3，925603至930229的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片 段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在一个实施例中，ATC中的TRAC多肽第47位氨基酸突变为半胱氨酸、第115位氨基酸突变为亮氨酸、第118位氨基酸突变为缬氨酸、第119位氨基酸突变为亮氨酸或其组合。In certain embodiments, the TRAC polypeptides included in the ATC polypeptides provided by the present invention have at least about 80% of the amino acid sequence encoded by the transcripts expressed by the genes of NCBI GenBank Gene ID: 28755, NG_001332.3, 925603 to 930229 , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to amino acid sequences or Fragments thereof, and/or may optionally contain up to one or up to two or up to three conservative amino acid substitutions. In one embodiment, amino acid 47 of the TRAC polypeptide in the ATC is mutated to cysteine, amino acid 115 to leucine, amino acid 118 to valine, and amino acid 119 to leucine acid or a combination thereof.
在一实施例中，本发明提供的ATC多肽所包含的TRBC多肽包含与SEQ ID NO：14所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在一个实施例中，ATC中的TRBC多肽第57位氨基酸突变为半胱氨酸。In one embodiment, the TRBC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions. In one embodiment, amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
在一实施例中，本发明提供的ATC多肽所包含的TRBC多肽具有与由NCBI GenBank Gene ID：28639，NC_000007.14，142791694至142793141(TRBC1，SEQ ID NO：15)，NCBI GenBank Gene ID：28638，NG_001333.2，655095至656583(TRBC2)的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在一个实施例中，ATC中的TRBC多肽第57位氨基酸突变为半胱氨酸。In one embodiment, the TRBC polypeptides contained in the ATC polypeptides provided by the present invention have the same properties as those provided by NCBI GenBank Gene ID: 28639, NC_000007.14, 142791694 to 142793141 (TRBC1, SEQ ID NO: 15), NCBI GenBank Gene ID: 28638 , NG_001333.2, 655095 to 656583 (TRBC2) genes expressed transcripts encoding amino acid sequences having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% %, at least about 98%, at least about 99%, or at least about 100% homology or identity to amino acid sequences or fragments thereof, and/or may optionally contain at most one or at most two or at most three conservative amino acid substitutions . In one embodiment, amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
在一实施例中，本发明提供的ATC多肽所包含的TRGC多肽包含与SEQ ID NO：20所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在一个实施例中，ATC中的TRBC多肽第57位氨基酸突变为半胱氨酸。In one embodiment, the TRGC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions. In one embodiment, amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
在一实施例中，本发明提供的ATC多肽所包含的TRGC多肽具有与由NCBI GenBank Gene ID：6966，NG_001336.2，108270至113860(TRGC1)，NCBI GenBank Gene ID：6967，NG_001336.2，124376至133924的基因表达的转录物编码的氨基酸序列具有至少约85％、约90％、约95％、约96％、约97％、约98％、约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。In one embodiment, the TRGC polypeptides contained in the ATC polypeptides provided by the present invention have the same properties as those provided by NCBI GenBank Gene ID: 6966, NG_001336.2, 108270 to 113860 (TRGC1), NCBI GenBank Gene ID: 6967, NG_001336.2, 124376 The amino acid sequence encoded by the transcript of the gene expression to 133924 has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homology or identity The amino acid sequence or fragment thereof, and/or may optionally contain up to one or up to two or up to three conservative amino acid substitutions.
在一实施例中，本发明提供的ATC多肽所包含的TRDC多肽包含与SEQ ID NO：23所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在一个实施例中，ATC中的TRBC多肽第57位氨基酸突变为半胱氨酸。In one embodiment, the TRDC polypeptide contained in the ATC polypeptide provided by the present invention comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, Amino acid sequences or fragments thereof that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical, and/or may optionally contain at most one or at most two one or up to three conservative amino acid substitutions. In one embodiment, amino acid 57 of the TRBC polypeptide in the ATC is mutated to cysteine.
在一实施例中，本发明提供的ATC多肽所包含的TRDC多肽具有与由NCBI GenBank Gene ID：28526，NG_001332.3，841011至844674的基因表达的转录物编码的氨基酸序 列具有至少约85％、约90％、约95％、约96％、约97％、约98％、约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。In one embodiment, the TRDC polypeptides contained in the ATC polypeptides provided by the present invention have at least about 85% of the amino acid sequences encoded by the transcripts expressed by NCBI GenBank Gene ID: 28526, NG_001332.3, 841011 to 844674, amino acid sequences or fragments thereof of about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homology or identity, and/or may optionally comprise at most one or Up to two or up to three conservative amino acid substitutions.
在一实施例中，本发明ATC包含与TCR亚基恒定区直接连接的抗原识别单元(也称为胞外抗原结合结构域)。在一实施例中，本发明ATC包含将抗原识别单元(也称为胞外抗原结合结构域)连接至TCR亚基恒定区的铰链/间隔区。铰链/间隔区可以是来自IgG1的铰链区，或者是免疫球蛋白的CH2CH3区和CD3的部分，CD28多肽的部分，CD8多肽的部分，与前述任一项具有至少约80％、至少约85％、至少约90％或至少约95％的同源性或同一性的变体，或合成的间隔序列。In one embodiment, the ATCs of the invention comprise an antigen recognition unit (also referred to as an extracellular antigen binding domain) directly linked to the constant region of the TCR subunit. In one embodiment, the ATCs of the invention comprise a hinge/spacer region linking an antigen recognition unit (also referred to as an extracellular antigen binding domain) to the constant region of the TCR subunit. The hinge/spacer region can be a hinge region from IgG1, or a CH2CH3 region of an immunoglobulin and a portion of CD3, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, having at least about 80%, at least about 85% of any of the foregoing , a variant of at least about 90% or at least about 95% homology or identity, or a synthetic spacer sequence.
在一实施例中，ATC包含TCRα恒定区(SEQ ID NO：5或9、11、13所示氨基酸序列)、TCRβ链恒定区(SEQ ID NO：14或18所示氨基酸序列)、TCRγ链恒定区(SEQ ID NO：20所示氨基酸序列)、和/或与TCRδ链恒定区(SEQ ID NO：23所示氨基酸序列)。在一实施例中，ATC不包含如SEQ ID NO：25和/或SEQ ID NO：28所示核苷酸序列。在一实施例中，ATC不包含如SEQ ID NO：41和/或SEQ ID NO：44所示序列。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：7、8、10或12所示核苷酸序列)、TCRβ突变恒定区(SEQ ID NO：16或17所示核苷酸序列)、TCRγ突变恒定区(SEQ ID NO:21所示核苷酸序列)、和/或TCRδ突变恒定区(SEQ ID NO：24所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：7、8、10或12所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：16或17所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：7所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：16所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：7所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：17所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：8所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：16所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：8所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：17所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：10所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：16所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：10所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：17所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：12所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：16所示核苷酸序列)。在一实施例中，ATC包括TCRα突变恒定区(SEQ ID NO：12所示核苷酸序列)和TCRβ突变恒定区(SEQ ID NO：17所示核苷酸序列)。在一实施例中，ATC包括TCRγ突变恒定区(SEQ ID NO:21 所示核苷酸序列)和TCRδ突变恒定区(SEQ ID NO：24所示核苷酸序列)。In one embodiment, the ATC comprises a TCRα constant region (amino acid sequence shown in SEQ ID NO: 5 or 9, 11, 13), a TCRβ chain constant region (amino acid sequence shown in SEQ ID NO: 14 or 18), a TCRγ chain constant region region (amino acid sequence shown in SEQ ID NO: 20), and/or with the TCRδ chain constant region (amino acid sequence shown in SEQ ID NO: 23). In one embodiment, the ATC does not comprise the nucleotide sequence set forth in SEQ ID NO:25 and/or SEQ ID NO:28. In one embodiment, the ATC does not comprise the sequence set forth in SEQ ID NO:41 and/or SEQ ID NO:44. In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12), a TCRβ mutated constant region (nucleotide sequence shown in SEQ ID NO: 16 or 17) ), TCRγ mutant constant region (nucleotide sequence shown in SEQ ID NO: 21), and/or TCRδ mutant constant region (nucleotide sequence shown in SEQ ID NO: 24). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12) and a TCRβ mutated constant region (nucleotide sequence shown in SEQ ID NO: 16 or 17) ). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 7) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 7) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 8) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 8) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 10) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 10) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 12) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 16). In one embodiment, the ATC comprises a TCRα mutated constant region (nucleotide sequence set forth in SEQ ID NO: 12) and a TCRβ mutated constant region (nucleotide sequence set forth in SEQ ID NO: 17). In one embodiment, the ATC includes a TCRγ mutated constant region (nucleotide sequence set forth in SEQ ID NO:21) and a TCRδ mutated constant region (nucleotide sequence set forth in SEQ ID NO:24).
本发明的ATC的胞外结构域可衍生自天然来源或重组来源。在天然来源的情况下，该结构域可衍生自任何蛋白质，但特别是膜结合蛋白质或跨膜蛋白质。在一个方面，胞外结构域能够与跨膜结构域缔合。在本发明中特别有用的胞外结构域可包括至少以下胞外区域：例如T细胞受体的α、β或γ、δ链，或者CD3ε、CD3γ或CD3δ，或者在替代实施方案中，包括CD28、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154。The extracellular domains of the ATCs of the present invention may be derived from natural or recombinant sources. In the case of natural sources, the domain may be derived from any protein, but in particular membrane-bound or transmembrane proteins. In one aspect, the extracellular domain is capable of associating with the transmembrane domain. Extracellular domains that are particularly useful in the present invention may include at least the following extracellular domains: for example, the alpha, beta or gamma, delta chains of T cell receptors, or CD3ε, CD3γ or CD3δ, or in alternative embodiments, CD28 , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
本发明的ATC的跨膜结构域可衍生自天然来源或重组来源。在天然来源的情况下，该结构域可衍生自任何膜结合蛋白质或跨膜蛋白质。在一个方面，每当ATC与靶抗原结合时，跨膜结构域能够向细胞内结构域发信号。在本发明中特别有用的跨膜结构域可包括至少以下跨膜区域：例如T细胞受体的α、β或γ、δ链，或者CD3ε、CD3γ、CD3δ、CD28、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154。在一些情况下，跨膜结构域可经由铰链(例如来自人类蛋白质的铰链)与ATC的胞外区域(例如ATC的抗原结合结构域)连接。例如，在一个实施方案中，该铰链可以是T细胞受体的α、β链的铰链。The transmembrane domains of the ATCs of the present invention can be derived from natural or recombinant sources. In the case of natural sources, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever an ATC binds to a target antigen. Transmembrane domains that are particularly useful in the present invention may include at least the following transmembrane regions: for example, the alpha, beta or gamma, delta chains of T cell receptors, or CD3ε, CD3γ, CD3δ, CD28, CD45, CD4, CD5, CD8 , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some cases, the transmembrane domain can be linked to the extracellular region of the ATC (eg, the antigen-binding domain of the ATC) via a hinge (eg, from a human protein). For example, in one embodiment, the hinge may be the hinge of the alpha, beta chain of the T cell receptor.
2.2胞外抗原结合结构域2.2 Extracellular antigen-binding domain
本发明提供的ATC包含胞外的抗原结合结构域(也称为抗原识别单元)和TCR亚基恒定区。抗原结合结构域特异性结合抗原，例如肿瘤抗原或病原体抗原。在一实施例中，抗原结合结构域包含抗体或其片段。在某些实施例中，抗原结合结构域包含抗体重链可变区和/轻链可变区；或者包含交联的Fab；或者包含F(ab) 2。在一实施例中，抗原结合结构域包含抗体重链可变区(VH)和轻链可变区(VL)，形成可变片段(Fv)。 The ATC provided by the present invention comprises an extracellular antigen binding domain (also referred to as an antigen recognition unit) and a TCR subunit constant region. The antigen binding domain specifically binds an antigen, such as a tumor antigen or a pathogen antigen. In one embodiment, the antigen binding domain comprises an antibody or fragment thereof. In certain embodiments, the antigen binding domain comprises an antibody heavy chain variable region and/or light chain variable region; or comprises a cross-linked Fab; or comprises F(ab) 2 . In one embodiment, the antigen binding domain comprises an antibody heavy chain variable region (VH) and light chain variable region (VL), forming a variable fragment (Fv).
在一个实施例中，ATC中的抗体重链可变区与TCR的α链恒定区(TRAC)直接连接，和/或抗体轻链可变区与TCR的β链恒定区(TRBC)直接连接；或抗体轻链可变区与TCR的α链恒定区直接连接，和/或抗体重链可变区与TCR的β链恒定区直接连接。在一实施例中，ATC中的抗体重链可变区与TCR的α链恒定区直接连接形成片段1，抗体轻链可变区与TCR的β链恒定区直接连接形成片段2，所述片段1和片段2由连接子连接，所述片段1和片段2可以交换相对于连接子的前后顺序。在一实施例中，ATC中的抗体轻链可变区与TCR的α链恒定区直接连接形成片段3，抗体重链可变区与TCR的β链恒定区直接连接形成片段4，所述片段3和片段4由连接子连接，所述片段3和片段4可以交换相对于连接子的前后顺序。在一实施例中，ATC中的抗体重链可变区与TCR的γ链恒定区直接连接形成片段5，抗体轻链可变区与TCR的δ链恒定区直接连接形成片段6，所述片段5和片段6由连接子连接，所述片段5和片段6可以交换相对于连接子的前后顺序。在一实施例 中，ATC中的抗体重链可变区与TCR的δ链恒定区直接连接形成片段7，抗体轻链可变区与TCR的γ链恒定区直接连接形成片段8，所述片段7和片段8由连接子连接，所述片段7和片段8可以交换相对于连接子的前后顺序。所述“连接子”包括编码自切割肽(例如，2A序列)或蛋白酶识别位点(例如，弗林蛋白酶)的序列。如本文所使用，“自切割肽”是指允许将多个蛋白编码为多蛋白的寡肽，其在翻译后解离为组分蛋白。本领域技术人员已知各种自切割肽，包括但不限于在小核糖核酸病毒科的成员中发现的那些病毒，如口蹄疫病毒(FMDV)，马鼻炎A病毒(ERAV0)、明脉扁刺蛾病毒(TaV)及猪铁士古病毒-1(PTV-1)、及心脏病毒诸如泰勒病毒(Theilovirus)及脑心肌炎病毒(encephalomyocarditis virus)。源自FMDV、ERAV、PTV-1和TaV的2A肽在本文中分别称为“F2A”、“E2A”、“P2A”和“T2A”。本领域技术人员将能够选择适合用于本发明的自切割肽。示例性，F2A包含SEQ ID NO：88或89所示序列，P2A包含SEQ ID NO：90或91所示序列，连接肽1包含SEQ ID NO：92所示序列。In one embodiment, the antibody heavy chain variable region in the ATC is directly linked to the alpha chain constant region (TRAC) of the TCR, and/or the antibody light chain variable region is directly linked to the TCR beta chain constant region (TRBC); Or the variable region of the antibody light chain is directly linked to the constant region of the alpha chain of the TCR, and/or the variable region of the heavy chain of the antibody is directly linked to the constant region of the beta chain of the TCR. In one embodiment, the variable region of the antibody heavy chain in the ATC is directly connected with the constant region of the α chain of the TCR to form fragment 1, and the variable region of the antibody light chain is directly connected with the constant region of the β chain of the TCR to form the fragment 2, the fragment Fragment 1 and Fragment 2 are joined by a linker, which can be swapped in order relative to the linker. In one embodiment, the variable region of the antibody light chain in the ATC is directly connected to the constant region of the α chain of the TCR to form fragment 3, and the variable region of the antibody heavy chain is directly connected to the constant region of the β chain of the TCR to form the fragment 4, the fragment Fragment 3 and Fragment 4 are joined by a linker, which can be swapped in order relative to the linker. In one embodiment, the variable region of the antibody heavy chain in the ATC is directly connected with the constant region of the γ chain of the TCR to form fragment 5, and the variable region of the antibody light chain is directly connected with the constant region of the δ chain of the TCR to form the fragment 6, the fragment Fragment 5 and Fragment 6 are linked by a linker, which can be swapped in the order of the front and rear relative to the linker. In one embodiment, the variable region of the antibody heavy chain in the ATC is directly linked to the constant region of the delta chain of the TCR to form fragment 7, and the variable region of the antibody light chain is directly linked to the constant region of the gamma chain of the TCR to form fragment 8, the fragment Fragment 7 and Fragment 8 are linked by a linker, which can be swapped in the order of the front and rear relative to the linker. The "linker" includes a sequence encoding a self-cleaving peptide (eg, 2A sequence) or a protease recognition site (eg, furin). As used herein, a "self-cleaving peptide" refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate post-translationally into component proteins. Various self-cleaving peptides are known to those of skill in the art, including but not limited to those viruses found in members of the Picornaviridae family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), Viruses (TaV) and porcine Texaco virus-1 (PTV-1), and cardioviruses such as Theilovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1 and TaV are referred to herein as "F2A", "E2A", "P2A" and "T2A", respectively. Those skilled in the art will be able to select self-cleaving peptides suitable for use in the present invention. Exemplarily, F2A comprises the sequence shown in SEQ ID NO: 88 or 89, P2A comprises the sequence shown in SEQ ID NO: 90 or 91, and connecting peptide 1 comprises the sequence shown in SEQ ID NO: 92.
本发明包括编码ATC的重组DNA分子(或称为构建体)，示例性，ATC包含与GPC3或Claudin18.2特异性结合的抗体片段，其中该抗体片段序列与编码TCR亚基或其部分的核酸序列连接并在同一开放阅读框(Open Reading Frame)中。The present invention includes recombinant DNA molecules (or constructs) encoding ATC, exemplarily, ATC comprising an antibody fragment that specifically binds to GPC3 or Claudin18.2, wherein the antibody fragment sequence is associated with a nucleic acid encoding a TCR subunit or portion thereof The sequences are linked and in the same Open Reading Frame.
在一实施例中，提供了识别GPC3的抗体，所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO：1、48、50、52、54、56、58、60、62或64所示氨基酸序列；和/或所述抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO：3、49、51、53、55、57、59、61、63或65所示氨基酸序列。在一实施例中，提供了识别CLDN18A2的抗体，所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO：66、68、70、72、74、76、78、80、82或84所示氨基酸序列；和/或所述抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO：67、69、71、73、75、77、79、81、83或85所示氨基酸序列。In one embodiment, there is provided an antibody recognizing GPC3, the antibody comprising a heavy chain variable region comprising SEQ ID NOs: 1, 48, 50, 52, 54, 56, 58, 60 , 62 or 64; and/or the antibody comprises a light chain variable region comprising SEQ ID NO: 3, 49, 51, 53, 55, 57, 59, 61, The amino acid sequence shown at 63 or 65. In one embodiment, there is provided an antibody recognizing CLDN18A2, the antibody comprising a heavy chain variable region comprising SEQ ID NOs: 66, 68, 70, 72, 74, 76, 78, 80 , 82 or 84; and/or the antibody comprises a light chain variable region comprising SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, The amino acid sequence shown at 83 or 85.
在一个方面，本发明考虑到产生功能上等同的分子的起始抗体或片段(例如，VH或VL)氨基酸序列的修饰。例如，可修饰ATC中包含的抗GPC3或Claudin18.2结合结构域例如VH或VL，以保留抗GPC3或Claudin18.2结合结构域例如VH或VL至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。In one aspect, the invention contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, VH or VL) that produces a functionally equivalent molecule. For example, an anti-GPC3 or Claudin18.2 binding domain, eg, VH or VL, contained in an ATC can be modified to retain at least about 70%, 71%, 72%, 73% of the anti-GPC3 or Claudin18.2 binding domain, eg, VH or VL , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
本发明考虑到整个ATC构建体的修饰，例如，ATC构建体的各个结构域的一个或多个氨基酸序列的修饰，以便产生功能上等同的分子。可修饰ATC构建体以保留起始ATC构建体的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93 ％、94％、95％、96％、97％、98％或99％的同一性。The present invention contemplates modification of the entire ATC construct, eg, modification of one or more amino acid sequences of individual domains of the ATC construct, in order to generate functionally equivalent molecules. The ATC construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the starting ATC construct , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity.
本领域普通技术人员将会理解，可进一步修饰本发明的抗体或抗体片段，使得它们在氨基酸序列上(例如，相对于野生型)有所变化，但在所需活性上没有变化。例如，可对蛋白质进行另外的核苷酸置换，导致“非必需”氨基酸残基处的氨基酸置换。例如，分子中的非必需氨基酸残基可被来自相同侧链家族的另一个氨基酸残基取代。在另一个实施方案中，氨基酸片段可被结构相似但在顺序和/或组成上与侧链家族成员不同的氨基酸片段取代，例如，可进行保守置换，其中氨基酸残基被具有相似侧链的氨基酸残基所取代。One of ordinary skill in the art will appreciate that the antibodies or antibody fragments of the invention can be further modified such that they vary in amino acid sequence (eg, relative to wild type), but not in the desired activity. For example, additional nucleotide substitutions can be made in the protein, resulting in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule can be substituted with another amino acid residue from the same side chain family. In another embodiment, amino acid fragments may be replaced by amino acid fragments that are structurally similar but differ in sequence and/or composition from side chain family members, eg, conservative substitutions may be made in which amino acid residues are replaced by amino acids with similar side chains residues replaced.
2.3.抗原2.3. Antigen
在一实施例中，ATC与肿瘤抗原结合。任何肿瘤抗原均可用于本发明所述的肿瘤相关的实施例中。抗原表达为多肽或完整蛋白或其部分。本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2 (OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。In one embodiment, the ATC binds to a tumor antigen. Any tumor antigen may be used in the tumor-related embodiments of the present invention. Antigens are expressed as polypeptides or intact proteins or parts thereof. The tumor antigens of the present invention include, but are not limited to: Thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); 11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2); Lewis (Y) Antigen; CD24; Platelet-Derived Growth Factor Receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4 , EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe ); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside ( OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integration B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR ; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid ; placenta specificity 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); epinephrine Receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma Cancer Testis Antigen-1 (MAD-CT-1); Melanoma Cancer Testis Antigen-2 (MAD-CT-2); Fos-Associated Antigen 1; p53 mutant; human telomerase reversed Transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transfer Enzyme V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase-anchored protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2) ; CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f(CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75 ); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLL1).
在一实施例中，ATC识别GPC3。在一实施例中，ATC识别Claudin18.2。在一实施例中，人GPC3多肽包含SEQ ID NO:86所示的氨基酸序列。在一实施例中，ATC结合至GPC3多肽的胞外结构域。在一实施例中，人Claudin18.2多肽包含SEQ ID NO:87所示的氨基酸序列。在一实施例中，ATC结合至Claudin18.2多肽的胞外结构域。In one embodiment, ATC identifies GPC3. In one embodiment, ATC recognizes Claudin18.2. In one embodiment, the human GPC3 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:86. In one embodiment, the ATC binds to the extracellular domain of a GPC3 polypeptide. In one embodiment, the human Claudin18.2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:87. In one embodiment, ATC binds to the extracellular domain of a Claudin18.2 polypeptide.
在一实施例中，ATC识别病原体抗原，例如用于治疗和/或预防病原体感染或其他感染性疾病，例如在免疫受损的受试者中。病原体抗原包括但不限于：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原包括但不限于：巨细胞病毒(CMV)抗原、爱泼斯坦-巴尔病毒(EBV)抗原、人类免疫缺陷病毒(HIV)抗原或流感病毒抗原。In one embodiment, the ATC recognizes pathogen antigens, eg, for the treatment and/or prevention of pathogen infections or other infectious diseases, eg, in immunocompromised subjects. Pathogen antigens include but are not limited to: antigens of viruses, bacteria, fungi, protozoa, or parasites; viral antigens include but are not limited to: cytomegalovirus (CMV) antigens, Epstein-Barr virus (EBV) antigens, human immune Defective virus (HIV) antigen or influenza virus antigen.
2.4 CD3复合物2.4 CD3 complex
本发明提供的ATC受体能够与CD3ζ多肽缔合。在一实施例中，ATC包含TCR亚基的恒定区与CD3ζ多肽缔合。CD3ζ多肽可以是内源性，也可以是外源性的。在一实施例中，ATC的胞外抗原结合结构域与抗原的结合能够激活与TCA亚基恒定区缔合的CD3ζ多肽。The ATC receptor provided by the present invention can associate with CD3ζ polypeptide. In one embodiment, the ATC comprises the constant region of the TCR subunit associated with a CD3ζ polypeptide. CD3ζ polypeptides can be endogenous or exogenous. In one embodiment, the binding of the extracellular antigen-binding domain of ATC to the antigen is capable of activating the CD3ζ polypeptide associated with the constant region of the TCA subunit.
激活的CD3ζ多肽可激活和/或刺激免疫效应细胞(例如，淋巴谱系的细胞，例如T细胞)。CD3ζ包含三个免疫受体酪氨酸激活基序(ITAM1、ITAM2和ITAM3)、三个富碱性拉伸区(BRS)(BRS1、BRS2和BRS3)，并在抗原与ATC的胞外抗原结合结构域结合后将激活信号传递至细胞(例如，淋巴谱系的细胞，例如T细胞)。CD3ζ链的细胞内信号传导结构域是TCR信号主要的传递者。Activated CD3ζ polypeptides can activate and/or stimulate immune effector cells (eg, cells of the lymphoid lineage, eg, T cells). CD3ζ contains three immunoreceptor tyrosine activation motifs (ITAM1, ITAM2, and ITAM3), three basic-rich stretch regions (BRS) (BRS1, BRS2, and BRS3), and binds to extracellular antigens of ATC at the Domain binding transmits an activation signal to cells (eg, cells of the lymphoid lineage, eg, T cells). The intracellular signaling domain of the CD3ζ chain is the primary transmitter of TCR signaling.
本发明提供的ATC受体能与CD3复合物(也称为“T细胞共受体”)缔合。在一实施例中，ATC和CD3复合物形成类似于天然TCR/CD3复合物的抗原识别受体复合物。在一实施例中，ATC与抗原结合后能激活与所述ATC缔合的CD3分子。本发明所述CD3分子包括CD3ζ、CD3γ、CD3δ和CD3ε。The ATC receptors provided herein are capable of associating with the CD3 complex (also referred to as "T cell co-receptor"). In one embodiment, the ATC and CD3 complex form an antigen-recognition receptor complex similar to the native TCR/CD3 complex. In one embodiment, the ATC can activate the CD3 molecule associated with the ATC upon binding to the antigen. The CD3 molecules of the present invention include CD3ζ, CD3γ, CD3δ and CD3ε.
CD3复合物可以是内源性的，也是外源性的。ATC受体替代了CD3/TCR复合物中的天然和/或内源性TCR。CD3复合物包含两条CD3ζ、CD3γ链、CD3δ链和两条CD3ε链。The CD3 complex can be endogenous as well as exogenous. ATC receptors replace native and/or endogenous TCRs in the CD3/TCR complex. The CD3 complex contains two CD3ζ, CD3γ, CD3δ and two CD3ε chains.
本发明提供的ATC受体比靶向相同抗原的CAR表现出更高的抗原敏感性。在某些实施例中，ATC在与肿瘤细胞表面上具有低密度的抗原结合时能够诱导免疫应答。在某些实施例中，包含本发明ATC的免疫效应细胞可以用于治疗具有表面抗原低表达水平的肿瘤细胞的受试者，例如由于疾病的复发，其中该受试者接受过导致残留的肿瘤细胞的治疗。The ATC receptor provided by the present invention exhibits higher antigen sensitivity than CAR targeting the same antigen. In certain embodiments, ATCs are capable of inducing an immune response when bound to antigens with low densities on the surface of tumor cells. In certain embodiments, immune effector cells comprising ATCs of the invention can be used to treat subjects with tumor cells that express low levels of surface antigens, such as due to relapse of the disease, where the subject has received a tumor that resulted in residual Cell therapy.
3.工程化细胞3. Engineered Cells
本发明提供的工程化细胞是包含表达ATC的免疫效应细胞。ATC能激活所述免疫效应细胞。本发明的工程化细胞在结合抗原后，对携带抗原的细胞表现出细胞溶解作用。The engineered cells provided by the present invention comprise immune effector cells expressing ATC. ATC can activate the immune effector cells. The engineered cells of the present invention exhibit cytolytic effects on antigen-bearing cells after binding to the antigen.
本发明提供了构建表达ATC的工程化细胞的技术平台，示例性，所述工程化细胞为T细胞，也称为ATCT细胞，将抗体/抗原识别单元的识别抗原的高亲和力和高度特异性，与T细胞的天然TCR信号传导能力相结合，在不减弱本发明所述工程化细胞杀伤作用的情况下可减少细胞因子分泌，提高临床安全性，同时ATCT细胞具有较低的分化程度和耗竭程度，提示本发明的细胞存活能力更强，在治疗实体瘤方面具有一定优势。The present invention provides a technical platform for constructing engineered cells expressing ATC. Exemplarily, the engineered cells are T cells, also known as ATCT cells, which combine the high affinity and high specificity of the antibody/antigen recognition unit to recognize antigens, Combined with the natural TCR signaling ability of T cells, it can reduce cytokine secretion and improve clinical safety without reducing the killing effect of the engineered cells of the present invention, and ATCT cells have a lower degree of differentiation and exhaustion. , suggesting that the cell survival ability of the present invention is stronger, and has certain advantages in the treatment of solid tumors.
在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或更好的治疗效力。在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或更好的细胞溶解作用。在一实施例中，本发明工程化细胞分泌抗肿瘤细胞因子。工程化细胞分泌的细胞因子包括但不限于TNFα、IFNγ和IL2。在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或更好的抗原结合后工程化细胞激活水平。在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或更低的分化程度，例如ATCT细胞的CCR7和CD45RA表达比例高。在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或接近天然状态的CD4/CD8表型。在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或更低的耗竭程度。在一实施例中，与包含靶向相同抗原CAR的细胞相比，本发明工程化细胞表现出相当或更接近天然状态的增殖能力。在一实施例中，与表达相同抗原识别单元的CAR的细胞相比，所述工程化细胞具有以下之一特点或其组合：1)对靶细胞的杀伤能力、和/或与靶细胞孵育后分泌IFN-γ、细胞增殖没有显著差异； 与靶细胞孵育后CD25、CD69表达水平高；2)原始T细胞比例大；3)CD4+/CD8+比例高；4)PD-1/TIM-3/LAG-3阳性率低。In one embodiment, the engineered cells of the invention exhibit comparable or better therapeutic efficacy compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention exhibit comparable or better cytolysis compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention secrete anti-tumor cytokines. Cytokines secreted by engineered cells include, but are not limited to, TNFα, IFNγ, and IL2. In one embodiment, the engineered cells of the invention exhibit comparable or better levels of activation of the engineered cells upon antigen binding compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention exhibit a comparable or lower degree of differentiation compared to cells comprising a CAR targeting the same antigen, eg, ATCT cells express a high ratio of CCR7 and CD45RA. In one embodiment, the engineered cells of the invention exhibit a CD4/CD8 phenotype comparable to or close to the native state compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention exhibit a comparable or lower degree of depletion compared to cells comprising a CAR targeting the same antigen. In one embodiment, the engineered cells of the invention exhibit a proliferative capacity comparable to or closer to the native state compared to cells comprising a CAR targeting the same antigen. In one embodiment, compared with cells expressing the CAR of the same antigen recognition unit, the engineered cells have one of the following characteristics or a combination thereof: 1) the ability to kill target cells, and/or after incubation with target cells There was no significant difference in the secretion of IFN-γ and cell proliferation; the expression levels of CD25 and CD69 were high after incubation with target cells; 2) the proportion of primitive T cells was large; 3) the ratio of CD4+/CD8+ was high; 4) PD-1/TIM-3/LAG -3 The positive rate is low.
在一个实施例中，与过表达ATC靶向抗原的肿瘤细胞共孵育，所述ATCT细胞能分泌大量IFN-γ、颗粒酶-B、IL2、TNF-α及GM-CSF；与同样抗原识别单元构建的CAR T细胞(嵌合抗原受体T细胞)相比，ATCT保持了同样显著的细胞毒性，但是细胞因子分泌量明显降低，这有效降低了细胞因子风暴的可能。本发明的平台将抗体识别的高亲和力和高度特异性与T细胞的天然TCR信号传导能力相结合，在不减弱杀伤作用的情况下可减少细胞因子分泌，提高临床安全性，同时ATCT细胞具有较低的分化程度和耗竭程度，提示本发明的细胞存活能力更强，在治疗实体瘤方面具有显著优势。In one example, co-incubated with tumor cells overexpressing ATC-targeted antigens that secrete large amounts of IFN-γ, granzyme-B, IL2, TNF-α and GM-CSF; with the same antigen recognition unit Compared with the constructed CAR T cells (chimeric antigen receptor T cells), ATCT maintained the same significant cytotoxicity, but the secretion of cytokines was significantly reduced, which effectively reduced the possibility of cytokine storm. The platform of the present invention combines the high affinity and high specificity of antibody recognition with the natural TCR signaling ability of T cells, can reduce cytokine secretion and improve clinical safety without weakening the killing effect, and ATCT cells have relatively The low degree of differentiation and depletion indicates that the cells of the present invention have stronger viability and have significant advantages in the treatment of solid tumors.
在另一个方面，本文所述的ATCT细胞可进一步表达另一种因子，例如细胞因子、转录因子、趋化因子、和/或其组合，来增加T细胞的增殖、细胞存活、抗凋亡作用、肿瘤浸润等作用来提高抗肿瘤活性。In another aspect, the ATCT cells described herein can further express another factor, such as a cytokine, transcription factor, chemokine, and/or a combination thereof, to increase T cell proliferation, cell survival, anti-apoptotic effects , tumor infiltration and other effects to enhance anti-tumor activity.
在一实施例中，本发明提供的工程化细胞中内源性TCR分子低表达或不表达，所包含的ATC能与内源性CD3形成复合体。所述ATC核酸分子在包含碱基突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子。在一实施例中，所述ATC核酸分子在包含碱基同义突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子，且ATC多肽可以与内源性CD3形成复合体。在一实施例中，所述ATC核酸分子在包含碱基同义突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子，且ATC氨基酸序列与野生型TCR亚基恒定区具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，且ATC多肽可以与内源性CD3形成复合体。In one embodiment, the engineered cells provided by the present invention have low expression or no expression of endogenous TCR molecules, and the contained ATC can form a complex with endogenous CD3. The ATC nucleic acid molecule is no longer the nucleic acid molecule of the target sequence targeted by the gene knockout technology and/or the gene silencing technology after including the base mutation. In one embodiment, the ATC nucleic acid molecule is no longer the nucleic acid molecule of the target sequence targeted by the gene knockout technology and/or gene silencing technology after comprising the base synonymous mutation, and the ATC polypeptide can interact with endogenous CD3 form a complex. In one embodiment, the ATC nucleic acid molecule is no longer the nucleic acid molecule of the target sequence targeted by the gene knockout technology and/or gene silencing technology after comprising the base synonymous mutation, and the ATC amino acid sequence is the same as that of the wild-type TCR subtype. The base constant region has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology amino acid sequences or fragments thereof, and ATC polypeptides can form complexes with endogenous CD3.
在一实施例中，本发明提供的工程化细胞中内源性αβTCR分子低表达或不表达，所表达的ATC核酸分子包含相对野生型TRAC核酸分子、TRBC核酸分子存在同义突变。在一实施例中，本发明提供的工程化细胞中内源性γδTCR分子低表达或不表达，所表达的ATC核酸分子包含相对野生型TRGC核酸分子、TRDC核酸分子存在同义突变。在一实施例中，与未基因工程化的细胞中内源性TCR的表达、活性和/或信号传导相比，所述工程化细胞中内源性TCR的表达、活性和/或信号传导减少大于约50％、60％、70％、80％、90％、95％或100％。在一实施例中，对工程化细胞的TCRα、β链恒定区相应编码基因的外显子用CRISPR/Cas技术敲除。在一实施例中，所述CRISPR/Cas技术所靶向的靶序列位于TCRα链和β链恒定区。在一实施例中，同时敲除工程化细胞中的内源性TRAC和内源性TRBC。在一个实施例中，工程化细胞包 含gRNA，序列如SEQ ID NO:25、28、33、34、35、36、37、38、39、40或其组合所示。在一实施例中，工程化细胞包含gRNA，序列如SEQ ID NO:41和/或44所示。在一实施例中，工程化细胞包含序列如SEQ ID NO:25和/或28所示gRNA。In one embodiment, the endogenous αβTCR molecules in the engineered cells provided by the present invention are underexpressed or not expressed, and the expressed ATC nucleic acid molecules contain synonymous mutations relative to wild-type TRAC nucleic acid molecules and TRBC nucleic acid molecules. In one embodiment, the endogenous γδ TCR molecule is underexpressed or not expressed in the engineered cell provided by the present invention, and the expressed ATC nucleic acid molecule contains a synonymous mutation relative to the wild-type TRGC nucleic acid molecule and TRDC nucleic acid molecule. In one embodiment, the expression, activity and/or signaling of an endogenous TCR in the engineered cell is reduced compared to the expression, activity and/or signaling of the endogenous TCR in the non-engineered cell Greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100%. In one embodiment, the exons of the genes encoding the constant regions of the TCRα and β chains of the engineered cells are knocked out using CRISPR/Cas technology. In one embodiment, the target sequences targeted by the CRISPR/Cas technology are located in the TCR alpha chain and beta chain constant regions. In one embodiment, both endogenous TRAC and endogenous TRBC are simultaneously knocked out in the engineered cells. In one embodiment, the engineered cell comprises a gRNA, the sequence of which is set forth in SEQ ID NO: 25, 28, 33, 34, 35, 36, 37, 38, 39, 40, or a combination thereof. In one embodiment, the engineered cell comprises a gRNA, the sequence of which is set forth in SEQ ID NO: 41 and/or 44. In one embodiment, the engineered cell comprises a gRNA having a sequence as set forth in SEQ ID NO: 25 and/or 28.
在一实施例中，与表达相同ATC但内源性TCR亚基的表达、活性和/或信号传导没有被降低或抑制的细胞相比，所述工程化细胞ATC阳性率和/或ATC和内源性CD3形成复合体比率提高,或提高约5％、10％、20％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。In one embodiment, the engineered cells have ATC positivity rates and/or ATC and endogenous TCR positivity rates compared to cells expressing the same ATC but without reduced or inhibited expression, activity and/or signaling of endogenous TCR subunits. The ratio of exogenous CD3 complex formation is increased, or about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95% or 100%.
本发明还提供了ATCT细胞，其被转导有编码所述ATC和靶向编码内源性TCR基因的抑制性核酸分子或gRNA的核酸、或被转导有包含所述核酸的重组质粒、或被转导包含所述质粒的病毒。在一个实施例中，ATCT细胞是指利用两组核苷酸片段修饰而得，第一组核苷酸片段包括抑制性核酸分子和/或gRNA，所述抑制性核酸分子的互补序列或gRNA靶序列位于野生型TCR亚基恒定区；第二组核苷酸片段包括编码识别抗原的抗原识别单元和包含核苷酸序列同义突变的TCR亚基恒定区的核苷酸片段，所述第二组核苷酸片段不包括所述第一组核苷酸片段中抑制性核酸分子的互补序列和/或gRNA靶序列。The present invention also provides ATCT cells transduced with a nucleic acid encoding the ATC and targeting an inhibitory nucleic acid molecule or gRNA encoding an endogenous TCR gene, or transduced with a recombinant plasmid comprising the nucleic acid, or The virus containing the plasmid is transduced. In one embodiment, ATCT cells are modified with two sets of nucleotide fragments, the first set of nucleotide fragments includes inhibitory nucleic acid molecules and/or gRNAs, the complementary sequences of the inhibitory nucleic acid molecules or gRNA targets The sequence is located in the constant region of the wild-type TCR subunit; the second group of nucleotide fragments includes an antigen recognition unit encoding an antigen and a nucleotide fragment comprising a TCR subunit constant region with a synonymous mutation of the nucleotide sequence, the second The set of nucleotide fragments does not include the complementary sequence and/or the gRNA target sequence of the inhibitory nucleic acid molecule in the first set of nucleotide fragments.
本发明所述的免疫效应细胞可以是淋巴谱系的细胞。包括B、T和自然杀伤(NK)细胞的淋巴谱系提供抗体的产生、细胞免疫系统的调节、血液中外源试剂的检测、宿主外源细胞的检测等。淋巴谱系的免疫效应细胞的非限制性实例包括T细胞、自然杀伤T(NKT)细胞及其前体，包括胚胎干细胞和多能干细胞(例如，分化成淋巴样细胞的干细胞或多能干细胞)。T细胞可以是在胸腺中成熟的淋巴细胞，主要负责细胞介导的免疫。T细胞参与适应性免疫系统。T细胞可以是任何类型的T细胞，包括但不限于辅助T细胞、细胞毒性T细胞、记忆T细胞(包括中央记忆T细胞、干细胞样记忆T细胞(或干样记忆T细胞)和两种效应记忆T细胞：例如TEM细胞和TEMRA细胞)、调节性T细胞(也称为抑制性T细胞)、自然杀伤T细胞、粘膜相关性不变T细胞、γδT细胞或αβT细胞。细胞毒性T细胞(CTL或杀伤性T细胞)是能够诱导被感染的体细胞或肿瘤细胞死亡的T淋巴细胞。受试者自身的T细胞可以被工程化改造以表达ATC靶向特定的抗原。在一实施例中，免疫效应细胞是T细胞。在一实施例中，T细胞可以是CD4+T细胞和/或CD8+T细胞。在一实施例中，免疫效应细胞是CD3+T细胞。在一实施例中，所述工程化细胞包括由PBMC细胞经CD3磁珠刺激后收集的细胞群。The immune effector cells of the present invention may be cells of the lymphoid lineage. The lymphatic lineage, including B, T, and natural killer (NK) cells, provides antibody production, regulation of the cellular immune system, detection of foreign agents in the blood, detection of foreign cells in the host, and the like. Non-limiting examples of immune effector cells of the lymphoid lineage include T cells, natural killer T (NKT) cells, and their precursors, including embryonic stem cells and pluripotent stem cells (eg, stem cells that differentiate into lymphoid cells or pluripotent stem cells). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. T cells can be any type of T cell including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-like memory T cells (or stem-like memory T cells), and both effector T cells Memory T cells: eg TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, γδ T cells or αβ T cells. Cytotoxic T cells (CTL or killer T cells) are T lymphocytes capable of inducing the death of infected somatic or tumor cells. A subject's own T cells can be engineered to express ATCs targeting specific antigens. In one embodiment, the immune effector cells are T cells. In one embodiment, the T cells may be CD4+ T cells and/or CD8+ T cells. In one embodiment, the immune effector cells are CD3+ T cells. In one embodiment, the engineered cells comprise a population of cells collected from PBMC cells stimulated with CD3 magnetic beads.
免疫效应细胞(例如，T细胞)可以是自体的、非自体的(例如，同种异体的)、或者是体外从工程化的祖细胞或干细胞衍生而来。可从许多来源获得，包括外周血单个核细胞(PBMC)、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。Immune effector cells (eg, T cells) can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells. It can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
在本发明的某些方面，可使用本领域技术人员已知的任意数量的技术如Ficoll TM分离技术从收集自受试者的血液样品中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体的循环血液的细胞。单采血液成分术产物通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个方面，可洗涤通过单采血液成分术收集的细胞以去除血浆部分并将细胞置于适当的缓冲液或培养基中以供后续处理步骤。在本发明的背景下还可使用多轮选择。在某些方面，可能需要进行选择程序并在激活和扩充过程中使用“未选择的”细胞。“未选择的”细胞也可以经受其他轮选择。 In certain aspects of the invention, T cells can be obtained from a blood sample collected from a subject using any number of techniques known to those of skill in the art, such as the Ficoll ™ separation technique. In a preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. Multiple rounds of selection can also be used in the context of the present invention. In some aspects, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "Unselected" cells can also undergo other rounds of selection.
本发明的工程化细胞能够调节肿瘤微环境。The engineered cells of the present invention are capable of modulating the tumor microenvironment.
未纯化的CTL来源可以是本领域已知的任何来源，例如骨髓、胎儿、新生儿或成年或其它造血细胞来源，例如胎儿肝、外周血或脐带血。可以采用各种技术来分离细胞。例如，阴性选择法可以最初去除非CTL。mAb对于鉴定与特定细胞谱系和/或阳性和阴性选择的分化阶段相关的标志物特别有用。The source of unpurified CTL can be any source known in the art, such as bone marrow, fetal, neonatal or adult or other source of hematopoietic cells, such as fetal liver, peripheral blood, or umbilical cord blood. Various techniques can be used to isolate cells. For example, negative selection can initially remove non-CTLs. mAbs are particularly useful for identifying markers associated with specific cell lineages and/or positively and negatively selected differentiation stages.
最初可以通过相对粗略的分离除去大部分末端分化的细胞。例如，最初可以使用磁珠分离来去除大量不相关的细胞。在某些实施方式中，在分离细胞之前将去除总造血细胞的至少约80％，通常至少约70％。Most terminally differentiated cells can initially be removed by relatively crude dissociation. For example, magnetic bead separation can initially be used to remove large numbers of irrelevant cells. In certain embodiments, at least about 80%, usually at least about 70%, of the total hematopoietic cells will be removed prior to isolating the cells.
分离的程序包括但不限于密度梯度离心；重沉(resetting)；偶联至改变细胞密度的颗粒；用抗体包被的磁珠进行磁分离；亲和色谱；与mAb结合或结合使用的细胞毒性剂，包括但不限于补体和细胞毒素；并用附着在固体基质(例如板、芯片、淘析)上的抗体淘选或任何其它方便的技术。Separation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; agents, including but not limited to complement and cytotoxins; and panning with antibodies attached to solid substrates (eg, plates, chips, panning, or any other convenient technique).
分离和分析的技术包括但不限于流式细胞术，其可以具有不同的复杂程度，例如多个颜色通道、低角度和钝角光散射检测通道、阻抗通道。Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low and obtuse angle light scatter detection channels, impedance channels.
通过使用与死细胞相关的染料，例如碘化丙啶(PI)，可以针对死细胞选择细胞。在某些实施方式中，将细胞收集在包含2％胎牛血清(FCS)或0.2％牛血清白蛋白(BSA)的培养基或任何其它合适的例如无菌等渗培养基中。Cells can be selected for dead cells by using dyes associated with dead cells, such as propidium iodide (PI). In certain embodiments, cells are collected in medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable eg sterile isotonic medium.
4.载体4. Carrier
工程化细胞(例如，T细胞或NKT细胞)的遗传修饰可以通过用重组DNA分子转导基本上均质的细胞群来完成。在某些实施方式中，逆转录病毒载体(γ-逆转录病毒或慢病毒)用于将DNA分子引入细胞。例如，可以将编码ATC的多核苷酸克隆到逆转录病毒载体。也可以使用非病毒载体。转导可以使用任何合适的病毒载体或非病毒递送系统。可以在单个多顺反子表达盒、单个载体的多个表达盒或多个载体中用辅助分子(例如细胞因子)构建ATC。产生多顺反子表达盒的元件的实例包括但不限于各种病毒和非病毒内部核糖体进入位点(IRES，例如，FGF-1IRES、FGF-2IRES、VEGF IRES、IGF-II IRES、NF-κB IRES、RUNX1 IRES、p53IRES、甲型肝炎IRES、丙型肝炎IRES、瘟病毒IRES、无杆状病毒IRES、小核糖核酸病毒IRES、脊髓灰质炎病毒IRES和脑心肌炎病毒IRES)和可切割的接头(例如2A肽，例如P2A、T2A、E2A和F2A肽)。Genetic modification of engineered cells (eg, T cells or NKT cells) can be accomplished by transduction of a substantially homogeneous population of cells with recombinant DNA molecules. In certain embodiments, retroviral vectors (gamma-retrovirus or lentivirus) are used to introduce DNA molecules into cells. For example, ATC-encoding polynucleotides can be cloned into retroviral vectors. Non-viral vectors can also be used. Transduction can use any suitable viral vector or non-viral delivery system. ATCs can be constructed with helper molecules (eg, cytokines) in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosomal entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-III IRES, NF- κB IRES, RUNX1 IRES, p53IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, baculovirus IRES, picornavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and cleavable linkers (eg 2A peptides such as P2A, T2A, E2A and F2A peptides).
可以使用的其它病毒载体包括，例如，腺病毒、慢病毒和与腺相关的病毒载体、牛痘病毒、牛乳头瘤病毒或疱疹病毒，例如爱泼斯坦-巴尔病毒。Other viral vectors that can be used include, for example, adenoviruses, lentiviruses and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus or herpesviruses such as Epstein-Barr virus.
非病毒方法也可以用于工程化细胞的遗传修饰。例如，可以通过在脂质转染，脱唾液酸血清类粘蛋白-聚赖氨酸偶联，或手术条件下的微注射的情况下施用核酸来将核酸分子引入免疫效应细胞中。其它非病毒的基因转移方法包括使用脂质体、磷酸钙、DEAE葡聚糖、电穿孔和原生质体融合的体外转染。也可以通过将核酸分子转移到可离体培养的细胞类型(例如，自体或同种异体原代细胞或其后代)中来完成将所述核酸分子移植到受试者体内，之后，将经所述核酸分子修饰后的细胞(或其后代)注射到受试者目标组织中或全身注射。Non-viral methods can also be used for genetic modification of engineered cells. For example, nucleic acid molecules can be introduced into immune effector cells by administering the nucleic acid in the context of lipofection, asialosomucoid-polylysine conjugation, or microinjection under surgical conditions. Other non-viral gene transfer methods include in vitro transfection using liposomes, calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Transplantation of the nucleic acid molecule into a subject can also be accomplished by transferring the nucleic acid molecule into a cell type that can be cultured ex vivo (eg, autologous or allogeneic primary cells or progeny thereof), after which the nucleic acid molecule is transferred to the subject. The nucleic acid molecule-modified cells (or progeny thereof) are injected into the target tissue of the subject or injected systemically.
采用基因敲除技术和/或基因沉默技术来制备内源性TCR分子低表达或不表达的工程化细胞。基因敲除技术包括Argonaute、CRISPR/Cas9技术、ZFN技术、TALE技术、TALE-CRISPR/Cas9技术、Base Editor技术、引导编辑技术和/或归巢核酸内切酶技术。基因沉默技术包括但不限于：反义RNA、RNA干扰、微小RNA介导的翻译抑制等。Gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR molecules. Gene knockout technologies include Argonaute, CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guide editing technology and/or homing endonuclease technology. Gene silencing techniques include, but are not limited to, antisense RNA, RNA interference, microRNA-mediated translational inhibition, and the like.
成簇的规律间隔的短回文重复序列(CRISPR)系统用于基因组编辑。该系统包括Cas9(一种能够使用crRNA作为其向导来修饰DNA的蛋白质)，CRISPR RNA(crRNA，包含Cas9用来引导其到达宿主DNA正确片段的RNA，以及与tracrRNA结合的区域(通常以发夹环形式)，与Cas9形成活性复合物)，反式激活crRNA(tracrRNA，与crRNA结合，与Cas9形成活性复合物)，以及DNA修复模板的可选片段(可指导细胞修复过程允许插入特定的DNA序列的DNA)。CRISPR/Cas9通常采用质粒、或电转方式传递核酸片段到靶细胞。crRNA需要针对每种应用进行设计，因为这是Cas9用来识别并直接结合细胞中靶DNA的序列。多个crRNA和tracrRNA可以包装在一起以形成指导RNA(gRNA)。该gRNA可以与Cas9基因连接在一起并制成质粒，以便被转染到细胞中。本发明凡涉及gRNA的序列时，其可以为靶向的DNA序列，亦可以为所述DNA对应的核糖核苷酸与crRNA、TracrRNA形成的完整Cas9引导序列。在进行基因编辑时，施用的gRNA、tracr配对序列及tracr序列可以单独施用，也可以一条完整的RNA序列施用。CRISPR/Cas9转基因可以通过载体(例如AAV、腺病毒、慢病毒)、和/或粒子和/或纳米粒子、和/或电转来递送。Clustered regularly interspaced short palindromic repeats (CRISPR) systems are used for genome editing. The system includes Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, the RNA that contains the Cas9 uses to guide it to the correct segment of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin) loop form), forms an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA, forms an active complex with Cas9), and an optional fragment of a DNA repair template (which directs cellular repair processes to allow insertion of specific DNA sequence of DNA). CRISPR/Cas9 usually uses plasmids or electroporation to deliver nucleic acid fragments to target cells. crRNA needs to be designed for each application, as this is the sequence Cas9 uses to recognize and bind directly to target DNA in cells. Multiple crRNAs and tracrRNAs can be packaged together to form guide RNAs (gRNAs). The gRNA can be linked to the Cas9 gene and made into a plasmid for transfection into cells. When the present invention relates to the sequence of gRNA, it can be a targeted DNA sequence, or a complete Cas9 guide sequence formed by the ribonucleotide corresponding to the DNA, crRNA and TracrRNA. When performing gene editing, the administered gRNA, tracr pairing sequence and tracr sequence can be administered alone, or a complete RNA sequence can be administered. CRISPR/Cas9 transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
锌指核酸酶(ZFN)是一种人工限制性酶，通过将锌指DNA结合结构域与DNA切割结构域结合而产生。锌指结构域可以被工程化以靶向特定的DNA序列，其允许锌指核酸酶靶向 基因组内的靶序列。Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by binding a zinc finger DNA binding domain to a DNA cleavage domain. Zinc finger domains can be engineered to target specific DNA sequences, which allow zinc finger nucleases to target target sequences within the genome.
转录激活因子样效应物核酸酶(TALEN)是限制性酶，可以工程化为切割DNA的特定序列。TALEN系统的工作原理几乎与ZFN相同。它们是通过将转录激活因子样效应物DNA结合结构域与DNA切割结构域结合而产生的。Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. The TALEN system works almost the same as the ZFN. They are generated by binding transcription activator-like effector DNA binding domains to DNA cleavage domains.
本发明还提供了编码本文所述的一种或多种ATC、靶向内源性TCR的核酸抑制分子或gRNA构建体的核酸分子。The invention also provides nucleic acid molecules encoding one or more of the ATCs described herein, nucleic acid inhibitory molecules targeting endogenous TCRs, or gRNA constructs.
5.工程化细胞制备方法5. Preparation method of engineered cells
在一实施例中，首先采用包含编码ATC的多核苷酸的病毒感染免疫效应细胞(例如T细胞或NKT细胞)，再利用CRISPR/Cas9技术敲除内源性TCR亚基，得到本发明的工程化细胞。在一实施例中，首先利用CRISPR/Cas9技术敲除免疫效应细胞内源性TCR亚基，再采用包含编码ATC的多核苷酸的病毒感染。在一实施例中，采用包含编码ATC的多核苷酸的病毒感染免疫效应细胞和利用CRISPR/Cas9技术敲除免疫效应细胞内源性TCR亚基同时进行。在一实施例中，编码ATC的多核苷酸片段不包括CRISPR/Cas9的靶序列。In one embodiment, firstly use a virus comprising a polynucleotide encoding ATC to infect immune effector cells (such as T cells or NKT cells), and then use CRISPR/Cas9 technology to knock out endogenous TCR subunits to obtain the engineering of the present invention. cells. In one embodiment, the endogenous TCR subunits of immune effector cells are first knocked out using CRISPR/Cas9 technology, and then a virus comprising a polynucleotide encoding ATC is used to infect. In one embodiment, infection of immune effector cells with a virus comprising a polynucleotide encoding ATC and knockout of endogenous TCR subunits in immune effector cells using CRISPR/Cas9 technology are performed simultaneously. In one embodiment, the ATC-encoding polynucleotide fragment does not include the target sequence of CRISPR/Cas9.
在T细胞激活第1，2，3，4，5，6，7，8，9或10天加入包含编码ATC的多核苷酸的病毒感染所述T细胞，感染后第1，2，3，4，5，6，7，8，9，10，15，20天采用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基，制备得到本发明的ATCT细胞。在T细胞激活第1，2，3，4，5，6，7，8，9或10天利用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基，敲除后1，2，3，4，5，6，7，8，9，10，15，20天内加入包含编码ATC的多核苷酸的病毒感染所述T细胞，制备得到本发明的ATCT细胞。Viruses containing ATC-encoding polynucleotides were added to infect the T cells on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th day after activation of the T cells, and the T cells were infected on the 1st, 2nd, 3rd, On days 4, 5, 6, 7, 8, 9, 10, 15, and 20, the endogenous TCR subunits of the T cells were knocked out by electroporation using the CRISPR/Cas9 technology to prepare the ATCT cells of the present invention. On the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th day of T cell activation, the T cell endogenous TCR subunit was knocked out by electroporation using CRISPR/Cas9 technology. Within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, and 20 days, a virus comprising a polynucleotide encoding ATC is added to infect the T cells to prepare the ATCT cells of the present invention.
在一实施例中，在T细胞激活1-5天内加入包含编码ATC的多核苷酸的病毒，感染后1-7天内采用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基。在一实施例中，在T细胞激活1-5天内利用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基，敲除后1-7天内加入包含编码ATC的多核苷酸的病毒感染所述T细胞。在一实施例中，在T细胞激活第2天加入包含编码ATC的多核苷酸的病毒，感染后第2天采用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基。在一实施例中，在T细胞激活第2天加入包含编码ATC的多核苷酸的病毒，感染后第2天采用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基。在一实施例中，在T细胞激活第2天利用CRISPR/Cas9技术用电转方法敲除所述T细胞内源性TCR亚基，敲除后第2天加入包含编码ATC的多核苷酸的病毒感染所述T细胞。In one embodiment, a virus comprising a polynucleotide encoding ATC is added within 1-5 days of T cell activation, and CRISPR/Cas9 technology is used within 1-7 days after infection to knock out the T cell endogenous TCR by electroporation subunit. In one embodiment, the T cell endogenous TCR subunit is knocked out by electroporation using CRISPR/Cas9 technology within 1-5 days of T cell activation, and a polynucleoside encoding ATC is added within 1-7 days after the knockout. Acidic virus infects the T cells. In one embodiment, a virus comprising a polynucleotide encoding ATC is added on the second day of T cell activation, and the T cell endogenous TCR subunit is knocked out by electroporation using CRISPR/Cas9 technology on the second day after infection . In one embodiment, a virus comprising a polynucleotide encoding ATC is added on the second day of T cell activation, and the T cell endogenous TCR subunit is knocked out by electroporation using CRISPR/Cas9 technology on the second day after infection . In one embodiment, the endogenous TCR subunit of the T cell is knocked out by electroporation using CRISPR/Cas9 technology on the 2nd day of T cell activation, and on the 2nd day after the knockout, a polynucleotide comprising a polynucleotide encoding ATC is added. The virus infects the T cells.
在一实施例中，为了减少T细胞中内源性TCR亚基与ATC亚基形成错配导致ATC低 表达，本发明敲除T细胞内源性TCR或对T细胞进行基因修饰使得内源性TCR分子低表达或不表达，并且对ATC中的TCR亚基的胞外恒定区进行基因修饰，使得在敲除T细胞内源性TCR或进行导致内源性TCR分子低表达或不表达的基因修饰时不影响T细胞中ATC表达和/或不影响T细胞中ATC与内源性CD3形成复合体。In one embodiment, in order to reduce the mismatch between the endogenous TCR subunit and the ATC subunit in T cells, resulting in low expression of ATC, the present invention knocks out the endogenous TCR in the T cell or genetically modifies the T cell to make the endogenous TCR subunit. TCR molecules are low or not expressed, and the extracellular constant regions of the TCR subunits in ATC are genetically modified, so that when the endogenous TCR in T cells is knocked out or genes that cause low or no expression of endogenous TCR molecules are performed Modification does not affect ATC expression in T cells and/or does not affect ATC complexing with endogenous CD3 in T cells.
在一个方面，将编码靶向靶抗原(示例性，GPC3或Claudin18.2肿瘤抗原)的ATC、靶向内源性TCR的核酸抑制分子或gRNA的核酸分子引入T细胞中以产生ATCT细胞。在一个实施方案中，体外转录的ATC核酸分子、靶向内源性TCR的核酸抑制分子或gRNA可作为瞬时转染的形式引入细胞中。示例性人工DNA序列是包含连接在一起以形成编码融合蛋白的开放阅读框的基因部分的序列。连接在一起的DNA部分可来自单个生物体或来自多于一个生物体。In one aspect, a nucleic acid molecule encoding an ATC targeting a target antigen (eg, GPC3 or Claudin 18.2 tumor antigen), a nucleic acid inhibitory molecule targeting an endogenous TCR, or a gRNA is introduced into T cells to generate ATCT cells. In one embodiment, in vitro transcribed ATC nucleic acid molecules, nucleic acid inhibitory molecules targeting endogenous TCRs, or gRNAs can be introduced into cells as a form of transient transfection. An exemplary artificial DNA sequence is a sequence comprising portions of a gene linked together to form an open reading frame encoding a fusion protein. The DNA portions that are linked together can be from a single organism or from more than one organism.
6.给药6. Administration
可以将包含本发明的工程化细胞的组合物系统地或直接提供给受试者，以诱导和/或增强对抗原的免疫应答和/或治疗和/或预防肿瘤、病原体感染或感染性疾病。在一实施例中，将本发明的工程化细胞或包含其的组合物直接注射到目的器官(例如，受肿瘤影响的器官)中。或者，例如通过向循环系统(例如，静脉、肿瘤脉管系统)给药，将本发明的工程化细胞或包含其的组合物间接地提供给目的器官。可以在施用细胞或组合物之前、同时或之后提供扩增和分化剂，以增加体外或体内T细胞、NKT细胞或CTL细胞的产生。Compositions comprising the engineered cells of the present invention can be provided to a subject systemically or directly to induce and/or enhance immune responses to antigens and/or to treat and/or prevent tumors, pathogen infections or infectious diseases. In one embodiment, the engineered cells of the invention or compositions comprising the same are injected directly into an organ of interest (eg, an organ affected by a tumor). Alternatively, the engineered cells of the invention, or compositions comprising the same, are provided indirectly to an organ of interest, eg, by administration to the circulatory system (eg, intravenous, tumor vasculature). Expansion and differentiation agents can be provided before, concurrently with, or after administration of the cells or compositions to increase the production of T cells, NKT cells, or CTL cells in vitro or in vivo.
本发明的工程化细胞可以包含纯化的细胞群。本领域技术人员可以使用各种众所周知的方法，例如荧光激活细胞分选(FACS)，容易地确定群体中本发明的工程化细胞的百分比。在包含本发明的工程化细胞的群体中，纯度的合适范围是约50％至约55％、约5％至约60％、以及约65％至约70％。在某些实施方式中，纯度为约70％至约75％、约75％至约80％或约80％至约85％。在某些实施方式中，纯度为约85％至约90％，约90％至约95％以及约95％至约100％。剂量可以由本领域技术人员容易地调节(例如，纯度降低可能需要增加剂量)。可以通过注射、导管等引入细胞。The engineered cells of the present invention may comprise purified cell populations. Those skilled in the art can readily determine the percentage of engineered cells of the invention in a population using a variety of well-known methods, such as fluorescence-activated cell sorting (FACS). In a population comprising the engineered cells of the present invention, suitable ranges of purity are from about 50% to about 55%, from about 5% to about 60%, and from about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Dosage can be easily adjusted by one skilled in the art (eg, a decrease in purity may require an increase in dose). Cells can be introduced by injection, catheter, and the like.
本发明的组合物可以是包含本发明的免疫效应细胞或其祖细胞和药学上可接受的载体的药物组合物。给药可以是自体的或异体的。例如，可以从一个受试者获得免疫效应细胞或祖细胞，并将其施用于相同受试者或不同的相容受试者。外周血来源的免疫效应细胞或其后代(例如，体内、离体或体外来源)可通过局部注射施用，包括导管给药、全身注射、局部注射、静脉内注射或肠胃外给药。当施用本发明的主题的治疗组合物(例如，包含本发明的免疫效应细胞的药物组合物)时，可以将其配制成单位剂量可注射形式(溶液剂、悬浮剂、乳剂等)。The composition of the present invention may be a pharmaceutical composition comprising the immune effector cell of the present invention or its progenitor cell and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. For example, immune effector or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived immune effector cells or progeny thereof (eg, in vivo, ex vivo, or in vitro sources) can be administered by local injection, including catheter, systemic, local, intravenous, or parenteral. When administering the therapeutic compositions of the present subject matter (eg, pharmaceutical compositions comprising immune effector cells of the present invention), they may be formulated in unit dose injectable forms (solutions, suspensions, emulsions, etc.).
7.剂型7. Dosage Form
包含本发明的工程化细胞的组合物可以方便地以无菌液体制剂的形式提供，例如等渗水溶液剂、悬浮液、乳剂、分散剂或粘性组合物，其可以缓冲至选定的pH。液体制剂通常比凝胶、其它粘性组合物和固体组合物更容易制备。另外，液体组合物在某种程度上更方便施用，尤其是通过注射。另一方面，可以在适当的粘度范围内配制粘性组合物以提供与特定组织的更长的接触时间。液体或粘性组合物可以包含载体，所述载体可以是溶剂或分散介质，其包含例如水、盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇等)及其合适的混合物。Compositions comprising the engineered cells of the invention can be conveniently provided in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, the viscous composition can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable suitable mixture.
可以通过将遗传修饰的工程化细胞掺入所需量的适当溶剂中，并根据需要掺入不同量的其它成分来制备无菌注射溶液。这样的组合物可以与合适的载体、稀释剂或赋形剂例如无菌水、生理盐水、葡萄糖、右旋糖等混合。组合物也可以冻干。所述组合物可包含辅助物质，例如润湿剂、分散剂或乳化剂(例如，甲基纤维素)、pH缓冲剂、胶凝剂或增粘剂、防腐剂、矫味剂、颜料等，这取决于给药途径和所需制剂。Sterile injectable solutions can be prepared by incorporating the genetically modified engineered cells in the required amount of the appropriate solvent with other ingredients in varying amounts as required. Such compositions may be admixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose, dextrose, and the like. Compositions can also be lyophilized. The compositions may contain auxiliary substances such as wetting agents, dispersing agents or emulsifying agents (eg, methyl cellulose), pH buffering agents, gelling or tackifying agents, preservatives, flavoring agents, pigments, and the like, This will depend on the route of administration and the desired formulation.
可以添加增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。可以通过各种抗细菌和抗真菌剂，例如对羟基苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等来确保防止微生物的作用。可通过使用延迟吸收的试剂例如单硬脂酸铝和明胶来延长可注射药物形式的吸收。然而，所使用的任何媒介物、稀释剂或添加剂将必须与遗传修饰的免疫效应细胞或其祖细胞相容。Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption such as aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immune effector cells or their progenitor cells.
该组合物可以是等渗的，即它们可以具有与血液和/或泪液相同的渗透压。组合物的所需等渗性可以使用氯化钠或其它药学上可接受的试剂例如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。氯化钠可以特别适用于含有钠离子的缓冲剂。The compositions may be isotonic, ie they may have the same osmotic pressure as blood and/or tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride can be particularly useful in buffers containing sodium ions.
如果需要，可使用药学上可接受的增稠剂将组合物的粘度保持在选定水平。例如，甲基纤维素容易且经济地获得并且易于使用。其它合适的增稠剂包括，例如，黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的浓度可以取决于选择的试剂。重要的是要使用能够达到所选粘度的用量。显然，合适载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型(例如，是否将组合物配制成溶液剂、悬浮液、凝胶剂或其它液体形式，例如定时释放形式或液体填充形式)。If desired, a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level. For example, methylcellulose is readily and economically available and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of thickening agent can depend on the agent chosen. It is important to use an amount that will achieve the chosen viscosity. Obviously, the selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, eg, liquid dosage form (eg, whether the composition is to be formulated as a solution, suspension, gel, or other liquid form, eg time-release form or liquid-filled form).
对于所治疗的受试者，要施用的细胞数量将有所不同。在一个实施方式中，向人受试者施用的本发明的免疫效应细胞为约10 4至约10 10之间、约10 5至约10 9之间、或约10 6至约10 9之间。可以更少的数量施用更有效的细胞。可以根据每个受试者的个体因素，包括其大小、年龄、性别、体重和受试者的状况，来确定有效剂量的精确确定。本领域技术人员从本发明和本领域知识中可以容易地确定剂量。 The number of cells to be administered will vary for the subject being treated. In one embodiment, the immune effector cells of the invention are administered to a human subject between about 10 4 to about 10 10 , between about 10 5 to about 10 9 , or between about 10 6 to about 10 9 . More potent cells can be administered in smaller numbers. The precise determination of the effective dose can be determined according to individual factors of each subject, including its size, age, sex, weight, and the condition of the subject. Dosages can be readily determined by those skilled in the art from the present invention and knowledge in the art.
本领域技术人员可以容易地确定组合物中和在方法中施用的细胞和任选的添加剂、媒 介物和/或载体的量。通常，任何添加剂(除一种或多种活性细胞和/或一种或多种试剂外)在磷酸盐缓冲盐水中的存在量为0.001％至50％(重量)溶液，并且活性成分按微克至毫克的顺序存在，例如约0.0001wt％至约5wt％、约0.0001wt％至约1wt％、约0.0001wt％至约0.05wt％或约0.001wt％至约20wt％、约0.01wt％至约10wt％或约0.05wt％至约5wt％。对于要施用于动物或人的任何组合物，可以确定以下结果：毒性，例如通过在合适的动物模型例如啮齿类动物如小鼠中确定致死剂量(LD)和LD50；组合物的剂量，其中的组分浓度和施用组合物的时间，引起合适的反应。The amount of cells and optional additives, vehicles and/or carriers to be administered in the composition and in the method can be readily determined by one skilled in the art. Typically, any additives (other than one or more active cells and/or one or more reagents) are present in phosphate buffered saline in an amount ranging from 0.001% to 50% by weight solution, and the active ingredient is in the range of micrograms to milligrams are present in the order of, for example, about 0.0001 wt% to about 5 wt%, about 0.0001 wt% to about 1 wt%, about 0.0001 wt% to about 0.05 wt%, or about 0.001 wt% to about 20 wt%, about 0.01 wt% to about 10 wt% % or about 0.05 wt % to about 5 wt %. For any composition to be administered to an animal or a human, the following results can be determined: toxicity, eg, by determining the lethal dose (LD) and LD50 in a suitable animal model, eg, rodents such as mice; the dosage of the composition, wherein Concentrations of components and timing of application of the composition, elicit an appropriate response.
8.治疗方法8. Treatment
本发明提供用于在需要所述工程化细胞的受试者中诱导和/或增加免疫应答的方法。本发明的工程化细胞和包含其的组合物可以用于治疗和/或预防受试者的肿瘤。本发明的工程化细胞和包含其的组合物可以用于延长患有肿瘤的受试者的存活。本发明的工程化细胞和包含其的组合物也可以用于治疗和/或预防诸如免疫功能低下的人受试者的病原体感染或其它感染性疾病。这种方法包括施用有效量的本发明的工程化细胞或包含其的组合物(例如药物组合物)以达到期望的效果，无论是减轻现有病症还是预防复发。为了治疗，施用的量是有效产生所需效果的量。可以一次或多次给药来提供有效量。可以大剂量或通过连续灌注来提供有效量。The present invention provides methods for inducing and/or increasing an immune response in a subject in need of such engineered cells. The engineered cells of the present invention and compositions comprising the same can be used to treat and/or prevent tumors in a subject. The engineered cells of the present invention and compositions comprising the same can be used to prolong the survival of subjects with tumors. The engineered cells of the present invention and compositions comprising the same can also be used to treat and/or prevent pathogenic infections or other infectious diseases, such as in immunocompromised human subjects. Such methods include administering an effective amount of an engineered cell of the invention or a composition comprising the same (eg, a pharmaceutical composition) to achieve the desired effect, whether reducing an existing condition or preventing relapse. For treatment, the amount administered is that amount effective to produce the desired effect. The effective amount can be provided in one or more administrations. Effective amounts can be provided in boluses or by continuous infusion.
在一实施例中，包含本发明的ATC的免疫效应细胞可以用于治疗具有表面抗原表达水平低的肿瘤细胞的受试者，例如由于疾病的复发，其中受试者接受过导致残留肿瘤细胞的治疗。在某些实施方式中，肿瘤细胞在肿瘤细胞表面上具有低密度的靶分子。In one embodiment, immune effector cells comprising ATCs of the invention can be used to treat subjects with tumor cells that express low levels of surface antigens, for example due to relapse of the disease, where the subject has received a treatment that results in residual tumor cells. treat. In certain embodiments, tumor cells have a low density of target molecules on the tumor cell surface.
在一实施例中，包含本发明的ATC的免疫效应细胞可以用于治疗患有疾病复发的受试者，其中该受试者接受过包含CAR的免疫效应细胞(例如，T细胞)，该CAR包含细胞内信号传导结构域，其包含含有共刺激性信号传导结构域(例如4-1BBz CAR)。在某些实施方式中，肿瘤细胞在肿瘤细胞表面上具有低密度的肿瘤特异性抗原。在某些实施方式中，该疾病是GPC3阳性肿瘤、Claudin18.2阳性肿瘤。在一实施例中，肿瘤细胞在肿瘤细胞上具有低密度的GPC3。在一实施例中，肿瘤细胞在肿瘤细胞上具有低密度的Claudin18.2。这种方法包括施用有效量的本发明的免疫效应细胞或包含其的组合物(例如药物组合物)以达到期望的效果，缓解现有病症或预防复发。In one embodiment, an immune effector cell comprising an ATC of the invention can be used to treat a subject suffering from disease relapse, wherein the subject has received an immune effector cell (eg, T cell) comprising a CAR, the CAR An intracellular signaling domain is included, which includes a co-stimulatory signaling domain (eg, a 4-1BBz CAR). In certain embodiments, tumor cells have a low density of tumor-specific antigens on the tumor cell surface. In certain embodiments, the disease is a GPC3 positive tumor, a Claudin18.2 positive tumor. In one embodiment, the tumor cells have a low density of GPC3 on tumor cells. In one embodiment, the tumor cells have a low density of Claudin 18.2 on the tumor cells. Such methods include administering an effective amount of an immune effector cell of the invention or a composition (eg, a pharmaceutical composition) comprising the same to achieve the desired effect, alleviation of an existing condition or prevention of relapse.
“有效量”(或“治疗有效量”)是足以在治疗后产生有益或期望的临床结果的量。可以以一剂或多剂剂量将有效量施用于受试者。就治疗而言，有效量是足以缓解、改善、稳定、逆转或减慢疾病进展或以其它方式减少疾病病理后果的量。有效量通常由医师根据具体情况确定，并且在本领域技术人员的能力范围内。当确定合适的剂量以达到有效量时，通常要考虑几个因素。这些因素包括受试者的年龄、性别和体重、所治疗的疾病、疾病的 严重程度以及所施用的免疫效应细胞的形式和有效浓度。An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result following treatment. An effective amount can be administered to a subject in one or more doses. For therapeutic purposes, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow disease progression or otherwise reduce the pathological consequences of the disease. Effective amounts are generally determined by the physician on a case-by-case basis and are within the capabilities of those skilled in the art. Several factors are generally considered when determining an appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of immune effector cells administered.
对于使用抗原特异性T细胞的过继免疫疗法，通常输注约10 6-10 10范围内的细胞剂量。在将本发明的工程化细胞施用于宿主并随后分化后，诱导特异性针对特定抗原的T细胞。工程化细胞可以通过本领域已知的任何方法施用，包括但不限于静脉内、皮下、结内、肿瘤内、鞘内、胸膜内、腹膜内和直接向胸腺施用。 For adoptive immunotherapy using antigen-specific T cells, cell doses in the range of about 106-1010 are typically infused. Following administration of the engineered cells of the invention to a host and subsequent differentiation, T cells specific for a particular antigen are induced. Engineered cells can be administered by any method known in the art, including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly to the thymus.
本发明提供用于治疗和/或预防受试者中的肿瘤的方法。该方法可以包括向患有肿瘤的受试者施用有效量的本发明的工程化或包含其的组合物。The present invention provides methods for treating and/or preventing tumors in a subject. The method can include administering to a subject having a tumor an effective amount of an engineered of the invention or a composition comprising the same.
肿瘤的非限制性实例包括血液癌症(例如白血病、淋巴瘤和骨髓瘤)、卵巢癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、前列腺癌、皮肤癌、胃癌、胶质母细胞瘤、喉癌、黑素瘤、神经母细胞瘤、腺癌、神经胶质瘤、软组织肉瘤和各种癌(包括前列腺癌和小细胞肺癌)。肿瘤的非限制性实例包括但不限于星形细胞瘤、纤维肉瘤、粘液肉瘤、脂肪肉瘤、少突胶质细胞瘤、室管膜瘤、髓母细胞瘤、原始神经外胚层肿瘤(PNET)、软骨肉瘤、成骨肉瘤、胰腺导管腺癌、小细胞和大细胞肺腺癌、脊索瘤、血管肉瘤、内皮肉瘤、鳞状细胞癌、支气管肺泡癌、上皮腺癌及其肝转移灶、淋巴管肉瘤、淋巴管内皮肉瘤、肝癌、胆管癌、滑膜瘤、间皮瘤、尤文氏瘤、横纹肌肉瘤、结肠癌、基底细胞癌、汗腺癌、乳头状癌、皮脂腺癌、状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、胆小管癌、绒毛膜癌、精原细胞瘤、胚胎癌、Wilms’肿瘤、睾丸肿瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质细胞瘤、脑膜瘤、神经母细胞瘤、视网膜母细胞瘤、白血病、多发性骨髓瘤、Waldenstrom’s巨球蛋白血症和重链疾病、诸如导管和小叶腺癌的乳腺肿瘤、子宫颈的鳞状和腺癌、子宫和卵巢上皮癌、前列腺腺癌、膀胱移行鳞状细胞癌、B和T细胞淋巴瘤(结节性和弥漫性)浆细胞瘤、急慢性白血病、恶性黑色素瘤、软组织肉瘤和平滑肌肉瘤。在某些实施方式中，肿瘤选自血液癌症(例如白血病、淋巴瘤和骨髓瘤)、卵巢癌、前列腺癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、前列腺癌、皮肤癌、胃癌、胶质母细胞瘤和喉癌。在一实施例中，本发明工程化细胞和包含其的组合物可以用于治疗和/或预防常规治疗措施不适合或复发难治性实体瘤，例如肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌。在一实施例中，肿瘤是血液肿瘤。Non-limiting examples of tumors include blood cancers (eg, leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer , gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma and various carcinomas (including prostate cancer and small cell lung cancer). Non-limiting examples of tumors include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), Chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphatic vessels Sarcoma, Lymphoendothelioma, Liver Cancer, Cholangiocarcinoma, Synovialoma, Mesothelioma, Ewing's Tumor, Rhabdomyosarcoma, Colon Cancer, Basal Cell Carcinoma, Sweat Gland Carcinoma, Papillary Carcinoma, Sebaceous Gland Carcinoma, Smoid Adenocarcinoma, Cystic Gland Carcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependyma tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia and severe chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinoma of the cervix, uterine and ovarian epithelial cancer, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and Diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the tumor is selected from hematological cancers (eg, leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer , prostate cancer, skin cancer, stomach cancer, glioblastoma and throat cancer. In one embodiment, the engineered cells of the present invention and compositions comprising the same can be used to treat and/or prevent solid tumors that are unsuitable or relapsed or refractory to conventional therapeutic measures, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, etc. cancer, thyroid cancer, stomach cancer, colorectal cancer. In one embodiment, the tumor is a hematological tumor.
本发明工程化细胞治疗目标可以包括缓解或逆转疾病进展和/或减轻副作用、或治疗目标包括降低或延迟复发风险。The therapeutic goals of engineered cells of the present invention may include alleviating or reversing disease progression and/or reducing side effects, or therapeutic goals including reducing or delaying the risk of relapse.
本发明提供用于在例如免疫受损的受试者中治疗和/或预防病原体感染(例如病毒感染、细菌感染、真菌感染、寄生虫感染或原生动物感染)的方法。该方法可以包括向患有病原体感染的受试者施用有效量的本发明工程化细胞或包含其的组合物。易于治疗的示例 性病毒感染包括但不限于巨细胞病毒、爱泼斯坦-巴尔病毒、人免疫缺陷病毒和流感病毒感染。The present invention provides methods for treating and/or preventing pathogenic infections (eg, viral, bacterial, fungal, parasitic, or protozoan infections) in, eg, immunocompromised subjects. The method may comprise administering to a subject suffering from a pathogen infection an effective amount of an engineered cell of the invention or a composition comprising the same. Exemplary viral infections that are amenable to treatment include, but are not limited to, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus infections.
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强的应答可以包含应答性中5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫效应细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％更多。The term "enhancing" refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein. For example, an enhanced response can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the responsiveness %, 75%, 80%, 85%, 90%, 95% or 98% or more increase. As used herein, "enhancing" can also refer to increasing the number of subjects that respond to treatment, eg, immune effector cell therapy. For example, an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentages are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
在本发明实例中，免疫效应细胞靶向GPC3表达阳性的肿瘤。在本发明实例中，免疫效应细胞靶向Claudin18.2表达阳性的肿瘤。在具体的实施方式中，所述肿瘤包括但不限于肝癌、胃癌、肺癌、食道癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤、食道癌。本领域技术人员知晓，有些肿瘤细胞，例如肝癌细胞对很多药物不敏感，因此，即便在体外有效的药物，有时候在体内的效果也不佳，甚至没有效果。因此，在优选的实施方式中，本文所述的GPC3表达阳性的肿瘤或GPC阳性肿瘤包括但不限于肝癌、胃癌、肺癌、食道癌。因此，在优选的实施方式中，本文所述的Claudin18.2表达阳性的肿瘤或Claudin18.2阳性肿瘤包括但不限于肝癌、胃癌、肺癌、食道癌、胰腺癌、胆囊瘤、胆管癌。In the examples of the present invention, immune effector cells target GPC3-positive tumors. In the examples of the present invention, immune effector cells target tumors that express positive Claudin18.2. In specific embodiments, the tumor includes, but is not limited to, liver cancer, gastric cancer, lung cancer, esophageal cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, Adrenal cancer, schwannoma, malignant fibrous histiocytoma, esophageal cancer. Those skilled in the art know that some tumor cells, such as liver cancer cells, are not sensitive to many drugs, so even drugs that are effective in vitro sometimes have poor or even no effect in vivo. Therefore, in preferred embodiments, GPC3 expression-positive tumors or GPC-positive tumors described herein include, but are not limited to, liver cancer, gastric cancer, lung cancer, and esophageal cancer. Thus, in preferred embodiments, Claudin18.2-positive tumors or Claudin18.2-positive tumors described herein include, but are not limited to, liver cancer, gastric cancer, lung cancer, esophageal cancer, pancreatic cancer, gallbladder tumor, bile duct cancer.
9.试剂盒9. Kit
本发明提供用于在受试者中诱导和/或增强免疫应答和/或治疗和/或预防肿瘤或病原体感染的试剂盒。在一实施例中，试剂盒包含有效量的本发明工程化细胞或包含其的药物组合物。在一实施例中，试剂盒包括无菌容器；这样的容器可以是盒子、安瓿、瓶、小瓶、管、袋、小袋、泡罩包装或本领域已知的其它合适的容器形式。这样的容器可以由塑料、玻璃、层压纸、金属箔或其它适合于容纳药物的材料制成。在一实施例中，试剂盒包括编码本发明的ATC的核酸分子，其以可表达的形式靶向目的抗原，可以任选地包含在一种或多种载体中。The present invention provides kits for inducing and/or enhancing an immune response and/or treating and/or preventing tumor or pathogen infection in a subject. In one embodiment, the kit comprises an effective amount of an engineered cell of the invention or a pharmaceutical composition comprising the same. In one embodiment, the kit includes a sterile container; such a container may be a box, ampule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments. In one embodiment, the kit includes a nucleic acid molecule encoding an ATC of the invention targeting an antigen of interest in an expressible form, optionally contained in one or more vectors.
在一实施例中，将本发明的工程化细胞和/或核酸分子，与将所述细胞或核酸分子施用于患有肿瘤或病原体或免疫疾病或有发展成肿瘤或病原体或免疫疾病的受试者的说明书一起提供。说明书通常包括有关组合物用于治疗和/或预防肿瘤或病原体感染的信息。在一实施例中，说明书包括以下至少一项：治疗剂的描述；用于治疗或预防肿瘤、病原体感染或免疫疾病或其症状的剂量表和给药；注意事项；警告；适应症；不适应症；用药信息；不良反应；动物药理学；临床研究；和/或参考。这些说明书可以直接打印在容器上， 或者作为粘贴在容器上的标签，或者作为单独的纸页、小册子、卡片或文件夹提供在容器内或与容器一起。In one embodiment, the engineered cells and/or nucleic acid molecules of the invention are administered to a subject having or developing a tumor or pathogen or immune disease. supplied with the user's manual. The instructions generally include information regarding the use of the composition for treating and/or preventing infection by a tumor or pathogen. In one embodiment, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for the treatment or prevention of tumors, pathogenic infections, or immune diseases or symptoms thereof; precautions; warnings; indications; indications Symptoms; Medication Information; Adverse Reactions; Animal Pharmacology; Clinical Studies; and/or References. These instructions can be printed directly on the container, or as labels affixed to the container, or provided in or with the container as separate sheets, brochures, cards or folders.
本发明的优点：Advantages of the present invention:
本发明所述ATCT细胞在不降低体外杀伤能力的同时可减少细胞因子的分泌，同时具有更持久的存活能力、且具有更高的抗原敏感性。本发明涉及ATCT细胞的设计和构建方法(包括但不限于GPC3、Claudin18.2靶点)。本发明还涉及ATCT细胞体外功能性实验的研究和应用(包括但不限于GPC3、Claudin18.2靶点)。The ATCT cells of the present invention can reduce the secretion of cytokines without reducing the killing ability in vitro, and at the same time have longer lasting survival ability and higher antigen sensitivity. The present invention relates to the design and construction method of ATCT cells (including but not limited to GPC3, Claudin18.2 target). The present invention also relates to the research and application of in vitro functional experiments of ATCT cells (including but not limited to GPC3 and Claudin18.2 targets).
本发明包括，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN109908176A、CN109880803A、CN110055275A、CN110123837A、CN110438082A、CN110468105A以及例如国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的那些CAR-T细胞及其制备方法、抗体。本发明包括，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、 CN109908176A、CN109880803A、CN110055275A、CN110123837A、CN110438082A、CN110468105A以及例如国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018 /219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/141270, WO2019/149279, WO2019/170147A1, WO 2019/210863, WO2019/29029 of those CAR-T cells disclosed in Methods, Antibodies.
下面结合具体实施例进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。本说明书中提到的所有出版物、专利和专利申请均通过引用并入本文，其程度如同特别地且单独地指出每一个单独的出版物、专利或专利申请均通过引用而并入本文。The present invention is further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
实施例1.T细胞内源性TCR敲除Example 1. Knockout of endogenous TCR in T cells
在实施过程中，由于内源性TCR会竞争外源性抗体-T细胞受体嵌合物(ATC)的表达或引起错配，进而影响ATC在细胞表面的表达，因此本实施例利用CRISPR技术将内源性TCR进行敲除。示例性，针对TCRα链恒定区(TRAC)和β链恒定区(TRBC)的靶序列及其对应的引物序列如表2所示，针对TCRγ链恒定区(TRGC)和δ链恒定区(TRDC)的 靶序列及其对应的引物序列如表3所示；gRNA的合成使用GeneArt TMPrecision gRNA Synthesis Kit(Invitrogen)，具体步骤参考其说明书。采用本领域常规分子生物学技术，通过电转将Cas 9酶、靶向TRAC的gRNA和靶向TRBC的gRNA共同电转至T细胞，最终成功获得了TCRα链和TCRβ链敲除的T细胞；通过电转将Cas 9酶、靶向TRGC的gRNA和靶向TRDC的gRNA共同电转至T细胞，最终成功获得了TCRγ链和TCRδ链敲除的T细胞。 During the implementation process, since the endogenous TCR will compete for the expression of the exogenous antibody-T cell receptor chimera (ATC) or cause mismatches, thereby affecting the expression of ATC on the cell surface, this example uses CRISPR technology. Endogenous TCR was knocked out. Exemplary, target sequences for TCR α chain constant region (TRAC) and β chain constant region (TRBC) and their corresponding primer sequences are shown in Table 2, for TCR γ chain constant region (TRGC) and δ chain constant region (TRDC) The target sequence and its corresponding primer sequence are shown in Table 3; GeneArt ™ Precision gRNA Synthesis Kit (Invitrogen) was used for the synthesis of gRNA, and the specific steps were referred to its instructions. Using conventional molecular biology techniques in the field, the Cas 9 enzyme, TRAC-targeting gRNA and TRBC-targeting gRNA were co-electrotransferred to T cells by electroporation, and finally TCRα chain and TCRβ chain knockout T cells were successfully obtained; The Cas 9 enzyme, TRGC-targeting gRNA and TRDC-targeting gRNA were co-electroporated into T cells, and finally TCRγ chain and TCRδ chain knockout T cells were successfully obtained.
表2内源性TCRα链和β链的gRNA及其引物序列Table 2 gRNA and primer sequences of endogenous TCR α chain and β chain
Figure PCTCN2022086816-appb-000002
Figure PCTCN2022086816-appb-000002
表3内源性TCRγ链和δ链的gRNA及其引物序列Table 3 gRNAs of endogenous TCR γ and δ chains and their primer sequences
Figure PCTCN2022086816-appb-000003
Figure PCTCN2022086816-appb-000003
实施例2.ATCT慢病毒载体构建Example 2. Construction of ATCT lentiviral vector
为了防止实施例1中对内源性TCR的敲除会影响到外源性ATC，因此本实施例对提供的ATC中TCR的恒定区进行了突变设计，保持对应的氨基酸不变但是碱基序列进行突变。示例性的，针对实施例1中的表2中所例举的gRNA所靶向的TCRα链恒定区和β链恒定区的序列进行突变设计，突变后序列如表4所示。示例性的，针对实施例1中的 表3中所例举的gRNA所靶向的TCRγ链恒定区和δ链恒定区的序列进行突变设计，突变后序列如表5所示。In order to prevent the knockout of the endogenous TCR in Example 1 from affecting the exogenous ATC, the constant region of the TCR in the provided ATC is mutated in this example, keeping the corresponding amino acid unchanged but the base sequence Make mutations. Exemplarily, mutation design is performed for the sequences of the TCR α chain constant region and β chain constant region targeted by the gRNAs exemplified in Table 2 in Example 1, and the sequences after mutation are shown in Table 4. Exemplarily, mutation design is performed for the sequences of the TCR γ chain constant region and δ chain constant region targeted by the gRNAs exemplified in Table 3 in Example 1, and the sequences after mutation are shown in Table 5.
表4外源性TCRα链和β链的恒定区突变位点(下划线标出)Table 4. Constant region mutation sites of exogenous TCR α chain and β chain (underlined)
位置Location 原序列original sequence 突变后序列mutated sequence
α链恒定区alpha chain constant region TGTACCAGCTGAGAGACTCTTGTACCAGCTGAGAGACTCT TGTA TCAGCTGAG GGA TTC C TGTA T CAGCTGAG G GA T TC C
β链恒定区beta chain constant region AGATCGTCAGCGCCGAGGCCAGATCGTCAGCGCCGAGGCC AGAT TGTC TCCGCCGA AGCC AGAT T GTC TC CGCCGA A GCC
表5外源性TCRγ链和δ链的恒定区突变位点(下划线标出)Table 5 Constant region mutation sites of exogenous TCR γ chain and δ chain (underlined)
位置Location 原序列original sequence 突变后序列mutated sequence
γ链恒定区gamma chain constant region GCCTTCTGGAGCTTTGTTTCGCCTTCTGGAGCTTTGTTTC GCCTTCTG CAGCTT GGT CTC GCCTTCTG C AGCTT G GT C TC
δ链恒定区delta chain constant region CTGGGAGAGATGACAATAGCCTGGGAGAGATGACAATAGC CTGGG TGAGAT CAC GAT GGC CTGGG T GAGAT C AC G AT G GC
本实施例以构建包括表4中TCRα链和β链恒定区突变序列的ATC、或构建包括表5中TCRγ链和δ链恒定区突变序列的ATC为例。示例性的，ATC含有两条链，链1包括识别靶抗原的抗体可变区VH或VL，与TCRα突变恒定区(SEQ ID NO：7、8、10、12)；链2包括识别靶抗原的抗体可变区VL或VH，与TCRβ突变恒定区(SEQ ID NO：16、17)；两条链基因序列通过连接肽(示例性的，F2A(SEQ ID NO：88)、P2A(SEQ ID NO：90)或连接肽1(SEQ ID NO：92)连接，构建于载体中。This example takes the construction of an ATC including the mutated sequences of the constant regions of the TCR α chain and β chain in Table 4, or the construction of an ATC including the mutated sequences of the constant regions of the TCR γ chain and δ chain in Table 5 as an example. Exemplarily, ATC contains two chains, chain 1 includes antibody variable region VH or VL that recognizes target antigen, and TCRα mutated constant region (SEQ ID NO: 7, 8, 10, 12); chain 2 includes antibody that recognizes target antigen The variable region VL or VH of the antibody, and the TCRβ mutated constant region (SEQ ID NO: 16, 17); the two chain gene sequences are linked by peptides (exemplary, F2A (SEQ ID NO: 88), P2A (SEQ ID NO: 88), P2A (SEQ ID NO: 88) NO: 90) or linker peptide 1 (SEQ ID NO: 92) and constructed in a vector.
示例性的，ATC含有两条链，链1包括识别靶抗原的抗体可变区VH或VL，与TCRγ突变恒定区(SEQ ID NO:21)串联；链2包括识别靶抗原的抗体可变区VL或VH，与TCRδ突变恒定区(SEQ ID NO：24)串联；两条链基因序列通过连接肽(示例性的，F2A(SEQ ID NO：88)、P2A(SEQ ID NO：90)或连接肽1(SEQ ID NO：92)连接，构建于载体中。Exemplarily, ATC contains two chains, chain 1 includes the antibody variable region VH or VL that recognizes the target antigen, in tandem with the TCRγ mutated constant region (SEQ ID NO: 21); chain 2 includes the antibody variable region that recognizes the target antigen VL or VH, in tandem with the TCRδ mutant constant region (SEQ ID NO:24); the two chain gene sequences are linked by a linker peptide (exemplarily, F2A (SEQ ID NO:88), P2A (SEQ ID NO:90) or Peptide 1 (SEQ ID NO: 92) was ligated and constructed in the vector.
示例性的，ATC的可变区靶向结合肿瘤抗原GPC3，具体而言，可变区包括靶向人GPC3抗体的轻链可变区及重链可变区。ATC含有两条链，链1包括抗GPC3抗体可变区VH(SEQ ID NO：1、2)，与TCRα突变恒定区(SEQ ID NO：7)串联；链2包括抗GPC3抗体可变区VL(SEQ ID NO：3、4)，与TCRβ突变恒定区(SEQ ID NO：16)串联；两条链基因序列通过连接肽F2A(SEQ ID NO：88)连接构建于pWPT慢病毒载体中，称为PWPT-ATC。Exemplarily, the variable region of ATC targets and binds to the tumor antigen GPC3. Specifically, the variable region includes a light chain variable region and a heavy chain variable region of an antibody targeting human GPC3. ATC contains two chains, chain 1 includes the anti-GPC3 antibody variable region VH (SEQ ID NO: 1, 2) in tandem with the TCRα mutated constant region (SEQ ID NO: 7); chain 2 includes the anti-GPC3 antibody variable region VL (SEQ ID NO: 3, 4), in series with the TCRβ mutant constant region (SEQ ID NO: 16); the gene sequences of the two chains were constructed in the pWPT lentiviral vector by linking the linker peptide F2A (SEQ ID NO: 88), called for PWPT-ATC.
示例性的，ATC的可变区靶向结合肿瘤抗原Claudin18.2，具体而言，可变区包括靶向人Claudin18.2抗体的轻链可变区及重链可变区。ATC含有两条链，链1包括抗Claudin18.2抗体可变区VH(SEQ ID NO：84)，与TCRα突变恒定区(SEQ ID NO：7)串联；链2包括抗Claudin18.2抗体可变区VL(SEQ ID NO：85)，与TCRβ突变恒定 区(SEQ ID NO：16)串联；两条链基因序列通过连接肽F2A(SEQ ID NO：88)连接构建于pWPT慢病毒载体中。Exemplarily, the variable region of ATC targets and binds to the tumor antigen Claudin18.2. Specifically, the variable region includes a light chain variable region and a heavy chain variable region of an antibody targeting human Claudin18.2. ATC contains two chains, chain 1 includes the anti-Claudin18.2 antibody variable region VH (SEQ ID NO: 84) in tandem with the TCRα mutated constant region (SEQ ID NO: 7); chain 2 includes the anti-Claudin18.2 antibody variable region Region VL (SEQ ID NO: 85), tandem with TCRβ mutant constant region (SEQ ID NO: 16); the two chain gene sequences are constructed in pWPT lentiviral vector by connecting peptide F2A (SEQ ID NO: 88).
慢病毒采用磷酸钙法进行包装，病毒上清用PEG8000/NaCl进行纯化，纯化后对病毒用流式细胞法进行滴度检测，即用稀释不同浓度(1:100、1:300、1:900、1:2700、1:8100)的病毒感染J.RT3-T3.5细胞(ATCC)，65小时后取细胞用5ug/ml抗原(生物素化的人GPC3蛋白)4度孵育45分钟，二抗采用SA-PE荧光抗体(eBioscience)1：300稀释使用，4度孵育45分钟后用流式细胞仪检测。The lentivirus was packaged by calcium phosphate method, and the virus supernatant was purified with PEG8000/NaCl. After purification, the titer of the virus was detected by flow cytometry. , 1:2700, 1:8100) virus infected J.RT3-T3.5 cells (ATCC), after 65 hours, the cells were taken and incubated with 5ug/ml antigen (biotinylated human GPC3 protein) at 4 degrees for 45 minutes, two Antibody was used at 1:300 dilution of SA-PE fluorescent antibody (eBioscience), and was detected by flow cytometer after incubation at 4 degrees for 45 minutes.
结果如图1所示，ATC病毒滴度为1.9E+09。The results are shown in Figure 1, and the ATC virus titer was 1.9E+09.
实施例3.ATCT细胞构建及鉴定Example 3. ATCT cell construction and identification
对于GPC3,采用常规生物学手段从健康人供主的血液中分离制备T细胞。T细胞复苏激活24-48小时加入病毒(示例性的，PWPT-ATC)体积(mL)＝细胞数x MOI值(MOI＝20)/滴度，感染后24-96小时采用参照实施例1所述的CRISPR/Cas9技术用电转方法敲除TCRα链和β链，gRNA在TRAC上的靶序列如SEQ ID NO：25所示，gRNA在TRBC上的靶序列如SEQ ID NO：28所示。随后在T细胞激活后第5-8天检测ATC阳性率和TCR敲除效率。ATC阳性率检测试剂为5ug/ml生物素化的人GPC3蛋白，然后加入SA-PE荧光抗体(eBioscience)1：300稀释进行标记；敲除效率通过细胞表面CD3的表达反映，用Anti-CD3-APC(Invitrogen)进行标记；最后通过流式细胞仪检测。UT组为未感染病毒T细胞。UT ko为未转导ATC载体，但敲除了内源性TCRα链和β链的T细胞。For GPC3, conventional biological methods were used to separate and prepare T cells from the blood of healthy human donors. T cell recovery and activation 24-48 hours after adding virus (exemplary, PWPT-ATC) volume (mL) = number of cells x MOI value (MOI = 20) / titer, 24-96 hours after infection using the method described in Reference Example 1 The described CRISPR/Cas9 technology uses electroporation to knock out TCR α chain and β chain, the target sequence of gRNA on TRAC is shown in SEQ ID NO: 25, and the target sequence of gRNA on TRBC is shown in SEQ ID NO: 28. The ATC positivity rate and TCR knockout efficiency were then detected on days 5-8 after T cell activation. The ATC positive rate detection reagent is 5ug/ml biotinylated human GPC3 protein, and then added with SA-PE fluorescent antibody (eBioscience) at a 1:300 dilution for labeling; the knockout efficiency is reflected by the expression of CD3 on the cell surface, using Anti-CD3- APC (Invitrogen) was used for labeling; finally detected by flow cytometry. UT group was uninfected virus T cells. UT ko was a T cell that was not transduced with ATC vector but knocked out endogenous TCR α and β chains.
结果如图2所示，UT ko组TCR敲除效率达到94.6％；ATCT组ATC阳性率达到68.5％；ATCT未敲除内源性TCR(ATCT w/o ko)组，由于存在内源性TCR的错配，ATCT阳性率为15.0％。作为对照的CAR-T组为anti-GPC3-CAR T细胞(按照常规CAR-T细胞制备方法制备，CAR的序列参见SEQ ID NO：47)，未敲除内源性TCR，其CAR阳性率为91.8％。The results are shown in Figure 2. The TCR knockout efficiency in the UT ko group reached 94.6%; the ATC positive rate in the ATCT group reached 68.5%; the ATCT did not knock out the endogenous TCR (ATCT w/o ko) group, due to the presence of endogenous TCR mismatch, the ATCT positive rate was 15.0%. The CAR-T group used as a control was anti-GPC3-CAR T cells (prepared according to the conventional CAR-T cell preparation method, the sequence of CAR is shown in SEQ ID NO: 47), and the endogenous TCR was not knocked out, and its CAR positive rate was 91.8%.
为了进一步证明在ATCT细胞中，ATC结构是否能与内源性CD3亚基整合表达于T细胞表面，形成完整的ATC/CD3复合体，我们采用免疫共沉淀方法进行了验证。收集慢病毒感染后的ATCT细胞，使用蛋白裂解液将细胞裂解后，采用生物素标记的GPC3抗原，链霉亲和素标记的磁珠将ATC及其结合蛋白免疫共沉淀后，进行蛋白质印迹实验，采用相应的抗体检测不同CD3亚基的表达。结果显示，ATC能与CD3ζ、CD3γ、CD3δ和CD3ε形成复合物。In order to further prove whether the ATC structure can integrate with the endogenous CD3 subunit and express on the surface of T cells in ATCT cells to form a complete ATC/CD3 complex, we used co-immunoprecipitation method to verify it. The ATCT cells infected with lentivirus were collected, and the cells were lysed with a protein lysis solution. The ATC and its binding proteins were co-immunoprecipitated with biotin-labeled GPC3 antigen and streptavidin-labeled magnetic beads, and then the western blotting experiment was performed. , and the corresponding antibodies were used to detect the expression of different CD3 subunits. The results showed that ATC could form complexes with CD3ζ, CD3γ, CD3δ and CD3ε.
另外，采用人Claudin18.2蛋白及相应对照进行了上述实验，获得了类似的结果。In addition, the above experiments were carried out using human Claudin18.2 protein and corresponding controls, and similar results were obtained.
实施例4.ATCT细胞对靶细胞的杀伤效果Example 4. Killing effect of ATCT cells on target cells
对于GPC3，对实施例3构建的ATCT、CAR-T细胞进行肿瘤细胞杀伤试验。For GPC3, tumor cell killing test was performed on ATCT and CAR-T cells constructed in Example 3.
靶细胞：表达GPC3的肝癌细胞Huh7(中国科学院细胞库)、不表达GPC3的肝癌细胞SK-hep-1(ATCC)Target cells: GPC3-expressing liver cancer cell Huh7 (Cell Bank of Chinese Academy of Sciences), liver cancer cell SK-hep-1 (ATCC) not expressing GPC3
效应细胞：参照实施例3制备的ATCT细胞、CAR-T细胞、UT细胞Effector cells: ATCT cells, CAR-T cells, UT cells prepared with reference to Example 3
首先将靶细胞分别用AIM-V培养基(AIM-V+2％ABS)调整密度至0.2x10 6/mL，96孔细胞培养板每孔加入10000个靶细胞，然后根据效靶比(E/T)3:1、1:1和1:3分别加入效应细胞，37度共孵育16小时后，取上清采用LDH试剂盒进行显色，最后用酶标仪进行OD490读数和数据分析。 First, the target cells were adjusted to a density of 0.2x10 6 /mL with AIM-V medium (AIM-V+2%ABS), and 10,000 target cells were added to each well of a 96-well cell culture plate. T) 3:1, 1:1 and 1:3 were added to effector cells respectively, and after co-incubating at 37 degrees for 16 hours, the supernatant was taken for color development with LDH kit, and finally OD490 reading and data analysis were carried out with a microplate reader.
结果如图3所示，ATCT组杀伤效果与CAR-T组较为一致：特异性杀伤表达GPC3的Huh7细胞，杀伤效率最高达到86.7％，且具有浓度依赖特性，而对无GPC3表达的SK-hep-1细胞则没有杀伤作用。The results are shown in Figure 3. The killing effect of the ATCT group was more consistent with that of the CAR-T group: it specifically killed Huh7 cells expressing GPC3, with the highest killing efficiency of 86.7%, and had a concentration-dependent characteristic, while the killing effect of SK-hep without GPC3 expression was the same as that of the CAR-T group. -1 cells have no killing effect.
另外，采用表达或不表达Claudin18.2的靶细胞进行了上述实验，获得了类似的结果。In addition, the above experiments were performed with target cells expressing or not expressing Claudin18.2, and similar results were obtained.
实施例5.ATCT细胞与靶细胞共孵育后IFN-γ分泌情况检测Example 5. Detection of IFN-γ secretion after co-incubation of ATCT cells and target cells
对于GPC3，将实施例4中效应细胞与靶细胞共孵育16小时后的上清进行细胞因子IFN-γ检测。实验采用Biolegend的检测试剂盒，按照试剂盒说明书的步骤进行。For GPC3, cytokine IFN-γ was detected in the supernatant after co-incubating effector cells with target cells in Example 4 for 16 hours. Experiments were carried out using Biolegend's detection kit according to the instructions of the kit.
结果如图4显示，ATCT与CAR-T细胞与表达GPC3的肝癌细胞Huh7共孵育后，IFN-γ分泌明显上调，显著高于UT组。当E/T＝1:1和1:3时，ATCT组分泌的IFN-γ低于CAR-T组；E/T＝3:1时，ATCT组分泌的IFN-γ略高于CAR-T组。The results are shown in Figure 4. After ATCT and CAR-T cells were co-incubated with GPC3-expressing liver cancer cells Huh7, the secretion of IFN-γ was significantly up-regulated, which was significantly higher than that in the UT group. When E/T=1:1 and 1:3, the IFN-γ secreted by the ATCT group was lower than that of the CAR-T group; when E/T=3:1, the IFN-γ secreted by the ATCT group was slightly higher than that of the CAR-T group Group.
此外，采用表达或不表达Claudin18.2的靶细胞进行了上述实验，获得了类似的结果。In addition, the above experiments were performed with target cells expressing or not expressing Claudin18.2, and similar results were obtained.
实施例6.ATCT细胞与靶细胞共孵育后CD25和CD69表达情况Example 6. Expression of CD25 and CD69 after ATCT cells co-incubated with target cells
对于GPC3，效应细胞(参照实施例3制备的ATCT、CAR-T细胞、UT细胞)与靶细胞(Huh7、SK-hep-1)按照效靶比为3:1进行共孵育，共孵育16小时后取细胞进行流式检测。实验将细胞分两组分别加入CD69-PE/CD3-APC(Invitrogen)和CD25-APC/CD3-FITC(Invitrogen)进行孵育，4度孵育45分钟后用流式细胞仪检测。For GPC3, effector cells (ATCT, CAR-T cells, and UT cells prepared in Example 3) were co-incubated with target cells (Huh7, SK-hep-1) at an effector-target ratio of 3:1 for 16 hours. Cells were then taken for flow cytometry. In the experiment, the cells were divided into two groups and respectively added to CD69-PE/CD3-APC (Invitrogen) and CD25-APC/CD3-FITC (Invitrogen) for incubation, and were detected by flow cytometry after incubation at 4 degrees for 45 minutes.
结果如图5所示，相较于肿瘤抗原GPC3表达阴性的SK-hep-1细胞，ATCT细胞与肿瘤抗原GPC3表达阳性的Huh7细胞共孵育后，细胞表面CD25和CD69的表达水平有明显提高，且相较于CAR-T细胞偏移更加明显，表明ATCT细胞受肿瘤抗原刺激后细胞 激活水平更高。The results are shown in Figure 5. Compared with SK-hep-1 cells with negative tumor antigen GPC3 expression, after co-incubating ATCT cells with tumor antigen GPC3 positive Huh7 cells, the expression levels of CD25 and CD69 on the cell surface were significantly increased. Compared with CAR-T cells, the offset is more obvious, indicating that ATCT cells have a higher level of cell activation after being stimulated by tumor antigens.
另外，采用表达或不表达Claudin18.2的靶细胞进行了上述实验，获得了类似的结果。In addition, the above experiments were performed with target cells expressing or not expressing Claudin18.2, and similar results were obtained.
实施例7.ATCT细胞分化情况检测Example 7. Detection of ATCT cell differentiation
对于GPC3，实施例3制备的ATCT细胞、CAR-T细胞进行分化指标检测，细胞分成4组分别与GranB-488、CCR7-FITC、CD45RA-FITC、CD45RO-APC抗体(BD Biosciences)共孵育，4度孵育45分钟后用流式细胞仪检测。For GPC3, the differentiation indicators of ATCT cells and CAR-T cells prepared in Example 3 were detected. The cells were divided into 4 groups and incubated with GranB-488, CCR7-FITC, CD45RA-FITC, and CD45RO-APC antibodies (BD Biosciences), respectively. After 45 minutes of incubation, the cells were detected by flow cytometry.
结果如图6所示，CAR-T细胞低表达CCR7和CD45RA以及高表达CD45RO，表明CAR-T组较ATCT和UT组分化程度更高，而ATCT细胞分化程度相对较低，原始T细胞更多。The results are shown in Figure 6. The CAR-T cells have low expression of CCR7 and CD45RA and high expression of CD45RO, indicating that the CAR-T group has a higher degree of differentiation than the ATCT and UT groups, while the ATCT cells have a relatively lower degree of differentiation and more primitive T cells. .
对于Clauding18.2，同样进行了上述实验，获得了类似的结果。For Clauding18.2, the above experiments were also carried out, and similar results were obtained.
实施例8.ATCT细胞CD4/CD8分型检测Example 8. Detection of CD4/CD8 typing of ATCT cells
对于GPC3，实施例3制备的ATCT细胞、CAR-T细胞进行CD4/CD8分型检测，抗体CD4-PE/CD8-APC(BD Biosciences)与细胞4度共孵育45分钟后用流式细胞仪检测。For GPC3, the ATCT cells and CAR-T cells prepared in Example 3 were subjected to CD4/CD8 typing detection, and the antibody CD4-PE/CD8-APC (BD Biosciences) was incubated with cells at 4 degrees for 45 minutes and detected by flow cytometry .
结果如图7所示，CAR-T组在病毒转导后一直保持较高比例的CD8+T细胞(大于85％)；而ATCT组CD8+T细胞、CD4+T细胞变化趋势与UT组一致。这说明ATCT细胞表型与未转染T细胞更相似，更接近天然状态。The results are shown in Figure 7. The CAR-T group maintained a high proportion of CD8+ T cells (greater than 85%) after viral transduction; while the changes of CD8+ T cells and CD4+ T cells in the ATCT group were consistent with those in the UT group. . This indicates that the ATCT cell phenotype is more similar to untransfected T cells and closer to the natural state.
对于Clauding18.2，同样进行了上述实验，获得了类似的结果。For Clauding18.2, the above experiments were also carried out, and similar results were obtained.
实施例9.ATCT细胞耗竭情况检测Example 9. Detection of ATCT cell exhaustion
对于GPC3，实施例3制备的细胞进行耗竭情况检测。细胞共分为三组，均先与抗原为5ug/ml生物素化的人GPC3蛋白共孵育，然后二抗均加入SA-APC-Cy7(BD Biosciences)进行阳性细胞检测，与二抗孵育的同时均加入CD4-BV510/CD8-APC(BD Biosciences)抗体进行共孵育，且与此同时三组细胞分别加入PD-1-BV421(BD Biosciences)、TIM-3-PE(BD Biosciences)和LAG-3-BV421(BD Biosciences)抗体在4度共孵育，45分钟后用流式细胞仪检测。For GPC3, the cells prepared in Example 3 were tested for depletion. The cells were divided into three groups, and they were first incubated with human GPC3 protein whose antigen was biotinylated at 5ug/ml, and then the secondary antibody was added with SA-APC-Cy7 (BD Biosciences) to detect positive cells. CD4-BV510/CD8-APC (BD Biosciences) antibodies were added for co-incubation, and at the same time, PD-1-BV421 (BD Biosciences), TIM-3-PE (BD Biosciences) and LAG-3 were added to the three groups of cells respectively. -BV421 (BD Biosciences) antibody was co-incubated at 4 degrees and detected by flow cytometry after 45 minutes.
结果如图8所示，CAR-T细胞表面PD-1/TIM-3/LAG-3表达有明显提高，ATCT组与UT组相似且表达水平较低。结果表明ATCT细胞的耗竭程度要低于CAR-T组，与未转染T细胞更相似。The results are shown in Figure 8, the expression of PD-1/TIM-3/LAG-3 on the surface of CAR-T cells was significantly increased, and the ATCT group was similar to the UT group with a lower expression level. The results showed that the depletion degree of ATCT cells was lower than that of the CAR-T group, which was more similar to that of untransfected T cells.
对于Clauding18.2，同样进行了上述实验，获得了类似的结果。For Clauding18.2, the above experiments were also carried out, and similar results were obtained.
实施例10.ATCT细胞增殖情况检测Example 10. Detection of ATCT cell proliferation
对于GPC3，分别取实施例3制备的1×10 6的CAR-T、ATCT、UT细胞在相同条件下培养(AIM-V+2％ABS+300U/ml IL-2)，之后每两天进行一次计数，检测细胞扩增情况。 For GPC3, 1×10 6 CAR-T, ATCT, and UT cells prepared in Example 3 were cultured under the same conditions (AIM-V+2%ABS+300U/ml IL-2), and then every two days. One count to detect cell expansion.
结果如图9所示，ATCT、CAR-T和UT组在T细胞活化10天后的细胞增殖情况相似，无明显区别。这说明ATCT细胞的体外增殖与未转染T细胞没有明显差异，接近天然状态。The results are shown in Figure 9. The cell proliferation of ATCT, CAR-T and UT groups after 10 days of T cell activation was similar, and there was no significant difference. This indicated that the in vitro proliferation of ATCT cells was not significantly different from that of untransfected T cells, and was close to the natural state.
对于Clauding18.2，同样进行了上述实验，获得了类似的结果。For Clauding18.2, the above experiments were also carried out, and similar results were obtained.
本发明所述实施例包括将该实施例作为任何单一实施例或与任何其他实施例或其部分相结合。此外应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明进行各种改动或修改，这些经过改动或修改的等价形式同样落入本申请所附权利要求书所限定的范围。The described embodiments of the invention include that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. In addition, it should be understood that after reading the above-mentioned content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms of these changes or modifications also fall within the scope of the appended claims of the application. scope.
序列信息sequence information
Figure PCTCN2022086816-appb-000004
Figure PCTCN2022086816-appb-000004
Figure PCTCN2022086816-appb-000005
Figure PCTCN2022086816-appb-000005
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Claims (40)

  1. 一种工程化细胞，其特征在于，所述工程化细胞表达T细胞受体(TCR)嵌合物(ATC)，所述ATC包含：An engineered cell, characterized in that the engineered cell expresses a T cell receptor (TCR) chimera (ATC), and the ATC comprises:
    (a)识别抗原的抗原识别单元，和(a) an antigen recognition unit that recognizes the antigen, and
    (b)核苷酸序列同义突变的TCR亚基恒定区；(b) a TCR subunit constant region with a synonymous mutation in the nucleotide sequence;
    所述工程化细胞的内源性TCR亚基的表达、活性和/或信号传导被降低或抑制，而所述ATC的表达、活性和/或信号传导不被降低或抑制。The expression, activity and/or signaling of the endogenous TCR subunit of the engineered cell is reduced or inhibited, while the expression, activity and/or signaling of the ATC is not reduced or inhibited.
  2. 如权利要求1所述的工程化细胞，其特征在于，相对于野生型TRAC核酸分子、TRBC核酸分子、TRGC核酸分子和/或TRDC核酸分子，所述ATC的TCR亚基恒定区的核酸分子有同义突变。The engineered cell of claim 1, wherein, relative to wild-type TRAC nucleic acid molecule, TRBC nucleic acid molecule, TRGC nucleic acid molecule and/or TRDC nucleic acid molecule, the nucleic acid molecule in the constant region of the TCR subunit of the ATC has Synonymous mutation.
  3. 如权利要求1或2所述的工程化细胞，其特征在于，所述ATC中的TCR亚基包括天然和/或修饰的TCRα链恒定区(TRAC)和β链恒定区(TRBC)；或包括天然和/或修饰的TCRγ链恒定区(TRGC)和δ链恒定区(TRDC)。The engineered cell of claim 1 or 2, wherein the TCR subunits in the ATC comprise native and/or modified TCR α chain constant region (TRAC) and β chain constant region (TRBC); or include Native and/or modified TCR gamma chain constant region (TRGC) and delta chain constant region (TRDC).
  4. 如权利要求1-3任一所述的工程化细胞，其特征在于，所述ATC的抗原识别单元包括一种或两种抗体；或所述ATC的抗原识别单元包括抗体重链可变区(VH)和/或轻链可变区(VL)。The engineered cell according to any one of claims 1-3, wherein the antigen recognition unit of the ATC comprises one or two antibodies; or the antigen recognition unit of the ATC comprises an antibody heavy chain variable region ( VH) and/or light chain variable region (VL).
  5. 如权利要求4所述的工程化细胞，其特征在于，所述ATC中的TRAC肽与VH直接连接或通过铰链区连接、TRBC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRAC肽与VL直接连接或通过铰链区连接、所述TRBC肽与VH直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VH直接连接或通过铰链区连接、TRGC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VL直接连接或通过铰链区连接、所述TRGC肽与VH直接连接或通过铰链区连接。The engineered cell of claim 4, wherein the TRAC peptide in the ATC is directly connected to VH or connected through a hinge region, the TRBC peptide is directly connected to VL or connected through a hinge region; or the ATC in The TRAC peptide is directly linked to the VL or through the hinge region, the TRBC peptide is directly linked to the VH or linked through the hinge region; or the TRDC peptide in the ATC is directly linked to the VH or is linked through the hinge region, the TRGC peptide is directly linked to the VL or through the hinge region; or the TRDC peptide in the ATC is directly linked to the VL or linked through the hinge region, the TRGC peptide is directly linked to the VH or linked through the hinge region.
  6. 如权利要求1-5任一所述的工程化细胞，其特征在于，所述ATC与抗原结合后能激活与所述ATC缔合的CD3分子。The engineered cell of any one of claims 1-5, wherein the ATC can activate the CD3 molecule associated with the ATC after binding to the antigen.
  7. 如权利要求1-6任一所述的工程化细胞，其特征在于，所述内源性TCR表达被降低或抑制是通过使用基因敲除技术和/或基因沉默技术包括：TALE核酸酶、巨核酸酶、锌指核酸酶、CRISPR/Cas9、Argonaute、引导编辑技术、归巢核酸内切酶技术或其组合。The engineered cell of any one of claims 1-6, wherein the endogenous TCR expression is reduced or inhibited by using gene knockout technology and/or gene silencing technology including: TALE nuclease, giant Nuclease, zinc finger nuclease, CRISPR/Cas9, Argonaute, guide editing technology, homing endonuclease technology, or a combination thereof.
  8. 如权利要求1-7任一所述的工程化细胞，其特征在于，所述ATC不包含基因敲除技术和/或基因沉默技术所靶向的核酸序列。The engineered cell of any one of claims 1-7, wherein the ATC does not comprise a nucleic acid sequence targeted by gene knockout technology and/or gene silencing technology.
  9. 如权利要求1-8任一所述的工程化细胞，其特征在于，所述ATC的核酸分子包含碱基同义突变后不再是基因敲除技术和/或基因沉默技术所针对的靶序列的核酸分子。The engineered cell according to any one of claims 1-8, wherein the nucleic acid molecule of the ATC is no longer a target sequence targeted by a gene knockout technology and/or a gene silencing technology after the nucleic acid molecule of the ATC contains a base synonymous mutation nucleic acid molecules.
  10. 如权利要求1-9任一所述的工程化细胞，其特征在于，所述ATC不包括gRNA靶序列。The engineered cell of any one of claims 1-9, wherein the ATC does not include a gRNA target sequence.
  11. 如权利要求10所述的工程化细胞，其特征在于，所述工程化细胞包含gRNA,序列分别如SEQ ID NO：25、28、33、34、35、36、37、38、39、40、41、44或其组合所示；或包含gRNA，序列分别如SEQ ID NO：25和SEQ ID NO：28所示；或包含gRNA，序列分别如SEQ ID NO：41和44所示。The engineered cell of claim 10, wherein the engineered cell comprises gRNA, the sequences of which are respectively as SEQ ID NOs: 25, 28, 33, 34, 35, 36, 37, 38, 39, 40, 41, 44 or a combination thereof; or a gRNA, the sequence of which is shown in SEQ ID NO: 25 and SEQ ID NO: 28, respectively; or a gRNA, the sequence of which is shown in SEQ ID NO: 41 and 44, respectively.
  12. 如权利要求1-11任一所述的工程化细胞，其特征在于，所述ATC包含SEQ ID NO：7、8、10或12所示的核苷酸序列，和SEQ ID NO：16或17所示的核苷酸序列；或所述ATC包含SEQ ID NO：5、9、11或13所示的氨基酸序列，和SEQ ID NO：14或18所示的氨基酸序列；或所述ATC包含SEQ ID NO：21和SEQ ID NO：24所示的核苷酸序列；或所述ATC包含SEQ ID NO：20和SEQ ID NO：23所示的氨基酸序列。The engineered cell of any one of claims 1-11, wherein the ATC comprises the nucleotide sequence shown in SEQ ID NO: 7, 8, 10 or 12, and the nucleotide sequence shown in SEQ ID NO: 16 or 17 The nucleotide sequence shown; or the ATC comprises the amino acid sequence shown in SEQ ID NO: 5, 9, 11 or 13, and the amino acid sequence shown in SEQ ID NO: 14 or 18; or the ATC comprises the SEQ ID NO: 14 or 18 The nucleotide sequences shown in ID NO: 21 and SEQ ID NO: 24; or the ATC comprises the amino acid sequences shown in SEQ ID NO: 20 and SEQ ID NO: 23.
  13. 如权利要求1-12任一所述的工程化细胞，其特征在于，所述工程化细胞选自T细胞、细胞毒性T淋巴细胞(CTL)、调节性T细胞、NK细胞、NKT细胞、人胚胎干细胞、和可从中分化出淋巴样细胞的多能干细胞。The engineered cell according to any one of claims 1-12, wherein the engineered cell is selected from the group consisting of T cells, cytotoxic T lymphocytes (CTL), regulatory T cells, NK cells, NKT cells, human Embryonic stem cells, and pluripotent stem cells from which lymphoid cells can be differentiated.
  14. 如权利要求1-13任一所述的工程化细胞，其特征在于，所述工程化细胞是自体或同种异体细胞。The engineered cell of any one of claims 1-13, wherein the engineered cell is an autologous or allogeneic cell.
  15. 如权利要求1-14任一所述的工程化细胞，其特征在于，所述抗原为肿瘤抗原和/或病原体抗原；The engineered cell of any one of claims 1-14, wherein the antigen is a tumor antigen and/or a pathogen antigen;
    优选地，所述抗原为肿瘤抗原；Preferably, the antigen is a tumor antigen;
    优选地，所述抗原为实体瘤抗原；Preferably, the antigen is a solid tumor antigen;
    优选地，所述抗原为GPC3、EGFR、Claudin18.2、BCMA、间皮素、CD19。Preferably, the antigen is GPC3, EGFR, Claudin18.2, BCMA, mesothelin, CD19.
  16. 如权利要求1-15任一所述的工程化细胞，其特征在于，所述抗原识别单元包含识别GPC3的抗原识别单元的氨基酸序列的VL选自SEQ ID NO：3、49、51、53、55、57、59、61、63或65或与之具有70-100％的序列同一性，和/或识别GPC3的抗原识别单元的氨基酸序列的VH选自SEQ ID NO：1、48、50、52、54、56、58、60、62或64或与之具有70-100％的序列同一性，或者，The engineered cell according to any one of claims 1-15, wherein the VL of the antigen recognition unit comprising the amino acid sequence of the antigen recognition unit recognizing GPC3 is selected from SEQ ID NOs: 3, 49, 51, 53, 55, 57, 59, 61, 63 or 65 or have 70-100% sequence identity therewith, and/or the VH that recognizes the amino acid sequence of the antigen recognition unit of GPC3 is selected from SEQ ID NO: 1, 48, 50, 52, 54, 56, 58, 60, 62 or 64 or have 70-100% sequence identity therewith, or,
    识别Claudin18.2的抗原识别单元的氨基酸序列的VL选自SEQ ID NO：67、69、71、73、75、77、79、81、83或85或与之具有70-100％的序列同一性，和/或识别Claudin18.2的抗原识别单元的氨基酸序列的VH选自SEQ ID NO：66、68、70、72、74、76、78、80、82或84或与之具有70-100％的序列同一性。The VL recognizing the amino acid sequence of the antigen recognition unit of Claudin18.2 is selected from or has 70-100% sequence identity with SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85 , and/or the VH that recognizes the amino acid sequence of the antigen recognition unit of Claudin18.2 is selected from or has 70-100% sequence identity.
  17. 如权利要求1-16任一所述的工程化细胞，其特征在于，所述工程化细胞具有以下特点之一或其组合：The engineered cell according to any one of claims 1-16, wherein the engineered cell has one of the following characteristics or a combination thereof:
    1)能杀伤携带所述抗原的靶细胞；1) can kill target cells carrying the antigen;
    2)与所述靶细胞孵育后分泌IFN；2) secrete IFN after incubation with the target cells;
    3)与所述靶细胞孵育后CD25、CD69阳性率提高；3) The positive rates of CD25 and CD69 are increased after incubation with the target cells;
    4)CD4+、CD8+阳性率与未转导编码ATC的多核苷酸片段的细胞接近；4) The positive rates of CD4+ and CD8+ are close to those of cells that have not been transduced with polynucleotide fragments encoding ATC;
    5)原始T细胞比例大；和/或5) A large proportion of naive T cells; and/or
    6)PD-1/TIM-3/LAG-3阳性率与未转导编码ATC的多核苷酸片段的细胞接近。6) The positive rate of PD-1/TIM-3/LAG-3 was close to that of cells not transduced with polynucleotide fragments encoding ATC.
  18. 如权利要求1-17任一所述的工程化细胞，其特征在于，与表达相同抗原识别单元的嵌合抗原受体的CAR的细胞相比，所述工程化细胞具有以下之一或其组合：The engineered cell of any one of claims 1-17, wherein the engineered cell has one of the following or a combination thereof compared to a cell expressing a CAR of a chimeric antigen receptor of the same antigen recognition unit :
    1)对靶细胞的杀伤能力、和/或与靶细胞孵育后分泌IFN-γ、细胞增殖没有显著差异；与靶细胞孵育后CD25、CD69表达水平高；1) There was no significant difference in the killing ability of target cells, and/or the secretion of IFN-γ and cell proliferation after incubation with target cells; the expression levels of CD25 and CD69 were high after incubation with target cells;
    2)原始T细胞比例大；2) The proportion of primitive T cells is large;
    3)CD4+/CD8+比例高；3) The ratio of CD4+/CD8+ is high;
    4)PD-1/TIM-3/LAG-3阳性率低。4) The positive rate of PD-1/TIM-3/LAG-3 is low.
  19. 如权利要求1-18任一所述的工程化细胞，其特征在于，与表达相同ATC但内源性TCR亚基的表达、活性和/或信号传导没有被降低或抑制的细胞相比，所述工程化细胞ATC阳性率提高或提高约5％、10％、20％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％。19. The engineered cell of any one of claims 1-18, wherein compared to a cell expressing the same ATC but not having reduced or inhibited expression, activity and/or signaling of endogenous TCR subunits The ATC positive rate of the engineered cells is increased or increased by about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.
  20. 如权利要求1-19任一所述工程化细胞中的ATC分子。The ATC molecule in the engineered cell of any of claims 1-19.
  21. 一种多核苷酸，其编码权利要求1-19任一所述的工程化细胞中的ATC或gRNA分子。A polynucleotide encoding the ATC or gRNA molecule in the engineered cell of any one of claims 1-19.
  22. 一种载体，其包含权利要求21所述的多核苷酸。A vector comprising the polynucleotide of claim 21.
  23. 一种药物组合物，其包含有效量的权利要求1-19任一所述的工程化细胞、权利要求20所述的ATC分子、权利要求21所述的多核苷酸或者权利要求22所述的载体和药学上可接受的赋形剂。A pharmaceutical composition comprising an effective amount of the engineered cell of any one of claims 1-19, the ATC molecule of claim 20, the polynucleotide of claim 21 or the polynucleotide of claim 22 Carriers and pharmaceutically acceptable excipients.
  24. 如权利要求23所述的药物组合物，其用于治疗肿瘤。The pharmaceutical composition of claim 23, which is used for the treatment of tumors.
  25. 一种试剂盒，其包含权利要求1-19任一所述的工程化细胞、权利要求20所述的ATC分子、权利要求21所述的多核苷酸、权利要求22所述的载体或者权利要求23或24所述的药物组合物。A kit comprising the engineered cell of any one of claims 1-19, the ATC molecule of claim 20, the polynucleotide of claim 21, the carrier of claim 22 or the claim The pharmaceutical composition of 23 or 24.
  26. 如权利要求25所述的试剂盒，其还包括用于治疗和/或预防肿瘤、病原体感染、自身免疫性疾病或同种异体移植的书面说明书。The kit of claim 25, further comprising written instructions for treating and/or preventing tumors, pathogen infections, autoimmune diseases, or allografts.
  27. 一种降低受试者肿瘤负荷的方法，其特征在于，包括向所述受试者施用有效量的权利要求1-19任一所述的工程化细胞、权利要求23或24所述的药物组合物。A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of the engineered cell described in any one of claims 1-19, the pharmaceutical combination described in claim 23 or 24 thing.
  28. 一种治疗或预防受试者肿瘤的方法，其特征在于，包括向所述受试者施用有效量的权利要求1-19任一所述的工程化细胞、权利要求23或24所述的药物组合物。A method for treating or preventing tumor in a subject, comprising administering to the subject an effective amount of the engineered cell described in any one of claims 1-19, the medicine described in claim 23 or 24 combination.
  29. 如权利要求27或28所述的方法，其特征在于，所述肿瘤选自肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、胰腺癌、多发性骨髓瘤、血液肿瘤。The method of claim 27 or 28, wherein the tumor is selected from the group consisting of liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, pancreatic cancer, multiple myeloma, blood tumor.
  30. 如权利要求27、28或29所述的方法，其特征在于，所述肿瘤是GPC3阳性肿瘤、或Claudin18.2阳性肿瘤。The method of claim 27, 28 or 29, wherein the tumor is a GPC3-positive tumor, or a Claudin18.2-positive tumor.
  31. 一种产生抗原特异性免疫效应细胞的方法，其特征在于，包括将编码权利要求20所述的ATC分子的多核苷酸、或将权利要求21所述的多核苷酸、或将权利要求22所述的载体导入免疫效应细胞。A method for producing antigen-specific immune effector cells, characterized in that it comprises encoding the polynucleotide of the ATC molecule of claim 20, or the polynucleotide of claim 21, or the polynucleotide of claim 22. The vector described above is introduced into immune effector cells.
  32. 一种延长患有肿瘤的受试者存活的方法，其特征在，包括向所述受试者施用有效量的权利要求1-19任一所述的工程化细胞、权利要求23或24所述的药物组合物。A method for prolonging the survival of a subject suffering from a tumor, comprising administering to the subject an effective amount of the engineered cell described in any of claims 1-19, the described engineered cell of claim 23 or 24 pharmaceutical composition.
  33. 权利要求1-19任一所述的工程化细胞、权利要求20所述的ATC分子、权利要求21所述的多核苷酸、权利要求22所述的载体、权利要求23或24所述的药物组合物在治疗中的用途。The engineered cell of any one of claims 1-19, the ATC molecule of claim 20, the polynucleotide of claim 21, the vector of claim 22, the drug of claim 23 or 24 Use of the composition in therapy.
  34. 权利要求1-19任一所述的工程化细胞、权利要求20所述的ATC分子、权利要求21所述的多核苷酸、权利要求22所述的载体、权利要求23或24所述的药物组合物用于减轻受试者的肿瘤负荷的用途。The engineered cell of any one of claims 1-19, the ATC molecule of claim 20, the polynucleotide of claim 21, the vector of claim 22, the drug of claim 23 or 24 Use of a composition for reducing tumor burden in a subject.
  35. 权利要求1-19任一所述的工程化细胞、权利要求20所述的ATC分子、权利要求21所述的多核苷酸、权利要求22所述的载体、权利要求23或24所述的药物组合物用于治疗或预防受试者肿瘤的用途。The engineered cell of any one of claims 1-19, the ATC molecule of claim 20, the polynucleotide of claim 21, the vector of claim 22, the drug of claim 23 or 24 Use of a composition for treating or preventing a tumor in a subject.
  36. 权利要求1-19任一所述的工程化细胞、权利要求20所述的ATC分子、权利要求21所述的多核苷酸、权利要求22所述的载体、权利要求23或24所述的药物组合物用于延长患有肿瘤的受试者存活的用途。The engineered cell of any one of claims 1-19, the ATC molecule of claim 20, the polynucleotide of claim 21, the vector of claim 22, the drug of claim 23 or 24 Use of a composition for prolonging the survival of a subject with a tumor.
  37. 靶向GPC3的ATC，其特征在于，所述ATC包含识别GPC3的抗原识别单元和TRAC形成的多肽，和识别GPC3的抗原识别单元和TRBC形成的多肽；或所述ATC包含识别GPC3的抗原识别单元和TRDC形成的多肽，和识别GPC3的抗原识别单元和TRGC形成的多肽。An ATC targeting GPC3, characterized in that the ATC comprises an antigen recognition unit that recognizes GPC3 and a polypeptide formed by TRAC, and an antigen recognition unit that recognizes GPC3 and a polypeptide formed by TRBC; or the ATC comprises an antigen recognition unit that recognizes GPC3 A polypeptide formed with TRDC, and a polypeptide formed by an antigen recognition unit that recognizes GPC3 and TRGC.
  38. 如权利要求37所述的ATC，其特征在于，所述识别GPC3的抗原识别单元包括抗GPC3抗体的抗体重链可变区(VH)和/或轻链可变区(VL)：The ATC of claim 37, wherein the antigen recognition unit recognizing GPC3 comprises an antibody heavy chain variable region (VH) and/or a light chain variable region (VL) of an anti-GPC3 antibody:
    所述TRAC肽与VH直接连接或通过铰链区连接、TRBC肽与VL直接连接或通过铰链区连接；或所述TRAC肽与VL直接连接或通过铰链区连接、所述TRBC肽与VH直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VH直接连接或通过铰链区连接、TRGC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VL直接连接或通过铰链区连接、所述TRGC肽与VH直接连接或通过铰链区连接。The TRAC peptide is directly linked to the VH or linked through the hinge region, the TRBC peptide is directly linked to the VL or linked through the hinge region; or the TRAC peptide is directly linked to the VL or linked through the hinge region, the TRBC peptide is directly linked to the VH or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VH or through the hinge region, the TRGC peptide is directly attached to the VL or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VL or through the hinge Region linkage, the TRGC peptide is linked directly to the VH or via a hinge region.
  39. 靶向Claudin18.2的ATC，其特征在于，所述ATC包含识别Claudin18.2的抗原识别 单元和TRAC形成的多肽，和识别Claudin18.2的抗原识别单元和TRBC形成的多肽；或所述ATC包含识别Claudin18.2的抗原识别单元和TRDC形成的多肽，和识别Claudin18.2的抗原识别单元和TRGC形成的多肽。An ATC targeting Claudin18.2, characterized in that the ATC comprises a polypeptide formed by an antigen recognition unit that recognizes Claudin18.2 and TRAC, and a polypeptide formed by an antigen recognition unit that recognizes Claudin18.2 and TRBC; or the ATC comprises A polypeptide that recognizes the antigen recognition unit of Claudin18.2 and TRDC, and a polypeptide that recognizes the antigen recognition unit of Claudin18.2 and TRGC.
  40. 如权利要求39所述的ATC，其特征在于，所述识别Claudin18.2的抗原识别单元包括抗Claudin18.2抗体的抗体重链可变区(VH)和/或轻链可变区(VL)：The ATC of claim 39, wherein the antigen recognition unit recognizing Claudin18.2 comprises an antibody heavy chain variable region (VH) and/or light chain variable region (VL) of an anti-Claudin18.2 antibody :
    所述TRAC肽与VH直接连接或通过铰链区连接、TRBC肽与VL直接连接或通过铰链区连接；或所述TRAC肽与VL直接连接或通过铰链区连接、所述TRBC肽与VH直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VH直接连接或通过铰链区连接、TRGC肽与VL直接连接或通过铰链区连接；或所述ATC中的TRDC肽与VL直接连接或通过铰链区连接、所述TRGC肽与VH直接连接或通过铰链区连接。The TRAC peptide is directly linked to the VH or linked through the hinge region, the TRBC peptide is directly linked to the VL or linked through the hinge region; or the TRAC peptide is directly linked to the VL or linked through the hinge region, the TRBC peptide is directly linked to the VH or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VH or through the hinge region, the TRGC peptide is directly attached to the VL or through the hinge region; or the TRDC peptide in the ATC is directly attached to the VL or through the hinge Region linkage, the TRGC peptide is linked directly to the VH or via a hinge region.
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