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WO2022068870A1 - 靶向egfr的嵌合抗原受体 - Google Patents

靶向egfr的嵌合抗原受体 Download PDF

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WO2022068870A1
WO2022068870A1 PCT/CN2021/121678 CN2021121678W WO2022068870A1 WO 2022068870 A1 WO2022068870 A1 WO 2022068870A1 CN 2021121678 W CN2021121678 W CN 2021121678W WO 2022068870 A1 WO2022068870 A1 WO 2022068870A1
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seq
cells
car
cancer
egfr
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PCT/CN2021/121678
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English (en)
French (fr)
Inventor
王皓毅
徐蓓蕾
李娜
唐娜
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中国科学院动物研究所
北京干细胞与再生医学研究院
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Application filed by 中国科学院动物研究所, 北京干细胞与再生医学研究院 filed Critical 中国科学院动物研究所
Priority to JP2023520016A priority Critical patent/JP2023545681A/ja
Priority to CN202180067431.8A priority patent/CN116601176A/zh
Priority to EP21874525.5A priority patent/EP4223779A4/en
Priority to US18/029,400 priority patent/US20240307539A1/en
Publication of WO2022068870A1 publication Critical patent/WO2022068870A1/zh

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    • A61K40/10—Cellular immunotherapy characterised by the cell type used
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to a chimeric antigen receptor (CAR) targeting EGFR, a CAR-T cell comprising the CAR, and a method for preparing and using the same.
  • CAR chimeric antigen receptor
  • Human epidermal growth factor receptor also known as HER-1 or Erb-B1, and referred to herein as "EGFR"
  • EGFR Human epidermal growth factor receptor
  • HER-1 or Erb-B1 is a 170 kDa transmembrane receptor encoded by the c-erbB proto-oncogene and exhibits intrinsic amino acid kinase activity.
  • EGFR regulates a variety of cellular processes through tyrosine kinase-mediated signal transduction pathways, including, but not limited to, activation of signal transduction pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitogenesis, and metastasis.
  • EGFR gene copy number can promote the malignant transformation of normal cells and the metastasis of malignant tumors, and the signaling network of EGFR plays an important role in the formation and development of tumors.
  • Overexpression of EGFR has been reported in various human malignancies, including lung, pancreatic, colorectal, gastric, and breast cancers. And clinical research results show that the overexpression of EGFR is associated with poor prognosis of patients. EGFR has become a specific target for anti-tumor therapy.
  • Some drugs targeting EGFR have been approved for clinical treatment of human malignancies. These drugs are mainly divided into two categories: one is the monoclonal antibody drugs that block the extracellular domain of EGFR, such as Cetuximab (Cetuximab), Panitumumab (Panitumumab) and Nimotuzumab (Nimotuzumab); Another class is the inhibitors of small molecule tyrosine kinases targeting the intracellular region of EGFR, such as gefitinib, erlotinib, and afatinib. Although the safety and clinical efficacy of these drugs have been proven, in many cases, their anti-tumor effects are not as effective as expected. EGFR mutations, etc.
  • the invention provides an EGFR-targeting chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain specifically targeting EGFR, the extracellular antigen binding domain comprising a heavy chain variable region (VH) and light chain variable region (VL), wherein
  • CAR EGFR-targeting chimeric antigen receptor
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, and VH-CDR3 shown in SEQ ID NO: 3, and the VL comprises SEQ ID NO: VL-CDR1 shown in 4, VL-CDR2 shown in SEQ ID NO:5, VL-CDR3 shown in SEQ ID NO:6;
  • VH comprises VH-CDR1 shown in SEQ ID NO: 10, VH-CDR2 shown in SEQ ID NO: 11, VH-CDR3 shown in SEQ ID NO: 12, and the VL comprises SEQ ID NO: VL-CDR1 shown in 13, VL-CDR2 shown in SEQ ID NO: 14, VL-CDR3 shown in SEQ ID NO: 16;
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 19, VH-CDR2 shown in SEQ ID NO: 20, and VH-CDR3 shown in SEQ ID NO: 21, and the VL comprises SEQ ID NO: VL-CDR1 shown in 22, VL-CDR2 shown in SEQ ID NO:23, VL-CDR3 shown in SEQ ID NO:24;
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 28, VH-CDR2 shown in SEQ ID NO: 29, and VH-CDR3 shown in SEQ ID NO: 30, and the VL comprises SEQ ID NO: VL-CDR1 shown in 31, VL-CDR2 shown in SEQ ID NO:32, VL-CDR3 shown in SEQ ID NO:33;
  • the VH comprises VH-CDR1 shown in SEQ ID NO:37, VH-CDR2 shown in SEQ ID NO:38, VH-CDR3 shown in SEQ ID NO:39, and the VL comprises SEQ ID NO: VL-CDR1 shown in 40, VL-CDR2 shown in SEQ ID NO:41, VL-CDR3 shown in SEQ ID NO:42; or
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 46, VH-CDR2 shown in SEQ ID NO: 47, VH-CDR3 shown in SEQ ID NO: 48, and the VL comprises SEQ ID NO: VL-CDR1 shown in 49, VL-CDR2 shown in SEQ ID NO:50, VL-CDR3 shown in SEQ ID NO:51.
  • VH comprises the amino acid sequence shown in SEQ ID NO:7
  • VL comprises the amino acid sequence shown in SEQ ID NO:8;
  • VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • VL comprises the amino acid sequence shown in SEQ ID NO: 17;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:25
  • the VL comprises the amino acid sequence shown in SEQ ID NO:26;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:34
  • the VL comprises the amino acid sequence shown in SEQ ID NO:35;
  • VH comprises the amino acid sequence set forth in SEQ ID NO:43 and the VL comprises the amino acid sequence set forth in SEQ ID NO:44; or
  • VH comprises the amino acid sequence shown in SEQ ID NO:52
  • VL comprises the amino acid sequence shown in SEQ ID NO:53.
  • the extracellular antigen binding domain comprises a single-chain Fv fragment (scFv).
  • scFv single-chain Fv fragment
  • the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 18, 27, 36, 45 and 54.
  • the CAR further comprises an N-terminal CD8 ⁇ signal peptide
  • the CD8 ⁇ signal peptide comprises the amino acid sequence of SEQ ID NO:55.
  • the CAR further comprises a transmembrane domain, such as a CD8 ⁇ transmembrane domain, for example, the CD8 ⁇ transmembrane region comprises the amino acid sequence of SEQ ID NO:57.
  • the CAR further comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain, eg, the hinge region is a CD8 ⁇ hinge region, eg, the CD8 ⁇ hinge region comprises Amino acid sequence of SEQ ID NO:56.
  • the CAR further comprises a signal transduction domain, such as a CD3 ⁇ signal transduction domain, such as a CD3 ⁇ signal transduction domain comprising the amino acid sequence set forth in SEQ ID NO:59.
  • the CAR further comprises one or more costimulatory domains, eg, a 4-1BB costimulatory domain, eg, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:58 .
  • the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 60-65.
  • the present invention provides a therapeutic T cell comprising a CAR of the present invention.
  • TGF ⁇ receptor eg, TGFBRI, TGFBRII, TGFBRIII
  • the therapeutic T cells are capable of in vitro from about 0.2:1 to about 0.00625:1, eg, about 0.2:1, about 0.1:1, about 0.05:1, about 0.025:1, about 0.0125 EGFR-expressing tumor cells were specifically lysed at an effector-target ratio of about 0.00625:1.
  • the present invention provides use of the therapeutic T cells of the present invention in the manufacture of a medicament for the treatment of EGFR-related cancers.
  • the present invention provides a pharmaceutical composition for treating EGFR-related cancer in a subject, comprising a therapeutically effective amount of a therapeutic T cell of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating EGFR-related cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutic T cell of the present invention or a pharmaceutical composition of the present invention.
  • the method further comprises administering to the subject radiation and/or chemotherapy and/or additional tumor-targeted drugs and/or immunotherapy.
  • the EGFR-related cancer is selected from the group consisting of esophageal cancer, gastric cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer NSCLC), breast cancer, Cervical cancer, endometrial cancer, endometrial cancer, ovarian cancer, bladder cancer, head and neck cancer (including head and neck squamous cell carcinoma SCCHN), osteosarcoma, prostate cancer, neuroblastoma, kidney cancer, glioma, Glioblastoma and skin cancer (including epithelial cancer).
  • the invention provides a polynucleotide comprising a nucleotide sequence encoding a CAR of the invention.
  • the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 66-71.
  • the present invention provides expression constructs comprising a polynucleotide of the present invention operably linked to regulatory sequences.
  • the present invention provides a method for preparing the therapeutic T cells of the present invention, the method comprising the steps of:
  • the method further comprises the step of
  • TGF ⁇ receptors eg TGFBRI, TGFBRII, TGFBRIII
  • Figure 1 Schematic diagram of the target gene sequence of the anti-EGFR CAR lentiviral vector.
  • FIG. 1 In vivo functional comparison of six humanized anti-EGFR CAR-T cells.
  • A. Experimental procedure of NPG mice. Tumor cell inoculation amount: 2 ⁇ 10 6 / mouse, CAR-T cell injection dose: 1 ⁇ 10 7 / mouse, 50% CAR positive, iv: tail vein injection, five NPG mice in each group.
  • FIG. 1 TGF- ⁇ Receptor II knockout hu806 CAR-T cells.
  • FIG. 8 Detection of killing function of hu806 CAR-T cells knocked out by TGF- ⁇ Receptor II.
  • FIG. 9 In vivo experiments in mice.
  • A. NPG mice were injected with different doses of CAR-T cells through the tail vein, and the tumor body changed. and tumor changes after tumor re-inoculation. Five mice in each group were injected with a 50% positive rate of CAR-T.
  • B. The content of human CD3 in peripheral blood of mice in each experimental group and PBS group was detected by flow cytometry.
  • FIG. 10 Analysis experiment of huCD3 subtypes in mice.
  • A. Tumor changes after NPG mice were injected with CAR-T cells (1 ⁇ 10 7 cells/mouse, 50% CAR positive) via tail vein.
  • CAR-TT cells were prepared from the peripheral blood of #4 donors with strong in vivo expansion ability. Five mice per group.
  • B. The content of human CD3 in peripheral blood of mice in each experimental group and PBS group was detected by flow cytometry.
  • C. The proportion of each subtype of T cells in the peripheral blood of mice, detected by flow cytometry.
  • D. The ratio of huCD4 and huCD8 to huCD3 in peripheral blood of mice, detected by flow cytometry.
  • Peripheral blood was collected from two groups of mice on days 21, 28, 36 and 42 for analysis.
  • FIG. 11 In vivo therapeutic dose experiment of Hu806-TKO CAR-T cells in tumor-bearing NPG mice.
  • A. NPG mice were injected with different doses of CAR-T cells in the tail vein (respectively: 2 ⁇ 10 6 CAR+ cells/a, 1 ⁇ 10 6 CAR+ cells/a, 0.5 ⁇ 10 6 CAR+ cells/a, and 0.25 ⁇ 10 6 CAR+ cells/only), tumor changes. After the tumor was completely removed, the tumor body was inoculated again. 5 mice per group.
  • B. The content of human CD3 in peripheral blood of mice in each experimental group and control group was detected by flow cytometry.
  • FIG. 1 Off-target safety detection of Hu806 CAR-T cells.
  • A. Flow cytometry of lung squamous cell line CRL-5826, human primary epidermal fibroblast Fibroblast and leukemia cell line K562. Staining antibodies: anti-EGFR antibody-PE and 806 antibody-PE.
  • Figure 13 Correspondence diagram of amino acid sequence and nucleotide sequence of m806 scFv and CAR.
  • the term “and/or” covers all combinations of the items linked by the term, as if each combination had been individually listed herein.
  • “A and/or B” covers “A”, “A and B", and “B”.
  • “A, B and/or C” encompasses "A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and "A and B and C”.
  • the protein or nucleic acid may consist of the sequence or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid Glycosides, but still have the activity described in the present invention.
  • the methionine encoded by the initiation codon at the N-terminus of the polypeptide is retained in some practical situations (eg, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide.
  • the present invention provides an EGFR-targeting chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain specifically targeting EGFR, the extracellular antigen-binding domain comprising a heavy chain variable region (VH) and light chain variable region (VL), wherein
  • CAR EGFR-targeting chimeric antigen receptor
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, and VH-CDR3 shown in SEQ ID NO: 3, and the VL comprises SEQ ID NO: VL-CDR1 shown in 4, VL-CDR2 shown in SEQ ID NO:5, VL-CDR3 shown in SEQ ID NO:6;
  • VH comprises VH-CDR1 shown in SEQ ID NO: 10, VH-CDR2 shown in SEQ ID NO: 11, VH-CDR3 shown in SEQ ID NO: 12, and the VL comprises SEQ ID NO: VL-CDR1 shown in 13, VL-CDR2 shown in SEQ ID NO: 14, VL-CDR3 shown in SEQ ID NO: 16;
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 19, VH-CDR2 shown in SEQ ID NO: 20, and VH-CDR3 shown in SEQ ID NO: 21, and the VL comprises SEQ ID NO: VL-CDR1 shown in 22, VL-CDR2 shown in SEQ ID NO:23, VL-CDR3 shown in SEQ ID NO:24;
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 28, VH-CDR2 shown in SEQ ID NO: 29, and VH-CDR3 shown in SEQ ID NO: 30, and the VL comprises SEQ ID NO: VL-CDR1 shown in 31, VL-CDR2 shown in SEQ ID NO:32, VL-CDR3 shown in SEQ ID NO:33;
  • the VH comprises VH-CDR1 shown in SEQ ID NO:37, VH-CDR2 shown in SEQ ID NO:38, VH-CDR3 shown in SEQ ID NO:39, and the VL comprises SEQ ID NO: VL-CDR1 shown in 40, VL-CDR2 shown in SEQ ID NO:41, VL-CDR3 shown in SEQ ID NO:42; or
  • the VH comprises VH-CDR1 shown in SEQ ID NO: 46, VH-CDR2 shown in SEQ ID NO: 47, VH-CDR3 shown in SEQ ID NO: 48, and the VL comprises SEQ ID NO: VL-CDR1 shown in 49, VL-CDR2 shown in SEQ ID NO:50, VL-CDR3 shown in SEQ ID NO:51.
  • the extracellular antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • VH comprises the amino acid sequence shown in SEQ ID NO:7
  • VL comprises the amino acid sequence shown in SEQ ID NO:8;
  • VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • VL comprises the amino acid sequence shown in SEQ ID NO: 17;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:25
  • the VL comprises the amino acid sequence shown in SEQ ID NO:26;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:34
  • the VL comprises the amino acid sequence shown in SEQ ID NO:35;
  • VH comprises the amino acid sequence set forth in SEQ ID NO:43 and the VL comprises the amino acid sequence set forth in SEQ ID NO:44; or
  • VH comprises the amino acid sequence shown in SEQ ID NO:52
  • VL comprises the amino acid sequence shown in SEQ ID NO:53.
  • the extracellular antigen binding domain comprises a single-chain Fv fragment (scFv).
  • scFv single-chain Fv fragment
  • the VH and the VL are linked by a linker.
  • the linker is a flexible peptide linker.
  • the linker comprises the amino acid sequence set forth in SEQ ID NO:72.
  • the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 18, 27, 36, 45, and 54.
  • the CAR further comprises an N-terminal CD8 ⁇ signal peptide.
  • the CD8 ⁇ signal peptide comprises the amino acid sequence of SEQ ID NO:55.
  • the CAR further comprises a transmembrane domain, such as a CD8 ⁇ transmembrane domain or a CD28 transmembrane domain.
  • the CAR comprises a CD8 ⁇ transmembrane region.
  • the CD8 ⁇ transmembrane region comprises the amino acid sequence of SEQ ID NO:57.
  • the CAR further comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain, eg, the hinge region is a CD8 ⁇ hinge region.
  • the CD8 ⁇ hinge region comprises the amino acid sequence of SEQ ID NO:56.
  • the CAR further comprises a signal transduction domain, eg, a signal transduction domain useful for T cell activation, eg, selected from TCR ⁇ , FcR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5 , the signal transduction domains of CD22, CD79a, CD79b and CD66d.
  • the CAR comprises a CD3 ⁇ signaling domain, eg, the CD3 ⁇ signaling domain comprises the amino acid sequence of SEQ ID NO:59.
  • the CAR further comprises one or more costimulatory domains, eg, a costimulatory domain selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, and 4-1BBL.
  • the CAR further comprises a 4-1BB costimulatory domain.
  • the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:58.
  • the CAR comprises an extracellular antigen binding domain for EGFR, a CD8 ⁇ hinge region, a CD8 ⁇ transmembrane region, a 4-1BB costimulatory domain, and a CD3 ⁇ signaling domain, and optionally an N-terminus CD8 ⁇ signal peptide.
  • the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 60-65.
  • the present invention provides a polynucleotide comprising a nucleotide sequence encoding a CAR of the present invention.
  • the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 66-71.
  • the present invention provides expression constructs comprising a polynucleotide of the present invention operably linked to regulatory sequences.
  • “Expression constructs” of the present invention may be linear nucleic acid fragments, circular plasmids, viral vectors, or may be RNA capable of translation (eg, mRNA).
  • the expression construct is a viral vector, such as a lentiviral vector.
  • regulatory sequence and “regulatory element” are used interchangeably and refer to a coding sequence upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) and affecting transcription, RNA processing or Stability or translated nucleotide sequence.
  • An expression regulatory element refers to a nucleotide sequence capable of controlling the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences.
  • operably linked refers to regulatory elements (eg, but not limited to, promoter sequences, transcription termination sequences, etc.) are linked to a nucleic acid sequence (eg, a coding sequence or open reading frame) such that nucleotides Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements.
  • a nucleic acid sequence eg, a coding sequence or open reading frame
  • the present invention provides a therapeutic T cell comprising the CAR of the present invention.
  • the CAR is expressed on the cell membrane surface of the T cell.
  • the TGF ⁇ receptor (eg, TGFBRI, TGFBRII, TGFBRIII) in the therapeutic T cell is knocked down or knocked out.
  • a TGF ⁇ receptor eg, TGFBRI, TGFBRII, TGFBRIII
  • TGF ⁇ receptor eg, TGFBRI, TGFBRII, TGFBRIII
  • the expression of TGFBRI, TGFBRII, TGFBRIII is down-regulated or not expressed, or the activity of TGF[beta] receptors (eg, TGFBRI, TGFBRII, TGFBRIII) is reduced or inactivated (eg, antagonized).
  • Knockdown or knockout as used herein can be at the genomic level, the transcriptional level, the translational level or the post-translational level.
  • the therapeutic T cells are derived from autologous cells of the subject.
  • autologous means that a cell, cell line, or population of cells used to treat a subject is derived from the subject.
  • the therapeutic T cells are derived from allogeneic cells, eg, from a donor subject compatible with the subject's human leukocyte antigen (HLA). Cells from a donor subject can be transformed into non-aloreactive cells using standard protocols and replicated as needed, resulting in cells that can be administered to one or more subjects.
  • HLA human leukocyte antigen
  • the T cells are derived from a healthy subject. In some embodiments, the T cells are derived from a subject with cancer.
  • T cells in the context of the present invention may be derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, and/or helper T lymphocytes.
  • T cells in the context of the present invention may be derived from CD4+ T lymphocytes and/or CD8+ T lymphocytes.
  • T cells in the context of the present invention can be obtained by various non-limiting methods from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, ascites, pleural effusion, spleen tissue, and tumors .
  • T cells in the context of the present invention may also be part of a mixed population of cells exhibiting different phenotypic characteristics.
  • the therapeutic T cells of the present invention can specifically lyse EGFR-expressing tumor cells in vitro.
  • the therapeutic T cells of the present invention can range from about 0.2:1 to about 0.00625:1, eg, about 0.2:1, about 0.1:1, about 0.05:1, about 0.025:1 1.
  • Effectively and specifically lyse EGFR-expressing tumor cells at an effector-to-target ratio (therapeutic T cells:EGFR-expressing tumor cells) of about 0.0125:1, about 0.00625:1, e.g., in co-cultures 1, 2, 3, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, even At least about 90% or more of the tumor cells expressing EGFR.
  • Subject refers to an organism suffering from or susceptible to a disease (eg, cancer, eg, EGFR-related cancer) that can be treated by the cells, pharmaceutical compositions, or methods of the present invention.
  • a disease eg, cancer, eg, EGFR-related cancer
  • Non-limiting examples include humans, cows, rats, mice, cats, dogs, monkeys, goats, sheep, and other non-mammals.
  • the subject is a human.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic T cell of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is used to treat EGFR-related cancer in a subject.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the present invention provides use of the therapeutic T cells of the present invention in the manufacture of a medicament for the treatment of EGFR-related cancers.
  • a method for treating EGFR-related cancer comprising administering to a subject in need thereof a therapeutically effective amount of the therapeutic T cells of the present invention or the pharmaceutical composition of the present invention.
  • the method further comprises administering to the subject radiotherapy and/or chemotherapy and/or additional tumor-targeting drugs (eg, monoclonal antibodies or small molecule compounds targeting other antigens) and/or immunization therapy (eg, immune checkpoint inhibitors).
  • tumor-targeting drugs eg, monoclonal antibodies or small molecule compounds targeting other antigens
  • immunization therapy eg, immune checkpoint inhibitors
  • a “therapeutically effective amount” or “therapeutically effective dose” or “effective amount” refers to an amount of a substance, compound, material, or cell that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • the treatment also encompasses preventing the recurrence of a disease such as cancer.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of injury or disability due to disease distress.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably, relative to an untreated subject. Preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors. Alternatively, it can also be assessed by examining the ability to inhibit tumor cell growth, which can be measured in vitro by assays well known to those skilled in the art.
  • the dosage level of cells in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response to a particular subject, composition and mode of administration, without toxicity to the patient.
  • the dose level selected depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the specificity of the
  • the other drugs, compounds and/or materials with which the composition is used in combination the age, sex, weight, condition, general health and medical history of the subject being treated, and similar factors well known in the medical arts.
  • a therapeutically effective amount of therapeutic T cells refers to the amount of therapeutic T cells capable of reducing tumor cell burden after use, eg, reducing tumor cell burden by at least about 5%, at least about 10%, at least about 20%, At least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or an amount that results in a complete remission of the cancer.
  • the effective amount of the therapeutic T cells is about 104 to about 109 cells, eg, about 104 , about 105, about 106 , about 107 , about 10 8 or about 10 9 cells.
  • the amount of therapeutic T cells administered is determined based on the body weight of the subject, eg, about 104 cells/kg body weight to about 109 cells/kg body weight, eg, about 104 , about 105, about 106 , about 107 , about 108 or about 109 cells/kg body weight.
  • the research results of the present inventors show that the therapeutic T cells in which the TGF ⁇ receptors (such as TGFBRI, TGFBRII, TGFBRIII) of the present invention are knocked down or knocked out are compared with control T cells (TGF ⁇ receptors (such as TGFBRI, TGFBRII, TGFBRIII) ) was not knocked down or knocked out), enabling better therapeutic effects at lower doses.
  • TGF ⁇ receptors of the invention eg, TGFBRI, TGFBRII, TGFBRIII
  • therapeutic T cells in which the TGF ⁇ receptors of the invention are knocked down or knocked out are able to obtain superior tumors at lower target ratios and/or longer periods of time than control T cells Killing effect. This is particularly beneficial in reducing preparation time and costs, while reducing side effects when high doses are administered.
  • therapeutic T cells of the invention in which the TGF ⁇ receptor (eg, TGFBRII) has been knocked down or knocked out are administered at a dose that is about lower than the dose administered to control T cells in which the TGF ⁇ receptor (eg, TGFBRII) is not knocked down or knocked out.
  • Administration of cells or compositions according to the present invention may be carried out in any convenient manner, including by injection, infusion, implantation or transplantation.
  • Administration of the cells or compositions described herein can be by intravenous, intralymphatic, intradermal, intratumoral, intramedullary, intramuscular, or intraperitoneal administration.
  • the cells or compositions of the invention are preferably administered by intravenous injection.
  • the EGFR-related cancer is a cancer in which tumor cells express EGFR, including but not limited to esophageal cancer, gastric cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, lung cancer (including non- Small cell lung cancer (NSCLC), breast cancer, cervical cancer, uterine body cancer, endometrial cancer, ovarian cancer, bladder cancer, head and neck cancer (including head and neck squamous cell carcinoma SCCHN), osteosarcoma, prostate cancer, neuroblastoma , kidney cancer, glioma, glioblastoma, and skin cancer (including epithelial cancer).
  • NSCLC non- Small cell lung cancer
  • SCCHN head and neck cancer
  • osteosarcoma including prostate cancer, neuroblastoma , kidney cancer, glioma, glioblastoma, and skin cancer (including epithelial cancer).
  • the present invention provides a method for preparing the therapeutic T cells of the present invention, the method comprising the steps of:
  • the step of providing isolated T cells can be performed by methods known in the art for isolating T cells.
  • T cells can be isolated from the peripheral blood of a subject using commercially available kits. Suitable kits include, but are not limited to, the EasySep human T cell enrichment kit (Stemcell Technologies).
  • the isolated T cells are not necessarily homogeneous, but can be a mixed population of different cells in which T cells are preferably enriched.
  • the method further comprises the step of
  • TGF ⁇ receptors eg TGFBRI, TGFBRII, TGFBRIII
  • step x) is performed before step b). In some embodiments, step x) is performed after step b).
  • the expression of a TGF ⁇ receptor (eg, TGFBRII) in the T cells is knocked down or knocked out by introduction of antisense RNA, antagomir, siRNA, shRNA.
  • the TGF ⁇ receptor in said T cells is knocked down or knocked out by means of gene editing, such as by introducing meganucleases, zinc finger nucleases, transcription activator-like effector nucleases or CRISPR systems (eg TGFBRII) expression.
  • the CRISPR system is used to knock down or knock out the expression of a TGF[beta] receptor (eg TGFBRII) in said T cells.
  • the nuclease (CRISPR nuclease) used by the CRISPR system can be selected from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5 , Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.
  • Polynucleotides, expression constructs and/or proteins can be introduced into cells by any suitable method, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or others; particle bombardment ; lipofection; and infection (eg, the expression construct is a virus).
  • T cells of the invention can be activated and expanded before or after any modification steps.
  • T cells can be expanded in vitro or in vivo.
  • the method further comprises the step of
  • step y) is performed before and/or after step b). In some embodiments, step y) is performed before and/or after step x).
  • T cells of the invention can be expanded, for example, by contact with an agent that stimulates the CD3TCR complex and co-stimulatory molecules on the surface of the T cell to generate a T cell activation signal.
  • an agent that stimulates the CD3TCR complex and co-stimulatory molecules on the surface of the T cell to generate a T cell activation signal.
  • chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitotic lectins such as phytohemagglutinin (PHA) can be used to generate activation signals for T cells .
  • T cells can be activated by in vitro contact with, eg, an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (eg, bryostatin ) in conjunction with calcium ionophore contact.
  • T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation.
  • Conditions suitable for T cell culture include suitable media (e.g. Minimal Essential Media or RPMI Media 1640, or X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g.
  • additives for cell growth include, but are not limited to, surfactants, human plasma protein powder, and reducing agents such as N-acetyl-cysteine and 2-mercaptoacetic acid.
  • Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, Amino Acids, Sodium Pyruvate and Vitamins, Serum Free or Supplemented Serum (or plasma) or a defined set of hormones, and/or an amount of cytokines sufficient to allow T cells to grow and expand.
  • T cells can be maintained under conditions necessary to support growth, such as an appropriate temperature (eg, 37°C) and environment (eg, air plus 5% CO2 ).
  • kits of the present invention include polynucleotides of the present invention, expression constructs of the present invention, and/or tools such as antisense RNA, antagomir, siRNA, shRNA for knocking down or knocking down the expression of TGF ⁇ receptors (eg, TGFBRII) , meganuclease, zinc finger nuclease, transcription activator-like effector nuclease or CRISPR system or its encoding nucleic acid or vector, etc.
  • the kit may also include reagents for isolating, culturing and/or expanding T cells, preparations for introducing polynucleotides or proteins into cells, and the like.
  • Mononuclear cells were isolated with human lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd.).
  • T cells were isolated using the EasySep human T cell enrichment kit (Stemcell Technologies), and anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) were added according to the instructions to activate the sorted T cells at a ratio of 1:1.
  • the medium for T cells was X-VIVO15 medium (Lonza) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (GIBCO) and 400 IU/mL recombinant human IL-2 (Sino-biologic Inc.).
  • the CD3 + T cell density was adjusted to 2 x 106 /ml.
  • co-transfection reagent Polybrene Sigma
  • the final concentration of Polybrene was 10 ⁇ g/ml.
  • the positive rate of CAR-T cells can be detected by flow cytometry 48h after infection.
  • Electroporation was performed using a 4D-Nucleofector System N (Lonza) electroporator under EO-115 electroporation conditions. After electroporation, the cell mixture was aspirated and transferred to pre-warmed T cell culture medium. After 48 hours, the electrotransfer efficiency was detected. Sanger sequencing was performed on the PCR products of the Surveyor assay (primers: TGFbR2-TIDE-F: 5'-cacatctggcccgcacatct-3'; TGFbR2-GT-R: 5'-gggtggctcagaaagagctg-3').
  • CRL-5826-Luci cells wild-type CRL-5826 cells were infected with lentivirus expressing luciferase and Puromycin resistance screening genes, and then screened with Puromycin for two weeks to obtain CRL-5826-Luci stably expressing luciferase cell. Killing experiment: The target cells CRL-5826-Luci were resuspended in 1640 complete medium to make the cell density 1 ⁇ 10 6 /ml. The target cell suspension was seeded in a 96-well plate with 100ul per well. According to different effector-target ratios, different numbers of effector CAR-T cells were added, and each effector-target ratio was repeated 4 times. The final volume per well was 200 ⁇ l.
  • CAR-T cells 2 ⁇ 10 5 CAR-T cells were co-cultured with CRL-5826 tumor cells at an effector-target ratio of 1:1. Two days later, all tumor cells were lysed, and new tumor cells were added after the CAR-T cells were counted. By analogy, new tumor cells were added every other day, and the effector-target ratio was maintained at 1:1 until the killing efficiency of CAR-T cells in different groups was significantly different. The concentration of TGF- ⁇ 1 in the added group was maintained at 5ng/ml.
  • mice used in the experiment were six-week-old NPG female mice (purchased from Weitongda Company).
  • the CRL-5826 cells were resuspended in DPBS, the cell density was 2 ⁇ 10 7 /ml, 100ul of the cell suspension was taken, 100 ⁇ l of Matrigel was added, and the mice were injected subcutaneously. Approximately 2 x 106 CRL-5826 cells were injected per mouse, and the tumor volume was approximately 300 mm3 after 4 weeks. Tumor-bearing mice were randomly assigned to 5 mice per experimental group according to tumor size.
  • CAR-T cells were injected into the tail vein once at different injection doses (about 50% of CAR + ).
  • Tumor volume human CD3 content in peripheral blood and proportion of T cell subtypes were measured weekly.
  • Re-inoculation of tumor mass The mice in the PBS group were sacrificed, and the tumor mass was taken out, divided into 200-300 mm3 tumor mass, and inoculated subcutaneously on the contralateral side of the mice with complete tumor clearance. Another four new NPG mice were taken and subcutaneously inoculated with the segmented tumor as a re-inoculation control.
  • Embodiment 1 anti-EGFR ScFv sequence synthesis and carrier construction
  • the seven CAR structures containing different ScFvs described in Example 1 were introduced into human primary T cells by lentivirus.
  • Human primary T cells were infected with the same virus titer, and the positive rate of CAR-T cells was detected after 5 days of infection ( Figure 2). From the results, even under the conditions of the same lentivirus titer, the positive rates of different CAR-T cells still have large differences and groups.
  • CRL-5826 cells formed tumors subcutaneously, and after 5 weeks, the same dose of 6 anti-EGFR CAR-T cells were injected into the tail vein. Thereafter, tumor volume changes were observed weekly (Fig. 5B). The results showed that hu806 CAR-T cells had the best tumor clearing effect in vivo.
  • Example 4 Comparison of killing function of human-derived hu806 CAR-T and mouse-derived m806 CAR-T cells in vitro
  • Anti-EGFR monoclonal antibody 806ScFv was originally derived from mouse IgG2b (m806), and the FR region sequence was humanized to become humanized 806 (hu806).
  • m806 mouse IgG2b
  • hu806 humanized 806
  • mice IgG2b(m806) CAR The amino acid sequence and nucleotide sequence of the mouse IgG2b(m806) CAR are shown in SEQ ID NOs: 81 and 82, respectively, and each part in the mouse IgG2b(m806) CAR corresponds to Table 1 below.
  • hu806 CAR-T cells with and without TGF- ⁇ Receptor II knockout The antitumor function of hu806 CAR-T cells with and without TGF- ⁇ Receptor II knockout was compared.
  • Human primary T cells were infected with lentivirus and electroporated with Cas9 RNP targeting TGFbR2 after 48 hours. Two days later, the genomic DNA of the knockout cells was extracted, and the TIDE method was used to detect the knockout efficiency (Fig. 7A) and the positive rate of CAR-T cells (Fig. 7B). After 7 days of in vitro culture, the in vitro killing of hu806 CAR-T cells and hu806-TKO CAR-T cells in the presence of TGF- ⁇ was observed.
  • hu806 CAR-T and 806-TKO CAR-T cells were injected, and the tumor volume changes of tumor-bearing NPG mice were observed (Fig. 9A).
  • the results of animal in vivo experiments showed that the larger the injection dose, the faster the tumor removal rate.
  • the therapeutic effect of hu806-TKO CAR-T cells was significantly better than that of hu806 CAR-T cells ( Figure 9A).
  • the mice in the hu806-TKO group who had completely cleared the tumor were re-inoculated with the tumor. After 3-4 weeks, the mice in the experimental group had the ability to clear the tumor again.
  • Example 7 The proportion of T cell subtypes in peripheral blood of hu806-TKO CAR-T cells in tumor-bearing NPG mice
  • hu806 and hu806-TKO cells were prepared from #4 donor CD3T cells with good in vivo expansion effect. Two kinds of CAR-T cells and PBS control were injected into the tail vein respectively, and the injection dose was 5e6 CAR+/cell. Blood was collected weekly to observe T cell subtypes. The results again showed that the knockout group had better tumor clearance ( Figure 10A).
  • the proportion of hCD3 in peripheral blood of mice first increased and then decreased. In the late stage of treatment, hCD3 still maintained a higher proportion in the hu806-TKO group than in the hu806 group (Fig. 10B).
  • the weight of the mouse is calculated as 20g, and the equivalent dose of the human body is converted by a factor of 10 (mice: human);
  • the lower dose groups were given multiple doses according to the tumor inhibition, and the dose interval was determined according to the results of peripheral blood flow.

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Abstract

提供针对EGFR的嵌合抗原受体(CAR)，包含所述CAR的CAR-T细胞，以及其制备方法和用途。

Description

靶向EGFR的嵌合抗原受体 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及靶向EGFR的嵌合抗原受体(CAR)，包含所述CAR的CAR-T细胞，以及其制备方法和用途。
发明背景
人表皮生长因子受体(也称为HER-1或Erb-B1，且在本文中称为“EGFR”)是由c-erbB原癌基因编码的170kDa跨膜受体，且表现出固有的酪氨酸激酶活性。EGFR通过酪氨酸激酶介导的信号转导途径调节多种细胞过程，包括但不限于控制细胞增殖、分化、细胞存活、凋亡、血管生成、有丝分裂发生和转移的信号转导途径的激活。
研究表明，EGFR基因拷贝数增加或者过度表达均能促进正常细胞的恶性转化和恶性肿瘤的转移，EGFR的信号传导网络在肿瘤的形成和发展过程中起着重要作用。EGFR的过表达已在多种人类恶性肿瘤研究中报道，包括肺癌、胰腺癌、结直肠癌、胃癌和乳腺癌等。且临床研究结果表明，EGFR的过表达与患者的不良预后相关。EGFR已经成为抗肿瘤治疗的特异性靶标。
针对EGFR的一些药物已经批准用于临床治疗人体恶性肿瘤。这些药物主要分为两大类：一是阻断EGFR胞外功能区的单抗药物，如西妥昔单抗(Cetuximab)、帕尼单抗(Panitumumab)和尼妥珠单抗(Nimotuzumab)；另一类是针对EGFR胞内区的小分子酪氨酸激酶的抑制剂，例如吉非替尼、厄洛替尼和阿法替尼等。尽管这些药物的安全性和临床疗效已经得到证明，然而在很多情况下，其抗肿瘤作用并不如预期那样有效，比如单抗靶向药物的血药浓度会随时间下降，目标反应率偏低和EGFR突变等问题。
因此，本领域仍然需要新的用于治疗EGFR相关恶性肿瘤的药物和疗法。
发明简述
在一方面，本发明提供一种靶向EGFR的嵌合抗原受体(CAR)，其包含特异性靶向EGFR的胞外抗原结合结构域，所述胞外抗原结合结构域包括重链可变区(VH)和轻链可变区(VL)，其中
i)所述VH包含SEQ ID NO:1所示的VH-CDR1、SEQ ID NO:2所示的VH-CDR2、SEQ ID NO:3所示的VH-CDR3，所述VL包含SEQ ID NO:4所示的VL-CDR1、SEQ ID NO:5所示的VL-CDR2、SEQ ID NO:6所示的VL-CDR3；
ii)所述VH包含SEQ ID NO:10所示的VH-CDR1、SEQ ID NO:11所示的VH-CDR2、SEQ ID NO:12所示的VH-CDR3，所述VL包含SEQ ID NO:13所示的VL-CDR1、SEQ ID NO:14所示的VL-CDR2、SEQ ID NO:16所示的VL-CDR3；
iii)所述VH包含SEQ ID NO:19所示的VH-CDR1、SEQ ID NO:20所示的VH-CDR2、SEQ ID NO:21所示的VH-CDR3，所述VL包含SEQ ID NO:22所示的VL-CDR1、SEQ ID NO:23所示的VL-CDR2、SEQ ID NO:24所示的VL-CDR3；
iv)所述VH包含SEQ ID NO:28所示的VH-CDR1、SEQ ID NO:29所示的VH-CDR2、SEQ ID NO:30所示的VH-CDR3，所述VL包含SEQ ID NO:31所示的VL-CDR1、SEQ ID NO:32所示的VL-CDR2、SEQ ID NO:33所示的VL-CDR3；
v)所述VH包含SEQ ID NO:37所示的VH-CDR1、SEQ ID NO:38所示的VH-CDR2、SEQ ID NO:39所示的VH-CDR3，所述VL包含SEQ ID NO:40所示的VL-CDR1、SEQ ID NO:41所示的VL-CDR2、SEQ ID NO:42所示的VL-CDR3；或
vi)所述VH包含SEQ ID NO:46所示的VH-CDR1、SEQ ID NO:47所示的VH-CDR2、SEQ ID NO:48所示的VH-CDR3，所述VL包含SEQ ID NO:49所示的VL-CDR1、SEQ ID NO:50所示的VL-CDR2、SEQ ID NO:51所示的VL-CDR3。
在一些实施方案中，其中
i)所述VH包含SEQ ID NO:7所示的氨基酸序列，所述VL包含SEQ ID NO:8所示的氨基酸序列；
ii)所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:17所示的氨基酸序列；
iii)所述VH包含SEQ ID NO:25所示的氨基酸序列，所述VL包含SEQ ID NO:26所示的氨基酸序列；
iv)所述VH包含SEQ ID NO:34所示的氨基酸序列，所述VL包含SEQ ID NO:35所示的氨基酸序列；
v)所述VH包含SEQ ID NO:43所示的氨基酸序列，所述VL包含SEQ ID NO:44所示的氨基酸序列；或
vi)所述VH包含SEQ ID NO:52所示的氨基酸序列，所述VL包含SEQ ID NO:53所示的氨基酸序列。
在一些实施方案中，其中所述胞外抗原结合结构域包括单链Fv片段(scFv)。
在一些实施方案中，其中所述scFv包含选自SEQ ID NO:9、18、27、36、45和54的氨基酸序列。
在一些实施方案中，其中所述CAR还包含N末端的CD8α信号肽，例如，所述CD8α信号肽包含SEQ ID NO:55的氨基酸序列。
在一些实施方案中，其中所述CAR还包含跨膜结构域，例如CD8α跨膜结构域，例如，所述CD8α跨膜区包含SEQ ID NO:57的氨基酸序列。
在一些实施方案中，所述CAR还包括位于胞外抗原结合结构域和所述跨膜结构域之间的铰链区，例如，所述铰链区为CD8α铰链区，例如，所述CD8α铰链区包含SEQ ID NO:56的氨基酸序列。
在一些实施方案中，所述CAR还包含信号转导结构域，例如CD3ζ信号转导结构 域，例如CD3ζ信号转导结构域包含SEQ ID NO：59所示氨基酸序列。
在一些实施方案中，所述CAR还包含一或多个共刺激结构域，例如，4-1BB共刺激结构域，例如，所述4-1BB共刺激结构域包含SEQ ID NO:58的氨基酸序列。
在一些实施方案中，所述CAR包含选自SEQ ID NO:60-65的氨基酸序列。
在一方面，本发明提供一种治疗性T细胞，其包含本发明的CAR。
在一些实施方案中，其中所述治疗性T细胞中的TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)被敲低或敲除。
在一些实施方案中，其中所述治疗性T细胞能够在体外在约0.2:1至约0.00625:1，例如约0.2:1、约0.1:1、约0.05:1、约0.025:1、约0.0125:1、约0.00625:1的效靶比下特异性裂解表达EGFR的肿瘤细胞。
在一方面，本发明提供本发明的治疗性T细胞在制备用于治疗EGFR相关癌症的药物中的用途。
在一方面，本发明提供一种用于在对象中治疗EGFR相关癌症的药物组合物，其包含治疗有效量的本发明的治疗性T细胞，以及药学上可接受的载体。
在一方面，本发明提供一种用于治疗EGFR相关癌症的方法，包括给有需要的对象施用治疗有效量的本发明的治疗性T细胞或本发明的药物组合物。
在一些实施方案中，所述方法还进一步包括给所述对象施用放疗和/或化疗和/或另外的肿瘤靶向药物和/或免疫疗法。
在本发明各个方面的一些实施方案中，其中所述EGFR相关癌症选自食管癌、胃癌、结肠癌、直肠癌、结直肠癌、胰腺癌、肺癌(包括非小细胞肺癌NSCLC)、乳腺癌、子宫颈癌、子宫体癌、子宫内膜癌、卵巢癌、膀胱癌、头颈癌(包括头颈鳞状细胞癌SCCHN)、骨肉瘤、前列腺癌、神经母细胞瘤、肾癌、神经胶质瘤、胶质母细胞瘤以及皮肤癌(包括上皮癌)。
在一方面，本发明提供多核苷酸，其包含编码本发明的CAR的核苷酸序列。在一些实施方案中，所述多核苷酸包含选自SEQ ID NO:66-71的核苷酸序列。
在一方面，本发明提供表达构建体，其包含与调控序列可操作连接的本发明的多核苷酸。
在一方面，本发明提供一种制备本发明的治疗性T细胞的方法，所述方法包括以下步骤：
a)提供分离的T细胞；
b)向所述T细胞导入本发明的多核苷酸或本发明的表达构建体，由此使所述T细胞表达本发明的CAR。
在一些实施方案中，所述方法还包括步骤
x)敲低或敲除所述T细胞中TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)。
附图简述
图1.抗-EGFR CAR的慢病毒载体目的基因序列示意图。
图2. 6种抗-EGFR CAR-T细胞阳性率检测。
图3. 6种人源化抗-EGFR CAR-T细胞与CRL-5826细胞体外长时间共培养，通过检测CRL-5826细胞中Luciferase的含量，定量肿瘤杀伤值(N＝4，SEM)。
图4. 6种人源化抗-EGFR CAR-T细胞体外功能比较：6种人源化抗-EGFR CAR-T细胞体外Stress Test实验。每隔一天，从杀伤样品孔取出CAR-T细胞，按E:T＝2:1的比例加入新的肿瘤靶细胞，并检测杀伤率(靶细胞：CRL-5826，E:T＝2:1，50％CAR-T阳性率，N＝4，SEM)。
图5. 6种人源化抗-EGFR CAR-T细胞体内功能比较。A。)NPG小鼠实验流程。肿瘤细胞接种量：2×10 6/只,CAR-T细胞注射剂量：1×10 7个/只,50％CAR阳性，i.v.:尾静脉注射，每组五只NPG鼠。B.)小鼠肿瘤体积变化(N＝5，SEM)。
图6.人源化hu806 CAR-T细胞和鼠源m806 CAR-T细胞体外功能比较。与CRL-5826细胞体外长时间共培养，通过检测CRL-5826细胞中Luciferase的含量，定量肿瘤杀伤值(N＝4，SEM)。
图7.TGF-β Receptor II敲除的hu806 CAR-T细胞。A.)TIDE法检测hu806-TKO CAR-T细胞的敲除效率。B.)流式细胞术检测hu806 CAR-T及hu806-TKO CAR-T细胞的阳性率。
图8.TGF-β Receptor II敲除的hu806 CAR-T细胞杀伤功能检测。A.)体外长时间杀伤CRL-5826细胞检测。TGF-β终浓度：5ng/μl，(N＝4，SEM)。B.)hu806 CAR-T及hu806-TKO CAR-T细胞第四轮及第五轮杀伤检测。添加TGF-β终浓度：5ng/μl。(N＝4，SEM)。C.)hu806及hu806-TKO CAR-T细胞在Stress-Test实验中细胞增殖统计。
图9.小鼠体内实验。A.)NPG小鼠尾静脉注射不同剂量CAR-T细胞，瘤体变化。及肿瘤再接种后瘤体变化。每组五只鼠，注射CAR-T阳性率50％。B.)各实验组及PBS组小鼠外周血中人CD3含量，流式检测。
图10.小鼠体内huCD3各亚型分析实验。A.)NPG小鼠尾静脉注射CAR-T细胞(1x10 7个/只,50％CAR阳性)后，瘤体变化。采用体内扩增能力强的#4供者外周血制备CAR-TT细胞。每组五只鼠。B.)各实验组及PBS组小鼠外周血中人CD3含量，流式检测。C.)小鼠外周血中T细胞各亚型的比例，流式检测。D.)小鼠外周血中huCD4和huCD8占huCD3的比例，流式检测。采集第21、28、36和42天两组小鼠的外周血进行分析。
图11.Hu806-TKO CAR-T细胞对荷瘤NPG小鼠体内治疗剂量实验。A.)NPG小鼠尾静脉注射不同剂量CAR-T细胞(分别为：2×10 6个CAR+细胞/只、1×10 6个CAR+细胞/只、0.5×10 6个CAR+细胞/只和0.25×10 6个CAR+细胞/只)，瘤体变化。及肿瘤完全清除后，再次接种瘤体变化。每组5只鼠。B.)各实验组及对照组小鼠外周血中人CD3含量，流式检测。
图12.Hu806 CAR-T细胞脱靶安全性检测。A.)肺鳞癌细胞系CRL-5826、人原代表皮成纤维细胞Fibroblast和白血病细胞系K562流式检测。染色抗体：anti-EGFR  antibody-PE及806 antibody-PE。B.)Hu806 CAR-T细胞对三种细胞体外杀伤功能检测。采用实时无标记细胞分析技术检测法(N＝2，SEM)。
图13.m806 scFv以及CAR的氨基酸序列与核苷酸序列对应关系图。
发明详述
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达系统表达时)会被保留，但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸的序列，相应地，其编码核苷酸序列也可以包含起始密码子；反之亦然。
在第一方面，本发明提供一种靶向EGFR的嵌合抗原受体(CAR)，其包含特异性靶向EGFR的胞外抗原结合结构域，所述胞外抗原结合结构域包括重链可变区(VH)和轻链可变区(VL)，其中
i)所述VH包含SEQ ID NO:1所示的VH-CDR1、SEQ ID NO:2所示的VH-CDR2、SEQ ID NO:3所示的VH-CDR3，所述VL包含SEQ ID NO:4所示的VL-CDR1、SEQ ID NO:5所示的VL-CDR2、SEQ ID NO:6所示的VL-CDR3；
ii)所述VH包含SEQ ID NO:10所示的VH-CDR1、SEQ ID NO:11所示的VH-CDR2、SEQ ID NO:12所示的VH-CDR3，所述VL包含SEQ ID NO:13所示的VL-CDR1、SEQ ID NO:14所示的VL-CDR2、SEQ ID NO:16所示的VL-CDR3；
iii)所述VH包含SEQ ID NO:19所示的VH-CDR1、SEQ ID NO:20所示的VH-CDR2、SEQ ID NO:21所示的VH-CDR3，所述VL包含SEQ ID NO:22所示的VL-CDR1、SEQ ID NO:23所示的VL-CDR2、SEQ ID NO:24所示的VL-CDR3；
iv)所述VH包含SEQ ID NO:28所示的VH-CDR1、SEQ ID NO:29所示的VH-CDR2、SEQ ID NO:30所示的VH-CDR3，所述VL包含SEQ ID NO:31所示的 VL-CDR1、SEQ ID NO:32所示的VL-CDR2、SEQ ID NO:33所示的VL-CDR3；
v)所述VH包含SEQ ID NO:37所示的VH-CDR1、SEQ ID NO:38所示的VH-CDR2、SEQ ID NO:39所示的VH-CDR3，所述VL包含SEQ ID NO:40所示的VL-CDR1、SEQ ID NO:41所示的VL-CDR2、SEQ ID NO:42所示的VL-CDR3；或
vi)所述VH包含SEQ ID NO:46所示的VH-CDR1、SEQ ID NO:47所示的VH-CDR2、SEQ ID NO:48所示的VH-CDR3，所述VL包含SEQ ID NO:49所示的VL-CDR1、SEQ ID NO:50所示的VL-CDR2、SEQ ID NO:51所示的VL-CDR3。
在一些实施方案中，所述胞外抗原结合结构域包括重链可变区(VH)和轻链可变区(VL)，其中
i)所述VH包含SEQ ID NO:7所示的氨基酸序列，所述VL包含SEQ ID NO:8所示的氨基酸序列；
ii)所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:17所示的氨基酸序列；
iii)所述VH包含SEQ ID NO:25所示的氨基酸序列，所述VL包含SEQ ID NO:26所示的氨基酸序列；
iv)所述VH包含SEQ ID NO:34所示的氨基酸序列，所述VL包含SEQ ID NO:35所示的氨基酸序列；
v)所述VH包含SEQ ID NO:43所示的氨基酸序列，所述VL包含SEQ ID NO:44所示的氨基酸序列；或
vi)所述VH包含SEQ ID NO:52所示的氨基酸序列，所述VL包含SEQ ID NO:53所示的氨基酸序列。
在一些实施方案中，所述胞外抗原结合结构域包括单链Fv片段(scFv)。
在一些实施方案中，所述VH和所述VL通过接头相连。在一些实施方案中，所述接头是柔性肽接头。在一些实施方案中，所述接头包含SEQ ID NO:72所示的氨基酸序列。
在一些实施方案中，所述scFv包括选自SEQ ID NO:9、18、27、36、45和54的氨基酸序列。
在一些实施方案中，所述CAR还包含N末端的CD8α信号肽。在一些实施方案中，所述CD8α信号肽包含SEQ ID NO:55的氨基酸序列。
在一些实施方案中，所述CAR还包含跨膜结构域，例如CD8α跨膜结构域或CD28跨膜结构域。在一些实施方案中，所述CAR包含CD8α跨膜区。在一些实施方案中，所述CD8α跨膜区包含SEQ ID NO:57的氨基酸序列。
在一些实施方案中，所述CAR还包括位于胞外抗原结合结构域和所述跨膜结构域之间的铰链区，例如，所述铰链区为CD8α铰链区。在一些实施方案中，所述CD8α铰链区包含SEQ ID NO:56的氨基酸序列。
在一些实施方式中，所述CAR还包含信号转导结构域，例如可用于T细胞活化的 信号转导结构域，例如选自TCRζ、FcRγ、FcRβ、FcRε、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、CD79a、CD79b和CD66d的信号转导结构域。在一些优选实施方式中，所述CAR包含CD3ζ信号转导结构域，例如所述CD3ζ信号转导结构域包含SEQ ID NO：59的氨基酸序列。
在一些实施方式中，所述CAR还包含一或多个共刺激结构域，例如选自CD3、CD27、CD28、CD83、CD86、CD127、4-1BB和4-1BBL的共刺激结构域。在一些实施方案中，所述CAR还包含4-1BB共刺激结构域。在一些实施方案中，所述4-1BB共刺激结构域包含SEQ ID NO:58的氨基酸序列。
在一些实施方式中，所述CAR包含针对EGFR的胞外抗原结合结构域、CD8α铰链区、CD8α跨膜区、4-1BB共刺激结构域和CD3ζ信号转导结构域，以及任选的N末端的CD8α信号肽。
在一些具体实施方案中，所述CAR包含选自SEQ ID NO:60-65的氨基酸序列。
在另一方面，本发明提供多核苷酸，其包含编码本发明的CAR的核苷酸序列。在一些实施方案中，所述多核苷酸包含选自SEQ ID NO:66-71的核苷酸序列。
在另一方面，本发明提供表达构建体，其包含与调控序列可操作连接的本发明的多核苷酸。
本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者可以是能够翻译的RNA(如mRNA)。在一些优选实施方案中，所述表达构建体是病毒载体，例如慢病毒载体。
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。表达调控元件指的是能够控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子、增强子和多腺苷酸化识别序列。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
在另一方面，本发明提供一种治疗性T细胞，其包含本发明的CAR。在一些实施方案中，所述CAR表达于所述T细胞的细胞膜表面。
在一些实施方案中，所述治疗性T细胞中的TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)被敲低或敲除。
如本文所用，“治疗性T细胞中的TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)被敲低或敲除”指的是相对于对照T细胞，本发明的治疗性T细胞中TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)的表达被下调或者不表达，或者，TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)的活性降低或者被失活(例如被拮抗)。本文所用的敲低或敲除可以 是基因组水平的、转录水平的、翻译水平的或翻译后水平的。
在本发明各方面的一些实施方案中，所述治疗性T细胞衍生自对象的自体细胞。如本文所用，“自体”是指用于治疗对象的细胞、细胞系或细胞群源自所述对象。在一些实施方案中，所述治疗性T细胞衍生自异体细胞，例如源自与所述对象人类白细胞抗原(HLA)相容的供体对象。可以使用标准方案将来自供体对象的细胞转化为非同种异体反应性细胞，并根据需要进行复制，从而产生可以施用至一个或多个对象的细胞。
在一些实施方案中，所述T细胞来源于健康对象。在一些实施方案中，所述T细胞来源于患有癌症的对象。
本发明上下文所述T细胞可以衍生自炎性T淋巴细胞、细胞毒性T淋巴细胞、调节性T淋巴细胞、和/或辅助性T淋巴细胞。在一些实施方案中，本发明上下文所述T细胞可以衍生自CD4+T淋巴细胞和/或CD8+T淋巴细胞。
本发明上下文的T细胞可以通过各种非限制性方法从许多非限制性来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、腹水、胸腔积液、脾组织和肿瘤。本发明上下文的T细胞还可以是呈现不同表型特征的细胞的混合群体的一部分。
本发明的治疗性T细胞能够在体外特异性裂解表达EGFR的肿瘤细胞。例如，与表达EGFR的肿瘤细胞体外共培养，本发明的治疗性T细胞能够在约0.2:1至约0.00625:1，例如约0.2:1、约0.1:1、约0.05:1、约0.025:1、约0.0125:1、约0.00625:1的效靶比(治疗性T细胞:表达EGFR的肿瘤细胞)下有效地特异性裂解表达EGFR的肿瘤细胞，例如，在共培养1、2、3、4、5、6或7天后裂解至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、甚至至少大约90％或更多的表达EGFR的肿瘤细胞。
如本申请上下文所用“对象”是指患有或者易于患有可以通过本发明的细胞、药物组合物、或方法治疗的疾病(如癌症，如EGFR相关癌症)的生物体。非限制性例子包括人、牛、大鼠、小鼠、猫、狗、猴、山羊、绵羊，及其它非哺乳动物。在优选实施方案中，对象是人。
在另一方面，本发明提供一种药物组合物，其包含治疗有效量的本发明的治疗性T细胞，以及药学上可接受的载体。在一些实施方案中，所述药物组合物用于在对象中治疗EGFR相关癌症。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。
在另一方面，本发明提供本发明的治疗性T细胞在制备用于治疗EGFR相关癌症的药物中的用途。
在本发明的另一方面，还提供一种用于治疗EGFR相关癌症的方法，包括给有需要的对象施用治疗有效量的本发明的治疗性T细胞或本发明的药物组合物。
在一些实施方式中，所述方法还进一步包括给所述对象施用放疗和/或化疗和/或另 外的肿瘤靶向药物(例如靶向其它抗原的单克隆抗体或小分子化合物)和/或免疫疗法(如免疫检查点抑制剂)。
如本文所用，“治疗有效量”或“治疗有效剂量”或“有效量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或细胞的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。如本文所用，所述治疗也涵盖防止疾病(如癌症)的复发。
例如，“有效量”的本发明的细胞或药物组合物优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的细胞或药物组合物优选地将肿瘤细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制肿瘤细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。
实际应用中，本发明药物组合物中细胞的剂量水平可能改变，以获得可有效实现对特定对象、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的对象的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
如本文所用，治疗性T细胞的治疗有效量是指使用后能够降低肿瘤细胞负荷的治疗性T细胞的量，例如使肿瘤细胞负荷降低至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％或者使癌症完全缓解的量。在本发明各方面的一些实施方案中，所述治疗性T细胞的有效量为约10 4至约10 9个细胞，例如约10 4、约10 5、约10 6、约10 7、约10 8或约10 9个细胞。在一些实施方案中，根据对象体重确定治疗性T细胞的施用量，例如约10 4细胞/kg体重至约10 9个细胞/kg体重，例如约10 4、约10 5、约10 6、约10 7、约10 8或约10 9个细胞/kg体重。
本发明人的研究结果表明，本发明的TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)被敲低或敲除的治疗性T细胞，相对于对照T细胞(TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)未被敲低或敲除)，能够以更低的剂量实现更优的治疗效果。例如，本发明的TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)被敲低或敲除的治疗性T细胞能够在低效靶比和/或更长的时间上获得比对照T细胞更优的肿瘤杀伤效果。这特别有利于减少制备时间和成本，同时能够减少高剂量施用时带来的副作用。
例如，本发明的TGFβ受体(如TGFBRII)被敲低或敲除的治疗性T细胞的施用剂量比TGFβ受体(如TGFBRII)未被敲低或敲除的对照T细胞的施用剂量低约2倍、低约3 倍、低约4倍、低约5倍、低约6倍、低约7倍、低约8倍、低约9倍、低约10倍、低约15倍、低约20倍、低约30倍、低约40倍、低约50倍、低约80倍、低约100倍、低约150倍、低约160倍、低约200倍或更低。
根据本发明的细胞或组合物的施用可以以任何方便的方式进行，包括通过注射、输注、植入或移植。本文所述的细胞或组合物施用可以通过静脉内、淋巴内、皮内、肿瘤内、髓内、肌内或腹膜内施用。在一个实施方案中，本发明的细胞或组合物优选通过静脉内注射施用。
在本发明各个方面的一些实施方案中，所述EGFR相关癌症是肿瘤细胞表达EGFR的癌症，包括但不限于食管癌、胃癌、结肠癌、直肠癌、结直肠癌、胰腺癌、肺癌(包括非小细胞肺癌NSCLC)、乳腺癌、子宫颈癌、子宫体癌、子宫内膜癌、卵巢癌、膀胱癌、头颈癌(包括头颈鳞状细胞癌SCCHN)、骨肉瘤、前列腺癌、神经母细胞瘤、肾癌、神经胶质瘤、胶质母细胞瘤以及皮肤癌(包括上皮癌)。
在另一方面，本发明提供一种制备本发明的治疗性T细胞的方法，所述方法包括以下步骤：
a)提供分离的T细胞；
b)向所述T细胞导入本发明的多核苷酸或本发明的表达构建体，由此使所述T细胞表达本发明的CAR。
提供分离的T细胞的步骤可以通过本领域已知的分离T细胞的方法来进行。例如，可以利用商品化试剂盒从对象的外周血中分离T细胞。合适的试剂盒包括但不限于EasySep human T cell enrichment kit(Stemcell Technologies)。如上所述，分离的T细胞并不一定是均质的，其可以是不同细胞的混合群体，优选地，在所述群体中T细胞被富集。
在一些实施方案中，所述方法还包括步骤
x)敲低或敲除所述T细胞中TGFβ受体(如TGFBRI、TGFBRII、TGFBRIII)的表达。
在一些实施方案中，所述步骤x)在步骤b)之前进行。在一些实施方案中，所述步骤x)在步骤b)之后进行。
本领域已知若干在细胞中敲低或敲除蛋白质表达的方法。在一些实施方案中，通过导入反义RNA、antagomir、siRNA、shRNA敲低或敲除所述T细胞中TGFβ受体(如TGFBRII)的表达。在另一些实施方式中，通过基因编辑的方法，例如通过导入大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶或CRISPR系统敲低或敲除所述T细胞中TGFβ受体(如TGFBRII)的表达。在本发明方法优选的实施方案中，使用CRISPR系统敲低或敲除所述T细胞中TGFβ受体(如TGFBRII)的表达。在一些实施方式中，CRISPR系统使用的核酸酶(CRISPR核酸酶)例如可以选自Cas3、Cas8a、Cas5、Cas8b、Cas8c、Cas10d、Cse1、Cse2、Csy1、Csy2、Csy3、GSU0054、Cas10、Csm2、Cmr5、Cas10、Csx11、Csx10、Csf1、Cas9、Csn2、Cas4、Cpf1、C2c1、C2c3或C2c2蛋白，或这些核酸酶的功能性变体。
多核苷酸、表达构建体和/或蛋白质可以通过任何适当的方法引入细胞，包括电穿孔； 使用氯化钙、氯化铷、磷酸钙、DEAE-葡聚糖或其他物质的转染；微粒轰击；脂质体转染；和感染(例如，表达构建体是病毒)。
本发明的T细胞可以在任何修饰步骤之前或之后被活化和扩增。T细胞可以在体外或体内扩增。
因此，在一些实施方案中，所述方法还包括步骤
y)扩增所述T细胞。
在一些实施方案中，所述步骤y)在在步骤b)之前和/或之后进行。在一些实施方案中，所述步骤y)在在步骤x)之前和/或之后进行。
通常，本发明的T细胞可以例如通过与刺激CD3TCR复合物和T细胞表面上的共刺激分子以产生T细胞活化信号的试剂接触来扩增。例如，可以使用诸如钙离子载体A23187、佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)、或有丝分裂凝集素如植物血凝素(PHA)的化学品来产生T细胞的活化信号。在一些实施方案中，T细胞可以通过在体外与例如抗CD3抗体或其抗原结合片段、或固定在表面上的抗CD2抗体接触被活化，或通过与蛋白激酶C激活剂(例如，苔藓抑素)连同钙离子载体的接触来活化。例如，在适合于刺激T细胞增殖的条件下，T细胞可与抗CD3抗体和抗CD28抗体接触。适用于T细胞培养的条件包括可能含有增殖和活力所必需的因子的合适培养基(例如Minimal Essential Media或RPMI Media 1640、或X-vivo 5、(Lonza))，其中必需的因子包括血清(例如胎牛或人类血清)、白介素-2(IL-2)、胰岛素、IFN-γ、IL-4、IL-7、GM-CSF、IL-10、IL-2、IL-15、TGFβ和TNF，或本领域技术人员已知的用于细胞生长的添加剂。其它用于细胞生长的添加剂包括但不限于表面活性剂、人血浆蛋白粉、和还原剂如N-乙酰-半胱氨酸和2-巯基乙酸。培养基可以包括RPMI 1640、A1M-V、DMEM、MEM、a-MEM、F-12、X-Vivo 1和X-Vivo 20、Optimizer、氨基酸、丙酮酸钠和维生素、无血清或适量补充的血清(或血浆)或一组明确的激素、和/或一定量的足以使T细胞生长和扩增的细胞因子。T细胞可以保持在支持生长所必需的条件下，例如适当的温度(例如37℃)和环境(例如，空气加5％CO 2)。
在另一方面，本发明还提供一种试剂盒，其用于制备本发明的治疗性T细胞。本发明的试剂盒包括本发明的多核苷酸、本发明的表达构建体和/或用于敲低或敲除TGFβ受体(如TGFBRII)的表达的工具如反义RNA、antagomir、siRNA、shRNA、大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶或CRISPR系统或其编码核酸或载体等。所述试剂盒还可以包含用于分离、培养和/或扩增T细胞的试剂、用于将多核苷酸或蛋白质导入细胞的制剂等。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实验材料和方法
1.CD3 +T细胞的分离、刺激及扩增
从北京脐带血库获取健康供者来源的新鲜脐带血，并已通过知情同意告知。用人淋巴细胞分离液(天津市灏洋生物制品科技有限责任公司)分离单个核细胞。使用EasySep human T cell enrichment kit(Stemcell Technologies)分离T细胞，并根据使用说明添加抗-CD3/CD28Dynabeads(Thermo Fisher Scientific)，以1:1的比例激活分选出的T细胞。T细胞的培养基为X-VIVO15培养基(Lonza)，并添加5％(v/v)热失活胎牛血清(GIBCO)和400IU/mL重组人IL-2(Sino-biologic Inc.)。
2.制备抗-EGFR CAR-T细胞
分别合成七个anti-EGFR ScFv基因片段(华大青兰生物科技有限公司)，通过BamH I和Mlu I两个酶切位点，分别将ScFv片段克隆进pRRLSIN慢病毒载体中。利用Lipo3000(Thermo Fisher Scientific)将三质粒系统载体质粒、pMD2.G和psPAX2共转染293T细胞，分别在48小时和72小时收集慢病毒培养上清，用浓缩柱(Millipore,Amicon Ultra-15 centrifuge Filters，Ultracel-100K)进行浓缩。人原代CD3 +T细胞经磁珠刺激24小时后，进行慢病毒感染。感染时，将CD3 +T细胞密度调整至2×10 6/ml。按MOI＝1比例，同时添加助转染试剂Polybrene(Sigma)，Polybrene的终浓度为10μg/ml。感染48h后可通过流式细胞仪检测CAR-T细胞的阳性率。
3.流式细胞检测
收集约1-10×10 5个细胞，根据抗体公司的推荐用量，进行细胞染色。使用CytoFLEX(Beckman Coulter Inc.)上机检测。该专利使用以下抗体：羊抗人IgG(H+L)流式抗体Alexa Fluor 647(109-606-003，Jackson)、鼠抗人CD3流式单克隆抗体Brilliant Violet 421(300434，BioLegend)、鼠抗人EGFR单克隆抗体PE(352903，BioLegend)、806重组单克隆抗体(金斯瑞合成)、鼠抗人CD4流式单克隆抗体PE(300508，BioLegend)、鼠抗人CD8α流式单克隆抗体APC(301014，BioLegend)鼠抗人CD45RO流式单克隆抗体PE(304205，BioLegend)和鼠抗人CCR7流式单克隆抗体APC(353213，BioLegend)。
4.电转CAR-T细胞及编辑效率检测
T细胞激活三天后，用磁铁去除Dynabeads。电转前准备RNP复合物，将6μg Cas9蛋白(深圳菲鹏生物科技有限公司)和6μg体外转录制备的sgRNA(靶序列：CCTGAGCAGCCCCCGACCCA)室温孵育20分钟。1×10 6个CAR-T细胞用20μl P3 Primary Cell 4D-NucleofectorX Kit电转液(V4XP-3024,Lonza)重悬，加入孵育好的RNP。使用4D-Nucleofector System N(Lonza)电转仪，EO-115电转条件进行电转。电转后，将细胞混合物吸出，转移至预热好的T细胞培养基中。48小时后，检测电转效率。对Surveyor assay的PCR产物(引物：TGFbR2-TIDE-F：5’-cacatctggcccgcacatct-3’；TGFbR2-GT-R：5’-gggtggctcagaaagagctg-3’)进行Sanger测序(测序引物：TGFbR2-TIDE-F：5’-cacatctggcccgcacatct-3’；TGFbR2-TIDE-R：5’-ggaaactttcctcgtttccgc-3’)。通过网站http://tide.nki.nl，对测序结果的进行分析。
5.CAR-T细胞体外杀伤实验(Luciferase检测法)
构建CRL-5826-Luci细胞：用表达荧光素酶及Puromycin抗性筛选基因的慢病毒侵染野生型CRL-5826细胞，然后用Puromycin筛选两周，获得稳定表达荧光素酶的CRL-5826-Luci细胞。杀伤实验：用1640完全培养基重悬靶细胞CRL-5826-Luci，使细胞密度为1×10 6/ml。将靶细胞悬液接种96孔板，每孔100ul。按不同的效靶比，分别加入不同数量的效应CAR-T细胞，每个效靶比做4个重复。每孔的终体积为200μl。置于培养箱，按不同时间点取出，检测杀伤效率。检测时，向每孔加入10μl Steady-glo荧光素底物(Promega)，反应5分钟，使用酶标仪PerinElmer VICTOR X3检测荧光值。基于每孔的荧光值计算效应细胞对靶细胞的杀伤效率：specific lysis(％)＝(1-RUL效应细胞加靶细胞/RUL靶细胞)x100(RUL：relative light unit)。
6.CAR-T细胞体外杀伤实验(RTCA检测法)
使用实时无标记细胞功能分析仪检测效应CAR-T细胞对靶细胞体外杀伤功能。分别将Fibroblast细胞、CRL-5826细胞和K562细胞种植于E-Plate16(ACEA)，每孔加入2500个细胞，进行两个重复。24小时后，依次按照不同效靶比(0.2:1、0.05:1、0.0125:1及0:1)分别加入效应CAR-T细胞。连续监测4天的Cell Index(CI)数值。基于每孔的CI值计算效应细胞对靶细胞的杀伤效率：specific lysis(％)＝(1-CI效应细胞加靶细胞/CI靶细胞)x100。
7.多轮抗原刺激(Stress-Test)实验
将2×10 5个CAR-T细胞与CRL-5826肿瘤细胞共培养，效靶比为1:1。两天后，肿瘤细胞全部裂解，将CAR-T细胞进行计数后，加入新的肿瘤细胞。以此类推，每隔一天加入新的肿瘤细胞，保持效靶比为1:1，直到不同组CAR-T细胞杀伤效率有明显区别。添加组TGF-β1浓度维持在5ng/ml。
8.荷瘤小鼠模型检测CAR-T细胞的功能
实验用小鼠为六周龄NPG雌鼠(购自维通达公司)。将CRL-5826细胞用DPBS重悬，细胞密度为2×10 7/ml，分别取100ul细胞悬液，加100μl Matrigel，对小鼠进行皮下注射。每只小鼠注射约2×10 6个CRL-5826细胞，4周后肿瘤体积约为300mm 3时。根据瘤体大小，荷瘤小鼠被随机分组，每个实验组分配5只小鼠。CAR-T细胞按不同注射剂量(CAR +约为50％)，进行尾静脉注射一次。每周测量肿瘤体积、外周血中人CD3含量及T细胞亚型比例。肿瘤块再接种：将PBS组小鼠处死，取出肿瘤块，分割成200-300mm3瘤块，分别接种于肿瘤完全清除小鼠对侧皮下。另取四只新NPG小鼠，将分割瘤块接种于皮下，作为再接种对照。
实施例1、抗-EGFR ScFv序列合成及载体构建
从已有的专利及NCBI中找到六个人源化anti-EGFR的ScFv序列及一个鼠源anti-EGFR的ScFv，分别是hu806(US 009493568B2)、E2(US 20150030599A1)、Pan(US 20150152184A1)、Nec(WO 2005/090407 A1)、Nimo(US 6506883 B2)、301(GeneBank  JQ306330.1)和m806(WO02092771A2)。经基因合成，分别将这七个ScFv插入到带有CAR骨架基因的慢病毒载体pRRLSIN质粒中(图1)。
实施例2、抗-EGFR CAR-T细胞的制备
将实施例1所述7个包含不同ScFv的CAR结构通过慢病毒分别导入人原代T细胞中。以相同病毒滴度感染人原代T细胞，感染第5天后，检测CAR-T细胞的阳性率(图2)。从结果来看，即使在相同慢病毒滴度的条件下，不同CAR-T细胞的阳性率仍有较大的差异及分群。
实施例3、抗-EGFR CAR-T细胞体外及体内杀伤功能比较
为了比较不同ScFv来源的anti-EGFR CAR-T细胞的杀伤功能，将CRL-5826细胞进行了体外低效靶比长时间杀伤实验及肿瘤抗原连续刺激杀伤Stress-Test实验(图3、图4)。从结果来看，hu806和Nimo CAR-T与其他4种ScFv CAR-T相比，具有更强的体外杀瘤功能。且Stress-Test结果表明，hu806 CAR-T的清瘤效果优于Nimo CAR-T细胞。接下来进行了NPG小鼠体内荷瘤及治疗实验(图5A)。CRL-5826细胞皮下成瘤，5周后，尾静脉分别注射相同剂量的6种anti-EGFR CAR-T细胞。之后，每周观察肿瘤体积变化(图5B)。结果表明，hu806 CAR-T细胞具有最优的体内清瘤效果。
实施例4、人源hu806 CAR-T和鼠源m806 CAR-T细胞体外杀伤功能比较
Anti-EGFR单克隆抗体806ScFv最初来源于鼠IgG2b(m806)，FR区序列经人源化后，成为人源化806(hu806)。本专利中，比较了人源和鼠源806ScFv的CAR-T细胞体外功能。实验结果表明，hu806 CAR-T细胞具有更强的体外抗肿瘤功能(图6)。
其中鼠IgG2b(m806)CAR的氨基酸序列和核苷酸序列分别如SEQ ID NO:81和82所示，鼠IgG2b(m806)CAR中各部分对应于下表1。
表1 鼠IgG2b(m806)CAR中各区域
Figure PCTCN2021121678-appb-000001
Figure PCTCN2021121678-appb-000002
实施例5、hu806 CAR-T细胞敲除TGF-β Receptor II后可增强其抗肿瘤能力
比较了敲除和未敲除TGF-β Receptor II的hu806 CAR-T细胞的抗肿瘤功能。慢病毒感染人原代T细胞，48小时后进行靶向TGFbR2的Cas9 RNP电转。两天后，提取敲除细胞的基因组DNA，TIDE法检测敲除效率(图7A)及CAR-T细胞的阳性率(图7B)。体外培养7天后，观察hu806 CAR-T细胞及hu806-TKO CAR-T细胞在TGF-β存在时，体外杀伤肿瘤的情况。
结果表明，TGF-β抑制了hu806 CAR-T细胞体外抗肿瘤功能，将TGF-β Receptor II敲除后，可以逆转TGF-β对CAR-T细胞功能的抑制作用(图8A)。Stress-Test实验结果也说明，经多轮肿瘤抗原连续刺激后，hu806-TKO相比hu806 CAR-T细胞更有抗肿瘤优势(图8B)。且TGF-β Receptor II敲除的hu806-TKO CAR-T细胞比hu806 CAR-T细胞更有增殖优势(图8C)。
实施例6、NPG小鼠体内实验显示hu806-TKO CAR-T具有更好的治疗效果
按不同剂量，注射hu806 CAR-T及806-TKO CAR-T细胞，观察荷瘤NPG小鼠肿瘤体积变化(图9A)。动物体内实验结果表明，注射剂量越大，清瘤速度越快。且相同剂量条件下，hu806-TKO CAR-T细胞的治疗效果明显优于hu806 CAR-T细胞(图9A)。对hu806-TKO组完全清除肿瘤的小鼠进行肿瘤再接种，3-4周后，实验组小鼠具有再次清除肿瘤的能力。分析小鼠外周血中人CD3的比例可知，hu806-TKO组hCD3的比例明显高于hu806组(图8B)，且其与清瘤效果成正相关。
实施例7、hu806-TKO CAR-T细胞在荷瘤NPG小鼠外周血中T细胞亚型比例
为了观察输注CAR-T细胞在动物体内扩增及亚型比例变化，用体内扩增效果好的#4供者CD3T细胞，制备hu806及hu806-TKO细胞。分别尾静脉注射两种CAR-T细胞及PBS对照，注射剂量按5e6 CAR+/只。每周采血观察T细胞亚型。结果再次表明，敲除组清除肿瘤效果更好(图10A)。小鼠外周血中hCD3比例先升高后降低。在治疗后期，hu806-TKO组相比hu806组，hCD3仍维持更高的比例(图10B)。进一步分析小鼠外周血中人CD3亚型，TKO组记忆状态T细胞比例更高，尤其是central memory T细胞比例更有优势(图10C)。从已发表的文献可知central memory T细胞比例和预后疗效成正相关。从CD4和CD8染色结果来看，治疗早期，CD8 T细胞的比例在TKO组较高。随时间延长，CD4 T细胞成为主要细胞亚群(图10D)。
实施例8、hu806-TKO CAR-T细胞的治疗剂量探索
为了给临床实验提供注射治疗剂量参考，我们对荷瘤NPG小鼠进行剂量分组实验。根据小鼠和人等效剂量换算，制定了不同的输注剂量(表2)。从动物体内分组治疗结果来看，hu806-TKO四个剂量组均能有效清除CDX模型肿瘤，且剂量与清瘤速度相关(图11A)。清瘤后进行了两个治疗剂量组的肿瘤再接种实验，均能观察到再接种瘤的体积的有效减小(图11A)。小鼠外周血中hCD3含量与输注剂量相关，随治疗时间，hCD3含量表现为先升高再降低(图11B)。
表2.Hu806-TKO CAR-T细胞对荷瘤NPG小鼠体内治疗分组剂量表。
NPG小鼠剂量实验分组
Figure PCTCN2021121678-appb-000003
a.小鼠体重以20g计，与人体等效剂量以系数10(小鼠：人)换算；
b.较低剂量各组视肿瘤抑制情况安排多次给药，给药间隔依外周血流式结果确定。
实施例9、hu806 CAR-T细胞的体内安全性
CAR-T细胞治疗的主要风险在于脱靶效应。为了检测本专利中抗EGFR hu806 ScFv的脱靶作用，明确其在体内的安全性。我们用hu806重组抗体对肺鳞癌细胞及人原代成纤维细胞进行染色。从流式结果可知，CRL-5826和成纤维细胞均有EGFR的表达，而成纤维细胞仅表达少量的hu806抗原。血液来源的白血病细胞K562不表达EGFR(图12A)。相应地，对这三种细胞的体外杀伤检测表明，hu806 CAR-T细胞对EGFR阳性806抗原阳性的CRL-5826细胞具有很强的杀伤功能，而对EGFR阳性806抗原阴性的成纤维细胞和EGFR阴性的K562细胞几乎不杀伤(图12B)。这提示hu806细胞作为药物注射体内后，脱靶副作用的风险较低。
序列表：
Figure PCTCN2021121678-appb-000004
Figure PCTCN2021121678-appb-000005
Figure PCTCN2021121678-appb-000006
Figure PCTCN2021121678-appb-000007
Figure PCTCN2021121678-appb-000008
Figure PCTCN2021121678-appb-000009
Figure PCTCN2021121678-appb-000010
Figure PCTCN2021121678-appb-000011
Figure PCTCN2021121678-appb-000012
Figure PCTCN2021121678-appb-000013

Claims (25)

  1. 一种靶向EGFR的嵌合抗原受体(CAR)，其包含特异性靶向EGFR的胞外抗原结合结构域，所述胞外抗原结合结构域包括重链可变区(VH)和轻链可变区(VL)，其中
    i)所述VH包含SEQ ID NO:1所示的VH-CDR1、SEQ ID NO:2所示的VH-CDR2、SEQ ID NO:3所示的VH-CDR3，所述VL包含SEQ ID NO:4所示的VL-CDR1、SEQ ID NO:5所示的VL-CDR2、SEQ ID NO:6所示的VL-CDR3；
    ii)所述VH包含SEQ ID NO:10所示的VH-CDR1、SEQ ID NO:11所示的VH-CDR2、SEQ ID NO:12所示的VH-CDR3，所述VL包含SEQ ID NO:13所示的VL-CDR1、SEQ ID NO:14所示的VL-CDR2、SEQ ID NO:16所示的VL-CDR3；
    iii)所述VH包含SEQ ID NO:19所示的VH-CDR1、SEQ ID NO:20所示的VH-CDR2、SEQ ID NO:21所示的VH-CDR3，所述VL包含SEQ ID NO:22所示的VL-CDR1、SEQ ID NO:23所示的VL-CDR2、SEQ ID NO:24所示的VL-CDR3；
    iv)所述VH包含SEQ ID NO:28所示的VH-CDR1、SEQ ID NO:29所示的VH-CDR2、SEQ ID NO:30所示的VH-CDR3，所述VL包含SEQ ID NO:31所示的VL-CDR1、SEQ ID NO:32所示的VL-CDR2、SEQ ID NO:33所示的VL-CDR3；
    v)所述VH包含SEQ ID NO:37所示的VH-CDR1、SEQ ID NO:38所示的VH-CDR2、SEQ ID NO:39所示的VH-CDR3，所述VL包含SEQ ID NO:40所示的VL-CDR1、SEQ ID NO:41所示的VL-CDR2、SEQ ID NO:42所示的VL-CDR3；或
    vi)所述VH包含SEQ ID NO:46所示的VH-CDR1、SEQ ID NO:47所示的VH-CDR2、SEQ ID NO:48所示的VH-CDR3，所述VL包含SEQ ID NO:49所示的VL-CDR1、SEQ ID NO:50所示的VL-CDR2、SEQ ID NO:51所示的VL-CDR3。
  2. 权利要求1的靶向EGFR的CAR，其中
    i)所述VH包含SEQ ID NO:7所示的氨基酸序列，所述VL包含SEQ ID NO:8所示的氨基酸序列；
    ii)所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:17所示的氨基酸序列；
    iii)所述VH包含SEQ ID NO:25所示的氨基酸序列，所述VL包含SEQ ID NO:26所示的氨基酸序列；
    iv)所述VH包含SEQ ID NO:34所示的氨基酸序列，所述VL包含SEQ ID NO:35所示的氨基酸序列；
    v)所述VH包含SEQ ID NO:43所示的氨基酸序列，所述VL包含SEQ ID NO:44所示的氨基酸序列；或
    vi)所述VH包含SEQ ID NO:52所示的氨基酸序列，所述VL包含SEQ ID NO:53所示的氨基酸序列。
  3. 权利要求1或2的靶向EGFR的CAR，其中所述胞外抗原结合结构域包括单链 Fv片段(scFv)。
  4. 权利要求3的靶向EGFR的CAR，其中所述scFv包含选自SEQ ID NO:9、18、27、36、45和54的氨基酸序列。
  5. 权利要求1-4中任一项的靶向EGFR的CAR，其中所述CAR还包含N末端的CD8α信号肽，例如，所述CD8α信号肽包含SEQ ID NO:55的氨基酸序列。
  6. 权利要求1-5中任一项的靶向EGFR的CAR，其中所述CAR还包含跨膜结构域，例如CD8α跨膜结构域，例如，所述CD8α跨膜区包含SEQ ID NO:57的氨基酸序列。
  7. 权利要求1-6中任一项的靶向EGFR的CAR，所述CAR还包括位于胞外抗原结合结构域和所述跨膜结构域之间的铰链区，例如，所述铰链区为CD8α铰链区，例如，所述CD8α铰链区包含SEQ ID NO:56的氨基酸序列。
  8. 权利要求1-7中任一项的靶向EGFR的CAR，所述CAR还包含信号转导结构域，例如CD3ζ信号转导结构域，例如CD3ζ信号转导结构域包含SEQ ID NO：59所示氨基酸序列。
  9. 权利要求1-8中任一项的靶向EGFR的CAR，所述CAR还包含一或多个共刺激结构域，例如，4-1BB共刺激结构域，例如，所述4-1BB共刺激结构域包含SEQ ID NO:58的氨基酸序列。
  10. 权利要求1-9中任一项的靶向EGFR的CAR，所述CAR包含选自SEQ ID NO:60-65的氨基酸序列。
  11. 一种治疗性T细胞，其包含权利要求1-10中任一项的CAR。
  12. 权利要求11的治疗性T细胞，其中所述治疗性T细胞中的TGFβ受体被敲低或敲除。
  13. 权利要求11或12的治疗性T细胞，其中所述治疗性T细胞能够在体外在约0.2:1至约0.00625:1，例如约0.2:1、约0.1:1、约0.05:1、约0.025:1、约0.0125:1、约0.00625:1的效靶比下特异性裂解表达EGFR的肿瘤细胞。
  14. 权利要求11-13中任一项的治疗性T细胞在制备用于治疗EGFR相关癌症的药物中的用途。
  15. 一种用于在对象中治疗EGFR相关癌症的药物组合物，其包含治疗有效量的权利要求11-13中任一项的治疗性T细胞，以及药学上可接受的载体。
  16. 一种用于治疗EGFR相关癌症的方法，包括给有需要的对象施用治疗有效量的权利要求11-13中任一项的治疗性T细胞或权利要求15的药物组合物。
  17. 权利要求16的方法，所述方法还进一步包括给所述对象施用放疗和/或化疗和/或另外的肿瘤靶向药物和/或免疫疗法。
  18. 权利要求14的用途、权利要求15的药物组合物或权利要求16-17中任一项的方法，其中所述EGFR相关癌症选自食管癌、胃癌、结肠癌、直肠癌、结直肠癌、胰腺癌、肺癌(包括非小细胞肺癌NSCLC)、乳腺癌、子宫颈癌、子宫体癌、子宫内膜癌、卵巢癌、膀胱癌、头颈癌(包括头颈鳞状细胞癌SCCHN)、骨肉瘤、前列腺癌、神经母细胞 瘤、肾癌、神经胶质瘤、胶质母细胞瘤以及皮肤癌(包括上皮癌)。
  19. 多核苷酸，其包含编码权利要求1-10中任一项的CAR的核苷酸序列。
  20. 权利要求19的多核苷酸，其包含选自SEQ ID NO:66-71的核苷酸序列。
  21. 表达构建体，其包含与调控序列可操作连接的权利要求19或20的多核苷酸。
  22. 一种制备权利要求11-13中任一项的治疗性T细胞的方法，所述方法包括以下步骤：
    a)提供分离的T细胞；
    b)向所述T细胞导入权利要求19或20的多核苷酸或权利要求21的表达构建体，由此使所述T细胞表达权利要求1-10中任一项的CAR。
  23. 权利要求22的方法，其中所述方法还包括步骤
    x)敲低或敲除所述T细胞中TGFβ受体。
  24. 权利要求22的方法，其中所述方法还包括步骤y)扩增所述T细胞。
  25. 一种试剂盒，其用于制备权利要求11-13中任一项的治疗性T细胞。
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