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WO2022007802A1 - T淋巴细胞及其应用 - Google Patents

T淋巴细胞及其应用 Download PDF

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Publication number
WO2022007802A1
WO2022007802A1 PCT/CN2021/104799 CN2021104799W WO2022007802A1 WO 2022007802 A1 WO2022007802 A1 WO 2022007802A1 CN 2021104799 W CN2021104799 W CN 2021104799W WO 2022007802 A1 WO2022007802 A1 WO 2022007802A1
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terminus
variable region
chain variable
light chain
connecting peptide
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PCT/CN2021/104799
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English (en)
French (fr)
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都晓龙
王保垒
彭亮
叶立军
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深圳市菲鹏生物治疗股份有限公司
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Priority to US18/014,753 priority Critical patent/US20230364136A1/en
Publication of WO2022007802A1 publication Critical patent/WO2022007802A1/zh

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K40/31—Chimeric antigen receptors [CAR]
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    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N2740/15021—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the present disclosure relates to the field of biopharmaceuticals, in particular, the present disclosure relates to T lymphocytes and applications thereof, and more particularly, the present disclosure relates to T lymphocytes, lentiviruses, transgenic lymphocytes, constructs, preparation of T lymphocytes or transgenic lymphocytes Methods, therapeutic compositions for treating cancer, and methods of increasing lymphocyte activity.
  • Hematological tumors are one of the ten most common malignant tumors in China, accounting for the sixth place in the incidence of tumors.
  • acute lymphoblastic leukemia mostly occurs in adolescents, and it is a malignant tumor with the highest morbidity and mortality among people under the age of 35.
  • B-ALL acute B lymphocytic leukemia
  • CAR-T19 treatment There are two main forms of relapse during CAR-T19 treatment. The first is that the patient loses CAR-T19 at an early stage and relapses. The other is that CAR-T19 is still present but CD19-leukemia appears. This one is treated with blinatumomab. It also appeared after that.
  • the present disclosure aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present disclosure proposes a T lymphocyte.
  • the T lymphocyte expresses a chimeric antigen receptor, and the chimeric antigen receptor includes: an extracellular region, and the extracellular region includes a first single-chain antibody, a second single-chain antibody, The first connecting peptide and the CD8 hinge region, the first single-chain antibody specifically recognizes the first antigen, the second single-chain antibody specifically recognizes the second antigen, and the first connecting peptide is arranged on the first single-chain antibody.
  • the first single chain antibody includes a first heavy chain variable region and a first light chain variable region and a second connecting peptide
  • the second connecting peptide is provided on the Between the first heavy chain variable region and the first light chain variable region
  • the second single chain antibody includes the second heavy chain variable region and the second light chain variable region and a third linking peptide
  • the first A triple linker peptide is disposed between the second heavy chain variable region and the second light chain variable region
  • the first linker peptide has a repeating amino acid sequence of GGGGS, the second linker peptide and the third linker peptide, respectively
  • the above-mentioned T lymphocytes may further include at least one of the following additional technical features:
  • the first single-chain antibody is CD19 single-chain antibody
  • the second single-chain antibody is CD22 single-chain antibody
  • the first antigen is CD19
  • the second antigen is CD22
  • the first heavy chain variable region is a CD19 heavy chain variable region
  • the first light chain variable region is a CD19 light chain variable region
  • the second heavy chain variable region is a CD22 heavy chain variable region
  • the second light chain variable region is the CD22 light chain variable region.
  • the first single-chain antibody is CD19 single-chain antibody
  • the second single-chain antibody is BCMA single-chain antibody
  • the first antigen is CD19
  • the second antigen is BCMA
  • the The first heavy chain variable region is a CD19 heavy chain variable region
  • the first light chain variable region is a CD19 light chain variable region
  • the second heavy chain variable region is a BCMA heavy chain variable region
  • the second light chain variable region is a BCMA light chain variable region.
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the second single-chain antibody, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first single-chain antibody , the C-terminus of the first single-chain antibody is connected to the N-terminus of the CD8 hinge region.
  • the inventors found that the second single-chain antibody, the first connecting peptide and the first single-chain antibody in the above-mentioned connecting sequence, the secretion of cytokines, such as IL-2, IFN- ⁇ , is lower, according to the embodiment of the present disclosure. In vivo treatment of T lymphocytes is safer.
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the CD22 single-chain antibody, and the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 single-chain antibody, so The C-terminus of the CD19 single chain antibody is linked to the N-terminus of the CD8 hinge region.
  • the above-mentioned T lymphocytes according to the embodiments of the present disclosure can specifically kill single/double positive tumor cells of CD19 and CD22, have lower cytokine secretion in vivo, and have a longer and more powerful effect in vivo compared with the prior art. Blood tumor cell killing and clearance effects.
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the CD19 single-chain antibody, and the C-terminus of the first connecting peptide is connected to the N-terminus of the CD22 single-chain antibody, so The C-terminus of the CD22 single-chain antibody is linked to the N-terminus of the CD8 hinge region.
  • the above-mentioned T lymphocytes according to the embodiments of the present disclosure can specifically kill single/double positive tumor cells of CD19 and CD22, and have a longer and more powerful killing and clearing effect of blood tumor cells in vivo compared with the prior art .
  • the N-terminus of the third linking peptide is connected to the C-terminus of the BCMA heavy chain variable region, and the C-terminus of the third linking peptide is connected to the N-terminus of the BCMA light chain variable region , the C-terminus of the BCMA light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region, and the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region.
  • the N-terminus of the double linking peptide is connected to the C-terminus of the CD19 light chain variable region
  • the C-terminus of the second linking peptide is connected to the N-terminus of the CD19 heavy chain variable region
  • the CD19 heavy chain variable region is linked to the N-terminus of the CD8 hinge region.
  • the N-terminus of the third linking peptide is connected to the C-terminus of the CD22 heavy chain variable region, and the C-terminus of the third linking peptide is connected to the N-terminus of the CD22 light chain variable region , the C-terminus of the CD22 light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region, and the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region.
  • the N-terminus of the double linking peptide is connected to the C-terminus of the CD19 light chain variable region
  • the C-terminus of the second linking peptide is connected to the N-terminus of the CD19 heavy chain variable region
  • the CD19 heavy chain variable region The C-terminus of the region is linked to the N-terminus of the CD8 hinge region.
  • the N-terminus of the second connecting peptide is connected to the C-terminus of the CD19 light chain variable region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the CD19 heavy chain variable region , the C-terminus of the CD19 heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD22 heavy chain variable region, and the first connecting peptide is connected to the N-terminus of the CD22 heavy chain variable region.
  • the N-terminus of the three-linking peptide is connected to the C-terminus of the CD22 heavy chain variable region
  • the C-terminus of the third linking peptide is connected to the N-terminus of the CD22 light chain variable region
  • the CD22 light chain variable region is linked to the N-terminus of the CD8 hinge region.
  • the second linking peptide and the third linking peptide independently have 3-5 repeated amino acid sequences of GGGGS.
  • the second linking peptide and the third linking peptide respectively have five repeats of the amino acid sequence of GGGGS.
  • the inventors found that when the second linking peptide and the third linking peptide respectively have 5 repeated GGGGS, the killing effect of T lymphocytes according to the embodiment of the present disclosure is stronger, and after being introduced into the body, the secretion of cytokines in the body is lower, Higher security.
  • the extracellular region has the amino acid sequences shown in SEQ ID NOs: 1-5.
  • the amino acid sequence shown in SEQ ID NO:1 is the sequence of the extracellular region of the chimeric antigen receptor expressed by Car-pCDHF32 (molecular structure shown in Figure 1)
  • the amino acid sequence shown in SEQ ID NO:2 is The sequence of the extracellular region of the chimeric antigen receptor expressed by Car-pCDHF34 (molecular structure shown in Figure 2)
  • the amino acid sequence shown in SEQ ID NO: 3 is the expression of Car-pCDHF31 (molecular structure shown in Figure 15)
  • the sequence of the extracellular region of the chimeric antigen receptor, the amino acid sequence shown in SEQ ID NO: 4 is Car-pCDHF58 (except that the second and third linking peptides are 5 repeating GGGGS, the rest of the structure is the same as the Car-pCDHF32 molecule.
  • sequence of the extracellular region of the expressed chimeric antigen receptor with the same structure is Car-pCDHF59 (except that the second and third linking peptides are 6-repeat GGGGS, the remaining structures are the same as The sequence of the extracellular region of the chimeric antigen receptor expressed by Car-pCDHF32 molecular structure.
  • the present disclosure proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule encoding a chimeric antigen receptor, and the chimeric antigen receptor includes: an extracellular region, the extracellular region includes a first single-chain antibody, a second single-chain antibody chain antibody, a first connecting peptide and a CD8 hinge region, the first single-chain antibody specifically recognizes the first antigen, the second single-chain antibody specifically recognizes the second antigen, and the first connecting peptide is arranged on the Between the first single-chain antibody and the second single-chain antibody, the first single-chain antibody includes a first heavy chain variable region and a first light chain variable region and a second connecting peptide, the second connecting peptide is provided Between the first heavy chain variable region and the first light chain variable region, the second single chain antibody comprises a second heavy chain variable region and a second light chain variable region and a third linking peptide, The third connecting peptide
  • the aforementioned T lymphocytes can be obtained, and the obtained T lymphocytes can specifically kill the single/double positive of the first antigen and the second antigen Compared with the existing technology, it has a longer and more powerful tumor cell killing and clearing effect in vivo.
  • the above lentivirus may further include at least one of the following additional technical features:
  • the first single-chain antibody is CD19 single-chain antibody
  • the second single-chain antibody is CD22 single-chain antibody
  • the first antigen is CD19
  • the second antigen is CD22
  • the first heavy chain variable region is a CD19 heavy chain variable region
  • the first light chain variable region is a CD19 light chain variable region
  • the second heavy chain variable region is a CD22 heavy chain variable region
  • the second light chain variable region is the CD22 light chain variable region.
  • the first single-chain antibody is CD19 single-chain antibody
  • the second single-chain antibody is BCMA single-chain antibody
  • the first antigen is CD19
  • the second antigen is BCMA
  • the The first heavy chain variable region is a CD19 heavy chain variable region
  • the first light chain variable region is a CD19 light chain variable region
  • the second heavy chain variable region is a BCMA heavy chain variable region
  • the second light chain variable region is a BCMA light chain variable region.
  • the N-terminus of the third linking peptide is connected to the C-terminus of the CD22 heavy chain variable region, and the C-terminus of the third linking peptide is connected to the N-terminus of the CD22 light chain variable region , the C-terminus of the CD22 light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region, and the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region.
  • the N-terminus of the double linking peptide is connected to the C-terminus of the CD19 light chain variable region
  • the C-terminus of the second linking peptide is connected to the N-terminus of the CD19 heavy chain variable region
  • the CD19 heavy chain variable region is linked to the N-terminus of the CD8 hinge region.
  • the above-mentioned lentivirus is introduced into T lymphocytes, and the obtained T lymphocytes can specifically kill CD19 and CD22 single/double positive tumor cells, and the secretion of cytokines in vivo is lower. Compared with the prior art, in It has a longer and more powerful blood tumor cell killing and clearing effect in the body.
  • the N-terminus of the second connecting peptide is connected to the C-terminus of the CD19 light chain variable region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the CD19 heavy chain variable region , the C-terminus of the CD19 heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD22 heavy chain variable region, and the first connecting peptide is connected to the N-terminus of the CD22 heavy chain variable region.
  • the N-terminus of the three-linking peptide is connected to the C-terminus of the CD22 heavy chain variable region
  • the C-terminus of the third linking peptide is connected to the N-terminus of the CD22 light chain variable region
  • the CD22 light chain variable region The C-terminus of the region is linked to the N-terminus of the CD8 hinge region.
  • the above lentivirus is introduced into T lymphocytes, and the obtained T lymphocytes can specifically kill CD19 and CD22 single/double positive tumor cells. Compared with the prior art, the obtained T lymphocytes have longer and more potent in vivo blood tumor cell killing and clearance effects.
  • the N-terminus of the third linking peptide is connected to the C-terminus of the BCMA heavy chain variable region, and the C-terminus of the third linking peptide is connected to the N-terminus of the BCMA light chain variable region , the C-terminus of the BCMA light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region, and the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region.
  • the N-terminus of the double linking peptide is connected to the C-terminus of the CD19 light chain variable region
  • the C-terminus of the second linking peptide is connected to the N-terminus of the CD19 heavy chain variable region
  • the CD19 heavy chain variable region is linked to the N-terminus of the CD8 hinge region.
  • the nucleic acid molecule encoding the extracellular region has the nucleotide sequence shown in any one of SEQ ID NOs: 6-10.
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:6 encodes the extracellular region of the chimeric antigen receptor with the amino acid sequence shown in SEQ ID NO:1; the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:7
  • the nucleic acid molecule of the sequence encodes the extracellular region of the chimeric antigen receptor with the amino acid sequence shown in SEQ ID NO:2;
  • the nucleic acid molecule encoding the nucleotide sequence shown in SEQ ID NO:8 has the nucleotide sequence shown in SEQ ID NO:3
  • the extracellular region of the chimeric antigen receptor of the amino acid sequence, the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:9 encodes the extracellular region of the chimeric antigen receptor with the amino acid sequence shown in SEQ ID NO:4,
  • the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:10 encodes
  • the nucleic acid molecule encoding the transmembrane region has the nucleotide sequence shown in SEQ ID NO: 11.
  • the nucleic acid molecule encoding the intracellular region has the nucleotide sequence shown in SEQ ID NO: 12.
  • the nucleic acid molecule encoding the chimeric antigen receptor has the nucleotide sequences shown in SEQ ID NOs: 13-17.
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:13 encodes the chimeric antigen receptor expressed by Car-pCDHF32 (molecular structure shown in Figure 1), and has the nucleoside shown in SEQ ID NO:14
  • the nucleic acid molecule of the acid sequence encodes the chimeric antigen receptor expressed by Car-pCDHF34 (molecular structure shown in Figure 2)
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO: 15 encodes Car-pCDHF31 (the molecular structure is shown in Figure 2).
  • nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO: 16 encodes the chimeric antigen receptor expressed by Car-pCDHF58, with the chimeric antigen receptor shown in SEQ ID NO: 17
  • the nucleic acid molecule of the nucleotide sequence encodes the chimeric antigen receptor expressed by Car-pCDHF59.
  • the present disclosure proposes a lentivirus.
  • the lentivirus carries nucleic acid molecules having the nucleotide sequences shown in SEQ ID NOs: 18-22. After the lentivirus according to the embodiment of the present disclosure is introduced into recipient cells-T lymphocytes, the obtained T lymphocytes can specifically kill the single/double positive tumor cells of the first antigen and the second antigen, and compared with the prior art , has a longer and more powerful tumor cell killing and clearing effect in vivo.
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:18 expresses the Car-pCDHF32 (molecular structure shown in Figure 1) chimeric antigen receptor, and has the nucleotide sequence shown in SEQ ID NO:19
  • the nucleic acid molecule expresses Car-pCDHF34 (molecular structure shown in Figure 2) chimeric antigen receptor
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO: 20 expresses Car-pCDHF31 (molecular structure shown in Figure 15 ) ) chimeric antigen receptor
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:21 expresses the Car-pCDHF58 chimeric antigen receptor
  • the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:22 expresses Car-pCDHF59 chimeric antigen receptor.
  • the present disclosure provides a transgenic lymphocyte.
  • the lymphocyte expresses a chimeric antigen receptor, and the chimeric antigen receptor includes: an extracellular region, and the extracellular region includes a first single-chain antibody, a second single-chain antibody, and a first single-chain antibody.
  • the first single-chain antibody specifically recognizes the first antigen
  • the second single-chain antibody specifically recognizes the second antigen
  • the first linking peptide is disposed between the first single-chain antibody and the second single-chain antibody
  • the first single-chain antibody includes a first heavy chain variable region and a first light chain variable region and a second connecting peptide
  • the second connecting peptide is disposed on the first heavy chain can be Between the variable region and the first light chain variable region
  • the second single-chain antibody includes a second heavy chain variable region and a second light chain variable region and a third connecting peptide
  • the third connecting peptide is provided in Between the second heavy chain variable region and the second light chain variable region
  • the first linking peptide has a repeating amino acid sequence of GGGGS
  • the second linking peptide and the third linking peptide independently have 2- The amino acid sequence of 6 repeated GGGGS; the transmembrane region, the transmembrane region is connected with the extracellular region, and is embedded in the cell
  • the above-mentioned transgenic lymphocytes may further include the following additional technical features:
  • the first single-chain antibody is CD19 single-chain antibody
  • the second single-chain antibody is CD22 single-chain antibody
  • the first antigen is CD19
  • the second antigen is CD22
  • the first heavy chain variable region is a CD19 heavy chain variable region
  • the first light chain variable region is a CD19 light chain variable region
  • the second heavy chain variable region is a CD22 heavy chain variable region
  • the second light chain variable region is the CD22 light chain variable region.
  • the first single-chain antibody is CD19 single-chain antibody
  • the second single-chain antibody is BCMA single-chain antibody
  • the first antigen is CD19
  • the second antigen is BCMA
  • the The first heavy chain variable region is a CD19 heavy chain variable region
  • the first light chain variable region is a CD19 light chain variable region
  • the second heavy chain variable region is a BCMA heavy chain variable region
  • the second light chain variable region is a BCMA light chain variable region.
  • the intracellular segment of the immunostimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28, and derivatives thereof.
  • the intracellular segment of the immune costimulatory molecule is the intracellular segment of 4-1BB, CD3.
  • the lymphocytes are CD3 + T lymphocytes.
  • the lymphocytes are CD8 + T lymphocytes.
  • the lymphocytes are natural killer cells.
  • the lymphocytes are natural killer T cells.
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the two single-chain antibody, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first single-chain antibody,
  • the C-terminus of the first single-chain antibody is linked to the N-terminus of the CD8 transmembrane region.
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the CD22 single-chain antibody, and the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 single-chain antibody, so The C-terminus of the CD19 single-chain antibody is connected to the N-terminus of the transmembrane region.
  • the above-mentioned transgenic lymphocytes according to the embodiments of the present disclosure can specifically kill single/double positive tumor cells of CD19 and CD22, have lower cytokine secretion in vivo, and have longer and more potent effects in vivo compared with the prior art. Blood tumor cell killing and clearance effects.
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the CD19 single-chain antibody, and the C-terminus of the first connecting peptide is connected to the N-terminus of the CD22 single-chain antibody, so The C-terminus of the CD22 single-chain antibody is connected to the N-terminus of the transmembrane region.
  • the above-mentioned transgenic lymphocytes according to the embodiments of the present disclosure can specifically kill single/double positive tumor cells of CD19 and CD22, and have a longer and more powerful killing and clearing effect of blood tumor cells in vivo compared with the prior art .
  • the N-terminus of the third linking peptide is connected to the C-terminus of the CD22 heavy chain variable region, and the C-terminus of the third linking peptide is connected to the N-terminus of the CD22 light chain variable region , the C-terminus of the CD22 light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region, and the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region.
  • the N-terminus of the double linking peptide is connected to the C-terminus of the CD19 light chain variable region
  • the C-terminus of the second linking peptide is connected to the N-terminus of the CD19 heavy chain variable region
  • the CD19 heavy chain variable region The C-terminus of the region is linked to the N-terminus of the transmembrane region.
  • the N-terminus of the second connecting peptide is connected to the C-terminus of the CD19 light chain variable region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the CD19 heavy chain variable region , the C-terminus of the CD19 heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD22 heavy chain variable region, and the first connecting peptide is connected to the N-terminus of the CD22 heavy chain variable region.
  • the N-terminus of the three-linking peptide is connected to the C-terminus of the CD22 heavy chain variable region
  • the C-terminus of the third linking peptide is connected to the N-terminus of the CD22 light chain variable region
  • the CD22 light chain variable region is linked to the N-terminus of the transmembrane region.
  • the N-terminus of the third linking peptide is connected to the C-terminus of the BCMA heavy chain variable region, and the C-terminus of the third linking peptide is connected to the N-terminus of the BCMA light chain variable region , the C-terminus of the BCMA light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region, and the first connecting peptide is connected to the N-terminus of the CD19 light chain variable region.
  • the N-terminus of the double linking peptide is connected to the C-terminus of the CD19 light chain variable region
  • the C-terminus of the second linking peptide is connected to the N-terminus of the CD19 heavy chain variable region
  • the CD19 heavy chain variable region is linked to the N-terminus of the CD8 hinge region.
  • the second linking peptide and the third linking peptide independently have 3-5 repeated amino acid sequences of GGGGS.
  • the second linker peptide and the third linker peptide have 5 repeats of the amino acid sequence of GGGGS.
  • the second linking peptide and the third linking peptide respectively have five repeats of the amino acid sequence of GGGGS.
  • the inventors found that when the second linking peptide and the third linking peptide respectively have 5 repeated GGGGS, the killing effect of the transgenic lymphocytes according to the embodiment of the present disclosure is stronger, and the secretion of cytokines in the body is lower after being introduced into the body, Higher security.
  • the extracellular region has the amino acid sequences shown in SEQ ID NOs: 1-3.
  • the present disclosure proposes a construct.
  • the construct comprises a nucleic acid molecule encoding a chimeric antigen receptor as previously defined.
  • the above-mentioned transgenic lymphocytes can be obtained by introducing the constructs according to the embodiments of the present disclosure into recipient cells-T lymphocytes, and the obtained transgenic lymphocytes can specifically kill the single/double positive of the first antigen and the second antigen Compared with the existing technology, it has a longer and more powerful tumor cell killing and clearing effect in vivo.
  • the above-mentioned construct may further include at least one of the following additional technical features:
  • the construct further comprises: a promoter operably linked to the nucleic acid molecule.
  • the promoter is a U6, H1, CMV, EF-1, LTR or RSV promoter.
  • the vector of the construct is a non-pathogenic viral vector
  • the viral vector includes at least one selected from a retroviral vector, a lentiviral vector, and an adeno-associated viral vector.
  • the present disclosure provides a method for preparing the aforementioned T lymphocytes or the aforementioned transgenic lymphocytes.
  • the method comprises: introducing the aforementioned construct or the aforementioned lentivirus into lymphocytes or T lymphocytes.
  • the T lymphocytes or transgenic lymphocytes prepared according to the methods of the embodiments of the present disclosure can specifically kill the single/double positive tumor cells of the first antigen and the second antigen, and have a longer duration in vivo compared with the prior art , More powerful tumor cell killing and removal effect.
  • the present disclosure proposes a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: the aforementioned construct, the aforementioned lentivirus, the aforementioned T lymphocyte, or the aforementioned transgenic lymphocyte.
  • the pharmaceutical compositions according to the embodiments of the present disclosure have longer and more powerful tumor cell killing and removal effects in vivo.
  • the cancer includes at least one selected from the group consisting of B-lymphocytic leukemia and B-cell lymphoma.
  • the pharmaceutical composition according to the embodiments of the present disclosure can be applied to the immunotherapy of patients with B lymphocytic leukemia and B cell lymphoma; for patients with B lymphocytic leukemia and B cell lymphoma, the immune cell therapy according to the embodiments of the present disclosure has excellent curative effect
  • the dual CART cells in the present disclosure greatly promote the possibility of relapse in patients with B lymphocytic leukemia and B cell lymphoma, and play a very important role in the treatment of B lymphocytic leukemia and B cell lymphoma. greatly promoted.
  • the present disclosure provides that the aforementioned T lymphocytes, the aforementioned lentiviruses, the aforementioned transgenic lymphocytes, the aforementioned constructs, or the aforementioned therapeutic compositions are in Use in the preparation of a medicament for the treatment of cancer.
  • the cancer includes at least one selected from the group consisting of B-lymphocytic leukemia and B-cell lymphoma.
  • the T lymphocytes, lentiviruses, transgenic lymphocytes, constructs or therapeutic compositions according to the embodiments of the present disclosure can be used as components of drugs for treating cancer, and can be used in the immunotherapy of patients with B lymphocytic leukemia and B cell lymphoma .
  • the present disclosure provides a method of treating cancer.
  • the method comprises administering to a subject with cancer at least one of:
  • T lymphocytes introduced with the aforementioned lentivirus
  • transgenic lymphocytes described above;
  • T lymphocytes introduced with the previously described construct
  • the cancer includes at least one selected from the group consisting of B-lymphocytic leukemia and B-cell lymphoma.
  • the present disclosure provides that the aforementioned T lymphocytes, the aforementioned lentiviruses, the aforementioned transgenic lymphocytes, the aforementioned constructs, or the aforementioned therapeutic compositions are in Use in the treatment of cancer.
  • the cancer includes at least one selected from the group consisting of B-lymphocytic leukemia and B-cell lymphoma.
  • the present disclosure provides a method for improving lymphocyte activity.
  • the method comprises: causing the lymphocyte to express a chimeric antigen receptor, the chimeric antigen receptor as defined above, the lymphocyte activity comprising the lymphocyte in a tumor At least one of the viability in the patient and the killing ability of the lymphocytes in the tumor patient.
  • Fig. 2 is a molecular structure diagram of Car-pCDHF34 according to an embodiment of the present disclosure
  • FIG. 3 is a map of the Car-pCDHF32 and Car-pCDHF34 plasmids according to an embodiment of the present disclosure
  • FIG. 4 is a schematic diagram of the working principle of pCDHF-32 cells according to an embodiment of the present disclosure
  • FIG. 5 is a result diagram of the positive rate of Car-pCDHF-32 and 34 detected by flow cytometry according to an embodiment of the present disclosure
  • FIG. 6 is a graph showing the kurtosis detection of CD19 and CD22 antigen expression on the surface of Nalm6 and Raji cells according to an embodiment of the present disclosure
  • A is the killing effect of Car-pCDHF32 and Car-pCDHF34 on K562/K562-CD19/K562-CD22/Nalm6/Raji cells;
  • Fig. 9 is a graph comparing the in vitro tumoricidal effect of Car-pCDHF32 with single-target CarT and Loop 6 Car according to an embodiment of the present disclosure
  • FIG. 10 is a molecular structure diagram of pCDHF-60 according to an embodiment of the present disclosure.
  • FIG. 11 is a graph showing the results of using fluorescent antigen staining to detect the positive rates of CarT-pCDHF32 and CarT-pCDHF60 obtained after lentivirus packaging and infecting T cells according to an embodiment of the present disclosure
  • Figure 12 is a graph of the results of the in vitro killing function of Car-pCDHF32 and Car-pCDHF60 according to an embodiment of the present disclosure, wherein,
  • A is the killing effect of Car-pCDHF32 and Car-pCDHF60 on K562/Nalm6 cells
  • B is the cytokine secretion in the killing experiment of Car-pCDHF32 and Car-pCDHF60 on K562/Nalm6 cells;
  • FIG. 13 is a schematic diagram of an in vivo functional comparison experiment scheme of CarT-pCDHF32 and CarT-pCDHF60 according to an embodiment of the present disclosure
  • Figure 14 is the results of the in vivo function comparison experiment of Car-pCDHF32 and Car-pCDHF60 according to the embodiment of the present disclosure, wherein, A is the monitoring data of body weight change of Car-pCDHF32 and Car-pCDHF60 in vivo experimental mice;
  • B is the survival curve of Car-pCDHF32 and Car-pCDHF60 mice in vivo;
  • Figure 15 is a molecular structure of Car-pCDHF31 according to an embodiment of the present disclosure.
  • 16 is a graph showing the killing effect of Car-pCDHF31 and Car-pCDHF60 on K562/K562-CD19/K562-BCMA/Daudi cells according to an embodiment of the present disclosure
  • Figure 17 shows the cytokine secretion in the killing experiment of K562/K562-CD19/K562-BCMA/Daudi cells by Car-pCDHF31 according to an embodiment of the present disclosure
  • Figure 18 is the in vitro killing effect of GGGGS in which the second and third linking peptides are 3-6 repeats in the bispecific CART structure according to an embodiment of the present disclosure.
  • T cell/T-cell refers to T cells; medium refers to culture medium; Linker refers to linker peptide; Linker annotation refers to linker peptide annotation.
  • Embodiments of the present disclosure are described in detail below.
  • the embodiments described below are exemplary and are only used to explain the present disclosure, and should not be construed as a limitation of the present disclosure. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used.
  • the reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless expressly and specifically defined otherwise.
  • a single-chain antibody is a genetically engineered antibody in which the VH and VL domains are linked to a flexible polypeptide linker. Compared to the Fab regions of monolithic antibodies, single-chain antibodies exhibit better tissue penetration pharmacokinetics and have full antigen-binding specificity due to the unaltered antigen-binding surface.
  • Immune costimulatory molecules refer to cell surface molecules and their ligands that provide costimulatory signals for the complete activation of T lymphocytes or B lymphocytes, such as 4-1BB, OX-40, CD40L, CD27, CD30, CD28, CD3 and their derivative.
  • the inventors insert the target nucleic acid into the viral genome at the position of certain viral sequences in order to construct a lentiviral vector, thereby producing a replication-deficient virus.
  • the inventors proceeded to construct a packaging cell line (containing the gag, pol and env genes, but excluding the LTR and packaging components).
  • the inventors introduced the recombinant plasmid containing the gene of interest, along with the lentiviral LTR and packaging sequence, into a packaging cell line.
  • Packaging sequences allow recombinant plasmid RNA transcripts to be packaged into viral particles and then secreted into the culture medium.
  • the inventors further collected the matrix containing the recombinant lentivirus, selectively concentrated, and used it for gene transfer.
  • Lentiviral vectors can infect a variety of cell types, including dividing and non-dividing cells.
  • the lentiviruses of the embodiments of the present disclosure are composite lentiviruses, in addition to the common lentiviral genes gag, pol and env, other genes with regulatory and structural functions are also included.
  • Lentiviral vectors are well known to those skilled in the art and include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors are produced by multiple attenuation of HIV pathogenic genes, such as all deletion of genes env, vif, vpr, vpu and nef, so that lentiviral vectors form biosafety vectors.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for gene transfer and nucleic acid sequence expression in vivo and in vitro.
  • suitable host cells together with two or more vectors with packaging functions (gag, pol, env, rev and tat)
  • non-dividing cells can be infected.
  • the targeting of recombinant viruses is achieved through the binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus enables the vector to have specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, together with another gene encoding a ligand for a receptor on a specific target cell.
  • Various useful lentiviral vectors, as well as vectors produced by various methods and manipulations, are used to alter expression in cells.
  • adeno-associated viral vectors of embodiments of the present disclosure can be constructed using the DNA of one or more well-known serotype adeno-associated viral vectors.
  • the embodiments of the present disclosure also include minigenes.
  • a minigene means that a combination (a selected nucleotide sequence and operably necessary associated linker sequences) is used to direct transformation, transcription and/or expression of a gene product in a host cell in vivo or in vitro.
  • An "operably linked" sequence is used to encompass expression control sequences that contiguously control the gene of interest, and expression control sequences that act to control the gene of interest in trans or at a distance.
  • the vectors of embodiments of the present disclosure also include conventional control elements in cell transfection with plasmid vectors or/and cell infection with viral vectors.
  • a wide variety of expression control sequences may be used.
  • the promoter is an RNA polymerase promoter selected from U6, H1, pol I, pol II and pol III.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from a selected vector-based promoter.
  • the promoter when a lentiviral vector is selected, the promoter is the U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C or phosphoglycerol kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance, and the like.
  • Other components of the vector include origins of replication.
  • compositions of the embodiments of the present disclosure provided to patients are preferably applied to biocompatible solutions or acceptable pharmaceutical carriers.
  • the various therapeutic compositions as prepared are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier such as physiological saline; isotonic saline or other formulations obvious to those skilled in the art.
  • a pharmaceutically or physiologically acceptable carrier such as physiological saline; isotonic saline or other formulations obvious to those skilled in the art.
  • physiological saline such as physiological saline; isotonic saline or other formulations obvious to those skilled in the art.
  • the appropriate carrier will largely depend on the route of administration.
  • Other aqueous and anhydrous isotonic sterile injectable solutions and aqueous and anhydrous sterile suspensions are pharmaceutically acceptable carriers.
  • a method of treatment includes the use of one or more drug therapies.
  • a novel dual-Car structure targeting CD19 and CD22 targets is obtained by optimizing the sequence of the light and heavy chains of the tandem CD19 and CD22 single-chain antibody sequences and the lengths of their interconnected linking peptides.
  • the present application can prepare CarT cells with a high positive rate, and has high specificity and good killing effect on CD19 and CD22 target cells. And this structure can be used for other targets to construct dual-target CarT, which has good versatility.
  • the B lymphocyte antigen CD19 also known as CD19, is a single-pass type I membrane protein containing two Ig-like C2-type (immunoglobulin-like) domains.
  • the scFv sequence-selecting antibody targeting CD19, FMC63-mIgG2a is a murine antibody obtained by immunizing animals in the last century against CD19, and its binding epitope is the Ig domain at the far membrane end of CD19.
  • FMC63-mIgG2a scFv has been successfully applied to anti-CD19 CAR constructs, such as Norvatis CTL019 and Juno Therapeutics JCAR015.
  • the clinical trials of CTL019 and JCAR015 in B-cell acute lymphoblastic leukemia achieved good results, and the proportion of patients with complete remission was >70%. Its light and heavy chain variable region sequences are as follows:
  • CD22 is a type I transmembrane glycoprotein and a member of the sialic acid-binding immunoglobulin-like lectin family. As an inhibitory co-receptor of the B-cell receptor (BCR), CD22 negatively regulates B-cell activation signaling, and its extracellular portion includes seven immunoglobulin domains.
  • BCR B-cell receptor
  • M971-mIgG is a humanized antibody obtained through antibody library screening and has been widely used in CarT targeting CD22. For example, the clinical data reported by Juno Therapeutics JCAR018 in ASH 2017 showed that the proportion of R/R ALL patients with complete remission was 78%. Its light and heavy chain variable region sequences are as follows:
  • the ratio of cells in the upper left quadrant is the positive rate of anti-CD19scFv and anti-CD22scFv, that is, the positive rate of pCDHF-32/pCDHF-34.
  • the flow cytometry results in the figure show that the positive rates of both pCDHF-32 and pCDHF-34 are over 70%, which indicates that the bispecific CART with this structure can produce cell preparations with high positive rates.
  • Nalm6 cells are acute lymphoblastic leukemia cells, and Raji cells are black Burkitt lymphoma cells.
  • the kurtosis of CD19 and CD22 antigen expression on the cell membrane surface was detected by flow cytometry.
  • the specific method is as follows: Take 5E+05 cancer cells and flow antibody at 4°C After incubation for 20 min (FITC anti-human CD22 (BD Pharmingen/555424) to detect the kurtosis of cell surface CD22 antigen expression, APC anti-human CD19 (Biolegend/302212) to detect the kurtosis of cell surface CD19 antigen expression), wash once with PBS and resuspend The cells were detected by flow cytometry, the results shown in Figure 6, Nalm6 cells were CD19 + /CD22 + cell line.
  • target cells Take the target cells as K562, K562-CD19, K562-CD22, Nalm6 (or Raji) 1E+07 cells for each of the four target cells, first use cytocalceinTM violet 550 to stain the target cells, 1*10E+05 cells/100 ⁇ l/well ; Effector cells (CarT V9/CarT M971/Car-pCDHF32/Car-pCDHF34, T cells as control) and target cells were added to 96-well plates at 0.25:1, 1:1, 5:1 and 10:1 and mixed. The final volume was 200 ⁇ l. After co-cultivation for 6 hours, the cells were mixed and centrifuged.
  • the supernatant was detected by Human IL-2 and Human IFN gamma ELISA kits to detect IL-2 and IFN- ⁇ , and the precipitated part was treated with 100 ⁇ l binding buffer. Resuspend, centrifuge at 300g for 5 min, add 2.0 ⁇ l APC-Annexin V and 1.5 ⁇ l PI dye, incubate in the dark for 15 min, add 100 ⁇ l binding buffer to resuspend, and detect the apoptosis ratio of each target cell by Beckmanc cou LTER flow cytometer as shown in Figure 7 As shown in Figure 8, the IL-2 and IFN- ⁇ concentrations in the supernatant of each well were detected by ELISA.
  • K562 is CD19 and CD22 negative cells
  • K562-CD19 and K562-CD22 are CD19 and CD22 single-positive cells, respectively
  • Raji and Nalm6 are both CD19 and CD22 double-positive cells.
  • K562-CD22, Raji and Nalm6 all have good killing effect, and the killing effect of Car-pCDHF32 is still slightly better than that of Car-pCDHF34 under the condition that Car-pCDHF34 has higher autocrine cytokines (see Figures 7 and 7). 8). It can be seen from the comparison of the in vitro tumor killing effect of Car-pCDHF32 and single-target CarT that the killing effect of Car-pCDHF32 is close to that of CD19 single-target CarT.
  • the positive rate of pCDHF60 was detected by fluorescent antigen staining as shown in Figure 11.
  • the CD19 and BCMA dual-target CarT was constructed with the linker structure in the present disclosure, wherein the anti-BCMA scFv sequence part uses the weight of the C11D5.3 antibody
  • the chain part whose light and heavy chain variable region sequences are as follows:
  • Daudi cells are human Burkitt's lymphoma cells (CD19 + /BCMA + ), K562/K562-CD19/K562-BCMA/Daudi cells were used as target cells, and the results of in vitro killing activity of CarT-pCDHF31 were verified by the method in Example 4, as shown in Figure 16 and Figure 17.
  • the double CarT using the linker structure in the present disclosure still retains good functional activity. Therefore, it can be proved that the double Car of the linker structure in the present disclosure has the universality and versatility of target replacement.
  • CarT-pCDHF32 is a double Car whose second and third linking peptides are the amino acid sequence of GGGGS with 3 repeats
  • CarT-pCDHF58 is a double Car whose second and third linking peptides are the amino acid sequence of GGGGS with 5 repeats
  • CarT - pCDHF59 is a double Car with the second and third linker peptides being the amino acid sequence of 6 repeats of GGGGS.

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Abstract

提供一种T淋巴细胞，该T淋巴细胞表达嵌合抗原受体，其包括：胞外区，所述胞外区包括第一单链抗体、第二单链抗体、第一连接肽以及CD8铰链区，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列。

Description

T淋巴细胞及其应用
优先权信息
本申请请求2020年07月07日向中国国家知识产权局提交的、专利申请号为202010647746.7的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开涉及生物制药领域，具体地，本公开涉及T淋巴细胞及其应用，更具体地，本公开涉及T淋巴细胞、慢病毒、转基因淋巴细胞、构建体、制备T淋巴细胞或者转基因淋巴细胞的方法、治疗癌症的治疗组合物以及提高淋巴细胞活性的方法。
背景技术
血液肿瘤是中国十大高发恶性肿瘤之一，占肿瘤发病率的第六位。尤其是急性淋巴细胞白血病多发于青少年，是35岁以下人群发病率、病死率最高的恶性肿瘤，其中B-ALL(急性B淋巴细胞白血病)最为常见。
有研究表明，大约90％的R/R B-ALL病人在接受CAR-T19(表达靶向CD19 CAR的淋巴细胞)后获得完全缓解(CR)，尽管有起始响应率很高，但是很多病人出现复发，超过30％的使用Blinatumomab治疗的复发的病人和超过60％的使用过CAR-T19的复发病人为CD19抗原靶点丢失，致使CD19特异性免疫疗法无法识别恶性细胞。这些现象同时也解释了抗原特异性免疫疗法的优势与劣势。
在CAR-T19治疗过程中复发主要有两种形式，第一种是病人早期丢失CAR-T19而出现复发，另一种是CAR-T19还在但出现了CD19-白血病，这一种在接受blinatumomab之后也出现了。
因此，CAR-T细胞仍需要进一步开发和改进。
发明内容
本公开旨在至少在一定程度上解决相关技术中的技术问题之一。
在本公开的第一方面，本公开提出了一种T淋巴细胞。根据本公开的实施例，所述T淋巴细胞表达嵌合抗原受体，所述嵌合抗原受体包括：胞外区，所述胞外区包括第一单链抗体、第二单链抗体、第一连接肽以及CD8铰链区，所述第一单链抗体特异性识别第一抗原，所述第二单链抗体特异性识别第二抗原，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连 接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列；跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；以及胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链。根据本公开实施例的T淋巴细胞，可特异性杀伤第一抗原和第二抗原的单/双阳性的肿瘤细胞，且相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，上述T淋巴细胞还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为CD22单链抗体，所述第一抗原为CD19，所述第二抗原为CD22，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为CD22重链可变区，所述第二轻链可变区为CD22轻链可变区。根据本公开实施例的T淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，
根据本公开的实施例，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为BCMA单链抗体，所述第一抗原为CD19，所述第二抗原为BCMA，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为BCMA重链可变区，所述第二轻链可变区为BCMA轻链可变区。
根据本公开的实施例，所述第一连接肽的N端与所述第二单链抗体的C端相连，所述第一连接肽的C端与所述第一单链抗体的N端相连，所述第一单链抗体的C端与所述CD8铰链区的N端相连。发明人发现，所述第二单链抗体、第一连接肽和第一单链抗体在上述连接顺序下，细胞因子，如IL-2，IFN-γ，分泌更低，根据本公开实施例的T淋巴细胞体内治疗更加安全。
根据本公开的实施例，所述第一连接肽的N端与所述CD22单链抗体的C端相连，所述第一连接肽的C端与所述CD19单链抗体的N端相连，所述CD19单链抗体的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述T淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，体内细胞因子分泌更低，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第一连接肽的N端与所述CD19单链抗体的C端相连，所述第一连接肽的C端与所述CD22单链抗体的N端相连，所述CD22单链抗体的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述T淋巴细胞，可特异性杀伤CD19和 CD22的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第三连接肽的N端与所述BCMA重链可变区的C端相连，所述第三连接肽的C端与BCMA轻链可变区的N端相连，所述BCMA轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述T淋巴细胞，可特异性杀伤BCMA和CD19的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述T淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，体内细胞因子分泌更低，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD22重链可变区的N端相连，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与所述CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述T淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第二连接肽和第三连接肽分别独立地具有3～5个重复的GGGGS的氨基酸序列。
根据本公开的实施例，所述第二连接肽和第三连接肽分别具有5个重复的GGGGS的氨基酸序列。发明人发现，当第二连接肽和第三连接肽分别具有5个重复的GGGGS时，根据本公开实施例的T淋巴细胞的杀伤效果更强，导入机体后，机体细胞因子分泌量更低，安全性更高。
根据本公开的实施例，所述胞外区具有SEQ ID NO:1～5所示的氨基酸序列。
Figure PCTCN2021104799-appb-000001
Figure PCTCN2021104799-appb-000002
Figure PCTCN2021104799-appb-000003
其中，SEQ ID NO:1所示的氨基酸序列是Car-pCDHF32(分子结构如图1所示)表达的嵌合抗原受体的胞外区的序列，SEQ ID NO:2所示的氨基酸序列是Car-pCDHF34(分子结构如图2所示)表达的嵌合抗原受体的胞外区的序列，SEQ ID NO:3所示的氨基酸序列是Car-pCDHF31(分子结构如图15所示)表达的嵌合抗原受体的胞外区的序列，SEQ ID NO:4所示的氨基酸序列是Car-pCDHF58(除第二和第三连接肽为5个重复GGGGS外，其余结构与Car-pCDHF32分子结构相同)表达的嵌合抗原受体的胞外区的序列，SEQ ID NO:5所示的氨基酸序列是Car-pCDHF59(除第二和第三连接肽为6个重复GGGGS外，其余结构与Car-pCDHF32分子结构相同)表达的嵌合抗原受体的胞外区的序列。
在本公开的第二方面，本公开提出了一种慢病毒。根据本公开的实施例，所述慢病毒携带编码嵌合抗原受体的核酸分子，所述嵌合抗原受体包括：胞外区，所述胞外区包括第一单链抗体、第二单链抗体、第一连接肽以及CD8铰链区，所述第一单链抗体特异性识别第一抗原，所述第二单链抗体特异性识别第二抗原，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列；跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；以及胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链。将根据本公开实施例的慢病毒导入受体细胞-T淋巴细胞，可获得前面所述的T淋巴细胞，所获得的T淋巴细胞可特异性杀伤第一抗原和第二抗原的单/双阳性的肿瘤细胞，且相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，上述慢病毒还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为CD22单链抗体，所述第一抗原为CD19，所述第二抗原为CD22，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为CD22重链 可变区，所述第二轻链可变区为CD22轻链可变区。
根据本公开的实施例，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为BCMA单链抗体，所述第一抗原为CD19，所述第二抗原为BCMA，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为BCMA重链可变区，所述第二轻链可变区为BCMA轻链可变区。
根据本公开的实施例，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述慢病毒导入T淋巴细胞，所获得的T淋巴细胞可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，体内细胞因子分泌更低，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD22重链可变区的N端相连，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与所述CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述慢病毒导入T淋巴细胞，所获得的T淋巴细胞可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第三连接肽的N端与所述BCMA重链可变区的C端相连，所述第三连接肽的C端与BCMA轻链可变区的N端相连，所述BCMA轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述慢病毒导入T淋巴细胞，所获得的T淋巴细胞可特异性杀伤CD19和BCMA的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，编码所述胞外区的核酸分子具有SEQ ID NO:6～10任一所示的核苷酸序列。
Figure PCTCN2021104799-appb-000004
Figure PCTCN2021104799-appb-000005
Figure PCTCN2021104799-appb-000006
Figure PCTCN2021104799-appb-000007
Figure PCTCN2021104799-appb-000008
其中，具有SEQ ID NO:6所示核苷酸序列的核酸分子编码具有SEQ ID NO:1所示氨基酸序列的嵌合抗原受体的胞外区；具有SEQ ID NO:7所示核苷酸序列的核酸分子编码具有SEQ ID NO:2所示氨基酸序列的嵌合抗原受体的胞外区；具有SEQ ID NO:8所示核苷酸序列的核酸分子编码具有SEQ ID NO:3所示氨基酸序列的嵌合抗原受体的胞外区，具有SEQ ID NO:9所示核苷酸序列的核酸分子编码具有SEQ ID NO:4所示氨基酸序列的嵌合抗原受体的胞外区，具有SEQ ID NO:10所示核苷酸序列的核酸分子编码具有SEQ ID NO:5所示氨基酸序列的嵌合抗原受体的胞外区。
根据本公开的实施例，编码所述跨膜区的核酸分子具有SEQ ID NO:11所示的核苷酸序列。
Figure PCTCN2021104799-appb-000009
根据本公开的实施例，编码所述胞内区的核酸分子具有SEQ ID NO:12所示的核苷酸序列。
Figure PCTCN2021104799-appb-000010
根据本公开的实施例，编码所述嵌合抗原受体的核酸分子具有SEQ ID NO:13～17所示的核苷酸序列。
Figure PCTCN2021104799-appb-000011
Figure PCTCN2021104799-appb-000012
Figure PCTCN2021104799-appb-000013
Figure PCTCN2021104799-appb-000014
Figure PCTCN2021104799-appb-000015
Figure PCTCN2021104799-appb-000016
其中，具有SEQ ID NO:13所示的核苷酸序列的核酸分子编码Car-pCDHF32(分子结构如图1所示)表达的嵌合抗原受体，具有SEQ ID NO:14所示的核苷酸序列的核酸分子编码Car-pCDHF34(分子结构如图2所示)表达的嵌合抗原受体，具有SEQ ID NO:15所示的核苷酸序列的核酸分子编码Car-pCDHF31(分子结构如图15所示)表达的嵌合抗原受体，具有SEQ ID NO:16所示的核苷酸序列的核酸分子编码Car-pCDHF58表达的嵌合抗原受体，具有SEQ ID NO:17所示的核苷酸序列的核酸分子编码Car-pCDHF59表达的嵌合抗原受体。
在本公开的第三方面，本公开提出了一种慢病毒。根据本公开的实施例，所述慢病毒携带具有SEQ ID NO：18～22所示的核苷酸序列的核酸分子。根据本公开实施例的慢病毒导入受体细胞-T淋巴细胞后，所获得的T淋巴细胞可特异性杀伤第一抗原和第二抗原的单/双阳性的肿瘤细胞，且相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
Figure PCTCN2021104799-appb-000017
Figure PCTCN2021104799-appb-000018
Figure PCTCN2021104799-appb-000019
Figure PCTCN2021104799-appb-000020
Figure PCTCN2021104799-appb-000021
Figure PCTCN2021104799-appb-000022
Figure PCTCN2021104799-appb-000023
其中，具有SEQ ID NO:18所示的核苷酸序列的核酸分子表达Car-pCDHF32(分子结构如图1所示)嵌合抗原受体，具有SEQ ID NO:19所示的核苷酸序列的核酸分子表达Car-pCDHF34(分子结构如图2所示)嵌合抗原受体，具有SEQ ID NO:20所示的核苷酸序列的核酸分子表达Car-pCDHF31(分子结构如图15所示)嵌合抗原受体，具有SEQ ID NO:21所示的核苷酸序列的核酸分子表达Car-pCDHF58嵌合抗原受体，具有SEQ ID NO:22所示的核苷酸序列的核酸分子表达Car-pCDHF59嵌合抗原受体。
在本公开的第四方面，本公开提出了一种转基因淋巴细胞。根据本公开的实施例，所述淋巴细胞表达嵌合抗原受体，所述嵌合抗原受体包括：胞外区，所述胞外区包括第一单链抗体、第二单链抗体以及第一连接肽，所述第一单链抗体特异性识别第一抗原，所述第二单链抗体特异性识别第二抗原，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述淋巴细胞的细胞膜中；以及胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段。根据本公开实施例的转基因淋巴细胞，可特异性杀伤第一抗原和第二抗原的单/双阳性的肿瘤细胞，且相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，上述转基因淋巴细胞还可以进一步包括如下附加技术特征：
根据本公开的实施例，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为CD22单链抗体，所述第一抗原为CD19，所述第二抗原为CD22，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为CD22重链可变区，所述第二轻链可变区为CD22轻链可变区。
根据本公开的实施例，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为BCMA单链抗体，所述第一抗原为CD19，所述第二抗原为BCMA，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为BCMA重链可变区，所述第二轻链可变区为BCMA轻链可变区。
根据本公开的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种。
根据本公开的实施例，所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段。
根据本公开的实施例，所述淋巴细胞是CD3 +T淋巴细胞。
根据本公开的实施例，所述淋巴细胞是CD8 +T淋巴细胞。
根据本公开的实施例，所述淋巴细胞是自然杀伤细胞。
根据本公开的实施例，所述淋巴细胞是自然杀伤T细胞。
根据本公开的实施例，所述第一连接肽的N端与所述二单链抗体的C端相连，所述第一连接肽的C端与所述第一单链抗体的N端相连，所述第一单链抗体的C端与所述CD8跨膜区的N端相连。发明人发现，所述第二单链抗体、第一连接肽和第一单链抗体在上述连接顺序下，细胞因子，如IL-2，IFN-γ，分泌更低，根据本公开实施例的转基因淋巴细胞体内治疗更加安全。
根据本公开的实施例，所述第一连接肽的N端与所述CD22单链抗体的C端相连，所述第一连接肽的C端与所述CD19单链抗体的N端相连，所述CD19单链抗体的C端与所述跨膜区的N端相连。根据本公开实施例的上述转基因淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，体内细胞因子分泌更低，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第一连接肽的N端与所述CD19单链抗体的C端相连，所述第一连接肽的C端与所述CD22单链抗体的N端相连，所述CD22单链抗体的C端与所述跨膜区的N端相连。根据本公开实施例的上述转基因淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述跨膜区的N端相连。根据本公开实施例的上述转基因淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿 瘤细胞，体内细胞因子分泌更低，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD22重链可变区的N端相连，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与所述CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述跨膜区的N端相连。根据本公开实施例的上述T淋巴细胞，可特异性杀伤CD19和CD22的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的血液肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第三连接肽的N端与所述BCMA重链可变区的C端相连，所述第三连接肽的C端与BCMA轻链可变区的N端相连，所述BCMA轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连。根据本公开实施例的上述转基因淋巴细胞，可特异性杀伤BCMA和CD19的单/双阳性的肿瘤细胞，相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述第二连接肽和第三连接肽分别独立地具有3～5个重复的GGGGS的氨基酸序列。
根据本公开的实施例，所述第二连接肽和第三连接肽具有5个重复的GGGGS的氨基酸序列。根据本公开的实施例，所述第二连接肽和第三连接肽分别具有5个重复的GGGGS的氨基酸序列。发明人发现，当第二连接肽和第三连接肽分别具有5个重复的GGGGS时，根据本公开实施例的转基因淋巴细胞的杀伤效果更强，导入机体后，机体细胞因子分泌量更低，安全性更高。根据本公开的实施例，所述胞外区具有SEQ ID NO:1～3所示的氨基酸序列。
在本公开的第五方面，本公开提出了一种构建体。根据本公开的实施例，所述构建体包括核酸分子，所述核酸分子编码嵌合抗原受体，所述嵌合抗原受体是如前面所限定的。将根据本公开实施例的构建体导入受体细胞-T淋巴细胞，可获得前面所述的转基因淋巴细胞，所获得的转基因淋巴细胞可特异性杀伤第一抗原和第二抗原的单/双阳性的肿瘤细胞，且相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，上述构建体还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述构建体进一步包括：启动子，所述启动子与所述核酸分子可 操作地连接。
根据本公开的实施例，所述启动子为U6，H1，CMV，EF-1，LTR或RSV启动子。
根据本公开的实施例，所述构建体的载体是非致病性病毒载体；
根据本公开的实施例，所述病毒载体包括选自反转录病毒载体、慢病毒载体和腺病毒相关病毒载体的至少之一。
在本公开的第六方面，本公开提出了一种制备前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞的方法。根据本公开实施例的方法，所述方法包括：将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。根据本公开实施例的方法所制备的T淋巴细胞或转基因淋巴细胞，可特异性杀伤第一抗原和第二抗原的单/双阳性的肿瘤细胞，且相比现有技术，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
在本公开的第七方面，本公开提出了一种用于治疗癌症的治疗组合物。根据本公开的实施例，所述治疗组合物包括：前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞。根据本公开实施例的药物组合物，在体内具有更长久、更有力地的肿瘤细胞杀伤和清除效果。
根据本公开的实施例，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。根据本公开实施例的药物组合物可以应用于对B淋巴细胞白血病及B细胞淋巴瘤患者的免疫治疗；针对B淋巴细胞白血病及B细胞淋巴瘤患者，根据本公开实施例的免疫细胞治疗疗效优于目前的治疗手段；另外，本公开中的双CART细胞的极大促进了B淋巴细胞白血病及B细胞淋巴瘤患者复发的可能性，对于B淋巴细胞白血病及B细胞淋巴瘤的治疗起到非常极大地推动作用。
在本公开的第八方面，本公开提出了前面所述的T淋巴细胞、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的构建体或前面所述的治疗组合物在制备治疗癌症的药物中的用途。
根据本公开的实施例，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。根据本公开实施例的T淋巴细胞、慢病毒、转基因淋巴细胞、构建体或治疗组合物作为治疗癌症的药物的组分，能够用于对B淋巴细胞白血病及B细胞淋巴瘤患者的免疫治疗中。
在本公开的第九方面，本公开提出了一种治疗癌症的方法。根据本公开的实施例，所述方法包括向患有癌症的受试者施用以下中的至少之一：
前面所述的T淋巴细胞；
导入前面所述的慢病毒的T淋巴细胞；
前面所述的转基因淋巴细胞；
导入前面所述的构建体的T淋巴细胞；
前面所述的治疗组合物。
根据本公开的实施例，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。
在本公开的第十方面，本公开提出了前面所述的T淋巴细胞、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的构建体或前面所述的治疗组合物在癌症的治疗中的用途。
根据本公开的实施例，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。
在本公开的第十一方面，本公开提出了一种提高淋巴细胞活性的方法。根据本公开的实施例，所述方法包括：使所述淋巴细胞表达嵌合抗原受体，所述嵌合抗原受体是如前面所限定的，所述淋巴细胞活性包括所述淋巴细胞在肿瘤病人体内的生存能力以及所述淋巴细胞在肿瘤病人体内的杀伤能力的至少一种。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本公开实施例的Car-pCDHF32分子结构图；
图2是根据本公开实施例的Car-pCDHF34分子结构图；
图3是根据本公开实施例的Car-pCDHF32和Car-pCDHF34质粒图谱；
图4是根据本公开实施例的pCDHF-32细胞工作原理图；
图5是根据本公开实施例的流式检测Car-pCDHF-32、34阳性率的结果图；
图6是根据本公开实施例的Nalm6和Raji细胞表面CD19及CD22抗原表达峰度检测图；
图7是根据本公开实施例的Car-pCDHF32和Car-pCDHF34体外杀伤功能图，其中，
A为Car-pCDHF32和Car-pCDHF34对K562/K562-CD19/K562-CD22/Nalm6/Raji细胞杀伤效果；
B为效靶比5:1时，Car-pCDHF32和Car-pCDHF34对K562/K562-CD19/K562-CD22/Raji细胞杀伤效果对比；
C为效靶比5:1时，Car-pCDHF32和Car-pCDHF34对K562/K562-CD19/K562-CD22/Raji 细胞杀伤实验中细胞因子分泌情况对比；
图8是根据本公开实施例的Car-pCDHF32和Car-pCDHF34对K562/K562-CD19/K562-CD22/Raji细胞杀伤实验中，细胞因子分泌情况图；
图9是根据本公开实施例的Car-pCDHF32与单靶点CarT、Loop 6 Car体外杀瘤效果比较图；
图10是根据本公开实施例的pCDHF-60分子结构图；
图11是根据本公开实施例的经过慢病毒包装并侵染T细胞后获得CarT-pCDHF32及CarT-pCDHF60，使用荧光抗原染色检测其阳性率的结果图；
图12是根据本公开实施例的Car-pCDHF32和Car-pCDHF60体外杀伤功能的结果图，其中，
A为Car-pCDHF32和Car-pCDHF60对K562/Nalm6细胞杀伤效果；
B为Car-pCDHF32和Car-pCDHF60对K562/Nalm6细胞杀伤实验中，细胞因子分泌情况；
图13是根据本公开实施例的进行CarT-pCDHF32及CarT-pCDHF60的体内功能对比实验方案图；
图14是根据本公开实施例的Car-pCDHF32和Car-pCDHF60体内功能对比实验结果，其中，A为Car-pCDHF32和Car-pCDHF60体内实验小鼠体重变化监测数据；
B为Car-pCDHF32和Car-pCDHF60体内实验小鼠生存曲线；
图15是根据本公开实施例的Car-pCDHF31分子结构；
图16是根据本公开实施例的Car-pCDHF31和Car-pCDHF60对K562/K562-CD19/K562-BCMA/Daudi细胞杀伤效果图；
图17是根据本公开实施例的Car-pCDHF31对K562/K562-CD19/K562-BCMA/Daudi细胞杀伤实验中，细胞因子分泌情况；
图18是根据本公开实施例的双特异性CART结构中第二和第三连接肽为3～6个重复的GGGGS的体外杀伤效果；以及
图19是根据本公开实施例的双特异性CART结构中第二和第三连接肽为3～6个重复的GGGGS的细胞因子检测；
在附图中，T cell/T-cell是指T细胞；medium是指培养基；Linker是指连接肽；Linker annotation是指连接肽注解。
发明详细描述
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开， 而不能理解为对本公开的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本公开的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。
单链抗体(scFv)是一种基因工程抗体，其中VH和VL域与柔性多肽连接体相连。与整体抗体的Fab区相比，单链抗体表现出更好的组织渗透药动学，并且由于抗原结合表面未被改变而具有完全的抗原结合特异性。
免疫共刺激分子是指为T淋巴细胞或B淋巴细胞完全活化提供共刺激信号的细胞表面分子及其配体，如4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物。
根据本公开的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因，但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。
另外，根据本公开的实施例，本公开实施例的慢病毒是复合慢病毒，除了常见的慢病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载 体，以及各种方法和操作等产生的载体，用于改变细胞的表达。
根据本公开的实施例，本公开实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。另外，根据本公开的实施例，本公开实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列，和作用于反式或远距离控制目的基因的表达控制序列。
另外，本公开实施例的载体还包括常规控制元素，在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本公开的实施例，启动子为选自U6、H1、pol I、pol II和pol III的RNA聚合酶启动子。根据本公开的实施例，启动子为组织特异型启动子。根据本公开的实施例，启动子为诱导型启动子。根据本公开的实施例，启动子为选自基于所选载体的启动子。根据本公开的实施例，当选择慢病毒载体时，启动子为U6、H1、CMV IE基因、EF-1α、泛素C或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素、潮霉素、氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术，
根据本公开的实施例，提供给患者的本公开实施例的组合物，较好的应用于生物兼容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体，如生理盐水；等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液，是医药上可接受的载体。
表达特有的针对抗原CD19和CD22嵌合抗原受体的这些方法是联合治疗的一部分。这些病毒载体和用于过继免疫治疗的抗肿瘤T细胞，可以被单独或结合其他治疗癌症的方法一起执行。在合适的条件下，一个治疗方法的包括使用一个或多个药物疗法。
下面将结合实施例对本公开的方案进行解释。
本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本公开通过优化串联CD19和CD22单链抗体序列的轻重链顺序及其相互连接的连接肽 的长短，获得了一种新型的针对CD19和CD22靶点的双Car结构。根据本公开的实施例，本申请可制备出高阳性率CarT细胞，且对于CD19和CD22靶点细胞具有高特异性和很好的杀伤效果。并且该结构可用于其他靶点构建双靶点CarT，具有良好的通用性。
实施例1靶向CD19和CD22的单链抗体(scFv)序列选择
B淋巴细胞抗原CD19也称为CD19，是一种单程I型膜蛋白，包含两个Ig样C2型(免疫球蛋白样)结构域。靶向CD19的scFv序列选择抗体FMC63-mIgG2a，FMC63-mIgG2a是上个世纪通过动物免疫获得抗CD19的鼠源抗体，其结合表位为CD19远膜端Ig结构域。FMC63-mIgG2a scFv已经成功被应用于抗CD19 CAR构造，比如Norvatis CTL019以及Juno Therapeutics JCAR015。CTL019以及JCAR015在B细胞急性淋巴细胞白血病的临床试验取得了较好的结果，完全缓解(complete remission)病人比例>70％。其轻重链可变区序列如下所示：
轻链可变区氨基酸序列：
Figure PCTCN2021104799-appb-000024
重链可变区氨基酸序列：
Figure PCTCN2021104799-appb-000025
CD22为Ⅰ型跨膜糖蛋白是唾液酸结合免疫球蛋白样凝集素家族成员。作为B细胞受体(BCR)的抑制性共受体，CD22对B细胞激活信号具有负性调节作用，胞外部分包括7个免疫球蛋白结构域。靶向CD22的scFv序列选择抗体M971，其结合表位为CD22近膜端Ig结构域5-7。M971-mIgG是通过抗体库筛选获得的人源化抗体，在靶向CD22的CarT中获得广泛应用。如Juno Therapeutics JCAR018在2017ASH报道的临床数据显示R/R ALL完全缓解(complete remission)病人比例为78％。其轻重链可变区序列如下所示：
轻链可变区氨基酸序列：
Figure PCTCN2021104799-appb-000026
重链可变区氨基酸序列：
Figure PCTCN2021104799-appb-000027
实施例2构建靶向CD19和CD22的双特异性CAR质粒(pCDHF-32、34)
Car-pCDHF32分子结构如图1所示。
Car-pCDHF34分子结构如图2所示。
Car-pCDHF32和Car-pCDHF34质粒图谱如图3所示。
其中，Car-pCDHF32细胞工作原理图图4所示。
实施例3 CD19和CD22的双特异性CART(pCDHF-32、34)阳性率检测
利用293T细胞包装慢病毒，获得的慢病毒按MOI＝10:1感染T细胞制备CART细胞(二抗APC Goat anti Mouse IgG(H+L)检测anti-CD19scFv阳性率，自产荧光标记抗原CD22-His-FITC检测anti-CD22scFv阳性率)，流式检测慢病毒滴度和慢病毒感染T细胞制备CART细胞方法可以通过公开途径获取)。流式检测结果如图5所示。左上象限中显示细胞比例为anti-CD19scFv和anti-CD22scFv的阳性率，即为pCDHF-32/pCDHF-34的阳性率。图中流式结果显示pCDHF-32和pCDHF-34阳性率均超过70％，该结果表明此结构的双特异性CART可制备出高阳性率的细胞制品。
实施例4 CD19和CD22的双特异性CART(pCDHF-32、34)体外杀瘤功能验证
Nalm6细胞为急性淋巴细胞白血病细胞，Raji细胞为黑人Burkitt淋巴瘤细胞，使用流式方式检测其细胞膜表面CD19及CD22抗原表达峰度，具体方法如下：取5E+05癌细胞与流式抗体4℃孵育20min(FITC anti-human CD22(BD Pharmingen/555424)检测细胞表面CD22抗原表达峰度，APC anti-human CD19(Biolegend/302212)检测细胞表面CD19抗原表达峰度)后，PBS清洗一次，重悬细胞上流式细胞仪检测，结果图6所示，Nalm6细胞为CD19 +/CD22 +细胞系。
取靶细胞为K562，K562-CD19，K562-CD22，Nalm6(or Raji)4种靶细胞各1E+07细胞，先利用cytocalceinTM violet 550对靶细胞进行染色，1*10E+05细胞/100μl/孔；效应细胞(CarT V9/CarT M971/Car-pCDHF32/Car-pCDHF34，T细胞为对照)与靶细胞按照0.25:1，1：1，5：1及10：1加入96孔板中混匀，终体积200μl，共培养6h后并将细胞混匀离心，上清利用Human IL-2与Human IFN gamma ELISA ELISA试剂盒检测IL-2及IFN-γ，沉淀部分用100μl结合缓冲液(binding buffer)重悬，300g离心5min，添加2.0μl APC-Annexin V和1.5μl PI染料，避光孵育15min，添加100μl binding buffer重悬，Beckmanc cou LTER流式细胞仪检测各靶细胞凋亡比例如图7所示，ELISA检测各孔上清IL-2及IFN-γ浓度如图8所示。其中K562为CD19和CD22阴性细胞，K562-CD19和K562-CD22分别为CD19和CD22单阳性细胞，Raji和Nalm6均为CD19和CD22双阳性细胞，结果显示Car-pCDHF32和Car-pCDHF34对K562-CD19、K562-CD22、Raji和Nalm6均有较好的杀伤效果，并且在Car-pCDHF34拥有较高自分泌细胞因子的条件下Car-pCDHF32的杀伤效果仍略优于Car-pCDHF34(见图7和图8)。Car-pCDHF32与单靶点CarT体外杀瘤效果对比中可以看出， Car-pCDHF32杀伤效果与CD19单靶点CarT杀伤效果接近，该结果与文献《Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22》报道中最优结构Loop 6 Car(本公开中其作为后续对照双Car，命名为pCDHF60)的体外杀瘤结果一致(见图9)。
实施例5本公开CD19和CD22的双特异性CART(pCDHF-32)与其他双特异性CART(pCDHF-60)体内外杀瘤功能对比
根据文献《Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22》中报道的最优双CarT结构构建双CarT质粒(pCDHF-60)，其结构如图10。
pCDHF60，使用荧光抗原染色检测其阳性率如图11。
使用Nalm6为靶细胞，以实施例4中方法比较pCDHF-32与pCDHF-60体外杀伤能力，结果图12所示。
使用NDG小鼠，以Nalm6细胞为靶细胞尾静脉注射方式，构建小鼠肿瘤模型。并根据图13中实验方案进行CarT-pCDHF32及CarT-pCDHF60的体内功能对比实验。实验过程中隔日记录小鼠体重，并绘制小鼠生存曲线(图14)以及定期采取小鼠尾血，使用流式方法监测尾血中肿瘤和CarT细胞比例(如表1)。
表1：
Figure PCTCN2021104799-appb-000028
综合以上实验数据可以看出，虽然体外杀伤实验结果显示Car-pCDHF32相较Car-pCDHF60的杀瘤效率略低，但体内功能实验显示出Car-pCDHF32相较Car-pCDHF60更持久的CarT续航能力以及更好的体内血液肿瘤细胞清除效果。
实施例6本公开双特异性CART结构与其他靶点配合的适用性(pCDHF-31)
为验证本公开中连接肽(linker)结构是否可以用于其他靶点，因此以本公开中的linker 结构构建CD19和BCMA双靶点CarT，其中anti-BCMA scFv序列部分使用C11D5.3抗体的轻重链部分，其轻重链可变区序列如下所示：
轻链可变区氨基酸序列：
Figure PCTCN2021104799-appb-000029
重链可变区氨基酸序列：
Figure PCTCN2021104799-appb-000030
Car-pCDHF31分子结构如图15所示。
Daudi细胞为人Burkitt's淋巴瘤细胞(CD19 +/BCMA +)，以K562/K562-CD19/K562-BCMA/Daudi细胞作为靶细胞，以实施例4中方法验证CarT-pCDHF31体外杀伤活性结果见图16和图17。
综合以上实验数据可以看出，替换靶点后，使用本公开中linker结构的双CarT仍保有良好的功能活性。因此可以证明本公开中linker结构的双Car具有靶点可替换的普适性和通用性。
实施例7本公开双特异性CART结构中长linker的长度对体外杀伤效果的影响
为验证本公开中CD19VH与CD19VL之间连接肽(linker)、CD22VH与CD22VL之间连接肽(linker)结构长度是否对其杀伤活性具有影响，本公开中将CD19VH与VL之间的linker长度、CD22VH与VL之间的linker长度分别构建了2～6个重复的GGGGS的氨基酸序列并进行了体外功能验证。
以K562/Raji细胞作为靶细胞，以实施例4中方法验证2～6个重复的GGGGS的氨基酸序列的双CART体外杀伤活性。图18和图19为部分实验结果。其中CarT-pCDHF32为第二和第三连接肽为3个重复的GGGGS的氨基酸序列的双Car，CarT-pCDHF58为第二和第三连接肽为5个重复的GGGGS的氨基酸序列的双Car，CarT-pCDHF59为第二和第三连接肽为6个重复的GGGGS的氨基酸序列的双Car。
综合以上实验数据可以看出，在E:T＝1:1和5:1时，CarT-pCDHF58和CarT-pCDHF59相较CarT-pCDHF32具有更强的体外杀伤效果。但细胞因子检测结果显示，IL-2的分泌能力在E:T＝0.25:1和1:1时，CarT-pCDHF32、CarT-pCDHF58和CarT-pCDHF59基本相当；在E:T＝5:1和10:1时，CarT-pCDHF59 IL-2的分泌较多。IFN-r的分泌能力除了在E:T＝5:1时，CarT-pCDHF58略低于CarT-pCDHF32外，其余CarT-pCDHF58和CarT-pCDHF59的IFN-r分泌多于CarT-pCDHF32。
综合实施例7的实验结果可以看出，第二和第三连接肽具有3～5个重复的GGGGS的氨 基酸序列的双CART引起细胞因子分泌量少于第二和第三连接肽具有6个重复的GGGGS的氨基酸序列的双CART引起细胞因子分泌量，第二和第三连接肽具有3～5个重复的GGGGS的氨基酸序列的双CART安全性更高。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

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  1. 一种T淋巴细胞，其中，所述T淋巴细胞表达嵌合抗原受体，所述嵌合抗原受体包括：
    胞外区，所述胞外区包括第一单链抗体、第二单链抗体、第一连接肽以及CD8铰链区，所述第一单链抗体特异性识别第一抗原，所述第二单链抗体特异性识别第二抗原，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，
    所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，
    所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，
    所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列；
    跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；以及
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链。
  2. 根据权利要求1所述的T淋巴细胞，其中，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为CD22单链抗体，所述第一抗原为CD19，所述第二抗原为CD22，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为CD22重链可变区，所述第二轻链可变区为CD22轻链可变区；
    任选地，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为BCMA单链抗体，所述第一抗原为CD19，所述第二抗原为BCMA，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为BCMA重链可变区，所述第二轻链可变区为BCMA轻链可变区。
  3. 根据权利要求2所述的T淋巴细胞，其中，所述第一连接肽的N端与所述二单链抗体的C端相连，所述第一连接肽的C端与所述第一单链抗体的N端相连，所述第一单链抗体的C端与所述CD8铰链区的N端相连；
    优选地，所述第一连接肽的N端与所述CD22单链抗体的C端相连，所述第一连接肽的C端与所述CD19单链抗体的N端相连，所述CD19单链抗体的C端与所述CD8铰链区的N端相连。
  4. 根据权利要求2所述的T淋巴细胞，其中，所述第一连接肽的N端与所述CD19单链抗体的C端相连，所述第一连接肽的C端与所述CD22单链抗体的N端相连，所述CD22 单链抗体的C端与所述CD8铰链区的N端相连。
  5. 根据权利要求3所述的T淋巴细胞，其中，所述第三连接肽的N端与所述BCMA重链可变区的C端相连，所述第三连接肽的C端与BCMA轻链可变区的N端相连，所述BCMA轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连；
    优选地，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连。
  6. 根据权利要求4所述的T淋巴细胞，其中，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD22重链可变区的N端相连，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与所述CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述CD8铰链区的N端相连。
  7. 根据权利要求1～6任一项所述的T淋巴细胞，其中，所述第二连接肽和第三连接肽分别独立地具有3～5个重复的GGGGS的氨基酸序列，优选地，所述第二连接肽和第三连接肽分别具有5个重复的GGGGS的氨基酸序列。
  8. 根据权利要求2所述的T淋巴细胞，其中，所述胞外区具有SEQ ID NO:1～5所示的氨基酸序列。
  9. 一种慢病毒，其中，所述慢病毒携带编码嵌合抗原受体的核酸分子，所述嵌合抗原受体包括：
    胞外区，所述胞外区包括第一单链抗体、第二单链抗体、第一连接肽以及CD8铰链区，所述第一单链抗体特异性识别第一抗原，所述第二单链抗体特异性识别第二抗原，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，
    所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，
    所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，
    所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列；
    跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；以及
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链。
  10. 根据权利要求9所述的慢病毒，其中，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为CD22单链抗体，所述第一抗原为CD19，所述第二抗原为CD22，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为CD22重链可变区，所述第二轻链可变区为CD22轻链可变区；
    任选地，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为BCMA单链抗体，所述第一抗原为CD19，所述第二抗原为BCMA，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为BCMA重链可变区，所述第二轻链可变区为BCMA轻链可变区。
  11. 根据权利要求10所述的慢病毒，其中，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连；
    任选地，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD22重链可变区的N端相连，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与所述CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述CD8铰链区的N端相连；
    任选地，所述第三连接肽的N端与所述BCMA重链可变区的C端相连，所述第三连接肽的C端与BCMA轻链可变区的N端相连，所述BCMA轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连；
    任选地，编码所述胞外区的核酸分子具有SEQ ID NO:6～10任一所示的核苷酸序列；任选地，编码所述跨膜区的核酸分子具有SEQ ID NO:11所示的核苷酸序列；任选地，编码所 述胞内区的核酸分子具有SEQ ID NO:12所示的核苷酸序列；
    任选地，编码所述嵌合抗原受体的核酸分子具有SEQ ID NO:13～17所示的核苷酸序列。
  12. 一种慢病毒，其中，携带具有SEQ ID NO：18～22所示的核苷酸序列的核酸分子。
  13. 一种转基因淋巴细胞，其中，所述淋巴细胞表达嵌合抗原受体，所述嵌合抗原受体包括：
    胞外区，所述胞外区包括第一单链抗体、第二单链抗体以及第一连接肽，所述第一单链抗体特异性识别第一抗原，所述第二单链抗体特异性识别第二抗原，所述第一连接肽设置于所述第一单链抗体和第二单链抗体之间，
    所述第一单链抗体包括第一重链可变区和第一轻链可变区以及第二连接肽，所述第二连接肽设置于所述第一重链可变区和第一轻链可变区之间，
    所述第二单链抗体包括第二重链可变区和第二轻链可变区以及第三连接肽，所述第三连接肽设置于第二重链可变区和第二轻链可变区之间，
    所述第一连接肽具有一个重复的GGGGS的氨基酸序列，所述第二连接肽和第三连接肽分别独立地具有2～6个重复的GGGGS的氨基酸序列；
    跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述淋巴细胞的细胞膜中；以及
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段。
  14. 根据权利要求13所述的转基因淋巴细胞，其中，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为CD22单链抗体，所述第一抗原为CD19，所述第二抗原为CD22，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为CD22重链可变区，所述第二轻链可变区为CD22轻链可变区；
    任选地，所述第一单链抗体为CD19单链抗体，所述第二单链抗体为BCMA单链抗体，所述第一抗原为CD19，所述第二抗原为BCMA，所述第一重链可变区为CD19重链可变区，所述第一轻链可变区为CD19轻链可变区，所述第二重链可变区为BCMA重链可变区，所述第二轻链可变区为BCMA轻链可变区。
  15. 根据权利要求13所述的转基因淋巴细胞，其中，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28以及他们的衍生物的至少一种；
    优选地，所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段；
    任选地，所述淋巴细胞是CD3 +T淋巴细胞；
    任选地，所述淋巴细胞是CD8 +T淋巴细胞；
    任选地，所述淋巴细胞是自然杀伤细胞；
    任选地，所述淋巴细胞是自然杀伤T细胞。
  16. 根据权利要求14所述的转基因淋巴细胞，其中，所述第一连接肽的N端与所述二 单链抗体的C端相连，所述第一连接肽的C端与所述第一单链抗体的N端相连，所述第一单链抗体的C端与所述CD8跨膜区的N端相连；
    任选地，所述第一连接肽的N端与所述CD22单链抗体的C端相连，所述第一连接肽的C端与所述CD19单链抗体的N端相连，所述CD19单链抗体的C端与所述跨膜区的N端相连；
    任选地，所述第一连接肽的N端与所述CD19单链抗体的C端相连，所述第一连接肽的C端与所述CD22单链抗体的N端相连，所述CD22单链抗体的C端与所述跨膜区的N端相连；
    优选地，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述跨膜区的N端相连；
    优选地，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD22重链可变区的N端相连，所述第三连接肽的N端与所述CD22重链可变区的C端相连，所述第三连接肽的C端与所述CD22轻链可变区的N端相连，所述CD22轻链可变区的C端与所述跨膜区的N端相连；
    任选地，所述第三连接肽的N端与所述BCMA重链可变区的C端相连，所述第三连接肽的C端与BCMA轻链可变区的N端相连，所述BCMA轻链可变区的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述CD19轻链可变区的N端相连，所述第二连接肽的N端与所述CD19轻链可变区的C端相连，所述第二连接肽的C端与所述CD19重链可变区的N端相连，所述CD19重链可变区的C端与所述CD8铰链区的N端相连；
    优选地，所述第二连接肽和第三连接肽分别独立地具有3～5个重复的GGGGS的氨基酸序列；
    优选地，所述第二连接肽和第三连接肽分别具有5个重复的GGGGS的氨基酸序列；
    任选地，所述胞外区具有SEQ ID NO:1～5所示的氨基酸序列。
  17. 一种构建体，其中，所述构建体包括核酸分子，所述核酸分子编码嵌合抗原受体，所述嵌合抗原受体是如权利要求1～11、13～16任一项中所限定的。
  18. 根据权利要求17所述的构建体，其中，进一步包括：启动子，所述启动子与所述核酸分子可操作地连接。
  19. 根据权利要求18所述的构建体，其中，所述启动子为U6，H1，CMV，EF-1，LTR 或RSV启动子。
  20. 根据权利要求19所述的构建体，其中，所述构建体的载体是非致病性病毒载体；
    任选地，所述病毒载体包括选自反转录病毒载体、慢病毒载体和腺病毒相关病毒载体的至少之一。
  21. 一种制备权利要求1～8任一项所述的T淋巴细胞或者权利要求13～16任一项所述的转基因淋巴细胞的方法，其中，包括：
    将权利要求17～20任一项所述的构建体或者权利要求9～12任一项所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。
  22. 一种用于治疗癌症的治疗组合物，其中，包括：
    权利要求17～20任一项所述的构建体、权利要求9～12任一项所述的慢病毒、权利要求1～8任一项所述的T淋巴细胞或者权利要求13～16任一项所述的转基因淋巴细胞。
  23. 根据权利要求22所述的治疗组合物，其中，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。
  24. 权利要求1～8任一项所述的T淋巴细胞、权利要求9～12任一项所述的慢病毒、权利要求13～16任一项所述的转基因淋巴细胞、权利要求17～20任一项所述的构建体或权利要求22或23所述的治疗组合物在制备治疗癌症的药物中的用途。
  25. 根据权利要求24所述的用途，其中，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。
  26. 一种治疗癌症的方法，所述方法包括向患有癌症的受试者施用以下中的至少之一：
    权利要求1～8任一项所述的T淋巴细胞；
    导入权利要求9～12任一项所述的慢病毒的T淋巴细胞；
    权利要求13～16任一项所述的转基因淋巴细胞；
    导入权利要求17～20任一项所述的构建体的T淋巴细胞；
    权利要求22或23所述的治疗组合物。
  27. 根据权利要求26所述的方法，其中，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。
  28. 权利要求1～8任一项所述的T淋巴细胞、权利要求9～12任一项所述的慢病毒、权利要求13～16任一项所述的转基因淋巴细胞、权利要求17～20任一项所述的构建体或权利要求22或23所述的治疗组合物在癌症的治疗中的用途。
  29. 根据权利要求28所述的用途，其中，所述癌症包括选自B淋巴细胞白血病及B细胞淋巴瘤的至少之一。
  30. 一种提高淋巴细胞活性的方法，其中，所述方法包括：使所述淋巴细胞表达嵌合 抗原受体，所述嵌合抗原受体是如权利要求1～11、13～16任一项中所限定的，
    所述淋巴细胞活性包括所述淋巴细胞在肿瘤病人体内的生存能力以及所述淋巴细胞在肿瘤病人体内的杀伤能力的至少一种。
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