WO2022116952A1 - 靶向cd70的抗原结合蛋白及其应用 - Google Patents

靶向cd70的抗原结合蛋白及其应用 Download PDF

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WO2022116952A1
WO2022116952A1 PCT/CN2021/134248 CN2021134248W WO2022116952A1 WO 2022116952 A1 WO2022116952 A1 WO 2022116952A1 CN 2021134248 W CN2021134248 W CN 2021134248W WO 2022116952 A1 WO2022116952 A1 WO 2022116952A1
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seq
cells
hcdr1
hcdr2
hcdr3
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French (fr)
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王文博
林彦妮
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苏州克睿基因生物科技有限公司
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Priority to EP21899983.7A priority Critical patent/EP4257617A1/en
Priority to US18/255,332 priority patent/US20240024475A1/en
Priority to CN202180081214.4A priority patent/CN116490608A/zh
Publication of WO2022116952A1 publication Critical patent/WO2022116952A1/zh

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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
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    • A61K39/46—Cellular immunotherapy
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/464438—Tumor necrosis factors [TNF], CD70
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
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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
    • C07K16/2875—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/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
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    • A61K2239/21—Transmembrane domain
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/56—Kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen binding protein targeting CD70 and its application.
  • CAR-T Chimeric Antigen Receptor-T cells
  • CD70 is a type II transmembrane protein, a member of the tumor necrosis factor (TNF) family, also known as a tumor necrosis factor receptor super-family (TNFSF) factor, which has the ability to regulate T cells and The ability of B cells to activate, proliferate and differentiate plays an important role in maintaining the body's immune response.
  • TNF tumor necrosis factor
  • TNFSF tumor necrosis factor receptor super-family
  • CD70 was highly expressed in primary and metastatic renal cancer, and the expression rate in renal clear cell carcinoma was 100%.
  • CD70 has also been reported to be highly expressed in acute myeloid leukemia stem cells.
  • the high-level expression of CD70 in tumor tissues can not only induce immune escape, but also activate some immune cells to kill tumor cells. It may serve as a potential tumor therapy target and bring a new direction to tumor immunotherapy.
  • CAR-T therapy usually uses the scFv segment derived from the antigen-binding region of monoclonal antibodies as the antigen-binding region.
  • the extracellular scFv domain can activate the co-stimulatory domain and the activation domain of the CAR structure after binding to the target protein expressed on the surface of the target cell.
  • the molecular weight of scFv is relatively large and it is easy to form multimers, which affects the function of CAR. Therefore, CARs containing novel structures of antigen-binding regions are needed.
  • the present application provides an antigen-binding protein targeting CD70, in particular to a chimeric antigen receptor comprising the antigen-binding protein and applications thereof, as well as cells comprising or expressing the antigen-binding protein, which have the following properties: One or more of: 1) strong specific recognition and binding ability to CD70 protein; and 2) better ability to secrete cytokines IL-2 and IFN- ⁇ .
  • the application provides a chimeric antigen receptor
  • the targeting moiety comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3)
  • the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 6, 9, 11, 14, 17, 20, 23, 26 and 29.
  • the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 15, 18, 21, 24, 27, 30, and 32.
  • the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31 and 33.
  • the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any of the following groups:
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 4;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 5;
  • HCDR1 SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 7
  • HCDR3 SEQ ID NO: 10;
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 12
  • HCDR3 SEQ ID NO: 13;
  • HCDR1 SEQ ID NO: 14
  • HCDR2 SEQ ID NO: 15
  • HCDR3 SEQ ID NO: 16;
  • HCDR1 SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 21
  • HCDR3 SEQ ID NO: 22;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25;
  • HCDR1 SEQ ID NO: 26
  • HCDR2 SEQ ID NO: 27
  • HCDR3 SEQ ID NO: 28;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, and HCDR3: SEQ ID NO: 31;
  • HCDR1 SEQ ID NO:23
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33.
  • the targeting moiety comprises VHH.
  • the targeting moiety comprises the amino acid set forth in any one of SEQ ID NOs: 39, 34, 35, 36, 37, 38, 44, 40, 41, 42, 43, 45, and 46 sequence.
  • the chimeric antigen receptor comprises a signal peptide.
  • the signal peptide comprises a signal peptide derived from a histone selected from the group consisting of CD8, 4-1BB, GM-CSF, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d, or a combination thereof.
  • the signal peptide comprises a signal peptide derived from CD8.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:88.
  • the C-terminus of the signal peptide is linked to the N-terminus of the targeting moiety.
  • the chimeric antigen receptor comprises a hinge region.
  • the hinge region comprises a hinge region derived from a protein selected from the group consisting of CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1 and CH2CH3, or a combination thereof.
  • the hinge region comprises a hinge region derived from CD8a in the CD8.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID NO:89.
  • the N-terminus of the hinge region is linked to the C-terminus of the targeting moiety.
  • the chimeric antigen receptor comprises a transmembrane domain.
  • the transmembrane domain comprises a transmembrane domain derived from a histone selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, CTLA- 4.
  • LAG-3 TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , cytokine receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM , DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • the transmembrane domain comprises a transmembrane domain derived from CD8a in the CD8.
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:90.
  • the N-terminus of the transmembrane domain is linked to the C-terminus of the hinge region.
  • the chimeric antigen receptor comprises a costimulatory signaling domain.
  • the costimulatory signaling domain comprises a costimulatory signaling domain derived from a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86 or a combination thereof , OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1 , PD-L2, 4-1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88.
  • a protein selected from the group consisting of CD28, CD137, CD27,
  • the costimulatory signaling domain comprises a costimulatory signaling domain derived from 4-1BB.
  • the costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:91.
  • the N-terminus of the costimulatory signaling domain is linked to the C-terminus of the transmembrane domain.
  • the chimeric antigen receptor comprises an intracellular signaling domain.
  • the intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5 , CD22, FcR ⁇ , FcR ⁇ , FcR ⁇ , FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus (BLV) gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus (SIV) PBj14 Nef, Kaposi sarcoma herpes virus (KSHV) ) K1, DAP10, DAP12, and a domain comprising at least one immunoreceptor tyrosine activation motif (ITAM).
  • a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5 , CD22,
  • the intracellular signaling domain comprises an intracellular signaling domain derived from CD3zeta.
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:92.
  • the N-terminus of the intracellular signaling domain is linked to the C-terminus of the costimulatory signaling domain.
  • the chimeric antigen receptor comprises any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 and 72 amino acid sequence.
  • the present application provides an isolated antigen binding protein that specifically binds to CD70 with a KD value of 1 nM or less.
  • the isolated antigen binding protein comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 heavy chain complementarity determining region 2
  • HCDR3 heavy chain complementarity determining region 3
  • the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 15, 18, 21, 24, 27, 30, and 32.
  • the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31 and 33.
  • the isolated antigen-binding protein is an antibody or an antigen-binding fragment thereof.
  • the isolated antigen-binding protein is VHH or an antigen-binding fragment thereof.
  • the antibody in the isolated antigen binding protein is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the HCDR1, HCDR2 and HCDR3 in the isolated antigen binding protein comprise an amino acid sequence selected from any one of the group consisting of:
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 4;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 5;
  • HCDR1 SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 7
  • HCDR3 SEQ ID NO: 10;
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 12
  • HCDR3 SEQ ID NO: 13;
  • HCDR1 SEQ ID NO: 14
  • HCDR2 SEQ ID NO: 15
  • HCDR3 SEQ ID NO: 16;
  • HCDR1 SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 21
  • HCDR3 SEQ ID NO: 22;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25;
  • HCDR1 SEQ ID NO: 26
  • HCDR2 SEQ ID NO: 27
  • HCDR3 SEQ ID NO: 28;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, and HCDR3: SEQ ID NO: 31;
  • HCDR1 SEQ ID NO:23
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33.
  • the isolated antigen binding protein comprises VHH.
  • the isolated antigen binding protein comprises any one of SEQ ID Nos: 39, 34, 35, 36, 37, 38, 44, 40, 41, 42, 43, 45 and 46 amino acid sequence.
  • the present application provides an isolated nucleic acid molecule encoding the chimeric antigen receptor and/or the isolated antigen binding protein.
  • the isolated nucleic acid molecule comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 47-59 and/or SEQ ID NOs: 73-85.
  • the application provides one or more vectors comprising the isolated nucleic acid molecules.
  • the vector comprises a viral vector.
  • the vector comprises a lentiviral vector.
  • the application provides one or more cells comprising the chimeric antigen receptor, the isolated antigen binding protein, the isolated nucleic acid molecule, and/or the vector .
  • the cells comprise immune cells.
  • the immune cells are selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes cells, leukocytes and/or peripheral blood mononuclear cells.
  • the cells comprise T cells.
  • said cells have reduced expression and/or activity of CD70 in said cells as compared to corresponding wild-type cells.
  • the cells are CD70 knocked out as compared to corresponding wild-type cells.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor, the isolated antigen-binding protein, the isolated nucleic acid molecule, the carrier, the cells, and/or pharmaceutically acceptable adjuvants and/or excipients.
  • the chimeric antigen receptor, the isolated antigen-binding protein, the isolated nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition is used to treat diseases or disorders associated with the expression of CD70.
  • the disease or disorder associated with the expression of CD70 comprises acute myeloid leukemia (AML) or renal cancer.
  • the chimeric antigen receptor, the isolated antigen binding protein, the isolated nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition are Use in the preparation of a medicament for treating a disease or condition related to the expression of CD70.
  • the use, wherein the disease or disorder associated with the expression of CD70 comprises acute myeloid leukemia (AML) or renal cancer.
  • AML acute myeloid leukemia
  • the present application provides a method of preventing and/or treating a disease or condition associated with the expression of CD70, comprising administering to a subject in need thereof an effective amount of the chimeric antigen receptor, wherein the The isolated antigen-binding protein, the isolated nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of CD70 comprises acute myeloid leukemia (AML) or renal cancer.
  • FIG 1 shows the structure of the CAR targeting CD70 described in this application.
  • Figures 2A-2D show the binding affinity of the CD70-targeting single domain antibodies described herein to cell line 786-0.
  • Figure 3 shows the expression efficiency of CD70-targeting CAR on the surface of CD70-knockout T cells detected by flow cytometry described in this application.
  • Figure 4 shows the detection of IL-2 and IFN- ⁇ cytokine secretion by Elisa method after co-culture of CD70-targeting CAR-T cells described in this application with target cells.
  • Figure 5 shows the killing rate of CD70-targeting CAR-T cells to target cells after co-culture with CD70-targeting CAR-T cells described in this application and luciferase-labeled target cells.
  • Figure 6 shows the anti-tumor effect of the CD70-targeting CAR-T cells described in this application in an acute myeloid leukemia animal model.
  • Figure 7 shows the anti-tumor activity of the CD70-targeting CAR-T cells described in this application in an animal model of renal cancer.
  • isolated antigen binding protein generally refers to a polypeptide polymer capable of specifically recognizing and/or neutralizing a particular antigen.
  • an isolated antigen binding protein can include a portion of a heavy chain.
  • an isolated antigen binding protein can include a heavy chain variable region.
  • isolated antigen binding protein may include single domain antibodies.
  • isolated antigen binding proteins can include, but are not limited to, human single domain antibodies.
  • single domain antibody or “sdAb” or “VHH” generally refers to a class of antibodies that lack the antibody light chain and only have the variable region of the heavy chain.
  • the single-domain antibody can be from a Bactrian camel, a dromedary, a llama, a llama, a nurse shark, a great star shark, or a ray (for example, see Kang Xiaozhen et al., Chinese Journal of Biological Engineering, 2018, 34 ( 12):1974-1984).
  • single domain antibodies can be from llamas.
  • Single domain antibodies may be composed of heavy chain variable regions (VH).
  • heavy chain variable region generally refers to the amino-terminal domain of the heavy chain of an antigen-binding fragment. Heavy chain variable regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs). Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The heavy chain variable region contains a binding domain that interacts with an antigen (eg, CD70).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the heavy chain variable region contains a binding domain that interacts with an antigen
  • CD70 antigen and “CD70 protein” are used interchangeably. Also included are any variants and homologues of CD70 that are naturally expressed by cells or expressed on cells transfected with the CD70 gene.
  • CD70 may be human CD70, whose accession number in GenBank is NM_001252.5, and whose accession number in UniProt/Swiss-Prot is P32970.
  • the CD70 protein can comprise a homologue of human CD70 protein.
  • the term “homolog” generally refers to amino acid sequences or nucleotide sequences that have certain homology to the amino acid sequence of human CD70 protein and the nucleotide sequence of human CD70 protein.
  • homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will contain the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • sequence alignments can be performed by various means known to those skilled in the art, eg, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • transmembrane domain generally refers to a sequence in a cell surface protein that spans the cell membrane, which may contain a hydrophobic alpha helix.
  • the transmembrane domain can be linked with the intracellular signaling domain to transmit signals.
  • the transmembrane domain may be derived from any type I, type II or type III transmembrane protein.
  • the transmembrane domain may comprise a transmembrane domain derived from a histone selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, T cell receptor Subunits, polypeptides constituting the CD3 complex, IL2 receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • a histone selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, T cell receptor Subunits, polypeptides constituting the CD3 complex, IL2 receptor, CD5, ICO
  • a subunit of a T cell receptor can include TCR ⁇ , TCR ⁇ , TCR ⁇ or TCR ⁇ .
  • the polypeptides that make up the CD3 complex can include CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the transmembrane domain may include a transmembrane domain derived from CD8a in the CD8.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • targeting moieties eg, targeting tumor-specific antigens and/or tumor-associated antigens
  • signal peptides eg, transmembrane domains
  • co- Stimulatory signaling domains e.g. CD70
  • intracellular signaling domains e.g. CD70
  • T cells expressing CAR can specifically recognize and eliminate malignant cells expressing the target antigen.
  • CAR and CAR-T cells see, e.g., Sadelain M, Brentjens R, Rivi'ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4): 388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. Curr Opin Immunol. 2012;24(5):633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev. 2014;257(1):107-126; and WO2013154760, WO2016014789.
  • the term "costimulatory signaling domain” generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective lymphocyte responses to antigens.
  • the costimulatory signaling domain can comprise a costimulatory signaling domain derived from a histone selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86, OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1, PD- Ligands for L2, 4-1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, LFA-1, L
  • the term "hinge region” generally refers to the connecting region between the targeting moiety (eg, targeting the extracellular domain of CD70) and the transmembrane domain.
  • the hinge region may comprise a hinge region derived from a protein selected from the group consisting of CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3, or a combination thereof.
  • the hinge region may include a hinge region derived from CD8a of the CD8.
  • intracellular signaling domain generally refers to a domain located inside a cell capable of transducing a signal.
  • the intracellular signaling domain can transmit a signal into a cell.
  • an intracellular signaling domain is any contiguous amino acid sequence used to direct protein targeting.
  • the intracellular signaling domain can comprise an intracellular signaling domain derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5, CD22 , FcR ⁇ , FcR ⁇ , FcR ⁇ , FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpes virus (HSKV), DAP10, DAP12, and at least Contains an ITAM domain.
  • the intracellular signaling domain can include an intracellular signaling domain derived from CD3zeta.
  • the term "signal peptide” generally refers to the amino-terminal (N-terminal) leader sequence of a chimeric antigen receptor (CAR), which guides the CAR molecule to the endoplasmic reticulum either during translation or after translation.
  • the signal peptide may comprise a signal peptide derived from a histone selected from the group consisting of CD8, 4-1BB, GM-CSF, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d, or a combination thereof.
  • the signal peptide can include a signal peptide derived from CD8.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (eg, scFvs), and antibodies with antigens Bound antibody fragments (eg, Fab, Fab' and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the term "antigen-binding fragment” generally refers to one or more fragments of an antibody that function to specifically bind an antigen.
  • the antigen-binding function of an antibody can be achieved by full-length fragments of the antibody.
  • Antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2, or alternatively, comprising Fv, ScFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2.
  • Fab fragment i.e.
  • a monovalent fragment consisting of VL, VH, CL and CH domains (2) F(ab')2 fragment, comprising two Fab fragments linked by a disulfide bond at the hinge region Bivalent fragments; (3) Fd fragments composed of VH and CH domains; (4) Fv fragments composed of VL and VH domains of the antibody one-arm; (5) dAb fragments composed of VH domains (Ward et al. , (1989) Nature 341:544-546); (6) a combination of separate complementarity determining regions (CDRs) and (7) two or more separate CDRs optionally linked by a linker.
  • CDRs complementarity determining regions
  • the "antigen-binding fragment” may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) with an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • the antigen-binding fragments may also include single-domain antibodies.
  • AML acute myeloid leukemia
  • Acute myeloid leukemia generally refers to a clonal malignant proliferative disorder of myeloid blasts of the hematopoietic system.
  • Acute myeloid leukemia can include all acute leukemias of non-lymphocytic origin.
  • kidney cancer generally refers to a disease in which kidney cells become diseased (cancerous) and grow out of control to form tumors.
  • symptoms such as varying degrees of anemia, blood in the urine, persistent pain on the side of the body, and swelling of the ankle or leg can be seen.
  • KD is used interchangeably with “KD” and generally refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, in M (mol/L).
  • the term "Raji cells” generally refers to a continuous human cell line capable of producing the Epstein-Barr strain.
  • the virus will transform umbilical cord lymphocytes and induce early antigens in Raji cells.
  • Raji cells are widely used as transfection hosts and are also used to understand hematopoietic and other cellular malignancies.
  • it is used to detect immune complexes because it possesses and expresses several receptors for certain complement components, as well as the Fc receptor for immunoglobulin G.
  • 786-O cells generally refers to human renal clear cell adenocarcinoma cells.
  • the cell line is derived from a primary clear cell carcinoma. This cell has microvilli and desmosomes.
  • THP-1 cells generally refers to the human acute monocytic leukemia cell line.
  • the cell line can phagocytose latex particles and activated erythrocytes, has no immunoglobulins in the cell membrane and cytoplasm, and express C3R and FcR; it can be induced by phorbol ester TPA to differentiate into mononuclear lineage.
  • K562 cells generally refers to the human immortalized myeloid leukemia cell line. K562 belongs to erythroleukemia cell line, which has the characteristics of high malignancy and fast proliferation. Originally isolated from the pleural effusion of a 53-year-old female chronic myelogenous leukemia (CML) patient in the acute phase.
  • CML chronic myelogenous leukemia
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host.
  • the vector can transfer the inserted nucleic acid molecule into and/or between cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector can produce the desired expression product by culturing suitable cells containing the vector.
  • the vector may comprise a lentiviral vector.
  • the term "cell” generally refers to a plasmid or vector that can or has contained a nucleic acid molecule described herein, or is capable of expressing a chimeric antigen receptor described herein or an antigen binding agent described herein individual cells, cell lines or cell cultures of the protein.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the daughter cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the chimeric antigen receptors or antigen-binding proteins described herein. .
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be immune cells.
  • the immune cells can be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and /or peripheral blood mononuclear cells.
  • the immune cells can be T cells.
  • treating generally refers to: (i) preventing the occurrence of a disease, disorder or condition in a patient who may be susceptible to a disease, disorder and/or condition but has not been diagnosed with the disease; (ii) inhibiting the disease , disease or condition, i.e. arresting its development; and (iii) alleviating the disease, disorder or condition, i.e. causing the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subsided.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (eg, binding to labeling components).
  • amino acid includes natural and/or non-natural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably and generally refer to nucleosides of any length Polymeric forms of acids, such as deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA Transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any sequence
  • a polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation to labeled components.
  • the present application may also include functional variants, derivatives, analogs, homologues, and fragments thereof.
  • a variant of any given sequence refers to one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one Sequence of endogenous functions.
  • Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.
  • the term "derivative" generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and /or addition, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analog generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie possessing at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics chemical compounds.
  • amino acid substitutions such as at least 1 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutions, can be made, so long as the modified sequence remains substantially as desired activity or ability.
  • Amino acid substitutions can include the use of non-naturally occurring analogs.
  • proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing uncharged polar headgroups with similar hydrophilicity values include amino acids Paraparagine, Glutamine, Serine, Threonine and Tyrosine.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen binding protein.
  • the isolated antigen binding protein may comprise a targeting moiety that can specifically bind to CD70.
  • the application provides a chimeric antigen receptor (CAR) comprising the isolated antigen binding protein (eg, the targeting moiety that can specifically bind to CD70).
  • CAR chimeric antigen receptor
  • the isolated antigen binding protein may comprise heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1 may comprise The amino acid sequence shown in any one of SEQ ID NOs: 1, 6, 9, 11, 14, 17, 20, 23, 26 and 29.
  • the HCDR1 can comprise at least 80% (eg, at least 85%) of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 6, 9, 11, 14, 17, 20, 23, 26, and 29 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 12, 15, 18, 21, 24, 27, 30 and 32.
  • the HCDR2 can comprise at least 80% (eg, at least 85%) of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 15, 18, 21, 24, 27, 30, and 32 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31 and 33.
  • the HCDR3 may comprise at least 80% of the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31 and 33 (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequences of sequence homology.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences selected from any one of the group, or the isolated antigen-binding protein may comprise at least 80% ( For example, amino acid sequences of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology:
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 4;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 5;
  • HCDR1 SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 7
  • HCDR3 SEQ ID NO: 10;
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 12
  • HCDR3 SEQ ID NO: 13;
  • HCDR1 SEQ ID NO: 14
  • HCDR2 SEQ ID NO: 15
  • HCDR3 SEQ ID NO: 16;
  • HCDR1 SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 21
  • HCDR3 SEQ ID NO: 22;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25;
  • HCDR1 SEQ ID NO: 26
  • HCDR2 SEQ ID NO: 27
  • HCDR3 SEQ ID NO: 28;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, and HCDR3: SEQ ID NO: 31;
  • HCDR1 SEQ ID NO:23
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33.
  • the antigen-binding protein may further include a framework region, and the framework region may be derived from human antibodies, or derived from the framework regions of single-domain antibodies of species such as alpaca and camel.
  • the framework regions of the antigen binding protein can be derived from humans.
  • the framework regions of the antigen binding protein can be derived from humans.
  • the antigen binding protein may comprise an Fc region. In certain instances, the antigen binding protein can comprise a single domain antibody.
  • the antigen binding protein may comprise a VHH.
  • the targeting moiety of the chimeric antigen receptor may comprise a VHH.
  • the VHH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 39, 34, 35, 36, 37, 38, 44, 40, 41, 42, 43, 45 and 46.
  • the VHH may comprise at least 80% of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 34, 35, 36, 37, 38, 44, 40, 41, 42, 43, 45 and 46 (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequences of sequence homology.
  • the chimeric antigen receptor may comprise an intracellular domain in addition to an extracellular targeting moiety (eg, targeting CD70).
  • the chimeric antigen receptor may comprise a signal peptide.
  • the signal peptide may include, but is not limited to, a signal peptide derived from a histone selected from the group consisting of CD8, 4-1BB, GM-CSF, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d, or a combination thereof.
  • the signal peptide can be the signal peptide from CD8.
  • the signal peptide may comprise the amino acid sequence set forth in SEQ ID NO:88.
  • the chimeric antigen receptor may comprise a hinge region.
  • the hinge region may include, but is not limited to, a hinge region derived from a histone selected from the group consisting of CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1 and CH2CH3, or a combination thereof.
  • the hinge region can be the hinge region from CD8a in the CD8.
  • the hinge region may comprise the amino acid sequence set forth in SEQ ID NO:89.
  • the chimeric antigen receptor may comprise a transmembrane domain.
  • the transmembrane domain may comprise a transmembrane domain derived from a histone selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, CTLA-4, LAG-3, TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , cytokine receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C , LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • the transmembrane domain may be the transmembrane
  • the chimeric antigen receptor can comprise a costimulatory signaling domain.
  • the costimulatory signaling domain may comprise a transmembrane domain derived from a histone selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86, OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, CD40 and MyD88.
  • the chimeric antigen receptor may comprise an intracellular signaling domain.
  • the intracellular signaling domain may comprise an intracellular signaling domain derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5, CD22, FcR ⁇ , FcR ⁇ , FcR ⁇ , FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus (BLV) gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus (SIV) PBj14 Nef, Kaposi's sarcoma herpes virus (KSHV) K1, DAP10 , DAP12 and a domain comprising at least one immunoreceptor tyrosine activation motif (ITAM).
  • the intracellular signaling domain can be the intracellular signaling domain from CD3zeta.
  • the intracellular signaling domain can be the
  • the C-terminus of the signal peptide may be linked to the N-terminus of the targeting moiety.
  • the N-terminus of the hinge region can be linked to the C-terminus of the targeting moiety.
  • the N-terminus of the transmembrane domain can be linked to the C-terminus of the hinge region.
  • the N-terminus of the costimulatory signaling domain can be linked to the C-terminus of the transmembrane domain.
  • the N-terminus of the intracellular signaling domain can be linked to the C-terminus of the costimulatory signaling domain.
  • the chimeric antigen receptor may comprise the following domains in order from N-terminal to C-terminal: signal peptide, targeting moiety, hinge region, transmembrane domain, costimulatory signaling domain and intracellular signaling domain.
  • the chimeric antigen receptor may comprise the following domains in order from N-terminal to C-terminal: human CD8a signal peptide, anti-human CD70 single-domain antibody VHH sequence, human CD8a hinge region, CD8a transmembrane domain, 4 -1BB costimulatory signaling domain and CD3zeta intracellular signaling domain (shown in Figure 1).
  • the chimeric antigen receptor may comprise any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 and 72. amino acid sequence.
  • the chimeric antigen receptor can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 and 72 Amino acids with at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology sequence.
  • the present application also provides isolated one or more nucleic acid molecules that encode the isolated antigen-binding proteins described herein.
  • each of the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode a portion thereof (eg, HCDR1-3, one of the heavy chain variable regions, or variety).
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
  • nucleic acids encoding the isolated antigen-binding proteins can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the isolated antigen-binding proteins described herein Fragments of nucleic acid molecules.
  • the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more of the nucleic acid molecules may be included in each vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc.
  • a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, the promoters for SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's A portion may be fused to a portion of the gene promoter for other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector can be an expression vector.
  • the vector can be a viral vector.
  • the viral vector can be administered directly to the patient (in vivo) or can be administered in an indirect form, eg, by treating cells with the virus in vitro, and then administering the treated cells to the patient (ex vivo).
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise long terminal repeats 5'LTR and truncated 3'LTRs, RREs, rev response elements (cPPT), central termination sequences (CTS) and/or post-translational regulatory elements (WPRE). The vectors described herein can be introduced into cells.
  • the application provides a cell.
  • the cells may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
  • the cells may also comprise a chimeric antigen receptor or antigen binding protein as described herein.
  • each or each cell may contain one or one nucleic acid molecule or vector described herein.
  • each or each cell can comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, such as prokaryotic cells (eg, bacterial cells), CHO cells, NS/0 cells, HEK293T cells, or HEK293A cells, or other eukaryotic cells, such as those from plants cells, fungi or yeast cells, etc.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the cells can include yeast cells.
  • the cells can include E. coli cells.
  • the cells can include mammalian cells.
  • the cells can include immune cells.
  • the cells may include immune cells.
  • the cells can include immune cells.
  • the cells can include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells cell.
  • NK natural killer
  • the cells can include T lymphocytes.
  • the T lymphocytes may include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • the T cells may be helper T cells (Th), such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • the T lymphocytes can be CD4+ helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+/ CD8+ T cells, CD4-/CD8- T cells or any other T lymphocyte subtype.
  • the modified T cells are human T cells.
  • a source of cells can be obtained from a subject, eg, a patient, by various non-limiting methods.
  • T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available and known to those of skill in the art can be used.
  • the cells can be derived from a healthy donor, from a patient diagnosed with cancer, or obtained from a patient diagnosed with an infection.
  • the cells are part of a mixed population of cells with different phenotypic properties.
  • the cells can include B cells.
  • the B cells can include effector B cells (plasma cells), memory B cells.
  • the B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, regulatory B cells.
  • the immune cells can include macrophages.
  • the B cells may include type I macrophages (M1), type II macrophages (eg, M2a, M2B, M2c).
  • the cells can include NK cells.
  • the NK cells can include CD56bright and CD56dim.
  • the NK cells can include NK1 and NK2.
  • the NK cells can include A-NK and NA-NK.
  • the cells can include leukocytes.
  • a leukocyte usually refers to a nucleated blood cell with active mobility that can migrate from within blood vessels to outside blood vessels, or from extravascular tissues into blood vessels.
  • white blood cells can be found in the lymphatic system, spleen, tonsils, and other tissues in the body.
  • the leukocytes may include granulocytes (such as neutrophils, eosinophils, basophils), agranulocytes (such as lymphocytes, monocytes, macrophages, phagocytes, Mast cells).
  • the cells can include lymphocytes, which can include any monocytes found in blood, lymph, and lymphoid tissues, non-phagocytic leukocytes, eg, B lymphocytes, T lymphocytes, natural Killer (NK) cells.
  • lymphocytes can include any monocytes found in blood, lymph, and lymphoid tissues, non-phagocytic leukocytes, eg, B lymphocytes, T lymphocytes, natural Killer (NK) cells.
  • the cells can include peripheral blood mononuclear cells, which can include any cell that has a single nucleus in peripheral blood.
  • the peripheral blood mononuclear cells may include T cells, B cells, NK cells, lymphocytes, monocytes and dendritic cells.
  • the cells can include macrophages.
  • a macrophage is a species that can engulf and digest cellular debris, microorganisms, cancer cells, and other substances that all lack surface markers expressed on the surface of normal cells, a process called phagocytosis. Macrophages are present in almost all tissues and search for possible pathogens through the movement of amoeba. In addition to their important role in nonspecific innate immune responses, they can also help initiate adaptive immunity by recruiting other immune cell types, such as lymphocytes.
  • the cells have reduced expression and/or activity of CD70 in the cells compared to wild-type cells.
  • the cells are CD70 knocked out as compared to wild-type cells.
  • CD70 can be knocked out using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), or the CRISPR-Cas9 system.
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CD70 knockdown using the Cas9-gRNA system can be included.
  • the "Cas9-gRNA system” which may also be referred to as the "CRISPR/Cas9 system” is a tool for site-specific genome targeting in an organism.
  • it could be the Type II CRISPR/Cas system, a prokaryotic adaptive immune response system that uses noncoding RNA to direct Cas9 nuclease to induce site-specific DNA cleavage.
  • the CRISPR/Cas9 system can be used to create a simple RNA-programmable method to mediate genome editing in mammalian cells and can be used to generate gene knockouts (via indels) or knock-ins (via HDR).
  • the application provides a pharmaceutical composition.
  • the pharmaceutical composition may comprise the chimeric antigen receptor described herein, the isolated antigen binding protein, the isolated nucleic acid molecule, the carrier, the cell, and/or the pharmaceutically acceptable Accepted adjuvants and/or excipients.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and /or nonionic surfactants. Unless incompatible with the immune cells of the present application and/or the cell populations of the present application, any conventional media or reagents are contemplated for use in the pharmaceutical compositions of the present application.
  • the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, and may also be referred to as excipients.
  • the excipients may include binders, fillers, disintegrants, lubricants in the tablet.
  • the excipients may include wine, vinegar, medicinal juice, etc. in Chinese medicine pills.
  • the excipient may comprise a base part of a semisolid formulation ointment, cream.
  • the excipients may include preservatives, antioxidants, flavors, fragrances, solubilizers, emulsifiers, solubilizers, osmo-regulators, colorants in liquid formulations.
  • the pharmaceutical composition can be used to treat a disease or disorder associated with the expression of CD70, eg, for the treatment of leukemia (eg, AML) or kidney cancer.
  • a disease or disorder associated with the expression of CD70 eg, for the treatment of leukemia (eg, AML) or kidney cancer.
  • the present application provides the use of the chimeric antigen receptor, the antigen-binding protein, the isolated nucleic acid molecule, the carrier, and/or the cell in preparing a medicine , the medicament is used to treat a disease or condition associated with the expression of CD70.
  • the present application provides a method of preventing or treating a disease or disorder associated with the expression of CD70, comprising administering to a subject in need thereof an effective amount of said chimeric antigen receptor, said antigen binding The protein, the isolated nucleic acid molecule, the vector, and/or the cell.
  • the diseases or disorders described herein that are associated with expression of CD70 include leukemia (eg, AML) or kidney cancer.
  • compositions and methods described herein can be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like.
  • the pharmaceutical compositions and methods described in this application can be used for other disease conditions that depend on an immune response, such as inflammation, immune diseases, and infectious diseases.
  • the present application provides methods of preparing the isolated antigen binding proteins described herein and the chimeric antigen receptors described herein.
  • the method may comprise culturing the host cell described herein under conditions such that the isolated antigen binding protein is expressed.
  • the method may further comprise the step of harvesting (eg, isolating and/or purifying) the isolated antigen binding protein described herein.
  • the isolated antigen-binding proteins described in this application can also be purified and separated by gel electrophoresis and/or high performance liquid chromatography, etc.
  • the fusion protein polypeptide bound to the affinity column can also be eluted by using a high salt buffer, changing the pH, and the like.
  • the subject may include a human or a non-human animal.
  • the non-human animal may be selected from the group consisting of monkey, chicken, goose, cat, dog, mouse and rat.
  • non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the person may be Caucasian, African, Asian, Semitic, or other race, or a hybrid of various races.
  • the human can be an elderly, adult, adolescent, child, or infant.
  • the effective amount in humans can be extrapolated from the effective amount in experimental animals.
  • Freireich et al. describe the correlation of doses (based on milligrams per square meter of body surface) in animals and humans (Freiheim et al., Cancer Chemother. Rep. 50, 219 (1966)).
  • Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
  • Example 1 Obtaining CD70 single domain antibody VHH gene sequence and detecting its affinity
  • Alpaca immunizations were performed using recombinant human CD70 antigen (Acrobiosystems: CDL-H82Q9) with a C-terminal human IgG Fc tag. A total of four immunizations were administered with an interval of about 3 weeks. The first immunization was subcutaneously injected with 0.5 mg of recombinant human CD70 antigen and CFA (complete Freund's adjuvant) 1:1 mixed and emulsified, and counted as the first day. For the next three immunizations, 0.25 mg of recombinant human CD70 antigen and IFA (incomplete Freund's adjuvant) were mixed 1:1 to be emulsified and injected subcutaneously.
  • CFA complete Freund's adjuvant
  • Single-domain antibody molecules are displayed on the surface of phage by phage display technology, and then antigen-specific single-domain antibodies are screened.
  • the human CD70His fusion protein (Acrobiosystems: CDL-H82Q9) was coated into a 96-well plate, and the Elisa experiment was screened for 3-5 rounds, and the CD70-specific phage was gradually enriched. A large number of positive clones were selected for Elisa detection, and positive clones were screened and sequenced. Unique clones were determined according to sequence alignment and their sequences were divided into framework region FR and complementarity determining region CDR. Through the above methods, a total of 13 single-domain antibodies (sdAbs) targeting human CD70 with high affinity were obtained.
  • sdAbs single-domain antibodies
  • the 13 antibodies were named as CD70-sdAb-1C (amino acid sequence shown in SEQ ID NO:34, nucleic acid sequence shown in SEQ ID NO:47), CD70-sdAb-1E (amino acid sequence shown in SEQ ID NO:35) shown, the nucleic acid sequence is shown in SEQ ID NO:48), CD70-sdAb-1F (amino acid sequence shown in SEQ ID NO:36, nucleic acid sequence shown in SEQ ID NO:49), CD70-sdAb-1G ( The amino acid sequence is shown in SEQ ID NO:37, the nucleic acid sequence is shown in SEQ ID NO:50), CD70-sdAb-1H (the amino acid sequence is shown in SEQ ID NO:38, and the nucleic acid sequence is shown in SEQ ID NO:51) ), CD70-sdAb-3H (amino acid sequence as shown in SEQ ID NO:39, nucleic acid sequence as shown in SEQ ID NO:52), CD70-sdAb-5A (amino acid
  • CD70-sdAb-1E Using different concentrations of CD70-sdAb-1E, CD70-sdAb-1G, CD70-sdAb-3H, CD70-sdAb-L2 corresponding sdAb-Fc antibody protein against renal cancer 786-O cell line (expressing CD70) and 786-O - KO (CD70) cell line (CD70 gene was knocked out with CRISPR, so it does not express CD70) was stained, the proportion of positive cells was analyzed by flow cytometry, and the curves shown in Figure 2A-2D were drawn respectively, and the four kinds of The corresponding EC50s of antibodies CD70-sdAb-1E, CD70-sdAb-1G, CD70-sdAb-3H, and CD70-sdAb-L2 were 14.14 ⁇ g/ml, 5.70 ⁇ g/ml, 10.76 ⁇ g/ml, and 11.65 ⁇ g/ml, respectively.
  • Example 2 Whole gene synthesis of CD70 chimeric antigen receptor molecule and construction of lentiviral expression vector
  • the obtained 13 sdAb strains were constructed into lentiviral vectors to screen out more effective CD70-targeting chimeric antigen receptors.
  • the chimeric antigen receptors targeting CD70 specifically include: human CD8 ⁇ signal peptide, single domain antibody VHH sequence targeting human CD70, human CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB costimulatory signaling domain and CD3zeta Intracellular signaling domains, which are connected in series in series ( Figure 1).
  • CD70-CAR-1C amino acid sequence shown in SEQ ID NO: 60, nucleic acid sequence shown in SEQ ID NO: 73
  • CD70-CAR- 1E amino acid sequence as shown in SEQ ID NO:61, nucleic acid sequence as shown in SEQ ID NO:74
  • CD70-CAR-1F amino acid sequence as shown in SEQ ID NO:62, nucleic acid sequence as shown in SEQ ID NO:75
  • CD70-CAR-1G amino acid sequence shown in SEQ ID NO:63, nucleic acid sequence shown in SEQ ID NO:76
  • CD70-CAR-1H amino acid sequence shown in SEQ ID NO:64, The nucleic acid sequence is shown in SEQ ID NO: 77), CD70-CAR-3H (the amino acid sequence is shown in SEQ ID NO: 65, and the nucleic acid sequence is shown in SEQ ID NO: 78), CD70-CAR-5A
  • CD70-targeting chimeric antigen receptor gene sequences were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and cloned into the pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • restriction endonucleases BamHI (NEB: R3136S) and SalI (NEB: R3138S) were added to the 5' and 3' ends of the gene when synthesizing the gene.
  • the chimeric antigen receptor gene sequence targeting CD70 was double-enzymatically cleaved from the pUC57 vector using the two restriction sites of BamHI and SalI, and the restriction bands were detected by agarose gel electrophoresis and recovered and purified respectively ( QIAGEN: 28706) to obtain a DNA fragment targeting the CD70 chimeric antigen receptor.
  • the DNA fragment targeting CD70 chimeric antigen receptor recovered by enzyme digestion was cloned into the lentiviral vector pCGK (PCT/CN2019/090605) by T4 ligase (NEB: M0202S).
  • Recombinant plasmids targeting CD70 chimeric antigen receptor gene sequences CD70-CAR-1C-pCGK, CD70-CAR-1E-pCGK, CD70-CAR-1F-pCGK, CD70-CAR-1G-pCGK, CD70-CAR -1H-pCGK, CD70-CAR-3H-pCGK, CD70-CAR-5A-pCGK, CD70-CAR-5B-pCGK, CD70-CAR-5H-pCGK, CD70-CAR-7D-pCGK, CD70-CAR-H3 -pCGK, CD70-CAR-L2-pCGK, CD70-CAR-L1-pCGK.
  • the 13 recombinant plasmids were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification.
  • the sequencing primers were:
  • Transformation of recombinant plasmids and shake bacteria The recombinant plasmids verified by sequencing were re-transformed into E. coli stbl3 (purchased from Beijing Cresibo Company). The next day, pick a single clone from the transformed plate into a 3 ml shake tube of liquid LB medium containing ampicillin, and cultivate for 8 hours at 30° C. at 220 rpm with a shaker shaker. The activated bacterial solution was inoculated into 250 ml of liquid LB medium containing ampicillin according to the inoculum amount of 1:500, and cultured at 30° C. at 220 rpm for 12-16 h with shaking on a shaker.
  • the cryopreserved 293T cells (purchased from ATCC) were taken out from liquid nitrogen and thawed by constant shaking in a 37°C water bath. After wiping the mouth of the cryopreservation tube with sterilized alcohol, transfer it to a 15ml centrifuge tube to which 10ml of pre-warmed DMEM complete medium (90%DMEM+10%FBS+1%penicillin/streptomycin) has been added in advance, and gently blow evenly. , 1000rpm centrifugation for 3min, suction and discard the supernatant. Add 10ml of DMEM complete medium, and then inoculate it into a 10cm dish, and inoculate it in a cell incubator at 37°C with 5% CO 2 .
  • pre-warmed DMEM complete medium 90%DMEM+10%FBS+1%penicillin/streptomycin
  • the 293T cells were passaged. First, remove the medium with a pipette, then wash the 293T cells once with 10 ml of PBS, add 3 ml of trypsin containing 0.25% EDTA, and put in Incubator for 1-2min (during this period, you need to take it out and observe whether the cells become round under the microscope). After the cells became round, 1 ml of DMEM complete medium was added to terminate the digestion, transferred to a 15 ml centrifuge tube, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. According to the needs of the experiment, the 293T cells were inoculated into a new 10cm culture dish for further culture or cryopreservation according to the ratio of 1:3 or 1:5.
  • the resuscitated 293T cells can be used to package lentivirus after being cultured for more than 2 passages. After the first cultured 293T cells were digested and mixed by trypsin, according to the counting results, 293T cells were inoculated according to about 1.7 ⁇ 10 7 cells/T175 flask (cultured in 30 ml medium), shaken gently, and then placed at 37°C with 5% CO. 2 in a cell incubator overnight to reach 70-80% cell density for transfection.
  • the medium before plasmid transfection should be changed to DMEM medium with 10% FBS but no dual antibody.
  • First prepare the plasmid complex add 1.5ml Opti-MEM (Thermo Fisher Scientific; 31985-070) to a 15ml centrifuge tube, then add viral vector plasmid: 18 ⁇ g, psPAX2 plasmid (Addgene; Cat. No.: 12260): 9 ⁇ g, pMD2.G Plasmid (Addgene; Cat. No.: 12259): 18 ⁇ g, after adding the above plasmids, mix gently and let stand for 5 min.
  • viral recombinant plasmids CD70-CAR-1C-pCGK, CD70-CAR-1E-pCGK, CD70-CAR-1F-pCGK, CD70-CAR-1G-pCGK, CD70-CAR-1H-pCGK, CD70-CAR -3H-pCGK, CD70-CAR-5A-pCGK, CD70-CAR-5B-pCGK, CD70-CAR-5H-pCGK, CD70-CAR-7D-pCGK, CD70-CAR-H3-pCGK, CD70-CAR-L2 -pCGK, CD70-CAR-L1-pCGK), 13 kinds of viruses need to be packaged, and each virus recombinant plasmid is added separately.
  • the transfection reagent complex prepares the transfection reagent complex: add 67.5 ⁇ l (2mg/ml) PEI (polysciences: 24765) to 1.5ml Opti-MEM and mix well, let stand for 5 minutes at room temperature, and then add the transfection reagent complex to the plasmid after the incubation. In the compound, let stand for 20min after mixing. Finally, the transfection complex was slowly added dropwise to the 293T cells, mixed gently, and cultured at 37°C in a cell incubator with 5% CO 2 .
  • PEI polysciences: 24765
  • the cell culture supernatant was collected into a 50ml centrifuge tube, and centrifuged at 2000rpm for 10min to remove cell debris.
  • the cell supernatant was filtered using a Millipore 0.45 ⁇ m filter, and the filtrate was transferred to a special centrifuge tube.
  • the filtrate was poured off after the ultra-centrifugation, the lentivirus was resuspended in serum-free medium, and the lentivirus was aliquoted and stored in an ultra-low temperature freezer at -80°C.
  • Example 4 Resuscitated human primary T cells knock out CD70 gene and then activate
  • PBMCs peripheral blood mononuclear cells
  • the human peripheral blood mononuclear cells were taken out of liquid nitrogen and counted after their recovery.
  • the recovered cells were electroporated with CRISPR/Cas9 to introduce CD70 sgRNA-19 into T cells to knock out T cells.
  • CD70 gene in cells The cell processing procedure is carried out with reference to Example 3 of patent WO2019/011118.
  • non-knockout T cells control T cells
  • CD70 knockout T cells were counted and the cell concentration was adjusted to 1 ⁇ 106/ml.
  • Each well was seeded with 500 ⁇ l of cells into 24 wells, containing CD70-CAR-1C-pCGK, CD70 -CAR-1E-pCGK, CD70-CAR-1F-pCGK, CD70-CAR-1G-pCGK, CD70-CAR-1H-pCGK, CD70-CAR-3H-pCGKCD70-CAR-5A-pCGK, CD70-CAR-5B - Lentiviruses of pCGK, CD70-CAR-5H-pCGK, CD70-CAR-7D-pCGKCD70-CAR-H3-pCGK, CD70-CAR-L2-pCGK, CD70-CAR-L1-pCGK were added to T-KO cell culture T cells were transfected in the wells, and untransfected T-KO cells were used as negative controls to
  • CAR-T cells corresponding to CD70-CAR-03 were prepared according to a similar process: CAR-T cells corresponding to CD70-CAR-03, named as: CD70KO-CAR-03.
  • CD70KO-CAR-03 Continue to culture T cells in each group to a resting state.
  • each group of CAR-T cells was taken, the genome of each group of cells was extracted, and it was confirmed that CD70 had been successfully knocked out in these CAR-T cells by PCR and TIDE analysis.
  • Control T cells CT-KO, CD70 KO-CAR-1C, CD70 KO-CAR-1E, CD70 KO-CAR-1F, CD70 KO-CAR-1G, CD70 KO-CAR-1H, CD70 KO-CAR-3H, CD70 KO-CAR-5A, CD70 KO-CAR-5B, CD70 KO-CAR-5H, CD70 KO-CAR-7D, CD70 KO-CAR-H3, CD70 KO-CAR-L2, CD70 KO-CAR-L1 cell knockout
  • Table 2 The efficiencies are shown in Table 2.
  • the expression of the targeted CD70 CAR-T cells prepared in Example 5 in each group of T cells was detected by fluorescent antibody staining and flow cytometry. The specific steps were: centrifuged to collect the targeted CD70 CAR-T cells and slow viral infection for 48h respectively. Virus-uninfected T cells and T-KO cells were added with biotinylated human CD70 protein (100 ⁇ g/ml) (Acrobiosystems: CDL-H82Q9), incubated at 4°C in the dark for 30 minutes, and washed once with PBS after the incubation.
  • biotinylated human CD70 protein 100 ⁇ g/ml
  • 7.1 Cell co-culture First, the expression of CD70 in 786-O (purchased from the cell bank of the Chinese Academy of Sciences), THP-1 cells, Raji cells, K562 cells and 293T cells was detected. A certain volume of cells was taken, stained with CD70 antibody and analyzed by flow cytometry. It was found that Raji, THP-1 and 786-O cells highly expressed CD70, while K562 and 293T cells did not express CD70. 293T cells were transfected with lentivirus expressing CD70 gene (Genebank: NM_001252.5) to prepare 293T.CD70 cell line expressing CD70 protein. See Example 3 for the lentivirus preparation process.
  • lentivirus expressing CD70 gene Genebank: NM_001252.5
  • CD70-targeting CAR-T cells CD70 KO-CAR-1C, CD70 KO-CAR-1E, CD70 KO-CAR-1F, CD70 KO-CAR-1G, CD70 KO-CAR-1F, CD70 KO-CAR-1G, CD70 KO-CAR -1H, CD70 KO-CAR-3H, CD70 KO-CAR-5A, CD70 KO-CAR-5B CD70 KO-CAR-5H, CD70 KO-CAR-7D, CD70 KO-CAR-H3, CD70 KO-CAR-L2 , CD70 KO-CAR-L1) and control T-KO cells were counted, the concentration was adjusted to 5 ⁇ 10 5 /ml, and then 100ul per well was seeded into a flat-bottom 96-well plate.
  • the target cells Raji purchased from the Cell Bank of the Chinese Academy of Sciences
  • 786-O THP-1
  • 293T.CD70 expressing CD70 protein negative cells K562 (Cell Bank of the Chinese Academy of Sciences)
  • 293T ATCC
  • the target ratio was 1:1, that is, the cell concentration was 5 ⁇ 10 5 /ml, and 100 ⁇ l/well was added to the CD70-targeting CAR-T cells or the control T-KO cells for co-culture.
  • Example 8 Detection of killing ability after co-culture of CD70-targeted CAR-T with different target cells
  • luciferase-labeled cell lines Use lentivirus with luciferase (GenBank: AAR29591.1) to transfect 786-O cells, THP-1 cells, K562 cells and 293T cells to prepare luciferase-labeled cell lines, respectively. Named: 780-O.luc cells, THP-1.luc cells, K562.luc cells and 293T.luc cells. See Example 3 for the lentivirus preparation process. The luciferase-labeled cell line was plated into a 96-well flat-bottom opaque white plate according to the cell concentration of 1 ⁇ 10 5 /ml and 50 ⁇ l/well.
  • CD70 CAR-T cells Three effector-to-target ratios of 2.5:1, 1:1 and 0.5:1 were set to co-culture targeting CD70 CAR-T cells with THP-1, and three effector-to-target ratios of 1:1, 0.5:1 and 0.25:1 were used to target CD70 CAR-T cells.
  • CD70 CAR-T cells were co-cultured with 786-O cells, with three effector-target ratios of 2.5:1, 1:1 and 0.5:1.
  • CD70-targeting CAR-T cells were co-cultured with K562 cells and 293T cells, respectively.
  • the CAR-T cells targeting CD70 are CD70 KO-CAR-1C, CD70 KO-CAR-1E, CD70 KO-CAR-1F, CD70 KO-CAR-1G, CD70 KO-CAR-1H, CD70 KO-CAR-1H 3H, CD70 KO-CAR-5A, CD70 KO-CAR-5B, CD70 KO-CAR-5H, CD70 KO-CAR-7D, CD70 KO-CAR-H3, CD70 KO-CAR-L2, CD70 KO-CAR-L1, Control T-KO cells were co-cultured with target cells, and each CD70-targeting CAR-T was replicated in 3 replicates. Place the 96-well plate in a 37°C 5% CO2 cell incubator overnight.
  • the remaining luciferase activity (relative light units, RLU) of the target cells was measured to detect the killing ability of each CD70-targeting CAR-T on different target cells.
  • the specific steps are as follows: directly add 100 ⁇ l of D-luciferin substrate (Thermo Fisher Scientific: 88293) to the cells after co-culture, mix well for 5 minutes in the dark, and use a microplate reader to detect the fluorescence intensity in chemiluminescence mode. Maximal luciferase activity was obtained as a control by adding medium to target cells in the absence of effector cells. The results are shown in Figure 5, the CAR-T targeting CD70 has killing effect on THP-1 (acute myeloid leukemia cell line) and 786-O (renal cancer cell line).
  • THP-1 Luc tumor cells are human acute myeloid leukemia cells expressing luciferase luciferase. They were expanded and cultured after routine resuscitation. After at least two passages, the cells in logarithmic growth phase were harvested and resuspended in serum-free medium. . 6-8 week old female NOG mice were taken, and 0.1 mL of a PBS suspension containing 5 ⁇ 10 6 cells was inoculated into the tail vein of all animals under sterile conditions.
  • THP-1Luc cells 7-11 days after inoculation of THP-1Luc cells in the tail vein, 33 animals were screened according to the tumor signal intensity and randomly divided into 11 groups, and the following cells were injected with a single intravenous injection: buffer group (PBS), control T cell group T-KO , CD70 KO-CAR-1C, CD70 KO-CAR-1E, CD70 KO-CAR-1F, CD70 KO-CAR-1G, CD70 KO-CAR-3H, CD70 KO-CAR-H3, CD70 KO-CAR-L2, CD70 KO-CAR-L1, CD70 KO-CAR-03 (positive control), 3 mice per group.
  • PBS buffer group
  • 786-O tumor cells are human renal cancer cells, which were expanded and cultured after routine resuscitation. After at least 2 passages, the cells in logarithmic growth phase were harvested and resuspended in serum-free medium to 1 ⁇ 10 7 cells/mL. Take 45 female NOG mice aged 6-8 weeks, resuspend 786-O cells in a 1:1 mixture of PBS and Matrigel matrix, and inoculate 0.2 mL of cell suspension (containing 5 ⁇ 10 cells) subcutaneously in all animals aseptically. 6 786-O cells), inoculated on the right back of mice.
  • control T cell group T-KO CD70 KO- CAR-1E, CD70 KO-CAR-1G, CD70 KO-CAR-3H, CD70 KO-CAR-L2, CD70 KO-CAR-03 (positive control), 5 mice per group.
  • Figure 7 can show that under the experimental conditions, all groups of CAR-T can significantly inhibit tumors compared with the control T-KO group. Compared with the positive control CD70 KO-CAR-03, the four groups of CD70 KO-CAR-1E, CD70 KO-CAR-1G, CD70 KO-CAR-3H and CD70 KO-CAR-L2 have stronger antitumor activity , consistent with the results in the above-mentioned acute myeloid leukemia animal model.

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Abstract

一种靶向CD70的抗原结合蛋白，尤其涉及一种包含该抗原结合蛋白的嵌合抗原受体以及其应用，以及包含或表达该抗原结合蛋白的细胞。所述抗原结合蛋白以1nM的K D值与CD70蛋白相结合。所述包含该抗原结合蛋白的嵌合抗原受体对于CD70蛋白的特异性识别结合能力强。所述包含或表达该抗原结合蛋白的细胞可分泌细胞因子IL-2和IFN-γ。

Description

靶向CD70的抗原结合蛋白及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向CD70的抗原结合蛋白及其应用。
背景技术
近年来，随着肿瘤免疫疗法的发展和临床研究的进展，嵌合抗原受体T细胞(Chimeric Antigen Receptor-T cells，CAR-T)免疫疗法以其独特的抗肿瘤优势，目前是最有发展前景的肿瘤免疫疗法之一。CAR-T细胞是由单克隆抗体的单链可变区和T细胞信号转导区连接而成，抗体与相应的肿瘤抗原结合后能以主要组织相容性复合体以非限制性方式使T细胞活化，进而发挥抗肿瘤效应。CAR的结构分为：胞外抗原结合区、铰链区、跨膜结构域和胞内信号传导结构域。目标抗原的选择对于CAR的特异性、有效性以及基因改造T细胞自身的安全性来讲都是关键的决定因素。
CD70是II型跨膜蛋白，是肿瘤坏死因子(tumor necrosis factor，TNF)家族成员，又称为肿瘤坏死因子受体超家族(tumor necrosis factor receptor super-family，TNFSF)因子，具有调控T细胞和B细胞活化、增殖及分化的能力，在维持机体免疫应答过程中起着重要的作用。目前，实体瘤治疗缺少有效的靶点是限制CAR-T的瓶颈之一。研究结果显示CD70高水平表达于原发性及转移性肾癌中，其中在肾透明细胞癌的表达率达100％。CD70也被报道在急性髓系白血病干细胞中高表达。CD70在肿瘤组织中的高水平表达，不仅可以诱导免疫逃逸，同时也可以激活部分免疫细胞杀伤肿瘤细胞，可能作为一种潜在的肿瘤治疗靶点，给肿瘤的免疫治疗带来了新的方向。
目前的CAR-T疗法通常以来源于单克隆抗体抗原结合区域的scFv段作为抗原结合区。细胞外的scFv结构域在结合表达于靶细胞表面的靶蛋白以后，可以激活CAR结构的共刺激域和激活域。但是scFv分子量比较大而且容易形成多聚体，影响CAR的功能。因此，需要包含新结构的抗原结合区的CAR。
发明内容
本申请提供了一种靶向CD70的抗原结合蛋白，尤其涉及一种包含该抗原结合蛋白的嵌合抗原受体以及其应用，以及包含或表达该抗原结合蛋白的细胞，其具有下列性质中的一种 或多种：1)对CD70蛋白的特异性识别结合能力强；和2)能表现出更好的分泌细胞因子IL-2和IFN-γ的能力。
一方面，本申请提供了一种嵌合抗原受体，所述靶向部分包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1包含SEQ ID NO:1、6、9、11、14、17、20、23、26和29中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:2、7、12、15、18、21、24、27、30和32中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:3、4、5、8、10、13、16、19、22、25、28、31和33中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR1、HCDR2和HCDR3包含选自下述任意一组的氨基酸序列：
a)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
b)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:4；
c)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:5；
d)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:8；
e)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:10；
f)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:13；
g)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:15，和HCDR3：SEQ ID NO:16；
h)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:18，和HCDR3：SEQ ID NO:19；
i)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:21，和HCDR3：SEQ ID NO:22；
j)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:24，和HCDR3：SEQ ID NO:25；
k)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:27，和HCDR3：SEQ ID NO:28；
l)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30，和HCDR3：SEQ ID NO:31；
m)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:32，和HCDR3：SEQ ID NO:33。
在某些实施方式中，所述靶向部分包括VHH。
在某些实施方式中，所述靶向部分包含SEQ ID NO:39、34、35、36、37、38、44、40、41、42、43、45和46中任一项所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体，其包含信号肽。
在某些实施方式中，所述信号肽包含源自选自下组蛋白的信号肽或其组合：CD8、4-1BB、GM-CSF、CD3γ、CD3δ、CD3ε、CD22、CD79a、CD79b和CD66d。
在某些实施方式中，所述信号肽包括源自CD8的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:88所示的氨基酸序列。
在某些实施方式中，所述信号肽的C端与所述靶向部分的N端连接。
在某些实施方式中，所述的嵌合抗原受体，其包含铰链区。
在某些实施方式中，所述铰链区包含源自选自下组的蛋白的铰链区或其组合：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。
在某些实施方式中，所述铰链区包括源自所述CD8中的CD8a的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:89所示的氨基酸序列。
在某些实施方式中，所述铰链区的N端与所述靶向部分的C端连接。
在某些实施方式中，所述的嵌合抗原受体，其包含跨膜结构域。
在某些实施方式中，所述跨膜结构域包含源自选自下组蛋白的跨膜结构域或其组合：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、CTLA-4、LAG-3、TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3δ、CD3γ、CD3ζ、细胞因子受体、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεR、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜结构域包括源自所述CD8中的CD8a的跨膜结构域。
在某些实施方式中，所述跨膜结构域包含SEQ ID NO:90所示的氨基酸序列。
在某些实施方式中，所述跨膜结构域的N端与所述铰链区的C端连接。
在某些实施方式中，所述的嵌合抗原受体，其包含共刺激信号传导结构域。
在某些实施方式中，所述共刺激信号传导结构域包含源自选自下组的蛋白的共刺激信号传导结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号传导结构域包括源自4-1BB的共刺激信号传导结构域。
在某些实施方式中，所述共刺激信号传导结构域包含SEQ ID NO:91所示的氨基酸序列。
在某些实施方式中，所述共刺激信号传导结构域的N端与跨膜结构域的C端连接。
在某些实施方式中，所述的嵌合抗原受体，其包含胞内信号传导结构域。
在某些实施方式中，所述胞内信号传导结构域包含源自选自下组的蛋白的胞内信号传导结构域或其组合：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、CD66d、CD5、CD22、FcRγ、FcRβ、FcRε、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒(BLV)gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒(SIV)PBj14 Nef、卡波西肉瘤疱疹病毒(KSHV)K1、DAP10、DAP12，和至少包含一个免疫受体酪氨酸激活基序(ITAM)的结构域。
在某些实施方式中，所述胞内信号传导结构域包括源自CD3zeta的胞内信号传导结构域。
在某些实施方式中，所述胞内信号传导结构域包含SEQ ID NO:92所示的氨基酸序列。
在某些实施方式中，所述胞内信号传导结构域的N端与所述共刺激信号传导结构域的C端连接。
在某些实施方式中，所述的嵌合抗原受体包含SEQ ID NO:60、61、62、63、64、65、66、67、68、69、70、71和72任一项所示的氨基酸序列。
另一方面，本申请提供了一种分离的抗原结合蛋白，其以1nM或更低的KD值特异性结合CD70。
在某些实施方式中，所述的分离的抗原结合蛋白包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，其中所述HCDR1包含SEQ ID NO:1、6、9、11、14、17、20、23、26和29中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:2、7、12、15、18、21、24、27、30和32中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:3、4、5、8、10、13、16、19、22、25、28、31和33中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白，其为抗体或其抗原结合片段。
在某些实施方式中，所述的分离的抗原结合蛋白，其为VHH或其抗原结合片段。
在某些实施方式中，所述的分离的抗原结合蛋白中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述的分离的抗原结合蛋白中所述HCDR1、HCDR2和HCDR3包含选自下组中任意一组的氨基酸序列：
a)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
b)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:4；
c)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:5；
d)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:8；
e)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:10；
f)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:13；
g)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:15，和HCDR3：SEQ ID NO:16；
h)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:18，和HCDR3：SEQ ID NO:19；
i)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:21，和HCDR3：SEQ ID NO:22；
j)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:24，和HCDR3：SEQ ID NO:25；
k)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:27，和HCDR3：SEQ ID NO:28；
l)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30，和HCDR3：SEQ ID NO:31；
m)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:32，和HCDR3：SEQ ID NO:33。
在某些实施方式中，所述的分离的抗原结合蛋白包括VHH。
在某些实施方式中，所述的分离的抗原结合蛋白包含SEQ ID NO:39、34、35、36、37、38、44、40、41、42、43、45和46任一项所示的氨基酸序列。
另一方面，本申请提供了一种分离的核酸分子，其编码所述的嵌合抗原受体和/或所述的分离的抗原结合蛋白。
在某些实施方式中，所述的分离的核酸分子包含SEQ ID NO:47-59和/或SEQ ID NO：73-85中任一项所示的核酸序列。
另一方面，本申请提供了一种或多种载体，其包含所述的分离的核酸分子。
在某些实施方式中，所述的载体包括病毒载体。
在某些实施方式中，所述的载体包括慢病毒载体。
另一方面，本申请提供了一种或多种细胞，其包含所述的嵌合抗原受体，所述的分离的抗原结合蛋白，所述的分离的核酸分子，和/或所述的载体。
在某些实施方式中，所述的细胞包括免疫细胞。
在某些实施方式中，所述免疫细胞选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些实施方式中，所述的细胞包括T细胞。
在某些实施方式中，所述的细胞与相应的野生型细胞相比，所述细胞中CD70的表达和/活性降低。
在某些实施方式中，所述的细胞与相应的野生型细胞相比，所述细胞敲除了CD70。
另一方面，本申请提供了一种药物组合物，其包含所述的嵌合抗原受体，所述的分离的抗原结合蛋白，所述的分离的核酸分子，所述的载体，所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
另一方面，所述的嵌合抗原受体，所述的分离的抗原结合蛋白，所述的分离的核酸分子，所述的载体，所述的细胞，和/或所述的药物组合物，其用于治疗与CD70的表达相关的疾病或病症。
在某些实施方式中，所述与CD70的表达相关的疾病或病症包括急性髓系白血病(AML)或肾癌。
另一方面，所述的嵌合抗原受体，所述的分离的抗原结合蛋白，所述的分离的核酸分子，所述的载体，所述的细胞，和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗与CD70的表达相关的疾病或病症。
在某些实施方式中，所述的用途，其中所述与CD70的表达相关的疾病或病症包括急性髓系白血病(AML)或肾癌。
另一方面，本申请提供了一种预防和/或治疗与CD70的表达相关的疾病或病症的方法，其包括向有需要的受试者施用有效量的所述的嵌合抗原受体，所述的分离的抗原结合蛋白，所述的分离的核酸分子，所述的载体，所述的细胞，和/或所述的药物组合物。
在某些实施方式中，所述与CD70的表达相关的疾病或病症包括急性髓系白血病(AML)或肾癌。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述靶向CD70的CAR的结构。
图2A-2D显示的是本申请所述靶向CD70的单域抗体与细胞系786-O结合的亲和性。
图3显示的是本申请所述流式细胞仪检测靶向CD70的CAR在敲除CD70的T细胞表面的表达效率。
图4显示的是本申请所述靶向CD70的CAR-T细胞与靶细胞共培养后用Elisa法检测IL-2和IFN-γ细胞因子的分泌。
图5显示的是本申请所述靶向CD70的CAR-T细胞与标记有荧光素酶的靶细胞共培养后检测靶向CD70的CAR-T细胞对靶细胞的杀伤率。
图6显示的是本申请所述靶向CD70的CAR-T细胞在急性髓系白血病动物模型上的抗肿瘤效果。
图7显示的是本申请所述靶向CD70的CAR-T细胞在肾癌动物模型上的抗肿瘤活性。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“分离的抗原结合蛋白”通常是指能够特异性识别和/或中和特定抗原的多肽聚合体。例如，分离的抗原结合蛋白可以包括重链的一部分。例如，分离的抗原结合蛋白可以包括重链可变区。术语“分离的抗原结合蛋白”可以包括单域抗体。例如，分离的抗原结合蛋白可以包括但不限于人单域抗体。
在本申请中，术语“单域抗体”或“sdAb”或“VHH”通常是指缺失抗体轻链而只有重链可变区的一类抗体。在某些情形中，单域抗体可以来自双峰驼、单峰驼、羊驼、美洲驼、护士鲨、大星鲨或鳐鱼(例如，可参见康晓圳等，生物工程学报,2018,34(12):1974-1984)。例如，单域抗体可以来自羊驼。单域抗体可由重链可变区(VH)构成。术语“重链可变区”通常是指抗原结合片段的重链的氨基末端结构域。重链可变区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在成为框架区(FR)的更保守的区域中。每个重链可变区可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链可变区含有与抗原(例如，CD70)相互作用的结合结构域。
在本申请中，术语“CD70抗原”和“CD70蛋白”可以互换使用。并且包括CD70的任何变体和同源物，其由细胞天然表达或在用CD70基因转染的细胞上表达。在本申请中，CD70可以为人CD70，其在GenBank中的登录号为NM_001252.5，其在UniProt/Swiss-Prot中的登录号为P32970。例如，所述CD70蛋白可包含人CD70蛋白的同源物。在本申请中，术语“同源物”通常是指与人CD70蛋白氨基酸序列和人CD70蛋白核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“跨膜结构域”通常是指细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。跨膜结构域可以与细胞内信号传导结构域相连接，起着传递信号的作用。在本申请中，跨膜结构域可以源自任意的I型、II型或III型跨膜蛋白。在本申请中，跨膜结构域可以包含源自选自下组蛋白的跨膜结构域或其组合：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、T细胞受体的亚基、构成CD3复合体的多肽、IL2受体、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεR、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，T细胞受体的亚基可以包括TCRα、TCRβ、TCRγ或TCRδ。例如，构成CD3复合体的多肽可以包括CD3ε、CD3δ、CD3γ或CD3ζ。例如，跨膜结构域可以包括源自所述CD8中的CD8a的跨膜结构域。
在本申请中，术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”通常是指包含能够结合抗原的靶向部分和至少一个胞内结构域的融合蛋白。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可包括靶向部分(例如，靶向肿瘤特异性抗原和/或肿瘤相关抗原)、信号肽、跨膜结构域、共刺激信号传导结构域和胞内信号传导结构域。在本申请中，所述CAR 可以基于抗体的抗原(例如CD70)特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。关于CAR和CAR-T细胞的描述，可参见例如Sadelain M,Brentjens R,Rivi`ere I.The basicprinciples of chimeric antigen receptor design.Cancer Discov.2013；3(4):388-398；Turtle CJ,Hudecek M,Jensen MC,Riddell SR.Engineered T cells for anti-cancer therapy.Curr Opin Immunol.2012；24(5):633-639；Dotti G,Gottschalk S,Savoldo B,Brenner MK.Design and development of therapies using chimeric antigen receptor-expressing T cells.Immunol Rev.2014；257(1):107-126；以及WO2013154760、WO2016014789。
在本申请中，术语“共刺激信号传导结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。在某些情形中，共刺激信号传导结构域可以包含源自选自下组蛋白的共刺激信号传导结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，共刺激信号传导结构域可以包括源自4-1BB的共刺激信号传导结构域。
在本申请中，术语“铰链区”通常是指靶向部分(例如，靶向CD70的胞外域)和跨膜结构域之间的连接区。在某些情形中，铰链区可以包含源自选自下组的蛋白的铰链区或其组合：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。例如，铰链区可以包括源自所述CD8中的CD8a的铰链区。
在本申请中，术语“胞内信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。通常，胞内信号传导结构域为用于指导蛋白质寻靶的任何一段连续的氨基酸序列。在某些情形中，胞内信号传导结构域可以包含源自选自下组的蛋白的胞内信号传导结构域或其组合：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、CD66d、CD5、CD22、FcRγ、FcRβ、FcRε、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP12，和至少包含一个ITAM的结构域。例如，胞内信号传导结构域可以包括源自CD3zeta的胞内信号传导结构域。
在本申请中，术语“信号肽”通常是指嵌合抗原受体(CAR)的氨基末端(N-末端)的 前导序列，其在翻译时或在翻译后将CAR分子引导到内质网。在某些情形中，信号肽可以包含包含源自选自下组蛋白的信号肽或其组合：CD8、4-1BB、GM-CSF、CD3γ、CD3δ、CD3ε、CD22、CD79a、CD79b和CD66d。例如，信号肽可以包括源自CD8的信号肽。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体(全人源抗体)、人源化抗体、嵌合抗体、单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本文所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。
在本申请中，术语“抗原结合片段”通常是指抗体中发挥特异性结合抗原功能的一个或多个片段。抗体的抗原结合功能可通过抗体的全长片段来实现。抗体的抗原结合功能也可通过以下来实现：包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的重链，或者，包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的轻链。(1)Fab片段，即由VL、VH、CL和CH结构域组成的一价片段；(2)F(ab’)2片段，包含通过铰链区处的二硫键连接的两个Fab片段的二价片段；(3)由VH和CH结构域组成的Fd片段；(4)由抗体单臂的VL和VH结构域组成的Fv片段；(5)由VH结构域组成的dAb片段(Ward等，(1989)Nature 341：544-546)；(6)分离的互补决定区(CDR)和(7)可任选地通过接头连接的两个或以上分离的CDR的组合。此外，还可包括由VL和VH配对形成的一价单链分子Fv(scFv)(参见Bird等(1988)Science 242：423-426；以及Huston等(1988)Proc.Natl.Acad.Sci.85：5879-5883)。所述“抗原结合片段”还可包括免疫球蛋白融合蛋白，所述融合蛋白包含选自以下的结合结构域：(1)与免疫球蛋白铰链区多肽融合的结合结构域多肽；(2)与铰链区融合的免疫球蛋白重链CH2恒定区；和(3)与CH2恒定区融合的免疫球蛋白重链CH3恒定区。例如，所述的抗原结合片段还可以包括单域抗体。
在本申请中，术语“急性髓系白血病(AML)”通常是指造血系统的髓系原始细胞克隆性恶性增殖性疾病。急性髓系白血病可以包括所有非淋巴细胞来源的急性白血病。
在本申请中，术语“肾癌”通常是指肾脏细胞发生病变(癌变)并生长失控进而形成肿瘤的疾病。临床可见不同程度的贫血、尿液带血、体侧持续性疼痛、脚踝或腿部肿胀等症状。
在本申请中，术语“K D”可与“KD”互换使用，通常是指特定的抗体-抗原相互作用的解离平衡常数，单位为M(mol/L)。KD可通过物质AB和其解离得到的物质A和物质B的浓度来计算：KD＝c(A)*c(B)/c(AB)。由该公式可知，KD值越大，说明解离越多，代表物质A、B之间的亲和力越弱；反之，KD值越小，说明解离越少，代表物质A、B之间的亲和力越强。
在本申请中，术语“Raji细胞”通常是指能产生Epstein-Barr病毒株的连续的人类细胞系。病毒将转化脐带淋巴细胞并且在Raji细胞中诱导早期抗原。Raji细胞被广泛用作转染宿主，也被用于了解造血细胞和其他细胞恶性肿瘤。此外，因为其具有和表达某些补体成分的几种受体以及免疫球蛋白G的Fc受体，因而还被用于检测免疫复合物。
在本申请中，术语“786-O细胞”通常是指人肾透明细胞腺癌细胞。该细胞系来源于一个原发性透明细胞癌。此细胞有微绒毛和桥粒。
在本申请中，术语“THP-1细胞”通常是指人急性单核细胞白血病细胞系。该细胞系可以吞噬乳胶颗粒和激活的红细胞，细胞膜和胞浆内均没有免疫球蛋白，表达C3R和FcR；可受佛波酯TPA诱导向单核系方向分化。
在本申请中，术语“K562细胞”通常是指人类永生化骨髓性白血病细胞系。K562属于红白血病细胞系，具有恶性程度高、增殖速度快的特点。最初分离自一名处于急性期的53岁女性慢性骨髓性白血病(CML)患者的胸水。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子。所述载体可将插入的核酸分子转移到细胞中和/或细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体可以是当引入合适的细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体可包含慢病毒载体。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒 或载体，或者能够表达本申请所述的嵌合抗原受体或本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的嵌合抗原受体或抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方式中，所述细胞可以是免疫细胞。例如，所述免疫细胞可以选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。例如，所述免疫细胞可以是T细胞。
在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。
在本申请中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换地使用，通常是指具有任何长度的氨基酸的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物。这些修饰可以包含：二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵(如与标记组分结合)。术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸，包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。
在本申请中，术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短干扰RNA(siRNA)、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进 一步修饰。
除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括其功能性变体、衍生物、类似物、同源物及其片段。
术语“功能性变体”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
发明详述
抗原结合蛋白和嵌合抗原受体
一方面，本申请提供了一种分离的抗原结合蛋白。在本申请中，所述分离的抗原结合蛋白可包含可特异性结合CD70的靶向部分。另一方面，本申请提供了一种包含所述分离的抗原结合蛋白(例如，所述可特异性结合CD70的靶向部分)的嵌合抗原受体(CAR)。
在本申请中，所述分离的抗原结合蛋白可包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，其中所述HCDR1可包含SEQ ID NO:1、6、9、11、14、17、20、23、26和29中任一项所示的氨基酸序列。例如，所述HCDR1可包含与SEQ ID NO:1、6、9、11、14、17、20、23、26和29中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述HCDR2可包含SEQ ID NO:2、7、12、15、18、21、24、27、30和32中任一项所示的氨基酸序列。例如，所述HCDR2可包含与SEQ ID NO:2、7、12、15、18、21、24、27、30和32中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述HCDR3可包含SEQ ID NO:3、4、5、8、10、13、16、19、22、25、28、31和33中任一项所示的氨基酸序列。例如，所述HCDR3可包含与SEQ ID NO:3、4、5、8、10、13、16、19、22、25、28、31和33中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3。例如，所述HCDR1、HCDR2和HCDR3可包含选自下组中任意一组的氨基酸序列，或者所述分离的抗原结合蛋白可包含与选自下组中任意一组的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列：
a)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
b)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:4；
c)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:5；
d)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:8；
e)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:10；
f)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:13；
g)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:15，和HCDR3：SEQ ID NO:16；
h)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:18，和HCDR3：SEQ ID NO:19；
i)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:21，和HCDR3：SEQ ID NO:22；
j)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:24，和HCDR3：SEQ ID NO:25；
k)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:27，和HCDR3：SEQ ID NO:28；
l)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30，和HCDR3：SEQ ID NO:31；
m)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:32，和HCDR3：SEQ ID NO:33。
在本申请中，所述抗原结合蛋白还可包括框架区，所述框架区可以来源于人的抗体，或者来源于羊驼、骆驼等物种的单域抗体的框架区。例如，所述抗原结合蛋白的框架区可以来源于人。例如，所述抗原结合蛋白的框架区可以来源于人。
在本申请中，所述抗原结合蛋白可以包括Fc区。在某些情形中，所述抗原结合蛋白可以包括单域抗体。
在某些情形中，所述抗原结合蛋白可以包括VHH。
在某些情形中，所述嵌合抗原受体的靶向部分可以包含VHH。
在本申请中，所述VHH可包含SEQ ID NO:39、34、35、36、37、38、44、40、41、42、43、45和46中任一项所示的氨基酸序列。例如，所述VHH可包含与SEQ ID NO:39、34、35、36、37、38、44、40、41、42、43、45和46中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述嵌合抗原受体除包含胞外的靶向部分(例如，靶向CD70)外，还可包含胞内结构域。
在某些情形中，所述嵌合抗原受体可包含信号肽。所述信号肽可以包括但不限于源自选自下组蛋白的信号肽或其组合：CD8、4-1BB、GM-CSF、CD3γ、CD3δ、CD3ε、CD22、CD79a、CD79b和CD66d。例如，所述信号肽可以是来自CD8的信号肽。例如，所述信号肽可包含SEQ ID NO:88所示的氨基酸序列。
在某些情形中，所述嵌合抗原受体可包含铰链区。所述铰链区可以包括但不限于源自选自下组蛋白的铰链区或其组合：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。例如，所述铰链区可以是来自所述CD8中的CD8a的铰链区。例如，所述铰链区可包含SEQ ID NO:89所示的氨基酸序列。
在某些情形中，所述嵌合抗原受体可包含跨膜结构域。所述跨膜结构域可包含源自选自 下组蛋白的跨膜结构域或其组合：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、CTLA-4、LAG-3、TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3δ、CD3γ、CD3ζ、细胞因子受体、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεR、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，所述跨膜结构域可以是来自所述CD8中的CD8a的跨膜结构域。例如，所述跨膜结构域可以包含SEQ ID NO:90所示的氨基酸序列。
在某些情形中，所述嵌合抗原受体可包含共刺激信号传导结构域。所述共刺激信号传导结构域可包含源自选自下组蛋白的跨膜结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，所述共刺激信号传导结构域可以是来自共刺激信号传导结构域。例如，所述共刺激信号传导结构域可包含SEQ ID NO:91所示的氨基酸序列。
在某些情形中，所述嵌合抗原受体可包含胞内信号传导结构域。例如，所述胞内信号传导结构域可包含源自选自下组的蛋白的胞内信号传导结构域或其组合：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、CD66d、CD5、CD22、FcRγ、FcRβ、FcRε、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒(BLV)gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒(SIV)PBj14 Nef、卡波西肉瘤疱疹病毒(KSHV)K1、DAP10、DAP12和至少包含一个免疫受体酪氨酸激活基序(ITAM)的结构域。例如，所述胞内信号传导结构域可以是来自CD3zeta的胞内信号传导结构域。例如，所述胞内信号传导结构域可以包含SEQ ID NO:92所示的氨基酸序列。
在本申请中，所述信号肽的C端可以与所述靶向部分的N端连接。
例如，所述铰链区的N端可以与所述靶向部分的C端连接。
例如，所述跨膜结构域的N端可以与所述铰链区的C端连接。
例如，所述共刺激信号传导结构域的N端可以与所述跨膜结构域的C端连接。
例如，所述胞内信号传导结构域的N端可以与所述共刺激信号传导结构域的C端连接。
在本申请中，所述嵌合抗原受体从N端到C端可以依次包含下述结构域：信号肽、靶向部分、铰链区、跨膜结构域、共刺激信号传导结构域和胞内信号传导结构域。
例如，所述嵌合抗原受体从N端到C端可以依次包含下述结构域：人CD8a信号肽、抗人CD70的单域抗体VHH序列、人CD8a铰链区、CD8a跨膜结构域、4-1BB共刺激信号传导结构域和CD3zeta胞内信号传导结构域(如图1所示)。
在本申请中，所述嵌合抗原受体可包含SEQ ID NO:60、61、62、63、64、65、66、67、68、69、70、71和72中任一项所示的氨基酸序列。例如，所述嵌合抗原受体可包含与SEQ ID NO:60、61、62、63、64、65、66、67、68、69、70、71和72中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
核酸、载体和细胞
另一方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述分离的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、重链可变区中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。例如，所述分离的核酸可以是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述分离的抗原结合蛋白的核酸，这些方法包括但不限于，采用逆转录PCR和PCR获得本申请所述分离的抗原结合片段的核酸分子。
另一方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动 子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体可为表达载体。例如，所述载体可为病毒载体。可以将病毒载体直接给予至患者(体内)或可以通过间接的形式，例如，在体外使用病毒处理细胞，然后将处理过的细胞给予至患者(离体)。病毒载体技术在本领域中是公知的，并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。常规的基于病毒的系统可以包括用于基因转移的逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体以及单纯疱疹病毒载体。在某些情形中，可以用逆转录病毒、慢病毒和腺相关病毒的方法将基因转移整合进宿主基因组中，使插入的基因长期表达。慢病毒载体是能够转导或感染非分裂细胞并典型地产生较高病毒效价的逆转录病毒载体。慢病毒载体可包含长末端重复序列5’LTR和截短的3’LTR、RRE、rev应答元件(cPPT)、中央终止序列(CTS)和/或翻译后调控元件(WPRE)。本申请所述的载体可以被引入细胞。
另一方面，本申请提供了一种细胞。所述细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。所述细胞还可包含本申请所述的嵌合抗原受体或抗原结合蛋白。例如，每种或每个细胞可包含一个或一种本申请所述的核酸分子或载体。例如，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293T细胞或HEK293A细胞，或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。例如，所述细胞可以包括酵母细胞。例如，所述细胞可以包括大肠杆菌细胞。例如，所述细胞可以包括哺乳动物细胞。例如，所述细胞可以包括免疫细胞。
所述细胞可以包括免疫细胞。在某些情形中，所述细胞可以包括免疫细胞。例如，所述细胞可包括T细胞、B细胞、天然杀伤(NK)细胞、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些情形中，所述细胞可包括T淋巴细胞。所述T淋巴细胞可包括胸腺细胞、天然T 淋巴细胞、未成熟T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。所述T细胞可以是辅助T细胞(Th)，例如辅助T细胞1(Th1)或辅助T细胞2(Th2)细胞。所述T淋巴细胞可以是CD4+辅助T细胞(HTL；CD4+T细胞)、细胞毒性T细胞(CTL；CD8+T细胞)、肿瘤浸润细胞毒性T细胞(TIL；CD8+T细胞)、CD4+/CD8+T细胞、CD4-/CD8-T细胞或任何其他T淋巴细胞亚型。在某些情形中，所述经修饰的T细胞是人类T细胞。在扩增和遗传修饰本申请的细胞之前，可以通过各种非限制性方法从受试者，例如患者，获得细胞来源。T细胞可以获自许多非限制性来源，包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、感染位点的组织、腹水、胸腔积液、脾脏组织和肿瘤。在某些情形中，可以使用本领域技术人员可利用的和已知的任何数量的T细胞系。在另一些情形中，所述细胞可以源自健康供体、源自确诊患有癌症的患者或获自确诊感染的患者。在另一些情形中，所述细胞是存在不同表型特性的细胞的混合群体的一部分。
在某些情形中，所述细胞可包括B细胞。在某些情形中，所述B细胞可包括效应B细胞(浆细胞)、记忆B细胞。所述B细胞可包括B2细胞、B1细胞、边缘区B细胞、滤泡B细胞、调节性B细胞。在某些情形中，所述免疫细胞可包括巨噬细胞。所述B细胞可包括I型巨噬细胞(M1)、II型巨噬细胞(如M2a、M2B、M2c)。
在某些情形中，所述细胞可包括NK细胞。在某些情形中，所述NK细胞可包括CD56bright和CD56dim。在某些情形中，所述NK细胞可包括NK1和NK2。在某些情形中，所述NK细胞可包括A-NK和NA-NK。
在某些情形中，所述细胞可包括白细胞。白细胞通常是指一种有核的血细胞，具有活跃的移动能力，可以从血管内迁移到血管外，或从血管外组织迁移到血管内。除了在血液外，白细胞还可以存在于淋巴系统、脾，扁桃腺以及身体的其他组织。在本申请中，所述白细胞可以包括粒细胞(如中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞)、无粒白细胞(如淋巴细胞、单核细胞、巨噬细胞、吞噬细胞、肥大细胞)。
在某些情形中，所述细胞可包括淋巴细胞，所述淋巴细胞可以包括在血液、淋巴和淋巴组织中发现的任何单核细胞、非吞噬白细胞，例如，B淋巴细胞、T淋巴细胞、天然杀伤(NK)细胞。
在某些情形中，所述细胞可包括外周血单个核细胞，其可包括在外周血中具有单个核的任何细胞。例如，在本申请中，所述外周血单个核细胞可以包括T细胞、B细胞、NK细胞、淋巴细胞、单核细胞和树突状细胞。
在某些情形中，所述细胞可包括巨噬细胞。巨噬细胞是一种可以吞噬和消化细胞碎片、 微生物、癌细胞和那些所有缺少正常细胞表面表达的表面标志的其他物质，这个过程叫做吞噬作用。巨噬细胞几乎存在于所有组织中，通过阿米巴运动寻找可能的病原物。它们除了在非专一的天然免疫反应中起重要作用外，还可以通过招募其他免疫细胞类型，如淋巴细胞，帮助启动获得性免疫。
在某些情形中，所述细胞与野生型细胞相比，所述细胞中CD70的表达和/或活性降低。
在某些情形中，所述细胞与野生型细胞相比，所述细胞敲除了CD70。
例如，可以利用锌指核酸酶(ZFNs)、转录激活样效应因子核酸酶(TALEN)或CRISPR-Cas9系统敲除CD70。例如，可以包括使用Cas9-gRNA系统敲除CD70。所述“Cas9-gRNA系统”也可称为“CRISPR/Cas9系统”，是一种用于生物体中位点特异性基因组靶向的工具。例如，它可以是II型CRISPR/Cas系统，它是一种原核适应性免疫反应系统，该系统使用非编码RNA指导Cas9核酸酶诱导位点特异性DNA切割。通过细胞DNA修复机制，可以通过非同源末端连接DNA修复途径(NHEJ)或同源性定向修复(HDR)途径修复这种DNA损伤。可以利用CRISPR/Cas9系统创建一种简单的RNA可编程方法来介导哺乳动物细胞中的基因组编辑，并可以用于生成基因敲除(通过插入/缺失)或敲入(通过HDR)。
药物组合物
另一方面，本申请提供了一种药物组合物。所述药物组合物可包含本申请所述的嵌合抗原受体、所述的分离的抗原结合蛋白、所述分离的核酸分子、所述的载体、所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。在本申请中，所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂。除非与本申请的免疫细胞和/或本申请的细胞群不相容，否则任何常规介质或试剂均可以考虑用于本申请的药物组合物中。在本申请中，所述药学上可接受的赋形剂可以包括在药物制剂中除主药以外的附加物，也可称为辅料。例如，所述赋形剂可以包括片剂中的粘合剂、填充剂、崩解剂、润滑剂。例如，所述赋形剂可以包括中药丸剂中的酒、醋、药汁等。例如，所述赋形剂可以包括半固体制剂软膏剂、霜剂中的基质部分。例如，所述赋形剂可以包括液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂。
所述药物组合物可以用来治疗与CD70的表达相关的疾病或病症，例如用于治疗白血病(例如，AML)或肾癌。
用途和方法
另一方面，本申请提供了所述的嵌合抗原受体，所述的抗原结合蛋白，所述的分离的核 酸分子，所述的载体，和/或所述的细胞在制备药物中的用途，所述药物用于治疗与CD70的表达相关的疾病或病症。
另一方面，本申请提供了预防或治疗与CD70的表达相关的疾病或病症的方法，其包括向有需要的受试者施用有效量的所述的嵌合抗原受体，所述的抗原结合蛋白，所述的分离的核酸分子，所述的载体，和/或所述的细胞。
在某些情形中，本申请所述与CD70的表达相关的疾病或病症包括白血病(例如，AML)或肾癌。
本申请所述药物组合物及方法可与其他类型的癌症疗法结合使用，诸如化学疗法、手术、放射、基因疗法等。本申请中所描述的药物组合物及方法可用于其他依赖于免疫反应的疾病病状，诸如炎症、免疫疾病及感染性疾病。
另一个方面，本申请提供了制备本申请所述分离的抗原结合蛋白和所述的嵌合抗原受体的方法。所述方法可包括，在使得所述分离的抗原结合蛋白表达的条件下，培养本申请所述的宿主细胞。
例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
在某些情形中，所述方法还可包括收获(例如分离和/或纯化)本申请所述分离的抗原结合蛋白的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述分离的抗原结合蛋白。例如，还可以通过使用高盐缓冲液、改变pH等方法洗脱结合在亲和柱上的融合蛋白多肽。
在本申请中，所述受试者可以包括人或非人动物。例如，所述非人动物可以选自下组：猴、鸡、鹅、猫、狗、小鼠和大鼠。此外，非人动物也可以包括任何除人以外的动物物种，例如家畜动物，或啮齿类动物，或灵长类动物，或家养动物，或家禽动物。所述人可以是高加索人、非洲人、亚洲人、闪族人，或其他种族，或各种种族的杂合体。又例如，所述人可以是老年、成年、青少年、儿童或者婴儿。
可以根据在实验动物中的有效量推测在人类中的有效量。例如，Freireich等人描述了动物和人的剂量的相互关系(基于每平方米身体表面的毫克数)(Freireich et al.，Cancer Chemother.Rep.50，219(1966))。身体表面积可以从患者的身高和体重近似确定。参见例如Scientific Tables，Geigy Pharmaceuticals，Ardsley，N.Y.，537(1970)。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和 用途等，而不用于限制本申请发明的范围。
实施例
实施例1获取CD70单域抗体VHH基因序列并检测其亲和力
1.1构建单域抗体免疫文库：
使用具有C-末端人IgG Fc标签的重组人CD70抗原(Acrobiosystems:CDL-H82Q9)进行羊驼免疫。总共免疫四次，免疫间隔时间约3周。第一次免疫使用0.5mg重组人CD70抗原与CFA(完全弗氏佐剂)1:1混匀乳化后皮下注射，计为第一天。后面三次免疫使用0.25mg重组人CD70抗原与IFA(不完全弗氏佐剂)1:1混匀乳化后皮下注射，期间通过取血采样评估免疫反应效果。在第七十天，取外周血通过梯度离心分离淋巴细胞，提取淋巴细胞RNA并反转录成cDNA，通过两轮聚合酶链式反应(PCR)扩增羊驼重链免疫球蛋白的可变区VHH序列，将扩增出来的VHH插入噬菌体展示载体，并通过电转化将携带有单域抗体VHH基因片段的产物转入感受态大肠杆菌，从而获得单域抗体免疫文库。
1.2筛选CD70特异性抗体序列：
利用噬菌体展示技术将单域抗体分子展示于噬菌体表面，进而筛选出抗原特异性的单域抗体。将人CD70His融合蛋白(Acrobiosystems:CDL-H82Q9)包被到96孔板中，通过Elisa实验筛选3-5轮，将CD70特异性的噬菌体逐步得到富集。挑选大量阳性克隆进行Elisa检测并对阳性克隆筛选和测序，根据序列比对确定独特的克隆并将其序列分为框架区FR和互补决定区CDR。通过以上方法，共获得13株亲和力较高的靶向人CD70的单域抗体(sdAb)。这13株抗体分别命名为CD70-sdAb-1C(氨基酸序列如SEQ ID NO:34所示，核酸序列如SEQ ID NO:47所示)、CD70-sdAb-1E(氨基酸序列如SEQ ID NO:35所示，核酸序列如SEQ ID NO:48所示)、CD70-sdAb-1F(氨基酸序列如SEQ ID NO:36所示、核酸序列如SEQ ID NO:49所示)、CD70-sdAb-1G(氨基酸序列如SEQ ID NO:37所示，核酸序列如SEQ ID NO:50所示)、CD70-sdAb-1H(氨基酸序列如SEQ ID NO:38所示，核酸序列如SEQ ID NO:51所示)、CD70-sdAb-3H(氨基酸序列如SEQ ID NO:39所示，核酸序列如SEQ ID NO:52所示)、CD70-sdAb-5A(氨基酸序列如SEQ ID NO:40所示，核酸序列如SEQ ID NO:53所示)、CD70-sdAb-5B(氨基酸序列如SEQ ID NO:41所示，核酸序列如SEQ ID NO:54所示)、CD70-sdAb-5H(氨基酸序列如SEQ ID NO:42所示，核酸序列如SEQ ID NO:55所示)、CD70-sdAb-7D(氨基酸序列如SEQ ID NO:43所示，核酸序列如SEQ ID NO:56所示)、CD70-sdAb-H3(氨基酸序列如SEQ ID NO:44所示，核酸序列如SEQ ID NO:57所示)、 CD70-sdAb-L2(氨基酸序列如SEQ ID NO:45所示，核酸序列如SEQ ID NO:58所示)、CD70-sdAb-L1(氨基酸序列如SEQ ID NO:46所示，核酸序列如SEQ ID NO:59所示)。
1.3检测抗体的亲和力
将CD70-sdAb-1E，CD70-sdAb-1G，CD70-sdAb-3H，CD70-sdAb-L2氨基酸序列的C端分别连接上人的Fc结构域(如SEQ ID NO:94所示)，并在HEK293中表达这4个融合了Fc的蛋白，纯化后使用Biacore T200(GE)，Protein A chip(GE,Cat#29127556)检测这4个sdAb-Fc与CD70蛋白(ACRO Biosystems，CDL-H82Q9)的亲和力，结果如表1所示。
从抗体的平衡解离常数KD可以看出，筛选到的这些抗体针对CD70都有较高的亲和力，KD值都在1nM以下。
表1不同单域抗体与CD70结合的亲和力
抗体 类型 靶蛋白 KD(M)
CD70-sdAb-1E VHH CD70 1.683E-10
CD70-sdAb-1G VHH CD70 2.65E-10
CD70-sdAb-3H VHH CD70 3.005E-10
CD70-sdAb-L2 VHH CD70 9.757E-10
使用不同浓度的CD70-sdAb-1E，CD70-sdAb-1G，CD70-sdAb-3H，CD70-sdAb-L2对应的sdAb-Fc抗体蛋白对肾癌786-O细胞系(表达CD70)和786-O-KO(CD70)细胞系(用CRISPR敲除了CD70基因，因此不表达CD70)进行染色，流式细胞术分析阳性细胞比例，分别绘制如图2A-2D所示的曲线，计算得出这4种抗体CD70-sdAb-1E，CD70-sdAb-1G，CD70-sdAb-3H，CD70-sdAb-L2对应的EC50分别是14.14μg/ml,5.70μg/ml,10.76μg/ml,11.65μg/ml。
实施例2全基因合成CD70嵌合抗原受体分子并构建慢病毒表达载体
将获得的13株sdAb都分别构建到慢病毒载体上来筛选出更有效的靶向CD70的嵌合抗原受体。
2.1基因合成含有不同sdAb的靶向CD70的嵌合抗原受体的基因序列：
靶向CD70的嵌合抗原受体具体包括：人CD8α信号肽、靶向人CD70的单域抗体VHH序列、人CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号传导结构域和CD3zeta胞内信号传导结构域，它们按照依次串联的方式连接(图1)。13种不同的嵌合抗原受体分子(CAR分子)分别命名为CD70-CAR-1C(氨基酸序列如SEQ ID NO:60所示，核酸序列如SEQ ID NO:73所示)、CD70-CAR-1E(氨基酸序列如SEQ ID NO:61所示，核酸序列如SEQ ID NO:74所示)、CD70-CAR-1F(氨基酸序列如SEQ ID NO:62所示，核酸序列如SEQ ID NO:75所 示)、CD70-CAR-1G(氨基酸序列如SEQ ID NO:63所示，核酸序列如SEQ ID NO:76所示)、CD70-CAR-1H(氨基酸序列如SEQ ID NO:64所示，核酸序列如SEQ ID NO:77所示)、CD70-CAR-3H(氨基酸序列如SEQ ID NO:65所示，核酸序列如SEQ ID NO:78所示)、CD70-CAR-5A(氨基酸序列如SEQ ID NO:66所示，核酸序列如SEQ ID NO:79所示)、CD70-CAR-5B(氨基酸序列SEQ ID NO:67所示，核酸序列如SEQ ID NO:80所示)、CD70-CAR-5H(氨基酸序列如SEQ ID NO:68所示，核酸序列如SEQ ID NO:81所示)、CD70-CAR-7D(氨基酸序列如SEQ ID NO:69所示，核酸序列如SEQ ID NO:82所示)、CD70-CAR-H3(氨基酸序列如SEQ ID NO:70所示，核酸序列如SEQ ID NO:83所示)、CD70-CAR-L2(氨基酸序列如SEQ ID NO:71所示，核酸序列如SEQ ID NO:84所示)、CD70-CAR-L1(氨基酸序列如SEQ ID NO:72所示，核酸序列如SEQ ID NO:85所示)。13种不同的靶向CD70的嵌合抗原受体基因序列由苏州金唯智生物科技有限公司合成并克隆至pUC57载体(苏州金唯智生物科技有限公司)上。为了便于后续构建到慢病毒载体上，在合成基因时分别在基因5’端和3’端添加限制性内切酶BamHI(NEB:R3136S)和SalI(NEB:R3138S)酶切位点。参考专利CN111909966A的制备方法，基于已有专利US20170369581中靶向CD70的单抗41D12的VL和VH序列，获取CD70-CAR-03核酸序列(SEQ ID NO:93)，制备对应的慢病毒和对应的CAR-T细胞作为实施例中的阳性对照。
2.2克隆靶向CD70的嵌合抗原受体基因序列至慢病毒载体：
利用BamHI和SalI两个酶切位点将靶向CD70的嵌合抗原受体基因序列分别从pUC57载体上双酶切下来，酶切条带经琼脂糖凝胶电泳检测后分别进行胶回收纯化(QIAGEN：28706)得到靶向CD70嵌合抗原受体DNA片段。将酶切回收的靶向CD70嵌合抗原受体DNA片段通过T4连接酶(NEB：M0202S)克隆至慢病毒载体pCGK(PCT/CN2019/090605)上，经转化酶切测序验证后得到13个含有靶向CD70的嵌合抗原受体基因序列的重组质粒:CD70-CAR-1C-pCGK、CD70-CAR-1E-pCGK、CD70-CAR-1F-pCGK、CD70-CAR-1G-pCGK、CD70-CAR-1H-pCGK、CD70-CAR-3H-pCGK、CD70-CAR-5A-pCGK、CD70-CAR-5B-pCGK、CD70-CAR-5H-pCGK、CD70-CAR-7D-pCGK、CD70-CAR-H3-pCGK、CD70-CAR-L2-pCGK、CD70-CAR-L1-pCGK。将13个重组质粒送苏州金唯智生物科技有限公司测序验证，测序引物为：
Lenti-For:TCAAGCCTCAGACAGTGGTTC(SEQ ID NO:86)
Lenti-Rev：CCTCATAAAGAGACAGCAACCAGG(SEQ ID NO:87)
测序验证13个重组质粒均构建正确。
实施例3制备含有靶向CD70嵌合抗原受体分子的慢病毒
3.1大提重组质粒：
(1)转化重组质粒和摇菌：将测序验证正确的重组质粒重新转化大肠杆菌stbl3(购自北京科瑞思博公司)。第二天从转化好的平板上挑取单克隆到3ml含有氨卞青霉素的液体LB培养基的摇菌管中，30℃220rpm，摇床振荡培养8h。按照1:500接种量将活化好的菌液接种到250ml含有氨卞青霉素的液体LB培养基中，30℃220rpm，摇床振荡培养12-16h。
(2)提取重组质粒：使用Qiagen HiSpeed Plasmid Maxi Kit试剂盒(货号：12662)，根据试剂盒提供的实验流程来提取质粒。
(3)检测抽提质粒的质量：提取的重组质粒使用Nanodrop(Thermo Fisher Scientific)测定重组质粒浓度进而判断重组质粒的纯度，并通过DNA琼脂糖凝胶电泳检测超螺旋质粒含量。
3.2培养293T细胞：
液氮中取出冻存的293T细胞(购自ATCC)，在37℃水浴锅内不断摇动促其融化。用消毒酒精擦拭冻存管口后，转移到提前已加入10ml预热的DMEM完全培养基(90％DMEM+10％FBS+1％青霉素/链霉素)的15ml离心管中，轻轻吹匀，1000rpm离心3min，吸弃上清。加入10ml DMEM完全培养基，轻轻吹匀后接种到10cm培养皿中，在37℃含5％CO 2的细胞培养箱中培养。第二天当细胞密度达到80％-90％时对293T细胞进行传代，先用移液管吸掉培养基接着用10ml PBS清洗1次293T细胞，加入3ml含0.25％EDTA的胰蛋白酶，放入培养箱1-2min(期间需要拿出在显微镜下观察细胞是否变圆)。细胞变圆后加1ml的DMEM完全培养基终止消化，转移到15ml离心管中，1000rpm离心3min，吸弃上清。根据实验需要，按照1:3或1:5的比例传代，将293T细胞接种到新的10cm培养皿中继续培养或者冻存。
3.3接种293T细胞到培养瓶中：
复苏的293T细胞一般培养传2代以上后，就可以用来包装慢病毒。首先培养的293T细胞经胰蛋白酶消化混匀后，根据计数结果，按照约1.7×10 7个/T175瓶(30ml培养基培养)接种293T细胞，轻轻摇匀后置于37℃含5％CO 2的细胞培养箱中培养过夜使其细胞密度达到70-80％时可转染。
3.4PEI法转染293T细胞：
第二天，质粒转染前培养基需换成有10％FBS但无双抗的DMEM培养基。首先准备质粒复合物：15ml离心管中加入1.5ml Opti-MEM(Thermo Fisher Scientific；31985-070)，再依次 加入病毒载体质粒：18μg，psPAX2质粒(Addgene；货号：12260)：9μg，pMD2.G质粒(Addgene；货号：12259)：18μg，以上质粒都加入后轻轻混匀后静置5min。病毒重组质粒共有13种(CD70-CAR-1C-pCGK、CD70-CAR-1E-pCGK、CD70-CAR-1F-pCGK、CD70-CAR-1G-pCGK、CD70-CAR-1H-pCGK、CD70-CAR-3H-pCGK、CD70-CAR-5A-pCGK、CD70-CAR-5B-pCGK、CD70-CAR-5H-pCGK、CD70-CAR-7D-pCGK、CD70-CAR-H3-pCGK、CD70-CAR-L2-pCGK、CD70-CAR-L1-pCGK)，需要包13种病毒，每个病毒重组质粒分别加入。再准备转染试剂复合物：将67.5μl(2mg/ml)PEI(polysciences：24765)加入到1.5ml Opti-MEM内混匀，室温静置5min，孵育结束再将转染试剂复合物加入到质粒复合物中，混匀后静置20min。最后将转染复合物慢慢逐滴加入到293T细胞中，轻轻混匀，37℃含5％CO 2的细胞培养箱中培养。
3.5收集慢病毒收集并浓缩：
重组质粒转染293T细胞48h后收取细胞培养基上清到50ml离心管中，2000rpm离心10min去除细胞碎片。使用Millipore的0.45μm滤膜过滤细胞上清，将滤液转移到专用离心管中，配平后使用超速离心机25000rpm超速离心2h。超离结束后倒掉滤液，使用无血清培养基重悬慢病毒，并将慢病毒分装后保存在-80℃超低温冰箱中。按照该流程分别制备含有CD70-CAR-1C-pCGK、CD70-CAR-1E-pCGK、CD70-CAR-1F-pCGK、CD70-CAR-1G-pCGK、CD70-CAR-1H-pCGK、CD70-CAR-3H-pCGK CD70-CAR-5A-pCGK、CD70-CAR-5B-pCGKCD70-CAR-5H-pCGK、CD70-CAR-7D-pCGK、CD70-CAR-H3-pCGK、CD70-CAR-L2-pCGK、CD70-CAR-L1-pCGK的慢病毒。
实施例4复苏人原代T细胞先敲除CD70基因再激活
将人外周血单个核细胞(PBMCs，购自上海妙顺生物)从液氮中取出对其复苏计数后，对复苏的细胞使用CRISPR/Cas9电转将CD70 sgRNA-19导入到T细胞中敲除T细胞中的CD70基因。细胞处理操作流程参考专利WO2019/011118实施例3进行。同时选择未敲除的T细胞(对照T细胞)做阴性对照。细胞敲除的第2天按照细胞与磁珠1:3的比例加入偶联有CD3/CD28抗体的磁珠(Thermo Fisher Scientific)激活敲除CD70的T细胞，同时培养基中添加300IU的IL-2(PeproTech：200-02)。
实施例5制备CD70敲除的靶向CD70的CAR-T细胞
细胞激活第2天，对敲除CD70的T细胞计数后将其细胞浓度调至为1×106/ml，每孔接种500μl细胞到24孔中，分别将含有CD70-CAR-1C-pCGK、CD70-CAR-1E-pCGK、 CD70-CAR-1F-pCGK、CD70-CAR-1G-pCGK、CD70-CAR-1H-pCGK、CD70-CAR-3H-pCGKCD70-CAR-5A-pCGK、CD70-CAR-5B-pCGK、CD70-CAR-5H-pCGK、CD70-CAR-7D-pCGKCD70-CAR-H3-pCGK、CD70-CAR-L2-pCGK、CD70-CAR-L1-pCGK的慢病毒加入到T-KO细胞培养孔内转染T细胞，未转染的T-KO细胞作为阴性对照，得到不同组别的可表达靶向人CD70抗原的嵌合抗原受体T细胞，命名为CD70 KO-CAR-1C、CD70 KO-CAR-1E、CD70 KO-CAR-1F、CD70 KO-CAR-1G、CD70 KO-CAR-1H、CD70 KO-CAR-3H、CD70 KO-CAR-5A、CD70 KO-CAR-5B CD70 KO-CAR-5H、CD70 KO-CAR-7D、CD70 KO-CAR-H3、CD70 KO-CAR-L2、CD70 KO-CAR-L1。同时按照相似流程制备阳性对照CAR：CD70-CAR-03对应的CAR-T细胞，命名为：CD70KO-CAR-03。继续培养各组T细胞至静息态。同时取各组CAR-T细胞，提取各组细胞的基因组，通过PCR和TIDE分析确定在这些CAR-T细胞中，CD70已经被成功敲除。对照T细胞CT-KO、CD70 KO-CAR-1C、CD70 KO-CAR-1E、CD70 KO-CAR-1F、CD70 KO-CAR-1G、CD70 KO-CAR-1H、CD70 KO-CAR-3H、CD70 KO-CAR-5A、CD70 KO-CAR-5B、CD70 KO-CAR-5H、CD70 KO-CAR-7D、CD70 KO-CAR-H3、CD70 KO-CAR-L2、CD70 KO-CAR-L1细胞敲除效率如表2所示。
表2不同组别CAR-T细胞的敲除效率
Figure PCTCN2021134248-appb-000001
Figure PCTCN2021134248-appb-000002
实施例6检测靶向CD70 CAR在T细胞内的表达情况
通过荧光抗体染色和流式细胞术检测实施例5制得的靶向CD70 CAR在各组T细胞内的表达情况，具体步骤：分别离心收集慢病毒感染48h的靶向CD70 CAR-T细胞和慢病毒未感染的T细胞和T-KO细胞，加入生物素化人CD70蛋白(100μg/ml)(Acrobiosystems:CDL-H82Q9)，4℃避光孵育30分钟，孵育结束用PBS清洗1次。使用适量体积PBS重悬后加入二抗PE-Streptavidin(BD Biosciences：554061)，4℃避光孵育20分钟，PBS清洗1次后再用适量的PBS重悬，最后使用流式细胞仪检测靶向CD70的CAR在敲除CD70的T细胞表面的表达效率。结果如图3所示，靶向CD70的CAR在敲除CD70的T细胞表面的表达效率较高。
实施例7靶向CD70 CAR-T与不同的靶细胞共培养后检测细胞因子的分泌
7.1细胞共培养：首先检测CD70在786-O(购自中国科学院细胞库)，THP-1细胞、Raji细胞、K562细胞和293T细胞内的表达情况。取一定体积的细胞，使用CD70抗体染色后流式细胞仪分析，发现Raji、THP-1和786-O细胞高表达CD70，K562和293T细胞不表达CD70。使用能表达CD70基因(Genebank：NM_001252.5)的慢病毒转染293T细胞，制备得到表达CD70蛋白的293T.CD70细胞系。慢病毒制备流程参见实施例3。对实施例5制得的13种靶向CD70的CAR-T细胞(CD70 KO-CAR-1C、CD70 KO-CAR-1E、CD70 KO-CAR-1F、CD70 KO-CAR-1G、CD70 KO-CAR-1H、CD70 KO-CAR-3H、CD70 KO-CAR-5A、CD70 KO-CAR-5B CD70 KO-CAR-5H、CD70 KO-CAR-7D、CD70 KO-CAR-H3、CD70 KO-CAR-L2、CD70 KO-CAR-L1)和对照T-KO细胞计数后将浓度调至5×10 5/ml，然后按照100ul每孔接种到平底96孔板中。每个靶向CD70的CAR-T细胞设置3个重复，将已接种好的靶向CD70的CAR-T和T-KO细胞暂时放置于37℃孵育。将靶细胞Raji(购自中国科学院细胞库)、786-O、THP-1、表达CD70蛋白的293T.CD70、不表达CD70蛋白的阴性细胞K562(中国科学院细胞库)和293T(ATCC)按照效靶比1:1即细胞浓度5×10 5/ml，100μl/孔，加入到靶向CD70的CAR-T细胞或者对照T-KO细胞中共培养。
7.2将上述细胞共培养24h后的上清转移到新的96孔板，使用ELISA试剂盒(Thermo Fisher Scientific；货号88-7316)按照试剂盒提供的标准流程检测CAR-T细胞IL-2和IFN-γ细胞因子的分泌。ELISA平板制备和细胞上清中细胞因子的检测按照试剂盒提供的标准流程 操作。结果如图4所示，靶向CD70的CAR-T细胞分别与Raji、THP-1和786-O细胞共培养，能够分泌白介素-2和干扰素γ。
实施例8靶向CD70 CAR-T与不同的靶细胞共培养后检测杀伤能力
8.1细胞铺板：使用带有荧光素酶(GenBank:AAR29591.1)的慢病毒转染786-O细胞、THP-1细胞、K562细胞和293T细胞，制备得到标记有荧光素酶的细胞系，分别命名为：780-O.luc细胞、THP-1.luc细胞、K562.luc细胞和293T.luc细胞。慢病毒制备流程参见实施例3。将标记有荧光素酶的细胞系，按照细胞浓度1×10 5/ml，50μl/孔铺至96孔平底不透明白板中。设置2.5：1、1：1和0.5：1三个效靶比将靶向CD70 CAR-T细胞与THP-1共培养，1：1、0.5：1和0.25：1三个效靶比将靶向CD70 CAR-T细胞与786-O细胞共培养，2.5:1、1:1和0.5:1共三个效靶比将靶向CD70的CAR-T细胞分别与K562细胞和293T细胞共培养，靶向CD70的CAR-T细胞分别是CD70 KO-CAR-1C、CD70 KO-CAR-1E、CD70 KO-CAR-1F、CD70 KO-CAR-1G、CD70 KO-CAR-1H、CD70 KO-CAR-3H、CD70 KO-CAR-5A、CD70 KO-CAR-5B CD70 KO-CAR-5H、CD70 KO-CAR-7D、CD70 KO-CAR-H3、CD70 KO-CAR-L2、CD70 KO-CAR-L1，并将对照T-KO细胞与靶细胞共培养，每个靶向CD70的CAR-T为3个重复。将96孔板放置于37℃5％CO 2细胞培养箱中培养过夜。
8.2细胞共培养24h后测定靶细胞剩余的荧光素酶活性(相对光单位，RLU)，来检测每种靶向CD70的CAR-T对不同靶细胞的杀伤能力。具体步骤为：共培养后的细胞直接加入100μl的D-luciferin底物(Thermo Fisher Scientific：88293)混匀避光显色5min，并用酶标仪以化学发光模式检测荧光强度。在不存在效应细胞的情况下，通过将培养基加入靶细胞来获得最大荧光素酶活性作为对照。结果如图5所示，靶向CD70的CAR-T对THP-1(急性髓系白血病细胞株)和786-O(肾癌细胞株)有杀伤作用。
实施例9靶向CD70 CAR-T在急性髓系白血病动物模型上的活性
THP-1 Luc肿瘤细胞为表达luciferase荧光素酶的人急性髓系白血病细胞，在常规复苏后扩增培养，至少传代2次后收获对数生长期的细胞，以不含血清的培养基重悬。取6-8周的雌性NOG小鼠，所有动物无菌条件尾静脉接种含有5×10 6个细胞的PBS悬液0.1mL。尾静脉接种THP-1Luc细胞后7-11天，根据肿瘤信号强度筛选33只动物随机分为11组，单次静脉给药注射如下细胞：缓冲液组(PBS)、对照T细胞组T-KO、CD70 KO-CAR-1C、CD70 KO-CAR-1E、CD70 KO-CAR-1F、CD70 KO-CAR-1G、CD70 KO-CAR-3H、CD70 KO-CAR-H3、CD70 KO-CAR-L2、CD70 KO-CAR-L1，CD70 KO-CAR-03(阳性对照)，每组3只小鼠。提前准备预热的1640完全培养基，转移到15mL离心管，每管5mL。将冻存管自低温保存场 所中取出后迅速置37℃水浴，水浴溶解后将细胞转移到15mL离心管中，与培养基混匀。取适量细胞，使用台盼蓝计数细胞密度和活力。细胞以1800rpm条件下离心5min，用PBS调整供试品细胞密度。供试品各组动物以3×10 6个T细胞/只(约1.5×10 6个CAR阳性细胞)给予细胞，在200μl PBS中注射。
在D7、D14、D21、D28、D35、D49天，所有动物腹腔注射150mg/kg的D-荧光素钾(Thermo Fisher Scientific)。注射后10～15min于异氟烷麻醉下以Bruker小动物成像仪拍摄化学发光信号，成像时间180s。
结果：实验结果见图6。图6可以显示，在本实验条件下，所有组别的CAR-T相比较于对照T-KO组和缓冲液组(PBS)都明显可以抑制肿瘤细胞的增殖。和阳性对照CD70 KO-CAR-03相比，CD70 KO-CAR-L2，CD70 KO-CAR-1E，CD70 KO-CAR-1G，CD70 KO-CAR-3H这4组具有更强的抗肿瘤活性(表3)。
表3小鼠CAR-T注射后第49天每组平均生物荧光值排序(从低到高)
CAR组别 生物荧光(photon/second)
CD70 KO-CAR-L2 800667
CD70 KO-CAR-1E 9273667
CD70 KO-CAR-1G 14689667
CD70 KO-CAR-3H 42893667
CD70 KO-CAR-03 193150333
CD70 KO-CAR-L1 258283667
CD70 KO-CAR-1C 340223667
CD70 KO-CAR-H3 430557000
CD70 KO-CAR-1F 2999083667
实施例10靶向CD70 CAR-T在肾癌动物模型上的活性
786-O肿瘤细胞为人肾癌细胞，在常规复苏后扩增培养，至少传代2次后收获对数生长期的细胞，以不含血清的培养基重悬至1×10 7个/mL。取45只6-8周的雌性NOG小鼠，将786-O细胞重悬在PBS和Matrigel基质1:1混合的溶液中，所有动物无菌条件皮下接种细胞悬液0.2mL(含5×10 6个786-O细胞)，接种于小鼠的右后背。接种786-O细胞后约33天，待肿瘤体积达到120-150mm 3时，筛选30只动物随机分为6组，单次静脉给药注射如下细胞：对照T细胞组T-KO、CD70 KO-CAR-1E，CD70 KO-CAR-1G，CD70 KO-CAR-3H，CD70 KO-CAR-L2，CD70 KO-CAR-03(阳性对照)，每组5只小鼠。提前准备预热的1640完全 培养基，转移到15mL离心管，每管5mL。将冻存管自低温保存场所中取出后迅速置37℃水浴，水浴溶解后将细胞转移到15mL离心管中，与培养基混匀。取适量细胞，使用台盼蓝计数细胞密度和活力。细胞以1800rpm条件下离心5min，用PBS调整供试品细胞密度。供试品各组动物以2×10 6个T细胞/只(约1×10 6个CAR阳性细胞)给予细胞，在200ul PBS中注射。给药后，每周三次测量肿瘤直径计算肿瘤体积，一直观察到给药后第41天。
结果：实验结果见图7。图7可以显示，在本实验条件下，所有组别的CAR-T相比较于对照T-KO组都明显可以抑制肿瘤。和阳性对照CD70 KO-CAR-03相比，CD70 KO-CAR-1E，CD70 KO-CAR-1G，CD70 KO-CAR-3H，CD70 KO-CAR-L2这4组都具有更强的抗肿瘤活性，与上述急性髓系白血病动物模型上的结果一致。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (60)

  1. 嵌合抗原受体，其包含靶向部分，所述靶向部分包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1包含SEQ ID NO:1、6、9、11、14、17、20、23、26和29中任一项所示的氨基酸序列。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述HCDR2包含SEQ ID NO:2、7、12、15、18、21、24、27、30和32中任一项所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的嵌合抗原受体，其中所述HCDR3包含SEQ ID NO:3、4、5、8、10、13、16、19、22、25、28、31和33中任一项所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的嵌合抗原受体，其中所述HCDR1、HCDR2和HCDR3包含选自下述任意一组的氨基酸序列：
    a)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
    b)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:4；
    c)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:5；
    d)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:8；
    e)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:10；
    f)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:13；
    g)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:15，和HCDR3：SEQ ID NO:16；
    h)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:18，和HCDR3：SEQ ID NO:19；
    i)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:21，和HCDR3：SEQ ID NO:22；
    j)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:24，和HCDR3：SEQ ID NO:25；
    k)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:27，和HCDR3：SEQ ID NO:28；
    l)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30，和HCDR3：SEQ ID NO:31；
    m)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:32，和HCDR3：SEQ ID NO:33。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，其中所述靶向部分包括VHH。
  6. 根据权利要求1-5中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:39、34、35、36、37、38、44、40、41、42、43、45和46中任一项所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的嵌合抗原受体，其包含信号肽。
  8. 根据权利要求7所述的嵌合抗原受体，其中所述信号肽包含源自选自下组蛋白的信号肽或其组合：CD8、4-1BB、GM-CSF、CD3γ、CD3δ、CD3ε、CD22、CD79a、CD79b和CD66d。
  9. 根据权利要求7-8中任一项所述的嵌合抗原受体，其中所述信号肽包括源自CD8的信号 肽。
  10. 根据权利要求7-9中任一项所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:88所示的氨基酸序列。
  11. 根据权利要求7-10中任一项所述的嵌合抗原受体，其中所述信号肽的C端与所述靶向部分的N端连接。
  12. 根据权利要求1-11中任一项所述的嵌合抗原受体，其包含铰链区。
  13. 根据权利要求12所述的嵌合抗原受体，其中所述铰链区包含源自选自下组的蛋白的铰链区或其组合：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。
  14. 根据权利要求12-13中任一项所述的嵌合抗原受体，其中所述铰链区包括源自所述CD8中的CD8a的铰链区。
  15. 根据权利要求12-14中任一项所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:89所示的氨基酸序列。
  16. 根据权利要求12-15中任一项所述的嵌合抗原受体，其中所述铰链区的N端与所述靶向部分的C端连接。
  17. 根据权利要求1-16中任一项所述的嵌合抗原受体，其包含跨膜结构域。
  18. 根据权利要求17所述的嵌合抗原受体，其中所述跨膜结构域包含源自选自下组蛋白的跨膜结构域或其组合：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、CTLA-4、LAG-3、TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3δ、CD3γ、CD3ζ、细胞因子受体、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεR、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  19. 根据权利要求17-18中任一项所述的嵌合抗原受体，其中所述跨膜结构域包括源自所述CD8中的CD8a的跨膜结构域。
  20. 根据权利要求17-19中任一项所述的嵌合抗原受体，其中所述跨膜结构域包含SEQ ID NO:90所示的氨基酸序列。
  21. 根据权利要求17-20中任一项所述的嵌合抗原受体，其中所述跨膜结构域的N端与所述铰链区的C端连接。
  22. 根据权利要求1-21中任一项所述的嵌合抗原受体，其包含共刺激信号传导结构域。
  23. 根据权利要求22所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含源自选自下组的蛋白的共刺激信号传导结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、 2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  24. 根据权利要求22-23中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包括源自4-1BB的共刺激信号传导结构域。
  25. 根据权利要求22-24中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含SEQ ID NO:91所示的氨基酸序列。
  26. 根据权利要求22-25中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域的N端与所述跨膜结构域的C端连接。
  27. 根据权利要求1-26中任一项所述的嵌合抗原受体，其包含胞内信号传导结构域。
  28. 根据权利要求27所述的嵌合抗原受体，其中所述胞内信号传导结构域包含源自选自下组的蛋白的胞内信号传导结构域或其组合：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、CD66d、CD5、CD22、FcRγ、FcRβ、FcRε、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒(BLV)gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒(SIV)PBj14Nef、卡波西肉瘤疱疹病毒(KSHV)K1、DAP10、DAP12和至少包含一个免疫受体酪氨酸激活基序(ITAM)的结构域。
  29. 根据权利要求27-28中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包括源自CD3zeta的胞内信号传导结构域。
  30. 根据权利要求27-29中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包含SEQ ID NO:92所示的氨基酸序列。
  31. 根据权利要求27-30中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域的N端与所述共刺激信号传导结构域的C端连接。
  32. 根据权利要求1-31中任一项所述的嵌合抗原受体，其包含SEQ ID NO:60、61、62、63、64、65、66、67、68、69、70、71和72任一项所示的氨基酸序列。
  33. 分离的抗原结合蛋白，其以1nM或更低的K D值特异性结合CD70。
  34. 根据权利要求33所述的分离的抗原结合蛋白，其包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，其中所述HCDR1包含SEQ ID NO:1、6、9、11、14、17、20、23、26和29中任一项所示的氨基酸序列。
  35. 根据权利要求34所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:2、7、12、15、18、21、24、27、30和32中任一项所示的氨基酸序列。
  36. 根据权利要求34-35中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:3、4、5、8、10、13、16、19、22、25、28、31和33中任一项所示的氨基酸序列。
  37. 根据权利要求33-36中任一项所述的分离的抗原结合蛋白，其为抗体或其抗原结合片段。
  38. 根据权利要求37所述的分离的抗原结合蛋白，其为VHH或其抗原结合片段。
  39. 根据权利要求37-38中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  40. 根据权利要求33-39中任一项所述的分离的抗原结合蛋白，其中所述HCDR1、HCDR2和HCDR3包含选自下组中任意一组的氨基酸序列：
    a)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
    b)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:4；
    c)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:5；
    d)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:8；
    e)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:7，和HCDR3：SEQ ID NO:10；
    f)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:13；
    g)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:15，和HCDR3：SEQ ID NO:16；
    h)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:18，和HCDR3：SEQ ID NO:19；
    i)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:21，和HCDR3：SEQ ID NO:22；
    j)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:24，和HCDR3：SEQ ID NO:25；
    k)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:27，和HCDR3：SEQ ID NO:28；
    l)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30，和HCDR3：SEQ ID NO:31；
    m)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:32，和HCDR3：SEQ ID NO:33。
  41. 根据权利要求33-40中任一项所述的分离的抗原结合蛋白，其包括VHH。
  42. 根据权利要求33-41中任一项所述的分离的抗原结合蛋白，其中所述抗原结合蛋白包含SEQ ID NO:39、34、35、36、37、38、44、40、41、42、43、45和46任一项所示的氨基酸序列。
  43. 分离的核酸分子，其编码权利要求1-32中任一项所述的嵌合抗原受体和/或权利要求33-42中任一项所述的分离的抗原结合蛋白。
  44. 根据权利要求43所述的分离的核酸分子，其包含SEQ ID NO:47-59和/或SEQ ID NO:73-85中任一项所示的核酸序列。
  45. 载体，其包含权利要求43-44中任一项所述的分离的核酸分子。
  46. 根据权利要求45所述的载体，其包括病毒载体。
  47. 根据权利要求45-46中任一项所述的载体，其包括慢病毒载体。
  48. 细胞，其包含权利要求1-32中任一项所述的嵌合抗原受体，权利要求33-42中任一项所述的分离的抗原结合蛋白，权利要求43-44中任一项所述的分离的核酸分子，和/或权利要求45-47中任一项所述的载体。
  49. 根据权利要求48所述的细胞，其包括免疫细胞。
  50. 根据权利要求48-49中任一项所述的细胞，其中所述免疫细胞选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  51. 根据权利要求48-50中任一项所述的细胞，其包括T细胞。
  52. 根据权利要求48-51中任一项所述的细胞，其与相应的野生型细胞相比，所述细胞中CD70的表达和/或活性降低。
  53. 根据权利要求48-52中任一项所述的细胞，其与相应的野生型细胞相比，所述细胞敲除了CD70。
  54. 药物组合物，其包含权利要求1-32中任一项所述的嵌合抗原受体，权利要求33-42中任一项所述的分离的抗原结合蛋白，权利要求43-44中任一项所述的分离的核酸分子，权利要求45-47中任一项所述的载体，权利要求48-53中任一项所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
  55. 权利要求1-32中任一项所述的嵌合抗原受体，权利要求33-42中任一项所述的分离的抗原结合蛋白，权利要求43-44中任一项所述的分离的核酸分子，权利要求45-47中任一项所述的载体，权利要求48-53中任一项所述的细胞，和/或权利要求54所述的药物组合物，其用于治疗与CD70的表达相关的疾病或病症。
  56. 根据权利要求55所述的用途，其中所述与CD70的表达相关的疾病或病症包括急性髓系白血病(AML)或肾癌。
  57. 权利要求1-32中任一项所述的嵌合抗原受体，权利要求33-42中任一项所述的分离的抗原结合蛋白，权利要求43-44中任一项所述的分离的核酸分子，权利要求45-47中任一项所述的载体，权利要求48-53中任一项所述的细胞，和/或权利要求54所述的药物组合物在制备药物中的用途，所述药物用于治疗与CD70的表达相关的疾病或病症。
  58. 根据权利要求57所述的用途，其中所述与CD70的表达相关的疾病或病症包括急性髓系白血病(AML)或肾癌。
  59. 预防和/或治疗与CD70的表达相关的疾病或病症的方法，其包括向有需要的受试者施用有效量的权利要求1-32中任一项所述的嵌合抗原受体，权利要求33-42中任一项所述的分离的抗原结合蛋白，权利要求43-44中任一项所述的分离的核酸分子，权利要求45-47中任一项所述的载体，权利要求48-53中任一项所述的细胞，和/或权利要求54所述的药物组合物。
  60. 根据权利要求59所述的方法，其中所述与CD70的表达相关的疾病或病症包括急性髓性白血病(AML)或肾癌。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063524A (zh) * 2022-11-21 2023-05-05 杭州荣谷生物科技有限公司 一种cd70纳米抗体的制备方法及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117844811A (zh) * 2024-03-08 2024-04-09 上海恒润达生生物科技股份有限公司 靶向敲除CD70基因的sgRNA组合物及其应用

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1252076A (zh) * 1997-04-14 2000-05-03 麦可麦脱生物药学研究有限公司 抗人抗原受体的新的生产方法及其用途
CN101370830A (zh) * 2005-09-26 2009-02-18 米德列斯公司 抗cd70的人单克隆抗体
CN101605906A (zh) * 2006-12-14 2009-12-16 梅达雷克斯公司 结合cd70的人类抗体及其用途
WO2013154760A1 (en) 2012-04-11 2013-10-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting b-cell maturation antigen
WO2015156673A1 (en) * 2014-04-10 2015-10-15 Stichting Vu-Vumc IMMUNOGLOBULINS BINDING HUMAN Vγ9Vδ2 T CELL RECEPTORS
WO2016014789A2 (en) 2014-07-24 2016-01-28 Bluebird Bio, Inc. Bcma chimeric antigen receptors
CN107207616A (zh) * 2014-12-08 2017-09-26 美国卫生和人力服务部 抗cd70嵌合抗原受体
US20170369581A9 (en) 2011-03-16 2017-12-28 Argen-X N.V. Antibodies to cd70
WO2018152181A1 (en) * 2017-02-14 2018-08-23 Kite Pharma, Inc. Cd70 binding molecules and methods of use thereof
WO2019011118A1 (zh) 2017-07-14 2019-01-17 苏州克睿基因生物科技有限公司 一种基因编辑系统及基因编辑的方法
CN109880802A (zh) * 2018-11-30 2019-06-14 北京美康基免生物科技有限公司 一种基于cd19和cd70的双重嵌合抗原受体基因修饰的免疫细胞及其应用
CN111909966A (zh) 2020-04-01 2020-11-10 苏州克睿基因生物科技有限公司 一种制备经修饰的免疫细胞的方法
CN111936158A (zh) * 2017-12-15 2020-11-13 艾丽塔生物治疗剂公司 Cd19变体
CN112739721A (zh) * 2019-09-26 2021-04-30 苏州克睿基因生物科技有限公司 一种单域抗体及包含抗体结构的嵌合抗原受体

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1252076A (zh) * 1997-04-14 2000-05-03 麦可麦脱生物药学研究有限公司 抗人抗原受体的新的生产方法及其用途
CN101370830A (zh) * 2005-09-26 2009-02-18 米德列斯公司 抗cd70的人单克隆抗体
CN101605906A (zh) * 2006-12-14 2009-12-16 梅达雷克斯公司 结合cd70的人类抗体及其用途
US20170369581A9 (en) 2011-03-16 2017-12-28 Argen-X N.V. Antibodies to cd70
WO2013154760A1 (en) 2012-04-11 2013-10-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting b-cell maturation antigen
WO2015156673A1 (en) * 2014-04-10 2015-10-15 Stichting Vu-Vumc IMMUNOGLOBULINS BINDING HUMAN Vγ9Vδ2 T CELL RECEPTORS
WO2016014789A2 (en) 2014-07-24 2016-01-28 Bluebird Bio, Inc. Bcma chimeric antigen receptors
CN107207616A (zh) * 2014-12-08 2017-09-26 美国卫生和人力服务部 抗cd70嵌合抗原受体
WO2018152181A1 (en) * 2017-02-14 2018-08-23 Kite Pharma, Inc. Cd70 binding molecules and methods of use thereof
WO2019011118A1 (zh) 2017-07-14 2019-01-17 苏州克睿基因生物科技有限公司 一种基因编辑系统及基因编辑的方法
CN111936158A (zh) * 2017-12-15 2020-11-13 艾丽塔生物治疗剂公司 Cd19变体
CN109880802A (zh) * 2018-11-30 2019-06-14 北京美康基免生物科技有限公司 一种基于cd19和cd70的双重嵌合抗原受体基因修饰的免疫细胞及其应用
CN112739721A (zh) * 2019-09-26 2021-04-30 苏州克睿基因生物科技有限公司 一种单域抗体及包含抗体结构的嵌合抗原受体
CN111909966A (zh) 2020-04-01 2020-11-10 苏州克睿基因生物科技有限公司 一种制备经修饰的免疫细胞的方法

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM_001252.5
"Scientific Tables, Geigy Pharmaceuticals", 1970, ARDSLEY, pages: 537
"UniProt", Database accession no. P32970
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
DOTTI GGOTTSCHALK SSAVOLDO BBRENNER MK: "Design and development of therapies using chimeric antigen receptor-expressing T cells", IMMUNOL REV., vol. 257, no. 1, 2014, pages 107 - 126, XP055552726, DOI: 10.1111/imr.12131
FREIREICH ET AL., CANCER CHEMOTHER. REP., vol. 50, 1966, pages 219
HUSTON ET AL., PROC.NATL.ACAD.SCI., vol. 85, 1988, pages 5879 - 5883
KANG, XIAOZHEN ET AL., CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 34, no. 12, 2018, pages 1974 - 1984
SADELAIN MBRENTJENS RRIVI 'ERE I: "The basicprinciples of chimeric antigen receptor design", CANCER DISCOV., vol. 3, no. 4, 2013, pages 388 - 398
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
TURTLE CJHUDECEK MJENSEN MCRIDDELL SR: "Engineered T cells for anti-cancer therapy", CURR OPIN IMMUNOL., vol. 24, no. 5, 2012, pages 633 - 639, XP055272888, DOI: 10.1016/j.coi.2012.06.004
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
YANG MEIJIA, TANG XIN, ZHANG ZONGLIANG, GU LEI, WEI HENG, ZHAO SHASHA, ZHONG KUNHONG, MU MIN, HUANG CHENG, JIANG CAIYING, XU JIANG: "Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors", THERANOSTICS, vol. 10, no. 17, 1 January 2020 (2020-01-01), AU , pages 7622 - 7634, XP055937499, ISSN: 1838-7640, DOI: 10.7150/thno.43991 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063524A (zh) * 2022-11-21 2023-05-05 杭州荣谷生物科技有限公司 一种cd70纳米抗体的制备方法及其应用

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