WO2024082178A1 - 靶向cd19和cd22的双特异性嵌合抗原受体 - Google Patents

靶向cd19和cd22的双特异性嵌合抗原受体 Download PDF

Info

Publication number
WO2024082178A1
WO2024082178A1 PCT/CN2022/126207 CN2022126207W WO2024082178A1 WO 2024082178 A1 WO2024082178 A1 WO 2024082178A1 CN 2022126207 W CN2022126207 W CN 2022126207W WO 2024082178 A1 WO2024082178 A1 WO 2024082178A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
sequence shown
antigen receptor
Prior art date
Application number
PCT/CN2022/126207
Other languages
English (en)
French (fr)
Inventor
廖雪梅
陈卓
谢曼曼
程晓翠
刘阳
马明柔
浦瞿津
刘婷婷
夏广新
柯樱
Original Assignee
上海医药集团股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海医药集团股份有限公司 filed Critical 上海医药集团股份有限公司
Priority to PCT/CN2022/126207 priority Critical patent/WO2024082178A1/zh
Publication of WO2024082178A1 publication Critical patent/WO2024082178A1/zh

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present application relates to the field of biomedicine, and specifically to a chimeric antigen receptor targeting CD19 and CD22 and its use.
  • B-ALL Acute B-lymphocytic leukemia
  • B-ALL Acute B-lymphocytic leukemia
  • the 5-year disease-free survival rate of relapsed or refractory childhood B-ALL patients is 27% and 15%.
  • patients who relapse after allogeneic hematopoietic stem cell transplantation are basically in a hopeless situation.
  • the chemotherapy survival rate is less than 10%, and the overall 20-year disease outcome of B-ALL has not been significantly improved. Therefore, there is a huge clinical need to develop effective treatments for relapsed or refractory B-ALL.
  • single-target CD19 CAR-T or CD22 CAR-T therapies have a high rate of disease recurrence after treatment, with a recurrence rate of about 40%-60%.
  • the main reasons for recurrence are the loss of CD19 antigen or the downregulation of CD19 after targeted immunotherapy and the reduction of cell surface CD22 expression. Therefore, it is urgent to design new CAR-T cells to overcome the shortcomings of currently available CAR-T therapies and improve the treatment effect.
  • the present application provides a bispecific chimeric antigen receptor targeting CD19 and CD22, and a cell comprising the chimeric antigen receptor, which can bind to CD19 and CD22 simultaneously and has a killing effect on target cells.
  • the CD19/CD22 CAR-T therapy provided by the present application is expected to overcome the shortcomings of the CAR-T therapy that has been on the market. Through the dual-targeting design, new recognition targets are added, the possibility of tumor escape through antigen loss is reduced, the probability of disease recurrence after treatment is reduced, and the proportion of memory T cells is increased. It is expected to greatly solve the many defects of existing CAR-T therapies and bring long-term and stable treatment effects to patients.
  • the present application provides a chimeric antigen receptor comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19, and the second antigen binding domain comprises a VHH targeting CD22.
  • first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19
  • second antigen binding domain comprises a VHH targeting CD22.
  • the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the second antigen binding domain comprises at least one CDR in the VHH sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.
  • the present application provides a chimeric antigen receptor, which comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can target CD19, the second antigen binding domain can target CD22, the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the VHH comprises the amino acid sequence shown in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.
  • the first antigen binding domain comprises an antibody or an antigen binding fragment thereof.
  • the second binding domain comprises an antibody or an antigen-binding fragment thereof.
  • the antigen binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the first antigen binding domain is a scFv.
  • the second antigen binding domain is a VHH.
  • the second antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59.
  • the HCDR3 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the second antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58.
  • the HCDR2 of the second antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12.
  • the second antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57.
  • HCDR1 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:11 or SEQ ID NO:12, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from the group consisting of:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:19;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:20;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:21.
  • the second antigen binding domain comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:60.
  • H-FR1 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
  • the second antigen binding domain comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:61.
  • H-FR2 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
  • the second antigen binding domain comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:62.
  • H-FR3 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.
  • the second antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:63.
  • H-FR4 of the second antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:60, H-FR2 comprises the amino acid sequence shown in SEQ ID NO:61, H-FR3 comprises the amino acid sequence shown in SEQ ID NO:62, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:63.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4,
  • the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10
  • the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from any one of the following groups:
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:15
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 16
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.
  • the second antigen binding domain comprises VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the VHH of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.
  • the first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:31, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:34, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:35, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:36, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.
  • the first antigen binding domain comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:37.
  • the first antigen binding domain comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:38, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:39 (HTS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:40.
  • the first antigen binding domain comprises L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:42, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:43, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:44.
  • the first antigen binding domain comprises VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:45.
  • the first antigen binding domain comprises VH and VL
  • the VH comprises the amino acid sequence shown in SEQ ID NO:37
  • the VL comprises the amino acid sequence shown in SEQ ID NO:45.
  • the VH and VL of the first antigen binding domain are connected by a linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:48.
  • the first antigen binding domain comprises a scFv
  • the scFv comprises the amino acid sequence shown in SEQ ID NO:46.
  • the first antigen binding domain and the second antigen binding domain are directly or indirectly linked.
  • the C-terminus of the first antigen binding domain is directly or indirectly connected to the N-terminus of the second antigen binding domain.
  • the N-terminus of the first antigen binding domain is directly or indirectly connected to the C-terminus of the second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain are connected by a linker.
  • the linker comprises a peptide linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:47.
  • the chimeric antigen receptor further comprises a co-stimulatory signal region, wherein the co-stimulatory signal region comprises an intracellular co-stimulatory signal region derived from one or more proteins selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.
  • the co-stimulatory signal region comprises an intracellular co-stimulatory signal region derived from one or more proteins selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the co-stimulatory signal region comprises the amino acid sequence shown in SEQ ID NO:52.
  • the chimeric antigen receptor includes an intracellular signaling domain, which comprises an intracellular signaling region derived from one or more proteins selected from the following groups: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.
  • an intracellular signaling domain which comprises an intracellular signaling region derived from one or more proteins selected from the following groups: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) L
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:53.
  • the chimeric antigen receptor comprises a transmembrane region, which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, 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 transmembrane region which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27,
  • the transmembrane region is a transmembrane region derived from CD8.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:51.
  • the chimeric antigen receptor further comprises a hinge region, which comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • a hinge region which comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region is a hinge region derived from CD8.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:50.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:49.
  • the chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, a second antigen binding domain, a linker, a first antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory domain, and an intracellular signaling domain.
  • the chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, a VHH targeting CD22, a linker 1, a VH targeting CD19, a linker 2, a VL targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:55.
  • the present application also provides one or more isolated nucleic acid molecules encoding the chimeric antigen receptor.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell, which comprises the chimeric antigen receptor, the nucleic acid molecule, and/or the vector.
  • the cell comprises an immune cell.
  • the immune cell is 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 cell comprises a T cell.
  • the present application provides a method for preparing the chimeric antigen receptor, the method comprising culturing the cell under conditions allowing the chimeric antigen receptor to be expressed.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable carrier.
  • the present application also provides a method for detecting CD19 and/or CD22, which comprises administering the cells.
  • the present application also provides a kit for detecting CD19 and/or CD22 proteins, which comprises the chimeric antigen receptor and/or the cell.
  • the present application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the vector, and the cell in preparing a drug, wherein the drug is used to prevent and/or treat a disease and/or condition.
  • the diseases and/or conditions include diseases and/or conditions associated with CD19 and/or CD22 expression.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a hematological tumor.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • the present application also provides a method for preventing and/or treating a disease and/or a condition, comprising administering the chimeric antigen receptor, the nucleic acid molecule, the vector, and/or the cell to a subject in need thereof.
  • the diseases and/or conditions include diseases and/or conditions associated with CD19 and/or CD22 expression.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a hematological tumor.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition, which are used for preventing and/or treating diseases and/or disorders.
  • the diseases and/or conditions include diseases and/or conditions associated with CD19 and/or CD22 expression.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a hematological tumor.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • Figure 1 shows the binding curve of the CD22 VHH described in this application and human CD22 expressed on the surface of K562 cells.
  • Figure 2 shows the results of flow cytometry detection of the positive rate of CD19/CD22 single and double target CART cells described in this application.
  • Figure 3 shows the results of flow cytometry detection of the affinity between the CD19/CD22 single and double target CART cells and antigens described in this application.
  • FIG4 shows the results of flow cytometry detection of changes in the proportion of TCM cells in the CD19/CD22 single- and double-target CART cells described in this application.
  • Figure 5 shows the killing effect of the CD19/CD22 single- and double-target CART cells described in this application on Nalm6-LUC cells.
  • Figure 6 shows the killing effect of the CD19/CD22 single and double target CART cells described in this application on the Nalm6-LUC single positive 1:1 mixed cell model.
  • Figure 7 shows the killing effect of the CD19/CD22 single and double target CART cells described in this application on the Nalm6-LUC wild type/single positive (8:1:1) mixed cell model.
  • Figure 8 shows the levels of cytokines secreted by the CD19/CD22 single- and double-target CART cells described in the present application after being activated by three types of Nalm6 model target cells.
  • FIG9 shows the fluorescence intensity detection results of in vivo imaging of Naml6-LUC mice.
  • FIG. 10 shows the fluorescence images of the in vivo imaging of Naml6-LUC mice.
  • FIG. 11 shows the results of body weight changes in Naml6-LUC mice.
  • FIG. 12 shows the fluorescence intensity detection results of in vivo imaging of Naml6-MIX-LUC mice.
  • FIG. 13 shows the fluorescence images of the in vivo imaging of Naml6-MIX-LUC mice.
  • FIG. 14 shows the results of body weight changes in Naml6-MIX-LUC mice.
  • single domain antibody or “sdAb” or “VHH” generally refers to a class of antibodies that lack antibody light chains and have only heavy chain variable regions.
  • single domain antibodies can be derived from Bactrian camels, dromedaries, alpacas, llamas, nurse sharks, white sharks or rays (for example, see Kang Xiaozhen et al., Journal of Biotechnology, 2018, 34 (12): 1974-1984).
  • single domain antibodies can be derived from alpacas.
  • Single domain antibodies can be composed of heavy chain variable regions (VH).
  • the term "heavy chain variable region” generally refers to the amino terminal domain of the heavy chain of an antigen binding fragment.
  • the heavy chain variable region can be further distinguished into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the heavy chain variable region contains a binding domain that interacts with an antigen (eg, CD22).
  • transmembrane domain generally refers to a sequence in a cell surface protein that spans the cell membrane, which may include a hydrophobic alpha helix.
  • the transmembrane domain may be connected to an intracellular signal transduction domain to play a role in transmitting signals.
  • the transmembrane domain may be derived from any type I, type II or type III transmembrane protein.
  • the transmembrane domain can include a transmembrane domain or a combination thereof derived from a histone selected from: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, a subunit of a T cell receptor, a polypeptide constituting a 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: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, a subunit of a T cell receptor, a polypeptide constituting a CD3 complex,
  • the subunit of a T cell receptor can include TCR ⁇ , TCR ⁇ , TCR ⁇ or TCR ⁇ .
  • the polypeptide constituting the CD3 complex may include CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or CD3 ⁇ .
  • the transmembrane domain may include a transmembrane domain derived from the CD8.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor immune cell
  • the CAR can be combined with an immune cell receptor activation intracellular domain based on the antigen (e.g., CD22) specificity of the antibody.
  • Immune cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing the target antigen.
  • CAR and CAR-T cells see, for example, 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 domain” generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for lymphocytes to effectively respond to antigens.
  • the costimulatory domain can comprise a costimulatory signaling domain or a combination thereof derived from a protein 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,
  • intracellular signaling domain generally refers to a domain located inside a cell that can transduce a signal.
  • the intracellular signaling domain can conduct a signal into the cell.
  • an intracellular signaling domain is any continuous amino acid sequence used to guide protein targeting.
  • the intracellular signaling domain may include an intracellular signaling domain or a combination thereof 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 PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP12, and a domain comprising at least one ITAM.
  • the intracellular signaling domain can include an intracellular signaling domain derived from CD3zeta.
  • signal peptide refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequent surface expression during or after translation.
  • an antibody generally refers to a polypeptide molecule that can specifically recognize and/or neutralize 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 (e.g., scFv), and antibody fragments that bind to antigens (e.g., Fab, VHH, Fab' and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens described herein.
  • Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain may be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the term "antigen binding fragment” generally refers to one or more fragments of an antibody that specifically binds to an antigen.
  • the antigen binding function of an antibody can be achieved by a full-length fragment of the antibody.
  • the 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 a light chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2.
  • Fab fragment usually a monovalent fragment composed of VL, VH, CL and CH domains;
  • F(ab')2 fragment a bivalent fragment that may include two Fab fragments connected by a disulfide bond at the hinge region;
  • Fd fragment composed of VH and CH domains;
  • Fv fragment composed of VL and VH domains of a single antibody arm;
  • dAb fragment composed of VH domain (Ward et al., (1989) Nature 341: 544-546);
  • the "antigen binding fragment” may also include an immunoglobulin fusion protein, wherein the fusion protein comprises a binding domain selected from the following: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) 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 fragment may also include a single domain antibody.
  • the term "scFv” generally refers to a single-chain antibody, which is an antibody formed by directly connecting the heavy chain variable region and the light chain variable region or connecting them through a connecting molecule (e.g., a connecting peptide).
  • the structure of the scFv from the N-terminus to the C-terminus can be a heavy chain variable region-light chain variable region, a light chain variable region-heavy chain variable region, a heavy chain variable region-connecting peptide-light chain variable region, or a light chain variable region-connecting peptide-heavy chain variable region.
  • the term "monoclonal antibody” generally refers to a group of substantially homologous antibodies, i.e., the individual antibodies comprising the group are identical except for possible naturally occurring mutations present in trace amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • the monoclonal antibodies can be prepared by hybridoma technology or produced in bacteria, eukaryotic animals or plant cells using recombinant DNA methods.
  • Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).
  • humanized antibody generally refers to a chimeric antibody that contains less sequence from non-human immunoglobulin, thereby reducing the immunogenicity of the xenoantibody when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.
  • CDR transplantation Jones et al., Nature 321:522 (1986)
  • its variants can be used.
  • the term "tumor” generally refers to a neoplasm formed by local tissue cell hyperplasia.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to a change in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22" may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with the protein expression of CD22 may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • nucleic acid molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule that can self-replicate in a suitable host.
  • the vector can transfer the inserted nucleic acid molecule into a cell and/or between cells.
  • the vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for the expression of transcription and/or translation of DNA or RNA.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector can produce a desired expression product by culturing a suitable cell containing the vector.
  • the vector may include a lentiviral vector.
  • the term "cell” generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector including a nucleic acid molecule described in the present application, or that can express a chimeric antigen receptor described in the present application or an antigen binding protein described in the present application.
  • the cell may include the offspring of a single cell. Due to natural, accidental or deliberate mutations, the offspring cells may not necessarily be completely identical in morphology or genome to the original parent cell, but can express the chimeric antigen receptor or antigen binding protein described in the present application.
  • the cell can be obtained by in vitro transfection of cells using the vector described in the present application.
  • the cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell).
  • the cell may be an immune cell.
  • the immune cell 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 cell can be a T cell.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a subject.
  • the pharmaceutical composition may include the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the cell, and an optional pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such preparations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the term "treat” generally refers to: (i) preventing a disease, disorder and/or condition from occurring in a patient who may be susceptible to the disease, but has not yet been diagnosed with the disease; (ii) inhibiting the disease, disorder or condition, i.e., curbing 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 to subside.
  • the present application also includes functional variants, derivatives, analogs, homologues and fragments thereof.
  • a variant of any given sequence refers to a sequence in which a specific sequence of residues (whether amino acids or nucleotide residues) has been modified so that the polypeptide or polynucleotide substantially retains at least one endogenous function.
  • Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, as long as the original functional activity is maintained.
  • the term “derivative” generally refers to any substitution, variation, modification, replacement, deletion and/or addition of one (or more) amino acid residues from/to the polypeptide or polynucleotide of the present application, as 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, a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.
  • amino acid substitutions such as at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 or more) amino acid substitutions, can be made, as long as the modified sequence substantially retains the desired activity or ability.
  • Amino acid substitutions may 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 may be made based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic properties of the residues, as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing non-electric polar head groups with similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.
  • CD22 generally refers to an important membrane antigen on human B lymphocytes that is associated with proliferation and differentiation.
  • the amino acid sequence of human CD22 can be found in UniProt/Swiss-Prot accession number P20273.
  • the antigen targeted by one of the antigen binding domains of the chimeric antigen receptor (CAR) can be CD22.
  • CD19 generally refers to a leukocyte differentiation antigen on the surface of B cells.
  • the CD19 may include human CD19.
  • the antigen targeted by one of the antigen binding domains of the chimeric antigen receptor may be CD19.
  • the term "about” generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 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% above or below a specified value.
  • the present application provides a chimeric antigen receptor comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19, and the second antigen binding domain comprises a VHH targeting CD22.
  • first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19
  • second antigen binding domain comprises a VHH targeting CD22.
  • the present application provides a chimeric antigen receptor, which comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is capable of targeting CD19, the second antigen binding domain is capable of targeting CD22, the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the CDR of an antibody is also called the complementary determining region, which is a part of the variable region.
  • the amino acid residues in this region can contact the antigen or antigen epitope.
  • the antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, North's, Kabat/Chothia, etc. These coding systems are known in the art, and specific references can be made to, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region.
  • the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids.
  • the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids.
  • the antigen-binding proteins described herein are defined by the IMGT coding system.
  • the second antigen binding domain of the chimeric antigen receptor targets CD22.
  • the second antigen binding domain of the chimeric antigen receptor may comprise an antigen binding protein targeting CD22.
  • the second antigen binding domain of the chimeric antigen receptor may include at least one CDR in the variable region of the antibody heavy chain.
  • the variable region of the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 64.
  • the variable region of the antibody heavy chain may include at least one CDR in the VHH sequence of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
  • the second antigen binding domain of the chimeric antigen receptor may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the second antigen binding domain of the chimeric antigen receptor may comprise an antibody or an antigen binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the second antigen binding domain of the chimeric antigen receptor may be VHH.
  • the HCDR3 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 59.
  • the HCDR3 sequence of the second antigen binding domain may be defined according to the IMGT coding system.
  • the second antigen-binding domain comprises HCDR3, which, compared with the sequence shown in SEQ ID NO: 59, has an amino acid substitution (eg, conservative amino acid substitution, etc.) at an amino acid selected from the following positions: X15 and X17 .
  • ATDPWTDCSLDGRYX 15 YX 17 Y (SEQ ID NO: 59), wherein X 15 can be E or R, and X 17 can be D, G or N.
  • the HCDR3 of the second antigen binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the HCDR2 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the HCDR2 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR2, and the HCDR2 has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at an amino acid selected from the following positions compared to the sequence shown in SEQ ID NO: 58: X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 58), wherein X 3 can be G or S.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12.
  • the HCDR2 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the HCDR1 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 57.
  • the HCDR1 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR1, and the HCDR1, compared with the sequence shown in SEQ ID NO: 57, has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at an amino acid selected from the following positions: X3 , X5 and X6 .
  • GFX3VX5X6YA (SEQ ID NO:57), wherein X3 may be P or S, X5 may be A or D, and X6 may be D or G.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • the HCDR1 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the second antigen binding domain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:57, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:58, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:59.
  • the second antigen binding domain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:11 or SEQ ID NO:12, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the HCDR1 of the second antigen binding domain may include the amino acid sequence shown in SEQ ID NO: 4, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.
  • the antigen binding protein may include antibody B010-B-22Nb-01 or an antigen binding fragment having the same HCDR3 therewith (e.g., having the same HCDR1-3 therewith).
  • the HCDR1 of the second antigen binding domain may include the amino acid sequence shown in SEQ ID NO:5
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO:12
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the antigen binding protein may include antibody B010-B-22Nb-02 or an antigen binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) therewith.
  • the antigen binding protein may include antibody B010-B-22Nb-03 or an antigen binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) therewith.
  • the HCDR1 of the second antigen binding domain may include the amino acid sequence shown in SEQ ID NO: 6
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 12
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 21.
  • the antigen binding protein may include antibodies B010-B-22Nb-04, B010-B-22Nb-04-H4, B010-B-22Nb-04-H5, B010-B-22Nb-04-H6, or an antigen binding fragment having the same HCDR3 therewith (e.g., having the same HCDR1-3 therewith).
  • the second antigen binding domain of the chimeric antigen receptor may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 60.
  • the H-FR1 of the antigen binding protein has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the following group: X1 and X16 .
  • X 1 VQLVESGGGLVQPGX 16 SLRLSCAAS (SEQ ID NO: 60), wherein X 1 may be A or E, and X 16 may be G or R.
  • H-FR1 of the second antigen binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
  • H-FR2 of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 61.
  • H-FR2 of the second antigen-binding domain has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X1 , X4 , X11 , X12 , X14 , X15 and X17 .
  • X1AWX4RQAPGKX11X12EX14X15SX17 (SEQ ID NO : 61 ) , wherein X1 may be I or M, X4 may be F or V, X11 may be E or G, X12 may be L or R, X14 may be G or W, X15 may be I or V, and X17 may be C or Y.
  • H-FR2 of the second antigen binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
  • H-FR3 of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 62.
  • H-FR3 of the second antigen-binding domain has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X2 , X3 , X7 , X12 , X20 , X21 , X27 , X29 and X30 .
  • YX2X3DSVX7GRFTX12SRDNAKNX20X21YLQMNX27LX29X30EDTAVYYC SEQ ID NO: 62 , wherein X2 may be D or Y, X3 may be A , Q or V, X7 may be E or K, X12 may be I or V, X20 may be S or T, X21 may be L or V, X27 may be D or S, X29 may be E, K or R, and X30 may be D or P.
  • H-FR3 of the second antigen binding domain may include the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.
  • the H-FR4 of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 63.
  • the H-FR4 of the second antigen-binding domain has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X3 and X6 .
  • WGX 3 GTX 6 VTVSS (SEQ ID NO: 63), wherein X 3 can be L or Q, and X 6 can be L or Q.
  • H-FR4 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the second antigen binding domain may include H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO:60
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO:61
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO:62
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO:63.
  • the second antigen binding domain of the chimeric antigen receptor may include H-FR1, H-FR2, H-FR3 and H-FR4,
  • the H-FR1 may include the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3
  • the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10
  • the H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 13
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-01 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 8
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-02 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 9
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 15
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-03 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 16
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-04 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 10
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the second antigen binding domain may include antibody B010-B-22Nb-04-H4 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the second antigen binding domain may include antibody B010-B-22Nb-04-H5 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 18,
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the antigen binding protein may include antibody B010-B-22Nb-04-H6 or an antigen binding fragment thereof having the same H-FR1-4.
  • the second antigen binding domain may comprise a VHH
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO: 64.
  • the VHH may comprise the amino acid sequence shown in any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
  • the first antigen binding domain of the chimeric antigen receptor may include an antibody or an antigen binding fragment thereof.
  • the antigen binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the first antigen binding domain of the chimeric antigen receptor may be scFv.
  • the first antigen binding domain of the chimeric antigen receptor can target CD19 protein.
  • the first antigen binding domain of the chimeric antigen receptor may include at least one CDR in the antibody heavy chain variable region.
  • the antibody heavy chain variable region may include the amino acid sequence shown in SEQ ID NO:37.
  • the first antigen binding domain of the chimeric antigen receptor may comprise HCDR3, for example, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen binding domain of the chimeric antigen receptor may comprise HCDR2, for example, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:32.
  • the first antigen binding domain of the chimeric antigen receptor may include HCDR1, for example, the HCDR1 may include the amino acid sequence shown in SEQ ID NO:31.
  • the first antigen binding domain of the chimeric antigen receptor may include HCDR1, HCDR2 and HCDR3.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:31
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:32
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen binding domain may include H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO:34.
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO:35.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO:36.
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO:23.
  • the first antigen binding domain may comprise an antibody heavy chain variable region VH.
  • the antibody heavy chain variable region VH may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the first antigen binding domain of the chimeric antigen receptor may include at least one CDR in the antibody light chain variable region.
  • the antibody light chain variable region may include the amino acid sequence shown in SEQ ID NO:45.
  • the first antigen binding domain of the chimeric antigen receptor may include LCDR3.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO:40.
  • the first antigen binding domain of the chimeric antigen receptor may include LCDR2.
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 39 (HTS).
  • the first antigen binding domain of the chimeric antigen receptor may comprise LCDR1.
  • the LCDR1 may comprise SEQ ID NO:38.
  • the first antigen binding domain of the chimeric antigen receptor may include LCDR1, LCDR2 and LCDR3.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:38
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:39 (HTS)
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:40.
  • the first antigen binding domain of the chimeric antigen receptor may include L-FR1, L-FR2, L-FR3 and L-FR4.
  • the L-FR1 may include the amino acid sequence shown in SEQ ID NO:41.
  • the L-FR2 may include the amino acid sequence shown in SEQ ID NO:42.
  • the L-FR3 may include the amino acid sequence shown in SEQ ID NO:43.
  • the L-FR4 may include the amino acid sequence shown in SEQ ID NO:44.
  • the first antigen binding domain of the chimeric antigen receptor may comprise an antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:45.
  • the first antigen binding domain of the chimeric antigen receptor may include VH and VL.
  • the VH includes the amino acid sequence shown in SEQ ID NO:37
  • the VL includes the amino acid sequence shown in SEQ ID NO:45.
  • the VH and the VL may be directly or indirectly connected.
  • the VH and the VL may be connected via a linker.
  • the linker may be a peptide linker.
  • the linker may include the amino acid sequence shown in SEQ ID NO:48.
  • the first antigen binding domain of the chimeric antigen receptor may comprise an scFv.
  • the scFv may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the first antigen binding domain may be directly or indirectly connected to the second antigen binding domain.
  • the indirect connection may include connection through a linker.
  • the linker may be a peptide linker.
  • the linker may include the amino acid sequence shown in SEQ ID NO:47.
  • the C-terminus of the first antigen-binding domain may be directly or indirectly connected to the N-terminus of the second antigen-binding domain.
  • the N-terminus of the first antigen-binding domain may be directly or indirectly connected to the C-terminus of the second antigen-binding domain.
  • the chimeric antigen receptor may include a transmembrane domain.
  • the transmembrane domain may include, but is not limited to, a transmembrane domain or a combination thereof derived from a protein selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, 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 include a transmembrane domain derived from CD8.
  • the transmembrane domain may include an amino acid sequence shown in SEQ ID NO:51.
  • the transmembrane domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO:51.
  • the chimeric antigen receptor may include a costimulatory signaling domain.
  • the costimulatory signaling domain may include, but is not limited to, a costimulatory signaling domain or a combination thereof derived from a protein selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.
  • the costimulatory signaling domain may include a costimulatory signaling domain derived from 4-1BB.
  • the costimulatory signaling domain may include an amino acid sequence as shown in SEQ ID NO: 52.
  • the co-stimulatory signaling domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO:52.
  • the chimeric antigen receptor may include an intracellular signaling domain.
  • the intracellular signaling domain may include an intracellular signaling domain or a combination thereof derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • EBV Epstein-Barr virus
  • HSKV Kaposi's sarcoma herpes virus
  • the intracellular signaling domain may include an intracellular signaling domain derived from CD3zeta.
  • the intracellular signaling domain may include an amino acid sequence as shown in SEQ ID NO:53.
  • the intracellular signaling domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO:53.
  • the chimeric antigen receptor may include a hinge region.
  • the hinge region includes a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region is a hinge region derived from CD8.
  • the hinge region may include the amino acid sequence shown in SEQ ID NO: 50.
  • the hinge region may include an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology with the amino acid sequence shown in SEQ ID NO: 50.
  • the chimeric antigen receptor may include a signal peptide.
  • the signal peptide may include an amino acid sequence shown in SEQ ID NO: 49.
  • the C-terminus of the signal peptide may be directly or indirectly connected to the N-terminus of the antigen binding domain.
  • the N-terminus of the transmembrane domain can be connected to the C-terminus of the antigen binding domain.
  • the C-terminus of the transmembrane domain can be connected to the N-terminus of the costimulatory signaling domain.
  • the C-terminus of the costimulatory signaling domain can be connected to the N-terminus of the intracellular signaling domain.
  • the chimeric antigen receptor may comprise the following domains from N-terminus to C-terminus: a signal peptide, a VHH targeting CD22, a linker 1, a VH targeting CD19, a linker 2, a VL targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor may include the following domains from N-terminus to C-terminus: a signal peptide, a VH targeting CD19, a linker 2, a VL targeting CD19, a linker 1, a VHH targeting CD22, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • the linker 1 may comprise the amino acid sequence shown in SEQ ID NO: 47.
  • the linker 2 may comprise the amino acid sequence shown in SEQ ID NO: 48.
  • the chimeric antigen receptor may comprise the following domains in sequence from N-terminus to C-terminus: an antigen binding protein of the present application (e.g., VHH), a transmembrane domain derived from CD8, a co-stimulatory signaling domain derived from CD137, and an intracellular signaling domain derived from CD3zeta.
  • an antigen binding protein of the present application e.g., VHH
  • a transmembrane domain derived from CD8 e.g., a transmembrane domain derived from CD8
  • co-stimulatory signaling domain derived from CD137
  • an intracellular signaling domain derived from CD3zeta.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO: 55.
  • the chimeric antigen receptor may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology with the amino acid sequence shown in SEQ ID NO: 55.
  • the present application also provides one or more isolated nucleic acid molecules, which can encode the chimeric antigen receptor described in the present application.
  • each of the one or more nucleic acid molecules can encode the complete chimeric antigen receptor, or a portion thereof (e.g., one or more of HCDR1-3 and heavy chain variable region).
  • the nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the chimeric antigen receptor can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the nucleic acid molecule of the chimeric antigen receptor described in the present application.
  • the present application provides one or more vectors, which contain one or more nucleic acid molecules described in the present application.
  • One or more nucleic acid molecules may be included in each vector.
  • other genes may also be included in the vector, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host.
  • control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is an adjustable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functionally connected to a nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a certain portion of the promoter may be fused to a certain portion of a promoter of a gene of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not include additional introns.
  • One or more nucleic acid molecules described herein may be operably connected to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector may be an expression vector.
  • the vector may be a viral vector.
  • the viral vector may be administered directly to the patient (in vivo) or may be administered indirectly, for example, by treating cells with a virus in vitro and then administering the treated cells to the patient (ex vivo).
  • Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex virus vectors for gene transfer.
  • gene transfer may be integrated into the host genome using retroviral, lentiviral, and adeno-associated viral methods to allow long-term expression of the inserted gene.
  • a lentiviral vector is a retroviral vector that is capable of transducing or infecting non-dividing cells and typically produces a higher viral titer.
  • the lentiviral vector may comprise a long terminal repeat sequence 5'LTR and a truncated 3'LTR, RRE, a rev response element (cPPT), a central termination sequence (CTS) and/or a post-translational regulatory element (WPRE).
  • the vector described in the present application may be introduced into a cell.
  • the present application provides a kind of cell.
  • the cell may include a chimeric antigen receptor described in the present application, one or more nucleic acid molecules and/or one or more vectors described in the present application.
  • each or each cell may include one or a nucleic acid molecule or vector described in the present application.
  • each or each cell may include multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in the present application may be introduced into the host cell, such as a prokaryotic cell (e.g., a bacterial cell), a CHO cell, a NS/0 cell, a HEK293T cell, a 293F cell or a HEK293A cell, or other eukaryotic cells, such as cells from plants, fungi or yeast cells, etc.
  • the vector described in the present application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.
  • the cell may include a yeast cell.
  • the cell may include an Escherichia coli cell.
  • the cell may include a mammalian cell.
  • the cell may include an immune cell.
  • the cell may include an immune cell.
  • the cell may include an immune cell.
  • the cell may include an immune cell.
  • the cell may include a T cell, a B cell, a natural killer (NK) cell, a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte and/or a peripheral blood mononuclear cell.
  • the cell may include a T cell.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may include the chimeric antigen receptor described in the present application, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex and/or a nonionic surfactant.
  • any conventional medium or reagent may be considered for use in the pharmaceutical composition of the present application.
  • the pharmaceutically acceptable excipient may include an additive other than the main drug in the pharmaceutical preparation, which may also be referred to as an excipient.
  • the excipient may include an adhesive, a filler, a disintegrant, and a lubricant in a tablet.
  • the excipient may include wine, vinegar, and a medicinal juice in a traditional Chinese medicine pill.
  • the excipient may include a matrix part in a semisolid preparation ointment or cream.
  • the excipients may include preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.
  • the present application provides a method for detecting or measuring CD22, which may include using the isolated antigen-binding protein or the polypeptide.
  • the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.
  • the method may include a method for detecting the presence and/or amount of CD22 for non-diagnostic purposes, which may include the following steps:
  • the present application provides a CD22 kit, which may include and use the isolated antigen-binding protein or the polypeptide.
  • the kit may further include instructions for use, which describe methods for detecting the presence and/or content of CD22.
  • the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.
  • the present application provides a use of the isolated antigen-binding protein or the polypeptide in preparing a kit, which can be used for a method for detecting the presence and/or content of CD22.
  • the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.
  • the present application provides an isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and the pharmaceutical composition for preventing, alleviating and/or treating a disease or condition.
  • the kit and/or the drug combination is used to prevent, alleviate and/or treat a disease or condition.
  • the disease or condition may include a tumor.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to changes in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22” may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with the protein expression of CD22 may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • the present application provides a use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and/or treating a disease or condition.
  • the present application provides a use of a drug combination in the preparation of a medicament for preventing, alleviating and/or treating a disease or condition.
  • the disease or condition may include a tumor.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to changes in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22” may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with CD22 protein expression may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • the present application provides a method for preventing and/or treating a disease or condition, which comprises administering the isolated antigen binding protein, the isolated nucleic acid molecule, the vector, the cell, or the pharmaceutical composition to a subject in need thereof.
  • the disease or condition may include a tumor.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to changes in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22” may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with CD22 protein expression may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • compositions and methods described herein can be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, etc.
  • the pharmaceutical compositions and methods described herein can be used for other disease conditions that rely on immune responses, such as inflammation, immune diseases, and infectious diseases.
  • the subject may include a human or a non-human animal.
  • the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats.
  • the non-human animal may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the human may be Caucasian, African, Asian, Semitic, or other races, or a hybrid of various races.
  • the human may be elderly, adult, adolescent, child or infant.
  • the effective amount in humans can be extrapolated from the effective amount in experimental animals.
  • Freireich et al. described the relationship between animal and human doses (based on milligrams per square meter of body surface) (Freiheim et al., Cancer Chemother. Rep. 50, 219 (1966)).
  • Body surface area can be approximately determined from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
  • a healthy female adult alpaca (Alpaca) was immunized with recombinant human CD22 protein (11958-H02, Beijing Sino Biological Technology Co., Ltd.).
  • 500 ⁇ g of recombinant human CD22 protein was emulsified with an equal volume of Freund's complete adjuvant and injected into the left and right sides near the cervical lymph nodes;
  • 500 ⁇ g of recombinant human CD22 protein was emulsified with an equal volume of Freund's complete or incomplete adjuvant and injected into the left and right sides near the cervical lymph nodes, and several booster immunizations were performed. Blood was collected one week after the last immunization to monitor the antiserum titer.
  • lymphocytes were separated by lymphocyte separation solution; total RNA was extracted by TRIzol TM reagent; cDNA was obtained by reverse transcription using PrimeScript TM II first-strand cDNA synthesis kit, and VHH gene was amplified by nested PCR.
  • the VHH gene fragment was recovered by gel purification kit and digested by restriction endonuclease Bgl 1, then cloned into phagemid vector pADL-10b, and the constructed cloned product was transformed into E.coli TG1 electroporation competent cells to construct VHH gene library; the library capacity was determined to be 2.2 ⁇ 10 9 pfu by plate gradient dilution method, and the colony PCR results showed that the library insertion rate was 97.9%. 10-100 times the library capacity of live cells was taken from the above gene library for inoculation and culture, and M13K07 phage was used for rescue after culture to logarithmic phase. After rescue culture, phage was collected by centrifugation, and PEG-NaCl was used to purify the phage to obtain phage display library, which can be directly used for subsequent screening.
  • the biologically active antibodies that have been screened can be optionally humanized.
  • the humanization of camel-derived monoclonal antibodies is carried out according to the methods disclosed in many documents in the art.
  • the constant domain of the parent can be replaced by the human antibody constant domain, and the human germline antibody sequence is selected according to the homology of the camel-derived antibody and the human antibody, and CDR transplantation is performed.
  • the amino acid residues of VH are reversely mutated, and the constant region of the camel-derived antibody is replaced by the human constant region to obtain the final humanized binding proteins B010-B-22Nb-04-H4, B010-B-22Nb-04-H5 and B010-B-22Nb-04-H6.
  • Table 1 shows the CDR, heavy chain variable region VH, and FR of CD22 VHH of this application.
  • Biacore was used to detect the binding affinity of different CD22 antigen binding proteins to antigen (CD22 protein, source: Beijing Sino Biological Technology Co., Ltd., catalog number: 11958-H08H1-B) protein.
  • CM5 chip 1-8 channels The surface of CM5 chip 1-8 channels was activated with a 1:1 mixture of 50 mM NHS and 200 mM EDC (NHS and EDC were from the amino coupling kit) at a flow rate of 10 ⁇ L/min for 420 seconds.
  • Anti-hFc or anti-mouse Fc antibody (diluted in sodium acetate solution at pH 4.5, concentration 20 ⁇ g/mL) was injected at a flow rate of 10 ⁇ L/min for 200 seconds, and finally the excess active carboxyl groups on the chip were blocked with 1 M ethanolamine hydrochloride (pH 8.5).
  • the chip surface was rinsed with 1 ⁇ HBS-EP+ at a flow rate of 10 ⁇ L/min for 2 hours to stabilize the baseline, and the instrument was set to 25°C.
  • the initial cycle consisting of two steps, sampling and regeneration, was repeated three times before measurement to stabilize the baseline.
  • Sample measurement Inject 1 ⁇ HBS-EP+ buffer into channels 1-8 at a flow rate of 30 ⁇ L/min for 120 seconds and dissociate for 60 seconds.
  • Regeneration Inject 10 mM glycine pH 1.5 into channels 1-8, 30 ⁇ L/min, 30 sec, stabilize for 30 sec.
  • the running buffer for kinetic determination is 1 ⁇ HBS-EP+ (pH7.4) solution.
  • Capture Different antibodies were injected into the test channels of channels 1-8 of the Anti-hFc or anti-mouse Fc chip, respectively, at a flow rate of 10 ⁇ L/min for 60s for capture.
  • the antigen CD22 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 11958-H08H1-B) was diluted to 100nM with 1 ⁇ HBS-EP+ (pH 7.4).
  • Sample measurement Inject into channels 1-8 at a flow rate of 30 ⁇ L/min, and a 0 concentration sample was used to remove background signals; the binding and dissociation times of the antigen and antibody were 180 and 400 seconds, respectively.
  • Regeneration Inject 10mM glycine pH1.5 into channels 1-8 at a flow rate of 30 ⁇ L/min for 30 seconds, and then stabilize for 60 seconds.
  • the equilibrium dissociation constant ( KD value) of each antigen binding protein of the present application was calculated using Biacore8K analysis software.
  • the reference channel (FC1) was used for background subtraction.
  • the binding ability of CD22 antigen binding proteins to human CD22 expressed on the surface of K562 cells was determined based on flow cytometry.
  • the binding ability of different CD22 antigen binding proteins of the present application to human CD22 expressed on the surface of K562 cells was determined by comparing the binding curves.
  • K562 cells were genetically modified to overexpress human CD22, and the cells were named K562-hCD22 cells.
  • K562-hCD22 cells were digested and plated in 96-well plates.
  • the above experiments show that the antigen-binding protein of the present application has binding activity with human CD22 on the surface of K562 cells and is superior to the positive control antibody.
  • the binding curve of the CD22 VHH of the present application and human CD22 on the surface of K562 cells was determined based on flow cytometry.
  • Example 7 CD19/CD22 bispecific CAR molecules and their corresponding single-target specific CAR molecules CD19 CAR, CD22 CAR plasmid construction and lentiviral packaging
  • the gene sequence of the gene-synthesized humanized chimeric antigen receptor molecule targeting CD19/CD22 specifically includes: a single-domain antibody VHH sequence targeting human CD22, Linker1, an antibody VH sequence targeting human CD19, Linker2, an antibody VL sequence targeting human CD19, a membrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3zeta intracellular signal transduction domain, which are connected in series in sequence.
  • the obtained chimeric antigen receptor molecule (CAR molecule) is named B010-B-03 (the amino acid sequence is shown in SEQ ID NO: 55).
  • the gene sequence of the gene-synthesized humanized chimeric antigen receptor molecule targeting CD22 specifically includes: a single-domain antibody VHH sequence targeting human CD22, a membrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3zeta intracellular signal transduction domain, which are connected in series in sequence, and the obtained chimeric antigen receptor molecule (CAR molecule) is named B010-B-22 (the amino acid sequence is shown in SEQ ID NO: 54).
  • the gene sequence of the gene-synthesized humanized chimeric antigen receptor molecule targeting CD19 specifically includes: antibody VH sequence targeting human CD19, Linker2, antibody VL sequence targeting human CD19, membrane domain, 4-1BB co-stimulatory signal transduction domain and CD3zeta intracellular signal transduction domain, which are connected in series in sequence.
  • the obtained chimeric antigen receptor molecule (CAR molecule) is named B010-B-19 (amino acid sequence as shown in SEQ ID NO:56). After synthesis, the chimeric antigen receptor gene sequence is cloned between the EcoRI and BamHI restriction sites on the pLVX-EF1a-IRES-Puro vector.
  • the constructed lentiviral core plasmid and packaging plasmid pMDLg, regulatory plasmid pRSV and envelope protein plasmid pMD2.G are transfected into 293T cells together with PEI transfection reagent, and the virus is collected after a certain period of culture and concentrated for collection.
  • Human peripheral blood T lymphocytes were collected and transfected with the lentivirus prepared in Example 1 to prepare CAR-T cells, which were named B010-B-03, B010-B-19, and B010-B-22 CART, respectively.
  • virus-transfected T lymphocytes obtained by preparing vector packaging using an unrelated sequence were used as the control group and defined as NT-T.
  • the humanized B010-B-03, B010-B-19, B010-B-22 CART cells and NT-T cells infected with the lentivirus in Example 2 were collected and expanded to day 6, day 8, day 10 and day 12.
  • B010-B-03 and B010-B-19 CART can bind to Protein L and CD19
  • B010-B-03 and B010-B-22 CART can bind to CD22.
  • the affinity ratio of Protein L and VHH CAR structure to CD22 protein was used as the CART cell positive rate
  • the binding of CD19 and CD22 was used as the affinity of CART cells to antigens
  • TCM Central Memory T cell, central memory T cell, CD45RO+/CD197+/CD62L+cells
  • the positivity rate and affinity of B010-B-03, B010-B-19, and B010-B-22 CART cells were high and stable, among which the positivity rate and affinity of dual-target CART were not lower than those of single-target CART.
  • the proportion of memory population of dual-target CART showed an upward trend, and the single-target CART reached a peak on Day 8 (Table 3, Figure 2, Figure 3, and Figure 4).
  • Firefly luciferase (Luciferase, abbreviated as LUC) was transferred into Nalm6 to obtain Nalm6-LUC cells.
  • LUC Firefly luciferase
  • Cas9 gene editing knockout technology Cas9 protein and specific sgRNA were transferred into Nalm6-LUC cell lines by electroporation to obtain Nalm6-CD19 - CD22 + -LUC and Nalm6-CD19 + CD22 -- LUC.
  • an in vitro killing assay was performed using an ELISA instrument to compare the killing effects of CART cells on three Nalm6 cell models at different effector-target ratios and compared with NT-T cells.
  • Example 11 Experiment on detecting the level of cytokine secretion by CART cells activated by target cells
  • the CART cells were mixed and co-incubated with the three Nalm6 cell models as the experimental group; the control group was NT-T.
  • B010-B-03 CART was activated to secrete tumor cell-killing-promoting cytokines IL-2, IFN- ⁇ , and TNF- ⁇ at higher levels than B010-B-19 and B010-B-22 CART in the three Nalm6 target cell models (Figure 8).
  • the target tumor cells Nalm6-LUC expressing luciferase constructed by us were inoculated into NSG mice (purchased from Shanghai Southern Model Organisms Technology Co., Ltd.) and injected into the tail vein to form tumors. 2.0 ⁇ 10 5 of the above target cells were resuspended in 200 ⁇ l serum-free culture medium and injected into the tail vein of mice.
  • the luciferase substrate D-Luciferin was injected intraperitoneally into the mouse at a dose of 3 mg/mouse.
  • the mouse was anesthetized and placed in a small animal in vivo imaging instrument for imaging.
  • B010-B-03, B010-B-19, and B010-B-22 CART cells were resuspended in serum-free culture medium and the cell density was adjusted to 2.5 ⁇ 10 7 cells/ml.
  • 5 ⁇ 10 6 CART cells were injected into the tail vein of each mouse, a total of 200 ⁇ l/mouse. Mice treated with non-transfected CAR T cells were set as the control group.
  • regular in vivo imaging tests were performed twice a week to collect the tumor elimination effect.
  • Figure 9 shows the results of in vivo imaging of Nalm6-LUC mice, and the imaging results reflect the number of tumor cells.
  • the results showed that compared with the non-CAR transfected T cell group and the single- and double-target CART cell groups with increasing bioluminescence intensity, the number of tumor cells in the dual-target CART administration group and the single-target CD19 CART administration group was significantly reduced before Day 15, but the CD19 CART administration group showed tumor cell recurrence and growth after Day 15, and the dual-target CART administration group did not recur and grow, and the tumor inhibition rate was 100.0% on the 32nd day after administration. ( Figure 10)
  • FIG11 shows the results of the body weight changes of Nalm6-LUC mice. The results show that the body weight fluctuations of the mice were stable after administration, and no animals died during the treatment period. There was no obvious drug toxicity, and the drug was safe and well tolerated.
  • the cells were detected and identified using a flow cytometer (Beckman Cytoflex). The ratio error was acceptable within ⁇ 10%.
  • Nalm6-MIX-LUC was inoculated into NSG mice (purchased from Shanghai South Model Organisms Science Co., Ltd.) and injected into the tail vein to form tumors. 2.0 ⁇ 10 5 of the above target cells were resuspended in 200 ⁇ l serum-free culture medium and injected into the tail vein of mice.
  • the luciferase substrate D-Luciferin was injected intraperitoneally into the mouse at a dose of 3 mg/mouse.
  • the mouse was anesthetized and placed in a small animal in vivo imaging instrument for imaging.
  • B010-B-03, B010-B-19, and B010-B-22 CART cells were resuspended in serum-free culture medium and the cell density was adjusted to 2.5 ⁇ 10 7 cells/ml.
  • 5 ⁇ 10 6 CART cells were injected into the tail vein of each mouse, a total of 200 ⁇ l/mouse. Mice treated with non-transfected CAR T cells were set as the control group.
  • regular in vivo imaging tests were performed twice a week to collect the tumor elimination effect.
  • Figure 12 shows the results of in vivo imaging of Nalm6-MIX-LUC mice, and the imaging results reflect the number of tumor cells.
  • the results show that compared with the non-CAR transfected T cell group and the single- and double-target CART cell groups with increasing bioluminescence intensity, the number of tumor cells in the dual-target CART administration group was significantly reduced and remained stable without tumor cell recurrence and growth.
  • the tumor inhibition rate was 100.0% on the 32nd day after administration.
  • Figure 14 shows the results of body weight changes in Nalm6-MIX-LUC mice. The results show that the body weight of mice fluctuated stably after administration, and no animals died during the treatment period. There was no obvious drug toxicity, and the drug was safe and well tolerated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Toxicology (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)

Abstract

提供一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，其中第一抗原结合域靶向CD19，第二抗原结合域靶向CD22。还提供了包含该嵌合抗原受体的细胞，以及该嵌合抗原受体和细胞的制备方法与用途。

Description

靶向CD19和CD22的双特异性嵌合抗原受体 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向CD19和CD22的嵌合抗原受体及其用途。
背景技术
急性B淋巴细胞白血病(B-ALL)是最常见的儿童恶性肿瘤，其中复发或难治性B-ALL缺少有效的治疗手段，是目前B-ALL治疗瓶颈。复发或难治性儿童B-ALL患者的5年无病生存率为27％和15％。其中异基因造血干细胞移植之后复发的患者基本处于无药可救的境地。初发或者复发难治性B-ALL，若不能完全清除微小残留病灶，化疗生存率低于10％，总体上B-ALL的20年疾病结局没有显著改善。因此，开发针对复发或难治性B-ALL的有效治疗有着巨大的临床需求。
目前，针对单靶点CD19 CAR-T或CD22 CAR-T疗法存在治疗后出现较高比例的疾病复发，复发率约为40％-60％。复发的主要原因是CD19抗原丢失或CD19靶向免疫治疗后的下调和细胞表面CD22表达减少。因此，亟需设计新型的CAR-T细胞，用于克服目前已上市的CAR-T疗法的不足之处，提高治疗效果。
发明内容
本申请提供了一种靶向CD19和CD22的双特异性嵌合抗原受体，以及包含所述嵌合抗原受体的细胞，其能够同时结合CD19和CD22，并对靶细胞具有杀伤作用。本申请所提供的CD19/CD22 CAR-T疗法有望克服已上市CAR-T疗法的不足之处，通过双靶向性设计，增加新的识别靶点，减少肿瘤通过抗原缺失逃脱的可能性，降低治疗后疾病复发的概率，增加记忆性T细胞比例，有望大大解决现有CAR-T疗法的诸多缺陷，为患者带来长期稳定的治疗效果。
一方面，本申请提供了一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域包含靶向CD19的单链抗体(scFv)，所述第二抗原结合域包含靶向CD22的VHH。
在某些实施方式中，所述第二抗原结合域包含抗体重链可变区VHH中的至少一个CDR，且所述VHH包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID  NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30的VHH序列中的至少一个CDR。
另一方面，本申请提供了一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域能够靶向CD19，所述第二抗原结合域能够靶向CD22，所述第二抗原结合域包含抗体重链可变区VHH中的至少一个CDR，且所述VHH包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述VHH包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含抗体或其抗原结合片段。
在某些实施方式中，所述第二结合域包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述第一抗原结合域为scFv。
在某些实施方式中，所述第二抗原结合域为VHH。
在某些实施方式中，所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的HCDR3包含SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的HCDR2包含SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:57所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的HCDR1包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:57所示的氨基酸序列，所述HCDR2包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11或SEQ ID NO:12中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下组的氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:19所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
(3)所述HCDR1包含SEQ ID NO:6所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的H-FR1包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:61所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的H-FR2包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:62所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的H-FR3包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:63所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的H-FR4包含SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:60所示的氨基酸序列，所述H-FR2包含SEQ ID NO:61所示的氨基酸序列，所述H-FR3包含SEQ ID NO:62所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:63所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18中任一项所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组的氨基酸序列：
(1)所述H-FR1包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
(2)所述H-FR1包含SEQ ID NO:2所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
(3)所述H-FR1包含SEQ ID NO:2所示的氨基酸序列，所述H-FR2包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
(4)所述H-FR1包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:16所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
(5)所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列；
(6)所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO: 7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列；以及
(7)所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域包含VHH，所述VHH包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合域的VHH包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:31所示的氨基酸序列，所述HCDR2包含SEQ ID NO:32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3包含SEQ ID NO:36所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含VH，所述VH包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:38所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39(HTS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:41所示的氨基酸序列，所述L-FR2包含SEQ ID NO:42所示的氨基酸序列，所述L-FR3包含SEQ ID NO:43所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:44所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含VL，所述VL包含SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含VH和VL，所述VH包含SEQ ID NO:37所示的氨基酸序列，所述VL包含SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域的VH和VL通过连接子连接。
在某些实施方式中，所述连接子包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域包含scFv，所述scFv包含SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合域和第二抗原结合域直接或间接相连。
在某些实施方式中，所述第一抗原结合域的C端与所述第二抗原结合域的N端直接或间接相连。
在某些实施方式中，所述第一抗原结合域的N端与所述第二抗原结合域的C端直接或间接相连。
在某些实施方式中，所述第一抗原结合域和所述第二抗原结合域通过连接子相连。
在某些实施方式中，所述连接子包含肽接头。
在某些实施方式中，所述连接子包含SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述共刺激信号区域包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:53所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、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，所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述跨膜区包含SEQ ID NO:51所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区为源自CD8的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:49所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体从N端到C端依次包含：信号肽、第二抗原结合域、连接子、第一抗原结合域、铰链区、跨膜区、共刺激域以及胞内信号传导域。
在某些实施方式中，所述嵌合抗原受体从N端到C端依次包含：信号肽、靶向CD22的VHH、连接子1、靶向CD19的VH、连接子2、靶向CD19的VL、CD8铰链区、CD8跨膜区、4-1BB共刺激域以及CD3ζ胞内信号传导域。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:55所示的氨基酸序列。
另一方面，本申请还提供了分离的一种或多种核酸分子，其编码所述嵌合抗原受体。
另一方面，本申请还提供了载体，其包含所述核酸分子。
另一方面，本申请还提供了细胞，其包含所述嵌合抗原受体，所述核酸分子，和/或所述载体。
在某些实施方式中，所述细胞包括免疫细胞。
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，所述细胞包括T细胞。
另一方面，本申请提供了制备所述嵌合抗原受体的方法，所述方法包括再使得所述嵌合抗原受体表达的条件下，培养所述细胞。
另一方面，本申请提供了药物组合物，其包括所述嵌合抗原受体，所述核酸分子，所述 载体，所述细胞，和/或药学上可接受的载剂。
另一方面，本申请还提供了一种用于检测CD19和/或CD22的方法，其包括施用所述细胞。
另一方面，本申请还提供了一种CD19和/或CD22蛋白检测的试剂盒，其包含所述嵌合抗原受体，和/或所述细胞。
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述载体，所述细胞在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括与CD19和/或CD22表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤选自下组：白血病和淋巴瘤。
另一方面，本申请还提供了一种预防和/或治疗疾病和/或病症的方法，其包括向有需要的受试者施用所述嵌合抗原受体，所述核酸分子，所述载体，和/或所述细胞。
在某些实施方式中，所述疾病和/或病症包括与CD19和/或CD22表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤选自下组：白血病和淋巴瘤。
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述载体，所述细胞，和/或所述药物组合物，其用于预防和/或治疗疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括与CD19和/或CD22表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤选自下组：白血病和淋巴瘤。
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。对附图简要说明如下：
图1显示的是本申请所述CD22 VHH与表达于K562细胞表面的人CD22的结合曲线。
图2显示的是流式检测本申请所述的CD19/CD22单双靶点CART细胞阳性率结果。
图3显示的是流式检测本申请所述的CD19/CD22单双靶点CART细胞与抗原亲和力结果。
图4显示的是流式检测本申请所述的CD19/CD22单双靶点CART细胞中TCM细胞比例变化结果。
图5显示的是本申请所述CD19/CD22单双靶点CART细胞对Nalm6-LUC细胞的杀伤作用。
图6显示的是本申请所述CD19/CD22单双靶点CART细胞对Nalm6-LUC单阳1:1混合细胞模型的杀伤作用。
图7显示的是本申请所述CD19/CD22单双靶点CART细胞对Nalm6-LUC野生型/单阳(8:1:1)混合细胞模型的杀伤作用。
图8显示的是本申请所述CD19/CD22单双靶点CART细胞受3种Nalm6模型靶细胞激活后分泌细胞因子水平。
图9显示的是Naml6-LUC小鼠活体成像荧光强度检测结果。
图10显示的是Naml6-LUC小鼠活体成像荧光图。
图11显示的是Naml6-LUC小鼠体重变化结果。
图12显示的是Naml6-MIX-LUC小鼠活体成像荧光强度检测结果。
图13显示的是Naml6-MIX-LUC小鼠活体成像荧光图。
图14显示的是Naml6-MIX-LUC小鼠体重变化结果。
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，本领域技术人员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“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，CD22)相互作用的结合结构域。
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是嵌合抗原受体免疫细胞(例如，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CD22)特异性与免疫细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的免疫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，共刺激域可以包括源自CD137(4-1BB)的共刺激信号传导结构域。
在本申请中，术语“胞内信号传导域”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，所述胞内信号传导域可以将信号传导至细胞内。通常，胞内信号传导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PBj14Nef、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”是指处于新生CAR蛋白的氨基末端(N-末端)的前导序列，其在翻译时或在翻译后将新生蛋白引导到内质网并后续表面表达。
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、VHH、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。
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，通常由VL、VH、CL和CH结构域组成的一价片段；(2)F(ab’)2片段，可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“scFv“通常是指单链抗体，是由重链可变区和轻链可变区直接连接或通过连接分子(例如，连接肽)连接而成的抗体。所述scFv的结构自N端至C端，可以是重链可变区-轻链可变区，轻链可变区-重链可变区，重链可变区-连接肽-轻链可变区，或轻链可变区-连接肽-重链可变区。
在本申请中，术语“单克隆抗体”通常是指一群基本同源的抗体，即包含该群的各个抗体除了可能的以微量存在的天然发生的突变之外是相同的。单克隆抗体是高度特异性的，直接针对单个抗原性位点。例如，所述单克隆抗体可以通过杂交瘤技术制备或者通过使用重组DNA方法在细菌、真核动物或植物细胞中产生。单克隆抗体也可以得自噬菌体抗体文库，使用例如Clackson etal.,Nature,352:624-628(1991)和Marks et al.,Mol.Biol.，222:581-597(1991)所述的技术进行。
在本申请中，术语“人源化抗体”通常是指一种嵌合抗体，其含有较少的来自非人免疫球蛋白的序列，从而降低异种抗体引入到人类中时的免疫原性，同时保持抗体的完全抗原结合亲和力和特异性。例如，可以使用CDR移植(Jones et al.,Nature 321:522(1986))及其变 体；包括“重塑”(reshaping)，(Verhoeyen,et al.,1988Science 239:1534-1536；Riechmann,et al.,1988Nature 332:323-337；Tempest,et al.,Bio/Technol 1991 9:266-271)，“高度加成”(hyperchimerization)，(Queen,et al.,1989Proc Natl Acad Sci USA 86:10029-10033；Co,et al.,1991Proc Natl Acad Sci USA 88:2869-2873；Co,et al.,1992J Immunol 148:1149-1154)和“贴面”(veneering)，(Mark,et al.,“Derivation of therapeutically active humanized and veneered anti-CD18antibodies.”In:Metcalf B W,Dalton B J,eds.Cellular adhesion:molecular definition to therapeutic potential.New York:Plenum Press,1994:291-312)、表面重建(美国专利US5639641)等技术手段，对非人源的结合域进行人源化。
在本申请中，术语“肿瘤”通常是指局部组织细胞增生所形成的赘生物。例如，所述肿瘤可以包括血液瘤。例如，所述肿瘤可包括淋巴瘤。例如，所述肿瘤可包括白血病。例如，所述肿瘤可包括与CD22的表达相关的肿瘤。术语“与CD22的表达相关的肿瘤”通常是指与正常细胞相比，所述肿瘤微环境中或肿瘤细胞表面CD22的表达改变。例如，所述“与CD22的表达相关的肿瘤”可以是与正常细胞相比，肿瘤微环境中或肿瘤细胞表面CD22的表达量上调的肿瘤。所述与CD22的蛋白表达相关的肿瘤可以是CD22阳性的肿瘤。在CD22阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的CD22的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。
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“药物组合物”通常指适合施用于受试者的化学或生物组合物。例如，所述药物组合物可以包括所述嵌合抗原受体，所述核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载剂。
在本申请中，术语“药学上可接受的载剂”通常是指不干扰活性成分的生物活性的有效性的一种或多种非毒性材料。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。
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。
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“CD22”通常是指人B淋巴细胞上与增殖和分化有关的重要膜抗原。人CD22的氨基酸序列可以见于UniProt/Swiss-Prot登录号P20273。本申请中，所述嵌合抗原受体(CAR)的其中一个抗原结合域靶向的抗原可以是CD22。
在本申请中，术语“CD19”通常是指B细胞表面的一种白细胞分化抗原。在本申请中，所述CD19可以包括人CD19。在本申请中，所述嵌合抗原受体的其中一个抗原结合域靶向的抗原可以是CD19。
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
嵌合抗原受体
一方面，本申请提供一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域包含靶向CD19的单链抗体(scFv)，所述第二抗原结合域包含靶向CD22的VHH。
另一方面，本申请提供一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域能够靶向CD19，所述第二抗原结合域能够靶向CD22，所述第二抗原结合域包含抗体重链可变区VHH中的至少一个CDR，且所述VHH包含SEQ ID NO:64所示的氨基酸序列。
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗 原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、North’s、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述的抗原结合蛋白通过IMGT编码系统定义。
在本申请中，所述嵌合抗原受体的第二抗原结合域靶向CD22。
在本申请中，所述嵌合抗原受体的第二抗原结合域可以包含靶向CD22的抗原结合蛋白。
在本申请中，所述嵌合抗原受体的第二抗原结合域可以包含抗体重链可变区中的至少一个CDR。例如，所述抗体重链可变区可包含SEQ ID NO:64所示的氨基酸序列。例如，所述抗体重链可变区可包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30的VHH序列中的至少一个CDR。
在本申请中，所述嵌合抗原受体的第二抗原结合域可包含重链可变区VH，所述VH可包含HCDR1，HCDR2和HCDR3中的至少一个、两个或三个。
在本申请中，所述嵌合抗原受体的第二抗原结合域可包含抗体或其抗原结合片段。
在本申请中，所述抗原结合片段可包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在本申请中，所述嵌合抗原受体的第二抗原结合域可以为VHH。
在本申请中，所述第二抗原结合域的HCDR3可包含SEQ ID NO:59所示的氨基酸序列。例如，所述第二抗原结合域的HCDR3序列可根据IMGT编码系统定义。
例如，所述第二抗原结合域包含HCDR3，所述HCDR3与SEQ ID NO:59所示的序列相比，在选自下述位置的氨基酸处存在氨基酸取代(例如，保守氨基酸取代等):X 15和X 17。
ATDPWTDCSLDGRYX 15YX 17Y(SEQ ID NO:59)，其中，X 15可以是E或R，X 17可以是D，G或N。
在本申请中，所述第二抗原结合域的HCDR3可包含SEQ ID NO:19、SEQ ID NO:20和 SEQ ID NO:21中任一项所示的氨基酸序列。
在本申请中，所述第二抗原结合域的HCDR2可包含SEQ ID NO:58所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR2序列可根据IMGT编码系统定义。例如，所述抗原结合蛋白包含HCDR2，所述HCDR2与SEQ ID NO:58所示的序列相比，在选自下述位置的氨基酸处存在氨基酸取代(例如，保守氨基酸取代等):X 3。
ISX 3RDGNT(SEQ ID NO:58)，其中，X 3可以是G或S。
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR2序列可根据IMGT编码系统定义。
在本申请中，所述第二抗原结合域的HCDR1可包含SEQ ID NO:57所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR1序列可根据IMGT编码系统定义。例如，所述抗原结合蛋白包含HCDR1，所述HCDR1与SEQ ID NO:57所示的序列相比，在选自下述位置的氨基酸处存在氨基酸取代(例如，保守氨基酸取代等):X 3，X 5和X 6。
GFX 3VX 5X 6YA(SEQ ID NO:57)，其中，X 3可以是P或S，X 5可以是A或D，X 6可以是D或G。
在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6中任一项所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR1序列可根据IMGT编码系统定义。
在本申请中，所述第二抗原结合域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:57所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:59所示的氨基酸序列。
在本申请中，所述第二抗原结合域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:11或SEQ ID NO:12中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21中任一项所示的氨基酸序列。
在本申请中，所述第二抗原结合域的所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:19所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体B010-B-22Nb-01或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。
在本申请中，所述第二抗原结合域的所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR3可包含SEQ ID  NO:20所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体B010-B-22Nb-02或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。例如，所述抗原结合蛋白可包括抗体B010-B-22Nb-03或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。
在本申请中，所述第二抗原结合域的所述HCDR1可包含SEQ ID NO:6所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体B010-B-22Nb-04、B010-B-22Nb-04-H4、B010-B-22Nb-04-H5、B010-B-22Nb-04-H6或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。
在本申请中，所述嵌合抗原受体的第二抗原结合域可包含框架区H-FR1，H-FR2，H-FR3和H-FR4。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:60所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR1与SEQ ID NO:60所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1和X 16。
X 1VQLVESGGGLVQPGX 16SLRLSCAAS(SEQ ID NO:60)，其中，X 1可以是A或E，X 16可以是G或R。
在本申请中，所述第二抗原结合域的H-FR1可包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中任一项所示的氨基酸序列。
在本申请中，所述第二抗原结合域的H-FR2可包含SEQ ID NO:61所示的氨基酸序列。例如，所述第二抗原结合域的H-FR2与SEQ ID NO:61所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1，X 4，X 11，X 12，X 14，X 15和X 17。
X 1AWX 4RQAPGKX 11X 12EX 14X 15SX 17(SEQ ID NO:61)，其中，X 1可以是I或M，X 4可以是F或V，X 11可以是E或G，X 12可以是L或R，X 14可以是G或W，X 15可以是I或V，X 17可以是C或Y。
在本申请中，所述第二抗原结合域的H-FR2可包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。
在本申请中，所述第二抗原结合域的H-FR3可包含SEQ ID NO:62所示的氨基酸序列。例如，所述第二抗原结合域的H-FR3与SEQ ID NO:62所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 2，X 3，X 7，X 12，X 20，X 21， X 27，X 29和X 30。
YX 2X 3DSVX 7GRFTX 12SRDNAKNX 20X 21YLQMNX 27LX 29X 30EDTAVYYC(SEQ ID NO:62)，其中，X 2可以是D或Y，X 3可以是A，Q或V，X 7可以是E或K，X 12可以是I或V，X 20可以是S或T，X 21可以是L或V，X 27可以是D或S，X 29可以是E，K或R，X 30可以是D或P。
在本申请中，所述第二抗原结合域的H-FR3可包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18中任一项所示的氨基酸序列。
在本申请中，所述第二抗原结合域的H-FR4可包含SEQ ID NO:63所示的氨基酸序列。例如，所述第二抗原结合域的H-FR4与SEQ ID NO:63所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 3和X 6。
WGX 3GTX 6VTVSS(SEQ ID NO:63)，其中，X 3可以是L或Q，X 6可以是L或Q。
在本申请中，所述第二抗原结合域的H-FR4可包含SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列。
在本申请中，所述第二抗原结合域可包含H-FR1，H-FR2，H-FR3和H-FR4。例如，所述H-FR1可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:61所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:62所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:63所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第二抗原结合域可包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中任一项所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18中任一项所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列。例如，所述第二抗原结合域可包括抗体B010-B-22Nb-01或与其具有相同H-FR1-4的抗原结合片段。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:2所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8所示的氨基酸序列，所述H-FR3包含SEQ ID NO: 14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列。例如，所述第二抗原结合域可包括抗体B010-B-22Nb-02或与其具有相同H-FR1-4的抗原结合片段。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:2所示的氨基酸序列，所述H-FR2包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列。例如，所述第二抗原结合域可包括抗体B010-B-22Nb-03或与其具有相同H-FR1-4的抗原结合片段。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:16所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列。例如，所述第二抗原结合域可包括抗体B010-B-22Nb-04或与其具有相同H-FR1-4的抗原结合片段。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。例如，所述第二抗原结合域可包括抗体B010-B-22Nb-04-H4或与其具有相同H-FR1-4的抗原结合片段。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。例如，所述第二抗原结合域可包括抗体B010-B-22Nb-04-H5或与其具有相同H-FR1-4的抗原结合片段。
例如，所述嵌合抗原受体的第二抗原结合域的所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体B010-B-22Nb-04-H6或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述第二抗原结合域可包含VHH，所述VHH可包含SEQ ID NO:64所示的氨基酸序列。例如，所述VHH可包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包括抗体或其抗原结合片段。例如，所述抗原结合片段可包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。例如，所述嵌合抗原受体的第一抗原结合域可以为scFv。
在本申请中，所述嵌合抗原受体的第一抗原结合域可靶向CD19蛋白。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含抗体重链可变区中的至少一个CDR。例如，所述抗体重链可变区可包含SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含HCDR3，例如，所述HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含HCDR2，例如，所述HCDR2可包含SEQ ID NO:32所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含HCDR1，例如，所述HCDR1可包含SEQ ID NO:31所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含HCDR1，HCDR2和HCDR3。例如，所述HCDR1包含SEQ ID NO:31所示的氨基酸序列，所述HCDR2包含SEQ ID NO:32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述第一抗原结合域可包含H-FR1，H-FR2，H-FR3和H-FR4。例如，所述H-FR1可包含SEQ ID NO:34所示的氨基酸序列。例如，所述H-FR2可包含SEQ ID NO:35所示的氨基酸序列。例如，所述H-FR3可包含SEQ ID NO:36所示的氨基酸序列。例如，所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。
在本申请中，所述第一抗原结合域可包含抗体重链可变区VH。例如，所述抗体重链可变区VH可包含SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含抗体轻链可变区中的至少一个CDR。例如，所述抗体轻链可变区可包含SEQ ID NO:45所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含LCDR3。例如，所述LCDR3可包含SEQ ID NO:40所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含LCDR2。例如，所述LCDR2可包含SEQ ID NO:39(HTS)所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含LCDR1。例如，所述LCDR1可包含SEQ ID NO:38。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含LCDR1，LCDR2和LCDR3。例如，所述LCDR1包含SEQ ID NO:38所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39(HTS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:40所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含L-FR1，L-FR2，L-FR3和L-FR4。例如，所述L-FR1可包含SEQ ID NO:41所示的氨基酸序列。例如，所述L-FR2可包 含SEQ ID NO:42所示的氨基酸序列。例如，所述L-FR3可包含SEQ ID NO:43所示的氨基酸序列。例如，所述L-FR4可包含SEQ ID NO:44所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含抗体轻链可变区VL，所述VL可包含SEQ ID NO:45所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可包含VH和VL。例如，所述VH包含SEQ ID NO:37所示的氨基酸序列，且所述VL包含SEQ ID NO:45所示的氨基酸序列。例如，所述VH和所述VL可以直接或间接相连。例如，所述VH和所述VL可以通过连接子相连。例如，所述连接子可以为肽接头。例如，所述连接子可包含SEQ ID NO:48所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的第一抗原结合域可以包含scFv。例如，所述scFv可包含SEQ ID NO:46所示的氨基酸序列。
在本申请中，所述第一抗原结合域可以和第二抗原结合域直接或间接相连。例如，所述间接相连可以包括通过连接子相连。例如，所述连接子可以为肽接头。例如，所述连接子可包含SEQ ID NO:47所示的氨基酸序列。
在本申请中，所述第一抗原结合域的C端可以与所述第二抗原结合域的N端直接或间接相连。
在本申请中，所述第一抗原结合域的N端可以与所述第二抗原结合域的C端直接或间接相连。
在本申请中，所述嵌合抗原受体可包含跨膜结构域。例如，所述跨膜结构域可以包含但不限于源自选自下述蛋白的跨膜结构域或其组合：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD5、ICOS、OX40、NKG2D、2B4、CD244、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，所述跨膜结构域可包括源自CD8的跨膜结构域。例如，所述跨膜结构域可包含SEQ ID NO:51所示的氨基酸序列。例如，所述跨膜结构域可包含与SEQ ID NO:51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，所述的嵌合抗原受体可包含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但不限于源自选自下述蛋白的共刺激信号传导结构域或其组合：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、 NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、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包括源自4-1BB的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如SEQ ID NO:52所示的氨基酸序列。例如，所述共刺激信号传导结构域可包含与SEQ ID NO:52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述蛋白的胞内信号传导结构域或其组合：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10和DAP-12。例如，所述胞内信号传导结构域可包括源自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包含如SEQ ID NO:53所示的氨基酸序列。例如，所述胞内信号传导结构域可包含与SEQ ID NO:53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，所述嵌合抗原受体可包含铰链区。例如，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。例如，所述铰链区为源自CD8的铰链区。例如，所述铰链区可包含SEQ ID NO:50所示的氨基酸序列。例如，所述铰链区可包含与SEQ ID NO:50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，所述嵌合抗原受体可以包含信号肽。例如，所述信号肽可包含SEQ ID NO:49所示的氨基酸序列。例如，所述信号肽的C端可以与所述抗原结合结构域的N端直接或间接相连。
例如，所述跨膜结构域的N端可以与所述抗原结合域的C端连接。例如，所述跨膜结构域的C端可以与所述共刺激信号传导结构域的N端连接。例如，所述共刺激信号传导结构域的C端可以与所述胞内信号传导结构域的N端连接。
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：信号肽、靶向CD22 的VHH、连接子1、靶向CD19的VH、连接子2、靶向CD19的VL、CD8铰链区、CD8跨膜区、4-1BB共刺激域以及CD3ζ胞内信号传导域。
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：信号肽、靶向CD19的VH、连接子2、靶向CD19的VL、连接子1、靶向CD22的VHH、CD8铰链区、CD8跨膜区、4-1BB共刺激域以及CD3ζ胞内信号传导域。
例如，所述连接子1可包含SEQ ID NO:47所示的氨基酸序列。例如，所述连接子2可包含SEQ ID NO:48所示的氨基酸序列。
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：本申请的抗原结合蛋白(例如，VHH)、源自CD8的跨膜结构域、源自CD137的共刺激信号传导结构域和源自CD3zeta的胞内信号传导结构域。
在本申请中，所述嵌合抗原受体可包含SEQ ID NO:55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:55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嵌合抗原受体。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嵌合抗原受体，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嵌合抗原受体的核酸，这些方法包括但不限于，采用逆转录PCR和PCR获得本申请所述嵌合抗原受体的核酸分子。
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嵌合抗原受体、一种或多种核酸分子和/或本申请所述的一种或多种载体。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、293F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。例如，所述细胞可包括T细胞。
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/或药学上可接受的载剂。在本申请中，所述药学上可接受的载剂可以包括缓冲剂、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。除非与本申请所述的细胞不相容，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。
试剂盒、用途和方法
另一方面，本申请提供了一种用于检测或测定CD22的方法，所述方法可包括使用所述分离的抗原结合蛋白或所述的多肽。
在本申请中，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。
例如，所述方法可包括用于非诊断目的的检测CD22的存在和/或含量的方法，其可包括下述步骤：
1)使样品与本申请的抗原结合蛋白接触；以及
2)检测样品结合的所述抗原结合蛋白的存在和/或含量来确定获自受试者的样品中CD22的存在和/或表达水平。
另一方面，本申请提供了一种CD22的试剂盒，其可包括使用所述分离的抗原结合蛋白或所述的多肽。
在本申请中，所述试剂盒还可包含使用说明，所述使用说明记载用于检测CD22的存在和/或含量的方法。例如，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。
另一方面，本申请提供了一种所述的分离的抗原结合蛋白或所述的多肽在制备试剂盒中的用途，所述试剂盒可用于检测CD22的存在和/或含量的方法。例如，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。
另一方面，本申请提供了分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、所述分离的核酸分子、所述的载体、所述的药物组合物用于预防、缓解和/或治疗疾病或病症。
另一方面，在本申请中，所述的试剂盒和/或所述的药物组合用于预防、缓解和/或治疗疾病或病症。
例如，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括与CD22的表达相关的肿瘤。术语“与CD22的表达相关的肿瘤”通常是指与正常细胞相比，所述肿瘤微环境中或肿瘤细胞表面CD22的表达改变。例如，所述“与CD22的表达相关的肿瘤”可以是与正常细胞相比，肿瘤微环境中或肿瘤细胞表面CD22的表达量上调的肿瘤。所述与CD22的蛋白表达相关的肿瘤可以是CD22阳性的肿瘤。在CD22阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的CD22的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。
例如，所述肿瘤可以包括血液瘤。例如，所述肿瘤可包括淋巴瘤。例如，所述肿瘤可包括白血病。
另一方面，本申请提供了一种所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、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，所述药物用于预防、缓解和/或治疗疾病或病症。
另一方面，本申请提供了一种药物组合在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病或病症。
例如，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括与CD22的表达相关的肿瘤。术语“与CD22的表达相关的肿瘤”通常是指与正常细胞相比，所述肿瘤微环境中或肿瘤细胞表面CD22的表达改变。例如，所述“与CD22的表达相关的肿瘤”可以是与正常细胞相比，肿瘤微环境中或肿瘤细胞表面CD22的表达量上调的肿瘤。所述与CD22蛋白表达相关的肿瘤可以是CD22阳性的肿瘤。在CD22阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的CD22的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。
例如，所述肿瘤可以包括血液瘤。例如，所述肿瘤可包括淋巴瘤。例如，所述肿瘤可包括白血病。
另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用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、所述的药物组合物。
例如，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括与CD22的表达相关的肿瘤。术语“与CD22的表达相关的肿瘤”通常是指与正常细胞相比，所述肿瘤微环境中或肿瘤细胞表面CD22的表达改变。例如，所述“与CD22的表达相关的肿瘤”可以是与正常细胞相比，肿瘤微环境中或肿瘤细胞表面CD22的表达量上调的肿瘤。所述与CD22蛋白表达相关的肿瘤可以是CD22阳性的肿瘤。在CD22阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的CD22的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。
例如，所述肿瘤可以包括血液瘤。例如，所述肿瘤可包括淋巴瘤。例如，所述肿瘤可包括白血病。
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。
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发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1 羊驼免疫
采用重组人CD22蛋白(11958-H02，北京义翘神州科技股份有限公司)免疫一只健康的雌性成年羊驼(Alpaca)。首次免疫将500μg重组人CD22蛋白与等体积弗氏完全佐剂乳化后颈部淋巴结附近左右两侧注射；加强免疫采用500μg重组人CD22蛋白与等体积弗氏完全或不完全佐剂乳化后颈部淋巴结附近左右两侧注射，并进行几次加强免疫，末次免疫一周后采 血，监测抗血清效价。
实施例2 羊驼免疫库构建
免疫结束后，收集羊驼外周血，采用淋巴细胞分离液分离得到淋巴细胞；采用TRIzol TM试剂提取总RNA；采用PrimeScript TMII第一链cDNA合成试剂盒反转录得到cDNA，巢式PCR扩增VHH基因。采用胶纯化试剂盒回收VHH基因片段后采用限制性内切酶Bgl 1酶切消化，随后将其克隆至噬菌粒载体pADL-10b，将构建好的克隆产物转化至E.coli TG1电转化感受态细胞，构建VHH基因文库；采用平板梯度稀释法测定库容量为2.2×10 9pfu，菌落PCR结果显示文库插入率为97.9％。从上述基因文库中取10-100倍库容量的活细胞进行接种培养，培养至对数期后采用M13K07噬菌体进行救援，救援培养后，离心收集噬菌体，采用PEG-NaCl纯化噬菌体，即得噬菌体展示文库，可直接用于后续筛选。
实施例3 噬菌体展示筛选本申请的CD22 VHH
将重组人CD22(11958-H08H1-B，北京义翘神州科技股份有限公司)和重组猴CD22(90246-C08H，北京义翘神州科技股份有限公司)包被到96孔板中，通过Elisa实验筛选3-5轮，将CD22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4株亲和力较高的靶向人CD22的单域抗体(sdAb)。这4株抗体分别命名为B010-B-22Nb-01、B010-B-22Nb-02、B010-B-22Nb-03和B010-B-22Nb-04。
实施例4 抗体的人源化设计
为了降低驼源抗体的免疫原性，任选地可以将已筛选出的有生物学活性的抗体进行人源化。驼源单克隆抗体的人源化根据本领域许多文献公示的方法进行。简言之，可以使用人抗体恒定结构域替代亲本(驼源抗体)恒定结构域，根据驼源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。然后可以以驼源抗体的三维结构为基础，通过VH的氨基酸残基进行回复突变，将驼源抗体的恒定区替换为人恒定区，得到最终的人源化结合蛋白B010-B-22Nb-04-H4、B010-B-22Nb-04-H5和B010-B-22Nb-04-H6。
表1为本申请CD22 VHH的CDR、重链可变区VH、FR。
表1本申请CD22 VHH的CDR、重链可变区VH、FR
Figure PCTCN2022126207-appb-000001
实施例5 CD22 VHH的结合亲和力
用Biacore检测不同CD22抗原结合蛋白与抗原(CD22蛋白，来源:北京义翘神州科技股份有限公司，货号:11958-H08H1-B)蛋白的结合亲和性。
混合100mL 10×HBS-EP+缓冲液和900mL Milli-Q水，得到1L 1×HBS-EP+缓冲液。
用1:1混合的50mM NHS和200mM EDC(NHS和EDC来自氨基偶联试剂盒)活化CM5芯片1-8通道的表面，流速为10μL/min，时间420秒。以10μL/min流速注射Anti-hFc或抗小鼠Fc抗体(稀释在pH4.5的醋酸钠溶液中，浓度20μg/mL)200秒，最后用1M乙醇胺盐酸盐(pH8.5)封闭芯片上多余的有活性的羧基。用1×HBS-EP+以10μL/min流速，冲洗芯片表面2小时，以稳定基线，仪器设定温度为25℃。
初始循环，由测样和再生两步组成，测量前重复3次，以稳定基线。
测样：以30μL/min流速向1-8通道注射1×HBS-EP+缓冲液120秒，解离60秒。
再生：向1-8通道注射10mM甘氨酸pH1.5，30μL/min，30秒，稳定30秒。
结合动力学参数测定的实验步骤:动力学测定的运行缓冲液是1×HBS-EP+(pH7.4)溶液。捕获：在Anti-hFc或抗小鼠Fc芯片第1-8通道的测试通道分别注射不同抗体，流速10μL/min，60s，进行捕获。用1×HBS-EP+(pH 7.4)将抗原CD22蛋白，(来源:北京义翘神州科技股份有限公司，货号:11958-H08H1-B)稀释至100nM。测样：以30μL/min流速向1-8通道注射，1个0浓度样品用于去除背景信号；抗原抗体的结合和解离时间分别为180和400秒。再生：以30μL/min流速向1-8通道注射10mM甘氨酸pH1.5，30秒，然后稳定60秒。使用Biacore8K分析软件计算每个本申请抗原结合蛋白的平衡解离常数(K D值)。参比通道(FC1)用于背景的扣减。
以上试验表明，本申请CD22 VHH具有与抗原的结合亲和力，SPH-B010-B-22PC-1(百英生物公司，货号B179301)为阳性对照抗体。
如表2所示，用Biacore检测不同抗原结合蛋白与抗原蛋白的结合亲和性。
表2不同CD22 VHH与抗原蛋白的结合亲和性
Figure PCTCN2022126207-appb-000002
实施例6 本申请的CD22 VHH与表达于细胞表面的人CD22的结合
基于流式细胞术测定法测定CD22抗原结合蛋白与表达于K562细胞表面的人CD22的结合能力。通过比较本申请不同CD22抗原结合蛋白与表达于K562细胞表面的人CD22的结合曲线来测定其结合能力。
6.1为了筛选人CD22结合蛋白，对K562细胞进行了基因改造，使其过量表达人源CD22，该细胞被命名为K562-hCD22细胞。将K562-hCD22细胞消化，铺于96孔板中。
6.2用含2％FBS的PBS分别配制阳性对照抗体(SPH-B010-B-22PC-1，百英生物，货号B179301)和本申请抗原结合蛋白抗体最大浓度为15μg/ml，3倍稀释，8个点，稀释好的样品加入96孔板中，4摄氏度孵育1小时。
6.3用含2％FBS的PBS洗板3次。
6.4将PE标记的-人IgG(Biolegend,货号410708)分别用2％FBS的PBS按产品说明书比例稀释，稀释好的样品加入96孔板中，4摄氏度孵育0.5小时。
6.5用含2％FBS的PBS洗板2次。
6.6用2％FBS的PBS重新悬浮细胞，使用流式细胞仪测定PE通道的中位荧光度值(MFI)。
在以上试验表明，本申请的抗原结合蛋白具有与K562细胞表面的人CD22的结合活性，并且优于阳性对照抗体。
如图1所示，基于流式细胞术测定本申请的CD22 VHH与K562细胞表面的人CD22的结合曲线。
实施例7 CD19/CD22双特异性CAR分子及其对应的单靶特异CAR分子CD19 CAR、CD22CAR质粒构建和慢病毒包装
基因合成人源化的靶向CD19/CD22嵌合抗原受体分子的基因序列具体包括：靶向人CD22的单域抗体VHH序列、Linker1、靶向人CD19的抗体VH序列、Linker2、靶向人CD19的抗体VL序列、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。获得的嵌合抗原受体分子(CAR分子)命名为B010-B-03(氨基酸序列如SEQ ID NO:55所示)。基因合成人源化的靶向CD22嵌合抗原受体分子的基因序列具体包括：靶向人CD22的单域抗体VHH序列、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，获得的嵌合抗原受体分子(CAR分子)命名为B010-B-22(氨基酸序列如SEQ ID NO:54所示)。基因合成人源化的靶向CD19嵌合抗原受体分子的基因序列具体包括：靶向人CD19的抗体VH序列、Linker2、 靶向人CD19的抗体VL序列、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，获得的嵌合抗原受体分子(CAR分子)命名为B010-B-19(氨基酸序列如SEQ ID NO:56所示)。嵌合抗原受体基因序列合成后克隆至pLVX-EF1a-IRES-Puro载体上EcoRI和BamHI两个酶切位点之间。将构建好的慢病毒核心质粒和包装质粒pMDLg，调节质粒pRSV和包膜蛋白质粒pMD2.G一起用PEI转染试剂转染293T细胞，培养一定时间后收集病毒并对病毒进行浓缩收集备用。
实施例8 制备CAR-T细胞
收集人外周血T淋巴细胞，用实施例1中制备的慢病毒的转染T淋巴细胞，制备得到CAR-T细胞，分别命名为B010-B-03、B010-B-19、B010-B-22 CART；同时采用无关序列制备载体包装获得的病毒转染后的T淋巴细胞作为对照组定义为NT-T。
1)取12孔板，配制含有抗CD3、抗CD28以及Retronectin抗体混合液，抗体混合液包被，室温孵育2h及以上。
2)复苏冻存的PBMC细胞1支。使用T细胞培养基洗一次，按照试剂盒(MojoSort TM人CD3T细胞分离试剂盒，biolegend，480022)使用说明实施分选步骤以获得CD3+T细胞。
3)取孵育完毕的12孔板，用PBS溶液清洗孔板，分选纯化T细胞液按照1E6/孔加入孔板中，补充T细胞培养基至1ml/孔，放入37℃，5％CO 2培养箱进行激活24小时。
4)取12孔板，加病毒，放入37℃，5％CO 2培养箱进行培养24h。补充T细胞培养基1ml/孔，放入37℃，5％CO 2培养箱进行培养48小时及以上。转移CART细胞悬液至T25培养瓶中，补充T细胞培养基，之后扩大培养获得所需的CART细胞。
5)转染慢病毒并扩大培养至第8天，将CART细胞冻存，液氮储存，用以冻存复苏后CART体内外杀伤数据分析验证。
6)留少量CART细胞继续培养用以后续评价。
实施例9 检测CART细胞阳性率，与抗原结合能力及记忆性群体比例
收集实施例2中感染慢病毒平扩大培养至第6天、第8天、第10天、第12天的人源化B010-B-03、B010-B-19、B010-B-22 CART细胞和NT-T细胞。
1)在相应标记的96孔圆底板内加入对应的细胞悬液，离心400xg，5分钟，室温。
2)弃上清，样本管加入Biotin-Protein L蛋白(1ug/test)或Biotin-CD19蛋白(1ug/test)或Biotin-CD22蛋白(1ug/test)，震荡96孔圆底板使充分混匀，放入4℃，避光孵育30分钟。
3)取出96孔圆底板，加入PBS溶液清洗，离心400xg，5分钟，室温。
4)重复步骤3)。
5)弃上清，加入混合抗体液(PE-Streptavidin+BV421-CD197+APC-CD62L+APC-Cy7-CD45RO)或PE-Streptavidin，震荡96孔圆底板使充分混匀，放入4℃，避光孵育30分钟。
6)取出96孔圆底板，加入PBS溶液清洗，离心400xg，5min，RT。
7)重复步骤6)。
8)弃上清，加入PBS溶液，上机检测。
结果：B010-B-03，B010-B-19 CART均能与Protein L蛋白和CD19蛋白结合，B010-B-03，B010-B-22 CART均能与CD22蛋白结合。以Protein L和VHH CAR结构以CD22蛋白亲和比例作为CART细胞阳性率，CD19和CD22结合为CART细胞与抗原亲和力，TCM(Central Memory T cell，中央记忆型T细胞，CD45RO+/CD197+/CD62L+cells)为CART细胞记忆性群体。B010-B-03，B010-B-19，B010-B-22 CART细胞阳性率及亲和力均较高且稳定，其中双靶点CART阳性率及亲和力均不低于单靶点CART，记忆性群体比例双靶点CART呈上升趋势，单靶点CART在Day8出现峰值(表3、图2、图3、图4)。
表3 CD19阳性细胞、CD22阳性细胞以及中央型T细胞比例检测
Figure PCTCN2022126207-appb-000003
实施例10 检测CART细胞对靶细胞的短效杀伤作用
对Nalm6转入萤火虫荧光素酶(Luciferase，简写为LUC)，获得的Nalm6-LUC细胞。使用Cas9基因编辑敲除技术，通过电转方式将Cas9蛋白和特异sgRNA转入Nalm6-LUC细胞株中，获得Nalm6-CD19 -CD22 +-LUC和Nalm6-CD19 +CD22 --LUC，将实施例8中冻存的CART细胞，复苏并恢复培养4天，以CART：Nalm6-LUC/Nalm6单阳1:1混合/Nalm6野生型单阳(8:1:1)混合细胞株＝10:1，5:1，2.5:1，1.25:1的不同比例，将CART细胞分别与3种Nalm6细胞模型混合和共孵育，作为试验组；对照组为NT-T。
在孵育4小时后通过酶标仪进行体外杀伤检测，比较不同效靶比下，CART细胞对3种Nalm6细胞模型的杀伤作用，并与NT-T细胞进行比较。
结果：B010-B-03相较于B010-B-19，B010-B-22 CART细胞对3种Nalm6靶细胞模型短效杀伤作用更好(图5、图6、图7)。
实施例11 检测CART细胞受靶细胞激活分泌细胞因子水平实验
将实施例10中的细胞，以CART：Nalm6-LUC/Nalm6单阳1:1混合/Nalm6野生型单阳(8:1:1)混合细胞株＝1.25:1的比例，同时设置相应的CART细胞空白孔，将CART细胞分别与3种Nalm6细胞模型混合和共孵育，作为试验组；对照组为NT-T。
在共孵育杀伤激活16-24小时后，离心400xg，5分钟，4℃，取上清。按照hu TH1/TH2CBA cytokines kit II试剂盒(供应商：BD Biosciences，货号：551809)使用说明实施细胞因子检测实验，细胞上清液稀释5倍，使用流式仪(Beckman Cytoflex)检测分析数据。比较B010-B-03，B010-B-19，B010-B-22 CART细胞被3种Nalm6细胞模型的激活后分泌细胞因子水平，并与NT-T细胞进行比较。
结果：B010-B-03CART对于3种Nalm6靶细胞模型被激活分泌促肿瘤细胞杀伤的细胞因子IL-2，IFN-γ，TNF-α水平均高于B010-B-19，B010-B-22 CART(图8)。
实施例12 CAR-T细胞Nalm6-LUC模型体内抑瘤实验
1.小鼠荷瘤模型构建
使用自行构建的表达有萤光素酶的靶肿瘤细胞Nalm6-LUC，接种于NSG小鼠(购自上海南方模式生物科技有限公司)，尾静脉脉注射成瘤。将2.0×10 5上述靶细胞重悬于200μl无血清培养液中，小鼠尾静脉注射。
2.活体成像
Nalm6-LUC小鼠模型成瘤3天后，在小鼠腹膜内注射萤光素酶底物D-Luciferin，注射用 量为3mg/只，将小鼠麻醉后置于小动物活体成像仪中成像。将B010-B-03，B010-B-19，B010-B-22 CART细胞用无血清培养液重悬，调整细胞密度为2.5×10 7个/ml。每只小鼠尾静脉注射5×10 6个CART细胞，共200μl/只。设置使用未转染CAR的T细胞处理的小鼠为对照组。实验完成后，每周进行两次定期活体成像检测，收集肿瘤消除效果。
3.肿瘤生物荧光测量
注射CAR-T细胞后，每周测量生物荧光两次，以注射细胞当天为第0天。
4.小鼠体重测量
注射CAR-T细胞后，每周测量体重两次，以注射细胞当天为第0天。
结果：图9显示的Nalm6-LUC小鼠活体成像结果，成像结果体现肿瘤细胞的数量。结果显示，与生物荧光强度不断增强的未转染CAR的T细胞组及单双靶点CART细胞组间比较，双靶点CART给药组和单靶点CD19 CART给药组在Day15之前肿瘤细胞数量显著减少，但CD19 CART给药组在Day15之后出现肿瘤细胞复发增长情况，双靶点CART给药组未复发增长，在给药后第32天抑瘤率为100.0％。(图10)
图11显示的是Nalm6-LUC小鼠体重变化结果，结果显示给药后小鼠体重波动稳定，且治疗期间均无动物死亡，没有表现明显的药物毒性，药物安全性及耐受良好。
实施例13 CAR-T细胞Nalm6-LUC复发模型体内抑瘤实验
1.小鼠荷瘤模型构建
使用自行构建的表达有萤光素酶的靶肿瘤细胞Nalm6-LUC，Nalm6-CD19 -CD22 +-LUC和Nalm6-CD19 +CD22 --LUC，按照复发模型理论比例Nalm6-LUC：Nalm6-CD19 -CD22 +-LUC：Nalm6-CD19 +CD22 --LUC＝8:1:1混合，定义为Nalm6-MIX-LUC，使用流式仪(Beckman Cytoflex)检测鉴定，比例误差在±10％内可接受，将Nalm6-MIX-LUC接种于NSG小鼠(购自上海南方模式生物科技股份有限公司)，尾静脉脉注射成瘤。将2.0×10 5上述靶细胞重悬于200μl无血清培养液中，小鼠尾静脉注射。
2.活体成像
Nalm6-MIX-LUC小鼠模型成瘤3天后，在小鼠腹膜内注射萤光素酶底物D-Luciferin，注射用量为3mg/只，将小鼠麻醉后置于小动物活体成像仪中成像。将B010-B-03，B010-B-19，B010-B-22 CART细胞用无血清培养液重悬，调整细胞密度为2.5×10 7个/ml。每只小鼠尾静脉注射5×10 6个CART细胞，共200μl/只。设置使用未转染CAR的T细胞处理的小鼠为对照组。实验完成后，每周进行两次定期活体成像检测，收集肿瘤消除效果。
3.肿瘤生物荧光测量
注射CAR-T细胞后，每周测量生物荧光两次，以注射细胞当天为第0天。
4.小鼠体重测量
注射CAR-T细胞后，每周测量体重两次，以注射细胞当天为第0天。
结果：图12显示的Nalm6-MIX-LUC小鼠活体成像结果，成像结果体现肿瘤细胞的数量。结果显示，与生物荧光强度不断增强的未转染CAR的T细胞组及单双靶点CART细胞组间比较，双靶点CART给药组肿瘤细胞数量显著减少且持续稳定未见肿瘤细胞复发增长，在给药后第32天抑瘤率为100.0％。(图13)
图14显示的是Nalm6-MIX-LUC小鼠体重变化结果，结果显示给药后小鼠体重波动稳定，且治疗期间均无动物死亡，没有表现明显的药物毒性，药物安全性及耐受良好。
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 (90)

  1. 嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域包含靶向CD19的单链抗体(scFv)，所述第二抗原结合域包含靶向CD22的VHH。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述第二抗原结合域包含抗体重链可变区VHH中的至少一个CDR，且所述VHH包含SEQ ID NO:64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，其中所述第二抗原结合域包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30的VHH序列中的至少一个CDR。
  4. 嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域能够靶向CD19，所述第二抗原结合域能够靶向CD22，所述第二抗原结合域包含抗体重链可变区VHH中的至少一个CDR，且所述VHH包含SEQ ID NO:64所示的氨基酸序列。
  5. 根据权利要求4所述的嵌合抗原受体，其中所述VHH包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30中任一项所示的氨基酸序列。
  6. 根据权利要求4-5中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含抗体或其抗原结合片段。
  7. 根据权利要求4-6中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含抗体或其抗原结合片段。
  8. 根据权利要求7所述的嵌合抗原受体，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  9. 根据权利要求4-8中任一项所述的嵌合抗原受体，其中所述第一抗原结合域为scFv。
  10. 根据权利要求4-9中任一项所述的嵌合抗原受体，其中所述第二抗原结合域为VHH。
  11. 根据权利要求1-10中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列。
  12. 根据权利要求11所述的嵌合抗原受体，其中所述第二抗原结合域的HCDR3包含SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21中任一项所示的氨基酸序列。
  13. 根据权利要求1-12中人任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:58所示的氨基酸序列。
  14. 根据权利要求13所述的嵌合抗原受体，其中所述第二抗原结合域的HCDR2包含SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所示的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:57所示的氨基酸序列。
  16. 根据权利要求15所述的嵌合抗原受体，其中所述第二抗原结合域的HCDR1包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6中任一项所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:57所示的氨基酸序列，所述HCDR2包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11或SEQ ID NO:12中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下组的氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:19所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
    (3)所述HCDR1包含SEQ ID NO:6所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:21所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:60所示的氨基酸序列。
  21. 根据权利要求20所述的嵌合抗原受体，其中所述第二抗原结合域的H-FR1包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:61所示的氨基酸序列。
  23. 根据权利要求22所述的嵌合抗原受体，其中所述第二抗原结合域的H-FR2包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:62所示的氨基酸序列。
  25. 根据权利要求24所述的嵌合抗原受体，其中所述第二抗原结合域的H-FR3包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18中任一项所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:63所示的氨基酸序列。
  27. 根据权利要求26所述的嵌合抗原受体，其中所述第二抗原结合域的H-FR4包含SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:60所示的氨基酸序列，所述H-FR2包含SEQ ID NO:61所示的氨基酸序列，所述H-FR3包含SEQ ID NO:62所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:63所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18中任一项所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组的氨基酸序列：
    1)所述H-FR1包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
    2)所述H-FR1包含SEQ ID NO:2所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8 所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
    3)所述H-FR1包含SEQ ID NO:2所示的氨基酸序列，所述H-FR2包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
    4)所述H-FR1包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:16所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:22所示的氨基酸序列；
    5)所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列；
    6)所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列；以及
    7)所述H-FR1包含SEQ ID NO:3所示的氨基酸序列，所述H-FR2包含SEQ ID NO:7所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含VHH，所述VHH包含SEQ ID NO:64所示的氨基酸序列。
  32. 根据权利要求31所述的嵌合抗原受体，其中所述第二抗原结合域的VHH包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30中任一项所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:31所示的氨基酸序列，所述HCDR2包含SEQ ID NO:32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3包含SEQ ID NO:36所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:23所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含VH， 所述VH包含SEQ ID NO:37所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:38所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39(HTS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:40所示的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:41所示的氨基酸序列，所述L-FR2包含SEQ ID NO:42所示的氨基酸序列，所述L-FR3包含SEQ ID NO:43所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:44所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含VL，所述VL包含SEQ ID NO:45所示的氨基酸序列。
  39. 根据权利要求1-38中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含VH和VL，所述VH包含SEQ ID NO:37所示的氨基酸序列，所述VL包含SEQ ID NO:45所示的氨基酸序列。
  40. 根据权利要求38-39中任一项所述的嵌合抗原受体，其中所述VH和VL通过连接子连接。
  41. 根据权利要求40所述的嵌合抗原受体，其中所述连接子包含SEQ ID NO:48所示的氨基酸序列。
  42. 根据权利要求1-41中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含scFv，所述scFv包含SEQ ID NO:46所示的氨基酸序列。
  43. 根据权利要求1-42中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和第二抗原结合域直接或间接相连。
  44. 根据权利要求1-43中任一项所述的嵌合抗原受体，其中所述第一抗原结合域的C端与所述第二抗原结合域的N端直接或间接相连。
  45. 根据权利要求1-44中任一项所述的嵌合抗原受体，其中所述第一抗原结合域的N端与所述第二抗原结合域的C端直接或间接相连。
  46. 根据权利要求1-45中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和所述第二抗原结合域通过连接子相连。
  47. 根据权利要求46所述的嵌合抗原受体，其中所述连接子包含肽接头。
  48. 根据权利要求46-47中任一项所述的嵌合抗原受体，其中所述连接子包含SEQ ID NO:47所示的氨基酸序列。
  49. 根据权利要求1-48中任一项所述的嵌合抗原受体，其还包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  50. 根据权利要求49所述的嵌合抗原受体，其中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
  51. 根据权利要求49-50中任一项所述的嵌合抗原受体，其中所述共刺激信号区域包含SEQ ID NO:52所示的氨基酸序列。
  52. 根据权利要求1-51中任一项所述的嵌合抗原受体，其包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
  53. 根据权利要求52所述的嵌合抗原受体，其中所述胞内信号传导域为源自CD3ζ的信号传导结构域。
  54. 根据权利要求52-53中任一项所述的嵌合抗原受体，其中所述胞内信号传导域包含SEQ ID NO:53所示的氨基酸序列。
  55. 根据权利要求1-54中任一项所述的嵌合抗原受体，其包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、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。
  56. 根据权利要求55所述的嵌合抗原受体，其中所述跨膜区为源自CD8的跨膜区。
  57. 根据权利要求55-56中任一项所述的嵌合抗原受体，其中所述跨膜区包含SEQ ID NO:51所示的氨基酸序列。
  58. 根据权利要求1-57中任一项所述的嵌合抗原受体，其还包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、 BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  59. 根据权利要求58所述的嵌合抗原受体，其中所述铰链区为源自CD8的铰链区。
  60. 根据权利要求58-59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:50所示的氨基酸序列。
  61. 根据权利要求1-60中任一项所述的嵌合抗原受体，其还包含信号肽。
  62. 根据权利要求61所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:49所示的氨基酸序列。
  63. 根据权利要求1-62中任一项所述的嵌合抗原受体，其从N端到C端依次包含：信号肽、第二抗原结合域、连接子、第一抗原结合域、铰链区、跨膜区、共刺激域以及胞内信号传导域。
  64. 根据权利要求1-63中任一项所述的嵌合抗原受体，其从N端到C端依次包含：信号肽、靶向CD22的VHH、连接子1、靶向CD19的VH、连接子2、靶向CD19的VL、CD8铰链区、CD8跨膜区、4-1BB共刺激域以及CD3ζ胞内信号传导域。
  65. 根据权利要求1-64中任一项所述的嵌合抗原受体，其包含SEQ ID NO:55所示的氨基酸序列。
  66. 分离的一种或多种核酸分子，其编码权利要求1-65中任一项所述的嵌合抗原受体。
  67. 载体，其包含权利要求66所述的核酸分子。
  68. 细胞，其包含权利要求1-65中任一项所述的嵌合抗原受体，权利要求66所述的核酸分子，和/或权利要求67所述的载体。
  69. 根据权利要求68所述的细胞，其包括免疫细胞。
  70. 根据权利要求69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。
  71. 根据权利要求68-70中任一项所述的细胞，其包括T细胞。
  72. 制备权利要求1-65中任一项所述的嵌合抗原受体的方法，所述方法包括在使得所述嵌合抗原受体表达的条件下，培养权利要求68-71中任一项所述的细胞。
  73. 药物组合物，其包含权利要求1-65中任一项所述的嵌合抗原受体，权利要求66所述的核酸分子，权利要求67所述的载体，权利要求68-71中任一项所述的细胞，和/或药学上可接受的载剂。
  74. 一种用于检测CD19和/或CD22蛋白的方法，其包括：施用权利要68-71中任一项所述的细胞。
  75. 一种CD19和/或CD22蛋白检测的试剂盒，其包含1-65中任一项所述的嵌合抗原受体，和/或权利要求68-71中任一项所述的细胞。
  76. 权利要求1-65中任一项所述的嵌合抗原受体，权利要求66所述的核酸分子，权利要求67所述的载体，权利要求68-71中任一项所述的细胞，和/或权利要求73所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  77. 根据权利要求76所述的用途，其中所述疾病和/或病症包括与CD19和/或CD22表达相关的疾病和/或病症。
  78. 根据权利要求76-77中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  79. 根据权利要求78所述的用途，其中所述肿瘤包括血液瘤。
  80. 根据权利要求78-79中任一项所述的用途，其中所述肿瘤选自下组：白血病和淋巴瘤。
  81. 一种预防和/或治疗疾病和/或病症的方法，其包括向有需要的受试者施用权利要求1-65中任一项所述的嵌合抗原受体，权利要求66所述的核酸分子，权利要求67所述的载体，权利要求68-71中任一项所述的细胞，和/或权利要求73所述的药物组合物。
  82. 根据权利要求81所述的方法，其中所述疾病和/或病症包括与CD19和/或CD22表达相关的疾病和/或病症。
  83. 根据权利要求81-82中任一项所述的方法，其中所述疾病和/或病症包括肿瘤。
  84. 根据权利要求83所述的方法，其中所述肿瘤包括血液瘤。
  85. 根据权利要求83-84中任一项所述的方法，其中所述肿瘤选自下组：白血病和淋巴瘤。
  86. 权利要求1-65中任一项所述的嵌合抗原受体，权利要求66所述的核酸分子，权利要求67所述的载体，权利要求68-71中任一项所述的细胞，和/或权利要求73所述的药物组合物，其用于预防和/或治疗疾病和/或病症。
  87. 根据权利要求86所述的嵌合抗原受体，所述核酸分子，所述载体和/或所述细胞，其中所述疾病和/或病症包括与CD19和/或CD22表达相关的疾病和/或病症。
  88. 根据权利要求86-87中任一项所述的嵌合抗原受体，所述核酸分子，所述载体和/或所述细胞，其中所述疾病和/或病症包括肿瘤。
  89. 根据权利要求88所述的嵌合抗原受体，所述核酸分子，所述载体和/或所述细胞，其中所述肿瘤包括血液瘤。
  90. 根据权利要求88-89中任一项所述的嵌合抗原受体，所述核酸分子，所述载体和/或所述细胞，其中所述肿瘤选自下组：白血病和淋巴瘤。
PCT/CN2022/126207 2022-10-19 2022-10-19 靶向cd19和cd22的双特异性嵌合抗原受体 WO2024082178A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/126207 WO2024082178A1 (zh) 2022-10-19 2022-10-19 靶向cd19和cd22的双特异性嵌合抗原受体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/126207 WO2024082178A1 (zh) 2022-10-19 2022-10-19 靶向cd19和cd22的双特异性嵌合抗原受体

Publications (1)

Publication Number Publication Date
WO2024082178A1 true WO2024082178A1 (zh) 2024-04-25

Family

ID=90736675

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/126207 WO2024082178A1 (zh) 2022-10-19 2022-10-19 靶向cd19和cd22的双特异性嵌合抗原受体

Country Status (1)

Country Link
WO (1) WO2024082178A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107383196A (zh) * 2017-08-30 2017-11-24 广州百暨基因科技有限公司 人源化抗cd19的抗原结合片段
CN110938148A (zh) * 2018-09-25 2020-03-31 上海恒润达生生物科技有限公司 靶向cd19和cd22嵌合抗原受体
CN112195157A (zh) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 Cd19和cd22双靶点嵌合抗原受体t细胞及其应用
CN113493516A (zh) * 2020-04-02 2021-10-12 重庆精准生物技术有限公司 靶向双特异性位点的嵌合抗原受体及其应用
CN114478803A (zh) * 2022-02-11 2022-05-13 北京大学深圳研究生院 一种新型双特异性嵌合抗原受体的构建及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107383196A (zh) * 2017-08-30 2017-11-24 广州百暨基因科技有限公司 人源化抗cd19的抗原结合片段
CN110938148A (zh) * 2018-09-25 2020-03-31 上海恒润达生生物科技有限公司 靶向cd19和cd22嵌合抗原受体
CN113493516A (zh) * 2020-04-02 2021-10-12 重庆精准生物技术有限公司 靶向双特异性位点的嵌合抗原受体及其应用
CN112195157A (zh) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 Cd19和cd22双靶点嵌合抗原受体t细胞及其应用
CN114478803A (zh) * 2022-02-11 2022-05-13 北京大学深圳研究生院 一种新型双特异性嵌合抗原受体的构建及其应用

Similar Documents

Publication Publication Date Title
JP7280828B2 (ja) Ｂｃｍａを標的とする抗体およびその使用
CN107406517B (zh) 包含cd19结合域的嵌合抗原受体(car)
CN113039205B (zh) 靶向cll1的抗体及其应用
CN113597433A (zh) Gprc5d嵌合抗原受体以及表达这些受体的细胞
CN109311999A (zh) 基于单结构域抗体的嵌合抗原受体及其使用方法
CN113412117A (zh) 嵌合抗原和t细胞受体及使用的方法
JP2022513383A (ja) 二重特異性抗体及びその作製方法と使用
CN111848809A (zh) 靶向Claudin18.2的CAR分子、其修饰的免疫细胞及用途
KR102316091B1 (ko) Bcma를 표적으로 하는 키메라 항원 수용체 및 이의 용도
CN111454358A (zh) 一种嵌合抗原受体及其应用
WO2022206975A1 (zh) Cldn18.2抗原结合蛋白及其用途
WO2023093811A1 (zh) 分子开关调控型嵌合抗原受体细胞和抗体的组合及其应用
CN113195542A (zh) Cd30-结合部分、嵌合抗原受体及其用途
WO2022116952A1 (zh) 靶向cd70的抗原结合蛋白及其应用
WO2023104099A1 (zh) 靶向bcma的p329g抗体及其与嵌合抗原受体细胞的组合和应用
WO2022206976A1 (zh) 靶向cldn18.2的抗原结合蛋白及其用途
CN116284389A (zh) 抗afp/hla02 tcr样抗体及其用途
WO2024082178A1 (zh) 靶向cd19和cd22的双特异性嵌合抗原受体
TW202229358A (zh) 前列腺癌嵌合抗原受體
WO2023016554A1 (zh) 靶向cd22的抗原结合蛋白及其用途
WO2024103251A1 (zh) 抗afp/hla02 tcr样抗体及其用途
CN114933654B (zh) 靶向cd123的抗体、嵌合抗原受体及其用途
CN115322257B (zh) Bcma靶向抗体、嵌合抗原受体及其应用
WO2022258011A1 (zh) 抗pd-1人源化抗体或其抗原结合片段及其应用
WO2024140641A1 (zh) 靶向ror1的抗原结合蛋白

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22962368

Country of ref document: EP

Kind code of ref document: A1