WO2023016554A1 - 靶向cd22的抗原结合蛋白及其用途 - Google Patents

靶向cd22的抗原结合蛋白及其用途 Download PDF

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WO2023016554A1
WO2023016554A1 PCT/CN2022/112171 CN2022112171W WO2023016554A1 WO 2023016554 A1 WO2023016554 A1 WO 2023016554A1 CN 2022112171 W CN2022112171 W CN 2022112171W WO 2023016554 A1 WO2023016554 A1 WO 2023016554A1
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seq
amino acid
acid sequence
sequence shown
binding protein
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PCT/CN2022/112171
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French (fr)
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廖雪梅
黄经纬
陈卓
谢曼曼
程晓翠
丁洁
夏广新
柯樱
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上海医药集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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

Definitions

  • This application relates to the field of biomedicine, in particular to an antigen-binding protein targeting CD22 and its application.
  • CAR-T Chimeric Antigen Receptor-T cells
  • the tumor surface antigen CD22 is a glycoprotein with a molecular weight of 140 kDa, a member of the sialic acid-binding immunoglobulin-like lectin family, and has several Ig-like extracellular domains. It is restrictedly expressed in B cell malignancies and normal B cells, with 60-80% of B cell malignancies expressing CD22. Almost all B precursor cell acute lymphoblastic leukemia (B-ALL) express CD22, more than 90% of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) express CD22; chronic lymphocytic leukemia (B- CLL), hairy cell leukemia (HCL) also have high levels of CD22 expression. Therefore, CD22 is another ideal target besides CD19 in CAR-T therapy for malignant hematological tumors, which brings a new direction for tumor immunotherapy.
  • B-ALL B precursor cell acute lymphoblastic leukemia
  • DLBCL diffuse large B cell lymphoma
  • FL
  • CAR-T therapies usually use scFv fragments derived from the antigen-binding region of monoclonal antibodies as the antigen-binding region. After the extracellular scFv domain binds the target protein expressed on the surface of the target cell, it can activate the costimulatory domain and activation domain of the CAR structure.
  • the molecular weight of scFv is relatively large and it is easy to form multimers, which affects the function of CAR. Therefore, there is an urgent need for CARs containing novel antigen-binding domains to treat tumors.
  • the present application provides an antigen-binding protein targeting CD22, and in particular relates to a chimeric antigen receptor comprising the antigen-binding protein.
  • the antigen-binding protein has one or more of the following properties: 1) It can specifically recognize human CD22.
  • the chimeric antigen receptor has one or more of the following properties: 1) can specifically recognize human CD22; 2) can effectively kill tumor cells, such as hematoma.
  • the chimeric antigen receptor comprises a targeting moiety comprising HCDR3 comprising the amino acid sequence shown in SEQ ID NO:48.
  • the HCDR3 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:21.
  • the targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 11 to SEQ ID NO: 12.
  • the targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:50.
  • the HCDR1 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:6.
  • the targeting moiety comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in SEQ ID NO:55.
  • the targeting moiety comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the targeting moiety comprises HCDR1, HCDR2, HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:48; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:49; and The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:50.
  • the HCDR3 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:21; the HCDR2 in the targeting moiety comprises SEQ ID NO: 11 to the amino acid sequence shown in any one of SEQ ID NO:12; and the HCDR1 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:6.
  • HCDR1, HCDR2 and HCDR3 in the targeting moiety comprise an amino acid sequence selected from any group consisting of:
  • HCDR1 SEQ ID NO:4, HCDR2: SEQ ID NO:11, and HCDR3: SEQ ID NO:19;
  • HCDR1 SEQ ID NO:5
  • HCDR2 SEQ ID NO:12
  • HCDR3 SEQ ID NO:20
  • HCDR1 SEQ ID NO:6, HCDR2: SEQ ID NO:12, and HCDR3: SEQ ID NO:21.
  • the targeting moiety 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 SEQ ID NO:51 Amino acid sequence shown.
  • the H-FR1 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3.
  • the targeting moiety comprises H-FR2, 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:52 .
  • the H-FR2 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:10.
  • the targeting moiety comprises H-FR3, 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:53 .
  • the H-FR3 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18.
  • the targeting moiety 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 SEQ ID NO:54 Amino acid sequence shown.
  • the H-FR4 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:51; the H- FR2 comprises the amino acid sequence shown in SEQ ID NO:52; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:53; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR1 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3; the H-FR2 in the targeting moiety comprises The amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:10; the H-FR3 in the targeting moiety comprises any one of SEQ ID NO:13 to SEQ ID NO:18 Amino acid sequence; and the H-FR4 in the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • H-FR1, H-FR2, H-FR3 and H-FR4 in the targeting moiety comprise any set of amino acid sequences selected from the group consisting of:
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:13
  • H-FR4 SEQ ID NO:22;
  • H-FR1 SEQ ID NO:2, H-FR2: SEQ ID NO:8, H-FR3: SEQ ID NO:14 and H-FR4: SEQ ID NO:22;
  • H-FR1 SEQ ID NO:2, H-FR2: SEQ ID NO:9, H-FR3: SEQ ID NO:15 and H-FR4: SEQ ID NO:22;
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:16
  • H-FR4 SEQ ID NO:22;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:10, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:7, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:18
  • H-FR4 SEQ ID NO:23.
  • the targeting moiety comprises an antibody or antigen-binding fragment.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the targeting moiety comprises a VHH.
  • the VHH targets CD22.
  • the targeting moiety comprises the amino acid sequence shown in SEQ ID NO:55.
  • the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the chimeric antigen receptor comprises a transmembrane domain.
  • the transmembrane domain comprises a transmembrane domain 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.
  • 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
  • the transmembrane domain comprises a CD8-derived transmembrane domain.
  • the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:45.
  • the N-terminus of the transmembrane domain is linked to the C-terminus of the targeting moiety.
  • the chimeric antigen receptor comprises a co-stimulatory signaling domain.
  • the costimulatory signaling domain comprises a costimulatory signaling domain derived from a protein selected from the group consisting of: CD28, CD137, CD27, CD2, CD7, CD8, 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, ligand for CD83, CD40 and MyD88.
  • a protein selected from the group consisting of: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10
  • the costimulatory signaling domain comprises a costimulatory signaling domain derived from CD137.
  • the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:46.
  • the N-terminus of the co-stimulatory signaling domain is linked to the C-terminus of the transmembrane domain.
  • the chimeric antigen receptor comprises an intracellular signaling domain.
  • the intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of: CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , FcyRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • a protein selected from the group consisting of: CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , FcyRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpe
  • said intracellular signaling domain in said chimeric antigen receptor comprises an intracellular signaling domain derived from CD3zeta.
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:47.
  • the N-terminus of the intracellular signaling domain is linked to the C-terminus of the co-stimulatory signaling domain.
  • the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:38 to SEQ ID NO:44.
  • the present application provides an isolated antigen-binding protein, which specifically binds to CD22 protein with a KD value of about 2E-08M or below.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:48.
  • the HCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:21.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 11 to SEQ ID NO: 12.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:50.
  • the HCDR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:6.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in SEQ ID NO:55.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the HCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:48; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:49; and the HCDR1 comprises Amino acid sequence shown in SEQ ID NO:50.
  • the HCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 19 to SEQ ID NO: 21; the HCDR2 comprises SEQ ID NO: 11 to The amino acid sequence shown in any one of SEQ ID NO:12; And the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:6.
  • the HCDR1, HCDR2 and HCDR3 of the isolated antigen binding protein comprise an amino acid sequence selected from any group consisting of:
  • HCDR1 SEQ ID NO:4, HCDR2: SEQ ID NO:11, and HCDR3: SEQ ID NO:19;
  • HCDR1 SEQ ID NO:5, HCDR2: SEQ ID NO:12, and HCDR3: SEQ ID NO:20; and c) HCDR1: SEQ ID NO:6, HCDR2: SEQ ID NO:12, and HCDR3: SEQ ID NO:12, and HCDR3: SEQ ID NO:20; ID NO: 21.
  • the isolated antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 51.
  • the H-FR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3.
  • said isolated antigen binding protein comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises SEQ ID NO:52 amino acid sequence.
  • the H-FR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:10.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:53 amino acid sequence.
  • the H-FR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 54.
  • the H-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the antigen binding protein of described separation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises the aminoacid sequence shown in SEQ ID NO:51; Said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:52; said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:53; and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3; the H-FR2 comprises SEQ ID The amino acid sequence shown in any one of NO:7 to SEQ ID NO:10;
  • the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18; and
  • the H -FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1, H-FR2, H-FR3 and H-FR4 in the isolated antigen binding protein comprise any set of amino acid sequences selected from the group consisting of:
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:13
  • H-FR4 SEQ ID NO:22;
  • H-FR1 SEQ ID NO:2, H-FR2: SEQ ID NO:8, H-FR3: SEQ ID NO:14 and H-FR4: SEQ ID NO:22;
  • H-FR1 SEQ ID NO:2, H-FR2: SEQ ID NO:9, H-FR3: SEQ ID NO:15 and H-FR4: SEQ ID NO:22;
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:16
  • H-FR4 SEQ ID NO:22;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:10, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:7, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:18
  • H-FR4 SEQ ID NO:23.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:55.
  • the VH in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment of the isolated antigen-binding protein is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di- scFv, VHH and dAb.
  • the isolated antigen binding protein comprises a VHH or an antigen binding fragment thereof.
  • said antibody in said isolated antigen binding protein is selected from the group consisting of monoclonal antibodies, humanized antibodies, chimeric antibodies and fully human antibodies.
  • the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the present application provides a polypeptide comprising said chimeric antigen receptor and/or said isolated antigen binding protein.
  • the present application provides an immunoconjugate comprising the isolated antigen-binding protein.
  • the application provides an isolated nucleic acid molecule encoding said chimeric antigen receptor, said isolated antigen binding protein, or said polypeptide.
  • the present application provides a vector comprising said isolated nucleic acid molecule.
  • the present application provides a cell comprising said chimeric antigen receptor, said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule and/or the carrier.
  • the cells include immune cells.
  • the immune cells are selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes cells, leukocytes, and/or peripheral blood mononuclear cells.
  • the cells comprise T cells.
  • the present application provides a method for preparing said chimeric antigen receptor, said isolated antigen binding protein and/or said polypeptide, said method comprising allowing said isolated antigen to bind
  • the cells are cultured under the condition that the protein and/or the polypeptide are expressed.
  • the present application provides a pharmaceutical composition, which comprises said chimeric antigen receptor, said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated Nucleic acid molecules, the carrier, the cells, and/or pharmaceutically acceptable adjuvants and/or excipients.
  • the application provides a method for detecting CD22 protein, which comprises:
  • the isolated antigen binding protein, the polypeptide or the immunoconjugate is administered.
  • the present application provides a CD22 protein detection kit, which comprises the isolated antigen-binding protein, the polypeptide or the immunoconjugate.
  • the present application provides the use of the isolated antigen-binding protein, the polypeptide or the immunoconjugate in the preparation of a kit for detecting the presence of CD22 protein and/or content.
  • the present application provides the chimeric antigen receptor, the isolated antigen-binding protein, the polypeptide and/or the immunoconjugate in the preparation of drugs for preventing and/or treating tumors the use of.
  • the tumor comprises a hematoma.
  • the tumor comprises a tumor associated with expression of CD22.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • the present application provides the chimeric antigen receptor, the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, And/or said cells, which are used for preventing and/or treating tumors.
  • the tumor comprises a hematoma.
  • the tumor comprises a tumor associated with expression of CD22.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • the present application provides a method for preventing and/or treating a disease or disorder, comprising administering to a subject in need an effective amount of the chimeric antigen receptor, the isolated antigen-binding protein , the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and/or the cell.
  • the tumor comprises a hematoma.
  • the tumor comprises a tumor associated with expression of CD22.
  • the tumor is selected from the group consisting of leukemia and hematoma.
  • Figure 1 shows the binding curve of the exemplary antigen-binding protein described in this application to human CD22 expressed on the surface of K562 cells.
  • Figure 2 shows the RLU detection results after the Jurkat-NFAT and Raji action of the exemplary transient CD22-CAR plasmid described in the present application.
  • Figure 3 shows the results of the flow cytometric detection of the binding of humanized CD22-CAR-04 CART cells to CD22 protein described in this application.
  • Figure 4 shows the killing effect of the exemplary humanized CD22-CAR-04 CART cells described in the present application on Raji-LUC cells.
  • Figure 5 shows the killing effect of the exemplary humanized CD22-CAR-04 CART cells described in the present application on Nalm6-LUC cells.
  • Figure 6 shows the detection results of fluorescence intensity in vivo imaging of Raji-LUC mice.
  • Figure 7 shows the fluorescence images of Raji-LUC mice in vivo imaging.
  • Figure 8 shows the results of body weight changes in Raji-LUC mice.
  • Figure 9 shows the detection results of fluorescence intensity in vivo imaging of Naml6-LUC mice.
  • Figure 10 shows the fluorescence images of Naml6-LUC mice in vivo imaging.
  • Figure 11 shows the results of body weight changes in Nalm6-LUC mice.
  • isolated antigen-binding protein generally refers to a polypeptide polymer capable of specifically recognizing and/or neutralizing a specific antigen.
  • an isolated antigen binding protein can include a portion of a heavy chain.
  • an isolated antigen binding protein can include a heavy chain variable region.
  • isolated antigen binding protein may include single domain antibodies.
  • an isolated antigen binding protein can include, but is not limited to, a human single domain antibody.
  • single domain antibody or “sdAb” or “VHH” generally refers to a class of antibodies lacking the light chain of the antibody and only having the variable region of the heavy chain.
  • the single domain antibody may be from a Bactrian camel, dromedary, alpaca, llama, nurse shark, great star shark, or ray (for example, see Kang Xiaozhen et al., Acta Biological Engineering, 2018, 34( 12): 1974-1984).
  • single domain antibodies can be from alpacas.
  • Single domain antibodies can be composed of a heavy chain variable region (VH).
  • 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 divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions known as the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each heavy chain variable region may consist of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the heavy chain variable region contains a binding domain that interacts with an antigen (eg, CD22).
  • transmembrane domain generally refers to a sequence in a cell surface protein that spans the cell membrane, which may contain a hydrophobic alpha helix.
  • the transmembrane domain can be connected with the intracellular signaling domain and 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 may comprise a transmembrane domain or a combination thereof derived from a histone selected from: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, T cell receptor Subunits, polypeptides constituting the CD3 complex, IL2 receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • a histone selected from: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, T cell receptor Subunits, polypeptides constituting the CD3 complex, IL2 receptor, CD5, ICOS,
  • a subunit of a T cell receptor can include TCR ⁇ , TCR ⁇ , TCR ⁇ , or TCR ⁇ .
  • polypeptides that make up the CD3 complex can include CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the transmembrane domain can include a transmembrane domain derived from said CD8.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • targeting moieties for example, targeting tumor-specific antigens and/or tumor-associated antigens
  • signal peptides for example, transmembrane domains
  • co- Stimulatory signaling domain for example, transmembrane domains
  • intracellular signaling domain for example, transmembrane domains
  • the CAR can be based on the antigen (eg CD22) specificity of the antibody combined with the T cell receptor activation intracellular domain.
  • T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • co-stimulatory signaling domain generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective response of lymphocytes to antigens.
  • the co-stimulatory signaling domain may comprise a co-stimulatory signaling domain derived from a histone selected from the group consisting of: CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86, OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1, PD- Ligands of L2, 4-1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta
  • intracellular signal transduction domain generally refers to a domain capable of transducing signals located inside a cell.
  • the intracellular signaling domain can transduce signals into cells.
  • an intracellular signaling domain is any contiguous amino acid sequence used to direct protein targeting.
  • the intracellular signaling domain may comprise an intracellular signaling domain derived from a protein selected from the group consisting of: CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5, CD22 , FcR ⁇ , FcR ⁇ , FcR ⁇ , FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP12, and at least An ITAM domain.
  • the intracellular signaling domain can include an intracellular signaling domain derived from CD3zeta.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected 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 antibodies associated with antigens. Bound antibody fragments (eg, Fab, Fab' and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the term "antigen-binding fragment” generally refers to one or more fragments of an antibody that specifically bind to an antigen.
  • the antigen-binding function of antibodies can be realized by full-length fragments of antibodies.
  • the antigen-binding function of an antibody can also be achieved by comprising a heavy chain of a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab'), or by comprising a Fv, ScFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2.
  • Fab fragments typically monovalent fragments consisting of VL, VH, CL, and CH domains
  • F(ab')2 fragments which may comprise two Fab fragments linked 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 antibody single arm
  • dAb fragment composed of VH domains (Ward et al., (1989) Nature 341:544-546)
  • CDR complementarity determining region
  • the "antigen-binding fragment” may also include an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; 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 "monoclonal antibody” generally refers to a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
  • Monoclonal antibodies are highly specific, directed against a single antigenic site.
  • the monoclonal antibodies can be produced by hybridoma technology or produced in bacterial, eukaryotic or plant cells by using recombinant DNA methods.
  • Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991) conduct.
  • chimeric antibody generally refers to an antibody in which a part of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class, and The remainder of the chain is then homologous to the corresponding sequence in another species.
  • the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human) .
  • B cells or hybridoma cells of non-human origin can be used to produce variable regions combined with constant regions of human origin.
  • variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region it is combined with.
  • the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody whose constant region is of non-human origin.
  • humanized antibody generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing the immunogenicity of a heterologous antibody when introduced into a human, while simultaneously Preserves the full antigen-binding affinity and specificity of the antibody.
  • CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof; including “reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann , et al., 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), "high addition” (hyperchimerization), (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154) and "veneering", (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.” In: Metcalf B W, Dalton B J, eds.
  • the term "tumor” generally refers to a neoplasm formed by the proliferation of local tissue cells.
  • the tumor can include a hematoma.
  • the tumor can include lymphoma.
  • the tumor can include leukemia.
  • the tumor can include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to the altered expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared with 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 up-regulated compared with 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 higher.
  • the term “immunoconjugate” generally refers to a conjugate formed by conjugating (for example, covalently linking through a linker molecule) the other therapeutic agent to the isolated antigen-binding protein, the conjugate
  • the other therapeutic agent can be delivered to a target cell (eg, a tumor cell) by specific binding of the isolated antigen binding protein to an antigen on the target cell.
  • the antigen may also be secreted by the target cell and located in the space outside the target cell.
  • K D (likewise, “K D " or “K D ”) generally refers to "affinity constant” or "equilibrium dissociation constant” and refers to a titration measurement at equilibrium, or Value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka). Binding of a binding protein (e.g., an isolated antigen-binding protein described herein) to an antigen (e.g., CD22 protein) is expressed using an on-rate constant (ka), an off-rate constant ( kd ), and an equilibrium dissociation constant (KD) affinity. Methods for determining association and dissociation rate constants are well known in the art.
  • the KD value can be determined by Biacore (Biomolecular Interaction Analysis) (for example, an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden), and other experimental approaches and instruments such as Octet can also be used.
  • Biacore Biomolecular Interaction Analysis
  • the KD value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho), or using a surface plasmon resonance (SPR) instrument.
  • K562 cells generally refers to human immortalized myeloid leukemia cell lines. K562 belongs to the erythroleukemia cell line, which has the characteristics of high malignancy and fast proliferation. Pleural effusion originally isolated from a 53-year-old female patient with chronic myelogenous leukemia (CML) in the acute phase.
  • CML chronic myelogenous leukemia
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host.
  • the vectors can transfer inserted nucleic acid molecules into and/or between cells.
  • the vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector produces the desired expression product by culturing appropriate cells containing the vector.
  • the vector may comprise a lentiviral vector.
  • the term "cell” generally refers to a plasmid or vector that can or has contained the nucleic acid molecule described in the application, or can express the chimeric antigen receptor described in the application or the antigen-binding protein described in the application.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the chimeric antigen receptor or antigen binding protein described in this application .
  • the cells can be obtained by transfecting cells in vitro with the vectors described in this application.
  • the cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • the cells can be immune cells.
  • the immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.
  • the immune cells can be T cells.
  • treating generally refers to: (i) preventing a disease, disorder, and/or condition from occurring in a patient who may be predisposed to it but has not been diagnosed with it; (ii) inhibiting the disease , disorder or condition, i.e. arresting its development; and (iii) relieving the disease, disorder or condition, i.e. making the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subside.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to a polymer of amino acids of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component).
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably and generally refer to nucleosides of any length
  • a polymeric form of an acid such as deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined by junctional analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Isolated DNA of any sequence, isolated RNA, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA Transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.
  • the present application may also include functional variants, derivatives, analogs, homologues and fragments thereof.
  • the term "functional variant” refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence.
  • a variant of any given sequence is one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one A sequence of endogenous functions.
  • Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.
  • derivative generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and and/or added, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analogue generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.
  • amino acid substitutions e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.
  • proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid.
  • Paragine, Glutamine, Serine, Threonine and Tyrosine are examples of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • CD22 antigen generally refers to an important membrane antigen related to proliferation and differentiation on human B lymphocytes.
  • the amino acid sequence of human CD22 can be found in UniProt/Swiss-Prot accession number P20273.
  • the antigen targeted by the antigen-binding domain of the nucleic acid molecule expressing at least two chimeric antigen receptors (CARs) may be CD22.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen binding protein, which can be measured in Biacore with a KD value of 2E-08M or below (for example, the KD is not higher than about 2E-08M, not higher than about 1.5E-08M, not higher than about 1E-08M, not higher than about 9E-09M, not higher than about 8E-09M, not higher than about 7E-09M, not higher than about 6E-09M, not higher than about 5E -09M, not higher than about 4E-09M, not higher than about 3E-09M, not higher than about 2E-09M, not higher than about 1E-09M, or not higher than 5E-09M or less) specific for human CD22 protein combined.
  • the KD is not higher than about 2E-08M, not higher than about 1.5E-08M, not higher than about 1E-08M, not higher than about 9E-09M, not higher than about 8E-09M, not higher than about 7E-09M, not higher than about 6E-
  • the application provides an isolated antigen-binding protein, which may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:55.
  • the VH may comprise the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the HCDR of the isolated antigen-binding protein can be divided in any form, as long as the VH is identical to the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30, it can be divided in any form to obtain All HCDRs can fall within the protection scope of the present application.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, North's, Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the isolated antigen binding proteins described herein are defined by the IMGT coding system.
  • the antigen binding protein 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 HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:48.
  • the HCDR3 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR3 having amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at amino acid positions selected from the group consisting of: X 15 and x17 .
  • ATDPWTDCSLDGRYX 15 YX 17 Y (SEQ ID NO: 48), wherein X 15 can be E or R, and X 17 can be D, G or N.
  • the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:21.
  • the HCDR3 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the antigen-binding protein comprises HCDR2 having an amino acid substitution (eg, a conservative amino acid substitution, etc.) at an amino acid selected from the following position compared to the sequence shown in SEQ ID NO: 49: X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 49), wherein X 3 can be G or S.
  • the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:11 to SEQ ID NO:12.
  • the HCDR2 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:50.
  • the HCDR1 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR1 having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at amino acids selected from the following positions compared to the sequence shown in SEQ ID NO: 50: X 3 , X5 and X6 .
  • GFX 3 VX 5 X 6 YA (SEQ ID NO:50), wherein X 3 can be P or S, X 5 can be A or D, and X 6 can 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 to SEQ ID NO:6.
  • the HCDR1 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:56.
  • the HCDR3 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the antigen-binding protein comprises HCDR3 having an amino acid substitution (eg, a conservative amino acid substitution, etc.) at an amino acid selected from the following position compared to the sequence shown in SEQ ID NO: 56: X 17 .
  • ATDPWTDCSLDGRYEYX 17 Y (SEQ ID NO: 56), wherein X 17 can be G or N.
  • the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:20.
  • the HCDR3 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the antigen-binding protein comprises HCDR2 having an amino acid substitution (eg, a conservative amino acid substitution, etc.) at an amino acid selected from the following position compared to the sequence shown in SEQ ID NO: 49: X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 49), wherein X 3 can be G or S.
  • the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:11 to SEQ ID NO:12.
  • the HCDR2 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:57.
  • the HCDR1 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR1 having amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at amino acid positions selected from the group consisting of: X5 and x6 .
  • GFPVX 5 X 6 YA (SEQ ID NO:57), wherein X 5 can be A or D, and X 6 can be D or G.
  • the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:4 to SEQ ID NO:5.
  • the HCDR1 sequence of the antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:4; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:19 The amino acid sequence shown.
  • the antigen binding protein may comprise antibody B010-B-22Nb-01 or an antigen binding fragment having the same HCDR3 (eg, having the same HCDR1-3) therewith.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20 The amino acid sequence shown.
  • the antigen binding protein may comprise antibody B010-B-22Nb-02 or an antigen binding fragment having the same HCDR3 as it (eg, having the same HCDR1-3 as it).
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20 The amino acid sequence shown.
  • the antigen binding protein may comprise antibody B010-B-22Nb-03 or an antigen binding fragment having the same HCDR3 (eg, having the same HCDR1-3) therewith.
  • the HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:21 The amino acid sequence shown.
  • the antigen binding protein may comprise or have a combination with antibody B010-B-22Nb-04, B010-B-22Nb-04-H4, B010-B-22Nb-04-H5, B010-B-22Nb-04-H6 Antigen-binding fragments of the same HCDR3 (eg, with the same HCDR1-3).
  • the VH of the antigen binding protein 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:51.
  • H-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the following group: X 1 and X16 .
  • X 1 VQLVESGGGLVQPGX 16 SLRLSCAAS (SEQ ID NO:51), wherein X 1 can be A or E, and X 16 can be G or R.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:52.
  • H-FR2 of the antigen-binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 4 , X 11 , X 12 , X 14 , X 15 and X 17 .
  • X 1 AWX 4 RQAPGKX 11 X 12 EX 14 X 15 SX 17 (SEQ ID NO:52), wherein, X 1 can be I or M, X 4 can be F or V, X 11 can be E or G, X 12 Can be L or R, X14 can be G or W, X15 can be I or V, X17 can be C or Y.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:10.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:53.
  • H-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 2 , X 3 , X 7 , X 12 , X 20 , X 21 , X 27 , X 29 and X 30 .
  • YX 2 X 3 DSVX 7 GRFTX 12 SRDNAKNX 20 X 21 YLQMNX 27 LX 29 X 30 EDTAVYYC (SEQ ID NO:53), wherein, X 2 can be D or Y, X 3 can be A, Q or V, and X 7 can be Is E or K, X 12 can be I or V, X 20 can be S or T, X 21 can be L or V, X 27 can be D or S, X 29 can be E, K or R, X 30 can is D or P.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18.
  • the H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:54.
  • H-FR4 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 3 and x6 .
  • WGX 3 GTX 6 VTVSS (SEQ ID NO:54), wherein X 3 can be L or Q, and X 6 can be L or Q.
  • the H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:58.
  • H-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 16 .
  • AVQLVESGGGLVQPGX16SLRLSCAAS (SEQ ID NO:58), wherein X16 can be G or R.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:2.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:59.
  • H-FR2 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 and X15 .
  • X 1 AWFRQAPGKEREGX 15 SC (SEQ ID NO:59), wherein X 1 can be I or M, and X 15 can be I or V.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:9.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:60.
  • H-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 2 , X 3 , X 7 , X 12 and X 27 .
  • YX 2 X 3 DSVX 7 GRFTX 12 SRDNAKNTVYLQMNX 27 LKPEDTAVYYC (SEQ ID NO:60), wherein, X 2 can be D or Y, X 3 can be A or V, X 7 can be E or K, X 12 can be I or V, X 27 can be D or S.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 13 to SEQ ID NO: 15.
  • the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:22.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:61.
  • H-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 .
  • X 1 VQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:61), wherein X 1 can be A or E.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:3.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:62.
  • H-FR2 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 4 , X 11 , X 12 , X 14 and X 17 .
  • IAWX 4 RQAPGKX 11 X 12 EX 14 VSX 17 (SEQ ID NO:62), wherein, X 4 can be F or V, X 11 can be E or G, X 12 can be L or R, X 14 can be G or W, X 17 can be C or Y.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:10.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:63.
  • H-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 3 , X 20 , X 21 , X 29 and X 30 .
  • YYX 3 DSVKGRFTISRDNAKNX 20 X 21 YLQMNSLX 29 X 30 EDTAVYYC (SEQ ID NO:63), wherein X 3 can be A or Q, X 20 can be S or T, X 21 can be L or V, and X 29 can be E or R, X 30 can be D or P.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 16 to SEQ ID NO: 18.
  • the H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:54.
  • H-FR4 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 3 and x6 .
  • WGX 3 GTX 6 VTVSS (SEQ ID NO:54), wherein X 3 can be L or Q, and X 6 can be L or Q.
  • the H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:51; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:52; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:53; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:58; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:59; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:60; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:61; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:62; the H-FR3 The amino acid sequence shown in SEQ ID NO:63 can be included; and the H-FR4 can include the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3; the H-FR2 may comprise SEQ ID NO: 7 to The amino acid sequence shown in any one of SEQ ID NO:10; the H-FR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18; and the H-FR4 can be Comprising the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:1; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:13; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the antigen-binding protein may include antibody B010-B-22Nb-01 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8; the H-FR3 The amino acid sequence shown in SEQ ID NO:14 can be included; and the H-FR4 can include the amino acid sequence shown in SEQ ID NO:22.
  • the antigen binding protein may comprise antibody B010-B-22Nb-02 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:9; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:15; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the antigen-binding protein may include antibody B010-B-22Nb-03 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:1; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:16; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:17; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04-H4 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:17; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04-H5 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:18; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04-H6 or an antigen binding fragment thereof having the same H-FR1-4.
  • the antigen-binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:55.
  • the antigen binding protein comprises a VH having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO: 55 compared to the VH: X1 , X16 , X28 , X30 , X31, X34 , X37 , X44 , X45 , X47 , X48 , X50 , X53 , X60 , X61 , X65 , X70 , X 78 , X 79 , X 85 , X 87 , X 88 , X 111 , X 113 , X 117 , X 120 .
  • the antigen-binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:64.
  • the antigen binding protein comprises a VH having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO: 64 compared to the VH: X16 , X30 , X31 , X34 , X48, X53 , X60 , X61 , X65 , X70 , X85 , X113 .
  • AVQLVESGGGLVQPGX 16 SLRLSCAASGFPVX 30 X 31 YAX 34 AWFRQAPGKEREGX 48 SCISX 53 RDGNTYX 60 X 61 DSVX 65 GRFTX 70 SRDNAKNTVYLQMNX 85 LKPEDTAVYYCATDPWTDCSLDGRYEY X 113 YWGLGTQVTVSS(SEQ ID NO:64), ⁇ ,X 16 ⁇ G ⁇ R,X 30 ⁇ A ⁇ D, X 31 can be D or G, X 34 can be I or M, X 48 can be I or V, X 53 can be G or S, X 60 can be D or Y, X 61 can be A or V, X 65 can be E or K, X 70 can be I or V, X 85 can be D or S, X 113 can be G or N.
  • the antigen-binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:65.
  • the antigen binding protein comprises a VH having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO: 65 compared to the VH: X 1 , X 37 , X 44 , X 45 , X 47 , X 50 , X 61 , X 78 , X 79 , X 87 , X 88 , X 117 and X 120 .
  • the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:19.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the antigen binding protein may include antibody B010-B-22Nb-01 or an antigen binding protein having the same heavy chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the antigen binding protein may include antibody B010-B-22Nb-02 or an antigen binding protein having the same heavy chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:9; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:15
  • the amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:26.
  • the antigen binding protein may include antibody B010-B-22Nb-03 or an antigen binding protein having the same heavy chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:27.
  • the antigen binding protein may include antibody B010-B-22Nb-04 or an antigen binding protein having the same heavy chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04-H4 or an antigen binding protein having the same heavy chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04-H5 or an antigen binding protein having the same heavy chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may comprise antibody B010-B-22Nb-04-H6 or an antigen binding protein having the same heavy chain variable region as it.
  • the isolated antigen binding protein can compete with the reference antibody for binding to human CD22.
  • the reference antibody may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:48.
  • the HCDR3 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the reference antibody comprises HCDR3 having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at amino acid positions selected from the group consisting of: X 15 and x17 .
  • ATDPWTDCSLDGRYX 15 YX 17 Y (SEQ ID NO: 48), wherein X 15 can be E or R, and X 17 can be D, G or N.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:21.
  • the HCDR3 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the reference antibody comprises HCDR2 having an amino acid substitution (eg, a conservative amino acid substitution, etc.) at an amino acid selected from the following position compared to the sequence shown in SEQ ID NO: 49: X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 49), wherein X 3 can be G or S.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:11 to SEQ ID NO:12.
  • the HCDR2 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:50.
  • the HCDR1 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the reference antibody comprises HCDR1 having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at amino acid positions selected from the following positions compared to the sequence shown in SEQ ID NO: 50: X 3 , X5 and X6 .
  • GFX 3 VX 5 X 6 YA (SEQ ID NO:50), wherein X 3 can be P or S, X 5 can be A or D, and X 6 can be D or G.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:6.
  • the HCDR1 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:56.
  • the HCDR3 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the reference antibody comprises HCDR3 having an amino acid substitution (eg, conservative amino acid substitution, etc.) at an amino acid selected from the following position compared to the sequence shown in SEQ ID NO: 56: X 17 .
  • ATDPWTDCSLDGRYEYX 17 Y (SEQ ID NO: 56), wherein X 17 can be G or N.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:20.
  • the HCDR3 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the reference antibody comprises HCDR2 having an amino acid substitution (eg, a conservative amino acid substitution, etc.) at an amino acid selected from the following position compared to the sequence shown in SEQ ID NO: 49: X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 49), wherein X 3 can be G or S.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:11 to SEQ ID NO:12.
  • the HCDR2 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:57.
  • the HCDR1 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the reference antibody comprises HCDR1 having amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at amino acid positions selected from the group consisting of: X5 and x6 .
  • GFPVX 5 X 6 YA (SEQ ID NO:57), wherein X 5 can be A or D, and X 6 can be D or G.
  • the HCDR1 of the reference antibody may comprise the amino acid sequences shown in SEQ ID NO:4 to SEQ ID NO:5.
  • the HCDR1 sequence of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:4; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:19 The amino acid sequence shown.
  • the reference antibody may comprise antibody B010-B-22Nb-01 or an antigen-binding fragment having the same HCDR3 (eg, having the same HCDR1-3) therewith.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20 The amino acid sequence shown.
  • the reference antibody can comprise antibody B010-B-22Nb-02 or an antigen-binding fragment having the same HCDR3 as it (eg, having the same HCDR1-3 as it).
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20 The amino acid sequence shown.
  • the reference antibody can comprise antibody B010-B-22Nb-03 or an antigen-binding fragment having the same HCDR3 as it (eg, having the same HCDR1-3 as it).
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21 The amino acid sequence shown.
  • the reference antibody may comprise or have a combination with antibody B010-B-22Nb-04, B010-B-22Nb-04-H4, B010-B-22Nb-04-H5, B010-B-22Nb-04-H6 Antigen-binding fragments of the same HCDR3 (eg, with the same HCDR1-3).
  • the chimeric antigen receptor comprises a targeting moiety.
  • the targeting moiety can specifically bind CD22.
  • the targeting portion 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 HCDR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:48.
  • the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the targeting moiety of the chimeric antigen receptor comprises HCDR3 having an amino acid substitution (eg, a conservative amino acid substitution) at an amino acid selected from the position of SEQ ID NO:48 compared to the sequence shown in SEQ ID NO:48 etc.): X 15 and X 17 .
  • ATDPWTDCSLDGRYX 15 YX 17 Y (SEQ ID NO: 48), wherein X 15 can be E or R, and X 17 can be D, G or N.
  • the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:21.
  • the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the targeting moiety of the chimeric antigen receptor comprises HCDR2 having an amino acid substitution (eg, a conservative amino acid substitution) at an amino acid selected from the position of SEQ ID NO:49 compared to the sequence shown in SEQ ID NO:49 etc.): X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 49), wherein X 3 can be G or S.
  • the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:11 to SEQ ID NO:12.
  • the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:50.
  • the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the targeting moiety of the chimeric antigen receptor comprises HCDR1 having an amino acid substitution (eg, a conservative amino acid substitution) at an amino acid selected from the position of SEQ ID NO: 50 compared to the sequence shown in SEQ ID NO: 50 etc.): X 3 , X 5 and X 6 .
  • GFX 3 VX 5 X 6 YA (SEQ ID NO:50), wherein X 3 can be P or S, X 5 can be A or D, and X 6 can be D or G.
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:6.
  • the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:56.
  • the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the targeting moiety of the chimeric antigen receptor comprises HCDR3 having amino acid substitutions (eg, conservative amino acid substitutions) at amino acids selected from the following positions compared to the sequence shown in SEQ ID NO: 56 etc.): X 17 .
  • ATDPWTDCSLDGRYEYX 17 Y (SEQ ID NO: 56), wherein X 17 can be G or N.
  • the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO:20.
  • the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the targeting moiety of the chimeric antigen receptor comprises HCDR2 having an amino acid substitution (eg, a conservative amino acid substitution) at an amino acid selected from the position of SEQ ID NO:49 compared to the sequence shown in SEQ ID NO:49 etc.): X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 49), wherein X 3 can be G or S.
  • the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:11 to SEQ ID NO:12.
  • the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:57.
  • the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the targeting moiety of the chimeric antigen receptor comprises HCDR1 having amino acid substitutions (eg, conservative amino acid substitutions) at amino acids selected from the following positions compared to the sequence shown in SEQ ID NO: 57 etc.): X 5 and X 6 .
  • GFPVX 5 X 6 YA (SEQ ID NO:57), wherein X 5 can be A or D, and X 6 can be D or G.
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequences shown in SEQ ID NO:4 to SEQ ID NO:5.
  • the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT coding system.
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:4; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11; and the HCDR3 may comprise Amino acid sequence shown in SEQ ID NO:19.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-01 or an antigen-binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise Amino acid sequence shown in SEQ ID NO:20.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-02 or an antigen-binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:5; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise Amino acid sequence shown in SEQ ID NO:20.
  • the targeting moiety of the chimeric antigen receptor can comprise antibody B010-B-22Nb-03 or an antigen-binding fragment having the same HCDR3 (eg, having the same HCDR1-3) therewith.
  • the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12; and the HCDR3 may comprise Amino acid sequence shown in SEQ ID NO:21.
  • the targeting moiety of the chimeric antigen receptor can 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 thereof having the same HCDR3 (eg, having the same HCDR1-3).
  • the VH of the targeting portion of the chimeric antigen receptor may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:51.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 1 and X 16 .
  • X 1 VQLVESGGGLVQPGX 16 SLRLSCAAS (SEQ ID NO:51), wherein X 1 can be A or E, and X 16 can be G or R.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:52.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 1 , X 4 , X 11 , X 12 , X 14 , X 15 and X 17 .
  • X 1 AWX 4 RQAPGKX 11 X 12 EX 14 X 15 SX 17 (SEQ ID NO:52), wherein, X 1 can be I or M, X 4 can be F or V, X 11 can be E or G, X 12 Can be L or R, X14 can be G or W, X15 can be I or V, X17 can be C or Y.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:10.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:53.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 2 , X 3 , X 7 , X 12 , X 20 , X 21 , X 27 , X 29 and X 30 .
  • YX 2 X 3 DSVX 7 GRFTX 12 SRDNAKNX 20 X 21 YLQMNX 27 LX 29 X 30 EDTAVYYC (SEQ ID NO:53), wherein, X 2 can be D or Y, X 3 can be A, Q or V, and X 7 can be Is E or K, X 12 can be I or V, X 20 can be S or T, X 21 can be L or V, X 27 can be D or S, X 29 can be E, K or R, X 30 can is D or P.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 3 and X 6 .
  • WGX 3 GTX 6 VTVSS (SEQ ID NO:54), wherein X 3 can be L or Q, and X 6 can be L or Q.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:58.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 16 .
  • AVQLVESGGGLVQPGX16SLRLSCAAS (SEQ ID NO:58), wherein X16 can be G or R.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:2.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:59.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 1 and X 15 .
  • X 1 AWFRQAPGKEREGX 15 SC (SEQ ID NO:59), wherein X 1 can be I or M, and X 15 can be I or V.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:9.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 2 , X 3 , X 7 , X 12 and X 27 .
  • YX 2 X 3 DSVX 7 GRFTX 12 SRDNAKNTVYLQMNX 27 LKPEDTAVYYC (SEQ ID NO:60), wherein, X 2 can be D or Y, X 3 can be A or V, X 7 can be E or K, X 12 can be I or V, X 27 can be D or S.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:15.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:22.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:61.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 1 .
  • X 1 VQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:61), wherein X 1 can be A or E.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:3.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:62.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 4 , X 11 , X 12 , X 14 and X 17 .
  • IAWX 4 RQAPGKX 11 X 12 EX 14 VSX 17 (SEQ ID NO:62), wherein, X 4 can be F or V, X 11 can be E or G, X 12 can be L or R, X 14 can be G or W, X 17 can be C or Y.
  • the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:10.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:63.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 3 , X 20 , X 21 , X 29 and X 30 .
  • YYX 3 DSVKGRFTISRDNAKNX 20 X 21 YLQMNSLX 29 X 30 EDTAVYYC (SEQ ID NO:63), wherein X 3 can be A or Q, X 20 can be S or T, X 21 can be L or V, and X 29 can be E or R, X 30 can be D or P.
  • the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:16 to SEQ ID NO:18.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor has amino acid substitutions at one or more amino acids selected from the following group (for example, conservative amino acids substitution etc.): X 3 and X 6 .
  • WGX 3 GTX 6 VTVSS (SEQ ID NO:54), wherein X 3 can be L or Q, and X 6 can be L or Q.
  • the H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:51; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:52 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:53; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:58; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:59
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:60; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:61; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:62 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:63; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3;
  • the H-FR2 may comprise The amino acid sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:10;
  • the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:18;
  • the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:1; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 22.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-01 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 14; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 22.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-02 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:15; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-03 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:1; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 16; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 22.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-04 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 17; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-04-H4 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7 ; The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 17; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-04-H5 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 18; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.
  • the targeting moiety of the chimeric antigen receptor may comprise antibody B010-B-22Nb-04-H6 or an antigen-binding fragment thereof having the same H-FR1-4.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:55.
  • the targeting portion of the chimeric antigen receptor comprises a VH having an amino acid substitution at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO: 55 (for example, Conservative amino acid substitutions, etc.): X 1 , X 16 , X 28 , X 30 , X 31 , X 34 , X 37 , X 44 , X 45 , X 47 , X 48 , X 50 , X 53 , X 60 , X 61 , X 65 , X 70 , X 78 , X 79 , X 85 , X 87 , X 88 , X 111 , X 113 , X 117 , X 120 .
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:64.
  • the targeting moiety of the chimeric antigen receptor may comprise a VH having an amino acid substitution at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO: 64 (e.g. , conservative amino acid substitution, etc.): X 16 , X 30 , X 31 , X 34 , X 48 , X 53 , X 60 , X 61 , X 65 , X 70 , X 85 , X 113 .
  • AVQLVESGGGLVQPGX 16 SLRLSCAASGFPVX 30 X 31 YAX 34 AWFRQAPGKEREGX 48 SCISX 53 RDGNTYX 60 X 61 DSVX 65 GRFTX 70 SRDNAKNTVYLQMNX 85 LKPEDTAVYYCATDPWTDCSLDGRYEY X 113 YWGLGTQVTVSS(SEQ ID NO:64), ⁇ ,X 16 ⁇ G ⁇ R,X 30 ⁇ A ⁇ D, X 31 can be D or G, X 34 can be I or M, X 48 can be I or V, X 53 can be G or S, X 60 can be D or Y, X 61 can be A or V, X 65 can be E or K, X 70 can be I or V, X 85 can be D or S, X 113 can be G or N.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:65.
  • the targeting moiety of the chimeric antigen receptor may comprise a VH having an amino acid substitution at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO: 65 (e.g. , conservative amino acid substitutions, etc.): X 1 , X 37 , X 44 , X 45 , X 47 , X 50 , X 61 , X 78 , X 79 , X 87 , X 88 , X 117 and X 120 .
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:24 to SEQ ID NO:30.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:19.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:24.
  • the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody B010-B-22Nb-01 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:5;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:25.
  • the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody B010-B-22Nb-02 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:5;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:26.
  • the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody B010-B-22Nb-03 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:27.
  • the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody B010-B-22Nb-04 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:28.
  • the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody B010-B-22Nb-04-H4 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:29.
  • the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody B010-B-22Nb-04-H5 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO:30.
  • the targeting moiety of the chimeric antigen receptor can comprise the targeting moiety of antibody B010-B-22Nb-04-H6 or a chimeric antigen receptor having the same heavy chain variable region as it.
  • the chimeric antigen receptor may comprise a transmembrane domain.
  • the transmembrane domain may comprise, but is not limited to, a transmembrane domain 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 can include a transmembrane domain derived from CD8.
  • the transmembrane domain can comprise the amino acid sequence shown in SEQ ID NO:45.
  • the transmembrane domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the chimeric antigen receptor may comprise a co-stimulatory signaling domain.
  • the co-stimulatory signaling domain may comprise, but is not limited to, a co-stimulatory signaling domain derived from a protein selected from the group consisting of: CD28, CD137, 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, ligands for CD83, CD40 and MyD88.
  • the costimulatory signaling domain may comprise a costimulatory signaling domain derived from CD137.
  • the co-stimulatory signaling domain may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the co-stimulatory signaling domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 46). %, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the chimeric antigen receptor may comprise an intracellular signaling domain.
  • the intracellular signaling domain may comprise an intracellular signaling domain derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia Viral gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • the intracellular signaling domain can comprise an intracellular signaling domain derived from CD3zeta.
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:47.
  • the intracellular signaling domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 47) %, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the N-terminus of the transmembrane domain can be linked to the C-terminus of the targeting moiety.
  • the C-terminus of the transmembrane domain can be linked to the N-terminus of the co-stimulatory signaling domain.
  • the C-terminus of the co-stimulatory signaling domain may be linked to the N-terminus of the intracellular signaling domain.
  • the chimeric antigen receptor may sequentially comprise the following domains from N-terminus to C-terminus: targeting moiety, transmembrane domain, co-stimulatory signaling domain and intracellular signaling domain.
  • the chimeric antigen receptor may comprise the following domains sequentially from the N-terminus to the C-terminus: the antigen binding protein of the present application (for example, VHH), the transmembrane domain derived from CD8, the co-stimulatory domain derived from CD137 Signaling domain and intracellular signaling domain derived from CD3zeta.
  • the antigen binding protein of the present application for example, VHH
  • the transmembrane domain derived from CD8 the co-stimulatory domain derived from CD137 Signaling domain
  • intracellular signaling domain derived from CD3zeta.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:38 to SEQ ID NO:44.
  • the chimeric antigen receptor can comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence set forth in SEQ ID NO: 38 to SEQ ID NO: 44 , 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the application provides one or more polypeptides, which may comprise an isolated antigen binding protein of the application.
  • the polypeptide can include a fusion protein.
  • the polypeptides can include multispecific antibodies (eg, bispecific antibodies).
  • the application provides one or more immunoconjugates, which may comprise an isolated antigen binding protein of the application.
  • the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, label and/or detection agent.
  • the present application also provides one or more isolated nucleic acid molecules that encode the isolated antigen-binding protein or polypeptide described herein.
  • each of the one or more nucleic acid molecules may encode the entirety of the antigen binding protein, or may encode a portion thereof (e.g., HCDR1-3, one of the heavy chain variable regions, or variety).
  • the one or more nucleic acid molecules may encode a chimeric antigen receptor described herein.
  • the nucleic acid molecule may comprise the nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 37.
  • the products encoded by the nucleic acid molecules together can form a functional (eg, CD22-binding) isolated antigen-binding protein of the present application.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
  • nucleic acid encoding the isolated antigen-binding protein can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the isolated antigen-binding protein described in the application. Nucleic acid molecule of protein or polypeptide.
  • the present application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • Each vector may contain one or more such nucleic acid molecules.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host.
  • control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequences are regulatable elements.
  • the specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may comprise a promoter region which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, CMV promoters, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's Portions may be fused to portions of gene promoters of other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector may be an expression vector.
  • the vector can be a viral vector.
  • Viral vectors may be administered directly to the patient (in vivo) or may be indirect, for example, in vitro by treating cells with virus and then administering the treated cells to the patient (ex vivo).
  • Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers.
  • Lentiviral vectors may contain long terminal repeat 5'LTR and truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and/or post-translational regulatory element (WPRE).
  • the vectors described herein can be introduced into cells.
  • the present application provides a cell.
  • the cells may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules and/or one or more carriers described herein .
  • each or each cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each cell may contain multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into said host cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293T cells, 293F cells or HEK293A cells, or other eukaryotic cells, Such as cells from plants, fungal or yeast cells, etc.
  • the vectors described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.
  • the cells can include yeast cells.
  • the cells may include E. coli cells.
  • the cells can include mammalian cells.
  • the cells can include immune cells.
  • the cells may include immune cells.
  • the cells may include immune cells.
  • the cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells cell.
  • the cells can include T cells.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and/or Or pharmaceutically acceptable adjuvants and/or excipients.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional media or reagents are contemplated for use in the pharmaceutical compositions of the present application.
  • the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, and may also be referred to as auxiliary materials.
  • the excipients may include binders, fillers, disintegrants, lubricants in tablets.
  • the excipients may include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills.
  • the excipient may comprise the base part of a semi-solid formulation ointment, cream.
  • the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.
  • the present application provides a method for detecting or measuring CD22, which may include using the isolated antigen-binding protein or the polypeptide.
  • the methods may include in vitro methods, ex vivo methods, methods 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 steps of:
  • kits for CD22 which may include the use of the isolated antigen-binding protein or the polypeptide.
  • the kit may further include instructions for use, which describe the method for detecting the presence and/or content of CD22.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the present application provides a use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit, and the kit can be used in a method for detecting the presence and/or content of CD22.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, and the pharmaceutical composition for preventing, alleviating and/or treat a disease or condition.
  • the kit and/or the drug combination are used to prevent, alleviate and/or treat diseases or conditions.
  • the disease or condition can include a tumor.
  • the tumor can include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to the altered expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared with 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 up-regulated compared with 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 higher.
  • the tumor can include a hematoma.
  • the tumor can include lymphoma.
  • the tumor can include leukemia.
  • the application provides a kind of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said carrier, said cell and /or the use of the pharmaceutical composition in the preparation of medicaments for preventing, alleviating and/or treating diseases or conditions.
  • the present application provides a use of a drug combination in the preparation of a drug for preventing, alleviating and/or treating a disease or condition.
  • the disease or condition can include a tumor.
  • the tumor can include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to the altered expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared with 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 up-regulated compared with normal cells.
  • the tumor associated with the expression of CD22 protein 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 higher.
  • the tumor can include a hematoma.
  • the tumor can include lymphoma.
  • the tumor can include leukemia.
  • the present application provides a method for preventing and/or treating a disease or disorder, comprising administering the isolated antigen-binding protein, the isolated nucleic acid molecule, the The carrier, the cell, the pharmaceutical composition.
  • the disease or condition can include a tumor.
  • the tumor can include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to the altered expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared with 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 up-regulated compared with normal cells.
  • the tumor associated with the expression of CD22 protein 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 higher.
  • the tumor can include a hematoma.
  • the tumor can include lymphoma.
  • the tumor can include leukemia.
  • compositions and methods described herein can be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like.
  • the pharmaceutical compositions and methods described in this application can be used in other disease conditions that depend on immune responses, such as inflammation, immune diseases and infectious diseases.
  • the subject may include humans or non-human animals.
  • the non-human animal can be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats.
  • non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the human can be Caucasian, African, Asian, Semitic, or other ethnicity, or a hybrid of various ethnicities.
  • the human can be elderly, adult, adolescent, child or infant.
  • the effective amount in humans can be inferred from the effective amount in experimental animals.
  • Freireich et al. describe the correlation of doses in animals and humans (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, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
  • the application may include the following implementations:
  • chimeric antigen receptor it comprises targeting part, and described targeting part comprises HCDR3, and described HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:21.
  • chimeric antigen receptor according to embodiment 1, wherein the targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:12.
  • chimeric antigen receptor according to any one of embodiments 1-2, wherein the targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:6.
  • chimeric antigen receptor according to any one of embodiments 1-3, wherein the targeting moiety comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:65.
  • chimeric antigen receptor according to any one of embodiments 1-4, wherein said targeting moiety comprises a heavy chain variable shown in any one of SEQ ID NO:27 to SEQ ID NO:30 HCDR1, HCDR2 and HCDR3 in the region.
  • chimeric antigen receptor according to any one of embodiments 1-5, wherein said targeting moiety comprises HCDR1, HCDR2, HCDR3, said HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 21;
  • chimeric antigen receptor according to any one of embodiments 1-6, wherein said targeting moiety comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly connected to the N-terminus of said HCDR1 ground connection, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:61.
  • H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:3.
  • chimeric antigen receptor according to any one of embodiments 1-8, wherein said targeting moiety comprises H-FR2, said H-FR2 being located between said HCDR1 and said HCDR2, and said Said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:62.
  • H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:10.
  • chimeric antigen receptor according to any one of embodiments 1-10, wherein said targeting moiety comprises H-FR3 located between said HCDR2 and said HCDR3, and said Said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:63.
  • H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 16 to SEQ ID NO: 18.
  • chimeric antigen receptor according to any one of embodiments 1-12, wherein said targeting moiety comprises H-FR4, the N-terminus of said H-FR4 is directly or indirectly connected to the C-terminus of said HCDR3 ground, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:54.
  • H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • H-FR1 comprising The amino acid sequence shown in SEQ ID NO:61;
  • the H-FR2 includes the amino acid sequence shown in SEQ ID NO:62;
  • the H-FR3 includes the amino acid sequence shown in SEQ ID NO:63;
  • the H- FR4 comprises the amino acid sequence shown in SEQ ID NO:54.
  • H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; said H-FR2 comprises SEQ ID NO: 1 The amino acid sequence shown in any one of ID NO:7 and SEQ ID NO:10; The H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:16 to SEQ ID NO:18; And the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • H-FR1, H-FR2, H-FR3 and H-FR4 comprise any set of amino acid sequences selected from the group consisting of :
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:16
  • H-FR4 SEQ ID NO:22;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:10, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:7, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:18
  • H-FR4 SEQ ID NO:23.
  • antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di - scFv, VHH and/or dAb.
  • transmembrane domain comprises a transmembrane domain 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.
  • 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, CD
  • co-stimulatory signaling domain comprises a co-stimulatory signaling domain derived from a protein selected from the group consisting of: CD28, CD137, 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, ligand of CD83, CD40 and MyD88.
  • a protein selected from the group consisting of: CD28, CD137, CD27, CD2 , CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, H
  • costimulatory signaling domain comprises a costimulatory signaling domain derived from CD137.
  • the intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of: CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , or a combination thereof , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10 and DAP-12.
  • a protein selected from the group consisting of: CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , or a combination thereof , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14
  • An isolated antigen binding protein that specifically binds a CD22 protein with a KD value of about 2E-08M or less.
  • the isolated antigen binding protein of embodiment 40 comprising HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:21.
  • the isolated antigen binding protein of any one of embodiments 40-44 comprising HCDR1 in the heavy chain variable region set forth in any one of SEQ ID NO:27 to SEQ ID NO:30, HCDR2 and HCDR3.
  • H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 1 and SEQ ID NO: 3.
  • H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:10.
  • the isolated antigen binding protein of any one of embodiments 40-50 comprising an H-FR3 located between said HCDR2 and said HCDR3, and said H-FR3 comprising Amino acid sequence shown in SEQ ID NO:63.
  • H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • H-FR1, H-FR2, H-FR3 and H-FR4 comprising SEQ ID NO:61
  • the amino acid sequence shown; the H-FR2 includes the amino acid sequence shown in SEQ ID NO:62; the H-FR3 includes the amino acid sequence shown in SEQ ID NO:63; and the H-FR4 includes the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 54.
  • H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:3; said H-FR2 comprises SEQ ID NO:1 The amino acid sequence shown in any one of ID NO:7 and SEQ ID NO:10;
  • the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:16 to SEQ ID NO:18;
  • the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:22 to SEQ ID NO:23.
  • H-FR1, H-FR2, H-FR3 and H-FR4 comprise any set of amino acid sequences selected from the group consisting of :
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:16
  • H-FR4 SEQ ID NO:22;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:10, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3, H-FR2: SEQ ID NO:7, H-FR3: SEQ ID NO:17 and H-FR4: SEQ ID NO:23;
  • H-FR1 SEQ ID NO:3
  • H-FR2 SEQ ID NO:7
  • H-FR3 SEQ ID NO:18
  • H-FR4 SEQ ID NO:23.
  • VH comprises the amino acid sequence set forth in any one of SEQ ID NO:27 to SEQ ID NO:30.
  • the isolated antigen binding protein of any one of embodiments 40-59 comprising an antibody or antigen binding fragment thereof.
  • antigen binding protein of embodiment 60 wherein said antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di - scFv, VHH and dAb.
  • the isolated antigen binding protein of any one of embodiments 40-61 comprising a VHH or an antigen binding fragment thereof.
  • the isolated antigen binding protein of any one of embodiments 40-63 comprising the amino acid sequence set forth in any one of SEQ ID NO:27 to SEQ ID NO:30.
  • An immunoconjugate comprising the isolated antigen binding protein of any one of embodiments 40-64.
  • a vector comprising the isolated nucleic acid molecule of embodiment 67.
  • a cell comprising the chimeric antigen receptor of any one of embodiments 1-39, the isolated antigen binding protein of any one of embodiments 40-64, the polypeptide of embodiment 65, The immunoconjugate of embodiment 66, the isolated nucleic acid molecule of embodiment 67 and/or the vector of embodiment 68.
  • the cell according to embodiment 69 comprising an immune cell.
  • the cell according to embodiment 70, wherein said 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.
  • NK cells natural killer cells
  • NKT cells monocytes, dendritic cells
  • monocytes monocytes
  • dendritic cells granulocytes
  • lymphocytes lymphocytes
  • leukocytes and/or peripheral blood mononuclear cells.
  • a pharmaceutical composition comprising the chimeric antigen receptor of any one of embodiments 1-39, the isolated antigen binding protein of any one of embodiments 40-64, the chimeric antigen binding protein of any one of embodiments 65
  • a method for detecting CD22 protein comprising:
  • the isolated antigen binding protein of any one of embodiments 40-64, the polypeptide of embodiment 65 or the immunoconjugate of embodiment 66 is administered.
  • a CD22 protein detection kit comprising the isolated antigen binding protein of any one of embodiments 40-64, the polypeptide of embodiment 65 or the immunoconjugate of embodiment 66.
  • the immunoconjugate described above, the isolated nucleic acid molecule described in embodiment 67, the carrier described in embodiment 68 and/or the cell described in any one of embodiments 69-72 are used in the preparation of a method for preventing and/or treating tumors. Uses in medicine.
  • a method of preventing and/or treating a disease or condition comprising administering to a subject in need thereof an effective amount of the chimeric antigen receptor of any one of embodiments 1-39, embodiment 40-
  • Embodiment 1 alpaca immunization
  • a healthy adult female 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 on the left and right sides of the cervical lymph nodes;
  • 500 ⁇ g of recombinant human CD22 protein was emulsified with an equal volume of Freund’s complete or incomplete adjuvant.
  • the left and right sides near the lymph nodes were injected, and several booster immunizations were carried out. Blood was collected one week after the last immunization to monitor the titer of antiserum.
  • the alpaca peripheral blood was collected, and lymphocytes were separated by lymphocyte separation medium; total RNA was extracted by TRIzol TM reagent; cDNA was obtained by reverse transcription using PrimeScript TM II first-strand cDNA synthesis kit, and nested PCR amplification VHH gene.
  • the VHH gene fragment was recovered with a gel purification kit, digested and digested with restriction endonuclease Bgl 1, and then cloned into the phagemid vector pADL-10b, and the constructed clone product was transformed into an E.coli TG1 electroporation State cells were used to construct a 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 insertion rate of the library was 97.9%. Take live cells with 10-100 times the library capacity from the above-mentioned gene library for inoculation and culture, and use M13K07 phage to rescue after culturing to the logarithmic phase. After the rescue culture, collect the phage by centrifugation, and use PEG-NaCl to purify the phage to obtain phage display. Libraries can be used directly for subsequent screening.
  • Embodiment 3 Phage display screens the antigen-binding protein of the present application
  • Recombinant human CD22 (11958-H08H1-B, Beijing Sino Biological Technology Co., Ltd.) and recombinant monkey CD22 (90246-C08H, Beijing Sino Biological Science and Technology Co., Ltd.) were coated into 96-well plates and screened by Elisa assay In 3-5 rounds, CD22-specific phages were gradually enriched. A large number of positive clones were selected for Elisa detection, and the positive clones were screened and sequenced. The unique clones were determined according to sequence alignment and their sequences were divided into framework region FR and complementarity determining region CDR. Through the above methods, a total of 4 high-affinity single-domain antibodies (sdAbs) targeting human CD22 were obtained. These four antibodies were named B010-B-22Nb-01, B010-B-22Nb-02, B010-B-22Nb-03 and B010-B-22Nb-04 respectively.
  • sdAbs high-affinity single-domain antibodies
  • the selected biologically active antibody can optionally be humanized.
  • the humanization of camel monoclonal antibodies is carried out according to the methods published in many documents in this field.
  • human antibody constant domains can be used to replace the parental (camel antibody) constant domains
  • human germline antibody sequences can be selected according to the homology of camel antibodies and human antibodies, and CDR grafting can be performed.
  • the constant region of the camel antibody can be replaced by a human constant region through the back mutation of the amino acid residues of VH to obtain the final humanized binding protein 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 the antigen-binding protein of the present application.
  • CD22 protein source: Beijing Sino Biological Science and Technology Co., Ltd., catalog number: 11958-H08H1-B
  • Inject Anti-hFc or anti-mouse Fc antibody (diluted in pH 4.5 sodium acetate solution, concentration 20 ⁇ g/mL) at a flow rate of 10 ⁇ L/min for 200 seconds, and finally block the chip with 1M ethanolamine hydrochloride (pH 8.5) Excess reactive carboxyl groups. Wash the surface of the chip with 1 ⁇ HBS-EP+ at a flow rate of 10 ⁇ L/min for 2 hours to stabilize the baseline, and set the temperature of the instrument at 25°C.
  • the initial cycle consisting of two steps of sampling and regeneration, was repeated 3 times before measurement to stabilize the baseline.
  • Sample measurement Inject 1 ⁇ HBS-EP+buffer solution 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 seconds, stabilize for 30 seconds.
  • the running buffer for kinetic determination is 1 ⁇ HBS-EP+(pH7.4) solution.
  • Capture Inject different antibodies into the test channels of channels 1-8 of the Anti-hFc or anti-mouse Fc chip respectively, and capture at a flow rate of 10 ⁇ L/min for 60 s.
  • Antigen CD22 protein (source: Beijing Sino Biological Technology Co., Ltd., catalog number: 11958-H08H1-B) was diluted to 100 nM 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 is used to remove background signals; the binding and dissociation times of antigen and antibody are 180 and 400 seconds, respectively.
  • Regeneration Inject 10 mM Glycine pH 1.5 into channels 1-8 at a flow rate of 30 ⁇ L/min for 30 seconds, 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) is used for background subtraction.
  • Biacore was used to detect the binding affinities of different antigen-binding proteins to antigen proteins.
  • the ability of the CD22 antigen binding protein to bind to human CD22 expressed on the surface of K562 cells was determined based on a flow cytometry assay.
  • the binding ability of different CD22 antigen binding proteins of the present application was determined by comparing the binding curves of human CD22 expressed on the surface of K562 cells.
  • 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 a 96-well plate.
  • the antigen-binding protein of the present application has binding activity to human CD22 on the surface of K562 cells, and is better than the positive control antibody.
  • the binding curve of the antigen-binding protein of the present application to human CD22 on the surface of K562 cells was determined based on flow cytometry.
  • chimeric antigen receptors targeting CD22 specifically includes: single domain antibody VHH sequences targeting human CD22, CD8 transmembrane domain, CD137 co-stimulatory signaling domain and CD3zeta intracellular signaling domain, which are sequentially connected in series way to connect.
  • CD22-CAR-01 amino acid sequence as shown in SEQ ID NO: 38, nucleic acid sequence as shown in SEQ ID NO: 31
  • CD22-CAR-02 Amino acid sequence as shown in SEQ ID NO:39, nucleic acid sequence as shown in SEQ ID NO:32
  • CD22-CAR-03 amino acid sequence as shown in SEQ ID NO:40, nucleic acid sequence as shown in SEQ ID NO:33
  • CD22-CAR-04 amino acid sequence as shown in SEQ ID NO: 41, nucleic acid sequence as shown in SEQ ID NO: 34.
  • m971 is the positive control.
  • luciferase detection reagent Promega, E6120
  • a microplate reader was used to detect the chemiluminescence value (RLU) of the 96-well plate.
  • Example 8 Whole gene synthesis of CD22 chimeric antigen receptor molecule and construction of lentiviral expression vector, packaging virus
  • the obtained humanized CD22 antigen-binding proteins were constructed on lentiviral vectors to screen for more effective chimeric antigen receptors targeting CD22.
  • the construction of a humanized chimeric antigen receptor targeting CD22 specifically includes: a single domain antibody VHH sequence targeting human CD22, a CD8 transmembrane domain, a CD137 co-stimulatory signaling domain and a CD3zeta intracellular signaling domain, They are connected sequentially in series.
  • the obtained chimeric antigen receptor molecules (CAR molecules) were named CD22-CAR-04-H4 (amino acid sequence as shown in SEQ ID NO:42, nucleic acid sequence as shown in SEQ ID NO:35), CD22-CAR-04-H4, respectively.
  • the chimeric antigen receptor gene sequence was synthesized by Jinweizhi Company and cloned into the pLVX-EF1a-IRES-Puro vector between EcoRI and BamHI restriction sites.
  • the constructed lentiviral plasmid and packaging plasmids Pspax2 and pMD2.G were transfected into 293T cells with PEI transfection reagent, and after culturing for a certain period of time, the virus was collected and concentrated for later use.
  • CAR-T cells which are named CD22-CAR-04-H4, CD22-CAR-04-H5, CD22-CAR-04-H5, CD22-CAR-04-H6.
  • Firefly luciferase was transferred to Raji and Nalm6 to obtain Raji-LUC and Nalm6-LUC cells.
  • Figure 3 shows the flow cytometric detection of the binding of humanized CD22-CAR-04 CART cells to CD22 protein
  • Figure 4 and Figure 5 show the killing effect of humanized CD22-CAR-04 CART cells on Raji-LUC and Nalm6-LUC cells.
  • CD22-CAR-04-H4 76.75 71,082.45 CD22-CAR-04-H5 77.86 114,189.71 CD22-CAR-04-H6 76.65 105,875.28 CD22-CAR-04 86.89 161,377.29
  • the self-constructed target tumor cells Raji-LUC expressing luciferase were inoculated into NDG mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), and injected into the tail vein to form tumors. 2.5 ⁇ 10 5 above-mentioned target cells were resuspended in 200 ⁇ l serum-free medium, and injected into the tail vein of mice.
  • the luciferase substrate D-Luciferin was injected intraperitoneally into the mice at a dosage of 3 mg/mouse. After the mice were anesthetized, they were placed in a small animal in vivo imager for imaging. After 5 days of tumor formation in the Raji-LUC mouse model, T lymphocytes expressing CD22-CAR-04, CD22-CAR-04-H4, CD22-CAR-04-H5, and CD22-CAR-04-H6 chimeric antigen receptors After the cells were washed with PBS, they were resuspended in serum-free medium, and the cell density was adjusted to 1.5 ⁇ 10 7 cells/ml.
  • mice were injected with 3 ⁇ 106 cells into the tail vein, 200 ⁇ l in total. Mice treated with T cells not transfected with CAR were set as the control group. After the experiment was completed, regular in vivo imaging tests were performed twice a week to collect tumor elimination effects.
  • Figures 6 and 7 show the results of in vivo imaging of Raji-LUC mice, and the imaging results reflect the number of tumor cells.
  • the results showed that compared with the non-transfected CAR T cell group with increasing bioluminescence intensity, those transfected with CD22-CAR-04, CD22-CAR-04-H4, CD22-CAR-04-H5, CD22-CAR-
  • the number of tumor cells in the mice in the 04-H6 T cell group was significantly reduced, and the CD22-CAR-04-H4 CAR-T group had the best tumor inhibition effect, and the tumor inhibition rate on Day 17 after administration was about 99.73%.
  • Figure 8 shows the results of the body weight changes of Raji-LUC mice. The results showed that the body weight of the mice fluctuated stably after administration, and no animals died during the treatment period. There was no obvious drug toxicity and the drug was well tolerated.
  • the self-constructed target tumor cell Nalm6-LUC expressing luciferase was inoculated into NSG mice (purchased from Shanghai Nguiding Model Biotechnology Co., Ltd.), and injected into the tail vein to form tumors. 2.5 ⁇ 10 5 above-mentioned target cells were resuspended in 200 ⁇ l serum-free medium, and injected into the tail vein of mice.
  • T lymphocytes expressing CD22-CAR-04, CD22-CAR-04-H4, CD22-CAR-04-H5, CD22-CAR-04-H6 chimeric antigen receptors After the cells were washed with PBS, they were resuspended in serum-free medium, and the cell density was adjusted to 1.5 ⁇ 10 7 cells/ml.
  • mice were injected with 3 ⁇ 106 cells into the tail vein, 200 ⁇ l in total. Mice treated with T cells not transfected with CAR were set as the control group. After the experiment was completed, regular in vivo imaging tests were performed twice a week to collect tumor elimination effects.
  • Figure 9 and Figure 10 show the results of in vivo imaging of Naml6-LUC mice.
  • the results showed that the fluorescence intensity of mice in the T cell group without CAR transfection increased rapidly, and the mice transfected with CD22-CAR-04, CD22-CAR-04-
  • Figure 11 shows the results of body weight changes in Nalm6-LUC mice. After administration, the body weight of the mice was normal, and no animal died during the treatment period, showing no obvious drug toxicity and being well tolerated.

Abstract

一种靶向CD22的抗原结合蛋白及其用途，尤其涉及一种包含该抗原结合蛋白的嵌合抗原受体及其应用，所述嵌合抗原受体包含靶向部分，所述靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列。还提供了包含或表达该嵌合抗原受体的细胞。所述抗原结合蛋白以约2E-08M或以下的K D值与CD22蛋白结合。所述嵌合抗原受体可以特异性结合CD22蛋白。

Description

靶向CD22的抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向CD22的抗原结合蛋白及其用途。
背景技术
近年来，随着肿瘤免疫疗法的发展和临床研究的进展，嵌合抗原受体T细胞(Chimeric Antigen Receptor-T cells，CAR-T)免疫疗法以其独特的抗肿瘤优势，目前是最有发展前景的肿瘤免疫疗法之一。CAR-T细胞是由单克隆抗体的单链可变区和T细胞信号转导区连接而成，抗体与相应的肿瘤抗原结合后能以主要组织相容性复合体以非限制性方式使T细胞活化，进而发挥抗肿瘤效应。CAR的结构分为：胞外抗原结合区、铰链区、跨膜结构域和胞内信号传导结构域。目标抗原的选择对于CAR的特异性、有效性以及基因改造T细胞自身的安全性来讲都是关键的决定因素。
肿瘤表面抗原CD22是分子量为140kDa的糖蛋白，是唾液酸结合免疫球蛋白样凝集素家族的一员，有几个Ig样的胞外区。它限制性表达在B细胞恶性肿瘤与正常B细胞中，60-80％的B细胞恶性肿瘤表达CD22。几乎所有的B前体细胞急性淋巴细胞白血病(B-ALL)表达CD22，90％以上的弥漫大B细胞淋巴瘤(DLBCL)和滤泡淋巴瘤(FL)表达CD22；慢性淋巴细胞白血病(B-CLL)、毛细胞白血病(HCL)也具有高水平的CD22表达。因此CD22是CAR-T治疗恶性血液肿瘤疗法中，继CD19之外的又一理想靶点，给肿瘤的免疫治疗带来了新的方向。
目前的CAR-T疗法通常以来源于单克隆抗体抗原结合区域的scFv段作为抗原结合区。细胞外的scFv结构域在结合表达于靶细胞表面的靶蛋白以后，可以激活CAR结构的共刺激域和激活域。但是scFv分子量比较大而且容易形成多聚体，影响CAR的功能。因此，亟需包含结构新颖的抗原结合区的CAR来***。
发明内容
本申请提供了一种靶向CD22的抗原结合蛋白，尤其涉及一种包含该抗原结合蛋白的嵌合抗原受体。所述抗原结合蛋白具有下述性质的一种或多种：1)能够特异性识别人CD22。所述嵌合抗原受体具有下述性质的一种或多种：1)能够特异性识别人CD22；2)能够有效杀伤肿瘤细胞，例如血液瘤。
在某些实施方式中，所述嵌合抗原受体包含靶向部分，所述靶向部分包含HCDR3，所述 HCDR3包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:49所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含SEQ ID NO:55所示的重链可变区中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述靶向部分包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的重链可变区中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述靶向部分包含HCDR1、HCDR2、HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；所述HCDR2包含SEQ ID NO:49所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列；所述靶向部分中的HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列；以及所述靶向部分中的HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的HCDR1、HCDR2和HCDR3包含选自下述的任意一组的氨基酸序列：
a)HCDR1：SEQ ID NO:4，HCDR2：SEQ ID NO:11，和HCDR3：SEQ ID NO:19；
b)HCDR1：SEQ ID NO:5，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:20；和
c)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:21。
在某些实施方式中，所述靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:51所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的H-FR1包含SEQ ID NO:1至SEQ ID NO:3中任 一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的H-FR2包含SEQ ID NO:7至SEQ ID NO:10中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:53所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的H-FR3包含SEQ ID NO:13至SEQ ID NO:18中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:51所示的氨基酸序列；所述H-FR2包含SEQ ID NO:52所示的氨基酸序列；所述H-FR3包含SEQ ID NO:53所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的H-FR1包含SEQ ID NO:1至SEQ ID NO:3中任一项所示的氨基酸序列；所述靶向部分中的H-FR2包含SEQ ID NO:7至SEQ ID NO:10中任一项所示的氨基酸序列；所述靶向部分中的H-FR3包含SEQ ID NO:13至SEQ ID NO:18中任一项所示的氨基酸序列；以及所述靶向部分中的H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分中的H-FR1、H-FR2、H-FR3和H-FR4包含选自下述的任意一组氨基酸序列：
a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:13和H-FR4：SEQ ID NO:22；
b)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:8，H-FR3：SEQ ID NO:14和H-FR4：SEQ ID NO:22；
c)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:9，H-FR3：SEQ ID NO:15和H-FR4：SEQ ID NO:22；
d)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22；
e)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
f)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
g)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:18和H-FR4：SEQ ID NO:23。
在某些实施方式中，所述靶向部分包括抗体或抗原结合片段。
在某些实施方式中，所述抗原结合片段选自下组：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，所述靶向部分包括VHH。
在某些实施方式中，所述VHH靶向CD22。
在某些实施方式中，所述靶向部分包含SEQ ID NO:55所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包含跨膜结构域。
在某些实施方式中，所述跨膜结构域包含源自选自下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。
在某些实施方式中，所述跨膜结构域包括源自CD8的跨膜结构域。
在某些实施方式中，所述跨膜结构域包含SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述跨膜结构域的N端与所述靶向部分的C端连接。
在某些实施方式中，所述嵌合抗原受体包含共刺激信号传导结构域。
在某些实施方式中，所述共刺激信号传导结构域包含源自选自下述蛋白的共刺激信号传导结构域或其组合：CD28、CD137、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，所述共刺激信号传导结构域包括源自CD137的共刺激信号传导结构域。
在某些实施方式中，所述共刺激信号传导结构域包含如SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，所述共刺激信号传导结构域的N端与所述跨膜结构域的C端连接。
在某些实施方式中，所述嵌合抗原受体包含胞内信号传导结构域。
在某些实施方式中，所述胞内信号传导结构域包含源自选自下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。
在某些实施方式中，所述嵌合抗原受体中所述胞内信号传导结构域包括源自CD3zeta的胞内信号传导结构域。
在某些实施方式中，所述胞内信号传导结构域包含如SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述胞内信号传导结构域的N端与所述共刺激信号传导结构域的C端连接。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:38至SEQ ID NO:44中任一项所示的氨基酸序列。
另一方面，本申请提供了一种分离的抗原结合蛋白，其以约2E-08M或以下的K D值与CD22蛋白特异性结合。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:49所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含SEQ ID NO:55所示的重链可变区中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述分离的抗原结合蛋白包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的重链可变区中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；所述HCDR2包含SEQ ID NO:49所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR1、HCDR2和HCDR3包含选自下述的任意一组的氨基酸序列：
a)HCDR1：SEQ ID NO:4，HCDR2：SEQ ID NO:11，和HCDR3：SEQ ID NO:19；
b)HCDR1：SEQ ID NO:5，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:20；和c)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:21。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:51所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR1包含SEQ ID NO:1至SEQ ID NO:3中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR2包含SEQ ID NO:7至SEQ ID NO:10中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:53所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR3包含SEQ ID NO:13至SEQ ID NO:18中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:51所示的氨基酸序列；所述H-FR2包含SEQ ID NO:52所示的氨基酸序列；所述H-FR3包含SEQ ID NO:53所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR1包含SEQ ID NO:1至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至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至SEQ ID NO:18中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述的任意一组氨基酸序列：
a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:13和H-FR4：SEQ ID NO:22；
b)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:8，H-FR3：SEQ ID NO:14和H-FR4：SEQ ID NO:22；
c)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:9，H-FR3：SEQ ID NO:15和H-FR4：SEQ ID NO:22；
d)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22；
e)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
f)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
g)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:18和H-FR4：SEQ ID NO:23。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ  ID NO:55所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述VH包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述分离的抗原结合蛋白中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和dAb。
在某些实施方式中，所述分离的抗原结合蛋白包括VHH或其抗原结合片段。
在某些实施方式中，所述分离的抗原结合蛋白中所述抗体选自下组：单克隆抗体、人源化抗体、嵌合抗体和全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
另一方面，本申请提供了一种多肽，其包含所述的嵌合抗原受体和/或所述分离的抗原结合蛋白。
另一方面，本申请提供了一种免疫缀合物，其包含所述分离的抗原结合蛋白。
另一方面，本申请提供了分离的核酸分子，其编码所述的嵌合抗原受体，所述的分离的抗原结合蛋白，或者所述的多肽。
另一方面，本申请提供了一种载体，其包含所述分离的核酸分子。
另一方面，本申请提供了一种细胞，其包含所述的嵌合抗原受体，所述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和/或所述的载体。
在某些实施方式中，所述的细胞包括免疫细胞。
在某些实施方式中，所述免疫细胞选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些实施方式中，所述细胞包括T细胞。
另一方面，本申请提供了一种制备所述的嵌合抗原受体，所述分离的抗原结合蛋白和/或所述的多肽的方法，所述方法包括在使得所述的分离的抗原结合蛋白和/或所述的多肽表达的条件下，培养所述的细胞。
另一方面，本申请提供了一种药物组合物，其包含所述的嵌合抗原受体，所述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，所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
另一方面，本申请提供了一种用于检测CD22蛋白的方法，其包括：
施用所述分离的抗原结合蛋白，所述的多肽或所述的免疫缀合物。
另一方面，本申请提供了一种CD22蛋白的检测试剂盒，其包含所述分离的抗原结合蛋白，所述的多肽或所述的免疫缀合物。
另一方面，本申请提供了所述分离的抗原结合蛋白，所述的多肽或所述的免疫缀合物在制备试剂盒中的用途，所述试剂盒用于检测CD22蛋白的存在和/或含量。
另一方面，本申请提供了所述的嵌合抗原受体，所述分离的抗原结合蛋白，所述的多肽和/或所述的免疫缀合物在制备预防和/或***的药物中的用途。
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，所述肿瘤包括与CD22的表达相关的肿瘤。
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，本申请提供了所述的嵌合抗原受体，所述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，和/或所述的细胞，其用于预防和/或***。
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，所述肿瘤包括与CD22的表达相关的肿瘤。
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有效量的所述的嵌合抗原受体，所述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，和/或所述的细胞。
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，所述肿瘤包括与CD22的表达相关的肿瘤。
在某些实施方式中，所述肿瘤选自下组：白血病和血液瘤。
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示例性的抗原结合蛋白与表达于K562细胞表面的人CD22的结合曲线。
图2显示的是本申请所述示例性的瞬转CD22-CAR质粒的Jurkat-NFAT与Raji作用后RLU检测结果。
图3显示的是本申请所述流式检测人源化CD22-CAR-04 CART细胞与CD22蛋白的结合结果。
图4显示的是本申请所述示例性的人源化CD22-CAR-04 CART细胞对Raji-LUC细胞的杀伤作用。
图5显示的是本申请所述示例性的人源化CD22-CAR-04 CART细胞对Nalm6-LUC细胞的杀伤作用。
图6显示的是Raji-LUC小鼠活体成像荧光强度检测结果。
图7显示的是Raji-LUC小鼠活体成像荧光图。
图8显示的是Raji-LUC小鼠体重变化结果。
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Nalm6-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，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“分离的抗原结合蛋白”通常是指能够特异性识别和/或中和特定抗原的多肽聚合体。例如，分离的抗原结合蛋白可以包括重链的一部分。例如，分离的抗原结合蛋白可以包括重链可变区。术语“分离的抗原结合蛋白”可以包括单域抗体。例如，分离的抗原结合蛋白可以包括但不限于人单域抗体。
在本申请中，术语“单域抗体”或“sdAb”或“VHH”通常是指缺失抗体轻链而只有重链可变区的一类抗体。在某些情形中，单域抗体可以来自双峰驼、单峰驼、羊驼、美洲驼、护士鲨、大星鲨或鳐鱼(例如，可参见康晓圳等，生物工程学报,2018,34(12):1974-1984)。 例如，单域抗体可以来自羊驼。单域抗体可由重链可变区(VH)构成。术语“重链可变区”通常是指抗原结合片段的重链的氨基末端结构域。重链可变区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在成为框架区(FR)的更保守的区域中。每个重链可变区可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链可变区含有与抗原(例如，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T细胞(CAR-T)的核心部件，其可包括靶向部分(例如，靶向肿瘤特异性抗原和/或肿瘤相关抗原)、信号肽、跨膜结构域、共刺激信号传导结构域和胞内信号传导结构域。在本申请中，所述CAR可以基于抗体的抗原(例如CD22)特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。关于CAR和CAR-T细胞的描述，可参见例如Sadelain M,Brentjens R,Rivi`ere I.The basicprinciples of chimeric antigen receptor design.Cancer Discov.2013；3(4):388-398；Turtle CJ,Hudecek M,Jensen MC,Riddell SR.Engineered T cells for anti-cancer therapy.Curr Opin Immunol.2012；24(5):633-639；Dotti G,Gottschalk S,Savoldo B,Brenner MK.Design and development of therapies using chimeric antigen receptor-expressing T cells.Immunol Rev.2014；257(1):107-126；以及WO2013154760、WO2016014789。
在本申请中，术语“共刺激信号传导结构域”通常是指可以提供免疫共刺激分子的胞内 结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。在某些情形中，共刺激信号传导结构域可以包含源自选自下组蛋白的共刺激信号传导结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，共刺激信号传导结构域可以包括源自CD137的共刺激信号传导结构域。
在本申请中，术语“胞内信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。通常，胞内信号传导结构域为用于指导蛋白质寻靶的任何一段连续的氨基酸序列。在某些情形中，胞内信号传导结构域可以包含源自选自下组的蛋白的胞内信号传导结构域或其组合：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、CD66d、CD5、CD22、FcRγ、FcRβ、FcRε、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒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，胞内信号传导结构域可以包括源自CD3zeta的胞内信号传导结构域。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体(全人源抗体)、人源化抗体、嵌合抗体、单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本文所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫***的多种细胞(例如，效应细胞)和经典补体***的第一成分(Clq)。
在本申请中，术语“抗原结合片段”通常是指抗体中发挥特异性结合抗原功能的一个或 多个片段。抗体的抗原结合功能可通过抗体的全长片段来实现。抗体的抗原结合功能也可通过以下来实现：包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的重链，或者，包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的轻链。(1)Fab片段，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“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“嵌合抗体”通常是指这样的抗体，其中每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别，而该链的其余区段则与另一物种中的相应序列同源。例如，轻链和重链的可变区均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。所述可变区具有易于制备的优点，并且其特异性不受与其组合的恒定区的来源的影响。同时，由于嵌合抗体的恒定区可来源于人类，因此嵌合在注射时抗体引发免疫应答的可能性会低于使用恒定区为非人来源的抗体。
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.,1988 Science 239:1534-1536；Riechmann,et al.,1988 Nature 332:323-337；Tempest,et al.,Bio/Technol 1991 9:266-271)，“高度加成”(hyperchimerization)，(Queen,et al.,1989 Proc Natl Acad Sci USA 86:10029-10033；Co,et al.,1991 Proc Natl Acad Sci USA 88:2869-2873；Co,et al.,1992 J Immunol 148:1149-1154)和“贴面”(veneering)，(Mark,et al.,“Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.”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。如果其他区域，例如铰链区和恒定区结构域也源自非人来源，则这些区域也可以被人源化。
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“免疫缀合物”通常是指所述其他治疗剂与所述分离的抗原结合蛋白缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述分离的抗原结合蛋白与靶细胞上的抗原的特异性结合，将所述其他治疗剂递送至靶细胞(例如，肿瘤细胞)。此外，所述抗原也可以由所述靶细胞分泌，并位于所述靶细胞外的间隙。
在本申请中，术语“K D”(同样地，“K D”或“K D”)通常指“亲和常数”或“平衡解离常数”，并指在滴定测量中在平衡时、或者通过将解离速率常数(kd)除以结合速率常数(ka)所获得的值。使用结合速率常数(ka)、解离速率常数(kd)和平衡解离常数(K D)表示结合蛋白(例如本申请所述的分离的抗原结合蛋白)对抗原(例如，CD22蛋白)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术提供了高灵敏度以及在生理缓冲液中在平衡时检查样品的能力。例如，可以通过Biacore(生物分子相互作用分析)测定(例如，可以从BIAcoreInternationalAB，aGEHealthcarecompany，Uppsala，瑞典获得的仪器)所述K D值，也可以使用其他实验途径和仪器例如Octet检测。另外，也可以使用可以从SapidyneInstruments(Boise，Idaho)获得的KinExA(动态排阻测定(KineticExclusionAssay)) 测定所述K D值，或者使用表面等离子共振仪(SPR)测定所述K D值。
在本申请中，术语“K562细胞”通常是指人类永生化骨髓性白血病细胞系。K562属于红白血病细胞系，具有恶性程度高、增殖速度快的特点。最初分离自一名处于急性期的53岁女性慢性骨髓性白血病(CML)患者的胸水。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子。所述载体可将***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***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体可以是当引入合适的细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体可包含慢病毒载体。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的嵌合抗原受体或本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的嵌合抗原受体或抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方式中，所述细胞可以是免疫细胞。例如，所述免疫细胞可以选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。例如，所述免疫细胞可以是T细胞。
在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。
在本申请中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换地使用，通常是指具有任何长度的氨基酸的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物。这些修饰可以包含：二 硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵(如与标记组分结合)。术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸，包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。
在本申请中，术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短干扰RNA(siRNA)、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进一步修饰。
除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括其功能性变体、衍生物、类似物、同源物及其片段。
术语“功能性变体”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、***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抗原”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，表达至少2种嵌合抗原受体(CAR)的核酸分子的抗原结合结构域靶向的抗原可以是CD22。
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
本申请所述分离的抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，其在Biacore测定中，可以以2E-08M或以下的K D值(例如，所述K D不高于约2E-08M、不高于约1.5E-08M、不高于约1E-08M、不高于约9E-09M、不高于约8E-09M、不高于约7E-09M、不高于约6E-09M、不高于约5E-09M、不高于约4E-09M、不高于约3E-09M、不高于约2E-09M、不高于约1E-09M或不高于5E-10M或以下)与人CD22蛋白特异性结合。
一方面，本申请提供一种分离的抗原结合蛋白，其可以包含抗体重链可变区VH中的至少一个CDR，所述VH可以包含SEQ ID NO:55所示的氨基酸序列。
例如，所述VH可以包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的HCDR可以通过任何形式划分，只要VH与SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列相同，以任何形式划分得到的HCDR都可落入本申请的保护范围内。
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***来确定，如CCG、Kabat、Chothia、IMGT、 AbM、North’s、综合考虑Kabat/Chothia等。这些编码***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***确定出CDR区。使用不同的编码***，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***；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，本申请所述分离的抗原结合蛋白通过IMGT编码***定义。
在本申请中，所述抗原结合蛋白可包含重链可变区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，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:48所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR3序列可根据IMGT编码***定义。例如，所述抗原结合蛋白包含HCDR3，所述HCDR3与SEQ ID NO:48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:48)，其中，X 15可以是E或R，X 17可以是D，G或N。
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR3序列可根据IMGT编码***定义。
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:49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***定义。例如，所述抗原结合蛋白包含HCDR2，所述HCDR2与SEQ ID NO:49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:49)，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至SEQ ID NO:12中任一项所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR2序列可根据IMGT编码***定义。
在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:50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***定义。例如，所述抗原结合蛋白包含HCDR1，所述HCDR1与SEQ ID NO:50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:50)，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至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***定义。
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:56所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR3序列可根据IMGT编码***定义。例如，所述抗原结合蛋白包含HCDR3，所述HCDR3与SEQ ID NO:56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 17。
ATDPWTDCSLDGRYEYX 17Y(SEQ ID NO:56)，其中，X 17可以是G或N。
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:19至SEQ ID NO:20中任一项所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR3序列可根据IMGT编码***定义。
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:49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***定义。例如，所述抗原结合蛋白包含HCDR2，所述HCDR2与SEQ ID NO:49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:49)，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至SEQ ID NO:12中任一项所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR2序列可根据IMGT编码***定义。
在本申请中，所述抗原结合蛋白的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***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 5和X 6。
GFPVX 5X 6YA(SEQ ID NO:57)，其中，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至SEQ ID NO:5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***定义。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:11所示的氨基酸序列；且所述HCDR3可包含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。
例如，所述抗原结合蛋白的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，所述抗原结合蛋白的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-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。
例如，所述抗原结合蛋白的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。
例如，所述抗原结合蛋白的VH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:51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:51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:51)，其中，X 1可以是A或E，X 16可以是G或R。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:1至SEQ ID NO:3中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR2可包含SEQ ID NO:52所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR2与SEQ ID NO:52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:52)，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，所述抗原结合蛋白的H-FR2可包含SEQ ID NO:7至SEQ ID NO:10中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR3可包含SEQ ID NO:53所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR3与SEQ ID NO:53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:53)，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，所述抗原结合蛋白的H-FR3可包含SEQ ID NO:13至SEQ ID NO:18中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR4可包含SEQ ID NO:54所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR4与SEQ ID NO:54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:54)，其中，X 3可以是L或Q，X 6可以是L或Q。
在本申请中，所述抗原结合蛋白的H-FR4可包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1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H-FR1与SEQ ID NO:58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 16。
AVQLVESGGGLVQPGX 16SLRLSCAAS(SEQ ID NO:58)，其中，X 16可以是G或R。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:1至SEQ ID NO:2中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR2可包含SEQ ID NO:59所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR2与SEQ ID NO:59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 15。
X 1AWFRQAPGKEREGX 15SC(SEQ ID NO:59)，其中，X 1可以是I或M，X 15可以是I 或V。
在本申请中，所述抗原结合蛋白的H-FR2可包含SEQ ID NO:7至SEQ ID NO:9中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR3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-FR3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 2，X 3，X 7，X 12和X 27。
YX 2X 3DSVX 7GRFTX 12SRDNAKNTVYLQMNX 27LKPEDTAVYYC(SEQ ID NO:60)，其中，X 2可以是D或Y，X 3可以是A或V，X 7可以是E或K，X 12可以是I或V，X 27可以是D或S。
在本申请中，所述抗原结合蛋白的H-FR3可包含SEQ ID NO:13至SEQ ID NO:15中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR4可包含SEQ ID NO:22中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:61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: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 1VQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:61)，其中，X 1可以是A或E。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:1和SEQ ID NO:3中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR2可包含SEQ ID NO:62所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR2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 4，X 11，X 12，X 14和X 17。
IAWX 4RQAPGKX 11X 12EX 14VSX 17(SEQ ID NO:62)，其中，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 17可以是C或Y。
在本申请中，所述抗原结合蛋白的H-FR2可包含SEQ ID NO:7和SEQ ID NO:10中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR3可包含SEQ ID NO:63所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR3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，X 20，X 21，X 29和X 30。
YYX 3DSVKGRFTISRDNAKNX 20X 21YLQMNSLX 29X 30EDTAVYYC(SEQ ID NO:63)，其 中，X 3可以是A或Q，X 20可以是S或T，X 21可以是L或V，X 29可以是E或R，X 30可以是D或P。
在本申请中，所述抗原结合蛋白的H-FR3可包含SEQ ID NO:16至SEQ ID NO:18中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR4可包含SEQ ID NO:54所示的氨基酸序列。例如，所述抗原结合蛋白的H-FR4与SEQ ID NO:54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:54)，其中，X 3可以是L或Q，X 6可以是L或Q。
在本申请中，所述抗原结合蛋白的H-FR4可包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:51所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:52所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:53所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:58所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:59所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:60所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:61所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:62所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:63所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:1至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至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至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至SEQ ID NO:23中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:13所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22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H-FR1-4的抗原结合片段。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:8所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:14所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22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H-FR1-4的抗原结合片段。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:15所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22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-03或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:16所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22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或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:10所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；且所述H-FR4可包含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-H4或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；且所述H-FR4可包含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-H5或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:18所示的氨基酸序列；且所述H-FR4可包含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，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO:55所示的氨基酸序列。例如，所述抗原结合蛋白包含VH，所述VH与SEQ ID NO:55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 16，X 28，X 30，X 31，X 34，X 37，X 44，X 45，X 47，X 48，X 50，X 53，X 60，X 61，X 65，X 70，X 78，X 79，X 85，X 87，X 88，X 111，X 113，X 117，X 120。
X 1VQLVESGGGLVQPGX 16SLRLSCAASGFX 28VX 30X 31YAX 34AWX 37RQAPGKX 44X 45EX 47X 48SX 50ISX 53RDGNTYX 60X 61DSVX 65GRFTX 70SRDNAKNX 78X 79YLQMNX 85LX 87X 88EDTAVYYCATDPWTDCSLDGRYX 111YX 113YWGX 117GTX 120VTVSS(SEQ ID NO：55)，其中，X 1可以是A或E，X 16可以是G或R，X 28可以是P或S，X 30可以是A或D，X 31可以是D或G， X 34可以是I或M，X 37可以是F或V，X 44可以是E或G，X 45可以是L或R，X 47可以是G或W，X 48可以是I或V，X 50可以是C或Y，X 53可以是G或S，X 60可以是D或Y，X 61可以是A，Q或V，X 65可以是E或K，X 70可以是I或V，X 78可以是S或T，X 79可以是L或V，X 85可以是D或S，X 87可以是E，K或R，X 88可以是D或P，X 111可以是E或R，X 113可以是D，G或N，X 117可以是L或Q，X 120可以是L或Q。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO:64所示的氨基酸序列。例如，所述抗原结合蛋白包含VH，所述VH与SEQ ID NO:64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 16，X 30，X 31，X 34，X 48，X 53，X 60，X 61，X 65，X 70，X 85，X 113。
AVQLVESGGGLVQPGX 16SLRLSCAASGFPVX 30X 31YAX 34AWFRQAPGKEREGX 48SCISX 53RDGNTYX 60X 61DSVX 65GRFTX 70SRDNAKNTVYLQMNX 85LKPEDTAVYYCATDPWTDCSLDGRYEY X 113YWGLGTQVTVSS(SEQ ID NO:64)，其中，X 16可以是G或R，X 30可以是A或D，X 31可以是D或G，X 34可以是I或M，X 48可以是I或V，X 53可以是G或S，X 60可以是D或Y，X 61可以是A或V，X 65可以是E或K，X 70可以是I或V，X 85可以是D或S，X 113可以是G或N。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO:65所示的氨基酸序列。例如，所述抗原结合蛋白包含VH，所述VH与SEQ ID NO:65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 37，X 44，X 45，X 47，X 50，X 61，X 78，X 79，X 87，X 88，X 117和X 120。
X 1VQLVESGGGLVQPGGSLRLSCAASGFSVDDYAIAWX 37RQAPGKX 44X 45EX 47VSX 50ISSRDGNTYYX 61DSVKGRFTISRDNAKNX 78X 79YLQMNSLX 87X 88EDTAVYYCATDPWTDCSLDGRYRYDYWGX 117GTX 120VTVSS(SEQ ID NO:65)，其中，X 1可以是A或E，X 37可以是F或V，X 44可以是E或G，X 45可以是L或R，X 47可以是G或W，X 50可以是C或Y，X 61可以是A或Q，X 78可以是S或T，X 79可以是L或V，X 87可以是E或R，X 88可以是D或P，X 117可以是L或Q，X 120可以是L或Q。
在本申请中，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:11所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:19所示的氨基酸 序列。例如，所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:13所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:24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或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:8所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:14所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:25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或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:15所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:26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-03或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:16所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:27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或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以 及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:10所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:28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-H4或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:29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-H5或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:18所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:30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或与其具有相同重链可变区的抗原结合蛋白。
在本申请中，所述分离的抗原结合蛋白可以与参比抗体竞争结合人CD22。
在本申请中，所述参比抗体可包含重链可变区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，所述参比抗体的HCDR3可包含SEQ ID NO:48所示的氨基酸序列。例如，所述参比抗体的HCDR3序列可根据IMGT编码***定义。例如，所述参比抗体包含HCDR3，所述HCDR3与SEQ ID NO:48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:48)，其中，X 15可以是E或R，X 17可以是D，G或N。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。例如，所述参比抗体的HCDR3序列可根据IMGT编码***定义。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO:49所示的氨基酸序列。例如，所述参比抗体的HCDR2序列可根据IMGT编码***定义。例如，所述参比抗体包含HCDR2，所述HCDR2与SEQ ID NO:49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:49)，其中，X 3可以是G或S。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。例如，所述参比抗体的HCDR2序列可根据IMGT编码***定义。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO:50所示的氨基酸序列。例如，所述参比抗体的HCDR1序列可根据IMGT编码***定义。例如，所述参比抗体包含HCDR1，所述HCDR1与SEQ ID NO:50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:50)，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，所述参比抗体的HCDR1可包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。例如，所述参比抗体的HCDR1序列可根据IMGT编码***定义。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO:56所示的氨基酸序列。例如，所述参比抗体的HCDR3序列可根据IMGT编码***定义。例如，所述参比抗体包含HCDR3，所述HCDR3与SEQ ID NO:56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 17。
ATDPWTDCSLDGRYEYX 17Y(SEQ ID NO:56)，其中，X 17可以是G或N。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO:19至SEQ ID NO:20中任一项所示的氨基酸序列。例如，所述参比抗体的HCDR3序列可根据IMGT编码***定义。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO:49所示的氨基酸序列。例如，所述参比抗体的HCDR2序列可根据IMGT编码***定义。例如，所述参比抗体包含HCDR2，所述HCDR2与SEQ ID NO:49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:49)，其中，X 3可以是G或S。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。例如，所述参比抗体的HCDR2序列可根据IMGT编码***定义。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO:57所示的氨基酸序列。例如，所述参比抗体的HCDR1序列可根据IMGT编码***定义。例如，所述参比抗体包含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 5和X 6。
GFPVX 5X 6YA(SEQ ID NO:57)，其中，X 5可以是A或D，X 6可以是D或G。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO:4至SEQ ID NO:5所示的氨基酸序列。例如，所述参比抗体的HCDR1序列可根据IMGT编码***定义。
例如，所述参比抗体的HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:11所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:19所示的氨基酸序列。例如，所述参比抗体可包括抗体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。
例如，所述参比抗体的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，所述参比抗体可包括抗体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，所述参比抗体的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，所述参比抗体可包括抗体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。
例如，所述参比抗体的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，所述参比抗体可包括抗体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。
嵌合抗原受体
在本申请中，所述嵌合抗原受体包含靶向部分。所述靶向部分可特异性结合CD22。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区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靶向部分的HCDR3可包含SEQ ID NO:48所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR3序列可根据IMGT编码***定义。例如，所述嵌合抗原受体的靶向部分包含HCDR3，所述HCDR3与SEQ ID NO:48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:48)，其中，X 15可以是E或R，X 17可以是D，G或N。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR3可包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR3序列可根据IMGT编码***定义。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR2可包含SEQ ID NO:49所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR2序列可根据IMGT编码***定义。例如，所述嵌合抗原受体的靶向部分包含HCDR2，所述HCDR2与SEQ ID NO:49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:49)，其中，X 3可以是G或S。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR2可包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR2序列可根据IMGT编码***定义。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR1可包含SEQ ID NO:50所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR1序列可根据IMGT编码***定义。例如，所述嵌合抗原受体的靶向部分包含HCDR1，所述HCDR1与SEQ ID NO:50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:50)，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，所述嵌合抗原受体的靶向部分的HCDR1可包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR1序列可根据IMGT编码***定义。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR3可包含SEQ ID NO:56所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR3序列可根据IMGT编码***定义。 例如，所述嵌合抗原受体的靶向部分包含HCDR3，所述HCDR3与SEQ ID NO:56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 17。
ATDPWTDCSLDGRYEYX 17Y(SEQ ID NO:56)，其中，X 17可以是G或N。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR3可包含SEQ ID NO:19至SEQ ID NO:20中任一项所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR3序列可根据IMGT编码***定义。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR2可包含SEQ ID NO:49所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR2序列可根据IMGT编码***定义。例如，所述嵌合抗原受体的靶向部分包含HCDR2，所述HCDR2与SEQ ID NO:49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:49)，其中，X 3可以是G或S。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR2可包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR2序列可根据IMGT编码***定义。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR1可包含SEQ ID NO:57所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR1序列可根据IMGT编码***定义。例如，所述嵌合抗原受体的靶向部分包含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 5和X 6。
GFPVX 5X 6YA(SEQ ID NO:57)，其中，X 5可以是A或D，X 6可以是D或G。
在本申请中，所述嵌合抗原受体的靶向部分的HCDR1可包含SEQ ID NO:4至SEQ ID NO:5所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的HCDR1序列可根据IMGT编码***定义。
例如，所述嵌合抗原受体的靶向部分的HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:11所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:19所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体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。
例如，所述嵌合抗原受体的靶向部分的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，所述嵌合抗原受体的靶向部分可包括抗体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，所述嵌合抗原受体的靶向部分的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，所述嵌合抗原受体的靶向部分可包括抗体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。
例如，所述嵌合抗原受体的靶向部分的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，所述嵌合抗原受体的靶向部分可包括抗体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。
例如，所述嵌合抗原受体的靶向部分的VH可包含框架区H-FR1，H-FR2，H-FR3和H-FR4。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:51所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR1与SEQ ID NO:51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:51)，其中，X 1可以是A或E，X 16可以是G或R。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:1至SEQ ID NO:3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靶向部分的H-FR2可包含SEQ ID NO:52所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR2与SEQ ID NO:52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:52)，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，所述嵌合抗原受体的靶向部分的H-FR2可包含SEQ ID NO:7至SEQ ID NO:10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靶向部分的H-FR3可包含SEQ ID NO:53所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR3与SEQ ID NO:53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:53)，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，所述嵌合抗原受体的靶向部分的H-FR3可包含SEQ ID NO:13至SEQ ID NO:18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靶向部分的H-FR4可包含SEQ ID NO:54所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR4与SEQ ID NO:54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:54)，其中，X 3可以是L或Q，X 6可以是L或Q。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR4可包含SEQ ID NO:22至SEQ ID NO:23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靶向部分的H-FR1可包含SEQ ID NO:58所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR1与SEQ ID NO:58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 16。
AVQLVESGGGLVQPGX 16SLRLSCAAS(SEQ ID NO:58)，其中，X 16可以是G或R。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:1至SEQ ID NO:2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靶向部分的H-FR2可包含SEQ ID NO:59所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR2与SEQ ID NO:59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 15。
X 1AWFRQAPGKEREGX 15SC(SEQ ID NO:59)，其中，X 1可以是I或M，X 15可以是I或V。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR2可包含SEQ ID NO:7至SEQ ID NO:9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靶向部分的H-FR3可包含SEQ ID NO:60所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR3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 2，X 3，X 7，X 12和X 27。
YX 2X 3DSVX 7GRFTX 12SRDNAKNTVYLQMNX 27LKPEDTAVYYC(SEQ ID NO:60)，其中，X 2可以是D或Y，X 3可以是A或V，X 7可以是E或K，X 12可以是I或V，X 27可以是D或S。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR3可包含SEQ ID NO:13至SEQ ID NO:15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靶向部分的H-FR4可包含SEQ ID NO:22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靶向部分的H-FR1可包含SEQ ID NO:61所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR1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 1VQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:61)，其中，X 1可以是A或E。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:1和SEQ ID NO:3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靶向部分的H-FR2可包含SEQ ID NO:62所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR2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 4，X 11，X 12，X 14和X 17。
IAWX 4RQAPGKX 11X 12EX 14VSX 17(SEQ ID NO:62)，其中，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 17可以是C或Y。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR2可包含SEQ ID NO:7和SEQ ID NO:10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靶向部分的H-FR3可包含SEQ ID NO:63所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR3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，X 20，X 21，X 29和X 30。
YYX 3DSVKGRFTISRDNAKNX 20X 21YLQMNSLX 29X 30EDTAVYYC(SEQ ID NO:63)，其中，X 3可以是A或Q，X 20可以是S或T，X 21可以是L或V，X 29可以是E或R，X 30可以是D或P。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR3可包含SEQ ID NO:16至SEQ ID NO:18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靶向部分的H-FR4可包含SEQ ID NO:54所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的H-FR4与SEQ ID NO:54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:54)，其中，X 3可以是L或Q，X 6可以是L或Q。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR4可包含SEQ ID NO:22至SEQ ID NO:23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靶向部分的H-FR1可包含SEQ ID NO:51所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:52所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:53所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:58所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:59所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:60所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:61所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:62所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:63所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:1至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至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至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至SEQ ID NO:23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靶向部分的H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:13所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-01或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:8所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:14所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-02或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:15所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-03或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:16所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:10所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04-H4或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04-H5或与其具有相同H-FR1-4的抗原结合片段。
在本申请中，所述嵌合抗原受体的靶向部分的H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:18所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体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，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:55所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分包含VH，所述VH与SEQ ID NO:55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 16，X 28，X 30，X 31，X 34，X 37，X 44，X 45，X 47，X 48，X 50，X 53，X 60，X 61，X 65，X 70，X 78，X 79，X 85，X 87，X 88，X 111，X 113，X 117，X 120。
X 1VQLVESGGGLVQPGX 16SLRLSCAASGFX 28VX 30X 31YAX 34AWX 37RQAPGKX 44X 45EX 47X 48SX 50ISX 53RDGNTYX 60X 61DSVX 65GRFTX 70SRDNAKNX 78X 79YLQMNX 85LX 87X 88EDTAVYYCATDPWTDCSLDGRYX 111YX 113YWGX 117GTX 120VTVSS(SEQ ID NO：55)，其中，X 1可以是A或E，X 16可以是G或R，X 28可以是P或S，X 30可以是A或D，X 31可以是D或G，X 34可以是I或M，X 37可以是F或V，X 44可以是E或G，X 45可以是L或R，X 47可以是G或W，X 48可以是I或V，X 50可以是C或Y，X 53可以是G或S，X 60可以是D或Y，X 61可以是A，Q或V，X 65可以是E或K，X 70可以是I或V，X 78可以是S或T，X 79可以是L或V，X 85可以是D或S，X 87可以是E，K或R，X 88可以是D或P，X 111可以是E或R，X 113可以是D，G或N，X 117可以是L或Q，X 120可以是L或Q。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:64所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包含VH，所述VH与SEQ ID NO:64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 16，X 30，X 31，X 34，X 48，X 53，X 60，X 61，X 65，X 70，X 85，X 113。
AVQLVESGGGLVQPGX 16SLRLSCAASGFPVX 30X 31YAX 34AWFRQAPGKEREGX 48SCISX 53RDGNTYX 60X 61DSVX 65GRFTX 70SRDNAKNTVYLQMNX 85LKPEDTAVYYCATDPWTDCSLDGRYEY X 113YWGLGTQVTVSS(SEQ ID NO:64)，其中，X 16可以是G或R，X 30可以是A或D，X 31可以是D或G，X 34可以是I或M，X 48可以是I或V，X 53可以是G或S，X 60可以是D或Y，X 61可以是A或V，X 65可以是E或K，X 70可以是I或V，X 85可以是D或S，X 113可以是G或N。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:65所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包含VH，所述VH与SEQ ID NO:65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 37，X 44，X 45，X 47，X 50，X 61，X 78，X 79，X 87，X 88，X 117和X 120。
X 1VQLVESGGGLVQPGGSLRLSCAASGFSVDDYAIAWX 37RQAPGKX 44X 45EX 47VSX 50ISSRDGNTYYX 61DSVKGRFTISRDNAKNX 78X 79YLQMNSLX 87X 88EDTAVYYCATDPWTDCSL DGRYRYDYWGX 117GTX 120VTVSS(SEQ ID NO:65)，其中，X 1可以是A或E，X 37可以是F或V，X 44可以是E或G，X 45可以是L或R，X 47可以是G或W，X 50可以是C或Y，X 61可以是A或Q，X 78可以是S或T，X 79可以是L或V，X 87可以是E或R，X 88可以是D或P，X 117可以是L或Q，X 120可以是L或Q。
在本申请中，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:24至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靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:11所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:19所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:13所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:24所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-01或与其具有相同重链可变区的嵌合抗原受体的靶向部分。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:8所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:14所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:25所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-02或与其具有相同重链可变区的嵌合抗原受体的靶向部分。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:2所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:15所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:26所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-03或与其具有相同重链可变区的嵌合抗原受体的靶向部分。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可 包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:16所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:22所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:27所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04或与其具有相同重链可变区的嵌合抗原受体的靶向部分。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:10所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:28所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04-H4或与其具有相同重链可变区的嵌合抗原受体的靶向部分。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:17所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:29所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04-H5或与其具有相同重链可变区的嵌合抗原受体的靶向部分。
在本申请中，所述嵌合抗原受体的靶向部分可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，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；所述HCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:18所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分的重链可变区可包含SEQ ID NO:30所示的氨基酸序列。例如，所述嵌合抗原受体的靶向部分可包括抗体B010-B-22Nb-04-H6或与其具有相同重链可变区的嵌合抗原受体的靶向部 分。
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:45所示的氨基酸序列。例如，所述跨膜结构域可包含与SEQ ID NO:45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、CD137、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CD137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:46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:46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:47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:47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。
例如，所述跨膜结构域的N端可以与所述靶向部分的C端连接。例如，所述跨膜结构域 的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：靶向部分、跨膜结构域、共刺激信号传导结构域和胞内信号传导结构域。
例如，所述嵌合抗原受体从N端到C端可以依次包含下述结构域：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:38至SEQ ID NO:44中任一项所示的氨基酸序列。例如，所述嵌合抗原受体可包含与SEQ ID NO:38至SEQ ID NO:44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。
多肽和免疫缀合物
另一方面，本申请提供了一种或多种多肽，其可包含本申请的分离的抗原结合蛋白。例如，所述多肽可包括融合蛋白。例如，所述多肽可包括多特异性抗体(例如，双特异性抗体)。
另一方面，本申请提供了一种或多种免疫缀合物，所述免疫缀合物可包含本申请的分离的抗原结合蛋白。在某些实施方式中，所述免疫缀合物还可包含药学上可接受的治疗剂、标记物和/或检测剂。
核酸、载体和细胞
另一方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述分离的抗原结合蛋白或多肽。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、重链可变区中的一种或多种)。所述一种或多种核酸分子可编码本申请所述的嵌合抗原受体。例如，所述核酸分子可包含SEQ ID NO:31至SEQ ID NO:37的核苷酸序列。
例如，当核酸分子分别编码所述抗原结合蛋白或多肽的一部分时，核酸分子编码的产物合在一起可以形成有功能的(例如，可结合CD22)的本申请的分离的抗原结合蛋白。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。例如，所述分离的核酸可以是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述分离的抗原结合蛋白的核 酸，这些方法包括但不限于，采用逆转录PCR和PCR获得本申请所述分离的抗原结合蛋白或多肽的核酸分子。
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***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***的基因长期表达。慢病毒载体是能够转导或感染非***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分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、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、所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。在本申请中，所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂。除非与本申请所述的细胞不相容，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)。
本申请可包括以下的实施方案：
1.嵌合抗原受体，其包含靶向部分，所述靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:21所示的氨基酸序列。
2.根据实施方案1所述的嵌合抗原受体，其中所述靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列。
3.根据实施方案1-2中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:6所示的氨基酸序列。
4.根据实施方案1-3中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:65所示的重链可变区中的HCDR1、HCDR2和HCDR3。
5.根据实施方案1-4中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:27至SEQ ID NO:30中任一项所示的重链可变区中的HCDR1、HCDR2和HCDR3。
6.根据实施方案1-5中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR1、HCDR2、HCDR3，所述HCDR3包含SEQ ID NO:21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；以及所述HCDR1包含SEQ ID NO:6所示的氨基酸序列。
7.根据实施方案1-6中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:61所示的氨基酸序列。
8.根据实施方案7所述的嵌合抗原受体，其中所述H-FR1包含SEQ ID NO:1和SEQ ID NO:3中任一项所示的氨基酸序列。
9.根据实施方案1-8中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:62所示的氨基酸序列。
10.根据实施方案9所述的嵌合抗原受体，其中所述H-FR2包含SEQ ID NO:7和SEQ ID NO:10中任一项所示的氨基酸序列。
11.根据实施方案1-10中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:63所示的氨基酸序列。
12.根据实施方案11所述的嵌合抗原受体，其中所述H-FR3包含SEQ ID NO:16至SEQ ID NO:18中任一项所示的氨基酸序列。
13.根据实施方案1-12中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR4，所述 H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
14.根据实施方案13所述的嵌合抗原受体，其中所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
15.根据实施方案1-14中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:61所示的氨基酸序列；所述H-FR2包含SEQ ID NO:62所示的氨基酸序列；所述H-FR3包含SEQ ID NO:63所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
16.根据实施方案15所述的嵌合抗原受体，其中所述H-FR1包含SEQ ID NO:1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:16至SEQ ID NO:18中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
17.根据实施方案15-16中任一项所述的嵌合抗原受体，其中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述的任意一组氨基酸序列：
a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22；
b)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
c)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
d)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:18和H-FR4：SEQ ID NO:23。
18.根据实施方案1-17中任一项所述的嵌合抗原受体，其中所述靶向部分包括抗体或抗原结合片段。
19.根据实施方案18所述的嵌合抗原受体，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
20.根据实施方案1-19中任一项所述的嵌合抗原受体，其中所述靶向部分包括VHH。
21.根据实施方案20所述的嵌合抗原受体，其中所述VHH靶向CD22。
22.根据实施方案1-21中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:65所示的氨基酸序列。
23.根据实施方案1-22中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:27至SEQ ID NO:30中任一项所示的氨基酸序列。
24.根据实施方案1-23中任一项所述的嵌合抗原受体，其包含跨膜结构域。
25.根据实施方案24所述的嵌合抗原受体，其中所述跨膜结构域包含源自选自下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。
26.根据实施方案24-25中任一项所述的嵌合抗原受体，其中所述跨膜结构域包括源自CD8的跨膜结构域。
27.根据实施方案24-26中任一项所述的嵌合抗原受体，其中所述跨膜结构域包含SEQ IDNO:45所示的氨基酸序列。
28.根据实施方案24-27中任一项所述的嵌合抗原受体，其中所述跨膜结构域的N端与所述靶向部分的C端连接。
29.根据实施方案1-28中任一项所述的嵌合抗原受体，其包含共刺激信号传导结构域。
30.根据实施方案29所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含源自选自下述蛋白的共刺激信号传导结构域或其组合：CD28、CD137、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。
31.根据实施方案29-30中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包括源自CD137的共刺激信号传导结构域。
32.根据实施方案29-31中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含如SEQ ID NO:46所示的氨基酸序列。
33.根据实施方案29-32中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域的N端与所述跨膜结构域的C端连接。
34.根据实施方案1-33中任一项所述的嵌合抗原受体，其包含胞内信号传导结构域。
35.根据实施方案34所述的嵌合抗原受体，其中所述胞内信号传导结构域包含源自选自下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。
36.根据实施方案34-35中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包括源自CD3zeta的胞内信号传导结构域。
37.根据实施方案34-36中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包含如SEQ ID NO:47所示的氨基酸序列。
38.根据实施方案34-37中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域的N端与所述共刺激信号传导结构域的C端。
39.根据实施方案1-38中任一项所述的嵌合抗原受体，其包含SEQ ID NO:41至SEQ ID NO:44中任一项所示的氨基酸序列。
40.分离的抗原结合蛋白，其以约2E-08M或以下的K D值与CD22蛋白特异性结合。
41.根据实施方案40所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:21所示的氨基酸序列。
42.根据实施方案40-41中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列。
43.根据实施方案40-42中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:6所示的氨基酸序列。
44.根据实施方案40-43中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:65所示的重链可变区中的HCDR1、HCDR2和HCDR3。
45.根据实施方案40-44中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:27至SEQ ID NO:30中任一项所示的重链可变区中的HCDR1、HCDR2和HCDR3。
46.根据实施方案44-45中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:21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；以及所述HCDR1包含SEQ ID NO:6所示的氨基酸序列。
47.根据实施方案40-46中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:61所示的氨基酸序列。
48.根据实施方案47所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:1和SEQ ID NO:3中任一项所示的氨基酸序列。
49.根据实施方案40-48中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:62所示的氨基 酸序列。
50.根据实施方案49所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:7和SEQ ID NO:10中任一项所示的氨基酸序列。
51.根据实施方案40-50中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:63所示的氨基酸序列。
52.根据实施方案51所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:16至SEQ ID NO:18中任一项所示的氨基酸序列。
53.根据实施方案40-52中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
54.根据实施方案53所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
55.根据实施方案40-54中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:61所示的氨基酸序列；所述H-FR2包含SEQ ID NO:62所示的氨基酸序列；所述H-FR3包含SEQ ID NO:63所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
56.根据实施方案55所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:1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:16至SEQ ID NO:18中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
57.根据实施方案55-56中任一项所述的分离的抗原结合蛋白，其中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述的任意一组氨基酸序列：
a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22；
b)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
c)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
d)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:18和H-FR4： SEQ ID NO:23。
58.根据实施方案40-57中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:65所示的氨基酸序列。
59.根据实施方案58所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:27至SEQ ID NO:30中任一项所示的氨基酸序列。
60.根据实施方案40-59中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
61.根据实施方案60所述的分离的抗原结合蛋白，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和dAb。
62.根据实施方案40-61中任一项所述的分离的抗原结合蛋白，其包括VHH或其抗原结合片段。
63.根据实施方案60-62中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、人源化抗体、嵌合抗体和全人源抗体。
64.根据实施方案40-63中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:27至SEQ ID NO:30中任一项所示的氨基酸序列。
65.多肽，其包含实施方案1-39中任一项所述的嵌合抗原受体和/或实施方案40-64中任一项所述的分离的抗原结合蛋白。
66.免疫缀合物，其包含实施方案40-64中任一项所述的分离的抗原结合蛋白。
67.分离的核酸分子，其编码实施方案1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白，或者实施方案65所述的多肽。
68.载体，其包含实施方案67所述的分离的核酸分子。
69.细胞，其包含实施方案1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽，实施方案66所述的免疫缀合物，实施方案67所述的分离的核酸分子和/或实施方案68所述的载体。
70.根据实施方案69中所述的细胞，其包括免疫细胞。
71.根据实施方案70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。
72.根据实施方案69-71中任一项所述的细胞，其中所述细胞包括T细胞。
73.制备实施方案1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白和/或实施方案65所述的多肽的方法，所述方法包括在使得所述的分离 的抗原结合蛋白和/或所述的多肽表达的条件下，培养实施方案69-72中任一项所述的细胞。
74.药物组合物，其包含实施方案1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽，实施方案66所述的免疫缀合物，实施方案67所述的分离的核酸分子，实施方案68所述的载体，实施方案69-72中任一项所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
75.一种用于检测CD22蛋白的方法，其包括：
施用实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽或实施方案66所述的免疫缀合物。
76.一种CD22蛋白的检测试剂盒，其包含实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽或实施方案66所述的免疫缀合物。
77.实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽或实施方案66所述的免疫缀合物在制备试剂盒中的用途，所述试剂盒用于检测CD22蛋白的存在和/或含量。
78.实施方案1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽，实施方案66所述的免疫缀合物，实施方案67所述的分离的核酸分子，实施方案68所述的载体和/或实施方案69-72中任一项所述的细胞在制备预防和/或***的药物中的用途。
79.根据实施方案78所述的用途，其中所述肿瘤包括血液瘤。
80.根据实施方案78-79中任一项所述的用途，其中所述肿瘤包括与CD22的表达相关的肿瘤。
81.根据实施方案78-80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。
82.实施方案1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽，实施方案66所述的免疫缀合物，实施方案67所述的分离的核酸分子，实施方案68所述的载体，和/或实施方案69-72中任一项所述的细胞，其用于预防和/或***。
83.根据实施方案82所述的用途，其中所述肿瘤包括血液瘤。
84.根据实施方案82-83中任一项所述的用途，其中所述肿瘤包括与CD22的表达相关的肿瘤。
85.根据实施方案82-84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。
86.一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用有效量的实施方案 1-39中任一项所述的嵌合抗原受体，实施方案40-64中任一项所述的分离的抗原结合蛋白，实施方案65所述的多肽，实施方案66所述的免疫缀合物，实施方案67所述的分离的核酸分子，实施方案68所述的载体，和/或实施方案69-72中任一项所述的细胞。
87.根据实施方案86所述的方法，其中所述肿瘤包括血液瘤。
88.根据实施方案86-87中任一项所述的方法，其中所述肿瘤包括与CD22的表达相关的肿瘤。
89.根据实施方案86-88中任一项所述的方法，其中所述肿瘤选自下组：白血病和血液瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、嵌合抗原受体、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1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***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***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***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抗原结合蛋白
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为本申请抗原结合蛋白的CDR、重链可变区VH、FR。
表1本申请抗原结合蛋白的CDR、重链可变区VH、FR
Figure PCTCN2022112171-appb-000001
实施例5抗原结合蛋白的结合亲和力
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抗原结合蛋白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不同抗原结合蛋白与抗原蛋白的结合亲和性
Figure PCTCN2022112171-appb-000002
Figure PCTCN2022112171-appb-000003
实施例6本申请的抗原结合蛋白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抗原结合蛋白与K562细胞表面的人CD22的结合曲线。
实施例7 CD22-CAR的生物学活性
构建靶向CD22的嵌合抗原受体具体包括：靶向人CD22的单域抗体VHH序列、CD8跨膜结构域、CD137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)分别命名为CD22-CAR-01(氨基酸序列如SEQ ID NO:38所示，核酸序列如SEQ ID NO:31所示)、CD22-CAR-02(氨基酸序列如SEQ ID NO:39所示，核酸序列如SEQ ID NO:32所示)、CD22-CAR-03(氨基酸序列如SEQ ID NO:40所示，核酸序列如SEQ ID NO:33所示)、CD22-CAR-04(氨基酸序列如SEQ ID NO:41所示，核酸序列如SEQ ID NO:34所示)。
通过荧光素酶报告基因***检测的荧光强度比较CD22-CAR-01、CD22-CAR-02、CD22- CAR-03、CD22-CAR-04瞬转至Jurkat-NFAT细胞中并在Raji细胞刺激下荧光素酶(Luciferase，简写为LUC)的表达。
1)使用电转缓冲液配制Jurkat-NFAT细胞分别与CD22-CAR-01、CD22-CAR-02、CD22-CAR-03、CD22-CAR-04、m971质粒混合的电转体系，并执行电转程序。其中m971为阳性对照。
2)电转后细胞放入37℃，5％CO 2培养箱中过夜培养并测定转染效率。
3)按照转染CD22-CAR-01、CD22-CAR-02、CD22-CAR-03、CD22-CAR-04后的Jurkat-NFAT:Raji＝1:1混合两种细胞，共同孵育作为实验组；另以未转染处理的Jurkat-NFAT细胞和Raji细胞共同孵育作为阴性对照组；转染m971的Jurkat-NFAT细胞和Raji细胞共同孵育作为阳性对照组。37℃，5％CO 2孵育4h。
4)使用荧光素酶检测试剂(Promega,E6120)，利用酶标仪检测96孔板的化学发光值(RLU)。
结果：(图2)转染CD22-CAR-01、CD22-CAR-02、CD22-CAR-03、CD22-CAR-04质粒的Jurkat-NFAT细胞与Raji细胞相互作用4h后均能够激活Jurkat-NFAT细胞，选择B010-B-22Nb-04抗原结合蛋白进行人源化设计，CAR构建和CART制备。
实施例8全基因合成CD22嵌合抗原受体分子并构建慢病毒表达载体，包装病毒
将获得的人源化CD22抗原结合蛋白都分别构建到慢病毒载体上来筛选出更有效的靶向CD22的嵌合抗原受体。
基因合成含有不同抗原结合蛋白的靶向CD22的嵌合抗原受体的基因序列：
构建人源化的靶向CD22的嵌合抗原受体具体包括：靶向人CD22的单域抗体VHH序列、CD8跨膜结构域、CD137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)分别命名为CD22-CAR-04-H4(氨基酸序列如SEQ ID NO:42所示，核酸序列如SEQ ID NO:35所示)、CD22-CAR-04-H5(氨基酸序列如SEQ ID NO:43所示，核酸序列如SEQ ID NO:36所示)、CD22-CAR-04-H6(氨基酸序列如SEQ ID NO:44所示，核酸序列如SEQ ID NO:37所示)。嵌合抗原受体基因序列由金维智公司合成并克隆至pLVX-EF1a-IRES-Puro载体上EcoRI和BamHI两个酶切位点之间。将构建好的慢病毒质粒和包装质粒Pspax2，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备用。
实施例9制备CAR-T细胞
收集人外周血T淋巴细胞，用实施例8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CD22-CAR-04-H4、CD22-CAR-04-H5、CD22-CAR-04-H6。
1)取12孔板，配制含有5ug/ml抗CD3、2.5ug/ml抗CD28以及20ug/ml Retronectin抗体混合液，抗体混合液以500ul/well包被，室温孵育2h及以上。
2)复苏冻存的PBMC细胞1支。使用5ml PBS洗一次，按照试剂盒(MojoSort TM人CD3 T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500ul/孔PBS溶液洗1次，PBMC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，加入病毒，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CAR T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。
实施例10检测CART细胞对靶细胞的杀伤作用
对Raji和Nalm6转入萤火虫荧光素酶，获得的Raji-LUC和Nalm6-LUC细胞。以CART：Raji-LUC/Nalm6-LUC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Raji-LUC/Nalm6-LUC细胞株混合和共孵育，作为试验组；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NC。
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Raji-LUC/Nalm6-LUC细胞的杀伤作用，并与无关序列制备载体包装获得的病毒转染后的T淋巴细胞进行比较。
结果：人源化CD22-CAR-04 CART(即，CD22-CAR-04-H4、CD22-CAR-04-H5和CD22-CAR-04-H6 CART)细胞均能与CD22蛋白结合。流式检测人源化CD22-CAR-04 CART细胞与CD22蛋白结合结果如表3所示。杀伤实验结果显示本申请的人源化CD22-CAR-04的CAR T(即，CD22-CAR-04-H4、CD22-CAR-04-H5和CD22-CAR-04-H6 CART)细胞对Raji-LUC/Nalm6-LUC细胞有明显的体外杀伤作用(图4图5)。
图3为流式检测人源化CD22-CAR-04 CART细胞与CD22蛋白结合，图4图5为人源化CD22-CAR-04 CART细胞对Raji-LUC和Nalm6-LUC细胞杀伤作用。
表3流式检测人源化CD22-CAR-04 CART细胞与CD22蛋白结合结果
组别 CD22结合率(％) MFI
CD22-CAR-04-H4 76.75 71,082.45
CD22-CAR-04-H5 77.86 114,189.71
CD22-CAR-04-H6 76.65 105,875.28
CD22-CAR-04 86.89 161,377.29
实施例11 CD22-CAR-T细胞Raji-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Raji-LUC，接种于NDG小鼠(购自百奥赛图江苏基因生物科技有限公司)，尾静脉脉注射成瘤。将2.5×10 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
Raji-LUC小鼠模型成瘤5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。Raji-LUC小鼠模型成瘤5天后，将表达CD22-CAR-04、CD22-CAR-04-H4、CD22-CAR-04-H5、CD22-CAR-04-H6嵌合抗原受体的T淋巴细胞用PBS洗涤后用无血清培养液重悬，调整细胞密度为1.5×10 7个/ml。每只小鼠尾静脉注射3×10 6个细胞，共200μl。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。
图6、图7显示的是Raji-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细胞组相比，转染了CD22-CAR-04、CD22-CAR-04-H4、CD22-CAR-04-H5、CD22-CAR-04-H6的T细胞组的小鼠体内的肿瘤细胞数量显著减少，其中CD22-CAR-04-H4 CAR-T组抑瘤效果最好，在给药后Day17抑瘤率约为99.73％。
图8显示的是Raji-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，耐受良好。
实施例12 CD22-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股份有限公司)，尾静脉脉注射成瘤。将2.5×10 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。Nalm6-LUC小鼠模型成瘤3天后，将表达CD22-CAR-04、CD22-CAR-04-H4、CD22-CAR-04-H5、CD22-CAR-04-H6嵌合抗原受体的T淋巴细胞用PBS洗涤后用无血清培养液重悬，调整细胞密度为1.5×10 7个/ml。每只小鼠尾静脉注射3×10 6个细胞，共200μl。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。
图9、图10显示的是Naml6-LUC小鼠活体成像结果，结果显示未转染CAR的T细胞组的小鼠荧光强度快速上升，转染了CD22-CAR-04、CD22-CAR-04-H4、CD22-CAR-04-H5、CD22-CAR-04-H6的T细胞组的小鼠体内均表现出显著的抑瘤效果，且各实验组间抑瘤效果未表现出明显差异。
图11显示的是Nalm6-LUC小鼠体重变化结果，给药后小鼠体重正常，且治疗期间均无动物死亡，没有表现明显的药物毒性，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 (99)

  1. 嵌合抗原受体，其包含靶向部分，所述靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:49所示的氨基酸序列。
  4. 根据权利要求3所述的嵌合抗原受体，其中所述HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
  6. 根据权利要求5所述的嵌合抗原受体，其中所述HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:55所示的重链可变区中的HCDR1、HCDR2和HCDR3。
  8. 根据权利要求1-7中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的重链可变区中的HCDR1、HCDR2和HCDR3。
  9. 根据权利要求1-8中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR1、HCDR2、HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；所述HCDR2包含SEQ ID NO:49所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
  10. 根据权利要求9所述的嵌合抗原受体，其中所述HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
  11. 根据权利要求9-10中任一项所述的嵌合抗原受体，其中所述HCDR1、HCDR2和HCDR3包含选自下述的任意一组的氨基酸序列：
    a)HCDR1：SEQ ID NO:4，HCDR2：SEQ ID NO:11，和HCDR3：SEQ ID NO:19；
    b)HCDR1：SEQ ID NO:5，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:20；和
    c)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:21。
  12. 根据权利要求1-11中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ  ID NO:51所示的氨基酸序列。
  13. 根据权利要求12所述的嵌合抗原受体，其中所述H-FR1包含SEQ ID NO:1至SEQ ID NO:3中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:52所示的氨基酸序列。
  15. 根据权利要求14所述的嵌合抗原受体，其中所述H-FR2包含SEQ ID NO:7至SEQ ID NO:10中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:53所示的氨基酸序列。
  17. 根据权利要求16所述的嵌合抗原受体，其中所述H-FR3包含SEQ ID NO:13至SEQ ID NO:18中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
  19. 根据权利要求18所述的嵌合抗原受体，其中所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:51所示的氨基酸序列；所述H-FR2包含SEQ ID NO:52所示的氨基酸序列；所述H-FR3包含SEQ ID NO:53所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:54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H-FR1包含SEQ ID NO:1至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至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至SEQ ID NO:18中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
  22. 根据权利要求20-21中任一项所述的嵌合抗原受体，其中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述的任意一组氨基酸序列：
    a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:13和H-FR4：SEQ ID NO:22；
    b)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:8，H-FR3：SEQ ID NO:14和H-FR4：SEQ ID NO:22；
    c)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:9，H-FR3：SEQ ID NO:15和H-FR4：SEQ ID NO:22；
    d)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22；
    e)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
    f)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
    g)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:18和H-FR4：SEQ ID NO:23。
  23. 根据权利要求1-22中任一项所述的嵌合抗原受体，其中所述靶向部分包括抗体或抗原结合片段。
  24. 根据权利要求23所述的嵌合抗原受体，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
  25. 根据权利要求1-24中任一项所述的嵌合抗原受体，其中所述靶向部分包括VHH。
  26. 根据权利要求25所述的嵌合抗原受体，其中所述VHH靶向CD22。
  27. 根据权利要求1-26中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:55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，其中所述靶向部分包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的嵌合抗原受体，其包含跨膜结构域。
  30. 根据权利要求29所述的嵌合抗原受体，其中所述跨膜结构域包含源自选自下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。
  31. 根据权利要求29-30中任一项所述的嵌合抗原受体，其中所述跨膜结构域包括源自CD8 的跨膜结构域。
  32. 根据权利要求29-31中任一项所述的嵌合抗原受体，其中所述跨膜结构域包含SEQ ID NO:45所示的氨基酸序列。
  33. 根据权利要求29-32中任一项所述的嵌合抗原受体，其中所述跨膜结构域的N端与所述靶向部分的C端连接。
  34. 根据权利要求1-33中任一项所述的嵌合抗原受体，其包含共刺激信号传导结构域。
  35. 根据权利要求34所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含源自选自下述蛋白的共刺激信号传导结构域或其组合：CD28、CD137、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。
  36. 根据权利要求34-35中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包括源自CD137的共刺激信号传导结构域。
  37. 根据权利要求34-36中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含如SEQ ID NO:46所示的氨基酸序列。
  38. 根据权利要求34-37中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域的N端与所述跨膜结构域的C端连接。
  39. 根据权利要求1-38中任一项所述的嵌合抗原受体，其包含胞内信号传导结构域。
  40. 根据权利要求39所述的嵌合抗原受体，其中所述胞内信号传导结构域包含源自选自下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。
  41. 根据权利要求39-40中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包括源自CD3zeta的胞内信号传导结构域。
  42. 根据权利要求39-41中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域包含如SEQ ID NO:47所示的氨基酸序列。
  43. 根据权利要求39-42中任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域的N端与所述共刺激信号传导结构域的C端连接。
  44. 根据权利要求1-43中任一项所述的嵌合抗原受体，其包含SEQ ID NO:38至SEQ ID NO:44中任一项所示的氨基酸序列。
  45. 分离的抗原结合蛋白，其以约2E-08M或以下的K D值与CD22蛋白特异性结合。
  46. 根据权利要求45所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列。
  47. 根据权利要求46所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列。
  48. 根据权利要求45-47中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:49所示的氨基酸序列。
  49. 根据权利要求48所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列。
  50. 根据权利要求45-49中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
  51. 根据权利要求50所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
  52. 根据权利要求45-51中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:55所示的重链可变区中的HCDR1、HCDR2和HCDR3。
  53. 根据权利要求45-52中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的重链可变区中的HCDR1、HCDR2和HCDR3。
  54. 根据权利要求45-53中任一项所述的分离的抗原结合蛋白，其包括重链可变区VH，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；所述HCDR2包含SEQ ID NO:49所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:50所示的氨基酸序列。
  55. 根据权利要求54所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:19至SEQ ID NO:21中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO:11至SEQ ID NO:12中任一项所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:4至SEQ ID NO:6中任一项所示的氨基酸序列。
  56. 根据权利要求54-55中任一项所述的分离的抗原结合蛋白，其中所述HCDR1、HCDR2和HCDR3包含选自下述的任意一组的氨基酸序列：
    a)HCDR1：SEQ ID NO:4，HCDR2：SEQ ID NO:11，和HCDR3：SEQ ID NO:19；
    b)HCDR1：SEQ ID NO:5，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:20；和
    c)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:12，和HCDR3：SEQ ID NO:21。
  57. 根据权利要求45-56中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:51所示的氨基酸序列。
  58. 根据权利要求57所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:1至SEQ ID NO:3中任一项所示的氨基酸序列。
  59. 根据权利要求45-58中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:52所示的氨基酸序列。
  60. 根据权利要求59所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:7至SEQ ID NO:10中任一项所示的氨基酸序列。
  61. 根据权利要求45-60中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:53所示的氨基酸序列。
  62. 根据权利要求61所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:13至SEQ ID NO:18中任一项所示的氨基酸序列。
  63. 根据权利要求45-62中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
  64. 根据权利要求63所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
  65. 根据权利要求45-64中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:51所示的氨基酸序列；所述H-FR2包含SEQ ID NO:52所示的氨基酸序列；所述H-FR3包含SEQ ID NO:53所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:54所示的氨基酸序列。
  66. 根据权利要求65所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:1至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至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至SEQ ID NO:18中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:22至SEQ ID NO:23中任一项所示的氨基酸序列。
  67. 根据权利要求65-66中任一项所述的分离的抗原结合蛋白，其中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述的任意一组氨基酸序列：
    a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:13和H-FR4：SEQ ID NO:22；
    b)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:8，H-FR3：SEQ ID NO:14和H-FR4：SEQ ID NO:22；
    c)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:9，H-FR3：SEQ ID NO:15和H-FR4：SEQ ID NO:22；
    d)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22；
    e)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
    f)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:17和H-FR4：SEQ ID NO:23；
    g)H-FR1：SEQ ID NO:3，H-FR2：SEQ ID NO:7，H-FR3：SEQ ID NO:18和H-FR4：SEQ ID NO:23。
  68. 根据权利要求45-67中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:55所示的氨基酸序列。
  69. 根据权利要求68所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
  70. 根据权利要求45-69中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  71. 根据权利要求70所述的分离的抗原结合蛋白，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和dAb。
  72. 根据权利要求45-71中任一项所述的分离的抗原结合蛋白，其包括VHH或其抗原结合片段。
  73. 根据权利要求70-72中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、人源化抗体、嵌合抗体和全人源抗体。
  74. 根据权利要求45-73中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:24至SEQ ID NO:30中任一项所示的氨基酸序列。
  75. 多肽，其包含权利要求1-44中任一项所述的嵌合抗原受体和/或权利要求45-74中任一项所述的分离的抗原结合蛋白。
  76. 免疫缀合物，其包含权利要求45-74中任一项所述的分离的抗原结合蛋白。
  77. 分离的核酸分子，其编码权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白，或者权利要求75所述的多肽。
  78. 载体，其包含权利要求77所述的分离的核酸分子。
  79. 细胞，其包含权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽，权利要求76所述的免疫缀合物，权利要求77所述的分离的核酸分子和/或权利要求78所述的载体。
  80. 根据权利要求79所述的细胞，其包括免疫细胞。
  81. 根据权利要求80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。
  82. 根据权利要求81所述的细胞，其中所述细胞包括T细胞。
  83. 制备权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白和/或权利要求75所述的多肽的方法，所述方法包括在使得所述的分离的抗原结合蛋白和/或所述的多肽表达的条件下，培养权利要求79-82中任一项所述的细胞。
  84. 药物组合物，其包含权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽，权利要求76所述的免疫缀合物，权利要求77所述的分离的核酸分子，权利要求78所述的载体，权利要求79-82中任一项所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
  85. 一种用于检测CD22蛋白的方法，其包括：
    施用权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽或权利要求76所述的免疫缀合物。
  86. 一种CD22蛋白的检测试剂盒，其包含权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽或权利要求76所述的免疫缀合物。
  87. 权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽或权利要求76所述的免疫缀合物在制备试剂盒中的用途，所述试剂盒用于检测CD22蛋白的存在和/或含量。
  88. 权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽，权利要求76所述的免疫缀合物，权利要求77所述的分离的核酸分子，权利要求78所述的载体和/或权利要求79-82中任一项所述的细胞在制备预防和/或***的药物中的用途。
  89. 根据权利要求88所述的用途，其中所述肿瘤包括血液瘤。
  90. 根据权利要求88-89中任一项所述的用途，其中所述肿瘤包括与CD22的表达相关的肿瘤。
  91. 根据权利要求88-90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。
  92. 权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽，权利要求76所述的免疫缀合物，权利要求77所述的分离的核酸分子，权利要求78所述的载体，和/或权利要求79-82中任一项所述的细胞，其用于预防和/或***。
  93. 根据权利要求92所述的用途，其中所述肿瘤包括血液瘤。
  94. 根据权利要求92-93中任一项所述的用途，其中所述肿瘤包括与CD22的表达相关的肿瘤。
  95. 根据权利要求92-94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。
  96. 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有效量的权利要求1-44中任一项所述的嵌合抗原受体，权利要求45-74中任一项所述的分离的抗原结合蛋白，权利要求75所述的多肽，权利要求76所述的免疫缀合物，权利要求77所述的分离的核酸分子，权利要求78所述的载体，和/或权利要求79-82中任一项所述的细胞。
  97. 根据权利要求96所述的方法，其中所述肿瘤包括血液瘤。
  98. 根据权利要求96-97中任一项所述的方法，其中所述肿瘤包括与CD22的表达相关的肿瘤。
  99. 根据权利要求96-98中任一项所述的方法，其中所述肿瘤选自下组：白血病和血液瘤。
PCT/CN2022/112171 2021-08-13 2022-08-12 靶向cd22的抗原结合蛋白及其用途 WO2023016554A1 (zh)

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