WO2022117032A1 - 抗cd22的纳米抗体及其用途 - Google Patents

抗cd22的纳米抗体及其用途 Download PDF

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WO2022117032A1
WO2022117032A1 PCT/CN2021/135058 CN2021135058W WO2022117032A1 WO 2022117032 A1 WO2022117032 A1 WO 2022117032A1 CN 2021135058 W CN2021135058 W CN 2021135058W WO 2022117032 A1 WO2022117032 A1 WO 2022117032A1
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seq
cdr2
cdr1
cdr3
antigen
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French (fr)
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王琼
杨翠青
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to US18/265,150 priority Critical patent/US20230416360A1/en
Priority to CN202180081208.9A priority patent/CN116685350A/zh
Publication of WO2022117032A1 publication Critical patent/WO2022117032A1/zh

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464413—CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the fields of bioengineering and biomedicine, and mainly relates to an anti-human CD22 nanobody or an antigen-binding fragment thereof, its encoding nucleic acid, an expression vector and expression cells, a preparation method, a pharmaceutical composition, and their use in the treatment of diseases. Uses such as the treatment of tumors and autoimmune diseases.
  • CD22 is a type I transmembrane glycoprotein, a member of the sialic acid-binding immunoglobulinlike lectins (Siglec, sialic acid-binding immunoglobulinlike lectins) family.
  • Siglec sialic acid-binding immunoglobulinlike lectins
  • B cell differentiation antigen it is specifically expressed in B cells, from the pre-B cell (pre-B cell) stage, and no longer express CD22 after B cells differentiate into plasma cells.
  • pre-B cell pre-B cell
  • the broad expression of CD22 in B cell development makes it an attractive molecule to target B cells.
  • the CD22 extracellular domain consists of 7 immunoglobulin-like domains (Ig-like domains) and 12 predicted N-linked glycosylation sites, and its N-terminal (ie, distal membrane end) domain domain 1 is V Type Ig-like domain, which can recognize ⁇ 2,6-conjugated sialic acid as a ligand binding site.
  • the intracellular domain of CD22 has tyrosine immunoreceptor-dependent inhibitory motifs (ITIMs, immunoreceptor tyrosine-based inhibitory motifs).
  • ⁇ 2,6-coupled sialoglycoprotein exists in hematopoietic cells, some endothelial cells and T cells and B cells, and CD22 protein itself also produces ⁇ 2,6-coupled sialic acid, so CD22 can interact with itself and It forms cis-interactions with other sialylglycoproteins on the surface of B cells, and forms trans-interactions with sialylglycoproteins on the surface of other cell types.
  • the cis-interaction between CD22 causes the ligand-binding site of CD22 to be obscured, but once the ligand is presented by adjacent cells, the obscured CD22 ligand-binding site is exposed to interact with Adjacent cellular ligands form trans interactions.
  • the cis-interaction between CD22 forms homo-oligomers on the surface of B cells that can form a dynamic nanocluster and generate an antigen-binding signal threshold that must be reached before B cell activation, thereby regulating B cell signaling pathways.
  • CD22 is expressed in 60% to 90% of B cell malignancies, but not in hematopoietic stem cells. In an early clinical study of acute lymphoblastic leukemia (ALL), 60% to 85% of ALL expressed CD22; in another study, the CD22-positive rate of B-lineage ALL patients was 93%. CD22 is expressed in more than 85% of patients with diffuse large B-cell lymphomas (DLBCLs).
  • Epratuzumab a CD22 monoclonal antibody, has a certain effect in B-ALL in adults and children; CD22 antibody-drug conjugates have a certain therapeutic effect on B-ALL.
  • Nanobodies are genetically engineered antibodies that contain only a single domain.
  • Belgian scientist Hamers-Casterman C discovered a natural heavy chain antibody in camel blood that only contains heavy chains and no light chains. Compared with ordinary antibodies, heavy chain antibodies lack light chains, but still retain the ability to bind antigens ability.
  • the obtained single domain antibody single domain antibody, sdAb
  • the obtained single domain antibody composed of only one heavy chain variable region is called nanobody or VHH antibody (variable heavy chain domain of a heavy chain antibody).
  • Nanobodies are not only 1/10 of the molecular weight of ordinary antibodies, but also have more flexible chemical properties, good stability, high solubility, easy expression, high tumor tissue penetration, and easy coupling to other molecules. Therefore, the application of nanobody technology to develop therapeutic antibodies against CD22 has broad prospects.
  • the present invention provides Nanobodies or antigen-binding fragments that specifically bind to CD22, nucleic acids encoding these antibodies and antigen-binding fragments, pharmaceutical compositions and kits comprising the antibodies and antigen-binding fragments, and its use in the treatment of tumors and self Preparation of medicines such as immune diseases.
  • a Nanobody or antigen-binding fragment that specifically binds CD22 comprises a combination of CDRs comprising: CDR1, CDR2 and CDR3; said CDR1, CDR2 and CDR3 have Any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence combination:
  • Each CDR1, CDR2 and CDR3 is encoded according to the pass analysis method of KABAT, Chothia or IMGT;
  • the substitutions are conservative amino acid substitutions.
  • Nanobodies or antigen-binding fragments of the invention wherein:
  • the CDR1, CDR2 and CDR3 are respectively shown in the sequence shown in SEQ ID NO.90, 91, 92;
  • the CDR1, CDR2 and CDR3 are respectively shown in the sequence shown in SEQ ID NO.99, 100, 101;
  • the CDR1, CDR2 and CDR3 are respectively as shown in SEQ ID NO.105, 106, 107 sequence;
  • the CDR1, CDR2 and CDR3 are respectively as shown in SEQ ID NO.147, 148, 149 sequence;
  • CDR1, CDR2 and CDR3 are respectively shown as the sequence shown in SEQ ID NO.207,208,209;
  • the CDR1, CDR2 and CDR3 are respectively the sequences shown in SEQ ID NO.216, 217, 218; or,
  • the CDR1, CDR2 and CDR3 are sequence combinations having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared with the above-mentioned (1)-(54) sequence combinations.
  • the present invention provides an antibody or antigen-binding fragment thereof comprising:
  • variable region has the sequence shown in SEQ ID NO: 21, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 23, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 25, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 27, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 29, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO:31, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO:33, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 35, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 37, or 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 39, or 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 41, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 43, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 45, or 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 47, or 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 49, or 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO:51, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity;
  • variable region has the sequence shown in SEQ ID NO: 53, or 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Sequences of 99% or greater identity; or,
  • variable region has the sequence shown in SEQ ID NO:55, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical sequences.
  • the antibodies of the invention or antigen-binding fragments thereof bind to human CD22 with a dissociation constant (KD) of no greater than 50 nM.
  • KD dissociation constant
  • the antibody or antigen-binding fragment thereof of the invention comprises the sequence of any one of the constant regions of human or murine antibodies IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably human or murine Sequences of the constant regions of antibodies IgG1, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region sequence in the absence of a CH1 fragment.
  • the antibody or antigen-binding fragment thereof of the invention further comprises heavy chain constant region sequences having CH2 and CH3 fragments.
  • the antibody or antigen-binding fragment thereof of the present invention is chimeric or humanized or fully human; preferably, the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies , natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g. bispecific antibodies), monovalent antibodies, multivalent antibodies, full length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies , fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody or single domain antibody.
  • the antibody or antigen-binding fragment thereof of the present invention is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents, and the tracer
  • the therapeutic agent is selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizers.
  • the present invention also provides a multispecific antigen binding molecule; preferably, the multispecific antigen binding molecule comprises a first antigen binding moiety and a second antigen binding moiety, the first antigen binding
  • the module comprises the antibody or antigen-binding fragment of any one of the above, and the second antigen-binding module specifically binds to other antigens other than CD22 or binds to a different CD22 antigenic epitope from the first antigen-binding module;
  • the other antigens are selected from CD3, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54 , CD66(a-d), CD74, CD80, CD126, CD138, B7, MUC, Ia, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, oncogene products, IL-2, IL-6 , TRAIL-R1 or TRAIL-R2;
  • the multispecific antibody is "bispecific", “trispecific” or “tetraspecific”.
  • the present invention provides a chimeric antigen receptor (CAR); preferably, the chimeric antigen receptor comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling structure domain, the extracellular antigen-binding domain comprises the CD22 antibody or antigen-binding fragment of any one of the above.
  • CAR chimeric antigen receptor
  • the present invention provides an immune effector cell; preferably, the immune effector cell comprises the above-mentioned chimeric antigen receptor or a nucleic acid fragment comprising the above-mentioned chimeric antigen receptor;
  • the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells;
  • the T cells Can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells;
  • the immune effector cells are allogeneic immune effector cells or autologous immune cells.
  • the present invention provides an isolated nucleic acid molecule encoding the Nanobody, antigen-binding fragment, or any combination thereof of any one of the above-described multispecific nucleic acid molecules of the present invention.
  • the present invention provides an expression vector comprising the isolated nucleic acid molecule of the present invention described above.
  • the present invention provides a host cell comprising the isolated nucleic acid molecule or expression vector of the present invention described above.
  • the host cells are eukaryotic cells or prokaryotic cells; more preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the The host cells are selected from HEK293E or Chinese hamster ovary cells (CHO).
  • the present invention provides a method for preparing an antibody or antigen-binding fragment or multispecific antigen-binding molecule, culturing the above-described host cell of the present invention under appropriate conditions, and isolating the antibody or antigen-binding fragment or Multispecific antigen binding molecules.
  • the present invention provides a method for preparing immune effector cells, wherein the nucleic acid fragment of the CAR described above is introduced into the immune effector cells, preferably, the method further comprises initiating the immune effector cells to express the above-mentioned immune effector cells. described CAR.
  • the present invention provides a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment of the present invention, the above-described multispecific antigen-binding molecule of the present invention, and the above-described embedded antigen-binding molecule of the present invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.
  • the present invention provides a method of preventing and/or treating a B cell disease, comprising administering to a patient in need thereof the above-described antibody or antigen-binding fragment of the present invention, the above-described multispecific antigen-binding molecule, the chimeric antigen receptor described above in the present invention, the immune effector cell described above in the present invention, the isolated nucleic acid molecule described above in the present invention, the expression vector described above in the present invention, the above-described in the present invention.
  • the B-cell disease is preferably a tumor or an autoimmune disease;
  • the tumor is selected from lymphoma or leukemia
  • the lymphoma or leukemia can be selected from B cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Primary mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphoblastic leukemia (pre-B ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, multiple myeloma;
  • the autoimmune disease is selected from systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, Sjogren's syndrome, Guillain-Barré syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, pulmonary hemorrhage-nephritic syndrome, glomerular Nephritis, primary biliary cirrhosis, Graves' disease, membranous nephropathy, autoimmune hepatitis, sprue, Addison's disease, polymyositis/dermatomyositis, monoclonal gamma globulin disease, factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and chronic
  • the present invention provides an antibody or antigen-binding fragment described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor described above of the invention, a chimeric antigen receptor described above of the invention, Immune effector cells, isolated nucleic acid molecules described above in the present invention, expression vectors described above in the present invention, cells described above in the present invention, products prepared by the methods described above in the present invention (eg, antibodies and antigen-binding fragments) , or the use of the above-mentioned pharmaceutical composition of the present invention in the preparation of a medicament for preventing and/or treating a B cell disease, the B cell disease is preferably a tumor or an autoimmune disease;
  • the tumor is selected from lymphoma or leukemia
  • the lymphoma or leukemia can be selected from B cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Primary mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphoblastic leukemia (pre-B ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, multiple myeloma;
  • the autoimmune disease is selected from systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, Sjogren's syndrome, Guillain-Barré syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, pulmonary hemorrhage-nephritic syndrome, glomerular Nephritis, primary biliary cirrhosis, Graves' disease, membranous nephropathy, autoimmune hepatitis, sprue, Addison's disease, polymyositis/dermatomyositis, monoclonal gamma globulin disease, factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and chronic
  • the present invention provides an antibody or antigen-binding fragment described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor described above of the invention, a chimeric antigen receptor described above of the invention, Immune effector cells, isolated nucleic acid molecules described above in the present invention, expression vectors described above in the present invention, cells described above in the present invention, products prepared by the methods described above in the present invention (eg, antibodies and antigen-binding fragments) , or the above-mentioned pharmaceutical composition of the present invention is used for the prevention and/or treatment of B cell diseases; the B cell diseases are preferably tumors or autoimmune diseases;
  • the tumor is selected from lymphoma or leukemia
  • the lymphoma or leukemia can be selected from B cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, primary Mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphoblastic leukemia (pre-B ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, multiple myeloma;
  • the autoimmune disease is selected from systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, Sjogren's syndrome, Guillain-Barré syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, pulmonary hemorrhage-nephritic syndrome, glomerular Nephritis, primary biliary cirrhosis, Graves' disease, membranous nephropathy, autoimmune hepatitis, sprue, Addison's disease, polymyositis/dermatomyositis, monoclonal gamma globulin disease, factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and chronic
  • the present invention provides a kit comprising the above-described antibody or antigen-binding fragment of the present invention, the above-described multispecific antigen-binding molecule of the present invention, and the above-described chimeric antigen of the present invention Receptors, immune effector cells described above in the present invention, isolated nucleic acid molecules described above in the present invention, expression vectors described above in the present invention, cells described above in the present invention, or prepared by methods described above in the present invention products (such as antibodies and antigen-binding fragments), or the above-described pharmaceutical compositions of the present invention, and instructions for use.
  • a kit comprising the above-described antibody or antigen-binding fragment of the present invention, the above-described multispecific antigen-binding molecule of the present invention, and the above-described chimeric antigen of the present invention Receptors, immune effector cells described above in the present invention, isolated nucleic acid molecules described above in the present invention, expression vectors described above in the present invention, cells described above in the
  • antibody refers to an immunoglobulin molecule that specifically binds or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugated antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies, antibody conjugates) and Antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab')2 fragments which lack the Fc fragment of an intact antibody (which clears faster from the animal's circulation) and thus lack Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).
  • an “antibody” herein can be derived from any animal, including, but not limited to, humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as camelid, llama , ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • natural antibody herein refers to an antibody that is produced and paired by the immune system of a multicellular organism.
  • the antibody of the term “engineered antibody” herein refers to a non-natural antibody obtained by genetic engineering, antibody engineering and other techniques.
  • engineered antibody includes humanized antibody, small molecule antibody (such as scFv, etc.), dual specific antibodies, etc.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen combine.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valency herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
  • the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.
  • Fully-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to mean that they have a structure that is substantially similar to that of a native antibody.
  • antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabodies, tribodies, affibodies, Nanobodies, aptamers or domain antibodies.
  • binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment comprising two Fab fragments linked at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of an antibody one-arm Constituent Fv fragments; (v) dAbs comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) consisting of VH or VL A dAb consisting of domains; (viii) discrete complementarity determining regions (CDRs); and (ix) a combination of two or more discrete CDRs, which may optionally be linked by synthetic
  • the two domains of the Fv fragment, VL and VH are encoded by separate genes, the two domains can be joined using recombinant methods by a linker that enables it to be made in which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883, 1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • CD22 refers to the molecule Siglec-2 belonging to the SIGLEC lectin family, which is present on the surface of mature B cells and to a lesser extent on certain immature B cells.
  • CD22 includes the CD22 protein of any human and non-human animal species, and specifically includes human CD22 as well as non-human mammalian CD22.
  • the term "bispecific antibody” refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificities for at least two different antigens.
  • one of the binding specificities can be detected against an epitope of CD22, the other can be against another epitope of CD22 or any other antigen other than CD22, such as for cell surface proteins, receptors, Receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins or bacterial surface proteins are detected.
  • chimeric antibody refers to an antibody having variable sequences of immunoglobulins derived from one source organism (eg, rat or mouse) and those derived from a different organism (eg, human). Constant regions of immunoglobulins.
  • Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, Bio Techniques 4: 214-221; Gillies et al, 1985 J Immunol Methods 125: 191-202; incorporated by reference above This article.
  • the term “heavy chain antibody” refers to an antibody that lacks the light chain of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising the VH antigen binding domain and the CH2 and CH3 constant domains in the absence of the CH1 domain.
  • the term "nanobody” refers to a natural heavy chain antibody lacking the light chain in camels, and the variable region of which can be cloned to obtain a single domain antibody composed of only the variable region of the heavy chain, also known as VHH (Variable domain). of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHHs and Nanobodies For a further description of VHHs and Nanobodies, reference is made to a review article by Muyldermans (2001, Reviews in Molecular Biotechnology 74: 277-302), and to the following patent applications mentioned as general background: WO 94/04678 of Vrije Universiteit Brussel; WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V.
  • Nanobodies can be characterized inter alia by the presence of one or more "characteristic residues" in one or more framework sequences.
  • Characteristic residues in one or more framework sequences.
  • Further descriptions of Nanobodies, including humanization and/or camelization of Nanobodies, and other modifications, parts or fragments, derivatives or "Nanobody fusions" can be found, for example, in WO 08/101985 and WO 08/142164. ", multivalent constructs (including some non-limiting examples of linker sequences) and various modifications that increase the half-life of Nanobodies and their formulations.
  • CDRs complementarity determining regions
  • FRs framework regions
  • amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within a variable domain can be considered as heterozygous hypervariable positions, as these positions can be considered to be within a hypervariable region under one set of criteria (such as IMGT or KABAT), but are considered to be within a different set of criteria (eg KABAT or IMGT) outside the hypervariable regions. One or more of these positions can also be found in extended hypervariable regions.
  • variable domains of native heavy and light chains each comprise four framework regions predominantly adopting a sheet configuration, connected by three CDRs (CDR1, CDR2 and CDR3) that form loops connecting the sheet structure , and in some cases form part of the lamellar structure.
  • CDR1, CDR2 and CDR3 The CDRs in each chain are held tightly together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, which constitute the heavy chain variable region (VH).
  • the CDR combination of the chain (or its variable region) (VHCDR combination);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the first CDR of the light chain variable region (VL), respectively
  • Three CDRs that make up the CDR combination of the light chain (or its variable region) (VLCDR combination).
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) without limitation to the method by which the antibody is produced.
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv or Fab.
  • VL refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • variable domains refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody
  • the variable domains have more conserved amino acid sequences.
  • a “heavy chain constant region” comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from a CH1, Fc or CH3 domain.
  • light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the papain hydrolyzed carboxy-terminal portion of an intact antibody, which typically comprises the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, thus, the Fc region can include or excluding Lys447.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.
  • Fully human antibody refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • naked antibody herein refers to an antibody that is not linked, fused or conjugated to another agent or molecule (eg, a label or drug), peptide or polypeptide.
  • naked antibodies expressed by mammalian host cells can be glycosylated by the host cell's glycosylation machinery (eg, glycosylase).
  • naked antibodies are not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (eg, glycosylase).
  • the naked antibody is an intact antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of an intact antibody, eg, a Fab antibody.
  • conjugated antibody refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal, chimeric, humanized, or human antibody.
  • diabody herein refers to bivalent bispecific antibodies that can bind to different epitopes on the same or different antigens.
  • percent (%) sequence identity refers to aligning sequences and introducing gaps, if necessary, for maximum percent sequence identity (eg, for optimal alignment, can be used between candidate and reference After the introduction of gaps in one or both of the sequences, and for comparison purposes, non-homologous sequences may be ignored), the amino acid (or nucleotide) residues of the candidate sequence differ from the amino acid (or nucleotide) residues of the reference sequence. ) residues that are identical.
  • alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence aligned for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence to 100% sequence identity.
  • the length of candidate sequences aligned for comparison purposes may be, for example, at least 30% (eg, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. .
  • amino acids herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids within each of the following groups belong to each other as conserved amino acid residues, and substitutions of amino acid residues within the groups belong to conservative amino acid substitutions:
  • Acidic amino acids Asp(D) and Glu(E);
  • Non-polar uncharged amino acids Cys(C), Met(M) and Pro(P);
  • Aromatic amino acids Phe(F), Tyr(Y) and Trp(W).
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; incorporated herein by reference).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883; incorporated herein by reference).
  • IMGT numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883; incorporated herein by reference).
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules such as antibodies or their antigens Binding fragment-specific recognition.
  • An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM).
  • Antibodies that do not exhibit specific binding to a particular antigen or epitope thereof or Antigen-binding fragments will exhibit a KD of greater than 100 nM (eg, greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for that particular antigen or epitope thereof.
  • immunoassay formats can be used to select for specificity for a particular protein or carbohydrate The antibody of sexual immune response.
  • solid-phase ELISA immunoassay to select the antibody that carries out specific immune response with protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • antibody conjugate refers to a conjugate/conjugate in which an antibody molecule is chemically bonded to another molecule, either directly or through a linker.
  • ADCs antibody-drug conjugates
  • chimeric antigen receptor herein refers to a recombinant protein comprising at least (1) an extracellular antigen-binding domain, such as a variable heavy or light chain of an antibody, and (2) anchoring the CAR into Transmembrane domains of immune effector cells, and (3) intracellular signaling domains.
  • the extracellular antigen binding domain of the CAR comprises an scFv.
  • the scFv can be derived from the variable heavy and light regions of fusion antibodies. Alternatively or additionally, scFvs can be derived from Fab's (rather than antibodies, eg from Fab libraries). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is generally represented as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as synthetic forms of DNA or RNA. A mixed polymer of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the invention in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg, cDNA
  • RNA eg, mRNA
  • the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823B1).
  • vector includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses, or other suitable replicons (eg, viral vectors).
  • DNA vectors eg, plasmids
  • RNA vectors eg. RNA vectors
  • viruses eg. viral vectors
  • viral vectors eg. viral vectors
  • Various vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • the expression vectors of the present invention contain polynucleotide sequences and additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences (eg, promoter and enhancer regions) that direct gene transcription.
  • Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on expression vectors.
  • Expression vectors of the present invention may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.
  • pharmaceutical composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain unacceptable toxicity to the subject to whom the pharmaceutical composition is administered of additional ingredients.
  • the terms "subject”, “subject” and “patient” refer to an organism receiving treatment for a particular disease or disorder (eg, cancer or infectious disease) as described herein.
  • subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, bovid family members (such as domestic cattle, bison, buffalo, elk and yak, etc.), sheep and horses, etc.
  • treatment refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) unwanted physiological changes or pathologies, such as cell proliferative disorders such as cancer, in the subject being treated or infectious disease).
  • beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
  • alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
  • an effective amount herein refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease.
  • Effective amount also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • appropriate conditions refers to conditions suitable for culturing various host cells, including eukaryotic cells and prokaryotic cells.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • anti-tumor agent refers to anti-tumor drugs, which are a class of drugs for the treatment of tumor diseases, including chemotherapeutic drugs, biological agents, and the like.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a half-way response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • EC80 refers to the concentration of antibody that elicits 80% of the maximal effect.
  • Figure 1A shows the antibody titer of alpaca serum after immunization with human CD22-ECD protein detected by ELISA
  • Figure 1B shows the antibody titer of alpaca serum after human CD22-ECD protein immunization detected by FACS.
  • Figure 2 shows the detection results of SDS-PAGE reducing gel and non-reducing gel of CD22-ECD-His, CD22domain1-4-His and CD22domain5-7-His protein samples.
  • Lane 1 is the protein band of hCD22-ECD-His under non-reducing conditions
  • lane 2 is the protein band of hCD22domain5-7-His under non-reducing conditions
  • lane 3 is the protein band of hCD22domain5-7-His under reducing conditions
  • Lane 4 is the protein band of hCD22-ECD-His under reducing conditions
  • lane 5 is the protein band of hCD22domain1-4-His under non-reducing conditions
  • lane 6 is the protein band of hCD22domain1-4-His under reducing conditions.
  • M is the protein maker band.
  • Figure 3A shows the binding reaction between the control antibody and human CD22-ECD-His protein detected by ELISA
  • Figure 3B is the binding reaction between the control antibody and human CD22domain1-4-His protein detected by ELISA
  • Figure 3C is the binding reaction between the control antibody and human CD22domain5-7 detected by ELISA - Binding reaction of His protein.
  • the anti-CD22 control antibodies were: HA22 and m971, and the negative control was hIgG1.
  • Figure 4A is the FACS result of detecting the expression of CD22 in Raji cells by HA22 antibody
  • Figure 4B is the FACS result of detecting the expression of CD22 in Raji cells by m971 antibody.
  • Figure 5 shows the results of FACS screening of CHO-K1 cells transfected with human CD22 protein.
  • Fig. 6 is the detection of the binding reaction of the VHH-Fc antibody of the present invention with human CD22-ECD-His protein by ELISA.
  • the anti-CD22 positive control antibodies were: HA22 and m971, and the negative control was hIgG1.
  • FIG. 7A is the FACS detection of the binding reaction of the VHH-Fc antibody of the present invention with CHO-K1-human CD22;
  • FIG. 7B is the FACS detection of the binding reaction of the VHH-Fc antibody of the present invention with Raji.
  • the anti-CD22 positive control antibodies are: HA22, m971 and hL22, and the negative control is hIgG1;
  • Figure 7C shows the binding reaction of 1nM and 10nM VHH-Fc antibody of the present invention with CHO-K1 cells and CHO-K1-human CD22 2C4 detected by FACS;
  • Figure 7C 7D is FACS to detect the binding reaction of 1 nM and 10 nM VHH-Fc antibody of the present invention to Raji cells and Jurkat cells.
  • Figure 8 shows the binding reaction between the VHH-Fc antibody of the present invention and the mouse CD22-ECD-His protein detected by ELISA; the positive control is 983; the negative control is hIgG1;
  • Figure 9 is a scatter plot of cynomolgus monkey peripheral blood mononuclear cells double-stained by FACS detection of CD20 antibody and 1 nM VHH-Fc antibody of the present invention
  • CD20 is a B cell marker
  • the ratio shown in the figure is the VHH-Fc antibody of the present invention positive cells
  • the proportion of CD20 positive cells, the anti-CD22 positive control antibody is: HA22, and the negative control is hIgG1.
  • Figure 10 shows the SPR detection of the affinity of the VHH-Fc antibody of the present invention to human CD22.
  • the anti-human CD22 positive control antibodies are: HA22 and m971.
  • Figure 11 shows the affinity of the VHH-Fc antibody of the present invention and cynomolgus monkey CD22 detected by SPR, and the anti-human CD22 positive control antibody is: HA22.
  • Fig. 12A shows the binding reaction of VHH-Fc of the present invention with human CD22domain1-4-His protein by ELISA
  • Fig. 12B is the binding reaction of VHH-Fc of the present invention with human CD22domain5-7-His protein detected by ELISA.
  • the anti-CD22 positive control antibodies were: HA22 and m971, and the negative control was hIgG1.
  • Figure 13 is a competitive ELISA method to detect the inhibition rate among the VHH antibodies of the present invention.
  • Figure 14 is a classification of the epitopes of the VHH antibodies of the present invention.
  • Human CD22(Asp20-Arg687)-His protein for immunization was purchased from ACRO Biosystems (Cat. No. CD2-H52H8). Two alpacas (Llama) were selected for immunization, and each alpaca was immunized four times with an interval of 3 weeks. After the third immunization and the fourth immunization, peripheral blood was collected and serum was separated. The titer and specificity of antibodies against human CD22 in serum were detected by ELISA) and flow cytometry (FACS), and the results are shown in Figures 1A-1B and Table 1.
  • Table 1 shows that the alpacas immunized with human CD22 have different degrees of binding to the immunogen after immunization, showing antigen-antibody reaction, and the highest dilution is about 5.9 million.
  • the blank control is 1% (w/w) BSA
  • the batch refers to the alpaca serum on the seventh day after the third (TB2) and fourth (TB3) immunizations
  • the data in the table are OD450nm values.
  • RNAiso Plus reagent (Takara, Cat. No.: #9108/9109), using PrimeScript TM II 1st Strand The cDNA Synthesis Kit (Takara, Cat. No. 6210A) reverse-transcribes the extracted RNA into cDNA.
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO.17)
  • Upstream primer CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO.18)
  • the target Nanobody nucleic acid fragment was recovered and cloned into the phage display vector pcomb3XSS (from Sichuan Apak Biotechnology Co., Ltd.) using the restriction enzyme SfiI (NEB, catalog number: R0123S).
  • the product was then electro-transformed into E. coli electro-competent cells TG1, and a nanobody phage display library against CD22 was constructed and assayed.
  • the size of the library volume was calculated to be 2.0 ⁇ 10 9 by serial dilution plating.
  • 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
  • the plate was coated with 0.5 ⁇ g/well of human CD22-llama Fc fusion protein (ACRO Biosystems, Cat. No.: SI2-H525a) and placed at 4°C overnight; the next day, after blocking with 3% BSA-PBS at 37°C for 1 h, 100 ⁇ l of phage display library was added , incubated at 37°C for 1 h; then washed 6 times with PBST and 2 times with PBS to wash off unbound phage. Finally, 100 ⁇ L of Gly-HCl eluate was added, and phages that specifically bind to CD22 were eluted to enrich positive clones.
  • human CD22-llama Fc fusion protein ACRO Biosystems, Cat. No.: SI2-H525a
  • CD22 binding-positive phages were infected with blank E. coli and plated. Then 96 single colonies were selected for expansion and culture. The plates were coated with human CD22-llama Fc and human CD22-His protein overnight at 4°C, respectively, and the phage culture supernatant was added, and incubated at 37°C for 1 hour. After washing, TMB color developing solution was added to develop color, and the optical density was measured at a wavelength of 450 nm. Human CD22-llama Fc and human CD22-His double-positive clones were selected for sequencing.
  • the MOE software was used to analyze the sequencing results, and the phylogenetic tree was constructed according to the amino acid sequence of the VHH-encoded protein. After removing the sequences with close distances on the phylogenetic tree according to the sequence similarity, 18 clones were obtained by screening, and the CDRs of the sequences were obtained by KABAT, Chothia Or IMGT software analysis, the corresponding sequence information is shown in Table 2-4 below, where Table 2 shows the antibody sequence represented by 18 Nanobody molecule amino acids, Table 3 shows the antibody sequence represented by 18 Nanobody molecule nucleotides, Table 4 shows the results of IMGT, Kabat and Chothia analysis of the CDRs of 18 Nanobody molecules. Production characterization of VHH Nanobody Fc fusion proteins was subsequently performed.
  • the VHH variable region sequence was recombined by Taizhou Baiying Biotechnology Co., Ltd. into the expression vector BI3 containing the signal peptide and human IgG1 Fc (human IgG1 Fc sequence such as SEQ ID NO: 14, hinge region sequence such as SEQ ID NO: 15) .4-huIgG1, and prepare plasmids according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition ( Plainview, New York: Cold Spring Harbor Laboratory Press).
  • the expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, Cat. No.: 24765-1), and used FreeStyle TM 293 (Thermofisher scientific, Cat. No. 12338018) at 37°C After continuous culture for 5 days, the cell components were removed by centrifugation, and the culture supernatant containing VHH antibody was obtained.
  • the culture supernatant was loaded onto a protein A chromatography column (Protein A packing AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borgron, catalog numbers: AA0273 and B-1620, respectively), using PBS phosphate After washing with buffer (pH 7.4), wash with 20 mM PB, 1 M NaCl (pH 7.2), and finally use pH 3.4 citrate buffer for elution, and collect the band eluted from the protein A chromatography column.
  • the Fc-tagged antibody was neutralized with 1/10 volume of 1M Tris, pH 8.0, and dialyzed overnight at 4°C with PBS. The dialyzed protein was sterile filtered through a 0.22-micron filter membrane and stored in aliquots at -80°C.
  • CD22 protein has 7 IgG-like extracellular domains, of which domain1 is located at the farthest membrane end and domain7 is at the nearest membrane end.
  • HA22, m971 and hL22 are antibodies that recognize human CD22, wherein the antigen-binding epitopes of HA22 and hL22 are located in domains 2-3, and the antigen-binding epitopes of m971 are located in domains 5-7.
  • the heavy chain variable region and light chain variable region sequences of HA22 were obtained according to patent US 9580461B (which is incorporated herein by reference), and the heavy chain variable region and light chain variable region sequences of m971 were according to patent US 8591889B (which was incorporated by reference).
  • the heavy chain variable region and light chain variable region amino acid sequences of hL22 were obtained according to patent US5789554B, which is incorporated herein by reference.
  • the VH and VL of the antibodies HA22, m971 and hL22 that recognize human CD22, and the human IgG1 Fc are connected in the sequence from the N-terminus to the C-terminus, wherein the VH and VL are connected by 3 GGGGS linkers to form the form of scFv-hFc , and the corresponding amino acid sequence information is shown in Table 5 below.
  • the corresponding nucleotide sequences were cloned into the pTT5 vector (completed by Universal Biosystems (Anhui) Co., Ltd.) and expressed in HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the method of Example 2.1. purification.
  • CD22 protein has 7 IgG-like extracellular domains, of which domain1 is located at the farthest membrane end, domain7 is at the nearest membrane end, the antigen-binding epitope of HA22 and hL22 is located in domain 2-3, and the antigen-binding epitope of m971 is located in domain5- 7.
  • amino acid sequence encoding human CD22 protein (NCBI: NP_001762.2, SEQ ID NO: 1), extracellular domain (ECD, extra-cellular domain) amino acid sequence Asp 20-Arg 687 (SEQ ID NO: 2), structure
  • the nucleotide sequences of domain 1-4 Asp 20-Val 425 amino acid sequence (SEQ ID NO: 3) and domain 5-7 Asp 414-Arg 687 amino acid sequence (SEQ ID NO: 4) were cloned into pTT5 vector (completed by General Biosystems (Anhui) Co., Ltd.) and plasmids were prepared according to established standard molecular biology methods, and the corresponding amino acid sequence information is shown in Table 6 below.
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • PI Polysciences, Cat. No. 24765-1
  • FreeStyle TM 293 Thermofisher scientific, Cat. No. 12338018
  • the culture supernatant was loaded on a nickel ion affinity chromatography column HisTrap TM Excel (GE Healthcare, Cat. No.: GE17-3712-06), and at the same time, the change of the ultraviolet absorption value (A280nm) was monitored with an ultraviolet (UV) detector.
  • HisTrap TM Excel GE Healthcare, Cat. No.: GE17-3712-06
  • the nickel ion affinity chromatography column was washed with 20mM PB, 0.5M NaCl (pH7.4) until the UV absorption value returned to the baseline, and then buffer A: 20mM PB, 0.5M NaCl (pH7.4) and buffer B : 20 mM PB, 0.5 M NaCl, 500 mM imidazole for gradient elution (2%, 4%, 8%, 16%, 50%, 100%), and the His-bands eluted from the nickel ion affinity chromatography column were collected
  • the tagged human CD22 protein was dialyzed against PBS phosphate buffered saline (pH 7.4) overnight in a refrigerator at 4°C.
  • the dialyzed protein was sterile filtered through a 0.22-micron filter membrane and stored at -80°C to obtain purified human CD22 protein.
  • the prepared CD22 protein was tested by ELISA using positive control antibodies that recognize different epitopes.
  • the negative control antibody hIgG1 was the antibody anti-hel-hIgG1 against chicken egg lysozyme (purchased from Baiying, article number: B117901).
  • the test results are as follows As shown in Figure 3A-3C, both HA22 and m971 can bind human CD22-ECD-His protein, HA22 can bind human CD22domain1-4-His protein, and m971 can bind human CD22domain5-7-His protein.
  • the detection results are consistent with those reported in the literature.
  • the binding epitopes of HA22 and m971 are the same, indicating that the protein with the above-mentioned binding activity has been prepared.
  • Raji cells (purchased from Wuhan University China Type Culture Collection Center) were expanded and cultured in a T-25 cell culture flask to logarithmic growth phase, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
  • HA22 and m971 antibodies were used as primary antibodies, and APC-labeled secondary antibodies (purchased from Biolegend, product number: 409306) were used to detect and analyze the results by FACS (FACS CantoTM, purchased from BD Company). The analysis results are shown in Table 7 and Figures 4A-4B. Raji cells can bind to both HA22 and m971.
  • the nucleotide sequence encoding the full-length amino acid sequence of human CD22 was cloned into the pcDNA3.1 vector and a plasmid was prepared (completed by General Biosystems (Anhui) Co., Ltd.).
  • CHO-K1 cell line purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
  • plasmids 3000 Transfection Kit, purchased from Invitrogen, Cat. No.: L3000-015
  • FITC-labeled anti-CD22 antibody (Thermofisher scientific, Cat. No.: 11-0229-42) was used to sort positive monoclonal cells on a flow cytometer FACS AriaII (BD Biosciences) into a 96-well plate, and placed at 37°C, 5% ( v/v) culture in a CO 2 cell incubator, and select some monoclonal wells for expansion after about 2 weeks.
  • the amplified clones were screened by flow cytometry. The monoclonal cell line with better growth and higher fluorescence intensity was selected to continue to expand the culture and cryopreserved in liquid nitrogen.
  • Table 8 illustrates that a series of CD22-positive CHO-K1 monoclonal cell lines have been generated.
  • the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells.
  • the results in FIG. 5 show that CHO-K1-human CD22 2C4, CHO-K1-human CD22 1G5 and CHO-K1-human CD22 1D9 are cell lines that express high levels of CD22.
  • the purified human CD22-ECD-His protein obtained in Example 2 was diluted with PBS to a final concentration of 2 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l/well. Cover with plastic film and incubate at 4°C overnight, wash the plate twice with PBS the next day, add blocking solution [PBS+2% (w/w) BSA] and block for 2 hours at room temperature. Pour off the blocking solution, and add 100 nM serially diluted VHH-Fc antibody or negative control antibody at 50 ⁇ l/well. After incubation at 37°C for 2 hours, the plate was washed 3 times with PBS.
  • the desired cells were expanded to the logarithmic growth phase in T-75 cell culture flasks.
  • adherent cells CHO-K1 the medium was aspirated, washed twice with PBS buffer, and then the cells were trypsinized. After terminating the digestion Cells were washed twice with PBS buffer; for suspension cells Raji was directly centrifuged to discard the medium supernatant, and the cell pellet was washed twice with PBS. After counting the cells in the previous step, resuspend the cell pellet with [PBS+2% (w/w) BSA] blocking solution to 2x10 6 cells/ml, and add 50 ⁇ l/well to a 96-well FACS reaction plate.
  • VHH-Fc antibody can bind to human CD22 protein on the surface of Raji cells and CHO-K1-human CD22 2C4 cells ( Figures 7A-7B).
  • Figures 7A-7B the binding of VHH-Fc antibody to endogenous CD22-negative cells Jurkat cells (purchased from ATCC, TIB-152) and CHO-K1 cells was simultaneously detected.
  • Figures 7C-7D all VHH-Fc None of the antibodies bind to Jurkat cells and CHO-K1 cells, with good specificity.
  • VHH-Fc In order to detect the species cross-activity of VHH-Fc antibody, commercial mouse CD22 (ACROBiosystems, product number: SI2-M52Ha) was coated on ELISA plate, and ELISA detection was carried out according to the method of Example 4.1.
  • the ELISA results of VHH-Fc and mouse CD22-ECD are shown in Figure 8 and Table 11.
  • Table 11 shows that only S002-NB151-51 of the purified antibody binds to mouse CD22-ECD at the ELISA level.
  • the IgG control is hIgG1
  • 983 is the serum from mice immunized with human CD22-ECD-His as a positive control.
  • the data in the table are OD450nm values.
  • VHH-Fc antibody Binding of VHH-Fc antibody to peripheral blood B cells of cynomolgus monkey (Latin name: Macaca fascicularis) detected by FACS
  • Monkey peripheral blood mononuclear cells were extracted from fresh cynomolgus monkey peripheral blood (purchased from Shanghai Medicilon Biopharmaceutical Co., Ltd.) according to the instructions of Ficoll-Paque Plus (purchased from GE Healthcare, Cat. No.: 171440-02). Cell suspension After centrifugation, the cells were resuspended in PBS containing 1% BSA and counted. At the same time, the murine antibody Brilliant Violet 605 anti-human CD20 (Cat. No.: 302334, purchased from Biolegend) with monkey CD20 cross-binding activity and the VHH-Fc antibody to be tested were added. (1 nM, 10 nM and 100 nM). Incubate for 1 hour at room temperature.
  • APC-labeled secondary antibody anti-human IgG Fc (Cat. No. 409306, purchased from Biolegend)
  • APC-labeled secondary antibody anti-human IgG Fc (Cat. No. 409306, purchased from Biolegend)
  • FACS FACS CantoTM, purchased from BD
  • CD20 is used as a marker of B cells
  • CD20-positive B cell population is gated to analyze the proportion of VHH-Fc positive cells, and calculate 100nM and 10nM respectively.
  • the ratio of VHH-Fc positive cell population to B cell population treated with VHH-Fc antibody at a concentration of 1 nM is shown in Table 12.
  • Figure 9 shows the double-stained cell scatter plot of Brilliant Violet 605 labeled CD20 and APC secondary antibody indirectly labeled VHH-Fc. It can be seen from the results that S002-NB151-2, S002-NB151-14, S002-NB151-23, S002-NB151-92, S002-NB151-51, and S002-NB150-2 still interact with crabs even at a low concentration of 1 nM. Monkey B cells have a higher proportion of binding, and have comparable or better binding activity than the positive antibody HA22; other antibodies have no or relatively weak binding to cynomolgus CD22.
  • Anti-human CD22 VHH-hFc antibody was captured using a Protein A chip (GE Helthcare; 29-127-558).
  • Sample and running buffer were HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25 °C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, then a single concentration of CD22 antigen protein was injected to record the binding and dissociation process of the antibody and antigen protein, and finally Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration. Binding was measured by injecting various concentrations of recombinant human CD22-ECD His in solution for 240 sec with a flow rate of 30 ⁇ L/min, starting at 200 nM (see detailed results for actual concentrations tested), diluted 1:1 for a total of 5 concentration. The dissociation phase was monitored for up to 600 seconds and triggered by switching from sample solution to running buffer.
  • Example 6.1 the affinity of VHH-Fc antibody and cynomolgus monkey CD22-ECD-His (purchased from R&D, product number: 9864-SL-050) protein was determined. The results are shown in Figure 11 and Table 14.
  • the antibody S002-NB151-51 which still has a higher ratio of binding with cynomolgus monkey B cells at the concentration, has a higher affinity with cynomolgus monkey CD22, which is 5.07E-10M.
  • CD22 protein has 7 IgG-like extracellular domains, of which domain1 is located at the farthest membrane end, domain7 is at the nearest membrane end, the antigen-binding epitope of HA22 and hL22 is located in domain 2-3, and the antigen-binding epitope of m971 is located in domain5- 7.
  • human CD22-domain1-4-His distal membrane end
  • human CD22domain5-7-His proximal membrane end
  • a competitive ELISA method was used for epitope sorting of VHH antibodies against control antibodies of known epitopes.
  • the ELISA plate was coated with 2 ⁇ g/mL antibody according to the method of Example 4.2, and the human CD22-ECD-his protein was serially diluted from 30 ⁇ g/mL, and the EC80 value was calculated (Table 16).
  • the ELISA plate was coated with 2 ⁇ g/mL of antibody, and after adding 25 ⁇ g/mL of the antibody to be detected, human CD22-ECD-his protein at the EC80 concentration corresponding to each antibody to be detected was added, incubated for 2 h, and washed with PBS for 5 times. Add HRP-labeled anti-His antibody for detection.
  • the coated antibody If there is no competition between the coated antibody and the antibody to be detected in the solution, it can bind to the antibody to be detected in the solution-human CD22-ECD-his antigen complex, and the OD450nm absorption is detected, according to the OD450nm absorbance value. Inhibition ratio between antibodies (FIG. 13).
  • each antibody epitope was classified as shown in Figure 14, S002-NB151-23, S002-NB151-64, S002-NB151-66, S002-NB150-2, S002-NB150-5, S002-NB150-40 , S002-NB150-42, S002-NB150-57, S002-NB150-152n compete with hL22 and HA22 and can be classified into one category; S002-NB151-82, S002-NB151-36, S002-NB151-14 There is competition between S002-NB151-30 and S002-NB151-51; S002-NB151-13 does not compete with any antibody; these antibodies can bind domain1-4 but not domain5- 7, can be classified into one category. There is competition among S002-NB151-2, S002-NB151-92, S002-NB150-159n; m971 does not compete with any antibodies, these antibodies can bind domain5-7 but not domain1-4, which can be classified as one category.

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Abstract

提供一种CD22的纳米抗体及其制备方法和应用，所述CD22纳米抗体与CD22蛋白具有高度亲和力，能够用于***和自身免疫性疾病等药物的制备。

Description

抗CD22的纳米抗体及其用途
本公开要求于2020年12月3日提交中国专利局、申请号为202011395861.6、发明名称为“抗CD22的纳米抗体及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本发明涉及生物工程、生物医药领域，主要涉及一种抗人CD22的纳米抗体或其抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及它们用于治疗疾病的用途，例如***和自身免疫病的用途。
背景技术
CD22是一个I型跨膜糖蛋白，属于唾液酸结合性免疫球蛋白样凝集素(Siglec,sialic acid-binding immunoglobulinlike lectins)家族中的一员，作为一种B细胞分化抗原，特异性表达于B细胞，从前B细胞(pre-B cell)时期开始表达，至B细胞分化为浆细胞后不再表达CD22。CD22在B细胞发育中的广谱表达，使其成为一个极具吸引力的靶向B细胞的分子。
CD22胞外区由7个免疫球蛋白样结构域(Ig-like domain)和12个预测的N-连接的糖基化位点组成，其N端(即远膜端)结构域domain 1是V型Ig-like domain，作为配体结合位点能够识别α2,6-偶联唾液酸。CD22胞内区具有酪氨酸免疫受体依赖的抑制结构(ITIMs，immunoreceptor tyrosine-based inhibitory motifs)，当ITIMs上的酪氨酸被Src家族蛋白激酶磷酸化后，会产生含有SH2(Src homology2)结构域分子的结合位点，随后招募SHP-1(Src homology region 2domain-containing phosphatase-1)从而抑制正常B细胞的BCR(B细胞受体，B-cell receptor)信号通路。
在造血细胞，某些内皮细胞以及T细胞和B细胞中都存在α2,6-偶联唾液酸糖蛋白，且CD22蛋白本身也会产生α2,6-偶联唾液酸，因此CD22能够与自身以及B细胞表面其它唾液酸糖蛋白之间形成顺式相互作用，与其它细胞类型细胞表面的唾液酸糖蛋白之间形成反式相互作用。在静息B细胞中，CD22之间的顺式相互作用使得CD22的配体结合位点被遮蔽，但是一旦临近细胞呈现出配体，被遮蔽的CD22配体结合位点便被暴露出来而与临近细胞配体形成反式相互作用。CD22之间的顺式相互作用会在B细胞表面形成同源寡聚体，这些同源寡聚体可以形成一个动态的纳米簇并产生B细胞激活之前必须达到的一个抗原结合信号阈值，从而调节B细胞信号通路。
CD22在60％～90％的B细胞恶性肿瘤中表达，在造血干细胞中不表达。在早期的一项急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)的临床研究中，60％到85％的ALL均表达CD22；另一项研究中B-lineage ALL病人的CD22阳性率达93％。在弥漫大B细胞淋巴瘤(DLBCLs)中85％以上的病人表达CD22。有许多临床试验研究了靶向CD22的药物的有效性。依他珠单抗(Epratuzumab)是一种CD22单克隆抗体，在成人和儿童B-ALL中具有一定的效果；CD22抗体偶联药物对B-ALL有一定的治疗作用。
纳米抗体(Nanobody，Nb)是一种只含有单个结构域的基因工程抗体。1993年比利时科学家Hamers-Casterman C在骆驼血液中发现一种只含重链不含轻链的天然重链抗体，重链抗体和普通的抗体相比虽然缺失了轻链，但是依然保留结合抗原的能力。克隆骆驼体内重链抗体的可变区后，得到的仅由一个重链可变区组成的单域抗体(single domain antibody，sdAb),称为纳米抗体或VHH抗体(variable heavy chain domain of a heavy chain antibody)。纳米抗体不仅分子量只是普通抗体的1/10，而且化学性质也更加灵活，稳定性好，可溶性高，表达容易，肿瘤组织穿透性高，且容易偶联其它分子。因此应用纳米抗体技术研发CD22的治疗性抗体具有广阔的前景。
发明内容
本发明提供特异性结合CD22的纳米抗体或抗原结合片段，编码这些抗体及抗原结合片段的核酸，包含所述抗体及抗原结合片段的药物组合物和试剂盒，以及它能够用于***和自身免疫病等药物的制备。
在一些实施方案中，特异性结合CD22的纳米抗体或抗原结合片段，所述纳米抗体或抗原结合片段包含CDRs组合，所述CDRs组合包含：CDR1、CDR2和CDR3；所述CDR1、CDR2和CDR3具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸***、缺失和/或替换的序列组合：
Figure PCTCN2021135058-appb-000001
Figure PCTCN2021135058-appb-000002
Figure PCTCN2021135058-appb-000003
各个CDR1、CDR2和CDR3为根据KABAT、Chothia或IMGT的通行分析方法编码；
优选地，所述替换为保守氨基酸的替换。
特别地，例如本发明的纳米抗体或抗原结合片段，其中：
(1)所述CDR1、CDR2和CDR3分别如SEQ ID NO.57、58、59所示序列；
(2)所述CDR1、CDR2和CDR3分别如SEQ ID NO.60、61、62所示序列；
(3)所述CDR1、CDR2和CDR3分别如SEQ ID NO.63、64、65所示序列；
(4)所述CDR1、CDR2和CDR3分别如SEQ ID NO.66、67、68所示序列；
(5)所述CDR1、CDR2和CDR3分别如SEQ ID NO.69、70、71所示序列；
(6)所述CDR1、CDR2和CDR3分别如SEQ ID NO.72、73、74所示序列；
(7)所述CDR1、CDR2和CDR3分别如SEQ ID NO.75、76、77所示序列；
(8)所述CDR1、CDR2和CDR3分别如SEQ ID NO.78、79、80所示序列；
(9)所述CDR1、CDR2和CDR3分别如SEQ ID NO.81、82、83所示序列；
(10)所述CDR1、CDR2和CDR3分别如SEQ ID NO.84、85、86所示序列；
(11)所述CDR1、CDR2和CDR3分别如SEQ ID NO.87、88、89所示序列；
(12)所述CDR1、CDR2和CDR3分别如SEQ ID NO.90、91、92所示序列；
(13)所述CDR1、CDR2和CDR3分别如SEQ ID NO.93、94、95所示序列；
(14)所述CDR1、CDR2和CDR3分别如SEQ ID NO.96、97、98所示序列；
(15)所述CDR1、CDR2和CDR3分别如SEQ ID NO.99、100、101所示序列；
(16)所述CDR1、CDR2和CDR3分别如SEQ ID NO.102、103、104所示序列；
(17)所述CDR1、CDR2和CDR3分别如SEQ ID NO.105、106、107所示序列；
(18)所述CDR1、CDR2和CDR3分别如SEQ ID NO.108、109、110所示序列；
(19)所述CDR1、CDR2和CDR3分别如SEQ ID NO.111、112、113所示序列；
(20)所述CDR1、CDR2和CDR3分别如SEQ ID NO.114、115、116所示序列；
(21)所述CDR1、CDR2和CDR3分别如SEQ ID NO.117、118、119所示序列；
(22)所述CDR1、CDR2和CDR3分别如SEQ ID NO.120、121、122所示序列；
(23)所述CDR1、CDR2和CDR3分别如SEQ ID NO.123、124、125所示序列；
(24)所述CDR1、CDR2和CDR3分别如SEQ ID NO.126、127、128所示序列；
(25)所述CDR1、CDR2和CDR3分别如SEQ ID NO.129、130、131所示序列；
(26)所述CDR1、CDR2和CDR3分别如SEQ ID NO.132、133、134所示序列；
(27)所述CDR1、CDR2和CDR3分别如SEQ ID NO.135、136、137所示序列；
(28)所述CDR1、CDR2和CDR3分别如SEQ ID NO.138、139、140所示序列；
(29)所述CDR1、CDR2和CDR3分别如SEQ ID NO.141、142、143所示序列；
(30)所述CDR1、CDR2和CDR3分别如SEQ ID NO.144、145、146所示序列；
(31)所述CDR1、CDR2和CDR3分别如SEQ ID NO.147、148、149所示序列；
(32)所述CDR1、CDR2和CDR3分别如SEQ ID NO.150、151、152所示序列；
(33)所述CDR1、CDR2和CDR3分别如SEQ ID NO.153、154、155所示序列；
(34)所述CDR1、CDR2和CDR3分别如SEQ ID NO.156、157、158所示序列；
(35)所述CDR1、CDR2和CDR3分别如SEQ ID NO.159、160、161所示序列；
(36)所述CDR1、CDR2和CDR3分别如SEQ ID NO.162、163、164所示序列；
(37)所述CDR1、CDR2和CDR3分别如SEQ ID NO.165、166、167所示序列；
(38)所述CDR1、CDR2和CDR3分别如SEQ ID NO.168、169、170所示序列；
(39)所述CDR1、CDR2和CDR3分别如SEQ ID NO.171、172、173所示序列；
(40)所述CDR1、CDR2和CDR3分别如SEQ ID NO.174、175、176所示序列；
(41)所述CDR1、CDR2和CDR3分别如SEQ ID NO.177、178、179所示序列；
(42)所述CDR1、CDR2和CDR3分别如SEQ ID NO.180、181、182所示序列；
(43)所述CDR1、CDR2和CDR3分别如SEQ ID NO.183、184、185所示序列；
(44)所述CDR1、CDR2和CDR3分别如SEQ ID NO.186、187、188所示序列；
(45)所述CDR1、CDR2和CDR3分别如SEQ ID NO.189、190、191所示序列；
(46)所述CDR1、CDR2和CDR3分别如SEQ ID NO.192、193、194所示序列；
(47)所述CDR1、CDR2和CDR3分别如SEQ ID NO.195、196、197所示序列；
(48)所述CDR1、CDR2和CDR3分别如SEQ ID NO.198、199、200所示序列；
(49)所述CDR1、CDR2和CDR3分别如SEQ ID NO.201、202、203所示序列；
(50)所述CDR1、CDR2和CDR3分别如SEQ ID NO.204、205、206所示序列；
(51)所述CDR1、CDR2和CDR3分别如SEQ ID NO.207、208、209所示序列；
(52)所述CDR1、CDR2和CDR3分别如SEQ ID NO.210、211、212所示序列；
(53)所述CDR1、CDR2和CDR3分别如SEQ ID NO.213、214、215所示序列；
(54)所述CDR1、CDR2和CDR3分别如SEQ ID NO.216、217、218所示序列；或，
所述CDR1、CDR2和CDR3为具有与上述(1)—(54)序列组合相比具有1、2、3或更多个氨基酸***、缺失和/或替换的序列组合。
在另一个具体实施方案中，本发明提供这样的抗体或其抗原结合片段，其包含：
(1)可变区具有SEQ ID NO:21所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(2)可变区具有SEQ ID NO:23所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(3)可变区具有SEQ ID NO:25所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(4)可变区具有SEQ ID NO:27所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(5)可变区具有SEQ ID NO:29所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(6)可变区具有SEQ ID NO:31所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(7)可变区具有SEQ ID NO:33所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(8)可变区具有SEQ ID NO:35所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(9)可变区具有SEQ ID NO:37所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(10)可变区具有SEQ ID NO:39所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(11)可变区具有SEQ ID NO:41所示序列，或者与上述所示序列具有70％、75％、80％、 85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(12)可变区具有SEQ ID NO:43所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(13)可变区具有SEQ ID NO:45所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(14)可变区具有SEQ ID NO:47所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(15)可变区具有SEQ ID NO:49所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(16)可变区具有SEQ ID NO:51所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
(17)可变区具有SEQ ID NO:53所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；或，
(18)可变区具有SEQ ID NO:55所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在一个优选实施方案中，本发明的抗体或其抗原结合片段与人CD22结合的解离常数(KD)不大于50nM。
在一个优选实施方案中，本发明的抗体或其抗原结合片段包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在一个优选实施方案中，本发明的抗体或其抗原结合片段进一步包含不存在CH1片段的重链恒定区序列。
在一个优选实施方案中，本发明的抗体或其抗原结合片段进一步包含具有CH2和CH3片段的重链恒定区序列。
在一个优选实施方案中，本发明的抗体或其抗原结合片段是嵌合的或人源化的或全人源的；优选地，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
在一个优选实施方案中，本发明的抗体或其抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
在一个优选实施方案中，本发明还提供一种多特异性抗原结合分子；优选地，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含上述任一项所述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合CD22以外的其他抗原或结合与第一抗原结合模块不同的CD22抗原表位；
优选地，所述其他抗原选自CD3、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、 CD19、CD20、CD21、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、B7、MUC、Ia、HLA-DR、腱生蛋白、VEGF、P1GF、ED-B纤连蛋白、癌基因产物、IL-2、IL-6、TRAIL-R1或TRAIL-R2；
优选地，所述多特异性抗体为“双特异性”、“三特异性”或“四特异性”。
在一个优选实施方案中，本发明提供一种嵌合抗原受体(CAR)；优选地，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含上述任一项所述CD22抗体或抗原结合片段。
在一个优选实施方案中，本发明提供一种免疫效应细胞；优选地，所述免疫效应细胞包含上述所述嵌合抗原受体或包含上述所述嵌合抗原受体的核酸片段；
优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
在一个优选实施方案中，本发明提供一种分离的核酸分子，所述核酸分子编码本发明上述任一项所述的纳米抗体、抗原结合片段、或其任意组合，上述所述的多特异性抗原结合分子或上述所述的嵌合抗原受体。
在一些实施方案中，本发明提供一种表达载体，其包含本发明上述所述分离的核酸分子。
在一些实施方案中，本发明提供一种宿主细胞，其包含本发明上述所述分离的核酸分子或表达载体。
在一个优选实施方案中，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自HEK293E或中国仓鼠卵巢细胞(CHO)。
在一些实施方案中，本发明提供一种抗体或抗原结合片段或多特异性抗原结合分子的制备方法，在适当的条件下培养本发明上述所述的宿主细胞，并分离抗体或抗原结合片段或多特异性抗原结合分子。
在一些实施方案中，本发明提供一种免疫效应细胞的制备方法，将上述所述CAR的核酸片段导入所述免疫效应细胞，优选地，所述方法还包括启动所述免疫效应细胞表达上述所述的CAR。
在一些实施方案中，本发明提供一种药物组合物，组合物包含本发明上述所述的抗体或抗原结合片段、本发明上述所述的多特异性抗原结合分子、本发明上述所述的嵌合抗原受体、本发明上述所述的免疫效应细胞、本发明上述所述分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞，或本发明上述所述方法制备的产品(例如抗体和抗原结合片段)，以及药学上可接受的载体。
在一个优选实施方案中，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；更优选地，所述药物组合物还包含额外的抗肿瘤剂。
在一些实施方案中，本发明提供一种预防和/或治疗B细胞疾病的方法，包含向有此需要的患者施用本发明上述所述的抗体或抗原结合片段、本发明上述所述的多特异性抗原结合分 子、本发明上述所述的嵌合抗原受体、本发明上述所述的免疫效应细胞、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本发明上述所述的方法制备的产品、或本发明上述所述的药物组合物；所述B细胞疾病优选肿瘤或自身免疫病；
优选地，所述肿瘤选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、多发性骨髓瘤；
优选地，所述自身免疫病选自***性红斑狼疮(SLE)、抗磷脂抗体综合征、多发性硬化症、溃疡性结肠炎、克罗恩病、类风湿性关节炎、斯耶格伦氏综合征、吉兰-巴雷综合征、重症肌无力、大血管血管炎、中血管血管炎、结节性多动脉炎、天疱疮、硬皮病、肺出血-肾炎综合征、肾小球肾炎、原发性胆汁性肝硬化、格雷夫斯氏病、膜性肾病、自身免疫性肝炎、口炎性腹泻、阿狄森氏病、多发性肌炎/皮肌炎、单克隆丙种球蛋白病、因子VIII缺乏、冷球蛋白血症、周围神经病变、IgM多神经病、慢性神经病和慢性淋巴细胞性甲状腺炎。
在一些实施方案中，本发明提供上述所述的抗体或抗原结合片段、本发明上述所述的多特异性抗原结合分子、本发明上述所述的嵌合抗原受体、本发明上述所述的免疫效应细胞、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述药物组合物在在制备预防和/或治疗B细胞疾病的药物中的用途，所述B细胞疾病优选肿瘤或自身免疫病；
优选地，所述肿瘤选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、多发性骨髓瘤；
优选地，所述自身免疫病选自***性红斑狼疮(SLE)、抗磷脂抗体综合征、多发性硬化症、溃疡性结肠炎、克罗恩病、类风湿性关节炎、斯耶格伦氏综合征、吉兰-巴雷综合征、重症肌无力、大血管血管炎、中血管血管炎、结节性多动脉炎、天疱疮、硬皮病、肺出血-肾炎综合征、肾小球肾炎、原发性胆汁性肝硬化、格雷夫斯氏病、膜性肾病、自身免疫性肝炎、口炎性腹泻、阿狄森氏病、多发性肌炎/皮肌炎、单克隆丙种球蛋白病、因子VIII缺乏、冷球蛋白血症、周围神经病变、IgM多神经病、慢性神经病和慢性淋巴细胞性甲状腺炎。
在一些实施方案中，本发明提供上述所述的抗体或抗原结合片段、本发明上述所述的多特异性抗原结合分子、本发明上述所述的嵌合抗原受体、本发明上述所述的免疫效应细胞、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述药物组合物用于预防和/或治疗B细胞疾病；所述B细胞疾病优选肿瘤或自身免疫病；
优选，所述肿瘤选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、多发性骨髓瘤；
优选地，所述自身免疫病选自***性红斑狼疮(SLE)、抗磷脂抗体综合征、多发性硬化症、溃疡性结肠炎、克罗恩病、类风湿性关节炎、斯耶格伦氏综合征、吉兰-巴雷综合征、重症肌无力、大血管血管炎、中血管血管炎、结节性多动脉炎、天疱疮、硬皮病、肺出血-肾炎综合征、肾小球肾炎、原发性胆汁性肝硬化、格雷夫斯氏病、膜性肾病、自身免疫性肝炎、口炎性腹泻、阿狄森氏病、多发性肌炎/皮肌炎、单克隆丙种球蛋白病、因子VIII缺乏、冷球蛋白血症、周围神经病变、IgM多神经病、慢性神经病和慢性淋巴细胞性甲状腺炎。
在一些实施方案中，本发明提供一种试剂盒，其包含本发明上述所述的抗体或抗原结合片段、本发明上述所述的多特异性抗原结合分子、本发明上述所述的嵌合抗原受体、本发明上述所述的免疫效应细胞、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、或本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述的药物组合物，以及使用说明。
术语定义和说明
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“天然抗体”是指通过多细胞生物体的免疫***制造和配对的抗体。本文术语“工程化抗体”的抗体是指通过基因工程、抗体工程等技术获得的非天然抗体，示例性地，“工程化抗体”包括人源化抗体、小分子抗体(例如scFv等)、双特异性抗体等等。
本文术语“单特异性”是指具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”可互换使用，是指其具有基本上与天然抗体结构相似的结构。
如本文所用，术语“抗原结合片段”是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature 341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“CD22”是指属于SIGLEC凝集素家族的分子Siglec-2，其存在于成熟B细胞的表面上，并且在某些未成熟的B细胞上存在程度较低。术语“CD22”包括任何人类和非人类动物物种的CD22蛋白，并且具体地包括人类CD22以及非人类哺乳动物的CD22。
如本文所用，术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对CD22的抗原表位而被检测，另一个可以针对CD22的另一个抗原表位或除CD22外的任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
如本文所用，术语“嵌合”抗体是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985J Immunol Methods 125:191-202；以上通过援引加入并入本文。
如本文所用，术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
如本文所用，术语“纳米抗体”是指骆驼体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain  of heavy chain antibody)，它是最小的功能性抗原结合片段。关于VHH和纳米抗体的进一步描述，参考Muyldermans的综述文章(2001，Reviews in Molecular Biotechnology 74：277-302)，以及参考作为一般背景技术提及的以下专利申请：布鲁塞尔自由大学的WO 94/04678、WO95/04079和WO 96/34103；联合利华的WO 94/25591、WO 99/37681、WO 00/40968、WO00/43507、WO 00/65057、WO 01/40310、WO 01/44301、EP 1134231和WO 02/48193；Vlaams Instituut voor Biotechnologie(VIB)的WO97/49805、WO 01/21817、WO 03/035694、WO03/054016和WO 03/055527；Algonomics N.V.和Ablynx N.V.的WO03/050531；加拿大国家研究理事会的WO 01/90190；Institute of Antibodies的WO 03/025020(＝EP 1433793)；以及Ablynx N.V.的WO 04/041867、WO 04/041862、WO 04/041865、WO 04/041863、WO 04/062551、WO 05/044858、WO 06/40153、WO 06/079372、WO 06/122786、WO 06/122787和WO06/122825，和Ablynx N.V.的进一步公开的专利申请。还参考了这些申请中提及的另外的现有技术，特别是国际申请WO 06/040153第41-43页上提及的参考文献列表，所述列表和参考文献通过引用并入本文。如这些参考文献中所述，纳米抗体(特别是VHH序列和部分人源化的纳米抗体)尤其可以通过在一个或更多个框架序列中存在一个或更多个“特征残基”来表征。可在例如WO 08/101985和WO 08/142164中发现纳米抗体的进一步描述，包括纳米抗体的人源化和/或骆驼源化，以及其他的修饰，部分或片段，衍生物或“纳米抗体融合”，多价构建体(包括接头序列的一些非限制性实例)和增加纳米抗体及其制剂的半衰期的不同修饰。对于纳米抗体的进一步的一般描述，参考本文引用的现有技术，例如WO 08/020079(第16页)中所述。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链) 的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含以下之一：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据EU编号***的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不意图包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“裸抗体”是指不与另一种作用剂或分子(例如标记或药物)、肽或多肽连接、融合或缀合的抗体。在具体的实施方案中，由哺乳动物宿主细胞表达的裸抗体可被宿主细胞的糖基化机器(例如糖基化酶)糖基化。在某些实施方案，当通过不具有其自身糖基化机器(例如糖基化酶)的宿主细胞表达时，裸抗体不被糖基化。在某些实施方案中，裸抗体是完整抗体，而在其它实施方案中，裸抗体是完整抗体的抗原结合片段，例如Fab抗体。
本文术语“缀合抗体”是指可与药学上可接受的载体或稀释剂缔合的抗体，其可为单克隆抗体、嵌合抗体、人源化抗体或人抗体。
本文术语“双抗体”是指二价的双特异性抗体，可以与相同或不同抗原上的不同表位结合。
如本文所用，术语“百分比(％)序列一致性”是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸彼此属于保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
(1)酸性氨基酸：Asp(D)和Glu(E)；
(2)碱性氨基酸：Lys(K)、Arg(R)和His(H)；
(3)亲水性不带电荷氨基酸：Ser(S)、Thr(T)、Asn(N)和Gln(Q)；
(4)脂肪族不带电荷氨基酸：Gly(G)、Ala(A)、Val(V)、Leu(L)和Ile(I)；
(5)非极性不带电荷的氨基酸：Cys(C)、Met(M)和Pro(P)；
(6)芳香族氨基酸：Phe(F)、Tyr(Y)和Trp(W)。
本文术语“Kabat编号***”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号***(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991；其通过援引加入并入本文)。
本文术语“Chothia编号***”通常是指由Chothia等人提出的免疫球蛋白编号***，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883；其通过援引加入并入本文)。
本文术语“IMGT编号***”通常是指由Chothia等人提出的免疫球蛋白编号***，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883；其通过援引加入并入本文)。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM((例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特 异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
如本文所用，术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
本文术语“嵌合抗原受体(CAR)”是指这样的重组蛋白，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。在某些实施方式中，CAR的细胞外抗原结合结构域包含scFv。scFv可以源自融合抗体的可变重和轻区。替代地或另外，scFv可以衍生自Fab’s(而不是抗体，例如获自Fab文库)。在某些实施方式中，将scFv融合至跨膜结构域，然后融合至细胞内信号传导结构域。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本发明的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823B1)。
如本文所用，术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码 用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、绵羊和马等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“适当的条件”指适合培养各种宿主细胞的条件，其中宿主细胞包括真核细胞和原核细胞。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。
本文术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“抗肿瘤剂”指抗肿瘤药物，其为***疾病的一类药物，有化疗药物、生物制剂等。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值 之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
本文术语“EC80”是指能引起80％最大效应的抗体浓度。
附图说明
除非本发明另外定义，与本发明相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
图1A为ELISA检测人CD22-ECD蛋白免疫后羊驼血清抗体效价情况；图1B为FACS检测人CD22-ECD蛋白免疫后羊驼血清抗体效价情况。
图2为CD22-ECD-His、CD22domain1-4-His和CD22domain5-7-His蛋白样品SDS-PAGE还原胶和非还原胶检测结果。泳道1为非还原条件下hCD22-ECD-His的蛋白条带，泳道2为非还原条件下hCD22domain5-7-His的蛋白条带，泳道3为还原条件下hCD22domain5-7-His的蛋白条带，泳道4为还原条件下hCD22-ECD-His的蛋白条带，泳道5为非还原条件下hCD22domain1-4-His的蛋白条带，泳道6为还原条件下hCD22domain1-4-His的蛋白条带，泳道M为蛋白maker条带。
图3A为ELISA检测对照抗体与人CD22-ECD-His蛋白的结合反应；图3B为ELISA检测对照抗体与人CD22domain1-4-His蛋白的结合反应；图3C为ELISA检测对照抗体与人CD22domain5-7-His蛋白的结合反应。抗CD22对照抗体为：HA22和m971，阴性对照为hIgG1。
图4A为HA22抗体检测Raji细胞CD22表达量的FACS结果；图4B为m971抗体检测Raji细胞CD22表达量的FACS结果。
图5为人CD22蛋白转染的CHO-K1细胞FACS筛选检测结果。
图6为ELISA检测本发明VHH-Fc抗体与人CD22-ECD-His蛋白的结合反应。抗CD22阳性对照抗体为：HA22和m971，阴性对照为hIgG1。
图7A为FACS检测本发明VHH-Fc抗体与CHO-K1-人CD22的结合反应；图7B为FACS检测本发明VHH-Fc抗体与Raji的结合反应。抗CD22阳性对照抗体为：HA22、m971和hL22，阴性对照为hIgG1；图7C为FACS检测1nM和10nM本发明VHH-Fc抗体与CHO-K1细胞和CHO-K1-人CD22 2C4的结合反应；图7D为FACS检测1nM和10nM本发明VHH-Fc抗体与Raji细胞和Jurkat细胞的结合反应。
图8为ELISA检测本发明VHH-Fc抗体与鼠CD22-ECD-His蛋白的结合反应；阳性对照为983；阴性对照为hIgG1；
图9为FACS检测CD20抗体和1nM本发明VHH-Fc抗体双染色食蟹猴外周血单核细胞散点图，CD20为B细胞标记物，图中所示比例为本发明VHH-Fc抗体阳性细胞占CD20阳性细胞比例，抗CD22阳性对照抗体为：HA22，阴性对照为hIgG1。
图10为SPR检测本发明VHH-Fc抗体与人CD22的亲和力，抗人CD22阳性对照抗体为：HA22和m971。
图11为SPR检测本发明VHH-Fc抗体与食蟹猴CD22的亲和力，抗人CD22阳性对照抗体为：HA22。
图12A为ELISA检测本发明VHH-Fc与人CD22domain1-4-His蛋白的结合反应；图12B为ELISA检测本发明VHH-Fc与人CD22domain5-7-His蛋白的结合反应。抗CD22阳性对照抗体为：HA22和m971，阴性对照为hIgG1。
图13为竞争性ELISA方法检测本发明VHH抗体之间的抑制率。
图14为本发明VHH抗体的抗原表位分类。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1针对人CD22的单域抗体的筛选
1.1羊驼的免疫和血清效价检测
免疫用的人CD22(Asp20-Arg687)-His蛋白购自ACRO Biosystems(货号：CD2-H52H8)。选取两只羊驼(Llama)进行免疫，每只羊驼免疫四次，每次间隔3周，第三次免疫后和第四次免疫后采集外周血并分离血清，用酶联免疫吸附实验(ELISA)和流式细胞实验(FACS)检测血清中针对人CD22的抗体效价和特异性，结果如图1A-1B和表1所示。表1说明，经人CD22免疫的羊驼，免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在五百九十万左右。其中空白对照为1％(w/w)BSA，其中批次指第三次(TB2)和第四次(TB3)免疫后第七天的羊驼血清，表中的数据为OD450nm值。
表1.ELISA检测人CD22蛋白免疫后羊驼血清抗体效价
Figure PCTCN2021135058-appb-000004
1.2文库的构建
采集三次免疫后和四次免疫后的羊驼外周血共100mL；使用淋巴细胞分离液分离PBMC， 并使用RNAiso Plus试剂(Takara，货号：#9108/9109)提取总RNA，使用PrimeScript TM II 1st Strand cDNA Synthesis Kit(Takara，货号：6210A)将提取的RNA反转录成cDNA。用巢式PCR扩增编码纳米抗体的可变区核酸片段：
第一轮PCR：
上游引物：CTTGGTGGTCCTGGCTGC(SEQ ID NO.16)
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.17)
第二轮PCR：
以第一轮PCR产物作为模板，
上游引物：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC(SEQ ID NO.18)
下游引物-1：
CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG(SEQ ID NO.19)
下游引物-2：
CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG(SEQID NO.20)
回收目标纳米抗体核酸片段，并使用限制性内切酶SfiI(NEB，货号：R0123S)将其克隆进入噬菌体展示用载体pcomb3XSS(来自四川阿帕克生物科技有限公司)中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对CD22的纳米抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为2.0×10 9。为检测文库的***率，随机选取48个克隆做菌落PCR。结果显示***率达到100％。
1.3针对CD22的纳米抗体VHH的淘选
用人CD22-llama Fc融合蛋白(ACRO Biosystems，货号：SI2-H525a)0.5μg/孔包被平板，4℃放置过夜；第二天用3％BSA-PBS 37℃封闭1h后，加入100μl噬菌体展示文库，37℃孵育1h；之后用PBST洗涤6次，PBS洗涤2次，以洗掉不结合的噬菌体。最后加入100μL Gly-HCl洗脱液，洗脱下来特异性结合CD22的噬菌体从而富集阳性的克隆。
1.4噬菌体酶联免疫方法筛选特异性单个阳性克隆
淘选后，将获得的CD22结合阳性的噬菌体感染空白大肠杆菌并铺板。随后挑选96个单菌落分别扩增培养。用人CD22-llama Fc和人CD22-His蛋白分别包被平板4℃过夜，将噬菌体培养上清加入，37℃孵育1小时。洗涤之后加入TMB显色液显色，于450nm波长测光密度。挑选人CD22-llama Fc和人CD22-His双阳性克隆进行测序。对测序结果使用MOE软件进行分析，根据VHH编码蛋白氨基酸序列构建进化树，根据序列相似性剔除在进化树上距离较近的序列后，筛选获得18个克隆，其序列的CDRs分别用KABAT、Chothia或IMGT软件分析，对应的序列信息如下表2-4所示，其中表2示出18个纳米抗体分子氨基酸表示的抗体序列，表3示出18个纳米抗体分子核苷酸表示的抗体序列，表4示出18个纳米抗体分子CDRs的IMGT、Kabat和Chothia分析结果。随后进行VHH纳米抗体Fc融合蛋白的生产鉴定。
表2.抗CD22抗体重链可变区的氨基酸具体序列信息
Figure PCTCN2021135058-appb-000005
Figure PCTCN2021135058-appb-000006
表3.抗CD22抗体重链可变区的核苷酸具体序列信息
Figure PCTCN2021135058-appb-000007
Figure PCTCN2021135058-appb-000008
Figure PCTCN2021135058-appb-000009
Figure PCTCN2021135058-appb-000010
表4.IMGT、KABAT和Chothia软件分析CD22纳米抗体的CDRs具体序列信息
Figure PCTCN2021135058-appb-000011
Figure PCTCN2021135058-appb-000012
Figure PCTCN2021135058-appb-000013
Figure PCTCN2021135058-appb-000014
实施例2 VHH抗体、对照抗体、多克隆抗体血清和CD22蛋白制备
2.1VHH抗体的表达纯化
由泰州市百英生物科技有限公司将VHH可变区序列重组到包含信号肽和人IgG1 Fc(人IgG1 Fc序列如SEQ ID NO：14，铰链区序列如SEQ ID NO：15)的表达载体BI3.4-huIgG1中，并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。将表达载体按照PEI(购自Polysciences，货号：24765-1)说明书瞬时转染HEK293E细胞(购自苏州益研生物科技有限公司)，并使用FreeStyle  TM 293(Thermofisher scientific，货号：12338018)在37℃下连续培养5天，离心去除细胞成分，获得含VHH抗体的培养上清液。将培养上清液上样到蛋白A层析柱(蛋白A填料AT Protein A Diamond和层析柱BXK16/26均购自博格隆，货号分别为：AA0273和B-1620)，使用PBS磷酸盐缓冲液(pH7.4)清洗后再用20mM PB,1M NaCl(pH 7.2)进行清洗，最后使用pH3.4的柠檬酸缓冲液进行洗脱，收集从蛋白A层析柱上洗脱下来的带Fc标签的抗体，用1/10体积的pH8.0的1M Tris中和，用PBS在4℃条件透析过夜，透析后的蛋白经0.22微米滤膜无菌过滤后分装于-80℃保存。
SEQ ID NO:14人IgG1 Fc的序列:
Figure PCTCN2021135058-appb-000015
SEQ ID NO:15铰链区序列:
EPKSADKTHTCPPCP
2.2对照抗体的制备
CD22蛋白胞外具有7个类IgG样结构域，其中domain1位于最远膜端，domain7位于最近膜端。HA22，m971和hL22是识别人CD22的抗体，其中HA22和hL22的抗原结合表位位于domain 2-3，m971的抗原结合表位位于domain5-7。HA22的重链可变区和轻链可变区序列根据专利US 9580461B(其通过援引加入并入本文)获得，m971的重链可变区和轻链可变区序列根据专利US 8591889B(其通过援引加入并入本文)获得，hL22的重链可变区和轻链可变区氨基酸序列根据专利US5789554B(其通过援引加入并入本文)获得。分别将识别人CD22的抗体HA22，m971和hL22的VH和VL以及人IgG1 Fc按照从N端到C端的顺序连接，其中VH和VL之间通过3个GGGGS连接子连接，形成scFv-hFc的形式，对应的氨基酸序列信息如下表5所示。将其对应的核苷酸序列分别克隆到pTT5载体(由通用生物***(安徽)有限公司完成)并按照实施例2.1的方法在HEK293E细胞(购自苏州益研生物科 技有限公司)中进行表达并纯化。
表5.抗人CD22的抗体HA22、m971和hL22的VH、VL及与人IgG1 Fc形成scFv-hFc形式的具体序列信息
Figure PCTCN2021135058-appb-000016
Figure PCTCN2021135058-appb-000017
2.3人CD22-his标签蛋白的制备
CD22蛋白胞外具有7个类IgG样结构域，其中domain1位于最远膜端，domain7位于最近膜端，HA22和hL22的抗原结合表位位于domain 2-3，m971的抗原结合表位位于domain5-7。将含有编码人CD22蛋白的氨基酸序列(NCBI：NP_001762.2,SEQ ID NO:1)、胞外区(ECD，extra-cellular domain)氨基酸序列Asp 20-Arg 687(SEQ ID NO：2)、结构域(domain)1-4Asp 20-Val 425氨基酸序列(SEQ ID NO：3)和结构域(domain)5-7Asp 414-Arg 687氨基酸序列(SEQ ID NO：4)的核苷酸序列分别克隆到pTT5载体(由通用生物***(安徽)有限公司完成)并按已建立的标准分子生物学方法制备质粒，对应的氨基酸序列信息如下表6所示。具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293E细胞(购自苏州益研生物科技有限公司)进行瞬时转染(PEI，Polysciences，货号：24765-1)并使用FreeStyle  TM 293(Thermofisher scientific，货号：12338018)在37℃下进行扩大培养。6天后收集细胞培养液，离心去除细胞成分，获得含人CD22蛋白胞外区的培养上清液。将培养上清液上样到镍离子亲和层析柱HisTrap TM Excel(GE Healthcare，货号：GE17-3712-06)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用20mM PB，0.5M NaCl(pH7.4)清洗镍离子亲和层析柱直到紫外吸收值回到基线，然后用buffer A：20mM PB，0.5M NaCl(pH7.4)和buffer B：20mM PB，0.5M NaCl，500mM咪唑进行梯度洗脱(2％,4％,8％,16％,50％,100％)，收集从镍离子亲和层析柱上洗脱下来的带His标签的人CD22蛋白，用PBS磷酸盐缓冲液(pH7.4)在4℃冰箱透析过夜。透析后的蛋白经0.22微米滤膜无菌过滤后分装于-80℃保存，即获得纯化的人CD22蛋白，SDS-PAGE还原胶和非还原胶检测样品目的条带如图2所示。对制备的上述CD22蛋白应用识别不同表位的阳性对照抗体进行ELISA检测，阴性对照抗体hIgG1为针对鸡卵溶菌酶的抗体anti-hel-hIgG1(购自百英，货号：B117901)，检测结果如图3A-3C所示，HA22和m971均能结合人CD22-ECD-His蛋白，HA22能结合人CD22domain1-4-His蛋白，而m971能结合人CD22domain5-7-His蛋白，检测结果与文献报道的HA22和m971的结合表位一致，说明已经制备获得具有上述结合活性的蛋白。
表6.人CD22蛋白及胞外区氨基酸序列
Figure PCTCN2021135058-appb-000018
实施例3内源表达细胞株的鉴定和过表达细胞株的制备
3.1内源性表达CD22细胞株的鉴定
将Raji细胞(购自武汉大学中国典型培养物保藏中心)在T-25细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。用HA22和m971抗体作为一抗，APC标记的二抗(购自Biolegend，货号：409306)，用FACS(FACS CantoTM，购自BD公司)检测和分析结果。分析结果如表7以及图4A-4B所示，Raji细胞均可与HA22和m971结合。
表7.内源细胞系Raji细胞的FACS检测结果
Figure PCTCN2021135058-appb-000019
3.2人CD22稳转CHO-K1单克隆细胞株的制备
编码人CD22全长氨基酸序列(NCBI：NP_001762.2，SEQ ID NO：1)的核苷酸序列被克隆到pcDNA3.1载体并制备质粒(由通用生物***(安徽)有限公司完成)。对CHO-K1细胞系(购自中国科学院上海生命科学研究院)进行质粒转染(
Figure PCTCN2021135058-appb-000020
3000 Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含10μg/ml嘌呤霉素和含10％(w/w)胎牛血清的DMEM/F12培养基中选择性培养2周，用FITC标记的抗CD22抗体(Thermofisher scientific，货号：11-0229-42)在流式细胞仪FACS AriaII(BD Biosciences)上分选阳性单克隆细胞到96孔板，并置于37℃，5％(v/v)CO 2细胞培养箱中培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较好、荧光强度较高的单克隆细胞系继续扩大培养并液氮冻存。
具体选择结果如表8和图5所示，IgG亚型对照为人IgG1对照。表8说明，已经制得一系列CD22阳性表达的CHO-K1单克隆细胞系。图5中，横坐标为细胞荧光强度，纵坐标为细胞数。图5的结果说明，CHO-K1-人CD22 2C4、CHO-K1-人CD22 1G5以及CHO-K1-人CD22 1D9为CD22高水平表达细胞株。
表8.人CD22蛋白的CHO-K1稳转细胞系FACS检测结果
Figure PCTCN2021135058-appb-000021
实施例4 VHH羊驼抗体的鉴定
4.1酶联免疫吸附实验(ELISA)检测VHH-Fc抗体与人CD22蛋白的结合
为了检测VHH-Fc与人CD22蛋白的结合活性，将实施例2获得的纯化的人CD22 -ECD-His蛋白用PBS稀释到终浓度2μg/mL，然后以100μl/孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS洗板2次，加入封闭液[PBS+2％(w/w)BSA]室温封闭2小时。倒掉封闭液，以50μl/孔加入100nM梯度稀释的VHH-Fc抗体或阴性对照抗体。37℃孵育2小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma，货号：A0170)，37℃孵育2小时后，用PBS洗板5次。加入TMB底物50μl/孔，室温孵育30分钟后，加入终止液(1.0N HCl)50μl/孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值，VHH-Fc与人CD22-ECD的ELISA结果如图6和表9所示，表9说明，纯化后的抗体均能与人CD22-ECD在ELISA水平结合。其中IgG对照为hIgG1，表中的数据为OD450nm值。
表9.ELISA检测VHH-Fc抗体与人CD22蛋白的结合反应
Figure PCTCN2021135058-appb-000022
4.2流式细胞实验(FACS)检测抗体与不同CD22表达细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，对于贴壁细胞CHO-K1吸除培养基，用PBS缓冲液洗涤2次，然后用胰酶消化细胞，终止消化后用PBS缓冲液洗涤细胞2次；对于悬浮细胞Raji直接离心弃去培养基上清，细胞沉淀用PBS洗涤2次。对上一步的细胞进行细胞计数后将细胞沉淀用[PBS+2％(w/w)BSA]封闭液重悬至2x10 6个细胞/毫升，按50μl/孔加入到96孔FACS反应板中，加入VHH-Fc抗体待测样品50μl/孔，冰上孵育2小时。用PBS缓冲液离心洗涤3次，加入50μl/孔Alexa Flour 488标记的二抗(购自Invitrogen，货号：A-11013)，冰上孵育1小时。用PBS缓冲液离心洗涤5次，用FACS(FACS CantoTM，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，得到细胞的平均荧 光密度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50值。分析结果如表10以及图7A-7B所示，VHH-Fc抗体均可结合Raji细胞和CHO-K1-人CD22 2C4细胞表面的人CD22蛋白(图7A-7B)。使用同样的方法同时检测了VHH-Fc抗体与内源CD22阴性的细胞Jurkat细胞(购自ATCC，TIB-152)以及CHO-K1细胞的结合，结果如图7C-7D所示，所有VHH-Fc抗体均不结合Jurkat细胞以及CHO-K1细胞，具有很好的特异性。
表10.FACS检测VHH-Fc抗体与Raji和CHO-K1-人CD22 2C4细胞的结合反应
Figure PCTCN2021135058-appb-000023
Figure PCTCN2021135058-appb-000024
实施例5 VHH-Fc抗体的种属交叉结合活性检测
5.1 ELISA检测VHH-Fc抗体与鼠CD22蛋白的结合
为检测VHH-Fc抗体的种属交叉活性，将商品化的鼠CD22(ACROBiosystems，货号：SI2-M52Ha)包被ELISA板，按照实施例4.1的方法进行ELISA检测。VHH-Fc与鼠CD22-ECD的ELISA结果如图8和表11所示，表11说明，纯化后的抗体只有S002-NB151-51与鼠CD22-ECD在ELISA水平有结合。其中IgG对照为hIgG1，983为人CD22-ECD-His免疫小鼠后的血清作为阳性对照，表中的数据为OD450nm值。
表11.ELISA检测VHH-Fc抗体与鼠CD22蛋白的结合反应
Figure PCTCN2021135058-appb-000025
Figure PCTCN2021135058-appb-000026
*：983血清从1：100开始进行5倍浓度梯度稀释。
5.2 FACS检测VHH-Fc抗体与食蟹猴(拉丁名：Macaca fascicularis)外周血B细胞的结合
从新鲜食蟹猴外周血(购自上海美迪西生物医药股份有限公司)中按照Ficoll-Paque Plus(购自GE Healthcae，货号:171440-02)说明书提取猴外周血单核细胞，细胞悬液离心后以含1％BSA的PBS重悬细胞后计数，同时加入有猴CD20交叉结合活性的鼠源抗体Brilliant Violet 605 anti-human CD20(货号:302334，购自Biolegend)和待测VHH-Fc抗体(1nM、10nM和100nM)。室温孵育1小时。清洗细胞三次后加入APC标记的二抗anti-human IgG Fc(货号：409306，购自Biolegend)，避光室温孵育30分钟后清洗细胞5次，用含1％BSA的PBS轻轻重悬细胞，用FACS(FACS CantoTM，购自BD公司)检测和分析，其中CD20作为B细胞的标记物，对CD20阳性的B细胞群进行圈门，分析其中VHH-Fc阳性细胞所占比例，分别计算100nM，10nM和1nM浓度下的VHH-Fc抗体处理的VHH-Fc阳性细胞群占B细胞群的比例，结果如表12所示。Brilliant Violet 605标记CD20和APC二抗间接标记VHH-Fc的双染细胞散点图见图9(1nM VHH-Fc抗体浓度条件下)。由结果可知，S002-NB151-2、S002-NB151-14、S002-NB151-23、S002-NB151-92、S002-NB151-51、S002-NB150-2即使在1nM低浓度条件下依然与食蟹猴B细胞有较高比例的结合，且相比于阳性抗体HA22有相当或者更好的结合活性；其它抗体与食蟹猴CD22无结合或者相对较弱结合。
表12.FACS检测VHH-Fc抗体与食蟹猴B细胞的结合反应
Figure PCTCN2021135058-appb-000027
Figure PCTCN2021135058-appb-000028
实施例6 CD22抗体亲和力测定
6.1 VHH-Fc与人CD22-ECD-His蛋白亲和力测定
使用Protein A芯片(GE Helthcare；29-127-558)捕获抗人CD22 VHH-hFc抗体。样品和运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃。样品块设置为16℃。两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的CD22抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Helthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的重组人CD22-ECD His持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1：1稀释，总共5个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5)以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异。也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1：1模型。VHH-Fc抗体与人CD22-His蛋白的结合速率(Ka)、解离速率(Kd)及结合亲和力(KD)如表13所示，其中抗体HA22作为对照。如图10和表13所示，VHH-Fc抗体与人CD22的亲和力都优于4.89E-8M
表13.SPR(biacore)检测VHH-Fc抗体与人CD22的亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
S002-NB151-2 1.69E+04 8.27E-04 4.89E-08
S002-NB151-13 5.98E+04 3.58E-05 5.98E-10
S002-NB151-14 1.60E+04 2.54E-04 1.59E-08
S002-NB151-23 2.04E+05 4.76E-04 2.33E-09
S002-NB151-30 8.43E+04 5.14E-04 6.09E-09
抗体名称 ka(1/Ms) kd(1/s) KD(M)
S002-NB151-36 8.73E+04 5.20E-04 5.97E-09
S002-NB151-51 1.42E+05 7.39E-05 5.20E-10
S002-NB151-64 2.39E+05 2.07E-03 8.68E-09
S002-NB151-66 2.49E+05 5.62E-04 2.25E-09
S002-NB151-82 6.00E+04 1.74E-03 2.90E-08
S002-NB151-92 4.40E+04 1.11E-03 2.52E-08
S002-NB150-2 3.17E+04 7.61E-05 2.40E-09
S002-NB150-5 8.32E+04 3.09E-04 3.71E-09
S002-NB150-40 8.63E+04 5.41E-04 6.27E-09
S002-NB150-42 1.96E+05 4.23E-04 2.16E-09
S002-NB150-57 1.10E+05 4.07E-04 3.70E-09
S002-NB150-152n 2.12E+05 4.42E-04 2.08E-09
S002-NB150-159n 2.89E+04 6.95E-05 2.40E-09
HA22 8.15E+04 1.12E-04 1.37E-09
m971 2.51E+05 7.36E-03 2.93E-08
6.2 VHH-Fc与食蟹猴CD22-ECD-His蛋白亲和力测定
按照实施例6.1的方法对VHH-Fc抗体与食蟹猴CD22-ECD-His(购自R&D，货号：9864-SL-050)蛋白进行亲和力测定，结果如图11和表14所示，在低浓度下与食蟹猴B细胞依然有较高比例结合的抗体S002-NB151-51，与食蟹猴CD22的亲和力较高，为5.07E-10M。
表14.SPR(biacore)检测VHH-Fc抗体与cyno CD22的亲和力
Figure PCTCN2021135058-appb-000029
实施例7抗体抗原结合表位(epitope)分析
7.1抗体抗原结合区域的鉴定
CD22蛋白胞外具有7个类IgG样结构域，其中domain1位于最远膜端，domain7位于最近膜端，HA22和hL22的抗原结合表位位于domain 2-3，m971的抗原结合表位位于domain5-7。为了鉴定VHH抗体的抗原结合表位分布，按照实施例4.1中的ELISA方法，分别包被人CD22-domain1-4-His(远膜端)和人CD22domain5-7-His(近膜端)，对VHH抗体进行远膜端和近膜端分类，如图12A-12B和表15所示，可以将VHH抗体分为两类：
表15.ELISA方法对VHH抗体进行远膜端和近膜端表位分类
Figure PCTCN2021135058-appb-000030
Figure PCTCN2021135058-appb-000031
7.2抗体抗原结合表位竞争实验(epitope binning)
采用竞争性ELISA方法对VHH抗体与已知表位的对照抗体进行表位分类。按照实施例4.2的方法将2μg/mL的抗体包被ELISA板，人CD22-ECD-his蛋白从30μg/mL开始进行梯度稀释，计算出EC80值(表16)。将2μg/mL的抗体包被ELISA板，加入25μg/mL待检测的抗体后，再加入每个待检测抗体对应的EC80浓度的人CD22-ECD-his蛋白，孵育2h，用PBS洗5次后加入HRP标记的anti-His抗体检测。如果包被抗体与溶液中的待检抗体不存在竞争关系，则能够与溶液中的待检抗体-人CD22-ECD-his抗原复合物结合，而检测到OD450nm吸收，根据OD450nm吸光值计算出每对抗体之间的抑制率(图13)。根据抑制率将各抗体表位进行如图14的分类，S002-NB151-23、S002-NB151-64、S002-NB151-66、S002-NB150-2、S002-NB150-5、S002-NB150-40、S002-NB150-42、S002-NB150-57、S002-NB150-152n与hL22和HA22之间存在竞争，可以归为一类；S002-NB151-82、S002-NB151-36、S002-NB151-14之间存在竞争关系；S002-NB151-30和S002-NB151-51之间存在竞争关系；S002-NB151-13不与任何抗体存在竞争关系；上述这些抗体都能结合domain1-4而不结合domain5-7，可以归为一大类。S002-NB151-2、S002-NB151-92、S002-NB150-159n之间存在竞争；m971不与任 何抗体存在竞争关系，这些抗体都能结合domain5-7而不结合domain1-4，可以归为一大类。
表16 VHH抗体对应的人CD22-ECD-his蛋白EC80值
Figure PCTCN2021135058-appb-000032

Claims (22)

  1. 特异性结合CD22的纳米抗体或抗原结合片段，其特征在于，所述纳米抗体或抗原结合片段包含CDRs组合，所述CDRs组合包含：CDR1、CDR2和CDR3；所述CDR1、CDR2和CDR3具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸***、缺失和/或替换的序列组合：
    Figure PCTCN2021135058-appb-100001
    Figure PCTCN2021135058-appb-100002
    Figure PCTCN2021135058-appb-100003
    各个CDR1、CDR2和CDR3为根据KABAT、Chothia或IMGT的通行分析方法编码；
    优选地，所述替换为保守氨基酸的替换。
  2. 权利要求1所述的纳米抗体或抗原结合片段，其特征在于，
    (1)所述CDR1、CDR2和CDR3分别如SEQ ID NO.57、58、59所示序列；
    (2)所述CDR1、CDR2和CDR3分别如SEQ ID NO.60、61、62所示序列；
    (3)所述CDR1、CDR2和CDR3分别如SEQ ID NO.63、64、65所示序列；
    (4)所述CDR1、CDR2和CDR3分别如SEQ ID NO.66、67、68所示序列；
    (5)所述CDR1、CDR2和CDR3分别如SEQ ID NO.69、70、71所示序列；
    (6)所述CDR1、CDR2和CDR3分别如SEQ ID NO.72、73、74所示序列；
    (7)所述CDR1、CDR2和CDR3分别如SEQ ID NO.75、76、77所示序列；
    (8)所述CDR1、CDR2和CDR3分别如SEQ ID NO.78、79、80所示序列；
    (9)所述CDR1、CDR2和CDR3分别如SEQ ID NO.81、82、83所示序列；
    (10)所述CDR1、CDR2和CDR3分别如SEQ ID NO.84、85、86所示序列；
    (11)所述CDR1、CDR2和CDR3分别如SEQ ID NO.87、88、89所示序列；
    (12)所述CDR1、CDR2和CDR3分别如SEQ ID NO.90、91、92所示序列；
    (13)所述CDR1、CDR2和CDR3分别如SEQ ID NO.93、94、95所示序列；
    (14)所述CDR1、CDR2和CDR3分别如SEQ ID NO.96、97、98所示序列；
    (15)所述CDR1、CDR2和CDR3分别如SEQ ID NO.99、100、101所示序列；
    (16)所述CDR1、CDR2和CDR3分别如SEQ ID NO.102、103、104所示序列；
    (17)所述CDR1、CDR2和CDR3分别如SEQ ID NO.105、106、107所示序列；
    (18)所述CDR1、CDR2和CDR3分别如SEQ ID NO.108、109、110所示序列；
    (19)所述CDR1、CDR2和CDR3分别如SEQ ID NO.111、112、113所示序列；
    (20)所述CDR1、CDR2和CDR3分别如SEQ ID NO.114、115、116所示序列；
    (21)所述CDR1、CDR2和CDR3分别如SEQ ID NO.117、118、119所示序列；
    (22)所述CDR1、CDR2和CDR3分别如SEQ ID NO.120、121、122所示序列；
    (23)所述CDR1、CDR2和CDR3分别如SEQ ID NO.123、124、125所示序列；
    (24)所述CDR1、CDR2和CDR3分别如SEQ ID NO.126、127、128所示序列；
    (25)所述CDR1、CDR2和CDR3分别如SEQ ID NO.129、130、131所示序列；
    (26)所述CDR1、CDR2和CDR3分别如SEQ ID NO.132、133、134所示序列；
    (27)所述CDR1、CDR2和CDR3分别如SEQ ID NO.135、136、137所示序列；
    (28)所述CDR1、CDR2和CDR3分别如SEQ ID NO.138、139、140所示序列；
    (29)所述CDR1、CDR2和CDR3分别如SEQ ID NO.141、142、143所示序列；
    (30)所述CDR1、CDR2和CDR3分别如SEQ ID NO.144、145、146所示序列；
    (31)所述CDR1、CDR2和CDR3分别如SEQ ID NO.147、148、149所示序列；
    (32)所述CDR1、CDR2和CDR3分别如SEQ ID NO.150、151、152所示序列；
    (33)所述CDR1、CDR2和CDR3分别如SEQ ID NO.153、154、155所示序列；
    (34)所述CDR1、CDR2和CDR3分别如SEQ ID NO.156、157、158所示序列；
    (35)所述CDR1、CDR2和CDR3分别如SEQ ID NO.159、160、161所示序列；
    (36)所述CDR1、CDR2和CDR3分别如SEQ ID NO.162、163、164所示序列；
    (37)所述CDR1、CDR2和CDR3分别如SEQ ID NO.165、166、167所示序列；
    (38)所述CDR1、CDR2和CDR3分别如SEQ ID NO.168、169、170所示序列；
    (39)所述CDR1、CDR2和CDR3分别如SEQ ID NO.171、172、173所示序列；
    (40)所述CDR1、CDR2和CDR3分别如SEQ ID NO.174、175、176所示序列；
    (41)所述CDR1、CDR2和CDR3分别如SEQ ID NO.177、178、179所示序列；
    (42)所述CDR1、CDR2和CDR3分别如SEQ ID NO.180、181、182所示序列；
    (43)所述CDR1、CDR2和CDR3分别如SEQ ID NO.183、184、185所示序列；
    (44)所述CDR1、CDR2和CDR3分别如SEQ ID NO.186、187、188所示序列；
    (45)所述CDR1、CDR2和CDR3分别如SEQ ID NO.189、190、191所示序列；
    (46)所述CDR1、CDR2和CDR3分别如SEQ ID NO.192、193、194所示序列；
    (47)所述CDR1、CDR2和CDR3分别如SEQ ID NO.195、196、197所示序列；
    (48)所述CDR1、CDR2和CDR3分别如SEQ ID NO.198、199、200所示序列；
    (49)所述CDR1、CDR2和CDR3分别如SEQ ID NO.201、202、203所示序列；
    (50)所述CDR1、CDR2和CDR3分别如SEQ ID NO.204、205、206所示序列；
    (51)所述CDR1、CDR2和CDR3分别如SEQ ID NO.207、208、209所示序列；
    (52)所述CDR1、CDR2和CDR3分别如SEQ ID NO.210、211、212所示序列；
    (53)所述CDR1、CDR2和CDR3分别如SEQ ID NO.213、214、215所示序列；
    (54)所述CDR1、CDR2和CDR3分别如SEQ ID NO.216、217、218所示序列；或，
    所述CDR1、CDR2和CDR3为具有与上述(1)—(54)序列组合相比具有1、2、3或更多个氨基酸***、缺失和/或替换的序列组合。
  3. 权利要求1-2任一项所述的纳米抗体或抗原结合片段，其特征在于，所述的纳米抗体或抗原结合片段包含：
    (1)可变区具有SEQ ID NO:21所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (2)可变区具有SEQ ID NO:23所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (3)可变区具有SEQ ID NO:25所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (4)可变区具有SEQ ID NO:27所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (5)可变区具有SEQ ID NO:29所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (6)可变区具有SEQ ID NO:31所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (7)可变区具有SEQ ID NO:33所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (8)可变区具有SEQ ID NO:35所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (9)可变区具有SEQ ID NO:37所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (10)可变区具有SEQ ID NO:39所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (11)可变区具有SEQ ID NO:41所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (12)可变区具有SEQ ID NO:43所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (13)可变区具有SEQ ID NO:45所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (14)可变区具有SEQ ID NO:47所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (15)可变区具有SEQ ID NO:49所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (16)可变区具有SEQ ID NO:51所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    (17)可变区具有SEQ ID NO:53所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；或，
    (18)可变区具有SEQ ID NO:55所示序列，或者与上述所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
  4. 权利要求1-3任一项所述的纳米抗体或抗原结合片段，其特征在于，其与人CD22结合的解离常数(KD)不大于50nM。
  5. 权利要求1-4任一项所述的纳米抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
  6. 权利要求1-4任一项所述的纳米抗体或抗原结合片段，其特征在于，其进一步包含不存在CH1片段的重链恒定区序列。
  7. 权利要求1-4任一项所述的纳米抗体或抗原结合片段，其特征在于，其进一步包含具有CH2和CH3片段的重链恒定区序列。
  8. 权利要求1-7任一项所述的纳米抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段为：
    (1)嵌合抗体或其片段；
    (2)人源化抗体或其片段；或，
    (3)全人源抗体或其片段；
    优选地，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  9. 根据权利要求1～8任一项所述的纳米抗体或抗原结合片段，其特征在于，所述纳米抗 体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
  10. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含权利要求1～9任一项所述的纳米抗体或抗原结合片段，所述第二抗原结合模块特异性结合CD22以外的其他抗原或结合与第一抗原结合模块不同的CD22抗原表位；
    优选地，所述其他抗原选自CD3、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、B7、MUC、Ia、HLA-DR、腱生蛋白、VEGF、P1GF、ED-B纤连蛋白、癌基因产物、IL-2、IL-6、TRAIL-R1或TRAIL-R2；
    优选地，所述多特异性抗体为双特异性抗体、三特异性抗体或四特异性抗体。
  11. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1～9任一项所述纳米抗体或抗原结合片段。
  12. 一种免疫效应细胞，其特征在于，所述免疫效应细胞包含权利要求11所述嵌合抗原受体或包含编码权利要求11所述嵌合抗原受体的核酸片段；
    优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自:炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
    优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
  13. 一种分离的核酸分子，其特征在于，所述核酸分子编码权利要求1-9任一项所述的纳米抗体或抗原结合片段、或其任意组合，权利要求10所述的多特异性抗原结合分子或权利要求11所述的嵌合抗原受体。
  14. 包含权利要求13所述分离的核酸分子的表达载体。
  15. 包含权利要求13所述的分离的核酸分子、或权利要求14所述的表达载体的分离的宿主细胞；优选，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自HEK293E或CHO细胞。
  16. 一种制备权利要求1～9任一项所述抗体或抗原结合片段或权利要求10所述多特异性抗原结合分子的方法，其特征在于，在适当的条件下培养权利要求15所述的宿主细胞，并分 离抗体或抗原结合片段或多特异性抗原结合分子。
  17. 一种制备权利要求12所述免疫效应细胞的方法，其特征在于，所述方法包括将编码权利要求11所述的嵌合抗原受体的核酸片段导入免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达权利要求11所述的嵌合抗原受体。
  18. 一种药物组合物，其特征在于，所述组合物包含权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的分离的核酸分子、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求16或17所述方法制备的产品；优选，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；优选，所述药物组合物还包含额外的抗肿瘤剂。
  19. 权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的分离的核酸分子、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求16或17所述方法制备的产品、或权利要求18所述的药物组合物在制备预防和/或治疗B细胞疾病的药物中的用途，所述B细胞疾病优选肿瘤或自身免疫病；
    优选，所述肿瘤选自淋巴瘤或白血病，更优选，所述淋巴瘤或白血病选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、多发性骨髓瘤；
    优选，其中所述自身免疫病选自***性红斑狼疮(SLE)、抗磷脂抗体综合征、多发性硬化症、溃疡性结肠炎、克罗恩病、类风湿性关节炎、斯耶格伦氏综合征、吉兰-巴雷综合征、重症肌无力、大血管血管炎、中血管血管炎、结节性多动脉炎、天疱疮、硬皮病、肺出血-肾炎综合征、肾小球肾炎、原发性胆汁性肝硬化、格雷夫斯氏病、膜性肾病、自身免疫性肝炎、口炎性腹泻、阿狄森氏病、多发性肌炎/皮肌炎、单克隆丙种球蛋白病、因子VIII缺乏、冷球蛋白血症、周围神经病变、IgM多神经病、慢性神经病和慢性淋巴细胞性甲状腺炎。
  20. 一种预防和/或治疗B细胞疾病的方法，包含向有此需要的患者施用有效量的权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的分离的核酸分子、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求16或17所述方法制备的产品、或权利要求18所述的药物组合物；所述B细胞疾病优选肿瘤或自身免疫病；
    优选，所述肿瘤选自淋巴瘤或白血病，更优选，所述淋巴瘤或白血病选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、多发性骨髓瘤；
    优选，其中所述自身免疫病选自***性红斑狼疮(SLE)、抗磷脂抗体综合征、多发性硬化症、溃疡性结肠炎、克罗恩病、类风湿性关节炎、斯耶格伦氏综合征、吉兰-巴雷综合征、重症肌无力、大血管血管炎、中血管血管炎、结节性多动脉炎、天疱疮、硬皮病、肺出血-肾炎综合征、肾小球肾炎、原发性胆汁性肝硬化、格雷夫斯氏病、膜性肾病、自身免疫性肝炎、口炎性腹泻、阿狄森氏病、多发性肌炎/皮肌炎、单克隆丙种球蛋白病、因子VIII缺乏、冷球蛋白血症、周围神经病变、IgM多神经病、慢性神经病和慢性淋巴细胞性甲状腺炎。
  21. 权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的分离的核酸分子、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求16或17所述方法制备的产品、或权利要求18所述的药物组合物，其特征在于，用于预防和/或治疗B细胞疾病；所述B细胞疾病优选肿瘤或自身免疫病；
    优选，所述肿瘤选自淋巴瘤或白血病，更优选，所述淋巴瘤或白血病选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、多发性骨髓瘤；
    优选，其中所述自身免疫病选自***性红斑狼疮(SLE)、抗磷脂抗体综合征、多发性硬化症、溃疡性结肠炎、克罗恩病、类风湿性关节炎、斯耶格伦氏综合征、吉兰-巴雷综合征、重症肌无力、大血管血管炎、中血管血管炎、结节性多动脉炎、天疱疮、硬皮病、肺出血-肾炎综合征、肾小球肾炎、原发性胆汁性肝硬化、格雷夫斯氏病、膜性肾病、自身免疫性肝炎、口炎性腹泻、阿狄森氏病、多发性肌炎/皮肌炎、单克隆丙种球蛋白病、因子VIII缺乏、冷球蛋白血症、周围神经病变、IgM多神经病、慢性神经病和慢性淋巴细胞性甲状腺炎。
  22. 一种试剂盒，其包含权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的分离的核酸分子、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求16或17所述方法制备的产品、或权利要求18所述的药物组合物，以及使用说明。
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