WO2022120943A1 - 抗cll1抗体及其应用 - Google Patents

抗cll1抗体及其应用 Download PDF

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WO2022120943A1
WO2022120943A1 PCT/CN2020/138286 CN2020138286W WO2022120943A1 WO 2022120943 A1 WO2022120943 A1 WO 2022120943A1 CN 2020138286 W CN2020138286 W CN 2020138286W WO 2022120943 A1 WO2022120943 A1 WO 2022120943A1
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seq
chain variable
variable region
cll1
antibody
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French (fr)
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罗敏
李光超
丁雯
周兆
王学俊
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Guangzhou Bio Gene Technology Co Ltd
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Guangzhou Bio Gene Technology Co Ltd
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Priority to AU2020480236A priority Critical patent/AU2020480236B2/en
Priority to KR1020227007231A priority patent/KR102712484B1/ko
Priority to JP2022529575A priority patent/JP7384493B2/ja
Priority to GB2207720.0A priority patent/GB2610467B/en
Priority to US17/609,340 priority patent/US12358990B2/en
Priority to EP20958916.7A priority patent/EP4039708A4/en
Publication of WO2022120943A1 publication Critical patent/WO2022120943A1/zh
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57505—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the blood, e.g. leukaemia
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    • G01—MEASURING; TESTING
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    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • 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]
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/624—Disulfide-stabilized antibody (dsFv)
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present application belongs to the technical field of biomedicine, and relates to anti-CLL1 antibodies and applications thereof.
  • CLL1 C-type lectin-like molecule 1
  • CLEC12A C-type lectin domain family 12 member A
  • DCAL-2 C-type lectin domain family 12 member A
  • CLL1 C-type lectin domain family 12 member A
  • the identification codes of CLL1 gene in some mainstream databases are as follows: Q5QGZ9 (UniprotKB), 31713 (HGNC), 160364 (Entrez Gene), ENSG00000172322 (Ensembl) and (612088) OMIM.
  • CLL1 is restricted to hematopoietic cells, including mainly myeloid-derived cells in peripheral blood and bone marrow, such as monocytes, dendritic cells, granulocytes, and most acute myeloid leukemia (AML) cells. It is worth noting that although CLL1 is abundantly expressed on myeloid cells in peripheral blood and bone marrow, it is not expressed on myeloid-derived cells in peripheral tissues, such as tissue macrophages and tissue dendritic cells. .
  • CLL1 is expressed on AML stem cells (CD34+/CD38-) and a small subset of hematopoietic progenitor cells (CD34+/CD38+ or CD34+/CD33+), but not on normal hematopoietic stem cells (CD34+/CD38- or CD34+/CD33-) not express. Due to this special expression pattern, CLL1 is expected to be a potential diagnostic and therapeutic target for AML.
  • the currently known human CLL1 gene includes 7 transcripts, 5 of which encode proteins.
  • Human CLL1 protein is a membrane receptor.
  • the extracellular segment of the classical structure has a C-type lectin domain, and the intracellular segment has an immune receptor.
  • Tyrosine motif (ITIM) phosphorylated ITIM binds to SH2 domain-containing phosphatase and negatively regulates the function of granulocytes and monocytes.
  • the application provides anti-CLL1 antibodies and uses thereof for the treatment of cancer and autoimmune diseases, alone and/or in combination with other drugs.
  • the application provides an anti-CLL1 antibody comprising a heavy chain variable region and a light chain variable region; wherein
  • the heavy chain variable region comprises the CDR3 shown in SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:14;
  • the light chain variable region includes the CDR3 shown in SEQ ID NO:6, SEQ ID NO:11 or SEQ ID NO:17.
  • the heavy chain variable region further comprises CDR1 as set forth in SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:12.
  • the heavy chain variable region further comprises the CDR2 set forth in SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:13.
  • the light chain variable region further comprises CDR1 as set forth in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:15.
  • the light chain variable region further comprises the CDR2 set forth in SEQ ID NO:5 or SEQ ID NO:16.
  • CDR1-3 of the heavy chain variable region of the antibody and CDR1-3 of the light chain variable region jointly determine the specific recognition and binding ability of the antibody to the antigen, including SEQ ID NO: 1-6, SEQ ID NO: 5 and the CDRs of SEQ ID NO: 7-11 or SEQ ID NO: 12-17 have significant binding ability to CLL1 protein.
  • the heavy chain variable region of the anti-CLL1 antibody 23D7 includes CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;
  • the light chain variable region of the anti-CLL1 antibody 23D7 includes CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; wherein
  • the anti-CLL1 antibody 23D7 comprising the heavy chain variable region CDRs of SEQ ID NOs: 1 to 3 and the light chain variable region CDRs of SEQ ID NOs: 4 to 6 has CLL1 protein binding activity.
  • the heavy chain variable region of the anti-CLL1 antibody 23D7 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; wherein
  • the heavy chain variable region of the anti-CLL1 antibody 19C1 includes CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;
  • the light chain variable region of the anti-CLL1 antibody 19C1 includes CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:11; wherein
  • the anti-CLL1 antibody 19C1 comprising the heavy chain variable region CDRs of SEQ ID NOs: 7 to 9 and the light chain variable region CDRs of SEQ ID NOs: 5 and 10 to 11 has CLL1 protein binding activity.
  • the heavy chain variable region of the anti-CLL1 antibody 19C1 comprises the amino acid sequence set forth in SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21; wherein
  • the heavy chain variable region of the anti-CLL1 antibody 27H4 includes CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:14;
  • the light chain variable region of the anti-CLL1 antibody 27H4 includes CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17; wherein
  • the anti-CLL1 antibody 27H4 comprising the heavy chain variable region CDRs of SEQ ID NOs: 12 to 14 and the light chain variable region CDRs of SEQ ID NOs: 15 to 17 has CLL1 protein binding activity.
  • the heavy chain variable region of the anti-CLL1 antibody 27H4 comprises the amino acid sequence set forth in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23; wherein
  • the anti-CLL1 antibody 27H4 is humanized, and the framework region of 27H4 is optimized to obtain a humanized H27H4 antibody that has a stronger affinity with CLL1, and the heavy chain variable region of the anti-CLL1 antibody H27H4 includes SEQ ID NO:
  • the amino acid sequence shown in 24, the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; wherein
  • the anti-CLL1 antibody is a monomer, formed by a set of heavy and light chains, and the variable regions of the heavy and light chains are linked by interchain disulfide bonds.
  • the anti-CLL1 antibody is a multimer, formed by multiple groups of heavy chains and light chains, and the variable regions of the heavy chain and the light chain are connected by interchain disulfide bonds, and different heavy chains The variable regions are linked by interchain disulfide bonds.
  • the anti-CLL1 antibody further comprises a constant region.
  • the anti-CLL1 antibody is modified with a glycosylation group.
  • the present application provides a nucleic acid molecule comprising a DNA fragment encoding the anti-CLL1 antibody of the first aspect.
  • the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
  • the present application provides a recombinant cell expressing the anti-CLL1 antibody of the first aspect.
  • the nucleic acid molecule of the second aspect is integrated into the genome of the recombinant cell.
  • the recombinant cell comprises the expression vector of the third aspect.
  • the application provides a preparation method of the anti-CLL1 antibody described in the first aspect, the preparation method comprising the following steps:
  • nucleic acid encoding the anti-CLL1 antibody is linked into a plasmid, transferred into competent cells, and cultured and then picked out of monoclonal cells for screening;
  • the present application provides a pharmaceutical composition comprising the anti-CLL1 antibody of the first aspect.
  • the pharmaceutical composition further includes an antitumor drug.
  • the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, diluents or excipients.
  • the present application provides the anti-CLL1 antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, or the sixth aspect.
  • the disease comprises acute myeloid leukemia.
  • the present application provides a method of treating cancer, the method comprising administering to a patient an effective dose of the anti-CLL1 antibody of the first aspect.
  • the method further comprises administering one or more anti-tumor drugs simultaneously, separately or sequentially with the anti-CLL1 antibody.
  • the cancer comprises acute myeloid leukemia.
  • the anti-CLL1 antibodies 23D7, 27H4 and 19C1 of the present application have significant binding ability to CLL1, and the affinities of ch23D7, ch27H4 and ch19C1 for binding to the antigen CLL1 are 2.19nM, 3.83nM and 10.9nM respectively, which are comparable to the control antibody 1075.7;
  • the anti-CLL1 antibodies 23D7, 27H4 and 19C1 of the present application can bind to the CLL1 protein on the cell surface, and the binding force increases with the increase of the antibody concentration;
  • the antibody of the present application and its humanized modified antibody have important application prospects in the treatment of CLL1 positive tumors.
  • Fig. 1 is the ForteBIO evaluation result of chimeric antibodies ch23D7, ch27H4 and ch19C1;
  • Figure 2 shows the binding of chimeric antibodies ch23D7, ch27H4 and ch19C1 to CLL1 antigen by flow cytometry
  • Figure 3 is a flow cytometry detection of the binding of humanized antibody hz27H4 to CLL1 antigen
  • Figure 4 shows the ability of the antibody to bind to CLL1 transient 293T cells by flow cytometry
  • FIG. 5A shows the results of MPA detection of the binding ability of 23D7-scFv-hFc to CLL1 antigen
  • Figure 5B shows the results of MPA detection of the binding ability of 27H4-scFv-hFc to CLL1 antigen
  • Figure 5C shows the results of MPA detection of the binding ability of 19C1-scFv-hFc to CLL1 antigen
  • FIG. 5D is the MPA detection result of the binding ability of Hz27H4-scFv-hFc and CLL1 antigen
  • FIG. 6A is a graph of the binding kinetics of recombinant human CLL-1
  • FIG. 6B is a graph of the binding kinetics of recombinant cynomolgus monkey CLL-1.
  • mice 10 BALB/C healthy female mice aged 7-8 weeks were selected, and the immunogen CLL1-mFc (fusion protein of CLL1 extracellular segment and mouse IgG1 Fc segment was used, and the amino acid sequence of CLL1 extracellular segment was as SEQ ID NO: 28) for immunization; two weeks after the second immunization, blood was collected from the tail vein of mice to separate serum, and ELISA was used to detect the antibody titer; two mice whose antibody titers reached the fusion requirement were selected three days before fusion.
  • CLL1-mFc fusion protein of CLL1 extracellular segment and mouse IgG1 Fc segment was used, and the amino acid sequence of CLL1 extracellular segment was as SEQ ID NO: 28
  • Blood was collected from the shock-immunized mice by cutting the neck. After sterilizing with 75% alcohol for 10 minutes, the spleen was removed, and the connective tissue was removed to prepare a spleen cell suspension. 5min, discard the supernatant, add RPMI1640 to 30mL, and count the cells; transfer the myeloma cells with good growth status (the number of viable cells>95%) to a 50mL centrifuge tube, add RPMI1640 to 30mL, centrifuge at 1000rpm for 5min, discard the supernatant , add RPMI1640 to 30mL, and count the cells;
  • the splenocytes and myeloma cells were mixed in a ratio of 4:1, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the precipitated cell mass was bounced into a paste and placed in a 37°C water bath, 1 mL of fusion agent was added within 1 min and stirred well. Place in a 37°C water bath for 45-60s, add RPMI1640 within 1min to stop the fusion of the fusion agent, centrifuge at 1000rpm for 5min, and discard the supernatant;
  • Antigen coating After diluting the pure antigen human CLL1 ECD-His (human CLL1 extracellular region with His tag) at a concentration of 50 ng/mL with the coating solution, take 100 ⁇ L and add it to the polystyrene enzyme-linked detection plate. In each well, overnight at 4°C;
  • Reading measure the OD value of each well with a single wavelength of 450 nm, and select multiple clones with high reading values according to the principle of high to low for the next functional screening.
  • the clones were sequenced to obtain the amino acid sequences of clones 23D7, 19C1, and 27H4, wherein the heavy chain variable region of 23D7 was shown in SEQ ID NO: 18, the light chain variable region was shown in SEQ ID NO: 19, and the variable region of 19C1 was shown in SEQ ID NO: 19.
  • the heavy chain variable region is shown in SEQ ID NO:20, the light chain variable region is shown in SEQ ID NO:21, the heavy chain variable region of 27H4 is shown in SEQ ID NO:22, and the light chain variable region is shown in SEQ ID NO:22. shown in SEQ ID NO:23.
  • the human-mouse chimeric antibody expression plasmid was transiently transfected into 293F cells, and chimeric antibodies ch23D7, ch19C1 and ch27H4 with 23D7, 19C1 and 27H4 as parents were obtained by transient expression and affinity purification.
  • the ForteBio affinity measurement method (P.Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):270-278.) was used to measure the chimeric antibodies ch23D7, ch19C1, ch27H4 Affinity testing was performed and a control antibody (Anti-CLL1-Ref) selected the VL and VL chains (SEQ ID NO: 29) of anti-human CLL-1 antibody 1075.7 (patent US8536310B2).
  • the antibody was loaded onto an anti-human IgG capture (AHC) biosensor, the sensor was equilibrated offline in assay buffer for 30 min, and monitored online for 60 s to establish a baseline; the antibody-loaded sensor was combined with 100 nM of the antigen human CLL1 ECD- His was incubated for 5 min, then transferred to assay buffer, and the dissociation rate was measured after 5 min; kinetic analysis was performed using a 1:1 binding model.
  • AHC anti-human IgG capture
  • flow cytometry was used to detect the binding of antibodies ch23D7, ch27H4, and ch19C1 to CLL1 on HEK293 cells, and the steps were as follows:
  • Control groups CLL1-RefAb (ch1075.7), Cell+secondary antibody and Blank were set.
  • 27H4 was humanized.
  • the gene sequence of the 27H4 antibody was compared with the human antibody Germline database to find sequences with high homology, while avoiding uncommon or small Germlines; after selecting a humanized template, rearranged
  • the frequency of the amino acid occurrence of the antibody at a specific FR site, and CDR transplantation is performed to avoid the introduction of protein modification sites such as glycosylation and sites that are prone to chemical degradation; if the affinity of the sequence decreases after CDR transplantation, perform back mutation.
  • the mutation principle is: Step-by-step single-point mutation is performed on the FR region; if there is a modification site or chemical degradation site in the CDR region, and the protein control is affected, the site is subjected to step-by-step single-point mutation; after each round of mutation, affinity (KD/Kon /Koff) detection; the finally obtained humanized sequence has the best performance in terms of affinity (within 3 times of the parental difference), stability, and protein quality (one-step PA>90%).
  • one heavy chain variable region hz27H4H1 (SEQ ID NO:24) and three light chain variable regions hz27H4L1 (SEQ ID NO:25), hz27H4L2 (SEQ ID NO:26) and hz27H4L3 (SEQ ID NO:27).
  • flow cytometry was used to detect the binding of antibodies hz27H4H1L1 and hz27H4H1L2 to CLL1 on HEK293 cells, and the steps were as follows:
  • Control groups ch27H4, CLL1-RefAb (ch1075.7) and NC-huIgG1 were set.
  • membrane proteome array (Membrane Proteome Array, MPA) was used to verify the non-target binding interaction of the antibody.
  • MPA Membrane proteome array
  • MPA is a platform for the analysis of specific antibodies and other ligands targeting human membrane proteins, which can be used to determine the specificity of antibody targets.
  • Plasmids containing about 6000 membrane protein clones were transfected into HEK-293T cells (ATCC, CRL-3216) or QT6 cells (ATCC, CRL-1708), respectively, according to 18000 The density of cells/well was seeded into 384-well cell culture plates (Corning, 3764); after 36 hours of incubation, the test antibodies were added to the membrane proteome array matrix plates at predetermined concentrations, and the antibody scFv was directly detected by flow cytometry Binding to cells expressing about 6000 membrane proteins. All target proteins have native conformation and appropriate post-translational modifications.
  • the mammalian-expressed single-chain antibody has a VL-(G4S) 3 -VH structure, and the C-terminal fusion expresses human hIgG1-Fc.
  • the specific information is shown in Table 4.
  • FCGR1A, FCGR2B, FCGR3B are IgG Fc receptors (IgG Fc receptors).
  • CLL-1 antigens recombinant human CLL-1, Acro, product number: CLA-H5245, batch number: 3413a-9B8F1-SQ; recombinant Cynomolgus monkey CLL-1, Acro, Cat. No.: CLA-H5263, Lot No.: 3765-2079F1-SS
  • scFv single chain antibody
  • scFv VL-(G4S) 3 -VH structure
  • C-terminal fusion Human hIgG1-Fc is expressed, and the sample information table is shown in Table 5.
  • Antibody Diluent (Ligand): Dilute the antibody to 5 ⁇ g/mL with 1 ⁇ HBS-EP+running buffer;
  • Recombinant human CLL-1 dilution solution (analyte 1): take recombinant human CLL-1 (250 ⁇ g/mL) and dilute it to 50nM with running buffer, 2-fold serial dilution to obtain 50nM, 25nM, 12.5nM, 6.25nM, 3.125 nM, 0nM recombinant human CLL-1 dilution;
  • Recombinant cynomolgus monkey CLL-1 dilution (analyte 2): take recombinant cynomolgus monkey CLL-1 (250 ⁇ g/mL) and dilute it to 50nM with running buffer, and obtain 50nM, 25nM, 12.5nM, 6.25nM by 2-fold serial dilution , 3.125nM, 0nM recombinant cynomolgus monkey CLL-1 dilution;
  • the protein A chip was used for detection. 5 ⁇ g/mL antibody diluent was passed through the experimental flow path (Fc2, Fc4) at a flow rate of 10 ⁇ L/min, and the capture volume was about 454RU for 20s. After that, the flow rate was adjusted to 30 ⁇ L/min.
  • Concentrations of recombinant human CLL-1 dilution (0, 3.125nM, 6.25nM, 12.5nM, 25nM, 50nM) passed through the surface of the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time, binding The time was 85s, the dissociation time was 70s, and finally glycine solution (Glycine, pH 1.5) was added for 60s to regenerate the chip and enter the next cycle.
  • the protein A chip was used for detection. 5 ⁇ g/mL antibody diluent was passed through the experimental flow path (Fc2, Fc4) at a flow rate of 10 ⁇ L/min. After capturing for 20s, the capture volume was about 454RU, and then the flow rate was adjusted to 30 ⁇ L/min.
  • Concentrations of recombinant cynomolgus monkey CLL-1 dilution (0, 3.125nM, 6.25nM, 12.5nM, 25nM, 50nM) passed through the surface of the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time, The binding time was 85s, the dissociation time was 70s, and finally glycine solution (Glycine, pH 1.5) was added for 60s to regenerate the chip and enter the next cycle.
  • the data analysis software Evaluation Software 3.1 was used to analyze the test results, and the sensing signal collected from the sample test flow path was double-deducted from the reference flow path and the sample blank, and the kinetic "1:1" model was used for fitting.
  • the kinetic parameters (ka: association rate; kd: dissociation rate; kD: binding dissociation equilibrium constant) of each batch of samples with shTNF- ⁇ were obtained.
  • the kinetic fitting results of the binding of 6 antibodies to recombinant human CLL-1 are shown in Table 6 and Figure 6A.
  • the kinetic fitting results of binding to recombinant cynomolgus CLL-1 are shown in Figure 6B.
  • the anti-CLL1 antibodies 23D7, 27H4, and 19C1 of the present application have significant binding ability to CLL1, and after humanization transformation, the affinity of the antibody and CLL1 is further improved, which is useful in the clinical diagnosis and/or treatment of tumors. It has important application prospects.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

提供抗CLL1抗体及其应用，所述抗CLL1抗体可变区包括SEQ ID NO:1～6、SEQ ID NO:5和SEQ ID NO:7～11或SEQ ID NO:12～17的CDR。所述抗CLL1抗体对游离和细胞表面CLL1均具有显著的结合能力，经过人源化改造后，进一步提高了抗体与CLL1的亲和力，在肿瘤的临床诊断和/或治疗方面具有重要的应用前景。

Description

抗CLL1抗体及其应用 技术领域
本申请属于生物医药技术领域，涉及抗CLL1抗体及其应用。
背景技术
C型凝集素样分子1(CLL1)，又称C型凝集素域家族12成员A(CLEC12A)，在文献和数据库中还有DCAL-2、MICL、CD371等名称，是一种II型跨膜蛋白，全长265个氨基酸，分子量为30762道尔顿，编码基因位于chr12:9,951,268～9,995,694(GRCh38/hg38)。目前一些主流数据库中CLL1基因的识别码如下：Q5QGZ9(UniprotKB)、31713(HGNC)、160364(Entrez Gene)、ENSG00000172322(Ensembl)和(612088)OMIM。
研究表明，CLL1限制性地表达于造血细胞上，主要包括外周血和骨髓中髓系来源的细胞，比如单核细胞、树突状细胞、粒细胞以及大多数急性髓细胞白血病(AML)细胞。值得注意的是，虽然CLL1大量表达于外周血和骨髓中的髓细胞上，但是在外周组织中的髓系来源的细胞上不表达，比如组织巨噬细胞和组织树突状细胞均不表达CLL1。研究还发现，CLL1表达于AML干细胞(CD34+/CD38-)以及一小部分造血祖细胞(CD34+/CD38+或者CD34+/CD33+)上，但是在正常的造血干细胞(CD34+/CD38-或者CD34+/CD33-)上不表达。由于这种特殊的表达模式，CLL1有望成为潜在的诊断和治疗AML的靶点。
目前已知的人CLL1基因包括7个转录本，其中5个编码蛋白质，人CLL1蛋白是一种膜受体，经典结构的胞外段具有C型凝集素结构域，胞内段具有免疫受体酪氨酸基序(ITIM)，磷酸化ITIM与含SH2结构域的磷酸酶结合，发挥负性调节粒细胞和单核细胞的功能。
发明内容
本申请提供了抗CLL1抗体及其应用，所述抗体单独和/或与其他药物联合用于癌症和自身免疫性疾病的治疗。
第一方面，本申请提供了抗CLL1抗体，所述抗CLL1抗体包括重链可变区和轻链可变区；其中
所述重链可变区包括SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:14所示的CDR3；并且
所述轻链可变区包括SEQ ID NO:6、SEQ ID NO:11或SEQ ID NO:17所示的CDR3。
在一些具体实施方案中，所述重链可变区还包括SEQ ID NO:1、SEQ ID NO:7或SEQ ID NO:12所示的CDR1。
在一些具体实施方案中，所述重链可变区还包括SEQ ID NO:2、SEQ ID NO:8或SEQ ID NO:13所示的CDR2。
在一些具体实施方案中，所述轻链可变区还包括SEQ ID NO:4、SEQ ID NO:10或SEQ ID NO:15所示的CDR1。
在一些具体实施方案中，所述轻链可变区还包括SEQ ID NO:5或SEQ ID NO:16所示的CDR2。
本申请中，抗体的重链可变区的CDR1～3和轻链可变区的CDR1～3共同决定抗体对抗原的特异性识别结合能力，含有SEQ ID NO:1～6、SEQ ID NO:5和SEQ ID NO:7～11或SEQ ID NO:12～17的CDR的抗体对CLL1蛋白具有显著的结合能力。
在一个具体实施例中，所述抗CLL1抗体23D7的重链可变区包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、SEQ ID NO:3所示的CDR3；
所述抗CLL1抗体23D7的轻链可变区包括SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:6所示的CDR3；其中
Figure PCTCN2020138286-appb-000001
本申请中，包含SEQ ID NO:1～3的重链可变区CDR和SEQ ID NO:4～6的轻链可变区CDR的抗CLL1抗体23D7具有CLL1蛋白结合活性。
在一些具体实施方案中，所述抗CLL1抗体23D7的重链可变区包括SEQ ID NO:18所示的氨基酸序列，轻链可变区包括SEQ ID NO:19所示的氨基酸序列；其中
Figure PCTCN2020138286-appb-000002
在一个具体实施例中，所述抗CLL1抗体19C1的重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、SEQ ID NO:9所示的CDR3；
所述抗CLL1抗体19C1的轻链可变区包括SEQ ID NO:10所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:11所示的CDR3；其中
Figure PCTCN2020138286-appb-000003
本申请中，包含SEQ ID NO:7～9的重链可变区CDR和SEQ ID NO:5、10～11的轻链可变区CDR的抗CLL1抗体19C1具有CLL1蛋白结合活性。
在一些具体实施方案中，所述抗CLL1抗体19C1的重链可变区包括SEQ ID NO:20所示的氨基酸序列，轻链可变区包括SEQ ID NO:21所示的氨基酸序列；其中
Figure PCTCN2020138286-appb-000004
在一个具体实施例中，所述抗CLL1抗体27H4的重链可变区包括SEQ ID NO:12所示的CDR1、SEQ ID NO:13所示的CDR2、SEQ ID NO:14所示的CDR3；
所述抗CLL1抗体27H4的轻链可变区包括SEQ ID NO:15所示的CDR1、SEQ ID NO:16所示的CDR2、SEQ ID NO:17所示的CDR3；其中
Figure PCTCN2020138286-appb-000005
本申请中，包含SEQ ID NO:12～14的重链可变区CDR和SEQ ID NO:15～17的轻链可变区CDR的抗CLL1抗体27H4具有CLL1蛋白结合活性。
在一些具体实施方案中，所述抗CLL1抗体27H4的重链可变区包括SEQ ID NO:22所示的氨基酸序列，轻链可变区包括SEQ ID NO:23所示的氨基酸序列；其中
Figure PCTCN2020138286-appb-000006
优选地，对抗CLL1抗体27H4进行人源化改造，优化27H4的框架区，获得人源化H27H4抗体具有与CLL1更强的亲和力，所述抗CLL1抗体H27H4的重链可变区包括SEQ ID NO:24所示的氨基酸序列，轻链可变区包括SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示的氨基酸序列；其中
Figure PCTCN2020138286-appb-000007
优选地，所述抗CLL1抗体为单体，由一组重链和轻链形成，所述重链可变区和轻链可变区之间通过链间二硫键连接。
优选地，所述抗CLL1抗体为多聚体，由多组重链和轻链形成，所述重链可变区和轻链可变区之间通过链间二硫键连接，不同的重链可变区之间通过链间二硫键连接。
优选地，所述抗CLL1抗体还包括恒定区。
优选地，所述抗CLL1抗体修饰有糖基化基团。
第二方面，本申请提供了核酸分子，所述核酸分子包括编码第一方面所述的抗CLL1抗体的DNA片段。
第三方面，本申请提供了表达载体，所述表达载体包括第二方面所述的核酸分子。
第四方面，本申请提供了重组细胞，所述重组细胞表达第一方面所述的抗CLL1抗体。
优选地，所述重组细胞的基因组中整合有第二方面所述的核酸分子。
优选地，所述重组细胞包括第三方面所述的表达载体。
第五方面，本申请提供了一种第一方面所述的抗CLL1抗体的制备方法，所述制备方法包括以下步骤：
(1)将抗CLL1抗体的编码核酸连接入质粒，转入感受态细胞，培养后挑取单克隆细胞进行筛选；
(2)提取筛选的阳性克隆的表达载体，转入宿主细胞，培养并收集上清液，分离纯化得到所述抗体。
第六方面，本申请提供了药物组合物，所述药物组合物包括第一方面所述的抗CLL1抗体。
优选地，所述药物组合物还包括抗肿瘤药物。
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
第七方面，本申请提供了第一方面所述的抗CLL1抗体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组细胞或第六方面所述的药物组合物在制备疾病检测试剂和/或疾病治疗药物中的应用。
优选地，所述疾病包括急性髓系白血病。
第八方面，本申请提供了一种治疗癌症的方法，所述方法包括向患者施用有效剂量的第一方面所述的抗CLL1抗体。
优选地，所述方法还包括与抗CLL1抗体同时、分开或依次施用一种或多种抗肿瘤药物。
优选地，所述癌症包括急性髓系白血病。
与现有技术相比，本申请具有如下有益效果：
(1)本申请的抗CLL1抗体23D7、27H4、19C1对CLL1具有显著的结合能力，ch23D7、ch27H4和ch19C1结合抗原CLL1的亲和力分别为2.19nM、3.83nM和10.9nM，与对照抗体1075.7相当；
(2)本申请的抗CLL1抗体23D7、27H4、19C1可以结合细胞表面的CLL1蛋白，结合力随着抗体浓度的升高而增大；
(3)本申请的抗体经过人源化改造后，进一步提高了抗体与游离和/或细胞表面CLL1的亲和力；
(4)本申请的抗体及其人源化改造抗体在治疗CLL1阳性肿瘤中具有重要的应用前景。
附图说明
图1为嵌合抗体ch23D7、ch27H4和ch19C1的ForteBIO评价结果；
图2为流式检测嵌合抗体ch23D7、ch27H4、ch19C1与CLL1抗原的结合；
图3为流式检测人源化抗体hz27H4与CLL1抗原的结合；
图4为流式检测抗体结合CLL1转瞬293T细胞的能力；
图5A为23D7-scFv-hFc与CLL1抗原的结合能力的MPA检测结果，图5B为27H4-scFv-hFc与CLL1抗原的结合能力的MPA检测结果，图5C为19C1-scFv-hFc与CLL1抗原的结合能力的MPA检测结果，图5D为Hz27H4-scFv-hFc与CLL1抗原的结合能力的MPA检测结果；
图6A为重组人CLL-1结合动力学曲线图；图6B为重组食蟹猴CLL-1结合动力学曲线图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常 规产品。
实施例1抗体的获取
选择10只鼠龄为7～8周的BALB/C健康雌性小鼠，采用免疫原CLL1-mFc(CLL1胞外段和鼠IgG1Fc段的融合蛋白，CLL1胞外段的氨基酸序列如SEQ ID NO:28所示)进行免疫；第二次免疫两周后，从小鼠尾静脉取血分离血清，ELISA检测抗体效价；选取抗体效价达到可融合要求的两只小鼠，在融合前三天腹腔注射抗原50μg/100μL/只进行冲击免疫；同时，效价检测前一周复苏骨髓瘤细胞SP2/0，37℃、5％CO 2培养箱培养，融合前一天对骨髓瘤细胞进行传代或更换新鲜培养基，使细胞保持最佳状态；
Figure PCTCN2020138286-appb-000008
对冲击免疫的小鼠进行断颈取血，75％酒精消毒10min后取脾脏，去除结缔组织，制备脾细胞悬液；将脾细胞悬液转移到50mL离心管中，加入RPMI1640至30mL，1000rpm离心5min，弃上清，加入RPMI1640至30mL，进行细胞计数；取生长状态良好的骨髓瘤细胞(活细胞数>95％)转移到50mL离心管中，加入RPMI1640至30mL，1000rpm离心5min，弃上清，加入RPMI1640至30mL，进行细胞计数；
将脾细胞和骨髓瘤细胞按照4:1的比例混合，1000rpm离心5min，弃上清，将沉淀细胞块弹成糊状后置于37℃水浴中，在1min内加入1mL融合剂并搅拌均匀，置于37℃水浴45～60s，在1min内加入RPMI1640终止融合剂的融合作用，1000rpm离心5min，弃上清；
轻轻将细胞弹匀，缓缓加入含HAT的完全培养液，将细胞悬液加入到预先准备好的完全培养基中，排枪滴加入96孔板中，每孔150～200μL，37℃、CO 2培养箱培养、观察；
从细胞融合后的第一天开始，观察细胞的生长状态、并确定培养液无污染，培养7～10天后将HAT培养液更换为HT培养液，继续培养3～4天，每孔取上清进行ELISA检测。
ELISA筛选步骤如下：
(1)抗原包被：将浓度为50ng/mL的纯抗原human CLL1 ECD-His(人CLL1胞外区连接His标签)用包被液稀释后，取100μL加入到聚苯乙烯酶联检测板的各孔中，4℃过夜；
(2)封闭：次日将酶标板平衡至室温，PBS清洗三次，每孔加入100μL封闭液，室温孵育一小时后PBS清洗3次，拍干；
(3)加待测样品：无菌条件下取融合细胞培养上清，35～50μL/孔加样到封闭好的酶标板中，同时，设置阴性对照孔(无细胞生长)和阳性对照孔(添加阳性血清)，室温孵育1h，PBST(0.05％吐温)清洗三次，PBS清洗两次；
(4)加二抗：以50μL/孔的添加量加入稀释的酶标二抗，37℃孵育30min，PBS清洗3次，拍干；
(5)显色：加入双组分TMB显色液终止液(Solarbio，Cat#PR1210)各50μL/孔，添加前混合均匀，37℃显色15～30min，随后加入50μL/孔终止液终止反应；
(6)读数：以450nm单波长测定各孔OD值，按从高到低原则选择读值高的多个克隆进行下一步功能筛选。
选取ELISA初筛的阳性孔，并对汇合率高的阳性孔细胞传代至24孔板，进一步进行功能实验，确定进行亚克隆的克隆编号；采用有限稀释法进行细胞亚克隆，同时冻存保种，具体如下：将细胞克隆稀释后铺96孔板，每个克隆铺1个板，于HT培养基中培养7～10天，第7天后镜下观察，挑选单克隆细胞上清用ELISA进一步筛选阳性克隆；
对克隆进行测序，获得克隆23D7、19C1、27H4的氨基酸序列，其中，23D7的重链可变区如SEQ ID NO:18所示、轻链可变区如SEQ ID NO:19所示，19C1的重链可变区如SEQ ID NO:20所示、轻链可变区如SEQ ID NO:21所示，27H4的重链可变区如SEQ ID NO:22所示，轻链可变区如SEQ ID NO:23所示。
实施例2抗体的表达与纯化
本实施例根据单克隆鉴定测序结果，设计特异性引物，PCR获得抗体23D7、19C1、27H4的基因，并将基因克隆至人IgG1重链恒定区Fc段编码基因上游，构建重组真核表达载体，获得人鼠嵌合抗体表达质粒；
将所述人鼠嵌合抗体表达质粒瞬时转染293F细胞，通过瞬时表达和亲和纯化获得以23D7、19C1、27H4为亲本的嵌合抗体ch23D7、ch19C1、ch27H4。
实施例3抗体的亲和力测试
本实施例采用ForteBio亲和力测量方法(P.Estep等，High throughput solution-based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):270-278.)对嵌合抗体ch23D7、ch19C1、ch27H4进行亲和力检测，对照抗体(Anti-CLL1-Ref)选择抗人CLL-1抗体1075.7(专利US8536310B2)的VL和VL链(SEQ ID NO:29)。
简言之，将抗体装载至抗人IgG捕获(AHC)生物传感器上，将传感器在测定缓冲液中离线平衡30min，在线监测60s用于确立基线；将装载抗体的传感器与100nM抗原human CLL1 ECD-His共孵育5min，随后转移至测定缓冲液中，5min后测定解离速率；动力学分析采用1:1结合模型进行。
结果如表1和图1所示，ch23D7、ch27H4和ch19C1结合抗原CLL1的亲和力分别为2.19nM、3.83nM和10.9nM，对照抗体1075.7结合抗原CLL1的亲和力为1.08nM。
表1
样品编号 浓度(nM) Response KD(M) Kon(1/Ms) Kdis(1/s) RMax
ch23D7 100 0.6536 2.19E-09 1.30E+05 2.84E-04 0.6693
ch27H4 100 0.6341 3.83E-09 2.65E+05 1.01E-03 0.6237
ch19C1 100 0.6194 1.09E-08 1.80E+05 1.97E-03 0.6574
ch1075.7 100 0.2361 4.14E-10 1.08E+05 4.48E-05 0.2384
Figure PCTCN2020138286-appb-000009
实施例4抗体与HEK293细胞上CLL1的结合
本实施例采用流式细胞术检测抗体ch23D7、ch27H4、ch19C1与HEK293细胞上CLL1的结合，步骤如下：
采用PBS+5％BSA重悬5×10 5个过表达CLL1的HEK293细胞，4℃孵育30min；加入不同浓度抗体(1μg/mL～0.01μg/mL，10倍梯度稀释)，4℃孵育60min；离心洗涤后加入含有FITC-标记羊抗人IgG-Fc二抗(1:200，sigma，F9512)的PBS+5％BSA溶液，冰上避光孵育30min；将细胞洗涤3次后进行流式细胞术分析；
设置对照组CLL1-RefAb(ch1075.7)、Cell+二抗和Blank。
结果如图2所示，嵌合抗体ch23D7、ch27H4、ch19C1、ch1075.7都能有效结合HEK293-CLL1细胞，并随着抗体浓度的提高，平均荧光强度(MFI)也随之升高。
实施例5抗体的人源化改造
本实施例对27H4进行人源化改造。简言之，将27H4抗体的基因序列与人抗体Germline数据库进行比对，找出同源性高的序列，同时避免不常用或小类Germline；选定人源化模板后，重排(rearranged)抗体在特定FR位点的氨基酸出现频率，并进行CDR移植，避免引入糖基化等蛋白修饰位点和易化学降解的位点；若CDR移植后序列的亲和力下降，进行回复突变，突变原则为对FR区进行逐级单点突变；若CDR区存在修饰位点或化学降解位点，且对蛋白质控有影响，对位点进行逐级单点突变；每轮突变后进行亲和力(KD/Kon/Koff)检测；最终获得的人源化序列在亲和力(与亲本比较相差3倍以内)、稳定性、蛋白质量(一步PA>90％)等方面均具有最优表现。
根据以上原则，获得人源化hz27H4抗体的一条重链可变区hz27H4H1(SEQ ID NO:24)和三条轻链可变区hz27H4L1(SEQ ID NO:25)、hz27H4L2(SEQ ID NO:26)和hz27H4L3(SEQ ID NO:27)。
实施例6 ForteBIo分析抗体人源化前后与CLL1的亲和力差异
简言之，将4μg/mL抗体装载至抗人IgG捕获(AHC)生物传感器上，将传感器在测定缓冲液中离线平衡30min，在线监测60s用于确立基线；将装载抗体的传感器与60nM抗原human CLL1 ECD-His共孵育3min，随后转移至测定缓冲液中，3min后测定解离速率；动力学分析采用1:1结合模型进行。
结果如表2所示，三个人源化抗体(hz27H4H1L1、hz27H4H1L2和hz27H4H1L3)与亲本抗体(ch27H4)相比，均保留了较高的亲和力。
表2 ForteBIo分析抗体人源化前后与CLL1的亲和力差异
样品编号 浓度(nM) Response KD(M) Kon(1/Ms) Kdis(1/s)
hz27H4H1L1 60 0.6123 2.29E-09 3.77E+05 8.62E-04
hz27H4H1L2 60 0.7255 2.21E-09 4.41E+05 9.76E-04
hz27H4H1L3 60 0.7224 2.49E-09 4.38E+05 1.09E-03
ch27H4 60 0.5972 3.88E-09 3.20E+05 1.24E-03
Anti-CLL1 Ref Ab 60 0.2189 6.76E-09 1.48E+05 9.97E-04
实施例7流式检测人源化抗体与CLL1的结合
本实施例采用流式细胞术检测抗体hz27H4H1L1、hz27H4H1L2与HEK293细胞上CLL1的结合，步骤如下：
采用PBS+5％BSA重悬2×10 5个过表达CLL1的HEK293细胞，4℃孵育30min；加入不同浓度抗体(5μg/mL～0.002286μg/mL，3倍梯度稀释)，4℃孵育60min；离心洗涤后加入含有FITC-标记羊抗人IgG-Fc二抗(1:200，sigma，F9512)的PBS+5％BSA溶液，冰上避光孵育30min；将细胞洗涤3次后进行流式细胞术分析；Graphpad软件计算得出EC50。
设置对照组ch27H4、CLL1-RefAb(ch1075.7)和NC-huIgG1。
结果如图3、图4和表3所示，ch27H4以剂量依赖的方式与CLL1结合，EC50(n＝1)为0.3583μg/mL，hz27H4H1L1以剂量依赖的方式与CLL1结合，EC50值(n＝1)为0.2681μg/mL，hz27H4H1L2以剂量依赖的方式与CLL1结合，EC50值(n＝1)为0.3214μg/mL，CLL1-refAb以剂量依赖的方式与CLL1结合，EC50值(n＝1)为0.2246μg/mL，ch27H4、hz27H4H1L1和hz27H4H1L2以与CLL1-refAb类似的EC50与人CLL1结合。
表3流式检测人源化抗体与HEK293-CLL1细胞的结合(EC50)
- ch27H4 hz27H4H1L1 hz27H4H1L2 CLL1-refAb
EC50(μg/mL) 0.3583 0.2618 0.3214 0.2446
实施例8膜蛋白质组阵列评估抗体特异性
本实施例采用膜蛋白质组阵列(Membrane Proteome Array，MPA)验证抗体的非靶点结合的相互作用。膜蛋白质组阵列(MPA)是一个分析特异性抗体和其他配体靶向人膜蛋白的平台，可用于确定抗体靶点的特异性。
将含有约6000个膜蛋白克隆(占人膜蛋白组的94％以上)的质粒分别转染到HEK-293T细胞(ATCC，CRL-3216)或QT6细胞(ATCC，CRL-1708)中，按18000个细胞/孔的密度接种至384孔细胞培养板(Corning，3764)；孵育36小时后，试验抗体以预先确定的浓度加入到膜蛋白组阵列基质板中，使用流式细胞仪直接检测抗体scFv与约6000种膜蛋白表达细胞的结合情况。所有靶蛋白都具有天然构象和适当的翻译后修饰，哺乳动物表达的单链抗体(scFv)为VL-(G4S) 3-VH结构，C端融合表达人hIgG1-Fc，具体信息见表4。
测试结果如图5A、图5B、图5C和图5D所示，23D7-scFv-hFc、27H4-scFv-hFc、 19C1-scFv-hFc、Hz27H4-scFv-hFc都能特异性结合CLL1靶标抗原，其中FCGR1A、FCGR2B、FCGR3B是IgG Fc受体(IgG Fc receptors)。
表4所用抗体信息
抗体 基本信息 靶抗原 Uniprot
23D7-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9
27H4-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9
19C1-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9
Hz27H4-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9
实施例9表面等离子体共振法测定抗体亲和力
本实施例采用表面等离子体共振技术(Surface Plasmon Resonance，SPR)检测并比较两种CLL-1抗原(重组人源CLL-1，Acro，货号：CLA-H5245，批号：3413a-9B8F1-SQ；重组食蟹猴CLL-1，Acro，货号：CLA-H5263，批号：3765-2079F1-SS)与6个抗体的亲和力，单链抗体(scFv)为VL-(G4S) 3-VH结构，C端融合表达人hIgG1-Fc，样品信息表见表5。
表5样品信息表
Figure PCTCN2020138286-appb-000010
(1)样品配制
抗体稀释液(配体)：将抗体用1×HBS-EP+运行缓冲液稀释至5μg/mL；
重组人源CLL-1稀释液(分析物1)：取重组人源CLL-1(250μg/mL)用运行缓冲液稀释至50nM，2倍梯度稀释得到50nM、25nM、12.5nM、6.25nM、3.125nM、0nM的重组人源CLL-1稀释液；
重组食蟹猴CLL-1稀释液(分析物2)：取重组食蟹猴CLL-1(250μg/mL)用运行缓冲液稀释至50nM，2倍梯度稀释得到50nM、25nM、12.5nM、6.25nM、3.125nM、0nM的重组食蟹猴CLL-1稀释液；
(2)重组人源CLL-1抗原分析
用Protein A芯片进行检测，5μg/mL抗体稀释液以10μL/min的流速通过实验流路(Fc2、 Fc4)，捕获20s使捕获量约为454RU，之后将流速调节为30μL/min，依次加入不同浓度的重组人源CLL-1稀释液(0、3.125nM、6.25nM、12.5nM、25nM、50nM)，同时经过实验流路(Fc2、Fc4)和参比流路(Fc1、Fc3)表面，结合时间为85s，解离时间为70s，最后加入甘氨酸液(Glycine，pH＝1.5)60s，对芯片进行再生并进入下一个循环。
(3)重组食蟹猴CLL-1抗原分析
用Protein A芯片进行检测，5μg/mL抗体稀释液以10μL/min的流速通过实验流路(Fc2、Fc4)，捕获20s使捕获量约为454RU，之后将流速调节为30μL/min，依次加入不同浓度的重组食蟹猴CLL-1稀释液(0、3.125nM、6.25nM、12.5nM、25nM、50nM)，同时经过实验流路(Fc2、Fc4)和参比流路(Fc1、Fc3)表面，结合时间为85s，解离时间为70s，最后加入甘氨酸液(Glycine，pH＝1.5)60s，对芯片进行再生并进入下一个循环。
(4)数据分析
用数据分析软件Evaluation Software3.1对试验结果进行分析，将样品试验流路采集所得传感信号进行参比流路、样品空白双扣减，并选用动力学“1:1”模型进行拟合，得出各批次样品同shTNF-α的动力学参数(ka：结合速率；kd：解离速率；kD：结合解离平衡常数)。6个抗体与重组人源CLL-1结合的动力学拟合结果如表6和图6A所示。与重组食蟹猴CLL-1结合的动力学拟合结果如图6B所示。
表6抗体同重组人CLL-1结合的动力学拟合结果统计表
样品 ka(1/Ms) kd(1/s) KD(M) Rmax(RU) Chi 2(RU 2)
27H4-scFv-hFc 4.11E+06 1.26E-02 3.06E-09 249.8 0.839
19C1-scFv-hFc 8.86E+05 5.09E-03 5.75E-09 273.8 0.11
23D7-scFv-hFc 2.93E+05 3.22E-02 1.10E-07 272.1 0.232
Hz27H4-scFv-hFc 4.12E+06 6.25E-03 1.52E-09 273.9 0.638
h27H4H1L1(全抗) 4.91E+06 5.49E-03 1.12E-09 256.3 0.67
h27H4H1L2(全抗) 5.04E+06 5.44E-03 1.08E-09 207.1 0.342
结果显示，除23D7-scFv-hFc与重组人源CLL-1的结合亲和力较低外，另外5个抗体与重组人源CLL-1结合的亲和力均在1nM～6nM之间；6个抗体与重组食蟹猴CLL-1均没有结合(图6B)。
综上所述，本申请的抗CLL1抗体23D7、27H4、19C1对CLL1具有显著的结合能力，经过人源化改造后，进一步提高了抗体与CLL1的亲和力，在肿瘤的临床诊断和/或治疗方面具有重要的应用前景。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 抗CLL1抗体，其包括重链可变区和轻链可变区；其中
    所述重链可变区包括SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:14所示的CDR3；并且
    所述轻链可变区包括SEQ ID NO:6、SEQ ID NO:11或SEQ ID NO:17所示的CDR3。
  2. 根据权利要求1所述的抗CLL1抗体，其中，所述重链可变区还包括SEQ ID NO:1、SEQ ID NO:7或SEQ ID NO:12所示的CDR1。
  3. 根据权利要求1或2所述的抗CLL1抗体，其中，所述重链可变区还包括SEQ ID NO:2、SEQ ID NO:8或SEQ ID NO:13所示的CDR2。
  4. 根据权利要求1-3任一项所述的抗CLL1抗体，其中，所述轻链可变区还包括SEQ ID NO:4、SEQ ID NO:10或SEQ ID NO:15所示的CDR1。
  5. 根据权利要求1-4任一项所述的抗CLL1抗体，其中，所述轻链可变区还包括SEQ ID NO:5或SEQ ID NO:16所示的CDR2。
  6. 根据权利要求1-5任一项所述的抗CLL1抗体，其中，
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、SEQ ID NO:3所示的CDR3；并且所述抗CLL1抗体的轻链可变区包括SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:6所示的CDR3；
    或者
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、SEQ ID NO:9所示的CDR3；并且所述抗CLL1抗体的轻链可变区包括SEQ ID NO:10所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:11所示的CDR3；
    或者
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:12所示的CDR1、SEQ ID NO:13所示的CDR2、SEQ ID NO:14所示的CDR3；并且所述抗CLL1抗体的轻链可变区包括SEQ ID NO:15所示的CDR1、SEQ ID NO:16所示的CDR2、SEQ ID NO:17所示的CDR3。
  7. 根据权利要求1-7任一项所述的抗CLL1抗体，其中，
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:18所示的氨基酸序列，轻链可变区包括SEQ ID NO:19所示的氨基酸序列；或者
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:20所示的氨基酸序列，轻链可变区包括SEQ ID NO:21所示的氨基酸序列；或者
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:22所示的氨基酸序列，轻链可变区包括SEQ ID NO:23所示的氨基酸序列；或者
    所述抗CLL1抗体的重链可变区包括SEQ ID NO:24所示的氨基酸序列，轻链可变区包括SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示的氨基酸序列。
  8. 根据权利要求1-7任一项所述的抗CLL1抗体，其中，所述抗CLL1抗体的重链可变区和轻链可变区之间通过链间二硫键连接；
    任选地，所述抗CLL1抗体的重链可变区之间通过链间二硫键连接；
    任选地，所述抗CLL1抗体还包括恒定区；
    任选地，所述抗CLL1抗体修饰有糖基化基团。
  9. 核酸分子，其包括编码权利要求1-8任一项所述的抗CLL1抗体的DNA片段。
  10. 表达载体，其包括权利要求9所述的核酸分子。
  11. 重组细胞，其表达权利要求1-8任一项所述的抗CLL1抗体。
  12. 根据权利要求11所述的重组细胞，其中，所述重组细胞的基因组中整合有权利要求6所述的核酸分子。
  13. 根据权利要求11所述的重组细胞，其中，所述重组细胞包括权利要求7所述的表达载体。
  14. 药物组合物，其包括权利要求1-8任一项所述的抗CLL1抗体；
    任选地，所述药物组合物还包括抗肿瘤药物；
    任选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
  15. 权利要求1-8任一项所述的抗CLL1抗体、权利要求9所述的核酸分子、权利要求10所述的表达载体、权利要求11-13任一项所述的重组细胞或权利要求14所述的药物组合物在制备疾病检测试剂和/或疾病治疗药物中的应用；
    任选地，所述疾病包括急性髓系白血病。
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