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WO2020083406A1 - 靶向cll1的抗体及其应用 - Google Patents

靶向cll1的抗体及其应用 Download PDF

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Publication number
WO2020083406A1
WO2020083406A1 PCT/CN2019/113767 CN2019113767W WO2020083406A1 WO 2020083406 A1 WO2020083406 A1 WO 2020083406A1 CN 2019113767 W CN2019113767 W CN 2019113767W WO 2020083406 A1 WO2020083406 A1 WO 2020083406A1
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Prior art keywords
antibody
seq
amino acid
acid sequence
chain variable
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PCT/CN2019/113767
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English (en)
French (fr)
Inventor
李宗海
王鹏
王华茂
Original Assignee
科济生物医药（上海）有限公司
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Application filed by 科济生物医药（上海）有限公司 filed Critical 科济生物医药（上海）有限公司
Priority to CN201980070975.2A priority Critical patent/CN113039205B/zh
Priority to CA3117759A priority patent/CA3117759A1/en
Priority to US17/288,189 priority patent/US20210324087A1/en
Priority to KR1020217016051A priority patent/KR20210089179A/ko
Priority to EP19877290.7A priority patent/EP3875484A4/en
Priority to SG11202104240TA priority patent/SG11202104240TA/en
Priority to JP2021523007A priority patent/JP2022505921A/ja
Publication of WO2020083406A1 publication Critical patent/WO2020083406A1/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
    • C07K16/2851—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 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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    • 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
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    • 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
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    • C07K—PEPTIDES
    • 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
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the field of tumor immunotherapy or diagnosis; more specifically, the invention relates to antibodies targeting CLL1 and their applications.
  • AML Acute myeloid leukemia
  • DNR daunorubicin
  • idarubicin deoxyrubicin
  • CLL1 Human C-type lectin-like molecule 1
  • LSCs leukemia stem cells
  • HSCs hematopoietic stem cells
  • the purpose of the present invention is to provide specific antibodies targeting CLL1.
  • the present invention provides an antibody targeting CLL1, the antibody having:
  • HCDR1 represented by SYX 1 MX 2
  • HCDR2 represented by X 3 X 4 X 5 X 6
  • SGGSTX 7 YAX 8 X 9 X 10 X 11 G
  • HCDR3 represented by SEQ ID NO: 40 or 41
  • LCDR2 shown by X 14 ASX 15 LX 16 S
  • RASQSISSX 12 LX 13 LCDR1 shown LCDR2 shown by X 14 ASX 15 LX 16 S
  • LCDR3 shown by QQX 17 YSX 18 PX 19 X 20 T
  • X 1 is selected from A or Y
  • X 2 is selected from S or H
  • X 3 is selected from A or I
  • X 4 is selected from I or F
  • X 5 is selected from S or N
  • X 6 is selected from G or P
  • X 7 is selected from Y or S
  • X 8 is selected from D or Q
  • X 9 is selected from S or K
  • X 10 is selected from V or F
  • X 11 is selected from K or Q
  • X 12 is selected from W or Y
  • X 13 is selected from A or N
  • X 14 is selected from D or V
  • X 15 is selected from N or S
  • X 16 is selected from E or Q
  • X 17 is selected from Y or S
  • X 18 is selected from Y or T
  • X 19 Selected from M or L
  • X 20 is I or not present.
  • the antibody is selected from the group consisting of:
  • the antibody LCDR1 is mutated from RASQSISSX 12 LX 13 to RASQWIARX 12 LX 13.
  • the sequence of the mutated LCDR1 is shown in SEQ ID NO: 44.
  • X 3 X 4 X 5 X 6 of the antibody HCDR2 is AISG or IFNP.
  • the antibody HCDR2 is mutated, and X 3 X 4 X 5 X 6 SGGSTX 7 YAX 8 X 9 X 10 X 11 G is mutated to X 3 X 4 X 5 X 6 GGGSTX 7 YAX 8 X 9 X 10 X 11 G, preferably, the sequence of the mutated HCDR2 is shown in SEQ ID NO: 39.
  • the antibody is selected from:
  • An antibody comprising HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 37, HCDR3 shown in SEQ ID NO: 40, and LCDR1 shown in SEQ ID NO: 42 and SEQ ID NO : LCDR2 shown in 45, LCDR3 shown in SEQ ID NO: 49;
  • the antibody is selected from:
  • Antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence shown in SEQ ID NO: 13, and SEQ The amino acid sequence shown in ID NO: 67, the amino acid sequence shown in SEQ ID NO: 68, and the amino acid sequence shown in SEQ ID NO: 69;
  • the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 11, SEQ The amino acid sequence shown in ID NO: 15;
  • An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
  • the antibody is selected from:
  • Antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 3;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 5 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 7;
  • Antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 11;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 13 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15;
  • Antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 5 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region of the antibody has LCDR1 shown in SEQ ID NO: 43, LCDR2 shown in SEQ ID NO: 48, and LCDR3 shown in SEQ ID NO: 50;
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain variable region of the antibody has SEQ ID NO: 5, 13, 67, 68, or 69.
  • the invention provides an antibody that recognizes the same epitope as the antibody of the first aspect of the invention.
  • the antibody of the first aspect or the second aspect provided by the present invention is a fully human antibody.
  • the antibodies of the first aspect or the second aspect provided by the present invention are whole antibodies, scFv, single domain antibodies, Fab fragments, Fv fragments, F (ab ') 2 fragments.
  • the invention provides a nucleic acid encoding the antibody of the first or second aspect of the invention.
  • the invention provides an expression vector comprising the nucleic acid of the third aspect of the invention.
  • the present invention provides a host cell comprising the expression vector or genome of the fourth aspect of the present invention integrated with the nucleic acid of the third aspect of the present invention.
  • the present invention provides the use of the antibody according to the first or second aspect of the present invention, for the preparation of a medicament for treating tumors, or for the preparation of reagents for diagnosing tumors.
  • the tumor is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), multiple bone marrow Tumor or bone marrow fibrosis.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • multiple bone marrow Tumor or bone marrow fibrosis multiple bone marrow Tumor or bone marrow fibrosis.
  • the tumor is a CLL1-positive tumor.
  • the present invention provides an immunoconjugate.
  • the immunoconjugate includes:
  • the tumor suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin.
  • the cytokine includes: IL-12, IL-15, type I interferon and TNF -One or more of alpha.
  • the molecule targeting the surface marker of immune cells is an antibody or ligand that binds to the surface marker of immune cells.
  • the surface markers of immune cells include one or more of CD3, CD16 and CD28. More preferably, the antibodies that bind to the surface markers of immune cells are anti-CD3 antibodies.
  • the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells, NK cells or NKT cells.
  • the present invention provides a nucleic acid encoding the multifunctional immunoconjugate according to the eighth aspect of the present invention.
  • the use of the immunoconjugate according to the eighth aspect of the present invention for preparing a medicine for treating tumors, or preparing an agent for diagnosing tumors.
  • the tumor is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), multiple bone marrow Tumor or bone marrow fibrosis.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • multiple bone marrow Tumor or bone marrow fibrosis multiple bone marrow Tumor or bone marrow fibrosis.
  • the tumor is a CLL1-positive tumor.
  • the present invention provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular signal domain.
  • the chimeric antigen receptor comprises the antibody of the first or second aspect of the present invention.
  • the antibody is a single chain antibody or a single domain antibody.
  • the intracellular signal domain of the chimeric antigen receptor comprises one or more costimulatory signal domains and / or primary signal domains.
  • the chimeric antigen receptor further includes a hinge domain.
  • the transmembrane domain of the chimeric antigen receptor is selected from one or more of the transmembrane regions of the following molecules: alpha, beta, zeta chain, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD1; and / or
  • the costimulatory signal domain is selected from one or more of the intracellular signal regions of the following molecules: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, Fc ⁇ RI ⁇ , MyD88 and 41BBL; and / or
  • the primary signal domain is selected from one or more of the following primary signal domains of molecules: TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278, CD66d, and CD3 ⁇ .
  • the transmembrane domain is selected from one or more of the following membrane transmembrane regions: CD8 ⁇ , CD4, CD45, PD1, CD154 and CD28; and / or
  • the costimulatory signal domain is selected from one or more of the intracellular signal regions of the following molecules: CD137, CD134, CD28 and OX40; and / or
  • the primary signal domain is selected from the primary signal domain of CD3 ⁇ .
  • the transmembrane domain is selected from the transmembrane domain of CD8 ⁇ or CD28
  • the costimulatory signal domain is selected from the intracellular signal domain of CD137 or CD28
  • the primary signal domain is selected from CD3 ⁇ Primary signal domain.
  • the chimeric antigen receptor includes the following sequentially linked antibodies, transmembrane region and intracellular signal region:
  • the antibody according to the first or second aspect of the present invention the transmembrane region of CD8 and the primary signal domain of CD3 ⁇ ;
  • the antibody according to the first or second aspect of the present invention the transmembrane region of CD8, the intracellular signal domain of CD137 and the primary signal domain of CD3 ⁇ ;
  • the antibody according to the first or second aspect of the present invention the transmembrane region of CD28, the intracellular signal domain of CD28 and the primary signal domain of CD3 ⁇ ; or
  • the antibody according to the first or second aspect of the present invention the transmembrane region of CD28, the intracellular signal domain of CD28, the intracellular signal domain of CD137, and the primary signal domain of CD3 ⁇ .
  • the chimeric antigen receptor has the sequence shown in SEQ ID NO: 9, 10, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66.
  • the present invention provides a nucleic acid encoding the chimeric antigen receptor of the tenth aspect of the present invention.
  • the present invention provides an expression vector comprising the nucleic acid according to the eleventh aspect of the present invention.
  • the present invention provides a virus, the virus comprising the vector of the twelfth aspect of the present invention.
  • the virus is a lentivirus or a retrovirus.
  • the present invention provides the chimeric antigen receptor of the tenth aspect of the present invention, or the nucleic acid of the eleventh aspect of the present invention, or the expression vector of the twelfth aspect of the present invention, or the present invention
  • the use of the virus according to the thirteenth aspect of the invention is for preparing a medicine for treating tumors.
  • the drug is a drug containing genetically modified immune cells.
  • the tumor is a specific embodiment, and the tumor is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), myeloproliferation Abnormal syndrome (MDS), multiple myeloma or myelofibrosis.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CMML chronic myelomonocytic leukemia
  • MDS myeloproliferation Abnormal syndrome
  • multiple myeloma or myelofibrosis multiple myeloma or myelofibrosis.
  • the tumor is a CLL1-positive tumor.
  • the present invention provides a genetically modified immune cell transduced with the nucleic acid according to the eleventh aspect of the invention, or the expression vector according to the twelfth aspect of the invention or the thirteenth aspect of the invention
  • the immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.
  • the genetically modified immune cell also expresses a second sequence other than the chimeric antigen receptor described in the tenth aspect of the present invention, the second sequence includes a cytokine, or another Chimeric antigen receptor, or chemokine receptor, or siRNA that reduces PD-1 expression, or a protein that blocks PD-L1, or TCR, or a safety switch;
  • the cytokines include one or more of IL-12, IL-15, IL-21 and type I interferon;
  • the chemokine receptor includes one or more of CCR2, CCR5, CXCR2 and CXCR4;
  • the safety switch includes one or more of iCaspase-9, Truancated EGFR and RQR8.
  • the present invention provides the use of the immune cell according to the seventeenth aspect of the present invention, and the genetically modified immune cell is used to prepare a medicine for treating tumors. .
  • the tumor is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), multiple bone marrow Tumor or bone marrow fibrosis.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • multiple bone marrow Tumor or bone marrow fibrosis multiple bone marrow Tumor or bone marrow fibrosis.
  • the tumor is a CLL1-positive tumor.
  • the present invention provides a pharmaceutical composition comprising:
  • the genetically modified immune cell according to the fifteenth aspect of the invention.
  • Figure 1 shows the results of ELISA assays for antibodies 4F2 and 25H8.
  • FIG. 7 ELISA results showing the binding of antibodies 7F8 and 12G4m to human CLL1.
  • FIG. 11 Shows the positive rate of CLL1-CAR T virus infection of T lymphocytes detected by FACs.
  • Fig. 12 shows the results of in vitro killing toxicity test of the antibody of the present invention and the prior art antibody
  • Fig. 13 shows the results of in vitro killing toxicity test of the antibody of the present invention.
  • CLL1 in the present invention refers to "C-Type Lectin-Like Molecule-1", which is a type II transmembrane protein.
  • CLL1 refers to human CLL1.
  • antibody herein refers to an antigen binding protein of the immune system.
  • antibody as mentioned herein includes a complete full-length antibody having an antigen-binding region and any fragments thereof retained by the "antigen-binding portion” or “antigen-binding region”, or single chains thereof such as single-chain variable fragments ( scFv).
  • a natural antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding fragments thereof interconnected by disulfide bonds.
  • antibody also includes all recombinant forms of antibodies (particularly the antibodies described herein), such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antibody fragments that bind to the antigen and derivatives described below.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), and they are interspersed in more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (C1q).
  • Antibody fragments include, but are not limited to: (i) Fab fragments composed of VL, VH, CL and CH1 domains, including Fab 'and Fab'-SH, (ii) Fd fragments composed of VH and CH1 domains, (iii) Fv fragment composed of VL and VH domains of a single antibody; (iv) dAb fragment composed of a single variable region (Ward et al., 1989, Nature 341: 544-546); (v) F (ab ') 2 fragment , A bivalent fragment containing 2 linked Fab fragments; (vi) single-chain Fv molecule antigen binding site (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc.
  • parent antibody or "parent immunoglobulin” as used herein includes unmodified antibodies that have been modified to produce variants.
  • the parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody.
  • Parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding it.
  • parent antibody or “parent immunoglobulin” includes murine or chimeric antibodies that are subsequently modified to produce humanized antibodies.
  • variant antibody or “antibody variant” as used herein includes antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent.
  • the variant antibody sequences herein preferably have at least about 80%, most preferably at least about 90%, and more preferably at least about 95% amino acid sequence identity with the parent antibody sequence.
  • Antibody variants can refer to the antibody itself, the composition comprising the parent antibody, or the amino acid sequence encoding it.
  • variant includes antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent.
  • the variant antibody sequences herein have at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98% of the parent antibody sequence. And most preferably at least about 99% amino acid sequence identity.
  • Antibody variants may refer to the antibody itself, the composition comprising the parent antibody, or the amino acid sequence encoding it.
  • amino acid modification includes amino acid substitutions, additions and / or deletions, and "amino acid substitution” means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • substitution R94K means that the arginine at position 94 is replaced with lysine
  • amino acid insertion means adding an amino acid at a specific position in the parent polypeptide sequence.
  • amino acid deletion or “deletion” means the removal of amino acids at specific positions in the parent polypeptide sequence.
  • antigenic determinant also known as an epitope
  • epitope can be composed of a continuous sequence of CLL1 protein sequences or a three-dimensional structure of CLL1 protein sequences that are not continuous.
  • single domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain of an antibody, or contains all or part of the light chain variable domain. For example, it contains only one heavy chain variable region (VHH).
  • VHH heavy chain variable region
  • immune cells also known as “immune effector cells” refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells , CIK cells, macrophages, mast cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cell may be an autologous T cell, a heterologous T cell, or an allogeneic T cell.
  • the NK cells may be allogeneic NK cells.
  • chimeric antigen receptor refers to: a polypeptide (ie, intracellular signal domain) that contains an extracellular domain capable of binding antigen, a transmembrane domain, and a cytoplasmic signal to the domain, intracellular
  • the signal domain refers to a protein that transmits information into the cell by generating a second messenger through a defined signaling pathway to regulate cell activity, or a protein that functions as an effector by corresponding to such messenger, and includes a primary signal domain, and Functional signaling domains (ie, costimulatory signaling domains) derived from stimulation molecules defined below may be included.
  • the intracellular signal domain generates signals that can promote the immune effector function of CAR cells (eg, CART cells). Examples of immune effector functions, such as in CART cells, include cell lysis and accessory activities, including cytokine secretion.
  • the term "primary signal domain” modulates the initial activation of the TCR complex in a stimulating manner.
  • the primary signal domain is triggered by, for example, the binding of the TCR / CD3 complex to peptide-loaded MHC molecules, thereby mediating T cell responses (including but not limited to proliferation, activation, differentiation, etc.).
  • the primary signaling domain that functions in a stimulating manner may contain the immunoreceptor tyrosine activation motif or the ITAM signaling motif.
  • ITAM-containing primary signal domains examples include, but are not limited to, derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS” )
  • the sequence of CD66d in a special case of the CAR of the present invention, includes an intracellular signaling sequence, such as the primary signaling domain of CD3 ⁇ .
  • costimulatory signal domain refers to a "costimulatory molecule", which is an associated binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of T cells, such as, but not limited to ,proliferation.
  • Costimulatory molecules are non-antigen receptor cell surface molecules or ligands required for effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) and 4-1BB (CD137) .
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain containing The functional signaling domain of the stimulating molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain contains functions derived from costimulatory molecules Sexual signaling domains and functional signaling domains derived from stimulating molecules.
  • the CAR comprises a chimeric fusion protein, the protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular conduction domain, the intracellular signaling domain comprising one or more costimulatory molecules At least two functional signaling domains and functional signaling domains derived from stimulating molecules.
  • CAR includes an optional leader sequence at the amino acid (ND end) of the CAR fusion protein.
  • the CAR further includes a leader sequence at the N-terminus of the extracellular antigen recognition domain, where the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during the cellular processing and localization of the CAR to the cell membrane.
  • CD3 ⁇ is defined herein as a protein provided by GenBan accession number BAG36664.1, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc.
  • CD3 ⁇ domain is defined as an amino acid residue from the cytoplasmic domain of the ⁇ chain, which is sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of ⁇ contains residues 52 to 164 of GenBan accession number BAG36664.1, its functional orthologs-from non-human species such as mice, rodents, monkeys, apes, etc. Valent residues.
  • 4-1BB refers to a member of the TNFR superfamily, which has the amino acid sequence of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • 4-1BB costimulatory domain is defined as the amino acid sequence 214-255 of GenBank ACC. No. AAA62478.2, or equivalent residues from non-classified species such as mice, rodents, monkeys, apes, etc.
  • the "4-1BB costimulatory domain” is the sequence provided in SEQ ID NO: 23, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc.
  • interferon refers to full-length interferon, or substantially maintains the biological activity of full-length wild-type interferon (eg, maintains at least 80% of the full-length, preferably at least 90%, more preferably at least 95%, 98% or 99%) of interferon fragments (truncated interferon) or interferon mutants.
  • Interferons include type I interferons (eg, interferon alpha and interferon beta) and type II interferons (eg, interferon gamma).
  • the "T cell” may be a pluripotent stem cell derived from bone marrow, which differentiates and matures into an immunologically active mature T cell in the thymus.
  • “T cells” may be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as “T cells” may be cells containing at least one T cell subpopulation: memory Stem cell-like memory cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and / or effector memory T cells ( Tem).
  • Tscm cells memory Stem cell-like memory cells
  • Tcm central memory T cells
  • effector T cells Tef, Teff
  • Tregs regulatory T cells
  • Tem effector memory T cells
  • Tem effector memory T cells
  • Tem effector memory T cells
  • Tem effector memory T cells
  • Tem effector memory T cells
  • Tem effector
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumor.
  • T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll TM isolation.
  • cells from circulating blood of the individual are obtained by a single blood collection.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from healthy donors from patients diagnosed with cancer.
  • the antibody or its variant of the present invention can be applied to the preparation of various targeted anti-tumor drugs and drugs for diagnosing tumors, especially for the preparation of immune effector cells targeting CLL1.
  • antigen binding proteins including antibodies, having scFv-based antigen binding regions are described.
  • the antibody can stably recognize HL60 and U937 cells, but not k562 cells.
  • the present invention includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies with human immunoglobulin Fc regions to produce bivalent proteins, thereby increasing the antibody's Overall affinity and stability.
  • the Fc portion allows direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) with antibodies used in antigen quantification studies, for example, to immobilize antibodies for affinity measurement, for targeted delivery therapy Drugs, use of immune effector cells to test Fc-mediated cytotoxicity, and many other applications.
  • the molecules of the present invention are based on the use of phage display to identify and select single-chain variable fragments (scFv) whose amino acid sequences confer molecular specificity to CLL1 and form the basis of all antigen-binding proteins of the present disclosure. Therefore, the scFv can be used to design a series of different "antibody” molecules, including, for example, full-length antibodies, fragments thereof such as Fab and F (ab ') 2, fusion proteins (including scFv_Fc), multivalent antibodies, ie, with targeting More than one specific antibody of the same antigen or different antigens, for example, bispecific T cell binding antibody (BiTE), tri-antibody, etc. (Cuesta et al., Multivalent antibodies: when design, surpasses evolution, Trends in Biotechnology 28: 355- 362,2010).
  • the heavy and light chains of the antibody of the invention may be full-length (eg, the antibody may include at least one and preferably two complete heavy chains, and at least one and preferably Two complete light chains) or may include an antigen binding moiety (Fab, F (ab ') 2, Fv, or scFv).
  • the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The choice of antibody type will depend on the immune effector function of the designed antibody. In constructing recombinant immunoglobulins, suitable amino acid sequences for the constant regions of various immunoglobulin isotypes and methods for generating a wide variety of antibodies are known to those skilled in the art.
  • the invention provides an antibody or fragment thereof that binds to CLL1, the antibody having:
  • HCDR1 represented by SYX 1 MX 2
  • HCDR2 represented by X 3 X 4 X 5 X 6 SGGSTX 7 YAX 8
  • X 9 X 10 X 11 G HCDR3 represented by SEQ ID NO: 40 or 41
  • RASQSISSX 12 LX 13 LCDR1 shown, LCDR2 shown as X 14 ASX 15 LX 16 S, LCDR3 shown as QQX 17 YSX 18 PX 19 X 20 T wherein X 1 is selected from A or Y, and X 2 is selected from S or H, X 3 is A or I, X 4 is selected from I or F, X 5 is selected from S or N, X 6 is selected from G or P, X 7 is selected from Y or S, X 8 is selected from D or Q, X 9 is selected from S or K, X 10 is selected from V or F, X 11 is selected from K or Q, X 12 is selected from W or Y, X 13 is selected from A or
  • the invention comprises a heavy chain CDR1 comprising any amino acid sequence of SEQ ID NO: 35, 36, and / or a heavy chain CDR2 comprising any amino acid sequence of SEQ ID NO: 37, 38, 39, and / or The heavy chain CDR3 containing any amino acid sequence of SEQ ID NO: 40, 41.
  • the invention provides an antibody or fragment thereof that binds to CLL1, which includes a light chain CDR1 comprising any amino acid sequence of SEQ ID NO: 42, 43, 44 and / or comprising SEQ ID NO: 45, 46, Light chain CDR2 of any amino acid sequence of 47, 48, and / or light chain CDR3 of any amino acid sequence of SEQ ID NO: 49, 50.
  • the invention provides an antibody or fragment thereof that binds to CLL1, which includes a heavy chain CDR1 comprising any amino acid sequence of SEQ ID NO: 35, 36, and / or comprising any of SEQ ID NO: 37, 38, 39
  • a heavy chain CDR2 of an amino acid sequence and / or a heavy chain CDR3 containing any amino acid sequence of SEQ ID NO: 40, 41, and / or a light chain CDR1 containing any amino acid sequence of SEQ ID NO: 42, 43, 44 , And / or light chain CDR2 containing any amino acid sequence of SEQ ID NO: 45, 46, 47, 48, and / or light chain CDR3 containing any amino acid sequence of SEQ ID NO: 49, 50.
  • the antibody or fragment thereof binding CLL1 comprises a heavy chain CDR1 including any amino acid sequence of SEQ ID NO: 35, 36, and a heavy chain CDR2 including any amino acid sequence of SEQ ID NO: 37, 38, 39, And a heavy chain CDR3 containing any amino acid sequence of SEQ ID NO: 40, 41, and / or a light chain CDR1 containing any amino acid sequence of SEQ ID NO: 42, 43, 44 and a light chain CDR1 containing SEQ ID NO: 45, 46, Light chain CDR2 of any amino acid sequence of 47, 48, and light chain CDR3 of any amino acid sequence of SEQ ID NO: 49, 50.
  • the antibody or fragment thereof that binds to CLL1 includes a heavy chain CDR1 containing any amino acid sequence of SEQ ID NO: 35, 36, and a heavy chain CDR2 containing any amino acid sequence of SEQ ID NO: 37, 38, 39 , And the heavy chain CDR3 containing any amino acid sequence of SEQ ID NO: 40, 41, and the light chain CDR1 containing any amino acid sequence of SEQ ID NO: 42, 43, 44 and the light chain CDR1 containing SEQ ID NO: 45, 46, Light chain CDR2 of any amino acid sequence of 47, 48, and light chain CDR3 of any amino acid sequence of SEQ ID NO: 49, 50.
  • the present invention provides an antibody or fragment thereof that binds to CLL1, which includes a heavy chain variable region sequence selected from SEQ ID NO: 1, 5, 13, 67, 68, 69.
  • the present invention provides an antibody or fragment thereof that binds to CLL1, which includes a light chain variable region sequence selected from SEQ ID NO: 3, 7, 11, 15.
  • the heavy chain and light chain variable region sequences can bind to CLL1
  • the heavy chain and light chain variable region sequences can be "mixed and matched" to produce an anti-CLL1 binding molecule of the present invention.
  • the invention provides variants of antibodies or fragments thereof that bind to CLL1.
  • the invention thus provides antibodies or fragments thereof having a heavy chain and / or light chain variable region that is at least 80% identical to the variable region sequence of the heavy chain or light chain.
  • the amino acid sequence identity of the heavy and / or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98% , Most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • Variants can be obtained by using the antibody described in this application as the parent antibody, by yeast library screening, phage library screening, point mutation and other methods.
  • the antibody LCDR1 provided by the present invention is mutated from RASQSISSX 12 LX 13 to RASQWIARX 12 LX 13.
  • the sequence of the mutated LCDR1 is shown in SEQ ID NO: 44.
  • the antibody HCDR2 “X 3 X 4 X 5 X 6 SGGSTX 7 YAX 8 X 9 X 10 X 11 G” of the present invention provides X 3 X 4 X 5 X 6 selected from AISG or IFNP.
  • the antibody HCDR2 is mutated from X 3 X 4 X 5 X 6 SGGSTX 7 YAX 8 X 9 X 10 X 11 G to X 3 X 4 X 5 X 6 GGGSTX 7 YAX 8 X 9 X 10 X 11 G, for example, the sequence of the mutated HCDR2 is shown in SEQ ID NO: 39.
  • the heavy chain variable region of the antibody of the present invention may be mutated, for example, the heavy chain variable region is mutated from SEQ ID NO: 13 to SEQ ID NO: 67, 68, or 69.
  • the invention provides antibodies that recognize the same epitope as the aforementioned anti-CLL1 antibodies.
  • Standard assays to assess the binding capacity of antibodies are known in the art and include, for example, ELISA, biacore, Western blot, and flow cytometry analysis. Suitable assays are described in detail in the examples.
  • the present invention also provides nucleic acids, vectors, and host cells containing the nucleic acids or vectors isolated from the antibodies and fragments thereof that bind to CLL1.
  • the nucleic acid can be located in whole cells, in cell lysates, or in partially purified or substantially purified form.
  • the nucleic acid of the present invention can be obtained using standard molecular biology techniques, for example, the light chain and heavy chain encoding antibodies or the cDNA encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • the cDNA encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • an immunoglobulin gene library for example, using phage display technology
  • one or more nucleic acids encoding the antibody can be recovered from the library.
  • Methods for introducing exogenous nucleic acids into host cells are generally known in the art and can vary with the host cell used.
  • Preferred nucleic acid molecules of the invention are those encoding heavy chain variable regions selected from SEQ ID NO: 2, 6, 14 and / or encoding light chain variable regions selected from SEQ ID NO: 4, 8, 12, 16 of those. More preferred are nucleic acid molecules comprising the SEQ ID NO: 2 sequence encoding the heavy chain, and the SEQ ID NO: 4 sequence encoding the light chain or the SEQ ID NO: 6 sequence encoding the heavy chain, And the SEQ ID NO: 8 sequence containing the encoded light chain or the SEQ ID NO: 2 sequence containing the heavy chain encoding, and the SEQ ID NO: 12 sequence containing the light chain encoding or the SEQ ID NO: 14 sequence containing the heavy chain encoding, And the SEQ ID NO: 16 sequence containing the encoded light chain.
  • the nucleic acid encoding the antibody of the present invention can be integrated into an expression vector.
  • Various expression vectors can be used for protein expression.
  • Expression vectors can include self-replicating extrachromosomal vectors, or vectors integrated into the host genome.
  • Expression vectors used in the present invention include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, various expression vectors are commercially or otherwise available. It can be used in the present invention to express antibodies.
  • the present invention also provides multifunctional immunoconjugates comprising the antibodies described herein and further comprising at least one other type of functional molecule.
  • the functional molecules are selected from but not limited to: molecules targeting tumor surface markers, tumor suppressing molecules, molecules targeting immune cell surface markers or detectable markers.
  • the antibody and the functional molecule may constitute a conjugate through covalent connection, coupling, attachment, cross-linking, and the like.
  • the immunoconjugate may include: the antibody of the present invention and at least one molecule targeting a tumor surface marker or a tumor suppressing molecule.
  • the tumor suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokine includes (but not limited to): IL-2, IL-7, IL-12, IL -15, type I IFN, TNF-alpha.
  • the molecule targeting the tumor surface marker is a molecule targeting the same tumor surface marker targeted by the antibody of the present invention.
  • the molecule targeting the tumor surface marker may be an antibody or ligand that binds to the tumor surface marker. For example, it may cooperate with the antibody of the present invention to more accurately target tumor cells.
  • the immunoconjugate may include: the antibody of the present invention and a detectable label.
  • the detectable labels include but are not limited to: fluorescent labels, chromogenic labels; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic Metal ion. It may also contain more than one marker. Labeling for labeling antibodies for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnostic techniques and / or methods used such as immunohistochemical staining (tissue) samples, flow cytometry, and the like. Suitable labels for detection / analysis / diagnostic techniques and / or methods known in the art are well known to those skilled in the art.
  • the immunoconjugate may include: the antibody of the present invention and a molecule targeting a surface marker of immune cells.
  • the molecules targeting the surface markers of immune cells can be antibodies or ligands that bind to the surface markers of immune cells, and can recognize immune cells, which carry the antibodies of the present invention to reach immune cells, and at the same time, the antibodies of the present invention can bind immune cells Targeting to tumor cells, thereby triggering immune cells to specifically kill the tumor.
  • the surface markers of immune cells may be selected from CD3, CD16, and CD28. More preferably, the antibody that binds to the surface markers of immune cells is an anti-CD3 antibody.
  • the immune cells can be selected from T cells, NK cells, NKT cells.
  • the immunoconjugate can be produced as a fusion protein comprising the antibody of the present invention and suitable other protein. Fusion proteins can be produced by methods known in the art, for example, by constructing a nucleic acid molecule and subsequently expressing the nucleic acid molecule to produce recombinantly, the nucleic acid molecule comprising an in-frame reading antibody-encoding nucleotide sequence and a suitable labeled nucleoside Acid sequence
  • Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate of the invention.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods.
  • the present invention also relates to vectors containing the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the present invention also provides a variety of chimeric antigen receptors (CARs), which include the antibodies or antibody fragments of the present invention, and the CAR-T cells exhibit anti-tumor properties.
  • CARs chimeric antigen receptors
  • the viral vector encoding CAR is used to transduce cells (eg, T cells).
  • the viral vector is a lentiviral vector.
  • the cells can stably express CAR.
  • the CLL1 binding portion of the CAR is an scFv antibody fragment, which maintains equivalent affinity binding compared to the IgG antibody from which it came from, for example, it binds the same antigen with comparable efficacy.
  • the antibody fragment is functional, and thus it provides biochemical reactions, such as activation of immune responses, inhibition of initiation of signal transduction from its target antigen, inhibition of kinase activity, etc. Therefore, the present invention provides a CLL1-CAR containing a WT1 binding domain that is engineered into T cells and a method for adopting it for adoptive immunotherapy.
  • the anti-CLL1 antigen-binding domain of CAR is a humanized scFv antibody fragment relative to the murine sequence scFv from which it is derived.
  • the CAR of the present invention combines the antigen binding domain of a specific antibody and intracellular signaling molecules.
  • intracellular signaling molecules include, but are not limited to, CD3 ⁇ chain, 4-1BB and CD28 signaling modules and combinations thereof.
  • CLL1-CAR contains at least one intracellular signaling domain, which selects CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3 ⁇ signaling domain, and any combination thereof. In one aspect, CLL1-CAR contains at least one intracellular signaling domain derived from one or more non-CD137 (4-1BB) or CD28 costimulatory molecules.
  • the sequence of CLL1-CAR can be 4F2 28Z (SEQ ID NO: 55), 4F2 BBZ (SEQ ID NO: 56), 4F2 28BBZ (SEQ ID NO: 57), 25H8 28Z (SEQ ID NO: 58 ), 25H8 BBZ (SEQ ID NO: 59), 25H8 28BBZ (SEQ ID NO: 60), 7F8 28Z (SEQ ID NO: 61), 7F8 BBZ (SEQ ID NO: 62), 7F8 28BBZ (SEQ ID NO: 63) ), 12G4m 28Z (SEQ ID NO: 64), 12G4m BBZ (SEQ ID NO: 65), 12G4m 28BBZ (SEQ ID NO: 66), 12G4m (M1331) 28Z (SEQ ID NO: 73), 12G4m (M1331) BBZ (SEQ ID NO: 9), 12G4m (M1331) 28BBZ (SEQ ID NO: 10), the transmembrane
  • the present invention also provides immune cells comprising the chimeric antigen receptor of the present invention.
  • the chimeric antigen receptor-modified T cells provided by the present invention also carry the coding sequence of exogenous cytokines; preferably, the cytokines include: IL-12, IL-15 or IL -twenty one.
  • the immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.
  • the chimeric antigen receptor-modified T cells provided by the present invention also carry a PD-L1 blocker or a protein that blocks PD-L1, such as natural PD-1, or a mutation capable of binding to PD-L1 PD-1, or a fragment of natural or mutant PD-1 capable of binding to PD-L1, or an antibody against PD-L1.
  • the antibody of the present invention an immunoconjugate containing the antibody, and genetically modified immune cells can be used to prepare a pharmaceutical composition or a diagnostic reagent.
  • the composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are properly administered to an animal or human, they will not produce adverse, allergic, or other adverse reactions.
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl fiber Sodium, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; Colorants; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers
  • composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient's type, age, weight, general disease status, and mode of administration.
  • the administration method can be, for example, injection or other treatment methods.
  • the present invention provides specific antibodies against CLL1;
  • the antibody of the present invention has a high affinity for CLL1, and after being prepared into chimeric antigen receptor modified T cells, it has a significant killing effect on cells expressing CLL1.
  • the present invention provides T cells modified with chimeric antigen receptors prepared using the antibodies, therefore, the antibodies and immune effector cells of the present invention can be safely and effectively applied to the treatment of acute myeloid leukemia.
  • Example 1 Screening of scFv specific for CLL1 using a fully human phage display library
  • the phage display library used in the present invention is a fully human natural scFv phage library constructed by the company, and the storage capacity is 1E + 11. ScFv fragments highly specific for CLL1 were obtained using screening methods known to those skilled in the art. In brief, 10ug / ml antigen CLL1-huFc (prepared by eukaryotic expression and protein purification using A filler to culture the supernatant for affinity purification) and human Fc segment were applied to the immune tube. In order to reduce the influence of the Fc segment, the phage library was added to the immune tube coated with human Fc segment for 1 hr.
  • the supernatant was taken and added to the CLL1-huFc-coated immune tube for 1.5 hours for binding, and then the non-specific phage was washed away, and the bound phage was eluted and infected with E. coli TG1 in the logarithmic growth phase.
  • the expanded culture eluted the phage and purified the expanded phage library using PEG / NaCl precipitation for the next round of screening.
  • the panning was performed for 3-4 cycles to enrich scFv phage clones that specifically bind to CLL1. Positive clones were determined by standard ELISA method against CLL1-huFc, and the ELISA detection pattern was shown in Figure 1.
  • ELISA uses human Fc segments as irrelevant antigens to verify the specificity of antibodies.
  • HL60 and U937 cells HL60 / U937 are CLL1 positive cells and k562 are CLL1 negative cells
  • Figure 3 The clone names are 4F2 and 25H8, and the Biacore maps of 4F2 and 25H8 are shown in Figure 2 As shown.
  • the heavy chain variable region of 4F2 is the amino acid sequence shown in SEQ ID NO: 1
  • the light chain variable region is the amino acid sequence shown in SEQ ID NO: 3
  • the heavy chain variable region of 25H8 is SEQ ID
  • the amino acid sequence shown in NO: 5 is the amino acid sequence shown in SEQ ID NO: 7.
  • the amino acid sequence of HCDR1 of 4F2 is shown in SEQ ID NO: 35
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 37
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 40
  • the amino acid sequence is shown in SEQ ID NO: 42
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 45
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 49.
  • the amino acid sequence of HCDR1 of 25H8 is shown in SEQ ID NO: 36
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 38
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 41
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 43
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 46
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 50.
  • LCDR1 and LCDR2 of the light chain variable region were randomly mutated to construct an affinity matured phage library. After two rounds of screening, the monoclonals were selected for ELISA detection, and the positive clones were sequenced and purified by expression. Based on the above experiments, two antibodies with high affinity and specific binding to human CLL1 were obtained, named 7F8 (from 4F2) and 12G4 ( From 25H8).
  • the heavy chain variable region of 7F8 is the amino acid sequence shown in SEQ ID NO: 1
  • the light chain variable region is the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region of 12G4 is SEQ ID The amino acid sequence shown in NO: 5
  • the light chain variable region is the amino acid sequence shown in SEQ ID NO: 15.
  • the sequence of LCDR1 of 7F8 is shown in SEQ ID NO: 44, the sequence of LCDR2 is shown in SEQ ID NO: 47, and the sequence of CDR regions of LCDR3 and the heavy chain is identical to the parent antibody (4F2).
  • the sequence of LCDR1 of 12G4 is the same as that of 25H8, the sequence of LCDR2 is shown in SEQ ID NO: 48, and the sequence of CDR regions of LCDR3 and the heavy chain are consistent with the parent antibody (25H8).
  • 12G4 contains a glycosylation site in the CDR2 region of the heavy chain, and a new antibody is obtained after mutating the serine to glycine.
  • the sequence is named 12G4m.
  • the sequence of 12G4m HCDR2 is shown in SEQ ID NO: 39.
  • the heavy chain variable region of 12G4m is the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 15.
  • ELISA uses human Fc segments as irrelevant antigens to verify the specificity of antibodies.
  • the scFv of antibodies 7F8 and 12G4m specifically binds to CLL1-expressing positive cells as shown in Figure 6. From the figure, it can be seen that the antibodies can specifically bind to positive cells but not to negative cells (HL60 and U937 are CLL1-expressing positive cells, k562 CLL1 does not express negative cells).
  • Example 3 ELISA detects the binding of 7F8 and 12G4m antibodies to different species of CLL1
  • Species specificity of antibodies 7F8 and 12G4m were detected by standard ELISA.
  • Human CLL1-hFc, mouse CLL1-hFc, monkey CLL1-hFc were coated with ug / ml at 4 degrees overnight, the ELISA plate was blocked at 2% MPBS for 1-2 hours at room temperature, 200nM antibody 7F8 / 12G4m (scFv) was added and incubated for 1 hour at room temperature
  • the secondary antibody is diluted with anti-His mouse monoclonal antibody 1: 1000, the third antibody is goat anti-mouse-HRP 1: 2000 dilution, and the color is developed after incubating for 1 hour at room temperature.
  • the reading is OD450 after the suspension of 1M concentrated sulfuric acid, Figure 7.
  • Antibodies 7F8 and 12G4m only bind human CLL1, but not mouse CLL1 and monkey CLL1.
  • Example 4 CLL1 antibody scFv-Fc format construction and monomer ratio detection
  • the anti-CLL1 antibodies 7F8 and 12G4m were fused and expressed with the Fc segment of human IgG, and 293F cells were transfected with 293Fectin transfection reagent. The supernatant was collected on the 6th day, and purified with Protein A filler. Collect the monomer peak and test the monomer ratio on a 40-air chromatography column. The monomer ratio of antibody 7F8 is 99.30%, and the monomer ratio of antibody 12G4m is 58.50%. The results are shown in Figure 8.
  • the antibody 7F8 protein was concentrated using millipore ultrafiltration tubes with a flow rate of 10KD, and the concentration was measured by OD280 / extinction coefficient. The results are shown in Table 1; the samples were run on SDS-PAGE, and the results are shown in Figure 9.
  • the 12G4m will be reduced and reconstructed.
  • Use Discovery software to build a 3D model of 12G4m, analyze the potential aggregation sites, and predict the software to perform point mutations at these sites, fuse the mutated antibody with the Fc segment of human IgG, in the form of scFv-Fc , Expressed by 293F cells, and after the purification of the expression product, the monomer rate was detected by SEC.
  • the mutation sites of 12G4m-A9P, 12G4m-V93I and 12G4m-M133I all occur in the heavy chain variable region, and the sequences of the heavy chain variable region are shown in SEQ ID NO: 67, 68, 69, respectively.
  • the scFv sequence of 12G4m-A9P is shown in SEQ ID NO: 70
  • the scFv sequence of 12G4m-V93I is shown in SEQ ID NO: 71
  • the scFv sequence of 12G4m-M133I is shown in SEQ ID NO: 72.
  • Example 5 Determination of EC50 of antibody binding to U937 cells using FACs
  • the EC50 of anti-CLL1 antibody was detected by FACs, the specific method is as follows: U937 cells were added to 96-well U-shaped bottom plate at 1 ⁇ 10 5 cells / well, and FcR blocking reagent was used for 4 degrees.
  • FITC-labeled Goat anti human Fc Jackson ImmunoResearch, Code number: 109-095-098 was added to cells by 200-fold dilution with 1% FBS, and after incubation at 4 ° C for 45 minutes, centrifugation was performed to remove the supernatant and 300 ⁇ L Wash cells twice with 1% FBS / well. Finally, the cells were resuspended in 200 ⁇ L / well 1% FBS and used for flow cytometry detection. As shown in FIG.
  • the EC50 values of the anti-human CLL1 antibodies 7F8, 12G4m, 4F2, and 25H8 were 0.06474nM, 0.4271nM, 0.7370nM, and 0.1638nM, respectively, and their EC50 values were less than 1nM, showing good cell killing effect.
  • PRRLSIN-cPPT.EF-1 ⁇ purchased from Addgene
  • a second generation chimeric antigen receptor lentiviral plasmid expressing antibody 7F8 was constructed, including PRRLSIN-cPPT.EF-1 ⁇ -7F8-28Z, PRRLSIN-cPPT .EF-1 ⁇ -7F8-BBZ and PRRLSIN-cPPT.EF-1 ⁇ -7F8-28BBZ. .
  • 7F8-28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 7F8scFV (SEQ ID NO: 31), CD8 hinge (SEQ ID NO: 19), CD28 transmembrane region (SEQ ID NO: 51) and intracellular signal Conduction domain (SEQ ID NO: 53) and CD3 ⁇ (SEQ ID NO: 25);
  • 7F8-BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 7F8scFV (SEQ ID NO: 31), CD8hinge (SEQ ID NO: 19) and transmembrane region (SEQ ID NO: 21), CD137 intracellular signaling domain (SEQ ID NO: 23) and CD3 ⁇ (SEQ ID NO: 25);
  • 7F8-28BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 7F8scFV (SEQ ID NO: 31), CD8 hinge (SEQ ID NO: 19), CD28 transmembrane region (
  • PRRLSIN-cPPT.EF-1 ⁇ a second-generation chimeric antigen receptor lentiviral plasmid expressing antibody 12G4m was constructed, including PRRLSIN-cPPT.EF-1 ⁇ -12G4m-28Z, PRRLSIN-cPPT.EF-1 ⁇ - 12G4m-BBZ and PRRLSIN-cPPT.EF-1 ⁇ -12G4m-28BBZ.
  • the 12G4m-28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 12G4m scFV (SEQ ID NO: 33), CD8 hinge (SEQ ID NO: 19), CD28 transmembrane region (SEQ ID NO: 51) and intracellular Signaling domain (SEQ ID NO: 53) and CD3 ⁇ (SEQ ID NO: 25); 12G4m-BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 12G4m scFV (SEQ ID NO: 33), CD8 hinge (SEQ ID NO: 19) and transmembrane region (SEQ ID NO: 21), CD137 intracellular signaling domain (SEQ ID NO: 23) and CD3 ⁇ (SEQ ID NO: 25); 12G4m-28BBZ sequence is composed of CD8 ⁇ Signal peptide (SEQ ID NO: 17), 12G4m scFV (SEQ ID NO: 33), CD8 hinge (SEQ ID NO: 19), CD137 intracellular signal trans
  • PRRLSIN-cPPT.EF-1 ⁇ a second-generation chimeric antigen receptor lentiviral plasmid expressing antibody 12G4m-M1331, namely PRRLSIN-cPPT.EF-1 ⁇ -12G4m (M1331) -BBZ, was constructed.
  • 12G4m (M1331) -28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 12G4m (M1331) scFV (SEQ ID NO: 72), CD8 hinge (SEQ ID NO: 19), CD28 transmembrane region (SEQ ID NO) : 51) and intracellular signaling domain (SEQ ID NO: 53) and CD3 ⁇ (SEQ ID NO: 25); 12G4m (M1331) -BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 12G4m (M1331 ) scFV (SEQ ID NO: 72), CD8 hinge (SEQ ID NO: 19) and transmembrane region (SEQ ID NO: 21), CD137 intracellular signaling domain (SEQ ID NO: 23) and CD3 ⁇ (SEQ ID NO: 25); 12G4m (M1331) -28BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 17), 12G4m (M1331)
  • Example 7 CLL1-specific CAR virus packaging and detection of CAR positive rate of infection
  • the calcium phosphate method was used to package the lentivirus.
  • the virus supernatant was purified with PEG8000 / NaCl. After purification, the virus was tested for titer by FACS method. According to the results of flow cytometry, 7F8-CART, 12G4m-CART and 12G4m -M1331 CART virus titers are 2.37E + 08, 1.41E + 08 and 7.99E + 07 respectively.
  • Example 8 In vitro killing toxicity test targeting CLL1 positive cells
  • the antibody M26 (CN104736562B) reported in the prior art was selected as a positive control, and M26-BBZ CAR T was prepared using a conventional protocol.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. Refer to CytoTox 96 Non-radioactive Cytotoxicity Test Kit for specific methods.
  • Effector cells seeded Untransduced (UTD) T cells, 7F8-BBZ CART cells, 12G4m-BBZ CART cells in 96-well plates according to the effective target ratio of 3: 1, 1: 1, 1: 3 and 1: 9.
  • Target cells Inoculate 50 ⁇ L 2 ⁇ 10 5 / mL of CLL1 positive HL-60 and U937 cells and CLL1 negative K562 cells into the corresponding 96-well plate.
  • mice 6-8 week-old female NPG mice were randomly divided into 4 groups, each with 6 mice, namely Untransduced T (UTD), 12G4m-BBZ CART group (1.0 ⁇ 10 6 / mouse), 12G4m-BBZ CART group (2.0 ⁇ 10 6 / mouse), 12G4m-BBZ CART (4.0 ⁇ 10 6 / mouse) cell treatment group.
  • UTD Untransduced T
  • 12G4m-BBZ CART group 1.0 ⁇ 10 6 / mouse
  • 12G4m-BBZ CART group 2.0 ⁇ 10 6 / mouse
  • 12G4m-BBZ CART 4.0 ⁇ 10 6 / mouse
  • Inoculation of subcutaneous transplanted tumors Collect HL-60 cells in logarithmic growth phase and in good growth state, adjust the cell density to 1.5 ⁇ 10 7 / mL, inoculate subcutaneously on the right armpit of NPG mice, and inoculate a volume of 200 ⁇ L cells The suspension, that is, each mouse was inoculated with 3.0 ⁇ 10 6 tumor cells, and the day of inoculation was recorded on day 0.
  • Example 10 In vitro killing results of 12G4m (M1331) -BBZ CART
  • Effector cells Inoculate Untransduced (UTD) T cells, 7F8-BBZ CART cells, 12G4m-BBZ CART cells, 12G4m (M1331) -BBZ CART cells in 96 wells according to the effective target ratio of 9: 1, 3: 1 and 1: 1 board.
  • Target cells Inoculate 50 ⁇ L0 2 ⁇ 10 5 / mL of CLL1 positive HL-60 and U937 cells and CLL1 negative K562 cells into corresponding 96-well plates.
  • the experimental results are shown in FIG. 13, and the experimental results are shown in FIG. 13.
  • the results show that the 7F8-BBZ CAR, T, 12G4m–BBZ CAR, T, 12G4m (M1331) -BBZ CART of the present invention are more effective than the control UTD.
  • Both HL-60 and U937 cells with positive expression had significant toxic killing effects, while K562 cells with negative CLL1 expression had almost no killing effect.

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Abstract

本发明提供了靶向CLL1的抗体及其应用。本发明提供的CLL1特异性抗体对于CLL1具有较高的亲和力，且在制备成嵌合抗原受体修饰的T细胞后，对表达CLL1的细胞具有明显的杀伤作用。

Description

靶向CLL1的抗体及其应用 技术领域
本发明属于肿瘤免疫治疗或诊断领域；更具体地，本发明涉及靶向CLL1的抗体及其应用。
背景技术
急性髓系白血病(AML)是成人急性白血病中最常见的类型。最传统的治疗方法是异源造血干细胞移植，然而存在早期或晚期移植相关的并发症，同时存在术后复发的几率。目前治疗AML的化疗药物主要有柔红霉素(DNR)、伊达比星(去甲氧柔红霉素)、阿糖胞苷等，化疗和放疗在一定程度上对AML产生明显抑制作用，但因副作用强、易复发而影响临床顺应性，因此急迫需要针对AML的新型治疗方法。
人C型凝集素样分子1(CLL1)是一类II型跨膜蛋白，它的表达受限于髓系细胞和大多数AML细胞。并且，CLL1表达在白血病干细胞(LSCs)上，但却不表达于造血干细胞(HSCs)，因此，CLL1是一种潜在的治疗AML的靶点。
虽然目前有研究人员在进行抗CLL1的抗体的研究，如CN104736562B公开了抗CLL1抗体，然而目前在市场上尚未有可以用于治疗AML的抗CLL1的抗体。
发明内容
本发明目的在于提供靶向CLL1的特异性抗体。
在第一方面，本发明提供一种靶向CLL1的抗体，所述抗体具有：
SYX 1MX 2所示的HCDR1，X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G所示的HCDR2，SEQ ID NO:40或41所示的HCDR3，及RASQSISSX 12LX 13所示的LCDR1，X 14ASX 15LX 16S所示的LCDR2，QQX 17YSX 18PX 19X 20T所示的LCDR3，
其中，所述的X 1选自A或者Y，X 2选自S或者H，X 3为A或I，X 4选自I或者F，X 5选自S或者N，X 6选自G或者P，X 7选自Y或者S，X 8选自D或者Q，X 9选自S或者K，X 10选自V或者F，X 11选自K或者Q，X 12选自W或者Y，X 13选自A或者N，X 14选自D或者V，X 15选自N或者S，X 16选自E或者Q，X 17选自Y或者S，X 18选自Y或者T，X 19选自M或者L，X 20为I或者不存在。
在一优选方案中，所述抗体选自下组：
(1)抗体，其重链可变区包含SEQ ID NO:35或36所示的HCDR1，和/或包含SEQ ID NO:37、38或39所示的HCDR2，和/或包含SEQ ID NO:40或41所示的HCDR3；
(2)抗体，其轻链可变区包含SEQ ID NO:42、43或44所示的LCDR1，和/或包含SEQ ID NO:45、46、47或48所示的LCDR2，和/或包含SEQ ID NO:49或50所示的LCDR3；
(3)抗体，包含(1)所述的重链可变区及(2)所述的轻链可变区；
(4)(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在一优选方案中，所述的抗体LCDR1发生了突变，由RASQSISSX 12LX 13突变为RASQWIARX 12LX 13，优选的，所述突变后的LCDR1的序列如SEQ ID NO:44所示。
在一优选方案中，所述的抗体HCDR2的X 3X 4X 5X 6为AISG或者IFNP。
在一优选方案中，所述的抗体HCDR2发生了突变，由X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G突变为X 3X 4X 5X 6GGGSTX 7YAX 8X 9X 10X 11G，优选的，所述突变后的HCDR2的序列如SEQ ID NO:39所示。
在一优选方案中，所述的抗体选自：
(1)抗体，其包含SEQ ID NO:35所示的HCDR1、SEQ ID NO:37所示的HCDR2、SEQ ID NO:40所示的HCDR3以及SEQ ID NO:42所示的LCDR1、SEQ ID NO:45所示的LCDR2、SEQ ID NO:49所示的LCDR3；
(2)抗体，其包含SEQ ID NO:36所示的HCDR1、SEQ ID NO:38所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:43所示的LCDR1、SEQ ID NO:46所示的LCDR2、SEQ ID NO:50所示的LCDR3；
(3)抗体，其包含SEQ ID NO:35所示的HCDR1、SEQ ID NO:37所示的HCDR2、SEQ ID NO:40所示的HCDR3以及SEQ ID NO:44所示的LCDR1、SEQ ID NO:47所示的LCDR2、SEQ ID NO:49所示的LCDR3；
(4)抗体，其包含SEQ ID NO:36所示的HCDR1、SEQ ID NO:38所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:43所示的LCDR1、SEQ ID NO:48所示的LCDR2、SEQ ID NO:50所示的LCDR3；
(5)抗体，其包含SEQ ID NO:36所示的HCDR1、SEQ ID NO:39所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:43所示的LCDR1、SEQ ID NO:48所示的LCDR2、SEQ ID NO:50所示的LCDR3；
(6)抗体，(1)～(5)中任一项所述的抗体的变体，且具备与(1)～(5)中任一项所述的抗体相同或相似的活性。
在一优选方案中，所述的抗体选自：
(1)抗体，所述的抗体的重链可变区具有SEQ ID NO:1所示的氨基酸序列、SEQ ID NO:5所示的氨基酸序列、SEQ ID NO:13所示的氨基酸序列、SEQ ID NO:67所示的氨基酸序列、SEQ ID NO:68所示的氨基酸序列、SEQ ID NO:69所示的氨基酸序列；
(2)抗体，所述的抗体的轻链可变区具有SEQ ID NO:3所示的氨基酸序列、SEQ ID NO:7所示的氨基酸序列、SEQ ID NO:11所示的氨基酸序列、SEQ ID NO:15所示的氨基酸序列；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
(4)(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在一优选方案中，所述的抗体选自：
(1)抗体，所述的抗体的重链可变区具有SEQ ID NO:1所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:3所示的氨基酸序列；
(2)抗体，所述的抗体的重链可变区具有SEQ ID NO:5所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:7所示的氨基酸序列；
(3)抗体，所述的抗体的重链可变区具有SEQ ID NO:1所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:11所示的氨基酸序列；
(4)抗体，所述的抗体的重链可变区具有SEQ ID NO:13所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:15所示的氨基酸序列；
(5)抗体，所述的抗体的重链可变区具有SEQ ID NO:5所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:15所示的氨基酸序列；
(6)(1)～(5)中任一项所述的抗体的变体，且具备(1)～(5)中任一项所述的抗体相同或相似的活性。
在一优选方案中，所述抗体的轻链可变区具有SEQ ID NO:43所示的LCDR1，SEQ ID NO:48所示的LCDR2，SEQ ID NO:50所示的LCDR3；
优选地，所述轻链可变区具有SEQ ID NO:15所示的氨基酸序列；
更优选地，所述抗体的轻链可变区具有SEQ ID NO:15所示的氨基酸序列，所述的抗体的重链可变区具有SEQ ID NO:5、13、67、68或69所示的氨基酸序列。
在第二方面，本发明提供一种抗体，其与本发明第一方面所述的抗体识别相同的抗原决定部位。
在一优选方案中，本发明提供的第一方面或第二方面的抗体是全人抗体。
在一优选方案中，本发明提供的第一方面或第二方面的抗体为全抗、scFv、单域抗体、Fab片段、Fv片段、F(ab’) 2片段。
在第三方面，本发明提供编码本发明第一或第二方面所述的抗体的核酸。
在第四方面，本发明提供一种表达载体，其包含本发明第三方面所述的核酸。
在第五方面，本发明提供一种宿主细胞，其包含本发明第四方面所述的表达载体或基因组中整合有本发明第三方面所述的核酸。
在第六方面，本发明提供本发明第一方面或第二方面所述抗体的用途，用于制备治疗肿瘤的药物，或制备诊断肿瘤的试剂。
在一具体实施例中，所述肿瘤是急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性粒-单核细胞白血病(CMML)、骨髓增生异常综合征(MDS)、多发性骨髓瘤或骨髓纤维化。
在一具体实施例中，所述肿瘤是CLL1阳性的肿瘤。
在第七方面，本发明提供一种免疫辍合物，所述的免疫辍合物包括：
本发明第一方面或第二方面所述的抗体，以及与之连接的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子，和可检测标记物。
在一优选方案中，所述的抑制肿瘤的分子为抗肿瘤的细胞因子或抗肿瘤的毒素，较佳地，所述的细胞因子包括：IL-12、IL-15、I型干扰素和TNF-alpha中的一种或多种。
在一优选方案中，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志 物的抗体或配体。较佳的，所述的免疫细胞表面标志物包括：CD3，CD16和CD28中的一种或多种，更佳地，所述的结合免疫细胞表面标志物的抗体是抗CD3的抗体。
在一优选方案中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞、NK细胞或NKT细胞表面标志物的抗体。
在第八方面，本发明提供编码本发明第八方面所述的多功能免疫辍合物的核酸。
在第九方面，提供了本发明第八方面所述的免疫辍合物的用途，用于制备治疗肿瘤的药物，或制备诊断肿瘤的试剂。
在一具体实施例中，所述肿瘤是急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性粒-单核细胞白血病(CMML)、骨髓增生异常综合征(MDS)、多发性骨髓瘤或骨髓纤维化。
在一具体实施例中，所述肿瘤是CLL1阳性的肿瘤。
在第十方面，本发明提供一种嵌合抗原受体，包含胞外域、跨膜域及胞内信号域，所述嵌合抗原受体包含本发明第一或第二方面所述的抗体。
在一优选方案中，所述的抗体为单链抗体或单域抗体。
在一优选方案中，所述嵌合抗原受体的胞内信号域包含一个或多个共刺激信号域和/或初级信号域。
在一优选方案中，所述嵌合抗原受体还包括铰链域。
在一优选方案中，所述嵌合抗原受体的跨膜域选自以下分子的跨膜区的一种或多种：TCR的alpha、beta、zeta链、CD3ε，CD3ζ、CD4、CD5，CD8α、CD9、CD16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD1；和/或
所述共刺激信号域选自以下分子的胞内信号区的一种或多种：CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54、CD83、OX40、CD137、CD134、CD150、CD152、CD223、CD270、PD-L2、PD-L1、CD278、DAP10、LAT、NKD2C SLP76、TRIM、FcεRIγ、MyD88和41BBL；和/或
所述初级信号域选自以下分子的初级信号域的一种或多种：TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278、CD66d和CD3ζ。
在一较佳的具体实施方式中，
所述的跨膜域选自以下分子的跨膜区的一种或多种：CD8α，CD4，CD45，PD1，CD154和CD28；和/或
所述共刺激信号域选自以下分子的胞内信号区的一种或多种：CD137，CD134，CD28和OX40；和/或
所述初级信号域选自CD3ζ的初级信号域。
在一更佳的具体实施方式中，所述跨膜域选自CD8α或CD28的跨膜区，所述共刺激信号域选自CD137或CD28的胞内信号区，所述初级信号域选自CD3ζ的初级信号域。
在一优选方案中，所述的嵌合抗原受体包括如下的顺序连接的抗体，跨膜区和胞内信 号区：
本发明第一或第二方面所述的抗体、CD8的跨膜区和CD3ζ的初级信号域；
本发明第一或第二方面所述的抗体、CD8的跨膜区、CD137的胞内信号域和CD3ζ的初级信号域；
本发明第一或第二方面所述的抗体、CD28的跨膜区、CD28的胞内信号域和CD3ζ的初级信号域；或
本发明第一或第二方面所述的抗体、CD28的跨膜区、CD28的胞内信号域、CD137的胞内信号区和CD3ζ的初级信号域。
在一优选方案中，所述的嵌合抗原受体具有SEQ ID NO:9、10、55、56、57、58、59、60、61、62、63、64、65或66所示序列。
在第十一方面，本发明提供编码本发明第十方面所述的嵌合抗原受体的核酸。
在第十二方面，本发明提供一种表达载体，其包含本发明第十一方面所述的核酸。
在第十三方面，本发明提供一种病毒，所述的病毒包含本发明第十二方面所述的载体。
在一优选方案中，所述病毒是慢病毒或逆转录病毒。
在第十四方面，本发明提供本发明第十方面所述的嵌合抗原受体、或本发明第十一方面所述的核酸、或本发明第十二方面所述的表达载体、或本发明第十三方面所述的病毒的用途，用于制备治疗肿瘤的药物。较佳地，所述药物为含基因修饰的免疫细胞的药物。
在一优选方案中，所述肿瘤是在一具体实施例中，所述肿瘤是急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性粒-单核细胞白血病(CMML)、骨髓增生异常综合征(MDS)、多发性骨髓瘤或骨髓纤维化。
在一优选方案中，所述肿瘤是CLL1阳性的肿瘤。
在第十五方面，本发明提供一种基因修饰的免疫细胞，其转导有本发明第十一方面所述的核酸，或本发明第十二方面所述的表达载体或本发明第十三方面所述的病毒；或表达有本发明第十方面所述的嵌合抗原受体。
在一优选方案中，所述的免疫细胞优选自T淋巴细胞，NK细胞或NKT细胞。
在一优选方案中，所述基因修饰的免疫细胞还表达有除本发明第十方面所述的嵌合抗原受体之外的第二序列，所述第二序列包括细胞因子、或另一种嵌合抗原受体、或趋化因子受体、或降低PD-1表达的siRNA、或阻断PD-L1的蛋白、或TCR、或安全开关；
较佳地，所述细胞因子包括IL-12、IL-15、IL-21和I型干扰素的一种或多种；
较佳地，所述趋化因子受体包括CCR2、CCR5、CXCR2和CXCR4的一种或多种；
较佳地，所述安全开关包括iCaspase-9、Truancated EGFR和RQR8的一种或多种。
在第十六方面，本发明提供本发明第十七方面所述的免疫细胞的用途，所述的基因修饰的免疫细胞用于制备治疗肿瘤的药物。。
在优选的实施方式中，所述肿瘤是急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性粒-单核细胞白血病(CMML)、骨髓增生异常综合征(MDS)、多发性骨髓瘤或骨髓纤维化。
在优选的实施方式中，所述肿瘤是CLL1阳性的肿瘤。
在第十七方面，本发明提供一种药物组合物，其包括：
本发明第一或第二方面所述的抗体或编码该抗体的核酸；或
本发明第七方面所述的免疫辍合物或编码该辍合物的核酸；或
本发明第十五方面所述的基因修饰的免疫细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一赘述。
附图说明
图1.显示了抗体4F2和25H8的ELISA测定结果。
图2.显示了抗体4F2和25H8的Biacore测定结果。
图3.显示了FACs检测的抗体4F2和25H8与HL60/U937细胞及k562的结合情况。
图4.显示了抗体7F8和12G4m的ELISA测定结果。
图5.显示了抗体7F8和12G4m的Biacore测定结果。
图6.显示了FACs检测的抗体7F8和12G4m与HL60/U937细胞及k562的结合情况。
图7.显示了抗体7F8和12G4m与人CLL1结合的ELISA测定结果。
图8.显示了抗体7F8和12G4m单体率检测结果。
图9.显示了SDS PAFE分析纯化的抗CLL1 scFv_Fc抗体(还原条件)。
图10.显示了FACs检测抗体7F8，12G4m，4F2，25H8结合U937细胞的EC50。
图11.显示了FACs检测的CLL1-CAR T病毒感染T淋巴细胞的阳性率。
图12.显示了本发明抗体和现有技术抗体的体外杀伤毒性试验结果
图13.显示了本发明抗体的体外杀伤毒性试验结果。
具体实施方式
发明人经过广泛而深入的研究，开发了特异性高，结合能力好的抗CLL1的抗体。在此基础上完成了本发明。
本文采用的科技术语具有与本领域技术人员常规理解的相同或相似的含义。为便于理解本发明，一些术语定义如下。
本发明的术语“CLL1”是指“C-Type Lectin-Like Molecule-1”，是一类II型跨膜蛋白。在具体实施例中，CLL1是指人CLL1。
本文中的术语“抗体”指免疫系统的抗原结合蛋白。如本文提到的术语“抗体”包括具有抗原结合区域的完整的全长抗体及其中“抗原结合部分”或“抗原结合区域”保留的其任何片段、或其单链例如单链可变片段(scFv)。天然抗体指包含通过二硫键互联的至少两条重(H)链和两条轻(L)链或其抗原结合片段的糖蛋白。术语“抗体”还包括抗体(特别是本文所述抗体)的所 有重组形式，例如在原核细胞中表达的抗体，未糖基化的抗体以及与抗原结合的抗体片段和下文所诉的衍生物。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区组成。VH和VL可进一步细分为称为互补性决定区(CDR)的高变区，他们散布在称为构架区(FR)的更保守区域中.每条VH和VL由三个CDR和四个FR组成，从氨基端至羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(如效应细胞)和经典补体系统的第一成分(C1q)。
抗体片段包括但不限于：(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341：544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点(Bird等，1988，Science 242：423-426；Huston等，1988，Proc.Natl.Acad.Sci.U.S.A 85:58795883)；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段(Tomlinson等，2000，Methods Enzymol.326:461-479；WO94/1380；Holliger等，1993,Proc.Natl.Acad.Sci.U.S.A 90:6444-6448)；和(ix)与相同或不同抗体遗传融合的scFv(Coloma&Morrison,1997,Nature Biotechnology 15,159163)。
本文中使用的术语“亲本抗体”或“亲本免疫球蛋白”包括未修饰的抗体，所述抗体之后经修饰产生变体。所述亲本抗体可以使天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。本文中使用的术语“亲本抗体”或“亲本免疫球蛋白”包括之后经修饰产生人源化抗体的鼠抗体或嵌合抗体。
本文中使用的术语“变体抗体”或“抗体变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％，最优选至少约90％，更优选至少约95％的氨基酸序列同一性。抗体变体可以指抗体本身，包含所述亲本抗体的组合物，或编码其氨基酸序列。
本文中使用的术语“变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。在具体的实施方式中，本文中的变体抗体序列具有与亲本抗体序列至少约80％、优选至少约90％、更优选至少约95％、更优选至少约97％、更优选至少约98％、最优选至少约99％的氨基酸序列同一性。抗体变体可以指抗体本身，包含所述亲本抗体的组合物，或编码其地氨基酸序列。术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代R94K指94位的精氨酸被赖氨酸替换，本文中使用的“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸。文中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。
本发明中讨论的所有免疫球蛋白重链恒定区位置，都根据Kabat的EU索引编号(Kabat等，1991，sequences of proteins of immunological interest，第5版，United States Public Health  Service,National Institutes of Health,Bethesda，通过引用全文整合)。“Kabat的EU索引”指人IgG1EU抗体的残基编号，如Edelman等人，1969，Biochemistry 63:78-85所述。
本文中使用的术语“抗原决定部位”又称抗原表位，可以由CLL1蛋白序列的连续序列组成，也可以由CLL1蛋白序列不连续的三维结构组成。
术语“单域抗体”为包含抗体的重链可变域的全部或一部分，或包含轻链可变域的全部或一部分的抗体片段。例如，只包含一个重链可变区(VHH)。
术语“免疫细胞”又称“免疫效应细胞”，是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。
本文中使用的术语“嵌合抗原受体”或“CAR”指：包含能够结合抗原的胞外域、跨膜域和使胞质信号传到结构域的多肽(即胞内信号域)，胞内信号域指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，包含初级信号域，还可以包括源自下文定义的刺激分子的功能信号传导结构域(即共刺激信号域)。胞内信号域产生可以促进CAR的细胞(例如CART细胞)的免疫效应子功能的信号，免疫效应子功能的例子，例如在CART细胞中，包括细胞裂解活性和辅助活性，包括细胞因子分泌。
术语“初级信号域”以刺激性方式调节TCR复合物的初始活化。一方面，初级信号域由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号域可以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本发明中尤其有用的包含ITAM的初级信号域的例子包括但不限于，源自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d的序列，在特例的本发明CAR中，在任何一个或多个本发明CAR中胞内信号传导结构域包含胞内信号传导序列，例如CD3ξ的初级信号域。
术语“共刺激信号域”指“共刺激分子”，为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，例如，但不限于，增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于，MHCI类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。
在本发明中，一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自刺激分子的功能信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自共刺激分子的功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域和胞内传导结构域，所述胞内信号传导结构 域包含源自一个或多个共刺激分子的至少两个功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR在CAR融合蛋白的氨基酸(ND端)包含可选的前导序列。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。
本文中术语“CD3ξ”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3ξ结构域”定义为来自ξ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，ξ的胞质结构域包含GenBan登录号BAG36664.1的残基52至164、其功能性直向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
本文中术语“4-1BB”指TNFR超家族的成员，其具有GenBankAcc.No.AAA62478.2的氨基酸序列、或来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基；“4-1BB共刺激结构域”定义为GenBankACC.No.AAA62478.2的氨基酸序列214-255，或来自非分类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。一方面，“4-1BB共刺激结构域”是SEQIDNO:23中提供的序列、或来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
本文中的术语“干扰素”是指全长干扰素，或者基本保持全长野生型干扰素的生物活性(例如保持全长至少80％、优选至少90％、更优选至少95％、98％或99％)的干扰素片段(截断的干扰素)或干扰素突变体。干扰素包括I型干扰素(例如，干扰素α和干扰素β)和II型干扰素(例如，干扰素γ)。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。或者，可以从健康供体，来自诊断患有癌症的患者衍生细胞。
本发明的抗体或其变体可以被应用于制备各种靶向性抗肿瘤药物以及诊断肿瘤的药物，特别是应用于制备靶向CLL1的免疫效应细胞。
抗CLL1的抗体
在本公开中，描述了具有基于scFv的抗原结合区域的抗原结合蛋白，包括抗体。从人scFv 噬菌体展示文库选择scFv。这些分子展示精细的特异性。例如，该抗体能稳定识别HL60及U937细胞，不识别k562细胞。
在一些实施方案中，本发明包括具有scFv序列的抗体，所述scFv序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。
本文提供的结果突出本发明抗体在靶向CLL1时的特异性、灵敏性和效用。
本发明的分子基于使用噬菌体展示鉴定和选择单链可变片段(scFv)，所述单链可变片段的氨基酸序列赋予分子针对CLL1的特异性并且形成本公开的全部抗原结合蛋白的基础。因此，所述scFv可以用来设计一系列不同“抗体”分子，包括例如全长抗体、其片段如Fab和F(ab’)2、融合蛋白(包括scFv_Fc)、多价抗体、即，具有针对相同抗原或不同抗原的多于一种特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(Cuesta等，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。
在抗原结合蛋白是全长抗体的一个实施方案中，本发明抗体的重链和轻链可以是全长(例如，抗体可以包括至少一条并优选地两条完整重链，和至少一条并优选地两条完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。
在第一方面，本发明提供了结合CLL1的抗体或其片段，所述抗体具有：
SYX 1MX 2所示的HCDR1，X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G所示的HCDR2，SEQ ID NO:40或41所示的HCDR3，及RASQSISSX 12LX 13所示的LCDR1，X 14ASX 15LX 16S所示的LCDR2，QQX 17YSX 18PX 19X 20T所示的LCDR3，其中，所述的X 1选自A或者Y，X 2选自S或者H，X 3为A或I，X 4选自I或者F，X 5选自S或者N，X 6选自G或者P，X 7选自Y或者S，X 8选自D或者Q，X 9选自S或者K，X 10选自V或者F，X 11选自K或者Q，X 12选自W或者Y，X 13选自A或者N，X 14选自D或者V，X 15选自N或者S，X 16选自E或者Q，X17选自Y或者S，X 18选自Y或者T，X 19选自M或者L，X 20选自I或者不存在。
在一优选方案中，其包括包含SEQ ID NO:35，36任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO:37，38，39任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO:40，41任一氨基酸序列的重链CDR3。在另一方面，本发明提供了结合CLL1的抗体或其片段，其包括包含SEQ ID NO:42，43，44任一氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:45，46，47，48任一氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:49，50任一氨基酸序列的轻链CDR3。在另一方面，本发明提供了结合CLL1的抗体或其片段，其包括包含SEQ ID NO:35，36任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO:37，38，39任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO:40，41任一氨基酸序列的重链CDR3，和/或包括包含SEQ ID NO: 42，43，44任一氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:45，46，47，48任一氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:49，50任一氨基酸序列的轻链CDR3。优选的，所述结合CLL1的抗体或其片段包含包括SEQ ID NO:35，36任一氨基酸序列的重链CDR1，和包含SEQ ID NO:37，38，39任一氨基酸序列的重链CDR2，和包含SEQ ID NO:40，41任一氨基酸序列的重链CDR3，和/或包含SEQ ID NO:42，43，44任一氨基酸序列的轻链CDR1，和包含SEQ ID NO:45，46，47，48任一氨基酸序列的轻链CDR2，和包含SEQ ID NO:49，50任一氨基酸序列的轻链CDR3。更优选的，所述结合CLL1的抗体或其片段包括包含SEQ ID NO:35，36任一氨基酸序列的重链CDR1，和包含SEQ ID NO:37，38，39任一氨基酸序列的重链CDR2，和包含SEQ ID NO:40，41任一氨基酸序列的重链CDR3，和包括包含SEQ ID NO:42，43，44任一氨基酸序列的轻链CDR1，和包含SEQ ID NO:45，46，47，48任一氨基酸序列的轻链CDR2，和包含SEQ ID NO:49，50任一氨基酸序列的轻链CDR3。
在另一方面，本发明提供了结合CLL1的抗体或其片段，其包括选自SEQ ID NO:1、5、13、67、68、69的重链可变区序列。
在另一方面，本发明提供了结合CLL1的抗体或其片段，其包括选自SEQ ID NO:3、7、11、15的轻链可变区序列。
考虑到这些重链和轻链可变区序列各自可以结合CLL1，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗CLL1的结合分子。
在另一个方面，本发明提供了结合CLL1的抗体或其片段的变体。因而本发明提供了抗体或其片段，具有与重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛选、点突变等方法得到。
在另一个方面，本发明提供了的抗体LCDR1发生了突变，由RASQSISSX 12LX 13突变为RASQWIARX 12LX 13，例如，突变后的LCDR1的序列如SEQ ID NO:44所示。
在另一个方面，本发明提供了的抗体HCDR2“X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G”的X 3X 4X 5X 6选自AISG或者IFNP。
在另一个方面，所述的抗体HCDR2发生了突变，由X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G突变为X 3X 4X 5X 6GGGSTX 7YAX 8X 9X 10X 11G，例如，突变后的HCDR2的序列如SEQ ID NO:39所示。
在另一个方面，可以针对本发明的抗体的重链可变区进行突变，如重链可变区由SEQ ID NO：13突变为SEQ ID NO：67、68或69。
在另一个方面，本发明提供了与前述的抗CLL1的抗体识别相同的抗原决定部位的抗体。
抗CLL1的抗体特性
评估抗体例如抗CLL1的抗体的结合能力的标准测定是本领域已知的，包括例如ELISA、biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。
核酸、载体和宿主细胞
本发明还提供了编码结合CLL1的抗体和其片段分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。
可以使用标准的分子生物学技术获得本发明的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变化。
优选的本发明核酸分子是编码重链可变区的选自SEQ ID NO:2,6,14的那些，和/或编码轻链可变区的选自SEQ ID NO:4,8,12,16的那些。更优选的是这样的核酸分子，所述核酸分子包含编码重链的SEQ ID NO:2序列，和包含编码轻链的SEQ ID NO:4序列或者包含编码重链的SEQ ID NO:6序列，和包含编码轻链的SEQ ID NO:8序列或者包含编码重链的SEQ ID NO:2序列，和包含编码轻链的SEQ ID NO:12序列或者包含编码重链的SEQ ID NO:14序列，和包含编码轻链的SEQ ID NO:16序列。
为了表达蛋白质，可以将编码本发明抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。用于本发明的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本发明中来表达抗体。
免疫辍合物
本发明还提供了多功能免疫缀合物，其包含本文所述抗体以及进一步包含至少一种其它类型的功能性分子。所述的功能性分子选自但不限于：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成辍合物。
作为一种优选方式，所述免疫缀合物可包含：本发明的抗体以及至少一种靶向肿瘤表面标志物的分子或抑制肿瘤的分子。所述的抑制肿瘤的分子可以是抗肿瘤的细胞因子，或抗肿瘤的毒素；较佳地，所述的细胞因子包括(但不限于)：IL-2、IL-7、IL-12、IL-15、I型IFN、TNF-alpha。在具体的实施方式中，所述的靶向肿瘤表面标志物的分子是靶向本发明抗体所靶向的相同肿瘤的表面标志物的分子。例如，所述的靶向肿瘤表面标志物的分子可以是结合肿瘤表面标志物的抗体或配体，例如可以与本发明的抗体协同作用，更精准地靶向肿瘤细胞。
作为一种优选方式，所述免疫缀合物可包含：本发明的抗体以及可检测标记物。所述 的可检测标记物包括但不限于：荧光标记物、显色标记物；如：酶、辅基、荧光材料、发光材料，生物发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子。也可包含一个以上的标记物。为了检测和/或分析和/或诊断目的用于标记抗体的标记依赖于使用的特定检测/分析/诊断技术和/或方法例如免疫组织化学染色(组织)样品、流式细胞计量术等。对于本领域已知的检测/分析/诊断技术和/或方法合适的标记为本领域技术人员所熟知。
作为一种优选方式，所述免疫缀合物可包含：本发明的抗体以及靶向免疫细胞的表面标志物的分子。所述靶向免疫细胞的表面标志物的分子可以是结合免疫细胞表面标志物的抗体或配体，能够识别免疫细胞，其携带本发明的抗体达到免疫细胞，同时本发明的抗体可将免疫细胞靶向于肿瘤细胞，从而引发免疫细胞特异性地杀伤肿瘤。所述的免疫细胞表面标志物可以选自CD3，CD16，CD28，更佳的，结合免疫细胞表面标志物的抗体是抗CD3抗体。免疫细胞可选自T细胞、NK细胞、NKT细胞。
作为通过直接或间接(例如通过接头)缀合而化学产生免疫缀合物的一种方式，所述免疫缀合物可以作为融合蛋白而产生，所述融合蛋白包含本发明的抗体及合适的其它蛋白。融合蛋白可以通过本领域已知方法产生，例如通过构建核酸分子以及随后表达所述核酸分子而重组产生，所述核酸分子包含符合读框的编码抗体的核苷酸序列以及编码合适标记的核苷酸序列
本发明另一方面提供了编码本发明的至少一种抗体、其功能变体或者免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
含有抗CLL1抗体的嵌合抗原受体
本发明还提供多种嵌合抗原受体(CAR)，其中包含本发明的抗体或抗体片段，该CAR-T细胞表现出抗肿瘤性质。一些实施案例中，用编码CAR的病毒载体转导细胞(例如T细胞)。在一些实施案例中，病毒载体是慢病毒载体。一些实施方案中，细胞可以稳定地表达CAR。
在一优选例中，CAR的CLL1结合部分是scFv抗体片段，与其所来自的IgG抗体相比，保持等价的亲和结合力，例如其以相当的功效结合相同抗原。该抗体片段是功能性的，由此其提供生物化学反应，例如激活免疫反应、抑制从其靶抗原的信号传导起始、抑制激酶活性等。因此，本发明提供，经工程化导入T细胞中的，包含WT1结合结构域的CLL1-CAR，以及将其用于过继免疫治疗的方法。
一方面，CAR的抗CLL1抗原结合结构域是，相对于其所源自的鼠序列scFv被人源化的scFv抗体片段。
一方面，本发明CAR将特定抗体的抗原结合结构域和胞内信号传导分子组合在一起。例如，一些方面，胞内信号传导分子包括但不限于，CD3ξ链、4-1BB和CD28信号传导模块 及其组合。
一方面，CLL1-CAR包含至少一个胞内信号传导结构域，其选择CD137(4-1BB)信号传导结构域、CD28信号传导结构域、CD3ξ信号结构域，及其任何组合。一方面，CLL1-CAR包含至少一个胞内信号传导结构域，其来自一个或多个非CD137(4-1BB)或CD28的共刺激分子。
作为示范性的，CLL1-CAR的序列可以是4F2 28Z(SEQ ID NO:55)、4F2 BBZ(SEQ ID NO:56)、4F2 28BBZ(SEQ ID NO:57)、25H8 28Z(SEQ ID NO:58)、25H8 BBZ(SEQ ID NO:59)、25H8 28BBZ(SEQ ID NO:60)、7F8 28Z(SEQ ID NO:61)、7F8 BBZ(SEQ ID NO:62)、7F8 28BBZ(SEQ ID NO:63)、12G4m 28Z(SEQ ID NO:64)、12G4m BBZ(SEQ ID NO:65)、12G4m 28BBZ(SEQ ID NO:66)、12G4m(M1331)28Z(SEQ ID NO:73)、12G4m(M1331)BBZ(SEQ ID NO:9)、12G4m(M1331)28BBZ(SEQ ID NO:10)，上述SEQ ID NO:55-66、SEQ ID NO:73、SEQ ID NO:9-10的跨膜域和胞内域本领域技术人员可以选择常规的跨膜域和胞内域进行替换，且均落入本申请的保护范围。
嵌合抗原受体修饰的T细胞
本发明还提供了包含有本发明所述的嵌合抗原受体的免疫细胞。
在另一方面，本发明提供的嵌合抗原受体修饰的T细胞其还携带外源的细胞因子的编码序列；较佳地，所述的细胞因子包括：IL-12，IL-15或IL-21。所述的免疫细胞优选自T淋巴细胞，NK细胞或NKT细胞。
在另一方面本发明提供的嵌合抗原受体修饰的T细胞其还携带PD-L1阻断剂或阻断PD-L1的蛋白，如天然PD-1，或能够与PD-L1结合的突变PD-1，或能够与PD-L1结合的天然或突变PD-1的片段、或抗PD-L1的抗体。
药物组合物
本发明的抗体、包含该抗体的免疫辍合物以及基因修饰的免疫细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物或免疫细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和 大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
本发明的优点：
1.本发明提供了针对CLL1的特异性抗体；
2.本发明的抗体对于CLL1具有较高的亲和力，且在制备成嵌合抗原受体修饰的T细胞后，对表达CLL1的细胞具有明显的杀伤作用。和
3.本发明提供了利用所述抗体制备的嵌合抗原受体修饰的T细胞，因此，本发明的抗体和免疫效应细胞能够安全有效地应用于急性髓系白血病的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编着，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1：使用全人噬菌体展示文库筛选针对CLL1特异的scFv
本发明使用的噬菌体展示文库为本公司构建的全人的天然的scFv噬菌体文库，库容为1E+11。利用本领域技术人员已知的筛选方法得到针对CLL1高度特异的scFv片段。简言之，分别包被10ug/ml抗原CLL1-huFc(由真核表达后采用protein A填料对培养上清进行亲和纯化制备而成)和人Fc段于免疫管。为了减少Fc段的影响，将噬菌体文库加入包被了人Fc段的免疫管中结合1hr。取上清加入包被CLL1-huFc的免疫管中结合1.5小时，随后将非特异性的噬菌体洗掉，将结合的噬菌体洗脱下来并感染对数生长期的大肠杆菌TG1。扩大培养洗脱下来得噬菌体并使用PEG/NaCl沉淀纯化扩大后的噬菌体文库用于下一轮的筛选。将淘选进行3-4个循环以富集与CLL1特异性结合的scFv噬菌体克隆。通过针对CLL1-huFc的标准ELISA方法确定阳性克隆，ELISA检测图谱，如图1所示。ELISA使用人Fc段做为无关抗原来验证抗体的特异性。
筛选后得到两个特异性结合的HL60和U937细胞(HL60/U937为CLL1阳性细胞，k562为CLL1阴性细胞)(图3)，克隆名称为4F2和25H8，4F2和25H8的Biacore图谱，如图2所示。经测序分析，4F2的重链可变区为SEQ ID NO:1所示的氨基酸序列，轻链可变区为SEQ ID NO:3所示的氨基酸序列；25H8的重链可变区为SEQ ID NO:5所示的氨基酸序列，轻链可变区为SEQ ID NO:7所示的氨基酸序列。
根据Kabat的EU索引分析，4F2的HCDR1的氨基酸序列如SEQ ID NO:35所示，HCDR2的氨基酸序列如SEQ ID NO:37所示，HCDR3的氨基酸序列如SEQ ID NO:40所示，LCDR1的氨基酸序列如SEQ ID NO:42所示，LCDR2的氨基酸序列如SEQ ID NO:45所示，LCDR3的氨基酸序列如SEQ ID NO:49所示。
25H8的HCDR1的氨基酸序列如SEQ ID NO:36所示，HCDR2的氨基酸序列如SEQ ID  NO:38所示，HCDR3的氨基酸序列如SEQ ID NO:41所示，LCDR1的氨基酸序列如SEQ ID NO:43所示，LCDR2的氨基酸序列如SEQ ID NO:46所示，LCDR3的氨基酸序列如SEQ ID NO:50所示。
实施例2：抗体的亲和力成熟
利用噬菌体展示技术进行亲和力成熟。
以4F2和25H8为亲本抗体，分别对轻链可变区的LCDR1和LCDR2进行随机突变，构建亲和力成熟的噬菌体文库。两轮筛选后挑选单克隆进行ELISA检测，并将阳性克隆进行测序和表达纯化，综合以上实验，共得到两个高亲和力且特异性结合人CLL1的抗体，命名7F8(来源于4F2)和12G4(来源于25H8)。
经测序分析，7F8的重链可变区为SEQ ID NO:1所示的氨基酸序列，轻链可变区为SEQ ID NO:11所示的氨基酸序列；12G4的重链可变区为SEQ ID NO:5所示的氨基酸序列，轻链可变区为SEQ ID NO:15所示的氨基酸序列。
7F8的的LCDR1的序列如SEQ ID NO:44所示，LCDR2的序列如SEQ ID NO:47所示，LCDR3及重链的CDR区序列与亲本抗体(4F2)一致。
12G4的LCDR1的序列与25H8相同，LCDR2的序列如SEQ ID NO:48所示，LCDR3及重链的CDR区序列与亲本抗体(25H8)一致。
其中12G4在重链的CDR2区含有一个糖基化位点，将其中的丝氨酸突变为甘氨酸后得到新的抗体，命名为12G4m，12G4m的HCDR2的序列如SEQ ID NO:39所示。
12G4m的重链可变区为SEQ ID NO:13所示的氨基酸序列，轻链可变区为SEQ ID NO:15所示的氨基酸序列。
通过ELISA检测7F8和12G4m的scF V与人CLL1的结合情况，显示有显著的结合(见图4)。
将7F8和12G4m的scFv进行Biacore检测，结果如图5所示，结果显示亲和力较亲本抗体提高了10倍左右)。
ELISA使用人Fc段做为无关抗原来验证抗体的特异性。抗体7F8和12G4m的scFv与CLL1表达阳性细胞特异性结合情况如图6所示，从图中看出抗体可以特异性结合阳性细胞，而不结合阴性细胞(HL60和U937为CLL1表达阳性细胞，k562为CLL1不表达阴性细胞)。
实施例3：ELISA检测7F8和12G4m抗体与不同种属CLL1的结合
通过标准的ELISA检测抗体7F8和12G4m的种属特异性。人CLL1-hFc，鼠CLL1-hFc，猴CLL1-hFc用1ug/ml于4度包被ELISA板过夜，2％MPBS室温封闭1-2小时，加入200nM抗体7F8/12G4m(scFv)室温孵育1小时，二抗采用抗His的鼠单抗1：1000稀释，三抗为羊抗鼠-HRP1:2000稀释使用，室温孵育1小时后显色，1M浓硫酸中止后读数OD450，图7。抗体7F8和12G4m只结合人CLL1，不结合鼠CLL1和猴CLL1。
实施例4：CLL1抗体scFv-Fc形式构建及其单体率检测
将抗CLL1抗体7F8和12G4m与人IgG的Fc段进行融合表达，通过293Fectin转染试剂转染293F细胞，第6天收集上清，采用Protein A填料进行亲和纯化，纯化后蛋白通过GE XK16/40空层析柱，收集单体峰，并进行单体率检测，其中抗体7F8的单体率为99.30％，抗体12G4m的单体率为58.50％，结果见图8。
抗体7F8蛋白用截流量为10KD的millipore超滤管进行浓缩，通过OD280/消光系数测浓度，结果见表1；取样跑SDS-PAGE，结果见图9。
表1
Figure PCTCN2019113767-appb-000001
对12G4m进行降聚改造。通过Discovery studio软件建立12G4m的3D模型，分析其中的潜在的聚集位点，通过软件预测，对这些位点进行点突变，将突变后的抗体与人IgG的Fc段进行融合，以scFv-Fc形式，通过293F细胞进行表达，表达产物纯化后通过SEC进行单体率检测，结果显示，得到3个单体率明显优于12G4m的突变体，分别为：将重链可变区第9位丙氨酸突变为脯氨酸(12G4m-A9P)，将第93位缬氨酸突变为异亮氨酸(12G4m-V93I)，将第113位甲硫氨酸突变为亮氨酸(12G4m-M133I)，其中，12G4m-M133I的单体率达到了95％以上。
12G4m-A9P、12G4m-V93I和12G4m-M133I的突变位点均发生在重链可变区，其重链可变区的序列分别如SEQ ID NO:67、68、69所示。
12G4m-A9P的scFv序列如SEQ ID NO:70所示，12G4m-V93I的scFv序列如SEQ ID NO:71所示，12G4m-M133I的scFv序列如SEQ ID NO:72所示。
实施例5：利用FACs测定抗体与U937细胞的结合EC50
利用实施例4获得的scFv-Fc融合蛋白，通过FACs检测抗CLL1抗体的EC50，具体做法如下：将U937细胞以1×10 5个/孔加入96孔U型底板中，用FcR blocking试剂4度封闭30分钟，用300uL/孔1％FBS清洗细胞两次，随后加入梯度稀释的100μL CLL1抗体7F8，12G4m，4F2，25H8(以1000nmol/mL为起始浓度，5倍稀释，7个浓度梯度)，混合后于4℃孵育45分钟，离心除去上清液，用300uL/孔1％FBS清洗细胞两次。将FITC标记Goat anti human Fc(JacksonImmunoResearch，Code number:109-095-098)用1％FBS稀释200倍添加到细胞中，于4℃孵育45分钟后，进行离心操作，除去上清液，用300μL/孔1％FBS清洗细胞两次。最终以200μL/孔1％FBS重悬细胞并用于流式细胞仪检测。如图10所示，抗人CLL1抗体7F8、12G4m、4F2和25H8的EC50值分别为0.06474nM、0.4271nM、0.7370nM和0.1638nM，其EC50值均小于1nM，表现出很好的细胞杀伤效果。
实施例6：抗CLL1嵌合抗原受体质粒(CAR)的构建
a.抗CLL1抗体7F8嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α(购自Addgene)为载体，构建了表达抗体7F8的二代嵌合抗原受体的慢病毒质粒，包括PRRLSIN-cPPT.EF-1α-7F8-28Z、PRRLSIN-cPPT.EF-1α-7F8-BBZ以及PRRLSIN-cPPT.EF-1α-7F8-28BBZ。。
7F8-28Z序列由CD8α信号肽(SEQ ID NO:17)、7F8scFV(SEQ ID NO:31)、CD8 hinge(SEQ ID NO:19)、CD28跨膜区(SEQ ID NO:51)和胞内信号传导结构域(SEQ ID NO:53)以及CD3ξ(SEQ ID NO:25)组成；7F8-BBZ序列由CD8α信号肽(SEQ ID NO:17)、7F8scFV(SEQ ID NO:31)、CD8 hinge(SEQ ID NO:19)和跨膜区(SEQ ID NO:21)、CD137胞内信号传导结构域(SEQ ID NO:23)以及CD3ξ(SEQ ID NO:25)组成；7F8-28BBZ序列由CD8α信号肽(SEQ ID NO:17)、7F8scFV(SEQ ID NO:31)、CD8 hinge(SEQ ID NO:19)、CD28跨膜区(SEQ ID NO:51)和胞内信号传导结构域(SEQ ID NO:53)、CD137胞内信号传导结构域(SEQ ID NO:23)以及CD3ξ(SEQ ID NO:25)组成。
b.抗CLL1抗体12G4m嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达抗体12G4m的二代嵌合抗原受体的慢病毒质粒，包括PRRLSIN-cPPT.EF-1α-12G4m-28Z、PRRLSIN-cPPT.EF-1α-12G4m-BBZ以及PRRLSIN-cPPT.EF-1α-12G4m-28BBZ。
12G4m-28Z序列由CD8α信号肽(SEQ ID NO:17)、12G4m scFV(SEQ ID NO:33)、CD8 hinge(SEQ ID NO:19)、CD28跨膜区(SEQ ID NO:51)和胞内信号传导结构域(SEQ ID NO:53)以及CD3ξ(SEQ ID NO:25)组成；12G4m-BBZ序列由CD8α信号肽(SEQ ID NO:17)、12G4m scFV(SEQ ID NO:33)、CD8 hinge(SEQ ID NO:19)和跨膜区(SEQ ID NO:21)、CD137胞内信号传导结构域(SEQ ID NO:23)以及CD3ξ(SEQ ID NO:25)组成；12G4m-28BBZ序列由CD8α信号肽(SEQ ID NO:17)、12G4m scFV(SEQ ID NO:33)、CD8 hinge(SEQ ID NO:19)、CD137胞内信号传导结构域(SEQ ID NO:23)以及CD3ξ(SEQ ID NO:25)组成。
c.抗CLL1抗体12G4m-M1331(在本文中又写作12G4m(M1331))嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达抗体12G4m-M1331的二代嵌合抗原受体的慢病毒质粒即PRRLSIN-cPPT.EF-1α-12G4m(M1331)-BBZ。12G4m(M1331)-28Z序列由CD8α信号肽(SEQ ID NO:17)、12G4m(M1331)scFV(SEQ ID NO:72)、CD8 hinge(SEQ ID NO:19)、CD28跨膜区(SEQ ID NO:51)和胞内信号传导结构域(SEQ ID NO:53)以及CD3ξ(SEQ ID NO:25)组成；12G4m(M1331)-BBZ序列由CD8α信号肽(SEQ ID NO:17)、12G4m(M1331)scFV(SEQ ID NO:72)、CD8 hinge(SEQ ID NO:19)和跨膜区(SEQ ID NO:21)、CD137胞内信号传导结构域(SEQ ID NO:23)以及CD3ξ(SEQ ID NO:25)组成；12G4m(M1331)-28BBZ序列由CD8α信号肽(SEQ ID NO:17)、12G4m(M1331)scFV(SEQ ID NO:72)、CD8 hinge(SEQ ID NO:19)、CD137胞内信号传导结构域(SEQ ID NO:23)以及CD3ξ(SEQ ID NO:25)组成。
实施例7：CLL1特异性CAR病毒包装以及感染CAR T阳性率的检测
首先用磷酸钙法进行慢病毒的包装，病毒上清用PEG8000/NaCl进行纯化，纯化后对病毒用FACS法进行滴度检测，根据流式细胞仪检测结果，7F8-CART、12G4m-CART和12G4m-M1331 CART病毒滴度分别为2.37E+08、1.41E+08和7.99E+07。
随后用病毒感染CD3/CD28磁珠活化48小时后的T细胞，加入病毒体积(mL)＝细胞数x MOI值(MOI＝10)/滴度，得到7F8-BBZ CAR T、12G4m-BBZ CAR T和12G4m(M1331)-BBZ-CART细胞，感染后第9天采用FACS方法检测CAR表达阳性率，结果见图11，7F8-BBZ CAR T的CAR阳性率为41.94％，12G4m-BBZ CAR T的CAR阳性率为60.14％，12G4m(M1331)-BBZ CAR T的CAR阳性率为55.64％。
实施例8：靶向CLL1阳性细胞的体外杀伤毒性检测
选择现有技术中报道的抗体M26(CN104736562B)作为阳性对照，采用常规的方案制备了M26-BBZ CAR T。采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。具体方法参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
效应细胞：按效靶比3:1、1:1、1:3及1:9分别接种Untransduced(UTD)T细胞、7F8-BBZ CART细胞、12G4m-BBZ CART细胞于96孔板。
靶细胞：分别接种50μL 2×10 5/mL的CLL1表达阳性的HL-60与U937细胞以及CLL1表达阴性的K562细胞到相应的96孔板。
实验结果如图12所示，本发明的7F8-BBZ CAR T、12G4m–BBZ CAR T与对照组UTD相比，对CLL1表达阳性的HL-60与U937细胞在效靶比为3:1、1:1和1:3时均有显著的毒性杀伤作用，而对CLL1表达阴性的K562细胞则几乎没有杀伤作用。此外，12G4m–BBZ CAR T对CLL1表达阳性的HL-60与U937细胞，均显示出比现有技术M26-BBZ CAR T有更好的杀伤作用，而7F8-BBZ CAR T对HL-60杀伤作用也优于M26-BBZ CAR T。
实施例9：小鼠体内细胞杀伤实验
1)实验分组：将6-8周龄雌性NPG小鼠随机分为4组，每组6只，分别为Untransduced T(UTD)、12G4m-BBZ CART组(1.0×10 6/只小鼠)、12G4m-BBZ CART组(2.0×10 6/只小鼠)、12G4m-BBZ CART(4.0×10 6/只小鼠)细胞治疗组。
2)皮下移植瘤的接种：收集对数生长期且生长状态良好的HL-60细胞，调整细胞密度为1.5×10 7/mL，接种于NPG小鼠右侧腋部皮下部位，接种体积200μL细胞悬液，即每只小鼠接种3.0×10 6的肿瘤细胞，接种日记为第0天。
3)CAR-T细胞回输：当肿瘤平均体积约为122mm 3时，即接种肿瘤后第8天，注射相应数量的12G4m BBZ细胞或未经转导的T细胞对照。结果显示，CAR T注射后14天，与UTD对照组相比，各组均见明显抑瘤效果，12G4m-BBZ细胞浓度为2.0×10 6时，肿瘤抑制率为57.35％；12G4m-BBZ细胞浓度为4.0×10 6时，肿瘤抑制率为72.89％。同时小鼠体重与UTD对照组相比无显著变化。
实施例10：12G4m(M1331)-BBZ CART的体外杀伤结果
参照实施例8的方法，检测12G4m(M1331)-BBZ CART的体外杀伤效果。
效应细胞：按效靶比9:1、3:1及1:1分别接种Untransduced(UTD)T细胞、7F8-BBZ CART细胞、12G4m-BBZ CART细胞、12G4m(M1331)-BBZ CART细胞于96孔板。
靶细胞：分别接种50μL0 2×10 5/mL的CLL1表达阳性的HL-60与U937细胞以及CLL1表达阴性的K562细胞到相应的96孔板。
实验结果如图13所示，实验结果如图13所示，结果表明，本发明的7F8-BBZ CAR T、12G4m–BBZ CAR T、12G4m(M1331)-BBZ CART与对照组UTD相比，对CLL1表达阳性的HL-60与U937细胞均有显著的毒性杀伤作用，而对CLL1表达阴性的K562细胞则几乎没有杀伤作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明所用的序列总结于下表：
Figure PCTCN2019113767-appb-000002
Figure PCTCN2019113767-appb-000003
Figure PCTCN2019113767-appb-000004
Figure PCTCN2019113767-appb-000005
Figure PCTCN2019113767-appb-000006
Figure PCTCN2019113767-appb-000007
Figure PCTCN2019113767-appb-000008

Claims (31)

  1. 靶向CLL1的抗体，其特征在于，所述抗体具有：
    SYX 1MX 2所示的HCDR1，X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G所示的HCDR2，SEQ ID NO:40或41所示的HCDR3，及RASQSISSX 12LX 13所示的LCDR1，X 14ASX 15LX 16S所示的LCDR2，QQX 17YSX 18PX 19X 20T所示的LCDR3，
    其中，所述的X 1选自A或者Y，X 2选自S或者H，X 3为A或I，X 4选自I或者F，X 5选自S或者N，X 6选自G或者P，X 7选自Y或者S，X 8选自D或者Q，X 9选自S或者K，X 10选自V或者F，X 11选自K或者Q，X 12选自W或者Y，X 13选自A或者N，X 14选自D或者V，X 15选自N或者S，X 16选自E或者Q，X 17选自Y或者S，X 18选自Y或者T，X 19选自M或者L，X 20为I或者不存在。
  2. 根据权利要求1所述的抗体，其特征在于，所述抗体选自以下任一组：
    (1)抗体，其重链可变区包含SEQ ID NO:35或36所示的HCDR1，和/或包含SEQ ID NO:37、38或39所示的HCDR2，和/或包含SEQ ID NO:40或41所示的HCDR3；
    (2)抗体，其轻链可变区包含SEQ ID NO:42、43或44所示的LCDR1，和/或包含SEQ ID NO:45、46、47或48所示的LCDR2，和/或包含SEQ ID NO:49或50所示的LCDR3；
    (3)抗体，包含(1)所述的重链可变区及(2)所述的轻链可变区；
    (4)(1)～(3)中任一项所述的抗体的变体，其具有与(1)～(3)中任一项所述的抗体相同或相似的活性。
  3. 根据权利要求1所述的抗体，其特征在于，所述的抗体LCDR1发生了突变，由RASQSISSX 12LX 13突变为RASQWIARX 12LX 13；
    优选的，所述突变后的LCDR1的序列如SEQ ID NO:44所示。
  4. 根据权利要求1所述的抗体，其特征在于，所述的抗体HCDR2的X 3X 4X 5X 6为AISG或者IFNP。
  5. 根据权利要求1或4所述的抗体，其特征在于，所述的抗体HCDR2发生了突变，由X 3X 4X 5X 6SGGSTX 7YAX 8X 9X 10X 11G突变为X 3X 4X 5X 6GGGSTX 7YAX 8X 9X 10X 11G；
    优选的，所述突变后的HCDR2的序列如SEQ ID NO:39所示。
  6. 根据权利要求2所述的抗体，其特征在于，所述的抗体选自以下任一组：
    (1)抗体，其包含SEQ ID NO:35所示的HCDR1、SEQ ID NO:37所示的HCDR2、SEQ ID NO:40所示的HCDR3，以及SEQ ID NO:42所示的LCDR1、SEQ ID NO:45所示的LCDR2、SEQ ID NO:49所示的LCDR3；
    (2)抗体，其包含SEQ ID NO:36所示的HCDR1、SEQ ID NO:38所示的HCDR2、SEQ ID NO:41所示的HCDR3，以及SEQ ID NO:43所示的LCDR1、SEQ ID NO:46所示的LCDR2、SEQ ID NO:50所示的LCDR3；
    (3)抗体，其包含SEQ ID NO:35所示的HCDR1、SEQ ID NO:37所示的HCDR2、SEQ ID NO:40所示的HCDR3，以及SEQ ID NO:44所示的LCDR1、SEQ ID NO:47所示的LCDR2、SEQ ID NO:49所示的LCDR3；
    (4)抗体，其包含SEQ ID NO:36所示的HCDR1、SEQ ID NO:38所示的HCDR2、SEQ ID  NO:41所示的HCDR3以及SEQ ID NO:43所示的LCDR1、SEQ ID NO:48所示的LCDR2、SEQ ID NO:50所示的LCDR3；
    (5)抗体，其包含SEQ ID NO:36所示的HCDR1、SEQ ID NO:39所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:43所示的LCDR1、SEQ ID NO:48所示的LCDR2、SEQ ID NO:50所示的LCDR3；
    (6)抗体，(1)～(5)中任一项所述的抗体的变体，且具备与(1)～(5)中任一项所述的抗体相同或相似的活性。
  7. 根据权利要求2所述的抗体，其特征在于，所述的抗体选自以下任一组：
    (1)抗体，所述的抗体的重链可变区具有SEQ ID NO:1所示的氨基酸序列、SEQ ID NO:5所示的氨基酸序列、SEQ ID NO:13所示的氨基酸序列、SEQ ID NO:67所示的氨基酸序列、SEQ ID NO:68所示的氨基酸序列、SEQ ID NO:69所示的氨基酸序列；
    (2)抗体，所述的抗体的轻链可变区具有SEQ ID NO:3所示的氨基酸序列、SEQ ID NO:7所示的氨基酸序列、SEQ ID NO:11所示的氨基酸序列、SEQ ID NO:15所示的氨基酸序列；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
    (4)(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
  8. 根据权利要求7所述的抗体，其特征在于，所述的抗体选自：
    (1)抗体，所述的抗体的重链可变区具有SEQ ID NO:1所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:3所示的氨基酸序列；
    (2)抗体，所述的抗体的重链可变区具有SEQ ID NO:5所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:7所示的氨基酸序列；
    (3)抗体，所述的抗体的重链可变区具有SEQ ID NO:1所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:11所示的氨基酸序列；
    (4)抗体，所述的抗体的重链可变区具有SEQ ID NO:13所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:15所示的氨基酸序列；
    (5)抗体，所述的抗体的重链可变区具有SEQ ID NO:5所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO:15所示的氨基酸序列；
    (6)(1)～(5)中任一项所述的抗体的变体，且具备(1)～(5)中任一项所述的抗体相同或相似的活性。
  9. 根据权利要求2所述的抗体，其特征在于，所述抗体的轻链可变区具有SEQ ID NO:43所示的LCDR1，SEQ ID NO:48所示的LCDR2，SEQ ID NO:50所示的LCDR3；
    优选地，所述轻链可变区具有SEQ ID NO:15所示的氨基酸序列；
    更优选地，所述抗体的轻链可变区具有SEQ ID NO:15所示的氨基酸序列，所述的抗体的重链可变区具有SEQ ID NO:5、13、67、68或69所示的氨基酸序列。
  10. 抗体，与权利要求1-9中任一项所述的抗体识别相同的抗原决定部位。
  11. 根据权利要求1-10任一项所述的抗体，其特征在于，所述抗体是全人抗体。
  12. 根据权利要求1-10任一项所述的抗体，其特征在于，所述抗体为全抗、scFv、单域抗体、Fab片段、Fv片段、F(ab’) 2片段。
  13. 编码权利要求1-12中任一项所述的抗体的核酸。
  14. 一种表达载体，其包含权利要求13所述的核酸。
  15. 一种宿主细胞，其包含权利要求14所述的表达载体或基因组中整合有权利要求13所述的核酸。
  16. 权利要求1-12任一项所述的抗体的用途，用于制备治疗肿瘤的药物，或制备诊断肿瘤的试剂。
  17. 一种免疫辍合物，其特征在于，所述的免疫辍合物包括：
    权利要求1-12任一项所述的抗体，以及与之连接的功能性分子；
    所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子，和可检测标记物。
  18. 根据权利要求17所述的免疫辍合物，其特征在于，其中，所述的抑制肿瘤的分子为抗肿瘤的细胞因子或抗肿瘤的毒素。
  19. 根据权利要求17所述的免疫辍合物，其特征在于，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体或配体。
  20. 根据权利要求19所述的免疫辍合物，其特征在于，所述的靶向免疫细胞的表面标志物的分子是结合T细胞、NK细胞或NKT细胞表面标志物的抗体。
  21. 编码权利要求17-20任一项所述的免疫辍合物的核酸。
  22. 嵌合抗原受体，其特征在于，所述嵌合抗原受体包含权利要求1-12任一项所述的抗体。
  23. 根据权利要求22所述的嵌合抗原受体，其特征在于，所述抗体为单链抗体或单域抗体。
  24. 编码权利要求22所述的嵌合抗原受体的核酸。
  25. 一种表达载体，其特征在于，其包含权利要求24所述的核酸。
  26. 一种病毒，其特征在于，所述的病毒包含权利要求25所述载体。
  27. 一种基因修饰的免疫细胞，其特征在于，其转导有权利要求24所述的核酸，或其表达有权利要求22所述的嵌合抗原受体。
  28. 如权利要求27所述的基因修饰的免疫细胞，其特征在于，所述免疫细胞还表达有除所述的嵌合抗原受体之外的第二序列。
  29. 如权利要求28所述的免疫细胞，其特征在于，所述第二序列包括细胞因子、或另一种嵌合抗原受体、或趋化因子受体、或降低PD-1表达的siRNA、或阻断PD-L1的蛋白、或TCR、或安全开关。
  30. 权利要求28所述的基因修饰的免疫细胞的用途，用于制备治疗肿瘤的药物。。
  31. 药物组合物，其特征在于，其包括：
    权利要求1-12中任一项所述的抗体或编码该抗体的核酸；或
    权利要求17-20中任一项所述的免疫辍合物或编码该辍合物的核酸；或
    权利要求27-29任一项所述的基因修饰的免疫细胞。
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WO2022074206A1 (en) 2020-10-08 2022-04-14 Affimed Gmbh Trispecific binders
US20220354890A1 (en) * 2020-12-11 2022-11-10 Guangchao LI Chimeric antigen receptor targeting cll1 and use thereof
WO2022214089A1 (zh) 2021-04-08 2022-10-13 克莱格医学有限公司 细胞免疫治疗的应用
WO2023007023A1 (en) 2021-07-30 2023-02-02 Affimed Gmbh Duplexbodies
WO2023078968A1 (en) 2021-11-03 2023-05-11 Affimed Gmbh Bispecific cd16a binders

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