WO2018233333A9 - 一种靶向人肿瘤干细胞的单克隆抗体及其应用 - Google Patents

一种靶向人肿瘤干细胞的单克隆抗体及其应用 Download PDF

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WO2018233333A9
WO2018233333A9 PCT/CN2018/079785 CN2018079785W WO2018233333A9 WO 2018233333 A9 WO2018233333 A9 WO 2018233333A9 CN 2018079785 W CN2018079785 W CN 2018079785W WO 2018233333 A9 WO2018233333 A9 WO 2018233333A9
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monoclonal antibody
cells
seq
amino acid
hetumomab
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PCT/CN2018/079785
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French (fr)
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WO2018233333A1 (zh
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沈敏
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苏州博聚华生物医药科技有限公司
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Priority claimed from CN201710485170.7A external-priority patent/CN107474139B/zh
Priority claimed from CN201710484849.4A external-priority patent/CN107163146B/zh
Priority claimed from CN201710485302.6A external-priority patent/CN107118276B/zh
Application filed by 苏州博聚华生物医药科技有限公司 filed Critical 苏州博聚华生物医药科技有限公司
Priority to US16/626,060 priority Critical patent/US20210147571A1/en
Priority to CA3068338A priority patent/CA3068338A1/en
Priority to JP2020520696A priority patent/JP2020524527A/ja
Priority to EP18820564.5A priority patent/EP3650469A4/en
Publication of WO2018233333A1 publication Critical patent/WO2018233333A1/zh
Publication of WO2018233333A9 publication Critical patent/WO2018233333A9/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/808—Materials and products related to genetic engineering or hybrid or fused cell technology, e.g. hybridoma, monoclonal products
    • Y10S530/809—Fused cells, e.g. hybridoma

Definitions

  • the invention relates to the field of biomedicine. Specifically, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof directed to human tumor stem cells, and the use of the antibody or fragment in tumor treatment and diagnosis.
  • Malignant tumors have become the "number one killer” that threatens the lives and health of people around the world. Every year, the number of tumors in the world exceeds 14 million, and in China alone, more than 3 million new cancer patients are added each year.
  • the underlying cause of high cancer mortality is the spread, metastasis of cancer cells, and most patients are prone to relapse and drug resistance after treatment.
  • the existing clinical treatment methods, surgery, radiation therapy, chemotherapy have little effect on cancer cell metastasis, recurrence, drug resistance, or only have short-term effects, which cannot change the long-term survival of patients.
  • surgical resection is effective for about 10-20% of early patients, but it is almost ineffective for patients who have spread and metastasized.
  • Radiotherapy can only treat local lesions, and is often used as an adjuvant treatment before and after surgery and as a radical treatment for a few types of cancer.
  • Chemotherapy can be used in patients with spread and metastasis, but because of its large toxic side effects, it is easy to produce short-term or long-term drug resistance, so it can only have obvious short-term effects on about 20-30% of patients. Even with the combined treatment of surgery, radiotherapy and chemotherapy, the long-term efficacy of 5 years of survival has been hovering at 20-30% for many years, and about 70-80% of patients have metastasis, recurrence and drug resistance after Died during the year. Even in patients with early cancers who did not have metastases at the time of consultation, some patients died of metastasis and recurrence after treatment.
  • New targeted tumor drugs developed in recent years including peptides, small molecules, protein factors, gene therapy, and antibody drugs, combined with chemotherapeutic drugs can usually only extend the survival of patients by 3-9 months compared to existing treatments There was no significant improvement in the 5-year survival rate for long-term patients.
  • Emerging tumor immunotherapy methods in the past two years such as PD-1 monoclonal antibody drugs for immune checkpoints and CAR-T cell therapy, have shown some promising long-term effects, but overall The effective rate of cancer patients can only reach about 20-30%, and there are still a large number of cancer patients who do not receive treatment with truly effective drugs. Therefore, the key to improving the long-term efficacy and prolonging the survival of cancer patients is to develop new drugs that inhibit tumor metastasis, recurrence and drug resistance.
  • the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof directed against a tumor stem cell, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,
  • the light chain variable region includes:
  • VLCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 2;
  • VLCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 3, and
  • VLCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 4;
  • the heavy chain variable region includes:
  • VH CDR1 which comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 6;
  • VHCDR2 which contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 7, such as the sequence shown in SEQ ID NO: 13, and
  • VHCDR3 which contains the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 8 such as the sequence shown in SEQ ID NO: 14.
  • the monoclonal antibody comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region includes:
  • VL CDR1 which contains the amino acid sequence shown in SEQ ID NO: 2,
  • VLCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3, and
  • VL CDR3 which contains the amino acid sequence shown in SEQ ID NO: 4;
  • the heavy chain variable region includes:
  • VH CDR1 which contains the amino acid sequence shown in SEQ ID NO: 6,
  • VH CDR2 which contains the amino acid sequence shown in SEQ ID NO: 13, and
  • VH CDR3 which contains the amino acid sequence shown in SEQ ID NO: 8.
  • the monoclonal antibody comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region includes:
  • VL CDR1 which contains the amino acid sequence shown in SEQ ID NO: 2,
  • VLCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3, and
  • VL CDR3 which contains the amino acid sequence shown in SEQ ID NO: 4;
  • the heavy chain variable region includes:
  • VH CDR1 which contains the amino acid sequence shown in SEQ ID NO: 6,
  • VH CDR2 which contains the amino acid sequence shown in SEQ ID NO: 7, and
  • VH CDR3 which contains the amino acid sequence shown in SEQ ID NO: 14.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid having at least 85%, at least 90%, at least 95%, or higher sequence identity with SEQ ID NO: 1. sequence.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 5. sequence.
  • the heavy chain variable region comprises the amino acid sequence shown in one of SEQ ID NOs: 15-19 (the variable regions corresponding to the humanized heavy chain versions H1-H5, respectively).
  • the light chain variable region comprises the amino acid sequence shown in one of SEQ ID NO: 20-31 (the variable regions corresponding to the humanized light chain versions L1-L12, respectively).
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30.
  • the monoclonal antibody comprises a human heavy chain constant region, for example, a human heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 11.
  • the monoclonal antibody comprises a human light chain constant region, for example, a human light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 12.
  • the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 9 and a light chain having the amino acid sequence shown in SEQ ID NO: 10.
  • the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in one of SEQ ID NO: 32-36 (corresponding to the humanized heavy chain version H1-H5, respectively) and having SEQ ID ID NO: 37- The light chain of the amino acid sequence shown in one of 48 (corresponding to the humanized light chain versions L1-L12, respectively).
  • the monoclonal antibody comprises a heavy chain shown in SEQ ID NO: 36 and a light chain shown in SEQ ID NO: 47 (corresponding to the humanized antibody H5L11).
  • the monoclonal antibody comprises a heavy chain shown in SEQ ID NO: 36 and a light chain shown in SEQ ID NO: 42 (corresponding to the humanized antibody H5L6).
  • the present invention provides a monoclonal antibody or an antigen-binding fragment thereof, which was deposited with the common microorganisms of the China Microbial Strain Collection Management Committee as the deposit number CGMCC No. 12251 on March 16, 2016. Central mouse hybridoma cells are produced.
  • the present invention provides a hybridoma cell, which was deposited at the General Microbiology Center of the China Microbial Strain Collection and Management Commission under the accession number CGMCC No. 12251 on March 16, 2016.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the monoclonal antibody or antigen-binding fragment thereof is conjugated to a therapeutic moiety selected from a cytotoxin, a radioisotope, or a biologically active protein.
  • the invention provides a method of treating a malignancy, preventing and / or treating malignant tumor metastasis or recurrence in a patient, said method comprising administering to said patient an effective amount of a monoclonal antibody of the invention or Its antigen-binding fragment or the pharmaceutical composition of the present invention.
  • the malignancy is selected from breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer, and gastric cancer.
  • the method further comprises administering to the patient another anti-tumor treatment, such as administering a chemotherapeutic agent, an antibody that targets other tumor-specific antigens, or radiation therapy.
  • another anti-tumor treatment such as administering a chemotherapeutic agent, an antibody that targets other tumor-specific antigens, or radiation therapy.
  • the present invention provides the use of a monoclonal antibody of the present invention or an antigen-binding fragment thereof or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating a malignant tumor, preventing and / or treating a metastatic or recurring malignant tumor .
  • the malignancy is selected from breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer, and gastric cancer.
  • the invention provides a method for detecting the presence of tumor stem cells in a biological sample, including:
  • the invention also provides a method for isolating tumor stem cells, the method comprising:
  • the tumor stem cells are selected from breast cancer stem cells, colorectal cancer stem cells, pancreatic cancer stem cells, prostate cancer stem cells, liver cancer stem cells, lung cancer stem cells, and gastric cancer stem cells.
  • the invention also provides a method for detecting the presence of a malignant tumor in a patient, comprising:
  • the present invention also provides a method for prognostic malignancy recurrence or progression in a patient, the method comprising:
  • prognosis is recurrence or progression of a malignancy in said patient.
  • the progression of the malignancy comprises metastasis of the malignancy in a patient.
  • the biological sample includes a blood sample, a lymph sample, or a component thereof.
  • the malignancy is selected from breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer, and gastric cancer.
  • the invention also provides an isolated nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof of the invention.
  • the nucleic acid molecule is operably linked to an expression control sequence.
  • the invention also provides an expression vector comprising a nucleic acid molecule of the invention.
  • the invention also provides a host cell transformed with a nucleic acid molecule of the invention or an expression vector of the invention.
  • the present invention also provides a method for producing a monoclonal antibody or an antigen-binding fragment thereof against human tumor stem cells, comprising:
  • FIG. Live cell immunofluorescence technology to detect the expression of monoclonal antibody Hetumomab target antigen on the surface of living cells of various tumor cells (some typical positive results).
  • FIG. 1 Immune flow cytometric detection. Cancer cells recognized by monoclonal antibody Hetumomab were significantly enriched in sphere cultured cells in a variety of human tumor cell lines (some typical flow cytometry).
  • FIG. 4 CCK8 method for detecting the drug resistance (IC50) of Hetumomab + cells of various human tumor cells (such as liver cancer, lung cancer, and gastric cancer) recognized by monoclonal antibody Hetumomab.
  • Monoclonal antibody Hetumomab significantly inhibits the self-renewal ability (spheroidization) of tumor stem cells in various tumors (such as liver cancer, lung cancer, and gastric cancer).
  • Monoclonal antibody Hetumomab significantly inhibits the invasion ability of tumor stem cells in various tumors (such as liver cancer, lung cancer, and gastric cancer).
  • Figure 8 In vivo tumor growth curve of monoclonal antibody Hetumomab and combined chemotherapy drugs in the treatment of human liver cancer transplantation tumor Be17402-V13.
  • Figure 9 In vivo tumor volume inhibition rate of human hepatocellular carcinoma be17402-V13 treated with monoclonal antibody Hetumomab and combined chemotherapy drugs (at the time of discontinuation).
  • Figure 10 In vivo tumor volume inhibition rate of monoclonal antibody Hetumomab and combined chemotherapy drugs in the treatment of human liver cancer xenograft Be17402-V13 (one month after drug withdrawal).
  • Figure 12 In vivo tumor growth curve of monoclonal antibody Hetumomab in the treatment of human lung cancer xenografts SPCA-1.
  • Figure 14 Shows that the chimeric antibody Hetuximab binds to the same antigen protein on tumor stem cells as the parent antibody Hetumomab.
  • Figure 15 Shows that the chimeric antibody Hetuximab competes with the parent antibody Hetumomab for antigen binding.
  • FIG. 16 Amino acid sequence alignments (parts) of Hetumomab humanized heavy chain versions are shown. The corresponding abbreviation for Hetumomab is E21.
  • FIG. 1 Electrophoretic analysis showing heterogeneity in glycosylation of H1 heavy chain series of expressions.
  • Figure 20 Shows that humanized antibody H5L11 with a purity greater than 95% was expressed and purified in CHO cells.
  • Figure 21 shows that the parental mouse monoclonal antibody Hetumomab, the chimeric antibody Hetuximab, and the humanized monoclonal antibody Hetuzumab compete for binding to the antigen.
  • A Different concentrations of parental mouse monoclonal antibody Hetumomab compete against three variants of Hetuzumab (H5L6, H5L11, H5L12), and the chimeric antibody Hetuximab binds to the antigen
  • B Three variants of Hetuzumab (H5L6, H5L11, H5L12) The chimeric antibody Hetuximab competes to inhibit the binding of the parental mouse monoclonal antibody Hetumomab to the antigen.
  • FIG. 22 Tumor growth curve in mice with monoclonal antibody Hetuzumab H5L11 and combined chemotherapy drugs in human liver cancer transplanted tumors.
  • antibody refers to immunoglobulins and immunoglobulin fragments, whether produced naturally or partially or wholly synthetically (e.g., recombinantly), including retained full-length immunity that includes at least a portion of the variable region of an immunoglobulin molecule Any fragment of the binding specificity of globulin.
  • antibodies include any protein that has a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site).
  • Antibodies include antibody fragments, such as anti-tumor stem cell antibody fragments.
  • the term antibody therefore includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments.
  • multispecific antibodies e.g., bispecific antibodies
  • human antibodies e.g., human antibodies
  • non-human antibodies humanized antibodies
  • chimeric antibodies intracellular antibodies
  • antibody fragments Such as, but not limited to, Fab fragments, Fab 'fragments, F (ab') 2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd 'fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabody, anti-idiotypic (anti-Id) antibody, or antigen-binding fragment of any of the above antibodies.
  • Antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses (e.g., IgG2a And IgG2b).
  • immunoglobulin type e.g., Ig., IgG, IgM, IgD, IgE, IgA, and IgY
  • any class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
  • subclasses e.g., IgG2a And IgG2b.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than the full-length, but contains at least a portion of a variable region (e.g., one or more) of the antibody that binds the antigen CDRs and / or one or more antibody binding sites), and thus retains the binding specificity and at least a portion of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived.
  • Antibody fragments include antibody derivatives produced by enzymatic processing of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd 'fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may include multiple chains linked together, for example by disulfide bonds and / or by peptide linkers.
  • Antibody fragments typically contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • An antigen-binding fragment includes any antibody fragment that, when inserted into an antibody framework (e.g., by replacing a corresponding region), obtains an antibody that specifically binds (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M -1 ) antigen .
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is the same as the other antibody molecules. This property is in contrast to the properties of a polyclonal population of antibodies, which contains antibodies with multiple different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117) .
  • monoclonal antibodies can be made by immortalizing B cells, for example by fusing with myeloma cells to produce a hybridoma cell line or by infecting B cells with a virus such as EBV.
  • Recombinant technology can also be used to prepare antibodies from a cloned population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibodies.
  • hybridomas refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by fusing antibody-producing lymphocytes and antibody-free cancer cells.
  • hybridomas can proliferate and are continuously supplied to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988).
  • hybridodoma or “hybridoma cell”
  • it also includes subclones and progeny cells of the hybridoma.
  • a "conventional antibody” refers to an antibody comprising two heavy chains (which can be labeled H and H ') and two light chains (which can be labeled L and L') and two antigen-binding sites, wherein Each heavy chain can be a full-length immunoglobulin heavy chain or any of its functional regions that retain antigen-binding capacity (e.g., heavy chains include, but are not limited to, V H chain, V H -C H 1 chain, and V H -C H 1- C H 2-C H 3 chain), and each light chain may be a full-length light chains, or any functional areas (e.g., but not limited to a light chain V L and V L -C L chain strand). Each heavy chain (H and H ') is paired with a light chain (L and L', respectively).
  • a full-length antibody is one having two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4 ) And two full-length light chain (V L -C L ) and hinge region antibodies, such as antibodies produced naturally by antibody-secreting B cells and synthetically produced antibodies with the same domain.
  • dsFv refers to Fv with an engineered intermolecular disulfide bond that stabilizes the V H -V L pair.
  • a Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment having the same structure produced, for example, by recombinant methods.
  • the Fab fragment contains a light chain (including V L and C L ) and another chain containing the variable domain (V H ) of the heavy chain and one constant region domain (C H 1) of the heavy chain.
  • an F (ab ') 2 fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure produced, for example, by recombinant methods.
  • the F (ab ') 2 fragment basically contains two Fab fragments, where each heavy chain portion contains several additional amino acids, including cysteine that forms a disulfide bond that connects the two fragments.
  • a Fab 'fragment is a fragment that contains half (one heavy and one light chain) of a F (ab') 2 fragment.
  • a scFv fragment refers to an antibody fragment comprising a variable light chain (V L ) and a variable heavy chain (V H ) covalently linked in any order by a polypeptide linker.
  • the linker length allows the two variable domains to bridge substantially undisturbed.
  • An exemplary linker is a (Gly-Ser) n residue dispersed with some Glu or Lys residues to increase solubility.
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as where the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody Antibodies.
  • a “humanized” antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab ', F (ab') 2 or Other antigen-binding subsequences of antibodies) contain minimal sequences derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody), wherein the residues of the complementarity determining region (CDR) of the recipient antibody are derived from a non-human species with the desired specificity, affinity and ability ( Donor antibody) such as mouse, rat or rabbit CDR residue replacement.
  • CDR complementarity determining region
  • the humanized antibody according to the present invention also encompasses antibodies that contain 1 or 2 amino acid mutations in the CDR.
  • epitope refers to any epitope on an antigen to which a paratope of an antibody binds.
  • Epitope determinants typically include chemically active surface typing of molecules, such as amino acid or sugar side chains, and often have specific three-dimensional structural characteristics and specific charge characteristics.
  • variable domain or variable region is a specific Ig domain of an antibody's heavy or light chain, which contains an amino acid sequence that varies between different antibodies.
  • Each light chain and each heavy chain has a variable domain domain, V L and V H, respectively .
  • the variable domain provides antigen specificity and is therefore responsible for antigen recognition.
  • Each variable region contains a CDR and a framework region (FR), which is part of an antigen binding site domain.
  • antigen-binding domain and "antigen-binding site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with a cognate antigen.
  • Natural conventional full-length antibody molecules have two conventional antigen-binding sites, each containing a heavy chain variable region portion and a light chain variable region portion.
  • Conventional antigen binding sites include loops that connect antiparallel beta chains within a variable region domain.
  • the antigen binding site may contain other parts of the variable region domain.
  • Each conventional antigen binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. Hypervariable regions are also called complementarity determining regions (CDRs).
  • variable region domain contains three CDRs, named CDR1, CDR2, and CDR3.
  • the light chain variable region domain contains three CDRs named VLCDR1, VLCDR2 and VLCDR3; the heavy chain variable region domain contains three CDRs named VHCDR1, VHCDR2 and VHCDR3.
  • the three CDRs in the variable region are discontinuous along the linear amino acid sequence, but are close in the folded polypeptide.
  • the CDRs are located within a loop of a ⁇ -sheet parallel strand that connects the variable domains.
  • those skilled in the art know and can identify CDRs based on Kabat or Chothia numbers (see, for example, Kabat, EAetal. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917).
  • the framework region is a domain within the variable region domain of an antibody located within the beta fold; the FR region is relatively more conserved than the hypervariable region in terms of amino acid sequence.
  • a "constant region” domain is a domain in the heavy or light chain of an antibody that contains an amino acid sequence that is relatively more conserved than the amino acid sequence of a variable region domain.
  • each light chain has a single light chain constant region (CL) domain
  • each heavy chain contains one or more heavy chain constant region (C H ) domains, including C H 1, C H 2, C H 3 and C H 4.
  • Full-length IgA, IgD and IgG isotypes comprising C H 1, C H 2, C H 3 and the hinge region, but contains IgE and IgM C H 1, C H 2, C H 3 and C H 4.
  • the C H 1 and C L domains extend the Fab arm of the antibody molecule and therefore help interact with the antigen and rotate the antibody arm.
  • the constant region of an antibody can serve effector functions such as, but not limited to, the elimination of antigens, pathogens, and toxins to which the antibody specifically binds, for example, by interacting with various cells, biomolecules, and tissues.
  • a functional region of an antibody comprising the antibody of at least V H, V L, C H ( e.g. C H 1, C H 2 or C H 3), C L or a hinge region domain or at least a ribbon Antibody section.
  • the ribbon V H domain is intact V H domain binding specificity at least a portion (e.g., by reserving one or more CDR domains of the complete V H) at least a portion of the complete V H domain,
  • the functional region of the V H domain binds the antigen alone or in combination with another antibody domain (eg, a V L domain) or a region thereof.
  • Exemplary functional regions of the V H domain comprising CDR1 V H domain, region CDR2 and / or CDR3, respectively.
  • the ribbon V L domains are intact V L domain binding specificity at least a portion (e.g. by intact V L domains of one or more CDR) at least a portion of the intact V L domain, whereby said ribbon V L domain, or a region alone or in combination with another antibody antigen binding domain (e.g., V H domain).
  • exemplary functional regions V L domain comprising a CDR1 V L domain, region CDR2 and / or CDR3, respectively.
  • telomere binding As used herein, "specific binding” or “immunospecific binding” with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the passage of an antibody or antigen-binding fragment between the antibody and the antibody's antibody-binding site The ability of non-covalent interactions to form one or more non-covalent bonds with the same antigen.
  • the antigen may be an isolated antigen or present in a tumor cell.
  • an antibody that specifically binds (or specifically binds) an antigen is an affinity constant Ka (or 1 x 10 -7 M or 1 x 10 7 M -1 or 1 x 10 8 M -1 or more).
  • Ka affinity constant
  • K d dissociation constant
  • Affinity constants can be determined by standard kinetic methods of antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), Isothermal Titration Calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also US Pat. No. 7,229,619, which describes an exemplary SPR and ITC method for calculating the binding affinity of antibodies.
  • SPR surface plasmon resonance
  • ITC Isothermal Titration Calorimetry
  • the term "competing" with respect to an antibody means that the first antibody or antigen-binding fragment thereof binds an epitope in a manner sufficiently similar to the second antibody or antigen-binding fragment thereof, whereby the first antibody is associated with the epitope.
  • the binding result is detectably reduced in the presence of the second antibody compared to the absence of the second antibody.
  • the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, this can be, but need not be, the case. That is, the first antibody can inhibit the binding of the second antibody to its epitope without using the second antibody to inhibit the binding of the first antibody to its respective epitope.
  • each antibody can detectably inhibit the binding of another antibody to its cognate epitope or ligand, whether to the same, higher or lower degree, the antibodies are said to "cross-compete" with each other for binding Their respective epitopes.
  • Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (such as steric hindrance, conformational changes, or binding to a common epitope or fragment thereof), those skilled in the art will be aware of such competition and / or cross-competitive antibodies based on the teachings provided by the present invention It is encompassed by the present invention and can be used in the methods disclosed by the present invention.
  • polypeptide refers to two or more amino acids covalently linked.
  • polypeptide and protein are used interchangeably herein.
  • isolated protein means that the protein, polypeptide or antibody (1) is not associated with its naturally associated components in its natural state, and (2) does not contain the same species Other proteins, (3) are expressed by cells from different species, or (4) do not occur in nature.
  • a chemically synthesized polypeptide or a polypeptide synthesized in a cell system other than the naturally-derived cell of the polypeptide will be “isolated” from its naturally-related components. It is also possible to isolate the protein so that it is substantially free of naturally related components, even using protein purification techniques well known in the art.
  • Suitable conservative amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule.
  • those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub .co., p. 224).
  • polynucleotide and “nucleic acid molecule” refer to oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, including those that are usually linked together by phosphodiester bonds.
  • DNA Deoxyribonucleic acid
  • RNA ribonucleic acid
  • an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibodies or antigen-binding fragments.
  • Sequence "identity” has art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule.
  • identity is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM, Applied Math. 48: 1073 (1988 )).
  • operably linked with respect to a nucleic acid sequence, region, element or domain means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
  • expression refers to the process by which a polypeptide is produced by the transcription and translation of a polynucleotide.
  • the expression level of a polypeptide can be evaluated using any method known in the art, including, for example, a method of determining the amount of a polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate, and expand a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • Codon optimization refers to replacing at least one codon of a natural sequence (e.g., about or more than about 1, 2, 3, 4, 5 by replacing codons more frequently or most frequently with genes in a host cell). , 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence while modifying the nucleic acid sequence to enhance expression in the host cell of interest. Different species have certain codes for specific amino acids Exhibit specific preferences. Codon preference (differences in codon usage between organisms) is often related to the translation efficiency of messenger RNA (mRNA), which is believed to be dependent on the codons being translated Properties and availability of specific transport RNA (tRNA) molecules. The advantages of selected tRNAs in cells generally reflect the codons most frequently used for peptide synthesis.
  • mRNA messenger RNA
  • tRNA transport RNA
  • genes can be tailored to codon-based optimization in a given organism the optimal gene expression codon usage table can be easily obtained, for example, in www.kazusa.orjp / codon / available on the codon usage database ( "codon usage database") in, and These tables may be adjusted in different ways applicable See, Nakamura Y. et, "Codon usage tabulated from the international DNA sequence databases:.. Status for the year2000.Nucl.Acids Res, 28: 292 (2000).
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell.
  • Concerning vectors include those in which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, usually by restriction digestion and ligation. Also included with respect to vectors are those comprising a nucleic acid encoding a polypeptide.
  • a vector is used to introduce a nucleic acid encoding a polypeptide into a host cell, to amplify a nucleic acid, or to express / display a polypeptide encoded by a nucleic acid.
  • Vectors generally remain episomal, but can be designed to integrate genes or parts of them into the genome's chromosomes.
  • Artificial chromosome vectors are also considered, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • vectors also include “viral vectors” or “viral vectors”.
  • a viral vector is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region, that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, and expression vectors that remain free of expression vectors or are integrated into the host cell genome.
  • treating means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment.
  • treatment includes prevention, treatment and / or cure.
  • Prevention refers to preventing underlying diseases and / or preventing worsening of symptoms or development of the disease.
  • Treatment also includes any of the antibodies or antigen-binding fragments thereof provided and any pharmaceutical use of the compositions provided herein.
  • effect refers to an effect resulting from the treatment of an individual that alters, generally ameliorates or improves the symptoms of a disease or disease state, or cures a disease or disease state.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to the amount of a substance, compound, material, or composition containing a compound that, when administered to a subject, has a desired preventive effect, for example, to prevent or delay a disease or symptom The occurrence or recurrence of the disease reduces the possibility of the occurrence or recurrence of the disease or symptoms.
  • a complete prophylactic effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
  • the term "patient” refers to a mammal, such as a human.
  • Tumor stem cells refer to a small portion of cancer cells with stem characteristics that exist in tumor tissues, which have self-renewal ability, strong invasion ability, tolerance to chemotherapy drugs, and strong tumorigenic ability compared with ordinary cancer cells.
  • a human tumor pluripotent stem cell line is used as an immunogen to immunize mice, and a monoclonal antibody Hetumomab is obtained by classical hybridoma fusion technology.
  • the mouse hybridoma cell line Hetumomab that produces the monoclonal antibody Hetumomab was deposited on March 16, 2016 under the accession number CGMCC No. 12251 at the General Microbial Center of the China Microbial Species Collection Management Committee. (Example 1)
  • the present invention provides a monoclonal antibody or an antigen-binding fragment thereof, which was deposited at the General Microbiology Center of the China Microbial Species Collection Management Committee under the accession number CGMCC No. 12251 on March 16, 2016. Murine hybridoma cells are produced.
  • the target antigen of the monoclonal antibody Hetumomab of the present invention is expressed on the surface of living cells of a variety of human tumor cells, and is specifically and highly expressed in a variety of tumor tissues (positive rate 79% -94%).
  • the Hetumomab monoclonal antibody of the present invention can be enriched in sphere culture cells of a variety of tumor cells and can recognize tumor stem cell markers such as ESA, CD90 and other tumor cells, suggesting that Hetumomab monoclonal antibodies are monoclonal antibodies specific to tumor stem cells (implementation Example 2).
  • Hetumomab target antigen-positive tumor cells have stronger self-renewal, invasion, drug resistance, and tumorigenicity in vivo than parent tumor cells and Hetumomab target antigen-negative tumor cells. It was demonstrated that Hetumomab mAb specifically targets tumor stem cells (Example 3).
  • the present invention further identified the light chain variable region and heavy chain variable region sequences of Hetumomab mAb and the corresponding CDR sequences (Example 6).
  • a human-mouse chimeric antibody Hetuximab was constructed by combining the variable region sequences with human light and heavy chain constant regions, respectively (Example 7).
  • the chimeric antibody Hetuximab and mouse antibody Hetumomab bind the same epitope on tumor stem cells and have similar pharmacodynamic effects on inhibiting tumor stem cells (Examples 8-9).
  • the present invention further humanized the Hetumomab monoclonal antibody to obtain various variants of the humanized antibody Hetuzumab (Example 10).
  • the humanized antibody Hetuzumab and the mouse antibody Hetumomab and the chimeric antibody Hetuximab bind the same epitope on tumor stem cells and have similar pharmacodynamic effects on inhibiting tumor stem cells (Examples 11-12).
  • the present invention provides an isolated monoclonal antibody or an antigen-binding fragment thereof directed against a tumor stem cell, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,
  • the light chain variable region includes:
  • VLCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 2;
  • VLCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 3, and
  • VLCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 4;
  • the heavy chain variable region includes:
  • VH CDR1 which comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 6;
  • VHCDR2 which contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 7, such as the sequence shown in SEQ ID NO: 13, and
  • VHCDR3 which contains the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions to SEQ ID NO: 8 such as the sequence shown in SEQ ID NO: 14.
  • the monoclonal antibody comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region includes:
  • VL CDR1 which contains the amino acid sequence shown in SEQ ID NO: 2,
  • VLCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3, and
  • VL CDR3 which contains the amino acid sequence shown in SEQ ID NO: 4;
  • the heavy chain variable region includes:
  • VH CDR1 which contains the amino acid sequence shown in SEQ ID NO: 6,
  • VH CDR2 which contains the amino acid sequence shown in SEQ ID NO: 13, and
  • VH CDR3 which contains the amino acid sequence shown in SEQ ID NO: 8.
  • the monoclonal antibody comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region includes:
  • VL CDR1 which contains the amino acid sequence shown in SEQ ID NO: 2,
  • VLCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3, and
  • VL CDR3 which contains the amino acid sequence shown in SEQ ID NO: 4;
  • the heavy chain variable region includes:
  • VH CDR1 which contains the amino acid sequence shown in SEQ ID NO: 6,
  • VH CDR2 which contains the amino acid sequence shown in SEQ ID NO: 7, and
  • VH CDR3 which contains the amino acid sequence shown in SEQ ID NO: 14.
  • the monoclonal antibody is a humanized antibody.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 1. sequence. In some embodiments, the light chain variable region comprises about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about Amino acid sequence with 97%, about 98%, or about 99% sequence identity.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 5. sequence. In some embodiments, the heavy chain variable region comprises SEQ ID NO: 5 with about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about Amino acid sequence with 97%, about 98%, or about 99% sequence identity.
  • the heavy chain variable region comprises the amino acid sequence shown in one of SEQ ID NOs: 15-19 (the variable regions corresponding to the humanized heavy chain versions H1-H5, respectively).
  • the light chain variable region comprises the amino acid sequence shown in one of SEQ ID NO: 20-31 (the variable regions corresponding to the humanized light chain versions L1-L12, respectively).
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30.
  • the monoclonal antibody comprises a human heavy chain constant region, for example, a human heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 11.
  • the monoclonal antibody comprises a human light chain constant region, for example, a human light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 12.
  • the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in one of SEQ ID NO: 32-36 (corresponding to the humanized heavy chain version H1-H5, respectively) and having SEQ ID ID NO: 37- The light chain of the amino acid sequence shown in one of 48 (corresponding to the humanized light chain versions L1-L12, respectively).
  • the monoclonal antibody comprises a heavy chain shown in SEQ ID NO: 36 and a light chain shown in SEQ ID NO: 47 (corresponding to the humanized antibody H5L11).
  • the monoclonal antibody comprises a heavy chain shown in SEQ ID NO: 36 and a light chain shown in SEQ ID NO: 42 (corresponding to the humanized antibody H5L6).
  • variable region has a glycosylation site
  • antibody molecules glycosylated isomers
  • Different glycosylated forms of antibody molecules may show different pharmacokinetic and pharmacodynamic effects in humans. Therefore, antibodies with different glycosylated forms are difficult to be approved for marketing by drug approval agencies in various countries. Eliminating the excess glycosylation site of the variable region will greatly facilitate the application of monoclonal antibodies in the preparation of human drugs.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention does not contain a glycosylation site, or the glycosylation site of the antibody is removed by mutation.
  • the heavy chain variable region shown in SEQ ID NO: 19 and the glycosylation site in the heavy chain shown in SEQ ID NO: 36 have been removed by mutation, and are therefore preferred in the present invention.
  • an isolated monoclonal antibody or antigen-binding fragment thereof of the invention is derived from Hetumomab. In some embodiments, an isolated monoclonal antibody or antigen-binding fragment thereof of the invention binds to the same antigen on tumor stem cells as Hetumomab. In some embodiments, an isolated monoclonal antibody or antigen-binding fragment thereof of the invention binds to the same epitope on a tumor stem cell as Hetumomab. In some embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof of the invention compete with Hetumomab for binding to tumor stem cells.
  • an isolated monoclonal antibody or antigen-binding fragment thereof of the invention specifically targets tumor stem cells.
  • the tumor stem cells specifically targeted by the isolated monoclonal antibodies or antigen-binding fragments thereof of the present invention include, but are not limited to, breast cancer stem cells, colorectal cancer stem cells, pancreatic cancer stem cells, prostate cancer stem cells, liver cancer stem cells, lung cancer stem cells, and gastric cancer stem cells.
  • the invention also encompasses an isolated monoclonal antibody or antigen-binding fragment thereof that binds to the same antigen on tumor stem cells as Hetumomab.
  • the invention also encompasses an isolated monoclonal antibody or antigen-binding fragment thereof that binds to the same epitope on a tumor stem cell as Hetumomab.
  • the invention also encompasses isolated monoclonal antibodies or antigen-binding fragments thereof that compete with Hetumomab for binding to tumor stem cells.
  • the tumor stem cells include, but are not limited to, breast cancer stem cells, colorectal cancer stem cells, pancreatic cancer stem cells, prostate cancer stem cells, liver cancer stem cells, lung cancer stem cells, and gastric cancer stem cells.
  • the present invention provides an isolated nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof of the present invention.
  • the nucleotide sequence of the nucleic acid molecule is codon optimized for the host cell used for expression.
  • a nucleic acid molecule of the invention is operably linked to an expression control sequence.
  • the invention also provides an expression vector comprising at least one of the aforementioned nucleic acid molecules of the invention.
  • the invention also provides a host cell transformed with at least one of the aforementioned nucleic acid molecules or expression vectors of the invention.
  • the invention provides a method for producing an antibody or antigen-binding fragment thereof of the invention, comprising:
  • the present invention also relates to the isolated antibody or antigen-binding fragment thereof obtained by the method of the present invention, which can specifically target tumor stem cells.
  • Tumor stem cells are a small number of cancer cells with stem characteristics that exist in tumor tissues. They have the following biological characteristics: they can self-renew, replicate, non-directional differentiation, high tumorigenicity, high ability to invade, spread and metastasize. Are not sensitive. Due to the existence of tumor stem cells, tumors continue to grow rapidly, spread, metastasize, and recur. What is more serious is that tumor stem cells are resistant to almost all traditional chemotherapy drugs, radiotherapy and targeted drugs (including antibody-targeted drugs) that have been marketed in recent years. Tumor stem cells are in a non-growth, non-proliferative state in the G0 phase of the cell cycle. Chemoradiation only affects cancer cells that are growing rapidly and proliferating, but cannot kill tumor stem cells in the G0 stage.
  • tumor stem cells resistant to radiotherapy and chemotherapy are screened and enriched, and the proportion is greatly increased.
  • tumor stem cells have a strong ability to self-replicate and spread and metastasize, these tumor stem cells will rapidly differentiate and proliferate, grow and spread, metastasize to various organs throughout the body to form new metastatic lesions, and the cancer cells of this metastatic lesion are resistant to chemoradiotherapy
  • the existence of tumor stem cells has been proven in breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer, gastric cancer and other malignant tumors. Cancers with a higher degree of malignancy have more tumor stem cells, and cancer patients with a higher proportion of tumor stem cells have more metastases, recurrences, and shorter survival times.
  • the present inventors found that the monoclonal antibody specifically recognizing tumor stem cells of the present invention can significantly inhibit the self-renewal, invasion, and drug resistance of a variety of tumor stem cells in vitro (Example 4). Further experiments show that the monoclonal antibody specifically recognizing tumor stem cells of the present invention can inhibit the growth, metastasis and drug resistance of various tumor xenografts in animal models (Example 5). Therefore, the monoclonal antibodies of the present invention can be used to treat malignant tumors, prevent or / or treat malignant tumor metastasis or recurrence by targeting tumor stem cells.
  • the present invention provides a method for treating a malignant tumor, preventing or / or treating malignant tumor metastasis or recurrence in a patient, the method comprising administering to the patient an effective amount of the antibody against tumor stem cells of the present invention or Its antigen-binding fragment.
  • Malignant tumors that can be treated and / or prevented by the methods of the present invention include, but are not limited to, breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer, and gastric cancer.
  • the present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention against tumor stem cells and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is used to treat a malignancy in a patient, prevent or / or treat malignant tumor metastasis or recurrence.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).
  • the active compound i.e., antibody molecule, immunoconjugate
  • the active compound can be encapsulated in a material to protect the compound from the effects of acids and other natural conditions that can inactivate the compound.
  • the pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc .; (2) oil-soluble antioxidants, such as palmitate ascorbic acid Esters, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, etc .; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA ), Sorbitol, tartaric acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc .
  • oil-soluble antioxidants such as palmitate ascorbic acid Esters, butylated
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the prevention of the presence of microorganisms can be ensured by sterilization procedures or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid and the like.
  • various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide in the composition.
  • Prolonged absorption of injectable drugs can be achieved by adding delayed absorption agents, such as monostearate and gelatin, to the composition.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the temporary preparation of sterile injectable solutions or dispersions.
  • sterile aqueous solutions or dispersions and powders for the temporary preparation of sterile injectable solutions or dispersions.
  • Conventional media or agents, other than to the extent incompatible with the active compound, are possible in the pharmaceutical composition of the invention.
  • Supplementary active compounds can also be incorporated into the composition.
  • compositions must generally be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.
  • the carrier can be a solvent or dispersant containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • proper fluidity can be maintained by using a coating, such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant.
  • a sterile injection solution can be prepared by mixing the active compound into a suitable solvent in a required amount, and adding one or a combination of the ingredients listed above as necessary, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred method of preparation is vacuum drying and freeze-drying (lyophilization), from which a previously sterile-filtered solution gives a powder of the active ingredient plus any additional required ingredients.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form is generally the amount of the composition which produces a therapeutic effect. Generally, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient, based on 100%, and pharmaceutically acceptable Combination of vectors.
  • the dosage regimen can be adjusted to provide the best desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as required by the emergency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions into dosage unit forms that are easy to administer and uniformly dosed.
  • the dosage unit form used herein refers to a physical discontinuous unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined amount of the active compound, which is calculated by combining the predetermined amount of the active compound with the required pharmaceutical carrier Desired therapeutic effect.
  • dosage unit form of the present invention is limited to and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) inherent in the art for formulating such sensitivities for treating individuals Restrictions on active compounds.
  • the dosage range is from about 0.0001 to 100 mg / kg, and more typically from 0.01 to 20 mg / kg of the recipient's body weight.
  • the dose can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, or 20 mg / kg body weight, or in the range of 1-20 mg / kg body weight.
  • Exemplary treatment regimens require dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or the initial dosing interval Slightly shorter (such as once a week to once every three weeks) and the interval of late administration is prolonged (such as once a month to once every 3-6 months).
  • antibody molecules directed against tumor stem cells can be administered as a sustained release formulation, in which case less frequent administration is required.
  • the dose and frequency will vary depending on the half-life of the antibody molecule in the patient.
  • human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies.
  • the dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are given at less frequent intervals over a long period of time. Some patients continue to be treated for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient shows partial or complete improvement in the symptoms of the disease. Patients can then be administered in a prophylactic regime.
  • the actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be changed to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response to a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention applied, the route of administration, the time of administration, the excretion rate of the particular compound applied, the duration of treatment, and the particular application Other drugs, compounds and / or materials in combination with the composition, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.
  • an "effective amount" of an antibody or antigen-binding fragment thereof of the present invention preferably results in a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of injury or disability due to the pain of the disease.
  • an "effective amount" of an antibody or antigen-binding fragment thereof of the present invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more than by no treatment. It is preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and still more preferably at least about 80%.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy in human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, and such inhibition can be determined in vitro by tests known to those skilled in the art.
  • An effective amount of an antibody or antigen-binding fragment thereof of the invention is capable of reducing tumor size, or otherwise alleviating a subject's symptoms such as preventing and / or treating metastasis or recurrence.
  • One skilled in the art can determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.
  • the antibody of the present invention or an antigen-binding fragment thereof or the pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or mode of administration will vary depending on the desired result. Preferred routes of administration of the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually injections, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions.
  • the antibody against the tumor stem cells of the present invention or an antigen-binding fragment thereof or the pharmaceutical composition of the present invention can also be administered by a parenteral route, such as a topical, epidermal, or mucosal route, for example, Vaginal, rectal, sublingual or local.
  • a parenteral route such as a topical, epidermal, or mucosal route, for example, Vaginal, rectal, sublingual or local.
  • the active compound can be prepared with carriers that will protect the compound against rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems.
  • controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the therapeutic composition can be administered using medical devices known in the art.
  • the therapeutic composition of the present invention can be administered using a needle-free subcutaneous injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • a needle-free subcutaneous injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • Examples of well-known implants and modules that can be used in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispersing drugs at a controlled rate; U.S. Patent No. 4,486,194, which The patent discloses a therapeutic device for administration through the skin; U.S. Patent
  • antibodies to tumor stem cells of the invention can be formulated to ensure proper distribution in the body.
  • the blood-brain barrier (BBB) prevents many highly hydrophilic compounds.
  • BBB blood-brain barrier
  • the therapeutic compounds of the invention can be formulated in, for example, liposomes.
  • liposomes For methods of making liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548 and 5,399,331. Liposomes contain one or more targeting moieties that can be selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29: 685 ).
  • targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); Mannoside (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PGBloeman et al. ( 1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273.
  • the antibody or antigen-binding fragment thereof of the present invention against tumor stem cells in the pharmaceutical composition may also be conjugated with a therapeutic moiety such as a cytotoxin, a radioisotope, or a biologically active protein.
  • a cytotoxin includes any agent that is harmful to the cells (e.g., kills cells). Examples include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, turpentine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thiophene glycoside, vincristine, vinblastine Base, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, radixomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, prolux Caine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs.
  • Therapeutic agents that can be used for conjugation also include, for example: antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, amimidinamine), alkylating agents (E.g.
  • cyclophosphamide busulfan, dibromomannitol, chain Pazomycin, Mitomycin C, and Cis-Dichlorodiamine Platinum (II) (DDP) Cisplatin), Anthromycins (for example, daunorubicin (formerly known as daunorubicin), and Amycin), antibiotics (e.g., actinomycin D (formerly known as actinomycin), bleomycin, bryomycin, and antramycin (AMC)), and antimitotic agents (e.g., vinpocetin And vinblastine).
  • daunorubicin formerly known as daunorubicin
  • AMC antramycin
  • antimitotic agents e.g., vinpocetin And vinblastine.
  • Cytotoxins can be conjugated to antibodies against tumor stem cells of the invention using linker technology used in the art.
  • linkers that have been used to conjugate cytotoxins to antibodies against tumor stem cells include, but are not limited to, hydrazone, thioethers, esters, disulfides, and peptide-containing linkers.
  • a linker in the lysosomal compartment that is easily cleaved by low pH or by a protease, such as a protease that is preferentially expressed in tumor tissues, such as cathepsins (e.g. cathepsins B, C, D) .
  • the antibody against tumor stem cells of the present invention can also be conjugated with a radioisotope to produce a cytotoxic radiopharmaceutical, which is also called a radioactive antibody conjugate.
  • a radioisotope that can be conjugated with antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90, and thallium 177. Methods for making radioactive antibody conjugates have been established in the art.
  • the antibody against tumor stem cells of the present invention can also be conjugated with a protein having the required biological activity and can be used to modify a specific biological response.
  • biologically active proteins include, for example: toxins or active fragments thereof having enzymatic activity, such as acacia toxin, ricin A, Pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon - ⁇ ; or biological response modifiers, such as lymphokine, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), interleukin-10 (“IL-10”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other immune factors such as IFN and the like.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • IL-10 interleukin-10
  • the antibody or pharmaceutical composition for tumor stem cells of the present invention can be administered in combination with a chemotherapeutic agent or an antibody that targets other tumor antigens.
  • Example 5 of the present application proves that the combination of the monoclonal antibody and the chemotherapeutic agent of the present invention has a synergistic effect in the treatment of tumors. Without being limited by any theory, it is believed that the antibody against tumor stem cells of the present invention can inhibit the drug resistance function of the tumor, and thus can achieve a synergistic effect when combined with a chemotherapeutic agent or an antibody targeting other tumor antigens.
  • a chemotherapeutic agent or an antibody that targets other tumor antigens that can be used in combination with the antibody of the present invention or the pharmaceutical composition of the present invention is not particularly limited.
  • the chemotherapeutic agent and antibodies that target other tumor antigens include, but are not limited to: ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enolta Bin, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, deoxyfluridine, nerabine, hydroxyurea, Fluorouracil, fludarabine, pemetrexed, pentamidine, thiopurine, methotrexate, irinotecan, etoposide, eribulin, sobutrazon, docetaxel, paclitaxel, vinca Ru
  • the antibodies of the present invention and the chemotherapeutic agents or antibodies targeting other tumor antigens may be administered all at once or separately. When applied separately (in the case of mutually different application schedules), they can be applied continuously without interruption or at predetermined intervals.
  • the combined dose of the antibody of the present invention and the chemotherapeutic agent or the antibody targeting other tumor antigens in the pharmaceutical composition of the present invention is not particularly limited. As described above, the dose of the antibody of the present invention can be determined by referring to the dose when the antibody is used alone. The chemotherapeutic agent and antibodies targeting other tumor antigens may be used according to the doses indicated by the respective drugs or may be reduced (considering the combined effect with the antibodies of the present invention).
  • the antibody of the invention or the pharmaceutical composition of the invention may also be combined with radiation therapy, for example, including the administration of ionizing radiation to a patient earlier, during, and / or later than the administration of the antibody or pharmaceutical composition of the invention.
  • the monoclonal antibodies of the invention specifically recognize tumor stem cells. Therefore, the present invention also provides a method for detecting the presence of tumor stem cells in a biological sample, including:
  • the tumor stem cells are selected from breast cancer stem cells, colorectal cancer stem cells, pancreatic cancer stem cells, prostate cancer stem cells, liver cancer stem cells, lung cancer stem cells, and gastric cancer stem cells.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention is further conjugated with a fluorescent dye, a chemical substance, a polypeptide, an enzyme, an isotope, which can be used for detection or can be detected by other reagents, Tags, etc.
  • the invention also provides a method for isolating tumor stem cells, the method comprising:
  • tumor stem cells can be isolated by flow cytometry.
  • the target antigen of the monoclonal antibody of the present invention is expressed on the living cell surface of a variety of human tumor cells, and is specifically and highly expressed in a variety of tumor tissues (positive rate 79% -94%).
  • the present invention also provides a method for detecting the presence of a malignant tumor in a patient, including:
  • the invention also provides a method for recurring or progressing a malignant tumor in a prognostic patient, the method comprising:
  • prognosis is recurrence or progression of a malignancy in said patient.
  • the progression of the malignancy comprises metastasis of the malignancy in a patient.
  • the presence of circulating cells identified to bind the monoclonal antibodies of the invention or antigen-binding fragments thereof is indicative of a high risk of recurrence or progression of a malignancy in said patient.
  • the biological sample includes a blood sample, a lymph sample, or a component thereof.
  • the malignancy is selected from breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, lung cancer, and gastric cancer.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention is further conjugated with a fluorescent dye, chemical substance, polypeptide, enzyme, isotope, tag that can be used for detection or can be detected by other reagents. Wait.
  • kit for use in the method of the present invention includes the monoclonal antibody or antigen-binding fragment thereof of the present invention, and instructions for use.
  • the kit may further include at least one additional detection reagent for detecting the presence of a monoclonal antibody of the invention.
  • a kit typically includes a label indicating the intended use and / or method of use of the contents of the kit.
  • the term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • a human tumor pluripotent stem cell line T3A-A3 is used as an immunogen (Liu H, et al. Cell Death and Disease. 2013, 4: e857).
  • the human tumor stem cell line is isolated from liver cancer tissues surgically removed from patients with primary liver cancer, and can be subcultured in vitro for a long time. .
  • the cell line has been passaged more than 100 times, and the cells still grow rapidly and retain stem cell properties.
  • This cell line not only expresses a variety of stem cell markers, has the ability to self-renew stem cells, has the potential to differentiate into different tumor cells, but also has the properties of tumors, and has the ability to form tumors and metastases.
  • the cell line has been cultured for a long time in vitro without changing its properties, and has strong tumorigenicity and metastasis ability in immunodeficient mice.
  • the liver cancer stem cell (human tumor pluripotent stem cell) line T3A-A3 obtained by expansion culture was fixed with paraformaldehyde and immunized ordinary Balb / c mice, once every 2-4 weeks, about 1 ⁇ 10 7 cells each time. Long-term immunization, until the titer of T3A-A3 of anti-human liver cancer stem cell (human tumor pluripotent stem cell) line of the immunized mouse serum is determined by conventional cellular immunochemical methods exceeds 1: 50000.
  • Mouse spleen cells and mouse myeloma cells SP2 / 0 were fused using conventional PEG-mediated fusion methods to form hybridomas that secreted mouse monoclonal antibodies.
  • Hybridoma monoclonals were prepared by the conventional methylcellulose plate method, and after each monoclonal was grown, they were picked to 96-well plates and cultured, thereby obtaining a line containing a large amount of human liver cancer stem cells (human tumor pluripotent stem cells).
  • Library of hybridoma clones of T3A-A3 monoclonal antibodies The culture supernatant of each hybridoma clone in a 96-well plate was collected and used for the next determination and screening process.
  • Serum-free suspension medium uses DMEM / F12 (1: 1) containing 20ng / mL EGF, 20ng / mL bFGF, B27 added at a ratio of 1:50, 10ng / mL LIF, 2mmol / mL glutamine, 1 ⁇ / mL Heparin Medium. Cells were washed once before incubation in serum-free medium. Human hepatocellular carcinoma stem cells (human tumor pluripotent stem cells) T3A-A3 spherical cells cultured in serum-free suspension were gently blown into single cells and seeded in a 96-well plate at 2000 cells / well.
  • wash cells with PBS containing 1% BSA add 100 ⁇ L of the culture supernatant of a hybridoma clone to each well, and incubate at room temperature for 2 hours; wash 5 times with PBS containing 1% BSA Add biotin-labeled anti-mouse secondary antibody at room temperature and react for 30 minutes. After washing 5 times with PBS containing 1% BSA, add Cy3-labeled Avidin and react at room temperature for 30 minutes.
  • a murine monoclonal antibody capable of binding to the surface antigen of the T3A-A3 membrane of human liver cancer stem cells can be obtained by preliminary screening.
  • human serum cancer cell lines (Yan Li, Zhao-You Tang, Sheng-Long Ye, Yin-Kun Liu, Jie CHEN, Qiong Xue, Jun-Chen, Dong-Mei Gao, Wei-Hua Bao) were selected.
  • a mouse monoclonal antibody, Hetumomab was screened from the anti-human tumor pluripotent stem cell mouse monoclonal antibody library, which not only binds to the human tumor pluripotent stem cell line T3A-A3 membrane surface antigen, but also simultaneously Stem cells (MHCC97L spheroid cells) membrane surface antigen binding, proved that mouse monoclonal antibody Hetumomab can recognize human liver cancer stem cells from different sources.
  • the mouse monoclonal antibody Hetumomab was selected for further identification and pharmacodynamic research.
  • Hetumomab monoclonal antibody-secreting mouse hybridoma cells were deposited under the accession number CGMCC No. 12251 on March 16, 2016 at the General Microbial Center (CGMCC) of the China Microbial Species Collection Management Committee No. 3 Institute of Microbiology, Chinese Academy of Sciences).
  • CGMCC General Microbial Center
  • Hetumomab hybridoma cells were expanded and the antibody-containing supernatant was collected.
  • the monoclonal antibody subclass detection kit from Southern Biotech was used to identify the class and subclass of the monoclonal antibody, and the antibody yield of the ELISA secondary antibody detection supernatant from Sigma.
  • the experimental results showed that the monoclonal antibody Hetumomab is an IgG1 heavy chain and a kappa light chain.
  • the hybridoma cells secreting monoclonal antibody Hetumomab were expanded in vitro. After the cells grew to 80%, the serum-free medium was replaced. After the culture continued for 4-5 days, the serum-free supernatants with secreted antibodies were collected. Monoclonal antibody Hetumomab was purified using an anti-Protein G purification column. The purity of the isolated and purified Hetumomab mAb was identified by Coomassie blue staining with 10% SDS-PAGE.
  • Hetumomab target antigen is expressed in a variety of human liver cancer, lung cancer, and gastric cancer cell lines and can be expressed on the surface of living cells.
  • the conventional live cell immunofluorescence staining technique was used to detect the expression of monoclonal antibody Hetumomab target antigen on the surface of living cells in human tumor cell lines such as liver cancer, lung cancer and gastric cancer cell lines.
  • the specific technical method is roughly as follows: cell climbing slides or inoculation of 96-well culture plates (4 ⁇ 10 3 cells / well). When the cells reach 60% to 70% full, wash the serum-free culture medium twice and wash the cells once with PBS.
  • the monoclonal antibody Hetumomab target antigen can be expressed on the surface of living cells of the following human liver, lung, and gastric cancer cell lines (purchased from the Cell Resource Center, Institute of Basic Medical Research, Chinese Academy of Medical Sciences):
  • Human liver cancer cell lines MHCC97L, Be17402-V13;
  • Human gastric cancer cell lines SNU-5, BGC-823.
  • the monoclonal antibody Hetumomab target antigen is expressed in a variety of human tumors, such as liver cancer, lung cancer, and gastric cancer cell lines, and can be expressed on the membrane surface of living cancer cells.
  • Hetumomab target antigen is specifically and highly expressed in human liver cancer, lung cancer, and gastric cancer tissues.
  • Hetumomab was used as the primary antibody, and anti-mouse antibody secondary antibody was used to detect the expression of monoclonal antibody Hetumomab target antigen in multiple cases of human liver, lung, and gastric cancer patients and other related tissues.
  • the specific technical method is roughly as follows: conventionally dewaxing the tissue piece; citric acid buffer solution (pH 6.0) is poured into the antigen repair box, the piece is placed, the repair box is placed in boiling water, the water bath is heated for 30 minutes, and the room temperature is naturally cooled for 2 hours; PBS Wash 3min ⁇ 3 times, dry the water on the tablet, and immediately circle the tissue with a tissue pen; add a drop of endogenous peroxidase blocking solution to each tissue, incubate at room temperature for 20min, and wash in PBS for 3min ⁇ 3 Then, shake off the wash solution, add a drop of normal animal serum-sheep serum, and incubate at room temperature for 20min; shake off the blocked serum, add primary antibodies to each tissue point, put in a humidifier, and incubate at 4 ° C overnight; shake off the primary antibody , Wash 3min ⁇ 3 times in PBS, shake off the wash solution, add secondary antibody biotin-anti-mouse antibody, and in
  • monoclonal antibody Hetumomab As the primary antibody, the expression of the monoclonal antibody Hetumomab antigen in 120 cases of human liver cancer tissues, 20 cases of adjacent tissues, 10 cases of normal liver tissues, 10 cases of hepatitis tissues, and 40 cases of liver cirrhosis was detected.
  • the experimental results (Table 1) showed that the target antigen of monoclonal antibody Hetumomab was specifically expressed in 77.17% (95/120) of human liver cancer tissues, but not in adjacent tissues, normal liver tissues, hepatitis tissues, and cirrhotic tissues. Seeing its expression, it was demonstrated that the monoclonal antibody Hetumomab target antigen was specifically and highly expressed in human liver cancer tissues.
  • the monoclonal antibody Hetumomab target antigen is specifically and highly expressed in the cancer tissues of most patients with human tumors, such as liver cancer, lung cancer, and gastric cancer.
  • the cancer cells recognized by monoclonal antibody Hetumomab were enriched in sphere culture cells of cancer cells.
  • serum-free suspension culture that is, sphere culture
  • can enrich tumor stem cells Reynolds, BAand S. Weiss, "Clonal and population analysis, demostrate strategy that EGF-responsive, mammalian, embryonic, CNS, precursor, etc.) Cell. "Dev Biol. 1996.175 (1): p.1-13 .; Fang, N., et al.," pH responsive adhesion of phospholipidvesicle on poly (acrylic acid) cushion grafted to poly (ethylene terephthalate) surface.
  • the human lung cancer cell lines SPCA-1 and A549 cells were serum-free cultured for 5 days, and then Hetumomab + cells in parental cells and sphere cultured cells were detected by live-cell flow fluorometry.
  • the experimental results (Table 5) showed that the proportion of Hetumomab + cells in SPCA-1 sphere cells was 7.34%, which was enriched 4.22 times than the ratio of 1.74% in the parent cells; the proportion of Hetumomab + cells in A549 sphere cells was 13.30 %, Which is 1.84 times more enriched than the ratio of 7.23% in the parental cells. That is, after serum-free culture, Hetumomab + cells of the lung cancer cell line were enriched.
  • cancer cells recognized by monoclonal antibody Hetumomab are significantly enriched in sphere culture cells of human tumors, such as liver cancer, lung cancer, and gastric cancer cell lines.
  • monoclonal antibody Hetumomab recognizes ESA and CD90 positive tumor stem cells.
  • Hetumomab hepatocellular carcinoma cells are also ESA, CD90 marker-positive liver cancer stem cells, we use two-color flow fluorometry to culture human liver cancer for 5 days in serum-free medium Be17402-V13 cells were stained.
  • the results (as shown in Table 7) showed that the proportion of cells recognized by monoclonal antibody Hetumomab was 8.52%, the expression ratio of ESA + stem cells was 9.02%, and the co-staining ratio of the two was 3.63%, that is, the monoclonal antibody Hetumomab recognized 40.2% of ESA + stem cells.
  • Human liver cancer MHCC97L cells cultured in serum-free medium for 5 days were stained.
  • the results (as shown in Table 7) showed that the proportion of cells recognized by monoclonal antibody Hetumomab was 2.38%, and the expression ratio of CD90 + stem cells was 4.54%.
  • the staining ratio was 2.01%, that is, monoclonal antibody Hetumomab recognized 44.3% of CD90 + stem cells.
  • Self-renewal ability, strong invasion ability, tolerance to chemotherapy drugs and strong tumorigenicity are the important basic characteristics of tumor stem cells different from ordinary progeny tumor cells. Therefore, in order to further verify whether the cells identified by the monoclonal antibody Hetumomab have the characteristics of tumor stem cells, Hetumomab + cells in a variety of human tumor cells were sorted and tested for their self-renewal, invasion, drug resistance, and tumorigenicity in vivo.
  • the self-renewal ability of tumor stem cells is mainly manifested in the ability to form spheroids in serum-free medium.
  • This method is called asymmetric division, that is, when one cell divides into two progeny cells, one of the progeny cells remains the same The cells have exactly the same characteristics, while the other progeny can continue to divide and form normal progeny. Therefore, the ability of Hetumomab + cells to self-renew can be determined by measuring their spheroidizing ability in serum-free medium.
  • the sorted cells were seeded at 500 cells / well in a semi-solid sphere medium containing 0.8% methylcellulose (the semi-solid sphere medium contains 0.8% methylcellulose, 20ng / mL EGF, 20ng / mL bFGF 1.
  • the semi-solid sphere medium contains 0.8% methylcellulose, 20ng / mL EGF, 20ng / mL bFGF 1.
  • Hetumomab + cells were seeded at 500 cells / well in a semi-solid sphere medium containing 0.8% methylcellulose, and cultured in an ultra-low-adhesion 24-well plate, and the number of spherules of each cell was observed.
  • the results show (Table 8), Hetumomab + cells, the parent cell and Hetumomab - number of cells into a ball under serum-free media conditions were 165.7 ⁇ 6.0,127 ⁇ 5.6,83.7 ⁇ 4.7, i.e.
  • Hetumomab + rate of the cells into a ball Significantly higher than the other two cells (p ⁇ 0.05).
  • Hetumomab + cells than the parental cells and Hetumomab - having stronger cell self-renewal capacity are considered.
  • Hetumomab + cells, parental cells, and Hetumomab - cells were isolated from human gastric cancer SNU-5 sphere cells using flow cytometry.
  • the sorted cells were seeded at 500 cells / well in a semi-solid sphere medium containing 0.8% methylcellulose, and cultured in an ultra-low-adhesion 24-well plate, and the number of spherules of each cell was observed.
  • the cancer cells recognized by monoclonal antibody Hetumomab have a stronger ability to invade
  • Self-renewal ability, strong invasion ability, tolerance to chemotherapy drugs and strong tumorigenicity are the important basic characteristics of tumor stem cells different from ordinary progeny tumor cells. Therefore, in order to further verify whether the cells identified by the monoclonal antibody Hetumomab have the characteristics of tumor stem cells, Hetumomab + cells in a variety of human tumor cells were sorted and tested for their self-renewal, invasion, drug resistance, and tumorigenicity in vivo.
  • cancer cells identified by monoclonal antibody Hetumomab have greater resistance to chemotherapy drugs
  • Self-renewal ability, strong invasion ability, tolerance to chemotherapy drugs and strong tumorigenicity are the important basic characteristics of tumor stem cells different from ordinary progeny tumor cells. Therefore, in order to further verify whether the cells identified by the monoclonal antibody Hetumomab have the characteristics of tumor stem cells, Hetumomab + cells in a variety of human tumor cells were sorted and tested for their self-renewal, invasion, drug resistance, and tumorigenicity in vivo.
  • Hetumomab + hepatocellular carcinoma cells In order to test the resistance of Hetumomab + hepatocellular carcinoma cells, Hetumomab + cells, parental cells and Hetumomab - cells obtained by flow sorting of human hepatocellular carcinoma Be17402-V13 sphere cells were seeded into 96-well plates at the number of 5000 / well.
  • Each group of cells was completely treated with 8 different concentrations of cisplatin containing 0 ⁇ g / mL, 0.0625 ⁇ g / mL, 0.125 ⁇ g / mL, 0.25 ⁇ g / mL, 0.5 ⁇ g / mL, 1 ⁇ g / mL, 2 ⁇ g / mL, and 4 ⁇ g / mL.
  • the medium was cultured. After 3 days, the medium was replaced once. After 7 days, the OD value was measured by the CCK8 method to determine the IC50 reflecting its drug resistance.
  • Hetumomab + cells, parental cells, and Hetumomab - cells obtained by flow sorting in human lung cancer cells SPCA-1 sphere cells were seeded in a 96-well plate at a number of 5000 / well.
  • Cells in each group were cultured in 7 different concentrations of cisplatin in 0 ⁇ g / mL, 0.2 ⁇ g / mL, 0.4 ⁇ g / mL, 0.6 ⁇ g / mL, 0.8 ⁇ g / mL, 1 ⁇ g / mL and 2 ⁇ g / mL.
  • Hetumomab + gastric cancer cells In order to test the resistance of Hetumomab + gastric cancer cells, we sorted Hetumomab + cells, parental cells, and Hetumomab - cells obtained by flow sorting from human gastric cancer cells SNU-5 sphere cells into 96-well plates at a rate of 5000 cells / well. For each group of cells, 8 different concentrations of 0 ⁇ g / mL, 0.0125 ⁇ g / mL, 0.025 ⁇ g / mL, 0.05 ⁇ g / mL, 0.1 ⁇ g / mL, 0.2 ⁇ g / mL, 0.4 ⁇ g / mL, and 0.8 ⁇ g / mL were used.
  • the cisplatin was cultured in a complete medium, and then the IC50 was measured by the CCK8 method to measure the OD value reflecting its drug resistance.
  • the experimental results showed (Table 16, Figure 4) that the IC50 of Hetumomab + cells, parent cells and Hetumomab - cells were 0.285 ⁇ mol / L, 0.155 ⁇ mol / L, and 0.094 ⁇ mol / L, respectively. That is, the drug resistance of Hetumomab + cells was significantly higher. There was a statistically significant difference between parental cells and Hetumomab - cells (p ⁇ 0.05). Ability to tolerate chemotherapy drugs having stronger cell - thus, parental cells and gastric cancer cells than Hetumomab Hetumomab +.
  • Self-renewal ability, strong invasion ability, tolerance to chemotherapy drugs and strong tumorigenicity are the important basic characteristics of tumor stem cells different from ordinary progeny tumor cells. Therefore, in order to further verify whether the cells identified by the monoclonal antibody Hetumomab have the characteristics of tumor stem cells, Hetumomab + cells in a variety of human tumor cells were sorted and tested for their self-renewal, invasion, drug resistance, and tumorigenicity in vivo.
  • the "gold standard" for testing whether a cell is a tumor stem cell is strong tumorigenicity in vivo.
  • the human hepatoma cells Be17402-V13 sphere flow fraction Hetumomab + cells selected from the obtained cells, the parent cell and Hetumomab - cells were inoculated subcutaneously into nude mice 4 weeks old size, long period of time to observe the in vivo tumorigenicity.
  • the results are shown in Table 17. 1 ⁇ 10 4 Hetumomab + cells can be tumorigenic in mice at 3 weeks of inoculation, while parental cells require 1 ⁇ 10 5 cells to emerge after 3 weeks of inoculation. Hetumomab - cells No tumors appeared during the observation period.
  • Hetumomab + Classic in vivo tumorigenicity of parental and more Hetumomab cells - cells significantly stronger. It is suggested that Hetumomab + cells have the highly tumorigenic characteristics of tumor stem cells and meet the "gold standard" of tumor stem cells. Therefore, the cells recognized by monoclonal antibody Hetumomab are liver cancer tumor stem cells.
  • Hetumomab + cells, parental cells and Hetumomab - cells obtained by flow sorting of human gastric cancer cells SNU-5 sphere cells were inoculated subcutaneously into nude mice at the age of 4 weeks, and the tumor formation in vivo was observed for a long time. The results are shown in Table 18. 2 ⁇ 10 3 Hetumomab + cells were tumorigenic in half of the mice at 3 months after inoculation, while 2 ⁇ 10 3 cells of the parent cells did not appear after 4 months of inoculation. Tumor, Hetumomab - cells did not develop tumors during the observation period.
  • Hetumomab + Classic in vivo tumorigenicity of parental and more Hetumomab cells - cells significantly stronger. It is suggested that Hetumomab + cells have the highly tumorigenic characteristics of tumor stem cells and meet the "gold standard" of tumor stem cells. Therefore, the cells recognized by monoclonal antibody Hetumomab are gastric cancer tumor stem cells.
  • Example 4 Monoclonal antibody Hetumomab can inhibit the self-renewal, invasion and drug resistance of tumor stem cells
  • monoclonal antibody Hetumomab significantly inhibits the self-renewal ability of tumor stem cells (one of the main characteristics of tumor stem cells) in a variety of tumors (liver cancer, gastric cancer, lung cancer).
  • the self-renewal ability of tumor stem cells is mainly manifested in the ability to form spheres in serum-free medium.
  • This method is called asymmetric division, that is, when a cell divides into two progeny cells, one of the progeny cells remains the same as the parent. The cells have exactly the same characteristics, while the other progeny can continue to divide and form normal progeny.
  • Hetumomab is a functional monoclonal antibody capable of directly inhibiting liver cancer stem cells, we prepared human hepatocellular carcinoma cell line Be17402-V13 sphere cells cultured in serum-free medium for 5 days into single cell suspensions, and purified them.
  • Monoclonal antibody Hetumomab 250 ⁇ g / mL was used as the experimental group, PBS was used as the negative control group, and the cells were incubated at 37 ° C for 2 hours. During the period, the cells were mixed with the antibody or the negative control every half an hour. 500 cells in each group were inoculated in semi-solid sphere medium (including EGF, LIF, bFGF, etc.) containing 0.8% methylcellulose, cultured in ultra-low adhesion 24-well plates, and rehydration the next day 1-1.5 mL. After 14 days, the number of cells formed in the two groups was observed.
  • semi-solid sphere medium including EGF, LIF, bFGF, etc.
  • the experimental results showed that the number of balls formed in the experimental group was 212 ⁇ 2.8, while the number of balls formed in the negative control group was 278.5 ⁇ 0.7, which was significantly higher than that in the experimental group.
  • the monoclonal antibody Hetumomab inhibited the spheroidization of Be17402-V13 cells by 23.9%, p ⁇ 0.05, which was statistically different.
  • the results show that monoclonal antibody Hetumomab can directly affect liver cancer stem cells and inhibit their self-renewal ability.
  • Hetumomab against SPCA The spheroidization inhibition rate of -1 cells reached 41.6%, p ⁇ 0.01, which was statistically different.
  • the results show that monoclonal antibody Hetumomab can directly affect lung cancer stem cells and inhibit their self-renewal ability.
  • monoclonal antibody Hetumomab is a functional monoclonal antibody capable of directly inhibiting gastric cancer stem cells
  • the same method was used to detect the inhibitory effect of monoclonal antibody Hetumomab on the tumor stem cell subpopulation formation of CD44 positive cells of human gastric cancer cell line SNU-5.
  • the results (as shown in Table 20 and Figure 5) showed that the number of spherules with the highest antibody concentration in the experimental group was 18 ⁇ 2.0, while the number of spherules in the negative control group was 128 ⁇ 4.0, which was significantly higher than the experimental group.
  • CD44 + cells had a spheroidification inhibition rate of 85.9%, p ⁇ 0.01, which was statistically different.
  • monoclonal antibody Hetumomab can directly affect gastric cancer stem cells and inhibit their self-renewal ability.
  • monoclonal antibody Hetumomab can directly inhibit the self-renewal ability of a variety of cancer cells (liver cancer, lung cancer, and gastric cancer). It proves that monoclonal antibody Hetumomab not only recognizes targeted tumor stem cells, but also directly inhibits the function of tumor stem cells ( Therapeutic) Anti-tumor stem cell monoclonal antibodies.
  • the monoclonal antibody Hetumomab significantly inhibited the invasion ability of tumor stem cells of a variety of tumors (liver cancer, gastric cancer, lung cancer) (the main characteristics of tumor stem cells).
  • the experimental results show that the cell invasion ability of monoclonal antibody Hetumomab after direct action is significantly weakened, and its inhibition rate on Be17402-V13 sphere cell invasion is 50.8%, p ⁇ 0.05, which is statistically different.
  • the experimental results show that monoclonal antibody Hetumomab can directly affect liver cancer stem cells and inhibit its invasion ability.
  • monoclonal antibody Hetumomab is a functional monoclonal antibody that can directly inhibit lung cancer stem cells
  • the same method was used to detect the inhibitory effect of monoclonal antibody Hetumomab on the invasion of human lung cancer cell line SPCA-1.
  • the experimental results showed (Table 21, Figure 6) that the number of invasive cells in the PBS negative control group was (232.3 ⁇ 3.1) / field, and the number of invasive cells in the monoclonal antibody Hetumomab group was (153.0 ⁇ 6.1) / field.
  • the experimental results showed that the cell invasion ability of monoclonal antibody Hetumomab was significantly weakened, and its inhibition rate on SPCA-1 sphere cell invasion was 34.1%, p ⁇ 0.05, which was statistically different.
  • the experimental results show that monoclonal antibody Hetumomab can directly affect lung cancer stem cells and inhibit their invasion ability.
  • monoclonal antibody Hetumomab is a functional monoclonal antibody capable of directly inhibiting gastric cancer stem cells
  • the same method was used to detect the inhibitory effect of monoclonal antibody Hetumomab on the invasion of tumor stem cell subsets of CD44 positive cells of the human gastric cancer cell line SNU-5.
  • the experimental results showed (Table 22, Figure 6) that the number of invasive cells in the PBS negative control group was (231 ⁇ 7.0) / field, and the number of invasive cells in the monoclonal antibody Hetumomab group was (56 ⁇ 4.0) / field.
  • the experimental results show that the cell invasion ability of monoclonal antibody Hetumomab after direct action is significantly reduced, and its inhibition rate on SNU-5 CD44 + cell invasion is 75.8%, p ⁇ 0.05, which is statistically different.
  • the experimental results show that monoclonal antibody Hetumomab can directly affect gastric cancer stem cells and inhibit their invasion ability.
  • monoclonal antibody Hetumomab can directly inhibit the invasion ability of a variety of cancer cells (liver cancer, lung cancer, and gastric cancer). It proves that monoclonal antibody Hetumomab not only recognizes targeted tumor stem cells, but also directly inhibits the function of tumor stem cells (treatment (Anti-tumor stem cell monoclonal antibodies).
  • monoclonal antibody Hetumomab significantly inhibits the ability of tumor stem cells of various tumors (liver cancer, lung cancer) to tolerate chemotherapy drugs (the main characteristics of tumor stem cells).
  • Drug resistance is one of the biological characteristics of tumor stem cells.
  • the monoclonal antibody Hetumomab is a functional monoclonal antibody that can directly inhibit liver cancer stem cells.
  • Be17402-V13 sphere cells cultured for 5 days were seeded in a 96-well plate at the number of 5000 / well, and the purified monoclonal antibody Hetumomab ( 0.5mg / mL) and PBS in each well. After incubating in a 5% CO 2 incubator at 37 ° C for 24 hours, remove the antibody-containing medium.
  • Each group of cells contains 0, 0.0625, 0.125, 0.25, 0.5 , 1, 2, 4, and 8 ⁇ g / mL of 9 different concentrations of cisplatin in culture cells, replace the complete medium containing cisplatin once every 48 hours, add CCK-8 reagent according to the instructions of the CCK-8 kit, and test OD450 absorbance value, and calculate the IC50 value of each group.
  • the experimental results showed ( Figure 7) that the cell resistance of Hetumomab was significantly reduced after the direct action of mAb. Its IC50 value was 0.334 ⁇ g / mL, while the IC50 value of the control group was 0.9 ⁇ g / mL. Cell resistance was significantly lower than the control group.
  • the results of this experiment show that monoclonal antibody Hetumomab can directly affect liver cancer stem cells and inhibit their ability to resist drug resistance.
  • monoclonal antibody Hetumomab can directly inhibit the ability of many cancer cells (liver cancer, lung cancer) to tolerate chemotherapy drugs. It proves that monoclonal antibody Hetumomab can not only target tumor stem cells, but also directly inhibit the function of tumor stem cells. (Therapeutic) Anti-tumor stem cell monoclonal antibody.
  • Example 5 The monoclonal antibody Hetumomab has the pharmacodynamic effect of inhibiting the growth of tumor transplanted tumors and synergistic chemotherapy in animals
  • monoclonal antibody Hetumomab is a monoclonal antibody that targets multiple tumor stem cells; and the results of in vitro pharmacodynamic studies show that monoclonal antibody Hetumomab can significantly inhibit the self-renewal, invasion and resistance of multiple tumor stem cells ability.
  • monoclonal antibody Hetumomab on the growth, metastasis, and resistance of multiple tumors in vivo
  • a variety of human tumor animal models were used to evaluate the pharmacodynamics of monoclonal antibody Hetumomab on the growth, metastasis, and resistance of multiple tumors in vivo. effect.
  • Hetumomab significantly inhibits the growth of human liver cancer xenografts in the body, can significantly enhance the efficacy of chemotherapy, significantly prolong the survival period, and has a significant antitumor pharmacological effect.
  • liver cancer An experimental study on the treatment of liver cancer with monoclonal antibody Hetumomab in nude mice was carried out. The efficacy of monoclonal antibody alone, chemotherapeutic drugs and monoclonal antibodies combined with chemotherapeutic drugs in the treatment of liver cancer was observed and compared. Growth and drug resistance, and explore the optimal treatment of liver cancer by targeting liver cancer stem cells.
  • the sphere cells of Be17402-V13 were seeded under the skin of nude mice at 30,000 per mouse, and the nude mice were randomly divided into 6 groups (6 per group), respectively: chemotherapy group alone (0.3mg / kg cisplatin, 6) ); High-dose antibody group (10mg / kg, 6 animals); High-dose antibody + chemotherapy group (6 animals); Low-dose antibody group (2.5mg / kg, 6 animals); Low-dose antibody + chemotherapy group (6 animals) ; PBS group (6 animals). The treatment was started the next day after cell inoculation, and treatment was performed twice a week, and treatment was terminated after 5 weeks.
  • Tumor growth rate (V t -V 0 ) / days, V t is the tumor volume at each measurement, and V 0 is the tumor volume before administration (V 0 refers to the tumor volume when the administration is stopped).
  • the growth curve of transplanted tumors in mice is shown in Figure 8.
  • Monoclonal antibody Hetumomab can significantly inhibit the growth of transplanted tumors in nude mice. And with the increase of the dose of antibody, the inhibition rate of xenograft tumors gradually increased, and there was a dose-dependent relationship.
  • the experimental results are shown in Figure 9.
  • the inhibition rates of high and low doses of monoclonal antibody Hetumomab on transplanted tumors were 71.5% and 54.4%, respectively.
  • the inhibition rate of the chemotherapy group was 83.5%.
  • the inhibition rates of the high and low dose combined chemotherapy groups were similar, both reaching about 97%.
  • the tumor growth rate of the monoclonal antibody combined chemotherapy group was 0.051 cm 3 / day, which was 4.7 lower than that of the PBS control group (0.239 cm 3 / day). times, and higher than the monoclonal antibody, tumor growth rate and low dose group of chemotherapeutic agents (0.148cm 3 / day, 0.185cm 3 / day and 0.154cm 3 / day), respectively, 2.9,3.6 and 3.1 times lower, the results show that The method of monoclonal antibody combined with chemotherapy can effectively inhibit tumor growth and recurrence.
  • the entire treatment and observation process lasted six months.
  • the six-month survival curve of the mice (Figure 11) shows that the distribution of the survival curve of the six groups of mice is statistically significant, P ⁇ 0.05. This indicates that the survival status of mice in the monoclonal antibody combination chemotherapy group is significantly better than that in the PBS control group, the monoclonal antibody group and the chemotherapy group. It is suggested that monoclonal antibody combined with chemotherapy can prolong the survival of mice.
  • the monoclonal antibody Hetumomab alone can significantly inhibit the growth of human liver cancer transplanted tumors, and has a significant pharmacodynamic effect on liver cancer.
  • Both the monoclonal antibody Hetumomab and the chemotherapeutic group showed a high rate of inhibition of transplanted tumors, which could significantly inhibit tumor growth, and the treatment effect was better than that of the antibody alone group and the chemotherapeutic group alone, indicating that the monoclonal antibody combined with the chemotherapeutic agent was effective Inhibits tumor growth and reduces chemotherapy resistance.
  • mice in the combination group was significantly longer than that in the antibody alone group and the chemotherapeutic group alone, indicating that the monoclonal antibody Hetumomab combined with chemotherapeutic drugs can not only treat tumor growth, but also prolong the survival time of the mice.
  • monoclonal antibody Hetumomab significantly inhibits the growth of human lung cancer xenografts in vivo, and has a significant antitumor pharmacodynamic effect.
  • spheroid cells of the human lung cancer cell line SPCA-1 were harvested, and nude mice were inoculated at 2.5 ⁇ 10 5 cells / head. Divided into 5 groups, 5 in each group: PBS control group, chemotherapy alone group (cisplatin 0.3mg / kg), Hetumomab antibody high dose group (40mg / kg), Hetumomab antibody medium dose group (10mg / kg), Hetumomab low Dose group (2.5mg / kg).
  • Antibody treatment was started on the second day after inoculation of lung cancer cells.
  • the experimental group and the control group were treated by intraperitoneal injection. The total treatment was 28 days after the inoculation, and the drug was discontinued.
  • the chemotherapy group was treated twice a week.
  • the long and short diameters of subcutaneously transplanted tumors were measured twice a week to calculate the tumor volume.
  • the tumor volume growth of the mice was observed and measured, and the inhibition rate was calculated.
  • the growth curve of transplanted tumors in mice is shown in Figure 12, and the tumor volume inhibition rate is shown in Table 22.
  • Monoclonal antibody Hetumomab can significantly inhibit the growth of transplanted tumors in nude mice. It also increased accordingly.
  • the inhibition rates of high, medium and low doses of monoclonal antibody Hetumomab on transplanted tumors were 55.97%, 43.56%, and 35.58%, respectively.
  • the inhibition rate of the chemotherapy group was only 24.91%.
  • Hetumomab significantly inhibits the growth of human gastric cancer xenografts in the body, and can significantly enhance the efficacy of chemotherapy in synergy, and has a significant anti-tumor pharmacodynamic effect.
  • spheroid cells of the human gastric cancer cell line SNU-5-V13 were harvested, and nude mice were inoculated at 2.5 ⁇ 10 5 cells / head.
  • Antibody treatment was started on the second day after inoculation of gastric cancer cells.
  • the experimental group and the control group were treated by intraperitoneal injection.
  • the chemotherapeutic group was treated for 4 weeks and discontinued after 2 times a week.
  • the long and short diameters of subcutaneously transplanted tumors were measured twice a week to calculate the tumor volume.
  • the growth curve of transplanted tumors in mice is shown in Figure 13.
  • the tumor volume inhibition rate is shown in Table 23.
  • Monoclonal antibody Hetumomab can significantly inhibit the growth of transplanted tumors in nude mice. It also increased accordingly.
  • the inhibition rates of high and low doses of monoclonal antibody Hetumomab on transplanted tumors were 57.62% and 30.68%, respectively.
  • the inhibition rate of the chemotherapy group was only 33.16%.
  • the combination of high and low doses of monoclonal antibodies reached 70.68% and 47.93%, respectively.
  • the monoclonal antibody Hetumomab alone can significantly inhibit the growth of human gastric cancer xenografts, and has a significant pharmacodynamic effect on gastric cancer.
  • Both the monoclonal antibody Hetumomab and the chemotherapeutic group showed a high rate of inhibition of transplanted tumors, which could significantly inhibit tumor growth, and the treatment effect was better than that of the antibody alone group and the chemotherapeutic group alone, indicating that the monoclonal antibody combined with the chemotherapeutic agent was effective Inhibits tumor growth and reduces chemotherapy resistance.
  • variable region primer combination was designed to perform subsequent routine PCR reactions to amplify the antibody variable region gene sequence, cloned into the ZT4-Blunt vector, transformed into E. coli DH5 ⁇ competent cells, selected positive clones and sequenced.
  • the amino acid sequence of the light chain variable region of Hetumomab monoclonal antibody cloned is shown in SEQ ID NO: 1
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5.
  • Amino acid sequence of light chain variable region of Hetumomab mAb (SEQ ID NO: 1):
  • VL-CDR1 SEQ ID ID: 2: RSSKSLLHSNGITYLY
  • VL-CDR2 SEQ ID ID: 3: QMSNLAS
  • VL-CDR3 SEQ ID ID: 4: AQNLELYT
  • Amino acid sequence of the heavy chain variable region of Hetumomab mAb (SEQ ID NO: 5):
  • VH-CDR1 SEQ ID NO: 6: TSGMGVS
  • VH-CDR2 SEQ ID ID: 7: HIYWDDDKRYNPSLKS
  • VH-CDR3 SEQ ID NO: 8: SFYYYANNSFAY
  • Example 7 Construction, expression and purification of a human-mouse chimeric antibody Hetuximab derived from the mouse monoclonal antibody Hetumomab
  • the light and heavy chain variable region gene fragments of the mouse monoclonal antibody Hetumomab were cloned into human IgG1CH (heavy chain constant region gene) and human IgG CK (light chain constant region gene) transient expression, respectively.
  • human IgG1CH heavy chain constant region gene
  • human IgG CK light chain constant region gene
  • a large number of heavy and light chain antibody variable region fragments of Hetumomab were amplified by PCR, and cloned into the corresponding cloning sites of pKN009 (containing human IgG1CH coding sequence) and pKN019 (containing human IgG and CK coding sequence) transient expression vectors. Transform E. coli and screen positive clones. Positive clones are further sequenced for identification. As a result, a human-mouse chimeric antibody Hetuximab derived from Hetumomab and its expression vector were obtained.
  • the specific protein sequence of the chimeric antibody is as follows:
  • Hetuximab chimeric antibody heavy chain amino acid sequence (SEQ ID NO: 9):
  • VH-CDR1 SEQ ID NO: 6
  • VH-CDR2 SEQ ID NO: 7
  • VH-CD3 SEQ ID NO: 8
  • human IgG1CH SEQ ID NO: 11
  • Hetuximab chimeric antibody light chain amino acid sequence (SEQ ID NO: 10):
  • VL-CDR1 SEQ ID NO: 2
  • VL-CDR2 SEQ ID NO: 3
  • VL-CDR3 SEQ ID NO: 4
  • human IgG SEQ ID NO: 12
  • the above-identified light and heavy chain chimeric antibody gene expression vectors were conventionally transfected into HEK-293 cells with liposomes to express the chimeric antibodies transiently.
  • the supernatant was collected for preliminary purification using a ProteinA affinity chromatography column, and then impurities were removed by cation exchange chromatography. The purity of the purified product was verified by SDS-PAGE electrophoresis. Results A human-mouse chimeric antibody Hetuximab derived from Hetumomab with a purity of> 95% and 2 mg / ml was obtained.
  • Example 8 The chimeric antibody Hetuximab recognizes the same epitope that binds to the same antigen protein as the parental mouse monoclonal antibody, and has the same specificity and consistent affinity.
  • the total protein of four human tumor cells SNU-5, BGC-823, MHCC-97L, BEL7402 and V13 positively expressed by Hetumomab target antigen was extracted by conventional methods, and the protein concentration of the extract solution was quantified by BCA kit.
  • An appropriate amount of protein sample (20 ⁇ g) was separated by SDS-polyacrylamide gel electrophoresis, and the protein was transferred to a PVDF membrane using a semi-dry electrokinetic instrument.
  • the PVDF membrane was then placed in a blocking solution (TBST / 5% skimmed milk powder) and blocked on a horizontal shaker at room temperature for 1 h.
  • Hetumomab was used as the primary antibody and the anti-mouse antibody secondary antibody was used; and the chimeric antibody Hetuximab was used as the primary antibody, and the anti-human antibody secondary antibody was used to detect the positive expression of the monoclonal antibody Hetumomab target antigen.
  • the competitive inhibitory cell ELISA was used to prove that the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab recognize the same epitope that binds to the same antigen protein, and have consistent affinity.
  • MHCC-97L cells were seeded in 96-well culture plates (4 ⁇ 10 3 cells / well), and experiments were performed when the cells reached 90% to 100% full. Discard the culture medium in the wells, wash with 0.05% Tween-20 in PBS, 300 ⁇ l / well, 1 min / times ⁇ 5 times; add samples as follows, add 100 ⁇ l / well, re-well, and incubate at 37 ° C for 1.5 h; discard Remove the liquid in the well, wash with 0.05% Tween-20 in PBS, 300 ⁇ l / well, 1 min / times ⁇ 5 times; add anti-human IgG Fc-HRP (does not react with mouse IgG Fc) or anti-mouse IgG Fc-HRP (Does not react with human IgG Fc) corresponding secondary antibodies, 1: 5000, 100 ⁇ l / well, incubate for 1 h at 37 ° C; discard the liquid in the wells, wash with PBS
  • Anti-human IgG Fc-HRP (which does not react with murine IgG Fc) was used as the secondary antibody. The results are shown in the following table:
  • Tables 24 and 25 are plotted and shown in FIG. 15. It can be seen from FIG. 15 that the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab can significantly compete with each other to inhibit the binding of each other with the target antigen on the surface of the target cell MHCC-97L cell, indicating that the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab Recognize the same epitope that binds to the same antigen protein; at the same time, the two show similar competitive inhibition efficiency, suggesting that the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab have comparable affinities and have consistent affinity.
  • Example 9 The chimeric antibody Hetuximab recognizes the same tumor stem cells as the parental monoclonal antibody, and has the same pharmacodynamic effect of inhibiting tumor stem cells.
  • the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab recognize the same group of tumor stem cells.
  • Table 26 Comparison of the positive rate of Hetuximab and Hetumomab in different tumor cells and the multiples of enrichment in sphere cells.
  • the chimeric antibody Hetuximab has the same pharmacodynamic effect in inhibiting tumor stem cells in vitro as the parental mouse monoclonal antibody Hetumomab.
  • the efficiency of inhibition of tumor stem cell spheroid formation by the antibodies of each concentration gradient in the tumor stem cell-rich sphere cells of the four tumor cell lines is very consistent. Therefore, the chimeric antibody Hetuximab and the parent mouse The monoclonal antibody Hetumomab has the same pharmacodynamic effect of inhibiting tumor stem cells in vitro.
  • the design, gene synthesis, cloning, and transfection of eukaryotic cells were used to perform the transformation and humanization of the glycosylation site of Hetumomab. Makeover.
  • H1 heavy chain sequence two other heavy chain mutants were designed for initial humanization: H2 and H3.
  • the degree of humanization was in order from high to low.
  • the specific sequence is shown in Figure 16.
  • H1, H2, H3 all three heavy chain mutants (H1, H2, H3) retained the affinity of the chimeric antibody Hetuximab. Since H1 is the most humanized, and the framework region is all humanized, H1 was finally determined as the preferred humanized molecular template.
  • the ELISA method was used to analyze the affinity and found that the phenylalanine Phe (F9 and F42) at positions 9 and 42 of the light chain must be restored, and the affinity of the sixth mutant (L6) of these two mouse-derived molecules was restored.
  • the affinity of L11 based on the deep humanized version of L6 is close to that of L6, and the affinity of humanized L12 based on the 11th Asn of the light chain is also similar to that of L11, but other mutant versions are different. Therefore, L6, L11, and L12 were finally selected for optimization screening of the best light chain humanized version.
  • glycosylated isomers were confirmed in Hetuzumab H1 heavy chain by antibody sequence analysis and reduction electrophoresis analysis.
  • H1L6, H1L11, and H1L12 all have visible weak sub-bands below the main chain of the heavy chain.
  • the analysis indicates that the heavy chain may have heterogeneous glycosylation and have glycosylation isomerism.
  • Body Figure 18. This will cause serious quality uniformity problems, which is very bad for drug development. Sequence analysis showed that N62 (located in CDR2) and N107 (located in CDR3) were two possible glycosylation sites.
  • N62A is H4 (the CDR2 sequence is shown in SEQ ID NO: 13), and N107A is H5 (the CDR3 sequence Shown in SEQ ID NO: 14).
  • H4 and H5 were combined with L11, respectively, and analyzed by electrophoresis after expression. It was found that the glycosylation isomerism of H5 (H1N107A) disappeared, and that H5L11 basically maintained approximately equivalent affinity by cell ELISA method, and the glycosylation isomerism of H4 (H1N62A) still existed and the affinity decreased ( Figure 19A).
  • H5 H1 N107A
  • H1 FR fully humanized heavy chain
  • H5L6, H5L11, H5L12 three candidate humanized combinations (H5L6, H5L11, H5L12) were selected to prepare purified antibodies.
  • the H5L6, H5L11, and H5L12 light and heavy chain coding sequences were cloned into a eukaryotic high-efficiency expression stable vector, and the CHO-K1 (ATCC CCL-64) cells that had been domesticated in suspension culture were electrotransfected.
  • the stable expression strains were obtained by MSX screening. Protein CD-CHO medium (Invitrogen) was fed-batch cultured.
  • the supernatant obtained by the culture was purified by a Protein A (GE, Mabselecture column) affinity chromatography column, and impurities and impurities were further removed by a cation exchange column.
  • GE Protein A
  • HPLC analysis the purity of the product was greater than 95% (the results are shown in Figure 20, shown with H5L11 as an example), and the endotoxin level was ⁇ 3EU / mg. It can be used for various pharmacodynamic experiments and monkey pharmacokinetic analysis tests.
  • Example 11 The three variants of the humanized antibody Hetuzumab (H5L6, H5L11, H5L12) all recognize the same epitope that binds to the same antigen protein as the parental mouse monoclonal antibody.
  • the experimental results showed that three variants of the humanized antibody Hetuzumab (H5L6, H5L11, H5L12), the chimeric antibody Hetuximab, and the parental mouse monoclonal antibody Hetumomab were able to stain the 10 cases of human liver cancer and lung cancer positively expressed by the monoclonal antibody Hetumomab Tissue sections of gastric cancer patients, and at the same time did not stain the negative sections of the monoclonal antibody Hetumomab target antigen in 10 cases of human liver cancer, lung cancer, and gastric cancer patients, illustrating the three variants of the humanized antibody Hetuzumab (H5L6, H5L11, H5L12) Both the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab recognize and bind to the same antigen protein, and do not recognize other proteins in human tissues, and have the same specificity.
  • Inoculate a 96-well culture plate (MHCC-97L cells, 4 ⁇ 10 3 cells / well), and perform experiments when the cells reach 90% to 100% full. Discard the culture medium in the wells, wash with 0.05% Tween-20 in PBS, 300 ⁇ l / well, 1 min / times ⁇ 5 times; add samples, 100 ⁇ l / well, re-well, and incubate at 37 ° C for 1.5 h; discard the wells Liquid, washed with PBS containing 0.05% Tween-20, 300 ⁇ l / well, 1 min / times ⁇ 5 times; added anti-human IgG Fc-HRP (does not react with mouse IgG Fc) or anti-mouse IgG Fc-HRP (not with Human IgG Fc reaction) Corresponding secondary antibody, 1: 5000, 100 ⁇ l / well, incubate for 1 h at 37 ° C; discard the liquid in the well, wash with PBS containing
  • Each humanized monoclonal antibody was constant at 0.25 ug / ml, and the murine monoclonal antibody concentration gradient was used to compete to inhibit the binding of each humanized monoclonal antibody to the antigen.
  • the calculated IC50 of the mouse monoclonal antibody is shown in Table 30 below.
  • the parental mouse monoclonal antibody Hetumomab can significantly inhibit the three variants of Hetuzumab (H5L6, H5L11, H5L12), the binding of the chimeric antibody Hetuximab to the target antigen on the surface of target cells MHCC-97L cells, indicating that The three variants (H5L6, H5L11, H5L12), the chimeric antibody Hetuximab, and the parental mouse monoclonal antibody Hetumomab recognize the same epitope that binds to the same antigenic protein.
  • the two variants of Hetuzumab showed similar degrees of competition inhibition to each other, which was slightly higher than the chimeric antibody Hetuximab, while one variant of Hetuzumab (H5L12) showed significantly higher High degree of competition suppression.
  • the affinity of the chimeric antibody Hetuximab and the parental mouse monoclonal antibody Hetumomab are comparable, while the affinity of the two variants of the humanized antibody Hetuzumab (H5L6, H5L11) is slightly reduced, but overall, they have the same affinity; and The affinity of the humanized antibody Hetuzumab variant H5L12 has decreased.
  • the murine monoclonal antibody was 0.25 ug / ml constant.
  • the concentration gradient of each humanized monoclonal antibody was used to compete to inhibit the binding of the murine monoclonal antibody to the antigen.
  • the IC50 of each humanized monoclonal antibody was calculated.
  • the three variants of Hetuzumab (H5L6, H5L11, H5L12) and the chimeric antibody Hetuximab can significantly compete to inhibit the binding of the parental mouse monoclonal antibody Hetumomab to the target antigen on the surface of target cells MHCC-97L cells, indicating that The three variants of Hetuzumab (H5L6, H5L11, H5L12), the chimeric antibody Hetuximab, and the parental mouse monoclonal antibody Hetumomab recognize the same epitope that binds to the same antigen protein; at the same time, the two variants of Hetuzumab (H5L6, H5L11) show each other The similar competitive inhibition efficiency was slightly lower than that of the chimeric antibody Hetuximab, while a variant of Hetuzumab (H5L12) showed a lower competitive inhibition efficiency.
  • the binding of the target antigen indicates that three variants of the humanized antibody Hetuzumab (H5L6, H5L11, H5L12), the chimeric antibody Hetuximab, and the parental mouse monoclonal antibody Hetumomab recognize the same epitope that binds the same antigen protein; at the same time (H5L6, H5L11, Hetuximab, Hetumomab) showed similar competitive inhibition relationship with each other, suggesting that H5L6, H5L11 and mouse monoclonal antibody Hetumomab have consistent affinity.
  • Example 12 The variants of the humanized antibody Hetuzumab all recognize the same tumor stem cells as the parental monoclonal antibody, and have the same pharmacodynamic effect of inhibiting tumor stem cells in vitro and in vivo.
  • Hetuzumab H5L6, H5L11, H5L12
  • Flow cytometry was used to demonstrate that the three variants of the humanized antibody Hetuzumab (H5L6, H5L11, H5L12) recognized the same group of tumor stem cells as the parental mouse monoclonal antibody Hetumomab.
  • the three variants of the humanized antibody Hetuzumab H5L6, H5L11, H5L12
  • the chimeric antibody Hetuximab the parental mouse monoclonal antibody Hetumomab were detected in four tumor cells and tumor stem cell-rich sphere cells.
  • the positive rate and enrichment were very consistent, especially Hetuzumab H5L6 and Hetuzumab H5L11. Therefore, the three variants of Hetuzumab (H5L6, H5L11, H5L12), the chimeric antibody Hetuximab, and the parental mouse monoclonal antibody Hetumomab recognized the same group of tumor stem cells.
  • tumor stem cell-rich sphere cells SNU-5, BGC-823, MHCC-97L, and BEL7402V13, were used.
  • different variants of Hetuzumab, Hetuximab, and Hetumomab were used for routine formation.
  • the sphere inhibition test was used to detect the pharmacological effects of tumor stem cells on sphere cells rich in tumor stem cells in the above four cell lines. The results of the four cell lines are as follows:
  • the variant of the humanized antibody Hetuzumab significantly inhibits the growth of human liver cancer transplanted tumors in the body, and can significantly enhance the efficacy of chemotherapy in synergy, and has a significant anti-tumor pharmacodynamic effect.
  • sphere cells of the human liver cancer cell line Be17402-V13 were harvested and subcutaneously in nude mice at 30,000 cells / cell.
  • Antibody treatment was started on the second day after the cancer cells were inoculated.
  • the experimental group and the control group were treated by intraperitoneal injection.
  • the chemotherapeutic group was treated for 4 weeks and discontinued after 2 times a week.
  • the long and short diameters of subcutaneously transplanted tumors were measured twice a week to calculate the tumor volume.
  • mice When the treatment was stopped 36 days after inoculation, the tumor volume growth of the mice was observed and measured, and the inhibition rate was calculated.
  • the growth curve of transplanted tumors in mice is shown in Fig. 22, and the tumor volume inhibition rate is shown in Table 34.
  • Humanized monoclonal antibody Hetuzumab H5L11 can significantly inhibit the growth of human liver cancer xenografts in nude mice, and the inhibition rate of xenografts also increases with the increase of antibody dose.
  • the inhibitory rates of high and low doses of humanized monoclonal antibody Hetuzumab H5L11 on transplanted tumors were 47.47% and 36.80%, respectively.
  • the inhibition rate of the chemotherapeutic group was only 14.52%, which was almost ineffective.
  • the inhibition rates of the humanized monoclonal antibody Hetuzumab H5L11 high and low dose combined chemotherapy group reached 65.08% and 32.66%, respectively.
  • the results suggest that the inhibitory rate of the humanized monoclonal antibody Hetuzumab H5L11 combined with the chemotherapeutic group on mouse transplantation tumors is higher than that of the chemotherapy alone and antibody alone treatment group, p ⁇ 0.05, and the humanized monoclonal antibody Hetuzumab combined with the H5L11 combined chemotherapy group In the treatment of transplanted tumors, a better therapeutic effect has been shown.
  • humanized monoclonal antibody Hetuzumab H5L11 alone can significantly inhibit the growth of human malignant tumors and has a significant pharmacodynamic effect on human malignant tumors.
  • the humanized monoclonal antibody Hetuzumab H5L11 combined with the chemotherapeutic group showed the highest inhibition rate of the transplanted tumor, which can significantly inhibit the growth of the tumor, and the treatment effect was better than that of the antibody alone group and the chemotherapeutic group alone, indicating that humanization Monoclonal antibody Hetuzumab H5L11 combined with chemotherapeutics can effectively inhibit tumor growth and reduce chemotherapy resistance.

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Abstract

提供一种针对人肿瘤干细胞的分离的单克隆抗体或其抗原结合片段，以及所述抗体或片段在肿瘤治疗和诊断中的用途。

Description

一种靶向人肿瘤干细胞的单克隆抗体及其应用 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及一种针对人肿瘤干细胞的分离的单克隆抗体或其抗原结合片段，以及所述抗体或片段在肿瘤治疗和诊断中的用途。
背景技术
恶性肿瘤(癌症)已成为威胁全世界人民生命与健康的“头号杀手”。全世界每年肿瘤的发病人数超过1400万，仅在我国每年新增的肿瘤患者超过300万。
导致癌症高死亡率的根本原因是癌细胞的扩散、转移和治疗后多数患者易复发和耐药。临床上现有的治疗手段，手术、放疗、化疗对癌细胞转移、复发、耐药疗效甚微，或只有近期疗效，并不能改变患者长期的存活情况。目前，手术切除对大约10-20％的早期患者效果好，但对已发生扩散转移的患者几乎无效。放疗只能治疗局部病灶，常作为术前、术后的辅助治疗和少数种类癌症的根治性治疗。化疗可用于已发生扩散转移的患者，但因毒副作用大，容易产生近期或远期的耐药，因而只能对大约20-30％的患者有明显的近期疗效。即使采用手术、放疗和化疗联合应用的综合治疗措施，其生存5年的远期疗效多年一直徘徊在20-30％，约70-80％的患者在治疗后因转移、复发和耐药在5年内死亡。即便是就诊时无转移的早期癌症患者，也还是有一部分在治疗后发生转移复发而死亡。近年发展起来的肿瘤的新型靶向药物，包括多肽、小分子、蛋白因子、基因治疗及抗体药物，与化疗药联合应用通常也只能相对现有治疗手段延长患者生存期3～9个月，对远期患者的5年生存率没有显著的提高。最近两年新兴起的肿瘤免疫治疗手段，例如针对免疫检查点的PD-1单抗药物以及CAR-T细胞疗法，虽然已经表现出可取得一些令人鼓舞的远期疗效的迹象，但总体对癌症患者的有效率仅能达到20-30％左右，仍然有大量的癌症患者得不到真正有效的药物的救治。因此提高肿瘤患者远期疗效、延长生存期的关键是开发抑制肿瘤转移、复发、耐药的新型药物。
发明概述
在一方面，本发明提供了一种针对肿瘤干细胞的分离的单克隆抗体或其抗原结合片段，其中所述单克隆抗体包含轻链可变区和重链可变区，
所述轻链可变区包含：
VL CDR1，其包含SEQ ID NO：2所示氨基酸序列或相对于SEQ ID NO：2具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，
VL CDR2，其包含SEQ ID NO：3所示氨基酸序列或相对于SEQ ID NO：3具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，和
VL CDR3，其包含SEQ ID NO：4所示氨基酸序列或相对于SEQ ID NO：4具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列；
所述重链可变区包含：
VH CDR1，其包含SEQ ID NO：6所示氨基酸序列或相对于SEQ ID NO：6具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，
VH CDR2，其包含SEQ ID NO：7所示氨基酸序列或相对于SEQ ID NO：7具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列例如SEQ ID NO：13所示序列，和
VH CDR3，其包含SEQ ID NO：8所示氨基酸序列或包含相对于SEQ ID NO：8具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列例如SEQ ID NO：14所示序列。
在一些实施方案中，其中所述单克隆抗体包含轻链可变区和重链可变区，
所述轻链可变区包含：
VL CDR1，其包含SEQ ID NO：2所示氨基酸序列，
VL CDR2，其包含SEQ ID NO：3所示氨基酸序列，和
VL CDR3，其包含SEQ ID NO：4所示氨基酸序列；
所述重链可变区包含：
VH CDR1，其包含SEQ ID NO：6所示氨基酸序列，
VH CDR2，其包含SEQ ID NO：13所示氨基酸序列，和
VH CDR3，其包含SEQ ID NO：8所示氨基酸序列。
在一些实施方案中，其中所述单克隆抗体包含轻链可变区和重链可变区，
所述轻链可变区包含：
VL CDR1，其包含SEQ ID NO：2所示氨基酸序列，
VL CDR2，其包含SEQ ID NO：3所示氨基酸序列，和
VL CDR3，其包含SEQ ID NO：4所示氨基酸序列；
所述重链可变区包含：
VH CDR1，其包含SEQ ID NO：6所示氨基酸序列，
VH CDR2，其包含SEQ ID NO：7所示氨基酸序列，和
VH CDR3，其包含SEQ ID NO：14所示氨基酸序列。
在一些实施方案中，所述轻链可变区包含SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1具有至少85％、至少90％、至少95％或更高序列相同性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO：5所示氨基酸序列或与SEQ ID NO：5具有至少85％、至少90％、至少95％或更高序列相同性的氨基酸序列。
在一些实施方式中，所述重链可变区包含SEQ ID NO：15-19之一所示氨基酸序列(分别对应人源化重链版本H1-H5的可变区)。
在一些实施方式中，所述轻链可变区包含SEQ ID NO：20-31之一所示的氨基酸序列(分别对应人源化轻链版本L1-L12的可变区)。
在一些实施方式中，所述重链可变区包含SEQ ID NO：19所示的氨基酸序列，且所 述轻链可变区包含SEQ ID NO：25所示氨基酸序列。
在一些实施方式中，所述重链可变区包含SEQ ID NO：19所示的氨基酸序列，且所述轻链可变区包含SEQ ID NO：30所示氨基酸序列。
在一些实施方式中，所述单克隆抗体包含人重链恒定区，例如，包含SEQ ID NO：11所示的氨基酸序列的人重链恒定区。
在一些实施方式中，所述单克隆抗体包含人轻链恒定区，例如，包含SEQ ID NO：12所示的氨基酸序列的人轻链恒定区。
在一些实施方案中，所述单克隆抗体包含具有SEQ ID NO：9所示氨基酸序列的重链和具有SEQ ID NO：10所示氨基酸序列的轻链。
在一些实施方式中，所述单克隆抗体包含具有SEQ ID NO：32-36之一所示的氨基酸序列重链(分别对应人源化重链版本H1-H5)和具有SEQ ID NO：37-48之一所示的氨基酸序列的轻链(分别对应人源化轻链版本L1-L12)。
在一些实施方式中，所述单克隆抗体包含SEQ ID NO：36所示的重链和SEQ ID NO：47所示的轻链(对应于人源化抗体H5L11)。
在一些实施方式中，所述单克隆抗体包含SEQ ID NO：36所示的重链和SEQ ID NO：42所示的轻链(对应于人源化抗体H5L6)。
在另一方面，本发明提供了一种单克隆抗体或其抗原结合片段，所述单克隆抗体由于2016年3月16日以保藏号CGMCC No.12251保藏于中国微生物菌种保藏管理委员会普通微生物中心的小鼠杂交瘤细胞产生。
在另一方面，本发明提供了一种杂交瘤细胞，其以保藏号CGMCC No.12251于2016年3月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
在另一方面，本发明提供了一种药物组合物，其包含本发明的单克隆抗体或其抗原结合片段以及药学上可接受的载体。
在一些实施方案中，所述单克隆抗体或其抗原结合片段与选自细胞毒素、放射性同位素或生物活性蛋白质的治疗性部分缀合。
在另一方面，本发明提供了一种在患者中治疗恶性肿瘤、预防和/或治疗恶性肿瘤转移或复发的方法，所述方法包括给所述患者施用有效量的本发明的单克隆抗体或其抗原结合片段或本发明的药物组合物。
在一些实施方案中，所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
在一些实施方案中，所述方法还包括给所述患者施用其它抗肿瘤治疗手段，例如施用化疗剂、靶向其它肿瘤特异性抗原的抗体或放疗。
在另一方面，本发明提供了本发明的单克隆抗体或其抗原结合片段或本发明的药物组合物在制备用于治疗恶性肿瘤、预防和/或治疗恶性肿瘤转移或复发的药物中的用途。
在一些实施方案中，所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
在另一方面，本发明提供了一种检测生物学样品中肿瘤干细胞存在的方法，包括：
a)使所述生物学样品与本发明的单克隆抗体或其抗原结合片段接触；
b)检测本发明的单克隆抗体或其抗原结合片段与所述生物学样品中的靶抗原的结合，其中检出所述结合代表所述生物学样品中存在肿瘤干细胞。
在另一方面，本发明还提供一种用于分离肿瘤干细胞的方法，所述方法包括：
(a)提供疑似包含肿瘤干细胞的细胞群；
(b)鉴定所述细胞的亚群，其结合本发明的单克隆抗体或其抗原结合片段；和
(c)分离所述亚群。
在前述各方面的一些实施方案中，所述肿瘤干细胞选自乳腺癌干细胞、大肠癌干细胞、胰腺癌干细胞、前列腺癌干细胞、肝癌干细胞、肺癌干细胞和胃癌干细胞。
在另一方面，本发明还提供了一种检测患者中恶性肿瘤的存在的方法，包括：
a)使获得自所述患者的生物学样品与本发明的单克隆抗体或其抗原结合片段接触；
b)检测本发明的单克隆抗体或其抗原结合片段与所述生物学样品中的靶抗原的结合，其中检出代表所述患者中存在恶性肿瘤。
在另一方面，本发明还提供一种用于预后患者中恶性肿瘤复发或进展的方法，所述方法包括：
(a)从所述患者中分离包含循环细胞的生物学样品；
(b)使所述包含循环细胞的生物学样品与本发明的单克隆抗体或其抗原结合片段接触；和
(c)鉴定结合本发明的单克隆抗体或其抗原结合片段的循环细胞的存在，
从而预后所述患者中恶性肿瘤的复发或进展。
在一些实施方案中，所述恶性肿瘤的进展包含所述恶性肿瘤在患者中的转移。
在前述各方面的一些实施方案中，所述生物学样品包括血液样品、淋巴样品或其组分。在一些实施方案中，所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
在另一方面，本发明还提供一种分离的核酸分子，其编码本发明的单克隆抗体或其抗原结合片段。
在一些实施方案中，所述核酸分子与表达调控序列可操作地连接。
在另一方面，本发明还提供一种表达载体，其包含本发明的核酸分子。
在另一方面，本发明还提供一种宿主细胞，其由本发明的核酸分子或本发明的表达载体转化。
在另一方面，本发明还提供一种生产针对人肿瘤干细胞的单克隆抗体或其抗原结合片段的方法，包括：
(i)在适合本发明的核酸分子或表达载体表达的情况下培养本发明的宿主细胞，和
(ii)分离并纯化由所述核酸分子或表达载体表达的抗体或其抗原结合片段。
附图说明
图1.活细胞免疫荧光技术检测单抗Hetumomab靶抗原在多种肿瘤细胞活细胞表面的表达(部分典型阳性结果)。
图2.免疫组化检测单抗Hetumomab靶抗原在人肝癌、肺癌、胃癌组织中特异高表达(部分典型阳性结果)。
图3.免疫流式荧光检测单抗Hetumomab识别的癌细胞在人多种肿瘤细胞系的sphere培养细胞中均显著富集(部分典型的流式荧光图谱)。
图4.CCK8法检测单抗Hetumomab识别的多种人肿瘤细胞(如肝癌、肺癌、胃癌)的Hetumomab+细胞的耐药能力(IC50)。
图5.单抗Hetumomab显著抑制多种肿瘤(如肝癌、肺癌、胃癌)的肿瘤干细胞的自我更新能力(成球)。
图6.单抗Hetumomab显著抑制多种肿瘤(如肝癌、肺癌、胃癌)的肿瘤干细胞的侵袭能力。
图7.单抗Hetumomab显著抑制多种肿瘤的肿瘤干细胞的侵袭能力。
图8.单抗Hetumomab及联合化疗药物治疗人肝癌移植瘤Be17402-V13的体内肿瘤生长曲线。
图9.单抗Hetumomab及联合化疗药物治疗人肝癌移植瘤Be17402-V13的体内肿瘤体积抑制率(停药时)。
图10.单抗Hetumomab及联合化疗药物治疗人肝癌移植瘤Be17402-V13的体内肿瘤体积抑制率(停药一个月后)。
图11.单抗Hetumomab及联合化疗药物治疗人肝癌移植瘤Be17402-V13的小鼠的生存曲线。
图12.单抗Hetumomab治疗人肺癌移植瘤SPCA-1的体内肿瘤生长曲线。
图13.单抗Hetumomab及联合化疗药物治疗人胃癌移植瘤SNU-5的体内肿瘤生长曲线。
图14.示出嵌合抗体Hetuximab与亲本抗体Hetumomab结合肿瘤干细胞上相同的抗原蛋白。
图15.示出嵌合抗体Hetuximab与亲本抗体Hetumomab相互竞争与抗原的结合。
图16.示出Hetumomab人源化重链各版本氨基酸序列比对(部分)，Hetumomab对应缩写为E21。
图17.示出Hetumomab人源化轻链各版本氨基酸序列比对(部分)，Hetumomab对应缩写为E21。
图18.电泳分析显示H1重链系列表达物存在糖基化不均一性。
图19.电泳分析显示A：H5的糖基化异构现象消失；B：H5与L11、L6组合糖基化异构现象均消失。
图20.示出在CHO细胞表达并纯化获得纯度大于95％的人源化抗体H5L11。A： 还原型SDS-PAGE电泳分析显示纯化抗体纯度大于95％；B：非还原型SDS-PAGE电泳分析显示纯化抗体纯度大于95％；C：HPLC分析显示纯化抗体纯度大于95％。
图21.示出亲本鼠单抗Hetumomab、嵌合抗体Hetuximab和人源化单抗Hetuzumab竞争结合抗原。A：不同浓度亲本鼠单抗Hetumomab竞争抑制Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab与抗原结合；B：不同浓度Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab竞争抑制亲本鼠单抗Hetumomab与抗原结合。
图22.单抗Hetuzumab H5L11及联合化疗药物治疗人肝癌移植瘤的小鼠体内肿瘤生长曲线。
发明详述
二、定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，“抗体”指免疫球蛋白和免疫球蛋白片段，无论天然的或者部分或全部合成(例如重组)产生的，包括其至少包含免疫球蛋白分子的部分可变区的保留全长免疫球蛋白的结合特异性能力的任何片段。因此，抗体包括具有与免疫球蛋白抗原结合结构域(抗体结合位点)同源或基本上同源的结合结构域的任何蛋白。抗体包括抗体片段，例如抗肿瘤干细胞抗体片段。如本文所用，因此术语抗体包括合成抗体、重组产生的抗体、多特异性抗体(例如双特异性抗体)、人抗体、非人抗体、人源化抗体、嵌合抗体、胞内抗体以及抗体片段，例如但不限于Fab片段、Fab′片段、F(ab’) 2片段、Fv片段、二硫键连接的Fv(dsFv)、Fd片段、Fd’片段、单链Fv(scFv)、单链Fab(scFab)、双抗体、抗独特型(抗Id)抗体、或者上述任何抗体的抗原结合片段。本文所提供的抗体包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)的成员。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab′、F(ab’) 2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd’片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207：Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25， Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M -1的Ka)抗原的抗体。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57，912-917；和Nelson et al.，J Clin Pathol(2000)，53，111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的(见例如，Harlow & Lane，1988)。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，“常规抗体”指包含两条重链(其可以标示为H和H’)和两条轻链(其可以标示为L和L’)和两个抗原结合位点的抗体，其中每条重链可以是全长免疫球蛋白重链或保留抗原结合能力的其任何功能区(例如重链包括但不限于V H链、V H-C H1链和V H-C H1-C H2-C H3链)，并且每条轻链可以是全长轻链或任何功能区(例如轻链包括但不限于V L链和V L-C L链)。每条重链(H和H’)与一条轻链(分别为L和L’)配对。
如本文所用，全长抗体是具有两条全长重链(例如V H-C H1-C H2-C H3或V H-C H1-C H2-C H3-C H4)和两条全长轻链(V L-C L)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
如本文所用，dsFv指具有稳定V H-V L对的工程化分子间二硫键的Fv。
如本文所用，Fab片段是用木瓜蛋白酶消化全长免疫球蛋白所获得的抗体片段，或者例如通过重组方法合成产生的具有相同结构的片段。Fab片段包含轻链(包含V L和C L)和另一条链，所述另一条链包含重链的可变结构域(V H)和重链的一个恒定区结构域(C H1)。
如本文所用，F(ab’) 2片段是在pH 4.0-4.5下用胃蛋白酶消化免疫球蛋白所导致的抗体片段，或者例如通过重组方法合成产生的具有相同结构的片段。F(ab’) 2片段基本上包含两个Fab片段，其中每个重链部分包含额外的几个氨基酸，包括形成连接两个片段的二硫键的半胱氨酸。
如本文所用，Fab’片段是包含F(ab’) 2片段的一半(一条重链和一条轻链)的片段。
如本文所用，scFv片段指包含通过多肽接头以任何顺序共价连接的可变轻链(V L)和 可变重链(V H)的抗体片段。接头长度使得两个可变结构域基本不干扰地桥接。示例性接头是分散有一些Glu或Lys残基以增加溶解性的(Gly-Ser) n残基。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab′、F(ab′) 2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本发明所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，可变结构域或可变区是抗体重链或轻链的特定Ig结构域，其包含在不同抗体之间变化的氨基酸序列。每条轻链和每条重链分别具有一个可变区结构域V L和V H。可变结构域提供抗原特异性，并且因此负责抗原识别。每个可变区包含CDR和框架区(FR)，CDR是抗原结合位点结构域的部分。
如本文所用，“抗原结合结构域”和“抗原结合位点(antigen-binding site)”同义地用来指识别并与同种(cognate)抗原物理相互作用的抗体内的结构域。天然的常规全长抗体分子具有两个常规抗原结合位点，每个包含重链可变区部分和轻链可变区部分。常规抗原结合位点包含连接可变区结构域内反向平行的β链的环。抗原结合位点可以包含可变区结构域的其他部分。每个常规抗原结合位点包含3个来自重链的高变区和3个来自轻链的高变区。高变区也称为互补决定区(CDR)。
如本文所用，“高变区”、“HV”、“互补决定区”和“CDR”和“抗体CDR”可交换地用来指一起形成抗体的抗原结合位点的每个可变区内的多个部分中的一个。每个可变区结构域包含3个CDR，命名为CDR1、CDR2和CDR3。例如，轻链可变区结构域包含3个CDR，命名为VL CDR1、VL CDR2和VL CDR3；重链可变区结构域包含3个CDR，命名为VH CDR1、VH CDR2和VH CDR3。可变区中的3个CDR沿线性氨基酸序列是不连续的，但是在折叠的多肽中接近。CDR位于连接可变结构域的β折叠的平行链的环内。如本文所述，本领域技术人员知道并且可以基于Kabat或Chothia编号鉴定CDR(参见例如，Kabat，E.A.et al.(1991)Sequences of Proteins of Immunological Interest，Fifth  Edition，U.S.Department of Health and Human Services，NIH Publication No.91-3242，和Chothia，C.et al.(1987)J.Mol.Biol.196：901-917)。
如本文所用，框架区(FR)是位于β折叠内的抗体可变区结构域内的结构域；在氨基酸序列方面，FR区比高变区相对更保守。
如本文所用，“恒定区”结构域是抗体重链或轻链中的结构域，其包含比可变区结构域的氨基酸序列相对更保守的氨基酸序列。在常规全长抗体分子中，每条轻链具有单个轻链恒定区(CL)结构域，而每条重链包含一个或多个重链恒定区(C H)结构域，包括C H1、C H2、C H3和C H4。全长IgA、IgD和IgG同种型包含C H1、C H2、C H3和铰链区，而IgE和IgM包含C H1、C H2、C H3和C H4。C H1和C L结构域延伸抗体分子的Fab臂，因此有助于与抗原相互作用以及转动抗体臂。抗体恒定区可以服务于效应子功能，例如但不限于清除该抗体特异性结合的抗原、病原体和毒素，例如通过与各种细胞、生物分子和组织相互作用。
如本文所用，抗体的功能区是包含该抗体的至少V H、V L、C H(例如C H1、C H2或C H3)、C L或铰链区结构域或者至少其功能区的抗体部分。
如本文所用，V H结构域的功能区是保留完整V H结构域的至少部分结合特异性(例如通过保留完整V H结构域的一个或多个CDR)的完整V H结构域的至少一部分，从而所述V H结构域的功能区单独地或者与另一抗体结构域(例如V L结构域)或其区域组合地结合抗原。示例性V H结构域的功能区是包含V H结构域的CDR1、CDR2和/或CDR3的区域。
如本文所用，V L结构域的功能区是保留完整V L结构域的至少部分结合特异性(例如通过保留完整V L结构域的一个或多个CDR)的完整V L结构域的至少一部分，从而所述V L结构域的功能区单独地或者与另一抗体结构域(例如V H结构域)或其区域组合地结合抗原。示例性V L结构域的功能区是包含V L结构域的CDR1、CDR2和/或CDR3的区域。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1x 10 8M -1或更大的亲和常数Ka(或者1x 10 -7M或1×10 -8M或更低的解离常数(K d))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11：54；Englebienne(1998)Analyst.123：1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定(参见，例如，Paul，ed.，Fundamental Immunology，2nd ed.，Raven Press，New York，pages 332-336(1989)；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7,229,619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000，Biacore AB，Upsala，Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27：335)。
如本文所用，关于抗体的术语“竞争”是指第一抗体或其抗原结合片段以与第二抗体或其抗原结合片段足够相似的方式结合一个表位，由此第一抗体与其关联表位的结合结果在存在第二抗体的条件下与不存在第二抗体的条件下相比可检测地降低。或者，在第二抗体与其表位的结合在存在第一抗体条件下也可检测地降低的情况中，可以但不必需是这种情况。也就是说，第一抗体可以抑制第二抗体与其表位的结合，而不用第二抗体抑制第一抗体与其各自表位的结合。然而，在每个抗体均可检测地抑制另一抗体与其关联表位或配体的结合的情况中，无论是相同、更高或更低程度，所述抗体被称为彼此“交叉竞争”结合其各自的表位。竞争及交叉竞争抗体均涵盖在本发明中。无论这种竞争或交叉竞争发生的机制如何(例如位阻、构象改变或者结合共同表位或其片段)，本领域技术人员基于本发明提供的教导将意识到这种竞争和/或交叉竞争抗体涵盖在本发明中且可用于本发明揭示的方法中。
如本文所用，“多肽”指共价连接的两个或更多个氨基酸。术语“多肽”和“蛋白质”在本文中可交换使用。
″分离的蛋白质″、″分离的多肽″或″分离的抗体″指所述蛋白质、多肽或抗体(1)不与在其天然状态下伴随其天然相关成分关联，(2)不含来自相同物种的其它蛋白质，(3)由来自不同物种的细胞表达，或(4)不在天然中发生。因此，经化学合成的多肽或在不同于多肽的天然来源细胞的细胞系统中合成的多肽将会与其天然相关成分″分离″。还可通过分离以使蛋白质实质上不含天然相关成分，即使用本领域众所周知的蛋白质纯化技术。
在肽或蛋白中，合适的保守氨基酸取代是本领域技术人员已知的，并且一般可以进行而不改变所得分子的生物活性。通常，本领域技术人员认识到多肽的非必需区中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson et al.，Molecular Biology of the Gene，4th Edition，1987，The Benjamin/Cummings Pub.co.，p.224)。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。
序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。(参见，例如：Computational Molecular Biology，Lesk，A.M.，ed.，Oxford University Press，New York，1988；Biocomputing：Informatics and Genome Projects，Smith，D.W.，ed.，Academic Press，New York，1993；Computer Analysis of Sequence Data，Part I，Griffin，A.M.，and Griffin，H.G.，eds.，Humana Press，New Jersey，1994；Sequence  Analysis in Molecular Biology，von Heinje，G.，Academic Press，1987；and Sequence Analysis Primer，Gribskov，M.and Devereux，J.，eds.，M Stockton Press，New York，1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo，H.& Lipman，D.，SIAM J Applied Math 48：1073(1988))。
如本文所用，关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而所述启动子调控或介导所述核酸的转录。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
“密码子优化”是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个或更多个密码子同时维持该天然氨基酸序列而修饰核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此，可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在 www.kazusa.orjp/codon/上可获得的密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。参见，Nakamura Y.等，“Codon usage tabulated from the international DNA sequence databases：status for the year2000.Nucl.Acids Res.，28：292(2000)。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”是指哺乳动物，例如人。
“肿瘤干细胞”是指存在于肿瘤组织中的一小部分具有干性特征的癌细胞，其与普通癌细胞相比具有自我更新能力、强侵袭能力、耐受化疗药物能力和强致瘤能力。
二、针对肿瘤干细胞的单克隆抗体
在本发明中，采用人肿瘤多能干细胞系作为免疫原免疫小鼠，通过经典的杂交瘤融合技术获得了单克隆抗体Hetumomab。产生单克隆抗体Hetumomab的小鼠杂交瘤细胞株Hetumomab于2016年3月16日以保藏号CGMCC No.12251保藏于中国微生物菌种保藏管理委员会普通微生物中心。(实施例1)
因此，本发明提供了一种单克隆抗体或其抗原结合片段，所述单克隆抗体由于2016年3月16日以保藏号CGMCC No.12251保藏于中国微生物菌种保藏管理委员会普通微生物中心的小鼠杂交瘤细胞产生。
本发明人发现，本发明的单克隆抗体Hetumomab的靶抗原表达于多种人肿瘤细胞的活细胞表面，并且在多种肿瘤组织中特异性高表达(阳性率79％-94％)。本发明的 Hetumomab单抗能够在多种肿瘤细胞的sphere培养细胞中富集且能够识别ESA、CD90等肿瘤干细胞标志物的肿瘤细胞，提示Hetumomab单抗是特异性针对肿瘤干细胞的单克隆抗体(实施例2)。
进一步的基于Hetumomab靶抗原阳性肿瘤细胞的研究显示，相对于亲本肿瘤细胞和Hetumomab靶抗原阴性肿瘤细胞，Hetumomab靶抗原阳性肿瘤细胞具有更强的自我更新、侵袭、耐药和体内致瘤能力，进一步证明Hetumomab单抗特异性靶向肿瘤干细胞(实施例3)。本发明进一步鉴定了Hetumomab单抗的轻链可变区和重链可变区序列以及相应的CDR序列(实施例6)。将所述可变区序列分别与人轻链和重链恒定区组合，构建了人-鼠嵌合抗体Hetuximab(实施例7)。实验证明，嵌合抗体Hetuximab和小鼠抗体Hetumomab结合肿瘤干细胞上相同的抗原表位，具有类似的抑制肿瘤干细胞的药效学作用(实施例8-9)。
本发明进一步对Hetumomab单抗进行人源化，获得人源化抗体Hetuzumab的多种变体(实施例10)。实验证明，人源化抗体Hetuzumab和小鼠抗体Hetumomab以及嵌合抗体Hetuximab结合肿瘤干细胞上相同的抗原表位，具有类似的抑制肿瘤干细胞的药效学作用(实施例11-12)。
因此，本发明提供针对肿瘤干细胞的分离的单克隆抗体或其抗原结合片段，其中所述单克隆抗体包含轻链可变区和重链可变区，
所述轻链可变区包含：
VL CDR1，其包含SEQ ID NO：2所示氨基酸序列或相对于SEQ ID NO：2具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，
VL CDR2，其包含SEQ ID NO：3所示氨基酸序列或相对于SEQ ID NO：3具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，和
VL CDR3，其包含SEQ ID NO：4所示氨基酸序列或相对于SEQ ID NO：4具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列；
所述重链可变区包含：
VH CDR1，其包含SEQ ID NO：6所示氨基酸序列或相对于SEQ ID NO：6具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，
VH CDR2，其包含SEQ ID NO：7所示氨基酸序列或相对于SEQ ID NO：7具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列例如SEQ ID NO：13所示序列，和
VH CDR3，其包含SEQ ID NO：8所示氨基酸序列或包含相对于SEQ ID NO：8具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列例如SEQ ID NO：14所示序列。
在一些实施方式中，其中所述单克隆抗体包含轻链可变区和重链可变区，
所述轻链可变区包含：
VL CDR1，其包含SEQ ID NO：2所示氨基酸序列，
VL CDR2，其包含SEQ ID NO：3所示氨基酸序列，和
VL CDR3，其包含SEQ ID NO：4所示氨基酸序列；
所述重链可变区包含：
VH CDR1，其包含SEQ ID NO：6所示氨基酸序列，
VH CDR2，其包含SEQ ID NO：13所示氨基酸序列，和
VH CDR3，其包含SEQ ID NO：8所示氨基酸序列。
在一些实施方式中，其中所述单克隆抗体包含轻链可变区和重链可变区，
所述轻链可变区包含：
VL CDR1，其包含SEQ ID NO：2所示氨基酸序列，
VL CDR2，其包含SEQ ID NO：3所示氨基酸序列，和
VL CDR3，其包含SEQ ID NO：4所示氨基酸序列；
所述重链可变区包含：
VH CDR1，其包含SEQ ID NO：6所示氨基酸序列，
VH CDR2，其包含SEQ ID NO：7所示氨基酸序列，和
VH CDR3，其包含SEQ ID NO：14所示氨基酸序列。
在一些实施方式中，所述单克隆抗体是人源化抗体。
在一些实施方式中，所述轻链可变区包含SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1具有至少85％、至少90％、至少95％或更高序列相同性的氨基酸序列。在一些实施方式中，所述轻链可变区包含与SEQ ID NO：1具有约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、或约99％序列相同性的氨基酸序列。
在一些实施方式中，所述重链可变区包含SEQ ID NO：5所示氨基酸序列或与SEQ ID NO：5具有至少85％、至少90％、至少95％或更高序列相同性的氨基酸序列。在一些实施方式中，所述重链可变区包含与SEQ ID NO：5具有约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、或约99％序列相同性的氨基酸序列。
在一些实施方式中，所述重链可变区包含SEQ ID NO：15-19之一所示氨基酸序列(分别对应人源化重链版本H1-H5的可变区)。
在一些实施方式中，所述轻链可变区包含SEQ ID NO：20-31之一所示的氨基酸序列(分别对应人源化轻链版本L1-L12的可变区)。
在一些实施方式中，所述重链可变区包含SEQ ID NO：19所示的氨基酸序列，且所述轻链可变区包含SEQ ID NO：25所示氨基酸序列。
在一些实施方式中，所述重链可变区包含SEQ ID NO：19所示的氨基酸序列，且所述轻链可变区包含SEQ ID NO：30所示氨基酸序列。
在一些实施方式中，所述单克隆抗体包含人重链恒定区，例如，包含SEQ ID NO：11所示的氨基酸序列的人重链恒定区。
在一些实施方式中，所述单克隆抗体包含人轻链恒定区，例如，包含SEQ ID NO：12所示的氨基酸序列的人轻链恒定区。
在一些实施方式中，所述单克隆抗体包含具有SEQ ID NO：32-36之一所示的氨基酸序列重链(分别对应人源化重链版本H1-H5)和具有SEQ ID NO：37-48之一所示的氨基酸 序列的轻链(分别对应人源化轻链版本L1-L12)。
在一些实施方式中，所述单克隆抗体包含SEQ ID NO：36所示的重链和SEQ ID NO：47所示的轻链(对应于人源化抗体H5L11)。
在一些实施方式中，所述单克隆抗体包含SEQ ID NO：36所示的重链和SEQ ID NO：42所示的轻链(对应于人源化抗体H5L6)。
本领域已知，在可变区具有糖基化位点的情况下，不同的糖基化形式的抗体分子(糖基化异构体)将在电泳中出现两条相邻的重链或轻链蛋白条带。不同的糖基化形式的抗体分子在人体内表现出的药代动力学和药效学效果均可能不同，因此，具有不同的糖基化形式的抗体很难被各国药物审批机构批准上市。去掉多余的可变区糖基化位点将十分有利于单抗在制备人用药物中的应用。因此，在一些优选实施方式中，本发明的分离的单克隆抗体或其抗原结合片段不包含糖基化位点，或者所述抗体的糖基化位点通过突变被去除。例如，SEQ ID NO：19所示的重链可变区和SEQ ID NO：36所示的重链中的糖基化位点已经通过突变而被去除，因而在本发明中是优选的。
在一些实施方式中，本发明的分离的单克隆抗体或其抗原结合片段衍生自Hetumomab。在一些实施方式中，本发明的分离的单克隆抗体或其抗原结合片段与Hetumomab结合肿瘤干细胞上的相同抗原。在一些实施方式中，本发明的分离的单克隆抗体或其抗原结合片段与Hetumomab结合肿瘤干细胞上的相同表位。在一些实施方式中，本发明的分离的单克隆抗体或其抗原结合片段与Hetumomab竞争结合肿瘤干细胞。
在一些实施方式中，本发明的分离的单克隆抗体或其抗原结合片段特异性靶向肿瘤干细胞。本发明的分离的单克隆抗体或其抗原结合片段特异性靶向的肿瘤干细胞包括但不限于乳腺癌干细胞、大肠癌干细胞、胰腺癌干细胞、前列腺癌干细胞、肝癌干细胞、肺癌干细胞和胃癌干细胞。
本发明还涵盖分离的单克隆抗体或其抗原结合片段，其与Hetumomab结合肿瘤干细胞上的相同抗原。本发明还涵盖分离的单克隆抗体或其抗原结合片段，其与Hetumomab结合肿瘤干细胞上的相同表位。本发明还涵盖分离的单克隆抗体或其抗原结合片段，其与Hetumomab竞争结合肿瘤干细胞。所述肿瘤干细胞包括但不限于乳腺癌干细胞、大肠癌干细胞、胰腺癌干细胞、前列腺癌干细胞、肝癌干细胞、肺癌干细胞和胃癌干细胞。
三、核酸、载体和抗体产生方法
在另一方面，本发明提供分离的核酸分子，其编码前述本发明的抗体或其抗原结合片段。在一些实施方式中，所述核酸分子的核苷酸序列针对用于表达的宿主细胞进行密码子优化。在一些实施方式中，本发明的核酸分子与表达调控序列可操作地连接。
本发明还提供表达载体，其包含至少一种前述本发明的核酸分子。
本发明还提供宿主细胞，其由至少一种前述本发明的核酸分子或表达载体转化。
在另一方面，本发明提供一种生产本发明的抗体或其抗原结合片段的方法，包括：
(i)在适合所述核酸分子或表达载体表达的情况下培养本发明的宿主细胞，和
(ii)分离并纯化由所述宿主细胞表达的抗体或其抗原结合片段。
本发明还涉及通过上述本发明的方法获得的分离的抗体或其抗原结合片段，其能够特异性靶向肿瘤干细胞。
四、疾病治疗和/或预防
肿瘤干细胞是存在于肿瘤组织中的一小部分具有干性特征的癌细胞，具有以下生物学特征：能够自我更新、复制，不定向分化，高致瘤性，高侵袭扩散转移能力，对放化疗均不敏感。由于存在肿瘤干细胞，使得肿瘤不断快速生长、扩散、转移、复发。更为严重的是肿瘤干细胞对几乎所有的传统化疗药物、放疗及近年上市的靶向药物(包括抗体靶向药物)均耐药。肿瘤干细胞均处于细胞周期的G0期不生长、不增殖状态。放化疗只对处于高度快速生长增殖的癌细胞有作用，而不能杀灭处于G0期的肿瘤干细胞。而且当放化疗将大量快速生长的癌细胞杀灭后，抵抗放化疗的肿瘤干细胞反而被筛选富集，比例大大提高。由于肿瘤干细胞具有极强的自我复制能力和扩散转移能力，这些肿瘤干细胞会快速分化增殖，生长并扩散，转移至全身各器官形成新的转移病灶，而这种转移病灶的癌细胞均抵抗放化疗，在乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌、胃癌等多种恶性肿瘤中已经证明了肿瘤干细胞的存在。恶性程度越高的癌症，肿瘤干细胞越多，而肿瘤干细胞比例越高的癌症患者转移、复发越多，生存期越短。
本发明人发现，本发明的特异性识别肿瘤干细胞的单克隆抗体能够在体外显著抑制多种肿瘤干细胞的自我更新、侵袭和耐药能力(实施例4)。进一步的实验表明，本发明的特异性识别肿瘤干细胞的单克隆抗体能够在动物模型中抑制多种肿瘤移植瘤的生长、转移和耐药(实施例5)。因此，本发明的单克隆抗体能够通过靶向肿瘤干细胞而用于治疗恶性肿瘤、预防或/或治疗恶性肿瘤转移或复发。
由此，本发明提供了一种在患者中治疗恶性肿瘤、预防或/或治疗恶性肿瘤转移或复发的方法，所述方法包括给所述患者施用有效量的本发明的针对肿瘤干细胞的抗体或其抗原结合片段。可以通过本发明的方法治疗和/或预防的恶性肿瘤包括但不限于乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
五、药物组合物
本发明还提供了一种药物组合物，其包含本发明的针对肿瘤干细胞的抗体或其抗原结合片段以及药学上可接受的载体。所述药物组合物用于在患者中治疗恶性肿瘤、预防或/或治疗恶性肿瘤转移或复发。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体分子、免疫缀合物包裹于一种材料中，以保护该化 合物免受可使该化合物失活的酸和其他天然条件的作用。
本发明的药物组合物也可含有药学上可接受的抗氧化剂。药学上可接受的抗氧化剂的例子包括：(1)水溶性抗氧化剂，如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠，亚硫酸钠等；(2)油溶性抗氧化剂，如棕榈酸抗坏血酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚等；和(3)金属螯合剂，如柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等。
这些组合物还可含有佐剂，如防腐剂、润湿剂、乳化剂和分散剂。
可以通过灭菌程序或通过包含诸如对羟基苯甲酸酯、氯代丁醇、苯酚山梨酸等各种抗细菌剂和抗真菌剂确保防止存在微生物。在很多情况下，组合物中优选包含等渗剂，例如，糖、多元醇例如甘露糖醇、山梨糖醇或氧化钠。通过在组合物中加入延迟吸收剂，例如单硬脂酸盐和明胶，可实现注射型药物延长的吸收。
药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射液或分散液的粉末剂。这些用于药学活性物质的介质和试剂的使用是本领域公知的。常规介质或试剂，除了任何与活性化合物不相容的范围外，都可能在本发明的药物组合物中。还可以向组合物中掺入补充的活性化合物。
治疗性组合物一般必须是无菌的并且在制备和贮存条件下稳定的。可以将组合物配制成溶液、微乳状液、脂质体或其他适合高药物浓度的有序结构。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液态聚乙二醇等)及其合适的混合物的溶剂或分散剂。例如，通过使用包衣，例如卵磷脂，在分散液的情况下通过保持所需的颗粒大小，以及通过使用表面活性剂，可以保持适当的流动性。
通过将活性化合物以需要的量混入合适的溶剂中，并且根据需要加入以上列举的成分中的一种或其组合，接着无菌微过滤，可制备无菌注射液。通常，通过将活性化合物掺入到含有基本分散介质和上面所列其他所需成分的无菌载体中制备分散剂。对于用于制备无菌注射液的无菌粉末剂，优选的制备方法是真空干燥和冷冻干燥(冻干)，由其预先无菌过滤的溶液得到活性成分加任何额外所需成分的粉末。
可以与载体材料组合制备单一剂量形式的活性成分的量根据所治疗的对象和特定给药方式而不同。可以与载体材料组合制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，优选大约0.1％至大约70％，最优选大约1％至大约30％的活性成分，与药学上可接受的载体相组合。
可以调节剂量方案以提供最佳的期望的反应(例如，治疗反应)。例如，可以施用单一推注，可以随时间施用几次分开的剂量，或者根据治疗状况的紧急情况所需，可以按比例减小或增加剂量。特别有利的是将肠胃外组合物配制成容易给药并且剂量均匀的剂量单位形式。此处使用的剂量单位形式是指适合作为单位剂量用于所治疗的对象的物理不连续单位；每个单位含有预定量的活性化合物，经计算该预定量的活性化合物与需要的药物载体组合产生所需的治疗效果。对本发明剂量单位形式 的具体说明限定于且直接依赖于(a)活性化合物的独特特性和要达到的特定治疗效果，和(b)本领域中固有的对于配制这种用于治疗个体敏感性的活性化合物的限制。
对于抗体分子的给药而言，剂量范围为约0.0001至100mg/kg，更通常为0.01至20mg/kg受者体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重，10mg/kg体重或20mg/kg体重，或在1-20mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。
或者，针对肿瘤干细胞的抗体分子也可以作为持续释放制剂来给药，在此情况中需要频率较低的给药。剂量和频率根据抗体分子在患者中的半衰期而不同。通常，人抗体表现出最长的半衰期，之后是人源化抗体、嵌合抗体和非人类抗体。给药剂量和频率根据处理是预防性的还是治疗性的而不同。在预防性应用中，在长时间内以较不频繁的间隔给予相对较低的剂量。有些患者在余生中持续接受处理。在治疗性应用中，有时需要以较短的间隔给予较高的剂量，直到疾病的进展减轻或停止，优选直到患者表现为疾病症状部分或完全改善。之后，可以以预防性方案给患者给药。
本发明药物组合物中活性成分的实际剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
“有效量”的本发明的抗体或其抗原结合片段优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的抗体或其抗原结合片段优选地将细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。有效量的本发明的抗体或其抗原结合片段能够减小肿瘤大小，或者以其他方式缓解对象的症状如预防和/或治疗转移或复发。本领域技术人员可以根据如下因素确定这种量，如对象的大小、对象症状的严重性和选择的特定组合物或给药途径。
本发明的抗体或其抗原结合片段或本发明的药物组合物可以利用本领域公知的一种或多种方法通过一种或多种给药途径给药。本领域技术人员应当理解，给药途径和/或方式根据期望的结果而不同。本发明的抗体优选给药途径包括静脉内、肌 肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外给药途径，例如注射或输注。本文使用的短语“肠胃外给药”是指除肠和局部给药以外的给药模式，通常是注射，包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
或者，本发明的针对肿瘤干细胞的抗体或其抗原结合片段或本发明的药物组合物也可以通过非肠胃外途径给药，如局部、表皮或粘膜途径给药，例如，鼻内、经口、阴道、直肠、舌下或局部。
活性化合物可以与保护化合物不被快速释放的载体一起制备，例如控释制剂，包括植入物、透皮贴剂和微胶囊递送系统。可以使用生物可降解的、生物相容的聚合物，例如乙烯乙酸乙烯酯、聚酐类、聚乙醇酸、胶原、聚原酸酯和聚乳酸。制备这样的制剂的很多方法受专利保护或者通常为本领域技术人员所公知。参见，例如，Sustainedand controlled Release Drug Delivery Systems，J.R.Robinson，ed.，Marcel Dekker，Inc.，New York，1978。
治疗性组合物可应用本领域公知的医疗装置给药。例如，在一个优选实施方案中，本发明的治疗组合物可用无针皮下注射装置给药，如在美国专利No.5,399,163；5,383,851；5,312,335；5,064,413；4,941,880；4,790,824；或4,596,556中公开的装置。可用于本发明的公知的植入物和模块的例子包括：美国专利No.4,487,603，该专利公开了用于以受控速率分散药物的可植入微量输注泵；美国专利No.4,486,194，该专利公开了用于通过皮肤给药的治疗装置；美国专利No.4,447,233，该专利公开了用于以精确的输注速率递送药物的医用输注泵；美国专利No.4,447,224，该专利公开了用于连续递送药物的变流可植入输注装置；美国专利No.4,439,196，该专利公开了具有多腔区室的渗透药物递送系统：和美国专利No.4,475,196，该专利公开了一种渗透药物递送系统。这些专利引入本文作为参考。本领域技术人员公知许多其他这样的植入物、递送系统和模块。
在某些实施方案中，本发明的针对肿瘤干细胞的抗体可配制为确保在体内的正确分布。例如，血-脑屏障(BBB)阻止了许多高度亲水性的化合物。为了确保本发明的治疗性化合物能够跨过BBB(如果需要时)，可将它们配制在如脂质体中。至于制备脂质体的方法，参见，例如，美国专利4,522,811；5,374,548和5,399,331。脂质体包含可被选择性地转运入特定细胞或器官内的一个或多个靶向部分，从而增强靶向药物递送(参见，例如，V.V.Ranade(1989)J.Clin.Pharmacol.29：685)。靶向部分的例子包括叶酸或生物素(参见，例如，Low等的美国专利5,416,016)；甘露糖苷(Umezawa等(1988)Biochem.Biophys.Res.Commun.153：1038)；抗体(P.G.Bloeman等(1995)FEBS Lett.357：140；M.Owais等(1995)Antimicrob.Agents Chemother.39：180)；表面活性剂蛋白A受体(Briscoe等(1995)Am.J.Physiol.1233：134)；p120(Schreier等(1994)J.Biol.Chem.269：9090)；也参见K.Keinanen；M.L.Laukkanen(1994) FEBS Lett.346：123；J.J.Killion；I.J.Fidler(1994)Immunomethods 4：273。
所述药物组合物中本发明的针对肿瘤干细胞的抗体或其抗原结合片段还可以与诸如细胞毒素、放射性同位素或生物活性蛋白质等治疗性部分缀合。
细胞毒素包括对细胞有害(例如杀伤细胞)的任何试剂。实例包括：紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙啶、吐根碱、丝裂霉素、表鬼臼毒吡喃葡糖苷、表鬼臼毒噻吩糖苷、长春新碱、长春碱、秋水仙素、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光辉霉素、放线菌素D、1-脱氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔和嘌呤霉素和它们的类似物或同系物。
可用于缀合的治疗剂还包括，例如：抗代谢物(例如，氨甲喋呤、6-巯基嘌呤、6-硫代鸟嘌呤、阿糖胞苷、5-氟尿嘧啶、氨烯咪胺)，烷化剂(例如，氮芥、苯丁酸氮芥、苯丙氨酸氮芥、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露糖醇、链唑霉素、丝裂霉素C和顺-二氯二胺合铂(II)(DDP)顺铂)，氨茴霉素类(例如，柔红菌素(以前称为道诺霉素)和阿霉素)，抗生素(例如，放线菌素D(以前称为放线菌素)、博来霉素、光辉霉素和安曲霉素(AMC))，和抗有丝分裂剂(例如，长春新碱和长春碱)。
能与本发明的针对肿瘤干细胞的抗体缀合的治疗性细胞毒素的其他优选例子包括倍癌霉素、刺孢霉素、美坦生、阿里他汀、和它们的衍生物。
可以利用本领域使用的接头技术将细胞毒素与本发明的针对肿瘤干细胞的抗体缀合。已经用于将细胞毒素与针对肿瘤干细胞的抗体缀合的接头类型的实例包括但不限于腙、硫醚、酯、二硫化物和含肽的接头。可以选择，例如，在溶酶体区室内易被低pH切割或易被蛋白酶切割的接头，该蛋白酶例如是在肿瘤组织中优先表达的蛋白酶，如组织蛋白酶(例如组织蛋白酶B、C、D)。
关于细胞毒素的类型、用于缀合治疗剂与抗体的接头和方法的进一步讨论，参见Saito，G.等(2003)Adv.Drug Deliv.Rev.55：199-215；Trail，P.A.等(2003)Cancer.Immunol.Immunother.52：328-337；Payne，G.(2003)Cancer Cell 3：207-212；Allen，T.M.(2002)Nat.Rev.Cancer 2：750-763；Pastan，I.和Kreitman，R.J.(2002)Curr.Opin.Investig.Drugs 3：1089-1091；Senter，P.D.和Springer，C.J.(2001)Adv.Drug Deliv.Rev.53：247-264。
本发明的针对肿瘤干细胞的抗体也可以与放射性同位素缀合，产生细胞毒性放射性药物，也被称作放射性抗体缀合物。能够与诊断或治疗性使用的抗体缀合的放射性同位素的例子包括但不限于碘131、铟111、钇90和镥177。制备放射性抗体缀合物的方法在本领域中已经建立。
本发明的针对肿瘤干细胞的抗体也可以与具有需要的生物活性的蛋白质缀合，可用于修饰特定的生物学反应。这样的生物活性蛋白质包括，例如：具有酶活性的毒素或其活性片段，如相思豆毒蛋白、蓖麻毒蛋白A、假单胞菌外毒素或白喉毒素；蛋白质，如肿瘤坏死因子或干扰素-γ；或生物学反应调节物，如淋巴因子、白介素-1(“IL-1”)、白介素-2(“IL-2”)、白介素-6(“IL-6”)、白介素-10(“IL-10”)、粒细胞巨噬细胞集落 刺激因子(“GM-CSF”)、粒细胞集落刺激因子(“G-CSF”)或其他免疫因子如IFN等。
将这种治疗性部分与抗体分子缀合的技术是众所周知的，参见，例如，Arnon等，“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”，Monoclonal Antibodies And Cancer Therapy，Reisfeld等(ed.)，pp.243-56(Alan R.Liss，Inc.1985)；Hellstrom等，“Antibodies For Drug Delivery”，Controlled Drug Delivery(2nd Ed.)，Robinson等(ed.)，pp.623-53(Marcel Dekker，Inc.1987)；Thorpe，“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy：A Review”，Monoclonal Antibodies’84：Biological And Clinical Applications，Pinchera等(ed.)，pp.475-506(1985)；“Analysis，Results，And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”，Monoclonal Antibodies For Cancer Detection And Therapy，Baldwin等(ed.)，pp.303-16(Academic Press 1985)，和Thorpe等，“The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates，”Immunol.Rev.，62：119-58(1982)。
六、联合治疗
本发明的针对肿瘤干细胞的抗体或药物组合物可以与化疗剂或靶向其他肿瘤抗原的抗体联合施用。本申请实施例5证明了本发明的单克隆抗体与化疗剂联合施用在肿瘤治疗中具有协同作用。不受任何理论的限制，认为本发明的针对肿瘤干细胞的抗体能够抑制肿瘤耐药功能，从而能够在与化疗剂或靶向其他肿瘤抗原的抗体组合施用时取得协同效果。
可以与本发明的抗体或本发明的药物组合物组合使用的化疗剂或靶向其他肿瘤抗原的抗体没有特别限制。所述化疗剂和靶向其他肿瘤抗原的抗体的实例包括但不限于：异环磷酰胺、环磷酰胺、达卡巴嗪、替莫唑胺、尼莫司汀、白消安、美法仑、依诺他宾、卡培他滨、卡莫氟、克拉屈滨、吉西他滨、阿糖胞苷、替加氟、替加氟-尿嘧啶、TS-1、去氧氟尿苷、奈拉滨、羟基脲、氟尿嘧啶、氟达拉滨、培美曲塞、喷司他丁、巯嘌呤、甲氨蝶呤、伊立替康、依托泊苷、艾立布林、索布佐生、多西他赛、紫杉醇、长春瑞滨、长春新碱、长春地辛、长春碱、放线菌素D、阿柔比星、氨柔比星、伊达比星、表柔比星、净司他丁酯、柔红霉素、多柔比星、吡柔比星、博来霉素、培洛霉素、丝裂霉素C、米托蒽醌、奥沙利铂、卡铂、顺铂、奈达铂、阿那曲唑、依西美坦、炔雌醇、氯地孕酮、戈舍瑞林、他莫昔芬、地塞米松、比卡鲁胺、托瑞米芬、氟他胺、泼尼松龙、磷雌酚、米托坦、甲睾酮、亮丙瑞林、来曲唑、甲基安宫黄体酮、替伊莫单抗、伊马替尼、依维莫司、厄洛替尼、吉非替尼、舒尼替尼、西妥昔单抗、索拉非尼、达沙替尼、他米巴罗汀、曲妥珠单抗、维甲酸、帕木单抗、贝伐单抗、硼替佐米、和拉帕替尼。在一个具体的实施方式中，所述化疗剂是含铂的化疗剂，例如顺铂。
本发明的抗体和所述化疗剂或靶向其他肿瘤抗原的抗体可以全部在一次施用或分开施用。当分开施用时(采用互相不同的施用方案的情况下)，它们可以连续施用而不中断或以预定的间隔施用。
本发明的抗体和所述化疗剂或靶向其他肿瘤抗原的抗体在本发明的药物组合物中的组合剂量没有特别限制。如上所述，本发明的抗体的剂量可以通过参考当所述抗体单独使用时的剂量来确定。所述化疗剂和靶向其他肿瘤抗原的抗体可以根据各自药物标明的剂量使用或可以减少(考虑到和本发明的抗体的组合效果)。
本发明的抗体或本发明的药物组合物还可以与放疗联合，例如包括向患者施用电离辐射，其早于、在其过程中和/或晚于本发明的抗体或药物组合物的施用过程。
七、肿瘤干细胞检测和纯化
如本文所述，本发明的单克隆抗体特异性识别肿瘤干细胞。因此，本发明还提供一种检测生物学样品中肿瘤干细胞存在的方法，包括：
a)使所述生物学样品与本发明的单克隆抗体或其抗原结合片段接触；
b)检测本发明的单克隆抗体或其抗原结合片段与所述生物学样品中的靶抗原的结合，其中检出代表所述生物学样品中存在肿瘤干细胞。
在一些实施方案中，所述肿瘤干细胞选自乳腺癌干细胞、大肠癌干细胞、胰腺癌干细胞、前列腺癌干细胞、肝癌干细胞、肺癌干细胞和胃癌干细胞。
在本发明上述检测方法的一些实施方案中，本发明的单克隆抗体或其抗原结合片段还缀合有可用于检测或可被其他试剂检测到的荧光染料、化学物质、多肽、酶、同位素、标签等。
用于检测抗体-抗原结合的方法是本领域已知的，例如ELISA等。
本发明还提供一种用于分离肿瘤干细胞的方法，所述方法包括：
(a)提供疑似包含肿瘤干细胞的细胞群；
(b)鉴定所述细胞的亚群，其结合本发明的单克隆抗体或其抗原结合片段；和
(c)分离所述亚群。
例如，可以通过流式细胞术分离肿瘤干细胞。
八、诊断和预后
如本文所述，本发明的单克隆抗体的靶抗原表达于多种人肿瘤细胞的活细胞表面，并且在多种肿瘤组织中特异性高表达(阳性率79％-94％)。
因此，本发明还提供了一种检测患者中恶性肿瘤的存在的方法，包括：
a)使获得自所述患者的生物学样品与本发明的单克隆抗体或其抗原结合片段接触；
b)检测本发明的单克隆抗体或其抗原结合片段与所述生物学样品中的靶抗原的结合，其中检出代表所述患者中存在恶性肿瘤。
本发明还提供一种用于预后患者中恶性肿瘤复发或进展的方法，所述方法包括：
(a)从所述患者中分离包含循环细胞的生物学样品；
(b)使所述包含循环细胞的生物学样品与本发明的单克隆抗体或其抗原结合片段接触；和
(c)鉴定结合本发明的单克隆抗体或其抗原结合片段的循环细胞的存在，
从而预后所述患者中恶性肿瘤的复发或进展。
在一些实施方案中，所述恶性肿瘤的进展包含所述恶性肿瘤在患者中的转移。
鉴定到结合本发明的单克隆抗体或其抗原结合片段的循环细胞的存在提示所述患者中恶性肿瘤复发或进展的高风险。
在一些实施方案中，所述生物学样品包括血液样品、淋巴样品或其组分。
在一些实施方案中，所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
在本发明上述方法的一些实施方案中，本发明的单克隆抗体或其抗原结合片段还缀合有可用于检测或可被其他试剂检测到的荧光染料、化学物质、多肽、酶、同位素、标签等。
用于检测抗体-抗原结合的方法是本领域已知的，例如ELISA等。
九、试剂盒
本发明的范围内还包括用于本发明的方法的试剂盒，该试剂盒包括本发明的单克隆抗体或其抗原结合片段，以及使用说明。该试剂盒可以进一步包括至少一种另外的检测试剂，其用于检测本发明的单克隆抗体的存在。试剂盒一般包括表明试剂盒内容物的预期用途和/或使用方法的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
通过参考在此给出的一些具体实施例可获得对本发明的进一步的理解，这些实施例仅用于说明本发明，其无意于对本发明的范围做出任何限制。显然，可以对本发明作出多种改动和变化而不脱离本发明的实质，因此，这些改动和变化同样在本申请要求保护的范围内。
实施例1 小鼠单克隆抗体Hetumomab的制备
一、抗人肿瘤多能干细胞小鼠单克隆抗体库的制备
本实施例采用一种人肿瘤多能干细胞系T3A-A3作为免疫原(Liu H，et al.Cell Death and Disease.2013，4：e857)。该人肿瘤干细胞系从原发肝癌患者手术切除的肝癌组织中分离获得，能够在体外长期传代培养。。该细胞系已传代超过100次，细胞仍然生长迅速，并保持干细胞性质。该细胞系既表达多种干细胞的标志物、具有干细胞的自我更新能力、具有向不同肿瘤细胞定向分化的潜能；同时也具有肿瘤性质，具有成瘤能力和转移能力。该细胞系在体外长期培养而保持性质不变，并且在免疫缺陷鼠体内成瘤能力和转移能力强。
将扩大培养获得的肝癌干细胞(人肿瘤多能干细胞)系T3A-A3采用多聚甲醛固定， 免疫普通Balb/c鼠，2-4周一次，每次约1×10 7细胞。长期免疫，直至采用常规细胞免疫化学方法测定免疫小鼠血清抗人肝癌干细胞(人肿瘤多能干细胞)系T3A-A3的滴度超过1∶50000。取小鼠脾细胞与小鼠骨髓瘤细胞SP2/0采用常规的PEG介导融合的方法，融合形成分泌小鼠单克隆抗体的杂交瘤。采用常规的甲基纤维素平板法制备杂交瘤单克隆，待各个单克隆生长后将其分别挑取至96孔板继续培养，由此获得含有大量抗人肝癌干细胞(人肿瘤多能干细胞)系T3A-A3的单克隆抗体的杂交瘤克隆的文库。收集96孔板中每个杂交瘤克隆的培养上清，用于下一步的测定和筛选过程。
二、抗人肿瘤多能干细胞小鼠单克隆抗体Hetumomab的筛选
无血清悬浮培养基采用含20ng/mL EGF、20ng/mL bFGF、按1∶50比例添加B27、10ng/mL LIF、2mmol/mL谷氨酰胺、1μ/mL Heparin的DMEM/F12(1∶1)培养液。使用无血清培养基培养前洗涤1次细胞。将经无血清悬浮培养的人肝癌干细胞(人肿瘤多能干细胞)系T3A-A3球形细胞，轻柔吹散成单细胞，以2000细胞/孔接种96孔板。用无血清培养基培养24h后，用含1％BSA的PBS洗涤细胞，每孔中分别加入一个杂交瘤克隆的培养上清100μL，室温孵育2小时；用含1％BSA的PBS洗涤5次后，加入生物素标记抗鼠二抗室温，反应30分钟；再用含1％BSA的PBS洗涤5次后，加入Cy3标记的Avidin，室温反应30分钟；含1％BSA的PBS洗涤5次后，在荧光显微镜下进行荧光镜检，判断单抗库中的各个单抗杂交瘤上清与人肝癌干细胞(人肿瘤多能干细胞)系T3A-A3的反应。能观察到部分细胞具有荧光染色即判断为阳性，初步筛选获得能与人肝癌干细胞(人肿瘤多能干细胞)系T3A-A3膜表面抗原结合的鼠单克隆抗体。
进一步，选择无血清悬浮培养的人肝癌细胞系(Yan Li，Zhao-You Tang，Sheng-Long Ye，Yin-Kun Liu，Jie CHEN，Qiong Xue，Jun Chen，Dong-Mei Gao，Wei-Hua Bao.Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97.世界胃肠病学杂志(英文版).2001，7(05)：630-636.)的球形细胞，同样进行以上程序，荧光显微镜镜检判断单抗库中的杂交瘤单抗上清与人肝癌干细胞(MHCC97L球形细胞富含肝癌干细胞)的反应，获得能与肝癌干细胞膜表面抗原结合的鼠单克隆抗体。
从抗人肿瘤多能干细胞小鼠单克隆抗体库筛选出1株小鼠单克隆抗体Hetumomab，其不仅与人肿瘤多能干细胞系T3A-A3膜表面抗原结合，也同时与人肝癌MHCC97L细胞系的干细胞(MHCC97L球形细胞)膜表面抗原结合，证明小鼠单克隆抗体Hetumomab能够识别不同来源的人肝癌干细胞。选择小鼠单克隆抗体Hetumomab进行进一步鉴定和药效研究。
分泌Hetumomab单抗的小鼠杂交瘤细胞以保藏号CGMCC No.12251于2016年3月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)。
将Hetumomab杂交瘤细胞进行扩大培养，收集含抗体上清。采用Southern Biotech 公司的单抗亚类检测试剂盒鉴定单抗的类和亚类以及Sigma公司的ELISA二抗检测上清的抗体产量。实验结果显示单抗Hetumomab为IgG1型重链、κ型轻链。
分泌单抗Hetumomab的杂交瘤细胞在体外扩大培养，待细胞长至80％，更换无血清培养基，继续培养4-5天后，收集分泌有抗体的无血清上清液。采用抗Protein G纯化柱纯化单抗Hetumomab。再用10％SDS-PAGE经考马斯亮兰染色鉴定分离纯化的Hetumomab单抗的纯度。结果显示，Hetumomab的重链分子量约在47kDa，轻链分子量约在26kDa，这与理论上一般IgG抗体重链和轻链的分子量相符合。扫描分析电泳目的条带的纯度在95％以上，纯化的Hetumomab单抗纯度满足后续实验的要求。
实施例2 Hetumomab靶抗原在多种肿瘤细胞及组织中特异表达
一、Hetumomab靶抗原在人多种肝癌、肺癌、胃癌细胞系中表达且可表达于活细胞表面。
采用常规活细胞免疫荧光染色技术检测了单抗Hetumomab靶抗原在人多种肿瘤细胞系如肝癌、肺癌、胃癌细胞系中在活细胞表面的表达情况。具体技术方法大致如下：细胞爬片或接种96孔培养板(4×10 3个/孔)，待细胞长到60％～70％满时，无血清培养液洗2次，PBS洗1次。加入一抗(杂交瘤上清或纯化抗体)，鼠抗α-tublin抗体(稀释度1∶1000)作为细胞是否通透的对照，正常鼠IgG、SP2/0上清、PBS作为阴性对照，室温孵育ih；活细胞洗液(含1％BSA的PBS)洗涤5次，每次5min；4％多聚甲醛室温下固定15min；固定细胞洗液(含0.1％BSA、0.05％tween-20的PBS)洗涤5次，每次5min；加入100μl的二抗，避光室温孵育30min；固定细胞洗液洗涤5次，每次5min；50μl含10μg/ml DAPI、50％甘油的PBS封闭；荧光显微镜下观察。以观察到部分细胞出现膜荧光染色为阳性结果。
结果发现，单抗Hetumomab靶抗原可表达于以下这些人肝癌、肺癌、胃癌细胞系(购自中国医学科学院基础医学研究所细胞资源中心)活细胞的表面：
人肝癌细胞系：MHCC97L、Be17402-V13；
人肺癌细胞系：A549、SPCA-1；
人胃癌细胞系：SNU-5、BGC-823。
部分典型的阳性结果照片如图1所示。
以上结果说明单抗Hetumomab靶抗原在人多种肿瘤，例如肝癌、肺癌、胃癌细胞系中均有表达且可表达于活的癌细胞的膜表面。
二、Hetumomab靶抗原在人肝癌、肺癌、胃癌组织中特异高表达。
采用传统的常规免疫组织化学技术，以Hetumomab为一抗，采用抗鼠抗体二抗，检测了单抗Hetumomab靶抗原在多例人肝癌、肺癌、胃癌患者及相关其他组织中的表达情况。
具体技术方法大致如下：将组织片子常规脱蜡；柠檬酸缓冲液(pH6.0)倒入抗原修复 盒，放入片子，将修复盒置于沸水中，水浴加热30min，室温自然冷却2h；PBS洗涤3min×3次，将片子上的水甩干，立即用组化笔沿组织划圈；每块组织上加一滴内源性过氧化物酶阻断溶液，室温孵育20min，PBS洗涤3min×3次，甩掉洗液，加一滴正常动物血清——羊血清封闭，室温孵育20min；甩掉封闭血清，各组织点上加一抗，放入保湿盒中，4℃过夜孵育；甩掉一抗，PBS洗涤3min×3次，甩掉洗液，加二抗biotin-抗鼠抗体二抗，室温孵育20min；PBS洗涤3min×5次，甩掉洗液，加一滴Avidin-HRP，室温孵育10min；PBS洗涤3min×3次，甩掉洗液，加一滴新鲜配制的DAB，显微镜下观察，同时严格计时，待组织呈阳性后，自来水冲洗终止；苏木素复染5min，1％盐酸-75％酒精分色2秒，自来水冲洗；片子脱水后用中性树胶封片，显微镜下观察。
以单抗Hetumomab为一抗，检测了120例人肝癌组织、20例癌旁组织、10例正常肝组织、10例肝炎组织、40例肝硬化组织中单抗Hetumomab靶抗原的表达情况。实验结果(表1)显示：单抗Hetumomab的靶抗原在79.17％(95/120)的人肝癌组织中特异性表达，而在癌旁组织、正常肝组织、肝炎组织及肝硬化组织中均未见其表达，说明了单抗Hetumomab靶抗原在人肝癌组织中特异高表达。
表1.免疫组化技术检测单抗Hetumomab靶抗原在人肝癌组织中表达的结果
Figure PCTCN2018079785-appb-000001
以单抗Hetumomab为一抗，检测了160例人肺癌组织、32例癌旁组织、3例正常肺组织中单抗Hetumomab靶抗原的表达情况。实验结果(表2)显示：单抗Hetumomab的靶抗原在82.5％(132/160)的人肺癌组织中特异性表达，而仅仅在6.25％的癌旁组织中表达，在正常肺组织中未见其表达，说明了单抗Hetumomab靶抗原在人肺癌组织中特异高表达。
表2.免疫组化技术检测Hetumomab靶抗原在人肺癌组织中的表达
Figure PCTCN2018079785-appb-000002
以单抗Hetumomab为一抗，检测了110例人胃癌组织、20例癌旁组织、3例正常胃组织中单抗Hetumomab靶抗原的表达情况。实验结果(表3)显示：单抗Hetumomab的靶抗原在93.64％(103/110)的人胃癌组织中特异性表达，而在癌旁组织和正常胃组织中 均未见其表达，说明了单抗Hetumomab靶抗原在人胃癌组织中特异高表达。
表3.免疫组化技术检测单抗Hetumomab靶抗原在人胃癌组织中表达的结果
Figure PCTCN2018079785-appb-000003
以上免疫组化检测结果的部分典型的阳性结果照片如图2所示。
以上这些结果说明单抗Hetumomab靶抗原在人多种肿瘤，例如肝癌、肺癌、胃癌的多数患者的癌组织中均有特异的高表达。
实施例3 单抗Hetumomab识别肿瘤干细胞
一、单抗Hetumomab识别的癌细胞在癌细胞的sphere培养细胞中富集。
根据现有大量文献报道，无血清悬浮培养即sphere(成球)培养可以富集肿瘤干细胞(Reynolds，B.A.and S.Weiss，″Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.″Dev Biol.1996.175(1)：p.1-13.；Fang，N.，et al.，″pH responsive adhesion of phospholipid vesicle on poly(acrylic acid)cushion grafted to poly(ethylene terephthalate)surface.″Colloids Surf B Biointerfaces，2005.42(3-4)：p.245-52.；和Tirino，V.，et al.，″The role of CD133in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer.″Eur J Cardiothorac Surg，2009.36(3)：p.446-53.)。所以根据单抗Hetumomab识别的癌细胞能否在sphere培养中得到富集来初步判断单抗Hetumomab识别的细胞是否与肿瘤干细胞相关。
对人肝癌细胞系Be17402-V13和MHCC97L细胞进行了无血清培养5天后，采用活细胞流式荧光术对亲本细胞和sphere培养细胞中Hetumomab +细胞进行了检测。实验结果(表4)显示Hetumomab +细胞在Be17402-V13 sphere细胞的比例为8.92％，比其在亲本细胞中的比例2.25％富集了3.96倍；Hetumomab +细胞在MHCC97L sphere细胞中的比例为7.51％，比其在亲本细胞中的比例3.45％富集了2.18倍。即经过无血清培养后，肝癌细胞系的Hetumomab +细胞得到了富集。
表4.免疫流式荧光技术检测单抗Hetumomab识别的肝癌细胞在肝癌细胞的sphere培养细胞中富集的结果
Figure PCTCN2018079785-appb-000004
对人肺癌细胞系SPCA-1和A549细胞进行了无血清培养5天后，采用活细胞流式荧光术对亲本细胞和sphere培养细胞中Hetumomab +细胞进行了检测。实验结果(表5)显示Hetumomab +细胞在SPCA-1 sphere细胞的比例为7.34％，比其在亲本细胞中的比例1.74％富集了4.22倍；Hetumomab +细胞在A549 sphere细胞中的比例为13.30％，比其在亲本细胞中的比例7.23％富集了1.84倍。即经过无血清培养后，肺癌细胞系的Hetumomab+细胞得到了富集。
表5.免疫流式荧光技术检测单抗Hetumomab识别的肺癌细胞在肺癌细胞的sphere培养细胞中富集的结果
Figure PCTCN2018079785-appb-000005
对人胃癌细胞系SNU-5和BGC-823细胞进行了无血清培养5天后，采用活细胞流式荧光术对亲本细胞和sphere培养细胞中Hetumomab +细胞进行了检测。实验结果(表6)显示Hetumomab +细胞在SNU-5 sphere细胞的比例为7.19％，比其在亲本细胞中的比例3.38％富集了2.13倍；Hetumomab +细胞在BGC-823 sphere细胞中的比例为7.45％，比其在亲本细胞中的比例2.95％富集了2.53倍。即经过无血清培养后，胃癌细胞系的Hetumomab +细胞得到了富集。
表6.免疫流式荧光技术检测单抗Hetumomab识别的胃癌细胞在胃癌细胞的sphere培养细胞中富集的结果
Figure PCTCN2018079785-appb-000006
以上免疫流式荧光检测结果的部分典型的图谱如图3所示。
以上这些结果说明单抗Hetumomab识别的癌细胞在人多种肿瘤，例如肝癌、肺癌、胃癌细胞系的sphere培养细胞中均显著富集。
二、单抗Hetumomab识别ESA、CD90阳性的肿瘤干细胞。
现已有多篇文献(Yamashita T，et al.EpCAMpositive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.Gastroenterology.2009，136(3)：1012-1024.；和Yang ZF.Identification of local and circulating cancer stem cells  in human liver cancer.Hepatology.2008，47(3)：919-928.)证明了ESA和CD90为一些肝癌细胞的肿瘤干细胞表面标志物。为了检测在人肝癌细胞Be17402-V13细胞中单抗Hetumomab识肝癌细胞同时也是ESA、CD90标志物阳性的肝癌干细胞，我们采用双色流式荧光术，对在无血清培养基中培养5天的人肝癌Be17402-V13细胞进行了染色。
结果(如表7)显示，单抗Hetumomab识别的细胞比例为8.52％，ESA +干细胞表达比例为9.02％，两者共染比例为3.63％，即单抗Hetumomab识别了40.2％的ESA +干细胞。对在无血清培养基中培养5天的人肝癌MHCC97L细胞进行了染色，结果(如表7)显示，单抗Hetumomab识别的细胞比例为2.38％，CD90 +干细胞表达比例为4.54％，两者共染比例为2.01％，即单抗Hetumomab识别了44.3％的CD90 +干细胞。
表7.双色流式荧光术检测单抗Hetumomab与ESA、CD90肝癌干细胞表面标志物在肝癌细胞中共染色的结果
Figure PCTCN2018079785-appb-000007
以上这些结果说明单抗Hetumomab识别ESA、CD90等标志物阳性的人肿瘤干细胞。
三、单抗Hetumomab识别的癌细胞具有更强的自我更新能力
自我更新能力、强侵袭能力、耐受化疗药物能力和强致瘤能力是肿瘤干细胞区别于普通子代肿瘤细胞的重要基本特征。因此，为了进一步验证单抗Hetumomab识别的细胞是否具有肿瘤干细胞特性，分选了多种人肿瘤细胞中的Hetumomab +细胞，检测其自我更新、侵袭、耐药和体内致瘤能力。
肿瘤干细胞的自我更新能力主要的表现形式为在无血清培养基中的成球能力，其方式称为不对称分裂，即一个细胞分裂成两个子代细胞时，其中一个子代细胞保持着与亲代细胞完全相同的特征，而另一个子代细胞可以继续分裂，形成正常的子代细胞。因此，可以通过检测Hetumomab +细胞在无血清培养基中的成球能力来确定其自我更新能力。
采用流式分选技术从培养5天的人肝癌Be17402-V13 sphere细胞中分离出Hetumomab +细胞、亲本细胞和Hetumomab -细胞。将分选得到的细胞以500个/孔接种于含0.8％甲基纤维素的半固态sphere培养基(半固态sphere培养基为含0.8％甲基纤维素、20ng/mL EGF、20ng/mL bFGF、按1∶50比例添加B27、10ng/mL LIF、2mmol/mL谷氨酰胺、lu/mL Heparin的DMEM/F12(1∶1)培养液)中，在超低粘附24孔板中培养，观察各细胞成球数量。结果显示(表8)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞在无血清培养基条件下的成球数分别为262±8.5、168±5.6、98±5.6，即Hetumomab +细胞的成球率明显高于其他两种细胞(p＜0.05)。因此，Hetumomab +细胞比亲本细胞和Hetumomab - 细胞具有更强的自我更新能力。
表8 肝癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞自我更新能力的比较
Figure PCTCN2018079785-appb-000008
采用流式分选技术从人肺癌SPCA-1 sphere细胞中分离出Hetumomab +细胞、亲本细胞和Hetumomab -细胞。将分选得到的细胞以500个/孔接种于含0.8％甲基纤维素的半固态sphere培养基中，在超低粘附24孔板板中培养，观察各细胞成球数量。结果显示(表8)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞在无血清培养基条件下的成球数分别为165.7±6.0、127±5.6、83.7±4.7，即Hetumomab +细胞的成球率明显高于其他两种细胞(p＜0.05)。因此，Hetumomab +细胞比亲本细胞和Hetumomab -细胞具有更强的自我更新能力。
表9 肺癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞自我更新能力的比较
Figure PCTCN2018079785-appb-000009
采用流式分选技术从人胃癌SNU-5 sphere细胞中分离出Hetumomab +细胞、亲本细胞和Hetumomab -细胞。将分选得到的细胞以500个/孔接种于含0.8％甲基纤维素的半固态sphere培养基中，在超低粘附24孔板板中培养，观察各细胞成球数量。结果显示(表10)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞在无血清培养基条件下的成球数分别为24±1.4、15.5±0.7、11.5±0.7，即Hetumomab +细胞的成球率明显高于其他两种细胞(p＜0.05)。因此，Hetumomab +细胞比亲本细胞和Hetumomab -细胞具有更强的自我更新能力。
表10 胃癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞自我更新能力的比较
Figure PCTCN2018079785-appb-000010
以上这些结果说明单抗Hetumomab识别的多种癌细胞(肝癌、肺癌、胃癌)均具有更强的自我更新能力，即具有肿瘤干细胞主要特征之一：高自我更新能力。
四、单抗Hetumomab识别的癌细胞具有更强的侵袭能力
自我更新能力、强侵袭能力、耐受化疗药物能力和强致瘤能力是肿瘤干细胞区别于普通子代肿瘤细胞的重要基本特征。因此，为了进一步验证单抗Hetumomab识别的细胞是否具有肿瘤干细胞特性，故分选了多种人肿瘤细胞中的Hetumomab +细胞，检测其自我更新、侵袭、耐药和体内致瘤能力。
采用流式分选技术从培养5天的人肝癌Be17402-V13 sphere细胞中分离出Hetumomab +细胞、亲本细胞和Hetumomab -细胞。将分选得到的细胞以相同数量接种于预先包被Matrigel胶的Transwell小室中，24h后固定，显微镜下观察穿膜的细胞数。结果显示(表11)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞穿膜的细胞数为分别为每视野308±9.5、210±10.7和132±14.7，即Hetumomab +细胞的侵袭穿过膜的数量明显高于其他两种细胞(p＜0.05)。因此，Hetumomab +细胞比亲本细胞和Hetumomab -细胞具有更强的侵袭能力。
表11 肝癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞侵袭能力的比较
Figure PCTCN2018079785-appb-000011
采用流式分选技术从培养的人肺癌SPCA-1 sphere细胞中分离出Hetumomab +细胞、亲本细胞和Hetumomab -细胞。将分选得到的细胞以相同数量接种于预先包被Matrigel胶的Transwell小室中，24h后固定，显微镜下观察穿膜的细胞数。结果显示(表12)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞穿膜的细胞数为分别为每视野222±11.5、193.7±5.7、154.3±12.1，即Hetumomab +细胞的侵袭穿过膜的数量明显高于其他两种细胞(p＜0.05)。因此，Hetumomab +细胞比亲本细胞和Hetumomab -细胞具有更强的侵袭能力。
表12 肺癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞侵袭能力的比较
Figure PCTCN2018079785-appb-000012
采用流式分选技术从培养的人胃癌SNU-5 sphere细胞中分离出Hetumomab +细胞、亲本细胞和Hetumomab -细胞。将分选得到的细胞以相同数量接种于预先包被Matrigel胶的Transwell小室中，24h后固定，显微镜下观察穿膜的细胞数。结果显示(表13)， Hetumomab +细胞、亲本细胞和Hetumomab -细胞穿膜的细胞数为分别为每视野247.5±19.1、142.5±9.2和145.0±11.3，即Hetumomab +细胞的侵袭穿过膜的数量明显高于其他两种细胞(p＜0.05)。因此，Hetumomab +细胞比亲本细胞和Hetumomab -细胞具有更强的侵袭能力。
表13 胃癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞侵袭能力的比较
Figure PCTCN2018079785-appb-000013
以上这些结果说明单抗Hetumomab识别的多种癌细胞(肝癌、肺癌、胃癌)均具有更强的侵袭能力，即具有肿瘤干细胞主要特征之一：高侵袭性。
五、单抗Hetumomab识别的癌细胞具有更强的耐受化疗药物的能力
自我更新能力、强侵袭能力、耐受化疗药物能力和强致瘤能力是肿瘤干细胞区别于普通子代肿瘤细胞的重要基本特征。因此，为了进一步验证单抗Hetumomab识别的细胞是否具有肿瘤干细胞特性，故分选了多种人肿瘤细胞中的Hetumomab +细胞，检测其自我更新、侵袭、耐药和体内致瘤能力。
为了检测Hetumomab +肝癌细胞的耐药能力，将人肝癌细胞Be17402-V13 sphere细胞流式分选获得的Hetumomab +细胞、亲本细胞和Hetumomab -细胞以5000个/孔的数量接种于96孔板中，每组细胞均用含0μg/mL、0.0625μg/mL、0.125μg/mL、0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL和4μg/mL 8种不同浓度的顺铂的完全培养基培养，3天后更换1次培养基，7天后用CCK8法检测OD值测定反映其耐药能力的IC50。实验结果显示(表14，图4)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞的IC50分别为0.739μg/mL、0.502μg/mL和0.313μg/mL，即Hetumomab +细胞的耐药性明显高于亲本细胞和Hetumomab -细胞，差异有统计学意义(p＜0.05)。因此，Hetumomab +肝癌细胞比亲本细胞和Hetumomab -细胞具有更强的耐受化疗药物的能力。
表14 肝癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞耐受化疗药物能力的比较
Figure PCTCN2018079785-appb-000014
为了检测Hetumomab +肺癌细胞的耐药能力，将人肺癌细胞SPCA-1 sphere细胞中流 式分选获得的Hetumomab +细胞、亲本细胞和Hetumomab -细胞以5000个/孔的数量接种于96孔板中，每组细胞均用含0μg/mL、0.2μg/mL、0.4μg/mL、0.6μg/mL、0.8μg/mL、1μg/mL和2μg/mL 7种不同浓度的顺铂的完全培养基培养，之后用CCK8法检测OD值测定反映其耐药能力的IC50。实验结果显示(表15，图4)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞的IC50分别为0.707μg/mL、0.513μg/mL和0.180μg/mL，即Hetumomab +细胞的耐药性明显高于亲本细胞和Hetumomab -细胞，差异有统计学意义(p＜0.05)。因此，Hetumomab +肺癌细胞比亲本细胞和Hetumomab -细胞具有更强的耐受化疗药物的能力。
表15 肺癌癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞耐受化疗药物能力的比较
Figure PCTCN2018079785-appb-000015
为了检测Hetumomab +胃癌细胞的耐药能力，我们将人胃癌细胞SNU-5 sphere细胞中流式分选获得的Hetumomab +细胞、亲本细胞和Hetumomab -细胞以5000个/孔的数量接种于96孔板中，每组细胞均用含0μg/mL、0.0125μg/mL、0.025μg/mL、0.05μg/mL、0.1μg/mL、0.2μg/mL、0.4μg/mL和0.8μg/mL 8种不同浓度的顺铂的完全培养基培养，之后用CCK8法检测OD值测定反映其耐药能力的IC50。实验结果显示(表16，图4)，Hetumomab +细胞、亲本细胞和Hetumomab -细胞的IC50分别为0.285μmol/L、0.155μmol/L和0.094μmol/L，即Hetumomab +细胞的耐药性明显高于亲本细胞和Hetumomab -细胞，差异有统计学意义(p＜0.05)。因此，Hetumomab +胃癌细胞比亲本细胞和Hetumomab -细胞具有更强的耐受化疗药物的能力。
表16 胃癌细胞中Hetumomab +细胞、亲本细胞和Hetumomab -细胞耐受化疗药物能力的比较
Figure PCTCN2018079785-appb-000016
以上这些结果说明单抗Hetumomab识别的多种癌细胞(肝癌、肺癌、胃癌)均具有更 强的耐受化疗药物能力，即具有肿瘤干细胞主要特征之一：强耐药性。
六、单抗Hetumomab识别的癌细胞具有更强的体内致瘤能力
自我更新能力、强侵袭能力、耐受化疗药物能力和强致瘤能力是肿瘤干细胞区别于普通子代肿瘤细胞的重要基本特征。因此，为了进一步验证单抗Hetumomab识别的细胞是否具有肿瘤干细胞特性，故分选了多种人肿瘤细胞中的Hetumomab +细胞，检测其自我更新、侵袭、耐药和体内致瘤能力。
检验一种细胞是否为肿瘤干细胞的“金标准”是体内的强致瘤性。因此，将人肝癌细胞Be17402-V13 sphere细胞流式分选获得的Hetumomab +细胞、亲本细胞和Hetumomab -细胞分别接种至4周龄大小的裸鼠皮下，长时间观察其体内成瘤的情况。结果如表17所示，1×10 4个Hetumomab +细胞在接种3周时就可在小鼠体内致瘤，而亲本细胞则需1×10 5个细胞在接种3周后才出瘤，Hetumomab -细胞在观察期间始终没有出瘤。这项经典实验的结果说明Hetumomab +细胞的体内致瘤性较亲本和Hetumomab -细胞显著强。提示Hetumomab +细胞具有肿瘤干细胞的高致瘤性特征，符合肿瘤干细胞的判定“金标准”。因此，单抗Hetumomab识别的细胞是肝癌肿瘤干细胞。
表17 肝癌细胞Be17402-V13中Hetumomab +细胞、亲本细胞和Hetumomab -细胞体内致瘤能力的比较(成瘤的动物只数)
Figure PCTCN2018079785-appb-000017
将人胃癌细胞SNU-5 sphere细胞流式分选获得的Hetumomab +细胞、亲本细胞和Hetumomab -细胞分别接种至4周龄大小的裸鼠皮下，长时间观察其体内成瘤的情况。结果如表18所示，2×10 3个Hetumomab +细胞在接种3月时就可在小鼠体内半数小鼠致瘤， 而亲本细胞2×10 3个细胞在接种4个月后仍未出瘤，Hetumomab -细胞在观察期间始终没有出瘤。这项经典实验的结果说明Hetumomab +细胞的体内致瘤性较亲本和Hetumomab -细胞显著强。提示Hetumomab +细胞具有肿瘤干细胞的高致瘤性特征，符合肿瘤干细胞的判定“金标准”。因此，单抗Hetumomab识别的细胞是胃癌肿瘤干细胞。
表18 胃癌细胞SNU-5中Hetumomab +细胞、亲本细胞和Hetumomab -细胞体内致瘤能力的比较(成瘤的动物只数)
Figure PCTCN2018079785-appb-000018
以上这些结果说明单抗Hetumomab识别的多种癌细胞(肝癌、胃癌)均具有更强的体内致瘤能力，即具有肿瘤干细胞主要特征之一高致瘤性。
实施例4 单抗Hetumomab可以抑制肿瘤干细胞的自我更新、侵袭和耐药功能
一、单抗Hetumomab显著抑制多种肿瘤(肝癌、胃癌、肺癌)的肿瘤干细胞的自我更新能力(肿瘤干细胞主要特征之一)。
肿瘤干细胞的自我更新能力主要的表现形式为在无血清培养基中的成球能力，其方式称为不对称分裂，即一个细胞分裂成两个子代细胞时，其中一个子代细胞保持着与亲代细胞完全相同的特征，而另一个子代细胞可以继续分裂，形成正常的子代细胞。为了证明单抗Hetumomab是否是能够直接抑制肝癌干细胞的功能性单抗，我们将在无血清培养基中培养5天的人肝癌细胞系Be17402-V13 sphere细胞制备成单细胞悬液后，以纯化的单抗Hetumomab(250μg/mL)为实验组，以PBS为阴性对照组，与细胞在37℃孵育2h，期间每隔半小时将细胞与抗体或阴性对照混匀一次。各组取500个细胞接种于含0.8％甲基纤维素的半固态sphere培养基(含EGF、LIF、bFGF等)中，于超低粘附24孔板板中培养，隔天补液1-1.5mL，14天后观察两组细胞成球数量。实验结果(图5)显示，实验组的成球数量为212±2.8，而阴性对照组的成球数量为278.5±0.7个，明显高于实验 组。单抗Hetumomab对Be17402-V13细胞的成球抑制率达23.9％，p＜0.05，有统计学差异。结果表明，单抗Hetumomab可直接作用于肝癌干细胞并抑制其自我更新能力。
为了证明单抗Hetumomab是否是能够直接抑制肺癌干细胞的功能性单抗，采用同样的方法检测了单抗Hetumomab对人肺癌细胞系SPCA-1成球的抑制作用。结果(如表19、图5)显示，实验组最高抗体浓度的成球数量为88.3±7.2，而阴性对照组的成球数量为151.3±9.1个，明显高于实验组，单抗Hetumomab对SPCA-1细胞的成球抑制率达41.6％，p＜0.01，有统计学差异。结果表明，单抗Hetumomab可直接作用于肺癌干细胞并抑制其自我更新能力。
表19 检测单抗Hetumomab抑制人肺癌细胞系SPCA-1成球的结果
Figure PCTCN2018079785-appb-000019
为了证明单抗Hetumomab是否是能够直接抑制胃癌干细胞的功能性单抗，采用同样的方法检测了单抗Hetumomab对人胃癌细胞系SNU-5的CD44阳性细胞肿瘤干细胞亚群成球的抑制作用。结果(如表20、图5)显示，实验组最高抗体浓度的成球数量为18±2.0，而阴性对照组的成球数量为128±4.0个，明显高于实验组，单抗Hetumomab对SNU-5 CD44 +细胞的成球抑制率达85.9％，p＜0.01，有统计学差异。结果表明，单抗Hetumomab可直接作用于胃癌干细胞并抑制其自我更新能力。
表20 检测单抗Hetumomab抑制人胃癌细胞系SNU-5 CD44 +细胞成球的结果
Figure PCTCN2018079785-appb-000020
以上这些结果说明单抗Hetumomab可以直接显著抑制多种癌细胞(肝癌、肺癌、胃癌)的自我更新能力，证明单抗Hetumomab不仅能识别靶向肿瘤干细胞，而且是可以直接抑制肿瘤干细胞的功能性(治疗性)抗肿瘤干细胞单抗。
二、单抗Hetumomab显著抑制多种肿瘤(肝癌、胃癌、肺癌)的肿瘤干细胞的侵袭能力(肿瘤干细胞主要特征之二)。
高侵袭性是肿瘤干细胞的另一个重要生物学特征。为了证明单抗Hetumomab是否是能够直接抑制肝癌干细胞的功能性单抗，采用Transwell侵袭实验对单抗Hetumomab抑制肝癌细胞的侵袭能力进行了分析。实验结果显示(图6)，PBS阴性对照组的侵袭细胞数为(301.0±16.3)/视野，单抗Hetumomab(0.5mg/ml)组的侵袭细胞数为(148±16.4)/视野。实验结果表明，单抗Hetumomab直接作用后的细胞侵袭能力明显减弱，其对 Be17402-V13 sphere细胞侵袭的抑制率为50.8％，p＜0.05，有统计学差异。该实验结果表明，单抗Hetumomab可直接作用于肝癌干细胞并抑制其侵袭能力。
为了证明单抗Hetumomab是否是能够直接抑制肺癌干细胞的功能性单抗，采用同样的方法检测了单抗Hetumomab对人肺癌细胞系SPCA-1侵袭的抑制作用。实验结果显示(表21、图6)，PBS阴性对照组的侵袭细胞数为(232.3±3.1)/视野，单抗Hetumomab组的侵袭细胞数最低为(153.0±6.1)/视野。实验结果表明，单抗Hetumomab直接作用后的细胞侵袭能力明显减弱，其对SPCA-1 sphere细胞侵袭的抑制率为34.1％，p＜0.05，有统计学差异。该实验结果表明，单抗Hetumomab可直接作用于肺癌干细胞并抑制其侵袭能力。
表21 检测单抗Hetumomab抑制人肺癌细胞系SPCA-1侵袭的结果
Figure PCTCN2018079785-appb-000021
为了证明单抗Hetumomab是否是能够直接抑制胃癌干细胞的功能性单抗，采用同样的方法检测了单抗Hetumomab对人胃癌细胞系SNU-5的CD44阳性细胞肿瘤干细胞亚群侵袭的抑制作用。实验结果显示(表22、图6)，PBS阴性对照组的侵袭细胞数为(231±7.0)/视野，单抗Hetumomab组的侵袭细胞数最低为(56±4.0)/视野。实验结果表明，单抗Hetumomab直接作用后的细胞侵袭能力明显减弱，其对SNU-5 CD44 +细胞侵袭的抑制率为75.8％，p＜0.05，有统计学差异。该实验结果表明，单抗Hetumomab可直接作用于胃癌干细胞并抑制其侵袭能力。
表21 检测单抗Hetumomab抑制人胃癌细胞系SNU-5 CD44 +细胞侵袭的结果
Figure PCTCN2018079785-appb-000022
以上这些结果说明单抗Hetumomab可以直接显著抑制多种癌细胞(肝癌、肺癌、胃癌)的侵袭能力，证明单抗Hetumomab不仅能识别靶向肿瘤干细胞，而且是可以直接抑制肿瘤干细胞的功能性(治疗性)抗肿瘤干细胞单抗。
三、单抗Hetumomab显著抑制多种肿瘤(肝癌、肺癌)的肿瘤干细胞的耐受化疗药物的能力(肿瘤干细胞主要特征之三)。
耐药是肿瘤干细胞的生物学特征之一。为了证明单抗Hetumomab是否是能够直接抑制肝癌干细胞的功能性单抗，将培养5天的Be17402-V13 sphere细胞以5000个/孔的 数量接种于96孔板中，并加入纯化的单抗Hetumomab(0.5mg/mL)和PBS于各孔中，在37℃，5％CO 2孵箱中培养24h后，移去含抗体的培养基，每组细胞均用含0、0.0625、0.125、0.25、0.5、1、2、4和8μg/mL共9不同浓度顺铂的培养基培养细胞，48h更换1次含顺铂的完全培养基，5天后按照CCK-8试剂盒说明书加入CCK-8试剂，检测OD450吸光度值，并计算各组IC50值。实验结果显示(图7)，单抗Hetumomab直接作用后的细胞的耐药能力明显下降，其IC50值为0.334μg/mL，而对照组的IC50值为0.9μg/mL，单抗Hetumomab直接作用的细胞耐药能力明显低于对照组。该实验结果表明，单抗Hetumomab可直接作用于肝癌干细胞并抑制其耐药的能力。
为了证明单抗Hetumomab是否是能够直接抑制肺癌干细胞的功能性单抗，采用同样的方法检测了单抗Hetumomab对人肺癌细胞系SPCA-1耐药的抑制作用。实验结果显示(图7)，单抗Hetumomab直接作用后的细胞的耐药能力明显下降，其IC50值最低为0.136μg/mL，而对照组的IC50值为0.351μg/mL，单抗Hetumomab直接作用的细胞耐药能力明显低于对照组。该实验结果表明，单抗Hetumomab可直接作用于肺癌干细胞并抑制其耐药的能力。
以上这些结果说明单抗Hetumomab可以直接显著抑制多种癌细胞(肝癌、肺癌)的耐受化疗药物的能力，证明单抗Hetumomab不仅能识别靶向肿瘤干细胞，而且是可以直接抑制肿瘤干细胞的功能性(治疗性)抗肿瘤干细胞单抗。
实施例5 单抗Hetumomab在动物体内具有抑制肿瘤移植瘤生长、协同化疗的药效学作用
前面的一系列实验结果证明，单抗Hetumomab是靶向多种肿瘤干细胞的单抗；并且体外药效学研究结果表明，单抗Hetumomab可以明显的抑制多种肿瘤干细胞的自我更新、侵袭和耐药能力。为了进一步明确单抗Hetumomab在体内对多种肿瘤生长、转移和耐药的影响，采用多种人肿瘤动物模型，评价了单抗Hetumomab在体内对多种肿瘤生长、转移和耐药的药效学作用。
一、单抗Hetumomab显著抑制人肝癌移植瘤在体内的生长，并能明显协同增强化疗的疗效、显著延长生存期，具有显著的抗肿瘤药效学作用。
进行了单抗Hetumomab在裸鼠体内治疗肝癌的实验研究，观察并比较单用单抗、单用化疗药及单抗联合化疗药治疗肝癌的疗效，分析比较单抗Hetumomab在体内能否治疗肝癌的生长和耐药，并探讨靶向肝癌干细胞治疗肝癌的最优方案。
将Be17402-V13的sphere细胞以3万/只接种至裸鼠皮下，将裸鼠随机分为6组(6只/组)，分别为：单独化疗药组(顺铂0.3mg/kg，6只)；抗体高剂量组(10mg/kg，6只)；抗体高剂量+化疗组(6只)；抗体低剂量组(2.5mg/kg，6只)；抗体低剂量+化疗组(6只)；PBS组(6只)。接种细胞后第二天开始治疗，每周治疗2次，5周后结束治疗。每周2次测量皮下移植瘤的长径和短径，用公式V＝(π/6)×(长径×短径×短径)计算瘤体积，观察各组的肿瘤体积、生长速率的变化。停药后，继续观察移植瘤的生长情况，并记录肿瘤 大小。肿瘤生长速率＝(V t-V 0)/天数，V t是每次测量时的肿瘤体积，V 0是给药前的肿瘤体积(V 0指停止给药时的肿瘤体积)。
小鼠体内移植瘤的生长曲线如图8所示，单抗Hetumomab能显著抑制裸鼠体内移植瘤的生长。并且随着抗体的剂量升高移植瘤抑制率也逐渐升高，存在着剂量依赖关系。实验结果如图9显示，在治疗5个星期停药时，高、低剂量的单抗Hetumomab对移植瘤的抑制率分别为71.5％、54.4％，化疗药组的抑制率为83.5％，单抗高、低剂量联合化疗药组的抑制率相似，都达到了97％左右。结果提示，与单用单抗和单用化疗药组相比，单抗联合化疗药组在移植瘤的治疗中显示出了较好的治疗效果。
停药一个月时，小鼠出现死亡的情况。这时各组的抑制率如图10所示，单抗高、低对移植瘤的抑制率分别为49.1％、34.4％，单抗高、低联合化疗药组的抑制率相似，约为84.5％左右，但单用化疗药组的抑制率为48.6％。该结果提示，单抗联合化疗药组对小鼠移植瘤的抑制率高于其他几种治疗方案组，P＜0.05，差异有统计学意义。停药后一个月时的移植瘤体积与停药时的相比，单抗联合化疗组的肿瘤生长速率为0.051cm 3/天，比PBS对照组肿瘤生长速率(0.239cm 3/天)低4.7倍，且比单抗高、低剂量组和化疗药组的肿瘤生长速率(0.148cm 3/天、0.185cm 3/天和0.154cm 3/天)分别低2.9、3.6和3.1倍，该结果表明单抗联合化疗药的方法可有效抑制肿瘤的生长和复发。
整个治疗和观察过程持续六个月。小鼠在六个月中生存曲线(图11)显示，这6组小鼠的生存曲线分布差别有统计学意义，P＜0.05。说明单抗联合化疗药组的小鼠生存状况明显好于PBS对照组、单抗组和化疗药组。提示，单抗联合化疗药治疗可以延长小鼠的生存期。
以上结果说明，单抗Hetumomab单独使用能够显著抑制人肝癌移植瘤的生长，具有显著的抑制肝癌的药效学作用。单抗Hetumomab联合化疗药组都显示出了对移植瘤高的抑制率，能显著抑制肿瘤的生长，且治疗效果优于单用抗体组和单用化疗药组，表明单抗联合化疗药可有效抑制肿瘤的生长，降低化疗耐药。联合用药组的小鼠生存期明显大于单用抗体组和单用化疗药组，表明单抗Hetumomab联合化疗药治疗肿瘤的方案不仅能治疗肿瘤的生长，还能延长小鼠的生存期，可能是显著抑制了肿瘤在体内的转移引起的小鼠死亡。
二、单抗Hetumomab显著抑制人肺癌移植瘤在体内的生长，具有显著的抗肿瘤药效学作用。
进行了单抗Hetumomab在裸鼠体内治疗肺癌的实验研究，并观察单用单抗、单用化疗药治疗肺癌的疗效。
具体地，收获人肺癌细胞系SPCA-1的球体细胞，以2.5×10 5个细胞/只接种裸小鼠。共分5组，每组5只：PBS对照组、单独化疗组(顺铂0.3mg/kg)、Hetumomab抗体高剂量组(40mg/kg)、Hetumomab抗体中剂量组(10mg/kg)、Hetumomab低剂量组(2.5mg/kg)。接种肺癌细胞后第2天开始抗体治疗，实验组以及对照组通过腹腔注射进行治疗，共治 疗至接种28天后，停药。化疗药组治疗每周2次。每周2次测量皮下移植瘤的长径和短径，计算瘤体积，公式V＝(π/6)×(长径×短径×短径)。观察各组的肿瘤体积变化。
接种28天后停止治疗时，观察并测量小鼠瘤体积生长情况，计算抑制率。小鼠体内移植瘤的生长曲线如图12所示，瘤体积抑制率如表22所示，单抗Hetumomab能显著抑制裸鼠体内移植瘤的生长，并随着抗体的剂量升高移植瘤抑制率也相应升高，在停药时，高、中、低剂量的单抗Hetumomab对移植瘤的抑制率分别为55.97％、43.56％、35.58％，化疗药组的抑制率仅为24.91％。
以上结果说明，单抗Hetumomab单独使用能够显著抑制人肺癌移植瘤的生长，具有显著的抑制肺癌的药效学作用。
表22 单抗Hetumomab抑制动物体内人肺癌SPCA-1移植瘤生长的结果
Figure PCTCN2018079785-appb-000023
三、单抗Hetumomab显著抑制人胃癌移植瘤在体内的生长，并能明显协同增强化疗的疗效，具有显著的抗肿瘤药效学作用。
进行了单抗Hetumomab在裸鼠体内治疗胃癌的实验研究，并观察比较单用单抗、单用化疗药及单抗联合化疗药治疗胃癌的疗效，分析比较单抗Hetumomab在体内能否治疗胃癌的生长和协同增强化疗疗效。
具体地，收获人胃癌细胞系SNU-5-V13的球体细胞，以2.5×10 5个细胞/只接种裸小鼠。共分7组，每组8只：PBS对照组、鼠IgG对照组、单独化疗组(顺铂0.3mg/kg)、Hetumomab抗体高剂量组(20mg/kg)、Hetumomab低剂量组(1.25mg/kg)、Hetumomab高剂量+化疗药、Hetumomab低剂量+化药。接种胃癌细胞后第2天开始抗体治疗，实验组以及对照组通过腹腔注射进行治疗，共治疗一个月后，停药。化疗药组治疗4周，每周2次后停药。每周2次测量皮下移植瘤的长径和短径，计算瘤体积，公式V＝(π/6)×(长径×短径×短径)。观察各组的肿瘤体积变化。
接种一个月后停止治疗时，观察并测量小鼠瘤体积生长情况，计算抑制率。小鼠体内移植瘤的生长曲线如图13所示，瘤体积抑制率如表23所示，单抗Hetumomab能显著抑制裸鼠体内移植瘤的生长，并随着抗体的剂量升高移植瘤抑制率也相应升高，在停药时，高、低剂量的单抗Hetumomab对移植瘤的抑制率分别为57.62％、30.68％，化疗药组的抑制率仅为33.16％，单抗高、低剂量联合化疗药组的抑制率分别达到了70.68％和47.93％，结果提示，单抗联合化疗药组对小鼠移植瘤的抑制率高于单独化疗和单独抗体治疗的治疗方案组，p＜0.05，单抗联合化疗药组在移植瘤的治疗中显示出了更好的 治疗效果。
以上结果说明，单抗Hetumomab单独使用能够显著抑制人胃癌移植瘤的生长，具有显著的抑制胃癌的药效学作用。单抗Hetumomab联合化疗药组都显示出了对移植瘤高的抑制率，能显著抑制肿瘤的生长，且治疗效果优于单用抗体组和单用化疗药组，表明单抗联合化疗药可有效抑制肿瘤的生长，降低化疗耐药。
表23 单抗Hetumomab抑制动物体内人胃癌SNU-5移植瘤生长的结果
Figure PCTCN2018079785-appb-000024
以上这一系列动物体内抑制肿瘤的实验结果证明，单抗Hetumomab在体内对多种人肿瘤(如肝癌、肺癌、胃癌)的生长和耐药具有显著的抑制作用(药效学作用)，对于治疗多种肿瘤生长、转移和耐药具有重要的应用价值。
实施例6 小鼠单克隆抗体Hetumomab可变区序列的克隆及互补决定区序列分析
本实施例采用亚型为IgG1，Kappa轻链的小鼠单克隆抗体Hetumomab的杂交瘤细胞，以Trizol试剂裂解细胞，提取杂交瘤细胞的总RNA，经异丙醇沉淀和乙醇洗涤后，通过RNA电泳以及紫外分光光度计检测，确定RNA的浓度、纯度和完整性。结果获得了来自Hetumomab杂交瘤细胞的浓度为2.5μg/μl、OD260/OD280＝1.8左右的总RNA。常规方法反转录成cDNA第一链，稀释2倍后当做用于PCR扩增的模板。
设计合适的可变区引物组合进行随后的常规的PCR反应扩增抗体可变区基因序列，克隆入ZT4-Blunt载体中，转化大肠杆菌DH5α感受态细胞，挑选阳性克隆并测序。克隆得到Hetumomab单抗的轻链可变区氨基酸序列示于SEQ ID NO：1，重链可变区氨基酸序列示于示于SEQ ID NO：5。
Hetumomab单抗的轻链可变区氨基酸序列(SEQ ID NO：1)：
Figure PCTCN2018079785-appb-000025
下划线分别依次标示VL-CDR1(SEQ ID NO：2：RSSKSLLHSNGITYLY)、VL-CDR2(SEQ ID NO：3：QMSNLAS)、VL-CDR3(SEQ ID NO：4：AQNLELYT)。
Hetumomab单抗的重链可变区氨基酸序列(SEQ ID NO：5)：
Figure PCTCN2018079785-appb-000026
Figure PCTCN2018079785-appb-000027
下划线分别依次标示VH-CDR1(SEQ ID NO：6：TSGMGVS)、VH-CDR2(SEQ ID NO：7：HIYWDDDKRYNPSLKS)、VH-CDR3(SEQ ID NO：8：SFYYYANNSFAY)。
实施例7 衍生自小鼠单克隆抗体Hetumomab的人-鼠嵌合抗体Hetuximab的构建、表达及纯化
本实施例将小鼠单克隆抗体Hetumomab的轻、重链的可变区基因片段，分别克隆入含有人IgG1CH(重链恒定区基因)和人IgG CK(轻链恒定区基因)的瞬转表达载体中，转染真核细胞，从转染细胞的培养上清中采用常规的Protein A亲和层析法纯化Hetumomab的人-鼠嵌合抗体变体，命名为Hetuximab。
采用PCR的方法大量扩增Hetumomab的重链和轻链抗体可变区片段，克隆入pKN009(含有人IgG1CH编码序列)和pKN019(含有人IgG CK编码序列)瞬时表达载体的相应酶切克隆位点，转化大肠杆菌并筛选阳性克隆，阳性克隆进一步测序进行鉴定。结果获得衍生自Hetumomab的人-鼠嵌合抗体Hetuximab及其表达载体。该嵌合抗体的具体蛋白序列如下：
Hetuximab嵌合抗体重链氨基酸序列(SEQ ID NO：9)：
Figure PCTCN2018079785-appb-000028
下划线分别依次标示VH-CDR1(SEQ ID NO：6)、VH-CDR2(SEQ ID NO：7)、VH-CD3(SEQ ID NO：8)、人IgG1CH(SEQ ID NO：11)。
Hetuximab嵌合抗体轻链氨基酸序列(SEQ ID NO：10)：
Figure PCTCN2018079785-appb-000029
下划线分别依次标示VL-CDR1(SEQ ID NO：2)、VL-CDR2(SEQ ID NO：3)、VL-CDR3(SEQ ID NO：4)、人IgG CK(SEQ ID NO：12)。
将上述鉴定正确的轻重链嵌合抗体基因表达载体采用脂质体常规转染真核细胞HEK-293细胞，瞬时表达嵌合抗体。收集上清先用ProteinA亲和层析柱进行初步纯化，再进一步采用阳离子交换层析除去杂质，纯化产物采用SDS-PAGE电泳鉴定纯度。结果 获得了纯度＞95％，2mg/ml的衍生自Hetumomab的人-鼠嵌合抗体Hetuximab。
实施例8 嵌合抗体Hetuximab与亲本鼠单抗识别结合同一抗原蛋白的相同表位，具有相同的特异性和一致的亲和性。
一、采用Western Blot实验证明嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别结合同一抗原蛋白，具有相同的特异性。
采用常规方法提取Hetumomab靶抗原阳性表达的4种人肿瘤细胞SNU-5、BGC-823、MHCC-97L、BEL7402 V13的细胞总蛋白，BCA试剂盒定量提取液蛋白浓度。采用SDS-聚丙烯酰胺凝胶电泳分离适量蛋白样品(20μg)，采用半干式电转仪将蛋白转移至PVDF膜。然后将PVDF膜放入封闭液中(TBST/5％脱脂奶粉)，于水平摇床上，室温封闭1h。加入一抗(嵌合抗体Hetuximab与亲本鼠单抗Hetumomab，均为2ug/ml，用封闭液稀释)，4℃孵育过夜。用TBST漂洗5次，每次5min。相应加入抗人IgG Fc-HRP或抗鼠IgG Fc-HRP二抗(1∶2000～1∶5000稀释)，室温孵育1h。用TBST漂洗3次，每次5min；再用PBS漂洗3次。显影照相。结果如图14所示。
结果表明嵌合抗体Hetuximab与亲本鼠单抗Hetumomab两者识别的蛋白抗原是同一蛋白分子。同时，因两者均特异地与该抗原蛋白结合，而不识别人真核细胞的其他任何蛋白染色其他带，证明嵌合抗体Hetuximab与亲本鼠单抗Hetumomab两者的特异性相同。
二、采用免疫组织化学实验证明嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别结合同一抗原蛋白，具有相同的特异性。
采用前述的传统的常规免疫组织化学技术，以Hetumomab为一抗，采用抗鼠抗体二抗；以及以嵌合抗体Hetuximab为一抗，采用抗人抗体二抗，检测了单抗Hetumomab靶抗原阳性表达的3例人肝癌、肺癌、胃癌患者组织切片及阴性表达的3例人肝癌、肺癌、胃癌患者组织切片。
实验结果显示：嵌合抗体Hetuximab与亲本鼠单抗Hetumomab均能阳性染色单抗Hetumomab靶抗原阳性表达的3例人肝癌、肺癌、胃癌患者组织切片，且同时均不染色单抗Hetumomab靶抗原阴性表达的3例人肝癌、肺癌、胃癌患者组织切片，说明了嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别结合同一抗原蛋白，且不识别人组织中的其他蛋白，具有相同的特异性。
三、采用竞争抑制细胞ELISA实验证明嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别结合同一抗原蛋白的相同表位，具有一致的亲和性。
MHCC-97L细胞接种96孔培养板(4×10 3个/孔)，待细胞长到90％～100％满时进行实验。弃去孔中的培养液，用含0.05％Tween-20的PBS洗涤，300μl/孔，1min/次×5次；按如下设计加入样品，100μl/孔，复孔，37℃孵育1.5h；弃去孔中的液体，用含 0.05％Tween-20的PBS洗涤，300μl/孔，1min/次×5次；加入抗人IgG Fc-HRP(不与鼠IgG Fc反应)或抗鼠IgG Fc-HRP(不与人IgG Fc反应)相应二抗，1∶5000，100μl/孔，37℃孵育1h；弃去孔中的液体，用含0.05％Tween-20的PBS洗涤，300μl/孔，1min/次×3次；纯水再洗涤2次；甩去纯水，加入TMB显色，100μl/孔，37℃孵育30min；加入2M H 2SO 4，50μl/孔；酶标仪测OD450。
采用抗人IgG Fc-HRP(不与鼠IgG Fc反应)作为二抗，结果如下表所示：
表24.
Figure PCTCN2018079785-appb-000030
当采用抗鼠IgG Fc-HRP(不与人IgG Fc反应)作为二抗，结果如下表所示：
表25.
Figure PCTCN2018079785-appb-000031
Figure PCTCN2018079785-appb-000032
将表24和25的原始结果作图，示于图15。从图15可以看到，嵌合抗体Hetuximab和亲本鼠单抗Hetumomab均可相互显著地竞争抑制对方与靶细胞MHCC-97L细胞表面的靶抗原的结合，表明嵌合抗体Hetuximab和亲本鼠单抗Hetumomab识别结合同一抗原蛋白的相同表位；同时两者相互显示出相近的竞争抑制效率，提示嵌合抗体Hetuximab和亲本鼠单抗Hetumomab亲合力相当，具有一致的亲和性。
实施例9 嵌合抗体Hetuximab与亲本鼠单抗识别相同的肿瘤干细胞，具有相同的抑制肿瘤干细胞的药效学作用。
一、嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别相同的一群肿瘤干细胞。
同前所述方法，采用SNU-5、BGC-823、MHCC-97L、BEL7402V13这4种细胞系，同时收获亲本细胞和富含肿瘤干细胞的sphere细胞，同时分别采用Hetuximab(采用抗人IgG Fc作为二抗)和Hetumomab(采用抗鼠IgG Fc作为二抗)进行流式免疫荧光实验，检测了上述4种细胞系亲本细胞和富含肿瘤干细胞的sphere细胞中的阳性细胞比例及在sphere细胞中富集倍数。结果如下：
表26.比较Hetuximab与Hetumomab在不同肿瘤细胞的阳性率以及在sphere细胞中的富集倍数。
Figure PCTCN2018079785-appb-000033
从以上结果可以看到，两者在4种肿瘤细胞及富含肿瘤干细胞的sphere细胞中的阳性率及富集情况均十分一致，因此，嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别相同的一群肿瘤干细胞。
二、嵌合抗体Hetuximab与亲本鼠单抗Hetumomab在体外具有相同的抑制肿瘤干细胞的药效学作用。
同前所述方法，采用SNU-5、BGC-823、MHCC-97L、BEL7402 V13这4种细胞系的富含肿瘤干细胞的sphere细胞，同时分别采用Hetuximab和Hetumomab进行常规的成球抑制实验，检测了Hetuximab和Hetumomab对上述4种细胞系富含肿瘤干细胞的sphere细胞的抑制肿瘤干细胞的药效学作用。结果如下表所示：
表27.Hetuximab和Hetumomab对上述4种细胞系的成球抑制率
Figure PCTCN2018079785-appb-000034
Figure PCTCN2018079785-appb-000035
Figure PCTCN2018079785-appb-000036
Figure PCTCN2018079785-appb-000037
从以上结果可以看到，两者在4种肿瘤细胞系的富含肿瘤干细胞的sphere细胞中的各浓度梯度的抗体抑制肿瘤干细胞成球的效率均十分一致，因此，嵌合抗体Hetuximab与亲本鼠单抗Hetumomab在体外具有相同的抑制肿瘤干细胞的药效学作用。
实施例10 小鼠单克隆抗体Hetumomab可变区序列的人源化改造和糖基化位点改造
本实施例在获得小鼠单克隆抗体Hetumomab的可变区蛋白序列的基础上，采用设计、基因合成、克隆、转染真核细胞的方法进行了Hetumomab的糖基化位点改造和人源化改造。
在人抗体种系(germline)基因序列库(http：//www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)，根据同源性比对结果，寻找最相似人源抗体模板。最终选择VH2(3-1)为基础模板进行CDR移植，将Hetumomab VH CDR-1、VH CDR-2、VH CDR-3移植入其FR框架中，并且根据Hetumomab VH CDR-3序列情况，选择人种系JH4(WGQGTLVTVSS)为J区序列，设计出Hetumomab的重链可变区FR全人源化的重链可变区蛋白序列，命名为Hetuzumab H1。H1版本实现了在Framework区的全人源化。
根据H1重链序列，初始人源化设计了另外两种重链突变体：H2、H3，人源化程度依次序由高到低，具体序列见图16。与嵌合抗体Hetuximab轻链组合，采用细胞ELISA法发现，三种重链突变体(H1、H2、H3)均保持了嵌合抗体Hetuximab的亲和力。由于H1是人源化程度最高的，框架区全部实现了人源化，所以H1被最终确定为优选的人源化分子模板。
轻链初始人源化也设计了三种突变体，根据同源性比对结果，在人抗体种系库(http：//www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板。选择VK II(O11)和JK1为基础模板，将Hetumomab VL CDR-1、VL CDR-2、VL CDR-3移植入其FR框架中，设计出Hetumomab的轻链可变区FR全人源化的轻链可变区蛋白序列，命名为Hetuzumab L1。版本L1实现了在框架区的全人源化。
在L1实现FR区全人源化基础上，设计了L2、L3，人源化程度依次序也是由高到低。然而，通过与嵌合抗体Hetuximab的重链组合，采用细胞ELISA法发现，这三种轻 链突变体的亲和力均大幅度降低。为了找出人源化过程中突变了哪些关键氨基酸，进行了回复突变(Back mutation)，制备了不同9种回复突变体(L4-L12)，具体序列见图17。
通过采用细胞ELISA法分析亲和力发现轻链第9位和第42位的苯丙氨酸Phe(F9和F42)必须回复，保持这两个鼠源分子第6个突变体(L6)亲和力得到回复。基于L6的深度人源化版本L11亲和力与L6接近，基于轻链第11位Asn人源化的L12亲和力也与L11有着一定的近似，其他突变版本则不同。因此L6、L11、L12被最终选定用于最佳轻链人源化版本的优化筛选。
然而，经过抗体序列分析以及在还原性电泳分析中确认Hetuzumab H1重链存在糖基化异构体现象。在电泳分析H1重链系列表达物中，H1L6、H1L11、H1L12均可在重链主带下方有可见的弱次带，分析表明重链可能存在糖基化不均一性，具有糖基化异构体(图18)。这将带来严重的质量均一性问题，非常不利于药物开发。序列分析认为N62(位于CDR2中)、N107(位于CDR3中)是两个可能的糖基化位点。分别对这两个位点进行了丙氨酸突变，设计了2个新的重链人源化版本：N62A为H4(其CDR2序列示于SEQ ID NO：13)，N107A为H5(其CDR3序列示于SEQ ID NO：14)。将H4、H5分别与L11组合，表达后进行电泳分析。结果发现，H5(H1 N107A)的糖基化异构现象消失，并且采用细胞ELISA法发现H5L11基本保持了大致相当的亲和力，H4(H1 N62A)的糖基化异构现象依旧存在并且亲和力降低(图19A)。进一步H5与L11、L6的组合均证实了糖基化异构现象消失，重链为均一条带(图19B)。因此，H5(H1 N107A)就成为基于H1的FR全人源化重链最终选定的可变区序列。
为确定最佳人源化版本，候选三种人源化组合(H5L6、H5L11、H5L12)分别制备纯化抗体。将H5L6、H5L11、H5L12轻重链编码序列克隆入真核高效表达稳定载体，并电转染已驯化悬浮培养CHO-K1(ATCC CCL-64)细胞，通过MSX筛选获得稳定表达株，在无血清无蛋白CD-CHO培养基(Invitrogen)进行流加(Fed-batch)培养。培养获得的上清通过Protein A(GE公司，Mabselect sure柱料)亲和层析柱纯化，进一步通过阳离子交换柱除去杂质和不纯物。通过SDS-PAGE和HPLC分析，产物纯度大于95％(结果见图20，以H5L11为例展示)，内毒素水平＜3EU/mg。可用于各种药效学实验和猴药代动力学分析试验。
实施例11 人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)均与亲本鼠单抗识别结合同一抗原蛋白的相同表位。
一、采用免疫组织化学实验证明人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)与嵌合抗体Hetuximab、亲本鼠单抗Hetumomab识别结合同一抗原蛋白，具有相同的特异性。
采用前述的传统的常规免疫组织化学技术，以Hetumomab为一抗，采用抗鼠抗体二抗；以嵌合抗体Hetuximab为一抗，采用抗人抗体二抗；以及分别以人源化抗体Hetuzumab的3种变体为一抗，采用抗人抗体二抗检测了单抗Hetumomab靶抗原阳性 表达的10例人肝癌、肺癌、胃癌患者组织切片及阴性表达的10例人肝癌、肺癌、胃癌患者组织切片。
实验结果显示：人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)与嵌合抗体Hetuximab、亲本鼠单抗Hetumomab均能阳性染色单抗Hetumomab靶抗原阳性表达的10例人肝癌、肺癌、胃癌患者组织切片，且同时均不染色单抗Hetumomab靶抗原阴性表达的10例人肝癌、肺癌、胃癌患者组织切片，说明了人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)与嵌合抗体Hetuximab、亲本鼠单抗Hetumomab均识别结合同一抗原蛋白，且不识别人组织中的其他蛋白，具有相同的特异性。
二、采用竞争抑制细胞ELISA实验证明人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)与亲本鼠单抗Hetumomab识别结合同一抗原蛋白的相同表位，其中H5L6、H5L11还与鼠单抗Hetumomab具有一致的亲和性。
接种96孔培养板(MHCC-97L细胞，4×10 3个/孔)，待细胞长到90％～100％满时，进行实验。弃去孔中的培养液，用含0.05％ Tween-20的PBS洗涤，300μl/孔，1min/次×5次；加入样品，100μl/孔，复孔，37℃孵育1.5h；弃去孔中的液体，用含0.05％Tween-20的PBS洗涤，300μl/孔，1min/次×5次；加入抗人IgG Fc-HRP(不与鼠IgG Fc反应)或抗鼠IgG Fc-HRP(不与人IgG Fc反应)相应二抗，1∶5000，100μl/孔，37℃孵育1h；弃去孔中的液体，用含0.05％Tween-20的PBS洗涤，300μl/孔，1min/次×3次；纯水再洗涤2次；甩去纯水，加入TMB显色(A液∶B液＝1∶1)，100μl/孔，37℃孵育30min；加入2M H 2SO 4，50μl/孔；酶标仪测OD450。
结果如下表28、29以及图21所示：
表28 当采用抗人IgG Fc-HRP(不与鼠IgG Fc反应)作为二抗时：
Figure PCTCN2018079785-appb-000038
表29 当采用抗鼠IgG Fc-HRP(不与人IgG Fc反应)作为二抗：
Figure PCTCN2018079785-appb-000039
Figure PCTCN2018079785-appb-000040
各人源化单抗0.25ug/ml恒定不变，采用鼠单抗浓度梯度去竞争抑制各人源化单抗与抗原结合时计算所得鼠单抗IC50的结果如下表30所示。
表30 鼠单抗对人源化单抗和嵌合单抗抑制的IC50
Figure PCTCN2018079785-appb-000041
从上述结果可以看到，亲本鼠单抗Hetumomab可显著竞争抑制Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab与靶细胞MHCC-97L细胞表面的靶抗原的结合，表明Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab和亲本鼠单抗Hetumomab识别结合同一抗原蛋白的相同表位。同时Hetuzumab的2种变体(H5L6、H5L11)两者相互显示出相近的被竞争抑制的程度，被抑制程度略高于嵌合抗体Hetuximab，而Hetuzumab的1种变体(H5L12)显示出显著较高的被竞争抑制的程度。这提示嵌合抗体Hetuximab和亲本鼠单抗Hetumomab亲合力相当，人源化抗体Hetuzumab的2种变体(H5L6，H5L11)亲合力虽略微下降，但总体相当，具有总体一致的亲和性；而人源化抗体Hetuzumab的变体H5L12亲合力有一定下降。
鼠单抗0.25ug/ml恒定不变，采用各人源化单抗浓度梯度去竞争抑制鼠单抗与抗原结合时计算所得各人源化单抗IC50的结果如下表31所示。
表31 人源化单抗和嵌合单抗对鼠单抗抑制的IC50
Figure PCTCN2018079785-appb-000042
从上述结果可以看到，Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab均可显著竞争抑制亲本鼠单抗Hetumomab与靶细胞MHCC-97L细胞表面的靶抗原的结合，表明Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab 和亲本鼠单抗Hetumomab识别结合同一抗原蛋白的相同表位；同时Hetuzumab的2种变体(H5L6、H5L11)两者相互显示出相近的竞争抑制效率，略低于嵌合抗体Hetuximab，而Hetuzumab的1种变体(H5L12)显示出较低的竞争抑制效率。这提示嵌合抗体Hetuximab和亲本鼠单抗Hetumomab亲合力相当，人源化抗体Hetuzumab的2种变体(H5L6，H5L11)亲合力虽略微下降，但总体相当，具有总体一致的亲和性；而人源化抗体Hetuzumab的1种变体(H5L12)亲合力有一定下降。
从以上这些结果说明，人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab和亲本鼠单抗Hetumomab均可相互显著地竞争抑制对方与靶细胞MHCC-97L细胞表面的靶抗原的结合，表明人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab和亲本鼠单抗Hetumomab识别结合同一抗原蛋白的相同表位；同时它们(H5L6、H5L11、Hetuximab、Hetumomab)相互显示出相近的竞争抑制关系，提示H5L6、H5L11与鼠单抗Hetumomab具有一致的亲和性。
实施例12 人源化抗体Hetuzumab的变体均与亲本鼠单抗识别相同的肿瘤干细胞，具有相同的抑制肿瘤干细胞的体内外药效学作用。
一、采用流式免疫荧光实验证明人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)与亲本鼠单抗Hetumomab识别相同的一群肿瘤干细胞。
同前所述方法，采用SNU-5、BGC-823、MHCC-97L、BEL7402V13这4种细胞系，同时收获亲本细胞和富含肿瘤干细胞的sphere细胞，同时分别采用人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)、Hetuximab(采用抗人IgG Fc作为二抗)和Hetumomab(采用抗鼠IgG Fc作为二抗)进行流式免疫荧光实验，检测上述4种细胞系亲本细胞和富含肿瘤干细胞的sphere细胞中的阳性细胞比例及在sphere细胞中富集倍数。结果如下：
表32 不同抗体在4种细胞系亲本细胞和sphere细胞中的阳性比例及对sphere细胞的富集倍数
Figure PCTCN2018079785-appb-000043
Figure PCTCN2018079785-appb-000044
从以上结果可以看到，人源化抗体Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab与亲本鼠单抗Hetumomab在4种肿瘤细胞及富含肿瘤干细胞的sphere细胞中的阳性率及富集情况均十分一致，尤其是Hetuzumab H5L6和Hetuzumab H5L11。因此，Hetuzumab的3种变体(H5L6、H5L11、H5L12)、嵌合抗体Hetuximab与亲本鼠单抗Hetumomab识别相同的一群肿瘤干细胞。
二、采用成球抑制实验证明人源化抗体Hetuzumab的变体、嵌合抗体Hetuximab与 亲本鼠单抗Hetumomab在体外具有相同的抑制肿瘤干细胞的药效学作用。
同前所述方法，采用SNU-5、BGC-823、MHCC-97L、BEL7402V13这4种细胞系的富含肿瘤干细胞的sphere细胞，同时分别采用Hetuzumab的不同变体、Hetuximab和Hetumomab进行常规的成球抑制实验，检测对上述4种细胞系富含肿瘤干细胞的sphere细胞的抑制肿瘤干细胞的药效学作用。4种细胞系结果分别如下：
表33 不同抗体对4种细胞系的成球抑制
Figure PCTCN2018079785-appb-000045
Figure PCTCN2018079785-appb-000046
Figure PCTCN2018079785-appb-000047
Figure PCTCN2018079785-appb-000048
Figure PCTCN2018079785-appb-000049
Figure PCTCN2018079785-appb-000050
从以上结果可以看到，人源化抗体Hetuzumab的2种变体(H5L6、H5L11)、嵌合抗体Hetuximab与亲本鼠单抗Hetumomab在4种肿瘤细胞系的富含肿瘤干细胞的sphere细胞中的各浓度梯度的抗体抑制肿瘤干细胞成球的效率均十分一致，因此，人源化抗体Hetuzumab的2种变体(H5L6、H5L11)与亲本鼠单抗Hetumomab在体外具有相同的抑制肿瘤干细胞的药效学作用。
三、人源化抗体Hetuzumab的变体显著抑制人肝癌移植瘤在体内的生长，并能明显协同增强化疗的疗效，具有显著的抗肿瘤药效学作用。
进行人源化抗体Hetuzumab在裸鼠体内治疗人肝癌的实验研究，并观察比较单用单抗、单用化疗药及单抗联合化疗药治疗肝癌的疗效，分析比较单抗Hetuzumab在体内能否治疗肝癌的生长和协同增强化疗疗效。
具体地，收获人肝癌细胞系Be17402-V13的sphere细胞以30000个细胞/只接种至裸鼠皮下。共分7组，每组8只：PBS对照组、鼠IgG对照组、单独化疗组(顺铂0.3mg/kg)、Hetuzumab H5L11高剂量组(20mg/kg)、Hetuzumab H5L11低剂量组(1.25mg/kg)、Hetuzumab H5L11高剂量+化疗药、Hetuzumab H5L11低剂量+化药。接种癌细胞后第2天开始抗体治疗，实验组以及对照组通过腹腔注射进行治疗，共治疗36d后，停药。化疗药组治疗4周，每周2次后停药。每周2次测量皮下移植瘤的长径和短径，计算瘤体积，公式V＝(π/6)×(长径×短径×短径)。观察各组的肿瘤体积变化。
接种36d后停止治疗时，观察并测量小鼠瘤体积生长情况，计算抑制率。小鼠体内移植瘤的生长曲线如图22所示，瘤体积抑制率如表34所示。人源化单抗Hetuzumab H5L11能显著抑制裸鼠体内人肝癌移植瘤的生长，并随着抗体的剂量升高移植瘤抑制率也相应升高。在停药时，高、低剂量的人源化单抗Hetuzumab H5L11对移植瘤的抑制率分别为47.47％、36.80％。化疗药组的抑制率仅为14.52％，几乎不起作用。但人源化单抗Hetuzumab H5L11高、低剂量联合化疗药组的抑制率分别达到了65.08％和32.66％。结果提示，人源化单抗Hetuzumab H5L11联合化疗药组对小鼠移植瘤的抑制率高于单独 化疗和单独抗体治疗的治疗方案组，p＜0.05，人源化单抗Hetuzumab H5L11联合化疗药组在移植瘤的治疗中显示出了更好的治疗效果。
以上结果说明，人源化单抗Hetuzumab H5L11单独使用能够显著抑制人恶性肿瘤移植瘤的生长，具有显著的抑制人恶性肿瘤的药效学作用。人源化单抗Hetuzumab H5L11联合化疗药组都显示出了对移植瘤最高的抑制率，能显著抑制肿瘤的生长，且治疗效果优于单用抗体组和单用化疗药组，表明人源化单抗Hetuzumab H5L11联合化疗药可有效抑制肿瘤的生长，降低化疗耐药。
表34 人源化单抗Hetuzumab H5L11抑制动物体内人肝癌细胞系Be17402-V13移植瘤生长的结果
Figure PCTCN2018079785-appb-000051
以上这动物体内抑制肿瘤的实验结果证明，人源化单抗Hetuzumab H5L11在体内对人肿瘤的生长和耐药具有显著的抑制作用(药效学作用)，对于治疗人肿瘤生长、转移和耐药具有重要的应用价值。
序列表
＞SEQ ID NO：1     Hetumomab轻链可变区
Figure PCTCN2018079785-appb-000052
＞SEQ ID NO：2     Hetumomab VL-CDR1
Figure PCTCN2018079785-appb-000053
＞SEQ ID NO：3     Hetumomab VL-CDR2
Figure PCTCN2018079785-appb-000054
＞SEQ ID NO：4     Hetumomab VL-CDR3
Figure PCTCN2018079785-appb-000055
＞SEQ ID NO：5     Hetumomab重链可变区
Figure PCTCN2018079785-appb-000056
＞SEQ ID NO：6     Hetumomab VH-CDR1
Figure PCTCN2018079785-appb-000057
＞SEQ ID NO：7     Hetumomab VH-CDR2
Figure PCTCN2018079785-appb-000058
＞SEQ ID NO：8       Hetumomab VH-CDR3
Figure PCTCN2018079785-appb-000059
＞SEQ ID NO：9       Hetuximab嵌合抗体重链氨基酸序列
Figure PCTCN2018079785-appb-000060
＞SEQ ID NO：10       Hetuximab嵌合抗体轻链氨基酸序列
Figure PCTCN2018079785-appb-000061
＞SEQ ID NO：11       人IgG1 CH
Figure PCTCN2018079785-appb-000062
＞SEQ ID NO：12       人IgG CK
Figure PCTCN2018079785-appb-000063
＞SEQ ID NO：13      突变的VH CDR2
Figure PCTCN2018079785-appb-000064
＞SEQ ID NO：14      突变的VH CDR3
Figure PCTCN2018079785-appb-000065
＞SEQ ID NO：15     人源化重链可变区H1
Figure PCTCN2018079785-appb-000066
＞SEQ ID NO：16     人源化重链可变区H2
Figure PCTCN2018079785-appb-000067
＞SEQ ID NO：17     人源化重链可变区H3
Figure PCTCN2018079785-appb-000068
＞SEQ ID NO：18     人源化重链可变区H4
Figure PCTCN2018079785-appb-000069
＞SEQ ID NO：19     人源化重链可变区H5
Figure PCTCN2018079785-appb-000070
＞SEQ ID NO：20     人源化轻链可变区L1
Figure PCTCN2018079785-appb-000071
＞SEQ ID NO：21     人源化轻链可变区L2
Figure PCTCN2018079785-appb-000072
＞SEQ ID NO：22     人源化轻链可变区L3
Figure PCTCN2018079785-appb-000073
＞SEQ ID NO：23     人源化轻链可变区L4
Figure PCTCN2018079785-appb-000074
＞SEQ ID NO：24     人源化轻链可变区L5
Figure PCTCN2018079785-appb-000075
＞SEQ ID NO：25     人源化轻链可变区L6
Figure PCTCN2018079785-appb-000076
＞SEQ ID NO：26     人源化轻链可变区L7
Figure PCTCN2018079785-appb-000077
＞SEQ ID NO：27     人源化轻链可变区L8
Figure PCTCN2018079785-appb-000078
＞SEQ ID NO：28     人源化轻链可变区L9
Figure PCTCN2018079785-appb-000079
＞SEQ ID NO：29     人源化轻链可变区L10
Figure PCTCN2018079785-appb-000080
＞SEQ ID NO：30     人源化轻链可变区L11
Figure PCTCN2018079785-appb-000081
＞SEQ ID NO：31     人源化轻链可变区L12
Figure PCTCN2018079785-appb-000082
＞SEQ ID NO：32     人源化重链H1
Figure PCTCN2018079785-appb-000083
＞SEQ ID NO：33     人源化重链H2
Figure PCTCN2018079785-appb-000084
Figure PCTCN2018079785-appb-000085
＞SEQ ID NO：34     人源化重链H3
Figure PCTCN2018079785-appb-000086
＞SEQ ID NO：35     人源化重链H4
Figure PCTCN2018079785-appb-000087
＞SEQ ID NO：36     人源化重链H5
Figure PCTCN2018079785-appb-000088
＞SEQ ID NO：37     人源化轻链L1
Figure PCTCN2018079785-appb-000089
＞SEQ ID NO：38     人源化轻链L2
Figure PCTCN2018079785-appb-000090
＞SEQ ID NO：39     人源化轻链L3
Figure PCTCN2018079785-appb-000091
＞SEQ ID NO：40     人源化轻链L4
Figure PCTCN2018079785-appb-000092
＞SEQ ID NO：41     人源化轻链L5
Figure PCTCN2018079785-appb-000093
＞SEQ ID NO：42     人源化轻链L6
Figure PCTCN2018079785-appb-000094
＞SEQ ID NO：43     人源化轻链L7
Figure PCTCN2018079785-appb-000095
＞SEQ ID NO：44    人源化轻链L8
Figure PCTCN2018079785-appb-000096
＞SEQ ID NO：45     人源化轻链L9
Figure PCTCN2018079785-appb-000097
＞SEQ ID NO：46     人源化轻链区L10
Figure PCTCN2018079785-appb-000098
＞SEQ ID NO：47     人源化轻链L11
Figure PCTCN2018079785-appb-000099
＞SEQ ID NO：48     人源化轻链L12
Figure PCTCN2018079785-appb-000100

Claims (35)

  1. 一种针对肿瘤干细胞的分离的单克隆抗体或其抗原结合片段，其中所述单克隆抗体包含轻链可变区和重链可变区，
    所述轻链可变区包含：
    VL CDR1，其包含SEQ ID NO：2所示氨基酸序列或相对于SEQ ID NO：2具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，
    VL CDR2，其包含SEQ ID NO：3所示氨基酸序列或相对于SEQ ID NO：3具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，和
    VL CDR3，其包含SEQ ID NO：4所示氨基酸序列或相对于SEQ ID NO：4具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列；
    所述重链可变区包含：
    VH CDR1，其包含SEQ ID NO：6所示氨基酸序列或相对于SEQ ID NO：6具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列，
    VH CDR2，其包含SEQ ID NO：7所示氨基酸序列或相对于SEQ ID NO：7具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列例如SEQ ID NO：13所示序列，和
    VH CDR3，其包含SEQ ID NO：8所示氨基酸序列或包含相对于SEQ ID NO：8具有1或2个氨基酸残基取代、缺失或添加的氨基酸序列例如SEQ ID NO：14所示序列。
  2. 权利要求1的分离的单克隆抗体或其抗原结合片段，其中所述单克隆抗体包含轻链可变区和重链可变区，
    所述轻链可变区包含：
    VL CDR1，其包含SEQ ID NO：2所示氨基酸序列，
    VL CDR2，其包含SEQ ID NO：3所示氨基酸序列，和
    VL CDR3，其包含SEQ ID NO：4所示氨基酸序列；
    所述重链可变区包含：
    VH CDR1，其包含SEQ ID NO：6所示氨基酸序列，
    VH CDR2，其包含SEQ ID NO：7所示氨基酸序列，和
    VH CDR3，其包含SEQ ID NO：14所示氨基酸序列。
  3. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述轻链可变区包含SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1具有至少85％、至少90％、至少95％或更高序列相同性的氨基酸序列。
  4. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述重链可变区包含SEQ ID NO：5所示氨基酸序列或与SEQ ID NO：5具有至少85％、至少90％、至少95％或更高序列相同性的氨基酸序列。
  5. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述重链可变区包含SEQ ID NO：15-19之一所示氨基酸序列。
  6. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述轻链可变区包含SEQ ID NO：20-31之一所示的氨基酸序列。
  7. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述重链可变区包含SEQ ID NO：19所示的氨基酸序列，所述轻链可变区包含SEQ ID NO：30所示氨基酸序列。
  8. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述单克隆抗体包含人重链恒定区，例如，包含SEQ ID NO：11所示的氨基酸序列的人重链恒定区。
  9. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述单克隆抗体包含人轻链恒定区，例如，包含SEQ ID NO：12所示的氨基酸序列的人轻链恒定区。
  10. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述单克隆抗体包含具有SEQ ID NO：9所示氨基酸序列的重链和具有SEQ ID NO：10所示氨基酸序列的轻链。
  11. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述单克隆抗体包含具有SEQ ID NO：32-36之一所示的氨基酸序列的重链和具有SEQ ID NO：37-48之一所示的氨基酸序列的轻链。
  12. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述单克隆抗体包含SEQ ID NO：36所示的重链和SEQ ID NO：47所示的轻链。
  13. 权利要求1的分离的单克隆抗体或其抗原结合片段，所述单克隆抗体包含SEQ ID NO：36所示的重链和SEQ ID NO：42所示的轻链。
  14. 一种单克隆抗体或其抗原结合片段，所述单克隆抗体由于2016年3月16日以保藏号CGMCC No.12251保藏于中国微生物菌种保藏管理委员会普通微生物中心的小鼠杂交瘤细胞产生。
  15. 一种杂交瘤细胞，其以保藏号CGMCC No.12251于2016年3月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
  16. 一种药物组合物，其包含权利要求1-14中任一项的单克隆抗体或其抗原结合片段以及药学上可接受的载体。
  17. 权利要求16的药物组合物，其中所述单克隆抗体或其抗原结合片段与选自细胞毒素、放射性同位素或生物活性蛋白质的治疗性部分缀合。
  18. 一种在患者中治疗恶性肿瘤、预防和/或治疗恶性肿瘤转移或复发的方法，所述方法包括给所述患者施用有效量的权利要求1-14中任一项的单克隆抗体或其抗原结合片段或权利要求16或17的药物组合物。
  19. 权利要求18的方法，其中所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
  20. 权利要求18或19的方法，还包括给所述患者施用其它抗肿瘤治疗手段，例如施用化疗剂、靶向其它肿瘤特异性抗原的抗体或放疗。
  21. 权利要求1-14中任一项的单克隆抗体或其抗原结合片段或权利要求16或17的药物组合物在制备用于治疗恶性肿瘤、预防和/或治疗恶性肿瘤转移或复发的药物中的用途。
  22. 权利要求21的用途，其中所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
  23. 一种检测生物学样品中肿瘤干细胞存在的方法，包括：
    a)使所述生物学样品与权利要求1-14中任一项的单克隆抗体或其抗原结合片段接触；
    b)检测所述单克隆抗体或其抗原结合片段与所述生物学样品中的靶抗原的结合，
    其中检出所述结合代表所述生物学样品中存在肿瘤干细胞。
  24. 一种用于分离肿瘤干细胞的方法，所述方法包括：
    (a)提供疑似包含肿瘤干细胞的细胞群；
    (b)鉴定所述细胞的亚群，其结合权利要求1-14中任一项的单克隆抗体或其抗原结合片段；和
    (c)分离所述亚群。
  25. 权利要求23或24的方法，其中所述肿瘤干细胞选自乳腺癌干细胞、大肠癌干细胞、胰腺癌干细胞、前列腺癌干细胞、肝癌干细胞、肺癌干细胞和胃癌干细胞。
  26. 一种检测患者中恶性肿瘤的存在的方法，包括：
    a)使获得自所述患者的生物学样品与权利要求1-14中任一项的单克隆抗体或其抗原结合片段接触；
    b)检测所述单克隆抗体或其抗原结合片段与所述生物学样品中的靶抗原的结合，其中检出所述结合代表所述患者中存在恶性肿瘤。
  27. 一种用于预后患者中恶性肿瘤复发或进展的方法，所述方法包括：
    (a)从所述患者中分离包含循环细胞的生物学样品；
    (b)使所述包含循环细胞的生物学样品与权利要求1-14中任一项的单克隆抗体或其抗原结合片段接触；和
    (c)鉴定结合所述单克隆抗体或其抗原结合片段的循环细胞的存在，
    从而预后所述患者中恶性肿瘤的复发或进展。
  28. 权利要求27的方法，其中所述恶性肿瘤的进展包含所述恶性肿瘤在患者中的转移。
  29. 权利要求26-28中任一项的方法，其中所述生物学样品包括血液样品、淋巴样品或其组分。
  30. 权利要求26-28中任一项的方法，其中所述恶性肿瘤选自乳腺癌、大肠癌、胰腺癌、前列腺癌、肝癌、肺癌和胃癌。
  31. 一种分离的核酸分子，其编码权利要求1-14中任一项的单克隆抗体或其抗原结合片段。
  32. 权利要求31的分离的核酸分子，所述核酸分子与表达调控序列可操作地连接。
  33. 一种表达载体，其包含权利要求31-32中任一项的核酸分子。
  34. 一种宿主细胞，其由权利要求31-32中任一项的核酸分子或权利要求31的表达 载体转化。
  35. 一种生产针对人肿瘤干细胞的单克隆抗体或其抗原结合片段的方法，包括：
    (i)在适合所述核酸分子或表达载体表达的情况下培养权利要求34的宿主细胞，和
    (ii)分离并纯化由所述核酸分子或表达载体表达的抗体或其抗原结合片段。
PCT/CN2018/079785 2017-06-23 2018-03-21 一种靶向人肿瘤干细胞的单克隆抗体及其应用 WO2018233333A1 (zh)

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