WO2021031930A1 - 抗体的突变体及其应用 - Google Patents

抗体的突变体及其应用 Download PDF

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WO2021031930A1
WO2021031930A1 PCT/CN2020/108434 CN2020108434W WO2021031930A1 WO 2021031930 A1 WO2021031930 A1 WO 2021031930A1 CN 2020108434 W CN2020108434 W CN 2020108434W WO 2021031930 A1 WO2021031930 A1 WO 2021031930A1
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antibody
fragment
seq
variant
amino acid
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PCT/CN2020/108434
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English (en)
French (fr)
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马宁宁
汪琳
宋春雨
李明莹
薛世平
刘永祥
徐伟伟
朱立峰
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沈阳药科大学
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Priority to KR1020227009204A priority Critical patent/KR20220045047A/ko
Priority to US17/636,818 priority patent/US20230211007A1/en
Priority to JP2022510797A priority patent/JP7448638B2/ja
Priority to EP20854930.3A priority patent/EP4019544A4/en
Priority to AU2020332026A priority patent/AU2020332026A1/en
Priority to CN202080008189.2A priority patent/CN113330029A/zh
Publication of WO2021031930A1 publication Critical patent/WO2021031930A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies. Specifically, it relates to mutants of antibodies, compositions and/or conjugates containing them, and uses thereof.
  • Antibody-drug conjugates are a type of targeted therapeutic drugs that combine the specificity of antibodies with the cytotoxicity of cytotoxic therapeutic agents.
  • ADC is mainly considered as a candidate for the treatment of various cancers.
  • ADC contains antibodies linked to therapeutic drugs.
  • bispecific antibodies can also be synthesized quickly and in large quantities from two existing antibodies by chemical coupling. For example, two IgG molecules or two Fab fragments are connected by a coupling reagent.
  • abzymes to prepare CovX-Bodies [1], connecting two polypeptides with target neutralization to the two Fab arms of an IgG molecule. Therefore, site-directed coupling technology can also be applied to prepare new bispecific antibodies [1].
  • Antibody molecules contain many groups that can be used to modify crosslinks, such as amino and carboxyl groups. However, these groups are abundant in antibody molecules, resulting in randomness of antibody coupling sites. There are many methods that rely on disulfide bonds on antibody molecules. Due to their limited number and relatively fixed positions, the antigen binding site will not be shielded after the drug is coupled. These characteristics make the disulfide bonds on the antibody become antibody coupling One of the suitable sites. However, the reduction of these endogenous disulfide bonds is non-specific, that is, the disulfide bonds in the hinge region and the disulfide bonds between the heavy and light chains are all reduced, resulting in the coupling product being a mixture of diverse components [2, 3] .
  • Cysteine thiols are reactive at neutral pH, which is different from most amines whose protonation and nucleophilicity decrease near pH 7. Due to the relative reactivity of free thiols (sulfhydryl groups), proteins with cysteine residues usually exist in their oxidized form as disulfide-linked oligomers or have internally bridged di Sulfide group. Cysteine sulfhydryl groups in antibodies are generally more reactive to electrophilic coupling reagents than antibody amines or hydroxyl groups, that is, more nucleophilic. Therefore, the amino acid residues in the antibody can be mutated to cysteine, and then the cysteine can be used for the conjugation reaction. Junutula et al.
  • the sulfhydryl group on the cysteine obtained by the mutation can always be kept free during the expression and purification process, without any pretreatment and directly applied to the downstream couple. Joint reaction. Therefore, the applicant has developed reasonable cysteine mutation sites, and the cysteine sulfhydryl groups obtained by mutating these sites are easier to maintain the free state and have higher reactivity. Then the antibody is coupled to another functional molecule (antibody fragment, polypeptide or small molecule drug) through a maleimide-containing linker.
  • the present invention mainly provides a site that can be mutated into cysteine in the constant region of an antibody.
  • the mutation is introduced into a selected site-specific conjugation site to allow the antibody, fragment or derivative to be effective Load conjugation, wherein the one or more mutations include a mutation at position 166 of the light chain (numbering according to the Kabat system position).
  • the present invention also provides an independent structure of a bispecific antibody with anti-angiogenesis function.
  • the present invention provides the following aspects:
  • a variant of an antibody or fragment thereof characterized in that the antibody or fragment thereof contains a light chain constant region, and according to the Kabat numbering system, the amino acid at position 166 is mutated to cysteine, preferably, the antibody is of human origin A modified antibody, a chimeric antibody, and more preferably an IgG antibody.
  • a variant of the antibody or fragment thereof of 1 or 2 the variant comprising a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the light chain is SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 9, the amino acid sequence of the light chain is shown in SEQ ID NO: 13; or the amino acid sequence of the heavy chain is SEQ ID NO: 17, the amino acid sequence of the light chain is shown in SEQ ID NO: 21.
  • the variant of the antibody or fragment thereof according to any one of the above 1-3 characterized in that the fragment is a Fab fragment, a Fab' fragment or a F(ab') 2 fragment, preferably the heavy chain of the Fab fragment
  • the amino acid sequence is shown in SEQ ID NO: 25, and the light chain amino acid sequence of the Fab fragment is shown in SEQ ID NO: 29.
  • a bispecific antibody or fusion protein characterized by comprising a variant of the antibody or fragment thereof described in any one of 1-4.
  • a conjugate characterized by comprising a variant of the antibody or fragment thereof described in any one of 1-4.
  • the conjugate of the above 6, the conjugate is selected from polyethylene glycol, cytotoxic agent, active peptide, nanobody, single domain antibody, Fab fragment, Fab' fragment, scFv, small molecule drugs, chemotherapy Or radiotherapy, preferably, the active peptide is Agn2 active peptide, more preferably the sequence is the Agn2 active peptide shown in SEQ ID NO: 5, more preferably, the Agn2 active peptide is connected to the Bevac of the present invention via a linker. Conjugation of monoclonal antibody variants, further preferably, the linker has an amide bond, specifically formamide-PEG 4 -NHS, and has the structure shown in structural formula I
  • polyethylene glycol is monomethoxy polyethylene glycol, preferably mPEG2000, mPEG5000, mPEG10000.
  • polyethylene glycol is further conjugated to a drug molecule, preferably the polyethylene glycol is a linear bifunctional polyethylene glycol, a linear heterofunctional polyethylene glycol Or multi-arm functionalized PEG.
  • a pharmaceutical composition characterized by comprising a variant of the antibody or fragment thereof according to any one of 1-4, the bispecific antibody or fusion protein according to 5, and any one of 6-9 above Conjugates, and optionally, a pharmaceutical carrier.
  • cancer preferably non-small cell lung cancer such as advanced, metastatic or recurrent non-squamous cell non-small cell lung cancer, colorectal cancer such as metastatic colorectal cancer, breast cancer, malignant glioma and renal cell carcinoma polymorphic Glioblastoma
  • method comprising administering a variant of the antibody or fragment thereof according to any one of 1-4 above, the bispecific antibody or fusion protein according to 5 above, any one of 6-9 above
  • the conjugate, or the pharmaceutical composition described in 10 above preferably non-small cell lung cancer such as advanced, metastatic or recurrent non-squamous cell non-small cell lung cancer, colorectal cancer such as metastatic colorectal cancer, breast cancer, malignant glioma and renal cell carcinoma polymorphic Glioblastoma
  • a variant of the antibody or fragment thereof of any one of the above 1-4, the bispecific antibody/fusion protein of the above 5 or the conjugate of any of the above 6-9, or the pharmaceutical composition of the above 10 In preparation for the treatment of cancer (preferably non-small cell lung cancer such as advanced, metastatic or recurrent non-squamous cell non-small cell lung cancer, colorectal cancer such as metastatic colorectal cancer, breast cancer, malignant glioma and renal cell carcinoma Glioblastoma multiforme) drugs or kits.
  • cancer preferably non-small cell lung cancer such as advanced, metastatic or recurrent non-squamous cell non-small cell lung cancer, colorectal cancer such as metastatic colorectal cancer, breast cancer, malignant glioma and renal cell carcinoma Glioblastoma multiforme
  • kits comprising a variant of the antibody or fragment of any one of the above 1-4, the bispecific antibody/fusion protein of the above 5 or the conjugate of any one of the above 6-9, or the above 10
  • the kit further comprises an agent for combined administration with the antibody or a fragment thereof, such as carboplatin or cisplatin.
  • FIG 1 shows the SDS-PAGE electrophoresis of wild-type (WT, also called “anti-VEGF antibody” in the present invention) antibody, 166 mutant antibody (166C), 124 mutant antibody (124C) in reduced and non-reduced states Figure.
  • WT wild-type
  • 166C 166 mutant antibody
  • 124C 124 mutant antibody
  • FIG. 1 SDS-PAGE electrophoresis image of anti-HER2 wild-type antibody and mutant after coupling with DC (polypeptide-linker).
  • FIG. 14 Inhibition of wild-type antibody (WT), mutants (124C and 166C) and conjugates (124ADC and 166ADC) on HUVEC proliferation (VEGF stimulated proliferation) of endothelial cells. It can be seen from the figure that the antibody mutant did not change the activity due to the cysteine mutation at the selected site, indicating that the selected site will not affect the biological activity of the antibody. Similarly, the conjugate produced after coupling DC has no effect on the activity of the antibody to inhibit cell proliferation.
  • WT wild-type antibody
  • mutants 124C and 166C
  • conjugates 124ADC and 166ADC
  • restriction endonucleases used in the examples were purchased from Thermo Fisher Scientific (China) Co., Ltd.
  • the reagents or materials used in the following examples, unless the source is explicitly mentioned, are all conventionally purchased in the field .
  • CHO-K1 cell culture medium was purchased from sigma company
  • the successfully constructed expression vector was transformed into DH5 ⁇ competent E. coli strain.
  • the transformed DH5 ⁇ strain was verified by double digestion with BstBI and PacI enzymes and HindIII and EcoRI enzymes, and sequencing, and positive clones were selected.
  • a plasmid extraction kit (purchased from Kangwei Century) was used to lyse the resulting E. coli and extract the plasmid to obtain an expression vector containing heavy and light chains.
  • the purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, spread on a 96-well plate, grown for 15 days, picked out a single clone, and measured the cell antibody production by ELISA. Before selection 20% was transferred to a 24-well plate. After 7 days of growth, the measured antibody production of the cells was selected from the first 5 to 6 strains and transferred to shake flask culture to achieve the expression of anti-VEGF antibody in CHO-K1 cells. After the serum-free culture is stable, the culture is continued for 7 days and then the material is collected for purification and subsequent experiments.
  • the variant 166C of the present invention was obtained by replacing the amino acid at position 166 of the light chain with cysteine, and the DNA of the control antibody 124C obtained by replacing the amino acid at position 124 of the light chain with cysteine was chemically synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.), then double-enzyme digestion of the antibody heavy chain gene with BstBI and PacI, double-enzyme digestion of the light chain gene with HindIII and EcoRI, and ligate the heavy chain gene to the BstBI and PacI treated with T4 ligase In the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.), after the ligation is successful, double enzyme digestion with HindIII and
  • the successfully constructed expression vector was transformed into DH5 ⁇ competent E. coli strain.
  • the transformed DH5 ⁇ strain was verified by double digestion with BstBI and PacI enzymes and HindIII and EcoRI enzymes, and sequencing, and positive clones were selected.
  • a plasmid extraction kit (purchased from Kangwei Century) was used to lyse the resulting E. coli and extract the plasmid to obtain an expression vector containing heavy and light chains.
  • the purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, spread on a 96-well plate, grown for 15 days, picked out a single clone, and measured the cell antibody production by ELISA. Before selection 20% was transferred to a 24-well plate. After 7 days of growth, the measured cell antibody yields were selected from the first 5 to 6 strains and transferred to shake flask culture to achieve the expression of anti-CD20 antibody in CHO-K1 cells. After the serum-free culture is stable, the culture is continued for 7 days and then the material is collected for purification and subsequent experiments.
  • the successfully constructed expression vector was transformed into DH5 ⁇ competent E. coli strain.
  • the transformed DH5 ⁇ strain was verified by double digestion with BstBI and PacI enzymes and HindIII and EcoRI enzymes, and sequencing, and positive clones were selected.
  • a plasmid extraction kit (purchased from Kangwei Century) was used to lyse the resulting E. coli and extract the plasmid to obtain an expression vector containing heavy and light chains.
  • the purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, spread on a 96-well plate, grown for 15 days, picked out a single clone, and measured the cell antibody production by ELISA. Before selection 20% was transferred to a 24-well plate. After 7 days of growth, the measured antibody yield of the cells was selected and transferred to shake flask culture by selecting the first 5 to 6 strains to realize the expression of anti-HER2 antibody in CHO-K1 cells. After the serum-free culture is stable, the culture is continued for 7 days and then the material is collected for purification and subsequent experiments.
  • Encoding the anti-VEGF antibody Fab fragment described herein (its corresponding wild-type sequence is from USP Medicines Compendium, the specific Fab fragment heavy chain sequence is shown in SEQ ID NO: 25, and the Fab fragment light chain sequence is shown in SEQ ID NO: 26 Show; on this basis, according to the kabat numbering, the 166th amino acid of the light chain of the Fab fragment is replaced with cysteine to obtain the variant 166C of the present invention, and the 124th amino acid of the light chain of the Fab fragment is replaced with cysteine to obtain The DNA of the control antibody 124C) was chemically synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.), and then the Fab fragment heavy chain gene was double digested with BstBI and PacI, and the Fab fragment light chain gene was double digested with HindIII and EcoRI.
  • T4 ligase connects the Fab fragment heavy chain gene to the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) that has been treated with BstBI and PacI. After the ligation is successful, double digestion with HindIII and EcoRI is performed, and then the T4 ligase The Fab fragment light chain gene is connected, that is, an expression vector containing both the Fab fragment heavy chain and light chain is successfully constructed.
  • the successfully constructed expression vector was transformed into DH5 ⁇ competent E. coli strain.
  • the transformed DH5 ⁇ strain was verified by double digestion with BstBI and PacI enzymes and HindIII and EcoRI enzymes, and sequencing, and positive clones were selected.
  • a plasmid extraction kit (purchased from Kangwei Century) was used to lyse the resulting E. coli and extract the plasmid to obtain an expression vector containing heavy and light chains.
  • the purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, spread on a 96-well plate, and grown for 15 days. Single clones were picked and the cell antibody production was determined by SDS-PAGE. Select the first 20% to transfer to a 24-well plate. After 7 days of growth, the measured cell antibody production, select the first 5 to 6 strains and transfer them to shake flask culture to achieve the expression of anti-antibody in CHO-K1 cells. After the serum-free culture is stable, the material is collected after continuous culture for 7 days, and the Ni affinity chromatography column purification and subsequent experiments are performed.
  • the present invention adopts Ellman's method (Thermo Fisher Technology (China) Co., Ltd.) to detect the free sulfhydryl content of the antibody product, thereby selecting a cell line suitable for further coupling.
  • the OD value was measured. Establish a standard curve based on the OD value, and calculate the sulfhydryl content of the sample.
  • WT basically does not contain free sulfhydryl groups, while about 100% of sulfhydryl groups in 166C and 124C are free.
  • conjugate polypeptide or mPEG2000-MAL
  • reaction buffer is PBS( pH7.2).
  • SDS-PAGE electrophoresis diagram of the coupling of anti-VEGF antibody and mPEG2000-MAL purchased from a chemical reagent company, such as Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) is shown in Figure 2.
  • Anti-VEGF antibody and DC (polypeptide-linker) coupling see the following formula II for the reaction, 166C mutant coupled with DC to obtain 166ADC, 124C mutant coupled with DC to obtain 124ADC
  • SDS-PAGE electrophoresis diagram see figure 3.
  • NIH Image J
  • the polypeptide is an active polypeptide with Ang2 neutralization effect, the sequence is Gln-Lys(Ac)-Tyr-Gln-Pro-Leu-Asp-Glu-Lys(Ac)-Asp- Lys- Thr-Leu-Tyr-Asp-Gln -Phe-Met-Leu-Gln-Gln-Gly-CONH 2 (SEQ ID NO: 9) comes from the reference Hanhua Huang, Jing-Yu Lai, Janet Do, Dingguo Liu, Lingna Li, Joselyn Del Rosario.
  • Figure 5 shows the coupling of anti-CD20 antibodies (WT, 124C, 166C) and DC (polypeptide-linker).
  • Figure 9 shows the coupling of anti-VEGF Fab antibodies (WT, 124C, 166C) and DC (polypeptide-linker).
  • the mass spectrometry system is the AB Sciex High Resolution Tandem Mass Spectrometry Triple TOF 4600 LC/MS/MS system, the electrospray ionization source, and the positive ion mode for analysis.
  • Mass spectrometry parameters GS1: 55; GS2: 55; CUR: 35; scanning range: 100-2000Da; atomization voltage (ISVF): 5500V; atomization temperature: 350°C; declustering voltage (DP): 150V; collision energy ( CE): 10eV. See Figure 10-13 for the results.
  • Biofilm layer interference technology BBI and Pro A probe (ForteBio) were used to determine the affinity of wild-type antibodies, antibody mutants, antibody polypeptide conjugates and VEGF.
  • the antibody samples were dissolved in PBS with a working concentration of 10 ⁇ g/mL; VEGF-165 (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was dissolved in PBS and diluted to concentrations of 50nM, 75nM, 100nM, 150nM, 200nM, respectively.
  • the working volume is 200 ⁇ L.
  • the data graph was fitted by the OCTET system and the data processing software, and the software calculated the intermolecular force between the antibody and VEGF or Ang2, expressed in KD value. The results are shown in Table 5.
  • HUVEC cells HUVEC cells (ScienCell Research Laboratories, Inc.). Cells were seeded in 96-well plates at a seeding density of 4000 cells/50 ⁇ L/well. Set up blank control wells, with no less than 6 control wells. Prepare antibody samples with gradient concentrations (0.001 ⁇ g/mL, 0.01 ⁇ g/mL, 0.05 ⁇ g/mL, 0.1 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL) according to the gradient dilution method. Dilute VEGF165 with test medium to a concentration of 110ng/mL.
  • FIG. 14 shows the inhibitory effect of wild-type antibodies (WT), mutants (124C and 166C) and conjugates (124ADC and 166ADC) on the proliferation of HUVEC endothelial cells (proliferation stimulated by VEGF). It can be seen from the figure that the antibody mutant did not change the activity due to the cysteine mutation at the selected site, indicating that the selected site will not affect the biological activity of the antibody. Similarly, the conjugate produced after coupling DC has no effect on the activity of the antibody to inhibit cell proliferation.
  • Biofilm layer interference technology BBI and Pro A probe (ForteBio) were used to determine the affinity of wild-type antibodies and antibody mutants to CD20.
  • Antibody samples were dissolved in PBS with a working concentration of 10 ⁇ g/mL; CD20 (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was dissolved in PBS and diluted to concentrations of 25nM, 50nM, 100nM, 150nM, 200nM, respectively.
  • the working volume is 200 ⁇ L.
  • the data graph was fitted by the OCTET system and the data processing software, and the software calculated the intermolecular force between the antibody, etc. and CD20, expressed as the K D value. The results are shown in Table 6.
  • Biofilm layer interference technology BBI
  • FormeBio Pro A probe
  • HER-FC Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • PBS PBS with a working concentration of 10 ⁇ g/mL
  • the antibody was dissolved in PBS2 and diluted to a concentration of 25nM, 50nM, 75nM, 150nM, 300nM, respectively.
  • the working volume is 200 ⁇ L.
  • the data graph was fitted by the OCTET system and the data processing software, and the software calculated the intermolecular force between the antibody, etc. and HER2, expressed as the K D value.
  • the results are shown in Table 7.
  • SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.Blood, (2013) 122: 1455-1463.

Abstract

抗体或其片段的突变体，其特征在于所述抗体或其片段包含轻链恒定区，并且按照Kabat编号系统，位置166的氨基酸突变为半胱氨酸。

Description

抗体的突变体及其应用 技术领域
本发明涉及抗体领域。具体地，涉及抗体的突变体，包含其的组合物和/或缀合物，及其用途。
背景技术
抗体-药物缀合物(Antibody-drug conjugates，ADC)是一类靶向治疗药物，其将抗体的特异性与细胞毒性治疗剂的细胞毒性相结合。ADC主要被认为是治疗各种癌症的候选物。ADC包含与治疗药物连接的抗体。此外，双特异性抗体也可以由两个现有的抗体通过化学偶联的方法，快速、大量的进行合成。比如，两个IgG分子或者两个Fab片段通过偶联试剂连接。还有报道利用抗体酶制备CovX-Bodies[1]，在一个IgG分子的两个Fab臂上分别连接两个具有靶位中和作用的多肽。因此定点偶联技术也可以应用于制备新型双特异性抗体[1]。
抗体分子含有很多可用来修饰交联的基团，比如氨基和羧基。但由于这些基团在抗体分子中大量存在，导致抗体偶联位点的随机性。还有很多方法借助于抗体分子上的二硫键，由于其数量有限且位置比较固定，偶联上药物后不会屏蔽抗原结合位点，这些特点使抗体上的二硫键成为抗体偶联的合适位点之一。但是这些内源二硫键的还原是非特异性的，即铰链区二硫键和重链与轻链间二硫键均被还原，造成偶联产物是一种成分多样的混合物[2，3]。
半胱氨酸硫醇在中性pH具有反应性，这与在接近pH7时质子化和亲核性降低的大部分胺类不同。由于游离硫醇(硫氢基)具有相对的反应性，所以带有半胱氨酸残基的蛋白质通常以其作为二硫化物-连接的寡聚体的氧化形式存在或具有内部桥连的二硫化物基团。抗体中的半胱氨酸巯基一般对亲电子偶联反应剂比对抗体胺或羟基更具反应性，即更具亲核性。因此，可以通过将抗体中的氨基酸残基突变成半胱氨酸，然后利用该半胱氨酸进行缀合反应。Junutula等[4]研究开发了PHESELECTOR(phage ELISA for selection of reactive thiols)技术，并应用此技术筛选得到含有反 应性Cys的IgG突变体THIOMAB，其与药物的偶联产物称为TDC(THIOMAB-drug conjugates)。与常规ADC相比，通过PHESELECTOR技术获得的相应TDC虽然表现出更好的治疗效应，但也存在缺陷：全长抗体在哺乳动物细胞中表达时，引入的Cys可以和培养基中谷胱甘肽或其它含巯基的物质形成二硫键，所以还需将其还原成游离的形式才能进行偶联，但与此同时，抗体的链间二硫键也会被打开，这又需再次氧化以恢复成完整的抗体；而且，在THIOMAB形成的整个过程中，这个额外引入的巯基也有可能在抗体的2个Fab间错误地形成二硫键[5-7]。
当选择适当的位点进行半胱氨酸突变，可以使其突变得到的半胱氨酸上的巯基在表达纯化过程中始终保持游离的状态，而不需要任何的预处理直接应用于下游的偶联反应。因此，本申请人开发了合理的半胱氨酸突变位点，针对这些位点进行突变后得到的半胱氨酸巯基更容易保持游离状态并具有较高反应性。然后通过含有马来酰亚胺的接头将抗体与另外一个功能分子(抗体片段，多肽或小分子药物)进行偶联。
发明内容
发明概述
本发明主要提供了抗体恒定区的可突变成半胱氨酸的位点，所述突变引入所选择的位点特异性的缀合位点，以允许所述抗体、片段或衍生物与有效负载缀合，其中所述一个或多个突变包括轻链位置166(按照Kabat系统位置编号)处的突变。同时，本发明也提供了一种具有抗血管生成功能的双特异性抗体的独立结构。
由此，本发明提供了以下方面：
1.抗体或其片段的变体，其特征在于所述抗体或其片段包含轻链恒定区，并且按照Kabat编号系统，位置166的氨基酸突变为半胱氨酸，优选地，所述抗体为人源化抗体，嵌合抗体，更优选为IgG抗体。
2.上述1的抗体或其片段的变体，其中所述轻链为λ或κ类型。
3.上述1或2的抗体或其片段的变体，所述变体包含重链和轻链，所述重链的氨基酸序列为SEQ ID NO：1所示，所述轻链的氨基酸序列为SEQ ID NO：7所示；所述重链的氨基酸序列为SEQ ID NO：9所示，所述 轻链的氨基酸序列为SEQ ID NO：13所示；或所述重链的氨基酸序列为SEQ ID NO：17所示，所述轻链的氨基酸序列为SEQ ID NO：21所示。
4.上述1-3任一项所述的抗体或其片段的变体，其特征在于所述片段是Fab片段，Fab’片段或F(ab’) 2片段，优选所述Fab片段的重链氨基酸序列为SEQ ID NO：25所示，所述所述Fab片段的轻链氨基酸序列为SEQ ID NO：29所示。
5.双特异性抗体或融合蛋白，其特征在于包含上述1-4任一项所述的抗体或其片段的变体。
6.缀合物，其特征在于包含上述1-4任一项所述的抗体或其片段的变体。
7.上述6的缀合物，所述缀合物选自聚乙二醇，细胞毒性剂，活性肽，纳米抗体，单结构域抗体，Fab片段，Fab’片段，scFv，小分子药物，化疗剂或放疗剂，优选地，所述活性肽为Agn2活性肽，更优选序列为SEQ ID NO：5所示的Agn2活性肽，更优选地，所述Agn2活性肽通过接头与本发明的贝伐单抗变体缀合，进一步优选地，所述接头具有酰胺键，具体为甲酰胺-PEG 4-NHS，具有结构式I所示的结构
Figure PCTCN2020108434-appb-000001
8.上述6的缀合物，其中所述聚乙二醇为单甲氧基聚乙二醇，优选mPEG2000，mPEG5000，mPEG10000。
9.上述7或8所述的缀合物，所述聚乙二醇进一步缀合药物分子，优选所述聚乙二醇为线性双官能化聚乙二醇，线性异官能化聚乙二醇或多臂官能化PEG。
10.药物组合物，其特征在于包含上述1-4任一项所述的抗体或其片段的变体，上述5所述的双特异性抗体或融合蛋白，上述6-9任一项所述的缀合物，和任选地，药用载体。
11.治疗癌症(优选非小细胞肺癌如晚期、转移性或复发性非鳞状细胞非小细胞肺癌，结直肠癌如转移性结直肠癌，乳腺癌、恶性胶质瘤和肾 细胞癌多形性胶质母细胞瘤)的方法，包含施用上述1-4任一项所述的抗体或其片段的变体，上述5所述的双特异性抗体或融合蛋白，上述6-9任一项所述的缀合物，或上述10所述的药物组合物。
12.上述1-4任一项的抗体或其片段的变体，上述5的双特异性抗体/融合蛋白或上述6-9任一项的缀合物，或上述10所述的药物组合物在制备用于治疗癌症(优选非小细胞肺癌如晚期、转移性或复发性非鳞状细胞非小细胞肺癌，结直肠癌如转移性结直肠癌，乳腺癌、恶性胶质瘤和肾细胞癌多形性胶质母细胞瘤)的药物或试剂盒中的应用。
13.试剂盒，其包含上述1-4任一项的抗体或其片段的变体，上述5的双特异性抗体/融合蛋白或上述6-9任一项的缀合物，或上述10所述的药物组合物，优选地，所述试剂盒进一步包含与所述抗体或其片段联合用药的药剂，例如卡铂或顺铂。
附图说明
图1，显示了野生型(WT，在本发明中也称为“抗VEGF抗体”)抗体，166突变抗体(166C)，124突变抗体(124C)在还原与非还原状态下的SDS-PAGE电泳图。
图2.抗VEGF野生型抗体及突变体与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，166C与124C均可与缀合物mPEG2000偶联成功。
图3.抗VEGF野生型抗体及突变体与DC(多肽-linker)偶联后的SDS-PAGE电泳图。利用灰度分析，85％的166C与DC偶联成功，而只有75％的124C与DC偶联成功。由此可见，同一条件下，166C抗体突变体表现出更好的缀合效率。
图4.抗CD20野生型抗体及突变体与mPEG2000-MAL偶联后的SDS-PAGE电泳图。
图5.抗CD20野生型抗体及突变体与DC(多肽-linker)偶联后的SDS-PAGE电泳图。
图6.抗HER2野生型抗体及突变体与mPEG2000-MAL偶联后的SDS-PAGE电泳图。
图7.抗HER2野生型抗体及突变体与DC(多肽-linker)偶联后的SDS-PAGE电泳图。
图8.抗VEGF抗体Fab片段野生型抗体及突变体与DC(多肽-linker)偶联后的SDS-PAGE电泳图。
图9.抗VEGF抗体Fab片段野生型抗体及突变体与mPEG2000-MAL偶联后的SDS-PAGE电泳图。
图10.突变体166C未进行偶联前的LC/MS谱图。
图11.突变体166C与DC偶联后的LC/MS谱图。
图12.突变体124C未进行偶联前的LC/MS谱图。
图13.突变体124C与DC偶联后的LC/MS谱图。
图14.野生型抗体(WT)，及突变体(124C和166C)及偶联物(124ADC和166ADC)对内皮细胞HUVEC增殖(VEGF刺激产生的增殖)的抑制作用。由图可知，抗体突变体没有因为所选位点的半胱氨酸突变而改变活性，说明所选位点不会对抗体生物活性造成影响。同样，偶联DC后产生的偶联物也没有对抗体抑制细胞增殖的活性产生影响。
具体实施方式
为使本发明的目的、技术方案和优点更加清晰，以下结合具体实施例，对本发明作进一步的详细说明。本领域的普通技术人员可以理解，以下实施例仅仅是举例说明的目的，本发明的保护以后附的权利要求书所记载的为准。
实施例中所用限制性内切核酸酶购自赛默飞世尔科技(中国)有限公司公司，以下实施例中所用的试剂或材料，如未明确提及来源，均是本领域可以常规购买的。CHO-K1细胞的培养基购自sigma公司
实施例1.半胱氨酸突变细胞株筛选和表达
(1)抗VEGF抗体及突变体表达载体的构建
编码本文所述的抗体(其对应的野生型序列来自于USP Medicines Compendium，具体重链序列为SEQ ID NO：1所示，轻链序列为SEQ ID NO：2所示；在此基础上，根据kabat编号，将轻链第166位氨基酸用半胱 氨酸置换获得本发明的变体166C，将轻链第124位氨基酸用半胱氨酸置换获得对照抗体124C)的DNA被化学合成(苏州金唯智生物科技有限公司)，然后将抗体重链基因用BstBI和PacI进行双酶切，轻链基因用HindIII和EcoRI进行双酶切，用T4连接酶将重链基因连接到经BstBI和PacI处理过的真核表达载体pCGS3(Biovector NTCC Inc.)中，连接成功后，用HindIII和EcoRI进行双酶切，之后通过T4连接酶将轻链基因连入，即构建成功同时含有重链和轻链的表达载体。
  WT贝伐单抗 166C 124C
重链序列的编码序列 SEQ ID NO：3 SEQ ID NO：3 SEQ ID NO：3
轻链序列的编码序列 SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6
重链的氨基酸序列 SEQ ID NO：1 SEQ ID NO：1 SEQ ID NO：1
轻链的氨基酸序列 SEQ ID NO：2 SEQ ID NO：7 SEQ ID NO：8
(2)抗VEGF抗体及突变体表达载体的筛选和纯化
将构建成功的表达载体转入DH5α感受态大肠杆菌菌株。转化后的DH5α菌株通过用BstBI和PacI两种酶和HindIII和EcoRI两种酶分别进行双酶切，并进行测序来验证，选取阳性克隆。使用质粒提取试剂盒(购自康为世纪)对所得的大肠杆菌裂解并提取质粒，获得含重链和轻链的表达载体。
(3)抗VEGF抗体及突变体在CHO细胞中的表达
纯化后的表达载体用电穿孔法转染到经CHO-K1(购自ATCC)细胞中，铺至96孔板，生长15天，挑取单克隆，通过ELISA的方式测定细胞抗体产量，选取前20％转入24孔板，生长7天后，测得的细胞抗体产量，选取前5到6株转至摇瓶培养，即实现抗VEGF抗体在CHO-K1细胞中的表达。无血清培养稳定后，连续培养7天后收料，进行纯化与后续实验。
(4)抗CD20抗体及突变体表达载体的构建
编码本文所述的抗体(其对应的野生型序列来自于USP Medicines Compendium，具体重链序列为SEQ ID NO：9所示，轻链序列为SEQ ID NO：10 所示；在此基础上，根据kabat编号，将轻链第166位氨基酸用半胱氨酸置换获得本发明的变体166C，将轻链第124位氨基酸用半胱氨酸置换获得对照抗体124C的DNA被化学合成(苏州金唯智生物科技有限公司)，然后将抗体重链基因用BstBI和PacI进行双酶切，轻链基因用HindIII和EcoRI进行双酶切，用T4连接酶将重链基因连接到经BstBI和PacI处理过的真核表达载体pCGS3(Biovector NTCC Inc.)中，连接成功后，用HindIII和EcoRI进行双酶切，之后通过T4连接酶将轻链基因连入，即构建成功同时含有重链和轻链的表达载体。
Figure PCTCN2020108434-appb-000002
(5)抗CD20抗体及突变体表达载体的筛选和纯化
将构建成功的表达载体转入DH5α感受态大肠杆菌菌株。转化后的DH5α菌株通过用BstBI和PacI两种酶和HindIII和EcoRI两种酶分别进行双酶切，并进行测序来验证，选取阳性克隆。使用质粒提取试剂盒(购自康为世纪)对所得的大肠杆菌裂解并提取质粒，获得含重链和轻链的表达载体。
(6)抗CD20抗体及突变体在CHO细胞中的表达
纯化后的表达载体用电穿孔法转染到经CHO-K1(购自ATCC)细胞中，铺至96孔板，生长15天，挑取单克隆，通过ELISA的方式测定细胞抗体产量，选取前20％转入24孔板，生长7天后，测得的细胞抗体产量，选取前5到6株转至摇瓶培养，即实现抗CD20抗体在CHO-K1细胞中的表达。无血清培养稳定后，连续培养7天后收料，进行纯化与后续实验。
(7)抗HER2抗体及突变体表达载体的构建
编码本文所述的抗体(其对应的野生型序列来自于USP Medicines Compendium，具体重链序列为SEQ ID NO：17所示，轻链序列为SEQ ID NO：19所示；在此基础上，根据kabat编号，将轻链第166位氨基酸用半胱氨酸置换获得本发明的变体166C，将轻链第124位氨基酸用半胱氨酸置换获得对照抗体124C)的DNA被化学合成(苏州金唯智生物科技有限公司)，然后将抗体重链基因用BstBI和PacI进行双酶切，轻链基因用HindIII和EcoRI进行双酶切，用T4连接酶将重链基因连接到经BstBI和PacI处理过的真核表达载体pCGS3(Biovector NTCC Inc.)中，连接成功后，用HindIII和EcoRI进行双酶切，之后通过T4连接酶将轻链基因连入，即构建成功同时含有重链和轻链的表达载体。
Figure PCTCN2020108434-appb-000003
(8)抗HER2抗体及突变体表达载体的筛选和纯化
将构建成功的表达载体转入DH5α感受态大肠杆菌菌株。转化后的DH5α菌株通过用BstBI和PacI两种酶和HindIII和EcoRI两种酶分别进行双酶切，并进行测序来验证，选取阳性克隆。使用质粒提取试剂盒(购自康为世纪)对所得的大肠杆菌裂解并提取质粒，获得含重链和轻链的表达载体。
(9)抗HER2抗体及突变体在CHO细胞中的表达
纯化后的表达载体用电穿孔法转染到经CHO-K1(购自ATCC)细胞中，铺至96孔板，生长15天，挑取单克隆，通过ELISA的方式测定细胞抗体产量，选取前20％转入24孔板，生长7天后，测得的细胞抗体产量，选取前5到6株转至摇瓶培养，即实现抗HER2抗体在CHO-K1细胞中的表达。无血清培养稳定后，连续培养7天后收料，进行纯化与后续实验。
(10)抗VEGF抗体Fab片段及突变体表达载体的构建
编码本文所述的抗VEGF抗体Fab片段(其对应的野生型序列来自于USP Medicines Compendium，具体Fab片段重链序列为SEQ ID NO：25所示，Fab片段轻链序列为SEQ ID NO：26所示；在此基础上，根据kabat编号，将Fab片段轻链第166位氨基酸用半胱氨酸置换获得本发明的变体166C，将Fab片段轻链第124位氨基酸用半胱氨酸置换获得对照抗体124C)的DNA被化学合成(苏州金唯智生物科技有限公司)，然后将Fab片段重链基因用BstBI和PacI进行双酶切，Fab片段轻链基因用HindIII和EcoRI进行双酶切，用T4连接酶将Fab片段重链基因连接到经BstBI和PacI处理过的真核表达载体pCGS3(Biovector NTCC Inc.)中，连接成功后，用HindIII和EcoRI进行双酶切，之后通过T4连接酶将Fab片段轻链基因连入，即构建成功同时含有Fab片段重链和轻链的表达载体。
Figure PCTCN2020108434-appb-000004
(11)抗VEGF抗体Fab片段及突变体表达载体的筛选和纯化
将构建成功的表达载体转入DH5α感受态大肠杆菌菌株。转化后的DH5α菌株通过用BstBI和PacI两种酶和HindIII和EcoRI两种酶分别进行双酶切，并进行测序来验证，选取阳性克隆。使用质粒提取试剂盒(购自康为世纪)对所得的大肠杆菌裂解并提取质粒，获得含重链和轻链的表达载体。
(12)抗VEGF抗体Fab片段及突变体在CHO细胞中的表达
纯化后的表达载体用电穿孔法转染到经CHO-K1(购自ATCC)细胞中，铺至96孔板，生长15天，挑取单克隆，通过SDS-PAGE的方式测定细胞抗体产量，选取前20％转入24孔板，生长7天后，测得的细胞抗体产量，选取前5到6株转至摇瓶培养，即实现抗抗体在CHO-K1细胞中的表达。无血清培养稳定后，连续培养7天后收料，进行Ni亲和层析柱纯化与后续实验。
实施例2.Pro A亲和层析柱(HiTrap FF)纯化
将细胞及培养基转移进50mL离心管，置于高速冷冻离心机中，8000r条件离心15min，保留上清液，弃去细胞碎片；将培养基用0.22μm微孔滤膜过滤一遍，收集滤液备用；用10倍柱体积超纯水、PBS清洗泵与进样器、纯化柱后等待上样；上样；用0.1M Gly-HCl洗脱并将纯化后的抗体蛋白溶液接取至EP管中，加1.5M Tris-HCl将pH调到7-8，置于2-8℃保存。结果见图1。
实施例3.抗体自由巯基占比检测
本发明采用Ellman’s方法(赛默飞世尔科技(中国)有限公司)检测抗体产物游离巯基含量，从而选择适合进行进一步偶联的细胞株。配置0.1M Na 2HPO 4溶液、DTNB(10mM)溶液，以及半胱氨酸标准溶液，使其终浓度为0.0059M～0.375M。同时准备浓度为5mg/mL的待测抗体样品。分别在装有半胱氨酸标准溶液以及待测样品的1.5mL EP管中加入250μL 0.1M Na 2HPO 4、5μL DTNB溶液，涡旋震荡混匀后，室温下避光孵育5min。在波长412nm处，检测OD值。根据OD值建立标准曲线，并计算样品巯基含量。
表1.使用Ellman’s方法检测VEGF抗体产物(WT，166C和124C)游离巯基的含量。其中WT基本不含有游离巯基，而166C和124C有约100％的巯基为游离状态。
Figure PCTCN2020108434-appb-000005
Figure PCTCN2020108434-appb-000006
表2.使用Ellman’s方法检测CD20抗体产物(WT，166C和124C)游离巯基的含量。其中WT基本不含有游离巯基，而166C和124C有约100％的巯基为游离状态。
Figure PCTCN2020108434-appb-000007
表3.使用Ellman’s方法检测HER2抗体产物(WT，166C和124C)游离巯基的含量。其中WT基本不含有游离巯基，而166C和124C有约100％的巯基为游离状态。
Figure PCTCN2020108434-appb-000008
表4.使用Ellman’s方法检测抗VEGF抗体Fab片段产物(WT，166C和124C)游离巯基的含量。其中WT基本不含有游离巯基，而166C和124C有约50％巯基为游离状态。
Figure PCTCN2020108434-appb-000009
Figure PCTCN2020108434-appb-000010
实施例4.抗体偶联物与检测
抗体与偶联物的偶联反应条件为抗体：偶联物(多肽或mPEG2000-MAL)＝1∶30(摩尔比)，反应温度为37℃，反应时长3-4h，反应缓冲液为PBS(pH7.2)。其中抗VEGF抗体与mPEG2000-MAL(购自化学试剂公司，如上海甄准生物科技有限公司，产品识别编号ZZP-MPEG-MAL-2K-01))偶联的SDS-PAGE电泳图参见图2。
抗VEGF抗体与DC(多肽-linker)偶联(反应参见下式II，166C突变体与DC偶联后得到166ADC，124C突变体与DC偶联后得到124ADC)后的SDS-PAGE电泳图参见图3。利用Image J(NIH)灰度分析可知，85％的166C与DC偶联成功，而只有75％的124C与DC偶联成功。由此可见，同一条件下，166C抗体突变体表现出更好的缀合效率。其中DC(多肽-linker)的构建信息如下：
多肽为具有Ang2中和作用的活性多肽，序列为Gln-Lys(Ac)-Tyr-Gln-Pro-Leu-Asp-Glu-Lys(Ac)-Asp- Lys-Thr-Leu-Tyr-Asp-Gln-Phe-Met-Leu-Gln-Gln-Gly-CONH 2(SEQ ID NO：9)来自于参考文献Hanhua Huang，Jing-Yu Lai，Janet Do，Dingguo Liu，Lingna Li，Joselyn Del Rosario.Specifically Targeting Angiopoietin-2 Inhibits Angiogenesis，Tie2-Expressing Monocyte Infiltration，and Tumor Growth.Clin Cancer Res；17(5)March 1，2011，1001-1011，下划线表示的Lys用于与linker连接。所述linker为甲酰胺-PEG 4-NHS，结构式如下式I所示：
Figure PCTCN2020108434-appb-000011
活性肽与linker反应路线如下
Figure PCTCN2020108434-appb-000012
抗CD20抗体(WT、124C、166C)与mPEG2000-MAL偶联见图4；
抗CD20抗体(WT、124C、166C)和DC(多肽-linker)偶联见图5。
抗HER2抗体(WT、124C、166C)与mPEG2000-MAL偶联见图6；
抗HER2抗体(WT、124C、166C)和DC(多肽-linker)偶联见图7。
抗VEGF Fab抗体(WT、124C、166C)与mPEG2000-MAL偶联见图8；
抗VEGF Fab抗体(WT、124C、166C)和DC(多肽-linker)偶联见图9。
质谱分析：
使用超滤管对样品进行前处理，溶解于双蒸水中。将所得样品进行质谱分析。质谱系统为AB Sciex公司高分辨串联质谱Triple TOF 4600 LC/MS/MS系统，电喷雾离子化源，采用正离子模式进行分析。质谱参数：GS1：55；GS2：55；CUR：35；扫描范围：100-2000Da；雾化电压(ISVF)：5500V；雾化温度：350℃；去簇电压(DP)：150V；碰撞能量(CE)：10eV。结果参见图10-13。从图10和图11所见，突变体166C与DC偶联后主峰右移了6478.8Da，为两分子的DC分子量。由此可知，166C上的两个巯基都与DC成功偶联。
从图12-13所见，突变体124C与DC进行偶联反应后，分别产生了偶联0分子DC、偶联1分子DC和2分子DC的峰，偶联均一性和效率都没有166C的高。
实施例5.抗VEGF抗体与抗原亲和力测定
采用生物膜层干涉技术(BLI)，Pro A探针(ForteBio)测定野生型抗体、抗体突变体、抗体多肽偶联物与VEGF的亲和力。抗体样品溶于PBS，工作浓度为10μg/mL；VEGF-165(北京义翘神州科技有限公司)溶于PBS中，梯度稀释至浓度分别为50nM、75nM、100nM、150nM、200nM。工作体积均为200μL。
采用生物膜层干涉技术(BLI)，Pro A探针测定野生型抗体、抗体突变体、抗体多肽偶联物与Ang2的亲和力。抗体样品溶于PBS，工作浓度为20μg/mL；Ang2(北京义翘神州科技有限公司)溶于PBS(0.02％tween-20，0.1％BSA)中，梯度稀释至浓度分别为25nM、50nM、75nM、100nM、200nM。工作体积均为200μL。
通过OCTET系统与数据处理软件拟合数据图，软件计算得出抗体等与VEGF或Ang2的分子间作用力，以KD值表示，结果见表5。
表5.野生型抗体、突变体及偶联物对于VEGF-165的结合力几乎没有影响。偶联物对Ang2的结合力也在抗体中和作用结合力的合理范围。
表5样品活性检测
Figure PCTCN2020108434-appb-000013
实施例7.抗VEGF抗体对细胞增殖活性测定
在HUVEC细胞(ScienCell Research Laboratories，Inc.)上来检测抗体对VEGF165的中和作用。按照4000细胞/50μL/孔的接种密度在96孔板中进行细胞接种。设置空白对照孔，对照孔不得少于6个。按照梯度稀 释方法准备梯度浓度的抗体样品(0.001μg/mL，0.01μg/mL，0.05μg/mL，0.1μg/mL，0.5μg/mL，1μg/mL，5μg/mL，10μg/mL)。用试验培养基稀释VEGF165至其浓度为110ng/mL。将准备好的VEGF165试剂与梯度浓度的样品混匀，使VEGF165终浓度为10ng/mL。将96孔板放置于CO 2培养箱培养48h后CCK-8计数。根据样品及其对照的OD值计算增值率。结果见图14，显示了野生型抗体(WT)，及突变体(124C和166C)及偶联物(124ADC和166ADC)对内皮细胞HUVEC增殖(VEGF刺激产生的增殖)的抑制作用。由图可知，抗体突变体没有因为所选位点的半胱氨酸突变而改变活性，说明所选位点不会对抗体生物活性造成影响。同样，偶联DC后产生的偶联物也没有对抗体抑制细胞增殖的活性产生影响。
实施例8.抗CD20抗体活性测定
采用生物膜层干涉技术(BLI)，Pro A探针(ForteBio)测定野生型抗体和抗体突变体与CD20的亲和力。抗体样品溶于PBS，工作浓度为10μg/mL；CD20(北京义翘神州科技有限公司)溶于PBS中，梯度稀释至浓度分别为25nM、50nM、100nM、150nM、200nM。工作体积均为200μL。
通过OCTET系统与数据处理软件拟合数据图，软件计算得出抗体等与CD20的分子间作用力，以K D值表示，结果见表6。
表6.野生型抗体和抗体突变体与CD20的亲和力
Figure PCTCN2020108434-appb-000014
实施例9.抗HER2抗体活性测定
采用生物膜层干涉技术(BLI)，Pro A探针(ForteBio)测定野生型抗体、抗体突变体、抗体多肽偶联物与HER-FC的亲和力。HER-FC(北京义翘神州科技有限公司)溶于PBS，工作浓度为10μg/mL；抗体溶于PBS2中，梯度稀释至浓度分别为25nM、50nM、75nM、150nM、300nM。工作体积均为200μL。
通过OCTET系统与数据处理软件拟合数据图，软件计算得出抗体等与HER2的分子间作用力，以K D值表示，结果见表7。
表7.野生型抗体和抗体突变体与HER2的亲和力
Figure PCTCN2020108434-appb-000015
参考文献
1.Doppalapudi VR，Huang J，Liu D，Jin P，Liu B，Li L，Chemical generation of bispecific antibodies.Proc Natl Acad Sci U S A.(2010)107：22611-22616.
2.Allen，T.M.，Ligand-targeted therapeutics in anticancer therapy，Nat.Rev.Cancer，(2002)2：750-763.
3.Hamblett，K.J.，Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate，Clin.Cancer Res.，(2004)10：7063-7070.
4.Junutula J R，Bhakta S，Raab H，et al.Rapid identifi cation of reactive cysteine residues for site-specific labeling of antibody-Fabs.J Immunol Methods，(2008)332：41-52.
5.Junutula J R，Flagella K M，Graham R A，et al.Engineered thiotrastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer.Clin Cancer Res，(2010)16：4769-4778.
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序列
SEQ ID NO：1
Figure PCTCN2020108434-appb-000016
SEQ ID NO：2
Figure PCTCN2020108434-appb-000017
Figure PCTCN2020108434-appb-000018
SEQ ID NO：3
Figure PCTCN2020108434-appb-000019
SEQ ID NO：4
Figure PCTCN2020108434-appb-000020
SEQ ID NO：5
Figure PCTCN2020108434-appb-000021
SEQ ID NO：6
Figure PCTCN2020108434-appb-000022
Figure PCTCN2020108434-appb-000023
SEQ ID NO：7
Figure PCTCN2020108434-appb-000024
SEQ ID NO：8
Figure PCTCN2020108434-appb-000025
SEQ ID NO：9
Figure PCTCN2020108434-appb-000026
SEQ ID NO：10
Figure PCTCN2020108434-appb-000027
SEQ ID NO：11
Figure PCTCN2020108434-appb-000028
Figure PCTCN2020108434-appb-000029
SEQ ID NO：12
Figure PCTCN2020108434-appb-000030
SEQ ID NO：13
Figure PCTCN2020108434-appb-000031
SEQ ID NO：14
Figure PCTCN2020108434-appb-000032
SEQ ID NO：15
Figure PCTCN2020108434-appb-000033
SEQ ID NO：16
Figure PCTCN2020108434-appb-000034
SEQ ID NO：17
Figure PCTCN2020108434-appb-000035
SEQ ID NO：18
Figure PCTCN2020108434-appb-000036
SEQ ID NO：19
Figure PCTCN2020108434-appb-000037
SEQ ID NO：20
Figure PCTCN2020108434-appb-000038
SEQ ID NO：21
Figure PCTCN2020108434-appb-000039
SEQ ID NO：22
Figure PCTCN2020108434-appb-000040
SEQ ID NO：23
Figure PCTCN2020108434-appb-000041
SEQ ID NO：24
Figure PCTCN2020108434-appb-000042
SEQ ID NO：25
Figure PCTCN2020108434-appb-000043
SEQ ID NO：26
Figure PCTCN2020108434-appb-000044
SEQ ID NO：27
Figure PCTCN2020108434-appb-000045
SEQ ID NO：28
Figure PCTCN2020108434-appb-000046
SEQ ID NO：29
Figure PCTCN2020108434-appb-000047
SEQ ID NO：30
Figure PCTCN2020108434-appb-000048
Figure PCTCN2020108434-appb-000049
SEQ ID NO：31
Figure PCTCN2020108434-appb-000050
SEQ ID NO：32
Figure PCTCN2020108434-appb-000051

Claims (13)

  1. 抗体或其片段的变体，其特征在于所述抗体或其片段包含轻链恒定区，并且按照Kabat编号系统，位置166的氨基酸突变为半胱氨酸，优选地，所述抗体为人源化抗体，嵌合抗体，更优选为IgG抗体。
  2. 权利要求1的抗体或其片段的变体，其中所述轻链为λ或κ类型。
  3. 权利要求1或2的抗体或其片段的变体，所述变体包含重链和轻链，所述重链的氨基酸序列为SEQ ID NO：1所示，所述轻链的氨基酸序列为SEQ ID NO：3所示；所述重链的氨基酸序列为SEQ ID NO：9所示，所述轻链的氨基酸序列为SEQ ID NO：13所示；或所述重链的氨基酸序列为SEQ ID NO：17所示，所述轻链的氨基酸序列为SEQ ID NO：21所示。
  4. 权利要求1-3任一项所述的抗体或其片段的变体，其特征在于所述片段是Fab片段，Fab’片段或F(ab’)2片段，优选所述Fab片段的重链氨基酸序列为SEQ ID NO：25所示，所述Fab片段的轻链氨基酸序列为SEQID NO：29所示。
  5. 双特异性抗体或融合蛋白，其特征在于包含权利要求1-4任一项所述的抗体或其片段的变体。
  6. 缀合物，其特征在于包含权利要求1-4任一项所述的抗体或其片段的变体。
  7. 权利要求6的缀合物，所述缀合物选自聚乙二醇，细胞毒性剂，活性肽，纳米抗体，单结构域抗体，Fab片段，Fab’片段，scFv，小分子药物，化疗剂或放疗剂，优选地，所述活性肽为Agn2活性肽，更优选序列为SEQ ID NO：5所示的Agn2活性肽，更优选地，所述Agn2活性肽通过接头与本发明的贝伐单抗变体缀合，进一步优选地，所述接头具有酰胺键，具体为甲酰胺-PEG 4-NHS，具有结构式I所示的结构
    Figure PCTCN2020108434-appb-100001
  8. 权利要求6的缀合物，其中所述聚乙二醇为单甲氧基聚乙二醇， 优选mPEG2000，mPEG5000，mPEG10000。
  9. 权利要求7或8所述的缀合物，所述聚乙二醇进一步缀合药物分子，优选所述聚乙二醇为线性双官能化聚乙二醇，线性异官能化聚乙二醇或多臂官能化PEG。
  10. 药物组合物，其特征在于包含权利要求1-4任一项所述的抗体或其片段的变体，权利要求5所述的双特异性抗体或融合蛋白，权利要求6-9任一项所述的缀合物，和任选地，药用载体。
  11. 治疗癌症(优选非小细胞肺癌如晚期、转移性或复发性非鳞状细胞非小细胞肺癌，结直肠癌如转移性结直肠癌，乳腺癌、恶性胶质瘤和肾细胞癌多形性胶质母细胞瘤)的方法，包含施用权利要求1-4任一项所述的抗体或其片段的变体，权利要求5所述的双特异性抗体或融合蛋白，权利要求6-9任一项所述的缀合物，或权利要求10所述的药物组合物。
  12. 权利要求1-4任一项所述的抗体或其片段的变体，权利要求5的双特异性抗体/融合蛋白或权利要求6-9任一项的缀合物，或权利要求10所述的药物组合物在制备用于治疗癌症(优选非小细胞肺癌如晚期、转移性或复发性非鳞状细胞非小细胞肺癌，结直肠癌如转移性结直肠癌，乳腺癌、恶性胶质瘤和肾细胞癌多形性胶质母细胞瘤)的药物或试剂盒中的应用。
  13. 试剂盒，其包含权利要求1-4任一项所述的抗体或其片段的变体，权利要求5的双特异性抗体/融合蛋白或权利要求6-9任一项的缀合物，或权利要求10所述的药物组合物，优选地，所述试剂盒进一步包含与所述抗体或其片段联合用药的药剂，例如卡铂或顺铂。
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