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WO2021259162A1 - 一种抗trop2抗体 - Google Patents

一种抗trop2抗体 Download PDF

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Publication number
WO2021259162A1
WO2021259162A1 PCT/CN2021/100903 CN2021100903W WO2021259162A1 WO 2021259162 A1 WO2021259162 A1 WO 2021259162A1 CN 2021100903 W CN2021100903 W CN 2021100903W WO 2021259162 A1 WO2021259162 A1 WO 2021259162A1
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Prior art keywords
antibody
seq
scfv
fab
sequence
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PCT/CN2021/100903
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English (en)
French (fr)
Inventor
朱义
张勇
卓识
丁沐然
王尊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Duote Antibody Pharmaceutical Co Ltd
Sichuan Baili Pharmaceutical Co Ltd
Original Assignee
Chengdu Duote Antibody Pharmaceutical Co Ltd
Sichuan Baili Pharmaceutical Co Ltd
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Priority to US18/011,175 priority Critical patent/US20230242665A1/en
Priority to CA3188014A priority patent/CA3188014A1/en
Priority to AU2021296205A priority patent/AU2021296205B2/en
Priority to IL299267A priority patent/IL299267A/en
Priority to EP21828738.1A priority patent/EP4169952A4/en
Priority to MX2022016300A priority patent/MX2022016300A/es
Application filed by Chengdu Duote Antibody Pharmaceutical Co Ltd, Sichuan Baili Pharmaceutical Co Ltd filed Critical Chengdu Duote Antibody Pharmaceutical Co Ltd
Priority to KR1020237001824A priority patent/KR20230026431A/ko
Priority to JP2022579053A priority patent/JP7586942B2/ja
Priority to BR112022025947A priority patent/BR112022025947A2/pt
Publication of WO2021259162A1 publication Critical patent/WO2021259162A1/zh
Anticipated expiration legal-status Critical
Priority to JP2024195200A priority patent/JP2025028871A/ja
Ceased legal-status Critical Current

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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2317/77—Internalization into the cell
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to antibodies that can specifically bind to human TROP2 protein, especially monoclonal antibodies of murine, chimeric and humanized origin, and the amino acid and nucleotide sequences encoding these antibodies.
  • the present invention also encompasses the use of these antibodies as diagnostic reagents or drugs in the diagnostic and/or therapeutic treatment of malignant tumors or any pathological changes related to the overexpression of the receptor.
  • Trop2 protein also known as type II trophoblast antigen (trophoblast antigen 2), tumor calcium signal transducer 2 (tumou associated calcium signal transducer 2), is encoded by the single-copy gene TACSTD2 at position 1p32 of chromosome.
  • the corresponding mRNA synthesizes a 36kDa nascent polypeptide, which forms a monomeric membrane protein with a single transmembrane domain after N-terminal glycosylation (Annie R.A. et al., 2015).
  • the extracellular domain of Trop2 has a domain capable of binding EGF growth factor, which can potentially intercept EGF down-regulating the activity of the IGF-1R/Akt signaling pathway. Therefore, the decrease in Trop2 expression can reversely activate the IGF-1R/Akt signaling pathway.
  • Trop2 has a significant role in promoting tumorigenesis. Overexpression of Trop2 can significantly enhance the tumorigenicity of NIH3T3 cells (Wang.et al., 2008). Trop2 can also affect epithelial-mesenchymal transition (EMT) and enhance the migration and invasion capabilities of cancer cells (Trerotola.et al. ,2013; Li.et al.,2017) This process may be achieved by affecting the PI3K/Akt signaling pathway: in gallbladder cancer cells that overexpress Trop2, the phosphorylation activation of Akt is significantly increased. On the contrary, knock down the expression of Trop2 It can inhibit the activity of this signaling pathway (Li. et al., 2017).
  • EMT epithelial-mesenchymal transition
  • Trop2 can also stimulate the activity of the MAPK/ERK signaling pathway, which leads to the up-regulation of the proliferation of pancreatic cancer cells and the increase in tumor deterioration in tumor-bearing mice (Cubas.et al., 2010).
  • Trop2 protein The expression of Trop2 protein is found in a series of epidermal-derived tissues, such as breast, kidney, and pancreas during embryonic development and adulthood (Annie R.A. et al., 2015). However, in tumor tissues corresponding to normal tissues, the expression level of Trop2 protein increased significantly indiscriminately, and the level of expression was positively correlated with the degree of tumor growth in animal models (Trerotola.et al., 2013), due to Trop2 The gene sequence itself has not been mutated or amplified, so the up-regulation of Trop2 expression in cancer is considered to be caused by stimulation at the level of transcriptional regulation (Trerotola. et al., 2013).
  • this protein In patients with different types of parenchymal tumors, the overexpression of this protein usually predicts the poor prognosis of the tumor (Zeng. et al., 2016). In gallbladder cancer (Chen.et al., 2014), gastrointestinal cancer (Muhlmann.et al., 2009), hilar cholangiocarcinoma (Ning.et al., 2013) and pancreatic cancer (Fong.et al., 2008) ) In the case of disease, the survival rate of patients with Trop2 high expression disease is significantly reduced. In view of the high expression of Trop2 in a variety of cancers and its significant impact on the survival rate of patients, this protein is considered to be a potential tumor treatment target.
  • ADC toxin-coupled antibody
  • IMMU-132 a toxin-coupled antibody against the Trop2 target: IMMU-132 (Goldenberg. et al., 2015).
  • the ADC loaded antibody RS7 was obtained by fusion screening of mouse spleen lymphocytes immunized with crude lung cancer cell membrane extracts and mouse myeloma cells through hybridoma preparation methods (Stein. et al., 1990). In the crude extract, the antigen bound by RS7 was later proved to be Trop2 (Stein. et al., 1994).
  • RS7 antibody itself can be endocytosed by a variety of cancer cells, so it has the potential to be prepared into ADC (Stein. et al., 1993).
  • IMMU-132 has clinical effectiveness in the treatment of a variety of cancers, especially triple-negative breast cancer (Starodub. et al., 2015; Sahota and Vahdat., 2017).
  • the chimeric antibody or humanized antibody described in this patent can bind to human type II trophoblast antigen protein (Trop2), and has the targeting antibody in antibody-conjugated drug therapy Features.
  • Trop2 human type II trophoblast antigen protein
  • the present invention does not involve natural forms of antibodies.
  • the antibodies involved in the present invention are all obtained through immunization of mice, identification and isolation methods, or through genetic recombination.
  • the object of protection is an antibody or a functional fragment or derivative, and the antibody is characterized by comprising at least one CDR whose amino acid sequence is derived from SEQ ID No. 1 to SEQ ID No. 12.
  • any antibody fragment or derivative contains at least one CDR, and the sequence of the CDR has at least 80% identity after optimized comparison with the sequence SEQ ID No. 1 to SEQ ID No. 12, or preferably 85 %, 90%, 95%, or 98% identity should be understood as equivalents of the present invention, and therefore also a part of the present invention.
  • one of the antibodies or functional fragments or derivatives of the present invention is characterized in that it comprises a heavy chain, and the heavy chain comprises at least one CDR, and the CDR is selected from SEQ ID No. containing the amino acid sequence. 1 to SEQ ID No. 6.
  • the antibody or one of its functional fragments or derivatives of the present invention is characterized in that it comprises a light chain, and the heavy chain comprises at least one CDR, and the CDR is selected from SEQ ID No. containing the amino acid sequence. 7 to SEQ ID No. 12.
  • the antibody or one of its functional fragments or derivatives of the present invention comprises a heavy chain comprising CDR-H1, CDR-H2, CDR-H3 CDR-H1 includes the amino acid sequence of SEQ ID No. 1, CDR-H2 includes the amino acid sequence of SEQ ID No. 2, and CDR-H3 includes the amino acid sequence of SEQ ID No. 3.
  • the antibody or one of its functional fragments or derivatives comprises a heavy chain, and in the sequence of the heavy chain, the chimeric antibody comprises the sequence SEQ ID No. 13, human origin
  • the antibody contains the sequence SEQ ID No.17.
  • the antibody or one of its functional fragments or derivatives of the present invention comprises a light chain comprising CDR-L1, CDR-L2, CDR-L3 CDR-L1 includes the amino acid sequence of SEQ ID No. 7, CDR-L2 includes the amino acid sequence of SEQ ID No. 8, and CDR-L3 includes the amino acid sequence of SEQ ID No. 9.
  • the antibody or one of its functional fragments or derivatives comprises a light chain, and in the sequence of the light chain, the chimeric antibody comprises the sequence SEQ ID No. 14, human origin
  • the antibody contains the sequence SEQ ID No. 18.
  • the antibody or one of its functional fragments or derivatives of the present invention comprises a heavy chain comprising CDR-H1, CDR-H2, CDR-H3 CDR-H1 includes the amino acid sequence of SEQ ID No. 4, CDR-H2 includes the amino acid sequence of SEQ ID No. 5, and CDR-H3 includes the amino acid sequence of SEQ ID No. 6.
  • the antibody or one of its functional fragments or derivatives comprises a heavy chain, and in the sequence of the heavy chain, the chimeric antibody comprises the sequence SEQ ID No. 15, human origin
  • the antibody contains the sequence SEQ ID No. 19.
  • the antibody or one of its functional fragments or derivatives of the present invention comprises a light chain comprising CDR-L1, CDR-L2, CDR-L3 CDR-L1 includes the amino acid sequence of SEQ ID No. 10, CDR-L2 includes the amino acid sequence of SEQ ID No. 11, and CDR-L3 includes the amino acid sequence of SEQ ID No. 12
  • the antibody or one of its functional fragments or derivatives comprises a light chain
  • the chimeric antibody in the sequence of the light chain comprises the sequence SEQ ID No. 16, humanized
  • the antibody contains the sequence SEQ ID No. 20
  • the present invention relates to isolated DNA, which features a nucleic acid selected from the following DNA sequences: the amino acid sequence encoded by each nucleic acid is the above amino acid sequence SEQ ID No. 1 to SEQ ID No. 20 One of them.
  • nucleic acid selected from the following DNA sequences:
  • the first specific embodiment includes the nucleic acid sequences of the sequences SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and the sequences SEQ ID No. 27, SEQ ID No. 28, and SEQ ID No. 29.
  • the second specific embodiment includes the nucleic acid sequences of the sequence SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and the sequence SEQ ID No. 30, SEQ ID No. 31, and SEQ ID No. 32.
  • the chimeric antibody sequence includes the sequences of SEQ ID No. 33 and SEQ ID No. 34
  • the humanized antibody sequence includes the nucleic acid sequences of the sequences of SEQ ID No. 37 and SEQ ID No. 38.
  • the chimeric antibody sequence includes the sequence SEQ ID No. 35 and SEQ ID No. 36
  • the humanized antibody sequence includes the nucleic acid sequence of the sequence SEQ ID No. 39 and SEQ ID No. 40.
  • Figure 1 is the CDR region identification and two-dimensional structure diagram of 4D3 light chain and heavy chain variable region sequence
  • Figure 2 is the CDR region identification and two-dimensional structure diagram of 7F11 light chain and heavy chain variable region sequence
  • Figure 3 is the ELISA binding curve of ch4D3 and ch7F11;
  • Figure 4 is a graph showing the cell surface binding activity of ch4D3 and ch7F11;
  • Figure 5 is a diagram showing the endocytosis activity of ch4D3 and ch7F11 antibodies on BXPC-3 living cells;
  • Fig. 6 is the light and heavy chain variable region sequence of the 4D3 murine antibody, and its humanness (Z-score) changes before and after humanization;
  • Figure 7 is the light and heavy chain variable region sequence of the 7F11 murine antibody, and its humanness (Z-score) changes before and after humanization;
  • Figure 8 is a graph showing the binding curves of 4D3 humanized antibody and chimeric antibody at the same dilution concentration
  • Figure 9 is a graph of the SEC detection results of the 4D3 humanized antibody on Day7 (Figure A), Day14 ( Figure B), and Day21 (Figure C);
  • Figure 10 is a graph showing the binding curves of 7F11 humanized antibody and chimeric antibody at the same dilution concentration
  • Figure 11 is a graph of the SEC detection results of the 7F11 humanized antibody on Day 7 (Figure A), Day 14 ( Figure B), and Day 21 (Figure C);
  • Figure 12 is the results of 4D3-humanized-biotin antibody and 0.02-50ug/ml ch4D3 and hu4D3 antibody binding epitope competition results;
  • Figure 13 shows the results of competition between 7F11-humanized-biotin antibody and 0.02-50ug/ml ch7F11 and hu7F11 antibody binding epitopes.
  • Figure 14 is the binding data of hu7F11 antibody in three kinds of cells.
  • Figure 15 is the binding data of Hu4D3 antibody in 3 kinds of cells.
  • Figure 16 is the endocytosis data of the hu7F11 antibody in three kinds of cells.
  • Figure 17 is the endocytosis data of Hu4D3 antibody in three kinds of cells.
  • Pre-culture mouse myeloma cells SP2/0 in DMEM+FBS 10% complete medium Before fusion, use a Pasteur pipette to blow out 5 x 10 7 SP2/0 cells, centrifuge at 1000 g, 5 min, and preheat at 37°C. hot rinse residual serum free DMEM serum, while acquiring the KM mouse feeder cells in the abdominal cavity, and in an amount of 5x10 3 cells th / 100ul / well to feeder cells were plated in 96 well plates. On the 3rd day after the final immunization, blood was collected from the eyeballs, and the final immunized mice were sacrificed. After being soaked in 75% alcohol, they were placed on a sterile operating table to cut the spleen tissue.
  • the medium was changed with DMEM+10% FBS medium one day before the supernatant test.
  • the ELISA plate was coated with TROP2 antigen at a concentration of 2ug/ml.
  • use a multi-channel electric pipette on a sterile operating table to suck the medium supernatant from the 96-well plate, and add it to the corresponding ELISA plate well.
  • the feeder cells were plated according to the method in Example 2, and the positive cells were blown evenly with a 200ul pipette tip. Take no more than 5ul cell suspension volume for subcloning, dilute it to 100ul, and add 96 that contains 100ul/well feeder cell suspension in advance. The first empty in the orifice plate. From A1 to H1, pipette evenly with a volume of 100ul to the last row, and then use a multi-channel electric pipette to pipette 100ul evenly from the direction of A1 to A12 to the last row. The well plates are cultured for 7-10 days, the wells formed by single clones are marked, and the supernatant positive detection is performed with reference to the method in the examples.
  • the Balb/c mice were injected intraperitoneally with paraffin oil of 1 ml per mouse. Subsequently, the monoclonal cells with a stable positive rate after the first subcloning were picked and expanded for culture. Collect the cells when they grow to at least one well size of a 6-well plate, and rinse the cells 3 times with phosphate buffer at 1000 g for 3 min centrifugation. It is prepared by injecting 1 ⁇ 2 ⁇ 10 6 per mouse per mouse. Feed the mice for 7-10 days and observe the abdominal cavity of the mice. Ascites was collected with an 18-gauge sterile needle, and the ascites fluid was collected at 14000 g at a rotation speed of 5 min. The obtained supernatant was purified by proteinA/G affinity column to obtain monoclonal antibodies for 4D3 and 7F11 cell lines.
  • This cDNA library is used as a PCR template after the 5'end of the terminal transferase TdT plus dGTP.
  • the upstream primer is oligo dC, and the downstream primer corresponds to the primer matching of the 5'end CH1 constant region of the antibody light and heavy chain, with the help of high-fidelity enzyme primerSTAR Perform 5'RACE amplification of variable region genes.
  • PCR products were analyzed by DNA agarose gel electrophoresis, and DNA fragments with a length of ⁇ 750bp were recovered for downstream TA cloning.
  • Strains that were identified as positive by colony PCR were sequenced. The obtained sequence was identified by the online IMGT database for sequence alignment and two-dimensional mapping of the variable region sequence.
  • Example 7 Obtaining 7F11 antibody light and heavy chain variable region coding sequence
  • Example 6 Refer to the operation process in Example 6 to obtain the light and heavy chain variable region sequence of the 7F11 clone, as shown in FIG. 2.
  • FreestyleTM 293-F (Invitrogen) suspension cells were used to express antibodies.
  • Inoculate cells at a density of 6 ⁇ 105 cells/mL in a 1L shake flask containing 300mL F17 complete medium (FreestyleTM F17 expression medium, Gibco), 37°C, 5% CO2, 120rpm cell culture shake Incubate in a bed overnight.
  • F17 complete medium FreestyleTM F17 expression medium, Gibco
  • the antibody expression plasmid was transfected with PEI, where the ratio of plasmid: PEI was 2:1.
  • TN1 feed medium was added at 2.5% (v/v), the culture was continued for 4 days, and the supernatant was collected by centrifugation.
  • the obtained expression supernatant was collected and passed through a Protein A affinity chromatography column (Mabselect Sure LX, GE), eluted with 0.1M citric acid (pH3.0), and the captured antibody was washed with 1M Tris-HCl (pH9.0) Adjust to pH 7.0 by 1/10 (v/v), and then pass the gel filtration chromatography column SEC (Superdex 200, GE) to remove impurities such as polymer and endotoxin, and at the same time replace the antibody buffer with PBS ( pH7.4), the antibody obtained by this method, the target antibody monomer (POI%) is greater than 99%, and is used for subsequent experiments.
  • variable region gene was cloned into an expression plasmid containing the constant region of a human antibody, and the eukaryotic cell 293F was transiently transfected according to the protocol of Example 8 and the secreted 4D3 and 7F11 chimeric antibodies were purified. Dilute the chimeric antibody to a concentration of 50ug/ml, add it to wells A1 ⁇ H1 on the TROP2-coated ELISA plate, and then perform a 3-fold horizontal dilution from A1 to A12; incubate at 37°C for 1hr, then rinse and add Mouse anti-human Fc HRP-labeled antibody was incubated at 37°C for color development.
  • Figure 3 shows that 4D3 and 7F11 have good relative affinity.
  • EC50(B4) 0.047ug/ml
  • EC50(B7) 0.071ug/ml.
  • the BXPC-3 cells were plated and grown for 24 to 48 hours, so that the cell confluence rate in the 96-well cell culture plate reached 40 to 50%. On the day of the experiment, the supernatant was aspirated and washed twice. Add 3% BSA content PBS solution and block at 37°C for 1 hr. Dilute the chimeric antibody and hRS7 antibody to 10ug/ml and incubate at 37°C for 1h, aspirate the primary antibody solution, wash 4 times, add 4% paraformaldehyde solution, 100 ⁇ L/well and stand at room temperature for 20min, repeat washing twice Then, according to the ratio of 1:800, dilute the secondary antibody with 1% BSA solution, 100ul/well.
  • Aspirate and discard the secondary antibody suspension repeat washing 4 times, add 100 ⁇ L/well of DAPI dye solution at a concentration of 2 ⁇ g/ml, and incubate for 5 min at room temperature in the dark.
  • Aspirate and discard the DAPI staining solution and after repeated washing 4 times, add 1 ⁇ DPBS solution, 100 ⁇ L/well, observe under a fluorescence microscope, and take pictures to record the experimental results.
  • Figure 4 Cell surface binding activity of ch4D3 and ch7F11 under the same antibody concentration and treatment conditions.
  • Example 10 Plate BXPC-3 cells.
  • aspirate the cell culture solution add PBS solution, and repeat the washing twice.
  • Dilute the chimeric antibody and hRS7 antibody to 10ug/ml in a medium containing 1% FBS and incubate at 4°C for 1 hour, aspirate the primary antibody solution, add complete medium for cell culture to be tested, and incubate at 37°C for 1 hour.
  • IMGT nucleic acid sequence sequencing results of the 4D3 variable region
  • sequence characteristics of the light and heavy chain variable regions including three CDR region sequences and four FR region sequences, as well as the most closely related germline gene family sequences.
  • IMGT-DomainGapAlign amino acid check window search for the human germline gene family sequence with the highest sequence similarity.
  • the 4D3 light chain corresponds to the human IGKV1-27*01+IGKJ2*02 family sequence
  • the 4D3 heavy chain corresponds to the human IGHV1-3* 01+IGHJ4*01 family sequence.
  • the 4D3-Hum version of the humanized sequence of light and heavy chains was obtained. Enter the 4D3 mouse sequence and humanized sequence into the online website to evaluate the similarity score of human antibodies.
  • the blue line represents the Z-score distribution range and frequency of the mouse antibody library
  • the green line represents the Z-score distribution range and frequency of the human antibody library.
  • the red straight line represents the Z-score obtained by the 4D3 light chain and heavy chain. After humanization, the Z-score of 4D3 increased significantly.
  • Example 12 Refer to the operation procedure in Example 12 to obtain the humanized sequence of the light and heavy chains of the 7F11 mouse antibody. Enter the 7F11 mouse sequence and humanized sequence into the online website to evaluate the similarity score of human antibodies.
  • the blue line in Figure 7 represents the Z-score distribution range and frequency of the mouse antibody library
  • the green line represents the Z-score distribution range and frequency of the human antibody library.
  • the red straight line represents the Z-score obtained by the 4D3 light chain and heavy chain. After humanization, the Z-score of 7F11 has increased significantly.
  • the 4D3 humanized antibody sequence was cloned into a eukaryotic expression vector, and the eukaryotic cell 293F was transiently transfected according to the protocol of Example 8.
  • the purified antibodies are uniformly diluted to 2ug/ml, and added to the wells A1 ⁇ H1 of the TROP2-coated ELISA plate together with the mouse antibody before humanization, and then diluted 3 times from the direction of A1 ⁇ A12; 37 After incubating at °C for 1hr, rinse, then add anti-human Fc HRP-labeled antibody, and incubate at 37°C for color development. Finally, the relative affinities between ch4D3 and hum4D3 antibodies were compared by EC50 and curve shape.
  • the purified hum4D3 antibody was dialyzed, dialyzed with PBS buffer and calibrated to a final concentration of 2mg/ml, and divided into two batches of 70ul/tube, 3 tubes in each batch.
  • the two batches of samples were placed at 4°C and 37°C, and the sample tubes were taken out according to the 0th day, the 7th day, and the 14th day.
  • the samples were used for SEC analysis to evaluate the degradation and aggregation of antibodies.
  • FIG 9 SEC detection results of the humanized 4D3 antibody on Day 7 ( Figure A), Day 14 ( Figure B), and Day 21 ( Figure C).
  • the monomers and aggregates of the 4D3-humanized antibody at each time point under the condition of 37°C, and the proportion (%) of the detection molecules in the 4D3-humanized antibody are shown in the table in Figure D.
  • Figure 10 Binding curve of 7F11 humanized antibody and chimeric antibody at the same dilution concentration, EC50 (7F11-chimeric): 0.061ug/ml; EC50 (7F11-humanized): 0.0601ug/ml.
  • FIG 11 SEC detection results of the 7F11 humanized antibody on Day7 ( Figure A), Day14 ( Figure B) and Day21 ( Figure C).
  • the monomers and aggregates of the 7F11-humanized antibody at each time point under the condition of 37°C, and the proportion (%) of the detection molecules in the 7F11-humanized antibody are shown in the table in Figure D.
  • Pall ForteBio Octet optical analysis technology platform is used to evaluate the absolute affinity of antibody-antigen binding.
  • the biotin-labeled antigen is immobilized on the surface of the streptavidin biosensor chip, the baseline is balanced for 180 seconds, and then it binds with the antibody diluted in the solution concentration gradient for 30 seconds, which increases the optical thickness of the chip, resulting in The wavelength shifts ( ⁇ ), and then enters the dissociation phase of 30sec.
  • the interaction between the Trop2 antigen and the corresponding antibody is measured in real time, and the specificity of binding, binding rate, dissociation rate or sample concentration are precisely and accurately detected at each concentration. After summarizing the k-on and k-off values under at least 5 concentration gradients, the KD binding constant is obtained.
  • Example 19 Activity analysis and binding epitope consistency analysis of 4D3 humanized antibody and chimeric antibody
  • the 4D3-humanized antibody was labeled with biotin, and the inflection point value of the binding curve was determined by ELISA to be 0.5ng/ml.
  • Figure 12 4D3-humanized-biotin antibodies compete with ch4D3 and hum4D3 antibodies at 0.02-50ug/ml, respectively.
  • the two competing antibodies show the same degree of competitive activity and bind the same epitope.
  • Example 20 Activity analysis and binding epitope consistency analysis of 7F11 humanized antibody, humanized antibody, and chimeric antibody
  • FIG. 13 The 7F11-humanized-biotin antibody competes with 0.02-50ug/ml ch7F11 and hum7F11 antibodies, respectively.
  • the two competing antibodies show the same degree of competitive activity and bind the same epitope.
  • HEK293 cells as negative cells and BXPC-3 and MCF-7 cells as positive cells to test the binding and endocytosis of each antibody at gradient concentrations.
  • BXPC-3 and MCF-7 cells as positive cells to test the binding and endocytosis of each antibody at gradient concentrations.
  • For cell binding test bind for 1 hr at 4°C, then add conventional FITC-labeled fluorescent secondary antibody, and collect data by flow cytometry.
  • the endocytosis test uses the acid-sensitive small molecule dye Phrodo-Red goat anti-human secondary antibody, which is incubated with each concentration of the primary antibody to form a complex, and then incubated with each cell line for 16 hours. After sampling, at least 10,000 cells are laid in the cell wells of each 96-well plate, and the data is collected and analyzed by a flow cytometer. Count the average fluorescence intensity values of cells at various concentrations in the far-red light channel, and plot the degree of endocytosis curve with the antibody concentration as the abscissa.
  • BxPC-3 cells Comparing the binding degree of hu7F11 and Hu4D3 antibodies in the three kinds of cells, BxPC-3 cells have the highest binding degree. Comparison of the degree of endocytosis of hu7F11 and Hu4D3 antibodies in three kinds of cells. BxPC-3 cells have the highest endocytosis.

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Abstract

单价和多价的抗TROP2抗体，该类抗体与TROP2蛋白特异性结合，相对亲和力强，内吞效率高，该特性抗体在ADC药物开发中具有广阔的应用前景。还涉及鼠单克隆抗体，人鼠嵌合抗体，人源化抗体以及该类抗体在肿瘤诊断和治疗中的用途。

Description

一种抗TROP2抗体 技术领域
本发明涉及能特异性结合人的TROP2蛋白的抗体，特别是鼠、嵌合和人源化来源的单克隆抗体，以及编码这些抗体的氨基酸和核苷酸序列。本发明也包含这些抗体作为诊断试剂或者药物在诊断性和/或治疗性处理恶性肿瘤或者所述受体过表达有关的任何病变的用途。
技术背景
Trop2蛋白，又名II类滋养层抗原(trophoblast antigen 2)、肿瘤钙信号转导因子2(tumou associated calcium signal transducer 2)，其由染色体1p32位的单拷贝基因TACSTD2编码。其对应的mRNA合成36kDa初生多肽，在经过N端糖基化后形成单跨膜域的单体膜蛋白(Annie R.A.et al.,2015)。
在TROP2的26氨基酸长度之胞内区序列中，存在第303位的丝氨酸位点，此位点在物种间的保守性较高(Basu et al.,1995；Annie R.A.et al.,2015)，且能被PKC激酶磷酸化，既成信号转导分子PIP 2的结合位点，并下游刺激胞质钙离子浓度的上升(Sewedy et al.,1998；Alberti.et al.,1999)。而在体外细胞系中过表达缺失胞质区的Trop2，此蛋白即失去生长刺激的功能，表明Trop2胞质区域的其它信号通路刺激作用(Guerra.et al)。Trop2的胞外区具有能够结合EGF生长因子的结构域，从而可潜在截获EGF以下调IGF-1R/Akt信号通路的活性，因此，Trop2表达量的下降可反向激活IGF-1R/Akt信号通路(Lin.et al.,2011；Annie R.A.et al.,2015)。
Trop2在肿瘤发生过程中具有明显促进作用。过表达Trop2可显著增强NIH3T3细胞的成瘤性(Wang.et al.,2008),Trop2亦可影响上皮-间质转化(EMT)，并增强癌细胞的迁徙与侵入能力(Trerotola.et al.,2013；Li.et al.,2017)此过程可能通过影响PI3K/Akt信号通路而实现：在过表达Trop2的胆囊癌细胞中，Akt的磷酸化激活程度明显提升，反之，敲低Trop2的表达则可抑制此信号通路的活性(Li.et al.,2017)。此外，Trop2过表达亦能刺激MAPK/ERK信号通路的活性，从而导致胰腺癌细胞的增殖上调，及荷瘤小鼠的肿瘤恶化程度增加(Cubas.et al.,2010)。
Trop2蛋白的表达见诸于一系列表皮源性组织，如胚胎发育和成体时期的乳腺、肾脏和胰腺等(Annie R.A.et al.,2015)。但在正常组织对应的肿瘤组织中，Trop2蛋白的表达量无差别地显著上升，且表达程度的强弱与动物模型中肿瘤生长程度呈现正相关性质(Trerotola.et al.,2013)，由于Trop2基因序列本身未发生突变或扩增，因此Trop2在癌症中的表达量上调被认为是转录调控层面的刺激所致(Trerotola.et al.,2013)。在不同种 类的实质瘤病患体内，此蛋白的过表达通常预示肿瘤的预后不良(Zeng.et al.,2016)。在胆囊癌(Chen.et al.,2014)、肠胃癌(Muhlmann.et al.,2009)、肝门部胆管癌(Ning.et al.,2013)和胰腺癌(Fong.et al.,2008)病症的案例中，Trop2高表达病患者的生存率明显下降。鉴于Trop2在多种癌症中的高表达性，以及其对病患生存率的显著影响，此蛋白被认为是一个潜在的肿瘤治疗靶点。
在已知的抗体药物种类中，一项针对Trop2靶点的毒素藕联抗体(ADC)：IMMU-132(Goldenberg.et al.,2015)。此ADC的装载抗体RS7是通过杂交瘤制备手段，经肺癌细胞膜粗提物免疫的小鼠脾脏淋巴细胞与鼠骨髓瘤细胞融合筛选获得(Stein.et al.,1990)。而在粗提物中，RS7所结合的抗原后被证明为Trop2(Stein.et al.,1994)。RS7抗体本身可被多种癌细胞所内吞，因而使其具有制备成ADC的潜力(Stein.et al.,1993)。在鼠源RS7经过序列人源化以后，人源化版本的RS7被用于制备抗体偶联药物，并以链间巯基藕联的方式装载SN-38药物、一种拓扑异构酶的抑制剂(Moon,et al.,2008；Sahota.et al.,2017)在最近的临床I期数据中，IMMU-132在多种癌症，尤其在三阴性乳腺癌的治疗中具有临床有效性(Starodub.et al.,2015；Sahota and Vahdat.,2017)。
此专利披露的信息涉及医药领域，具体而言，本专利所述嵌合抗体或人源化抗体可结合人II类滋养层抗原蛋白(Trop2)，且具备抗体偶联药物疗法中的靶向抗体功能。
发明内容
本发明不涉及天然形式的抗体，发明中所涉及抗体均通过免疫小鼠以及鉴定和分离手段获得，或者通过基因重组获得。根据本发明所述，要求保护对象为抗体或功能片段或衍生物，所述抗体的特征在于包含至少一个CDR，其氨基酸序列来源于SEQ ID No.1至SEQ ID No.12。
任何一个抗体片段或者衍生物若含有至少一个CDR，且该CDR的序列在与序列SEQ ID No.1至SEQ ID No.12进行优化比对后，至少具有80％的同一性，或优选的85％、90％、95％或者98％同一性的，都应该理解为本发明的等同物，因此也是本发明的一部分。
更具体的，本发明所述的抗体或者起功能片段之一或者衍生物的特征在于其包含重链，所述的重链包含至少一个CDR，所述的CDR选自含有氨基酸序列SEQ ID No.1至SEQ ID No.6的序列。
更具体的，本发明所述的抗体或者其功能片段之一或者衍生物的特征在于其包含轻链，所述的重链包含至少一个CDR，所述的CDR选自含有氨基酸序列SEQ ID No.7至SEQ ID No.12的序列。
根据所述方面，更具体的第一个具体实施方式，本发明所述的抗体或者其功能片段之一 或者衍生物包含重链，所述重链包含CDR-H1、CDR-H2、CDR-H3，其中CDR-H1包含氨基酸序列SEQ ID No.1,CDR-H2包含氨基酸序列SEQ ID No.2,CDR-H3包含氨基酸序列SEQ ID No.3.
更具体的,根据第一个具体实施方式,所述的抗体或者其功能片段之一或者衍生物包含重链，所述重链的序列中，嵌合抗体包含序列SEQ ID No.13,人源化抗体包含序列SEQ ID No.17。
根据所述方面，更具体的第一个具体实施方式,本发明所述的抗体或者其功能片段之一或者衍生物包含轻链，所述轻链包含CDR-L1、CDR-L2、CDR-L3，其中CDR-L1包含氨基酸序列SEQ ID No.7，CDR-L2包含氨基酸序列SEQ ID No.8，CDR-L3包含氨基酸序列SEQ ID No.9。
更具体的,根据第一个具体实施方式,所述的抗体或者其功能片段之一或者衍生物包含轻链，所述轻链的序列中，嵌合抗体包含序列SEQ ID No.14，人源化抗体包含序列SEQ ID No.18。
根据所述方面，更具体的第二个具体实施方式,本发明所述的抗体或者其功能片段之一或者衍生物包含重链，所述重链包含CDR-H1、CDR-H2、CDR-H3，其中CDR-H1包含氨基酸序列SEQ ID No.4，CDR-H2包含氨基酸序列SEQ ID No.5,CDR-H3包含氨基酸序列SEQ ID No.6。
更具体的,根据第二个具体实施方式,所述的抗体或者其功能片段之一或者衍生物包含重链，所述重链的序列中，嵌合抗体包含序列SEQ ID No.15,人源化抗体包含序列SEQ ID No.19.
根据所述方面，更具体的第二个具体实施方式,本发明所述的抗体或者其功能片段之一或者衍生物包含轻链，所述轻链包含CDR-L1、CDR-L2、CDR-L3，其中CDR-L1包含氨基酸序列SEQ ID No.10,CDR-L2包含氨基酸序列SEQ ID No.11,CDR-L3包含氨基酸序列SEQ ID No.12
更具体的,根据第二个具体实施方式,所述的抗体或者其功能片段之一或者衍生物包含轻链，所述轻链的序列中嵌合抗体包含序列SEQ ID No.16,人源化抗体包含序列SEQ ID No.20
作为发明的另一方面，本发明涉及分离的DNA，其特征包含选自下述DNA序列的核酸：其每一条核酸编码的氨基酸序列为上述氨基酸序列SEQ ID No.1至SEQ ID No.20中的一条。
更为具体的，包括选自下述的DNA序列的核酸：
第一个具体实施方式包含序列SEQ ID No.21、SEQ ID No.22、SEQ ID No.23和序列SEQ ID No.27、SEQ ID No.28、SEQ ID No.29的核酸序列。
第二个具体实施方式包含序列SEQ ID No.24、SEQ ID No.25、SEQ ID No.26和序列SEQ ID No.30、SEQ ID No.31、SEQ ID No.32的核酸序列。
再进一步具体说明
第一个具体实施方式嵌合抗体序列包含序列SEQ ID No.33和SEQ ID No.34，人源化抗体序列包含序列SEQ ID No.37和SEQ ID No.38的核酸序列。
第二个具体实施方式嵌合抗体序列包含序列SEQ ID No.35和SEQ ID No.36，人源化抗体 序列包含序列SEQ ID No.39和SEQ ID No.40的核酸序列。
附图说明
图1是4D3轻链、重链可变区序列CDR区识别以及二维结构图；
图2是7F11轻链、重链可变区序列CDR区识别以及二维结构图；
图3是ch4D3、ch7F11的ELISA结合曲线；
图4是ch4D3与ch7F11的细胞表面结合活性图；
图5是ch4D3与ch7F11抗体在BXPC-3活细胞上的内吞活性图；
图6是4D3鼠源抗体的轻重链可变区序列，其在人源化前后的类人分值(humanness；Z-score)变化图；
图7是7F11鼠源抗体的轻重链可变区序列，其在人源化前后的类人分值(humanness；Z-score)变化图；
图8是4D3人源化抗体与嵌合抗体在同等稀释浓度下的结合曲线图；
图9是4D3人源化抗体在Day7(图A)，Day14(图B)，Day21(图C)时刻的SEC检测结果图；
图10是7F11人源化抗体与嵌合抗体在同等稀释浓度下的结合曲线图；
图11是7F11人源化抗体在Day7(图A)，Day14(图B)，Day21(图C)时刻的SEC检测结果图；
图12是4D3-humanized-biotin抗体分别与0.02～50ug/ml的ch4D3与hu4D3抗体结合表位竞争结果；
图13是7F11-humanized-biotin抗体分别与0.02～50ug/ml的ch7F11与hu7F11抗体结合表位竞争结果。
图14是hu7F11抗体在3种细胞中的结合数据。
图15是Hu4D3抗体在3种细胞中的结合数据。
图16是hu7F11抗体在3种细胞中的内吞数据。
图17是Hu4D3抗体在3种细胞中的内吞数据。
具体实施方式
实施例1 TROP2抗原免疫6～8周龄的Balb/c小鼠
构建TROP2胞外区表达载体，用悬浮细胞293F瞬时表达毫克级的TROP2蛋白。选择6-8周龄的小鼠，按如下表格中的免疫剂量和时间点进行TROP2抗原的皮下免疫，终免选择三免后血浆效价最高的小鼠，免疫流程如表1所示。
程序 免疫进程(天数) 途径、剂量等
初免 0 100ug/0.25ml/每只鼠，完全佐剂，皮下免疫
二免 14 100ug/0.25ml/每只鼠，不完全佐剂，皮下免疫
三免 35 100ug/0.25ml/每只鼠，不完全佐剂，皮下免疫
三免采血 42 TROP2抗原包板的ELISA检测尾静脉血浆效价
终免 56 50ug/0.25ml/每只鼠，磷酸盐缓冲液，腹腔免疫
表1
实施例2免疫小鼠脾脏细胞体外融合
预先培养小鼠骨髓瘤细胞SP2/0于DMEM+FBS 10％完全培养基中，融合前使用巴斯德吸管吹取5 x 10 7个SP2/0细胞，以1000g，5min转速离心并用37℃预热的无血清DMEM润洗残留血清，同时采集KM鼠腹腔内的饲养细胞，并以5x10 3个/100ul/孔的细胞量将饲养细胞铺于96孔板。在终免后第3天眼球采血、并处死终免疫小鼠，75％酒精浸泡消毒后置于无菌操作台剪取脾脏组织。用预热的无血清DMEM吹取脾细胞，取1/2个脾脏的细胞进行计数，以脾细胞:SP2/0细胞数量比例1:1～10:1混合，离心后吸干残留DMEM。加入1ml体积的预热PEG-1450并均匀混合，3min后加入35ml预热的DMEM培养基稀释终止。1000rpm，5min离心细胞，后用HAT筛选培养基重悬细胞，铺板于10块96孔板中。
实施例3杂交瘤细胞上清阳性检测
融合后7～10天，观察细胞形成克隆的情况。在上清检测前一天用DMEM+10％FBS培养基换液一次。同时以2ug/ml浓度的TROP2抗原包被ELISA酶标板。在检测当天，在无菌操作台上用多通道电动移液器吸取96孔板中的培养基上清，悬空加入对应的ELISA酶标板孔中。将ELISA板置于37℃孵育1hr，后PBST洗涤孔板3次，加入1:5000稀释的HRP标记羊抗小鼠抗体。37℃孵育1hr后，PBST洗涤3次。配置TMB底物显色液，每孔添加50ul，于室温条件反应5～10min。随后加入50ul/孔2M硫酸溶液终止显色。根据酶标板上的OD450读值筛选阳性克隆。
实施例4阳性细胞株亚克隆筛选
标记OD450值较高的融合细胞板孔，持续培养不超过2天。按照实施例2中方法铺板饲养细胞，并用200ul枪头吹匀阳性细胞，取不超过5ul细胞悬液体积进行亚克隆，将其稀 释至100ul，并加入预先含有100ul/孔饲养细胞悬液的96孔板中的第一个空。从A1到H1的方向以100ul体积均匀吹打至最后一排，再用多通道电动移液器吸取100ul从A1～A12的方向均匀吹打至最后一列。将孔板培养7～10天，标记单一克隆形成的板孔，并参照实施例中的方法进行上清阳性检测。
实施例5腹水制备单克隆抗体
制备腹水前7日，向Balb/c小鼠腹腔注射1ml/每只用量的石蜡油。随后挑取第一次亚克隆后阳性率稳定的单克隆细胞，进行扩大培养。在细胞长至至少一个6孔板板孔规模时收取细胞，以1000g，3min离心条件，用磷酸缓冲液润洗细胞3次。按每只小鼠注射1～2x10 6个/每只的量制备。饲养小鼠7～10天并观察小鼠腹腔。用18号无菌针头采集腹水，并以14000g，5min转速收集腹水上清。所得上清经过proteinA/G亲和柱纯化后即得到4D3和7F11细胞株的单克隆抗体。
实施例6获取4D3抗体轻重链可变区编码序列
培养单克隆细胞株至6孔板，其汇合率达到90～100％时用trizol收集细胞。在无RNase环境中提取总RNA，使用oligo dT作为逆转录引物合成cDNA文库。此cDNA文库经过末端转移酶TdT的5’端加dGTP反应后用作PCR模板，使用上游引物为oligo dC,下游引物对应抗体轻重链的5’端CH1恒定区的引物搭配，借助高保真酶primerSTAR进行可变区基因5’RACE扩增。将PCR产物进行DNA琼脂糖凝胶电泳分析，回收位于～750bp长度的DNA片段，进行下游TA克隆。将菌落PCR鉴定为阳性的菌株实施测序。所得序列由在线IMGT数据库进行序列比对鉴定以及可变区序列二维作图。
实施例7获取7F11抗体轻重链可变区编码序列
参照实施案例6中的操作过程，得到7F11克隆的轻重链可变区序列，如图2所示。
实施例8抗体的表达与纯化
使用FreestyleTM 293-F(Invitrogen)悬浮细胞表达抗体。转染前一天，以6×105个/mL密度将细胞接种于含300mL F17完全培养基(FreestyleTM F17表达培养基，Gibco公司)的1L摇瓶中，37℃，5％CO2，120rpm细胞培养摇床过夜培养。次日，用PEI进行抗体表达质粒的转染，其中质粒:PEI比例为2:1。转染后一天，按2.5％(v/v)加入TN1补料培养基，继续培养4天后离心收集上清。收集得到的表达上清，经Protein A亲和层析柱(Mabselect Sure LX，GE公司)，以0.1M柠檬酸(pH3.0)洗脱，捕获的抗体用1M Tris-HCl(pH9.0)按1/10(v/v)调节至pH7.0，再通过凝胶过滤层析柱SEC(Superdex 200，GE公司)去除多聚体和内毒素等杂质，同时将抗体缓冲液置换成PBS(pH7.4)，通过此方法获得的抗体，目标抗体 单体(POI％)大于99％，用于后续试验。
实施例9 ELISA方法评估抗体亲和性
将可变区基因克隆至含有人抗体恒定区的表达质粒上，参照实施例8方案瞬时转染真核细胞293F并纯化分泌的4D3与7F11嵌合抗体。将嵌合抗体稀释为50ug/ml浓度，添加至TROP2包被的ELISA酶标板上的A1～H1孔，再从A1至A12方向进行3倍体积横向稀释；37℃孵育1hr后润洗，加入小鼠抗人Fc的HRP标记抗体，37℃孵育后进行显色。
图3表明4D3与7F11均具有较好的相对亲和力。EC50(B4)：0.047ug/ml；EC50(B7)：0.071ug/ml。实施例10细胞免疫荧光方法评估抗体生物学活性
将BXPC-3细胞铺板并生长24～48hr，使96孔细胞培养板内的细胞汇合率达到40～50％。实验当天吸弃上清并洗涤2次。添加3％BSA含量之PBS溶液，37℃封闭1hr。将嵌合抗体以及hRS7抗体稀释至10ug/ml于37℃孵育1h，吸弃一抗溶液，洗涤4次后，加入4％多聚甲醛溶液，100μL/孔并室温静置20min，重复洗涤2次后，按1:800比例，用1％BSA溶液稀释二抗，100ul/孔。吸弃二抗悬液，重复洗涤4次，将2μg/ml浓度的DAPI染液按100μL/孔添加，室温避光孵育5min。吸弃DAPI染液，重复洗涤4次后，加入1×DPBS溶液，100μL/孔，于荧光显微镜下观察，并拍照记录实验结果。
附图4：同等抗体浓度及处理条件下，ch4D3与ch7F11的细胞表面结合活性。
实施例11细胞内吞方法评估抗体生物学活性
参照实施例10中的方法铺板BXPC-3细胞。实验当天，吸弃细胞培养液，加入PBS溶液，重复洗涤2次。用含1％FBS之培养基，将嵌合抗体以及hRS7抗体稀释至10ug/ml于4℃孵育1h，吸弃一抗溶液，加入待检细胞培养用的完全培养基，37℃孵育1hr。吸弃细胞培养基，重复洗涤4次后，加入4％多聚甲醛溶液，100μL/孔并静置20min。重复洗涤2次后，添加含0.5％Triton-X100的3％BSA封闭液，100μL/孔，室温静置1h。吸弃打孔液，重复洗涤2次。按1:800比例稀释抗人IgG荧光二抗；100μL/孔，37℃孵育1h。吸弃荧光二抗溶液，重复洗涤4次后进行DAPI染色，室温避光静置15min。重复洗涤4次，加入1×DPBS溶液，100μL/孔，于荧光显微镜下观察荧光染色结果，如图4、5所示。
实施例12 4D3人源化序列改造
获得4D3可变区的核酸序列测序结果，将序列输入网站IMGT所提供的V-QUEST序列查对窗口(http://www.imgt.org/IMGT_vquest/vquest)。以得到轻重链可变区的序列特征，包括三个CDR区序列和四个FR区序列，以及亲缘关系最相近的胚系基因家族序列。在IMGT-DomainGapAlign氨基酸查对窗口中，查找序列相似度最高的人源胚系基因家族序列， 4D3轻链对应人IGKV1-27*01+IGKJ2*02家族序列，4D3重链对应人IGHV1-3*01+IGHJ4*01家族序列。将此人源胚系基因序列上的CDR区替换为4D3轻重链的CDR，再使用IMGT-Structural query查对抗体结构信息。最终获得4D3-Hum版本的轻重链人源化序列。将4D3鼠源序列及人源化序列输入线上网站评估人类抗体相似性分值。
图6蓝色线代表鼠源抗体库的Z-score分布范围和频率，绿色线代表人抗体库的Z-score分布范围和频率。红色直线代表4D3轻链重链所获得的Z-score分值。人源化后，4D3的Z-score分值明显上升。
实施例13 7F11人源化序列改造
参照实施案例12中的操作过程，得到7F11鼠抗体的轻重链人源化序列。将7F11鼠源序列，人源化序列输入线上网站评估人类抗体相似性分值。
图7中蓝色线代表鼠源抗体库的Z-score分布范围和频率，绿色线代表人抗体库的Z-score分布范围和频率。红色直线代表4D3轻链重链所获得的Z-score分值。在人源化以后，7F11的Z-score分值明显上升。
实施例14 4D3人源化抗体相对亲和力分析
将4D3人源化抗体序列克隆至真核表达载体，参照实施例8方案瞬时转染真核细胞293F。纯化后的抗体统一稀释至2ug/ml，与人源化之前的鼠源抗体一同添加至TROP2包被的ELISA酶标板上的A1～H1孔，再从A1～A12方向进行3倍稀释；37℃孵育1hr后润洗，再加入抗人Fc的HRP标记抗体，37℃孵育后进行显色。最终通过EC50和曲线形态来比较ch4D3和hum4D3抗体之间的相对亲和力。
图8，EC50(4D3-chimeric)：0.056ug/ml；EC50(4D3-humanized)：0.0502ug/ml。
实施例15 4D3人源化抗体热稳定性分析
将纯化后的hum4D3抗体透析，以PBS缓冲液透析并标定终浓度为2mg/ml，并按70ul/管分两批，每批3管。将两批样品各放置于4℃和37℃，并按照第0天，第7天，第14天分别取出样品管。将样品用于SEC分析以评估抗体的降解及聚集情况。
图9：4D3人源化抗体在Day7(图A)，Day14(图B)，Day21(图C)时刻的SEC检测结果。4D3-humanized抗体在37℃条件下各时间点的单体和聚集体，以及其所占检测分子的比例(％)显示于图D表格。
实施例16 7F11人源化抗体相对亲和力及结合表位一致性分析
参照实施例14中的操作过程进行7F11人源化抗体的相对亲和力评估
图10：7F11人源化抗体与嵌合抗体在同等稀释浓度下的结合曲线，EC50(7F11-chimeric)： 0.061ug/ml；EC50(7F11-humanized)：0.0601ug/ml。
实施例17 7F11人源化抗体热稳定性分析
参照实施例15中的操作过程进行7F11人源化抗体的热稳定性分析。
附图11：7F11人源化抗体在Day7(图A)，Day14(图B)，Day21(图C)时刻的SEC检测结果。7F11-humanized抗体在37℃条件下各时间点的单体和聚集体，以及其所占检测分子的比例(％)显示于图D表格。
实施例18人源化抗体与亲本抗体的抗原亲和力分析
采用Pall ForteBio Octet光学分析技术平台，评估抗体-抗原结合的绝对亲和力。在该方法中，生物素标记的抗原被固定在链霉亲和素生物传感器芯片表面，基线平衡180sec，后与溶液中的浓度梯度稀释之抗体发生30sec的结合，使得芯片的光学厚度增加，导致波长发生漂移(Δλ)，随后进入30sec的解离阶段。Trop2抗原和相应的抗体相互作用被实时测定，在各浓度下对结合的特异性、结合速率、解离速率或者样品浓度进行精密准确的检测。汇总至少5个浓度梯度下的k-on和k-off值后，到KD结合常数。
Ab Code KD(M) ka(1/Ms) kd(1/s) R 2
ch4D3 6.89E-09 2.35E+05 1.62E-03 0.9935
Hu4D3 3.07E-08 1.39E+05 4.28E-03 0.9916
ch7F11 6.11E-11 2.43E+05 1.45E-05 0.9925
Hu7F11 9.94E-11 1.86E+05 1.85E-05 0.9966
实施例19 4D3人源化抗体与嵌合抗体的活性分析及结合表位一致性分析
生物素标记4D3-humanized抗体，并通过ELISA测定结合曲线的拐点值为0.5ng/ml。配置含0.5ng/ml 4D3生物素标记抗体的ELISA封闭液，在此溶液基础上配置50ug/ml的竞争抗体4D3-chimeric与4D3-humanized。添加含生物素标记抗体与竞争抗体的溶液于A1～A12，每孔150ul，后吸出50ul添加至B2～B12中，与预先添加的体积为100ul之生物素抗体溶液充分混合，再依次稀释3倍至H1～H12，37℃孵育1hr，后洗涤并孵育anti-human IgG Fc二抗，37℃孵育1hr后洗涤3次，进行25min时长的显色并读值。
附图12：4D3-humanized-biotin抗体分别与0.02～50ug/ml的ch4D3与hum4D3抗体相竞争。两种竞争抗体表现同等程度竞争活性，结合表位一致。EC50(ch4D3)：0.336ug/ml。EC50(hum4D3)：0.326ug/ml
实施例20 7F11人源化抗体与人源化抗体与嵌合抗体的活性分析及结合表位一致性分析
参照实施例18中的操作流程，分析7F11的表位竞争活性及结合表位一致性。
附图13：7F11-humanized-biotin抗体分别与0.02～50ug/ml的ch7F11与hum7F11抗体相竞 争。两种竞争抗体表现同等程度竞争活性，结合表位一致。EC50(ch7F11)：0.732ug/ml。EC50(hum7F11)：0.856ug/ml。
实施例21人源化抗体hu7F11&hu4D3的细胞结合和内吞实验
使用HEK293细胞作为阴性细胞，BXPC-3和MCF-7细胞作为阳性细胞，测试梯度浓度下的各个抗体结合与内吞情况。细胞结合测试在4℃下结合1hr，后添加常规FITC标记的荧光二抗，流式收集数据。
内吞测试使用标记有酸敏感型的小分子染料Phrodo-Red羊抗人二抗，先与各个浓度的一抗共孵育形成复合物后，再与各细胞系共孵育16hr。后取样，每个96孔板的细胞孔铺设至少10000个细胞，采用流式细胞仪收集并分析数据。统计各个浓度下的细胞在远红光通道中的平均荧光强度值，以抗体浓度为横坐标描绘内吞程度曲线。
hu7F11和Hu4D3抗体在3种细胞中的结合程度比较，BxPC-3细胞结合程度最大。hu7F11和Hu4D3抗体在3种细胞中的内吞程度比较。BxPC-3细胞内吞程度最大。
Figure PCTCN2021100903-appb-000001
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Figure PCTCN2021100903-appb-000009
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Claims (20)

  1. 一种抗TROP2的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其特征在于所述抗体由重链和轻链组成，其特征在于所述抗体由氨基酸序列SEQ ID No.13的重链和由氨基酸序列SEQ ID No.14轻链组成，或者是由氨基酸序列SEQ ID No.15的重链和由氨基酸序列SEQ ID No.16轻链组成。
  2. 权利要求1所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其重链衍生物序列包含SEQ ID No.17和SEQ ID No.18。
  3. 权利要求1所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其重链衍生物序列包含SEQ ID No.19和SEQ ID No.20。
  4. 权利要求1所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，与权利要求1中所述序列有95％以上同源性都应该理解为本发明的等同物，因此也是本发明的一部分。
  5. 权利要求1所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其所述的重链所包含的氨基酸分别由SEQ ID No.1、2和3的CDRH1、CDRH2和CDRH3组成，或者由SEQ ID No.4、5和6的CDRH1、CDRH2和CDRH3组成，所述的轻链所包含的氨基酸分别由SEQ ID No.7、8和9的CDRL1、CDRL2和CDRL3，或者由SEQ ID No.10、11和12的CDRL1、CDRL2和CDRL3组成。
  6. 任何一个抗体片段或者衍生物若含有至少一个CDR，且该CDR的序列在与序列SEQ ID No.1至SEQ ID No.12进行优化比对后，至少具有80％的同一性，或优选的85％、90％、95％或者98％同一性的，都应该理解为本发明的等同物，因此也是本发明的一部分。
  7. 权利要求1-3任一项所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其特征在于其是单克隆抗体或者其衍生物。
  8. 权利要求1所述抗体的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其特征在于是鼠源的。
  9. 权利要求2、3所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一，其特征在于其为权利要求1所述序列的人源化序列之一。
  10. 权利要求1所述抗体的编码核酸，其特征在于所述核酸编码如权利要求1-3所述的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一的核酸。
  11. 根据权利要求9所分离的核酸，其特征在于所述核酸包含重链的序列SEQ ID No.21、22、23或者SEQ ID No.24、25、26，所述核酸包含轻链的序列SEQ ID No.27、28、29或者SEQ ID No.30、31、32。
  12. 根据权利要求10所述的核酸，其特征在于所述核酸包含SEQ ID No.33和SEQ ID No.34,或者SEQ ID No.35和SEQ ID No.36。
  13. 根据权利要求10所述的核酸，其特征在于所述核酸包含SEQ ID No.37和SEQ ID No.38,或者SEQ ID No.39和SEQ ID No.34。
  14. 与权利要求10-12中限定的核酸相对应的RNA核酸，或者与权利要求10-12中限定的核酸互补的核酸。
  15. 一种包含权利要求10-12任一项所述核酸的表达载体。
  16. 一种与权利要求14相关的表达宿主。
  17. 权利要求14所述的表达载体与权利要求15所述的表达宿主，可以产生如权利要求1-3所述的氨基酸序列。
  18. 权利要求1-6所述的、或通过权利要求16所述方法获得的的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一用于诊断或治疗TROP2靶点相关恶性肿瘤。
  19. 权利要求17所述的用途的抗体或者其Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段之一偶联相关细胞毒性化合物或者放射性元素。
  20. 权利要求18所述的细胞毒性化合物选自烷化剂、抗代谢物、抗肿瘤药物、有丝分裂抑制剂、染色质功能抑制剂、抗血管生成剂、抗雄激素、抗雌激素或者免疫调节剂等。
PCT/CN2021/100903 2020-06-22 2021-06-18 一种抗trop2抗体 Ceased WO2021259162A1 (zh)

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KR1020237001824A KR20230026431A (ko) 2020-06-22 2021-06-18 항-trop2 항체
CA3188014A CA3188014A1 (en) 2020-06-22 2021-06-18 Anti-trop2 antibody
AU2021296205A AU2021296205B2 (en) 2020-06-22 2021-06-18 Anti-TROP2 antibody
IL299267A IL299267A (en) 2020-06-22 2021-06-18 Anti-trop2 antibody
EP21828738.1A EP4169952A4 (en) 2020-06-22 2021-06-18 Anti-trop2 antibody
US18/011,175 US20230242665A1 (en) 2020-06-22 2021-06-18 Anti-trop2 Antibody
BR112022025947A BR112022025947A2 (pt) 2020-06-22 2021-06-18 Anticorpo anti-trop2, sequência de ácidos nucleicos, rna, vetor de expressão, hospedeiro, conjugado imune e usos do anticorpo
MX2022016300A MX2022016300A (es) 2020-06-22 2021-06-18 Anticuerpo anti-trop2.
JP2022579053A JP7586942B2 (ja) 2020-06-22 2021-06-18 抗ｔｒｏｐ２抗体
JP2024195200A JP2025028871A (ja) 2020-06-22 2024-11-07 抗ｔｒｏｐ２抗体

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TWI903241B (zh) 2022-09-30 2025-11-01 美商西雅圖免疫公司 一種帶有高穩定性親水連接單元的奧瑞他汀類藥物及其偶聯物

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