WO2020248938A1 - 抗cd25抗体及其应用 - Google Patents

抗cd25抗体及其应用 Download PDF

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WO2020248938A1
WO2020248938A1 PCT/CN2020/094919 CN2020094919W WO2020248938A1 WO 2020248938 A1 WO2020248938 A1 WO 2020248938A1 CN 2020094919 W CN2020094919 W CN 2020094919W WO 2020248938 A1 WO2020248938 A1 WO 2020248938A1
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Prior art keywords
antibody
antigen
binding fragment
igg1
cancer
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PCT/CN2020/094919
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English (en)
French (fr)
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宋德勇
刘秀
韩静
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Shandong Boan Biotechnology Co Ltd
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Shandong Boan Biotechnology Co Ltd
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Priority to AU2020293160A priority Critical patent/AU2020293160B2/en
Priority to US17/601,321 priority patent/US12325751B2/en
Priority to BR112021024826A priority patent/BR112021024826A2/pt
Priority to ES20822215T priority patent/ES3037019T3/es
Priority to KR1020217040156A priority patent/KR102789146B1/ko
Priority to CN202080000963.5A priority patent/CN113924316B/zh
Priority to PL20822215.8T priority patent/PL3932949T3/pl
Priority to CA3135992A priority patent/CA3135992C/en
Priority to JP2021560469A priority patent/JP7245358B2/ja
Priority to EP20822215.8A priority patent/EP3932949B1/en
Publication of WO2020248938A1 publication Critical patent/WO2020248938A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/50—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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Definitions

  • the invention relates to the field of biomedicine, in particular to an antibody that binds to CD25 and its application.
  • Treg Regulatory T cells
  • Treg tumor invasion is also related to several human cancers with poor prognosis (Shang B et al., 2015, Sci Rep. 5: 15179). It has been shown that Treg cells contribute to the establishment and progression of tumors in mouse models and their absence leads to delayed tumor progression. In humans, a high proportion of tumor Treg cells infiltrate, and more importantly, the low ratio of effector T cells (Teff) to Treg cells, which is related to the poor prognosis of many human cancers (Shang et al., 2015).
  • CD25 is one of the potential molecular targets to achieve Treg depletion.
  • CD25 is also known as interleukin-2 high affinity receptor alpha chain (IL-2R ⁇ ).
  • IL-2R ⁇ interleukin-2 high affinity receptor alpha chain
  • CD25 is expressed at high levels on Treg, but CD25 is absent or expressed at low levels on Teff.
  • VL light chain variable region
  • VH heavy chain variable region
  • LCDR light chain complementarity determining region
  • HCDR heavy chain complementarity determining region
  • LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 The various embodiments of HCDR3 and HCDR3 can be implemented individually or in any combination.
  • the present invention relates to an antibody or antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions, wherein the HCDR1 amino acid sequence is shown in SEQ ID NO: 14, and the HCDR2 amino acid sequence is shown in SEQ ID NO: 15
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 13 is shown.
  • the antibody or antigen-binding fragment thereof provided by the present invention includes the heavy chain variable region shown in SEQ ID NO: 4; preferably, it also contains the light chain variable region shown in SEQ ID NO: 3.
  • the present invention relates to an antibody or antigen-binding fragment thereof, comprising three light chain complementarity determining regions, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 6
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 7; and/or three heavy chain complementarity determining regions, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 8, and the amino acid sequence of HCDR2 is shown in SEQ ID NO: 9, HCDR3
  • the amino acid sequence is shown in SEQ ID NO: 10.
  • the present invention relates to an antibody or an antigen-binding fragment thereof comprising the light chain variable region of the amino acid sequence shown in SEQ ID NO: 1, and/or the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the light chain constant region of the antibody or antigen-binding fragment thereof in any of the foregoing aspects is SEQ ID NO: 20.
  • sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof in any one of the foregoing aspects is SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof provided by the present invention preferably comprises the light chain variable region of the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO The light chain constant region of the amino acid sequence shown in: 20 and the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof provided by the present invention preferably comprises the light chain variable region of the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: The light chain constant region of the amino acid sequence shown in: 20 and the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof of the present invention binds to CD25, preferably to human CD25.
  • the present invention relates to the antibody or antigen-binding fragment thereof according to any one of the foregoing aspects, including monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab' )2, Fv, scFv or dsFv fragments, etc.
  • the present invention relates to a nucleic acid that encodes the antibody or antigen-binding fragment thereof according to any one of the foregoing aspects.
  • the present invention relates to a vector comprising the nucleic acid described in the previous aspect, or the antibody or antigen-binding fragment thereof described in any one of the foregoing aspects can be expressed.
  • the vector can be a viral vector; preferably, the viral vector includes, but is not limited to, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector; preferably, the vector can be a non-viral vector Vector;
  • the vector can be a mammalian expression vector; preferably, the expression vector can be a bacterial expression vector; preferably, the expression vector can be a fungal expression vector.
  • the present invention relates to a cell that can express the antibody or antigen-binding fragment thereof according to any one of the foregoing aspects.
  • the cell is a bacterial cell; preferably, the bacterial cell is an E. coli cell, etc.; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast
  • the cells are Pichia pastoris cells, etc.; preferably, the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells, etc.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid, carrier or cell according to any one of the preceding aspects.
  • the pharmaceutical composition further comprises a pharmaceutical Acceptable excipients, preferably, the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, plasticizers and the like.
  • the pharmaceutical composition may also include other therapeutic agents.
  • other therapeutic agents include chemotherapeutic agents, immunotherapeutic agents, or hormone therapy agents. The combined administration of the antibody or antigen-binding fragment thereof and other therapeutic agents can enhance the therapeutic effects of these therapeutic agents.
  • the "enhancement of the therapeutic effect” refers to enhancing the therapeutic effect of other therapeutic agents or therapies.
  • the antibody or antigen-binding fragment provided by the present invention can be administered alone or in combination with other therapeutic agents or therapies.
  • other therapeutic agents or therapies include chemotherapeutic agents, immunotherapeutic agents, hormone therapy agents, radiotherapy, surgical treatment, and the like.
  • kits comprising the antibody or antigen-binding fragment thereof of the present invention, or a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the present invention relates to the application of the antibody or antigen-binding fragment, nucleic acid, carrier or cell of any one of the foregoing aspects in the preparation of drugs for treating or preventing diseases.
  • the present invention relates to the use of the antibody or antigen-binding fragment or nucleic acid of any one of the foregoing aspects in the preparation of a diagnostic or detection kit.
  • the present invention provides a method for treating or preventing diseases, comprising administering the antibody or antigen-binding fragment, nucleic acid, vector, cell or pharmaceutical composition of the present invention to a subject in need.
  • the present invention provides a method of diagnosis or detection, which includes administering the antibody or antigen-binding fragment, nucleic acid or kit of the present invention to a subject or sample in need.
  • the method is a method of diagnosing or detecting a disease.
  • the present invention relates to the use of the antibody or antigen-binding fragment, nucleic acid, carrier, cell or pharmaceutical composition of any one of the foregoing aspects for the treatment or prevention of diseases.
  • the present invention relates to the use of the antibody or antigen-binding fragment, nucleic acid or kit of any one of the foregoing aspects for detection or diagnosis.
  • the use is for diagnosing or detecting diseases.
  • the disease is cancer.
  • the cancer includes gastric cancer, esophageal cancer, head and neck cancer, bladder cancer, cervical cancer, sarcoma, cell tumor, lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer , Melanoma, breast cancer, myeloma, glioma, leukemia and lymphoma.
  • the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the foregoing aspects, comprising transfecting cells with the above-mentioned vector, and expressing the antibody or antigen-binding fragment by the transfected cells ; Or include using the above-mentioned cells to express the antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof provided by the present invention has one or more of the following advantages: enhanced CD25 protein binding ability, enhanced CD25 protein affinity, enhanced CD25 expressing cell killing ability, weakened PBMC activation inhibition, enhanced tumor growth inhibition ability in vivo, enhanced tumor killing ability in vivo, enhanced ability to reduce the number of Treg cells, or enhanced ability to increase the number of effector T cells.
  • Figure 1 shows the serum titers of BoAn-hMab1 mice after seven immunization in the immunization protocol in Example 1 (62500 dilution);
  • 2A-2E show the sensitivity of ELSIA to detect the binding of CD25 antibody to CD25 in Example 3;
  • 3A-3B show the test results of the simulated killing activity of the candidate antibody in Example 3.
  • Figure 4 shows the detection results of candidate antibody cell blocking activity in Example 3.
  • Figure 5 shows the effect of CD25Q2-BA9-IgG1 in Example 4 on reducing the content of rhesus monkey Treg cells
  • Figure 6 shows the drug metabolism of the candidate antibody in Example 4 in cynomolgus monkeys
  • Figures 7A-7B show the results of the efficacy of the candidate antibody in Example 5.1 in the B-hIL2R ⁇ humanized mouse MC38 colon cancer animal model, where Figure 7A shows the weight data of the MC38 tumor model mouse, and Figure 7B shows The tumor volume data of the MC38 tumor model is presented.
  • Figure 8A shows the content of CD8+ cells (Teff) in CD3 in Example 5.2.
  • Figure 8B shows the content of CD25+Foxp3+ cells (Treg) in CD3 in Example 5.2.
  • Figure 8C shows the content of Foxp3+ cells (Treg) in CD4 in Example 5.2.
  • the scFv is digested with the plasmid pCOMB3x, and then the ligation product is electrotransfected into the large intestine
  • TG1 is cultured and then infected with phage, and the culture supernatant is concentrated to become the phage library of the present invention.
  • CD25 protein (Yiqiao Shenzhou, 10165-H08H) coated the plate at 1 ⁇ g/well, placed overnight at 4°C, blocked the plate with 2% BSA the next day for 1h, and added the phage library (2 ⁇ 10 12 ) Incubate for 2h, wash 4-10 times and use elution buffer (add 4.2ml concentrated hydrochloric acid (Komeo) to 500ml ultrapure water, adjust pH to 2.2 with glycine powder (Biotopped, BG0617-500)) or 15 ⁇ g/ mL MA251 elutes phages that specifically bind to CD25.
  • elution buffer add 4.2ml concentrated hydrochloric acid (Komeo) to 500ml ultrapure water, adjust pH to 2.2 with glycine powder (Biotopped, BG0617-500)
  • MA251 elutes phages that specifically bind to CD25.
  • CD25-Fc protein (Yiqiao Shenzhou, 10165-H02H) is biotinylated according to conventional procedures, and then combined with Thermo magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, 00355871) and incubated with the phage library After washing 4-10 times, use elution buffer (pH 2.2) or 15 ⁇ g/mL MA251 to elute phages that specifically bind to CD25.
  • elution buffer pH 2.2
  • MA251 15 ⁇ g/mL MA251
  • the selected phage clones express scFv, detect the binding of scFv to CD25, and detect the blocking of IL2/CD25 binding by scFv, and select scFv that binds well to CD25 and does not block for subsequent construction.
  • HRP-66008 anti-flag secondary antibody
  • ELISA detection of scFv blocking CD25/IL2 binding Dilute CD25 (10165-H08H, Yiqiao Shenzhou) protein to 0.5 ⁇ g/mL with pH9.6 CBS, coat the microtiter plate, 100 ⁇ L/well, incubate overnight at 4°C; wash After the plate, it was sealed with 3% skimmed milk powder at 37°C for 1 hour. After washing the plate, add 50 ⁇ L of scFv periplasm to each well.
  • biotin-labeled IL2 protein final concentration 0.02 ⁇ g/mL
  • 50 ⁇ L/well 50 ⁇ L/well
  • PBST diluted STREP/HRP 100 ⁇ L/well
  • incubate at 37°C for 1h After washing the plate, add 100 ⁇ L of TMB to each well for color development.
  • 50 ⁇ L of 2M H 2 SO 4 50 ⁇ L of 2M H 2 SO 4 to each well to stop the color development, and read the OD450 with a microplate reader.
  • MA251 antibody is an anti-human CD25 antibody that does not block the binding of IL2 and CD25 in the prior art. It has a high affinity for human CD25 and has a good performance of not blocking the binding of IL2 and CD25.
  • MA251 antibody is a classic antibody that studies the binding of IL2 to CD25.
  • the nucleotide sequence encoding the variable region of the MA251 antibody was fully synthesized and inserted into the vector pCDNA3.4 and expressed by HEK293 cells.
  • the produced antibody was named CD25-MA251-IgG1 (the heavy chain variable region sequence is SEQ ID NO: 18.
  • the light chain variable region sequence is SEQ ID NO: 19
  • the light chain constant region sequence is SEQ ID NO: 20
  • the heavy chain constant region sequence is SEQ ID NO: 17).
  • the candidate antibody CD25Q2-BA9-IgG1 and CD25 antigen binding EC 50 value is 3.328, which is significantly lower than the control group CD25-MA251-IgG1 EC 50 value 10.63, indicating that the antigen binding ability of the candidate antibody is significantly better than the control Group CD25-MA251-IgG1.
  • the EC 50 value of the candidate antibody CD25Q8-BT942-IgG1 binding to CD25 antigen is 2.26, which is significantly lower than the EC 50 value of 24.5 of the control group CD25-MA251-IgG1, indicating that the antigen binding ability of the candidate antibody is better than the control group CD25-MA251-IgG1.
  • candidate antibodies CD25Q2-BA9-IgG1, CD25Q8-BT942-IgG1 have stronger targeting and binding to Treg cells expressing CD25 than the control group CD25-MA251-IgG1, have better killing effect, and reduce Treg cells Inhibition of Teff cells has a better pharmaceutical effect.
  • Antibody name EC 50 (ng/mL) CD25Q2-BA3-IgG1 7.375 CD25Q5-BA303-IgG1 1.202 CD25Q2-BA9-IgG1 3.328 CD25Q11-BA402-IgG1 0.6743 CD25Q3-BA125-IgG1 1.608 CD25-MA251-IgG1 10.63
  • Antibody name EC 50 (ng/mL)
  • Antibody name EC 50 (ng/mL)
  • CD25Q11-BA406-IgG1 0.6515
  • CD25Q11-BA415-IgG1 0.9405
  • CD25Q11-BA410-IgG1 16.82
  • CD25Q14-CA705-IgG1 4.773
  • CD25Q11-CA36-IgG1 2.41 CD25-MA251-IgG1 14.06
  • Antibody name EC 50 (ng/mL) CD25Q11-BA422-IgG1 1.304 CD25Q14-BA448-IgG1 2.684 CD25Q11-BA428-IgG1 0.5974 CD25Q14-BA458-IgG1 1.003 CD25Q14-BA443-IgG1 1.06 CD25-MA251-IgG1 16.98
  • Antibody name EC 50 (ng/mL)
  • Antibody name EC 50 (ng/mL)
  • CD25Q14-CA707-IgG1 6.415 CD25-MA251-IgG1 12.09 CD25Q14-CA721-IgG1 3.252 / /
  • CD25Q11-CT848-IgG1 9.162
  • CD25-MA251-IgG1 24.5
  • CD25Q8-BT942-IgG1 2.26 / /
  • FBS (Gibco, catalog number: 10091-148) and RPMI-1640 (Gibco, catalog number: 11875-093) were mixed according to 1:99 to prepare 1% FBS RPMI-1640, and SU-DHL-1 target cells were collected and used 1% FBS RPMI-1640 is diluted to 1.2 ⁇ 10 6 cells/mL, and the appropriate candidate antibody is diluted with 1% FBS RPMI-1640 to 25 ⁇ g/mL, and this concentration is used as the starting concentration; a total of 8 points are sequentially diluted by 4 times , For subsequent use; collect effector cells Jurkat (G7011, Promega), dilute with 1% FBS RPMI-1640 to 2.4 ⁇ 10 6 cells/mL, add 25 ⁇ L/well of target cells to a white 96-well plate; Add 25 ⁇ L/well of serially diluted antibody to the wells; add 25 ⁇ L/well of effector cells Jurkat, put the 96-well plate in the cell incubator for 5h; take out the 96-
  • Antibody name EC 50 ( ⁇ g/mL)
  • Antibody name EC 50 ( ⁇ g/mL)
  • CD25-MA251-IgG1 0.3606 CD25Q11-BA410-IgG1 0.2149 / /
  • Antibody name EC 50 ( ⁇ g/mL)
  • Antibody name EC 50 ( ⁇ g/mL)
  • CD25Q14-BA443-IgG1 0.3604 CD25Q11-BA35-IgG1 0.5040
  • CD25Q14-CA707-IgG1 0.2476 CD25-MA251-IgG1 0.2546 CD25Q14-CA721-IgG1 0.2879 / /
  • candidate antibodies CD25Q2-BA9-IgG1 anti analog detection activity EC 50 value of 0.2356, lower than the control group CD25-MA251-IgG1 EC 50 value of 0.3606, indicating that the candidate antibody SU-DHL-1 cytolytic activity Better than the control group CD25-MA251-IgG1.
  • the antibody binding kinetics was measured using a BIAcore8K instrument based on surface plasmon resonance (SRP) technology.
  • SRP surface plasmon resonance
  • GE anti Human IgG Fc amino coupling kit GE, cat#BR-1008-39
  • the anti-human IgG antibody amino group was coupled to the CM5 biosensor chip to obtain approximately 1000 response units (RU) .
  • the CD25 protein (Yiqiao Shenzhou, 10165-H08H) was serially diluted 2 times with HBS-EP+1 ⁇ (GE, BR-1008-26) buffer, starting at 50 nM, and 2 times diluted 4 concentrations Gradient and set 0 concentration.
  • Antibody to be tested 2 ⁇ g/mL, injection time 70s, flow rate 5 ⁇ L/min, stable for 5s; CD25 protein: binding 60s, flow rate 30 ⁇ L/min, dissociation 450s; Regeneration: Regenerate with 3M MgCl 2 buffer for 30s, Startup 3 times.
  • a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2) was used to calculate the binding constant (ka) and dissociation constant (kd), and the equilibrium dissociation constant (KD) was calculated as the ratio kd/ka. See Table 9 for the affinity data of each antibody.
  • the equilibrium dissociation constant KD value of the candidate antibody CD25Q2-BA9-IgG1 is 8.43E-10 lower than the KD value of the control CD25-MA251-IgG1 5.19E-09, indicating that the candidate antibody CD25Q2-BA9-IgG1
  • the affinity of CD25 protein is better than that of the control group CD25-MA251-IgG1.
  • the equilibrium dissociation constant KD value of the candidate antibody CD25Q8-BT942-IgG1 is 3.79E-09 lower than the KD value of the control CD25-MA251-IgG1 5.19E-09, indicating that the candidate antibody CD25Q8-BT942-IgG1
  • the affinity of CD25 protein is better than that of the control group CD25-MA251-IgG1.
  • PBMC peripheral blood mononuclear cells
  • ALLCELLS manufacturer: ALLCELLS, article number: PB003F-C
  • PB003F-C peripheral blood mononuclear cells
  • No antibody was added to the control group, labeled as NoAb, and then added IL2 (0.1U/mL, 1U/mL, 10U/mL) culture for 10 min (working medium: 1640+10% FBS, containing 2mM L-glutamine and 10000U/mL Pen-Strep), prepare cell suspension: finally After one wash, discard the supernatant and pulse vortex the sample to completely dissociate the precipitate; add 200 ⁇ L Foxp3 fixation/breaking membrane working solution to each well.
  • IL2 0.1U/mL, 1U/mL, 10U/mL
  • working medium 1640+10% FBS, containing 2mM L-glutamine and 10000U/mL Pen-Strep
  • the %PSTAT5 value of the candidate antibody CD25Q2-BA9-IgG1 is 26.09, which is significantly higher than the %PSTAT5 value of 18.52 of the control group CD25-MA251-IgG1, indicating that the candidate antibody does not block IL2 binding to PBMC.
  • This indicates that the candidate antibody CD25Q2-BA9-IgG1 can inhibit the activation of PBMC less than the control group CD25-MA251-IgG1, indicating that the candidate antibody CD25Q2-BA9-IgG1 can better exert the PBMC immune effect and have better pharmaceutical effects/ Anti-tumor effect.
  • the %PSTAT5 value of the candidate antibody CD25Q8-BT942-IgG1 is 16.46
  • the %PSTAT5 value of CD25-MA251-IgG1 is 18.52, indicating that the candidate antibody does not block the binding of IL2 to PBMC.
  • MA251-IgG1 is basically equivalent. It indicates that the candidate antibody CD25Q8-BT942-IgG1 can play a good PBMC immune effect, and has a good pharmaceutical effect/tumor inhibitory effect.
  • the CD25Q2-BA9-IgG1 antibody was administered intravenously to 3 rhesus monkeys at a dose of 10 mg/kg.
  • Flow cytometry (CytomicsTM FC500) was used to detect Treg cells (CD3+) at different time points before and after the administration.
  • the content of CD4+CD25+FoxP3+), the detection time points are: the end of the administration ( ⁇ 1 minute) before the administration, 0.5 hour ( ⁇ 1 minute), 3 hours ( ⁇ 2 minutes), 6 hours ( ⁇ 5 minutes), 24 hours ( ⁇ 10 minutes), 48 hours ( ⁇ 20 minutes, 2 days), 96 hours ( ⁇ 30 minutes, 4 days), 168 hours ( ⁇ 1 hour, 7 days), 336 hours ( ⁇ 1 Hours, 14 days), the experimental results are shown in Figure 5.
  • CD25Q2-BA9-IgG1 can significantly reduce the content of Treg cells in rhesus monkeys and can effectively regulate the immune microenvironment.
  • CD25Q2-BA9-IgG1, CD25Q8-BT942-IgG1, CD25Q11-CT848-IgG1 can significantly inhibit the growth of mouse MC38 tumors, and CD25Q8-BT942-IgG1 shows better The anti-tumor effect.
  • mice were taken from the negative control group and 3 mice from each group of the treatment group. The mice were killed and the tumors were cut into pieces and added to digest Enzyme, 37°C, incubate and digest for 40 min, filter, wash, and resuspend into a single cell suspension.
  • CD25Q2-BA9-IgG1, CD25Q8-BT942-IgG1, CD25Q11-CT848-IgG1 can reduce the proportion of Treg cells in mouse MC38 tumors and increase Infiltration of Teff cells, thereby enhancing tumor killing ability.

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Abstract

本发明提供一种结合CD25的抗体或其抗原结合片段及其在制备癌症治疗药物中的用途，该抗体或其抗原结合片段包含含有特定互补决定区序列的轻链可变区和重链可变区。该抗体或抗原结合片段具有以下一种或多种优势：增强的CD25蛋白结合能力、增强的CD25蛋白亲和力、增强的CD25表达细胞杀伤能力、减弱的PBMC的激活抑制性、增强的体内肿瘤生长抑制能力、增强的体内肿瘤杀伤能力、增强的减少Treg细胞数量的能力，或增强的增加效应T细胞数量的能力。

Description

抗CD25抗体及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种结合CD25的抗体及其应用。
背景技术
调节性T细胞(Treg)在介导免疫稳态中起着至关重要的作用，可以促进外周耐受的建立和维持。但是，在癌症的背景下他们的角色更复杂。由于癌细胞表达自身和肿瘤相关的抗原，因此抑制效应细胞反应的Treg的存在可以促进肿瘤进展。因此，已建立的肿瘤中Treg的浸润是有效的主要障碍之一。使用抑制机制的Treg被认为对当前疗法的局限性甚至失败有重大贡献，特别是依赖诱导或增强抗肿瘤反应的免疫疗法(Onishi H et al,2012 Anticanc.Res.32,997-1003)。Treg的肿瘤浸润也与几种预后较差的人类癌症有关(Shang B et al.,2015,Sci Rep.5:15179)。已经证明Treg细胞有助于小鼠模型中肿瘤的建立和进展以及它们的缺失导致肿瘤进展延迟。在人类中，高比例肿瘤Treg细胞浸润，更重要的是效应T细胞(Teff)与Treg细胞的比例较低，与多种人类癌症的不良预后相关(Shang et al.,2015)。
CD25是实现Treg消耗的潜在分子靶标之一。CD25也称为白细胞介素-2高亲和力受体α链(IL-2Rα)，CD25在Treg上高水平表达，但CD25在Teff上不存在或以低水平表达。
现有技术中存在结合CD25但不阻断IL2与CD25结合的抗体，如MA251(Rubin et al,1985,Hybridoma 4(2)91-102,Tanaka et al,1986,Microbiol.Immunol 30(4),373-388)，但其仍然存在CD25结合活性不够高、抑制PBMC激活、药代动力学表现一般等缺陷。
面对患者对于疾病治疗的药物需求，尤其是抗体药物的需求，临床上仍然亟待提供一种结合活性更高的抗CD25抗体。
发明内容
本发明全文中关于VL(轻链可变区)、VH(重链可变区)、LCDR(轻链互补决定区)、HCDR(重链互补决定区)、LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3的各个实施方式可以各自单独实施，也可以任意组合实施。
在本发明的一方面，本发明涉及抗体或其抗原结合片段，包含3个重链互补决定区，其中HCDR1氨基酸序列如SEQ ID NO:14所示，HCDR2氨基酸序列如SEQ ID NO:15所示，HCDR3氨基酸序列如SEQ ID NO:16所示。进一步地，所述抗体或其抗原结合片段还包含3个轻链互补决定区，其中LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:12所示，LCDR3氨基酸序列如SEQ ID NO:13所示。
在本发明的一方面，本发明提供的抗体或其抗原结合片段包含SEQ ID NO:4所示的重链可变区；优选还含有SEQ ID NO:3所示的轻链可变区。
在本发明的一方面，本发明涉及抗体或其抗原结合片段，包含3个轻链互补决定区，其中LCDR1氨基酸序列如SEQ ID NO:5所示，LCDR2氨基酸序列如SEQ ID NO:6所示，LCDR3氨基酸序列如SEQ ID NO:7所示；和/或3个重链互补决定区，其中HCDR1氨基酸序列如SEQ ID NO:8所示，HCDR2氨基酸序列如SEQ ID NO:9所示，HCDR3氨基酸序列如SEQ ID NO:10所示。
在另一方面，本发明涉及抗体或其抗原结合片段包含SEQ ID NO:1所示氨基酸序列的 轻链可变区，和/或SEQ ID NO:2所示氨基酸序列的重链可变区。
根据本发明的一方面，前述任一方面所述抗体或其抗原结合片段的轻链恒定区的序列为SEQ ID NO:20。
根据本发明的一方面，前述任一方面所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:17。
具体地，本发明提供的抗体或其抗原结合片段优选包含SEQ ID NO:3所示氨基酸序列的轻链可变区，SEQ ID NO:4所示氨基酸序列的重链可变区，SEQ ID NO:20所示氨基酸序列的轻链恒定区和SEQ ID NO:17所示氨基酸序列的重链恒定区。
具体地，本发明提供的抗体或其抗原结合片段优选包含SEQ ID NO:1所示氨基酸序列的轻链可变区，SEQ ID NO:2所示氨基酸序列的重链可变区，SEQ ID NO:20所示氨基酸序列的轻链恒定区和SEQ ID NO:17所示氨基酸序列的重链恒定区。
根据本发明的一方面，本发明的抗体或其抗原结合片段结合CD25，优选结合人CD25。
根据本发明的一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
根据本发明的一方面，本发明涉及一种核酸，其编码前述任一方面所述的抗体或其抗原结合片段。
根据本发明的一方面，本发明涉及一种载体，其包含前一方面所述的核酸，或其可表达前述任一方面所述的抗体或其抗原结合片段。优选地，所述载体可以是病毒载体；优选地，所述病毒载体包含但不限于慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体等；优选地，所述载体可以是非病毒载体；优选地，所述载体可以是哺乳细胞表达载体；优选地，所述表达载体可以是细菌表达载体；优选地，所述表达载体可以是真菌表达载体。
根据本发明的一方面，本发明涉及一种细胞，其可表达前述任一方面所述的抗体或其抗原结合片段的细胞。优选地，所述细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌细胞等；优选地，所述细胞为真菌细胞；优选地，所述真菌细胞为酵母细胞；优选地，所述酵母细胞为毕赤酵母细胞等；优选地，所述细胞为哺乳动物细胞；优选地，所述哺乳动物细胞为中国仓鼠卵巢细胞(CHO)、人胚胎肾细胞(293)、B细胞、T细胞、DC细胞或NK细胞等。
根据本发明的一方面，本发明涉及一种药物组合物，其包含前述任一方面所述的抗体或其抗原结合片段、核酸、载体或细胞，优选地，所述药物组合物还包含药学上可接受的赋形剂，优选地，所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂等。
在一些实施方式中，所述药物组合物还可以包含其他治疗剂。在一些实施方式中，其他治疗剂包括化疗剂、免疫治疗剂或激素治疗剂。所述抗体或其抗原结合片段与其他治疗剂的联合施用能够增强这些治疗剂的治疗效果。
在一些实施方式中，所述“增强治疗效果”是指增强其他治疗剂或疗法的治疗效果。本发明提供的所述抗体或抗原结合片段可以单独施用，也可以与其他治疗剂或疗法联合施用。在一些实施方式中，其他治疗剂或疗法包括化疗剂、免疫治疗剂、激素治疗剂、放射治疗、手术治疗等。
根据本发明的一方面，提供一种试剂盒，其包含本发明所述的抗体或其抗原结合片段，或包含编码抗体或其抗原结合片段的核酸。
根据本发明的一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸、载体或细胞在制备治疗或预防疾病的药物中的应用。
根据本发明的一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段或核酸在制备诊断或检测试剂盒中的应用。
根据本发明的一方面，本发明提供一种治疗或预防疾病的方法，包括将本发明的抗体或抗原结合片段、核酸、载体、细胞或药物组合物给予有需要的受试者。
根据本发明的一方面，本发明提供一种诊断或检测的方法，包括将本发明的抗体或抗原结合片段、核酸或试剂盒给予有需要的受试者或样本。优选地，所述方法是诊断或检测疾病的方法。
根据本发明的一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸、载体、细胞或药物组合物用于治疗或预防疾病的用途。
根据本发明的一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸或试剂盒用于检测或诊断的用途。优选地，所述用途是诊断或检测疾病的用途。
根据本发明的一方面，所述疾病是癌症。
根据本发明的一方面，所述癌症包括胃癌、食道癌、头颈癌、膀胱癌、宫颈癌、肉瘤、细胞瘤、肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤等。
根据本发明的一方面，本发明涉及制备前述任一方面所述的抗体或其抗原结合片段的方法，包括使用上述载体转染细胞，并由转染的细胞表达所述抗体或其抗原结合片段；或者包括用上述细胞表达所述抗体或其抗原结合片段。
根据本发明的一方面，本发明提供的抗体或其抗原结合片段具有以下中的一种或多种优势：增强的CD25蛋白结合能力、增强的CD25蛋白亲和力、增强的CD25表达细胞杀伤能力、减弱的PBMC的激活抑制性、增强的体内肿瘤生长抑制能力、增强的体内肿瘤杀伤能力、增强的减少Treg细胞数量的能力，或增强的增加效应T细胞数量的能力。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1示出实施例1中免疫方案的BoAn-hMab1小鼠七免后血清滴度(62500倍稀释)；
图2A-图2E示出实施例3中ELSIA检测CD25抗体与CD25的结合灵敏度；
图3A-图3B示出实施例3中候选抗体模拟杀伤活性检测结果；
图4示出实施例3中候选抗体细胞阻断活性检测结果；
图5示出实施例4中CD25Q2-BA9-IgG1对降低恒河猴Treg细胞含量的作用；
图6示出实施例4中候选抗体在食蟹猴体内的药物代谢情况；
图7A-图7B示出实施例5.1中候选抗体在B-hIL2Rα人源化小鼠MC38结肠癌动物模型中的药效结果，其中，图7A示出MC38肿瘤模型小鼠体重数据，图7B示出MC38肿瘤模型肿瘤体积数据。
图8A示出实施例5.2中CD3中的CD8+细胞(Teff)含量。
图8B示出实施例5.2中CD3中的CD25+Foxp3+细胞(Treg)含量。
图8C示出实施例5.2中CD4中的Foxp3+细胞(Treg)含量。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明全文所描述的不同实施方式中所涉及的技术特征均可以相互组合实施。
实施例1 抗CD25单克隆抗体的产生
1.1 免疫方案
用CD25(义翘神州，目录号10165-H08H)与弗氏佐剂乳化后免疫博安生物全人抗体转基因小鼠BoAn-hMab1(按照中国专利CN103571872B记载的方法制备)。首免使用弗氏完全佐剂(Sigma，目录号：F5881-10ML)，二免至六免使用弗氏不完全佐剂(Sigma，目录号：F5506-10ML)，本次共免疫14只小鼠。选取血清滴度较高的7只小鼠加强免疫，3天后处死小鼠取出脾脏用于后续实验。小鼠血清滴度(62500倍稀释)见图1。
1.2 噬菌体库的建立
取1.1中所述免疫小鼠的脾脏细胞，提取RNA后反转录获得cDNA，噬菌体库的建立步骤参照Carlos F.BarbasⅢ,Phage display：A laboratory manual中记载的方法进行，用PCR的方法从cDNA中获得重链和轻链的可变区，再将重链和轻链的可变区通过重叠延伸PCR的方法获得scFv，scFv酶切后与质粒pCOMB3x连接，然后将连接产物电转染至大肠杆菌TG1感受态细胞中，TG1经培养后加入噬菌体侵染，培养物上清浓缩后即为本发明的噬菌体库。
1.3 噬菌体库筛选(两种方法)
(1)平板筛选：CD25蛋白(义翘神州，10165-H08H)以1μg/孔包被平板，4℃放置过夜，第二天通过2％BSA封闭平板1h，加入噬菌体库(2×10 12)孵育2h，洗涤4-10次后用洗脱缓冲液(向500ml超纯水中加入4.2ml浓盐酸(科密欧)，用甘氨酸粉末(Biotopped，BG0617-500)调节pH至2.2)或15μg/mL MA251洗脱与CD25特异性结合的噬菌体。
(2)磁珠筛选：CD25-Fc蛋白(义翘神州，10165-H02H)按照常规步骤进行生物素化，再与Thermo的磁珠(Invitrogen Dynabeads M-280 Streptavidin，00355871)结合后与噬菌体库孵育，洗涤4-10次后用洗脱缓冲液(pH 2.2)或15μg/mL MA251洗脱与CD25特异性结合的噬菌体。
筛选的噬菌体克隆表达scFv，检测scFv与CD25的结合，并检测scFv对IL2/CD25结合的阻断，选择与CD25结合好并且不阻断的scFv进行后续构建。
ELISA检测scFv与CD25的结合：CBS缓冲液的制备：称取Na 2CO 3(国药，10019260)1.59g、NaHCO 3 2.93g加蒸馏水至1L配制成CBS缓冲液。用pH 9.6 CBS稀释CD25(10165-H08H，义翘神州)蛋白至0.2μg/mL，包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉于37℃封闭1h；洗板后加入80μL PBST(PBS+0.05％Tween20)，然后加入20μL ScFv周质，37℃孵育1h。洗板后加入anti-flag二抗(Proteintech，目录号：HRP-66008)，37℃孵育1h。洗板后每孔加入100μL TMB(梅科万德，目录号1001)底物显色，10min后每孔加入50μL 2M H 2SO 4终止显色，用酶标仪读取OD450。
ELISA检测scFv阻断CD25/IL2的结合：用pH9.6 CBS稀释CD25(10165-H08H，义 翘神州)蛋白至0.5μg/mL，包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉于37℃封闭1h。洗板后每孔加入scFv周质50μL。然后加入生物素标记的IL2蛋白(终浓度0.02μg/mL)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H 2SO 4终止显色，用酶标仪读取OD450。
实施例2 候选抗体分子的构建与生产
将磁珠筛选的克隆CD25Q2-BA3\BA9\BA125、CD25Q8-BT942、CD25Q11-BA402\BA406\BA410\BA415\BA422\BA428、CD25Q14-BA443\BA448\BA458和平板筛选的克隆CD25Q11-CA35\CA36\CT848、CD25Q14-CA705\CA707\CA721送Invitrogen生物技术有限公司测序。各克隆的轻链可变区和重链可变区氨基酸序列如表1所示。
表1.克隆的轻链可变区和重链可变区的氨基酸序列
Figure PCTCN2020094919-appb-000001
Figure PCTCN2020094919-appb-000002
Figure PCTCN2020094919-appb-000003
通过常规的分子生物学技术进行可变区基因扩增(2*Phanta Max Master Mix厂家：Vazyme货号：P515-AA批号：TE211GB)、信号肽与可变区重叠延伸、同源重组(ClonExpress Ⅱ One Step Cloning Kit厂家：Vazyme货号：C112-01批号：TE211L8)等，最终把编码VH的核苷酸序列片段插入带有编码抗体重链恒定区氨基酸序列SEQ ID NO:17的核苷酸序列的载体pCDNA3.4(Life Technology)，把编码VL的核苷酸序列片段插入带有编码抗体轻链恒定区氨基酸序列(SEQ ID NO:20)的核苷酸序列的载体pCDNA3.4(Life Technology)，将连接好的载体转染进入HEK293细胞在37℃\8％CO 2\125rpm摇床中培养，瞬时表达6-7天后，上清通过Protein A亲和层析，纯化获得抗CD25抗体，并通过UV280结合消光系数确定抗体浓度。
对照抗体生产：MA251抗体是现有技术中不阻断IL2与CD25结合的抗人CD25抗体，对人CD25有较高亲和力，且具有良好的不阻断IL2与CD25结合的表现。MA251抗体是研究IL2与CD25结合的经典抗体。将编码MA251抗体可变区的核苷酸序列经全基因合成后插 入载体pCDNA3.4并通过HEK293细胞表达，生产的抗体命名为CD25-MA251-IgG1(重链可变区序列为SEQ ID NO:18，轻链可变区序列为SEQ ID NO:19，轻链恒定区序列为SEQ ID NO:20，重链恒定区序列为SEQ ID NO:17)。
实施例3 候选抗体的表征
3.1 ELISA检测候选抗体与CD25蛋白的结合活性
用CBS稀释CD25蛋白(10165-H08H，义翘神州)至不同浓度(0.08μg/mL、0.02μg/mL、0.005μg/mL、0.00125μg/mL、0.0003125μg/mL、0.000078125μg/mL)，包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉37℃封闭1h；每孔加入用PBST(PBS+0.05％Tween20)稀释至1μg/mL的候选抗体各100μL，37℃孵育1h；然后加入山羊抗人IgG/HRP(KPL，目录号：5450-0009)，37℃孵育1h，显色10min后，酶标仪上读取OD450，计算得到EC 50。结果见图2A-2E和表2-表6。
如表2所示，候选抗体CD25Q2-BA9-IgG1与CD25抗原结合EC 50值为3.328，显著低于对照组CD25-MA251-IgG1的EC 50值10.63，说明候选抗体的抗原结合能力显著优于对照组CD25-MA251-IgG1。
如表6所示，候选抗体CD25Q8-BT942-IgG1与CD25抗原结合EC 50值为2.26，显著低于对照组CD25-MA251-IgG1的EC 50值24.5，说明候选抗体的抗原结合能力优于对照组CD25-MA251-IgG1。
预示候选抗体CD25Q2-BA9-IgG1、CD25Q8-BT942-IgG1相较对照组CD25-MA251-IgG1对表达CD25的Treg细胞有更强的靶向性和结合性，有更好杀伤效果，减少Treg细胞对Teff细胞的抑制，有更好的药学效果。
表2.ELISA检测候选抗体与CD25蛋白的结合活性数据(对应图2A)
抗体名称 EC 50(ng/mL) 抗体名称 EC 50(ng/mL)
CD25Q2-BA3-IgG1 7.375 CD25Q5-BA303-IgG1 1.202
CD25Q2-BA9-IgG1 3.328 CD25Q11-BA402-IgG1 0.6743
CD25Q3-BA125-IgG1 1.608 CD25-MA251-IgG1 10.63
表3.ELISA检测候选抗体与CD25蛋白的结合活性数据(对应图2B)
抗体名称 EC 50(ng/mL) 抗体名称 EC 50(ng/mL)
CD25Q11-BA406-IgG1 0.6515 CD25Q11-BA415-IgG1 0.9405
CD25Q11-BA410-IgG1 16.82 CD25Q14-CA705-IgG1 4.773
CD25Q11-CA36-IgG1 2.41 CD25-MA251-IgG1 14.06
表4.ELISA检测候选抗体与CD25蛋白的结合活性数据(对应图2C)
抗体名称 EC 50(ng/mL) 抗体名称 EC 50(ng/mL)
CD25Q11-BA422-IgG1 1.304 CD25Q14-BA448-IgG1 2.684
CD25Q11-BA428-IgG1 0.5974 CD25Q14-BA458-IgG1 1.003
CD25Q14-BA443-IgG1 1.06 CD25-MA251-IgG1 16.98
表5.ELISA检测候选抗体与CD25蛋白的结合活性数据(对应图2D)
抗体名称 EC 50(ng/mL) 抗体名称 EC 50(ng/mL)
CD25Q14-CA707-IgG1 6.415 CD25-MA251-IgG1 12.09
CD25Q14-CA721-IgG1 3.252 / /
表6.ELISA检测候选抗体与CD25蛋白的结合活性数据(对应图2E)
抗体名称 EC 50(ng/mL) 抗体名称 EC 50(ng/mL)
CD25Q11-CT848-IgG1 9.162 CD25-MA251-IgG1 24.5
CD25Q8-BT942-IgG1 2.26 / /
3.2 候选抗体的模拟杀伤活性检测
用FBS(Gibco，目录号：10091-148)和RPMI-1640(Gibco，目录号：11875-093)按照1:99混合以配制1％FBS RPMI-1640，收集SU-DHL-1靶细胞，用1％FBS RPMI-1640稀释至1.2×10 6细胞/mL，取适当候选抗体用1％FBS RPMI-1640稀释至25μg/mL，以此浓度为起始浓度；依次4倍梯度稀释共8个点，备后续使用；收集效应细胞Jurkat(G7011，Promega)，用1％FBS RPMI-1640稀释至2.4×10 6细胞/mL，在白色96孔板中加入靶细胞25μL/孔；在铺有靶细胞的孔中加入梯度稀释的抗体25μL/孔；加入效应细胞Jurkat 25μL/孔，将96孔板放入细胞培养箱培养5h；取出96孔板，放置室温，使其温度平衡到室温；加入Bio-Gl显色液(G7940，Promega)75μL/孔，反应15min，Tecan酶标仪读取Luminescense得值。结果见图3A-图3B及表7-表8。
表7.候选抗体模拟杀伤活性检测结果(对应图3A)
抗体名称 EC 50(μg/mL) 抗体名称 EC 50(μg/mL)
CD25Q2-BA3-IgG1 0.3310 CD25Q14-CA705-IgG1 0.4094
CD25Q2-BA9-IgG1 0.2356 CD25Q11-BA422-IgG1 0.8362
CD25Q3-BA125-IgG1 0.2512 CD25Q11-BA428-IgG1 0.2170
CD25Q11-BA402-IgG1 0.4429 CD25-MA251-IgG1 0.3606
CD25Q11-BA410-IgG1 0.2149 / /
表8.候选抗体模拟杀伤活性检测结果(对应图3B)
抗体名称 EC 50(μg/mL) 抗体名称 EC 50(μg/mL)
CD25Q14-BA443-IgG1 0.3604 CD25Q11-BA35-IgG1 0.5040
CD25Q14-BA448-IgG1 0.2756 CD25Q11-CA36-IgG1 0.8569
CD25Q14-BA458-IgG1 0.2656 CD25Q11-BA415-IgG1 0.2332
CD25Q14-CA707-IgG1 0.2476 CD25-MA251-IgG1 0.2546
CD25Q14-CA721-IgG1 0.2879 / /
如表7所示，候选抗体CD25Q2-BA9-IgG1模拟杀伤活性检测EC 50值为0.2356，低于对照组CD25-MA251-IgG1的EC 50值0.3606，说明候选抗体对SU-DHL-1的杀伤能力优于对照组CD25-MA251-IgG1。
以上结果说明候选抗体CD25Q2-BA9-IgG1对表达CD25的细胞有良好杀伤效果，预示候选抗体能够减少表达CD25的Treg细胞及其对Teff细胞的抑制，从而具有更好的药学效果。
3.3 BiaCore检测抗体亲和力
抗体结合动力学使用基于表面等离振子共振(surface plasmon resonance，SRP)技术的BIAcore8K仪器测量。通过GE anti Human IgG Fc氨基偶联试剂盒(GE，cat#BR-1008-39)，将anti-human IgG抗体氨基偶联到CM5生物传感器芯片上以获得大约1000应答单位(response units，RU)。对于动力学测量，将CD25蛋白(义翘神州，10165-H08H)用HBS-EP+1×(GE，BR-1008-26)缓冲液2倍连续稀释，50nM起始，2倍稀释4个浓度梯度，并设置0浓度。待测抗体：2μg/mL，进样时间70s，流速5μL/min，稳定5s；CD25蛋白：结合60s，流速30μL/min，解离450s；再生：用3M MgCl 2buffer再生30s，Startup 3次。使用简单一对一Languir结合模型(BIAcore Evaluation Software version 3.2)计算结合常数(ka)和解离常数(kd)，平衡解离常数(KD)以比率kd/ka计算。各抗体亲和力数据见表9。
表9.BiaCore检测候选抗体结合动力学数据
抗体名称 ka(1/Ms) kd(1/s) KD(M)
CD25Q2-BA3-IgG1 4.56E+05 1.36E-03 2.98E-09
CD25Q2-BA9-IgG1 6.96E+05 5.86E-04 8.43E-10
CD25Q11-CA36-IgG1 6.02E+04 9.66E-05 1.61E-09
CD25Q3-BA125-IgG1 6.99E+05 3.01E-04 4.31E-10
CD25Q11-BA402-IgG1 3.50E+05 2.12E-04 6.07E-10
CD25Q11-BA410-IgG1 1.40E+06 6.74E-03 4.81E-09
CD25Q11-BA415-IgG1 3.23E+05 1.99E-04 6.14E-10
CD25Q11-BA428-IgG1 5.55E+05 2.10E-04 3.79E-10
CD25Q14-BA443-IgG1 1.36E+05 2.01E-04 1.48E-09
CD25Q14-BA448-IgG1 5.87E+05 5.31E-04 9.04E-10
CD25Q14-BA458-IgG1 3.62E+05 2.16E-04 5.96E-10
CD25Q14-CA705-IgG1 3.81E+05 7.52E-04 1.97E-09
CD25Q14-CA707-IgG1 3.31E+05 9.44E-04 2.85E-09
CD25Q14-CA721-IgG1 6.80E+05 3.65E-04 5.36E-10
CD25Q11-CT848-IgG1 7.05E+05 2.18E-03 3.09E-09
CD25Q8-BT942-IgG1 8.96E+04 3.39E-04 3.79E-09
CD25-MA251-IgG1 1.76E+05 9.12E-04 5.19E-09
如表9所示，候选抗体CD25Q2-BA9-IgG1的平衡解离常数KD值为8.43E-10低于对照组CD25-MA251-IgG1的KD值5.19E-09，说明候选抗体CD25Q2-BA9-IgG1的CD25蛋白亲和力优于对照组CD25-MA251-IgG1。
如表9所示，候选抗体CD25Q8-BT942-IgG1的平衡解离常数KD值为3.79E-09低于对照组CD25-MA251-IgG1的KD值5.19E-09，说明候选抗体CD25Q8-BT942-IgG1的CD25蛋白亲和力优于对照组CD25-MA251-IgG1。
以上结果预示候选抗体CD25Q2-BA9-IgG1、CD25Q8-BT942-IgG1相较对照组CD25-MA251-IgG1对表达CD25的Treg细胞有更强的靶向性和结合性，有更好杀伤效果，减少Treg细胞对Teff细胞的抑制，有更好的药学效果。
3.4 候选抗体的细胞阻断活性
冻存的PBMC(外周血单核细胞，厂家：ALLCELLS货号：PB003F-C)复苏后与10μg/mL的CD25抗体在96-U底板共培养30min，对照组不加抗体，标记为NoAb，然后加入IL2(0.1U/mL、1U/mL、10U/mL)培养10min(工作培养基：1640+10％FBS，含2mM L-谷氨酰胺以及10000U/mL Pen-Strep)，制备细胞悬液：最后一次洗涤后，弃上清并脉冲涡旋样品以完全离解沉淀；每孔加入200μL Foxp3固定/破膜工作溶液。于2-8℃或室温避光孵育30-60分钟；在室温下400-600g离心样品5分钟，弃上清；每孔加入200μL 1X破膜液，室温下400-600g离心样品5分钟，弃上清，洗涤2遍；(预先将BD Phosflow TM Perm BufferⅢ放入-20℃预冷)PBS洗涤1次，离心弃上清；涡旋同时缓慢加入冰冷的Phosflow TM Perm BufferⅢ，冰上孵育30分钟；PBS洗涤细胞2次，250g离心10分钟弃上清；PBS重悬细胞至10 7个/mL，分装100μL/孔，继续荧光标记抗体染色并流式检测；划门区分活细胞，进一步区分CD3阳性T细胞。磷酸化信号转导子与转录激活子5(PSTAT5)百分比越高，说明阻断率越低。结果见图4及表10。
如表10所示，候选抗体CD25Q2-BA9-IgG1的％PSTAT5值为26.09，显著高于对照组CD25-MA251-IgG1的％PSTAT5值18.52，说明候选抗体在不阻断IL2与PBMC结合的表现上显著优于对照组CD25-MA251-IgG1。说明候选抗体CD25Q2-BA9-IgG1相较对照组CD25-MA251-IgG1能更少的抑制PBMC的激活，预示候选抗体CD25Q2-BA9-IgG1能更好的发挥PBMC免疫效果，有更好的药学效果/抑瘤效果。
如表10所示，候选抗体CD25Q8-BT942-IgG1的％PSTAT5值为16.46，CD25-MA251-IgG1的％PSTAT5值18.52，说明候选抗体在不阻断IL2与PBMC结合的表现上与对照组CD25-MA251-IgG1基本相当。预示候选抗体CD25Q8-BT942-IgG1能良好的发挥PBMC免疫效果，有良好的药学效果/抑瘤效果。
表10.候选抗体细胞阻断活性数据(对应图4)
抗体名称 ％PSTAT5 抗体名称 ％PSTAT5
CD25Q2-BA9 26.09 CD25Q11-CT848-IgG1 15.76
CD25Q11-Q36 23.08 CD25Q8-BA747-IgG1 8.1
NoAb 22.38 CD25Q11-BT957-IgG1 6.72
CD25Q-MA251 18.52 CD25Q14-BT819-IgG1 5.3
CD25Q11-BA422-IgG1 18.09 CD25Q11-CT847-IgG1 4.41
CD25Q11-BA410 16.84 CD25Q11-BT956-IgG1 3.78
CD25Q8-BT942-IgG1 16.46 CD25Q2-CT805-IgG1 1.68
实施例4 候选抗体的体内活性
4.1 CD25Q2-BA9-IgG1在健康恒河猴体内的PD活性
将CD25Q2-BA9-IgG1抗体以10mg/kg的给药剂量静脉给药3只恒河猴，采用流式细胞仪(CytomicsTM FC500)按照流式细胞术检测给药前后不同时间点Treg细胞(CD3+CD4+CD25+FoxP3+)的含量，检测时间点为：给药前给药结束(±1分钟)，给药结束后0.5小时(±1分钟)、3小时(±2分钟)、6小时(±5分钟)、24小时(±10分钟)、48小时(±20分钟，2天)、96小时(±30分钟，4天)、168小时(±1小时，7天)、336小时(±1小时，14天)，实验结果见图5。
由图5可见，CD25Q2-BA9-IgG1给药后，可以显著降低恒河猴体内Treg细胞含量，能够有效调节免疫微环境。
4.2 候选抗体在食蟹猴体内的PK活性
每组两只食蟹猴，静脉注射不同CD25抗体后，并于给药前(0h)及给药后1min，30min，3h，6h，1d，2d，4d，7d，10d，14d经静脉取出全血样品，置血样收集管中，冰盒内让其自然凝固，于血样取出后8h内置离心机中，1000～3000g离心10min，分离血清，置样本保存管中，第14天再次静脉注射不同CD25抗体，并于给药前(0h)及给药后1min(14d+1m)，30min(14d+30m)，3h(14d+3h)，6h(14d+6h)，1d(15d)，2d(16d)，4d(18d)和7d(21d)经静脉取出全血样品，置血样收集管中，冰盒内让其自然凝固，于血样取出后8h内置离心机中，1000～3000g离心10min，分离血清，置样本保存管中，用ELISA的方法检测抗体在食蟹猴体内的代谢情况，结果见图6和表11。
结果显示：CD25Q2-BA9-IgG1与CD25Q8-BT942-IgG1的生物利用度高于对照抗体CD25-MA251-IgG1，有良好的药代动力学表现。
表11.候选抗体在食蟹猴体内代谢
Figure PCTCN2020094919-appb-000004
Figure PCTCN2020094919-appb-000005
实施例5 候选抗体的体内肿瘤模型药效
5.1 候选抗体在B-hIL2Rα人源化小鼠MC38结肠癌动物模型中的药效测试
根据体重将B-hIL2Rα人源化小鼠(百奥赛图)分为5组，其中G1阴性对照组10只，G2-G4治疗组每组8只。分组当天开始给药(10mg/kg，I.P.，BIW)(10mg/kg，腹腔注射，每周两次)，第二天将PBS重悬的MC38细胞以5×10 5个/0.1mL浓度，0.1mL/只，接种于B-hIL2Rα人源化小鼠的右侧皮下。每周测量两次肿瘤体积及动物体重，并记录测量值，肿瘤体积(mm3)＝0.5×长径×短径 2。结果见图7A、7B及表12、表13。
如图7A所示，小鼠体重稳定增长，说明CD25Q2-BA9-IgG1、CD25Q8-BT942-IgG1、CD25Q11-CT848-IgG1(10mg/kg，I.P.，BIW)对小鼠没有毒副作用。
如图7B所示，与对照组相比CD25Q2-BA9-IgG1、CD25Q8-BT942-IgG1、CD25Q11-CT848-IgG1均可显著抑制小鼠MC38肿瘤的生长，其中CD25Q8-BT942-IgG1显示出了更好的抑瘤效果。
表12.MC38肿瘤模型小鼠体重数据(对应图7A)
Figure PCTCN2020094919-appb-000006
表13.MC38肿瘤体积数据(对应图7B)
Figure PCTCN2020094919-appb-000007
Figure PCTCN2020094919-appb-000008
5.2 FACS检测给药后免疫细胞对MC38肿瘤的浸润效果
5.1试验小鼠在第5次给药后(分组后第16天)，阴性对照组取4只小鼠，治疗组每组取3只小鼠，杀死小鼠并将肿瘤剪碎，加入消化酶，37℃，孵育消化40min，过滤，洗涤，重悬为单细胞悬液。96孔圆底板每孔加入25μL封闭与死活染液和25μL的细胞悬液，混匀，4℃，避光孵育15min；每孔加入50μL表面染料，混匀，4℃，避光孵育30min；每孔加入150μL FACS液洗2次，4℃，500g，离心5min，弃上清；每孔加入200μL固定液重悬细胞，混匀后，室温固定30min；固定结束后4℃，1400g，离心5min，弃上清；每孔加入200μL穿膜液，4℃，1400g，离心5min，弃上清；每孔加入100μL胞内染液，室温，避光孵育30min；每孔加150μL穿膜液洗涤2次，1400g，离心5min，弃上清；250μL PBS重悬细胞，上机检测CD3中的CD8+细胞(Teff)含量、CD3中的CD25+Foxp3+细胞(Treg)含量及CD4中的Foxp3+细胞(Treg)含量。结果见图8A-图8C。
如图8A-图8C所示，与对照组Human IgG1 Kappa Isotype相比，CD25Q2-BA9-IgG1、CD25Q8-BT942-IgG1、CD25Q11-CT848-IgG1均可降低小鼠MC38肿瘤内的Treg细胞比例，增加Teff细胞的浸润，从而提高肿瘤杀伤能力。

Claims (10)

  1. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:14所示，HCDR2氨基酸序列如SEQ ID NO:15所示，HCDR3氨基酸序列如SEQ ID NO:16所示。
  2. 根据权利要求1所述的抗体或其抗原结合片段，还包含3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:12所示，LCDR3氨基酸序列如SEQ ID NO:13所示。
  3. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含SEQ ID NO:4所示的重链可变区；优选还含有SEQ ID NO:3所示的轻链可变区。
  4. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，所述抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合CD25，优选结合人CD25。
  6. 一种核酸，所述核酸编码根据权利要求1-5任一项所述的抗体或其抗原结合片段。
  7. 一种细胞，所述细胞表达根据权利要求1-5任一项所述的抗体或其抗原结合片段。
  8. 一种药物组合物，其包含根据权利要求1-5中任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的核酸，或根据权利要求7所述的细胞。
  9. 一种试剂盒，其包含根据权利要求1-5中任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的核酸。
  10. 根据权利要求1-5中任一项所述的抗体或其抗原结合片段或根据权利要求6所述的核酸用于治疗、预防、检测或诊断疾病的用途，优选地，所述疾病是癌症；更优选为胃癌、食道癌、头颈癌、膀胱癌、宫颈癌、肉瘤、细胞瘤、肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
PCT/CN2020/094919 2019-06-10 2020-06-08 抗cd25抗体及其应用 Ceased WO2020248938A1 (zh)

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JP2025502379A (ja) * 2022-01-17 2025-01-24 諾納生物（蘇州）有限公司 Ｃｄ２５を標的とする抗体及びその製造方法と応用
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