WO2021129775A1 - 抗ctla-4单克隆抗体及其制备方法与应用 - Google Patents

抗ctla-4单克隆抗体及其制备方法与应用 Download PDF

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WO2021129775A1
WO2021129775A1 PCT/CN2020/139205 CN2020139205W WO2021129775A1 WO 2021129775 A1 WO2021129775 A1 WO 2021129775A1 CN 2020139205 W CN2020139205 W CN 2020139205W WO 2021129775 A1 WO2021129775 A1 WO 2021129775A1
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antibody
ctla
seq
sequence
amino acid
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French (fr)
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苏紫琪
秦超
俞金泉
黄丹丹
吴用
曹迪
刘翠华
宋述强
李胜峰
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Bio Thera Solutions Ltd
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Bio Thera Solutions Ltd
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Priority claimed from CN201911363904.XA external-priority patent/CN113024670A/zh
Priority claimed from CN202010430026.5A external-priority patent/CN113712906A/zh
Application filed by Bio Thera Solutions Ltd filed Critical Bio Thera Solutions Ltd
Priority to CN202080086339.1A priority Critical patent/CN114793422A/zh
Priority to EP20907897.1A priority patent/EP4082572A4/en
Priority to US17/788,330 priority patent/US20230203164A1/en
Publication of WO2021129775A1 publication Critical patent/WO2021129775A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C07K2317/70—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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention belongs to the technical field of biomedicine, and relates to an anti-CTLA-4 monoclonal antibody and a preparation method and application thereof.
  • the immune system is an important defense against cancer development and progression.
  • Immunotherapy such as the adoptive transfer of IL-2 and autologous tumor-infiltrating lymphocytes, can induce sustained tumor responses in a subgroup of patients, thereby enabling patients with poor prognosis to achieve long-term survival (Atkins MB, et al. (1999), J Clin Oncol) .17(7):2105–2116).
  • treatment methods based on checkpoint blockade have recently begun to provide patients with long-lasting benefits and seem to be applicable to a wide range of malignancies.
  • T cells are one of the key mediators of the immune response, which require costimulation to activate, and can be inactivated by inhibiting signals (Sharpe AH, Abbas AK. (2006), N Engl J Med.355(10):973-975 ).
  • Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) is such a key inhibitory receptor that affects T cell function, and plays a key role in the initiation of the immune response (Scalapino KJ1, Daikh DI. (2008), Immunol Rev. .223:143-155).
  • TCR T cell receptor
  • B7-1 CD80
  • B7-2 CD86
  • CTLA-4 When CD28 binds and causes T cell proliferation, CTLA-4 is transported and expressed on the surface of T cells (Linsley PS, et al. (1996), Immunity. 4(6): 535-543). The stronger the activation signal through TCR, the more CTLA-4 is transported and expressed. When on the cell surface, the inhibitory signal of CTLA-4 is transmitted (Egen JG, Allison JP. (2002), Immunity. 16(1): 23-25). Compared with CD28, CTLA-4 has a higher affinity with B7 and can prevent further co-activation (Krummel MF, Allison JP. (1995), J Exp Med. 182(2): 458-465). In addition, cells expressing CTLA-4 can capture and degrade B7-1 and B7-2 through endocytosis (Qureshi OS, et al. (2011), Science. 332(6029): 600-603).
  • Stimulatory receptors include inducible T cell costimulator (ICOS) (Tremble LF, et al. (2016), Cancer Lett. 420: 109-115), glucocorticoid-induced TNF receptor (GITR) (Schaer DA ,et al. (2013), Cancer Immunol Res. 1(5): 320-331), CD27(Buchan SL, et al. (2016), Blood.131(1): 39-48), 4-1BB( Eun SY, et al. (2015), J Immunol.
  • IGITR glucocorticoid-induced TNF receptor
  • inhibitory receptors include programmed cell death receptor-1 (PD-1) (Dong H, et al. (1999), Nat Med. 5(12): 1365-1369), lymphocyte activation gene-3 (LAG-3) (Di CE, et al. (2005), J Pathol. 205:82 -91), T cell immunoglobulin and mucin domain 3 (TIM3) (Sánchez-Fueyo A, et al.
  • PD-1 programmed cell death receptor-1
  • LAG-3 lymphocyte activation gene-3
  • TIM3 T cell immunoglobulin and mucin domain 3
  • T cell immunoglobulin Protein and ITIM domain (TIGIT) (Johnston RJ, et al. (2014), Cancer Cell. 26:923-937).
  • TAGIT T cell immunoglobulin Protein and ITIM domain
  • over-activation In cancer, the use of agonists to target stimulatory receptors or inhibitors to block inhibitory receptors can regulate the immune response, allowing the immune system to recognize and attack tumor cells to achieve therapeutic effects.
  • some therapeutic drugs have been developed in clinical or on the market (Elad S, et al. (2014), Chin J Cancer. 33(9): 434-444), of which CTLA-4 blocks The drug is the first monoclonal antibody drug used in clinical cancer treatment.
  • the more well-known anti-CTLA-4 monoclonal antibody is Ipilimumab from Bristol-Myers Squibb And AstraZeneca's Tremelimumab (Tremelimumab), which is still in the clinical stage.
  • Their mechanism is to block the binding of CTLA-4 and B7, liberate the B7 protein to bind to CD28, thereby enhancing the proliferation response of T cells.
  • Blocking the CTLA-4/B7 interaction in several established preclinical mouse models has shown rejection of transplantable tumors, including colon cancer, prostate cancer, lymphoma and kidney cancer (Korman AJ, et al . (2006), Adv Immunol. 90:297-339).
  • the present invention provides an anti-CTLA-4 antibody with low content of high mannose glycoforms and/or low content of sialylated glycoforms, which helps to extend the half-life of the antibody and/or reduce the immunogenicity.
  • the antibody is an ADCC-enhanced anti-CTLA-4 antibody, which is used to target patient cells or tissues to increase the anti-tumor effect of the drug.
  • the present invention provides a monoclonal antibody against CTLA-4, the monoclonal antibody comprising
  • Heavy chain CDR1 which comprises the sequence shown in SEQ ID NO: 12, and has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, About 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two values (including endpoints) or any value therein) sequence identity, or The sequence has, or consists of, one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) of amino acid sequences,
  • Heavy chain CDR2 which includes the sequence shown in SEQ ID NO: 13, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% with the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of them,
  • the heavy chain CDR3 which comprises the sequence shown in SEQ ID NO: 14, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% with the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of them,
  • Light chain CDR1 which includes the sequence shown in SEQ ID NO: 15, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% with the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with or consisting of one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) of amino acid sequences,
  • Light chain CDR2 (LCDR2), which includes the sequence shown in SEQ ID NO: 16, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% of the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with or consists of one or more (preferably about 1, about 2, or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) of amino acid sequences, and
  • Light chain CDR3 which includes the sequence shown in SEQ ID NO: 17, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% of the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of them;
  • the heavy chain variable region which comprises the sequence shown in SEQ ID NO: 18, has at least about 80% of the sequence, preferably at least about 85%, about 90%, about 91%, about 92 %, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two values (including endpoints) or any value therein) sequence identity
  • a sexual sequence or an amino acid sequence with one or more (preferably about 1, about 2, or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence, or consisting of, with
  • the light chain variable region (VL), which comprises the sequence shown in SEQ ID NO: 19, is at least about 80%, preferably at least about 85%, about 90%, about 91%, about 92%, about A sequence of 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity , Or an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence, or consisting of them;
  • the monoclonal antibody has a reduced level of fucosylation.
  • the monoclonal antibody comprises a heavy chain and a light chain
  • the heavy chain comprises the amino acid sequence of SEQ ID NO:1, which has at least about 80% of the amino acid sequence of SEQ ID NO:1, preferably at least About 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93 %, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two values (including endpoints) or any value therein) homology sequence, Or consist of it;
  • the light chain includes the amino acid sequence shown in SEQ ID NO: 2, which has at least about 80%, preferably at least about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% , About 98% or about 99% (or the range between any two values (including endpoints) or any value therein) homology sequence, or consisting of the sequence.
  • the heavy chain has at least about 80% of the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 3, preferably at least about 81%, about 82%. %, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, About 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two numerical values (including endpoints) or any value therein) homology nucleotide sequence encoding, said The light chain has at least about 80%, preferably at least about 81%, about 82%, about 83%, or about 84% of the nucleotide sequence shown in SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 4 , About 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%
  • the fucosylation level is 0%-20%, such as about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6% , About 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20%, or a range between any two values (including endpoints) or any value therein.
  • the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is ⁇ 5% and/or the total amount of sialylated glycoforms is ⁇ 3%. In some embodiments, the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is about 0.1%, about 0.3%, about 0.9%, about 1.18%, about 1.7%, about 2.6%, about 3.3%, About 4.1%, about 4.9%, about 4.99%, or a range between any two values (including the endpoints) or any value therein.
  • the total amount of sialylated glycoforms in the Fc region of the anti-monoclonal antibody is about 0.1%, about 0.2%, about 0.36%, about 0.8%, about 1.5%, about 2.2%, about 2.7%, Approximately 2.9%, 2.99%, or any range (including endpoints) between two values or any value therein.
  • the monoclonal antibody is IgG1, and the total amount of high mannose glycoforms in the CH2 region of the monoclonal antibody is ⁇ 5% and/or the total amount of sialylated glycoforms is ⁇ 3%.
  • the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is less than 3% and/or the total amount of sialylated glycoforms is less than 2%.
  • the monoclonal antibody is IgG1, and the total amount of high mannose glycoforms in the CH2 region of the monoclonal antibody is ⁇ 3% and/or the total amount of sialylated glycoforms is ⁇ 2%.
  • the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is less than 2% and/or the total amount of sialylated glycoforms is less than 1%.
  • the monoclonal antibody is IgG1, and the total amount of high mannose glycoforms in the CH2 region of the monoclonal antibody is less than 2% and/or the total amount of sialylated glycoforms is less than 1%.
  • the fucosylation level of the Fc region of the monoclonal antibody is 0%-10%.
  • the monoclonal antibody is IgG1, and the fucosylation level of the CH2 region of the monoclonal antibody is 0%-10%.
  • the fucosylation level of the Fc region of the monoclonal antibody is 0%-5%. In some embodiments, the fucosylation level of the Fc region of the anti-monoclonal antibody is about 0, about 0.1%, about 0.3%, about 0.4%, about 0.6%, about 1.3%, about 1.9%, about 2.2%, about 2.8%, about 3.3%, about 3.7%, about 4.1%, about 4.5%, about 5%, or a range between any two values (including endpoints) or any value therein. In some embodiments, the monoclonal antibody is IgG1, and the fucosylation level of the CH2 region of the monoclonal antibody is 0%-5%.
  • the level of glycation is achieved by:
  • the monoclonal antibody against the light chain CDR2 of SEQ ID NO: 17 and the light chain CDR3 of SEQ ID NO: 17 are expressed in CHO host cells that knock down or knock down the FUT8 gene ( ⁇ -(1,6)-fucosyltransferase gene), The high mannose glycoform, sialylated glycoform and/or fucose content of the monoclonal antibody is lower than the content of the monoclonal antibody expressed in normal CHO host cells;
  • the high mannose glycoform, sialylated glycoform and/or fucose content of the monoclonal antibody is compared with that without glycosyltransferase inhibitor such as 2F-peracetyl-fucose defucosylation
  • the content of monoclonal antibodies is low.
  • the monoclonal antibody is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line, such as the CHO-BAT-KF cell line disclosed in PCT/CN2018/100008.
  • the present invention provides a pharmaceutical composition or fusion protein, which comprises the monoclonal antibody of the present invention.
  • the pharmaceutical composition is in a form suitable for oral, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, nasal, abdominal, intraarterial, and subcutaneous absorption administration. In some embodiments, the pharmaceutical composition is in a form suitable for systemic administration.
  • the present invention provides an antibody conjugate, which includes the monoclonal antibody of the present invention, and a coupling part coupled to the monoclonal antibody, and the coupling part is a purification tag (such as a His tag). ), a cytotoxic agent, a structural part that prolongs the half-life of the monoclonal antibody, or a detectable label, preferably, the coupling part is a radioisotope, a chemiluminescent agent, an enzyme, a serum albumin (such as human or mouse serum Albumin), or polyethylene glycol.
  • a purification tag such as a His tag
  • the coupling part is a radioisotope, a chemiluminescent agent, an enzyme, a serum albumin (such as human or mouse serum Albumin), or polyethylene glycol.
  • the anti-CTLA-4 antibodies of the invention are monovalent antibodies or multivalent antibodies.
  • the present invention provides multispecific antibodies (such as bispecific antibodies), which comprise the monoclonal antibodies of the present invention, and antibodies or antigen-binding fragments directed against other antigens and/or other epitopes.
  • the present invention provides a kit, which includes the monoclonal antibody, antibody conjugate, multispecific antibody or fusion protein of the present invention.
  • the kit further includes a second antibody, a cytotoxic agent, a chemotherapeutic agent, and a radiotherapy agent.
  • the second antibody specifically recognizes the monoclonal antibody; optionally, the second antibody It also includes detectable labels, such as fluorescein, metal ions, biotin, radioisotopes, chemiluminescent substances, enzymes, or polyethylene glycols.
  • the present invention provides the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody, which is used for the treatment of cancer or for rejection of transplantable tumors, such as melanoma, non-small cell Lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • transplantable tumors such as melanoma, non-small cell Lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • the present invention provides a method for preparing a monoclonal antibody with enhanced ADCC activity, comprising:
  • the monoclonal antibody against the light chain CDR2 of SEQ ID NO: 17 and the light chain CDR3 of SEQ ID NO: 17 knocks down or knocks out the FUT8 gene ( ⁇ -(1,6)-fucosyltransferase gene) in CHO host cells (e.g. CHO -BAT-KF cells);
  • the monoclonal antibody against the light chain CDR2 of SEQ ID NO: 17 and the light chain CDR3 of SEQ ID NO: 17 is expressed in CHO-DG44 cells knocked down GDP-fucose 4,6-dehydratase (GMD); or
  • the present invention provides a method for treating cancer or for rejecting transplantable tumors, such as melanoma, non-small cell lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma, or kidney cancer, which includes giving The patient is given an effective amount of the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody.
  • transplantable tumors such as melanoma, non-small cell lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma, or kidney cancer
  • the present invention provides a method for enhancing the proliferation response of T cells, which comprises using the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody of the present invention to block CTLA-4 and B7 Combine.
  • the present invention provides a method for reducing tumor infiltration of CTLA-4 expressing Treg cells in a patient, comprising administering to the patient a therapeutically effective amount of the monoclonal antibody, fusion protein, antibody conjugate or multispecific Antibody.
  • the present invention provides that the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody is prepared for the treatment of cancer or for rejection of transplantable tumors, such as melanoma, non-small cell Application in medicines for lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • transplantable tumors such as melanoma, non-small cell Application in medicines for lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • the present invention provides methods or uses of anti-CTLA-4 antibodies or antigen-binding fragments for the treatment of tumors or cancers.
  • anti-CTLA-4 antibodies or antigen-binding fragments are used to treat tumors or cancers.
  • the anti-CTLA-4 antibody or antigen-binding fragment at least comprises HCDR1 shown in SEQ ID NO: 12, HCDR2 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 14
  • the anti-CTLA-4 antibody or antigen-binding fragment includes HCDR1 shown in SEQ ID NO: 12, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 14, and SEQ ID LCDR1 shown in NO:15, LCDR2 shown in SEQ ID NO:16, and LCDR3 shown in SEQ ID NO:17.
  • the heavy chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 18, which is at least 80% identical to the sequence shown in SEQ ID NO: 18 Sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 18; and/or
  • the light chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 19, or a sequence with SEQ ID NO: 19 Compared with the sequence shown in NO:19, the amino acid sequence has one or more conservative amino acid substitutions.
  • the heavy chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment The region contains the sequence shown in SEQ ID NO: 19.
  • the heavy chain of the anti-CTLA-4 antibody comprises a sequence shown in SEQ ID NO:1, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:1, or a sequence with SEQ ID NO: :1 Amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in 1; and/or
  • the light chain of the anti-CTLA-4 antibody includes the sequence shown in SEQ ID NO: 2, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2, or is similar to the sequence shown in SEQ ID NO: 2. Than an amino acid sequence with one or more conservative amino acid substitutions.
  • the anti-CTLA-4 antibody is antibody 1 or antibody 2
  • the heavy chains of antibody 1 and antibody 2 both include the sequence shown in SEQ ID NO:1
  • the light chains of antibody 1 and antibody 2 Both contain the sequence shown in SEQ ID NO: 2
  • Antibody 1 and Antibody 2 contain two heavy chains with the same sequence and two light chains with the same sequence.
  • the total amount of high mannose glycoforms in the Fc region of the anti-CTLA-4 antibody is ⁇ 5% and/or the total amount of sialylated glycoforms is ⁇ 3%.
  • the total amount of high mannose glycoforms in the Fc region of the anti-CTLA-4 antibody is about 0.1%, about 0.3%, about 0.9%, about 1.18%, about 1.7 %, about 2.6%, about 3.3%, about 4.1%, about 4.9%, about 4.99%, or a range between any two values (including endpoints) or any value therein.
  • the total amount of sialylated glycoforms in the Fc region of the anti-CTLA-4 antibody is about 0.1%, about 0.2%, about 0.36%, about 0.8%, about 1.5% , About 2.2%, about 2.7%, about 2.9%, 2.99%, or any range between two values (including endpoints) or any value therein.
  • the total amount of high mannose glycoforms in the Fc region of the anti-CTLA-4 antibody is ⁇ 3% and/or the total amount of sialylated glycoforms is ⁇ 2%.
  • the total amount of high mannose glycoforms in the Fc region of the anti-CTLA-4 antibody is less than 2% and/or the total amount of sialylated glycoforms is less than 1%.
  • the fucosylation level of the anti-CTLA-4 antibody (eg, antibody 2) or antigen-binding fragment is 0%-20%, such as about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% , About 16%, about 17%, about 18%, about 19%, or about 20%, or a range between any two values (including endpoints) or any value therein.
  • the fucosylation level of the anti-CTLA-4 antibody (eg, antibody 2) or antigen-binding fragment is 0-10%.
  • the fucosylation level of the anti-CTLA-4 antibody (eg, antibody 2) or antigen-binding fragment is 0-5%. In some embodiments, the fucosylation level of the anti-CTLA-4 antibody (eg, antibody 2) or antigen-binding fragment is 0-1%.
  • the fucosylation level of the anti-CTLA-4 antibody (eg, Antibody 2) or antigen-binding fragment is about 0, about 0.1%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.8%, about 1%, about 1.3%, about 1.6%, about 2.1%, about 2.2%, about 2.8%, about 2.9%, about 3%, about 3.3%, 3.8%, about 4%, About 4.1%, about 4.2%, 4.3%, about 4.6%, about 5%, or a range between any two of these values (including endpoints) or any value therein.
  • the anti-CTLA-4 antibody (eg, Antibody 2) or antigen-binding fragment does not bind fucose.
  • the anti-CTLA-4 antibody (eg, antibody 2) or antigen-binding fragment has enhanced ADCC (antibody-dependent cell-mediated cytotoxicity).
  • Anti-CTLA-4 antibodies or antigen-binding fragments can be expressed in CHO cells or 293 cells by genetic engineering and obtained by purification; purification can be carried out by conventional methods, such as first centrifuging the cell suspension and collecting the supernatant, and then centrifuging it for further removal Impurities. Methods such as Protein A affinity column and ion exchange column can be used to purify antibody proteins.
  • the anti-CTLA-4 antibody e.g., antibody 2 or antigen-binding fragment is expressed by a cell line in which the ⁇ -(1,6)-fucosyltransferase gene is knocked out, as disclosed in PCT/CN2018/100008 CHO-BAT-KF cell line.
  • Antibody 2 is expressed by the CHO-BAT-KF cell line.
  • the method or use includes administering an effective amount of an anti-CTLA-4 antibody or antigen-binding fragment to a patient in need.
  • the anti-CTLA-4 antibody is antibody 1 or antibody 2.
  • an anti-CTLA-4 antibody e.g., antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the effective dose of anti-CTLA-4 antibody administered is about 7 mg to 180 mg per dose.
  • the patient has a tumor or cancer.
  • tumors and cancers include, but are not limited to, blood cancers, solid tumors.
  • blood cancers include, but are not limited to, leukemia, lymphoma, and myeloma.
  • leukemias include acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and myeloproliferative disease/tumor (MPDS ).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MPDS myeloproliferative disease/tumor
  • lymphomas include Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).
  • myeloma includes multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bens-Jones myeloma.
  • solid tumors include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer.
  • tumors and cancers are locally late or metastatic malignant solid tumors that are pathologically diagnosed for which there is no effective treatment.
  • the present invention discloses a method for treating tumor or cancer in a patient in need, which comprises administering an effective amount of anti-CTLA-4 antibody, wherein the effective amount of anti-CTLA-4 antibody administered is about 7 mg To 180mg per treatment cycle.
  • a treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including Endpoint) or any of them.
  • the anti-CTLA-4 antibody is Antibody 1, Antibody 2.
  • Antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the anti-CTLA-4 antibody can be formulated into a pharmaceutical composition and administered to the patient in a variety of forms suitable for the chosen route of administration, such as parenteral, intravenous (iv), Intramuscular, local or subcutaneous.
  • the anti-CTLA-4 antibody can be infused intravenously.
  • the amount of anti-CTLA-4 antibody administered will depend on the nature of the drug, the degree of internalization, transport, and release of the drug triggered by the cell surface, as well as the disease to be treated and the patient's condition (such as age, gender, weight, etc.).
  • the anti-CTLA-4 antibody (such as antibody 1 or 2) administered per administration is about 0.1 mg/kg to 16 mg/kg or a formulation containing this dose of anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody administered per administration is about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.9 mg/kg.
  • the anti-CTLA-4 antibody (such as antibody 1 or 2) administered per administration is about 0.1 mg/kg to 2.5 mg/kg or a formulation containing this dose of anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody administered per administration is about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg.
  • the administration is once about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, or The range (including endpoints) between any two of these values or any value therein.
  • it is administered once every about 3 weeks, and the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles. Cycles or 10 cycles.
  • the administration is once about 4 weeks, and the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 Cycles or 10 cycles.
  • it is administered once every about 5 weeks, and the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles. Cycles or 10 cycles.
  • the present invention discloses a method for treating tumor or cancer, the method comprising: administering about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg to a patient in need every 3 weeks /kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, About 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, about 1.9mg/kg, about 2mg /kg, about 2.1mg/kg, about 2.2mg/kg, about 2.3mg/kg, about 2.4mg/kg, about 2.5mg/kg of anti-CTLA-4 antibody, or a preparation containing this dose of anti-CTLA-4 antibody .
  • the present invention discloses a method for treating tumor or cancer, the method comprising: administering about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg to a patient in need every 3 weeks /kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, About 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, about 1.9mg/kg, about 2mg /kg, about 2.1mg/kg, about 2.2mg/kg, about 2.3mg/kg, about 2.4mg/kg, about 2.5mg/kg of antibody 1 or 2, or preparations containing this dose of antibody 1 or 2; treatment
  • the cycle is 4 cycles.
  • the present invention discloses a method for treating tumor or cancer, which comprises administering an effective amount of anti-CTLA-4 antibody (or preparation) to a patient in need; wherein, the effective amount of anti-CTLA-4 antibody For a single administration of about 7mg to 180mg (or a preparation containing this dose of anti-CTLA-4 antibody).
  • the dosage schedule and mode of administration depend on the benefit and risk assessment of anti-CTLA-4 antibodies (or preparations) in certain patient groups and general clinical practice guidelines.
  • the effective amount of anti-CTLA-4 antibody administered to the patient per treatment cycle is about 7 mg to 180 mg (or a formulation containing this dose of anti-CTLA-4 antibody).
  • a treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including endpoints) or where Any value.
  • the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, or 6 cycles.
  • the effective amount of an anti-CTLA-4 antibody (such as antibody 1 or 2) administered to a patient per treatment cycle is about 6 mg, about 10 mg, about 12 mg, about 18 mg, about 24 mg, about 30 mg, about 40 mg, About 50mg, about 60mg, about 100mg, about 120mg, about 180mg, about 200mg, about 250mg, about 290mg, about 300mg, about 330mg, about 380mg, about 400mg, about 434mg, about 480mg, about 500mg, about 567mg, about 580mg , About 600 mg, about 700 mg, about 800 mg, about 900 mg, about 960 mg, or the range between any two of these values (including endpoints) or any value therein, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 7 mg, about 8 mg, about 10 mg, about 12 mg, about 18 mg, about 24 mg, about 30 mg, About 40 mg, about 50 mg, about 60 mg, about 68 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, or these The range between any two of the values (including endpoints) or any value therein, or a preparation containing this dose of anti-CTLA-4 antibody.
  • one treatment cycle is one administration every 1 week to 7 weeks.
  • the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, or a range between any two of these values (including endpoints) Or any of them.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 7 mg to about 120 mg, or a preparation containing this dose of anti-CTLA-4 antibody; of which, one
  • the treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of an anti-CTLA-4 antibody (such as antibody 1 or 2) administered to a patient per treatment cycle is about 7 mg, about 10 mg, about 12 mg, about 18 mg, about 20 mg, about 24 mg, about 28 mg, About 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, or a range between any two of these values (including endpoints) or any value therein, or A preparation containing this dose of anti-CTLA-4 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles Cycle or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient during each treatment cycle is about 120 mg to 180 mg, or a preparation containing this dose of anti-CTLA-4 antibody; among them, one treatment
  • the cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, Or the range between any two of these values (including endpoints) or any value therein, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, or about 3 weeks. It is administered once every week or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 6.9 mg to 7.2 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 7 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 10 mg to 13 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 12 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 20 mg to 24 mg, or a formulation containing this dose of anti-CTLA-4 antibody. In some embodiments, the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 22 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 28 mg to 33 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 30 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 59 mg to 64 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 60 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 95 mg to 113 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 100 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 110 mg to 123 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient during each treatment cycle is about 120 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 127 mg to 133 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 130 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 132 mg to 144 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient during each treatment cycle is about 140 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 140 mg to 155 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 (such as antibody 1 or 2) antibody administered to the patient during each treatment cycle is about 150 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 177 mg to 180 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 180 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody administered is about 7 mg to 120 mg once every 3 weeks for 4 treatment cycles. In some embodiments, the effective amount of anti-CTLA-4 antibody administration is about 7 mg, about 10 mg, about 12 mg, about 20 mg, about 26 mg, about 30 mg, about 35 mg, about 40 mg, about 44 mg, about 50 mg, about 58 mg, about 60 mg, about 69 mg, about 70 mg, about 77 mg, about 85 mg, about 99 mg, about 110 mg, about 114 mg, or about 120 mg once every 3 weeks for 4 treatment cycles. In some embodiments, the effective amount of anti-CTLA-4 antibody administered is about 7 mg, about 12 mg, about 30 mg, about 60 mg, or about 120 mg once every 3 weeks for 4 treatment cycles.
  • the effective amount of anti-CTLA-4 antibody administered is about 120 mg to 180 mg once every 3 weeks. In some embodiments, the effective amount of anti-CTLA-4 antibody administered is about 120 mg, about 130 mg, about 134 mg, about 140 mg, about 146 mg, about 150 mg, about 154 mg, about 160 mg, about 164 mg, about 170 mg, about 173 mg, about 1760mg, about 180mg once every 3 weeks, 4 treatment cycles. In some embodiments, the effective amount of anti-CTLA-4 antibody administered is about 130 mg, 140 mg, about 160 mg, or about 180 mg once every 3 weeks for 4 treatment cycles.
  • the patient is administered the anti-CTLA-4 antibody (or formulation) once per treatment cycle.
  • the anti-CTLA-4 antibody (or formulation) is administered multiple times during each treatment cycle, for example, 2 times, 3 times, 4 times, or 5 times.
  • the patient can only be administered 1 or 4 times per treatment cycle.
  • the patient receives treatment for one treatment cycle. In some embodiments, the patient receives multiple (e.g., 2, 3, 4, 5, or 6) treatment cycles. In some embodiments, the patient is treated for 4 treatment cycles. In some embodiments, the patient receives treatment until the condition is relieved and treatment is no longer needed.
  • the present invention discloses a method for treating tumor or cancer, the method comprising: administering about 7 mg to 30 mg, about 30 mg to 60 mg, about 60 mg to 120 mg, every 3 weeks to a patient in need, About 120 mg to 180 mg, such as about 7 mg, about 10 mg, about 12 mg, about 18 mg, about 30 mg, 60 mg, about 80 mg, about 100 mg, about 120 mg, about 150 mg, about 160 mg, about 180 mg of anti-CTLA-4 antibody, or containing it Dose anti-CTLA-4 antibody preparation.
  • the anti-CTLA-4 antibody is Antibody 1.
  • the anti-CTLA-4 antibody is antibody 2.
  • the patient's symptoms are relieved after a single dose is administered. In some embodiments, after a single dose administration, the patient's symptoms do not get the expected alleviation, and then about 7 mg to 180 mg of anti-CTLA-4 antibody is administered to the patient until the patient's symptoms are alleviated.
  • the anti-CTLA-4 antibody (or formulation) is administered by subcutaneous (s.c.) injection, intraperitoneal (i.p.) injection, parenteral injection, intraarterial injection, or intravenous (i.v.) injection.
  • the anti-CTLA-4 antibody (or formulation) is administered by infusion.
  • the anti-CTLA-4 antibody (or formulation) is administered as a bolus injection.
  • the anti-CTLA-4 antibody (or formulation) is administered by intravenous (i.v.) infusion (ie, intravenous infusion).
  • intravenous infusion ie, intravenous infusion
  • the duration of intravenous infusion is about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 81 minutes, about 87 minutes, about 90 minutes, about 95 minutes. Minutes, or the range (including endpoints) between any two of these values, or any value therein.
  • the duration of intravenous infusion is> 60 minutes.
  • anti-CTLA-4 antibodies are used in combination with other treatment methods to treat tumors or cancers, such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted therapy, biological therapy, and surgical therapy.
  • anti-CTLA-4 antibodies are combined with other tumor or cancer therapeutic agents to treat tumors or cancers, such as hormones, antibodies for the treatment of tumors or cancers, and the like.
  • the present invention provides the use of anti-CTLA-4 antibodies in the preparation of drugs for the treatment of tumors or cancers.
  • the drugs used to treat tumors or cancers include anti-CTLA-4 antibodies.
  • the anti-CTLA-4 antibody is antibody 1 or 2.
  • Antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the present invention also provides a kit comprising an anti-CTLA-4 antibody (or preparation) and instructions for instructing patients in need to administer the anti-CTLA-4 antibody (or preparation).
  • the anti-CTLA-4 antibody is antibody 1 or 2.
  • Antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the present invention also provides a pharmaceutical composition suitable for injection containing an anti-CTLA-4 antibody, such as a bolus injection type pharmaceutical composition or an infusion (drip) type pharmaceutical composition.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water or phosphate buffered saline (PBS), ethanol, solvents or dispersion media of polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and Suitable mixture.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include antibacterial and/or antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • the pharmaceutically acceptable carrier may include isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride.
  • the pharmaceutical composition contains at least 0.1% anti-CTLA-4 antibodies. The percentage of antibody can vary and is between about 2% and 90% of the weight of a given dosage form. The amount of anti-CTLA-4 antibody in this therapeutically useful pharmaceutical composition can be an effective amount for administration.
  • the present invention also provides a method for preparing the above-mentioned pharmaceutical composition: separately mixing the anti-CTLA-4 antibody described herein with a pharmaceutically acceptable carrier suitable for injection (such as water for injection, physiological saline, etc.) .
  • a pharmaceutically acceptable carrier suitable for injection such as water for injection, physiological saline, etc.
  • the mixing method of the above-mentioned anti-CTLA-4 antibody and a pharmaceutically acceptable carrier is generally known in the art.
  • the present invention also provides a liquid preparation containing anti-CTLA-4 antibody that is stable during storage and delivery, which comprises the following components: 4-80 mg/mL anti-CTLA-4 antibody or fragments thereof, buffer Agents, stabilizers, chelating agents, surfactants, water; the pH value of liquid preparations is 5-7.
  • the liquid formulation includes the following components:
  • the pH of the liquid formulation is 5.2-6.5.
  • the liquid formulation includes the following components:
  • the pH of the liquid formulation is 5.4-6.2.
  • the buffer does not include Tris buffer.
  • the buffer is selected from a histidine buffer, an acetate buffer, or a combination thereof.
  • the histidine buffer comprises histidine and histidine hydrochloride (such as L-histidine and L-histidine hydrochloride), and the acetate buffer comprises acetic acid and Sodium acetate.
  • the buffer is selected from a histidine buffer or a combination of histidine buffer and acetate buffer.
  • the stabilizer is selected from trehalose, sucrose, mannitol, or a combination thereof.
  • the stabilizer is selected from trehalose or sucrose.
  • the chelating agent comprises disodium edetate.
  • the chelating agent is edetate disodium.
  • the surfactant is selected from polysorbate 20, polysorbate 80 or a combination thereof. In some embodiments, the surfactant is polysorbate 80.
  • the concentration of the anti-CTLA-4 antibody or fragment thereof is about 5 mg/mL, about 9 mg/mL, about 13 mg/mL, about 17 mg/mL, about 24 mg/mL, about 29 mg/mL, about 31 mg/mL. mL, about 36 mg/mL, about 40 mg/mL, or the range between any two of these values (including the end point) or any value therein.
  • the concentration of the buffer is about 10 mM, about 14 mM, about 17 mM, about 21 mM, about 25 mM, about 29 mM, about 30 mM, or a range between any two of these values (including the end point) or Any of these values.
  • the concentration of the stabilizer is about 200 mM, about 210 mM, about 219 mM, about 227 mM, about 235 mM, about 244 mM, about 251 mM, about 260 mM, or a range between any two of these values (including End point) or any of these values.
  • the concentration of the chelating agent is about 0.02 mg/mL, about 0.09 mg/mL, about 0.12 mg/mL, about 0.17 mg/mL, about 0.2 mg/mL, or any two of these values The range between (including the end point) or any value in it.
  • the concentration of the surfactant is about 0.1 mg/mL, about 0.18 mg/mL, about 0.24 mg/mL, about 0.3 mg/mL, about 0.36 mg/mL, about 0.4 mg/mL, or these The range (including the end point) between any two values in the numerical value or any value therein.
  • the buffer is a histidine buffer or a combination of histidine buffer and acetate buffer
  • the pH of the liquid formulation is about 5.4, about 5.5, about 5.7, about 5.9, or about 6.1. , About 6.2, or the range between any two of these values (including the end point) or any value therein.
  • the pH of the liquid formulation is about 5.7, about 5.74, about 5.81 About 5.86, about 5.9, or the range between any two of these values (including the end point) or any value therein.
  • the liquid formulation contains 7-15 mg/mL anti-CTLA-4 antibody or fragments thereof.
  • the concentration of the anti-CTLA-4 antibody or fragment thereof in the liquid formulation is about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, About 13 mg/mL, about 14 mg/mL, about 15 mg/mL, or the range between any two of these values (including the end point) or any value therein.
  • a 17-23 mM histidine buffer is included in the liquid formulation.
  • the concentration of the histidine buffer in the liquid formulation is about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, or between any two of these values. Range (including end point) or any value in it.
  • the liquid formulation contains 220-250 mM sucrose.
  • the concentration of sucrose in the liquid formulation is about 220 mM, about 224 mM, about 228 mM, about 232 mM, about 237 mM, about 239 mM, about 241 mM, about 243 mM, about 247 mM, about 250 mM, or any two of these values. The range (including the end point) or any value between the values.
  • the liquid formulation contains 0.02-0.05 mg/mL edetate disodium.
  • the concentration of edetate disodium in the liquid formulation is about 0.02 mg/mL, about 0.03 mg/mL, about 0.032 mg/mL, about 0.033 mg/mL, about 0.038 mg/mL, about 0.04 mg /mL, about 0.043 mg/mL, about 0.048 mg/mL, about 0.05 mg/mL, or the range between any two of these values (including the end point) or any value therein.
  • the liquid formulation contains 0.1-0.3 mg/mL polysorbate 80.
  • the concentration of polysorbate 80 in the liquid formulation is about 0.1 mg/mL, about 0.15 mg/mL, about 0.19 mg/mL, about 0.21 mg/mL, about 0.26 mg/mL, about 0.3 mg/mL. mL, or the range between any two of these values (including the end point) or any value therein.
  • the pH of the liquid formulation is 5.7-5.9. In some embodiments, the pH of the liquid formulation is about 5.7, about 5.73, about 5.75, about 5.78, about 5.8, about 5.83, about 5.87, about 5.9, or a range between any two of these values ( Including the end point) or any of these values.
  • the liquid formulation contains the following components:
  • the pH of the liquid formulation is 5.8.
  • the liquid formulation contains sterile water or water for injection.
  • the anti-CTLA-4 antibody or fragment thereof comprises HCDR1 shown in SEQ ID NO: 12, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 14 : LCDR1 shown in 15 and LCDR2 shown in SEQ ID NO: 16 and LCDR3 shown in SEQ ID NO: 17.
  • the heavy chain variable region of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 18 or is at least about 90% identical to the SEQ ID NO: 18
  • the amino acid sequence, the light chain variable region of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least about 90% identity with the SEQ ID NO: 19.
  • the heavy chain variable region of the anti-CTLA-4 antibody or fragment thereof has a sequence that is at least about 90%, about 92%, about 94%, or about 95% with the SEQ ID NO: 18 , About 97%, about 99%, about 100% identity, or the range (including the end point) between any two of these values, or any value therein, the light chain of the anti-CTLA-4 antibody or fragment thereof
  • the sequence contained in the variable region has at least about 90%, about 92%, about 94%, about 95%, about 97%, about 99%, about 100% identity with the SEQ ID NO: 19, or among these values The range (including the end point) between any two values of, or any value in it.
  • the heavy chain variable region of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region of the anti-CTLA-4 antibody or fragment thereof Contains the amino acid sequence shown in SEQ ID NO: 19.
  • the heavy chain of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least about 90% identity with the SEQ ID NO:1
  • the light chain of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least about 90% identity with the SEQ ID NO: 2.
  • the heavy chain of the anti-CTLA-4 antibody or fragment thereof has a sequence that is at least about 90%, about 92%, about 94%, about 95%, about 97% with the SEQ ID NO:1. %, about 99%, about 100% identity, or the range between any two of these values (including the end point) or any value therein, the sequence contained in the light chain of the anti-CTLA-4 antibody or fragment thereof It is at least about 90%, about 92%, about 94%, about 95%, about 97%, about 99%, about 100% identical to the SEQ ID NO: 2, or any two of these values The range between (including the end point) or any value in it.
  • the heavy chain of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain of the anti-CTLA-4 antibody or fragment thereof comprises SEQ ID NO: 2. The amino acid sequence shown.
  • the anti-CTLA-4 antibody or fragment thereof is a monoclonal antibody. In some embodiments, the anti-CTLA-4 antibody or fragment thereof is a fully human monoclonal antibody. In some embodiments, the anti-CTLA-4 antibody or fragment thereof is expressed by CHO cells. In some embodiments, the anti-CTLA-4 antibody or fragment thereof is expressed by CHO cells knocked out of ⁇ -(1,6)-fucose transferase. In some embodiments, the anti-CTLA-4 antibody or fragments thereof are expressed by CHO-BAT-KF fut8(-/-) cells (the aforementioned cells have been disclosed in the WO2019029713A1 patent). The anti-CTLA-4 antibody or its fragments are purified by conventional methods, such as low-speed centrifugation to separate cells and culture medium, high-speed centrifugation of the supernatant, and subsequent protein A affinity purification and ion exchange purification.
  • the level of fucosylation in the anti-CTLA-4 antibody or fragment thereof is ⁇ 5%. In some embodiments, the level of fucosylation in the anti-CTLA-4 antibody or fragment thereof is about 0, about 0.1%, about 0.3%, about 0.4%, about 0.6%, about 1.3%, about 1.9% , About 2.2%, about 2.8%, about 3.3%, about 3.7%, about 4.1%, about 4.5%, about 5%, or the range between any two of these values (including the end point) or any value therein .
  • the total amount of high mannose glycoforms in the anti-CTLA-4 antibody or fragment thereof is ⁇ 5%. In some embodiments, the total amount of high mannose glycoforms in the anti-CTLA-4 antibody or fragment thereof is about 0.1%, about 0.3%, about 0.9%, about 1.18%, about 1.7%, about 2.6%, about 3.3 %, about 4.1%, about 4.9%, about 4.99%, or the range between any two of these values (including the end point) or any value therein.
  • the total amount of sialylated glycoforms in the anti-CTLA-4 antibody or fragment thereof is ⁇ 3%. In some embodiments, the total amount of sialylated glycoforms in the anti-CTLA-4 antibody or fragment thereof is about 0.1%, about 0.2%, about 0.36%, about 0.8%, about 1.5%, about 2.2%, about 2. %, about 2.9%, about 2.99%, or the range between any two of these values (including the end point) or any value therein.
  • the present invention also provides the application of the liquid preparation in the preparation of medicines for treating CTLA-4 related diseases.
  • Another aspect of the present invention provides a method for treating CTLA-4 related diseases, the method comprising administering an effective dose of the liquid formulation to a patient.
  • CTLA-4 related diseases include cancer, and the liquid formulation can be used to treat cancer such as rejection of transplantable tumors.
  • the CTLA-4 related disease is melanoma, non-small cell lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma, or kidney cancer.
  • the liquid formulation can be administered by injection or infusion; the site of administration is parenteral, intravenous, mucosal, sublingual, muscle, intradermal, nasal, abdominal, intraarterial, or subcutaneous.
  • the patient is a mammal. In some embodiments, the patient is a human.
  • the present invention also discloses a preparation method of the liquid preparation, which comprises mixing the components in a solvent to prepare a liquid preparation.
  • the method includes the following steps:
  • step (3) Prepare the auxiliary material mother liquor containing buffers, stabilizers, surfactants and chelating agents, and add the aseptically filtered auxiliary material mother liquor to the antibody concentrate prepared in step (2) to obtain a liquid preparation.
  • the present invention develops a liquid formulation that can significantly inhibit the freezing/rejuvenation cycle by selecting an appropriate buffer system, optimizing stabilizers, chelating agents, and adding surfactants.
  • the formation of acid peaks, dimers, polymers, degradation products and insoluble particles during long-term storage and temperature changes.
  • the anti-CTLA-4 antibody or fragment thereof of the present invention has good stability after repeated freezing and thawing for at least 5 times in the above preparation, and can be stored stably for at least 6 months at room temperature.
  • the liquid preparation of the present invention can be used to stably store the anti-CTLA-4 antibody or its fragments for clinical treatment, and is of great significance for the treatment of CTLA-4 related diseases.
  • the anti-CTLA-4 antibody (or preparation) of the present invention can be used for the treatment of tumor or cancer.
  • Figure 1 shows the comparison of the glycoforms of Antibody 2 and Antibody 1; the triangles represent fucose, and Antibody 2 contains substantially no fucose.
  • Figure 2 shows the ELISA binding curve of Antibody 2, Antibody 1 and CTLA-4 antigen; in the figure, sample represents an antibody sample.
  • Figure 3 shows The binding curve of Antibody 2, Antibody 1 and CTLA-4 antigen on Biacore.
  • Figure 4 shows a schematic diagram of the in vitro biological activity model of anti-CTLA-4 antibody; in the figure, promotor is represented as a promoter, and luciferase is represented as a luciferase gene.
  • Figure 5 shows the plasmid map of pCMV2-OKT3.
  • Figure 6 shows that Antibody 2 and Antibody 1 both show activity to enhance T cell function on the activity model; the sample in the figure represents an antibody sample.
  • Figure 7 shows the binding and dissociation sensing curves of Antibody 2, Antibody 1 and Fc ⁇ RIIIa 158V.
  • Figure 8 shows the plasmid map of pCDH-Fc ⁇ RIIIa 158V FL-Puro.
  • Figure 9 shows that antibody 2 has higher in vitro ADCC activity than antibody 1 on the ADCC reporter gene model; in the figure, sample represents an antibody sample.
  • Figure 10A and Figure 10B respectively show the weight change trend and tumor inhibition effect of Antibody 2 and Antibody 1 on CTLA-4 humanized MC38 tumor-bearing mice after administration; the arrow in the figure represents the time of each administration.
  • Figure 11A, Figure 11B, Figure 11C and Figure 11D respectively show that on CTLA-4 humanized MC38 tumor-bearing mice, The ratio of CD4 + , CD8 + , and Treg cells in peripheral blood mononuclear cells to CD45 cells and the ratio of CD8 + /Treg after the administration of antibody 1 and antibody 2.
  • Antibody should include but is not limited to immunoglobulin, which is a large "Y"-shaped protein produced mainly by plasma cells, which is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses.
  • the antibody recognizes a unique molecule called an "antigen" on the pathogen through the variable region of the Fab.
  • Each tip of the "Y” structure of an antibody contains a paratope to a specific epitope on the antigen, allowing the two structures to be accurately joined together.
  • antibodies can mark microorganisms or infected cells so that the immune system can attack them, or they can directly neutralize the target.
  • the ability of an antibody to communicate with other components of the immune system is mediated through its Fc region, which contains conserved glycosylation sites that participate in these interactions.
  • the production of antibodies is the main function of the humoral immune system.
  • Antibody fragment includes (comprises) a portion of a full-length antibody, usually including its antigen binding region.
  • antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Identity and “homology” refer to the sequence similarity between two peptides or between two nucleic acid molecules.
  • the homology can be determined by comparing the positions that can be aligned in each sequence. When a position in the sequence being compared is occupied by the same base or amino acid, then the molecules are homologous at that position.
  • the degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • At least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, About 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these The range (including endpoints) between any two values in the numerical value or any value therein.
  • amino acid refers to an organic compound containing both amino and carboxyl groups, such as ⁇ -amino acids, which can be encoded by nucleic acids directly or in the form of precursors.
  • a single amino acid is encoded by a nucleic acid composed of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The same amino acid encoded by different codons is called “the degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine Acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
  • Constant amino acid substitution refers to the substitution of an amino acid residue by another amino acid residue containing a side chain (R group) that is chemically similar (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions are unlikely to substantially change the functional properties of the protein.
  • amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: serine and threon Acid; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.
  • the number of "conservative amino acid substitutions of VL and VH” can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15 conservative amino acid substitutions, or the range (including endpoints) between any two of these values, or any value therein.
  • the number of “conservative amino acid substitutions in the heavy or light chain” can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, or about 10. One, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, About 38, about 41, about 45 conservative amino acid substitutions, or the range (including endpoints) between any two of these values, or any value therein.
  • Antibodies can be of different types, called isotypes or classes. In placental mammals, there are five antibody isotypes, called IgA, IgD, IgE, IgG, and IgM. They are each named with the "Ig” prefix, representing immunoglobulins (sometimes used interchangeably with “antibodies"), and their biological properties, functional positions, and ability to handle different antigens are different. The different suffixes of the antibody isotype indicate the different types of heavy chains contained in the antibody. Each heavy chain category is named in alphabetical order: ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , corresponding to the production of IgA, IgG, IgD, IgE and IgM. . There are two types of immunoglobulin light chains in mammals, called ⁇ ( ⁇ ) and ⁇ ( ⁇ ). Generally, each antibody contains two identical light chains; in mammals, only one type of light chain ⁇ or ⁇ exists for each antibody.
  • Immunoglobulin G is a type of antibody. IgG is about 75% of the serum antibodies in the human body and is the most common type of antibody in the blood circulation. There are four IgG subclasses in the human body (IgG1, 2, 3, and 4), named in order of their abundance in the serum (IgG1 is the most abundant).
  • the arms of "Y" contain sites that can bind to antigens to recognize specific foreign objects.
  • This antibody region is called the Fab region. It consists of a constant region and a variable region of each heavy and light chain of an antibody.
  • the variable domain is also called the FV region, and is the most important region for antigen binding.
  • each of the three regions on the light chain variable region (VL) and the heavy chain variable region (VH) is responsible for binding to the antigen, and these regions are called complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • the base of "Y” plays a role in regulating the activity of immune cells.
  • This region is called the Fc region and consists of two heavy chains, which contribute two or three constant regions according to the class of the antibody.
  • the Fc region ensures that each antibody produces an appropriate immune response to a given antigen by binding to a specific class of Fc receptors and other immune molecules (such as complement proteins). In this way, it mediates different physiological effects, including recognition of opsonized particles (binding to Fc ⁇ R), cell lysis (binding to complement), and degranulation of mast cells, basophils and eosinophils (binding to Fc ⁇ R) .
  • the CDRs defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared with each other. Nevertheless, it is within the scope of the present invention to apply any definition to refer to the CDRs of antibodies or variants thereof.
  • the exact residue number that contains a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDR contains based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those of ordinary skill in the art can apply the "Kabat numbering" system to any variable region sequence without relying on other experimental data other than the sequence itself. "Kabat number” refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest” (1983). Antibodies can also use the EU or Chothia numbering system.
  • antigen refers to a substance that specifically binds to antibodies or T lymphocyte antigen receptors. They are substances that stimulate the production of antibodies or are recognized by antibodies. Sometimes the antigen is part of the host itself in autoimmune diseases.
  • antigen originally described a structural molecule that specifically binds to an antibody. It was later expanded to refer to any molecule or linear molecular fragment that can be recognized by the highly variable antigen receptors (B cell receptors or T cell receptors) of the adaptive immune system.
  • a “monoclonal antibody” is an antibody made from the same immune cell, which is all clones of a single parent cell.
  • Monoclonal antibodies can have monovalent affinity because they bind to the same epitope (the part of the antigen recognized by the antibody).
  • polyclonal antibodies bind to multiple epitopes and are usually secreted by several different plasma cells. It can also be engineered into a bispecific monoclonal antibody by increasing the therapeutic target of a single monoclonal antibody to two epitopes.
  • Monoclonal antibodies can be produced by hybridomas, recombination, transgenics, or other techniques known to those skilled in the art.
  • Humanized antibodies refer to antibodies from non-human species whose protein sequences have been modified to increase their similarity to naturally-occurring human antibody variants.
  • the "humanization” process is generally applied to monoclonal antibodies developed for human administration (for example, antibodies developed as anti-cancer drugs).
  • humanization is also required.
  • Humanized mice are mice that carry functional human genes, cells, tissues and/or organs. Humanized mice are commonly used as small animal models in biological and medical research for human therapy. Immunodeficient mice are often used as recipients of human cells or tissues because they can accept heterologous cells relatively easily due to lack of host immunity. This humanized mouse model can be used to simulate the human immune system under healthy and pathological conditions, and can evaluate therapeutic candidates in an in vivo environment related to human physiology.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC is independent of the immune complement system, which also dissolves the target but does not require any other cells.
  • ADCC requires an effector cell, which is known to be a natural killer (NK) cell that normally interacts with IgG antibodies.
  • NK natural killer
  • macrophages, neutrophils, and eosinophils can also mediate ADCC.
  • eosinophils use IgE antibodies to kill certain parasites called worms.
  • ADCC is part of the adaptive immune response because it relies on previous antibody responses.
  • Fc region is the tail region of an antibody, which interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
  • Fc region is composed of two identical protein fragments, derived from the second and third constant regions of the two heavy chains of the antibody; IgM and IgE Fc regions are in each polypeptide chain It contains three heavy chain constant domains (CH domains 2-4).
  • the Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of Fc fragments is necessary for Fc receptor-mediated activity, and different glycotypes have different effects on the pharmaceutical properties of therapeutic antibodies.
  • Fc receptor or “FcR” is a protein found on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, Basophils, human platelets and mast cells, etc., all help protect the function of the immune system.
  • the name of the Fc receptor is derived from its binding specificity to the Fc region of an antibody.
  • Fc receptors bind to antibodies attached to infected cells or invading pathogens. Their activity works through phagocytes and cytotoxic cells destroying microorganisms, or through antibody-mediated phagocytosis to infect cells, or through antibody-dependent cell-mediated cytotoxicity.
  • Some viruses, such as flavivirus use Fc receptors to help them infect cells through a mechanism called antibody-dependent infection enhancement.
  • Fc receptors There are several different types of Fc receptors, which are classified based on the type of antibody they recognize.
  • the Latin alphabet used to identify the antibody type is converted to the corresponding Greek alphabet, which is located after the "Fc" part of the name.
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • Fc ⁇ R Fc- ⁇ receptors
  • the types of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signaling properties of each receptor.
  • Fc ⁇ R All Fc ⁇ receptors (Fc ⁇ R) belong to the immunoglobulin superfamily and are the most important Fc receptors that induce microbial phagocytosis. This family includes several members, FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). Because of their different molecular structures, their antibody affinities are different. For example, FcyRI binds IgG more strongly than FcyRII or FcyRIII. FcyRI also has an extracellular part composed of three immunoglobulin (Ig)-like domains, and has more domains than FcyRII or FcyRIII.
  • Ig immunoglobulin
  • Fc ⁇ RI Fc ⁇ RI to bind to a single IgG molecule (or monomer), but all Fc ⁇ receptors must bind to multiple IgG molecules in the immune complex to be activated.
  • Fc- ⁇ receptors have different affinities for IgG, and different IgG subclasses have unique affinities for each Fc ⁇ receptor.
  • These interactions are further regulated by the glycans (oligosaccharides) at positions CH2-84.4 of IgG. For example, by creating steric hindrance, fucose containing CH2-84.4 glycans reduces the IgG affinity for Fc ⁇ RIIIA.
  • Cancer refers to a group of diseases involving abnormal cell growth, which may invade or spread to other parts of the body. Cancer is in contrast to benign tumors, which do not spread to other parts of the body. Possible signs and symptoms include lumps, abnormal bleeding, long-term coughing, unexplained weight loss, and changes in bowel movements.
  • Tumor-bearing mice are divided into primary models and transplanted tumor models.
  • the most commonly used in research is the xenograft model, which mainly includes orthotopic xenografts and subcutaneous xenografts. In short, it is to inject relevant tumor cells into mice by in situ injection or subcutaneous injection to make them tumorigenic.
  • administering means that the drug may be absorbed by oral, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, nasal, abdominal, intraarterial, subcutaneous, or through any other combination with existing technology The way of administration is completed. In one embodiment of the invention, the administration is systemic.
  • Effective amount refers to the amount needed to achieve the therapeutic goal.
  • the effective amount is related to the binding affinity of the antibody to its specific antigen, the severity of the disease, disorder or condition, the route of administration, and the rate at which the antibody administered in the subject is depleted from the free volume.
  • the therapeutically effective amount of the antibody of the present invention can be from about 0.01 mg/kg to about 15 mg/kg of the patient’s body weight, from about 0.1 mg/kg to about 10 mg/kg of the patient’s body weight, or from about 1 mg/kg to about 1 mg/kg.
  • the frequency of administration may be, for example, once a week to once a month, such as once every three weeks.
  • Patient generally refers to any patient in need of diagnosis, prognosis or treatment, especially mammalian patients, including humans, cats, guinea pigs, rabbits, rats, mice, horses, cows, and the like.
  • glycotype is an isotype of a protein, which differs only in the number or type of attached glycans.
  • Glycoproteins are usually composed of many different sugar forms, in which the attached sugars or oligosaccharides are changed. These modifications may be due to differences in biosynthesis in the glycosylation process, or due to the action of glycosidases or glycosyltransferases.
  • the glycoform can be detected by detailed chemical analysis of the separated glycoform or by lectin affinity chromatography and lectin affinity electrophoresis.
  • glycosylation is a reaction in which a carbohydrate (ie, a glycosyl donor) is attached to a hydroxyl group or other functional group of another molecule (the glycosyl acceptor).
  • glycosylation mainly refers specifically to the enzymatic process of attaching glycans to proteins or other organic molecules. This enzymatic process produces a basic biopolymer (as well as DNA, RNA and protein) found in cells.
  • Glycosylation is a form of co-translation and post-translational modification. Glycans play a variety of structural and functional roles in membranes and secreted proteins. Most proteins synthesized in the rough endoplasmic reticulum undergo glycosylation.
  • glycosylation is also present in the cytoplasm and nucleus as an O-GlcNAc modification. Common types of glycosylation include O-glycosylation and N-glycosylation.
  • the term "in need” means that the patient has been identified as in need of a particular method or treatment. In some embodiments, identification can be performed by any diagnostic method. In any of the methods and treatments described herein, the patient may require it.
  • the amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system constituting the buffer.
  • the molar concentration is used as the unit of the amount of the buffer, and its value refers to the molar concentration of the buffer pair in the buffer system of the buffer.
  • a histidine buffer composed of histidine and histidine hydrochloride is used as a buffer
  • a given concentration of histidine buffer is the amount of histidine and histidine hydrochloride Combination concentration (e.g.
  • histidine is 8mM, histidine hydrochloride is 12mM; or histidine is 6mM, histidine hydrochloride is 14mM; or histidine is 8.46mM, histidine hydrochloride is 11.54mM etc.).
  • histidine hydrochloride also known as histidine hydrochloride
  • histidine hydrochloride can be anhydrous histidine hydrochloride or histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate
  • "12mM histidine hydrochloride” can be a 1L liquid preparation containing 12mmol histidine hydrochloride or 12mmol histidine hydrochloride monohydrate; for example, adding "2.52g histidine hydrochloride” to the preparation "Monohydrate” is equivalent to adding 2.3g of histidine hydrochloride to the formulation.
  • both trehalose and corresponding amounts of trehalose dihydrate can be added to the preparation; for example, "225mM trehalose” can be a 1L liquid preparation containing 225mmol trehalose or 225mmol trehalose dihydrate; for example, Adding "85g of trehalose dihydrate” to the formulation is equivalent to adding 76.9g of trehalose to the formulation.
  • edetate disodium can be added to the preparation, and a corresponding amount of edetate disodium dihydrate or edetate can be added; for example, "0.033mg/mL edetate disodium” can be added Add 0.037g edetate disodium dihydrate or 0.033g edetate disodium or 0.029g edetate to 1L liquid preparation.
  • Treatment of a patient's disease refers to (1) preventing the disease from appearing in patients who are predisposed or have not yet shown symptoms of the disease; (2) inhibiting the disease or symptoms or preventing their development; or (3) reducing or eliminating the disease or symptoms Or cause its degradation.
  • Stability means that in the liquid preparation containing the antibody, the antibody or its fragments do not aggregate, degrade or fragment under given production, preparation, transportation and/or storage conditions, or only rarely undergo aggregation, degradation or fragmentation.
  • “Stable” formulations maintain biological activity under given conditions of production, preparation, transportation, and/or storage. The degree of aggregation, degradation or fragmentation of the preparation can be measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS (NR), lamp detection and turbidity, insoluble particles, DLS detection particle size, etc., thereby The stability of the antibody is evaluated.
  • a “stable" formulation means that the formulation is stable for storage at room temperature for at least 3 months, at least 6 months, or at least 12 months.
  • the present invention provides a monoclonal antibody against CTLA-4, the monoclonal antibody comprising
  • Heavy chain CDR1 which comprises the sequence shown in SEQ ID NO: 12, and has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, About 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two values (including endpoints) or any value therein) sequence identity, or The sequence has, or consists of, one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) of amino acid sequences,
  • Heavy chain CDR2 which includes the sequence shown in SEQ ID NO: 13, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% with the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of them,
  • the heavy chain CDR3 which comprises the sequence shown in SEQ ID NO: 14, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% with the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of them,
  • Light chain CDR1 which includes the sequence shown in SEQ ID NO: 15, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% with the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with or consisting of one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) of amino acid sequences,
  • Light chain CDR2 (LCDR2), which includes the sequence shown in SEQ ID NO: 16, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% of the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with or consists of one or more (preferably about 1, about 2, or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) of amino acid sequences, and
  • Light chain CDR3 which includes the sequence shown in SEQ ID NO: 17, has at least about 80%, about 90%, preferably at least about 91%, about 92%, about 93%, about 94% of the sequence , About 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity, or a sequence with said sequence Compared with an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of them;
  • the heavy chain variable region which comprises the sequence shown in SEQ ID NO: 18, has at least about 80% of the sequence, preferably at least about 85%, about 90%, about 91%, about 92 %, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two values (including endpoints) or any value therein) sequence identity
  • a sexual sequence or an amino acid sequence with one or more (preferably about 1, about 2, or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence, or consisting of, with
  • the light chain variable region (VL), which comprises the sequence shown in SEQ ID NO: 19, is at least about 80%, preferably at least about 85%, about 90%, about 91%, about 92%, about A sequence of 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) sequence identity , Or an amino acid sequence with one or more (preferably about 1, about 2 or about 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence, or consisting of them;
  • the monoclonal antibody has a reduced level of fucosylation.
  • the monoclonal antibody comprises a heavy chain and a light chain
  • the heavy chain comprises the amino acid sequence of SEQ ID NO:1, which has at least about 80% of the amino acid sequence of SEQ ID NO:1, preferably at least About 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93 %, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or the range between any two values (including endpoints) or any value therein) homology sequence, Or consist of it;
  • the light chain includes the amino acid sequence shown in SEQ ID NO: 2, which has at least about 80%, preferably at least about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% , About 98% or about 99% (or the range between any two values (including endpoints) or any value therein) homology sequence, or consisting of the sequence.
  • the heavy chain has at least about 80% of the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 3, preferably at least about 81%, about 82%. %, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, A nucleotide sequence encoding about 95%, about 96%, about 97%, about 98%, or about 99% (or a range between any two values (including endpoints) or any value therein) homology, said The light chain has at least about 80%, preferably at least about 81%, about 82%, about 83%, or about 84% of the nucleotide sequence shown in SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 4 , About 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 9
  • the fucosylation level is 0%-20%, such as about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6% , About 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20%, or a range between any two values (including endpoints) or any value therein.
  • the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is ⁇ 5% and/or the total amount of sialylated glycoforms is ⁇ 3%. In some embodiments, the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is about 0.1%, about 0.3%, about 0.9%, about 1.18%, about 1.7%, about 2.6%, about 3.3%, About 4.1%, about 4.9%, about 4.99%, or a range between any two values (including the endpoints) or any value therein.
  • the total amount of sialylated glycoforms in the Fc region of the anti-monoclonal antibody is about 0.1%, about 0.2%, about 0.36%, about 0.8%, about 1.5%, about 2.2%, about 2.7%, Approximately 2.9%, 2.99%, or any range (including endpoints) between two values or any value therein.
  • the monoclonal antibody is IgG1, and the total amount of high mannose glycoforms in the CH2 region of the monoclonal antibody is ⁇ 5% and/or the total amount of sialylated glycoforms is ⁇ 3%.
  • the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is less than 3% and/or the total amount of sialylated glycoforms is less than 2%.
  • the monoclonal antibody is IgG1, and the total amount of high mannose glycoforms in the CH2 region of the monoclonal antibody is ⁇ 3% and/or the total amount of sialylated glycoforms is ⁇ 2%.
  • the total amount of high mannose glycoforms in the Fc region of the monoclonal antibody is less than 2% and/or the total amount of sialylated glycoforms is less than 1%.
  • the monoclonal antibody is IgG1, and the total amount of high mannose glycoforms in the CH2 region of the monoclonal antibody is less than 2% and/or the total amount of sialylated glycoforms is less than 1%.
  • the fucosylation level of the Fc region of the monoclonal antibody is 0%-10%.
  • the monoclonal antibody is IgG1, and the fucosylation level of the CH2 region of the monoclonal antibody is 0%-10%.
  • the fucosylation level of the Fc region of the monoclonal antibody is 0%-5%. In some embodiments, the fucosylation level of the Fc region of the anti-monoclonal antibody is about 0, about 0.1%, about 0.3%, about 0.4%, about 0.6%, about 1.3%, about 1.9%, about 2.2%, about 2.8%, about 3.3%, about 3.7%, about 4.1%, about 4.5%, about 5%, or a range between any two values (including endpoints) or any value therein. In some embodiments, the monoclonal antibody is IgG1, and the fucosylation level of the CH2 region of the monoclonal antibody is 0%-5%.
  • the monoclonal antibody is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line, such as the CHO-BAT-KF cell line disclosed in PCT/CN2018/100008.
  • the present invention provides a pharmaceutical composition or fusion protein, which comprises the monoclonal antibody of the present invention.
  • the pharmaceutical composition is in a form suitable for oral, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, nasal, abdominal, intraarterial, and subcutaneous absorption administration. In some embodiments, the pharmaceutical composition is in a form suitable for systemic administration.
  • the present invention provides an antibody conjugate, which includes the monoclonal antibody of the present invention, and a coupling part coupled to the monoclonal antibody, and the coupling part is a purification tag (such as a His tag). ), a cytotoxic agent, a structural part that prolongs the half-life of the monoclonal antibody, or a detectable label, preferably, the coupling part is a radioisotope, a chemiluminescent agent, an enzyme, a serum albumin (such as human or mouse serum Albumin), or polyethylene glycol.
  • a purification tag such as a His tag
  • the coupling part is a radioisotope, a chemiluminescent agent, an enzyme, a serum albumin (such as human or mouse serum Albumin), or polyethylene glycol.
  • the anti-CTLA-4 antibodies of the invention are monovalent antibodies or multivalent antibodies.
  • the present invention provides multispecific antibodies (such as bispecific antibodies), which comprise the monoclonal antibodies of the present invention, and antibodies or antigen-binding fragments directed against other antigens and/or other epitopes.
  • the present invention provides a kit, which includes the monoclonal antibody, antibody conjugate, multispecific antibody or fusion protein of the present invention.
  • the kit further includes a second antibody, a cytotoxic agent, a chemotherapeutic agent, and a radiotherapy agent.
  • the second antibody specifically recognizes the monoclonal antibody; optionally, the second antibody It also includes detectable labels, such as fluorescein, metal ions, biotin, radioisotopes, chemiluminescent substances, enzymes, or polyethylene glycols.
  • the present invention provides the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody, which is used for the treatment of cancer or for rejection of transplantable tumors, such as melanoma, non-small cell Lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • transplantable tumors such as melanoma, non-small cell Lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • the present invention provides a method for treating cancer or for rejecting transplantable tumors, such as melanoma, non-small cell lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma, or kidney cancer, which includes giving The patient is given an effective amount of the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody.
  • transplantable tumors such as melanoma, non-small cell lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma, or kidney cancer
  • the present invention provides a method for enhancing the proliferation response of T cells, which comprises using the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody of the present invention to block CTLA-4 and B7 Combine.
  • the present invention provides a method for reducing tumor infiltration of CTLA-4 expressing Treg cells in a patient, comprising administering to the patient a therapeutically effective amount of the monoclonal antibody, fusion protein, antibody conjugate or multispecific Antibody.
  • the present invention provides that the monoclonal antibody, fusion protein, antibody conjugate or multispecific antibody is prepared for the treatment of cancer or for rejection of transplantable tumors, such as melanoma, non-small cell Application in medicines for lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • transplantable tumors such as melanoma, non-small cell Application in medicines for lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma or kidney cancer.
  • the present invention discloses methods or uses of anti-CTLA-4 antibodies or antigen-binding fragments for treating tumors or cancers.
  • the anti-CTLA-4 antibody or antigen-binding fragment agent is used to treat tumors or cancer.
  • the anti-CTLA-4 antibody e.g., antibody 2 or antigen-binding fragment is expressed by a cell line in which the ⁇ -(1,6)-fucosyltransferase gene is knocked out, as disclosed in PCT/CN2018/100008 CHO-BAT-KF cell line.
  • Antibody 2 is expressed by the CHO-BAT-KF cell line.
  • the method or use includes administering an effective amount of an anti-CTLA-4 antibody or antigen-binding fragment to a patient in need.
  • the anti-CTLA-4 antibody is antibody 1 or antibody 2.
  • an anti-CTLA-4 antibody e.g., antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the effective dose of anti-CTLA-4 antibody administered is about 7 mg to 180 mg per dose.
  • the patient has a tumor or cancer.
  • tumors and cancers include, but are not limited to, blood cancers, solid tumors.
  • blood cancers include, but are not limited to, leukemia, lymphoma, and myeloma.
  • leukemias include acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and myeloproliferative disease/tumor (MPDS ).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MPDS myeloproliferative disease/tumor
  • lymphomas include Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).
  • myeloma includes multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bens-Jones myeloma.
  • solid tumors include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer.
  • tumors and cancers are locally advanced or metastatic malignant solid tumors that are pathologically diagnosed for which there is no effective treatment.
  • the present invention discloses a method for treating tumor or cancer in a patient in need, which comprises administering an effective amount of anti-CTLA-4 antibody, wherein the effective amount of anti-CTLA-4 antibody administered is about 7 mg To 180mg per treatment cycle.
  • a treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including Endpoint) or any of them.
  • the anti-CTLA-4 antibody is Antibody 1, Antibody 2.
  • Antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the anti-CTLA-4 antibody can be formulated into a pharmaceutical composition and administered to the patient in a variety of forms suitable for the chosen route of administration, such as parenteral, intravenous (iv), Intramuscular, local or subcutaneous.
  • the anti-CTLA-4 antibody can be infused intravenously.
  • the amount of anti-CTLA-4 antibody administered will depend on the nature of the drug, the degree of internalization, transport, and release of the drug triggered by the cell surface, as well as the disease to be treated and the patient's condition (such as age, gender, weight, etc.).
  • the anti-CTLA-4 antibody (such as antibody 1 or 2) administered per administration is about 0.1 mg/kg to 16 mg/kg or a formulation containing this dose of anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody administered per administration is about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.9 mg/kg.
  • the anti-CTLA-4 antibody (such as antibody 1 or 2) administered per administration is about 0.1 mg/kg to 2.5 mg/kg or a formulation containing this dose of anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody administered per administration is about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg.
  • the administration is once about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, or The range (including endpoints) between any two of these values or any value therein.
  • it is administered once every about 3 weeks, and the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles. Cycles or 10 cycles.
  • the administration is once about 4 weeks, and the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 Cycles or 10 cycles.
  • it is administered once every about 5 weeks, and the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles. Cycles or 10 cycles.
  • the present invention discloses a method for treating tumor or cancer, the method comprising: administering about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg to a patient in need every 3 weeks /kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, About 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, about 1.9mg/kg, about 2mg /kg, about 2.1mg/kg, about 2.2mg/kg, about 2.3mg/kg, about 2.4mg/kg, about 2.5mg/kg of anti-CTLA-4 antibody, or a preparation containing this dose of anti-CTLA-4 antibody .
  • the present invention discloses a method for treating tumor or cancer, the method comprising: administering about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg to a patient in need every 3 weeks /kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, About 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, about 1.9mg/kg, about 2mg /kg, about 2.1mg/kg, about 2.2mg/kg, about 2.3mg/kg, about 2.4mg/kg, about 2.5mg/kg of antibody 1 or 2, or preparations containing this dose of antibody 1 or 2; treatment
  • the cycle is 4 cycles.
  • the present invention discloses a method for treating tumor or cancer, which comprises administering an effective amount of anti-CTLA-4 antibody (or preparation) to a patient in need; wherein, the effective amount of anti-CTLA-4 antibody For a single administration of about 7mg to 180mg (or a preparation containing this dose of anti-CTLA-4 antibody).
  • the dosage schedule and mode of administration depend on the benefit and risk assessment of anti-CTLA-4 antibodies (or preparations) in certain patient groups and general clinical practice guidelines.
  • the effective amount of anti-CTLA-4 antibody administered to the patient per treatment cycle is about 7 mg to 180 mg (or a formulation containing this dose of anti-CTLA-4 antibody).
  • a treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including endpoints) or where Any value.
  • the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, or 6 cycles.
  • the effective amount of an anti-CTLA-4 antibody (such as antibody 1 or 2) administered to a patient per treatment cycle is about 6 mg, about 10 mg, about 12 mg, about 18 mg, about 24 mg, about 30 mg, about 40 mg, About 50mg, about 60mg, about 100mg, about 120mg, about 180mg, about 200mg, about 250mg, about 290mg, about 300mg, about 330mg, about 380mg, about 400mg, about 434mg, about 480mg, about 500mg, about 567mg, about 580mg , About 600 mg, about 700 mg, about 800 mg, about 900 mg, about 960 mg, or the range between any two of these values (including endpoints) or any value therein, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 7 mg, about 8 mg, about 10 mg, about 12 mg, about 18 mg, about 24 mg, about 30 mg, About 40 mg, about 50 mg, about 60 mg, about 68 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, or these The range between any two of the values (including endpoints) or any value therein, or a preparation containing this dose of anti-CTLA-4 antibody.
  • one treatment cycle is one administration every 1 week to 7 weeks.
  • the treatment cycle is 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, or a range between any two of these values (including endpoints) Or any of them.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 7 mg to about 120 mg, or a preparation containing this dose of anti-CTLA-4 antibody; of which, one The treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of an anti-CTLA-4 antibody (such as antibody 1 or 2) administered to a patient per treatment cycle is about 7 mg, about 10 mg, about 12 mg, about 18 mg, about 20 mg, about 24 mg, about 28 mg, About 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, or a range between any two of these values (including endpoints) or any value therein, or A preparation containing this dose of anti-CTLA-4 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles Cycle or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 120 mg to 180 mg, or a preparation containing this dose of anti-CTLA-4 antibody; of which, one treatment The cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, Or the range between any two of these values (including endpoints) or any value therein, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, or about 3 weeks. It is administered once every week or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 6.9 mg to 7.2 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 7 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 10 mg to 13 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 12 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 20 mg to 24 mg, or a formulation containing this dose of anti-CTLA-4 antibody. In some embodiments, the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 22 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 28 mg to 33 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 30 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 59 mg to 64 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 60 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 95 mg to 113 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 100 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 110 mg to 123 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient during each treatment cycle is about 120 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 127 mg to 133 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 130 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 132 mg to 144 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient during each treatment cycle is about 140 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 140 mg to 155 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective dose of anti-CTLA-4 (such as antibody 1 or 2) antibody administered to the patient during each treatment cycle is about 150 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient per treatment cycle is about 177 mg to 180 mg, or a formulation containing this dose of anti-CTLA-4 antibody.
  • the effective amount of anti-CTLA-4 antibody (such as antibody 1 or 2) administered to the patient in each treatment cycle is about 180 mg, or a preparation containing this dose of anti-CTLA-4 antibody; wherein, one treatment cycle is It is administered once in about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, and the treatment cycle is 2 cycles, 3 cycles, 4 cycles, or 5 cycles.
  • the effective amount of anti-CTLA-4 antibody administered is about 7 mg to 120 mg once every 3 weeks for 4 treatment cycles. In some embodiments, the effective amount of anti-CTLA-4 antibody administration is about 7 mg, about 10 mg, about 12 mg, about 20 mg, about 26 mg, about 30 mg, about 35 mg, about 40 mg, about 44 mg, about 50 mg, about 58 mg, about 60 mg, about 69 mg, about 70 mg, about 77 mg, about 85 mg, about 99 mg, about 110 mg, about 114 mg, or about 120 mg once every 3 weeks for 4 treatment cycles. In some embodiments, the effective amount of anti-CTLA-4 antibody administered is about 7 mg, about 12 mg, about 30 mg, about 60 mg, or about 120 mg once every 3 weeks for 4 treatment cycles.
  • the effective amount of anti-CTLA-4 antibody administered is about 120 mg to 180 mg once every 3 weeks. In some embodiments, the effective amount of anti-CTLA-4 antibody administered is about 120 mg, about 130 mg, about 134 mg, about 140 mg, about 146 mg, about 150 mg, about 154 mg, about 160 mg, about 164 mg, about 170 mg, about 173 mg, about 1760mg, about 180mg once every 3 weeks, 4 treatment cycles. In some embodiments, the effective amount of anti-CTLA-4 antibody administered is about 130 mg, 140 mg, about 160 mg, or about 180 mg once every 3 weeks for 4 treatment cycles.
  • the patient is administered the anti-CTLA-4 antibody (or formulation) once per treatment cycle.
  • the anti-CTLA-4 antibody (or formulation) is administered multiple times during each treatment cycle, for example, 2 times, 3 times, 4 times, or 5 times.
  • the patient can only be administered 1 or 4 times per treatment cycle.
  • the patient receives treatment for one treatment cycle. In some embodiments, the patient receives multiple (e.g., 2, 3, 4, 5, or 6) treatment cycles. In some embodiments, the patient is treated for 4 treatment cycles. In some embodiments, the patient receives treatment until the condition is relieved and treatment is no longer needed.
  • the present invention discloses a method for treating tumor or cancer, the method comprising: administering about 7 mg to 30 mg, about 30 mg to 60 mg, about 60 mg to 120 mg, every 3 weeks to a patient in need, About 120 mg to 180 mg, such as about 7 mg, about 10 mg, about 12 mg, about 18 mg, about 30 mg, 60 mg, about 80 mg, about 100 mg, about 120 mg, about 150 mg, about 160 mg, about 180 mg of anti-CTLA-4 antibody, or containing it Dose anti-CTLA-4 antibody preparation.
  • the anti-CTLA-4 antibody is Antibody 1.
  • the anti-CTLA-4 antibody is antibody 2.
  • the patient's symptoms are relieved after a single dose is administered. In some embodiments, after a single dose administration, the patient's symptoms do not get the expected alleviation, and then about 7 mg to 180 mg of anti-CTLA-4 antibody is administered to the patient until the patient's symptoms are alleviated.
  • the anti-CTLA-4 antibody (or formulation) is administered by subcutaneous (s.c.) injection, intraperitoneal (i.p.) injection, parenteral injection, intraarterial injection, or intravenous (i.v.) injection.
  • the anti-CTLA-4 antibody (or formulation) is administered by infusion.
  • the anti-CTLA-4 antibody (or formulation) is administered as a bolus injection.
  • the anti-CTLA-4 antibody (or formulation) is administered by intravenous (i.v.) infusion (ie, intravenous infusion).
  • intravenous infusion ie, intravenous infusion
  • the duration of intravenous infusion is about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 81 minutes, about 87 minutes, about 90 minutes, about 95 minutes. Minutes, or the range (including endpoints) between any two of these values, or any value therein.
  • the duration of intravenous infusion is> 60 minutes.
  • anti-CTLA-4 antibodies are used in combination with other treatment methods to treat tumors or cancers, such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted therapy, biological therapy, and surgical therapy.
  • anti-CTLA-4 antibodies are combined with other tumor or cancer therapeutic agents to treat tumors or cancers, such as hormones, antibodies for the treatment of tumors or cancers, and the like.
  • the present invention discloses the application of anti-CTLA-4 antibodies in the preparation of drugs for the treatment of tumors or cancers.
  • the drugs used to treat tumors or cancers include anti-CTLA-4 antibodies.
  • the anti-CTLA-4 antibody is antibody 1 or 2.
  • Antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the present invention also discloses a kit, which contains an anti-CTLA-4 antibody (or preparation) and instructions for instructing patients in need to administer the anti-CTLA-4 antibody (or preparation).
  • the anti-CTLA-4 antibody is antibody 1 or 2.
  • Antibody 2 is expressed by an ⁇ -(1,6)-fucosyltransferase gene knockout cell line.
  • the present invention provides a liquid formulation containing an anti-CTLA-4 antibody or a fragment thereof.
  • the liquid formulation contains the following components: 4-80 mg/mL anti-CTLA-4 antibody or fragments thereof, buffer, stabilizer, chelating agent, surfactant, water; the pH of the liquid formulation is 5- 7.
  • the buffer does not include Tris buffer.
  • the buffer is selected from a histidine buffer, an acetate buffer, or a combination thereof.
  • the stabilizer is selected from trehalose, sucrose, or mannitol.
  • the chelating agent comprises disodium edetate.
  • the chelating agent is edetate disodium.
  • the surfactant is selected from polysorbate 20, polysorbate 80 or a combination thereof.
  • the anti-CTLA-4 antibody or fragment thereof comprises HCDR1 shown in SEQ ID NO: 12, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 14 : LCDR1 shown in 15 and LCDR2 shown in SEQ ID NO: 16 and LCDR3 shown in SEQ ID NO: 17.
  • the heavy chain variable region of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 18 or is at least about 90% identical to the SEQ ID NO: 18
  • the amino acid sequence, the light chain variable region of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least about 90% identity with the SEQ ID NO: 19.
  • the heavy chain of the anti-CTLA-4 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain of the anti-CTLA-4 antibody or fragment thereof comprises SEQ ID NO: 2.
  • the anti-CTLA-4 antibody or fragment thereof is a fully human monoclonal antibody.
  • the anti-CTLA-4 antibody or fragment thereof is expressed by CHO cells.
  • the anti-CTLA-4 antibody or fragment thereof is produced by Chinese hamster ovary cells (CHO fut8(-/-), such as CHO-(1,6)-fucose transferase gene knockout. BAT-KF) expression.
  • the level of fucosylation in the anti-CTLA-4 antibody or fragment thereof is ⁇ 5%, the total amount of high mannose glycoforms is ⁇ 5%, and the total amount of sialylated glycoforms is ⁇ 3%.
  • the level of fucosylation in the anti-CTLA-4 antibody or fragment thereof is about 0, about 0.1%, about 0.3%, about 0.4%, about 0.6%, about 1.3%, about 1.9% , About 2.2%, about 2.8%, about 3.3%, about 3.7%, about 4.1%, about 4.5%, about 5%, or the range between any two of these values (including the end point) or any value therein .
  • the total amount of high mannose glycoforms in the anti-CTLA-4 antibody or fragment thereof is about 0.1%, about 0.3%, about 0.9%, about 1.18%, about 1.7%, about 2.6%, about 3.3 %, about 4.1%, about 4.9%, about 4.99%, or the range between any two of these values (including the end point) or any value therein.
  • the total amount of sialylated glycoforms in the anti-CTLA-4 antibody or fragment thereof is about 0.1%, about 0.2%, about 0.36%, about 0.8%, about 1.5%, about 2.2%, about 2. %, about 2.9%, about 2.99%, or the range between any two of these values (including the end point) or any value therein.
  • the liquid formulation comprises the following components: 5-40 mg/mL anti-CTLA-4 antibody or fragments thereof, 10-30 mM buffer, 200-260 mM stabilizer, 0.02-0.2 mg/mL chelating agent, 0.1-0.4mg/mL surfactant, water; the pH of the liquid formulation is 5.4-6.2.
  • the liquid formulation comprises the following components: 5-40 mg/mL anti-CTLA-4 antibody or fragments thereof, 10-30 mM acetate buffer, 200-260 mM trehalose, 0.02-0.2 mg/mL Yidi Disodium acid, 0.1-0.4mg/mL polysorbate 80, water; the pH of the liquid preparation is 5.7-5.9.
  • the stabilizer is sucrose or mannitol.
  • the surfactant is polysorbate 20.
  • the liquid formulation comprises the following components: 5-40 mg/mL anti-CTLA-4 antibody or fragments thereof, 10-30 mM histidine buffer, 200-260 mM sucrose, 0.02-0.2 mg/mL Disodium oleate, 0.1-0.4 mg/mL polysorbate 80, water; the pH of the liquid preparation is 5.4-6.2.
  • the stabilizer is trehalose or mannitol.
  • the surfactant is polysorbate 20.
  • the liquid formulation includes the following components: 5-40 mg/mL anti-CTLA-4 antibody or fragments thereof, a combination of 10-30 mM histidine buffer and acetate buffer, 200-260 mM sucrose, 0.02-0.2 mg/mL edetate disodium, 0.1-0.4 mg/mL polysorbate 20, water; the pH of the liquid preparation is 5.7-5.9.
  • the stabilizer is trehalose or mannitol.
  • the surfactant is polysorbate 80.
  • the liquid formulation comprises the following components: about 5 mg/mL anti-CTLA-4 antibody or fragments thereof, about 10 mM histidine buffer, about 200 mM trehalose, about 0.02 mg/mL edetate Sodium, about 0.1 mg/mL polysorbate 20, water; the pH of the liquid formulation is about 5.4.
  • the liquid formulation comprises the following components: about 9 mg/mL anti-CTLA-4 antibody or fragments thereof, about 14 mM acetate buffer, about 219 mM trehalose, about 0.09 mg/mL disodium edetate, About 0.24 mg/mL polysorbate 20, water; the pH of the liquid formulation is about 5.7.
  • the liquid formulation comprises the following components: about 24 mg/mL anti-CTLA-4 antibody or fragments thereof, about 17 mM acetate buffer, about 227 mM sucrose, about 0.12 mg/mL disodium edetate, about 0.3 mg/mL Polysorbate 20, water; the pH of the liquid formulation is about 5.81.
  • the liquid formulation comprises the following components: about 29 mg/mL anti-CTLA-4 antibody or fragments thereof, about 21 mM histidine buffer, about 235 mM sucrose, about 0.17 mg/mL disodium edetate , About 0.3mg/mL polysorbate 80, water; the pH of the liquid formulation is about 5.7.
  • the liquid formulation comprises the following components: about 31 mg/mL anti-CTLA-4 antibody or fragments thereof, about 25 mM histidine buffer, about 244 mM sucrose, about 0.11 mg/mL disodium edetate , About 0.3mg/mL polysorbate 80 and polysorbate 20 composition, water; the pH of the liquid formulation is about 5.9.
  • the liquid formulation comprises the following components: about 36 mg/mL anti-CTLA-4 antibody or fragments thereof, about 29 mM histidine buffer, about 251 mM mannitol, about 0.16 mg/mL edetate Sodium, about 0.36 mg/mL polysorbate 80, water; the pH of the liquid formulation is about 6.1.
  • the liquid formulation comprises the following components: about 40 mg/mL anti-CTLA-4 antibody or fragments thereof, a combination of about 30 mM histidine buffer and acetate buffer, about 260 mM mannitol, about 0.2 mg/mL disodium edetate, about 0.4 mg/mL polysorbate 80, water; the pH of the liquid preparation is about 5.9.
  • the liquid formulation comprises the following components: about 40 mg/mL anti-CTLA-4 antibody or fragments thereof, about 30 mM acetate buffer, about 248 mM mannitol, about 0.2 mg/mL disodium edetate, About 0.3 mg/mL polysorbate 80, water; the pH of the liquid formulation is about 5.9.
  • the liquid formulation contains the following components: 7-15 mg/mL of anti-CTLA-4 antibody or fragments thereof, 17-23 mM histidine buffer, 220-250 mM sucrose, 0.02-0.05 mg/mL Edetate disodium, 0.1-0.3 mg/mL polysorbate 80, water; the pH of the liquid preparation is 5.7-5.9.
  • the liquid formulation comprises the following components: about 7 mg/mL anti-CTLA-4 antibody or fragments thereof, about 17 mM histidine buffer, about 220 mM sucrose, about 0.02 mg/mL disodium edetate , About 0.1mg/mL polysorbate 80, water; the pH of the liquid formulation is about 5.7.
  • the liquid formulation comprises the following components: about 9 mg/mL anti-CTLA-4 antibody or fragments thereof, about 20 mM histidine buffer, about 228 mM sucrose, about 0.032 mg/mL disodium edetate, about 0.19 mg/mL Polysorbate 80, water; the pH of the liquid formulation is about 5.75.
  • the liquid formulation comprises the following components: about 10 mg/mL anti-CTLA-4 antibody or fragments thereof, about 20 mM histidine buffer, about 240 mM sucrose, about 0.033 mg/mL disodium edetate, about 0.2 mg/mL Polysorbate 80, water; the pH of the liquid formulation is about 5.8.
  • the liquid formulation comprises the following components: about 14 mg/mL anti-CTLA-4 antibody or fragments thereof, about 22 mM histidine buffer, about 243 mM sucrose, about 0.043 mg/mL disodium edetate, About 0.26 mg/mL polysorbate 80, water; the pH of the liquid formulation is about 5.87.
  • the liquid formulation comprises the following components: about 15 mg/mL of anti-CTLA-4 antibody or fragments thereof, about 23 mM histidine buffer, about 250 mM sucrose, and about 0.05 mg/mL disodium edetate , About 0.3mg/mL polysorbate 80, water; the pH of the liquid formulation is about 5.9.
  • the water contained in the liquid formulation is sterile water or water for injection.
  • Another aspect of the present invention provides the application of the liquid preparation in the preparation of a medicine for treating CTLA-4 related diseases.
  • Another aspect of the present invention provides a method for treating CTLA-4 related diseases, the method comprising administering an effective dose of the liquid formulation to a patient.
  • CTLA-4 related diseases include cancer or transplantable tumors for rejection.
  • the CTLA-4 related disease is melanoma, non-small cell lung cancer, bladder cancer, liver cancer, colon cancer, prostate cancer, lymphoma, or kidney cancer.
  • the liquid formulation can be administered by injection or infusion; the site of administration is parenteral, intravenous, mucosal, sublingual, muscle, intradermal, nasal, abdominal, intraarterial, or subcutaneous.
  • the anti-CTLA-4 antibody-containing solution is diluted with an isotonic solution (such as 0.9% NaCl solution for injection), and the diluted solution is then administered by intravenous infusion.
  • the solution containing 10 mg/mL anti-CTLA-4 antibody is diluted with an isotonic solution (such as 0.9% NaCl solution for injection), and the diluted solution is administered by intravenous infusion immediately.
  • the concentration of the anti-CTLA-4 antibody after dilution is about 0.05 mg/mL to about 5 mg/mL or about 0.5 mg/mL to about 1 mg/mL.
  • the concentration of the anti-CTLA-4 antibody after dilution is about 0.05 mg/mL, about 0.07 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL mL, about 0.5mg/mL, about 0.7mg/mL, about 0.8mg/mL, about 0.9mg/mL, about 1mg/mL, about 1.7mg/mL, about 2.4mg/mL, about 2.9mg/mL, about 3.1 mg/mL, about 3.8 mg/mL, 4.1 mg/mL, about 4.6 mg/mL, or about 5 mg/mL.
  • the effective amount is the dosage described herein.
  • the effective amount may be 0.1-100 mg/kg patient body weight. In some embodiments, the effective amount is 0.1-20 mg/kg patient body weight. In other embodiments, the effective amount is about 2 mg/kg patient body weight. In other embodiments, the effective amount is about 3 mg/kg patient body weight. In other embodiments, the effective amount is about 10 mg/kg patient body weight. In other embodiments, the effective amount is about 20 mg/kg patient body weight. In other embodiments, the effective amount is about 30 mg/kg patient body weight. In other embodiments, the effective amount is about 50 mg/kg patient body weight. In other embodiments, the effective amount is about 100 mg/kg patient body weight. In some embodiments, an effective amount of anti-CTLA-4 antibody or fragments thereof can be administered to the patient daily, weekly, monthly, and/or yearly as needed, in single or multiple times per hour/day/week/month.
  • Another aspect of the present invention provides a method for preparing the liquid formulation, which includes the following steps:
  • CHO cells eg, Chinese hamster ovary cells cultured to adapt to suspension growth from the CHO-K1 cell line
  • CHO-BAT-KF eg, Chinese hamster ovary cells cultured to adapt to suspension growth from the CHO-K1 cell line
  • the invention patent CN109096399A has disclosed the above cells, which will contain the coding anti-CTLA-4 antibody by the electroporation method
  • the linearized plasmids of the light chain/heavy chain nucleotide sequence of the anti-CTLA-4 antibody were transfected into two cell lines respectively.
  • the heavy chain CDR1 of the anti-CTLA-4 antibody is SEQ ID NO: 12, and the heavy chain CDR2 is SEQ ID NO: 13,
  • the heavy chain CDR3 is SEQ ID NO: 14, the light chain CDR1 is SEQ ID NO: 15, the light chain CDR2 is SEQ ID NO: 16, the light chain CDR3 is SEQ ID NO: 17, and the heavy chain variable region sequence is SEQ ID NO: 18, the light chain variable region sequence is SEQ ID NO: 19, the heavy chain sequence is SEQ ID NO: 1, the light chain sequence is SEQ ID NO: 2, the heavy chain coding sequence is SEQ ID NO: 3, the light chain The coding sequence of is SEQ ID NO:4.
  • the electroporation conditions are: voltage 300V, 1500 ⁇ F, 4mm electroporation cup, 40 ⁇ g linearized plasmid, 10 million cells.
  • BAT1 which is a cell expressing "antibody 1"herein
  • BAT2 which is a cell expressing "antibody 2" herein.
  • the two cells were resuspended in CHO-AGT (Gibco, product number: 12490025) medium containing 1 ⁇ GS (Specialty Media, product number: GSS1016-C), and divided evenly at a density of 1500 cells per well.
  • each well has a volume of 100 ⁇ L. Put the cell culture plate into 8% CO 2 , 37°C incubator for culture. After 48 hours, 100 ⁇ L of CHO-AGT medium (1 ⁇ GS and 50 ⁇ M final concentration of MSX (methionine sulfoximine)) was added to continue the culture.
  • MSX methionine sulfoximine
  • the detection step is to thaw the ELISA plate coated with goat anti-human Fc antibody, wash the plate 3 times with PBST, apply a 40-fold diluted 96-well cell culture supernatant, incubate at 37°C for 2 hours, wash the plate 5 times with PBST, and add 1: 10000 times diluted goat anti-human Kappa light chain-secondary antibody coupled with peroxidase (Sigma, catalog number: A7164), incubated at 37°C for 0.5 hours, washed with PBST 7 times, added TMB single component for color development
  • the solution Huzhou Yingchuang Biology, Item No.
  • TMB-S-001 TMB-S-001 was developed in the dark for 10-15 minutes, and the reaction was terminated with 0.1M sulfuric acid. The reading was performed at a wavelength of 450nm to mark positive clones with higher expression levels.
  • the positive clones were transferred to a 24-well plate and cultured for 5-7 days, and the culture supernatant was diluted 1000 times for the above-mentioned ELISA test. Positive clones with higher expression and good cell status are transferred to a 6-well plate and cultured for 5-7 days, and the culture supernatant is diluted 2000 times for the above-mentioned ELISA detection.
  • the positive clones with higher expression and good cell status were transferred to a 125 mL small shake flask and cultured in a shaker.
  • the cells When the cell growth status is stable and good, the viability is above 95%, and the cells reach a certain number, they are inoculated into the CHO-AGT medium at a density of 30-50 ⁇ 10 5 cells/mL and a volume of 30 mL, and then cultured in a shake flask for 12 days. During the cultivation process, 0.5ml of the culture was taken to measure the cell density and viability on the 3rd, 6th, and 9th days. After sampling, 3ml Feed C (CD EfficientFeed C AGT, manufacturer: Gibco) was added. The 6th and 9th days were cultured. Centrifuge the supernatant and freeze it. Take the 10th, 11th, and 12th day cultures to measure cell density and viability.
  • CD EfficientFeed C AGT manufacturer: Gibco
  • the antibody purified from BAT1 cells is antibody 1
  • the antibody purified from BAT2 cells is antibody 2.
  • the antibody 1 and antibody 2 were sequenced.
  • the heavy chain sequence of antibody 1 and antibody 2 is SEQ ID NO: 1 (each heavy chain contains 448 amino acids and the molecular weight is 51kDa), and the light chain sequence is SEQ ID NO: 2 (each Each light chain contains 215 amino acids and has a molecular weight of 23 kDa).
  • the obtained protein was subjected to various quality analysis.
  • antibody 2 was expressed in the cell line CHO-BAT-KF which knocked out ⁇ -(1,6)-fucosyltransferase, so The glycotype is different from that of antibody 1 ( Figure 1 and Table 1).
  • the main glycotype of antibody 2 is defucosylated glycoform: the fucosylation level is about zero.
  • the frozen supernatant on the 6th, 9th, 10th, 11th, and 12th days described above was subjected to HPLC to detect the protein expression level.
  • the glycoforms that affect the risk of antibody immunogenicity are sialylated glycoforms: the content of sialylated glycoforms from high to low is Antibody 1, antibody 2, the low content of sialylated glycoforms in antibody 2 of the present invention is beneficial to reduce the risk of immunogenicity; the content of high mannose glycoforms from high to low is Antibody 2, Antibody 1, and the low content of high mannose glycoforms in Antibody 2 of the present invention are beneficial to prolong the half-life of the antibody.
  • the plasmid used to express the extracellular region of the CTLA-4 antigen and the sequence of the Fc segment of the human IgG1 antibody heavy chain (SEQ ID NO: 5, whose amino acid sequence is SEQ ID NO: 6) was transiently transfected into 293F cells.
  • 293F cells were cultured with CD 293 TGE Medium (BPM Cell Culture, article number: CM-1156). When the cell viability was above 95%, the cells were subcultured to 80-100 ⁇ 10 5 cells/mL with fresh medium, and the cells were subcultured after 24 hours. When the density is about 150-200 ⁇ 10 5 cells/mL, transfection can be carried out. Firstly, the plasmid was placed in a water bath at 65°C for 30 minutes, 1 ⁇ g of plasmid per 100 ⁇ 10 5 cells, and 3 ⁇ L of PEI (Polysciences, catalog number: 24765-2) per 1 ⁇ g of plasmid.
  • PEI Polysciences, catalog number: 24765-2
  • CTLA-4-ECD-Fc antigen protein was coated on the ELISA plate at 4°C overnight. The next day, it was blocked with 5% BSA blocking solution at 37°C for 2 hours. After washing the plate, add antibody 2 and antibody 1 protein in 3-fold dilutions starting from 1 ⁇ g/mL, and dilute 12 concentrations in total (the last one is blank 0 concentration), make 3 replicate wells for each concentration, and incubate 2 at 37°C hour. After washing the plate, a 1:10000-fold diluted goat anti-human Kappa light chain secondary antibody (Sigma, catalog number A7164) coupled with peroxidase was added, and incubated at 37°C for 0.5 hours.
  • antibody 2 antibody 1 protein and CTLA-4-His (Yiqiao Shenzhou, 11159-H08H) antigen was tested on BIAcore (T200, GE Healthcare).
  • the antibody to be tested antibody 2, antibody 1, Dilute with HBS-EP (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.005% P20, pH 7.4) to 5 ⁇ g/mL, and dilute antigen CTLA-4-His to 100nM with HBS-EP, and use this as the starting concentration Make 2-fold gradient dilutions to obtain 100, 50, 25, 12.5, 6.25, 3.125nM CTLA-4-His dilutions.
  • the data analysis software Evaluation Software 3.1 was used to analyze the test results.
  • the sensor signal collected from the sample test flow path was subtracted from the reference flow path and the sample blank, and the kinetic "1:1" model was selected for fitting.
  • the kinetic parameters of the affinity binding of each antibody sample to CTLA-4 were obtained, as shown in Table 2.
  • the Biacore curve fitting is shown in Figure 3 (where Yervoy_930395 is Y20180901 is antibody 1, 181110-B14-2-CEX is antibody 2).
  • ka binding rate
  • kd dissociation rate
  • KD binding and dissociation equilibrium constant, that is, affinity
  • pCMV2-OKT3 (where the OKT3 part is the ScFv form of the antibody, its nucleotide sequence is shown in SEQ ID NO: 7, the heavy chain amino acid sequence of OKT3 is shown in SEQ ID NO: 8, and the light chain amino acid sequence is shown in SEQ ID NO: 9) Plasmid, the plasmid map is shown in Figure 5.
  • Raji-OKT3 cells and Jurkat-IL2-luciferase-CTLA4FL cells were respectively diluted to a density of 400 ⁇ 10 5 cells/mL.
  • a 96-well white board (Corning, catalog number: 3917) was added with 25 ⁇ L of Raji-OKT3 and 25 ⁇ L of Jurkat-IL2-luciferase-CTLA4FL to each well.
  • Antibody 2 and Antibody 1 both show similar activity to enhance T cell function in the activity model. It shows that the enhancement of ADCC on anti-CTLA-4 antibody did not change its biological activity with CTLA-4 antigen in vitro.
  • the SA sensor (Fertebio) was pre-wet in PBS for 10 minutes for later use, and the biotin-labeled Fc ⁇ RIIIa 158V (ACRO Biosystems) and Fc ⁇ RIIIa 158F (ACRO Biosystems) were used with pH 6.8 containing 0.5% BSA in PBST (hereinafter referred to as dilution buffer) ) Dilute to 1 ⁇ g/mL, and solidify the SA sensor in Fc ⁇ RIIIa 158-Biotin to a signal of about 0.6nm.
  • dilution buffer hereinafter referred to as dilution buffer
  • the plasmid map is shown in Figure 8.
  • the construction process is as follows: Synthesize the Fc ⁇ RIIIa 158V FL cDNA sequence shown in SEQ ID NO: 10, and the amino acid sequence is shown in SEQ ID NO: 11, placed in the pUC-57 vector, and named Puc-57-Fc ⁇ RIIIa 158V FL cDNA. Synthesize the forward and reverse PCR primers shown in SEQ ID NO: 20 and SEQ ID NO: 21 (used to amplify the Fc ⁇ RIIIa 158VFL cDNA fragment with EcoRI and NotI restriction sites on both ends of the fragment).
  • the lentivirus packaging system pLP1, pLP2, pLP/VSV-G (for example, available from invitrogen) and pCDH-Fc ⁇ RIIIa 158V FL-Puro were used to package the virus, and then the Jurkat cells were infected. After 48h, 0.3 ⁇ g/mL puromycin was added as the selection pressure. Two weeks after transfection, when the cells grow up, FITC-labeled anti-Fc ⁇ RIIIa antibody (Invitrogen, catalog number: MHCD1601) was used to detect the expression of Fc ⁇ RIIIa 158V by flow cytometry, and a positive single clone was screened out.
  • FITC-labeled anti-Fc ⁇ RIIIa antibody Invitrogen, catalog number: MHCD1601
  • the pGL4.30-luc2P-NFAT-RE-Hygro plasmid (purchased from Promega) was transfected into the aforementioned Fc ⁇ RIIIa 158V-positive Jurkat monoclonal cells by electroporation, and selected with hygromycin under pressure. Stimulate with PMA (50 ⁇ g/mL) and ionomycin (1 ⁇ g/mL) for 6 hours and add luciferase substrate, and use a microplate reader to detect autofluorescence to screen out that it can be stimulated by PMA and ionomycin to emit spontaneous
  • the fluorescent positive single clone was named Jurkat-NFAT-luciferase-Fc ⁇ RIIIa 158V cells.
  • Jurkat-NFAT-luciferase-Fc ⁇ RIIIa 158V cells and Jurkat-IL2-luciferase-CTLA4FL cells were diluted to a density of 200 ⁇ 10 5 cells/mL, respectively.
  • MC38 cells were inoculated subcutaneously on the right side of B-hCTLA4 humanized female mice at a concentration of 5 ⁇ 10 5 cells/0.1 mL .
  • the tumor grew to about 125 mm 3 , they were randomly divided into groups according to the tumor volume, 8 in each group, a total of 8 Groups, respectively: G1 control group (0.9% sodium chloride injection solvent), G2 group ( 1mg/kg), G3 group (antibody 1, 0.3mg/kg), G4 group (antibody 1, 1mg/kg), G5 group (antibody 1, 3mg/kg), G6 group (antibody 2, 0.3mg/kg) , G7 group (antibody 2, 1mg/kg) group and G8 group (antibody 2, 3mg/kg).
  • G1 control group (0.9% sodium chloride injection solvent
  • G2 group 1mg/kg
  • G3 group antibody 1, 0.3mg/kg
  • G4 group antibody 1, 1mg/kg
  • G5 group antibody 1, 3m
  • the route of administration for all groups was intraperitoneal injection, once every 3 days, for 6 consecutive times, and the experiment ended 12 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded.
  • the animals were euthanized, the tumors were stripped and weighed, photographed, and the relative tumor inhibition rate (TGI%) was calculated.
  • a mean ⁇ standard error
  • b statistical comparison between the body weight of the administration group and the body weight of the solvent control group 27 days after administration, t-test.
  • a mean ⁇ standard error
  • b statistical comparison between the body weight of the administration group and the body weight of the solvent control group 27 days after administration, t-test.
  • test antibody antibody 2 and test antibody: antibody 1 have a significant effect on inhibiting tumor growth, and it is comparable to the positive control drug commercial CTLA-4 antibody
  • the anti-tumor effect is equivalent.
  • the tested drug has no obvious toxic effect on animals, and its safety is good.
  • the efficacy seems to have reached saturation, so there is no difference, but in the low dose group at 0.3 mg/kg, the ADCC-enhanced antibody 2 is significantly better than that without ADCC.
  • the antibody 1 has a better anti-tumor effect.
  • Example 7 The effect of antibody 2 on CD4 + , CD8 + , and Treg cells in mouse blood
  • Example 6 In the drug efficacy experiment mentioned in Example 6, after 21 days of administration, 150 ⁇ L of anticoagulant blood was collected from each mouse through the orbital intracanthal venous plexus, and the CD4, CD8 positive cells and Treg cells were detected by flow cytometry. Proportion of CD45 positive cells.
  • the antibodies used include anti-CD45 (Biolegend, article number: 103138), anti-CD4 (Biolegend, article number: 100438), anti-CD8 (Biolegend, article number: 100759), anti-Foxp3 (eBioscience, article number: 17-5773) -82) Antibodies.
  • the experiment selected 24 cynomolgus monkeys, half male and half male, and randomly divided into 3 groups according to gender: low, medium, and high-dose groups were given 1 mg/kg antibody 2, 3 mg/kg antibody 2, and 10 mg/kg antibody by a single intravenous infusion respectively. .
  • low, medium, and high-dose groups were given 1 mg/kg antibody 2, 3 mg/kg antibody 2, and 10 mg/kg antibody by a single intravenous infusion respectively.
  • the validated ELISA method analyzes the concentration of drugs in each sample, and the lower limit of quantification is 15ng/mL.
  • 40 cynomolgus monkeys were selected and randomly divided into 4 groups according to sex, with 5 males and 5 males in each group. They were the vehicle control group, the low-dose group (antibody 2, 0.2mg/kg), and the medium-dose group (antibody 2, 0.6mg). /kg) and high-dose group (antibody 2, 2mg/kg); intravenous infusion of the test product or vehicle reference product once every 3 weeks, continuous administration for 13 weeks, that is, the first day, the 22nd day, and the 43rd day It was administered 5 times on day, 64th and 85th day; the administration rate was about 0.2mL/min/kg.
  • the blood collection time points of the control group were: 1st and 64th days before the drug and 2h after the start of the administration, and the blood collection time points of the administration group were: the 1st and 64th days before the drug and immediately after the end of the administration ( ⁇ 1min) , 2h, 6h, 24h, 72h, 120h, 168h, 336h, 504h after the start of administration, collect blood and separate serum.
  • a validated ELISA method was used to detect the concentration of the test substance in the serum, and the lower limit of quantification of the method was 15ng/mL.
  • WinNonlin 8.0 non-compartmental model was used to calculate the toxicokinetic parameters of the test product.
  • 8 cynomolgus monkeys were randomly divided into 4 groups (1/gender/group), the first group was the vehicle control group, the second, third, and fourth groups were the 10, 30, and 60 mg/kg test product groups (antibody 2).
  • the animals were given a single intravenous infusion on the first day, the administration volume was 6 mL/kg, and the infusion rate was 0.5 mL/min/kg.
  • a total of 40 cynomolgus monkeys (20/sex) were randomly divided into 4 groups, each with 5 animals per sex; the first group was the vehicle control group, and the second, third, and fourth groups were given 0.2 and 0.6 respectively.
  • This test is a combination of repeated intravenous infusion and a long-term toxicological study during the recovery period of 4 weeks. It mainly investigates the effects of the provided product (antibody 2) on the T lymphocyte subsets, complement, cytokines, and immunity of cynomolgus monkeys under the test conditions.
  • test results show that the test products with concentrations of 10.1mg/mL and 1mg/mL do not cause hemolysis of rabbit red blood cells in vitro and have no aggregation effect, and are suitable for clinically safe injection.
  • This study is a multi-center, open, dose-escalating phase I clinical trial evaluating the safety, tolerability and pharmacokinetic characteristics of antibody 2 injection in the treatment of patients with advanced solid tumors, exploring the maximum tolerated dose (MTD) ), to provide recommended doses for subsequent clinical trials; and to preliminarily evaluate the immunogenicity and anti-tumor efficacy of Antibody 2 injection.
  • MTD maximum tolerated dose
  • the "3+3" dose escalation rule was adopted to explore the safe dose range.
  • the doses were 0.1mg/kg, 0.2mg/kg, 0.5mg/kg, 1mg/kg and 2mg/kg; 5 dose groups were entered in sequence Antibody 2 injection was given to the group. Only when all subjects in the same dose group have completed the DLT assessment and determined that the Maximum Tolerated Dose (MTD) has not been explored, the next higher dose group can be carried out. Explore until the MTD is explored or until the tolerance study of the highest dose group is completed.
  • MTD Maximum Tolerated Dose
  • the second stage after the dose escalation is completed, 1-2 tolerated doses are selected for extended research on melanoma (20-40 cases), and recommended doses for subsequent clinical trials.
  • the drug is administered by intravenous infusion every 3 weeks, the treatment cycle is 4 cycles, and the drug is administered on the first day of each cycle.
  • the following situations should be used with caution/appropriate use as appropriate: 1) Prevent infusion reactions: If the nth subject has an infusion reaction, in the subsequent administration, the nth, n+1, n+ can be given as appropriate 2.
  • Preventive medication for each subject limited to steroid hormones, analgesics, and antihistamines alone or in combination; 2) Treatment of infusion reactions: if infusion reactions occur, steroid hormones and other drugs can be used as appropriate; 3 ) Treatment adverse reactions: researchers can use infliximab, methimazole, carmidazole, levothyroxine, steroid hormones and other treatments for adverse reactions that occur during the study as appropriate; 4) local treatment of tumors, such as palliative bone pain Radiotherapy. DLT evaluation period: 21 days, that is, the first treatment cycle. After the patient completes the visit at the end of the fourth treatment cycle, continue the observation visit for 6 weeks to complete the study. Based on the data collected in the experiment, analyze and summarize the safety, tolerability, and pharmacokinetic characteristics of antibody 2 injection , Immunogenicity and preliminary efficacy.
  • Safety indicators include: vital signs, physical examination, laboratory tests (blood routine, blood biochemistry, thyroid function, coagulation routine, urine routine, stool routine (including occult blood test)), electrocardiogram, cardiac color Doppler ultrasound, adverse events (including immune-related Adverse events) and so on.
  • PK blood samples were collected from the subjects before and after each administration in the first to fourth cycles.
  • the first cycle and the third cycle are intensive sampling to study the PK characteristics of single-dose and multiple-dose steady-state studies, respectively.
  • Specific PK parameters include: single administration: C max , T max , T 1/2 , CL, Vd, Ke, MRT, AUC (0- ⁇ ) , AUC (0- ⁇ ) ; multiple administration: C max ,ss , C avg,ss , C min,ss , AUC (0- ⁇ ),ss , AUC (0- ⁇ ),ss , T max,ss , T 1/2,ss , CL, V ss , Ke, MRT, accumulation index (Rac), volatility index DF.
  • Subjects in all dose groups need to collect blood samples at specific time points during the treatment period. Each time point is planned to collect 3.5mL blood samples to detect anti-drug antibodies and neutralizing antibodies in the serum. Each dose is administered during the first to fourth cycles. Blood samples were collected for immunogenicity testing before and 21 days after the end of the last administration of the fourth cycle.
  • the immunogenicity evaluation indicators include: the positive rate of anti-drug antibody (ADA) samples and the positive rate of individuals, the titer of ADA-positive samples, and whether the ADA-positive samples are neutralizing antibodies (Nab).
  • RECIST1.1 and iRECIST1.1 were used to evaluate the anti-tumor efficacy of antibody 2.
  • Tumor imaging examination CT or MRI of the chest, abdomen, pelvis, etc.
  • the same imaging examination technique was used for the same patient and the same site during the entire study period.
  • Tumor evaluation after administration will be performed in the 6th week, 12th week, and 18th week. Evaluation of clinical effectiveness includes:
  • ORR Objective Response Rate
  • Duration of Response The time from the first assessment of the tumor as objective remission to the first assessment as PD (Progressive Disease) or death from any cause before PD, reflecting the duration of ORR;
  • PFS Progression-Free Survival
  • Dose-Limited Toxicity is as follows: It refers to the following events related to the study drug in the patient during the first day to the 21st day after the first cycle of administration:
  • Eye AEs ⁇ Grade 2 (adverse events) that have not been reduced to Grade 1 within 2 weeks of receiving local treatment; or eye AEs ⁇ Grade 2 that require systemic treatment;
  • Non-hematological toxicity of grade ⁇ 3 (nausea, vomiting, etc. can be relieved within 3 days of supportive treatment, except for infusion reactions of grade ⁇ 3 that occur without preventive administration);
  • auxiliary material mother liquor of surfactant and/or chelating agent is adjusted to a suitable pH value; an appropriate amount of aseptically filtered auxiliary material mother liquor is added to an appropriate amount of antibody concentrate to obtain a liquid preparation.
  • the solvent used in the examples is water for injection.
  • buffer systems are used for antibody stability studies.
  • the present invention determines the following buffer systems after preliminary screening: His, HAc, CB, Sua, Tris.
  • HAc 20mM acetate buffer, pH 5.8;
  • CB 20mM citrate buffer, pH 5.8;
  • Tris 20mM Tris Hydrochloride, pH 7.0.
  • the antibody 2 contained in His, HAc, CB, and Sua was 10 mg/mL, the buffer was 20 mM, and the pH of the buffer was 5.8.
  • SEC-HPLC, SEC for short monomer purity
  • IEC-HPLC, IEC for short charge isomers
  • histidine buffer is the best buffer, followed by acetate buffer, and Tris buffer is inferior: the purity of SEC-HPLC monomer in histidine buffer and the main peak content of IEC-HPLC are higher than others. Buffer system. It can be seen that the histidine buffer has the best protective effect on antibody 2.
  • the pH range was finely screened in the histidine buffer system.
  • the antibodies, buffers, stabilizers, metal ion chelating agents, and surfactants contained in each system are shown in Table 11; the molar concentration of 82mg/mL sucrose is 240mM.
  • the liquid preparation of the present invention can stably store the active ingredient antibody 2, and the formulations with better stability are such as the liquid preparations A, B, C, D, E, F, G, H, I of the present invention; among them, the J preparation is used as the comparison preparation, As shown in the table below.
  • liquid preparation of the present invention has good compatibility with the medicine bottle and the rubber stopper, and meets the requirements of packaging.
  • SEQ ID NO: 1 anti-CTLA-4 antibody heavy chain amino acid sequence
  • SEQ ID NO: 4 the nucleic acid sequence of the light chain of the anti-CTLA-4 antibody
  • SEQ ID NO: 18 Amino acid sequence of heavy chain variable region of anti-CTLA-4 antibody

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Abstract

抗CTLA-4单克隆抗体及该抗体的制备和用途，抗CTLA-4抗体具有毒性更低和活性更佳的优点；另外，抗CTLA-4抗体还具有低含量的高甘露糖糖型和/或低含量的唾液酸化糖型，有助于延长抗体的半衰期和/或降低免疫原性。特别地，含有该抗体的制剂具有良好的稳定性。

Description

抗CTLA-4单克隆抗体及其制备方法与应用 技术领域
本发明属于生物医药技术领域，涉及一种抗CTLA-4单克隆抗体及其制备方法与应用。
背景技术
免疫系统是防止癌症发展和进展(progression)的重要防御手段。免疫疗法，如IL-2和自体肿瘤浸润淋巴细胞的过继转移，可以诱导患者亚组持续的肿瘤反应，从而使得预后不良的患者获得长期存活(Atkins MB,et al.(1999),J Clin Oncol.17(7):2105–2116)。随着对于抗肿瘤免疫的启动和效应阶段的重要性的进一步理解，基于检查点阻断的治疗方法最近开始为患者提供持久的益处，并似乎适用于广泛的恶性肿瘤。
免疫系统受到严格的调节，以响应适当的抗原，而不会对自身产生反应(Bretscher P,Cohn M.(1970),Science.169(3950):1042-1049)。T细胞是免疫反应的关键介质之一，需要共刺激才能激活，并且可以通过抑制信号使其失活(Sharpe AH,Abbas AK.(2006),N Engl J Med.355(10):973-975)。细胞毒性T淋巴细胞抗原-4(CTLA-4)就是这样一种影响T细胞功能的关键抑制性受体，在免疫应答的启动阶段发挥关键作用(Scalapino KJ1,Daikh DI.(2008),Immunol Rev.223:143-155)。当抗原被MHC-I类或II类抗原提呈细胞运输到T细胞受体(TCR)时，TCR上的信号就会放大，并被共刺激分子抵消。T细胞上的CD28与抗原提呈细胞上的B7-1(CD80)和B7-2(CD86)结合后，会发出一种需要T细胞全激活的信号。这种CD28/B7的结合导致白介素2及其他刺激性的细胞因子产生的增加，提高代谢，促进细胞周期进程，上调细胞存活基因，最终使T细胞得到增殖和分化。当CD28结合并引起T细胞增殖后，CTLA-4被运送并表达在T细胞表面(Linsley PS,et al.(1996),Immunity.4(6):535-543)。通过TCR的激活信号越强烈，就越多CTLA-4运送并表达。当在细胞表面上时，CTLA-4的抑制信号就被传播(Egen JG,Allison JP.(2002),Immunity.16(1):23-25)。与CD28相比，CTLA-4与B7有更高的亲和力，并能阻碍更进一步的共激活(Krummel MF,Allison JP.(1995),J Exp Med.182(2):458-465)。并且，CTLA-4表达的细胞能通过内吞作用捕获和降解B7-1和B7-2(Qureshi OS,et al.(2011),Science.332(6029):600-603)。
除了CTLA-4，目前已经报道了其他一些可以调节T细胞应答的刺激性或抑制性的受体和配体。刺激性受体包括诱导性T细胞共刺激子(ICOS)(Tremble LF,et al.(2018),Cancer Lett.420:109-115)、糖皮质激素诱导的TNF受体(GITR)(Schaer DA,et al.(2013),Cancer Immunol Res.1(5):320-331)、CD27(Buchan SL,et al.(2018),Blood.131(1):39-48)、4-1BB(Eun SY,et al.(2015),J Immunol.194(1):134-141)、CD40(Richman LP,Vonderheide RH.(2014),Cancer Immunol Res.2(1):19-26)、DNAX配件分子-1(DNAM-1)(Kearney CJ,et al.(2016),Oncoimmunology.5(8):e1196308)，抑制性受体包括细胞程序性死亡受体-1(PD-1)(Dong H,et al.(1999),Nat Med.5(12):1365-1369)、淋巴细胞激活基因-3(LAG-3)(Di CE,et al.(2005),J Pathol.205:82–91)、T细胞免疫球蛋白和粘蛋白结构域3(TIM3)(Sánchez-Fueyo A,et al.(2003),Nat Immunol.4(11):1093-1101)、T细胞活化的V结构域Ig抑制剂(VISTA)(Lines JL,et al.(2014),Cancer Res.74(7):1924-1932)、CD47(Liu X,et al.(2015),Nat Med.21(10):1209-1215)、吲哚胺-2,3-双加氧酶(IDO)(Friberg M,et al.(2002),Cancer.101:151–155)、CD94/NKG2A (Muntasell A,et al.(2017),Curr Opin Immunol.45:73-81)、杀伤性免疫球蛋白样受体(KIR)(Tu MM,et al.(2016),Front Immunol.7:116)、T细胞免疫球蛋白和ITIM结构域(TIGIT)(Johnston RJ,et al.(2014),Cancer Cell.26:923-937)。这些刺激性的受体和配体，即目前研究领域热论的“免疫检查点”，在免疫系统的自我调节中起到至关重要的作用，使免疫系统的功能保持在正常范围内而不至于过度活化。在癌症机体中，利用激动剂靶向刺激性受体或利用抑制剂阻断抑制性受体，就能调节免疫应答，使免疫系统识别和攻击肿瘤细胞，达到治疗的效果。对于上述免疫检查点，目前有一部分已开发出临床中或上市的治疗性药物(Elad S,et al.(2014),Chin J Cancer.33(9):434–444)，其中CTLA-4阻断剂就是第一个应用于临床癌症治疗的单克隆抗体药物。
目前较为人所知的抗CTLA-4单克隆抗体是百时美施贵宝公司的伊匹单抗
Figure PCTCN2020139205-appb-000001
以及阿斯利康公司尚处于临床阶段的曲美母单抗(Tremelimumab)。它们的机理都是通过阻断CTLA-4与B7的结合，解放B7蛋白使其能跟CD28结合，从而增强T细胞的增殖反应。在临床前几种已建立的小鼠模型中阻断CTLA-4/B7相互作用，已经显示出对可移植肿瘤的排斥，包括结肠癌，前列腺癌，淋巴瘤和肾癌(Korman AJ,et al.(2006),Adv Immunol.90:297-339)。然而，这种作用机制推广到人体上，仅对一小部分治疗患者提供了有限的临床益处。因此，人们认为应该存在与宿主依赖因子相关的未知机制(Romano E,et al.(2015),Proc Natl Acad Sci USA.112(19):6140-6145)。
发明内容
基于此，有必要提供一种毒性更低、活性更佳的新型抗CTLA-4单克隆抗体药物。本发明的上述目的通过以下技术手段实现：
本发明提供一种低含量的高甘露糖糖型和/或低含量的唾液酸化糖型的抗CTLA-4抗体，有助于延长抗体的半衰期和/或降低免疫原性。在一些实施方案中，所述抗体为ADCC增强的抗CTLA-4抗体，用于靶向病患细胞或组织，增大药物的抗肿瘤效果。
具体地，一方面，本发明提供了针对CTLA-4的单克隆抗体，所述单克隆抗体包含
(1)重链CDR1(HCDR1)，其包含SEQ ID NO:12所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
重链CDR2(HCDR2)，其包含SEQ ID NO:13所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
重链CDR3(HCDR3)，其包含SEQ ID NO:14所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
轻链CDR1(LCDR1)，其包含SEQ ID NO:15所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
轻链CDR2(LCDR2)，其包含SEQ ID NO:16所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，和
轻链CDR3(LCDR3)，其包含SEQ ID NO:17所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成；
或
(2)重链可变区(VH)，其包含SEQ ID NO:18所示的序列，与所述序列具有至少约80％，优选至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，和
轻链可变区(VL)，其包含SEQ ID NO:19所示的序列，与所述序列具有至少约80％，优选至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成；
所述单克隆抗体具有降低的岩藻糖基化水平。
在具体的实施方案中，所述单克隆抗体包含重链和轻链，所述重链包含SEQ ID NO:1的氨基酸序列，与SEQ ID NO:1的氨基酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的序列，或由其组成；
所述轻链包含SEQ ID NO:2所示的氨基酸序列，与SEQ ID NO:2的氨基酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的序列，或由其组成。
在具体的实施方案中，所述重链由SEQ ID NO:3所示的核苷酸序列或与SEQ ID NO:3的核苷酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、 约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的核苷酸序列编码，所述轻链由SEQ ID NO:4所示的核苷酸序列或与SEQ ID NO:4的核苷酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的核苷酸序列编码。
在具体的实施方案中，所述岩藻糖基化水平为0％-20％，比如约0％，约1％，约2％，约3％，约4％，约5％，约6％，约7％，约8％，约9％，约10％，约11％，约12％，约13％，约14％，约15％，约16％，约17％，约18％，约19％或约20％，或在其中任意两个数值之间的范围(包括端点)或其中任何值。
在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量<5％和/或唾液酸化糖型总量<3％。在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量为约0.1％、约0.3％、约0.9％、约1.18％、约1.7％、约2.6％、约3.3％、约4.1％、约4.9％、约4.99％，或任何两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗单克隆抗体Fc区的唾液酸化糖型总量为约0.1％、约0.2％、约0.36％、约0.8％、约1.5％、约2.2％、约2.7％、约2.9％、2.99％，或任何两个数值之间的范围(包括端点)或其中任何值。
在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的高甘露糖糖型总量<5％和/或唾液酸化糖型总量<3％。
在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量<3％和/或唾液酸化糖型总量<2％。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的高甘露糖糖型总量<3％和/或唾液酸化糖型总量<2％。
在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量<2％和/或唾液酸化糖型总量<1％。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的高甘露糖糖型总量<2％和/或唾液酸化糖型总量<1％。
在一些实施方案中，所述单克隆抗体Fc区的岩藻糖基化水平为0％-10％。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的岩藻糖基化水平为0％-10％。
在一些实施方案中，所述单克隆抗体Fc区的岩藻糖基化水平为0％-5％。在一些实施方案中，所述抗单克隆抗体Fc区的岩藻糖基化水平为约0、约0.1％、约0.3％、约0.4％、约0.6％、约1.3％、约1.9％、约2.2％、约2.8％、约3.3％、约3.7％、约4.1％、约4.5％、约5％，或任何两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的岩藻糖基化水平为0％-5％。
在一些实施方案中，所述糖化水平是通过以下方式实现的：
(1)将包含SEQ ID NO:12的重链CDR1，SEQ ID NO:13的重链CDR2，SEQ ID NO:14的重链CDR3，SEQ ID NO:15的轻链CDR1，SEQ ID NO:16的轻链CDR2和SEQ ID NO:17的轻链CDR3的单克隆抗体在敲降或敲除FUT8基因(α-(1,6)-岩藻糖基转移酶基因)的CHO宿主细胞中表达，所述单克隆抗体的高甘露糖糖型、唾液酸化糖型和/或岩藻糖型含量相比于在正常CHO宿主细胞中表达的单克隆抗体的含量低；
(2)将包含SEQ ID NO:12的重链CDR1，SEQ ID NO:13的重链CDR2，SEQ ID NO:14的重链CDR3，SEQ ID NO:15的轻链CDR1，SEQ ID NO:16的轻链CDR2和SEQ ID NO:17的轻链CDR3的单克隆抗体在敲降GDP-岩藻糖4,6-脱水酶(GMD)的CHO-DG44细胞中表 达，所述单克隆抗体的高甘露糖糖型、唾液酸化糖型和/或岩藻糖型含量相比于在正常CHO-DG44宿主细胞中表达的单克隆抗体的含量低；或
(3)将包含SEQ ID NO:12的重链CDR1，SEQ ID NO:13的重链CDR2，SEQ ID NO:14的重链CDR3，SEQ ID NO:15的轻链CDR1，SEQ ID NO:16的轻链CDR2和SEQ ID NO:17的轻链CDR3的单克隆抗体在CHO细胞中表达，然后用糖基转移酶抑制剂如2F-过乙酰基-岩藻糖去岩藻糖基化，所述单克隆抗体的高甘露糖糖型、唾液酸化糖型和/或岩藻糖型含量相比于未用糖基转移酶抑制剂如2F-过乙酰基-岩藻糖去岩藻糖基化的单克隆抗体的含量低。
在一些实施方案中，所述单克隆抗体由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达，如PCT/CN2018/100008公开的CHO-BAT-KF细胞系。
另一方面，本发明提供了药物组合物或融合蛋白，其包含本发明所述的单克隆抗体。
在一些实施方案中，所述药物组合物为适于通过口服、注射、输液、肠外、静脉、黏膜、舌下、肌肉、皮内、鼻腔、腹腔、动脉内、皮下吸收给药的形式。在一些实施方案中，所述药物组合物为适于全身给药的形式。
另一方面，本发明提供了抗体偶联物，其包括本发明所述的单克隆抗体，以及与所述单克隆抗体偶联的偶联部分，所述偶联部分为纯化标签(如His标签)、细胞毒性剂，延长所述单克隆抗体半衰期的结构部分，或可检测的标记，优选地，所述偶联部分为放射性同位素、化学发光剂、酶、血清白蛋白(例如人或鼠血清白蛋白)、或聚乙二醇。
在一些实施方案中，本发明抗CTLA-4抗体为单价抗体或多价抗体。在另一方面，本发明提供了多特异性抗体(例如双特异性抗体)，其包含本发明所述的单克隆抗体，以及针对其他抗原和/或其他抗原表位的抗体或抗原结合片段。
在又一个方面，本发明提供了试剂盒，其包括本发明所述的单克隆抗体，抗体偶联物、多特异性抗体或融合蛋白。
在具体的实施方案中，所述试剂盒还包括第二抗体，细胞毒性剂，化疗剂，放疗剂，所述第二抗体特异性识别所述单克隆抗体；任选地，所述第二抗体还包括可检测的标记，例如荧光素、金属离子、生物素、放射性同位素、化学发光物质、酶或聚乙二醇。
在又一个方面，本发明提供了所述的单克隆抗体，融合蛋白，抗体偶联物或多特异性抗体，其用于治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤，非小细胞肺癌，膀胱癌，肝癌，结肠癌，前列腺癌，淋巴瘤或肾癌。
在又一个方面，本发明提供了制备具有提高ADCC活性的单克隆抗体的方法，包含：
(1)将包含SEQ ID NO:12的重链CDR1，SEQ ID NO:13的重链CDR2，SEQ ID NO:14的重链CDR3，SEQ ID NO:15的轻链CDR1，SEQ ID NO:16的轻链CDR2和SEQ ID NO:17的轻链CDR3的单克隆抗体在敲降或敲除FUT8基因(α-(1,6)-岩藻糖基转移酶基因)的CHO宿主细胞(例如CHO-BAT-KF细胞)中表达；
(2)将包含SEQ ID NO:12的重链CDR1，SEQ ID NO:13的重链CDR2，SEQ ID NO:14的重链CDR3，SEQ ID NO:15的轻链CDR1，SEQ ID NO:16的轻链CDR2和SEQ ID NO:17的轻链CDR3的单克隆抗体在敲降GDP-岩藻糖4,6-脱水酶(GMD)的CHO-DG44细胞中表达；或
(3)将包含SEQ ID NO:12的重链CDR1，SEQ ID NO:13的重链CDR2，SEQ ID NO:14的重链CDR3，SEQ ID NO:15的轻链CDR1，SEQ ID NO:16的轻链CDR2和SEQ ID NO:17的轻链CDR3的单克隆抗体在CHO细胞中表达，然后用糖基转移酶抑制剂(如2F-过乙酰基 -岩藻糖)去岩藻糖化。
在又一个方面，本发明提供了治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤、非小细胞肺癌、膀胱癌、肝癌、结肠癌、前列腺癌、淋巴瘤或肾癌的方法，包括给患者给予有效量的所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体。
在又一个方面，本发明提供了增强T细胞的增殖反应的方法，其包含利用本发明所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体阻断CTLA-4与B7的结合。
在又一个方面，本发明提供了降低患者体内肿瘤浸润表达CTLA-4的Treg细胞的方法，包括给患者给予治疗有效量的所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体。
在又一个方面，本发明提供了所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体在制备用于治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤、非小细胞肺癌、膀胱癌、肝癌、结肠癌、前列腺癌、淋巴瘤或肾癌的药物中的应用。
在又一个方面，本发明提供了抗CTLA-4抗体或抗原结合片段用于治疗肿瘤或癌症的方法或用途。在一些实施方案中，抗CTLA-4抗体或抗原结合片段用于治疗肿瘤或癌症。
在一些实施方案中，所述抗CTLA-4抗体或抗原结合片段至少包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2、SEQ ID NO:14所示的HCDR3、SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2、SEQ ID NO:17所示的LCDR3中一个或多个。
在一些实施方案中，所述抗CTLA-4抗体或抗原结合片段包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2、SEQ ID NO:14所示的HCDR3、SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3。
在一些实施方案中，所述抗CTLA-4抗体或抗原结合片段的重链可变区包含SEQ ID NO:18所示的序列，与SEQ ID NO:18所示序列具有至少80％同一性的序列，或与SEQ ID NO:18所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
所述抗CTLA-4抗体或抗原结合片段的轻链可变区包含SEQ ID NO:19所示的序列，与SEQ ID NO:19所示序列具有至少80％同一性的序列，或与SEQ ID NO:19所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体或抗原结合片段的重链可变区包含SEQ ID NO:18所示的序列，所述抗CTLA-4抗体或抗原结合片段的轻链可变区包含SEQ ID NO:19所示的序列。
在一些实施方案中，所述抗CTLA-4抗体的重链包含SEQ ID NO:1所示的序列，与SEQ ID NO:1所示序列具有至少80％同一性的序列，或与SEQ ID NO:1所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
所述抗CTLA-4抗体的轻链包含SEQ ID NO:2所示的序列，与SEQ ID NO:2所示序列具有至少80％同一性的序列，或与SEQ ID NO:2所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体为抗体1或抗体2，抗体1和抗体2的重链均包含如SEQ ID NO:1所示序列，所述抗体1和抗体2的轻链均包含如SEQ ID NO:2所示序列；抗体1和抗体2含有两条序列相同的重链和两条序列相同的轻链。
在一些实施方案中，所述抗CTLA-4抗体(例如抗体1或2)中Fc区的高甘露糖糖型总量<5％和/或唾液酸化糖型总量<3％。在一些实施方案中，所述抗CTLA-4抗体(例如抗体1或2)中Fc区的高甘露糖糖型总量为约0.1％、约0.3％、约0.9％、约1.18％、约1.7％、约 2.6％、约3.3％、约4.1％、约4.9％、约4.99％，或任何两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体(例如抗体1或2)中Fc区的唾液酸化糖型总量为约0.1％、约0.2％、约0.36％、约0.8％、约1.5％、约2.2％、约2.7％、约2.9％、2.99％，或任何两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体(例如抗体1或2)中Fc区的高甘露糖糖型总量<3％和/或唾液酸化糖型总量<2％。在一些实施方案中，所述抗CTLA-4抗体(例如抗体1或2)中Fc区的高甘露糖糖型总量<2％和/或唾液酸化糖型总量<1％。
在一些实施方案中，所述抗CTLA-4抗体(例如抗体2)或抗原结合片段的岩藻糖基化水平为0％-20％，比如约0％、约1％、约2％、约3％、约4％、约5％、约6％、约7％、约8％、约9％、约10％、约11％、约12％、约13％、约14％、约15％、约16％、约17％、约18％、约19％或约20％，或在其中任意两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体(例如抗体2)或抗原结合片段的岩藻糖基化水平为0-10％。在一些实施方案中，所述抗CTLA-4抗体(例如抗体2)或抗原结合片段的岩藻糖基化水平为0-5％。在一些实施方案中，所述抗CTLA-4抗体(例如抗体2)或抗原结合片段的岩藻糖基化水平为0-1％。在一些实施方案中，所述抗CTLA-4抗体(例如抗体2)或抗原结合片段的岩藻糖基化水平为约0、约0.1％、约0.3％、约0.4％、约0.5％、约0.6％、约0.8％、约1％、约1.3％、约1.6％、约2.1％、约2.2％、约2.8％、约2.9％、约3％、约3.3％、3.8％、约4％、约4.1％、约4.2％、4.3％、约4.6％、约5％，或这些数值中任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，抗CTLA-4抗体(例如抗体2)或抗原结合片段没有结合岩藻糖。在一些实施方案中，抗CTLA-4抗体(例如抗体2)或抗原结合片段具有增强的ADCC效应(antibody-dependent cell-mediated cytotoxicity)。
抗CTLA-4抗体或抗原结合片段可以通过基因工程在CHO细胞或293细胞中表达，并通过纯化获得；纯化可以采用常规方法进行，例如先离心细胞悬液并收集上清液，再次离心进一步去除杂质。Protein A亲和柱和离子交换柱等方法可以用于纯化抗体蛋白。
在一些实施方案中，抗CTLA-4抗体(例如抗体2)或抗原结合片段由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达，如PCT/CN2018/100008公开的CHO-BAT-KF细胞系。在一些实施方案中，抗体2由CHO-BAT-KF细胞系表达。
在一些实施方案中，所述方法或用途包括：向有需要的患者施用有效量的抗CTLA-4抗体或抗原结合片段。在一些实施方案中，所述抗CTLA-4抗体为抗体1或抗体2。在一些实施方案中，抗CTLA-4抗体(例如抗体2)由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。在一些实施方案中，抗CTLA-4抗体施用的有效剂量为每剂约7mg至180mg。
在一些实施方案中，患者患有肿瘤或癌症。在一些实施方案中，肿瘤和癌症包括但不限于血液癌症、实体瘤。在一些实施方案中，血液癌症包括但不限于白血病、淋巴瘤和骨髓瘤。在一些实施方案中，白血病包括急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)和骨髓性增生疾病/肿瘤(MPDS)。在一些实施方案中，淋巴瘤包括霍奇金淋巴瘤、无痛性和侵袭性非霍奇金淋巴瘤、伯基特淋巴瘤和滤泡性淋巴瘤(小细胞和大细胞)。在一些实施方案中，骨髓瘤包括多发性骨髓瘤(MM)、巨细胞骨髓瘤、重链骨髓瘤和轻链或本斯-琼斯骨髓瘤。在一些实施方案中，实体瘤包括乳腺癌、卵巢癌、肺癌、胰腺癌、前列腺癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌和肾癌。在一些实施方案中，肿瘤和癌症为尚无有效治疗手段的经病理学确诊的局部晚 期或转移性恶性实体肿瘤。
在一些实施方案中，本发明公开了一种用于治疗有需要患者的肿瘤或癌症的方法，其包括施用有效量的抗CTLA-4抗体，其中抗CTLA-4抗体施用的有效量为约7mg至180mg每个治疗周期。在一些实施方案中，一个治疗周期为1周、2周、3周、4周、1个月、5周、6周、7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体为抗体1、抗体2。在一些实施方案中，抗体2由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。
在一些实施方案中，可以将抗CTLA-4抗体配制成药物组合物，并以适合于所选给药途径的多种形式向患者给药，给药途径例如肠胃外、静脉内(iv)、肌肉内、局部或皮下。在一些实施方案中，可以将抗CTLA-4抗体静脉输注。抗CTLA-4抗体的给药量将取决于药物的性质、细胞表面触发药物的内在化、运输和释放的程度，以及所治疗的疾病和患者的状况(如年龄、性别、体重等)。
在一些实施方案中，每次施用的抗CTLA-4抗体(如抗体1或2)约0.1mg/kg至16mg/kg或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，每次施用的抗CTLA-4抗体为约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.9mg/kg、约1mg/kg、约1.3mg/kg、约1.5mg/kg、约1.8mg/kg、约2mg/kg、约2.5mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg、约10mg/kg、约11mg/kg、约12mg/kg、约13mg/kg、约14mg/kg、约15mg/kg、约16mg/kg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，每次施用的抗CTLA-4抗体(如抗体1或2)约0.1mg/kg至2.5mg/kg或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，每次施用的抗CTLA-4抗体为约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约1.1mg/kg、约1.2mg/kg、约1.3mg/kg、约1.4mg/kg、约1.5mg/kg、约1.6mg/kg、约1.7mg/kg、约1.8mg/kg、约1.9mg/kg、约2mg/kg、约2.1mg/kg、约2.2mg/kg、约2.3mg/kg、约2.4mg/kg、约2.5mg/kg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，约1周、约2周、约3周或约4周给药1次。在一些实施方案中，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期、10个周期，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，约3周给药1次，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期或10个周期。在一些实施方案中，约4周给药1次，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期或10个周期。在一些实施方案中，约5周给药1次，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期或10个周期。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：每3周向有需要的患者给予约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约1.1mg/kg、约1.2mg/kg、约1.3mg/kg、约1.4mg/kg、约1.5mg/kg、约1.6mg/kg、约1.7mg/kg、约1.8mg/kg、约1.9mg/kg、约2mg/kg、约2.1mg/kg、约2.2mg/kg、约2.3mg/kg、约2.4mg/kg、约2.5mg/kg的抗CTLA-4抗体，或含此剂量抗CTLA-4抗体的制剂。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：每3周向有需要的患者给予约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约1.1mg/kg、约1.2mg/kg、约1.3mg/kg、约1.4mg/kg、约1.5mg/kg、约1.6mg/kg、约1.7mg/kg、约1.8mg/kg、约1.9mg/kg、约2mg/kg、约2.1mg/kg、约2.2mg/kg、约2.3mg/kg、约2.4mg/kg、约2.5mg/kg的抗体1或2，或含此剂量抗体1或2的制剂；治疗周期为4个周期。
在一些实施方案中，本发明公开了一种治疗肿瘤或癌症的方法，其包括向有需要的患者施用有效量的抗CTLA-4抗体(或制剂)；其中，抗CTLA-4抗体的有效量为单次给药约7mg至180mg(或含此剂量抗CTLA-4抗体的制剂)。剂量时间表和给药方式取决于某些患者群中的抗CTLA-4抗体(或制剂)的获益风险评估和一般临床实践指南。
在一些实施方案中，患者每个治疗周期内抗CTLA-4抗体施用的有效量为约7mg至180mg(或含此剂量抗CTLA-4抗体的制剂)。在一些实施方案中，一个治疗周期为1周、2周、3周、4周、5周、6周、7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期或6个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约6mg、约10mg、约12mg、约18mg、约24mg、约30mg、约40mg、约50mg、约60mg、约100mg、约120mg、约180mg、约200mg、约250mg、约290mg、约300mg、约330mg、约380mg、约400mg、约434mg、约480mg、约500mg、约567mg、约580mg、约600mg、约700mg、约800mg、约900mg、约960mg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg、约8mg、约10mg、约12mg、约18mg、约24mg、约30mg、约40mg、约50mg、约60mg、约68mg、约70mg、约80mg、约90mg、约100mg、约110mg、约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，一个治疗周期为1周至7周给药1次。在一些实施方案中，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg至约120mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg、约10mg、约12mg、约18mg、约20mg、约24mg、约28mg、约30mg、约40mg、约50mg、约60mg、约70mg、约80mg、约90mg、约100mg、约120mg，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约120mg至180mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1 周、约2周、约3周、或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约6.9mg至7.2mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约10mg至13mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约12mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约20mg至24mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约22mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约28mg至33mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约30mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约59mg至64mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约60mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约95mg至113mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约100mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约110mg至123mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约120mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次， 治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约127mg至133mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约130mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约132mg至144mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约140mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约140mg至155mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4(如抗体1或2)抗体的有效量为约150mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约177mg至180mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约180mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，抗CTLA-4抗体施用的有效量为约7mg至120mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约7mg、约10mg、约12mg、约20mg、约26mg、约30mg、约35mg、约40mg、约44mg、约50mg、约58mg、约60mg、约69mg、约70mg、约77mg、约85mg、约99mg、约110mg、约114mg或约120mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约7mg、约12mg、约30mg、约60mg或约120mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约120mg至180mg每3周一次。在一些实施方案中，抗CTLA-4抗体施用的有效量为约120mg、约130mg、约134mg、约140mg、约146mg、约150mg、约154mg、约160mg、约164mg、约170mg、约173mg、约1760mg、约180mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约130mg、140mg、约160mg或约180mg每3周一次，4个治疗周期。
在一些实施方案中，患者每个治疗周期内给药一次抗CTLA-4抗体(或制剂)。在一些实施方案中，每个治疗周期内多次给药抗CTLA-4抗体(或制剂)，例如2次、3次、4次或5次。在一些实施方案中，患者每个治疗周期只能给药1次或4次。
在一些实施方案中，患者接受一个治疗周期治疗。在一些实施方案中，患者接受多个(例如2个、3个、4个、5个或6个)治疗周期治疗。在一些实施方案中，患者接受4个治疗周期治疗。在一些实施方案中，患者接受治疗直至病症得到缓解而不再需要治疗。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：每3周向有需要的患者给予约7mg至30mg、约30mg至60mg、约60mg至120mg、约120mg 至180mg，比如约7mg、约10mg、约12mg、约18mg、约30mg、60mg、约80mg、约100mg、约120mg、约150mg、约160mg、约180mg的抗CTLA-4抗体，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，抗CTLA-4抗体为抗体1。在一些实施方案中，抗CTLA-4抗体为抗体2。
在一些实施方案中，单剂量给药后，患者的症状得到缓解。在一些实施方案中，单剂量给药后，患者后的症状未得到预期缓解，再对患者给药约7mg至180mg抗CTLA-4抗体，直至患者的症状得到缓解。
在一些实施方案中，抗CTLA-4抗体(或制剂)是通过皮下(s.c.)注射、腹膜内(i.p.)注射、肠胃外注射、动脉内注射或静脉内(i.v.)注射等方式进行给药。在一些实施方案中，抗CTLA-4抗体(或制剂)是输液方式进行给药。在一些实施方案中，抗CTLA-4抗体(或制剂)是推注方式进行给药。
在一些实施方案中，抗CTLA-4抗体(或制剂)是通过静脉内(i.v.)输液方式(即静脉输注)进行给药。在一些实施方案中，静脉内输液持续时间为约50分钟、约55分钟、约60分钟、约65分钟、约70分钟、约75分钟、约81分钟、约87分钟、约90分钟、约95分钟，或这些数值中任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，静脉输液持续时间≥60分钟。
在一些实施方案中，抗CTLA-4抗体(或制剂)与其他治疗方法联合用于治疗肿瘤或癌症，例如化疗、放疗、免疫治疗、激素治疗、靶向治疗、生物治疗和手术治疗等。在一些实施方案中，抗CTLA-4抗体(或制剂)与其他肿瘤或癌症治疗剂联合治疗肿瘤或癌症，如激素、治疗肿瘤或癌症的抗体等。
在又一方面，本发明提供了抗CTLA-4抗体在制备用于治疗肿瘤或癌症的药物中的应用。在一些实施方案中，用于治疗肿瘤或癌症的药物包括抗CTLA-4抗体。在一些实施方案中，抗CTLA-4抗体为抗体1或2。在一些实施方案中，抗体2由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。
在又一方面，本发明还提供了一种试剂盒，试剂盒包含抗CTLA-4抗体(或制剂)和用于指导有需要患者给药抗CTLA-4抗体(或制剂)的说明书。在一些实施方案中，抗CTLA-4抗体为抗体1或2。在一些实施方案中，抗体2由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。
在又一方面，本发明还提供了包含抗CTLA-4抗体的适合注射用的药物组合物，如推注型药物组合物或输液(滴注)型药物组合物。适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的载体包括生理盐水、抑菌水或磷酸盐缓冲盐水(PBS)、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。在一些实施方案中，药物组合物还包括药学可接受的载体。在一些实施方案中，药学上可接受的载体可以包含抗细菌剂和/或抗真菌剂，如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在一些实施方案中，药学上可接受的载体可以包含等渗剂，如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。在一些实施方案中，药物组合物至少包含0.1％的抗CTLA-4抗体。抗体的百分比可以变化，并且为给定剂型重量的约2％至90％之间。这种治疗上有用的药物组合物中抗CTLA-4抗体的量可以为给药的有效量。
在又一方面，本发明还提供了上述药物组合物的制备方法：分别将本文所述的抗CTLA-4 抗体与药学上可接受的适合注射用的载体(例如注射用水，生理盐水等)混合。上述抗CTLA-4抗体与药学上可接受的载体的混合方法是本领域通常已知的。
在又一方面，本发明还提供了一种储藏和递送中保持稳定的含抗CTLA-4抗体的液体制剂，其包含以下组分：4-80mg/mL抗CTLA-4抗体或其片段、缓冲剂、稳定剂、螯合剂、表面活性剂、水；液体制剂的pH值为5-7。
在一些实施方案中，所述液体制剂包含以下组分：
(1)4-80mg/mL抗CTLA-4抗体或其片段，
(2)8-45mM缓冲剂，
(3)180-290mM稳定剂，
(4)0.01-0.3mg/mL螯合剂，
(5)0.1-0.5mg/mL表面活性剂，
(6)水；
液体制剂的pH值为5.2-6.5。
在一些实施方案中，所述液体制剂包含以下组分：
(1)5-40mg/mL抗CTLA-4抗体或其片段，
(2)10-30mM缓冲剂，
(3)200-260mM稳定剂，
(4)0.02-0.2mg/mL螯合剂，
(5)0.1-0.4mg/mL表面活性剂，
(6)水；
液体制剂的pH值为5.4-6.2。
在一些实施方案中，缓冲剂不包括Tris缓冲剂。
在一些实施方案中，所述缓冲剂选自组氨酸缓冲剂、醋酸缓冲剂或其组合。在一些实施方案中，所述组氨酸缓冲剂包含组氨酸和组氨酸盐酸盐(如L-组氨酸和L-组氨酸盐酸盐)，所述醋酸缓冲剂包含醋酸和醋酸钠。在一些实施方案中，所述缓冲剂选自组氨酸缓冲剂或组氨酸缓冲剂与醋酸缓冲剂的组合物。在一些实施方案中，所述稳定剂选自海藻糖、蔗糖、甘露醇或其组合物。在一些实施方案中，所述稳定剂选自海藻糖或蔗糖。在一些实施方案中，所述螯合剂包含依地酸二钠。在一些实施方案中，所述螯合剂为依地酸二钠。在一些实施方案中，所述表面活性剂选自聚山梨酯20、聚山梨酯80或其组合物。在一些实施方案中，所述表面活性剂为聚山梨酯80。
在一些实施方案中，抗CTLA-4抗体或其片段的浓度为约5mg/mL、约9mg/mL、约13mg/mL、约17mg/mL、约24mg/mL、约29mg/mL、约31mg/mL、约36mg/mL、约40mg/mL，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，缓冲剂的浓度为约10mM、约14mM、约17mM、约21mM、约25mM、约29mM、约30mM，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，稳定剂的浓度为约200mM、约210mM、约219mM、约227mM、约235mM、约244mM、约251mM、约260mM，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，螯合剂的浓度为约0.02mg/mL、约0.09mg/mL、约0.12mg/mL、约0.17mg/mL、约0.2mg/mL，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，表面活性剂的浓度为约0.1mg/mL、约0.18mg/mL、约0.24mg/mL、约 0.3mg/mL、约0.36mg/mL、约0.4mg/mL，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，当缓冲剂为组氨酸缓冲剂或组氨酸缓冲剂与醋酸缓冲剂的组合物时，液体制剂的pH值为约5.4、约5.5、约5.7、约5.9、约6.1、约6.2，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，当缓冲剂为醋酸缓冲剂、组氨酸缓冲剂、或组氨酸缓冲剂与醋酸缓冲剂的组合物时，液体制剂的pH值为约5.7、约5.74、约5.81、约5.86、约5.9，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂包含7-15mg/mL抗CTLA-4抗体或其片段。在一些实施方案中，液体制剂中抗CTLA-4抗体或其片段的浓度为约7mg/mL、约8mg/mL、约9mg/mL、约10mg/mL、约11mg/mL、约12mg/mL、约13mg/mL、约14mg/mL、约15mg/mL，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂中包含17-23mM组氨酸缓冲剂。在一些实施方案中，液体制剂中组氨酸缓冲剂的浓度为约17mM、约18mM、约19mM、约20mM、约21mM、约22mM、约23mM，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂包含220-250mM蔗糖。在一些实施方案中，液体制剂中蔗糖的浓度为约220mM、约224mM、约228mM、约232mM、约237mM、约239mM、约241mM、约243mM、约247mM、约250mM，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂包含0.02-0.05mg/mL依地酸二钠。在一些实施方案中，液体制剂中依地酸二钠的浓度为约0.02mg/mL、约0.03mg/mL、约0.032mg/mL、约0.033mg/mL、约0.038mg/mL、约0.04mg/mL、约0.043mg/mL、约0.048mg/mL、约0.05mg/mL，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂包含0.1-0.3mg/mL聚山梨酯80。在一些实施方案中，液体制剂中聚山梨酯80的浓度为约0.1mg/mL、约0.15mg/mL、约0.19mg/mL、约0.21mg/mL、约0.26mg/mL、约0.3mg/mL，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂的pH值为5.7-5.9。在一些实施方案中，液体制剂的pH值为约5.7、约5.73、约5.75、约5.78、约5.8、约5.83、约5.87、约5.9，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，液体制剂包含以下组分：
(1)10mg/mL抗CTLA-4抗体或其片段，
(2)20mM组氨酸缓冲剂，
(3)240mM蔗糖，
(4)0.033mg/mL依地酸二钠，
(5)0.2mg/mL聚山梨酯80，
(6)水；
液体制剂的pH值为5.8。
在一些实施方案中，所述液体制剂含无菌水或注射用水。
在一些实施方案中，所述抗CTLA-4抗体或其片段包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2、SEQ ID NO:14所示的HCDR3、SEQ ID NO:15所示的LCDR1、 SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链可变区包含SEQ ID NO:18所示的氨基酸序列或与所述SEQ ID NO:18具有至少约90％同一性的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链可变区包含SEQ ID NO:19所示的氨基酸序列或与所述SEQ ID NO:19具有至少约90％同一性的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链可变区包含的序列与所述SEQ ID NO:18具有至少约90％、约92％、约94％、约95％、约97％、约99％、约100％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值，所述抗CTLA-4抗体或其片段的轻链可变区包含的序列与所述SEQ ID NO:19具有至少约90％、约92％、约94％、约95％、约97％、约99％、约100％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链可变区包含SEQ ID NO:18所示的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链可变区包含SEQ ID NO:19所示的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链包含SEQ ID NO:1所示的氨基酸序列或与所述SEQ ID NO:1具有至少约90％同一性的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链包含SEQ ID NO:2所示的氨基酸序列或与所述SEQ ID NO:2具有至少约90％同一性的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链包含的序列与所述SEQ ID NO:1具有至少约90％、约92％、约94％、约95％、约97％、约99％、约100％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值，所述抗CTLA-4抗体或其片段的轻链包含的序列与所述SEQ ID NO:2具有至少约90％、约92％、约94％、约95％、约97％、约99％、约100％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链包含SEQ ID NO:1所示的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链包含SEQ ID NO:2所示的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体或其片段为单克隆抗体。在一些实施方案中，所述抗CTLA-4抗体或其片段为全人源单克隆抗体。在一些实施方案中，所述抗CTLA-4抗体或其片段由CHO细胞表达。在一些实施方案中，所述抗CTLA-4抗体或其片段由敲除了α-(1,6)-岩藻糖转移酶的CHO细胞表达。在一些实施方案中，所述抗CTLA-4抗体或其片段由CHO-BAT-KF fut8(-/-)细胞(WO2019029713A1专利已公开上述细胞)表达。抗CTLA-4抗体或其片段通过常规方法进行纯化，如低速离心使细胞和培养基分离，高速离心上清液，并依次进行蛋白A亲和纯化和离子交换纯化。
在一些实施方案中，所述抗CTLA-4抗体或其片段中岩藻糖基化水平≤5％。在一些实施方案中，所述抗CTLA-4抗体或其片段中岩藻糖基化水平为约0、约0.1％、约0.3％、约0.4％、约0.6％、约1.3％、约1.9％、约2.2％、约2.8％、约3.3％、约3.7％、约4.1％、约4.5％、约5％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗CTLA-4抗体或其片段中高甘露糖糖型总量<5％。在一些实施方案中，所述抗CTLA-4抗体或其片段中高甘露糖糖型总量为约0.1％、约0.3％、约0.9％、约1.18％、约1.7％、约2.6％、约3.3％、约4.1％、约4.9％、约4.99％，或这些数值中的任何 两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗CTLA-4抗体或其片段中唾液酸化糖型总量<3％。在一些实施方案中，所述抗CTLA-4抗体或其片段中唾液酸化糖型总量为约0.1％、约0.2％、约0.36％、约0.8％、约1.5％、约2.2％、约2.7％、约2.9％、约2.99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在又一方面，本发明还提供了所述液体制剂在制备用于治疗与CTLA-4相关疾病的药物中的应用。本发明另一方面提供了一种治疗与CTLA-4相关疾病的方法，所述方法包括对患者施用有效剂量的所述液体制剂。
在一些实施方案中，与CTLA-4相关疾病包括癌症，所述液体制剂可用于治疗癌症例如排斥可移植的肿瘤。在一些实施方案中，与CTLA-4相关疾病为黑色素瘤，非小细胞肺癌，膀胱癌，肝癌，结肠癌，前列腺癌，淋巴瘤或肾癌。
在一些实施方案中，所述液体制剂的给药方式可为注射或输液；给药的部位为肠外、静脉、黏膜、舌下、肌肉、皮内、鼻腔、腹腔、动脉内或皮下。
在一些实施方案中，所述患者是哺乳动物。在一些实施方案中，所述患者是人。
在又一方面，本发明还公开了所述液体制剂的制备方法，包括将各组分在溶剂中混合制成液体制剂。在一些实施方案中，所述方法包括以下步骤：
(1)配置缓冲液，无菌过滤；
(2)通过UF/DF超滤，采用步骤(1)制备的缓冲液对抗体溶液进行超滤换液，然后进行浓缩得到抗体浓缩物；
(3)配制含有缓冲剂、稳定剂、表面活性剂、螯合剂的辅料母液，将无菌过滤后的辅料母液添加到步骤(2)制备的抗体浓缩物中，得到液体制剂。
为了保持抗CTLA-4抗体或其片段的稳定性，本发明通过选择适当的缓冲体系、优化稳定剂、螯合剂和添加表面活性剂，开发得到的液体制剂可以显著抑制冷冻/复融循环期间、长期存储和温度变化过程中酸峰、二聚物、多聚物、降解物和不溶性微粒的形成。本发明抗CTLA-4抗体或其片段在上述制剂中，经过至少5次反复冻融后稳定性良好，在室温可稳定保存至少6个月。本发明的液体制剂可用于稳定地保存临床治疗用的抗CTLA-4抗体或其片段，对于治疗与CTLA-4相关疾病有重大意义。
本发明抗CTLA-4抗体(或制剂)可用于肿瘤或癌症的治疗。
附图说明
图1显示了抗体2与抗体1糖型的对比图；其中三角形代表岩藻糖，抗体2中基本不含岩藻糖。
图2显示了抗体2、抗体1与CTLA-4抗原的ELISA结合曲线；图中sample表示为抗体样品。
图3显示了
Figure PCTCN2020139205-appb-000002
抗体2、抗体1与CTLA-4抗原在Biacore上的结合曲线。
图4显示了抗CTLA-4抗体的体外生物学活性模型示意图；图中promotor表示为启动子，luciferase表示为荧光素酶基因。
图5显示了pCMV2-OKT3的质粒图谱。
图6显示了抗体2、抗体1均在活性模型上表现出增强T细胞功能的活性作用；图中sample表示为抗体样品。
图7显示了抗体2、抗体1与FcγRIIIa 158V的结合解离传感曲线。
图8显示了pCDH-FcγRIIIa 158V FL-Puro的质粒图谱。
图9显示了抗体2在ADCC报告基因模型上比抗体1有更高的体外ADCC活性；图中sample表示为抗体样品。
图10A和图10B分别显示了抗体2、抗体1在CTLA-4人源化MC38荷瘤小鼠上给药后小鼠体重变化趋势和抑制肿瘤效果；图中箭头代表各次给药时间。
图11A、图11B、图11C和图11D分别显示了在CTLA-4人源化MC38荷瘤小鼠上，
Figure PCTCN2020139205-appb-000003
抗体1、抗体2给药后外周血单个核细胞中的CD4 +、CD8 +、Treg细胞占CD45细胞的比例以及CD8 +/Treg的比值。
术语
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。
“抗体”(Ab)，应包括但不限于免疫球蛋白，是主要由浆细胞产生的大的“Y”形蛋白，其被免疫系统用于中和病原体如病原细菌和病毒。抗体通过Fab的可变区识别病原体上的一种称为“抗原”的独特分子。抗体的“Y”结构的每个尖端含有对抗原上一个特定表位的互补位，允许这两个结构精确地结合在一起。使用这种结合机制，抗体可以标记微生物或受感染的细胞，使免疫系统对其进行攻击，或者可以直接中和目标。抗体与免疫系统的其他组分通信的能力是通过其Fc区介导的，Fc区包含参与这些相互作用的保守糖基化位点。抗体的产生是体液免疫系统的主要功能。
“抗体片段”包括(comprise)全长抗体的一部分，通常包括其抗原结合区。抗体片段的实例包括Fab，Fab’，F(ab’)2和Fv片段；双抗体(diabodies)；线性抗体(linear antibody)；单链抗体分子；和由抗体片段形成的多特异性抗体。
“同一性”和“同源性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
“至少80％同一性”为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、 丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
“VL、VH的保守氨基酸取代”的氨基酸数目可为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。“重链或轻链的保守氨基酸取代”的氨基酸数目可为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约18个、约19个、约22个、约24个、约25个、约29个、约31个、约35个、约38个、约41个、约45个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
抗体可以有不同的种类，称为同种型或类别。在胎盘哺乳动物中，有五种抗体同种型，称为IgA，IgD，IgE，IgG和IgM。它们各自以“Ig”前缀命名，代表免疫球蛋白(有时可与“抗体”互换使用)，其生物学特性、功能位置和处理不同抗原的能力不同。抗体同种型的不同后缀表示抗体含有的不同类型的重链，每个重链类别按字母顺序命名：α、γ、δ、ε、和μ，分别对应产生IgA，IgG，IgD，IgE和IgM。而在哺乳动物中有两种类型的免疫球蛋白轻链，称为λ(λ)和κ(κ)。一般来说，每种抗体含有两条相同的轻链；在哺乳动物中每种抗体仅存在一种类型的轻链κ或λ。
免疫球蛋白G(IgG)是抗体的一种。IgG是人体中大约75％的血清抗体，是血液循环中最常见的抗体类型。人体中有四种IgG亚类(IgG1、2、3和4)，按其在血清中的丰度顺序命名(IgG1是最丰富的)。
抗体的某些部分具有相同的功能。例如，“Y”的臂包含可以与抗原结合的位点以识别特定的外来物体。该抗体区域称为Fab区域。它由抗体的每条重链和轻链的一个恒定区和一个可变区组成。可变结构域也称为FV区，并且是结合抗原的最重要区域。具体而言，轻链可变区(VL)和重链可变区(VH)上的各三个区域负责与抗原结合，这些区域称为互补决定区(CDR)。“Y”的基部在调节免疫细胞活性中起作用。该区域称为Fc区，由两条重链组成，根据抗体的类别，它们贡献两个或三个恒定区域。因此，Fc区通过结合特定类别的Fc受体和其他免疫分子(例如补体蛋白)确保每种抗体对给定抗原产生适当的免疫应答。通过这样，它介导不同的生理效应，包括识别调理颗粒(与FcγR结合)，细胞裂解(与补体结合)，以及肥大细胞，嗜碱性粒细胞和嗜酸性粒细胞(与FcεR结合)的脱粒。
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在“Sequence of Proteinsof Immunological Interest”(1983)提出的编号系统。抗体还可以用EU或Chothia编号系统。
“抗原”在免疫学中，是指与抗体或T淋巴细胞抗原受体特异性结合的物质。它们是刺激抗体产生或被抗体识别的物质。有时抗原是自身免疫性疾病中宿主自身的一部分。术语抗原最初描述的是与抗体特异性结合的结构分子。后它被扩展为指可被适应性免疫系统的高度可 变抗原受体(B细胞受体或T细胞受体)识别的任何分子或线性分子片段。
“单克隆抗体”(mAb)是由相同的免疫细胞制备的抗体，所述免疫细胞是单一亲本细胞的所有克隆。单克隆抗体可以具有单价亲和力，因为它们结合相同的表位(抗体识别的抗原部分)。相反，多克隆抗体与多个表位结合，并且通常由几种不同的浆细胞分泌。还可以通过将一种单一单克隆抗体的治疗靶标增加至两个表位来改造成双特异性单克隆抗体。单克隆抗体可以通过杂交瘤、重组、转基因或本领域技术人员已知的其他技术制造。
“人源化”抗体是指来自非人类物种的抗体，其蛋白质序列已被修饰以增加它们与人类天然产生的抗体变体的相似性。“人源化”过程通常应用于为人类施用而开发的单克隆抗体(例如，作为抗癌药物开发的抗体)。当开发特异性抗体的过程涉及在非人免疫系统(例如小鼠中)中产生时，也需要进行人源化。人源化小鼠是携带功能性人类基因、细胞、组织和/或器官的小鼠。人源化小鼠通常用作人类治疗的生物学和医学研究中的小动物模型。免疫缺陷小鼠通常用作人细胞或组织的接受者，因为它们由于缺乏宿主免疫力而可以相对容易地接受异源细胞。这种人源化小鼠模型可用于在健康和病理情况下模拟人体免疫系统，并且可以在与人体生理学相关的体内环境中评估治疗候选物。
“抗体依赖性细胞介导的细胞毒性”(ADCC)是细胞介导的免疫防御机制，其中免疫系统的效应细胞主动裂解靶细胞，其膜表面抗原被特异性抗体结合。它是抗体作为体液免疫反应的一部分，可以通过其作用来限制和控制感染。
ADCC独立于免疫补体系统，该系统也溶解靶标但不需要任何其他细胞。ADCC需要一种效应细胞，其已知是通常与IgG抗体相互作用的天然杀伤(NK)细胞。然而，巨噬细胞、中性粒细胞和嗜酸性粒细胞也可以介导ADCC，例如嗜酸性粒细胞通过IgE抗体杀死某些被称为蠕虫的寄生虫。另外，ADCC是适应性免疫应答的一部分，因为它依赖于先前的抗体应答。
“Fc区”，是抗体的尾区，其与称为Fc受体的细胞表面受体和补体系统的一些蛋白质相互作用。该特性允许抗体激活免疫系统。在IgG，IgA和IgD抗体同种型中，Fc区由两个相同的蛋白质片段组成，衍生自抗体的两条重链的第二和第三恒定区；IgM和IgE Fc区在每条多肽链中含有三个重链恒定结构域(CH结构域2-4)。IgG的Fc区具有高度保守的N-糖基化位点。Fc片段的糖基化对于Fc受体介导的活性是必需的，且不同的糖型对治疗性抗体的药学性质有不同的影响。高甘露糖糖型会导致抗体在血液中快速清除，半衰期缩短；G0F促进补体通路作用，加快清除速率；唾液酸修饰对静脉注射免疫球蛋白的炎症作用影响明显；岩藻糖基化的降低导致ADCC活性明显增强。因此，根据治疗性抗体的主要作用机制和药物用途，设计和优化抗体的糖链是有必要的。本专利中，“Fc”或“Fc段”或“Fc结构域”的含义等同于“Fc区”。
“Fc受体”或“FcR”是在某些细胞表面发现的蛋白质，这些细胞包括B淋巴细胞、滤泡树突细胞、自然杀伤细胞、巨噬细胞、中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、人血小板和肥大细胞等，它们都有助于保护免疫系统的功能。Fc受体的名称来源于其对抗体的Fc区域的结合特异性。Fc受体与附着于受感染细胞或入侵病原体的抗体结合。它们的活性通过吞噬细胞和细胞毒性细胞破坏微生物、或通过抗体介导的吞噬作用去感染细胞、或通过抗体依赖性细胞介导的细胞毒性去发挥作用。一些病毒如黄病毒通过称为抗体依赖性感染增强的机制使用Fc受体来帮助它们感染细胞。
有几种不同类型的Fc受体，其基于它们识别的抗体类型进行分类。用于识别抗体类型的 拉丁字母被转换为相应的希腊字母，该字母位于名称的“Fc”部分之后。例如，结合最常见类别的抗体IgG的那些称为Fc-γ受体(FcγR)，结合IgA的那些称为Fc-α受体(FcαR)，结合IgE的那些称为Fc-ε受体(FcεR)。FcR的类别还通过表达它们的细胞(巨噬细胞、粒细胞、天然杀伤细胞、T细胞和B细胞)和每种受体的信号传导特性来区分。
所有Fcγ受体(FcγR)都属于免疫球蛋白超家族，是诱导微生物吞噬作用的最重要的Fc受体。该家族包括几个成员，FcγRI(CD64)、FcγRIIA(CD32)、FcγRIIB(CD32)、FcγRIIIA(CD16a)、FcγRIIIB(CD16b)，由于它们的分子结构不同，它们的抗体亲和力不同。例如，FcγRI比FcγRII或FcγRIII更强地结合IgG。FcγRI还具有由三个免疫球蛋白(Ig)样结构域组成的细胞外部分，比FcγRII或FcγRIII具有更多的结构域。这种性质允许FcγRI结合唯一的IgG分子(或单体)，但所有Fcγ受体必须结合免疫复合物中的多个IgG分子才能被激活。Fc-γ受体对IgG的亲和力不同，而不同的IgG亚类对每种Fcγ受体具有独特的亲和力。这些相互作用通过IgG的位置CH2-84.4处的聚糖(寡糖)进一步调节。例如，通过产生空间位阻，含有CH2-84.4聚糖的岩藻糖降低了对FcγRIIIA的IgG亲和力。
“癌症”是指一组涉及异常细胞生长的疾病，有可能侵入或扩散到身体的其他部位。癌症与良性肿瘤形成对比，良性肿瘤不会扩散到身体的其他部位。可能的体征和症状包括肿块，异常出血，长期咳嗽，原因不明的体重减轻和排便改变。
“荷瘤小鼠”分为原发模型和移植瘤模型。研究中最常使用的是移植瘤模型，主要包括原位移植瘤和皮下移植瘤。简而言之，就是将相关的肿瘤细胞通过原位注射或者通过皮下注射入小鼠体内，使其致瘤。
“给药”指的是药物可能通过口服、注射、输液、肠外、静脉、黏膜、舌下、肌肉、皮内、鼻腔、腹腔、动脉内、皮下吸收或通过任意其他与现有技术相结合的给药方式完成。在本发明的一个实施方案中，给药是全身性的。
“有效量”指达到治疗目标所需的量。有效量与抗体对其特异抗原的结合亲和力，疾病、紊乱或病症的严重程度、给药途径、在接受给药对象中给予的抗体从自由体积耗尽的速率等有关。作为非限制性示例，本发明的抗体的治疗有效量的可以为从约0.01mg/kg到约15mg/kg患者体重，从约0.1mg/kg到约10mg/kg患者体重，或从约1mg/kg到约5mg/kg患者体重，比如约0.3mg/kg，约1mg/kg，或约3mg/kg患者体重。给药频率可以是例如每周一次到每月一次，比如每三周一次。
“患者”通常指需要诊断、预后或治疗的任何患者，特别是哺乳动物患者，包括人类、猫、豚鼠、兔子、大鼠、小鼠、马、牛等。
“糖型”是蛋白质的同种型，其仅在附着的聚糖的数量或类型方面不同。糖蛋白通常由许多不同的糖形式组成，其中附着的糖或寡糖发生改变。这些修饰可能是由于糖基化过程中生物合成的差异，或者是由于糖苷酶或糖基转移酶的作用。可以通过对分离的糖型的详细化学分析或通过凝集素亲和层析和凝集素亲和电泳等来检测糖型。
“糖基化”是碳水化合物(即糖基供体)与另一分子(糖基受体)的羟基或其他官能团连接的反应。在生物学中，糖基化主要特别指将聚糖附着到蛋白质或其他有机分子上的酶促过程。这种酶促过程产生了一种在细胞中发现的基本生物聚合物(以及DNA，RNA和蛋白质)。糖基化是共翻译和翻译后修饰的一种形式。聚糖在膜和分泌蛋白中起着多种结构和功能作用。在粗面内质网中合成的大多数蛋白质经历糖基化。它是一种酶导向的位点特异性过程，与糖化的非酶促化学反应相反。糖基化也作为O-GlcNAc修饰存在于细胞质和细胞核中。常见的 糖基化类型包括O-糖基化和N-糖基化。
如本文所用，术语“有需要”是指已将患者鉴定为需要特定方法或治疗。在一些实施例中，可以通过任何诊断方式进行识别。在本文描述的任何方法和治疗中，患者可能需要。
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
本发明中缓冲剂的量，是指组成缓冲剂的缓冲体系中缓冲对的总量。在一些实施方式中，采用摩尔浓度作为缓冲剂的量的单位，其数值指缓冲剂的缓冲体系中缓冲对的摩尔浓度。如，由组氨酸和组氨酸盐酸盐组成的组氨酸缓冲剂作为缓冲剂时，给定浓度的组氨酸缓冲剂(如20mM)是组氨酸和组氨酸盐酸盐的组合浓度(如组氨酸为8mM，组氨酸盐酸盐为12mM；或者组氨酸为6mM，组氨酸盐酸盐为14mM；或者组氨酸为8.46mM，组氨酸盐酸盐为11.54mM等)。
本发明所述的制剂可以用所述辅料或其水合物或盐类配制。比如组氨酸盐酸盐，又称盐酸组氨酸，可以是无水组氨酸盐酸盐，也可以是组氨酸盐酸盐水合物，如组氨酸盐酸盐一水合物；如，“12mM组氨酸盐酸盐”可以为1L液体制剂中含有12mmol组氨酸盐酸盐或12mmol组氨酸盐酸盐一水合物；如，制剂中加入“2.52g组氨酸盐酸盐一水合物”等同于制剂中加入2.3g组氨酸盐酸盐。又比如，制剂中既可以加入海藻糖，又可以加入对应量的海藻糖二水合物；如，“225mM海藻糖”可以为1L液体制剂中含有225mmol海藻糖或225mmol海藻糖二水合物；如，制剂中加入“85g海藻糖二水合物”等同于制剂中加入76.9g海藻糖。又比如，制剂中既可以加入依地酸(EDTA)二钠，又可以加入对应量的依地酸二钠二水合物或依地酸；如，“0.033mg/mL依地酸二钠”可以为1L液体制剂中加入0.037g依地酸二钠二水合物或0.033g依地酸二钠或0.029g依地酸。
患者疾病“治疗”指的是(1)阻止疾病在有倾向性或还没表现疾病症状的患者中出现；(2)抑制疾病或症状或阻止其发展；或(3)减轻或消除疾病或症状或致其退化。
“稳定性”是指包含抗体的液体制剂中，抗体或其片段在给定的生产、制备、运输和/或贮存条件下不发生、或仅极少地发生聚集、降解或片段化。“稳定”制剂在给定的生产、制备、运输和/或贮存条件下保持生物学活性。可通过例如SEC-HPLC、IEC-HPLC、CE-SDS(NR)、灯检及浑浊度、不溶性颗粒、DLS检测粒子粒径等技术测量的所述制剂的聚集、降解或片段化程度等，从而评估所述抗体的稳定性。在一些实施方案中，“稳定”制剂指制剂在室温可稳定保存至少3个月、至少6个月或至少12个月。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
一方面，本发明提供了针对CTLA-4的单克隆抗体，所述单克隆抗体包含
(1)重链CDR1(HCDR1)，其包含SEQ ID NO:12所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
重链CDR2(HCDR2)，其包含SEQ ID NO:13所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
重链CDR3(HCDR3)，其包含SEQ ID NO:14所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
轻链CDR1(LCDR1)，其包含SEQ ID NO:15所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，
轻链CDR2(LCDR2)，其包含SEQ ID NO:16所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，和
轻链CDR3(LCDR3)，其包含SEQ ID NO:17所示的序列，与所述序列具有至少约80％、约90％、优选至少约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成；
或
(2)重链可变区(VH)，其包含SEQ ID NO:18所示的序列，与所述序列具有至少约80％，优选至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成，和
轻链可变区(VL)，其包含SEQ ID NO:19所示的序列，与所述序列具有至少约80％，优选至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)序列同一性的序列，或与所述序列相比具有一个或多个(优选约1个、约2个或约3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，或由其组成；
所述单克隆抗体具有降低的岩藻糖基化水平。
在具体的实施方案中，所述单克隆抗体包含重链和轻链，所述重链包含SEQ ID NO:1的氨基酸序列，与SEQ ID NO:1的氨基酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约 93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的序列，或由其组成；
所述轻链包含SEQ ID NO:2所示的氨基酸序列，与SEQ ID NO:2的氨基酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的序列，或由其组成。
在具体的实施方案中，所述重链由SEQ ID NO:3所示的核苷酸序列或与SEQ ID NO:3的核苷酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的核苷酸序列编码，所述轻链由SEQ ID NO:4所示的核苷酸序列或与SEQ ID NO:4的核苷酸序列具有至少约80％，优选至少约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％(或任何两个数值之间的范围(包括端点)或其中任何值)同源性的核苷酸序列编码。
在具体的实施方案中，所述岩藻糖基化水平为0％-20％，比如约0％，约1％，约2％，约3％，约4％，约5％，约6％，约7％，约8％，约9％，约10％，约11％，约12％，约13％，约14％，约15％，约16％，约17％，约18％，约19％或约20％，或在其中任意两个数值之间的范围(包括端点)或其中任何值。
在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量<5％和/或唾液酸化糖型总量<3％。在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量为约0.1％、约0.3％、约0.9％、约1.18％、约1.7％、约2.6％、约3.3％、约4.1％、约4.9％、约4.99％，或任何两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗单克隆抗体Fc区的唾液酸化糖型总量为约0.1％、约0.2％、约0.36％、约0.8％、约1.5％、约2.2％、约2.7％、约2.9％、2.99％，或任何两个数值之间的范围(包括端点)或其中任何值。
在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的高甘露糖糖型总量<5％和/或唾液酸化糖型总量<3％。
在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量<3％和/或唾液酸化糖型总量<2％。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的高甘露糖糖型总量<3％和/或唾液酸化糖型总量<2％。
在一些实施方案中，所述单克隆抗体Fc区的高甘露糖糖型总量<2％和/或唾液酸化糖型总量<1％。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的高甘露糖糖型总量<2％和/或唾液酸化糖型总量<1％。
在一些实施方案中，所述单克隆抗体Fc区的岩藻糖基化水平为0％-10％。在一些实施方案中，所述单克隆抗体为IgG1，所述单克隆抗体CH2区的岩藻糖基化水平为0％-10％。
在一些实施方案中，所述单克隆抗体Fc区的岩藻糖基化水平为0％-5％。在一些实施方案中，所述抗单克隆抗体Fc区的岩藻糖基化水平为约0、约0.1％、约0.3％、约0.4％、约0.6％、约1.3％、约1.9％、约2.2％、约2.8％、约3.3％、约3.7％、约4.1％、约4.5％、约5％，或任何两个数值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述单克隆抗体为 IgG1，所述单克隆抗体CH2区的岩藻糖基化水平为0％-5％。
在一些实施方案中，所述单克隆抗体由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达，如PCT/CN2018/100008公开的CHO-BAT-KF细胞系。
另一方面，本发明提供了药物组合物或融合蛋白，其包含本发明所述的单克隆抗体。
在一些实施方案中，所述药物组合物为适于通过口服、注射、输液、肠外、静脉、黏膜、舌下、肌肉、皮内、鼻腔、腹腔、动脉内、皮下吸收给药的形式。在一些实施方案中，所述药物组合物为适于全身给药的形式。
另一方面，本发明提供了抗体偶联物，其包括本发明所述的单克隆抗体，以及与所述单克隆抗体偶联的偶联部分，所述偶联部分为纯化标签(如His标签)、细胞毒性剂，延长所述单克隆抗体半衰期的结构部分，或可检测的标记，优选地，所述偶联部分为放射性同位素、化学发光剂、酶、血清白蛋白(例如人或鼠血清白蛋白)、或聚乙二醇。
在一些实施方案中，本发明抗CTLA-4抗体为单价抗体或多价抗体。在另一方面，本发明提供了多特异性抗体(例如双特异性抗体)，其包含本发明所述的单克隆抗体，以及针对其他抗原和/或其他抗原表位的抗体或抗原结合片段。
在又一个方面，本发明提供了试剂盒，其包括本发明所述的单克隆抗体，抗体偶联物、多特异性抗体或融合蛋白。
在具体的实施方案中，所述试剂盒还包括第二抗体，细胞毒性剂，化疗剂，放疗剂，所述第二抗体特异性识别所述单克隆抗体；任选地，所述第二抗体还包括可检测的标记，例如荧光素、金属离子、生物素、放射性同位素、化学发光物质、酶或聚乙二醇。
在又一个方面，本发明提供了所述的单克隆抗体，融合蛋白，抗体偶联物或多特异性抗体，其用于治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤，非小细胞肺癌，膀胱癌，肝癌，结肠癌，前列腺癌，淋巴瘤或肾癌。
在又一个方面，本发明提供了治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤、非小细胞肺癌、膀胱癌、肝癌、结肠癌、前列腺癌、淋巴瘤或肾癌的方法，包括给患者给予有效量的所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体。
在又一个方面，本发明提供了增强T细胞的增殖反应的方法，其包含利用本发明所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体阻断CTLA-4与B7的结合。
在又一个方面，本发明提供了降低患者体内肿瘤浸润表达CTLA-4的Treg细胞的方法，包括给患者给予治疗有效量的所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体。
在又一个方面，本发明提供了所述的单克隆抗体、融合蛋白、抗体偶联物或多特异性抗体在制备用于治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤、非小细胞肺癌、膀胱癌、肝癌、结肠癌、前列腺癌、淋巴瘤或肾癌的药物中的应用。
在又一个方面，本发明公开了抗CTLA-4抗体或抗原结合片段用于治疗肿瘤或癌症的方法或用途。在一些实施方案中，抗CTLA-4抗体或抗原结合片段剂用于治疗肿瘤或癌症。
在一些实施方案中，抗CTLA-4抗体(例如抗体2)或抗原结合片段由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达，如PCT/CN2018/100008公开的CHO-BAT-KF细胞系。在一些实施方案中，抗体2由CHO-BAT-KF细胞系表达。
在一些实施方案中，所述方法或用途包括：向有需要的患者施用有效量的抗CTLA-4抗体或抗原结合片段。在一些实施方案中，所述抗CTLA-4抗体为抗体1或抗体2。在一些实施方案中，抗CTLA-4抗体(例如抗体2)由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。 在一些实施方案中，抗CTLA-4抗体施用的有效剂量为每剂约7mg至180mg。
在一些实施方案中，患者患有肿瘤或癌症。在一些实施方案中，肿瘤和癌症包括但不限于血液癌症、实体瘤。在一些实施方案中，血液癌症包括但不限于白血病、淋巴瘤和骨髓瘤。在一些实施方案中，白血病包括急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)和骨髓性增生疾病/肿瘤(MPDS)。在一些实施方案中，淋巴瘤包括霍奇金淋巴瘤、无痛性和侵袭性非霍奇金淋巴瘤、伯基特淋巴瘤和滤泡性淋巴瘤(小细胞和大细胞)。在一些实施方案中，骨髓瘤包括多发性骨髓瘤(MM)、巨细胞骨髓瘤、重链骨髓瘤和轻链或本斯-琼斯骨髓瘤。在一些实施方案中，实体瘤包括乳腺癌、卵巢癌、肺癌、胰腺癌、前列腺癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌和肾癌。在一些实施方案中，肿瘤和癌症为尚无有效治疗手段的经病理学确诊的局部晚期或转移性恶性实体肿瘤。
在一些实施方案中，本发明公开了一种用于治疗有需要患者的肿瘤或癌症的方法，其包括施用有效量的抗CTLA-4抗体，其中抗CTLA-4抗体施用的有效量为约7mg至180mg每个治疗周期。在一些实施方案中，一个治疗周期为1周、2周、3周、4周、1个月、5周、6周、7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体为抗体1、抗体2。在一些实施方案中，抗体2由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。
在一些实施方案中，可以将抗CTLA-4抗体配制成药物组合物，并以适合于所选给药途径的多种形式向患者给药，给药途径例如肠胃外、静脉内(iv)、肌肉内、局部或皮下。在一些实施方案中，可以将抗CTLA-4抗体静脉输注。抗CTLA-4抗体的给药量将取决于药物的性质、细胞表面触发药物的内在化、运输和释放的程度，以及所治疗的疾病和患者的状况(如年龄、性别、体重等)。
在一些实施方案中，每次施用的抗CTLA-4抗体(如抗体1或2)约0.1mg/kg至16mg/kg或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，每次施用的抗CTLA-4抗体为约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.9mg/kg、约1mg/kg、约1.3mg/kg、约1.5mg/kg、约1.8mg/kg、约2mg/kg、约2.5mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg、约10mg/kg、约11mg/kg、约12mg/kg、约13mg/kg、约14mg/kg、约15mg/kg、约16mg/kg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，每次施用的抗CTLA-4抗体(如抗体1或2)约0.1mg/kg至2.5mg/kg或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，每次施用的抗CTLA-4抗体为约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约1.1mg/kg、约1.2mg/kg、约1.3mg/kg、约1.4mg/kg、约1.5mg/kg、约1.6mg/kg、约1.7mg/kg、约1.8mg/kg、约1.9mg/kg、约2mg/kg、约2.1mg/kg、约2.2mg/kg、约2.3mg/kg、约2.4mg/kg、约2.5mg/kg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，约1周、约2周、约3周或约4周给药1次。在一些实施方案中，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期、10个周期，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，约3周给药1次，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个 周期、9个周期或10个周期。在一些实施方案中，约4周给药1次，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期或10个周期。在一些实施方案中，约5周给药1次，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期、7个周期、8个周期、9个周期或10个周期。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：每3周向有需要的患者给予约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约1.1mg/kg、约1.2mg/kg、约1.3mg/kg、约1.4mg/kg、约1.5mg/kg、约1.6mg/kg、约1.7mg/kg、约1.8mg/kg、约1.9mg/kg、约2mg/kg、约2.1mg/kg、约2.2mg/kg、约2.3mg/kg、约2.4mg/kg、约2.5mg/kg的抗CTLA-4抗体，或含此剂量抗CTLA-4抗体的制剂。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：每3周向有需要的患者给予约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约1.1mg/kg、约1.2mg/kg、约1.3mg/kg、约1.4mg/kg、约1.5mg/kg、约1.6mg/kg、约1.7mg/kg、约1.8mg/kg、约1.9mg/kg、约2mg/kg、约2.1mg/kg、约2.2mg/kg、约2.3mg/kg、约2.4mg/kg、约2.5mg/kg的抗体1或2，或含此剂量抗体1或2的制剂；治疗周期为4个周期。
在一些实施方案中，本发明公开了一种治疗肿瘤或癌症的方法，其包括向有需要的患者施用有效量的抗CTLA-4抗体(或制剂)；其中，抗CTLA-4抗体的有效量为单次给药约7mg至180mg(或含此剂量抗CTLA-4抗体的制剂)。剂量时间表和给药方式取决于某些患者群中的抗CTLA-4抗体(或制剂)的获益风险评估和一般临床实践指南。
在一些实施方案中，患者每个治疗周期内抗CTLA-4抗体施用的有效量为约7mg至180mg(或含此剂量抗CTLA-4抗体的制剂)。在一些实施方案中，一个治疗周期为1周、2周、3周、4周、5周、6周、7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期或6个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约6mg、约10mg、约12mg、约18mg、约24mg、约30mg、约40mg、约50mg、约60mg、约100mg、约120mg、约180mg、约200mg、约250mg、约290mg、约300mg、约330mg、约380mg、约400mg、约434mg、约480mg、约500mg、约567mg、约580mg、约600mg、约700mg、约800mg、约900mg、约960mg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg、约8mg、约10mg、约12mg、约18mg、约24mg、约30mg、约40mg、约50mg、约60mg、约68mg、约70mg、约80mg、约90mg、约100mg、约110mg、约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，一个治疗周期为1周至7周给药1次。在一些实施方案中，治疗周期为1个周期、2个周期、3个周期、4个周期、5个周期、6个周期，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效 量为约7mg至约120mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg、约10mg、约12mg、约18mg、约20mg、约24mg、约28mg、约30mg、约40mg、约50mg、约60mg、约70mg、约80mg、约90mg、约100mg、约120mg，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约120mg至180mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约6.9mg至7.2mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约7mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约10mg至13mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约12mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约20mg至24mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约22mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约28mg至33mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约30mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约59mg至64mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约60mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期 为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约95mg至113mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约100mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约110mg至123mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约120mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约127mg至133mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约130mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约132mg至144mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约140mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约140mg至155mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4(如抗体1或2)抗体的有效量为约150mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约177mg至180mg，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，患者每个治疗周期内施用抗CTLA-4抗体(如抗体1或2)的有效量为约180mg，或含此剂量抗CTLA-4抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周给药1次，治疗周期为2个周期、3个周期、4个周期或5个周期。
在一些实施方案中，抗CTLA-4抗体施用的有效量为约7mg至120mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约7mg、约10mg、约12mg、约20mg、约26mg、约30mg、约35mg、约40mg、约44mg、约50mg、约58mg、约60mg、约69mg、约70mg、约77mg、约85mg、约99mg、约110mg、约114mg或约120mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约7mg、约12mg、约30mg、约60mg或约120mg每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约120mg至180mg每3周一次。在一些实施方案中，抗CTLA-4抗体施用的有效量为约120mg、约130mg、约134mg、约140mg、约146mg、约150mg、约154mg、约160mg、约164mg、约170mg、约173mg、约1760mg、约180mg 每3周一次，4个治疗周期。在一些实施方案中，抗CTLA-4抗体施用的有效量为约130mg、140mg、约160mg或约180mg每3周一次，4个治疗周期。
在一些实施方案中，患者每个治疗周期内给药一次抗CTLA-4抗体(或制剂)。在一些实施方案中，每个治疗周期内多次给药抗CTLA-4抗体(或制剂)，例如2次、3次、4次或5次。在一些实施方案中，患者每个治疗周期只能给药1次或4次。
在一些实施方案中，患者接受一个治疗周期治疗。在一些实施方案中，患者接受多个(例如2个、3个、4个、5个或6个)治疗周期治疗。在一些实施方案中，患者接受4个治疗周期治疗。在一些实施方案中，患者接受治疗直至病症得到缓解而不再需要治疗。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：每3周向有需要的患者给予约7mg至30mg、约30mg至60mg、约60mg至120mg、约120mg至180mg，比如约7mg、约10mg、约12mg、约18mg、约30mg、60mg、约80mg、约100mg、约120mg、约150mg、约160mg、约180mg的抗CTLA-4抗体，或含此剂量抗CTLA-4抗体的制剂。在一些实施方案中，抗CTLA-4抗体为抗体1。在一些实施方案中，抗CTLA-4抗体为抗体2。
在一些实施方案中，单剂量给药后，患者的症状得到缓解。在一些实施方案中，单剂量给药后，患者后的症状未得到预期缓解，再对患者给药约7mg至180mg抗CTLA-4抗体，直至患者的症状得到缓解。
在一些实施方案中，抗CTLA-4抗体(或制剂)是通过皮下(s.c.)注射、腹膜内(i.p.)注射、肠胃外注射、动脉内注射或静脉内(i.v.)注射等方式进行给药。在一些实施方案中，抗CTLA-4抗体(或制剂)是输液方式进行给药。在一些实施方案中，抗CTLA-4抗体(或制剂)是推注方式进行给药。
在一些实施方案中，抗CTLA-4抗体(或制剂)是通过静脉内(i.v.)输液方式(即静脉输注)进行给药。在一些实施方案中，静脉内输液持续时间为约50分钟、约55分钟、约60分钟、约65分钟、约70分钟、约75分钟、约81分钟、约87分钟、约90分钟、约95分钟，或这些数值中任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，静脉输液持续时间≥60分钟。
在一些实施方案中，抗CTLA-4抗体(或制剂)与其他治疗方法联合用于治疗肿瘤或癌症，例如化疗、放疗、免疫治疗、激素治疗、靶向治疗、生物治疗和手术治疗等。在一些实施方案中，抗CTLA-4抗体(或制剂)与其他肿瘤或癌症治疗剂联合治疗肿瘤或癌症，如激素、治疗肿瘤或癌症的抗体等。
在又一方面，本发明公开了抗CTLA-4抗体在制备用于治疗肿瘤或癌症的药物中的应用。在一些实施方案中，用于治疗肿瘤或癌症的药物包括抗CTLA-4抗体。在一些实施方案中，抗CTLA-4抗体为抗体1或2。在一些实施方案中，抗体2由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。
在又一方面，本发明还公开了一种试剂盒，试剂盒包含抗CTLA-4抗体(或制剂)和用于指导有需要患者给药抗CTLA-4抗体(或制剂)的说明书。在一些实施方案中，抗CTLA-4抗体为抗体1或2。在一些实施方案中，抗体2由α-(1,6)-岩藻糖基转移酶基因敲除的细胞系表达。
在又一方面，本发明提供一种含抗CTLA-4抗体或其片段的液体制剂。在一些实施方案中，液体制剂包含以下组分：4-80mg/mL抗CTLA-4抗体或其片段，缓冲剂，稳定剂，螯合 剂，表面活性剂，水；液体制剂的pH值为5-7。
在一些实施方案中，缓冲剂不包括Tris缓冲剂。在一些实施方案中，所述缓冲剂选自组氨酸缓冲剂、醋酸缓冲剂或其组合。在一些实施方案中，所述稳定剂选自海藻糖、蔗糖或甘露醇。在一些实施方案中，所述螯合剂包含依地酸二钠。在一些实施方案中，所述螯合剂为依地酸二钠。在一些实施方案中，所述表面活性剂选自聚山梨酯20、聚山梨酯80或其组合物。
在一些实施方案中，所述抗CTLA-4抗体或其片段包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2、SEQ ID NO:14所示的HCDR3、SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链可变区包含SEQ ID NO:18所示的氨基酸序列或与所述SEQ ID NO:18具有至少约90％同一性的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链可变区包含SEQ ID NO:19所示的氨基酸序列或与所述SEQ ID NO:19具有至少约90％同一性的氨基酸序列。
在一些实施方案中，所述抗CTLA-4抗体或其片段的重链包含SEQ ID NO:1所示的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链包含SEQ ID NO:2所示的氨基酸序列。在一些实施方案中，所述抗CTLA-4抗体或其片段为全人源单克隆抗体。在一些实施方案中，所述抗CTLA-4抗体或其片段由CHO细胞表达。在一些实施方案中，所述抗CTLA-4抗体或其片段由敲除了α-(1,6)-岩藻糖转移酶基因的中国仓鼠卵巢细胞(CHO fut8(-/-)，如CHO-BAT-KF)表达。在一些实施方案中，所述抗CTLA-4抗体或其片段中岩藻糖基化水平≤5％，高甘露糖糖型总量<5％，唾液酸化糖型总量<3％。
在一些实施方案中，所述抗CTLA-4抗体或其片段中岩藻糖基化水平为约0、约0.1％、约0.3％、约0.4％、约0.6％、约1.3％、约1.9％、约2.2％、约2.8％、约3.3％、约3.7％、约4.1％、约4.5％、约5％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体或其片段中高甘露糖糖型总量为约0.1％、约0.3％、约0.9％、约1.18％、约1.7％、约2.6％、约3.3％、约4.1％、约4.9％、约4.99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，所述抗CTLA-4抗体或其片段中唾液酸化糖型总量为约0.1％、约0.2％、约0.36％、约0.8％、约1.5％、约2.2％、约2.7％、约2.9％、约2.99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述液体制剂包含以下组分：5-40mg/mL抗CTLA-4抗体或其片段，10-30mM缓冲剂，200-260mM稳定剂，0.02-0.2mg/mL螯合剂，0.1-0.4mg/mL表面活性剂，水；液体制剂的pH值为5.4-6.2。
在一些实施方案中，所述液体制剂包含以下组分：5-40mg/mL抗CTLA-4抗体或其片段，10-30mM醋酸缓冲剂，200-260mM海藻糖，0.02-0.2mg/mL依地酸二钠，0.1-0.4mg/mL聚山梨酯80，水；液体制剂的pH值为5.7-5.9。在一些实施方案中，所述稳定剂为蔗糖或甘露醇。在一些实施方案中，所述表面活性剂为聚山梨酯20。
在一些实施方案中，所述液体制剂包含以下组分：5-40mg/mL抗CTLA-4抗体或其片段，10-30mM组氨酸缓冲剂，200-260mM蔗糖，0.02-0.2mg/mL依地酸二钠，0.1-0.4mg/mL聚山梨酯80，水；液体制剂的pH值为5.4-6.2。在一些实施方案中，所述稳定剂为海藻糖或甘露醇。在一些实施方案中，所述表面活性剂为聚山梨酯20。
在一些实施方案中，所述液体制剂包含以下组分：5-40mg/mL抗CTLA-4抗体或其片段，10-30mM组氨酸缓冲剂和醋酸缓冲剂的组合物，200-260mM蔗糖，0.02-0.2mg/mL依地酸二钠，0.1-0.4mg/mL聚山梨酯20，水；液体制剂的pH值为5.7-5.9。在一些实施方案中，所述稳定剂为海藻糖或甘露醇。在一些实施方案中，所述表面活性剂为聚山梨酯80。
在一些实施方案中，所述液体制剂包含以下组分：约5mg/mL抗CTLA-4抗体或其片段，约10mM组氨酸缓冲剂，约200mM海藻糖，约0.02mg/mL依地酸二钠，约0.1mg/mL聚山梨酯20，水；液体制剂的pH值为约5.4。
在一些实施方案中，所述液体制剂包含以下组分：约9mg/mL抗CTLA-4抗体或其片段，约14mM醋酸缓冲剂，约219mM海藻糖，约0.09mg/mL依地酸二钠，约0.24mg/mL聚山梨酯20，水；液体制剂的pH值为约5.7。
在一些实施方案中，所述液体制剂包含以下组分：约24mg/mL抗CTLA-4抗体或其片段，约17mM醋酸缓冲剂，约227mM蔗糖，约0.12mg/mL依地酸二钠，约0.3mg/mL聚山梨酯20，水；液体制剂的pH值为约5.81。
在一些实施方案中，所述液体制剂包含以下组分：约29mg/mL抗CTLA-4抗体或其片段，约21mM组氨酸缓冲剂，约235mM蔗糖，约0.17mg/mL依地酸二钠，约0.3mg/mL聚山梨酯80，水；液体制剂的pH值为约5.7。
在一些实施方案中，所述液体制剂包含以下组分：约31mg/mL抗CTLA-4抗体或其片段，约25mM组氨酸缓冲剂，约244mM蔗糖，约0.11mg/mL依地酸二钠，约0.3mg/mL聚山梨酯80与聚山梨酯20的组合物，水；液体制剂的pH值为约5.9。
在一些实施方案中，所述液体制剂包含以下组分：约36mg/mL抗CTLA-4抗体或其片段，约29mM组氨酸缓冲剂，约251mM甘露醇，约0.16mg/mL依地酸二钠，约0.36mg/mL聚山梨酯80，水；液体制剂的pH值为约6.1。
在一些实施方案中，所述液体制剂包含以下组分：约40mg/mL抗CTLA-4抗体或其片段，约30mM组氨酸缓冲剂与醋酸缓冲剂的组合物，约260mM甘露醇，约0.2mg/mL依地酸二钠，约0.4mg/mL聚山梨酯80，水；液体制剂的pH值为约5.9。
在一些实施方案中，所述液体制剂包含以下组分：约40mg/mL抗CTLA-4抗体或其片段，约30mM醋酸缓冲剂，约248mM甘露醇，约0.2mg/mL依地酸二钠，约0.3mg/mL聚山梨酯80，水；液体制剂的pH值为约5.9。
在一些实施方案中，液体制剂包含以下组分：7-15mg/mL的抗CTLA-4抗体或其片段，17-23mM的组氨酸缓冲剂，220-250mM蔗糖，0.02-0.05mg/mL的依地酸二钠，0.1-0.3mg/mL的聚山梨酯80，水；液体制剂的pH值为5.7-5.9。
在一些实施方案中，液体制剂包含以下组分：约7mg/mL的抗CTLA-4抗体或其片段，约17mM的组氨酸缓冲剂，约220mM蔗糖，约0.02mg/mL依地酸二钠，约0.1mg/mL聚山梨酯80，水；液体制剂的pH值为约5.7。
在一些实施方案中，液体制剂包含以下组分：约9mg/mL抗CTLA-4抗体或其片段，约20mM组氨酸缓冲剂，约228mM蔗糖，约0.032mg/mL依地酸二钠，约0.19mg/mL聚山梨酯80，水；液体制剂的pH值为约5.75。
在一些实施方案中，液体制剂包含以下组分：约10mg/mL抗CTLA-4抗体或其片段，约20mM组氨酸缓冲剂，约240mM蔗糖，约0.033mg/mL依地酸二钠，约0.2mg/mL聚山梨酯80，水；液体制剂的pH值为约5.8。
在一些实施方案中，液体制剂包含以下组分：约14mg/mL抗CTLA-4抗体或其片段，约22mM的组氨酸缓冲剂，约243mM蔗糖，约0.043mg/mL依地酸二钠，约0.26mg/mL聚山梨酯80，水；液体制剂的pH值为约5.87。
在一些实施方案中，液体制剂包含以下组分：约15mg/mL的抗CTLA-4抗体或其片段，约23mM组氨酸缓冲剂，约250mM蔗糖，约0.05mg/mL的依地酸二钠，约0.3mg/mL聚山梨酯80，水；液体制剂的pH值为约5.9。
在一些实施方案中，所述液体制剂含的水为无菌水或注射用水。
本发明另一面提供了所述液体制剂在制备用于治疗与CTLA-4相关疾病的药物中的应用。本发明另一方面提供了一种治疗与CTLA-4相关疾病的方法，所述方法包括对患者施用有效剂量的所述液体制剂。
在一些实施方案中，与CTLA-4相关疾病包括癌症或用于排斥可移植的肿瘤。在一些实施方案中，与CTLA-4相关疾病为黑色素瘤，非小细胞肺癌，膀胱癌，肝癌，结肠癌，前列腺癌，淋巴瘤或肾癌。
在一些实施方案中，所述液体制剂的给药方式可以为注射、输液；给药的部位为肠外、静脉、黏膜、舌下、肌肉、皮内、鼻腔、腹腔、动脉内或皮下。
在一些实施方案中，采用等渗溶液(如0.9％注射用NaCl溶液)对所述含抗CTLA-4抗体的溶液进行稀释，经稀释后随即以静脉输注的方式给药。在一些实施方案中，采用等渗溶液(如0.9％注射用NaCl溶液)对所述含10mg/mL抗CTLA-4抗体的溶液进行稀释，经稀释后随即以静脉输注的方式给药。在一些实施方案中，稀释后抗CTLA-4抗体的浓度为约0.05mg/mL-约5mg/mL或约0.5mg/mL-约1mg/mL。在一些实施方案中，稀释后抗CTLA-4抗体的浓度为约0.05mg/mL、约0.07mg/mL、约0.09mg/mL、约0.1mg/mL、约0.2mg/mL、约0.3mg/mL、约0.5mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL、约1mg/mL、约1.7mg/mL、约2.4mg/mL、约2.9mg/mL、约3.1mg/mL、约3.8mg/mL、4.1mg/mL、约4.6mg/mL或约5mg/mL。
在一些实施方案中，有效量为本文所述剂量。
在一些实施方案中，有效量可为0.1-100mg/每kg患者体重。在一些实施方案中，有效量为0.1-20mg/每kg患者体重。在另一些实施方案中，有效量为约2mg/每kg患者体重。在另一些实施方案中，有效量为约3mg/每kg患者体重。在另一些实施方案中，有效量为约10mg/每kg患者体重。在另一些实施方案中，有效量为约20mg/每kg患者体重。在另一些实施方案中，有效量为约30mg/每kg患者体重。在另一些实施方案中，有效量为约50mg/每kg患者体重。在另一些实施方案中，有效量为约100mg/每kg患者体重。在一些实施方案中，可根据需要每日、每周、每月和/或每年以每小时/天/周/月单次或多次给予患者有效量的抗CTLA-4抗体或其片段。
本发明另一方面提供了所述液体制剂的制备方法，其包括以下步骤：
(1)配置缓冲液，无菌过滤；
(2)通过UF/DF超滤，采用步骤(1)制备的缓冲液对抗体进行超滤换液，然后进行浓缩，得到抗体浓缩物；
(3)配制含有缓冲剂、稳定剂、表面活性剂和螯合剂的辅料母液，调节到合适的pH值；将无菌过滤后的辅料母液添加到步骤(2)制备的抗体浓缩物中，得到液体制剂。
实施例1.抗体1、抗体2的制备
准备CHO细胞(如，由CHO-K1细胞株经培养适应悬浮生长的中国仓鼠卵巢细胞)和CHO-BAT-KF，发明专利CN109096399A已公开上述细胞，通过电穿孔方法将含有编码抗CTLA-4抗体的轻链/重链的核苷酸序列的线性化后的质粒分别转染两株细胞，该抗CTLA-4抗体重链CDR1为SEQ ID NO:12，重链CDR2为SEQ ID NO:13，重链CDR3为SEQ ID NO:14，轻链CDR1为SEQ ID NO:15，轻链CDR2为SEQ ID NO:16，轻链CDR3为SEQ ID NO:17，重链可变区序列为SEQ ID NO:18，轻链可变区序列为SEQ ID NO:19，重链序列为SEQ ID NO:1，轻链序列为SEQ ID NO:2，重链的编码序列为SEQ ID NO:3，轻链的编码序列为SEQ ID NO:4。电转条件为：电压300V，1500μF，4mm电转杯，40μg线性化质粒，1000万细胞。转染CHO细胞的，命名为BAT1(其为表达本文中“抗体1”的细胞)；转染CHO-BAT-KF细胞的，命名为BAT2(其为表达本文中“抗体2”的细胞)。电转染之后分别将两种细胞重悬入含有1×GS(Specialty Media，货号：GSS1016-C)的CHO-AGT(Gibco，货号：12490025)培养基，以每孔1500个细胞的密度均匀分到40块96孔板中，每孔体积为100μL。将细胞培养板放入8％CO 2，37℃培养箱培养。48小时后补加100μL CHO-AGT培养基(1×GS和50μM终浓度MSX(甲硫氨酸亚砜胺))继续培养。
提前准备好筛选阳性克隆用的ELISA板(Corning，货号：9018)。将Sigma公司的羊抗人Fc抗体(Sigma，货号：I2136)用PBS稀释至浓度为2μg/mL，每孔100μL加入ELISA板中，37℃孵育2h后，甩掉板中液体并拍干，每孔加入200μL封闭液(含有5％BSA的PBS)，37℃孵育2h后，直接放进-20℃保存备用。96孔细胞培养板中的细胞约16-20天后形成肉眼可见的细胞克隆团。BAT1共挑出1389个克隆，BAT2共挑出852个克隆。检测步骤为解冻包被好羊抗人Fc抗体的ELISA板，用PBST洗板3次，敷上稀释40倍的96孔细胞培养上清，37℃孵育2小时，用PBST洗板5次，加入1:10000倍稀释的耦联过氧化物酶的山羊抗人Kappa轻链–二抗(Sigma，货号：A7164)，37℃孵育0.5小时，用PBST洗板7次，加入TMB单组分显色液(湖州英创生物，货号：TMB-S-001)避光显色10-15分钟，用0.1M硫酸终止反应，在450nm波长下读数，标记出表达量较高的阳性克隆。阳性克隆转移到24孔板中培养5-7天，培养上清稀释1000倍进行上述ELISA检测。表达量较高且细胞状态良好的阳性克隆转移到6孔板中培养5-7天，培养上清稀释2000倍进行上述ELISA检测。表达量较高且细胞状态良好的阳性克隆转移到125mL小摇瓶，在摇床中培养。待细胞生长状态稳定良好，活力95％以上，且细胞达到一定数量时，以30-50×10 5细胞/mL密度、30mL体积接种到CHO-AGT培养基中，进行12天的摇瓶培养。培养过程中在第3、6、9天分别取0.5ml培养物测细胞密度及活力，取样后分别加入3ml Feed C(CD EfficientFeed C AGT，生产商：Gibco)，其中第6、9天的培养物离心取上清冻存，取第10、11、12天的培养物测细胞密度及活力，培养物离心取上清冻存。第12天收集所有细胞上清，Protein A纯化。从BAT1细胞纯化得到的抗体为抗体1，从BAT2细胞纯化得到的抗体为抗体2。对抗体1和抗体2进行测序，抗体1和抗体2的重链序列为SEQ ID NO:1(每条重链含有448个氨基酸，分子量为51kDa)，轻链序列为SEQ ID NO:2(每条轻链含有215个氨基酸，分子量为23kDa)。得到的蛋白进行各项质量分析，其中在蛋白糖型的分析上，由于抗体2在敲除了α-(1,6)-岩藻糖基转移酶的细胞系CHO-BAT-KF中表达，因此糖型与抗体1有差异(如图1和表1)，抗体2主要糖型为去岩藻糖基化糖型：岩藻糖糖基化水平约为0。前面所述冻存的第6、9、10、11、12天上清则进行HPLC检测蛋白表达量。
另外，测定
Figure PCTCN2020139205-appb-000004
(伊匹单抗，购自百时美施贵宝公司，在本发明中，
Figure PCTCN2020139205-appb-000005
也表示为_930395)、抗体1和抗体2的CH2区中糖型含量，结果是高甘露糖糖型总量<5％，唾液酸化糖型总量<3％；其中，一组高甘露糖糖型、唾液酸化糖型的测定结果如表1所示(表1中，/表示未检测到或含量约为0)：
表1.部分糖型百分含量
Figure PCTCN2020139205-appb-000006
影响抗体免疫原性风险的糖型有唾液酸化糖型：唾液酸化糖型含量从高到低依次是
Figure PCTCN2020139205-appb-000007
抗体1、抗体2，本发明抗体2中低含量的唾液酸化糖型有利于降低免疫原性的风险；高甘露糖糖型含量从高到低依次是
Figure PCTCN2020139205-appb-000008
抗体2、抗体1，本发明抗体2中低含量的高甘露糖糖型有利于延长抗体的半衰期。
实施例2.抗体2蛋白与CTLA-4抗原在体外的结合活性
把用于表达CTLA-4抗原的胞外区和人类IgG1抗体重链Fc段的序列(SEQ ID NO:5，其氨基酸序列为SEQ ID NO:6)的质粒瞬时转染到293F细胞中。
293F细胞用CD 293 TGE Medium(BPM Cell Culture，货号：CM-1156)培养，待细胞活力在95％以上时，用新鲜培养基将细胞传代到80-100×10 5细胞/mL，24h后细胞密度大概在150-200×10 5细胞/mL时，可进行转染。先将质粒在65℃水浴30分钟，每100×10 5细胞用1μg质粒，每1μg质粒用3μL的PEI(Polysciences，货号：24765-2)。分别将质粒和PEI用培养基稀释(两个溶液的体积加起来是总体积的5％)，室温静置5分钟。然后把PEI溶液加到质粒溶液中，混匀，室温静置20分钟。将复合物溶液加入到细胞中，37℃摇床培养24小时后， 加入总体积比为10％的CD Feed X Supplement(BPM Cell Culture，货号：CF-1116)到细胞中。5天后收获细胞上清，上清用Protein A进行纯化，得到CTLA-4-ECD-Fc抗原蛋白。
CTLA-4-ECD-Fc抗原蛋白包被ELISA板，4℃过夜。第二天用5％BSA封闭液于37℃封闭2小时。洗板后分别加上从1μg/mL开始3倍梯度稀释的抗体2、抗体1蛋白，一共稀释12个浓度(最后一个为空白0浓度)，每个浓度做3个复孔，37℃孵育2小时。洗板后加入1:10000倍稀释的耦联过氧化物酶的山羊抗人Kappa轻链二抗(Sigma，货号A7164)，37℃孵育0.5小时。洗板后加入TMB单组分显色液避光显色10-15分钟，用0.1M硫酸终止反应，在450nm波长下读数，抗体2、抗体1与CTLA-4抗原的ELISA结合曲线如图2。
同时在BIAcore(T200，GE Healthcare)上也进行了抗体2、抗体1蛋白与CTLA-4-His(义翘神州，11159-H08H)抗原在体外的结合活性检测。将待测抗体：抗体2、抗体1、
Figure PCTCN2020139205-appb-000009
用HBS-EP(0.01M HEPES,0.15M NaCl,3mM EDTA,0.005％P20,pH 7.4)稀释至5μg/mL，抗原CTLA-4-His用HBS-EP稀释至100nM，并以此为起始浓度作2倍梯度稀释，得100、50、25、12.5、6.25、3.125nM的CTLA-4-His稀释液。用Protein A芯片(GE Healthcare)进行检测，5μg/ml药物稀释液以10μl/min的流速通过实验流路(Fc2、Fc4)，捕获15s使捕获量约为600RU；之后流速调为30μL/min，依次进分析物不同浓度的CTLA-4-His稀释液(0、3.125、6.25、12.5、25、50、100nM)，同时经过实验流路(Fc2、Fc4)和参比流路(Fc1、Fc3)表面，结合时间180s，解离时间600s，最后进Glycine 1.5缓冲液60s对芯片进行再生并进入下一个循环。用数据分析软件Evaluation Software3.1对试验结果进行分析，将样品试验流路采集所得传感信号进行参比流路、样品空白双扣减，并选用动力学“1:1”模型进行拟合，得出各抗体样品对CTLA-4亲和结合的动力学参数，见表2。Biacore曲线拟合见图3(其中Yervoy_930395为
Figure PCTCN2020139205-appb-000010
Y20180901为抗体1，181110-B14-2-CEX为抗体2)。
表2.抗体2、抗体1、
Figure PCTCN2020139205-appb-000011
对CTLA-4亲和结合的动力学参数
Figure PCTCN2020139205-appb-000012
注：ka：结合速率；kd：解离速率；KD：结合解离平衡常数，也即亲和力。
结论：抗体2、抗体1蛋白与CTLA-4抗原的结合，在ELISA和BIAcore上均显示出相似的结合活性。说明在抗CTLA-4抗体上进行ADCC的增强并没有改变其与CTLA-4抗原的结合能力。
实施例3.抗体2和抗体1蛋白在体外的生物学活性
抗CTLA-4抗体的体外生物学活性模型示意图如图4。
构建pCMV2-OKT3(其中OKT3部分为抗体的ScFv形式，其核苷酸序列为SEQ ID NO:7所示，OKT3的重链氨基酸序列为SEQ ID NO:8所示，轻链氨基酸序列为SEQ ID NO:9所示)质粒，质粒图谱如图5。
通过电转的方法，把pCMV2-OKT3转染到Raji细胞中，用潮霉素加压筛选，通过与Jurkat-IL2-luciferase细胞相互混合并刺激6个小时，加入荧光素酶底物用酶标仪检测自发荧光的方法，筛选出能刺激Jurkat-IL2-luciferase细胞(Promega，JA3005)产生自发荧光的阳性单克隆，命名为Raji-OKT3细胞。
用含有1％FBS的1640培养基作为该实施例中的Buffer，把Raji-OKT3细胞与 Jurkat-IL2-luciferase-CTLA4FL细胞(Promega，JA3005)分别稀释成400×10 5细胞/mL的密度，于96孔白板(Corning，货号：3917)每孔各加入25μL的Raji-OKT3与25μL的Jurkat-IL2-luciferase-CTLA4FL。把抗体2和抗体1分别用Buffer分别稀释到100μg/mL，以此为起始浓度，3倍梯度稀释，一共稀释10个浓度(最后一个为空白0浓度)。往上述已加入两种细胞的孔中，分别加入不同浓度的抗体溶液，每孔加入25μL，每个浓度做3个复孔。此时每孔中共有75μL的细胞抗体混合液。把白板放入37℃培养箱孵育6个小时后，取出白板，于室温放置10分钟。每孔加入75μL已平衡至室温的萤火虫荧光素酶底物(Promega)，将白板放入酶标仪，震荡5s，10分钟后读取荧光值。结果见图6。
结论：抗体2、抗体1在活性模型上均表现出相似的增强T细胞功能的活性作用。说明在抗CTLA-4抗体上进行ADCC的增强并没有改变其与CTLA-4抗原在体外的生物学活性。
实施例4.抗体2和抗体1蛋白与FcγRIIIa的结合
SA传感器(Fertebio)置于PBS中预湿10分钟备用，将生物素标记的FcγRIIIa 158V(ACRO Biosystems)、FcγRIIIa 158F(ACRO Biosystems)用pH为6.8的含有0.5％BSA的PBST(以下简称稀释缓冲液)稀释至1μg/mL，将SA传感器在FcγRIIIa 158-Biotin中固化至信号约为0.6nm。将待测抗体2、抗体1用稀释缓冲液稀释至500nM，并以此为起始浓度作2倍梯度稀释，一共准备7个浓度点(500、250、125、62.5、31.25、15.62、7.812nM)。将稀释缓冲液、梯度浓度的药物抗体稀释液、再生缓冲液(pH为8.4的1M MgCl 2)、中和缓冲液(PBST)依次加入96孔板相应列中，运行如下步骤：①Baseline：在稀释缓冲液中检测基线60秒；②Association：在抗体梯度稀释液及样品空白(稀释缓冲液)中结合90秒；③Dissociation：在稀释缓冲液中解离90秒；④Regeneration：在再生缓冲液中再生5秒；⑤Neutralization：在中和缓冲液中中和5秒；⑥Regeneration和Neutralization循环进行3次。
用Data Analysis 8.2对数据进行处理分析，样品数据经参比(样品空白)信号扣减后进行拟合，得到亲和常数KD。与FcγRIIIa 158F的亲和力结果统计见表3，与FcγRIIIa 158V的亲和力结果统计见表4，其中抗体2、抗体1与FcγRIIIa 158V的结合解离传感图见图7(0-240s为抗体1与FcγRIIIa 158V的结合解离，270-510s为抗体2与FcγRIIIa 158V的结合解离)。
表3.抗体2、抗体1对FcγRIIIa 158F亲和结合的动力学参数
Figure PCTCN2020139205-appb-000013
注：kon：结合速率；kdis：解离速率；KD：结合解离平衡常数，也即亲和力。
表4.抗体2、抗体1对FcγRIIIa 158V亲和结合的动力学参数
Figure PCTCN2020139205-appb-000014
注：kon：结合速率；kdis：解离速率；KD：结合解离平衡常数，也即亲和力。
结论：结果表明，经过ADCC增强的抗体2，其与FcγRIIIa的亲和力比抗体1的更强。
实施例5.抗体2和抗体1蛋白在ADCC报告基因模型上的体外生物学活性
构建pCDH-FcγRIIIa 158V FL-Puro质粒，质粒图谱如图8。构建过程如下：合成SEQ ID NO:10所示FcγRIIIa 158V FL cDNA序列，氨基酸序列如SEQ ID NO:11所示，放于pUC-57载体中，命名为Puc-57-FcγRIIIa 158V FL cDNA。合成SEQ ID NO:20和SEQ ID NO:21所示的正反向PCR引物(用于扩增FcγRIIIa 158V FL cDNA片段并在片段两端带上EcoRI和NotI酶切位点)。以Puc-57-FcγRIIIa 158V FL cDNA为模板，用上述正反向引物进行PCR，得到扩增后的FcγRIIIa 158V FL cDNA片段。用EcoRI和NotI分别对pCDH-EF1-MCS-T2A-Puro质粒(常规质粒，例如可以购自优宝生物编号VT1482)和引物扩增后的FcγRIIIa 158V FL cDNA进行双酶切，酶切产物进行胶回收，胶回收的产物进行连接、转化大肠杆菌、测序，正确克隆命名为pCDH-FcγRIIIa 158V FL-Puro。
用慢病毒包装系统pLP1、pLP2、pLP/VSV-G(例如可以购自invitrogen)及pCDH-FcγRIIIa 158V FL-Puro进行病毒的包装，然后感染Jurkat细胞。48h后加入0.3μg/mL嘌呤霉素作为筛选压。转染两周后待细胞长起来，用FITC标记的抗FcγRIIIa抗体(Invitrogen，货号：MHCD1601)通过流式细胞仪检测FcγRIIIa 158V的表达情况，筛选出阳性单克隆。
通过电转的方法，把pGL4.30-luc2P-NFAT-RE-Hygro质粒(购自Promega)转染到上述FcγRIIIa 158V表达阳性的Jurkat单克隆细胞中，用潮霉素加压筛选。通过PMA(50μg/mL)和离子霉素(1μg/mL)刺激6个小时并加入荧光素酶底物，采用酶标仪检测自发荧光的方法筛选出能被PMA和离子霉素刺激而发出自发荧光的阳性单克隆，命名为Jurkat-NFAT-luciferase-FcγRIIIa 158V细胞。
用含有1％FBS的1640培养基作为该实施例中的缓冲液，把Jurkat-NFAT-luciferase-FcγRIIIa 158V细胞与Jurkat-IL2-luciferase-CTLA4FL细胞分别稀释成200×10 5细胞/mL的密度，于96孔白板(Corning，货号：3917)每孔各加入25μL的Jurkat-NFAT-luciferase-FcγRIIIa 158V与25μL的Jurkat-IL2-luciferase-CTLA4FL。把抗体2和抗体1用缓冲液分别稀释到100ng/mL，以此为起始浓度，3倍梯度稀释，一共稀释10个浓度(最后一个为空白0浓度)。往上述已加入两种细胞的孔中，加入不同浓度的抗体溶液，每孔加入25μL，每个浓度做3个复孔。此时每孔中共有75μL的细胞抗体混合液。把白板放入37℃培养箱孵育6个小时后，取出白板，于室温放置10分钟。每孔加入75μL已平衡至室温的萤火虫荧光素酶底物，将白板放入酶标仪，震荡5s，10分钟后读取荧光值。结果见图9。
结论：在ADCC报告基因模型上，抗体2比抗体1显示出更强的体外ADCC活性，从EC50值上看，抗体2在体外模型上的ADCC活性约为抗体1的12倍。
实施例6.抗体2和抗体1蛋白在小鼠体内的抗肿瘤药效
将MC38细胞以5×10 5个/0.1mL浓度接种于B-hCTLA4人源化雌性小鼠的右侧皮下，待肿瘤生长到约125mm 3时按肿瘤体积随机分组，每组8只，共8组，分别为：G1对照组(0.9％氯化钠注射液溶剂)、G2组(
Figure PCTCN2020139205-appb-000015
1mg/kg)、G3组(抗体1，0.3mg/kg)、G4组(抗体1，1mg/kg)、G5组(抗体1，3mg/kg)、G6组(抗体2，0.3mg/kg)、G7组(抗体2，1mg/kg)组和G8组(抗体2，3mg/kg)。所有组给药途径均为腹腔注射，每3天给药1次，连续给药6次，末次给药12天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％)。
整个实验过程中，所有实验动物在给药期间活动和进食状态良好，实验动物体重均有一定程度的上升。实验过程中无小鼠死亡。抗体2、抗体1对MC38细胞移植B-hCTLA4小鼠体重的影响见表5，给药后动物体重变化趋势图见图10A。而抗体2、抗体1对MC38细胞移 植B-hCTLA4小鼠肿瘤体积的影响见表6，给药后肿瘤体积增长趋势图见图10B。
表5.抗体2、抗体1对MC38细胞移植B-hCTLA4小鼠体重的影响
Figure PCTCN2020139205-appb-000016
注：a：平均数±标准误；b：给药组体重与溶剂对照组体重在给药27天后统计学比较，t-test。
表6.抗体2、抗体1对MC38细胞移植B-hCTLA4小鼠肿瘤体积的影响
Figure PCTCN2020139205-appb-000017
注：a：平均数±标准误；b：给药组体重与溶剂对照组体重在给药27天后统计学比较，t-test。
结论：在本实验药效模型上，受试抗体：抗体2和受试抗体：抗体1均有显著抑制肿瘤生长效果，且与阳性对照药品市售CTLA-4抗体
Figure PCTCN2020139205-appb-000018
的抑瘤效果相当。并且在发挥抗肿瘤生长作用的同时受试药物未对动物产生明显毒性作用，安全性较好。值得注意的是，在相同剂量中、高剂量组，药效似已达到饱和，因此比较不出差异，但在低剂量组0.3mg/kg下，经过ADCC增强的抗体2明显比未经ADCC增强的抗体1有更优的抑瘤效果。
实施例7.抗体2对小鼠血液中CD4 +、CD8 +、Treg细胞的影响
在实施例6提到的药效实验中，给药21天后，对每只小鼠通过眼眶内眦静脉丛采集150μL抗凝血，流式检测其中CD4、CD8阳性细胞及Treg细胞，统计各自占CD45阳性细胞比例。其中用到的抗体包括anti-CD45(Biolegend，货号：103138)、anti-CD4(Biolegend，货 号：100438)、anti-CD8(Biolegend，货号：100759)、anti-Foxp3(eBioscience，货号：17-5773-82)抗体。
结果显示，给药后21天，G4组和G5组小鼠血液中CD4阳性细胞比例显著增加(P<0.05,P<0.01)，G6组、G7组和G8组小鼠血液中CD4阳性细胞比例极显著增加(P<0.01)；抗体1各给药组小鼠血液中CD8阳性细胞比例无显著变化，而G6组、G7组和G8组小鼠血液中CD8阳性细胞比例极显著增加(P<0.01)；G3组、G4组、G5组、G6组、G7组和G8组小鼠血液中Treg细胞比例显著降低(P<0.05,P<0.01)；G3组、G4组、G5组小鼠血液中CD8细胞/Treg细胞比值显著增加(P<0.05,P<0.01)。各组比较的示意图见图11A-11D。
结论：ADCC增强后，抗体2的CD4、CD8阳性细胞比例极显著增加，Treg细胞比例显著降低，CD8细胞/Treg细胞比值显著增加。提示在ADCC增强后的CTLA-4抗体下，T细胞得到更强的激活，从而可对CTLA-4人源化荷瘤小鼠产生更强的抗肿瘤效力。
实施例8.药代动力学研究
1)单次静脉输注
试验选用食蟹猴24只，雌雄各半，按性别随机分成3组：低、中、高剂量组分别单次静脉输注给予1mg/kg抗体2、3mg/kg抗体2、10mg/kg抗体2。于给药前、给药结束后即刻，给药开始后1小时、6小时、24小时、48小时、3天、4天、5天、7天、9天、11天、14天、17天、21天、28天采集全血，分离血清。经验证的ELISA法分析各样本中药物的浓度，定量下限为15ng/mL。
试验期间各动物均未见异常临床表现，各组动物血清中药物代谢动力学参数见表7；结果显示，各组不同性别动物间血清药物浓度变化趋势基本一致，低、中及高剂量组动物血清药物浓度与给药剂量正相关；各组各药代动力学参数均无性别差异(p>0.05)。
表7.单次给药的药代动力学参数
Figure PCTCN2020139205-appb-000019
Figure PCTCN2020139205-appb-000020
2)多次静脉输注
试验选用食蟹猴40只，按性别随机分成4组，每组雌雄各5只，分别为溶媒对照组、低剂量组(抗体2，0.2mg/kg)、中剂量组(抗体2，0.6mg/kg)和高剂量组(抗体2，2mg/kg)；每3周静脉输注给予供试品或溶媒对照品1次，连续给药13周，即第1天、第22天、第43天、第64天、第85天共给药5次；给药速度约为0.2mL/min/kg。对照组采血时间点为：第1天和第64天药前及给药开始后2h，给药组采血时间点为：第1天和第64天药前、给药结束后即刻(±1min)、给药开始后2h、6h、24h、72h、120h、168h、336h、504h，采血并分离血清。采用经验证的ELISA分析方法检测血清中供试品浓度，方法的定量下限为15ng/mL。用WinNonlin 8.0非房室模型(NCA)计算供试品的毒代动力学参数。
在0.2～2mg/kg范围内，血清药物暴露量随时间变化趋势基本一致；第4次给药后各剂量组的暴露量均有一定程度的增加。
表8.多次给药的药代动力学参数
Figure PCTCN2020139205-appb-000021
实施例9.毒理学研究
1)单次静脉输注的急性毒性试验
8只食蟹猴随机分为4个组(1只/性别/组)，第1组为溶媒对照组，第2、3、4组分别为10、30和60mg/kg供试品组(抗体2)。动物于第1天单次静脉输注给药，给药容量为6mL/kg，输注速度为0.5mL/min/kg。
结果显示，试验期间各组动物均未见死亡或濒死现象，各组动物、体温、心电、血生化和尿液均未见明确与供试品(抗体2)相关的异常改变。在本试验条件下，30和60mg/kg组动物可见胸腺轻度至重度萎缩，最大耐受剂量(MTD)为60mg/kg。
2)重复静脉输注的毒性试验
本试验共使用40只食蟹猴(20只/性别)随机分为4组，每组每性别5只动物；第1组为溶媒对照组，第2、3、4组动物分别给予0.2、0.6、2mg/kg的供式品(抗体2)；每3周给药1次，连续给药13周，共给药5次；静脉输注给药，给药容量为2mL/kg，给药速度约为0.2mL/min/kg；恢复期4周。
高剂量组动物可见胃肠道反应，停药后可见恢复或恢复趋势，各剂量组动物均未见毒性靶器官，未观察到临床不良反应的剂量水平(NOAEL)为2mg/kg。
3)重复静脉输注的免疫毒性试验
本试验是结合重复静脉输注和恢复期4周的长期毒理研究进行，主要考察该试验条件下供式品(抗体2)对食蟹猴的T淋巴细胞亚群、补体、细胞因子、免疫球蛋白的影响；其中，第1组为溶媒对照组，第2、3、4组动物分别给予0.2、0.6、2mg/kg的供式品(抗体2)；每3周给药1次，连续给药13周，共给药5次
试验期间，3种剂量组食蟹猴的细胞因子(TNF-α、IFN-γ、IL-2、IL-4、IL-5、IL-6)均未见与抗体2相关的异常改变；与对照组相比，各剂量组动物免疫球蛋白(IgG、IgM、IgA)未见统计学意义的改变；食蟹猴重复静脉输注给予抗体2后，低、中、高剂量组分别有0/10、1/10、3/10的食蟹猴检测出ADA(抗药抗体)呈阳性。
实施例10.体外溶血试验
将氯化钠注射液和供试品(抗体2)，分别加入到含有2％兔红细胞混悬液的玻璃试管中，置于37℃电热恒温箱中温育3小时，观察红细胞的溶解或凝聚情况。
试验结果显示，浓度为10.1mg/mL和1mg/mL的供试品，在体外不引起兔红细胞溶血，无凝聚作用，适合临床安全注射。
实施例11.抗体2的临床研究
本研究是一项评价抗体2注射液治疗晚期实体瘤患者的安全性、耐受性和药代动力学特征的多中心、开放性、剂量递增的Ⅰ期临床试验，探索最大耐受剂量(MTD)，为后续临床试验提供推荐剂量；并初步评价抗体2注射液的免疫源性和抗肿瘤疗效。
第一阶段，采用“3+3”方式剂量递增规则探索安全剂量范围，剂量为0.1mg/kg、0.2mg/kg、0.5mg/kg、1mg/kg和2mg/kg；5个剂量组依次入组给予抗体2注射液，只有当同一剂量组的所有受试者均已完成DLT评估，确定没有探索到最大耐受剂量(Maximum Tolerated Dose，MTD)后，才可以开展下一较高剂量组的探索，直至探索到MTD或直至完成最高剂量组的耐受性研究不再递增。第二阶段，剂量递增完成后，选择1-2个耐受剂量针对黑色素瘤(20-40例)进行扩展研究，为后续临床试验提供推荐剂量。
每3周以静脉输注给药1次，治疗周期为4个周期，每周期第1天进行给药。研究期间，发生以下情况，慎用/可酌情使用：1)预防输液反应：若第n个受试者出现输液反应，在后续的给药中，可酌情给予第n、n+1、n+2……个受试者预防用药，仅限于类固醇激素、 镇痛药、抗组胺药的单用或组合方案；2)治疗输液反应：若发生输液反应可以酌情使用类固醇激素及其他药物；3)治疗不良反应：研究者可酌情使用英夫利昔单抗、甲氧咪唑、卡咪唑、左旋甲状腺素、类固醇激素等治疗研究期间发生的不良反应；4)肿瘤的局部治疗，如骨痛的姑息放疗。DLT评估期：21天，即第1个治疗周期。当患者完成第4个治疗周期结束访视后，继续观察访视6周，即完成该研究，根据试验收集的数据，分析总结抗体2注射液的安全性、耐受性、药代动力学特征、免疫原性以及初步疗效。
安全性指标包括：生命体征、体格检查、实验室检查(血常规、血生化、甲状腺功能、凝血常规、尿常规、便常规(含隐血试验))、心电图、心脏彩超、不良事件(包括免疫相关不良事件)等。
所有剂量组的受试者在治疗期间的特定时间点需要收集血样，每个时间点计划采2mL血样，检测血清中抗体2的浓度水平，研究抗体2注射液的药代动力学(PK)特征。在第1至第4周期每次给药前和给药结束后对受试者采集PK血样。其中，第1周期和第3周期密集采样，分别研究单次给药和多次给药稳态研究的PK特征。具体PK参数包括：单次给药：C max、T max、T 1/2、CL、Vd、Ke、MRT、AUC (0-τ)、AUC (0-∞)；多次给药：C max,ss、C avg,ss、C min,ss、AUC (0-τ),ss、AUC (0-∞),ss、T max,ss、T 1/2,ss、CL、V ss、Ke、MRT、蓄积指数(Rac)、波动指数DF。
所有剂量组的受试者在治疗期间的特定时间点需要收集血样，每个时间点计划采集3.5mL血样，检测血清中抗药抗体与中和抗体，于第1至第4周期每次给药前及第4周期末次给药结束后21天采集免疫原性检测血样。免疫原性评价指标包括：抗药抗体(ADA)的样品阳性率和个体阳性率，ADA阳性样品的滴度，ADA阳性的样本是否为中和抗体(Nab)。
整个试验过程中，采用RECIST1.1和iRECIST1.1评估抗体2的抗肿瘤疗效。肿瘤影像学检查(进行胸、腹、骨盆等的CT或MRI)在首次给药前28天内进行，在整个研究期间同一患者同一部位均采用同一种影像学检查技术。给药后的肿瘤评估将在第6周、第12周、第18周进行。临床有效性评价包括：
客观缓解率(Objective Response Rate,ORR)：为完全缓解(CR)和部分缓解(PR)的受试者的比例，第6、12、18周的ORR分别为ORR6w、ORR12w、ORR18w；
最佳总体缓解率(Best Overall Response Rate,BORR)：为第6、12、18周肿瘤疗效评估中出现的最佳ORR；
缓解持续时间(Duration of Response,DOR)：肿瘤第一次评估为客观缓解至第一次评估为PD(Progressive Disease)或PD前任何原因死亡的时间，反映ORR的持续时间；
无进展生存期(Progression-Free Survival：PFS)：为从首次给药至出现肿瘤客观进展或全因死亡的时间(以先发生者为准)。
剂量限制性毒性(DLT)的定义如下：指患者在第1周期给药后第1天至第21天内出现以下与研究药物相关的以下事件：
■≥2级的眼部AE(不良事件)，在接受局部治疗的2周内没有降低到1级的；或≥2级的眼部AE需要全身治疗的；
■≥3级的非血液学毒性(恶心、呕吐等进行支持治疗3天内能缓解，未预防给药发生的≥3级的输液反应除外)；
■≥4级的血液学毒性(包括≥3级的中性粒细胞减少伴发热；但4级中性粒细胞减少需 持续时间≥7天才可判断DLT)；≥4级血小板减少或3级血小板减少伴有出血。
实施例12.抗体2制剂的制备
配置缓冲液，通过UF/DF超滤采用过滤后的缓冲液对抗体溶液进行超滤换液，然后进行浓缩得到抗体浓缩物；按照配方比例配制适当的含有缓冲剂和/或稳定剂和/或表面活性剂和/或螯合剂的辅料母液，调节到合适的pH值；将适当量的无菌过滤后的辅料母液添加到适当量的抗体浓缩物中，得到液体制剂。实施例中采用的溶剂为注射用水。
1)缓冲剂筛选
采用多种缓冲剂体系进行抗体稳定性研究。鉴于抗CTLA-4抗体的性质，本发明初步筛选后确定以下多种缓冲剂体系：His、HAc、CB、Sua、Tris。
上述各英文缩写的含义：
His：20mM组氨酸缓冲剂，pH 5.8；
HAc：20mM醋酸缓冲剂，pH 5.8；
CB：20mM柠檬酸缓冲剂，pH 5.8；
Sua：20mM琥珀酸缓冲剂，pH 5.8；
Tris：20mM三羟甲基氨基甲烷盐酸盐，pH 7.0。
进行稳定性实验的抗体-缓冲剂体系中，His、HAc、CB、Sua所含有的抗体2为10mg/mL，缓冲剂为20mM，缓冲液的pH值为5.8。研究各种配方在高温40℃的条件下放置4周，单体纯度(SEC-HPLC，简称SEC)和电荷异构体(IEC-HPLC，简称IEC)变化的趋势，见表9。
表9缓冲剂筛选
Figure PCTCN2020139205-appb-000022
Figure PCTCN2020139205-appb-000023
根据以上实验结果，组氨酸缓冲剂为最优缓冲剂，醋酸缓冲剂次之，Tris缓冲剂较差：组氨酸缓冲液中SEC-HPLC单体纯度和IEC-HPLC主峰含量都高于其他缓冲体系。可见，组氨酸缓冲液对抗体2的保护作用最优。
2)pH范围初步筛选
进行pH范围筛选的体系中，所含有的抗体、缓冲剂见表10。
表10 pH范围筛选
Figure PCTCN2020139205-appb-000024
根据以上结果，当抗体加入缓冲液高温(40℃)测试中(缓冲液中未加入其他成分)，当缓冲体系是pH值为5.5-6.5(不包括6.5)的组氨酸缓冲液时，其SEC-HPLC聚体和IEC-HPLC酸峰含量相对较低，SEC-HPLC单体纯度和IEC-HPLC主峰含量相对较高。
3)pH精细筛选
在组氨酸缓冲体系中进行pH范围精细筛选，各体系中所含有的抗体、缓冲液、稳定剂、金属离子螯合剂、表面活性剂见表11；82mg/mL蔗糖的摩尔浓度为240mM。
表11 pH精细筛选
Figure PCTCN2020139205-appb-000025
根据以上结果，当抗体2中加入的缓冲液中还加入稳定剂、金属离子螯合剂、表面活性 剂时，当缓冲体系是pH值为5.4-6.2的组氨酸缓冲液时，其稳定性较好，以pH值为5.8更好。
4)表面活性剂振荡稳定性研究
进行表面活性剂筛选的体系中，所含有的抗体、缓冲液、稳定剂、金属离子螯合剂、表面活性剂，见表12。
在抗体制剂中，在剧烈的振荡下，容易发生聚集，产生不溶性颗粒，可以通过添加一定量的表面活性剂来防止。在本实验中，向含有缓冲液、稳定剂、金属离子螯合剂的配方中分别添加0.1mg/mL聚山梨酯20、0.2mg/mL聚山梨酯20、0.4mg/mL聚山梨酯20、0.1mg/mL聚山梨酯80、0.2mg/mL聚山梨酯80、0.4mg/mL聚山梨酯80，观察在振荡72h后样品的质量与浊度。实验条件：把不同表面活性剂组合的制剂，在200rpm下，平放，常温振荡72h，在振荡0h及72h检测溶液性状、SEC、IEC、浊度，结果见表12。
表12表面活性剂筛选
Figure PCTCN2020139205-appb-000026
Figure PCTCN2020139205-appb-000027
结果显示，在振荡的过程中，加入的0.1-0.4mg/mL聚山梨酯20或聚山梨酯80表面活性剂都能对抗体蛋白起到保护作用，浊度没有明显变化。
5)液体制剂配方
本发明液体制剂能够稳定保存活性成分抗体2，稳定性较好的制剂配方如本发明液体制剂A、B、C、D、E、F、G、H、I；其中，J制剂作为对比制剂，如下表。
表13A 液体制剂A配方
Figure PCTCN2020139205-appb-000028
表13B 液体制剂B配方
Figure PCTCN2020139205-appb-000029
表13C 液体制剂C配方
Figure PCTCN2020139205-appb-000030
表13D 液体制剂D配方
Figure PCTCN2020139205-appb-000031
表13E 液体制剂E配方
Figure PCTCN2020139205-appb-000032
表13F 液体制剂F配方
Figure PCTCN2020139205-appb-000033
表13G 液体制剂G配方
Figure PCTCN2020139205-appb-000034
表13H 液体制剂H配方
Figure PCTCN2020139205-appb-000035
表13I 液体制剂I配方
Figure PCTCN2020139205-appb-000036
Figure PCTCN2020139205-appb-000037
表13J 液体制剂J配方
Figure PCTCN2020139205-appb-000038
6)冻融实验
在抗体制剂中，经过冷冻/复融循环时，容易产生不溶性颗粒，可以通过添加稳定剂、表面活性剂来稳定抗体。本发明液体制剂B的反复冻融结果见表14。
表14液体制剂B的冻融实验结果
Figure PCTCN2020139205-appb-000039
结果表明，本发明液体制剂B从-80℃冻结，37℃解冻，反复冻融五次，澄清度、颜色、不溶性微粒、SEC-HPLC、IEC-HPLC均没有明显变化，说明该制剂在反复冻融实验中性质稳定，无析出物，不影响抗体2的性状。
7)加速稳定性研究
将上述制剂A、制剂B、制剂C、制剂D、制剂E、制剂F、制剂G、制剂H、制剂I和制剂J放置于恒温箱中，在25±2℃的条件下放置6个月，分别于第0、2、3、6个月末取样。按稳定性重点考察项目进行检测，考察抗体2的单体纯度(SEC-HPLC)、电荷异构体(IEC-HPLC)、非还原毛细管凝胶电泳(CE-SDS(NR))，见表15、表16。
表15加速稳定性研究的SEC-HPLC结果
Figure PCTCN2020139205-appb-000040
表16加速稳定性研究的IEC-HPLC结果
Figure PCTCN2020139205-appb-000041
Figure PCTCN2020139205-appb-000042
结果表明，制剂J在SEC-HPLC中，单体下降较其他制剂快，表现为聚体的增加；IEC-HPLC主峰下降迅速，表现为酸峰的快速增加；这意味着对比制剂J不能稳定地保存抗体2，而本发明制剂A-I能稳定地保存抗体2，尤其是本发明制剂B。
8)相容性检测
通过加速稳定性实验以及长期稳定性实验，得出本发明液体制剂与药瓶和胶塞具有良好的相容性，符合包装的要求。
SEQ ID NO:1，抗CTLA-4抗体重链氨基酸序列
Figure PCTCN2020139205-appb-000043
SEQ ID NO:2，抗CTLA-4抗体轻链氨基酸序列
Figure PCTCN2020139205-appb-000044
SEQ ID NO:3，抗CTLA-4抗体重链核酸序列
Figure PCTCN2020139205-appb-000045
Figure PCTCN2020139205-appb-000046
SEQ ID NO:4，抗CTLA-4抗体轻链核酸序列
Figure PCTCN2020139205-appb-000047
SEQ ID NO:5，CTLA-4-ECD-Fc核酸序列
Figure PCTCN2020139205-appb-000048
Figure PCTCN2020139205-appb-000049
SEQ ID NO:6，CTLA-4-ECD-Fc氨基酸序列
Figure PCTCN2020139205-appb-000050
SEQ ID NO:7，OKT3ScFv核酸序列
Figure PCTCN2020139205-appb-000051
SEQ ID NO:8，OKT3重链氨基酸序列
Figure PCTCN2020139205-appb-000052
SEQ ID NO:9，OKT3轻链氨基酸序列
Figure PCTCN2020139205-appb-000053
SEQ ID NO:10，FcγRIIIa 158V FL cDNA核酸序列
Figure PCTCN2020139205-appb-000054
SEQ ID NO:11，FcγRIIIa 158V FL氨基酸序列
Figure PCTCN2020139205-appb-000055
SEQ ID NO:12，抗CTLA-4抗体重链CDR1氨基酸序列
SYTMH
SEQ ID NO:13，抗CTLA-4抗体重链CDR2氨基酸序列
FISYDGNNKYYADSVKG
SEQ ID NO:14，抗CTLA-4抗体重链CDR3氨基酸序列
TGWLGPFDY
SEQ ID NO:15，抗CTLA-4抗体轻链CDR1氨基酸序列
RASQSVGSSYLA
SEQ ID NO:16，抗CTLA-4抗体轻链CDR2氨基酸序列
GAFSRAT
SEQ ID NO:17，抗CTLA-4抗体轻链CDR3氨基酸序列
QQYGSSPWT
SEQ ID NO:18，抗CTLA-4抗体重链可变区氨基酸序列
Figure PCTCN2020139205-appb-000056
SEQ ID NO:19，抗CTLA-4抗体轻链可变区氨基酸序列
Figure PCTCN2020139205-appb-000057
SEQ ID NO:20，FcγRIIIa 158V FL cDNA正向引物(下划线为限制性内切酶的酶切位点)
5’-GC GAATTCATGTGGCAGCTGCTCCTCCC-3’
SEQ ID NO:21，FcγRIIIa 158V FL cDNA反向引物(下划线为限制性内切酶的酶切位点)
5’-CA GCGGCCGCTCATTTGTCTTGAGGGTCCTTTCTC-3’

Claims (36)

  1. 针对CTLA-4的单克隆抗体，所述单克隆抗体包含
    (1)重链HCDR1，其包含SEQ ID NO:12所示的序列，或由其组成，
    重链HCDR2，其包含SEQ ID NO:13所示的序列，或由其组成，
    重链HCDR3，其包含SEQ ID NO:14所示的序列，或由其组成，
    轻链LCDR1，其包含SEQ ID NO:15所示的序列，或由其组成，
    轻链LCDR2，其包含SEQ ID NO:16所示的序列，或由其组成，和
    轻链LCDR3，其包含SEQ ID NO:17所示的序列，或由其组成；
    或
    (2)重链可变区，其包含SEQ ID NO:18所示的序列，与所述序列具有至少80％、90％、优选91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个保守氨基酸突变的氨基酸序列，或由其组成，和
    轻链可变区，其包含SEQ ID NO:19所示的序列，与所述序列具有至少80％、90％、优选91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个保守氨基酸突变的氨基酸序列，或由其组成；
    其中，所述单克隆抗体Fc区的高甘露糖糖型总量<5％和/或唾液酸化糖型总量<3％。
  2. 根据权利要求1所述的单克隆抗体，其包含重链和轻链，
    所述重链包含SEQ ID NO:1的氨基酸序列，与SEQ ID NO:1的氨基酸序列具有至少80％，至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的序列，或由其组成；
    所述轻链包含SEQ ID NO:2所示的氨基酸序列，与SEQ ID NO:2的氨基酸序列具有至少80％，至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的序列，或由其组成。
  3. 根据权利要求1或2所述的单克隆抗体，其中所述重链由SEQ ID NO:3所示的核苷酸序列或与SEQ ID NO:3的核苷酸序列具有至少80％，至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的核苷酸序列编码，所述轻链由SEQ ID NO:4所示的核苷酸序列或与SEQ ID NO:4的核苷酸序列具有至少80％，至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的核苷酸序列编码。
  4. 根据权利要求1-3任一项所述的单克隆抗体，其中所述单克隆抗体Fc区的岩藻糖基化水平为0％-5％。
  5. 根据权利要求1-4任一项所述的单克隆抗体，其由敲除α-(1,6)-岩藻糖基转移酶基因的细胞表达。
  6. 药物组合物或融合蛋白，其包含权利要求1-5任一项所述的单克隆抗体。
  7. 多特异性抗体(例如双特异性抗体)，其包含权利要求1-5任一项所述的单克隆抗体，以及针对其他抗原和/或其他抗原表位的抗体或抗原结合片段。
  8. 试剂盒，其包括权利要求1-5任一项所述的单克隆抗体，权利要求7所述的多特异性抗体或权利要求6所述的融合蛋白。
  9. 根据权利要求8所述的试剂盒，其中所述试剂盒还包括第二抗体、细胞毒性剂、化疗剂或放疗剂，所述第二抗体特异性识别所述单克隆抗体；任选地，所述第二抗体还包括可检测的标记，例如荧光素、金属离子、生物素、放射性同位素、化学发光物质、酶或聚乙二醇。
  10. 权利要求1-5任一项所述的单克隆抗体，权利要求6所述的融合蛋白或权利要求7所述的多特异性抗体在制备用于治疗癌症或用于排斥可移植的肿瘤，例如黑色素瘤、非小细胞肺癌、膀胱癌、肝癌、结肠癌、前列腺癌、淋巴瘤或肾癌的药物中的应用。
  11. 根据权利要求10所述的应用，其中所述药物配制成用于每个治疗周期内将抗CTLA-4抗体以7mg至180mg的剂量施用。
  12. 根据权利要求11所述的应用，其中一个治疗周期为1周、2周、3周或4周。
  13. 根据权利要求10所述的应用，其中所述药物配制成用于每次抗CTLA-4抗体给药量为0.1mg/kg-2.5mg/kg。
  14. 根据权利要求13所述的应用，其中每1周、2周、3周或4周给药1次。
  15. 根据权利要求10-14任一项所述的应用，其中所述药物配制成用于静脉注射或皮下注射。
  16. 一种液体制剂，其包含以下组分：
    (1)5-40mg/mL抗CTLA-4抗体或其片段，
    (2)10-30mM缓冲剂，
    (3)200-260mM稳定剂，
    (4)0.02-0.2mg/mL螯合剂，
    (5)0.1-0.4mg/mL表面活性剂，
    (6)水；
    液体制剂的pH值为5.4-6.2。
  17. 根据权利要求16所述的制剂，其中所述缓冲剂选自组氨酸缓冲剂、醋酸缓冲剂中的至少一种，所述稳定剂选自海藻糖、蔗糖和甘露醇中的至少一种，所述螯合剂包含依地酸二钠，所述表面活性剂选自聚山梨酯20和聚山梨酯80中的至少一种。
  18. 根据权利要求16或17所述的液体制剂，其中所述抗CTLA-4抗体或其片段包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2、SEQ ID NO:14所示的HCDR3、SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3；或
    其中所述抗CTLA-4抗体或其片段的重链可变区包含SEQ ID NO:18所示的氨基酸序列或与所述SEQ ID NO:18具有至少90％同一性的氨基酸序列，所述抗CTLA-4抗体或其片段的轻链可变区包含SEQ ID NO:19所示的氨基酸序列或与所述SEQ ID NO:19具有至少90％同一性的氨基酸序列。
  19. 根据权利要求16-18任一项所述的制剂，其中所述抗CTLA-4抗体的重链包含SEQ ID NO:1所示的序列，与SEQ ID NO:1所示序列具有至少80％同一性的序列，或与SEQ ID NO:1所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或，
    所述抗CTLA-4抗体的轻链包含SEQ ID NO:2所示的序列，与SEQ ID NO:2所示序列具有至少80％同一性的序列，或与SEQ ID NO:2所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  20. 根据权利要求16-19任一项所述的制剂，其中所述抗CTLA-4抗体或其片段为单克隆抗体，所述抗CTLA-4抗体或其片段中岩藻糖基化水平≤5％，高甘露糖糖型总量<5％，唾液酸化糖型总量<3％。
  21. 根据权利要求16-20任一项所述的制剂，其中所述抗CTLA-4抗体由α-(1,6)-岩藻糖基转移酶基因敲除的细胞表达。
  22. 根据权利要求16-21任一项所述的液体制剂，其中所述液体制剂包含7-15mg/mL抗CTLA-4抗体或其片段。
  23. 根据权利要求16-22任一项所述的液体制剂，其中所述液体制剂包含17-23mM组氨 酸缓冲剂。
  24. 根据权利要求16-23任一项所述的液体制剂，其中所述液体制剂包含220-250mM蔗糖。
  25. 根据权利要求16-24任一项所述的液体制剂，其中所述液体制剂包含0.02-0.05mg/mL依地酸二钠。
  26. 根据权利要求16-25任一项所述的液体制剂，其中所述液体制剂包含0.1-0.3mg/mL聚山梨酯80。
  27. 一种液体制剂，其包含以下组分：
    (1)10mg/mL抗CTLA-4抗体或其片段，
    (2)20mM组氨酸缓冲剂，
    (3)240mM蔗糖，
    (4)0.033mg/mL依地酸二钠，
    (5)0.2mg/mL聚山梨酯80，
    (6)水；
    液体制剂的pH值为5.8。
  28. 一种用于治疗肿瘤或癌症的方法，所述方法包括：向有需要的患者给药有效量的抗CTLA-4抗体；
    所述抗CTLA-4抗体包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2、SEQ ID NO:14所示的HCDR3、SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3。
  29. 根据权利要求28所述的方法，其中所述抗CTLA-4抗体的重链包含SEQ ID NO:1所示的序列，与SEQ ID NO:1所示序列具有至少80％同一性的序列，或与SEQ ID NO:1所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗CTLA-4抗体的轻链包含SEQ ID NO:2所示的序列，与SEQ ID NO:2所示序列具有至少80％同一性的序列，或与SEQ ID NO:2所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  30. 根据权利要求28或29所述的方法，其中所述抗CTLA-4抗体的岩藻糖基化水平为0-10％。
  31. 根据权利要求28或29所述的方法，其中所述抗CTLA-4抗体由α-(1,6)-岩藻糖基转移酶基因敲除的细胞表达。
  32. 根据权利要求28-31任一项所述的方法，其中所述肿瘤或癌症包括急性淋巴细胞性白血病、急性骨髓性白血病、慢性淋巴细胞性白血病、慢性骨髓性白血病、骨髓性增生疾病/肿瘤、霍奇金淋巴瘤、无痛性和侵袭性非霍奇金淋巴瘤、伯基特淋巴瘤、滤泡性淋巴瘤、多发性骨髓瘤、巨细胞骨髓瘤、重链骨髓瘤、轻链或本斯-琼斯骨髓瘤、乳腺癌、卵巢癌、肺癌、胰腺癌、前列腺癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌和肾癌。
  33. 根据权利要求28-31任一项所述的方法，其中每个治疗周期内抗CTLA-4抗体给药的有效量为7mg至180mg。
  34. 根据权利要求28-31任一项所述的方法，其中一个治疗周期为1周、2周、3周或4周。
  35. 根据权利要求28-31任一项所述的方法，其中所述抗CTLA-4抗体的每次给药量为0.1mg/kg-2.5mg/kg。
  36. 根据权利要求28-31任一项所述的方法，其中给药方式为静脉注射或皮下注射。
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