WO2021190582A1 - 一种抗ox40抗体药物组合物及其用途 - Google Patents

一种抗ox40抗体药物组合物及其用途 Download PDF

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WO2021190582A1
WO2021190582A1 PCT/CN2021/082855 CN2021082855W WO2021190582A1 WO 2021190582 A1 WO2021190582 A1 WO 2021190582A1 CN 2021082855 W CN2021082855 W CN 2021082855W WO 2021190582 A1 WO2021190582 A1 WO 2021190582A1
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seq
antibody
antigen
cancer
pharmaceutical composition
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PCT/CN2021/082855
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English (en)
French (fr)
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吴婷婷
颜贞
刘洵
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江苏恒瑞医药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

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  • the present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing an anti-OX40 antibody and its use as a medicine.
  • Tumor immunotherapy is a continuous hot spot in the field of tumor therapy. Recent studies have proven that enhancing the function of anti-tumor T cells can be used to fight cancer. There is a lot of evidence that tumor cells “escape” the immune system by inducing active immune tolerance mediated mainly by regulatory T lymphocytes (Treg; Quezda et al. Immunol Rev 2011; 241:104-118). Therefore, the balance between effector T lymphocytes (Teff) and tolerogenic Treg is essential for effective anti-tumor immunotherapy. Therefore, an effective anti-tumor immune response can be obtained by enhancing the effector function of tumor-specific Teff and/or by weakening the inhibitory function of tumor-specific Treg.
  • Treg regulatory T lymphocytes
  • the CD134 (OX40) receptor has been shown to be a key receptor that mediates these responses (Sugamura, K, Ishii, N, Weinberg, A. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nature Rev Imm 2004; 4:420-431).
  • OX40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is a glycoprotein with a molecular weight of about 50kDa expressed on the cell surface.
  • the extracellular ligand binding domain of OX40 is composed of 4 cysteine-rich domains (CRD).
  • the natural ligand of OX40 is OX40L (CD252), and the two form an OX40-OX40L complex.
  • OX40 is mainly expressed on activated T cells, and OX40 is a secondary costimulatory molecule that is expressed 24-72 hours after activation.
  • the ligand OX40L of OX40 is mainly expressed on activated antigen presenting cells.
  • T lymphocytes expressing OX40 have been confirmed to exist in the draining lymph nodes of various human malignant tumors and cancer patients.
  • SCID severe combined immunodeficiency
  • the interaction of OX40 and OX40L binding domains can enhance anti-tumor immunity, resulting in a variety of human malignant tumor cell lines, such as lymphoma, prostate cancer, colon cancer, and breast cancer tumors Growth inhibition.
  • Anti-OX40 antibodies can activate immunity through specific stimulation, improve the patient's own immune system response to tumors, and achieve the purpose of killing tumor cells.
  • Anti-OX40 related antibodies have been published, such as WO2013038191, WO2015153513, WO2016179517, WO2017096182, CN110078825A, WO2016196228 and so on.
  • the present disclosure provides an anti-OX40 antibody pharmaceutical composition and its use.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof, and a buffer
  • the buffer being a histidine buffer or an acetate buffer
  • the buffer is The pH is about 5.0 to about 6.5, preferably the pH is about 5.5 to about 6.5, more preferably the pH is about 6.0
  • the histidine salt buffer is preferably histidine hydrochloride buffer
  • the acetate buffer is preferably Sodium acetate buffer (referred to as sodium acetate buffer).
  • the pH of the buffer in the aforementioned pharmaceutical composition is about 5.0 to about 6.5, preferably 5.0 to 6.5; in some embodiments, the pH of the buffer is about 5.5 to about 6.5, Preferably it is 5.5 to 6.5; in other embodiments, the buffer has a pH of about 6.0, preferably 6.0; in other embodiments, the buffer is histidine with a pH of about 5.5 to about 6.5 Salt buffer; in other embodiments, the buffer is a histidine hydrochloride buffer with a pH of 6.0; in other embodiments, the buffer is a vinegar with a pH of about 5.0 to about 6.0 In other embodiments, the buffer is a sodium acetate buffer with a pH of about 5.0 to about 5.5; in other embodiments, the buffer is a sodium acetate buffer with a pH of about 5.5.
  • non-limiting examples of the pH value of the buffer include about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0. , About 6.1, about 6.2, about 6.3, about 6.4, about 6.5, and any range between these points.
  • the pH value of the aforementioned pharmaceutical composition is the same or almost the same as the pH value of its buffer (it is well known to those skilled in the art that in the process of preparing pharmaceutical formulations, sometimes there is a pH drift (the pH of the formulation and the pH of the buffer are present). Some differences are called pH drift values).
  • the pH drift value is usually within ⁇ 0.3. Unless otherwise specified, the pH drift of the pharmaceutical composition of the present disclosure is within ⁇ 0.3, preferably within ⁇ 0.2, and more Preferably in the range of ⁇ 0.1).
  • the buffer concentration in the aforementioned pharmaceutical composition is about 5 mM to about 30 mM, preferably 5 mM to 30 mM; in some embodiments, the buffer concentration is about 5 mM to about 15 mM, preferably 5 mM to 15 mM ; In some embodiments, the buffer concentration is about 10 mM, preferably 10 mM.
  • Non-limiting examples of buffer concentrations include about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 18mM, about 20mM , About 25mM, about 30mM, and any range between these points.
  • the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition has a concentration of about 1 mg/ml to about 100 mg/ml, preferably 1 mg/ml to 100 mg/ml; in some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof The concentration of the OX40 antibody or antigen-binding fragment thereof is about 30 mg/ml to about 70 mg/ml, preferably 30 mg/ml to 70 mg/ml; in some embodiments, the concentration of the anti-OX40 antibody or antigen-binding fragment thereof is about 50 mg/ml, preferably 50mg/ml.
  • Non-limiting examples of the concentration of anti-OX40 antibodies or antigen-binding fragments thereof include: about 1 mg/ml, about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, Approximately 50 mg/ml, approximately 55 mg/ml, approximately 60 mg/ml, approximately 65 mg/ml, approximately 70 mg/ml, approximately 80 mg/ml, approximately 90 mg/ml, approximately 100 mg/ml, and any range between these points.
  • the aforementioned pharmaceutical composition further includes sugars.
  • the sugars include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like.
  • the sugar is selected from: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbose Alcohol, mannitol, miribiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, and iso-maltulose.
  • the sugar is a non-reducing disaccharide; in some embodiments, the sugar is preferably trehalose or sucrose, most preferably sucrose.
  • the sugar concentration in the aforementioned pharmaceutical composition is about 50 mg/ml to about 90 mg/ml, preferably 50 mg/ml to 90 mg/ml; in some embodiments, the sugar concentration is about 75 mg/ml to about 90 mg/ml; About 85 mg/ml, preferably 75 mg/ml to 85 mg/ml; in some embodiments, the sugar concentration is about 80 mg/ml, preferably 80 mg/ml.
  • Non-limiting examples include about 50 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, and values between these points Any range of.
  • the aforementioned pharmaceutical composition further includes a surfactant, which may be selected from polysorbate 20 (also known as polysorbate 20, Tween 20, PS20), polysorbate 80 (also known as polysorbate 80, Tween 80, PS80), polyhydroxyalkene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfo Betaine, linoleyl-sultaine, stearyl-sultaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine Acid, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropy
  • the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.1 mg/ml to about 1.0 mg/ml, preferably 0.1 mg/ml to 1.0 mg/ml; in some embodiments, the surfactant The concentration of the surfactant is about 0.2 mg/ml to about 0.6 mg/ml, preferably 0.2 mg/ml to 0.6 mg/ml; in some embodiments, the concentration of the surfactant is about 0.4 mg/ml, preferably 0.4 mg/ml.
  • Non-limiting examples include about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, 0.4 mg/ml, about 0.45 mg/ml, about 0.5 mg/ml, about 0.55 mg/ml, about 0.6 mg/ml ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, and any range between these points.
  • the aforementioned pharmaceutical composition comprises: a) an anti-OX40 antibody or antigen-binding fragment thereof at a concentration of about 1 mg/ml to about 100 mg/ml, b) histidine with a pH of about 5.0 to about 6.5 Salt buffer or acetate buffer, c) sugar with a concentration of about 50 mg/ml to about 90 mg/ml, and d) polysorbate 80 with a concentration of about 0.1 mg/ml to about 1.0 mg/ml.
  • the aforementioned pharmaceutical composition comprises: a1) an anti-OX40 antibody or antigen-binding fragment thereof at a concentration of about 30 mg/ml to about 70 mg/ml, b1) a histidine salt with a pH of about 5.5 to about 6.5 Buffer, c1) sucrose with a concentration of about 75 mg/ml to about 85 mg/ml, and d1) polysorbate 80 with a concentration of about 0.2 mg/ml to about 0.6 mg/ml.
  • the aforementioned pharmaceutical composition comprises: about 10 mM histidine hydrochloride buffer with a pH of about 6.0, an anti-OX40 antibody or antigen-binding fragment thereof with a concentration of about 50 mg/ml, and a concentration of about 80 mg/ml Sucrose, and polysorbate 80 at a concentration of about 0.4 mg/ml.
  • the aforementioned pharmaceutical composition comprises: 10 mM histidine hydrochloride buffer with a pH of 6.0, an anti-OX40 antibody or antigen-binding fragment thereof at a concentration of 50 mg/ml, and a concentration of 80 mg/ml Sucrose, and polysorbate 80 at a concentration of 0.4 mg/ml.
  • the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 3, 4, and 5, and the light chain variable region includes SEQ ID NOs: 6, 7, and 5, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 11, 12, and 13, and the light chain variable region includes SEQ ID NOs: 14, 15, and 13, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 11, 33, and 13, respectively, and the light chain variable region includes SEQ ID NO: 14, 15, and HCDR3, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 11, 34, and 13, respectively, and the light chain variable region includes SEQ ID NO: 14, 15, and HCDR3, respectively.
  • 16 shows the light chain LCDR1, LCDR2, LCDR3.
  • the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises:
  • (E) A heavy chain variable region as shown in SEQ ID NO:1 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1, and as shown in SEQ ID NO: 2 or a light chain variable region with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2;
  • (F) A heavy chain variable region as shown in SEQ ID NO: 9 or having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9, and as shown in SEQ ID NO: 10 or a light chain variable region that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10;
  • the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises: a heavy chain variable region as shown in SEQ ID NO: 31 and a light chain as shown in SEQ ID NO: 19 Variable region. In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises: a heavy chain variable region as shown in SEQ ID NO: 29 and a light chain as shown in SEQ ID NO: 23 Variable region.
  • the anti-OX40 antibody in the aforementioned pharmaceutical composition comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is derived from human IgG1, IgG2, IgG3 or IgG4 or a mutant sequence thereof ;
  • the light chain constant region is derived from human kappa, lambda chains or their mutant sequences; more preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 35 or has at least 90% of SEQ ID NO: 35, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 36 Show or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 36.
  • the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises:
  • (K) As shown in SEQ ID NO: 39 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 39, 95%, 96%, 97%, 98%, 99% or 100% sequence identity heavy chain, and/or as shown in SEQ ID NO: 40 or having at least 85%, 86% with SEQ ID NO: 40, Light chains with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises: a heavy chain as shown in SEQ ID NO: 37 and a light chain as shown in SEQ ID NO: 38; or The heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40.
  • the aforementioned pharmaceutical composition comprises: 10 mM histidine hydrochloride buffer with a pH of 6.0, an anti-OX40 antibody at a concentration of 50 mg/ml, a concentration of 80 mg/ml sucrose, and a concentration of 0.4mg/ml polysorbate 80, the anti-OX40 antibody comprises a heavy chain as shown in SEQ ID NO: 37 and a light chain as shown in SEQ ID NO: 38.
  • the present disclosure provides a method for preparing the aforementioned pharmaceutical composition, the method comprising the step of replacing the stock solution of the anti-OX40 antibody or antigen-binding fragment thereof with a buffer.
  • the present disclosure also provides a freeze-dried preparation, which can be reconstituted to form the pharmaceutical composition described in any one of the foregoing.
  • the present disclosure provides a freeze-dried preparation containing an anti-OX40 antibody or an antigen-binding fragment thereof, which is obtained by freeze-drying the aforementioned pharmaceutical composition.
  • the freeze-drying includes the steps of pre-freezing, primary drying and secondary drying in sequence.
  • the present disclosure provides a lyophilized preparation containing an anti-OX40 antibody or an antigen-binding fragment thereof, which can be reconstituted to form the aforementioned pharmaceutical composition.
  • the lyophilized preparation of any one of the foregoing, wherein the amount ratio of the anti-OX40 antibody or antigen-binding fragment thereof to the substance of the buffer is: 0.007 to 0.685 mole parts of anti-OX40 antibody or Its antigen-binding fragments (non-limiting examples include: about 0.007 mole parts, about 0.068 mole parts, about 0.137, about 0.206 mole parts, about 0.240 mole parts, about 0.274 mole parts, about 0.308 mole parts, about 0.343 mole parts, About 0.377 mole parts, about 0.411 mole parts, about 0.446 mole parts, about 0.480 mole parts, about 0.548 mole parts, about 0.617 mole parts, about 0.685 mole parts, and any range between these points): 5 mole parts to 30 mole parts of histidine or acetate buffer (non-limiting examples include: about 5 mole parts, about 6 mole parts, about 7 mole parts, about 8 mole parts,
  • the lyophilized formulation comprises an anti-OX40 antibody or antigen-binding fragment thereof, sugar (e.g., trehalose or sucrose), surfactant (e.g., polysorbate 80 or polysorbate 20), and histidine.
  • sugar e.g., trehalose or sucrose
  • surfactant e.g., polysorbate 80 or polysorbate 20
  • histidine e.g., histidine
  • the weight ratio of the anti-OX40 antibody or its antigen-binding fragment, sugar and surfactant is: 1 part by weight to 100 parts by weight of anti-OX40 antibody or antigen-binding fragment thereof: 50 parts by weight to 90 parts by weight of sugar: 0.1 part by weight to 1.0 part by weight of surfactant, said anti-OX40 antibody or antigen-binding fragment thereof and buffer
  • the ratio of the amount of substances in the solution is: 0.007 to 0.685 mole parts of anti-OX40 antibody or antigen-binding fragment thereof: 5 to 30 mole parts of histidine or acetate buffer.
  • the lyophilized preparation comprises an anti-OX40 antibody with a heavy chain as shown in SEQ ID NO: 37 and a light chain as shown in SEQ ID NO: 38, sucrose, polysorbate 80, and histidine Hydrochloride buffer solution, wherein the weight ratio of the anti-OX40 antibody, sucrose and polysorbate 80 is: 50 parts by weight of anti-OX40 antibody: 80 parts by weight of sucrose: 0.4 parts by weight of polysorbate 80
  • the amount ratio of the anti-OX40 antibody to the histidine hydrochloride buffer is 0.343 mole parts of the anti-OX40 antibody: 10 mole parts of the histidine hydrochloride buffer.
  • the aforementioned lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable for at least 7 days, at least 14 days, or at least 28 days under high temperature conditions of 40°C.
  • the present disclosure provides a reconstitution solution containing an anti-OX40 antibody or an antigen-binding fragment thereof, the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation.
  • the solution used for reconstitution includes but is not limited to water for injection, physiological saline or glucose solution, preferably water for injection.
  • the components and content of the reconstituted solution are the same as the aforementioned pharmaceutical composition.
  • the present disclosure provides an article of manufacture, which includes a container containing the pharmaceutical composition, lyophilized formulation, or reconstitution solution as described above.
  • the container is a neutral borosilicate glass control injection bottle.
  • the present disclosure provides a method for treating or preventing a disease or condition, the method comprising administering to a subject a therapeutically effective amount or a preventively effective amount of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product; in some implementations
  • the disease or condition may be cancer or a cell proliferative disease.
  • the cancer is lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, Melanoma, kidney cancer, squamous cell carcinoma, blood system cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • the blood system cancer includes, but is not limited to, acute and chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, bone marrow tissue proliferative disease, multiple myeloma, Hodgkin Hodgkin’s disease, non-Hodgkin’s lymphoma, B cell lymphoma, T cell lymphoma, follicular center cell lymphoma, chronic myeloid leukemia.
  • the present disclosure provides a method for enhancing the immune response in a human subject, comprising: administering to the subject a therapeutically effective amount or a prophylactically effective amount of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product; preferably
  • the enhanced immune response includes an increase in the immune stimulatory/effector function of T effector cells, and/or a down-regulation of the immune suppressive function of T regulatory cells.
  • the increase may be the result of cell proliferation
  • the down-regulation may be the result of no increase in the number of cells (or decrease in the number of cells).
  • the present disclosure provides the use of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product in the preparation of a medicament for enhancing the immune response in a human subject.
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product in the preparation of a medicament for the treatment/prevention of diseases or disorders.
  • the disease or condition can be cancer or a cell proliferative disease.
  • the cancer is lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, Melanoma, kidney cancer, squamous cell carcinoma, blood system cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • the blood system cancer includes, but is not limited to, acute and chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, bone marrow tissue proliferative disease, multiple myeloma, Hodgkin Hodgkin’s disease, non-Hodgkin’s lymphoma, B cell lymphoma, T cell lymphoma, follicular center cell lymphoma, chronic myeloid leukemia.
  • the present disclosure also provides the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product for the treatment/prevention of diseases or conditions.
  • the disease or condition may be cancer or a cell proliferative disease.
  • the cancer is lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, Melanoma, kidney cancer, squamous cell carcinoma, blood system cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • the blood system cancer includes, but is not limited to, acute and chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, bone marrow tissue proliferative disease, multiple myeloma, Hodgkin Hodgkin’s disease, non-Hodgkin’s lymphoma, B cell lymphoma, T cell lymphoma, follicular center cell lymphoma, chronic myeloid leukemia.
  • the present disclosure provides the aforementioned pharmaceutical composition, lyophilized preparation, reconstitution solution or product for one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Treg), killing OX40-expressing cells (e.g., expressing Cells with high levels of OX40), improve effector T cell function and/or improve memory T cell function, reduce tumor immunity, enhance T cell function and/or reduce OX40-expressing cells.
  • Treg function e.g., inhibiting the suppressive function of Treg
  • OX40-expressing cells e.g., expressing Cells with high levels of OX40
  • improve effector T cell function and/or improve memory T cell function reduce tumor immunity, enhance T cell function and/or reduce OX40-expressing cells.
  • the present disclosure provides the aforementioned pharmaceutical compositions, lyophilized preparations, reconstituted solutions or products for the following purposes: treating cancer, stimulating immune responses in subjects, stimulating antigen-specific T cell responses, activating or co-stimulating T cells , Increasing the production of cytokines (such as IL-2 and/or IFN- ⁇ ) in T cells, and/or the proliferation of T cells, reducing or depleting the number of T regulatory cells in the tumor, and/or inhibiting the growth of tumor cells.
  • cytokines such as IL-2 and/or IFN- ⁇
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product to prepare a medicament for: stimulating an immune response in a subject, stimulating an antigen-specific T cell response, activating or co-activating Stimulate T cells, increase IL-2 and/or IFN- ⁇ production in T cells and/or T cell proliferation, reduce or deplete the number of T regulatory cells in tumors, and/or inhibit tumor cell growth.
  • Figure 1A to Figure 1B ELISA test results of the affinity of murine antibody and chimeric antibody to human OX40.
  • Figure 1A is the affinity results of the murine antibody m2G3 and the chimeric antibody ch2G3, m2G3-NC and ch2G3-NC are negative controls
  • Figure 1B is the affinity results of the murine antibody m4B5 and the chimeric antibody ch4B5, m4B5-NC and ch4B5 -NC is a negative control.
  • FIG. 2 Experiment of anti-OX40 antibody stimulating T cells to secrete IFN- ⁇ . The results show that the test OX40 antibody can reach the maximum stimulating effect at a concentration of 10ng/mL antibody.
  • Figure 3A and Figure 3B Anti-OX40 antibody anti-tumor effect in mice
  • Figure 3A shows the changes in tumor volume in mice at different days after administration
  • Figure 3B shows the effect of different anti-OX40 antibodies in mice on the 20th day after administration
  • the impact of tumor quality The results showed that on the 20th day after treatment, when the dose was 3 mg/kg, the tumor inhibition rate of 2G3 antibody was as high as 97%.
  • Figure 4 DOE fitting diagram of anti-OX40 antibody formulation prescription
  • buffer refers to a buffer that withstands changes in pH through the action of its acid-base conjugated components.
  • buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, and fumarate. , Glycylglycine and other organic acid buffers.
  • the term "histidine salt buffer” or “histidine buffer” is a buffer containing histidine ions.
  • histidine salt buffers include buffers such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like.
  • the histidine salt buffer is selected from histidine hydrochloride buffer.
  • succinate buffer or “succinate buffer” is a buffer that includes succinate ions.
  • succinate buffer include sodium succinate succinate, potassium succinate succinate, calcium succinate succinate, and the like.
  • succinic acid buffer is sodium succinate succinate.
  • acetate buffer or "acetate buffer” is a buffer that includes acetate ions.
  • acetate buffers include sodium acetate acetate, histidine acetate, potassium acetate, calcium acetate, magnesium acetate, and the like.
  • the acetate buffer is sodium acetate acetate.
  • phosphate buffer or "phosphate buffer” is a buffer that includes phosphate ions.
  • examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. In some embodiments of the present disclosure, disodium hydrogen phosphate-citric acid.
  • the term "pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiological /Pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical composition maintains the stability of the active ingredient of the antibody, promotes the administration to the organism, and facilitates the absorption of the active ingredient to exert its biological activity.
  • pharmaceutical composition and “preparation” are not mutually exclusive.
  • buffer replacement refers to replacing the solvent system that dissolves the antibody protein with the buffer system of the stable formulation described herein, for example, using the stable formulation described herein.
  • the buffer system of the preparation is physically operated to replace the high salt or hypertonic solvent system containing the antibody protein, so that the antibody protein exists in the stable preparation.
  • the so-called physical operation methods include but are not limited to ultrafiltration, dialysis or centrifugation followed by reconstitution.
  • the pharmaceutical composition or formulation of the present disclosure can be prepared by methods well known in the art.
  • the preparation of antibody pharmaceutical compositions or preparations the first step: take a certain amount of purified antibody solution and use a buffer without antibody (such as a 10 mM histidine hydrochloride buffer at pH 6.0) Perform solvent replacement (preferably ultrafiltration), and at least 6 times the volume of the ultrafiltration membrane is replaced, and the antibody is concentrated to about 70 mg/mL.
  • a buffer without antibody such as a 10 mM histidine hydrochloride buffer at pH 6.0
  • solvent replacement preferably ultrafiltration
  • the volume of the ultrafiltration membrane is replaced, and the antibody is concentrated to about 70 mg/mL.
  • the solvent therein is water.
  • lyophilized formulation means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.
  • the lyophilized preparation can be obtained by freeze-drying the pharmaceutical composition or preparation in liquid or solution form. Freeze drying is performed by freezing the preparation and then sublimating water at a temperature suitable for one-time drying. Under this condition, the product temperature is lower than the eutectic point or decomposition temperature of the formulation. Under a suitable pressure usually in the range of about 50-250 millitorr, generally, the storage temperature of the primary drying is in the range of about -30 to 25°C (assuming that the product remains frozen during the primary drying process).
  • the size and type of the formulation, the container (for example, a glass vial) holding the sample, and the volume of the liquid determine the time required for drying, which may range from a few hours to a few days (for example, 40-60 hours).
  • the secondary drying stage can be carried out at about 0-40°C, depending on the type and size of the container and the type of protein used.
  • the secondary drying time is determined by the desired residual humidity level in the product, and usually requires at least about 5 hours.
  • the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%.
  • the pressure may be the same as the pressure applied in the primary drying step.
  • the pressure of the secondary drying is lower than that of the primary drying. Freeze-drying conditions can vary with the formulation and vial size.
  • the "sugar” in the present disclosure includes conventional compositions (CH2O)n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and so on.
  • the preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.
  • the surfactant of the present disclosure can be selected from polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl -Sultaine, Myristyl-Sultaine, Linoleyl-Sultaine, Stearyl-Sultaine, Lauryl-Sultaine, Myristyl-Sultaine, Linoleum Alkyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocaamidopropyl -Betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl
  • “about”, “approximately” or “substantially comprising” may mean up to 20% of the range, for example, may mean ⁇ 20%, ⁇ 19%, ⁇ 18%, ⁇ 17%, ⁇ 16%, ⁇ 15%, ⁇ 14%, ⁇ 13%, ⁇ 12%, ⁇ 11%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4 %, ⁇ 3%, ⁇ 2%, or ⁇ 1%.
  • the term can mean at most an order of magnitude or at most 5 times the value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about”, “approximately” or “substantially encompassing” should be assumed to be within the acceptable error range of the specific value.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody basically retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, the drug
  • the composition basically retains its physical and chemical stability and its biological activity after storage.
  • the shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition.
  • analytical techniques for measuring protein stability which can measure the stability after storage at a selected temperature for a selected period of time.
  • a stable antibody pharmaceutical preparation is a preparation in which no significant changes are observed under the following conditions: storage at refrigerated temperature (2-8°C), for example, at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably at most Up to 2 years.
  • stable liquid formulations include liquid formulations that exhibit desired characteristics after storage at a temperature of, for example, 25°C and/or 40°C, for example, for a period of 1 month, 3 months, or 6 months.
  • the stable formulation for example, by visual analysis, the drug antibody formulation is colorless, or clear to slightly milky white.
  • the concentration, pH and osmolality of the preparation have no more than ⁇ 10% change.
  • a truncation of no more than about 10%, preferably no more than about 5% is generally observed. Generally, no more than about 10%, preferably no more than about 5% of aggregates are formed.
  • the pharmaceutical composition or lyophilized formulation of the present disclosure is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months; °C can be stable for at least 3 months, or at least 6 months.
  • the antibody does not show a significant increase in aggregation, precipitation and/or denaturation , Then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).
  • the antibody does not show a significant chemical change, then the antibody "retains its chemical stability" in the pharmaceutical formulation.
  • chemical stability can be assessed.
  • Degradation processes that often change the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS, etc.) Method to evaluate), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.) and isomerization (by measuring isoaspartic acid content, Peptide mapping etc. to evaluate).
  • the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical preparation is prepared, then the antibody "retains its biological activity" in the pharmaceutical preparation.
  • the biological activity of an antibody can be determined, for example, by an antigen binding assay.
  • the term "enhance T cell function” means inducing, causing or stimulating effector or memory T cells to enable them to have renewal, sustained or amplified biological functions.
  • Examples of enhanced T cell function include: increased secretion of ⁇ -interferon from CD8+ effector T cells, increased secretion of ⁇ -interferon from CD4+ memory and/or effector T cells, CD4+ effect and /Or the proliferation of memory T cells is increased, the proliferation of CD8+ effector T cells is increased, and the antigen responsiveness (e.g., clearance) is increased.
  • the term "enhance the immune response” refers to stimulating, arousing, increasing, improving or enhancing the response of the mammalian immune system.
  • the immune response can be a cellular response (ie, cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (ie, antibody-mediated response), and can be a primary or secondary immune response. Examples of enhanced immune response include increased CD4+ helper T cell activity and production of cytotoxic T cells.
  • the enhancement of immune response can be assessed by some in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assay, cytokine release (such as IL-2 production), tumor regression, and tumor-carrying animals Survival, antibody production, immune cell proliferation, cell surface marker expression and cytotoxicity.
  • the method enhances cellular immune responses, particularly cytotoxic T cell responses.
  • tumor immunity refers to the process by which tumors evade immune recognition and clearance.
  • tumor immunity when tumor immunity is weakened in its ability to evade immune recognition and elimination, the tumor is recognized and attacked by the immune system, and the patient is treated.
  • tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • Teff T effector cell
  • T cells for example, CD4+ and CD8+ T cells
  • Th T helper cells that have cytolytic activity. Teff secretes cytokines and activates Guides other immune cells, but does not include regulatory T cells (Treg cells).
  • the anti-OX40 antibodies described in the present disclosure can activate Teff cells, such as CD4+ and CD8+ Teff cells.
  • Treg cell means a specialized type of CD4+ T cell that can suppress the response of other T cells.
  • Treg cells are characterized by expressing CD4, the ⁇ subunit of IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), and they are inducing and maintaining peripheral autologous A vital role is played in tolerance, which targets antigens expressed by tumors.
  • OX40 refers to a receptor that binds to OX40 ligand (OX40-L), which is a member of the TNF-receptor superfamily. OX40 is also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), ACT35, IMD16, TXGP1L and CD134.
  • TNFRSF4 tumor necrosis factor receptor superfamily member 4
  • ACT35 tumor necrosis factor receptor superfamily member 4
  • IMD16 TXGP1L
  • CD134 CD134
  • OX40 includes any OX40 variant or isotype that is naturally expressed by the cell. Therefore, the OX40 antibodies or fragments described in the present disclosure can cross-react with OX40 from species other than humans (for example, cynomolgus monkey OX40). Alternatively, the OX40 antibody or fragment may be specific to human OX40 and does not exhibit cross-reactivity with other species.
  • OX40 or its variants and isoforms are isolated from the cells or tissues in which it is naturally expressed, or produced recombinantly using techniques well known in the art and/or the techniques described in this disclosure.
  • OX40 can be derived from any vertebrate source, including natural OX40 from mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term encompasses "full length", unprocessed OX40 and any form of OX40 due to processing in the cell.
  • the term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.
  • OX40 activation refers to the activation of the OX40 receptor. Normally, OX40 activation leads to signal transduction.
  • anti-OX40 antibody or “an antibody that binds to OX40” or “an antibody that binds to OX40” refers to an antibody that can bind to OX40 with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent for targeting OX40 .
  • the anti-OX40 antibody in some embodiments of the present disclosure is the anti-OX40 antibody described in the international patent application PCT/CN2019/107787 (publication number WO2020063660A1), such as the "2G3" antibody. This disclosure introduces the entire content of the international patent application PCT/CN2019/107787 into this application.
  • the term "decrease OX40-expressing cells” refers to the killing or depletion of OX40-expressing cells by anti-OX40 antibodies or fragments thereof. Depletion of OX40-expressing cells can be achieved through a variety of mechanisms, such as antibody-dependent cell (ADCC)-mediated cytotoxicity and/or phagocytosis.
  • ADCC antibody-dependent cell
  • cytokine is a general term for a type of protein that is released by a cell population and acts as an intercellular mediator on another cell.
  • cytokines are lymphokines, monocytes; interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7.
  • IL-8, IL-9, IL-11, IL-12, IL-15 tumor necrosis factor, such as TNF- ⁇ or TNF- ⁇
  • tumor necrosis factor such as TNF- ⁇ or TNF- ⁇
  • KL kit ligand
  • cytokine includes proteins from natural sources or from recombinant cell culture and their biologically active equivalents, including small molecular entities produced by artificial synthesis, and pharmaceutically acceptable derivatives thereof And salt.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • variable region The sequence of about 110 amino acids near the N-terminus in the heavy and light chains of an antibody varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1 FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known Kabat numbering rules (LCDR1-3, HCDR1-3).
  • recombinant human antibody includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies isolated from host cells transformed to express antibodies such as transfectionomas
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • they are humanized antibodies.
  • the term "murine antibody” is a monoclonal antibody against human OX40 prepared according to the knowledge and skills in the art.
  • the test subject is injected with the OX40 antigen during preparation, and then spleen cells (B lymphocytes) expressing antibodies with the desired sequence or functional characteristics are isolated, and then the B lymphocytes are fused with myeloma cells to obtain corresponding hybridoma cells.
  • B lymphocytes spleen cells
  • the murine OX40 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1, IgG2, IgG3 or IgG4 Or the constant region of the heavy chain of a variant thereof.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • humanized antibody refers to an antibody produced by grafting non-human species CDR sequences into a human antibody variable region framework, that is, different types of human germline antibody framework sequences. It can overcome the heterologous reaction induced by the chimeric antibody carrying a large number of heterologous protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be obtained in the "VBase" human germline sequence database, and can be found in Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition .
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further obtained after affinity maturation of CDRs by phage display or yeast display.
  • the affinity of the produced OX40 antibody (or antigen-binding fragment thereof) to the antigen is weakened due to changes in the framework residues in contact with the antigen. Such interactions can be the result of hypermutation of somatic cells. Therefore, there is still a need to transplant such donor framework amino acids into the framework of humanized antibodies.
  • the amino acid residues involved in antigen binding from the non-human OX40 antibody or its antigen-binding fragment can be identified by examining the sequence and structure of the variable region of the non-human monoclonal antibody. Residues in the CDR donor framework that are different from the germline can be considered related.
  • the antibody light chain of the OX40 humanized antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the OX40 humanized antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, specifically, the heavy chain constant region of human IgG1.
  • back mutation refers to the mutation of amino acid residues in the FR region derived from a human antibody into amino acid residues in the corresponding position of the original source antibody, usually to avoid the decrease in immunogenicity caused by the humanized antibody. , The activity is reduced, the variable region of the humanized antibody can be subjected to minimal back mutation to maintain the activity of the antibody.
  • chimeric antibody is an antibody formed by fusing the variable region of a non-human antibody with the constant region of a human antibody, which can reduce the immune response induced by the non-human antibody.
  • a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody according to the need.
  • variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, specifically, it comprises a human IgG1 heavy chain constant region.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments in an antibody that retain the ability to specifically bind to an antigen (e.g., OX40). It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of antibodies. Examples of the binding fragment contained in the term "antigen-binding fragment" of the antibody include:
  • Fab fragment a monovalent fragment composed of VL, VH, CL and CH1 domains
  • F(ab')2 fragment a bivalent fragment formed by connecting two Fab fragments through a disulfide bridge on the hinge region;
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to connect them through a synthetic linker, so that it can produce a single protein chain (called single-chain Fv (scFv), In which the VL and VH regions are paired to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Science USA 85: 5879-5883).
  • single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment” refers to Fab, Fv, sFv, F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody (peptibody) with antigen-binding activity. ), domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv).
  • the term "Fab" can be obtained by treating an IgG antibody molecule with the protease papain (cutting the amino acid residue at position 224 of the H chain) to obtain an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, wherein the H chain N About half of the end side and the entire L chain are joined together by disulfide bonds.
  • the Fab of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure that specifically recognizes human OX40 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain.
  • the Fab can be produced by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express the Fab.
  • F(ab')2 is an antibody fragment obtained by digesting the downstream part of two disulfide bonds in the hinge region of IgG with pepsin, which has a molecular weight of about 100,000 and has antigen-binding activity. It also contains two Fab regions connected at the hinge position.
  • the F(ab')2 of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure with pepsin.
  • the F(ab')2 can be produced by linking Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.
  • the Fab' of the present disclosure can be produced by treating the F(ab') 2 of the present disclosure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting the DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab' .
  • single chain antibody means a variable domain comprising an antibody heavy chain (or region; VH) and an antibody light chain variable domain (or Region; VL) molecule.
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • Other linkers that can be used in this disclosure are described by Alfthan et al.
  • the scFv of the present disclosure can be produced by the following steps: obtaining cDNA encoding the monoclonal antibodies VH and VL of the present disclosure, constructing the DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then adding The expression vector is introduced into prokaryotes or eukaryotes to express scFv.
  • diabodies are antibody fragments in which scFv is dimerized, and are antibody fragments with bivalent antigen-binding activity. In the bivalent antigen binding activity, the two antigens can be the same or different.
  • the diabody of the present disclosure can be produced by the following steps: obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure, constructing the DNA encoding the scFv so that the amino acid sequence of the peptide linker is 8 residues or less, and The DNA is inserted into a prokaryote expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryote to express the diabody.
  • dsFv can be obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (Protein Engineering, 7,697 (1994)).
  • the dsFv of the present disclosure can be produced by the following steps: obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure, constructing the DNA encoding the dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then The expression vector is introduced into prokaryotes or eukaryotes to express dsFv.
  • antibody framework (FR) used in this disclosure refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • amino acid difference refers to the difference between a polypeptide and its variants at a certain or certain amino acid position on the polypeptide fragment, wherein the variant can be determined by a certain position or certain positions on the polypeptide. Obtained by substitution, insertion or deletion of amino acids.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods for determining epitopes are well known in the art, including immunoblotting and immunoprecipitation detection and analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described in this disclosure, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • the term "specific binding” as used in the present invention refers to the binding of an antibody to an epitope on a predetermined antigen.
  • a predetermined antigen when recombinant human OX40 is used as an analyte and an antibody is used as a ligand, when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody has an equilibrium dissociation constant of approximately lower than 10 -7 M or even smaller (KD) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen (or a closely related antigen).
  • SPR surface plasmon resonance
  • KD surface plasmon resonance
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” in this disclosure.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibodies of the present disclosure bind to OX40 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, or 10 -10 M or less, for example, as using a surface Plasma resonance (SPR) technique is measured in BIACORE instrument.
  • SPR surface Plasma resonance
  • the term “competition” in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen binding protein to be detected for example, an antibody or a functional fragment thereof
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J. Immunol.
  • solid Phase direct labeling assay solid-phase direct labeling sandwich assay (see for example Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press); direct solid phase labeling with I-125 label RIA (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82).
  • the assay involves the use of purified antigens that can bind to an unlabeled test antigen binding protein and a labeled reference antigen binding protein (the antigen is on a solid surface or on a cell surface).
  • the antigen-binding protein identified by the competitive assay includes: the antigen-binding protein that binds to the same epitope as the reference antigen-binding protein; and the table that is sufficiently close to the epitope bound to the reference antigen-binding protein The two epitopes sterically hinder the occurrence of binding to each other.
  • additional detailed information on the method for determining competitive binding is provided.
  • the competitive antigen binding protein when it is present in excess, it will inhibit (eg reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or more.
  • cross-reactivity refers to the ability of the antibodies of the present invention to bind to OX40 from different species.
  • the antibody of the present invention that binds to human OX40 can also bind to OX40 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express OX40.
  • Methods of determining cross-reactivity include standard binding assays as described in this disclosure, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • inhibitor As used herein, the terms “inhibit” or “block” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the term “inhibition of growth” is intended to include any measurable decrease in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of a specific antigen by the immune response.
  • induction for inducing CDC or ADCC refers to the stimulation of a specific direct cell killing mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NK cells The main cells that mediate ADCC (NK cells) only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII.
  • an in vitro ADCC assay can be performed, such as described in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta).
  • Useful effector cells for such assays include PBMC and NK cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • the ADCC effect function of antibodies can be reduced or eliminated by modifying the Fc segment on IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody, such as mutations selected from N297A, L234A, L235A of IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutations.
  • complement-dependent cytotoxicity refers to the lysis of target cells in the presence of complement.
  • the activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), which has bound to its cognate antigen.
  • C1q the first component of the complement system
  • an antibody of the appropriate subclass
  • a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • Polypeptide variants with altered Fc region amino acid sequence polypeptide with variant Fc region
  • increased or decreased C1q binding ability are described in, for example, US Patent No. 6,194,551B1 and WO 1999/51642. See also, for example, Idusogie et al., J. Immunol. 164: 4178-4184 (2000).
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but specifically, is double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • the term "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors in the present disclosure can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • mice can be immunized with human OX40 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the website of ImmunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • IMGT ImmunoGeneTics
  • the antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • mAb refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include microorganisms (e.g. bacteria), plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), NSO cells, and 293 cells.
  • the term "conservative modification” or “conservative substitution or substitution” refers to the substitution of other amino acids in proteins with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) Amino acids allow frequent changes without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity.
  • the term "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • exogenous refers to a substance that is produced outside an organism, cell, or human body according to circumstances.
  • endogenous refers to a substance produced in an organism, cell, or human body according to circumstances.
  • mutant sequence refers to the nucleotide sequence and amino acid sequence of the present disclosure with appropriate substitutions, insertions or deletions and other mutation modifications, which are different from the nucleotide sequence and amino acid sequence of the present disclosure.
  • the nucleotide sequence and amino acid sequence of percent sequence identity may be at least 85%, 90% or 95%, non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and determination of the percent identity between two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the total number of positions compared ⁇ 100.
  • the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homology, then the two sequences are 95% homologous.
  • the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • PCR polymerase chain reaction
  • oligonucleotide primers are identical or similar in sequence to the corresponding strands of the template to be amplified.
  • the 5'terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from the total genome, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA.
  • RNA sequences specific DNA sequences from the total genome
  • cDNA, phage, or plasmid sequences transcribed from total cellular RNA.
  • the PCR used in the present disclosure is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, and the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify Or produce a specific part of the nucleic acid.
  • the term “optional” or “optionally” means that the event or environment described later can but does not necessarily occur, and the description includes occasions where the event or environment occurs or does not occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.
  • cancer refers to or describes a physiological condition in mammals that is characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, liver Cell cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, rectal cancer , Colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigines Melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphoma, chronic
  • cancers suitable for treatment by the OX40 antibodies or fragments of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin’s lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple bone marrow tumor.
  • the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g.
  • the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (e.g., triple negative breast cancer), including those metastatic forms of cancer.
  • cell proliferative disorder and “proliferative disorder” refer to disorders related to a certain degree of abnormal cell proliferation.
  • the cell proliferative disorder refers to cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • administering when applied to animals, humans, subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or Contact of the composition with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering also mean treating cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • treatment means the administration of an internal or external therapeutic agent to a patient, such as a composition comprising any one of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
  • a patient has one or more diseases or symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more diseases or symptoms in the treated patient or population, to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of therapeutic agent (also referred to as "therapeutically effective amount") that is effective to alleviate any specific disease or symptom can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • the term "preventing cancer” refers to delaying, inhibiting or preventing the onset of cancer in mammals where the onset of cancer or tumorigenesis has not been confirmed, but is determined by, for example, genetic screening or other methods , Has been identified as having cancer susceptibility.
  • the term also includes the treatment of a mammal with a precancerous condition to stop the progression of the precancerous condition to a malignant tumor or cause its regression.
  • the following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure.
  • the experimental methods that do not specify specific conditions in the examples of this disclosure usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the library of anti-human OX40 monoclonal antibodies was generated by immunizing mice.
  • Experimental mice of BalB/C and A/J strains Comparative Medicine Center of Yangzhou University, Animal Production License Number; SCXK ( ⁇ )2017-007), female, 10 weeks old.
  • Immune antigen is human OX40 recombinant protein with Fc tag (OX40-Fc: OX40 Leu 29-Ala 216 (Accession#NP_003318) fused with Fc), purchased from Acro Biosystems, catalog number #OX40-H5255, expressed in HEK293 as usual Method purification.
  • OX40-Fc OX40 Leu 29-Ala 216 (Accession#NP_003318) fused with Fc
  • Emulsify OX40-Fc with Freund's adjuvant Freund's complete adjuvant (sigma-aldrich, F5881-10ML) for the first immunization, and Freund's incomplete adjuvant (sigma-aldrich, F5506-10ML) for the rest of the booster immunization.
  • Freund's complete adjuvant Sigma-aldrich, F5881-10ML
  • Freund's incomplete adjuvant Sigma-aldrich, F5506-10ML
  • the ratio of antigen to adjuvant is 1:1, and 25 ⁇ g protein/200 ⁇ l/mouse is injected per immunization. See details as follows:
  • the mouse serum was detected by the ELISA method in Example 2, to determine the antibody titer of the mouse serum and the neutralizing activity of blocking the binding of OX40/OX40L.
  • the mice with strong serum titer, affinity and ligand binding blocking ability were selected for a final immunization and then sacrificed.
  • the target hybridomas were selected by the indirect ELISA, capture ELISA and cell-based functional screening of Example 2, and monoclonal antibodies were established by the limiting dilution method.
  • the 19 established strains of OX40 mouse monoclonal antibodies were produced by serum-free expression, and purified mouse monoclonal antibodies were obtained by protein A affinity chromatography technology. Through indirect ELISA, capture ELISA and cell functional activity detection, the hybridoma cells secreting activated anti-OX40 antibody were screened.
  • the brief procedure of functional screening is as follows: culture GS-H2/OX40 stable cell line (purchased from genscript, cat#M00608). Prepare the diluted test antibody and OX40L (Sino Biological, 13127-H04H) solution and add them to the GS-H2/OX40 cells in the logarithmic growth phase.
  • the cell supernatant is collected and the IL-8 content in the supernatant is determined (See human IL-8kit, cisbio, cat#62IL8PEB).
  • the antibody GPX4 prepared with reference to 1A7.gr.1 in the patent WO2015153513 patent
  • hIgG prepared in the laboratory
  • the total cell RNA can be obtained by conventional RNA extraction technology, and then the PCR product of the variable region of the monoclonal antibody can be obtained by reverse transcription polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription polymerase chain reaction
  • the PCR products were separated and recovered by agarose gel, then cloned into gene vector and transformed into Escherichia coli.
  • Several transformed colonies were randomly selected, and the variable regions of monoclonal antibodies were amplified by PCR for gene sequencing.
  • the corresponding sequence of the obtained exemplary murine monoclonal antibody is shown below.
  • the heavy and light variable region sequences of the murine monoclonal antibody m4B5 are as follows:
  • the heavy and light variable region sequences of the murine monoclonal antibody m2G3 are as follows:
  • Example 3 Construction and expression of anti-OX40 recombinant chimeric antibody
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain vector is designed as follows: signal peptide + heavy chain variable region sequence + human IgG1 constant region sequence.
  • the light chain vector is designed as follows: signal peptide + light chain variable region sequence + human Kappa constant region sequence.
  • the above sequences were inserted into the pCEP4 vector (Thermofisher, V04450). After obtaining the vector plasmid, extract the plasmid and send the plasmid to sequencing for verification.
  • the qualified plasmids were transfected into human 293F cells with PEI and cultured continuously, and the 293F cells were cultured with serum-free medium (Shanghai Optima Biotech, OPM-293 CD03) to the logarithmic growth phase for cell transfection.
  • m2G3-NC means negative control
  • Ch2G3-NC, m4B5-NC, Ch4B5-NC are negative controls, which are the same constant regions used by m2G3, Ch2G3, m4B5, and Ch4B5, but the variable regions are different.
  • Example 5 In vitro cell reporter gene experiment of anti-OX40 antibody
  • the murine antibody was humanized.
  • the heavy chain variable region (VH) and light chain variable region (VL) of the chimeric antibody were respectively subjected to site-directed amino acid mutations in the FR (framework region) region. According to different combinations of amino acid mutations, different humanized antibody heavy and light chains are designed, and plasmids encoding different light/heavy chain combinations can be transfected into cells to produce humanized antibodies.
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain vector is designed as follows: signal peptide + mutant heavy chain variable region sequence + human IgG1 constant region sequence.
  • the light chain vector is designed as follows: signal peptide + mutated light chain variable region sequence + human Kappa constant region sequence.
  • the above sequences were inserted into the pCEP4 vector (Thermofisher, V04450). Ask a third-party gene synthesis company to synthesize the expression vector according to the above design, and after obtaining the vector plasmid, extract the plasmid and send the plasmid to sequencing for verification.
  • the qualified plasmids were transfected into human 293F cells with PEI and cultured continuously, and the 293F cells were cultured with serum-free medium (Shanghai Optima Biotech, OPM-293 CD03) to the logarithmic growth phase for cell transfection.
  • Cell culture conditions 5% CO 2 , 37°C, 125 rpm/min. During the culture period, feed was added on the 1st and 3rd day until the cell viability was less than 70%, and the cell supernatant was collected and centrifuged. Load the cell culture fluid after centrifugation and filtration on the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, And phosphate buffer dialysis, and finally obtain purified humanized antibody.
  • glycine hydrochloric acid buffer pH 2.7 0.1M Gly-HCl
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence, and the underline is the CDR sequence.
  • 2G3-hu1 in Table 4 indicates that the variable region of the heavy chain of the humanized antibody 2G3-hu1 is hu2G3 VH1, and the variable region of the light chain is hu2G3 VL1, and so on.
  • the sequence of the humanized variable region of m4B5 is as follows:
  • Hu4B5 V1 means that the variable region of the heavy chain of the humanized antibody hu4B5 V1 is hu4B5 VH0, and the variable region of the light chain is hu4B5 VLO, and so on.
  • the HCDR2 sequence obtained after mutation is as follows:
  • the heavy chain variable regions hu2G3 VH1.1, hu2G3 VH1.2 and the light chain variable region hu2G3 VL are combined to form a new optimized humanized antibody, see Table 7.
  • the above-mentioned humanized antibody was subjected to affinity evaluation (refer to the capture ELISA in Example 2), and the experimental results showed that the humanized molecule can bind to OX40.
  • the light chain variable region and the light chain constant region sequence in the above sequence are combined to form the final light chain sequence, and the heavy chain variable region and the heavy chain constant region in the above sequence are combined to form the final heavy chain sequence.
  • the specific light and heavy chain constant regions are not a limitation of the antibody constant regions of the present disclosure, and other light and heavy chain constant regions and their mutants known in the art can also be used to increase the performance of the antibody.
  • IgG1 heavy chain constant region 1 + IgG1 heavy chain constant region:
  • Example 7 In vitro binding affinity and kinetic test of humanized antibody
  • the Biacore method is a recognized method for objectively detecting the affinity and kinetics between proteins.
  • the recombinant anti-OX40 antibody to be tested of the present invention is covalently connected to the CM5 (GE) chip using the NHS standard amino coupling method. Then, a series of concentration gradients of human OX40-His protein (Yiqiao Shenzhou #10481-H08H) diluted in the same buffer were injected into each cycle at a flow rate of 30 ⁇ L/min. After injection, all were regenerated in the kit. Reagent regeneration. Track the antigen-antibody binding kinetics for 3 minutes and track the dissociation kinetics for 10 minutes.
  • Antibody to be tested ka(1/Ms) kd(1/s) K D (M) 2G3 4.371E+05 2.023E-03 4.628E-09 4B5 1.314E+05 3.694E-04 2.812E-09
  • Example 8 ELISA test for blocking the binding of OX40 and OX40L by antibodies
  • Antibody 2G3 4B5 EC 50 (nM) 2.294 0.3411
  • Isolate CD4+ memory T cells add the antibody to be tested to a 96-well plate coated with anti-CD3 antibody (Chempartner, A05-001), incubate at 37°C for 72 hours, take the supernatant to detect IFN- ⁇ , the results are shown in Figure 2 and Table 13 shown.
  • the results show that GPX4, 4B5 and 2G3 can significantly enhance the release of IFN- ⁇ , and 2G3 can achieve the maximum stimulating effect at 10ng/mL.
  • Example 11 Inhibition of anti-OX40 antibody on tumor cell growth
  • B-hTNFRSF4 (OX40) humanized mouse B-hTNFRSF4 (OX40) humanized mouse, Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), female, 17 to 20 g, 6 to 7 weeks.
  • Collect logarithmic growth phase MC38 tumor cells 7 (purchased from Nanjing Yinhe Biomedicine Co., Ltd.), adjust the cell concentration to 5 ⁇ 10 6 /mL with PBS buffer, and inoculate 0.1 mL of cell suspension to the flanks of OX40 mice. Observe the mice after inoculation and monitor the growth of tumors. On the 7th day after inoculation, the average tumor volume on the flank of the tumor-bearing mice reached 102.5 mm 3. Grouping and drug administration were performed according to the tumor size. The grouping information is shown in Table 14.
  • Dosage (mg/kg) Route of administration Dosing frequency Control (IgG1) 3 i.p. Q3D ⁇ 6 GPX4 3 i.p. Q3D ⁇ 6 2G3 0.3 i.p. Q3D ⁇ 6 2G3 1 i.p. Q3D ⁇ 6 2G3 3 i.p. Q3D ⁇ 6
  • the OX40 humanized antibody was tested for its inhibitory effect on the growth of mouse MC38 colon cancer cell xenograft tumors.
  • the average tumor volume of the IgG1 control group reached 1732.593 mm 3
  • the average tumor volume of the test compound 2G3 low-dose administration group (0.3 mg/kg) mice reached 930.37 mm 3
  • the average tumor volume of tumor-bearing mice in the drug group (2G3, 1mg/kg) and the high-dose administration group (2G, 3mg/kg) were 303.49mm 3 and 155.79mm 3 , respectively.
  • the medium and high-dose group inhibited tumor growth compared with the control group. Obvious (**P ⁇ 0.01), and showed a preliminary dose-dependent relationship, and its tumor growth inhibition rates reached 49%, 88% and 97.0%, respectively.
  • the average tumor volume in the 3mg/kg GPX4 group was 362.47mm 3 , which was significantly different from the control group. It also showed significant tumor growth inhibition (*P ⁇ 0.05), and its tumor growth inhibition rate reached 84%.
  • the average tumor mass weight in the control group was 1.568g, and the test compound 2G3 in the low-dose group (0.3mg/kg), medium-dose group (1mg/kg) and high-dose group (3mg/kg), the average tumor weight was 0.926, respectively g, 0.251g and 0.181g, among which the medium and high-dose group is significantly different from the control group, and the tumor growth inhibition effect is obvious (**P ⁇ 0.01).
  • the average tumor weight in the administration group of GPX4 3mg/kg was 0.372g, which was significantly different from the control group, and it also showed a significant inhibitory effect on the growth of MC38 tumor cells (**P ⁇ 0.01).
  • SEC Size Exclusion Chromatography A method of separation and analysis of solutes based on the relative relationship between the pore size of the gel pore and the coil size of the polymer sample molecule.
  • SEC monomer content percentage A monomer/A total * 100% ("A monomer” is the peak area of the main peak monomer in the sample, and "A total” is the sum of all peak areas).
  • SEC measurement equipment Agilent 1260; column: waters, XBrige SEC (300 ⁇ 7.8mm3.5 ⁇ m).
  • CE capillary gel electrophoresis A method of electrophoresis in which the gel is moved to a capillary as a support medium and separated according to the molecular weight of the sample under a certain voltage.
  • Non-reducing CE (NR-CE) purity percentage A main peak/A total * 100% ("A main peak” is the peak area of the main peak in the sample, and "A total” is the sum of all peak areas).
  • CE measuring instrument Beckman, model plus800.
  • iCIEF imaging capillary isopoint focusing electrophoresis (iCE): A technique for separating proteins based on the pI of their isoelectric points.
  • iCIEF neutral peak content percentage neutral peak area/total area*100% (total area is the sum of acidic peak, neutral peak and basic peak area).
  • Osmotic pressure The freezing point method is used to determine osmotic pressure, based on the direct proportional relationship between the freezing point drop and the molar concentration of the solution.
  • a high-sensitivity temperature sensor is used to determine the freezing point of the solution, which is converted into osmotic pressure through electricity.
  • a 2G3 preparation containing 0.1 mg/ml polysorbate 80 (PS80) and a protein concentration of 50 mg/mL was prepared.
  • the buffer system was 10mM sodium acetate (abbreviated as AA) at pH 5.0, pH 5.5, 10 mM sodium succinate (abbreviated as SA) at pH 5.0, pH 5.5, and pH 6.0, pH 5.5, pH 6.0, 10mM citrate sodium salt (abbreviated as CA) at pH 6.5, 10 mM histidine hydrochloride (abbreviated as His) at pH 5.5, pH 6.0, and pH 6.5, pH 6.0, pH 6.5, pH 7.0 , 10mM sodium phosphate pH 7.5 (abbreviated as PB).
  • Each preparation is filtered, filled, stoppered, and capped.
  • the samples were placed under high temperature conditions of 40°C for 14 days or subjected to a forced degradation experiment with shaking (25°C, 300rpm), and the appearance, SEC, IEC, and non-reducing CE-SDS were used as evaluation indicators to investigate the stability of the formulation.
  • the experimental results are shown in Table 16.
  • the appearance data shows that the protein preparations of the AA and His buffer system have a better appearance under the forced degradation conditions of 40 °C high temperature or shaking
  • SEC data show that AA, His, The protein preparation of CA buffer system has better stability
  • CE-SDS data shows that under 40°C high temperature or shaking conditions, pH 5.0 or pH 5.5 AA, pH 5.0 SA, pH 6.0 His and other histone preparations The stability is relatively good
  • IEC data shows that the protein preparations of pH 5.5 AA, pH 6.0/pH 6.5 His, and pH 6.0 SA are slightly better at 40°C.
  • 2G3 protein preparations are preferably histidine or acetate buffer systems, with pH 6.0 His or pH 5.5 AA, and pH 6.0 His.
  • D in the table means days, for example, D14 means 14 days; T0 means the beginning of the experiment; "+” means the degree of opalescence and granularity, the more “+” means the heavier opalescence or more granules, and vice versa ; N/A means not detected.
  • Example 13 pH value screening of anti-OX40 antibody preparation buffer system
  • His histidine hydrochloride
  • Each preparation is filtered, filled, stoppered, and capped.
  • the samples were subjected to a forced degradation experiment under 40°C high temperature conditions for 1 month, and the stability of the preparation was investigated using SEC, IEC, and non-reducing CE-SDS as evaluation indicators.
  • the experimental results are shown in Table 17.
  • the experimental results showed that there was no significant difference between the SEC/CE/IEC groups for the histone preparations of His buffer at pH 5.5, pH 6.0, and pH 6.5 when placed at a high temperature of 40°C for 1 month.
  • the pH of the His group at pH 5.5 IEC is slightly lower.
  • the pH range of the optimal buffer for the preparation is 5.5-6.5, and a pH of 6.0 is selected for further experiments.
  • T0 means at the beginning of the experiment, "M” means month, for example, M1 means one month.
  • Each preparation is filtered, filled, stoppered, and capped.
  • the samples were placed under 40°C high temperature conditions for 13 days or subjected to a forced degradation experiment with shaking (25°C, 300rpm, 13 days).
  • the appearance, SEC, IEC, and non-reducing CE-SDS were used as evaluation indicators to investigate the stability of the formulation.
  • the experimental results are shown in Table 18.
  • D in the table means days, for example, D13 means 13 days; T0 means the beginning of the experiment.
  • the experimental results show that there is no significant difference in the CE and IEC data between the groups for the types and concentrations of surfactants.
  • the SEC of the 0.1mg/ml PS80 group is slightly lower, and the appearance of the 0.1mg/ml PS20 group and the 0.1mg/ml PS80 group after forced degradation Opalescence or particles appear.
  • the 0.4mg/ml PS80 group looks good under all conditions.
  • the compulsory degradation sample is placed at 4°C for a period of time to review the appearance, 0.4mg/ml PS20 group shakes and 40°C samples Particles appear; therefore, 0.4mg/ml PS80 can be selected; for sugar type and concentration: there is no significant difference between groups under various conditions, and the stability of sucrose and trehalose in the 50mg/mL-90mg/mL group is better.
  • sucrose concentration is 80mg/ml and the osmotic pressure of the sample is 304mosm, which is close to the osmotic pressure in the human body, the sucrose concentration is 75-80mg/ml for the following experiments.
  • M means month, such as M3 means 3 months; T0 means the beginning of the experiment.
  • the experimental results showed that the IEC of the 2G3 preparation decreased slightly at 25°C, but the decline was within the acceptable range, and there was no significant change in other test items; under the condition of 4°C, there was no significant change in all test items.
  • the prescription preparation has good stability.
  • the DOE experiment was designed with 10mM histidine hydrochloride (His) buffer pH value, protein concentration and polysorbate 80 concentration as variables.
  • DOE experiment factors and levels were set to pH5.5 ⁇ 6.5, PS80 concentration 0.2 ⁇ 0.6 mg/mL, protein concentration 30 ⁇ 70mg/mL, design a series of prescriptions (see Table 20), pass the compulsory degradation experiment under 40°C high temperature conditions for 13 days, take appearance, SEC, IEC as evaluation indicators, and adopt the least square method
  • the results are statistically analyzed, and the results are shown in Table 21 and Figure 4.
  • D means days, such as D13 means 13 days; D0 means the beginning of the experiment, N/A means not tested.
  • the pH drift fitting results show that the pH drift is related to the protein concentration.
  • the protein concentration is higher than 50mg/ml, the pH drifts 0.2, and the protein concentration is 30mg/ml.
  • the decrease in IEC is within the acceptable range, and the formulation stability is good, so the preparation protein concentration is 30-70mg/ml, 0.2-0.6mg/ml PS80, pH 5.5-6.5.
  • the IEC with a pH of 5.9-6.2 is the best. Based on the above data, the following protein concentration is 50mg/ml, 0.4mg/ml PS80, 10mM His pH 6.0 prescription for further experiments.
  • D stands for days, such as D13 stands for 13 days; T0 stands for the beginning of the experiment; M stands for months, such as M1 stands for 1 month.
  • the experimental results show that the protein preparation has little change in SEC/CE/IEC under freezing and thawing, high temperature of 40°C or shaking (25°C, 300rpm), and the stability of the preparation is good.
  • the sample After being lyophilized, the sample is out of the box, and the appearance of the sample: white powder cake, full appearance, no collapse. Moisture ⁇ 1.0%. Therefore, the freeze-drying procedure is good.
  • the lyophilized preparation was reconstituted with water for injection to obtain a protein concentration of 50mg/ml, 10mM His pH6.0, 80mg/ml sucrose, 0.4mg/ml PS80 2G3 reconstituted solution, the samples were reconstituted at room temperature and 2 ⁇ Place it at 8°C for 24 hours, and use appearance, SEC, non-reducing CE-SDS, IEC as evaluation indicators to investigate the quality changes of the preparations.
  • the experimental results are shown in Table 24. The results show that the stability of the reconstituted solution is better when placed at room temperature or 2-8°C for 24 hours.

Abstract

本申请公开一种抗OX40抗体药物组合物及其用途。具体地，本申请公开一种药物组合物，其包含抗OX40抗体或其抗原结合片段以及缓冲液。进一步地，该药物组合物还包含糖和表面活性剂。本申请中的药物组合物用于治疗癌症。

Description

一种抗OX40抗体药物组合物及其用途 技术领域
本披露属于药物制剂领域，具体涉及一种包含抗OX40抗体的药物组合物，以及其作为药物的用途。
背景技术
这里的陈述仅是提供与本披露有关的背景信息，而不必然地构成现有技术。
肿瘤免疫治疗是肿瘤治疗领域一个持续热点。最近研究已证明，增强抗肿瘤T细胞功能可以用于抵抗癌症。有大量证据表明，肿瘤细胞通过诱导主要由调节T淋巴细胞(Treg；Quezda et al.Immunol Rev 2011；241:104-118)介导的主动免疫耐受，而“逃避”免疫系统。因此，效应T淋巴细胞(Teff)与耐受原性(tolerogenic)Treg之间的平衡，对于有效抗肿瘤免疫治疗至关重要。因此，可以通过增强肿瘤特异性Teff的效应功能和/或通过减弱肿瘤特异性Treg的抑制功能，获得有效的抗肿瘤免疫应答。CD134(OX40)受体已被证明是介导这些应答的一种关键受体(Sugamura,K,Ishii,N,Weinberg,A.Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40.Nature Rev Imm 2004；4:420-431)。
OX40是肿瘤坏死因子受体(TNFR)超家族的成员之一，是一种在细胞表面表达的分子量约50kDa的糖蛋白。OX40的胞外配体结合结构域由4个富含半胱氨酸的结构域(CRD)组成。OX40的天然配体是OX40L(CD252)，两者形成OX40-OX40L复合物。OX40主要表达在激活的T细胞上，OX40是次级共刺激分子，在激活24-72小时后表达。OX40的配体OX40L主要表达在激活的抗原递呈细胞上。表达OX40的T淋巴细胞已被证实存在于各种人恶性肿瘤及癌症患者的引流淋巴结中。在严重联合免疫缺陷(SCID)小鼠模型中，OX40与OX40L结合结构域相互作用可增强抗肿瘤免疫，导致各种人恶性肿瘤细胞系，如淋巴瘤、前列腺癌、结肠癌及乳腺癌的肿瘤生长抑制。
抗OX40抗体可通过特异性刺激，激活免疫，提高患者自身对肿瘤的免疫系统反应，达到对肿瘤细胞进行杀伤的目的。抗OX40相关抗体已见公开，例如WO2013038191、WO2015153513、WO2016179517、WO2017096182、CN110078825A、WO2016196228等。
发明内容
本披露提供一种抗OX40抗体药物组合物及其用途。
一方面，本披露提供一种药物组合物，其包含抗OX40抗体或其抗原结合片段，以及缓冲液，所述缓冲液为组氨酸盐缓冲液或醋酸盐缓冲液，所述缓冲液的pH为 约5.0至约6.5，优选pH为约5.5至约6.5，更优选pH为约6.0；所述组氨酸盐缓冲液优选组氨酸盐酸盐缓冲液，所述醋酸盐缓冲液优选醋酸钠盐缓冲液(简称醋酸钠缓冲液)。
在一些实施方案中，前述药物组合物中所述缓冲液的pH值为约5.0至约6.5，优选为5.0至6.5；在一些实施方案中，所述缓冲液pH值为约5.5至约6.5，优选为5.5至6.5；在另一些实施方案中，所述缓冲液pH值为约6.0，优选为6.0；在另一些实施方案中，所述缓冲液为pH为约5.5至约6.5的组氨酸盐缓冲液；在另一些实施方案中，所述缓冲液为pH为6.0的组氨酸盐酸盐缓冲液；在另一些实施方案中，所述缓冲液为pH为约5.0至约6.0的醋酸盐缓冲液；在另一些实施方案中，所述缓冲液为pH约5.0至约5.5的醋酸钠缓冲液；在另一些实施方案中，所述缓冲液为pH约5.5的醋酸钠缓冲液。在另一些实施方案中，缓冲液的pH值非限制的实施例包括约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0、约6.1、约6.2、约6.3、约6.4、约6.5以及这些点值之间的任意范围。在一些实施方案中，前述药物组合物的pH值与其缓冲液pH值一致或几乎一致(本领域技术人员公知，在制备药物制剂的过程中，有时会存在pH漂移(制剂pH与缓冲液pH存在一些差值，称为pH漂移值)，pH的漂移值通常在±0.3范围内，如无特别说明，本披露药物组合物的pH漂移值在±0.3范围内，优选在±0.2范围内，更优选在±0.1范围内)。
在一些实施方案中，前述的药物组合物中所述缓冲液浓度为大约5mM至大约30mM，优选为5mM至30mM；在一些实施方案中，缓冲液浓度为大约5mM至大约15mM，优选5mM至15mM；在一些实施方案中，缓冲液浓度为大约10mM，优选10mM。缓冲液浓度的非限制性实例包括约5mM、约6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、约15mM、约16mM、约18mM、约20mM、约25mM、约30mM以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物中所述抗OX40抗体或其抗原结合片段浓度为大约1mg/ml至大约100mg/ml，优选1mg/ml至100mg/ml；在一些实施方案中，抗OX40抗体或其抗原结合片段浓度为大约30mg/ml至大约70mg/ml，优选30mg/ml至70mg/ml；在一些实施方案中，抗OX40抗体或其抗原结合片段浓度为大约50mg/ml，优选50mg/ml。抗OX40抗体或其抗原结合片段浓度非限制性实例包括：大约1mg/ml、大约10mg/ml、大约20mg/ml、大约30mg/ml、大约35mg/ml、大约40mg/ml、大约45mg/ml、大约50mg/ml、大约55mg/ml、大约60mg/ml、大约65mg/ml、大约70mg/ml、大约80mg/ml、大约90mg/ml、大约100mg/ml以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物还包括糖，在一些实施方案中，所述糖包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。在一些实 施方案中、所述糖选自：葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、木糖醇、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇和异-麦芽酮糖。在一些实施方案中，糖是非还原性二糖；在一些实施方案中，所述的糖优选为海藻糖或蔗糖，最优选为蔗糖。
在一些实施方案中，前述的药物组合物中所述糖浓度为大约50mg/ml至大约90mg/ml，优选50mg/ml至90mg/ml；在一些实施方案中，糖浓度为大约75mg/ml至大约85mg/ml，优选75mg/ml至85mg/ml；在一些实施方案中，糖浓度为大约80mg/ml，优选80mg/ml。非限制性实例包括约50mg/ml、约60mg/ml、约65mg/ml、约70mg/ml、约75mg/ml、约80mg/ml、约85mg/ml、约90mg/ml以及这些点值之间的任意范围。
在一些实施方案中，前述药物组合物还包括表面活性剂，可选自聚山梨醇酯20(也称聚山梨酯20、吐温20、PS20)、聚山梨醇酯80(也称聚山梨酯80、吐温80、PS80)、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇和乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯80。
在一些实施方案中，前述的药物组合物中表面活性剂的浓度为大约0.1mg/ml至大约1.0mg/ml，优选0.1mg/ml至1.0mg/ml；在一些实施方案中，表面活性剂的浓度为大约0.2mg/ml至大约0.6mg/ml，优选0.2mg/ml至0.6mg/ml；在一些实施方案中，表面活性剂的浓度为大约0.4mg/ml，优选0.4mg/ml。非限制性实例包括约0.1mg/ml、约0.2mg/ml、约0.3mg/ml、0.4mg/ml、约0.45mg/ml、约0.5mg/ml、约0.55mg/ml、约0.6mg/ml、约0.7mg/ml、约0.8mg/ml、约0.9mg/ml、约1.0mg/ml以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其包含：a)浓度大约1mg/ml至大约100mg/ml的抗OX40抗体或其抗原结合片段，b)pH为约5.0至约6.5的组氨酸盐缓冲液或醋酸盐缓冲液，c)浓度为大约50mg/ml至大约90mg/ml的糖，和d)浓度为大约0.1mg/ml至大约1.0mg/ml的聚山梨醇酯80。
在一些实施方案中，前述的药物组合物包含：a1)浓度为大约30mg/ml至大约70mg/ml的抗OX40抗体或其抗原结合片段，b1)pH为约5.5至约6.5的组氨酸盐缓冲液，c1)浓度为大约75mg/ml至大约85mg/ml的蔗糖，和d1)浓度为 大约0.2mg/ml至大约0.6mg/ml的聚山梨醇酯80。
在一些实施方案中，前述的药物组合物包含：约10mM的pH为约6.0的组氨酸盐酸盐缓冲液，浓度约50mg/ml的抗OX40抗体或其抗原结合片段，浓度约80mg/ml蔗糖，以及浓度约0.4mg/ml聚山梨酯80。
在一些实施方案中，前述的药物组合物，其包含：10mM的pH为6.0的组氨酸盐酸盐缓冲液，浓度为50mg/ml的抗OX40抗体或其抗原结合片段，浓度为80mg/ml蔗糖，以及浓度为0.4mg/ml聚山梨酯80。
在一些实施方案中，前述的药物组合物中的抗OX40抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(A)所述重链可变区包含分别如SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3；
(B)所述重链可变区包含分别如SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；
(C)所述重链可变区包含分别如SEQ ID NO：11、33、13所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；或
(D)所述重链可变区包含分别如SEQ ID NO：11、34、13所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3。
在一些实施方案中，前述的药物组合物中所述抗OX40抗体或其抗原结合片段包含：
(E)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少95％，96％，97％，98％，99％或100％序列同一性的重链可变区，和如SEQ ID NO：2所示或与SEQ ID NO：2具有至少95％，96％，97％，98％，99％或100％序列同一性的轻链可变区；
(F)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少95％，96％，97％，98％，99％或100％序列同一性的重链可变区，和如SEQ ID NO：10所示或与SEQ ID NO：10具有至少95％，96％，97％，98％，99％或100％序列同一性的轻链可变区；
(G)如SEQ ID NO：17、18、31或32所示或与SEQ ID NO：17、18、31或32具有至少95％，96％，97％，98％，99％或100％序列同一性的重链可变区，和如SEQ ID NO：19所示或与SEQ ID NO：19具有至少95％，96％，97％，98％，99％或100％序列同一性的轻链可变区；
(H)如SEQ ID NO：17、18、31或32所示或与SEQ ID NO：17、18、31 或32具有至少95％，96％，97％，98％，99％或100％序列同一性的重链可变区，和如SEQ ID NO：20所示或与SEQ ID NO：20具有至少95％，96％，97％，98％，99％或100％序列同一性的轻链可变区；或
(I)如SEQ ID NO：26、27、28、29或30所示或与SEQ ID NO：26、27、28、29或30具有至少95％，96％，97％，98％，99％或100％序列同一性的重链可变区，和如SEQ ID NO：21、22、23、24或25所示或与SEQ ID NO：21、22、23、24或25具有至少95％，96％，97％，98％，99％或100％序列同一性的轻链可变区。
在一些实施方案中，前述的药物组合物中所述抗OX40抗体或其抗原结合片段包含：如SEQ ID NO：31所示的重链可变区和如SEQ ID NO：19所示的轻链可变区。在一些实施方案中，前述的药物组合物中所述抗OX40抗体或其抗原结合片段包含：如SEQ ID NO：29所示的重链可变区和如SEQ ID NO：23所示的轻链可变区。
在一些实施方案中，前述的药物组合物中所述的抗OX40抗体包含重链恒定区和轻链恒定区；优选地，重链恒定区源自人IgG1、IgG2、IgG3或IgG4或其突变序列；轻链恒定区源自人κ、λ链或其突变序列；更优选地，所述重链恒定区的氨基酸序列如SEQ ID NO：35所示或与SEQ ID NO：35具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性，所述轻链恒定区的氨基酸序列如SEQ ID NO：36所示或与SEQ ID NO：36具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性。
在一些实施方案中，前述的药物组合物中所述的抗OX40抗体或其抗原结合片段包含：
(J)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的重链，和/或如SEQ ID NO：38所示或与SEQ ID NO：38具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的轻链；或
(K)如SEQ ID NO：39所示或与SEQ ID NO：39具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的重链，和/或如SEQ ID NO：40所示或与SEQ ID NO：40具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的轻链。
在一些实施方案中，前述的药物组合物中所述的抗OX40抗体或其抗原结合片段包含：如SEQ ID NO：37所示的重链和如SEQ ID NO：38所示轻链；或如SEQ ID NO：39所示的重链和如SEQ ID NO：40所示轻链。
在一些实施方案中，前述的药物组合物，其包含：10mM的pH为6.0的组氨 酸盐酸盐缓冲液，浓度为50mg/ml的抗OX40抗体，浓度为80mg/ml蔗糖，以及浓度为0.4mg/ml聚山梨酯80，所述抗OX40抗体包含如SEQ ID NO：37所示的重链和如SEQ ID NO：38所示轻链。
在一些实施方案中，本披露提供一种制备前述的药物组合物的方法，所述方法包括将抗OX40抗体或其抗原结合片段原液经缓冲液置换的步骤。
本披露还提供一种冻干制剂，其复溶后可形成前述任一项所述的药物组合物。
在一些实施方案中，本披露提供一种含抗OX40抗体或其抗原结合片段的冻干制剂，所述冻干制剂通过将前述的药物组合物经冷冻干燥获得。在可选的实施方案中，所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
在一些实施方案中，本披露提供一种含抗OX40抗体或其抗原结合片段的冻干制剂，所述冻干制剂经复溶可形成前述的药物组合物。
在一些实施方案中，前面任意一项所说的冻干制剂，其中所述抗OX40抗体或其抗原结合片段与缓冲液的物质的量比为：0.007摩尔份至0.685摩尔份的抗OX40抗体或其抗原结合片段(非限制性实施例包括：约0.007摩尔份、约0.068摩尔份、约0.137、约0.206摩尔份、约0.240摩尔份、约0.274摩尔份、约0.308摩尔份、约0.343摩尔份、约0.377摩尔份、约0.411摩尔份、约0.446摩尔份、约0.480摩尔份、约0.548摩尔份、约0.617摩尔份、约0.685摩尔份，以及这些点值之间的任意范围)：5摩尔份至30摩尔份的组氨酸盐或醋酸盐缓冲液(非限制性实例包括：约5摩尔份、约6摩尔份、约7摩尔份、约8摩尔份、约9摩尔份、约10摩尔份、约11摩尔份、约12摩尔份、约13摩尔份、约14摩尔份、约15摩尔份、约16摩尔份、约18摩尔份、约20摩尔份、约25摩尔份、约30摩尔份，以及这些点值之间的任意范围)。在一些实施方案中，所述冻干制剂包含抗OX40抗体或其抗原结合片段、糖(例如海藻糖或蔗糖)、表面活性剂(例如聚山梨醇酯80或聚山梨醇酯20)和组氨酸盐(例如组氨酸盐酸盐缓冲液)或醋酸盐缓冲液(醋酸钠缓冲液)，其中，所述抗OX40抗体或其抗原结合片段、糖和表面活性剂的重量份比为：1重量份至100重量份抗OX40抗体或其抗原结合片段：50重量份至90重量份的糖：0.1重量份至1.0重量份的表面活性剂，所述抗OX40抗体或其抗原结合片段与缓冲液的物质的量比为：0.007摩尔份至0.685摩尔份的抗OX40抗体或其抗原结合片段：5摩尔份至30摩尔份的组氨酸盐或醋酸盐缓冲液。在一些实施方案中，所述冻干制剂包含如SEQ ID NO：37所示的重链和如SEQ ID NO：38所示的轻链的抗OX40抗体、蔗糖、聚山梨醇酯80和组氨酸盐酸盐缓冲液，其中，所述抗OX40抗体、蔗糖和聚山梨醇酯80的重量份比为：50重量份抗OX40抗体：80重量份的蔗糖：0.4重量份的聚山梨醇酯80，所述抗OX40抗体与组氨酸盐酸盐缓冲液的物质的量比为：0.343摩尔份的抗OX40抗体：10摩尔份的组氨酸盐酸盐缓冲液。
在一些实施方案中，前述冻干制剂于2-8℃稳定至少3个月，至少6个月，至 少12个月，至少18个月或至少24个月。在一些实施方案中，该冻干制剂于40℃高温条件下稳定至少7天，至少14天或至少28天。
在一些实施方案中，本披露提供一种含抗OX40抗体或其抗原结合片段的复溶溶液，所述复溶溶液通过将前述的冻干制剂经复溶制备获得。复溶所用溶液包括但不限于注射用水、生理盐水或葡萄糖溶液，优选注射用水。在一些实施方案中，所述复溶溶液组分和含量与前述的药物组合物相同。
在一些实施方案中，本披露提供一种制品，其包括容器，该容器中装有如前所述的药物组合物、冻干制剂或复溶溶液。在一些实施方案中，该容器为中性硼硅玻璃管制注射剂瓶。
本披露提供一种治疗或预防疾病或病症的方法，所述方法包括向受试者施用治疗有效量或预防有效量的前述的药物组合物、冻干制剂、复溶溶液或制品；在一些实施方案中，所述疾病或病症可以为癌症或细胞增殖性疾病。在一些具体实施方案中，所述癌症为肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症或者任何特征在于不受控细胞生长的其它疾病或病症。在一些具体实施方案中，所述的血液系统癌症包括但不限于急性及慢性骨髓性白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、骨髓组织增殖性疾病、多发性骨髓瘤、何杰金氏(Hodgkin)疾病、非何杰金氏淋巴瘤、B细胞淋巴瘤、T细胞淋巴瘤、滤泡中心细胞淋巴瘤、慢性粒细胞白血病。
本披露提供一种增强人受试者中的免疫应答的方法，包括：向受试者施用治疗有效量或预防有效量的前述的药物组合物、冻干制剂、复溶溶液或制品；优选地，所述增强的免疫应答包括T效应细胞的免疫刺激/效应功能的增加，和/或T调节细胞的免疫抑制功能的下调。其中，所述增加可以是细胞增殖的结果，所述下调可以是细胞数量不增加(或细胞数量降低)的结果。
本披露提供前述的药物组合物、冻干制剂、复溶溶液或制品在制备用于增强人类对象中的免疫应答的药物中的用途。
本披露还提供前述的药物组合物、冻干制剂、复溶溶液或制品在制备用于治疗/预防疾病或病症的药物中的用途。疾病或病症可以为癌症或细胞增殖性疾病。在一些具体实施方案中，所述癌症为肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症或者任何特征在于不受控细胞生长的其它疾病或病症。在一些具体实施方案中，所述的血液系统癌症包括但不限于急性及慢性骨髓性白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、骨髓组织增殖性疾病、多发性骨髓瘤、何杰金氏(Hodgkin)疾病、非何杰金氏淋巴瘤、B细胞淋巴瘤、T细胞淋巴瘤、滤泡中心细胞淋巴瘤、慢性粒细胞白血病。
本披露还提供用于治疗/预防疾病或病症的前述的药物组合物、冻干制剂、复溶溶液或制品。在一些实施方案中，疾病或病症可以为癌症或细胞增殖性疾病。在一些具体实施方案中，所述癌症为肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症或者任何特征在于不受控细胞生长的其它疾病或病症。在一些具体实施方案中，所述的血液系统癌症包括但不限于急性及慢性骨髓性白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、骨髓组织增殖性疾病、多发性骨髓瘤、何杰金氏(Hodgkin)疾病、非何杰金氏淋巴瘤、B细胞淋巴瘤、T细胞淋巴瘤、滤泡中心细胞淋巴瘤、慢性粒细胞白血病。
本披露提供前述的药物组合物、冻干制剂、复溶溶液或制品用于下述一项或多项：抑制Treg功能(例如抑制Treg的遏制性功能)、杀死表达OX40的细胞(例如表达高水平OX40的细胞)、提高效应T细胞功能和/或提高记忆T细胞功能、降低肿瘤免疫、增强T细胞功能和/或消减表达OX40的细胞。
本披露提供前述的药物组合物、冻干制剂、复溶溶液或制品用于以下的用途：治疗癌症、刺激受试者中的免疫反应、刺激抗原特异性T细胞反应、激活或共刺激T细胞、增加T细胞中的细胞因子(例如IL-2及/或IFN-γ)产生、和/或T细胞的增殖、减少或耗竭肿瘤中T调控性细胞的数目、和/或抑制肿瘤细胞生长。
本披露亦提供前述的药物组合物、冻干制剂、复溶溶液或制品制备药剂的用途，所述药剂用于：刺激受试者中的免疫反应、刺激抗原特异性T细胞反应、激活或共刺激T细胞、增加T细胞中的IL-2和/或IFN-γ产生和/或T细胞的增殖、减少或耗竭肿瘤中T调控性细胞的数目和/或抑制肿瘤细胞生长。
附图说明
图1A至图1B：鼠源抗体和嵌合抗体与人OX40的亲和力ELISA检测结果。其中，图1A是鼠源抗体m2G3和嵌合抗体ch2G3的亲和力结果，m2G3-NC和ch2G3-NC为阴性对照；图1B是鼠源抗体m4B5和嵌合抗体ch4B5的亲和力结果，m4B5-NC和ch4B5-NC为阴性对照。
图2：抗OX40抗体刺激T细胞分泌IFN-γ的实验。结果显示，待测OX40抗体在10ng/mL抗体浓度时即可达到最大刺激效果。
图3A和图3B：抗OX40抗体小鼠体内抑瘤效果，图3A显示给药后不同天数小鼠体内肿瘤体积的变化；图3B显示给药后第20天，不同抗OX40抗体对小鼠体内肿瘤质量的影响。结果显示给药治疗后第20天，在给药量为3mg/kg时，2G3抗体的肿瘤抑制率高达97％。
图4：抗OX40抗体制剂处方DOE拟合图
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
如本文所用，术语“缓冲液”或“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲液。将pH控制在适当范围中的缓冲液的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、柠檬酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲液。
如本文所用，术语“组氨酸盐缓冲剂”或“组氨酸缓冲液”，是包含组氨酸离子的缓冲液。组氨酸盐缓冲液的示例包括组氨酸盐酸盐，组氨酸醋酸盐，组氨酸磷酸盐，组氨酸硫酸盐等缓冲液。本披露的一些实施方式中，组氨酸盐缓冲液选自组氨酸盐酸盐缓冲液。
如本文所用，术语“琥珀酸盐缓冲剂”或“琥珀酸盐缓冲液”，是包括琥珀酸离子的缓冲液。琥珀酸盐缓冲液的示例包括琥珀酸琥珀酸钠、琥珀酸琥珀酸钾、琥珀酸琥珀酸钙盐等。本披露在一些实施方式中，琥珀酸缓冲液是琥珀酸琥珀酸钠。
如本文所用，术语“醋酸盐缓冲剂”或“醋酸盐缓冲液”，是包括醋酸根离子的缓冲液。醋酸盐缓冲液的示例包括醋酸醋酸钠、醋酸组氨酸盐、醋酸醋酸钾、醋酸醋酸钙、醋酸醋酸镁等。本披露在一些实施方式中，醋酸缓冲液是醋酸醋酸钠。
如本文所用，术语“磷酸盐缓冲剂”或“磷酸盐缓冲液”，是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。本披露在一些实施方式中，磷酸氢二钠-枸橼酸。
如本文所用，术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物保持抗体活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本文中，“药物组合物”和“制剂”并不互相排斥。
在制备包含本文所述的抗体的药物组合物的上下文中，术语“缓冲液置换”是指用本文所述的稳定制剂的缓冲体系置换溶解抗体蛋白的溶剂体系，例如，使用本文所述的稳定制剂的缓冲体系经物理操作方式将含抗体蛋白的高盐或高渗溶剂体系置换，从而使抗体蛋白存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
本披露的药物组合物或制剂可通过本领域公知的方法制备。示例性的，抗体药物组合物或制剂的制备：第一步：取一定量的纯化的抗体溶液，用不含抗体的缓冲剂(如pH6.0的10mM的组氨酸盐酸盐缓冲液)进行溶剂置换(优选超滤)，经超滤膜至少6倍体积置换，抗体浓缩到约70mg/mL。加入一定体积的蔗糖母液，混匀，使最终蔗糖浓度为80mg/mL。加入一定体积的聚山梨醇酯80母液，混匀， 使最终聚山梨醇酯-80浓度为0.4mg/mL。加pH6.0的10mM组氨酸盐酸盐缓冲液定容，使抗体浓度为50mg/mL(其他待测试制剂或稳定性制剂可参照相似步骤进行配制)。产品经过滤后中控取样检测无菌。将原液过0.22μm滤芯，收集滤液。第二步：调节装量，将滤液灌装于西林瓶中，加塞，分别于灌装开始、灌装中间、灌装结束时取样中控检测装量差异。第三步：开启轧盖机，加铝盖，进行轧盖。第四步：目检，确认产品无装量不准等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
本披露中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂为水。
如本文所用，术语“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。冻干制剂可通过将液体或溶液形式的药物组合物或制剂经冷冻干燥获得。通过冷冻制剂和随后在适于一次干燥的温度使水升华，进行冷冻干燥。在此条件下，产物温度低于制剂的低共熔点或分解温度。在通常约50-250毫托范围的合适压力下，通常，一次干燥的存放温度范围为约-30至25℃(假设产物在一次干燥过程中保持冷冻)。制剂、容纳样品的容器(例如，玻璃小瓶)的大小和类型以及液体的体积决定了干燥所需的时间，所述时间的范围可为几小时至几天(例如40-60小时)。二次干燥阶段可在约0-40℃进行，这主要取决于容器的类型和大小以及采用的蛋白的类型。二次干燥时间由产物中的期望残余湿度水平决定，通常需要至少约5小时。通常，低压冻干的制剂的含水量小于约5％，优选小于约3％。压力可与在一次干燥步骤中应用的压力相同，优选的，二次干燥的压力低于一次干燥。冷冻干燥条件可以随制剂和小瓶大小而变化。
本披露的“糖”包含常规组合物(CH2O)n及其衍生物，包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等。可选自葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、木糖醇、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等等。优选的糖是非还原性二糖，更优选为海藻糖或蔗糖，最优选为蔗糖。
本披露的表面活性剂可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇和乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨醇酯80或聚山 梨醇酯20，更优选为聚山梨醇酯80。
本文所用术语“约”、“大约”或“基本上包含”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”、“大约”或“基本上包含”可意味着至多20％的范围，例如，可意味着在其后数值的±20％、±19％、±18％、±17％、±16％、±15％、±14％、±13％、±12％、±11％、±10％、±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％或±1％、的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”、“大约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本披露所述的药物组合物能够达到一种稳定的效果：其中的抗体在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的抗体药物制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存例如至少3个月、优选6个月、更优选1年，且甚至更优选地最多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在，例如25℃和/或40℃的温度保存，例如1个月、3个月、或6个月的时段后表现出期望的特征。稳定制剂，例如，通过视觉分析，药物抗体制剂是无色的，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的截短。通常形成不超过约10％、优选不超过约5％的聚集。在一些实施方式中，本披露中的药物组合物或冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月；在25℃可稳定存在至少3个月，或至少6个月。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱 法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。抗体的生物活性可以例如通过抗原结合测定来确定。
如本文所用，术语“增强T细胞功能”意指诱导、引起或刺激效应或记忆T细胞，使其具有更新、持续或放大的生物学功能。增强T细胞功能的例子包括：相对于干预前的水平，来自CD8+效应T细胞的γ-干扰素分泌升高、来自CD4+记忆和/或效应T细胞的γ-干扰素分泌升高、CD4+效应和/或记忆T细胞增殖升高、CD8+效应T细胞增殖升高、抗原响应性(例如清除)增强。
如本文所用，术语“增强免疫应答”是指刺激、激起、增加、改善或增强哺乳动物免疫系统的应答。免疫应答可以是细胞应答(即细胞介导的，如细胞毒性T淋巴细胞介导的)或者体液应答(即抗体介导的应答)，并且可以是初次或再次免疫应答。增强免疫应答的例子包括增加的CD4+辅助T细胞活性及产生细胞毒性T细胞。免疫应答的增强可以用本领域技术人员已知的一些体外或体内测量进行评估，包括但不限于细胞毒性T淋巴细胞测定、细胞因子释放(例如IL-2的产生)、肿瘤消退、携带肿瘤动物的存活、抗体产生、免疫细胞增殖、细胞表面标记表达及细胞毒性。在一个实施方案中，所述方法增强细胞免疫应答，特别是细胞毒性T细胞应答。
如本文所用，术语“肿瘤免疫”指肿瘤逃避免疫识别和清除的过程。作为治疗概念，肿瘤免疫在其逃避免疫识别和清除的能力被减弱时，肿瘤被免疫系统识别并攻击，患者得到治疗。肿瘤识别的例子包括肿瘤结合、肿瘤收缩和肿瘤清除。
如本文所用，术语“T效应细胞”(“Teff”)，指具有细胞溶解活性的T细胞(例如，CD4+及CD8+T细胞)以及T辅助(Th)细胞，Teff分泌细胞因子、且激活并引导其他免疫细胞，但不包括调控性T细胞(Treg细胞)。本披露所述抗OX40抗体可激活Teff细胞，例如CD4+及CD8+Teff细胞。
如本文所用，术语“调节性T细胞”或“Treg细胞”意指专门化类型的CD4+T细胞，其能阻抑其它T细胞的应答。Treg细胞特征在于表达CD4、IL-2受体的α亚基(CD25)、和转录因子forkhead box P3(FOXP3)(Sakaguchi,Annu Rev Immunol 22,531-62(2004))，且在诱导和维持外周自体耐受中发挥至关重要作用，所述耐受针对肿瘤表达的抗原。
如本文所用，术语“OX40”是指一种结合OX40配体(OX40-L)的受体，它是TNF-受体超家族的成员。OX40亦称作肿瘤坏死因子受体超家族成员4(TNFRSF4)、ACT35、IMD16、TXGP1L及CD 134。术语“OX40”包括由细胞天然表达的任何OX40变体或同种型。因此，本披露所述OX40抗体或片段可与来自除人类外的物种的OX40(例如，食蟹猴OX40)交叉反应。或者，所述OX40抗体或片段可对人OX40 具有特异性，且不展现与其他物种的交叉反应性。
OX40或其变体及同种型从其天然表达的细胞或组织中分离，或使用本领域熟知的技术和/或本披露所述的技术以重组方式产生。除非另有说明，否则“OX40”可以来自任何脊椎动物来源，包括哺乳动物诸如灵长类(例如人)和啮齿类(例如小鼠和大鼠)的天然OX40。该术语涵盖“全长”，未加工的OX40以及因细胞中的加工所致的任何形式的OX40。该术语还涵盖OX40的天然发生变体，例如剪接变体或等位变体。
如本文所用，术语“OX40激活”指OX40受体的激活。通常，OX40激活导致信号转导。
如本文所用，术语“抗OX40抗体”或“结合OX40的抗体”或“OX40的抗体”指能够以足够亲和力结合OX40，使得该抗体可作为诊断剂和/或治疗剂用于靶向OX40的抗体。本披露的一些实施例中所述抗OX40抗体为国际专利申请PCT/CN2019/107787(公开号WO2020063660A1)中所述的抗OX40抗体，例如“2G3”抗体。本披露将国际专利申请PCT/CN2019/107787的全部内容引入本申请。
如本文所用，术语“消减表达OX40的细胞”，是指抗OX40抗体或其片段杀死或消减表达OX40的细胞。消减表达OX40的细胞可以通过多种机制来实现，诸如抗体依赖性细胞(ADCC)介导的细胞毒性和/或吞噬作用。
如本文所用，术语“细胞因子”是由一类蛋白质的通称，其由细胞群释放，并作为细胞间介质作用于另一细胞。此类细胞因子的例子有淋巴因子、单核因子；白介素(IL)，诸如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-11、IL-12、IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本披露中使用的，术语细胞因子包括来自天然来源或来自重组细胞培养物的蛋白质及其生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。
本披露所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。
如本文所用，术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
抗体重链和轻链中靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL或LCVR)和重链可变区(VH或HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本披露所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
如本文所用，术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞(如转染瘤)中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
本披露的抗体包括鼠源抗体、嵌合抗体、人源化抗体、人抗体。
一些实施方案中为人源化抗体。
如本文所用，术语“鼠源抗体”为根据本领域知识和技能制备的针对人OX40的单克隆抗体。例如，制备时用OX40抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的脾细胞(B淋巴细胞)，再将B淋巴细胞与骨髓瘤细胞融合，获得相应杂交瘤细胞。
在一些实施方案中，所述的鼠源OX40抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。
如本文所用，术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本披露的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
如本文所用，术语“人源化抗体(humanized antibody)”，是指将非人物种CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服由于嵌合抗体携带大量异源蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库中获得，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本披露的人源化抗体也包括进一步由噬菌体展示或酵母菌展示对CDR进行亲和力成熟后获得的人源化抗体。
在涉及CDR移植的情况中，由于与抗原接触的构架残基的变化而导致产生的OX40抗体(或其抗原结合片段)对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人OX40抗体或其抗原结合片段的参与抗原结合的氨基酸残基，可通过检查非人单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。在本披露一些实施方案中，所述的OX40人源化抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的OX40人源化抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，具体地，包含人源IgG1重链恒定区。
如本文所用，术语“回复突变”是指将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基，通常是为了避人源化抗体引起的免疫原性下降的同时，引起的活性下降，对所述的人源化抗体可变区可进行最少的回复突变，以保持抗体的活性。
如本文所用，术语“嵌合抗体(chimeric antibody)”，是将非人抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻非人抗体诱发的免疫应答反应。例如，建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，具体地，包含人源IgG1重链恒定区。
如本文所用，术语抗体的“抗原结合片段”或“功能片段”是指抗体中的一个或多个片段，其保持特异性结合抗原(例如，OX40)的能力。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括：
(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；
(ii)F(ab')2片段，通过铰链区上的二硫桥连接两个Fab片段形成的二价片段；
(iii)由VH和CH1结构域组成的Fd片段；
(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；和
(v)单结构域或dAb片段(Ward等人，(1989)Nature，341：544-546)，其由VH结构域组成。
此外，虽然Fv片段的两个结构域VL和VH由单独的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生单个蛋白质链(称为单链Fv(scFv)，在其中VL和VH区配对形成单价分子；参见，例如，Bird等人 (1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性能筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
一些实施方案中，术语“抗原结合片段”指具有抗原结合活性的Fab、Fv、sFv、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、双抗体(diabody)、三抗(triabody)和四抗(tetrabody)、串联二-scFv、串联三-scFv)。
如本文所用，术语“Fab”可以通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子，获得具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。本披露的Fab可以通过用木瓜蛋白酶处理本披露的特异性识别人OX40并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
如本文所用，术语“F(ab')2”是通过用胃蛋白酶消化IgG铰链区中两个二硫键的下游部分，而获得的抗体片段，其分子量为约100,000，并具有抗原结合活性，并包含铰链位置相连的两个Fab区。
本披露的F(ab')2可以通过用胃蛋白酶处理本披露的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
如本文所用，术语“Fab'”是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本披露的Fab'可以通过用还原剂例如二硫苏糖醇处理本披露的的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中，并将载体导入到原核生物或真核生物中，以表达Fab'来生产所述Fab'。
如本文所用，术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本披露的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)， Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本披露的scFv可以通过以下步骤来生产：获得编码本披露单克隆抗体VH和VL的cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
如本文所用，术语“双抗体”是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本披露的双抗体可以通过以下步骤来生产：获得编码本披露单克隆抗体的VH和VL的cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
如本文所用，术语“dsFv”可以通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本披露的dsFv可以通过以下步骤来生产：获得编码本披露单克隆抗体的VH和VL的cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
本披露中使用的术语“抗体框架(FR)”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
如本文所用，术语“氨基酸差异”是指多肽与其变体之间，在多肽片段上某个或某些氨基酸位点之间的差异，其中变体可以由多肽上某个或某些位点经替换、插入或缺失氨基酸获得。
如本文所用，术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定表位的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本披露所述的技术，例如X射线晶体分析法和二维核磁共振等。
本发明所用的术语“特异性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人OX40作为分析物并使用抗体作为配体，在仪器中通过表面等离子体 共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(KD)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原(或紧密相关的抗原)之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本披露中可以与术语“特异性结合的抗体”互换使用。
如本文所用，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合OX40，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如OX40抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用纯化抗原，其能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞表面的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争性抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与参考抗原结合蛋白结合的表位所充分接近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。在本披露实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争性抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或更多。
如本文所用，术语“交叉反应”是指本发明的抗体与来自不同物种的OX40结合的能力。例如，结合人OX40的本发明的抗体也可以结合另一物种的OX40。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达OX40的细胞的结合或功能性相互作用来测量。确定交叉反应性 的方法包括如本披露所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
如本文所用，术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。
如本文所用，术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
如本文所用，术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的刺激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指刺激特定的直接细胞杀伤机制。
如本文所用，“抗体依赖性细胞介导的细胞毒性”或“ADCC”指其中结合到某些细胞毒性细胞(例如NK细胞，嗜中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)上的分泌型免疫球蛋白使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞的细胞毒性形式。介导ADCC的主要细胞(NK细胞)只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch和Kinet,Annu.Rev.Immunol.9:457-92(1991)第464页表3总结了造血细胞上的FcR表达。为了评估目的分子的ADCC活性，可进行体外ADCC测定法，诸如美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。或者，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes等人PNAS(USA)95:652-656(1998)中所披露的。此外，可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合体，IgG4的F235E、或L234A/E235A突变。
如本文所用，术语“补体依赖性细胞毒性”或“CDC”指存在补体时对靶细胞的溶解。经典补体途径的激活是由补体系统第一组分(C1q)结合抗体(适宜亚类的)起始的，该抗体已结合至其关联抗原。为了评估补体激活，可进行CDC测定法，例如Gazzano-Santoro等人,J.Immunol.Methods 202:163(1996)中所记载的。具有更改的Fc区氨基酸序列(具有变异Fc区的多肽)及提高或降低的C1q结合能力的多肽变体记载于例如美国专利No.6,194,551B1和WO 1999/51642。还可参见例如Idusogie等人,J.Immunol.164:4178-4184(2000)。
本披露中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但具体地，是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
如本文所用，术语“载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区 段连接至病毒基因组中。本披露中的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人OX40或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本披露的抗体或抗原结合片段可用常规方法制备和纯化。比如，将编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
如本文所用，术语“单克隆抗体”(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
如本文所用，术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(例如细菌)、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、NS0细胞、293细胞。
如本文所用，术语“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings  Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
如本文所用，术语“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
如本文所用，术语“外源性”指根据情况在生物、细胞或人体外产生的物质。
如本文所用，术语“内源性”指根据情况在生物、细胞或人体内产生的物质。
如本文所用，术语“突变序列”是指对本披露的核苷酸序列和氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本披露的核苷酸序列和氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和氨基酸序列。所述的序列同一性可以至少为85％、90％或95％，非限制性实例包括85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
本披露中“同源性”、“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的全部位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本披露使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本披露使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989) PCR TECHNOLOGY(Stockton Press,N.Y.)。本披露使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
如本文所用，术语“任选”或“任选地”意味着随后所描述地事件或环境可以但不必然发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
如本文所用，术语“癌症”指向或描述哺乳动物中特征为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤、母细胞瘤、肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、腺癌、和肺的鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠基质癌)、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、尿道癌、肝瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肛门癌、阴茎癌、黑素瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端黑素瘤、结节性黑素瘤、多发性骨髓瘤和B细胞淋巴瘤、慢性淋巴细胞性白血病(CLL)、急性成淋巴细胞性白血病(ALL)、毛细胞性白血病、慢性成髓细胞性白血病、和移植后淋巴增殖性病症(PTLD)、以及与瘢痣病(phakomatoses)、水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖、脑瘤和脑癌、以及头颈癌、及相关转移。
在某些实施方案中，适合于通过本披露的OX40抗体或片段来治疗的癌症包括乳腺癌、结肠直肠癌、直肠癌、非小细胞肺癌、成胶质细胞瘤、非何杰金氏淋巴瘤(NHL)、肾细胞癌、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西(Kaposi)氏肉瘤、类癌癌(carcinoid carcinoma)、头颈癌、卵巢癌、间皮瘤、和多发性骨髓瘤。在一些实施方案中，癌症选自：非小细胞肺癌、成胶质细胞瘤、成神经细胞瘤、黑素瘤、乳腺癌(例如三重阴性乳腺癌)、胃癌、结肠直肠癌(CRC)、和肝细胞癌。还有，在一些实施方案中，癌症选自：非小细胞肺癌、结肠直肠癌、成胶质细胞瘤和乳腺癌(例如三重阴性乳腺癌)，包括那些癌症的转移性形式。
如本文所用，术语“细胞增殖性病症”和“增殖性病症”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，细胞增殖性病症指癌症。
如本文所用，语术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。
如本文所用，术语“癌症”，“癌性”，“细胞增殖性病症”，“增殖性病症”和“肿瘤”在本披露中提到时并不互相排斥。
如本文所用，术语“给予”、“施用”和“处理”当应用于动物、人、受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以 指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
如本文所用，术语“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病或症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病或症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
如本文所用，术语“预防癌症”是指在哺乳动物中延迟、抑制或防止癌症发作，所述哺乳动物中癌发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定其具有癌症易感性。该术语还包括治疗具有癌变前病症的哺乳动物以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
以下结合实施例用于进一步描述本披露，但这些实施例并非限制着本披露的范围。本披露实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：抗体的制备
抗人OX40单抗库通过免疫小鼠产生。实验用BalB/C和A/J品系小鼠(扬州大学比较医学中心，动物生产许可证号；SCXK(苏)2017-007)，雌性，10周龄。
免疫抗原为带Fc标签的人OX40重组蛋白(OX40-Fc：OX40 Leu 29-Ala 216(Accession#NP_003318)与Fc融合)，购自Acro Biosystems公司，货号#OX40-H5255，使用HEK293表达后按常规方法纯化。
将OX40-Fc用弗氏佐剂乳化：首次免疫用弗氏完全佐剂(sigma-aldrich，F5881-10ML)，其余加强免疫用弗氏不完全佐剂(sigma-aldrich，F5506-10ML)。抗原与佐剂比例为1:1，每次免疫注射25μg蛋白/200μl/只小鼠。详细见如下：
Figure PCTCN2021082855-appb-000001
用实施例2中的ELISA方法检测小鼠血清，确定小鼠血清的抗体滴度和阻断OX40/OX40L结合的中和活性。选择血清效价、亲和力和配体结合阻断能力强的小鼠进行一次终免疫后处死小鼠。取脾细胞和SP2/0骨髓瘤细胞(
Figure PCTCN2021082855-appb-000002
CRL-1581 TM)融合后铺板获得杂交瘤，通过实施例2的间接ELISA，捕获ELISA以及基于细胞的功能性筛选选择目标杂交瘤，并通过有限稀释法建株单克隆抗体。
把已建株的19株OX40小鼠单抗进行无血清表达生产，并用protein A亲和层析技术得到纯化的小鼠单抗。通过间接ELISA，捕获ELISA及细胞功能学活性检测筛选其中的分泌激活型抗OX40抗体的杂交瘤细胞。功能性筛选的简要步骤如下：培养GS-H2/OX40稳定细胞系(购自genscript，cat#M00608)。制备稀释的待测抗体及OX40L(Sino Biological，13127-H04H)溶液添加到处于对数生长期的GS-H2/OX40细胞中，培养后收集细胞上清，测定上清中的IL-8含量(使用human IL-8kit，cisbio，cat#62IL8PEB)。实验中用抗体GPX4(参照专利WO2015153513专利中1A7.gr.1制备)做阳性参照，hIgG(实验室制备)做阴性参照。
根据细胞功能性活性，选择活性高的10株进行基因克隆和测序。通过常规RNA抽提技术即可获得细胞总RNA，然后通过逆转录聚合酶链式反应(RT-PCR)获得单克隆抗体可变区的PCR产物。PCR产物经琼脂糖凝胶分离回收，然后克隆到基因载体，转化大肠杆菌。随机挑选数个转化菌落，PCR扩增单克隆抗体可变区，用于基因测序。获得的示例性的鼠源单克隆抗体的相应序列如下所示。
鼠单克隆抗体m4B5的重链和轻链可变区序列如下：
m4B5重链可变区：
Figure PCTCN2021082855-appb-000003
m4B5轻链可变区：
Figure PCTCN2021082855-appb-000004
鼠单克隆抗体m4B5的CDR区序列如表1：
表1.鼠单克隆抗体m4B5的CDR区序列
名称 序列 编号
HCDR1 SYGLH SEQ ID NO：3
HCDR2 VIWSGGSTDYNAAFIS SEQ ID NO：4
HCDR3 EEYDV SEQ ID NO：5
LCDR1 RASQDISNYLN SEQ ID NO：6
LCDR2 YTSRLHS SEQ ID NO：7
LCDR3 QQGNTLPWT SEQ ID NO：8
鼠单克隆抗体m2G3的重链和轻链可变区序列如下：
m2G3重链可变区：
Figure PCTCN2021082855-appb-000005
m2G3轻链可变区：
Figure PCTCN2021082855-appb-000006
鼠单克隆抗体m2G3的CDR区序列如表2：
表2.鼠单克隆抗体m2G3的CDR区序列
名称 序列 编号
HCDR1 RYSVH SEQ ID NO：11
HCDR2 MIWDGGNTDYNSALKS SEQ ID NO：12
HCDR3 NPLYFSYAMDY SEQ ID NO：13
LCDR1 RASQDISNYLN SEQ ID NO：14
LCDR2 YTSRLQS SEQ ID NO：15
LCDR3 QQVNTFPFT SEQ ID NO：16
实施例2：抗体的ELISA鉴定与筛选方法
间接ELISA法：
将20×包被缓冲液用去离子水稀释至1×，用1×包被液(碳酸盐缓冲液)配制抗原人OX40-His(Acro biosytems,OXL-H52Q8)，使其终浓度为2μg/mL，每孔加液100μL，4℃过夜或37℃孵育2h。PBST洗板1次，每孔加入200μL封闭液(含5％脱脂牛奶的PBST)，37℃孵育2h，PBST洗板4次。每孔加入稀释好的一抗100μL(待测抗体浓度从10000ng/ml起5倍系列稀释，7个梯度，即10000ng/ml，2000ng/ml，400ng/ml，80ng/ml，16ng/ml，3.2ng/ml，0.64ng/ml，空白孔为纯稀释液即2.5％脱脂牛奶的PBST)，37℃孵育40min。PBST洗板4次，用PBST缓冲液稀释酶标二抗(HRP标记山羊抗小鼠IgG，购自Jackson Immunoresearch,Cat#115036071或HRP标记山羊抗人IgG，购自Jackson Immunoresearch,Cat#109036098)，每孔加100μL，37℃孵育40min。PBST洗板4次，每孔加100μL TMB显色液，室温避光孵育3-15分钟，每孔加入50μl终止液(1M硫酸)。设置酶标仪参数，在450-630nm处读取OD值，保存实验数据。
捕获ELISA：
将20×PBS缓冲液用去离子水稀释至1×，用1×PBS配制GAM二抗(Jackson Immunoresearch,115-006-071)，使其终浓度为2μg/mL，每孔加入100μL，4℃过夜或37℃孵育2h；PBST洗板1次，每孔加入200μL封闭液(含5％脱脂牛奶的PBST)，37℃孵育2h，PBST洗板4次。每孔加入稀释好的一抗100μL(待测抗体浓度从10000ng/ml起5倍系列稀释，7个梯度，即10000ng/ml，2000ng/ml，400ng/ml，80ng/ml，16ng/ml，3.2ng/ml，0.64ng/ml，空白孔为纯稀释液即2.5％脱脂牛奶的PBST)，37℃孵育40min。PBST洗板4次，用2.5％脱脂牛奶的PBST稀释人OX40-FC-生物素(Acro biosystem，OX0-H5255，标记生物素)，每孔加100μl，37℃孵育40min，PBST洗板4次。每孔加100μL TMB显色液，室温避光孵育3-15分钟，每孔加入50μl终止液(1M硫酸)，设置酶标仪参数，在450-630nm处读取OD值，保存实验数据。
配体结合阻断ELISA：
将20×包被缓冲液用去离子水稀释至1×，用1×包被液(碳酸盐缓冲液)配置抗原OX40L-His(Acro biosytems,OXL-H52Q8)，使其终浓度为2μg/mL，每孔加入100μL，4℃过夜或37℃孵育2h；PBST洗板1次；每孔加入200μL封闭液(含5％脱脂牛奶的PBST)，37℃孵育2h；PBST洗板4次；用预先配制好的200ng/ml的人OX40-Fc溶液(配制在2.5％的脱脂牛奶中)梯度稀释小鼠血清/抗体，而后室温预孵育40min后，加到已封闭好的OX40L板子上，100μL/孔，孵育40min；PBST洗板4次；用PBST缓冲液稀释HRP标记的羊抗人二抗(GAH-HRP，Jackson Immunoresearch,109-035-006)，每孔加100μl，37℃孵育40min；PBST洗板4次；每孔加100μL TMB显色液，室温避光孵育3-15分钟；每孔加入50μl终止液(1M硫酸)，设置酶标仪参数，450-630nm处读取OD值，保存实验数据。
实施例3：抗OX40重组嵌合抗体的构建表达
将鼠源抗体m2G3和m4B5的重链可变区(VH)加上人免疫球蛋白重链恒定区，轻链可变区(VL)加上人免疫球蛋白Kappa轻链恒定区，分别克隆到真核表达载体上，通过转染细胞生产出鼠-人嵌合抗体。
重链载体设计如下：信号肽+重链可变区序列+人的IgG1恒定区序列。
轻链载体设计如下：信号肽+轻链可变区序列+人的Kappa恒定区序列。
分别将上述序列插入pCEP4载体(Thermofisher，V04450)。得到载体质粒后，提取质粒，将质粒送测序验证。将验证合格的质粒用PEI转染至人293F细胞中，连续培养，将293F细胞用无血清培养液(上海奥浦迈生物，OPM-293 CD03)培养至对数生长期用于细胞转染。将21.4μg嵌合抗体轻链质粒和23.6μg嵌合抗体重链质粒溶解在10ml
Figure PCTCN2021082855-appb-000007
I Reduced Serum Medium(GIBCO，31985-070)中混匀，然后加入200μg PEI，混匀，室温孵育15min，加入50mL细胞中。细胞培养条 件：5％CO 2，37℃，125rpm/min。培养期间，第1天和第3天加补料，直到细胞活率低于70％，收取细胞上清，离心过滤。将离心过滤后的细胞培养液上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，最终获得纯化的嵌合抗体Ch2G3和Ch4B5。
实施例4：体外结合亲和力和动力学实验
对鼠源抗体和嵌合抗体进行人OX40的亲和力进行检测(方法步骤同实施例2)，结果见图1A和图1B。其中，m2G3-NC(-NC表示negative control)、Ch2G3-NC、m4B5-NC、Ch4B5-NC为阴性对照，其分别与m2G3、Ch2G3、m4B5、Ch4B5使用的相同的恒定区，但可变区不识别OX40。
结果显示，嵌合抗体Ch2G3和Ch4B5与人OX40具有很高的亲和力。
实施例5：抗OX40抗体的体外细胞报告基因实验
用抗CD3抗体(Chempartner,A05-001)包被孔板，4度放置过夜，PBS洗3次；收获Jurkat-NF-Kb luc-hOX40细胞(ATCC,TIB-152稳定细胞系由上海睿智化学构建)和Raji细胞(ATCC,CCL-86)，重悬然后混合两种细胞。添加50μl/孔稀释的待测抗体和50μl/孔已混合的两种细胞到细胞板中，细胞板放在37度5％CO 2孵育箱中孵育5小时。添加100μl one-Glo TM luciferase reagent(Promega,Cat#E6120)到每一孔中，室温孵育3min以上。上机检测发光信号，记录RLU读值，结果见表3。
表3.抗OX40抗体的体外激活报告基因结果
抗体 EC 50(nM)
Ch2G3 0.6371
Ch4B5 0.5589
结果显示，嵌合抗体Ch2G3和Ch4B5能有效的激活报告基因。
实施例6：小鼠抗体人源化实验
为了降低可能的免疫原性，将鼠源抗体进行人源化改造。将嵌合抗体的重链可变区(VH)和轻链可变区(VL)分别在FR(framework region)区域进行定点氨基酸突变。根据不同的氨基酸突变组合，设计不同的人源化抗体重链和轻链，将编码不同轻/重链组合的质粒转染细胞可以生产出人源化抗体。简述如下：
首先设计表达载体：
重链载体设计如下：信号肽+突变的重链可变区序列+人的IgG1恒定区序列。
轻链载体设计如下：信号肽+突变的轻链可变区序列+人的Kappa恒定区序列。
分别将上述序列插入pCEP4载体(Thermofisher，V04450)。请第三方基因 合成公司按照上述设计合成表达载体，得到载体质粒后，提取质粒，将质粒送测序验证。将验证合格的质粒用PEI转染至人293F细胞中，连续培养，将293F细胞用无血清培养液(上海奥浦迈生物，OPM-293 CD03)培养至对数生长期用于细胞转染。将21.4μg编码人源化抗体轻链的质粒和23.6μl编码人源化抗体重链的质粒溶解在10ml
Figure PCTCN2021082855-appb-000008
I Reduced Serum Medium(GIBCO,31985-070)中混匀，然后加入200μg PEI，混匀，室温孵育15min，加入50mL细胞中。
细胞培养条件：5％CO 2，37℃，125rpm/min。培养期间，第1天和第3天加补料，直到细胞活率低于70％，收取细胞上清，离心过滤。将离心过滤后的细胞培养液上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH9.0中和、以及磷酸缓冲液透析，最终获得纯化的人源化抗体。
人源化可变区序列如下：
>hu2G3 VH1
Figure PCTCN2021082855-appb-000009
>hu2G3 VH2
Figure PCTCN2021082855-appb-000010
>hu2G3 VL1
Figure PCTCN2021082855-appb-000011
>hu2G3 VL2
Figure PCTCN2021082855-appb-000012
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
将设计的人源化抗体的可变区按下表组合成不同的抗体：
表4.m2G3人源化抗体及其重轻链可变区
抗体 VH VL
2G3-hu1 hu2G3 VH1 hu2G3 VL1
2G3-hu2 hu2G3 VH2 hu2G3 VL1
2G3-hu3 hu2G3 VH1 hu2G3 VL2
2G3-hu4 hu2G3 VH2 hu2G3 VL2
注：表4中2G3-hu1表示，人源化的抗体2G3-hu1的重链可变区为hu2G3 VH1，轻链可变区为hu2G3 VL1，其他类推。
m4B5的人源化可变区序列如下：
hu4B5 VL0：
Figure PCTCN2021082855-appb-000013
hu4B5 VL1：
Figure PCTCN2021082855-appb-000014
hu4B5 VL2：
Figure PCTCN2021082855-appb-000015
hu4B5 VL3：
Figure PCTCN2021082855-appb-000016
hu4B5 VL4：
Figure PCTCN2021082855-appb-000017
hu4B5 VH0：
Figure PCTCN2021082855-appb-000018
hu4B5 VH1：
Figure PCTCN2021082855-appb-000019
hu4B5 VH2：
Figure PCTCN2021082855-appb-000020
hu4B5 VH3：
Figure PCTCN2021082855-appb-000021
hu4B5 VH4：
Figure PCTCN2021082855-appb-000022
将设计的人源化抗体的可变区按下表组合成不同的抗体：
表5.m4B5人源化抗体及其重轻链可变区的对应关系
Figure PCTCN2021082855-appb-000023
注：Hu4B5 V1表示，人源化的抗体hu4B5 V1的重链可变区为hu4B5 VH0，轻链可变区为hu4B5 VL0，其他类推。
为了获得更好的m2G3的人源化改造抗体，对hu2G3 VH1的氨基酸序列进行突变，突变后的重链可变区序列如下：
hu2G3 VH1.1
Figure PCTCN2021082855-appb-000024
>hu2G3 VH1.2
Figure PCTCN2021082855-appb-000025
突变后获得的HCDR2序列如下：
表6.突变后获得的HCDR2序列
名称 序列 编号
HCDR2 V1 MIWDGGNTDYNAALKS SEQ ID NO：33
HCDR2 V2 MIWDGGNTDYAAPVKG SEQ ID NO：34
将重链可变区hu2G3 VH1.1、hu2G3 VH1.2与轻链可变区hu2G3 VL组合形成新的优化的人源化抗体，见表7。
表7.突变后，获得的新的人源化hu2G3抗体的轻/重链可变区
抗体 VH VL
2G3 hu2G3 VH1.1 hu2G3 VL1
2G3-hu5 hu2G3 VH1.1 hu2G3 VL2
2G3-hu6 hu2G3 VH1.2 hu2G3 VL1
2G3-hu7 hu2G3 VH1.2 hu2G3 VL2
将上述人源化抗体进行亲和力测评(参照实施例2中的捕获ELISA)，实验结果表明人源化后的分子可同OX40结合。
示例性m2G3及其人源化抗体亲和力测评(捕获ELISA)结果如表8所示：
表8.抗体亲和力
Figure PCTCN2021082855-appb-000026
示例性的m4B5及其改造抗体的捕获ELISA检测结果如表9所示：
表9.抗体亲和力
Figure PCTCN2021082855-appb-000027
将上述序列中的轻链可变区与轻链恒定区序列组合形成最终的轻链序列，将上述序列中的重链可变区与重链恒定区组合形成最终的重链序列。具体轻、重链恒定区并非作为本披露抗体恒定区的限制，也可选用本领域其它已知的轻、重链恒定区及其突变体来增加抗体的性能。
示例性的恒定区如下所示：
IgG1重链恒定区：
Figure PCTCN2021082855-appb-000028
Figure PCTCN2021082855-appb-000029
kappa轻链恒定区：
Figure PCTCN2021082855-appb-000030
示例性的2G3、hu4B5-V7(又称4B5)抗体全长氨基酸序列如下：
2G3重链：
Figure PCTCN2021082855-appb-000031
2G3轻链：
Figure PCTCN2021082855-appb-000032
4B5重链：
Figure PCTCN2021082855-appb-000033
4B5轻链：
Figure PCTCN2021082855-appb-000034
阳性参照GPX4的制备参照专利申请WO2015153513中1A7.gr.1制备，其重轻链氨基酸序列如下所示：
GPX4重链：
Figure PCTCN2021082855-appb-000035
GPX4轻链：
Figure PCTCN2021082855-appb-000036
实施例7：人源化抗体体外结合亲和力和动力学试验
Biacore方法是公认的客观检测蛋白相互间亲和力和动力学的检测方法。我们通过Biacore T200(GE)分析本发明待测OX40抗体表征亲和力及结合动力学。
利用由Biacore提供的试剂盒，采用NHS标准氨基偶联法将本发明待测重组抗OX40抗体共价连接至CM5(GE)芯片上。然后将稀释于同样缓冲液中的一系列浓度梯度的人OX40-His蛋白(义翘神州#10481-H08H)于前后各个循环进样，流速30μL/min，进样后均以试剂盒内配再生试剂再生。追踪抗原-抗体结合动力学3分钟并追踪解离动力学10分钟。使用GE的BIAevaluation软件以1:1(Langmuir)结合模型分析所得数据，以此法测定的嵌合抗体ka(k on)、kd(k off)和K D值显示于下表。
表10.人源化抗体的体外结合亲和力结果
待测抗体 ka(1/Ms) kd(1/s) K D(M)
2G3 4.371E+05 2.023E-03 4.628E-09
4B5 1.314E+05 3.694E-04 2.812E-09
结果显示，2G3和4B5可有效结合OX40。
实施例8：抗体阻断OX40与OX40L结合ELISA试验
用配体OX40配体(Acrobiosystem，OXL-H52Q8)包被孔板，封闭后，加入 梯度稀释待测抗体(抗体稀释在含人Bio-OX40-FC(Acrobiosystem，OX40-H5255，标记生物素)的溶液中预孵育40min后加板)，孵育40min后，洗板。再加入SA-HRP(Jackson Immunoresearch,016-030-084)，孵育40min后，加入显色液和终止液，测OD值，结果见下表。
表11.抗体阻断OX40与OX40L结合实验结果
抗体 2G3 4B5
IC 50(nM) 1.88 0.725
结果显示，2G3和4B5均可阻断OX40同OX40L的结合。
实施例9:人源化抗体活性数据
用抗CD3抗体(Chempartner,A05-001)包被孔板，4℃放置过夜，PBS洗3次；收获Jurkat-NF-Kb luc-hOX40细胞(ATCC,TIB-152(稳定细胞系由睿智化学公司构建)和Raji细胞(ATCC,CCL-86)，重悬然后混合两种细胞。添加50μl/孔稀释的待测抗体，和50μl/孔已混合的两种细胞到孔板，细胞板放在37℃ 5％CO 2孵育箱中孵育5小时。添加one-Glo TM荧光素酶试剂(Promega,Cat#E6120)到每一孔中，室温孵育3min以上。上机检测发光信号，记录RLU读值。结果见表12，结果表明2G3和4B5可有效激活报告基因。
表12.人源化抗体激活报告基因实验结果
抗体 2G3 4B5
EC 50(nM) 2.294 0.3411
实施例10：体外细胞功能试验
分离CD4+记忆T细胞，同待测抗体加入到抗CD3抗体(Chempartner,A05-001)包被的96孔板中，37℃共孵育72h，取上清检测IFN-γ，结果如图2和表13所示。结果显示，GPX4，4B5及2G3可显著增强IFN-γ的释放，其中2G3在10ng/mL即可达到最大刺激效果。
表13.不同抗体对应的IFNγ(pg/mL)数值
Figure PCTCN2021082855-appb-000037
注：数值计算方法是：均值+/-SEM(平均值+/-平均值标准误差)，N＝3(三次重复)。
实施例11:抗OX40抗体对肿瘤细胞生长的抑制
B-hTNFRSF4(OX40)人源化小鼠(B-hTNFRSF4(OX40)人源化小鼠，百奥赛图江苏基因生物技术有限公司)，雌性，17至20g，6至7周。
收集对数生长期MC38肿瘤细胞7(购买于南京银河生物医药有限公司)，用PBS缓冲液调整细胞浓度为5×10 6/mL，接种0.1mL细胞悬液至OX40小鼠胁腹部。观察接种后小鼠并监测肿瘤的生长，在接种后第7天时荷瘤小鼠胁腹部平均肿瘤体积达到102.5mm 3，按照肿瘤体积大小进行分组和给药观察，分组信息见表14。
表14.小鼠分组及给药剂量表
给药组 给药剂量(mg/kg) 给药途径 给药频率
对照(IgG1) 3 i.p. Q3D×6
GPX4 3 i.p. Q3D×6
2G3 0.3 i.p. Q3D×6
2G3 1 i.p. Q3D×6
2G3 3 i.p. Q3D×6
检测OX40人源化抗体对小鼠MC38结肠癌细胞移植瘤的生长抑制作用。
肿瘤体积和荷瘤鼠体重测量：使用游标卡尺每周两次测量，肿瘤体积计算公式为V＝0.5a×b 2，a，b分别代表肿瘤的长径和宽径；肿瘤生长移植瘤TGI(％)＝[1-T/C]×100。所有荷瘤鼠体重每周测量两次。
给药治疗后第20天，IgG1对照组的平均肿瘤体积达到了1732.593mm 3，受试化合物2G3低剂量给药组(0.3mg/kg)小鼠平均肿瘤体积达到了930.37mm 3，中剂量给药组(2G3，1mg/kg)和高剂量给药组(2G，3mg/kg)组内荷瘤鼠平均肿瘤体积分别为303.49mm 3和155.79mm 3，中高剂量组抑制肿瘤生长与对照组差异明显(**P<0.01)，并表现出初步的剂量依赖关系，其肿瘤生长抑制率分别达到了49％、88％和97.0％。3mg/kg GPX4组中平均肿瘤体积分别为362.47mm 3，与对照组比较差异显著，也表现有明显的抑制肿瘤生长的作用(*P<0.05)，其肿瘤生长抑制率分别达到了84％。
给药后第20天结束实验，结果见表15和图3A和图3B。所有治疗小鼠被安乐死处理，剥离荷瘤鼠皮下移植瘤块并进行称量。其中对照组平均肿瘤块重量为1.568g,受试化合物2G3在低剂量组(0.3mg/kg)、中剂量组(1mg/kg)和高剂量组(3mg/kg)，平均肿瘤重量分别为0.926g、0.251g和0.181g，其中中高剂量组与对照组比较差异显著，抑制肿瘤生长作用明显(**P<0.01)。在同一时间，给药组GPX4 3mg/kg中，平均肿瘤重量为0.372g，与对照组比较差异明显，也表现出明显的抑制MC38肿瘤细胞生长的作用(**P<0.01)。
在实验过程中，所有治疗荷瘤小鼠体重变化无明显异常，同时在药物治疗过程中未见明显异常行为和其他表现。
表15.小鼠体内抑瘤效果
Figure PCTCN2021082855-appb-000038
Figure PCTCN2021082855-appb-000039
以下通过试验配制抗OX40抗体稳定制剂(示例性的，以下制剂中的抗OX40抗体为前述的2G3)
抗体制剂的检测中所使用的方法和设备和具体如下：
SEC分子排阻色谱法：根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。SEC单体含量百分比＝A单体/A总*100％(“A单体”为样品中主峰单体的峰面积，“A总”为所有峰面积之和)。SEC测定用仪器：安捷伦1260；柱子：waters，XBrige
Figure PCTCN2021082855-appb-000040
SEC(300×7.8mm3.5μm)。
CE毛细管凝胶电泳：将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。非还原CE(NR-CE)纯度百分比＝A主峰/A总*100％(“A主峰”为样品中主峰的峰面积，“A总”为所有峰面积之和)。CE测定用仪器：Beckman，型号plus800。
iCIEF成像毛细管等点聚焦电泳(简称iCE)：根据蛋白质等电点pI不同进行分离的技术。iCIEF中性峰含量百分比＝中性峰面积/总面积*100％(总面积为酸性峰、中性峰和碱性峰面积之和)。iCIEF测定所用仪器：simple protein，型号muarice。
渗透压：冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM815。
实施例12:抗OX40抗体制剂缓冲体系筛选
在pH5.0-7.5一系列缓冲液中，配制含0.1mg/ml聚山梨酯80(PS80)，蛋白浓度为50mg/mL的2G3制剂，其中。本实验中缓冲体系分别为pH5.0、pH 5.5的10mM醋酸钠(简写AA)，pH5.0、pH 5.5、pH 6.0的10mM琥珀酸钠盐(简写SA)、pH5.5、pH6.0、pH6.5的10mM枸橼酸钠盐(简写CA)、pH5.5、pH6.0、pH6.5的10mM组氨酸盐酸盐(简写His)、pH6.0、pH6.5、pH7.0、pH7.5的10mM磷酸钠(简写PB)。将每种制剂过滤，灌装，加塞，轧盖。将样品进行40℃高温条件下放置14天或进行振摇(25℃，300rpm)强制降解实验，并以外观、SEC、IEC、非还原CE-SDS为评价指标，考察制剂稳定性。
实验结果见表16，其中，外观数据显示，在40℃高温或振摇的强制降解条件下，AA、His缓冲体系的蛋白制剂外观较好；SEC数据显示，40℃高温条件下AA、His、CA缓冲体系的蛋白制剂稳定性较好；CE-SDS数据显示，40℃高温或振摇条件下pH5.0或pH5.5的AA、pH 5.0的SA、pH6.0的His等各组蛋白制剂稳定性相对较好；IEC数据显示，40℃高温条件下pH 5.5的AA、pH6.0/pH 6.5的His、 pH6.0的SA等组的蛋白制剂略优。综合考虑，2G3蛋白制剂优选组氨酸盐或醋酸盐缓冲体系，可选pH 6.0的His或pH 5.5的AA，可选pH 6.0的His。
表16.pH和缓冲体系筛选实验结果
Figure PCTCN2021082855-appb-000041
Figure PCTCN2021082855-appb-000042
备注：表中“D”表示天，例如D14表示14天；T0表示实验开始时；“+”表示乳光及颗粒程度，“+”越多代表乳光越重或颗粒越多，反之亦然；N/A表示未检测。
实施例13:抗OX40抗体制剂缓冲体系pH值筛选
在不同pH的10mM组氨酸盐酸盐(简写His)缓冲液中，制备蛋白浓度为50mg/mL，含0.1mg/ml PS80的2G3制剂。具体如下：
1)10mM His(pH5.5)
2)10mM His(pH6.0)
3)10mM His(pH6.5)
将每种制剂过滤，灌装，加塞，轧盖。将样品进行40℃高温条件下放置1个月强制降解实验，以SEC、IEC、非还原CE-SDS为评价指标，考察制剂稳定性。实验结果见表17。实验结果显示，40℃高温放置1个月，pH5.5、pH6.0、pH6.5的His缓冲液的各组蛋白制剂，SEC/CE/IEC组间无显著差异，pH 5.5的His组的IEC略低。综合考虑，制剂较优缓冲液pH范围为5.5-6.5，选pH为6.0进行进一步实验。
表17.制剂缓冲体系pH值筛选结果
Figure PCTCN2021082855-appb-000043
Figure PCTCN2021082855-appb-000044
备注：T0表示实验开始时，“M”表示月，例如M1表示一个月。
实施例14:抗OX40抗体制剂中其它辅料的筛选
在含下列1)-7)不同种类辅料及pH6.0的10mM组氨酸盐酸盐(简写His)缓冲液中，制备蛋白浓度为50mg/mL的2G3制剂。具体辅料如下：
1)50mg/mL蔗糖，0.1mg/mL聚山梨酯20(PS20)；
2)50mg/mL蔗糖，0.4mg/mL PS20；
3)50mg/mL蔗糖，0.1mg/mL聚山梨酯80(PS80)；
4)50mg/mL蔗糖，0.4mg/mL PS80；
5)50mg/mL海藻糖，0.4mg/mL PS80；
6)90mg/mL蔗糖，0.4mg/mL PS80；
7)90mg/mL海藻糖，0.4mg/mL PS80。
将每种制剂过滤，灌装，加塞，轧盖。将样品进行40℃高温条件下放置13天或进行振摇(25℃，300rpm，13天)强制降解实验，以外观、SEC、IEC、非还原CE-SDS为评价指标，考察制剂稳定性。实验结果见表18。
表18.制剂中表面活性剂和糖的筛选实验结果
Figure PCTCN2021082855-appb-000045
Figure PCTCN2021082855-appb-000046
备注：表中“D”表示天，例如D13表示13天；T0表示实验开始时。
实验结果显示，对于表面活性剂种类及浓度，各组间CE、IEC数据无显著差异，0.1mg/ml PS80组SEC略低，0.1mg/ml PS20组和0.1mg/ml PS80组强制降解后外观出现乳光或颗粒，0.4mg/ml PS80组在各条件下外观均良好，另外，将强制降解样品置于4℃一段时间后复看外观，0.4mg/ml PS20组振摇和40℃的样品均出现颗粒；因此，可选0.4mg/ml PS80；对于糖种类及浓度：各条件下各组间无显著性差异，50mg/mL-90mg/mL组蔗糖和海藻糖稳定性均较好。另外，当蔗糖浓度为80mg/ml，样品渗透压为304mosm，与人体内渗透压接近，以下选择蔗糖浓度75-80mg/ml进行以下的实验。
实施例15:抗OX40抗体制剂稳定性实验
制备含蛋白浓度50mg/ml，10mM组氨酸盐酸盐(His)pH6.0，75mg/ml蔗糖，0.4mg/ml PS80的2G3制剂。
将样品分别置于25℃及4℃条件下长期放置，以SEC、IEC、非还原CE-SDS为评价指标，考察制剂稳定性，结果见表19。
表19.制剂稳定性实验结果
Figure PCTCN2021082855-appb-000047
备注，M表示月，如M3表示3个月；T0表示实验开始时。
实验结果显示：在25℃条件下，2G3制剂IEC略有下降，但下降幅度均在可接受范围，其他检测项均无显著变化；在4度条件下，各检测项均无显著变化。该处方制剂稳定性较好。
实施例16:抗OX40抗体制剂处方优化实验
以10mM组氨酸盐酸盐(His)缓冲液pH值、蛋白浓度和聚山梨醇酯80浓度为变量进行DOE实验设计，DOE实验因子及水平设为pH5.5～6.5、PS80浓度0.2～0.6mg/mL、蛋白浓度30～70mg/mL，设计一系列处方(见表20)，通过40℃高温条件下放置13天的强制降解实验，以外观、SEC、IEC为评价指标，采用最小二乘法对结果进行统计分析，结果见表21和附图4。
表20.DOE处方筛选实验处方设计
Figure PCTCN2021082855-appb-000048
注：为增加制剂稳定性所有处方均加入80mg/ml蔗糖。
表21.DOE筛选实验结果
Figure PCTCN2021082855-appb-000049
Figure PCTCN2021082855-appb-000050
备注，D表示天，如D13表示13天；D0表示实验开始时，N/A表示未检测。
将各强制降解数据进行拟合，其中pH漂移值(制剂pH与缓冲液差值)及SEC、IEC下降值拟合较好，模型有效，拟合结果见图4。
pH漂移拟合结果显示，pH偏移与蛋白浓度相关，蛋白浓度高于50mg/ml时pH偏移0.2，蛋白浓度30mg/ml时偏移0.1。40℃高温条件下放置13天，制剂SEC、IEC下降幅度均在可接受范围内，处方稳定性较好，因此制剂蛋白浓度30-70mg/ml，0.2-0.6mg/ml PS80，pH5.5-6.5。另外，40℃高温条件下放置13天，SEC及IEC略有差异，pH越低SEC越好，pH 5.9-6.2的IEC最优，综合上述数据，以下选蛋白浓度50mg/ml，0.4mg/ml PS80，10mM His pH 6.0的组方进行进一步实验。
实施例17:抗OX40抗体制剂稳定性检测
制备蛋白浓度50mg/ml，10mM组氨酸盐酸盐(简称His)pH6.0，80mg/ml蔗糖，0.4mg/ml PS80的2G3制剂。
将样品置于40℃高温条件下放置或进行振摇(25℃，300rpm)强制降解实验，以外观、SEC、IEC、非还原CE-SDS为评价指标，考察制剂稳定性，实验结果见表22-1。
表22-1.蛋白制剂稳定性结果
Figure PCTCN2021082855-appb-000051
Figure PCTCN2021082855-appb-000052
备注，D表示天，如D13表示13天；T0表示实验开始时；M表示月，如M1表示1个月。
实验结果显示：蛋白制剂在冻融、40℃高温或振摇(25℃，300rpm)条件下，SEC/CE/IEC变化较小，制剂稳定性良好。
另外，将样品置于4℃条件下放置1年，以外观、SEC、IEC、非还原CE-SDS为评价指标，考察制剂稳定性，实验结果见表22-2，实验结果显示，该溶液制剂在4℃条件下放置1年，制剂的外观、SEC、IEC、非还原CE-SDS与开始时相比无显著差异，制剂溶液状态下稳定性很好。
表22-2.蛋白制剂稳定性结果
Figure PCTCN2021082855-appb-000053
备注：M12表示12个月
实施例18:抗OX40抗体制剂冻干与复溶实验
一、抗OX40抗体制剂冻干制剂
制备蛋白浓度50mg/ml，10mM His pH6.0，80mg/ml蔗糖，0.4mg/ml PS80的2G3的冻干制剂，冻干制备过程：按上述处方制备样品，将样品无菌过滤，灌装于西林瓶中，半加塞，按下表程序进行预冻、一次干燥和二次干燥，冻干程序结束后，真空加塞。
表23.蛋白制剂冻干步骤
Figure PCTCN2021082855-appb-000054
冻干后出箱，样品外观：白色粉饼，外观饱满，无塌陷。水分<1.0％。因此该冻干程序良好。
二、抗OX40抗体制剂冻干制剂的复溶
将冻干制剂用注射用水复溶获得蛋白浓度为50mg/ml，10mM His pH6.0，80mg/ml蔗糖，0.4mg/ml PS80的2G3的复溶溶液，复溶后样品分别在室温及2～ 8℃放置24小时，以外观、SEC、非还原CE-SDS、IEC为评价指标，考察制剂质量变化。实验结果见表24，结果表明，在室温或2～8℃条件放置24h，复溶溶液稳定性较好。
表24.复溶溶液稳定性实验结果
Figure PCTCN2021082855-appb-000055

Claims (20)

  1. 一种药物组合物，其包含抗OX40抗体或其抗原结合片段，以及缓冲液，所述缓冲液为组氨酸盐缓冲液或醋酸盐缓冲液，所述缓冲液的pH为约5.0至约6.5，优选pH为约5.5至约6.5，更优选pH为约6.0；所述组氨酸盐缓冲液优选为组氨酸盐酸盐缓冲液，所述醋酸盐缓冲液优选为醋酸钠缓冲液。
  2. 根据权利要求1所述的药物组合物，其中所述缓冲液浓度为大约5mM至大约30mM，优选为大约5mM至大约15mM，更优选为大约10mM。
  3. 根据权利要求1或2所述的药物组合物，其中所述抗OX40抗体或其抗原结合片段浓度为大约1mg/ml至大约100mg/ml，优选为大约30mg/ml至大约70mg/ml，更优选为大约50mg/ml。
  4. 根据权利要求1至3中任一项所述的药物组合物，其还包括糖，所述糖优选为蔗糖或海藻糖。
  5. 根据权利要求4所述的药物组合物，其中所述糖浓度为大约50mg/ml至大约90mg/ml，优选为大约75mg/ml至大约85mg/ml，更优选为大约80mg/ml。
  6. 根据权利要求1至5中任一项所述的药物组合物，其还包括表面活性剂，所述表面活性剂优选为聚山梨醇酯，更优选为聚山梨醇酯80。
  7. 根据权利要求6所述的药物组合物，其中表面活性剂的浓度为大约0.1mg/ml至大约1.0mg/ml，优选为大约0.2mg/ml至大约0.6mg/ml，更优选为大约0.4mg/ml。
  8. 根据权利要求1至7中任一项所述的药物组合物，其包含：
    a)蛋白浓度大约1mg/ml至大约100mg/ml的抗OX40抗体或其抗原结合片段，
    b)pH为约5.0至约6.5的组氨酸盐缓冲液或pH为约5.0至约5.5的醋酸盐缓冲液，
    c)浓度为大约50mg/ml至大约90mg/ml的糖，和
    d)浓度为大约0.1mg/ml至大约1.0mg/ml的聚山梨醇酯80；
    优选的，所述的药物组合物包含：
    a1)浓度为大约30mg/ml至大约70mg/ml的抗OX40抗体或其抗原结合片段，
    b1)pH为约5.5至约6.5的组氨酸盐缓冲液，
    c1)浓度为大约75mg/ml至大约85mg/ml的蔗糖，和
    d1)浓度为大约0.2mg/ml至大约0.6mg/ml的聚山梨醇酯80。
  9. 一种药物组合物，其包含：约10mM的pH为约6.0的组氨酸盐酸盐缓冲液，浓度约50mg/ml的抗OX40抗体或其抗原结合片段，浓度约80mg/ml蔗糖，以及浓度约0.4mg/ml聚山梨醇酯80。
  10. 根据权利要求1至9中任一项所述的药物组合物，所述抗OX40抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (A)所述重链可变区包含分别如SEQ ID NO：11、33、13所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；
    (B)所述重链可变区包含分别如SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；
    (C)所述重链可变区包含分别如SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3；或
    (D)所述重链可变区包含分别如SEQ ID NO：11、34、13所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3。
  11. 根据权利要求10所述的药物组合物，其中所述抗OX40抗体或其抗原结合片段包含：
    (E)如SEQ ID NO：17、18、31或32所示或与SEQ ID NO：17、18、31或32具有至少95％序列同一性的重链可变区，和如SEQ ID NO：19所示或与SEQ ID NO：19具有至少95％序列同一性的轻链可变区；
    (F)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少95％序列同一性的重链可变区，和如SEQ ID NO：10所示或与SEQ ID NO：10具有至少95％序列同一性的轻链可变区；
    (G)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少95％序列同一性的重链可变区，和如SEQ ID NO：2所示或与SEQ ID NO：2具有至少95％序列同一性的轻链可变区；
    (H)如SEQ ID NO：17、18、31或32所示或与SEQ ID NO：17、18、31或32具有至少95％序列同一性的重链可变区，和如SEQ ID NO：20所示或与SEQ ID NO：20具有至少95％序列同一性的轻链可变区；或
    (I)如SEQ ID NO：26、27、28、29或30所示或与SEQ ID NO：26、27、28、29或30具有至少95％序列同一性的重链可变区，和如SEQ ID NO：21、22、23、24或25所示或与SEQ ID NO：21、22、23、24或25具有至少95％序列同一性的轻链可变区；
    优选地，所述抗OX40抗体或其抗原结合片段包含：如SEQ ID NO：31所示的重链可变区和如SEQ ID NO：19所示的轻链可变区。
  12. 根据权利要求11所述的药物组合物，其中所述的抗OX40抗体包含重链恒定区和/或轻链恒定区；优选地，所述重链恒定区源自人IgG1、IgG2、IgG3或IgG4或其突变序列；轻链恒定区源自人κ、λ链或其突变序列；更优选地，所述重链恒定区的氨基酸序列如SEQ ID NO：35所示或与SEQ ID NO：35具有至少90％的序列同一性，所述轻链恒定区的氨基酸序列如SEQ ID NO：36所示或与SEQ ID NO：36具有至少90％的序列同一性。
  13. 根据权利要求12所述的药物组合物，其中所述的抗OX40抗体或其抗原结合片段包含：
    (J)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少85％序列同一性的重链，和/或如SEQ ID NO：38所示或与SEQ ID NO：38具有至少85％序列同一性的轻链；或
    (K)如SEQ ID NO：39所示或与SEQ ID NO：39具有至少85％序列同一性的重链，和/或如SEQ ID NO：40所示或与SEQ ID NO：40具有至少85％序列同一性的轻链；
    优选地，所述抗OX40抗体或其抗原结合片段包含如SEQ ID NO：37所示的重链和如SEQ ID NO：38所示的轻链。
  14. 制备如权利要求1至13中任一项所述的药物组合物的方法，所述方法包括将抗OX40抗体或其抗原结合片段原液经缓冲液置换的步骤。
  15. 一种含抗OX40抗体或其抗原结合片段的冻干制剂，所述冻干制剂通过将权利要求1至13中任一项所述的药物组合物经冷冻干燥获得。
  16. 一种含抗OX40抗体或其抗原结合片段的复溶溶液，所述复溶溶液通过将权利要求15所述的冻干制剂经复溶制备获得。
  17. 一种含抗OX40抗体或其抗原结合片段的冻干制剂，所述冻干制剂经复溶可形成如权利要求1至13中任一项所述的药物组合物：
    优选地，所述冻干制剂包含抗OX40抗体或其抗原结合片段、糖、表面活性 剂和组氨酸盐或醋酸盐缓冲液，其中，所述抗OX40抗体或其抗原结合片段、糖和表面活性剂的重量份比为：1重量份至100重量份抗OX40抗体或其抗原结合片段：50重量份至90重量份的糖：0.1重量份至1.0重量份的表面活性剂，所述抗OX40抗体或其抗原结合片段与缓冲液的物质的量比为：0.007摩尔份至0.685摩尔份的抗OX40抗体或其抗原结合片段：5摩尔份至30摩尔份的组氨酸盐或醋酸盐缓冲液。
  18. 一种制品，其包括容器，该容器中装有如权利要求1至13中任一项所述的药物组合物或如权利要求15或17所述的冻干制剂或如权利要求16所述的复溶溶液。
  19. 一种治疗或预防疾病或病症的方法，所述方法包括向受试者施用治疗有效量或预防有效量的如权利要求1至13中任一项所述的药物组合物，或如权利要求15或17所述的冻干制剂，或如权利要求16所述的复溶溶液，或如权利要求18所述的制品；优选地，所述疾病或病症为癌症；更优选地，所述癌症选自：肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌和血液系统癌症。
  20. 一种增强人受试者中的免疫应答的方法，所述方法包括：
    向受试者施用治疗有效量或预防有效量的如权利要求1至13中任一项所述的药物组合物，或如权利要求15或17所述的冻干制剂，或如权利要求16所述的复溶溶液，或如权利要求18所述的制品；
    优选地，所述增强的免疫应答包括T效应细胞的免疫刺激/效应功能的增加，和/或T调节细胞的免疫抑制功能的下调。
PCT/CN2021/082855 2020-03-25 2021-03-25 一种抗ox40抗体药物组合物及其用途 WO2021190582A1 (zh)

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