TW202144005A - An anti-ox40 antibody pharmaceutical composition and use thereof - Google Patents

Abstract

The disclosure relates to an anti-OX40 antibody pharmaceutical composition and use thereof. Specifically, the disclosure relates to a pharmaceutical composition comprises an anti-OX40 antibody or antigen-binding fragment thereof and a buffer. Further, the pharmaceutical composition also comprises a sugar and a surfactant. The pharmaceutical composition of the disclosure is used to treat cancer.

Description

A kind of anti-OX40 antibody pharmaceutical composition and use thereof

The present disclosure belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising an anti-OX40 antibody and its use as a medicine.

The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

Tumor immunotherapy is a continuous hot spot in the field of tumor therapy. Recent studies have demonstrated that enhancing anti-tumor T cell function can be used to fight cancer. There is substantial evidence that tumor cells "evade" the immune system by inducing active immune tolerance mediated primarily by regulatory T lymphocytes (Treg; Quezda et al. Immunol Rev 2011;241:104-118). Therefore, the balance between effector T lymphocytes (Teff) and tolerogenic Tregs is crucial for effective antitumor immunotherapy. Therefore, an effective anti-tumor immune response can be obtained by enhancing the effector function of tumor-specific Teff and/or by attenuating the suppressive function of tumor-specific Treg. The CD134 (OX40) receptor has been shown to be a key receptor mediating 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, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a glycoprotein with a molecular weight of about 50 kDa expressed on the cell surface. The extracellular ligand-binding domain of OX40 consists of four cysteine-rich domains (CRDs). The natural ligand of OX40 is OX40L (CD252), and the two form the 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. OX40-expressing T lymphocytes have been shown to be present in the draining lymph nodes of various human malignancies and cancer patients. In a mouse model of severe combined immunodeficiency (SCID), OX40 interacts with the OX40L-binding domain to enhance antitumor immunity, resulting in tumor growth in various human malignancy cell lines, such as lymphoma, prostate, colon, and breast cancer 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 disclosed, for example, WO2013038191, WO2015153513, WO2016179517, WO2017096182, CN110078825A, WO2016196228 and the like.

The present disclosure provides an anti-OX40 antibody pharmaceutical composition and uses thereof.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof, and a buffer, wherein the buffer is a histamine buffer or an acetate buffer, and the buffer has a pH of about 5.0 to about 6.5, preferably pH is about 5.5 to about 6.5, more preferably pH is about 6.0; the histamine salt buffer is preferably histamine hydrochloride buffer, the acetate buffer is preferably sodium acetate buffer solution (referred to as sodium acetate buffer).

In some embodiments, 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 pH of the buffer is about 6.0, Preferably it is 6.0; in other embodiments, the buffer is histamine salt buffer at a pH of about 5.5 to about 6.5; in other embodiments, the buffer is histamine salt at pH 6.0 In other embodiments, the buffer is an acetate buffer 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 solution; in other embodiments, the buffer is sodium acetate buffer at a pH of about 5.5. In other embodiments, non-limiting examples of buffer pH values 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, about 6.0 , about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, and any range between these point values. In some embodiments, the pH value of the aforementioned pharmaceutical composition is the same or nearly the same as the pH value of its buffer (as is known to those with ordinary knowledge in the art, in the process of preparing pharmaceutical formulations, there is sometimes a pH drift (formulations). There is some difference between pH and buffer pH, which is called pH drift value), and the pH drift value is usually within the range of ±0.3. Unless otherwise specified, the pH drift value of the pharmaceutical composition of the present disclosure is within the range of ±0.3, preferably within ±0.2, more preferably within ±0.1).

In some embodiments, the buffer concentration in the aforementioned pharmaceutical composition is about 5mM to about 30mM, preferably 5mM to 30mM; in some embodiments, the buffer concentration is about 5mM to about 15mM, preferably 5mM to 15 mM; in some embodiments, the buffer concentration is about 10 mM, preferably 10 mM. Non-limiting examples of buffer concentrations include about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 18 mM, about 20 mM , about 25 mM, about 30 mM, and any range between these point values.

In some embodiments, the concentration of the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition is 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 is at a concentration of about 30 mg/ml to about 70 mg/ml, preferably 30 mg/ml to 70 mg/ml; The OX40 antibody or antigen-binding fragment thereof is at a concentration of about 50 mg/ml, preferably 50 mg/ml. Non-limiting examples of concentrations 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, About 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml and any range between these point values.

In some embodiments, the aforementioned pharmaceutical compositions further include sugars, in some embodiments, the sugars include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. In some embodiments, the sugar is selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol , mannitol, milibiose, melanitol, raffinose, mannose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol and iso-maltulose. In some embodiments, the sugar is a non-reducing disaccharide; in some embodiments, the sugar is preferably trehalose or sucrose, most preferably sucrose.

In some embodiments, 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 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 points in between any range of .

In some embodiments, the aforementioned pharmaceutical composition further includes a surfactant, which can be selected from polysorbate 20 (also known as polysorbate 20, Tween 20, PS20), polysorbate 80 (also known as polysorbate 20) 80, Tween 80, PS80), polyhydroxyalkene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfo Betaine, Linoleyl-Sulfobetaine, Stearyl-Sulfobetaine, Lauryl-sarcosine, Myristyl-sarcosine, Linoleyl-sarcosine Amino acid, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroaminopropyl-betaine, cocalamidopropyl- Betaine, Linoleamidopropyl-Betaine, Myristamidopropyl-Betaine, Palmamidopropyl-Betaine, Isostearamidopropyl-Betaine, Myristate Aminopropyl-dimethylamine, palmamidopropyl-dimethylamine, isostearylaminopropyl-dimethylamine, sodium methyl cocoamide, methyl oleyl taurine Sodium, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, among others. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In some embodiments, 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 point values.

In some embodiments, the aforementioned pharmaceutical composition comprising: 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 at a pH of about 5.0 to about 6.5 Salt buffer or acetate buffer, c) sugar at a concentration of about 50 mg/ml to about 90 mg/ml, and d) polysorbate 80 at a concentration of about 0.1 mg/ml to about 1.0 mg/ml.

In some embodiments, 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 histamine salt at a pH of about 5.5 to about 6.5 Buffer, c1) sucrose at a concentration of about 75 mg/ml to about 85 mg/ml, and d1) polysorbate 80 at a concentration of about 0.2 mg/ml to about 0.6 mg/ml.

In some embodiments, the aforementioned pharmaceutical composition comprises: about 10 mM histamine hydrochloride buffer at a pH of about 6.0, and an anti-OX40 antibody or antigen-binding fragment thereof at a concentration of about 50 mg/ml at a concentration of about 80 mg/ml Sucrose, and polysorbate 80 at a concentration of about 0.4 mg/ml.

In some embodiments, the aforementioned pharmaceutical composition comprises: 10 mM histamine hydrochloride buffer with a pH of 6.0, an anti-OX40 antibody or an antigen-binding fragment thereof at a concentration of 50 mg/ml, at a concentration of 80 mg/ml sucrose, and polysorbate 80 at a concentration of 0.4 mg/ml.

In some embodiments, 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:

(A) The heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 3, 4, 5, respectively, and the light chain variable region comprises HCDR1, SEQ ID NOs: 6, 7, 8, respectively LCDR1, LCDR2, LCDR3 shown;

(B) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 11, 12, 13, respectively, and the light chain variable region comprises as SEQ ID NOs: 14, 15, 16, respectively The light chain LCDR1, LCDR2, LCDR3 shown;

(C) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 11, 33, 13, respectively, and the light chain variable region comprises SEQ ID NOs: 14, 15, 16, respectively light chain LCDR1, LCDR2, LCDR3 shown; or

(D) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 11, 34, 13, respectively, and the light chain variable region comprises SEQ ID NOs: 14, 15, 16, respectively The light chains LCDR1, LCDR2, LCDR3 shown.

In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises:

(E) a heavy chain variable region as set forth in SEQ ID NO: 1 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, and as SEQ ID NO: 1 ID A light chain variable region set forth in NO:2 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:2;

(F) a heavy chain variable region as set forth in SEQ ID NO:9 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9, and as SEQ ID NO:9 A light chain variable region set forth in ID NO: 10 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10;

(G) having at least 95%, 96%, 97%, 98%, 99% or 100% sequence as set forth in or with SEQ ID NO: 17, 18, 31 or 32 A heavy chain variable region of identity, and a light chain as set forth in SEQ ID NO: 19 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19 variable region;

(H) having at least 95%, 96%, 97%, 98%, 99% or 100% sequence as set forth in SEQ ID NO: 17, 18, 31 or 32 or with SEQ ID NO: 17, 18, 31 or 32 A heavy chain variable region of identity, and a light chain as set forth in SEQ ID NO:20 or having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20 variable region; or

(I) as set forth in SEQ ID NO: 26, 27, 28, 29 or 30 or having at least 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 26, 27, 28, 29 or 30 or a heavy chain variable region of 100% sequence identity, and at least 95% as shown in SEQ ID NO: 21, 22, 23, 24 or 25 or with SEQ ID NO: 21, 22, 23, 24 or 25, Light chain variable regions of 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises: a heavy chain variable region as set forth in SEQ ID NO:31 and a light chain as set forth 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 set forth in SEQ ID NO:29 and a light chain as set forth in SEQ ID NO:23 variable region.

In some embodiments, the anti-OX40 antibody described 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 mutation thereof sequence; the light chain constant region is derived from human kappa, lambda chain or a mutant sequence thereof; more preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 35 or has at least 90% of the same as 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 as shown in SEQ ID NO : 36 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:36.

In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises:

(J) as set forth in SEQ ID NO: 37 or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, A heavy chain of 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and/or as set forth in SEQ ID NO:38 or having at least 85%, 86%, A light chain of 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or

(K) as set forth in SEQ ID NO:39 or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, A heavy chain of 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and/or as set forth in SEQ ID NO:40 or having at least 85%, 86%, Light chains of 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, 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 as SEQ ID NO: 38 The heavy chain shown in NO:39 and the light chain shown in SEQ ID NO:40.

In some embodiments, the aforementioned pharmaceutical composition comprising: 10 mM histamine hydrochloride buffer at pH 6.0, anti-OX40 antibody at a concentration of 50 mg/ml, sucrose at a concentration of 80 mg/ml, and a concentration of 0.4 mg/ml polysorbate 80, the anti-OX40 antibody comprises a heavy chain as set forth in SEQ ID NO:37 and a light chain as set forth in SEQ ID NO:38.

In some embodiments, the present disclosure provides a method for preparing the aforementioned pharmaceutical composition, the method comprising the step of buffer-exchanging a stock solution of an anti-OX40 antibody or an antigen-binding fragment thereof.

The present disclosure also provides a freeze-dried preparation, which can form any one of the aforementioned pharmaceutical compositions after reconstitution.

In some embodiments, the present disclosure provides a lyophilized formulation containing an anti-OX40 antibody or an antigen-binding fragment thereof, the lyophilized formulation obtained by lyophilizing the aforementioned pharmaceutical composition. In an alternative embodiment, the freeze-drying comprises the steps of pre-freezing, primary drying and secondary drying in sequence.

In some embodiments, 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.

In some embodiments, any one of the aforementioned lyophilized preparations, wherein the amount ratio of the anti-OX40 antibody or its antigen-binding fragment to the buffer is: 0.007 mol to 0.685 mol of the anti-OX40 antibody or an antigen-binding fragment thereof (non-limiting examples include: about 0.007 mol, about 0.068 mol, about 0.137, about 0.206 mol, about 0.240 mol, about 0.274 mol, about 0.308 mol mol, about 0.343 mol, about 0.377 mol, about 0.411 mol, about 0.446 mol, about 0.480 mol, about 0.548 mol, about 0.617 mol, about 0.685 mol, and any range between these points): 5 mol to 30 mol of histidine or acetate buffer (non-limiting examples include: about 5 mol, about 6 mol, about 7 moles, about 8 moles, about 9 moles, about 10 moles, about 11 moles, about 12 moles, about 13 moles, about 14 moles, about 15 moles ear, about 16 moles, about 18 moles, about 20 moles, about 25 moles, about 30 moles, and any range between these point values). In some embodiments, the lyophilized formulation comprises an anti-OX40 antibody or antigen-binding fragment thereof, a saccharide (eg, trehalose or sucrose), a surfactant (eg, polysorbate 80 or polysorbate 20), and histidine Salt (such as histamine hydrochloride buffer) or acetate buffer (sodium acetate buffer), wherein 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 the anti-OX40 antibody or its antigen-binding fragment: 50 to 90 parts by weight of sugar: 0.1 to 1.0 part by weight of a surfactant, the anti-OX40 antibody or its antigen-binding fragment and the substance of the buffer The amount ratio is: 0.007 mol to 0.685 mol of anti-OX40 antibody or antigen-binding fragment thereof: 5 to 30 mol of histamine or acetate buffer. In some embodiments, the lyophilized formulation comprises an anti-OX40 antibody with a heavy chain as set forth in SEQ ID NO: 37 and a light chain as set forth in SEQ ID NO: 38, sucrose, polysorbate 80, and histidine Hydrochloride buffer, wherein the ratio of the anti-OX40 antibody, sucrose and polysorbate 80 by weight is: 50 parts by weight of anti-OX40 antibody: 80 parts by weight of sucrose: 0.4 part by weight of polysorbate 80, the The substance ratio of anti-OX40 antibody to histamine hydrochloride buffer is: 0.343 mol parts of anti-OX40 antibody: 10 mol parts of histamine hydrochloride buffer.

In some embodiments, the aforementioned lyophilized formulations are 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 at a high temperature of 40°C for at least 7 days, at least 14 days or at least 28 days.

In some embodiments, the present disclosure provides a reconstituted solution containing an anti-OX40 antibody or an antigen-binding fragment thereof, the reconstituted solution being prepared by reconstituting the aforementioned lyophilized preparation. Solutions for reconstitution include but are not limited to water for injection, physiological saline or glucose solution, preferably water for injection. In some embodiments, the composition and content of the reconstituted solution are the same as the aforementioned pharmaceutical compositions.

In some embodiments, the present disclosure provides an article of manufacture comprising a container containing the pharmaceutical composition, lyophilized formulation, or reconstitution solution as before. In some embodiments, the container is a neutral borosilicate glass vial for injection.

The present disclosure provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the aforementioned pharmaceutical composition, lyophilized formulation, reconstituted solution or product; in some embodiments , the disease or disorder may be cancer or a cell proliferative disease. In some specific embodiments, 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 tumor, kidney cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell growth. In some embodiments, the hematological cancer includes, but is not limited to, acute and chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, Hodgkin's ( 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 an 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 immunostimulatory/effector function of T effector cells, and/or a down-regulation of the immunosuppressive function of T regulatory cells. Among other things, the increase may be the result of cell proliferation, and the down-regulation may be the result of no increase in the number of cells (or a decrease in the number of cells).

The present disclosure provides use of the aforementioned pharmaceutical compositions, lyophilized formulations, reconstituted solutions or preparations in the manufacture of a medicament for enhancing an immune response in a human subject.

The present disclosure also provides the use of the aforementioned pharmaceutical compositions, lyophilized preparations, reconstituted solutions or preparations in the preparation of medicaments for the treatment/prevention of diseases or disorders. The disease or disorder may be cancer or a cell proliferative disease. In some specific embodiments, the cancer is lung cancer, prostate cancer, breast cancer, Cancer of the head and neck, esophagus, stomach, colon, colorectum, bladder, cervix, uterus, ovary, liver, melanoma, kidney, squamous cell, blood, or any Other diseases or conditions in which cell growth is controlled. In some embodiments, the hematological cancer includes, but is not limited to, acute and chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, Hodgkin's ( 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 compositions, lyophilized formulations, reconstituted solutions or articles of manufacture for use in the treatment/prevention of a disease or disorder. In some embodiments, the disease or disorder may be cancer or a cell proliferative disease. In some specific embodiments, 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 tumor, kidney cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell growth. In some embodiments, the hematological cancer includes, but is not limited to, acute and chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, Hodgkin's ( 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 compositions, lyophilized preparations, reconstituted solutions or preparations for use in one or more of the following: inhibiting Treg function (eg inhibiting the suppressive function of Treg), killing cells expressing OX40 (eg expressing high OX40 levels), increase effector T cell function and/or increase memory T cell function, decrease tumor immunity, enhance T cell function and/or deplete OX40 expressing cells.

The present disclosure provides the aforementioned pharmaceutical compositions, lyophilized preparations, reconstituted solutions or preparations for use in treating cancer, stimulating an immune response in a subject, stimulating antigen-specific T cells Responding to, activating or costimulating T cells, increasing production of cytokines (eg, IL-2 and/or IFN-γ) in T cells, and/or proliferation of T cells, reducing or depleting the number of T regulatory cells in tumors, and/or inhibit tumor cell growth.

The present disclosure also provides the use of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product for preparing a medicament for: stimulating an immune response in a subject, stimulating an antigen-specific T cell response, activating or costimulating T cells, increase IL-2 and/or IFN-γ production in T cells and/or proliferation of T cells, reduce or deplete the number of T regulatory cells in a tumor and/or inhibit tumor cell growth.

Figure 1A to Figure 1B : ELISA results of the affinity of murine and chimeric antibodies to human OX40. Among them, Figure 1A shows the affinity results of murine antibody m2G3 and chimeric antibody ch2G3, m2G3-NC and ch2G3-NC are negative controls; Figure 1B shows the affinity results of murine antibody m4B5 and chimeric antibody ch4B5, m4B5-NC and ch4B5 -NC is a negative control.

Figure 2: Experiment in which anti-OX40 antibody stimulates T cells to secrete IFN-γ. The results show that the OX40 antibody to be tested can achieve the maximum stimulation effect at the 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 on different days after administration; Figure 3B shows the 20th day after administration, different anti-OX40 antibodies in vivo in mice effect on tumor mass. The results showed that the tumor inhibition rate of 2G3 antibody was as high as 97% when the dose was 3 mg/kg on the 20th day after administration.

Figure 4: Fitted graph of DOE for the formulation of anti-OX40 antibody formulations

the term

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the term "buffer" or "buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugated component. Examples of buffers that control pH in the appropriate range include acetate, succinate, gluconate, histamine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycerol Aminoglycine and other organic acid buffers.

As used herein, the term "histidine buffer" or "histidine buffer", is a buffer containing histidine ions. Examples of histidine buffers include histamine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. In some embodiments of the present disclosure, the histamine hydrochloride buffer is selected from histamine hydrochloride buffer.

As used herein, the term "succinate buffer" or "succinate buffer", is a buffer that includes succinate ions. Examples of succinate buffers include sodium succinate succinate, potassium succinate succinate, calcium succinate succinate, and the like. In some embodiments of the present disclosure, the succinate buffer is sodium succinate succinate.

As used herein, the term "acetate buffer" or "acetate buffer", is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, histidine acetate, potassium acetate, calcium acetate, magnesium acetate, and the like. In some embodiments of the present disclosure, the acetate buffer is sodium acetate acetate.

As used herein, the term "phosphate buffer" or "phosphate buffer", is a buffer that includes phosphate ions. Examples of phosphate buffers 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.

As used herein, the term "pharmaceutical composition" means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiological/pharmaceutically acceptable salts or prodrugs. Pharmaceutically acceptable carriers and excipients. The pharmaceutical composition maintains the stability of the active ingredient of the antibody, promotes the administration to the organism, facilitates the absorption of the active ingredient and exerts biological activity. Herein, "pharmaceutical composition" and "preparation" are not mutually exclusive.

In the context of preparing a pharmaceutical composition comprising the antibody herein, the term "buffer exchange" refers to replacing the solvent system that dissolves the antibody protein with the buffer system of the stabilized formulation described herein, eg, using the stabilized formulation described herein. The buffer system is physically manipulated to displace the antibody protein-containing high-salt or hypertonic solvent system so that the antibody protein is present in a stable formulation. The so-called physical manipulations include, but are not limited to, ultrafiltration, dialysis or reconstitution after centrifugation.

The pharmaceutical compositions or formulations of the present disclosure can be prepared by methods known in the art. Exemplary, preparation of antibody pharmaceutical composition or preparation: the first step: take a certain amount of purified antibody solution, use buffer without antibody (such as 10mM histamine hydrochloride buffer at pH 6.0) Solvent replacement (preferably ultrafiltration) is performed, and the antibody is concentrated to about 70 mg/mL by at least 6 volumes of ultrafiltration membrane replacement. A certain volume of sucrose stock solution was added and mixed to make the final sucrose concentration 80 mg/mL. A certain volume of the polysorbate 80 mother solution was added and mixed to make the final polysorbate-80 concentration 0.4 mg/mL. Add pH 6.0 10mM histamine hydrochloride buffer to make up the volume to make the antibody concentration 50mg/mL (other preparations to be tested or stable preparations can be prepared by referring to similar steps). The product is filtered and tested for sterility by central control sampling. Pass the stock solution through a 0.22 μm filter and collect the filtrate. Step 2: Adjust the filling volume, fill the filtrate in vials, add stoppers, and take samples at the beginning of filling, in the middle of filling, and at the end of filling to detect the difference in filling volume. Step 3: Open the capping machine, add aluminum caps, and perform capping. Step 4: Visual inspection to confirm that the product has no defects such as inaccurate loading. Printing and pasting vial labels; printing carton labels, folding carton boxes, packing boxes, sticker box labels.

In the solution form of the pharmaceutical composition in this disclosure, unless otherwise specified, the solvent therein is water.

As used herein, the term "lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation is subjected to a vacuum freeze-drying step. Lyophilized formulations can be obtained by lyophilizing pharmaceutical compositions or formulations in liquid or solution form. Freeze drying is performed by freezing the formulation and then subliming the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or decomposition temperature of the formulation. At suitable pressures, typically in the range of about 50-250 mTorr, the storage temperature for primary drying is typically in the range of about -30 to 25°C (assuming the product remains frozen during the primary drying). The formulation, the size and type of container (eg, glass vial) holding the sample, and the volume of the liquid determine the time required for drying, which can range from hours to days (eg, 40-60 hours). The secondary drying stage can be carried out at about 0-40°C, depending mainly on the type and size of the vessel and the type of protein employed. The secondary drying time is determined by the desired residual moisture level in the product, and typically requires at least about 5 hours. Typically, the moisture 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, preferably, the pressure for the secondary drying is lower than that for the primary drying. Freeze drying conditions can vary with formulation and vial size.

The present disclosure "sugar" includes general composition (CH ₂ O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars and the like. Can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, miribiose , melanose, raffinose, mannose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose and so on. Preferably the sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, most preferably sucrose.

The surfactant of the present disclosure may be selected from polysorbate 20, polysorbate 80, polyhydroxyalkene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl -Sulfobetaine, Myristyl-Sulfobetaine, Linole-Sulfobetaine, Stearyl-Sulfobetaine, Moon Cum-sarcosine, myristyl-sarcosine, linole-sarcosine, stearyl-sarcosine, linole-betaine, myristyl-betaine, cetyl-beet Base, Lauramidopropyl-Betaine, Cocalamidopropyl-Betaine, Linoleamidopropyl-Betaine, Myristamidopropyl-Betaine, Palmamidopropyl Betaine-Betaine, Isostearamidopropyl-Betaine, Myristamidopropyl-Dimethylamine, Palmamidopropyl-Dimethylamine, Isostearamidopropyl - Dimethylamine, sodium methyl cocoamide, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol and copolymers of ethylene and propylene glycol, and the like. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

As used herein, the terms "about", "approximately" or "substantially comprising" mean that the index value is within an acceptable error range of the specific value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined ( i.e. the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, "about", "approximately" or "substantially comprising" may mean a range of up to 20%, for example, may mean ±20%, ±19%, ±18%, ±17%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4 %, ±3%, ±2% or ±1%, range. Furthermore, particularly with respect to biological systems or processes, the term can mean at most one order of magnitude or at most five times the value. Unless otherwise stated, when a specific value appears in this application and the claimed scope, the meanings of "about", "approximately" or "substantially comprising" should be assumed to be within an acceptable error range of the specific value.

The pharmaceutical composition of the present disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, a pharmaceutical composition Its physical and chemical stability and its biological activity are substantially retained after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

Stable antibody pharmaceutical formulations are those in which no significant change is observed when stored at refrigerated temperatures (2-8°C), for example, for at least 3 months, preferably 6 months, more preferably 1 year, and even more. Good land for up to 2 years. Additionally, stable liquid formulations include those that exhibit desirable characteristics after storage at, eg, 25°C and/or 40°C for a period of, eg, 1 month, 3 months, or 6 months. Stable formulations, eg, by visual analysis, drug antibody formulations are colorless or clear to slightly opalescent. The formulations had no more than ±10% variation in concentration, pH and osmolality. Typically, no more than about 10%, preferably no more than about 5% truncation is observed. Typically no more than about 10% aggregates are formed, preferably no more than about 5% aggregates. In some embodiments, the pharmaceutical compositions or lyophilized formulations of the present disclosure are 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; at 25 °C is stable for at least 3 months, or at least 6 months.

If the antibody does not show a significant increase in aggregation, precipitation and/or after visual inspection for color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS) Denatured, then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently change the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by peptide mapping such as in combination with mass spectrometry or MALDI/TOF/MS) method, etc.), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.), and isomerization (by measuring isoaspartic acid) The content of paraffinic acid, peptide mapping, etc. to evaluate).

An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by antigen binding assays.

As used herein, the term "enhancing T cell function" means inducing, eliciting, or stimulating effector or memory T cells with renewed, sustained or amplified biological function. Examples of enhanced T cell function include: increased secretion of interferon-gamma from CD8+ effector T cells relative to pre-intervention levels, increased secretion of interferon-gamma from CD4+ memory and/or effector T cells, CD4+ effector and Increased proliferation of memory T cells, increased proliferation of CD8+ effector T cells, enhanced antigen responsiveness (eg, clearance).

As used herein, the term "enhancing an immune response" refers to stimulating, eliciting, increasing, improving or enhancing the response of the immune system of a mammal. An immune response can be a cellular response (ie, cell-mediated, eg, mediated by cytotoxic T lymphocytes) or a humoral response (ie, an antibody-mediated response), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+ helper T cell activity and generation of cytotoxic T cells. Enhancement of the immune response can be assessed using a number of in vitro or in vivo measures known to those of ordinary skill in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (eg, IL-2 production), tumor regression , survival of tumor-bearing animals, antibody production, immune cell proliferation, cell surface marker expression and cytotoxicity. In one embodiment, the method enhances cellular immune responses, particularly cytotoxic T cell responses.

As used herein, the term "tumor immunity" refers to the process by which tumors evade immune recognition and clearance. As a therapeutic concept, tumor immunity is when its ability to evade immune recognition and clearance is weakened, the tumor is recognized and attacked by the immune system, and the patient is treated. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

As used herein, the term "T effector cells" ("Teff") refers to T cells (eg, CD4+ and CD8+ T cells) and T helper (Th) cells with cytolytic activity that secrete cytokines, activate and direct Other immune cells, but not regulatory T cells (Treg cells). The anti-OX40 antibodies of the present disclosure can activate Teff cells, such as CD4+ and CD8+ Teff cells.

As used herein, the term "regulatory T cell" or "Treg cell" means a specialized type of CD4+ T cell that is capable of suppressing the response of other T cells. Treg cells are characterized by the expression of CD4, the alpha subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), and are It plays a crucial role in in vivo tolerance against tumor-expressed antigens.

As used herein, the term "OX40" refers to a receptor that binds the 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. The term "OX40" includes any OX40 variant or isoform that is naturally expressed by a cell. Accordingly, the OX40 antibodies or fragments of the present disclosure can cross-react with OX40 from species other than humans (eg, cynomolgus OX40). Alternatively, the OX40 antibody or fragment may be specific for human OX40 and not exhibit cross-reactivity with other species.

OX40 or variants and isoforms thereof are isolated from the cells or tissues in which it is naturally expressed, or recombinantly produced using techniques well known in the art and/or described in this disclosure. Unless otherwise stated, "OX40" can be from any vertebrate source, including native OX40 of mammals such as primates (eg, humans) and rodents (eg, mice and rats). The term encompasses "full-length", unprocessed OX40 as well as any form of OX40 that results from processing in the cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.

As used herein, the term "OX40 activation" refers to the activation of the OX40 receptor. Typically, OX40 activation results in signal transduction.

As used herein, the term "anti-OX40 antibody" or "antibody that binds OX40" or "antibody to OX40" refers to an antibody capable of binding OX40 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting OX40 . In some embodiments of the present disclosure, the anti-OX40 antibody is the anti-OX40 antibody described in the international patent application PCT/CN2019/107787 (Publication No. WO2020063660A1). OX40 antibody, eg "2G3" antibody. The present disclosure incorporates the entire content of international patent application PCT/CN2019/107787 into the present application.

As used herein, the term "deplete OX40-expressing cells" refers to the killing or depletion of OX40-expressing cells by an anti-OX40 antibody or fragment thereof. Depletion of OX40-expressing cells can be achieved by a variety of mechanisms, such as antibody-dependent cell (ADCC)-mediated cytotoxicity and/or phagocytosis.

As used herein, the term "cytokine" is a generic term for a class of proteins that are released by a population of cells and act on another cell as an intercellular mediator. Examples of such cytokines are lymphokines, monokines; interleukins (IL) such as 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; tumor necrosis factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and Gamma-interferon. As used in this disclosure, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents thereof, including small molecular entities produced by artificial synthesis, and pharmaceutically acceptable derivatives thereof matter and salt.

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

As used herein, the term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

The sequence of about 110 amino acids near the N-terminus in the antibody heavy and light chains varies greatly, which 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 three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). 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 order from the amino end to the carboxyl end is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

The number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering convention (LCDR1-3, HCDR1-3).

As used herein, the term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, involving techniques and methods well known in the art, such as:

(1) Antibodies isolated from transgenic human immunoglobulin genes, transchromosomic animals (eg, mice) or fusion tumors prepared therefrom;

(2) An antibody isolated from a host cell (such as a transfectoma) transformed to express the antibody;

(3) antibodies isolated from recombinant combinatorial human antibody libraries; and

(4) Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that 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.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

In some embodiments, a humanized antibody.

As used herein, the term "murine antibody" is a monoclonal antibody directed against human OX40 prepared according to the knowledge and skill in the art. For example, the test object is injected with OX40 antigen during preparation, and then splenocytes (B lymphocytes) expressing antibodies with desired sequences or functional properties are isolated, and then B lymphocytes are fused with myeloma cells to obtain corresponding fusion tumor cells.

In some embodiments, the murine OX40 antibody or antigen-binding fragment thereof may comprise a light chain constant region of a murine kappa, lambda chain or a variant thereof, or may comprise a murine IgG1, IgG2, IgG3 or IgG4 or its Variant heavy chain constant regions.

As used herein, the term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the present disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "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 backbone sequences (ie, "humanized antibodies").

The term "humanized antibody", as used herein, refers to an antibody produced by grafting non-human species CDR sequences into a human antibody variable region framework, ie, a different type of human germline antibody framework sequence. The heterologous response induced by the chimeric antibody carrying a large amount of heterologous protein components can be overcome. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained in the "VBase" human germline sequence database, and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th ed. . In order to avoid a decrease in activity while reducing immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies obtained after affinity maturation of CDRs by phage display or yeast display.

In cases involving CDR grafting, the resulting OX40 antibody (or antigen-binding fragment thereof) has reduced affinity for the antigen due to changes in the framework residues in contact with the antigen. Such interactions can be the result of somatic hypermutation. Therefore, there remains a need to graft such donor framework amino acids into the framework of a humanized antibody. Amino acid residues from the non-human OX40 antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by examining the variable region sequence and structure of the non-human monoclonal antibody. CDRs Residues in the donor framework that differ from germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subtype consensus sequence or a consensus sequence of murine sequences with a high percentage of similarity. Rare framework residues are thought to be likely the result of somatic hypermutation and thus play an important role in binding. In some embodiments of the present disclosure, 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 a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, specifically, a human IgG1 heavy chain constant region.

As used herein, the term "backmutation" refers to the mutation of amino acid residues in the FR region of a human antibody to the amino acid residues in the corresponding positions of the antibody of the original source, usually to avoid immunogenicity caused by the humanized antibody At the same time as the decrease in activity is caused, the variable region of the humanized antibody can be subjected to minimal back-mutation to maintain the activity of the antibody.

As used herein, the term "chimeric antibody" is an antibody obtained by fusing the variable region of a non-human antibody to the constant region of a human antibody, which can reduce the immune response induced by the non-human antibody. For example, to create a chimeric antibody, we need to select a fusion tumor that secretes a mouse-specific monoclonal antibody, then select the variable region gene from the mouse fusion tumor cells, and then select the constant region gene of the human antibody as needed. The mouse 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 the human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, in particular, comprising the heavy chain constant regions of human IgG1.

As used herein, the term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, OX40). It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of antibodies. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include:

(i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains;

(ii) F(ab')2 fragment, a bivalent fragment formed by connecting two Fab fragments via a disulfide bridge on the hinge region;

(iii) Fd fragments consisting of VH and CH1 domains;

(iv) Fv fragments consisting of the VH and VL domains of the one-armed antibody; and

(v) Single domain or dAb fragments (Ward et al. (1989) Nature, 341:544-546), which consist of VH domains.

Furthermore, although the two domains of Fv fragments, VL and VH, are encoded by separate genes, recombinant methods can be used to connect them by synthetic linkers, allowing them to generate a single protein chain (called a single-chain Fv (scFv)) , in which the VL and VH regions pair to form a monovalent molecule; see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using general techniques known to those of ordinary skill in the art, and the fragments are screened for functional properties in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

In some embodiments, the term "antigen-binding fragment" refers to a Fab, Fv, sFv, F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptidobody ( peptibody), domain antibodies and multispecific antibodies (bispecifics, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv).

As used herein, the term "Fab" can be obtained by treating an IgG antibody molecule with the protease papain (which cleaves 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 About half of the N-terminal side of the H chain and the entire L chain are held 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. In addition, the Fab can be produced by inserting DNA encoding the Fab 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.

As used herein, the term "F(ab')2" is an antibody fragment obtained by pepsin digestion of the downstream portion of the two disulfide bonds in the hinge region of IgG, having a molecular weight of about 100,000 and having antigen-binding activity , and contains two Fab regions linked 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. Furthermore, the F(ab')2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.

As used herein, the term "Fab'" is an antibody fragment having a molecular weight of about 50,000 and having 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.

In addition, the Fab' can be produced by inserting 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' .

As used herein, the terms "single-chain antibody", "single-chain Fv" or "scFv" are meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain ( or region; VL) molecules. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amines The amino acid sequence or variants thereof consist of, for example, 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol.31:94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

The scFv of the present disclosure can be produced by the following steps: obtaining cDNAs encoding VH and VL of the monoclonal antibodies of the present disclosure, constructing 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 the scFv.

As used herein, the term "diabody" is an antibody fragment in which the scFv is dimerized, an antibody fragment having bivalent antigen binding activity. In bivalent antigen binding activity, the two antigens can be the same or different.

The diabodies of the present disclosure can be produced by obtaining cDNAs encoding the VH and VL of the monoclonal antibodies of the present disclosure, and constructing DNA encoding the scFv such that the amino acid sequence of the peptide linker is 8 residues or less in length , insert the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introduce the expression vector into a prokaryotic or eukaryotic organism to express the diabody.

As used herein, the term "dsFv" may be linked via a disulfide bond between cysteine residues by a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue and obtained. Amino acid residues substituted with cysteine residues can be selected according to known methods (Protein Engineering, 7, 697 (1994)) based on the prediction of the three-dimensional structure of the antibody.

The dsFv of the present disclosure can be produced by the following steps: obtaining cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure, constructing a DNA encoding the dsFv, and inserting the DNA into a prokaryotic into a biological expression vector or a eukaryotic expression vector, which is then introduced into a prokaryotic or eukaryotic organism to express the dsFv.

The term "antibody framework (FR)", as used in this disclosure, refers to the portion of the VL or VH of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

As used herein, the term "amino acid difference" refers to the difference between a polypeptide and a variant thereof at one or more amino acid sites on a polypeptide fragment, wherein the variant may be derived from one or more on the polypeptide These sites are obtained by substitution, insertion or deletion of amino acids.

As used herein, the term "epitope" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from adjacent amino acids, or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in unique spatial conformations. Methods for determining epitopes are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques of the present disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The term "specifically binds" as used in the present invention refers to the binding of an antibody to a predetermined epitope on an antigen. Typically, when using recombinant human OX40 as the analyte and the antibody as the ligand, the antibody ^exhibits an equilibrium dissociation constant of approximately less than 10-7 M or even less when measured in an instrument by surface plasmon resonance (SPR) techniques (KD) binds to a predetermined antigen with at least twice the affinity for binding to a non-specific antigen (eg, BSA, etc.) other than the predetermined antigen (or closely related antigen). The term "antibody that recognizes an antigen" is used interchangeably with the term "antibody that specifically binds" in this disclosure.

As used herein, the term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure bind OX40 with a dissociation equilibrium constant (KD) of ^{less than about 10-7} M, eg, less than about ^10-8 M, ^10-9 M, or ^{10-10 M or less, eg, as using surface plasmons} The bulk resonance (SPR) technique was measured in a BIACORE instrument.

When the term "compete" is used in the context of antigen-binding proteins that compete for the same epitope (eg, neutralizing antigen-binding proteins or neutralizing antibodies), it means competition between antigen-binding proteins, as determined by the following assay : The antigen-binding protein (eg, antibody or functional fragment thereof) to be detected prevents or inhibits (eg, reduces) the specific binding of the reference antigen-binding protein (eg, ligand or reference antibody) to a common antigen (eg, OX40 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Assays (see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, eg, Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeling Assays, solid phase direct labeling sandwich assay (see eg Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling of RIA with I-125 label (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). Typically the assay involves the use of purified antigen that binds to an unlabeled test antigen binding protein and a labeled reference antigen binding protein (either on a solid surface or on a cell surface). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell surface in the presence of the antigen binding protein to be tested. Typically, the antigen binding protein to be tested is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and tables that are sufficiently close to the epitope to which the reference antigen-binding protein binds. An antigen-binding protein that binds to an epitope, and the two epitopes sterically prevent the binding from occurring. Additional details regarding methods for determining competitive binding are provided in the disclosed examples. Usually when a competing antigen-binding protein is present in excess, it will inhibit (e.g. decrease) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more specificity of the reference antigen binding protein to a common antigen combine. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or more.

As used herein, the term "cross-reactivity" refers to the ability of an antibody of the invention to bind to OX40 from a different species. For example, an antibody of the invention that binds human OX40 can also bind OX40 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express OX40. Methods for determining cross-reactivity include standard binding assays such as surface plasmon resonance (SPR) analysis, or flow cytometry, as disclosed herein.

As used herein, the terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking.

As used herein, the term "inhibition of growth" (eg, in relation to a cell) is intended to include any measurable reduction in cell growth.

As used herein, the terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a specific antigen. The term "induction" with respect to induction of CDC or ADCC refers to stimulation of a specific mechanism of direct cell killing.

As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to Fc receptors ( Secreted immunoglobulins on FcR) enable these cytotoxic effector cells to specifically bind antigen-bearing target cells, followed by a cytotoxic form of killing the target cells with cytotoxins. The primary cells that mediate ADCC (NK cells) express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, in vitro ADCC assays such as US Pat. No. 5,500,362 or 5,821,337 or US Pat. Described in No. 6,737,056 (Presta). Useful effector cells for such assays include PBMC and NK cells. Alternatively, the ADCC activity of the molecule of interest can be assessed in vivo, eg, in animal models such as those disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). In addition, the ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc region of IgG. The modification refers to a mutation in the heavy chain constant region of the antibody, such as a mutation selected from N297A, L234A, L235A of IgG1; IgG2/4 chimera, F235E, or L234A/E235A of IgG4.

As used herein, the term "complement-dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of an antibody (of the appropriate subclass) by the first component of the complement system (Clq), which has bound to its cognate antigen. To assess complement activation, a CDC assay can be performed, eg, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding capacity are described, for example, in US Patent No. 6,194,551 B1 and WO 1999/51642. See also, eg, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

The term "nucleic acid molecule" as used in this disclosure refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are specifically double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vectors of the present disclosure are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomals with a bacterial origin of replication mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome (eg, a non-episomal mammalian vector).

Methods of producing and purifying antibodies and antigen-binding fragments are well known in the art, eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15. For example, mice can be immunized with human OX40 or a fragment thereof, and the resulting antibody can be coated, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen-binding fragment of the invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) by aligning the IMGT human antibody variable region germline gene database with MOE software, or from J. Immunoglobulins, 2001 ISBN012441351.

The antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using general methods. For example, 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 stably transfect CHO cells. As a more preferred prior art, mammalian-like expression systems lead to glycosylation of the antibody, 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 were expanded in serum-free medium in bioreactors for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.

As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a single pure cell line, which is not limited to eukaryotic, prokaryotic or phage pure cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant methods such as fusion tumor technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include microorganisms (eg, bacteria), plant or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; 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, 293 cells.

As used herein, the term "conservative modification" or "conservative substitution or substitution" refers to amino acid replacement in other proteins with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). of amino acids, allowing frequent changes without altering the biological activity of the protein. It is known to those of ordinary skill in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, eg, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/ Cummings Pub. Co., p. 224, (4th ed.). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

As used herein, the term "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition 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, for example, the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of the side effect. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

As used herein, the term "exogenous" refers to a substance produced outside an organism, cell or human as the case may be.

As used herein, the term "endogenous" refers to a substance produced in an organism, cell or human body as the case may be.

As used herein, the term "mutated sequence" refers to the nucleotide sequence and amino acid sequence of the present disclosure obtained under the condition of appropriate mutation modification such as substitution, insertion or deletion of the nucleotide sequence and amino acid sequence of the present disclosure. Acid sequences Nucleotide sequences and amino acid sequences with varying degrees of percent sequence identity. The 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 percent identity between two sequences can be performed with the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.

"Homology" and "identity" in the present disclosure refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if every position in two DNA molecules is occupied by an adenine, then the molecules are homologous at that position of. The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of total positions compared x 100. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 matches or homology at 10 positions in the two sequences; if 95 matches at 100 positions in the two sequences or homologous, then the two sequences are 95% homologous. In general, comparisons are made when the two sequences are aligned for the greatest percent homology.

As used in this disclosure, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.

"Polymerase chain reaction" or "PCR" as used in the present disclosure refers to a process in which a specified portion of nucleic acid, RNA and/or DNA is amplified as in, eg, US Pat. No. 4,683,195 or Technology. Generally, it is desirable to obtain sequence information from the end of the target region or beyond, so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand 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, and the like. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich eds. (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used in the present disclosure is considered to be an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample that includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Generate a specific portion of nucleic acid.

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance can, but does not necessarily, occur, and that the description includes instances where the event or circumstance occurs or instances where it does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

As used herein, the term "cancer" refers to or describes a physiological disorder 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. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (eg, 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 carcinoma, 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, colon cancer Rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, lentigo malignant melanoma , acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastosis cellular leukemia, and post-transplant lymphoproliferative disorders disease (PTLD), and abnormal vascular proliferation associated with phakomatoses, edema (such as associated with brain tumors), and Meigs' syndrome, brain tumors and brain cancer, and head and neck cancer, and related transfers.

In certain embodiments, 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 myeloma . In some embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (eg, triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocytes cancer. Also, in some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (eg, triple negative breast cancer), including metastatic forms of those cancers.

As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder refers to cancer.

As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

As used herein, the terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to in this disclosure.

As used herein, the terms "administer," "administer," and "treating," when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or Contact of a composition with an animal, human, subject, cell, tissue, organ or biological fluid. "Administering," "administering," and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contacting of reagents with cells, and contact of reagents with fluids, wherein the fluids contact with cells. "Administering," "administering," and "treating" also mean treating a cell in vitro and ex vivo by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

As used herein, the term "treating" means administering an internal or external therapeutic agent, eg, a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure, or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, to a patient who has One or more diseases or conditions for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more diseases or symptoms in a patient or population to be treated, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular disease or symptom (also referred to as a "therapeutically effective amount") can vary depending on factors such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by physicians or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method according to any statistical test known in the art such as Student's t-test, Chi-square test, according to Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.

As used herein, the term "preventing cancer" refers to delaying, inhibiting, or preventing the onset of cancer in a mammal in which carcinogenesis or initiation of tumorigenesis has not been demonstrated, but has been determined, for example, by genetic screening or other methods , which have been identified as having cancer susceptibility. The term also includes treatment of a mammal having a precancerous condition to stop the progression of the precancerous condition to a malignancy 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. In the embodiments of the present disclosure, the experimental methods that do not specify specific conditions are usually based on a General conditions, such as Cold Spring Harbor's Antibody Technology Laboratory Manual, Molecular Cloning Manual; or as recommended by the manufacturer of raw materials or commodities. The reagents that do not indicate the specific source are general reagents purchased in the market.

Example 1: Preparation of Antibodies

A library of anti-human OX40 mAbs was generated by immunizing mice. The mice of BalB/C and A/J strains (Yangzhou University Comparative Medical Center, animal production license number; SCXK (Su) 2017-007) were used in the experiment, female, 10 weeks old.

The immunization antigen is human OX40 recombinant protein with Fc tag (OX40-Fc: OX40 Leu 29-Ala 216 (Accession # NP_003318) is fused to Fc), purchased from Acro Biosystems, Cat. No. #OX40-H5255, expressed by HEK293 as usual method purification.

OX40-Fc was emulsified with Freund's adjuvant: complete Freund's adjuvant (sigma-aldrich, F5881-10ML) for the first immunization and incomplete Freund's adjuvant (sigma-aldrich, F5506-10ML) for the remaining booster immunizations. The ratio of antigen to adjuvant was 1:1, and each immunization was injected with 25 μg protein/200 μl/mouse. See below for details:

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 OX40/OX40L binding. Mice with strong serum titer, affinity and ligand binding blocking ability were selected for one final immunization and then sacrificed. Spleen cells and SP2/0 myeloma cells (ATCC® CRL-1581 ^™ ) were fused and plated to obtain fusion tumors. The target fusion tumors were selected by indirect ELISA, capture ELISA and cell-based functional screening in Example 2. The monoclonal antibody was constructed by limiting dilution method.

The 19 strains of OX40 mouse monoclonal antibodies that have been established are produced by serum-free expression, and purified mouse monoclonal antibodies are obtained by protein A affinity chromatography technology. The fusion tumor cells secreting activated anti-OX40 antibody were screened by indirect ELISA, capture ELISA and cell functional activity assay. The brief steps for functional screening are as follows: Cultivate the GS-H2/OX40 stable cell line (purchased from genscript, cat#M00608). Prepare diluted test antibody and OX40L (Sino Biological, 13127-H04H) solution and add it to GS-H2/OX40 cells in logarithmic growth phase, collect the cell supernatant after culture, and measure the IL-8 content in the supernatant (using human IL-8 kit, cisbio, cat#62IL8PEB). In the experiment, the antibody GPX4 (prepared with reference to 1A7.gr.1 in the patent WO2015153513) was used as a positive reference, and hIgG (prepared in a laboratory) was used as a negative reference.

According to the functional activity of cells, 10 strains with high activity were selected for gene selection and sequencing. Total cellular RNA can be obtained by general RNA extraction techniques, and then the PCR product of the variable region of the monoclonal antibody can be obtained by reverse transcription polymerase chain reaction (RT-PCR). The PCR product was separated and recovered by agarose gel, and then cloned into the gene vector and transformed into E. coli. Several transformed colonies were randomly selected, and the variable region of the monoclonal antibody was amplified by PCR for gene sequencing. The corresponding sequences of the obtained exemplary murine monoclonal antibodies are shown below.

The heavy and light chain variable region sequences of murine monoclonal antibody m4B5 are as follows:

m4B5 heavy chain variable region:

SEQ ID NO：1

SEQ ID NO: 1

m4B5 light chain variable region:

SEQ ID NO：2

SEQ ID NO: 2

The CDR region sequence of mouse monoclonal antibody m4B5 is shown in Table 1:

Table 1. CDR region sequences of murine monoclonal antibody m4B5

The heavy and light chain variable region sequences of the murine monoclonal antibody m2G3 are as follows:

m2G3 heavy chain variable region:

SEQ ID NO：9

SEQ ID NO: 9

m2G3 light chain variable region:

SEQ ID NO：10

SEQ ID NO: 10

The CDR region sequence of mouse monoclonal antibody m2G3 is shown in Table 2:

Table 2. CDR region sequences of murine monoclonal antibody m2G3

Example 2: ELISA identification and screening method of antibodies

Indirect ELISA method:

Dilute 20× coating buffer to 1× with deionized water, and prepare antigen human OX40-His (Acro biosytems, OXL-H52Q8) with 1× coating buffer (carbonate buffer) to make the final concentration 2 μg/ mL, add 100 μL of liquid to each well, and incubate at 4 °C overnight or 37 °C for 2 h. The plate was washed once with PBST, 200 μL of blocking solution (PBST containing 5% skim milk) was added to each well, incubated at 37°C for 2 h, and the plate was washed four times with PBST. Add 100 μL of the diluted primary antibody to each well (the concentration of the antibody to be tested is 5-fold serial dilution from 10000ng/ml, 7 gradients, namely 10000ng/ml, 2000ng/ml, 400ng/ml, 80ng/ml, 16ng/ml, 3.2 ng/ml, 0.64ng/ml, blank wells are pure diluent (ie 2.5% skim milk in PBST), incubated at 37°C for 40min. The plate was washed 4 times with PBST, and the enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG, purchased from Jackson Immunoresearch, Cat#115036071 or HRP-labeled goat anti-human IgG, purchased from Jackson Immunoresearch, Cat#109036098) was diluted with PBST buffer, Add 100 μL to each well and incubate at 37°C for 40min. PBST wash plate 4 times, add 100 μL of TMB chromogenic solution to each well, incubate at room temperature for 3-15 minutes in the dark, and add 50 μL of stop solution (1M sulfuric acid) to each well. Set the microplate reader parameters, read the OD value at 450-630nm, and save the experimental data.

Capture ELISA:

Dilute 20×PBS buffer to 1× with deionized water, prepare GAM secondary antibody (Jackson Immunoresearch, 115-006-071) with 1×PBS to make the final concentration 2 μg/mL, add 100 μL to each well, 4°C Incubate overnight or at 37°C for 2h; wash the plate once with PBST, add 200 μL of blocking solution (PBST containing 5% skim milk) to each well, incubate at 37°C for 2h, and wash the plate four times with PBST. Add 100 μL of the diluted primary antibody to each well (the concentration of the antibody to be tested is 5-fold serial dilution from 10000ng/ml, 7 gradients, namely 10000ng/ml, 2000ng/ml, 400ng/ml, 80ng/ml, 16ng/ml, 3.2 ng/ml, 0.64ng/ml, blank wells are pure diluent (ie 2.5% skim milk in PBST), incubated at 37°C for 40min. The plate was washed 4 times with PBST, human OX40-FC-biotin (Acro biosystem, OX0-H5255, labeled with biotin) was diluted with 2.5% skim milk in PBST, 100 μl was added to each well, incubated at 37°C for 40 min, and the plate was washed 4 times with PBST. Add 100 μL of TMB color developing solution to each well, incubate at room temperature for 3-15 minutes in the dark, add 50 μL of stop solution (1M sulfuric acid) to each well, set the microplate reader parameters, read the OD value at 450-630 nm, and save the experimental data.

Ligand binding blocking ELISA:

Dilute 20× coating buffer to 1× with deionized water, and prepare antigen OX40L-His (Acro biosytems, OXL-H52Q8) with 1× coating buffer (carbonate buffer) to make the final concentration 2 μg/mL , add 100 μL to each well, incubate overnight at 4°C or incubate at 37°C for 2 hours; wash the plate once with PBST; add 200 μL of blocking solution (PBST containing 5% skim milk) to each well, incubate at 37°C for 2 hours; wash the plate 4 times with PBST; The prepared 200ng/ml human OX40-Fc solution (prepared in 2.5% nonfat milk) was used to serially dilute mouse serum/antibody, and then pre-incubated at room temperature for 40min, then added to the blocked OX40L plate, 100μL/ Well, incubate for 40min; wash plate 4 times with PBST; dilute HRP-labeled goat anti-human with PBST buffer Secondary antibody (GAH-HRP, Jackson Immunoresearch, 109-035-006), add 100 μl to each well, incubate at 37°C for 40 min; wash the plate 4 times with PBST; add 100 μL of TMB chromogenic solution to each well, incubate at room temperature in the dark for 3-15 minutes; add 50 μl of stop solution (1M sulfuric acid) to each well, set the parameters of the microplate reader, read the OD value at 450-630 nm, and save the experimental data.

Example 3: Construction and expression of anti-OX40 recombinant chimeric antibody

The heavy chain variable region (VH) of the murine antibodies m2G3 and m4B5 was added to the human immunoglobulin heavy chain constant region, and the light chain variable region (VL) was added to the human immunoglobulin Kappa light chain constant region. To eukaryotic expression vectors, murine-human chimeric antibodies are produced by transfecting cells.

The heavy chain vector was designed as follows: signal peptide+heavy chain variable region sequence+human IgG1 constant region sequence.

The light chain vector was 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), respectively. After obtaining the vector plasmid, extract the plasmid and send the plasmid for sequencing verification. The qualified plasmid was transfected into human 293F cells with PEI, and cultured continuously. The 293F cells were cultured with serum-free medium (Shanghai Opmax Bio, OPM-293 CD03) to logarithmic growth phase for cell transfection. Dissolve 21.4μg of chimeric antibody light chain plasmid and 23.6μg of chimeric antibody heavy chain plasmid in 10ml Opti-MEM® I Reduced Serum Medium (GIBCO, 31985-070) and mix well, then add 200μg PEI, mix well, room temperature Incubate for 15min and add to 50mL cells. Cell culture conditions: 5% CO ₂ , 37° C., 125 rpm/min. During the culture, feeding was added on the 1st and 3rd days until the cell viability was lower than 70%, and the cell supernatant was collected and filtered by centrifugation. The cell culture solution after centrifugal filtration was loaded onto the antibody purification affinity column, and the column was washed with phosphate buffer, eluted with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), and 1M Tris hydrochloric acid pH 9.0 After neutralization and phosphate buffer dialysis, purified chimeric antibodies Ch2G3 and Ch4B5 were finally obtained.

Example 4: In vitro binding affinity and kinetic experiments

The affinity of human OX40 was detected for the mouse-derived antibody and the chimeric antibody (the method steps are the same as those in Example 2), and the results are shown in Fig. 1A and Fig. 1B . Among them, m2G3-NC (-NC means negative control), Ch2G3-NC, m4B5-NC, and Ch4B5-NC are negative controls, which are the same constant regions used in m2G3, Ch2G3, m4B5, and Ch4B5, respectively, but the variable regions are not Identify OX40.

The results showed that the chimeric antibodies Ch2G3 and Ch4B5 had high affinity to human OX40.

Example 5: In vitro cell reporter gene assay of anti-OX40 antibody

The plate was coated with anti-CD3 antibody (Chempartner, A05-001), placed overnight at 4 degrees, and washed 3 times with PBS; Jurkat-NF-Kb luc-hOX40 cells were harvested (ATCC, TIB-152 stable cell line was constructed by Shanghai Ruizhi Chemical) ) and Raji cells (ATCC, CCL-86), resuspended and mixed. Add 50 μl/well of the diluted antibody to be tested and 50 μl/well of the mixed cells to the cell plate, and place the cell plate in a 37-degree 5% CO ₂ incubator for 5 hours. 100 μl one-Glo™ luciferase reagent (Promega, Cat#E6120) was added to each well and incubated at room temperature for more than 3 min. The luminescent signal was detected on the machine, and the RLU reading was recorded. The results are shown in Table 3.

Table 3. In vitro activation reporter gene results for anti-OX40 antibodies

The results showed that the chimeric antibodies Ch2G3 and Ch4B5 could effectively activate the reporter gene.

Example 6: Mouse Antibody Humanization Experiment

To reduce possible immunogenicity, murine antibodies were 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 mutation in the FR (framework region) region. According to different amino acid mutation combinations, different humanized antibody heavy and light chains are designed, and humanized antibodies can be produced by transfecting cells with plasmids encoding different light/heavy chain combinations. It is briefly described as follows:

First design the expression vector:

The heavy chain vector was designed as follows: signal peptide + mutated heavy chain variable region sequence + human IgG1 constant region sequence.

The light chain vector was 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), respectively. 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 be sequenced for verification. The qualified plasmid was transfected into human 293F cells with PEI, and cultured continuously. The 293F cells were cultured with serum-free medium (Shanghai Opmax Bio, OPM-293 CD03) to logarithmic growth phase for cell transfection. Dissolve 21.4μg of plasmid encoding humanized antibody light chain and 23.6μl of plasmid encoding humanized antibody heavy chain in 10ml Opti-MEM® I Reduced Serum Medium (GIBCO, 31985-070) and mix well, then add 200μg PEI , Mix well, incubate at room temperature for 15 min, and add to 50 mL of cells.

Cell culture conditions: 5% CO ₂ , 37° C., 125 rpm/min. During the culture, feeding was added on the 1st and 3rd days until the cell viability was lower than 70%, and the cell supernatant was collected and filtered by centrifugation. The cell culture solution after centrifugal filtration was loaded onto the antibody purification affinity column, and the column was washed with phosphate buffer, eluted with glycine hydrochloride buffer (pH2.7 0.1M Gly-HCl), and 1M Tris hydrochloric acid pH9. 0 neutralization and phosphate buffer dialysis to finally obtain purified humanized antibodies.

The humanized variable region sequences are as follows:

>hu2G3 VH1

SEQ ID NO：17

SEQ ID NO: 17

>hu2G3 VH2

SEQ ID NO：18

SEQ ID NO: 18

>hu2G3 VL1

SEQ ID NO：19

SEQ ID NO: 19

>hu2G3 VL2

SEQ ID NO：20

SEQ ID NO: 20

Note: 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.

The variable regions of the designed humanized antibodies were combined into different antibodies according to the following table:

Table 4. m2G3 humanized antibody and its heavy and light chain variable regions

Note: 2G3-hu1 in Table 4 indicates that the variable region of the heavy chain of the humanized antibody 2G3-hu1 is hu2G3 VH1, the variable region of the light chain is hu2G3 VL1, and so on.

The humanized variable region sequence of m4B5 is as follows:

hu4B5 VL0:

SEQ ID NO：21

SEQ ID NO: 21

hu4B5 VL1:

SEQ ID NO：22

SEQ ID NO: 22

hu4B5 VL2:

SEQ ID NO：23

SEQ ID NO: 23

hu4B5 VL3:

SEQ ID NO：24

SEQ ID NO: 24

hu4B5 VL4:

SEQ ID NO：25

SEQ ID NO: 25

hu4B5 VH0:

SEQ ID NO：26

SEQ ID NO: 26

hu4B5 VH1:

SEQ ID NO：27

SEQ ID NO: 27

hu4B5 VH2:

SEQ ID NO：28

SEQ ID NO: 28

hu4B5 VH3:

SEQ ID NO：29

SEQ ID NO: 29

hu4B5 VH4:

SEQ ID NO：30

SEQ ID NO: 30

The variable regions of the designed humanized antibodies were combined into different antibodies according to the following table:

Table 5. The correspondence between m4B5 humanized antibodies and their heavy and light chain variable regions

Note: Hu4B5 V1 indicates that the variable region of the heavy chain of the humanized antibody hu4B5 V1 is hu4B5 VH0, the variable region of the light chain is hu4B5 VL0, and so on.

In order to obtain a better humanized antibody of m2G3, the amino acid sequence of hu2G3 VH1 was mutated. The mutated heavy chain variable region sequence is as follows:

hu2G3 VH1.1

SEQ ID NO：31

SEQ ID NO: 31

>hu2G3 VH1.2

SEQ ID NO：32

SEQ ID NO: 32

The HCDR2 sequence obtained after mutation is as follows:

Table 6. HCDR2 sequences obtained after mutation

The heavy chain variable regions hu2G3 VH1.1, hu2G3 VH1.2 were combined with the light chain variable region hu2G3 VL to form new optimized humanized antibodies, see Table 7.

Table 7. Light/heavy chain variable regions of novel humanized hu2G3 antibodies obtained after mutation

The above-mentioned humanized antibodies were subjected to affinity evaluation (refer to the capture ELISA in Example 2), and the experimental results showed that the humanized molecules could bind to OX40.

Exemplary m2G3 and its humanized antibody affinity assessment (capture ELISA) results are shown in Table 8:

Table 8. Antibody Affinities

The capture ELISA detection results of exemplary m4B5 and its engineered antibodies are shown in Table 9:

Table 9. Antibody Affinities

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 limited to the antibody constant regions of the present disclosure, and other known light and heavy chain constant regions and their mutants in the art can also be used to increase the performance of the antibody.

An exemplary constant region is shown below:

IgG1 heavy chain constant region:

SEQ ID NO：35

SEQ ID NO: 35

kappa light chain constant region:

SEQ ID NO：36

SEQ ID NO: 36

An exemplary 2G3, hu4B5-V7 (also known as 4B5) antibody full-length amino acid sequence is as follows:

2G3 heavy chain:

SEQ ID NO：37

SEQ ID NO: 37

2G3 light chain:

SEQ ID NO：38

SEQ ID NO: 38

4B5 heavy chain:

SEQ ID NO：39

SEQ ID NO: 39

4B5 light chain:

SEQ ID NO：40

SEQ ID NO: 40

The positive reference GPX4 was prepared with reference to 1A7.gr.1 in the patent application WO2015153513, and its heavy and light chain amino acid sequences are as follows: GPX4 heavy chain:

SEQ ID NO：41

SEQ ID NO: 41

GPX4 light chain:

SEQ ID NO：42

SEQ ID NO: 42

Example 7: In vitro binding affinity and kinetic assays of humanized antibodies

The Biacore method is a recognized method for objectively detecting the affinity and kinetics of proteins. We characterized the affinity and binding kinetics of the tested OX40 antibodies of the present invention by Biacore T200 (GE) analysis.

Using the kit provided by Biacore, the recombinant anti-OX40 antibody to be tested of the present invention was covalently linked to a CM5 (GE) chip by NHS standard amine coupling method. Then, a series of concentration gradients of human OX40-His protein (Yiqiao Shenzhou #10481-H08H) diluted in the same buffer were injected in each cycle at a flow rate of 30 μL/min. Reagent regeneration. Antigen-antibody binding kinetics were tracked for 3 minutes and dissociation kinetics were tracked for 10 minutes. GE's BIAevaluation software used to 1: 1 (Langmuir) binding model resulting data analysis, determination of this chimeric antibody _{ka (k on), kd (} k off) , and K _D values are shown in the following table.

Table 10. In vitro binding affinity results for humanized antibodies

The results showed that 2G3 and 4B5 could effectively bind OX40.

Example 8: Antibody blocking OX40 and OX40L binding ELISA test

The well plate was coated with the ligand OX40 ligand (Acrobiosystem, OXL-H52Q8), and after blocking, the antibody to be tested was added in a series of dilutions (antibody diluted in human Bio-OX40-FC (Acrobiosystem, OX40-H5255, labeled with biotin) ). Add the plate after pre-incubating in the solution for 40 min), and wash the plate after incubating for 40 min. Then SA-HRP (Jackson Immunoresearch, 016-030-084) was added, and after 40 minutes of incubation, chromogenic solution and stop solution were added, and the OD value was measured. The results are shown in the following table.

Table 11. Results of antibody blocking the binding of OX40 and OX40L

The results showed that both 2G3 and 4B5 could block the binding of OX40 to OX40L.

Example 9: Humanized Antibody Activity Data

Plates were coated with anti-CD3 antibody (Chempartner, A05-001), placed overnight at 4°C, and washed 3 times with PBS; Jurkat-NF-Kb luc-hOX40 cells (ATCC, TIB-152 (stable cell line by Wisdom Chemical Company) were harvested. Construct) and Raji cells (ATCC, CCL-86), resuspend and mix the two cells. Add 50 μl/well of the diluted antibody to be tested, and 50 μl/well of the mixed cells to the well plate, and the cell plate is placed at 37 Incubate for 5 hours in a 5% CO ₂ incubator at ℃. Add one-Glo TM luciferase reagent (Promega, Cat#E6120) to each well and incubate for more than 3 min at room temperature. Detect the luminescent signal on the computer and record the RLU reading The results are shown in Table 12, the results show that 2G3 and 4B5 can effectively activate the reporter gene.

Table 12. Results of humanized antibody activation reporter gene experiments

Example 10: In vitro cell function test

The CD4+ memory T cells were isolated, added to the 96-well plate coated with anti-CD3 antibody (Chempartner, A05-001) with the antibody to be tested, incubated at 37°C for 72h, and the supernatant was collected to detect IFN-γ. The results are shown in Figure 2 and Table 2. 13 shown. The results show that GPX4, 4B5 and 2G3 can significantly enhance the release of IFN-γ, and 2G3 can achieve the maximum stimulation effect at 10ng/mL.

Table 13. IFNγ (pg/mL) values corresponding to different antibodies

Note: The numerical calculation method is: mean +/- SEM (mean +/- standard error of the mean), N=3 (three replicates).

Example 11: Inhibition of tumor cell growth by anti-OX40 antibodies

B-hTNFRSF4(OX40) humanized mouse (B-hTNFRSF4(OX40) humanized mouse, Biositu Jiangsu Gene Biotechnology Co., Ltd.), female, 17 to 20 g, 6 to 7 weeks old.

Collect logarithmic growth phase MC38 tumor cells 7 (purchased from Nanjing Yinhe Biopharmaceutical Co., Ltd.), adjust the cell concentration to 5×10 ⁶ /mL with PBS buffer, and inoculate 0.1 mL of cell suspension into the flank of OX40 mice. The mice after inoculation were observed and tumor growth was monitored. On the 7th day after inoculation, the average tumor volume in the flank of tumor-bearing mice reached 102.5 mm ³ .

Table 14. Mice grouping and dosing table

To detect the growth inhibitory effect of OX40 humanized antibody on mouse MC38 colon cancer cell xenografts.

Measurement of tumor volume and body weight of tumor-bearing mice: use vernier calipers to measure twice a week, the formula for tumor volume is V=0.5 a×b ² , a, b represent the long and wide diameters of the tumor, respectively; tumor growth and transplanted tumor TGI (% )=[1-T/C]×100. Body weights of all tumor-bearing mice were measured twice a week.

On the 20th day after administration, the average tumor volume of the IgG1 control group reached 1732.593 mm ³ , and the average tumor volume of the test compound 2G3 low-dose administration group (0.3 mg/kg) reached 930.37 mm ³ . The average tumor volume of tumor-bearing mice in the drug group (2G3, 1 mg/kg) and high-dose administration group (2G, 3 mg/kg) were 303.49 mm ³ and 155.79 mm ³ , respectively. The middle and high dose groups inhibited tumor growth differently from the control group. Significant (** P < 0.01 ), and showed a preliminary dose-dependent relationship, the tumor growth inhibition rate reached 49%, 88% and 97.0%, respectively. The average tumor volume in the 3 mg/kg GPX4 group was 362.47 mm ³ , which was significantly different from that in the control group. It also showed a significant inhibitory effect on tumor growth (* P < 0.05 ), and the tumor growth inhibition rate reached 84%.

The experiment was terminated on the 20th day after administration, and the results are shown in Table 15 and Figures 3A and 3B. All treated mice were euthanized, and tumor-bearing mice were excised and weighed subcutaneously. The average tumor mass weight of the control group was 1.568 g, and the average tumor weight of the test compound 2G3 in the low-dose group (0.3 mg/kg), medium-dose group (1 mg/kg) and high-dose group (3 mg/kg) was 0.926, respectively. g, 0.251g and 0.181g, among which the middle and high dose groups were significantly different from the control group, and the tumor growth inhibition effect was obvious (** P < 0.01) . At the same time, the average tumor weight of GPX4 3 mg/kg in the administration group was 0.372 g, which was significantly different from that in the control group, and also showed a significant inhibitory effect on the growth of MC38 tumor cells (** P < 0.01 ).

During the experiment, there was no obvious abnormality in the body weight of all the tumor-bearing mice treated, and no obvious abnormal behavior and other manifestations were found during the drug treatment.

Table 15. Antitumor effect in mice

The following is a stable preparation of anti-OX40 antibody prepared by experiments (exemplarily, the anti-OX40 antibody in the following preparation is the aforementioned 2G3)

The methods and equipment used in the detection of antibody preparations are as follows:

SEC Size Exclusion Chromatography: An analytical method for the separation of solutes based on the relative relationship between the pore size of the gel pores and the coil size of the polymer sample molecules. SEC monomer content percentage = A monomer / A total * 100% ("A monomer" is the peak area of the main peak monomer in the sample, "A total" is the sum of all peak areas). Instrument for SEC measurement: Agilent 1260; tube column: waters, XBrige BEH200Å SEC (300×7.8 mm 3.5 μm).

CE capillary gel electrophoresis: a method in which the gel is moved into a capillary as a support medium for electrophoresis, and is separated according to the molecular weight of the sample under a certain voltage. Non-reduced 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, "A total" is the sum of all peak areas). Instrument for CE determination: Beckman, model plus800.

iCIEF Imaging Capillary Isofocus Electrophoresis (iCE): A technique for separating proteins based on their isoelectric point pI. iCIEF neutral peak content percentage = neutral peak area / total area * 100% (total area is the sum of the areas of acidic peaks, neutral peaks and basic peaks). Instrument used for iCIEF assay: simple protein, model muarice.

Osmotic pressure: The freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point drop value and the molar concentration of the solution, a high-sensitivity temperature sensing element is used to measure the freezing point of the solution, and the electricity is converted into osmotic pressure. Instrument manufacturer Roser Loser, model OM815.

Example 12: Screening of anti-OX40 antibody preparation buffer system

A 2G3 formulation containing 0.1 mg/ml polysorbate 80 (PS80) and a protein concentration of 50 mg/mL was prepared in a series of buffers at pH 5.0-7.5, wherein. The buffer systems in this experiment were 10 mM sodium acetate (abbreviated AA) at pH 5.0, pH 5.5, 10 mM sodium succinate (abbreviated SA) at pH 5.0, pH 5.5, pH 6.0, 10 mM citric acid at pH 5.5, pH 6.0, pH 6.5 Citrate sodium salt (abbreviated CA), pH5.5, pH6.0, 10 mM histamine hydrochloride (abbreviated His), pH5.5, pH6.0, pH6.5, 10 mM histamine hydrochloride (abbreviated His), pH6.0, pH6.5, pH7.0, pH7.5 Sodium Phosphate (abbreviated PB). Each formulation was filtered, filled, stoppered, and capped. The samples were placed under high temperature conditions of 40°C for 14 days or subjected to forced degradation experiments by shaking (25°C, 300 rpm), and the stability of the formulations was investigated with appearance, SEC, IEC, and non-reduced CE-SDS as evaluation indicators.

The experimental results are shown in Table 16. Among them, the appearance data shows that under the forced degradation conditions of 40℃ high temperature or shaking, the appearance of the protein preparation of AA and His buffer system is better; SEC data shows that AA, His, His, etc. The stability of the protein preparation in the CA buffer system is better; CE-SDS data show that the histone preparations such as pH 5.0 or pH 5.5 AA, pH 5.0 SA, pH 6.0 His and other histone preparations under high temperature or shaking conditions at 40 °C The stability is relatively good; IEC data shows that the protein preparations of AA with pH 5.5, His with pH 6.0/pH 6.5, and SA with pH 6.0 under the high temperature condition of 40°C are slightly better. Considering comprehensively, the 2G3 protein preparation is preferably a histamine or acetate buffer system, His pH 6.0 or pH 5.5 AA can be selected, and pH 6.0 His can be selected.

Table 16. Results of pH and buffer system screening experiments

Remarks: "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 the opalescence or the more granularity, and vice versa ; N/A means not detected.

Example 13: pH value screening of anti-OX40 antibody preparation buffer system

2G3 formulations containing 0.1 mg/ml PS80 were prepared at a protein concentration of 50 mg/mL in 10 mM histamine hydrochloride (abbreviated His) buffer at various pHs. details as follows:

1) 10mM His (pH 5.5)

2) 10mM His (pH 6.0)

3) 10mM His (pH 6.5)

Each formulation was filtered, filled, stoppered, and capped. The samples were subjected to forced degradation experiments under the high temperature condition of 40 ℃ for 1 month, and the stability of the preparations was investigated with SEC, IEC and non-reducing CE-SDS as evaluation indicators. The experimental results are shown in Table 17. The experimental results showed that the protein preparations of each group of His buffer solutions with pH 5.5, pH 6.0 and pH 6.5, placed at a high temperature of 40 °C for 1 month, had no significant difference between the SEC/CE/IEC groups. difference, the IEC of the His group at pH 5.5 was slightly lower. Taking into account comprehensively, the optimal buffer pH range of the formulation is 5.5-6.5, and pH 6.0 is selected for further experiments.

Table 17. Screening results of pH value of formulation buffer system

Remarks: T0 means the beginning of the experiment, "M" means month, for example M1 means one month.

Example 14: Screening of other excipients in anti-OX40 antibody preparations

2G3 formulations with a protein concentration of 50 mg/mL were prepared in 10 mM histamine hydrochloride (abbreviated as His) buffer containing the following 1)-7) different kinds of excipients and pH 6.0. The specific accessories are as follows:

1) 50mg/mL sucrose, 0.1mg/mL polysorbate 20 (PS20);

2) 50mg/mL sucrose, 0.4mg/mL PS20;

3) 50mg/mL sucrose, 0.1mg/mL polysorbate 80 (PS80);

4) 50mg/mL sucrose, 0.4mg/mL PS80;

5) 50mg/mL trehalose, 0.4mg/mL PS80;

6) 90mg/mL sucrose, 0.4mg/mL PS80;

7) 90 mg/mL trehalose, 0.4 mg/mL PS80.

Each formulation was filtered, filled, stoppered, and capped. The samples were placed under high temperature conditions of 40°C for 13 days or subjected to forced degradation experiments by shaking (25°C, 300 rpm, 13 days), and the stability of the formulation was investigated with appearance, SEC, IEC, and non-reduced CE-SDS as evaluation indicators. The experimental results are shown in Table 18.

Table 18. Results of screening experiments for surfactants and sugars in formulations

Remarks: "D" in the table represents days, for example, D13 represents 13 days; T0 represents the beginning of the experiment.

The experimental results show that there is no significant difference in CE and IEC data among the groups for the type and concentration of surfactants, the SEC of the 0.1mg/ml PS80 group is slightly lower, and the appearance of the 0.1mg/ml PS20 and 0.1mg/ml PS80 groups after forced degradation. Opalescence or particles appeared. The 0.4mg/ml PS80 group had a good appearance under all conditions. In addition, the forced degradation samples were placed at 4°C for a period of time to check the appearance. The 0.4mg/ml PS20 group was shaken and 40°C samples. Particles appeared in all cases; therefore, 0.4mg/ml PS80 was selected; for sugar type and concentration: there was no significant difference between the groups under each condition, and the 50mg/mL-90mg/mL groups had better stability of sucrose and trehalose. In addition, when the sucrose concentration is 80 mg/ml, the sample osmotic pressure is 304 mosm, which is close to the osmotic pressure in the human body.

Example 15: Stability test of anti-OX40 antibody preparations

A 2G3 formulation was prepared containing protein concentration 50 mg/ml, 10 mM histamine hydrochloride (His) pH 6.0, 75 mg/ml sucrose, 0.4 mg/ml PS80.

The samples were placed under the conditions of 25°C and 4°C for a long time, and SEC, IEC, and non-reduced CE-SDS were used as evaluation indicators to investigate the stability of the formulation. The results are shown in Table 19.

Table 19. Results of formulation stability experiments

Remarks, M means month, such as M3 means 3 months; T0 means the beginning of the experiment.

The experimental results showed that: under the condition of 25℃, the IEC of 2G3 preparation decreased slightly, but the decrease range 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 each test item. The formulation has good stability.

Example 16: Prescription optimization experiment of anti-OX40 antibody preparations

The DOE experimental design was carried out with 10mM histamine hydrochloride (His) buffer pH value, protein concentration and polysorbate 80 concentration as variables. mg/mL, protein concentration of 30~70mg/mL, a series of formulations were designed (see Table 20), and the forced degradation experiment was placed under 40 high temperature conditions for 13 days. Appearance, SEC, IEC were used as evaluation indicators, and the least squares method was used. Statistical analysis was performed on the results, and the results are shown in Table 21 and FIG. 4 .

Table 20. Prescription Design of DOE Prescription Screening Experiment

Note: 80mg/ml sucrose was added to all formulations to increase the stability of the preparation.

Table 21. DOE Screening Experiment Results

Remarks, D means days, such as D13 means 13 days; D0 means the beginning of the experiment, N/A means no detection.

The forced degradation data were fitted, and the pH drift value (the difference between the pH of the preparation and the buffer) and the SEC and IEC drop values were well fitted, and the model was valid. The fitting results are shown in Figure 4.

The pH drift fitting results show that the pH shift is related to the protein concentration. When the protein concentration is higher than 50 mg/ml, the pH shift is 0.2, and when the protein concentration is 30 mg/ml, the pH shift is 0.1. The decrease of IEC is within the acceptable range, and the formulation stability is good. Therefore, the protein concentration of the preparation is 30-70 mg/ml, 0.2-0.6 mg/ml PS80, and pH 5.5-6.5. In addition, the SEC and IEC are slightly different after being placed under high temperature conditions of 40 °C for 13 days. The lower the pH, the better the SEC. The IEC with pH 5.9-6.2 is the best. Based on the above data, the following protein concentrations are selected as 50 mg/ml and 0.4 mg/ml. The formulation of PS80, 10 mM His pH 6.0 was further tested.

Example 17: Stability test of anti-OX40 antibody preparations

A 2G3 formulation with a protein concentration of 50 mg/ml, 10 mM histamine hydrochloride (abbreviated as His) pH 6.0, 80 mg/ml sucrose, and 0.4 mg/ml PS80 was prepared.

The samples were placed under a high temperature condition of 40 °C or subjected to a forced degradation test by shaking (25 °C, 300 rpm), and the appearance, SEC, IEC, and non-reducing CE-SDS were used as evaluation indicators to investigate the stability of the preparation. The experimental results are shown in Table 22. -1.

Table 22-1. Protein formulation stability results

Remarks, D means days, such as D13 means 13 days; T0 means the beginning of the experiment; M means months, such as M1 means 1 month.

The experimental results showed that the SEC/CE/IEC changed little and the preparation stability was good under the conditions of freezing and thawing, high temperature at 40°C or shaking (25°C, 300rpm).

In addition, the samples were placed at 4°C for 1 year, and the stability of the preparation was investigated with appearance, SEC, IEC, and non-reduced CE-SDS as evaluation indicators. The experimental results are shown in Table 22-2. The experimental results show that the solution preparation After being placed at 4°C for 1 year, the appearance, SEC, IEC, and non-reduced CE-SDS of the preparation were not significantly different from those at the beginning, and the stability of the preparation solution was very good.

Table 22-2. Protein formulation stability results

Remarks: M12 means 12 months

Example 18: Lyophilization and reconstitution experiments of anti-OX40 antibody preparations

1. Lyophilized preparation of anti-OX40 antibody preparation

Prepare a lyophilized preparation of 2G3 with a protein concentration of 50 mg/ml, 10 mM His pH 6.0, 80 mg/ml sucrose, and 0.4 mg/ml PS80. The lyophilized preparation process: prepare the sample according to the above prescription, filter the sample aseptically, and fill it with cillin In the bottle, half stopper, pre-freeze, primary drying and secondary drying according to the procedure in the table below, after the freeze-drying procedure, vacuum stopper.

Table 23. Protein formulation lyophilization steps

Out of the box after lyophilization, the appearance of the sample: white powder, full appearance, no collapse. Moisture <1.0%. Therefore the lyophilization procedure is good.

2. Reconstitution of lyophilized preparations of anti-OX40 antibody preparations

The lyophilized preparation was reconstituted with water for injection to obtain a reconstituted solution of 2G3 with a protein concentration of 50 mg/ml, 10 mM His pH 6.0, 80 mg/ml sucrose, and 0.4 mg/ml PS80. After being placed at ~8°C for 24 hours, the appearance, SEC, non-reduced CE-SDS and IEC were used as evaluation indicators to examine the quality change of the preparation. The experimental results are shown in Table 24. The results show that the stability of the reconstituted solution is good when placed at room temperature or 2~8 ℃ for 24 hours.

Table 24. Results of the stability test of the reconstituted solution

<110> JIANGSU HENGRUI MEDICINE CO.,LTD.

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<221> Domain

<222> (1)..(119)

<223> Heavy chain variable region VH1.2 of humanized monoclonal antibody hu2G3

<400> 32

<210> 33

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<222> (1)..(16)

<223> HCDR2V1 of monoclonal antibody hu2G3

<400> 33

<210> 34

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<222> (1)..(16)

<223> HCDR2V2 of monoclonal antibody hu2G3

<400> 34

<210> 35

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<222> (1)..(330)

<223> IgG1 heavy chain constant region

<400> 35

<210> 36

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<222> (1)..(107)

<223> kappa light chain constant region

<400> 36

<210> 37

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<222> (1)..(449)

<223> Heavy chain of antibody 2G3

<400> 37

<210> 38

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<222> (1)..(214)

<223> Light chain of antibody 2G3

<400> 38

<210> 39

<211> 443

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<222> (1)..(443)

<223> Heavy chain of antibody 4B5

<400> 39

<210> 40

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Domain

<222> (1)..(214)

<223> Light chain of antibody 4B5

<400> 40

<210> 41

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<222> (1)..(447)

<223> Heavy chain of control antibody GPX4

<400> 41

<210> 42

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Chain

<222> (1)..(214)

<223> Light chain of control antibody GPX4

<400> 42

Claims (20)

A pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof, and a buffer, the buffer being a histamine buffer or an acetate buffer, and the buffer has a pH of about 5.0 to about 6.5, preferably The pH is about 5.5 to about 6.5, preferably about 6.0; the histamine hydrochloride buffer is preferably a histamine hydrochloride buffer, and the acetate buffer is preferably a sodium acetate buffer. The pharmaceutical composition of claim 1, wherein the buffer concentration is about 5 mM to about 30 mM, preferably about 5 mM to about 15 mM, more preferably about 10 mM. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the anti-OX40 antibody or its antigen-binding fragment is about 1 mg/ml to about 100 mg/ml, preferably about 30 mg/ml to about 70 mg/ml, more Preferably about 50 mg/ml. The pharmaceutical composition according to any one of claims 1 to 3, further comprising sugar, and the sugar is preferably sucrose or trehalose. The pharmaceutical composition of claim 4, wherein the sugar concentration is about 50 mg/ml to about 90 mg/ml, preferably about 75 mg/ml to about 85 mg/ml, more preferably about 80 mg/ml. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a surfactant, and the surfactant is preferably polysorbate, more preferably polysorbate 80. The pharmaceutical composition of claim 6, wherein the concentration of the surfactant is about 0.1 mg/ml to about 1.0 mg/ml, preferably about 0.2 mg/ml to about 0.6 mg/ml, more preferably about 0.4mg/ml. The pharmaceutical composition according to any one of claims 1 to 7, comprising: a) an anti-OX40 antibody or antigen-binding fragment thereof at a protein concentration of about 1 mg/ml to about 100 mg/ml; b) a histidine buffer having a pH of about 5.0 to about 6.5 or an acetate buffer having a pH of about 5.0 to about 5.5; c) sugar at a concentration of from about 50 mg/ml to about 90 mg/ml; and d) polysorbate 80 at a concentration of from about 0.1 mg/ml to about 1.0 mg/ml; Preferably, the 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 buffer at a pH of from about 5.5 to about 6.5; c1) sucrose at a concentration of about 75 mg/ml to about 85 mg/ml; and d1) Polysorbate 80 at a concentration of about 0.2 mg/ml to about 0.6 mg/ml. A pharmaceutical composition comprising: about 10 mM histamine hydrochloride buffer at a pH of about 6.0, an anti-OX40 antibody or antigen-binding fragment thereof at a concentration of about 50 mg/ml, sucrose at a concentration of about 80 mg/ml, and a concentration of about 0.4 mg/ml polysorbate 80. The pharmaceutical composition according to any one of claims 1 to 9, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein, (A) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 11, 33, 13, respectively, and the light chain variable region comprises as SEQ ID NOs: 14, 15, 16, respectively The light chain LCDR1, LCDR2, LCDR3 shown; (B) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 11, 12, 13, respectively, and the light chain variable region comprises as SEQ ID NOs: 14, 15, 16, respectively Show light chain LCDR1, LCDR2, LCDR3; (C) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 3, 4, and 5, respectively, and the light chain variable region comprises HCDR1, as shown in SEQ ID NOs: 6, 7, and 8, respectively LCDR1, LCDR2, LCDR3 shown; or (D) the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 11, 34, 13, respectively, and the light chain variable region comprises SEQ ID NOs: 14, 15, 16, respectively The light chains LCDR1, LCDR2, LCDR3 shown. The pharmaceutical composition of claim 10, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises: (E) a heavy chain variable region as set forth in SEQ ID NO: 17, 18, 31 or 32 or having at least 95% sequence identity to SEQ ID NO: 17, 18, 31 or 32, and as in SEQ ID NO: A light chain variable region shown in 19 or having at least 95% sequence identity with SEQ ID NO: 19; (F) a heavy chain variable region as set forth in SEQ ID NO:9 or having at least 95% sequence identity to SEQ ID NO:9, and a heavy chain variable region as set forth in SEQ ID NO:10 or having at least 95% sequence identity to SEQ ID NO:10 A light chain variable region of 95% sequence identity; (G) a heavy chain variable region as set forth in SEQ ID NO: 1 or having at least 95% sequence identity to SEQ ID NO: 1, and a heavy chain variable region as set forth in SEQ ID NO: 2 or having at least 95% sequence identity to SEQ ID NO: 2 A light chain variable region of 95% sequence identity; (H) a heavy chain variable region as set forth in SEQ ID NO: 17, 18, 31 or 32 or having at least 95% sequence identity with SEQ ID NO: 17, 18, 31 or 32, and as in SEQ ID NO: A light chain variable region shown in 20 or having at least 95% sequence identity with SEQ ID NO: 20; or (I) a heavy chain variable region as set forth in SEQ ID NO: 26, 27, 28, 29 or 30 or having at least 95% sequence identity with SEQ ID NO: 26, 27, 28, 29 or 30, and as in A light chain variable region set forth in SEQ ID NO: 21, 22, 23, 24 or 25 or having at least 95% sequence identity to SEQ ID NO: 21, 22, 23, 24 or 25; Preferably, the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:31 and a light chain variable region as shown in SEQ ID NO:19. The pharmaceutical composition of claim 11, wherein the anti-OX40 antibody comprises a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region is derived from human IgG1, IgG2, IgG3 or IgG4 or Its mutated sequence; the light chain constant region is derived from human kappa, lambda chain or its mutated sequence; more preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 35 or with SEQ ID NO: 35 At least 90% sequence identity, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:36 or has at least 90% sequence identity with SEQ ID NO:36. The pharmaceutical composition of claim 12, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises: (J) a heavy chain as set forth in SEQ ID NO:37 or having at least 85% sequence identity to SEQ ID NO:37, and/or as set forth in SEQ ID NO:38 or having at least 85% sequence identity to SEQ ID NO:38 % sequence identity of the light chain; or (K) a heavy chain as set forth in SEQ ID NO:39 or having at least 85% sequence identity to SEQ ID NO:39, and/or as set forth in SEQ ID NO:40 or having at least 85% sequence identity to SEQ ID NO:40 % sequence identity of the light chain; Preferably, the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:38. A method for preparing the pharmaceutical composition according to any one of claims 1 to 13, the method comprising the step of buffer-replacing a stock solution of an anti-OX40 antibody or an antigen-binding fragment thereof. A freeze-dried preparation containing an anti-OX40 antibody or an antigen-binding fragment thereof, the freeze-dried preparation obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 13. A reconstituted solution containing an anti-OX40 antibody or an antigen-binding fragment thereof, the reconstituted solution is prepared by reconstituting the lyophilized preparation described in claim 15. A lyophilized preparation containing an anti-OX40 antibody or an antigen-binding fragment thereof, the lyophilized preparation can be reconstituted to form the pharmaceutical composition described in any one of claims 1 to 13, Preferably, the lyophilized preparation comprises an anti-OX40 antibody or an antigen-binding fragment thereof, a saccharide, a surfactant, and a histamine or acetate buffer, wherein the anti-OX40 antibody or an antigen-binding fragment thereof, a saccharide and a surface active agent The weight ratio of the agent is 1 to 100 parts by weight of the anti-OX40 antibody or its antigen-binding fragment: 50 to 90 parts by weight of sugar: 0.1 to 1.0 parts by weight of the surfactant, the anti-OX40 antibody or its Anti-OX40 antibody or antigen-binding fragment thereof whose substance ratio of antigen-binding fragment to buffer is 0.007 mol to 0.685 mol: 5 mol to 30 mol of histamine or acetate buffer . A product comprising a container containing the pharmaceutical composition as claimed in any one of claims 1 to 13 or the lyophilized preparation as claimed in claim 15 or 17 or the reconstitution as claimed in claim 16 solution. A method of treating or preventing a disease or disorder, the method comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the pharmaceutical composition according to any one of claims 1 to 13, or as claimed in 15 or 17 The lyophilized preparation, or the reconstituted solution as claimed in claim 16, or the product as claimed in claim 18; preferably, the disease or condition is cancer; more preferably, the cancer is selected from lung cancer, Prostate, breast, head and neck, esophagus, stomach, colon, colorectal, bladder, cervix, uterus, ovary, liver, melanoma, kidney, squamous cell and blood system cancer. A method of enhancing an immune response in a human subject, the method comprising: Administer to a subject a therapeutically effective amount or a prophylactically effective amount of the pharmaceutical composition as claimed in any one of claims 1 to 13, or the lyophilized preparation as claimed in claim 15 or 17, or as claimed in claim 16 The reconstituted solution described, or the product of claim 18; Preferably, the enhanced immune response includes an increase in the immunostimulatory/effector function of T effector cells, and/or a down-regulation of the immunosuppressive function of T regulatory cells.

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