TWI726426B - Formulation comprising anti-ox40 antibody, preparation method and use thereof - Google Patents

Abstract

The present invention relates to a formulation comprising an anti-OX40 antibody, in particular to a pharmaceutical formulation comprising an antibody specifically binding to an OX40 molecule and/or an antigen-binding fragment thereof, a buffer, a stabilizer, and a surfactant. Furthermore, the invention relates to a use of the formulation for the treatment or prophylaxis of diseases.

Description

Preparation containing anti-OX40 antibody, its preparation method and its use

The present invention relates to the field of antibody preparations. More specifically, the present invention relates to a pharmaceutical preparation comprising an antibody that specifically binds to OX40 (hereinafter also referred to as "anti-OX40 antibody") and/or an antigen-binding fragment thereof, a method for preparing the pharmaceutical preparation, and The therapeutic and/or preventive use of the pharmaceutical preparation.

Costimulatory molecules are a class of cell surface molecules that are required for lymphocytes to make an effective immune response to antigens, except for antigen receptors or antigen ligands. By specifically binding with costimulatory ligands, lymphocytes mediate co-stimulatory molecules. Stimulus response. It has been confirmed that costimulatory molecules enhance T cell expansion, effector function and survival in vitro; and enhance human T cell retention and anti-tumor activity in vivo.

OX40 (also known as CD134, TNFRSF4 and ACT35) is a costimulatory molecule and it has been shown that OX40 signaling can promote costimulatory signals to T cells, leading to enhanced cell proliferation, survival, effector function and migration (Gramaglia I et al. , Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol. 1998; 161: 6510-6517; Gramaglia I et al., The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion. J Immunol. 2000; 165: 3043-3050).

Anti-OX40 antibodies as OX40 agonists that specifically bind to OX40 are described in, for example, WO 2012/027328, US Patent No. 7,959,925, PCT Publication No. WO 2006/121810, and Chinese Patent Application Nos. 201710185399.9, 201710185400.8. There is a need in the art for anti-OX40 antibody preparations that can be used to treat, prevent or delay various cancers, immune-related diseases, and T cell dysfunction diseases.

The antibody preparation must be formulated in a way that not only makes the antibody suitable for administration to the subject, but also in a way that maintains its stability during storage and subsequent use. For example, if the antibody is not properly formulated in the liquid, the antibody in the liquid solution tends to decompose, aggregate, or undergo undesirable chemical modification. The stability of antibodies in antibody preparations depends on the buffers, stabilizers and surfactants used in the preparations.

Antibodies against OX40 are an example of the need to be appropriately formulated to treat or prevent diseases. Although some anti-OX40 antibodies are known, there is still a need in the art for novel pharmaceutical formulations containing anti-OX40 antibodies that are sufficiently stable and suitable for administration to a subject.

The present invention meets the above-mentioned needs by providing a pharmaceutical preparation containing an antibody that specifically binds to OX40.

In one aspect, the present invention provides a liquid antibody preparation comprising (i) an anti-OX40 antibody or an antigen-binding fragment thereof; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

In one embodiment, the concentration of the anti-OX40 antibody or antigen-binding fragment thereof in the liquid antibody preparation of the present invention is about 1-150 mg/mL. In another embodiment, the concentration of the anti-OX40 antibody or antigen-binding fragment thereof in the liquid antibody preparation of the present invention is about 10 mg/mL to about 100 mg/mL. In other embodiments, the concentration of the anti-OX40 antibody or antigen-binding fragment thereof in the liquid antibody preparation of the present invention is about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mg/mL.

In one embodiment, the anti-OX40 antibody is any antibody that binds to an OX40 molecule (such as a human OX40 molecule), such as a multi-strain antibody, a monoclonal antibody, or a combination of the two. Preferably, in one embodiment, the anti-OX40 antibody is a monoclonal antibody. In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof is an anti-OX40 antibody or antigen-binding fragment thereof as defined herein.

In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 0.1-50 mg/ml. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 1-20 mg/ml, for example, about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 , 7, 7.5, 8mg/ml.

In one embodiment, the buffer is selected from phosphate, citrate, citrate solvate, succinic acid, trimethylolaminomethane and combinations thereof, more preferably citrate, lemon Salt hydrates, for example, sodium citrate, sodium citrate dihydrate.

In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 10-200 mg/ml. In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 20-100 mg/ml, for example, about 30, 40, 50, 60, 70, 80, 90 mg/ml.

In one embodiment, the stabilizer is selected from sucrose, trehalose, mannitol and combinations thereof, more preferably sucrose.

In one embodiment, the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.01 to 5 mg/ml. In one embodiment, the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.1-2 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/ml.

In one embodiment, the surfactant is a nonionic surfactant. In one embodiment, the surfactant is, for example, pluronics, polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate-20, and the like.

In one embodiment, the pH of the liquid formulation is about 5.0-8.0. In one embodiment, the pH value of the liquid formulation is about 5.0, 6.0, 7.0, 8.0.

In one embodiment, the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection.

In a preferred embodiment, the liquid antibody preparation of the present invention contains (i) about 1-150 mg/mL of anti-OX40 antibody or antigen-binding fragment thereof; (ii) about 0.1-50 mg/ml of citrate, lemon Salt hydrates, for example, sodium citrate, sodium citrate dihydrate, as a buffer; (iii) about 10-200 mg/ml sucrose as a stabilizer, and (iv) about 0.01-5 mg/ml polysorbate Ester-80 acts as a surfactant; wherein the pH of the liquid formulation is about 5.0-8.0.

In a preferred embodiment, the liquid antibody preparation of the present invention contains (i) about 10-100 mg/mL anti-OX40 antibody or antigen-binding fragment thereof; (ii) about 1-20 mg/ml sodium citrate or two Sodium citrate water as a buffer; (iii) about 20-100 mg/ml sucrose as a stabilizer, and (iv) about 0.1-2 mg/ml polysorbate-80 as a surfactant; The pH of the liquid formulation is about 5.0-8.0.

In a preferred embodiment, the liquid antibody preparation of the present invention contains (i) about 20-30 mg/mL anti-OX40 antibody or antigen-binding fragment thereof; (ii) about 4-6 mg/ml sodium citrate or two Sodium citrate water as a buffer; (iii) about 40-60 mg/ml sucrose as a stabilizer, and (iv) about 0.6-0.8 mg/ml polysorbate-80 as a surfactant; wherein the liquid The pH of the formulation is about 6.0-7.0.

On the other hand, the present invention provides a solid antibody preparation, which is obtained by curing the liquid antibody preparation of the present invention. The curing treatment is performed by, for example, a crystallization method, a spray drying method, or a freeze drying method. In a preferred embodiment, the solid antibody preparation is, for example, in the form of a lyophilized powder injection.

The solid antibody preparation can be reconstituted in a suitable solvent before use to form the reconstituted preparation of the present invention. The reconstituted preparation is also a liquid antibody preparation of the present invention. In one embodiment, the appropriate solvent is selected from water for injection, organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.

In one embodiment, the liquid preparation or solid preparation containing the anti-OX40 antibody of the present invention is at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C. Store at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 under the conditions of ℃, about 25℃, about 35℃, about 38℃, about 40℃, about 42℃ or about 45℃ Months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 Month, at least 24 months, at least 36 months, or longer, use size exclusion high performance liquid chromatography to detect The purity of the OX40 antibody or antigen-binding fragment thereof is reduced by no more than 10%, for example, no more than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.

In one embodiment, the liquid preparation or solid preparation containing the anti-OX40 antibody of the present invention is at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C. Store at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 under the conditions of ℃, about 25℃, about 35℃, about 38℃, about 40℃, about 42℃ or about 45℃ Months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 Months, at least 24 months, at least 36 months, or longer, use non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method and/or reduced CE-SDS method to detect, anti-OX40 antibody The purity of the antigen-binding fragment thereof is reduced by no more than 10%, for example, no more than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.

In one embodiment, the liquid preparation or solid preparation containing the anti-OX40 antibody of the present invention is at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C. Store at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 under the conditions of ℃, about 25℃, about 35℃, about 38℃, about 40℃, about 42℃ or about 45℃ Months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 Months, at least 24 months, at least 36 months, or longer, after detection by cation exchange high performance liquid chromatography (CEX-HPLC), the changes in the charge variants of the anti-OX40 antibody or its antigen-binding fragment did not exceed 10%, such as not exceeding 5%, 4%, 3%, 2%.

In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof in the liquid formulation of the present invention can have a high affinity, such as 10 ^-7 M or less, preferably 10 ^-8 M to 10 ^-12 M. The K _D specifically binds to OX40, and thereby mediates the costimulatory response of the anti-OX40 antibody or antigen-binding fragment thereof.

In a preferred embodiment, the anti-OX40 antibody or antigen-binding fragment thereof in the liquid formulation of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein (a) the VH comprises ( i) As shown in SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 in the VH 3 complementarity determining region CDRs, (ii) selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17 The HCDR1 of the amino acid sequence is selected from SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36 The combination of the HCDR2 of the amino acid sequence of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43 and 44 ; or (iii) with respect to (i) or The sequence of (ii) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) sequence in the three CDRs; And/or (b) the VL comprises (i) three CDRs in the VL shown in SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123 and 124, (ii) LCDR1 selected from the amino acid sequence of SEQ ID NO: 45, 46, 47, 48, 49 and 50, LCDR2 selected from the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55 and 56, And a combination of LCDR3 with amino acid sequences selected from SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, 65, and 66; or (iii) relative to (i) or (ii) The sequence includes at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) sequence in the three CDRs.

In yet another preferred embodiment, the anti-OX40 antibody in the liquid formulation of the present invention comprises heavy chain variable region VH and/or light chain variable region VL, wherein (a) heavy chain variable region VH contains and The amino acid sequence selected from SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical amino acid sequences or consisting of them; and/or ( b) The light chain variable region VL contains at least 90%, 91%, 92% with an amino acid sequence selected from SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123 and 124 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity of the amino acid sequence or composed thereof.

In yet another preferred embodiment, the anti-OX40 antibody in the liquid formulation of the present invention comprises a heavy chain and/or a light chain, wherein the heavy chain comprises and is selected from the group consisting of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, The amino acid sequence of 154, 155, 156, 157, 158, 159, 160, 161 and 162 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or consisting of, the light chain comprises an amino acid sequence selected from SEQ ID NO: 163, 164, 165, 166, 167, 168, 169, 170, 171, and 172 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical amino acid sequences or consist of them.

In one aspect, the present invention provides a delivery device comprising the liquid antibody preparation or solid antibody preparation of the present invention described in the various embodiments of this specification.

In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe containing the liquid antibody preparation or solid antibody preparation of the present invention described in each embodiment of this specification, for example, for intravenous injection or intramuscular injection .

In one embodiment, the present invention relates to a method of delivering an anti-OX40 antibody to a subject, such as a mammal, comprising administering to the subject, such as a mammal, the liquid of the present invention as described in the various embodiments of this specification In the step of antibody preparation or solid antibody preparation, the delivery is performed by, for example, a delivery device using a pre-filled syringe.

The present invention further provides the use of the liquid antibody preparation or solid antibody preparation of the present invention to prepare a delivery device (such as a delivery device that activates T cells or induces T cell-mediated anti-tumor activity or enhances the body’s immune response in a subject). , Pre-filled syringes) or drugs, particularly for the treatment of a subject’s disease, such as cancer, such as lung cancer (e.g. non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer.

The present invention further provides a liquid antibody preparation or solid antibody preparation of the present invention, or a delivery device (eg, a pre-filled syringe) or a drug containing the liquid antibody preparation or solid antibody preparation, to a subject. The method of activating T cells or inducing T cell-mediated anti-tumor activity or enhancing the body’s immune response.

The present invention further provides a method for treating a subject by administering the liquid antibody preparation or solid antibody preparation of the present invention, or a delivery device (eg, a pre-filled syringe) or drug containing the liquid antibody preparation or solid antibody preparation, to a subject Patients’ diseases, such as cancer, such as lung cancer (e.g. non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer.

Other embodiments of the present invention will be made clear by referring to the detailed description hereinafter.

When read together with the following drawings, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the figure shows the currently preferred embodiment. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 shows the charge variant map of anti-OX40 antibody placed 10 days under heat stress at pH 5.0.

Figure 2 shows the charge variant map of the anti-OX40 antibody placed under heat stress for 10 days at pH 6.0.

Figure 3 shows the charge variant map of anti-OX40 antibody placed 10 days under heat stress at pH 7.0.

Figure 4 shows the charge variant map of anti-OX40 antibody placed 10 days under heat stress at pH 8.0.

Figure 5 shows the turbidity change graph of the anti-OX40 antibody preparation of the present invention after being placed under the temperature condition of 40°C±2°C.

Figure 6 shows the purity change graph of the anti-OX40 antibody preparation of the present invention as measured by the SEC-HPLC method after being placed under the temperature condition of 40°C±2°C.

Figure 7 shows the change in the acidic composition of the charge variant of the anti-OX40 antibody measured by the CEX-HPLC method after the anti-OX40 antibody preparation of the present invention is placed under the temperature condition of 40°C±2°C.

Figure 8 shows the change in the main components of the charge variant of the anti-OX40 antibody determined by the CEX-HPLC method after the anti-OX40 antibody preparation of the present invention is placed under the temperature condition of 40°C±2°C.

Figure 9 shows the change in the basic composition of the charge variant of the anti-OX40 antibody measured by the CEX-HPLC method after the anti-OX40 antibody preparation of the present invention is placed under the temperature condition of 40°C±2°C.

Detailed description of the invention

Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because the methods and conditions can be changed. In addition, the terms used herein are only for describing specific embodiments, and are not intended to be limiting.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those of ordinary skill in the art to which the present invention belongs. As used herein, the term "about" when used in conjunction with a numerical value means to encompass a numerical value within a range that has a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value. The term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.

I. Antibody preparations

The term "antibody preparation" refers to a preparation in a form that allows the biological activity of the antibody as an active ingredient to be effective, and does not contain unacceptably toxic extras to the subject to which the preparation will be administered Components. Such antibody preparations are usually sterile. "Pharmaceutically acceptable" excipients are those that can be reasonably administered to the mammal under test to provide an effective dose of the active ingredients used.

As used herein, the term "anti-OX40 antibody preparation" means a combination of at least one anti-OX40 antibody as an active ingredient and at least one inactive ingredient. After the combination, the anti-OX40 antibody as the active ingredient is suitable for therapeutic or prophylactic administration to humans or non-human animals. According to the present invention, the anti-OX40 antibody as the active ingredient in the preparation specifically binds to the OX40 molecule, and the generated signal transduction promotes the costimulatory signal to T cells, leading to enhanced cell proliferation, survival, effector function and migration.

The antibody preparation of the present invention can be sterile, homogeneous and/or isotonic, and the antibody preparation can be directly prepared as a liquid preparation in an aqueous form, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized preparation, Reconstitution (ie, reconstitution) by dissolving and/or suspending in a physiologically acceptable solution immediately before use. In some embodiments, the anti-OX40 antibody formulation is in the form of a liquid formulation, a lyophilized formulation, or a reconstituted formulation.

Antibodies are now widely used as active ingredients of drugs, including products HERCEPTIN ^TM (trastuzumab), RITUXAN ^TM (rituximab), SYNAGIS ^TM (palivizumab) and so on. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art.

A "sterile" preparation is a preparation that is sterile or free or essentially free of live microorganisms and their spores.

The terms "lyophilization process" and the term "freeze-drying process" are used interchangeably herein and should be considered synonymous.

The term "lyophilized preparation" refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

The term "reconstituted preparation" refers to a liquid preparation obtained by dissolving and/or suspending a solid preparation (for example, a lyophilized preparation) in a physiologically acceptable solution.

In a specific embodiment, the anti-OX40 antibody preparation of the present invention exhibits low to undetectable levels of antibody aggregation or degradation or chemical modification during manufacture, preparation, transportation and long-term storage, so that there is little or no anti-OX40. The biological activity of the antibody is lost, showing a high degree of stability. As used herein, the term "stable" antibody preparation is that the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation does not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time. Or about 99% of the structure or function of the antibody, the antibody preparation is considered "stable." In some embodiments, the anti-OX40 antibody formulation of the present invention substantially retains its physical and chemical stability after storage. The storage time is usually selected based on the expected shelf life of the formulation.

In some embodiments, the stability test is performed by performing various stress tests on the antibody preparation. These tests can indicate the extreme conditions that may be encountered during the manufacture, storage or transportation of the formulated antibody preparations, and can also indicate the acceleration of the instability of the antibodies in the antibody preparations under extreme conditions other than those during manufacture, storage or transportation. condition. For example, the formulated anti-OX40 antibody preparation is filled into a 5 mL glass bottle for shaking stress, freeze/thaw cycle (ie, freeze-thaw cycle) stress; the formulated anti-OX40 antibody preparation is filled into a glass vial to test Antibody stability under high temperature stress.

The term "room temperature" as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.

The term "high temperature stress" refers to the effect on the stability of the antibody preparation after a long storage time at room temperature or even higher temperature (for example, 40°C).

After a long storage period, if the appearance, color and/or clarity of the preparation is checked, or for example, when measured by UV light scattering or by size exclusion chromatography, the antibody in the preparation does not show aggregation, precipitation and / Or turbidity; or shows very little aggregation, precipitation and/or turbidity, the antibody "maintains its physical stability" in the formulation. Since the aggregation of antibodies in the preparation can potentially lead to an increased immune response in patients, it leads to safety issues. Therefore, there is a need to minimize or prevent aggregation of the antibody in the formulation.

In one embodiment, the stability of the formulation is measured by determining the percentage of non-aggregated and non-decomposed antibodies in the formulation after storage at a specific temperature for a specific time, wherein the percentage of non-aggregated and non-decomposed antibodies in the formulation The higher the stability, the higher the stability of the formulation. The percentage of non-aggregated and non-decomposed antibodies can be determined by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography).

When the phrase "acceptable degree of stability" is used herein, it means that after storage at a specific temperature for a specific time, at least about 92% of non-aggregated and non-degradable anti-OX40 antibodies are detected in the formulation. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable degree of stability means at least About 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-degradable anti-OX40 antibodies. When evaluating the stability, the specific temperature for storage of the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C. °C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if stored at about 40°C for 3 months, at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-degradable anti-OX40 antibodies are detected Form, the pharmaceutical preparation is considered stable. If stored at about 25°C for 3 months, at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-degradable anti-OX40 antibody forms are detected, The pharmaceutical preparation is considered stable. If stored at about 5°C for 9 months, at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-degradable anti-OX40 antibody forms are detected, The pharmaceutical preparation is considered stable.

In addition, after a long storage period, if the chemical structure of the antibody in the preparation is complete, the antibody "maintains its chemical stability" in the preparation. Most chemical instabilities result from the formation of covalently modified forms of antibodies (for example, charge variants of antibodies). In some embodiments, after the antibody preparation is stored, changes in the components of each charge variant of the anti-OX40 antibody are detected.

In one embodiment, the charge variant of the anti-OX40 antibody in the antibody preparation is determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, the peaks that eluted from the CEX-HPLC column earlier than the retention time of the main peak are marked as "acidic peaks", and those that eluted from the CEX-HPLC column later than the retention time of the main peak The dropped peaks are labeled as "basic peaks". In the CEX-HPLC method, the percentage of acidic component is determined by the ratio of the area of the acidic peak to the sum of the area of the main peak, acidic peak and the basic peak; It is determined by the ratio of the sum of the area of the alkaline peak; the percentage of the alkaline component is determined by the ratio of the area of the alkaline peak to the sum of the area of the main peak, acidic peak and alkaline peak. Without being bound by theory, it is believed that the deamidation of an antibody can make the antibody more negatively charged and thus more acidic relative to non-deamidated antibodies (see, for example, Robinson, N., Protein Deamidation, PNAS , April 16, 2002, 99(8): 5283-5288). When the term "acceptable degree of stability" is used herein, it means that after storage at a specific temperature for a specific period of time, up to about 25% of the antibody in the formulation is in a more acidic form. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, the acceptable degree of stability is expressed in After storage at a specific temperature for a specific time, up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in acidic form in the formulation. When evaluating the stability, the temperature for storing the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, or about 4°C. ℃-8℃, about 5℃, about 25℃ or about 45℃. For example, if stored at -80℃, -30℃ or -20℃ for 3 months, less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% antibody is in a more acidic form, the pharmaceutical preparation can be Considered to be stable. If stored at 5℃ for 9 months, less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% antibodies are in a more acidic form, then Pharmaceutical formulations can also be considered stable. If stored at 25℃ for 28 days, less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13 %, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% antibody is in a more acidic form, then the drug The formulation can also be considered stable. If after 28 days of storage at 37℃, less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% are detected , 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% antibodies are in a more acidic form, Then the pharmaceutical preparation can also be considered stable. If stored at 40℃ for 28 days, less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% are detected , 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% antibodies are in a more acidic form, Then the pharmaceutical preparation can also be considered stable.

In some embodiments, the stable anti-OX40 antibody formulations of the invention are administered to the subject parenterally. As used herein, the term "parenteral administration" means a mode of administration other than enteral and local administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, and intrasaccular , Intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions. In a specific embodiment, the anti-OX40 antibody formulation of the present invention is administered to a subject subcutaneously.

In some embodiments, the stable anti-OX40 antibody formulation of the present invention comprises: (i) an anti-OX40 antibody or antigen-binding fragment thereof that specifically binds to an OX40 molecule; (ii) a buffer; (iii) a stabilizer, and ( iv) Surfactant, the pH of the anti-OX40 antibody preparation is about 5.0-8.0.

(i). Anti-OX40 antibody or antigen-binding fragment thereof that specifically binds to OX40 molecule

The antibody preparation of the present invention may comprise an anti-OX40 antibody or antigen-binding fragment thereof that specifically binds to an OX40 molecule. As used herein, the term "OX40" is known to be a costimulatory molecule whose activation can lead to enhanced cell proliferation, survival, effector function and migration. The prior art discloses anti-OX40 antibodies as OX40 agonists. For example, WO 2012/027328 discloses the amino acid sequences of the heavy chain variable region and light chain variable region of anti-OX40 antibody mAb 106-222 and humanized 106-222 (Hu106); anti-OX40 antibody mAb 119- 122 and humanized 119-122 (Hu119) heavy chain variable region and light chain variable region amino acid sequences. In addition, the United States Lee No. 7,959,925, PCT Publication No. WO 2006/121810, and Chinese Patent Application Nos. CN201710185399.9 and CN201710185400.8 also disclose anti-OX40 antibodies as OX40 agonists. The anti-OX40 antibody can activate OX40, thereby inducing effector T lymphocyte proliferation, and promoting the immune response against tumor cells expressing tumor-associated antigen (TAA). The full text of said document is incorporated herein as a reference.

The term "costimulatory molecule" refers to a corresponding binding partner on T cells that specifically binds to a costimulatory ligand to mediate a costimulatory response (such as but not limited to proliferation) of the T cell. Co-stimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to antigen receptors or their ligands. In an embodiment of the invention, the costimulatory molecule is an OX40 molecule.

1μM，

100nM，

10nM，

1nM，

0.1nM，

0.01nM，或

0.001nM。又在一些實施方案中，所述抗OX40抗體能夠以高的親和力，例如以10^-7M或更小、較佳地以10^-8M至10^-12M的K_D特異性結合OX40，並由此介導共刺激反應。在本文中，當談及“抗OX40抗體”時，也包含抗OX40抗體的抗原結合片段。 The terms "anti-OX40 antibody", "anti-OX40", "OX40 antibody" or "OX40-binding antibody" refer to antibodies that are capable of binding to OX40 molecules with sufficient affinity so that the antibody can be used as a target Therapeutic and/or prophylactic agent of OX40 molecule. In one embodiment, the degree of binding of an anti-OX40 antibody to an unrelated, non-OX40 protein is less than about 10% of the binding of said antibody to OX40, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (K _D ) of the anti-OX40 antibody

1μM,

100nM,

10nM,

1nM,

0.1nM,

0.01nM, or

0.001nM. In some embodiments, the anti-OX40 antibody can specifically bind to OX40 with a high affinity, for example, with a K _{D of} ^{10 -7} M or less, preferably 10 ^-8 M to 10 ^-12 M, and This mediates the costimulatory response. In this article, when talking about "anti-OX40 antibody", it also includes antigen-binding fragments of anti-OX40 antibody.

An "antigen-binding fragment" of an antibody refers to a molecule different from a complete antibody, which contains a part of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibodies Body (e.g. scFv); single domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antigen-binding fragments.

As used herein, the term "epitope" refers to the part of an antigen (for example, OX40) that specifically interacts with an antibody molecule.

A "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point") area. CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundary of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. Assignment systems include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", based on the three-dimensional structure of antibodies and the topology of CDR loops. Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987) )), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (on the World Wide Web imgt.cines.fr/on), and affinity propagation clustering based on the use of a large number of crystal structures Clustering) North CDR definition.

However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the same antibody defined under different assignment systems can be variable The CDR sequences of the regions are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-part of the CDR. As those skilled in the art will understand, the residues of the rest of the CDR sequence can be determined by the structure of the antibody and protein folding. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residue of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.

The term "conservative amino acid residue substitutions" are those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains has been defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamine), and uncharged polar side chains (e.g., Glycine, aspartame, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, Leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. , Tyrosine, phenylalanine, tryptophan, histidine) of the amino acid. Therefore, one or more amino acid residues of the anti-OX40 antibody can be replaced by other amino acid residues from the same side chain family. Acid residues are replaced, and the function of the modified antibody can be tested, especially the same binding properties as the OX40 molecule. The change in the charge performance of the CDR surface is expected to affect the interface between the antibody and the solvent. Therefore, the substitution of non-conservative amino acid residues has an unpredictable effect on maintaining or improving the stability of the antibody in solution.

The "antibody or antigen-binding fragment thereof" applicable to the present invention includes, but is not limited to, multi-strain, mono-strain, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterozygous, chimeric , Humanized (especially grafted with CDR), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, fragments produced by Fab expression libraries, Fd, Fv, two Sulfide-linked Fv (dsFv), single-chain antibodies (e.g. scFv), diabodies or tetrabodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA90 (14), 6444-6448), nano Antibodies (nanobodies) (also called single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against the antibodies of the present invention), and epitope binding fragments of any of the above.

"Human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally speaking, the choice of human immunoglobulin VL or VH sequence is selected from the subtypes of variable domain sequences.

"Antibody in the form of IgG" refers to the form of IgG to which the heavy chain constant region of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in the form of IgG1 means that the Ig domain of its heavy chain constant region is the Ig domain of IgG1.

"Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source, which utilizes Human antibody library or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDR and amino acid residues derived from human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDRs correspond to those of the non-human antibody, and all or substantially all of the FR corresponds to those of human antibodies. The humanized antibody optionally may comprise at least a portion of the constant region of an antibody derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

The term "isolated antibody", as used herein, is intended to mean an antibody that is substantially free of other antibodies with different antigen specificities, for example, an isolated antibody that specifically binds to OX40 is substantially free of antibodies that specifically bind to antigens other than OX40 .

The term "specific binding" or similar terms means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. The specific binding is characterized by a dissociation constant of at least about 10 ^-7 M or less, preferably with a K _{D of} ^{10 -8} to 10 ^-12 M that specifically binds to OX40, and thereby mediates a costimulatory response.

The "percent (%) amino acid sequence identity" relative to the reference polypeptide sequence is defined as when the sequences are aligned (and gaps are introduced when necessary) to obtain the maximum percent sequence identity without any conservative The percentage of amino acid residues in the candidate sequence with the same amino acid residues in the reference polypeptide sequence after substitutions are considered part of sequence identity. Various methods in the art can be used for sequence alignment to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the measurement alignment, including any algorithm required to obtain the maximum alignment over the entire length of the sequence being compared.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.

The amount of the antibody or antigen-binding fragment thereof contained in the antibody preparation of the present invention may vary according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used. In some embodiments, the antibody preparation is a liquid preparation, which may contain about 1-150 mg/mL, preferably about 10-100 mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mg/mL anti-OX40 antibody or antigen-binding fragment thereof.

In one embodiment, the present invention relates to a formulation having a high concentration of anti-OX40 antibody, for example, containing 40-150 mg/mL of anti-OX40 antibody. It is known in the art that such high-concentration antibody preparations can be diluted before injection, for example, if a lower antibody concentration is required for a specific therapeutic or prophylactic intervention or when treating smaller weight patients including children. A suitable concentration can be 25 mg/mL or 10 mg/mL. Alternatively, the original formulation can be produced at such a low concentration.

In addition, the anti-OX40 antibody preparations of the present invention described in the various embodiments herein are stable. In one embodiment, after storage at about -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 40°C, or 45°C for 6 months, the anti-OX40 in the antibody preparation of the present invention The antibody purity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99%, as determined by size exclusion chromatography. In one embodiment, after storage at about -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 40°C, or 45°C for 6 months, the anti-OX40 in the antibody preparation of the present invention At least 50% of the antibody is in the non-basic and non-acidic form (ie, the main peak or the main charge form), as determined by cation exchange chromatography.

Exemplary anti-OX40 antibodies that can be included in the antibody preparations of the present invention are, for example, the anti-OX40 antibodies disclosed in CN201710185399.9 and CN201710185400.8, ADI-20057, ADI-23504, ADI-23507, ADI-23509, ADI-20112, ADI-25650, ADI-25651, ADI-25652, ADI-25653, ADI-25654, ADI-20078, ADI-23515, ADI-23518, ADI-23519, ADI-20048, ADI-20096, ADI-20051, ADI-20065, ADI- 20066, ADI-20118 or ADI-20113, which respectively contain the sequences shown in the table below.

In a preferred embodiment, the anti-OX40 antibody in the antibody preparation of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein (a) the VH comprises (i) as SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 show three complementary determining regions in VH CDR, (ii) selected from the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17 HCDR1, selected from the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36 A combination of HCDR2 and an HCDR3 selected from the amino acid sequence of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43 and 44; or (iii) a sequence relative to (i) or (ii) , Each of the three CDRs contains at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) sequence; and/or (b ) The VL comprises (i) three CDRs in the VL shown in SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123 and 124, and (ii) is selected from SEQ ID NO : LCDR1 of the amino acid sequence of 45, 46, 47, 48, 49 and 50, LCDR2 of the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55 and 56, and selected from SEQ ID NO: a combination of LCDR3 with amino acid sequences of 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66; or (iii) Relative to the sequence of (i) or (ii), each of the three CDRs contains at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution , Preferably conservative substitution) sequence.

In yet another preferred embodiment, the anti-OX40 antibody in the antibody preparation of the present invention comprises heavy chain variable region VH and/or light chain variable region VL, wherein (a) heavy chain variable region VH contains and The amino acid sequence selected from SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical amino acid sequences or consisting of them; and/or ( b) The light chain variable region VL contains at least 90%, 91%, 92% with an amino acid sequence selected from SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123 and 124 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity of the amino acid sequence or composed thereof.

In yet another preferred embodiment, the anti-OX40 antibody in the antibody preparation of the present invention comprises a heavy chain and/or a light chain, wherein the heavy chain comprises and is selected from the group consisting of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, The amino acid sequence of 154, 155, 156, 157, 158, 159, 160, 161 and 162 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or 99% identity or consisting of, the light chain comprises an amino acid sequence selected from SEQ ID NO: 163, 164, 165, 166, 167, 168, 169, 170, 171, and 172 that has at least 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical amino acid sequences or consist of them.

(ii). Buffer

Suitable buffers for use in the present invention include, but are not limited to, salts of organic acid salts such as citric acid, ascorbic acid, gluconic acid, succinic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Methane, or phosphate buffer, or a combination thereof.

In one embodiment, the antibody formulation of the present invention contains such a buffering agent or pH adjusting agent to provide improved pH control. The liquid formulation of the present invention has a pH between 5.0 and 8.0, between 5.0 and 7.0, between 5.5 and 7.0, or between 6.5 and 7.0. In a specific embodiment, the antibody of the invention has a pH of about 5.0, 6.0, 7.0, 8.0.

In one embodiment, the concentration of the buffer contained in the antibody preparation of the present invention is about 0.1-50 mg/ml, preferably about 1-20 mg/ml, for example, about 1.5, 2, 2.5, 3, 3.5, 4. 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8mg/ml.

(iii). Stabilizer

Suitable stabilizers used in the present invention can function as, for example, viscosity enhancers, fillers, solubilizers, and the like. The stabilizer can be ionic or non-ionic. For example, the stabilizer may be a non-ionic stabilizer, for example, sugar.

For sugars as stabilizers, it includes, but is not limited to, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides, such as lactose, sucrose, trehalose, cellobiose, etc. ; Polysaccharides, such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol and sorbitol (Glucitol), etc., and their combinations. For example, the sugar may be sucrose, trehalose, raffinose, maltose, sorbitol or mannitol. Preferably, the sugar is sucrose.

For ionic stabilizers, it includes salts, for example, NaCl.

(iv). Surfactant

As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually oil-soluble hydrocarbon chains and water-soluble ionic groups. group.

In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, for example, alkyl poly(ethylene oxide). Specific nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates, such as polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate -20; Plunik et al.

The amount of the nonionic surfactant contained in the antibody preparation of the present invention can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used. In some embodiments, the formulation may contain a concentration of about 0.01-5 mg/ml, preferably about 0.1-2 mg/ml, such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg /ml of polysorbate-80 or plonic.

Other excipients contemplated that can be utilized in the antibody liquid formulations of the present invention include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, gelatin, and the like. These and other known pharmaceutical excipients and/or additives suitable for the formulation of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, Rowe et al., eds., American Pharmaceuticals Association (2003); And Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)".

In addition, the antibody preparation according to the present invention as described in the various embodiments herein is stable, so that even after storage at 25°C or 40°C for 4 weeks, 1 month, or 3 months, measured by SEC-HPLC, the antibody The purity of OX40 antibody is greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

II. Target diseases and conditions

The antibody preparation of the present invention comprising the anti-OX40 antibody of the present invention can be used to treat, ameliorate or prevent various diseases or disorders. Preparations containing anti-OX40 antibodies are particularly useful for treating, improving or preventing cancer, immune-related diseases and T cell dysfunction diseases.

For example, the antibody preparation of the present invention containing an anti-OX40 antibody can be used to treat, ameliorate, or prevent cancer. The cancer includes but is not limited to B-cell lymphoma (including low-grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate-grade diffuse NHL, High-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small nonnucleoblastic NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's ( Waldenstrom) macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD) , And abnormal blood vessel proliferation associated with phakomatoses, edema (such as those associated with brain tumors), B-cell proliferative disorders, and Meigs syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, front line low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphoma Cellular lymphoma, B Cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoma Plasma cell lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasma Cell tumor and/or plasma cell myeloma, low-grade/follicular lymphoma, intermediate/follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma Tumor, aggressive NHL (including aggressive frontline NHL and aggressive recurrent NHL), recurrent or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary exudative lymphoma Tumors, high-grade immunoblasts NHL, high-grade lymphoblasts NHL, high-grade small nonnuclear cleft cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytes Leukemia, granuloma fungoides and/or Sezary syndrome, skin lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

In some embodiments, the antibody formulations of the present invention are used to treat, ameliorate, or prevent lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer.

III. Antibody preparations are administered to subjects or patients

The antibody preparation of the present invention can be administered to a subject or patient. Administration is usually by infusion or by syringe. Therefore, the present invention provides a delivery device (e.g., a syringe) that includes the antibody preparation of the present invention (e.g., a pre-filled syringe). The patient will receive an effective amount of anti-OX40 antibody as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.

The therapeutic effect can also include reducing physical symptoms. The optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the age, weight, health and/or gender of the patient, the nature and extent of the disease, the activity of the particular antibody, and the body’s Clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation. For specific situations, the effective amount delivered can be determined within the judgment of the clinician. For the purpose of the present invention, the effective dose may be about 0.005 mg/kg body weight to about 50 mg/kg body weight, or about 0.05 mg/kg body weight to about 10 mg/kg body weight. Known antibody-based drugs provide guidance in this regard. For example, HERCEPTIN ^TM is administered at an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; RITUXAN ^TM is administered at 375 mg/m ² every week; SYNAGIS ^TM is administered intramuscularly at 15 mg/kg body weight; and so on.

The present invention also provides the preparation of the present invention as described in the various embodiments herein for use as a medicament, for example, for the delivery of anti-OX40 antibodies to mammals, or for the treatment, prevention or amelioration of one of the above-mentioned diseases and disorders or Many kinds.

The mammal is preferably a human, but can also be, for example, a horse or cow or a dog or cat. These antibodies are ideally antibodies that match the target species, for example, human anti-OX40 antibodies are used for humans, horse anti-OX40 antibodies are used for horses, and dog anti-OX40 antibodies are used for dogs. If the natural host antibody is not available, the transfer from the CDR residues (usually, one or more framework residues) from the donor antibody to the recipient framework from the host species can be used to achieve the transition from one species to The other antibody is specifically transferred, for example, humanized. Equine, bovine, canine and feline antibodies are known in the art. The antibody will bind to OX40 from the target species, but it can also cross-react with OX40 from other species.

The dosage can be a single-dose schedule or a multiple-dose schedule.

The following examples are described to assist the understanding of the present invention. The examples are not intended and should not be construed in any way to limit the scope of protection of the present invention.

Example

Materials and Method

1.1. Chemicals used in the study of freeze-dried preparations

1.2. Equipment used

1.3. Test items and test methods for the stability of the preparation

The following items were tested for antibody preparations: (1) Check the appearance and presence of visible foreign matter; (2) Determine the protein content in the preparation by ultraviolet method (UV method); (3) Use a pH meter to determine the pH value; (4) Detect the turbidity of the preparation; (5) Determine the non-aggregated and non-decomposed anti-antibody in the antibody preparation by size exclusion chromatography, for example, size-exclusion high performance liquid chromatography (size-exclusion chromatography-HPLC; SEC-HPLC) Percentage of OX40 antibody; (6) Determination of charge variants of anti-OX40 antibody in antibody preparations by cation exchange chromatography, for example, cation-exchange chromatography-HPLC (CEX-HPLC); (7) The purity of the antibody preparation was determined by sodium dodecyl sulfate capillary electrophoresis (CE-SDS).

Size exclusion high performance liquid chromatography (SEC-HPLC) method

In the SEC-HPLC method for analyzing antibody preparations, the following parameters are used:

Chromatographic column: TSK-gel SuperSW mAb HR (7.8×300mm, 4μm) type analytical column, TSK-gel SuperSW (6.0×40mm, 4μm) protection column

Mobile phase: 20mmol/L phosphate (PB)+150mmol/L NaCl+200mmol/L Arg, pH 6.8

Flow rate: 0.5ml/min

Column temperature: 25℃

Detection wavelength: 280nm

Injection volume: 50μl

Sample tray temperature: about 10℃

Running time: 30 minutes

Cation exchange high performance liquid chromatography (CEX-HPLC) method

In the CEX-HPLC method for analyzing antibody preparations, the following parameters are used:

Chromatographic column: Thermo Scientific ProPacTM WCX-10 weak cation analysis column

Mobile phase A: 10mmol/L PB

Mobile phase B: 10mmol/L PB, 200mmol/L NaCl

Gradient: 25min from 100%A to 60%A

Column temperature: 35℃

Injection volume: 50μl

Detection wavelength: 280nm

Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method

Dilute the antibody preparation sample to 10.0 mg/ml with ultrapure water, take 10 μl of the diluted sample, and add pH 6.5 sample buffer to it in turn (the sample buffer is obtained by drawing 200 μl of pH 6.5 citric acid-phosphate buffer Solution, add 47μl 10% SDS, add ultrapure water to 1000μl to prepare) 85μl, 10kDa internal standard (Beckman, United States, catalog number 390953) 2μl, 250mmol/L N-ethylmaleimide (NEM) 5μl, mix well, heat at 70°C for 10 minutes, and transfer to the sample tube after cooling. The CE-SDS analysis was performed with a Beckman PA800 Plus capillary electrophoresis instrument. The sampling voltage was -5kV, the sampling time was 20 seconds, the separation voltage was -15kV and -16.5kV, and the analysis time was 35 minutes and 36 minutes, respectively.

Judgment standard of formulation stability

Example 1. Preparation and purification of anti-OX40 antibodies

According to CN201710185399.9 and CN201710185400.8, a novel anti-OX40 antibody that specifically binds to OX40 was prepared and purified. The antibodies have the antibody names shown in Table 6 below, and their sequence characteristics are summarized in Table 1 to Table 5 above. .

Example 2. The effect of buffer pH on the stability of anti-OX40 antibody

This example evaluated the effect of the pH of the buffer on the stability of the anti-OX40 antibody. Prepare buffer solutions at different pH values according to Table 7 below. The concentration of each of the above-mentioned anti-OX40 antibodies in the buffer at each pH value is 15 mg/ml. After filtration, it is aliquoted, and the following measurements are performed.

A. Heat stress (40℃±2℃) experiment

Place the above-mentioned samples containing 15 mg/ml anti-OX40 antibody in a constant temperature and humidity cabinet at 40°C±2°C, and take samples on day 0, day 1, 5, and 10. Observed at the sampling time, the appearance of each sample, whether there is visible foreign matter; using an ultraviolet visible spectrophotometer (Japan Shimadzu production, model UV-1800) to determine the protein content in each sample; by size exclusion high-efficiency liquid The purity (%) of each sample was determined by SEC-HPLC; the charge variant (%) of each sample was determined by cation exchange high performance liquid chromatography (CEX HPLC). The results are as follows.

(1) Appearance, whether there are visible foreign objects

After being placed for 1 day or 5 days under the conditions of pH 5.0, 6.0, 7.0, 8.0 at 40°C±2°C, the appearance and visible foreign matter of each group of samples were all qualified.

After being placed for 10 days under the conditions of pH 5.0, 6.0, 7.0 at 40°C±2°C, the appearance and visible foreign matter of each group of samples were all qualified.

After standing for 10 days under the condition of 40℃±2℃ and pH about 8.0, the samples of each group appeared slightly turbid.

(2) Protein content

Under the condition of 40℃±2℃, the protein content of each group of samples did not change. Table 8 lists the protein content determination results of the antibody ADI-20112 (IgG1 type) samples.

N/A表示未設置該項。

N/A means that this item is not set.

(3) Purity

Under the condition of 40±2℃, the purity of each sample (SEC-HPLC method) only slightly decreased.

Under pH5.0 conditions, the main reason for the change in sample purity is that the antibody protein degrades after high temperature acceleration; under pH7.0 conditions, the main reason for the change in sample purity is that the antibody protein aggregates after high temperature acceleration; at pH8 Under .0 conditions, the initial purity of the antibody protein in the sample is slightly lower, mainly because the protein aggregates.

Table 9 lists the protein content determination results of samples of the antibody ADI-20112 (IgG1 type). When the anti-OX40 antibody ADI-20112 was formulated in the buffer at pH 6.0 or pH 7.0, higher purity was observed, indicating that the anti-OX40 antibody had better stability in the buffer at pH 6.0 or pH 7.0.

(4) Charge variant

The charge variant of each anti-OX40 antibody sample changes under high temperature (40±2℃). Among them, for the acidic variant of the antibody, at pH 8.0, the acidic component of the anti-OX40 antibody ADI-20112 (IgG1 type) sample Significant increase, followed by samples with pH 7.0, and samples with pH 5.0 and pH 6.0 have the smallest changes; for the main component of antibodies, the main component of samples with pH 7.0 has the least change; for basic variants of antibodies, each The alkaline component of the sample under pH conditions decreased consistently with respect to the alkaline component on day 0. For the results, refer to Figures 1 to 4 of the specification.

B. Oscillation stress experiment

The above-mentioned samples containing 15 mg/ml anti-OX40 antibody were shaken at 650 rpm at 25° C. and protected from light, and samples were taken on day 0, day 1, day 3, and day 5. Observed at the sampling time, the appearance of each sample, whether there is visible foreign matter; using an ultraviolet visible spectrophotometer (Japan Shimadzu, model UV-1800) to determine the protein content in each sample; by SEC-HPLC method The purity (%) of each sample is shown.

The results are as follows.

(1) Appearance, whether there are visible foreign objects

When shaking for up to 5 days under the conditions of pH 5.0, 6.0, 7.0, 8.0, the appearance of each group of samples and the detection of visible foreign matter were all qualified.

(2) Protein content

Under shaking conditions, the protein content of each group of samples was not significantly affected. Table 10 lists the protein content determination results of the antibody ADI-20112 (IgG1 type) samples.

N/A表示未設置該項。

N/A means that this item is not set.

(3) Purity

After shaking for 5 days under the conditions of pH 5.0, 6.0, 7.0, 8.0, the purity of each group of samples was determined. The results showed that the purity of each group of samples was not significantly affected under shaking conditions.

Table 11 lists the purity determination results of the antibody ADI-20112 (IgG1 type) samples.

N/A表示未設置該項。

N/A means that this item is not set.

C. Freeze-thaw stress experiment

The above-mentioned samples containing 15 mg/ml anti-OX40 antibody were subjected to cycles of freezing (below -30°C) and thawing (using a water bath at a temperature of 25°C), and sampling at the 0th, 3rd, and 6th cycles. Observed at the sampling time, the appearance of each sample, whether there is visible foreign matter; using an ultraviolet visible spectrophotometer (Japan Shimadzu, model UV-1800) to determine the protein content in each sample; by SEC-HPLC method The purity (%) of each sample is shown.

The results are as follows.

(1) Appearance, whether there are visible foreign objects

After freezing and thawing up to 6 cycles under the conditions of pH 5.0, 6.0, and 7.0, the appearance of each group of samples and the detection of visible foreign matter were all qualified.

After three cycles of freezing and thawing under the condition of pH 8.0, the appearance of each group of samples and the detection of visible foreign matter were all qualified. After 6 cycles of freezing and thawing at pH 8.0, the sample began to appear turbid.

(2) Protein content

Under freezing and thawing conditions, the protein content of each group of samples was not significantly affected. Table 12 lists the protein content determination results of the antibody ADI-20112 (IgG1 type) samples.

N/A表示未設置該項。

N/A means that this item is not set.

(3) Purity

Freeze-thaw cycles were performed under the conditions of pH 5.0, 6.0, 7.0, 8.0, and the purity of each group of samples was determined. The results showed that under freezing and thawing conditions, the purity of each group of samples was not significantly affected.

Table 13 lists the purity determination results of the antibody ADI-20112 (IgG1 type) samples.

N/A表示未設置該項。

N/A means that this item is not set.

It can be seen from the above experimental results that when the anti-OX40 antibody sample is in a buffer with a pH of about 5.0, 6.0, 7.0, 8.0, no obvious changes in appearance, protein content, and purity are observed after various stress conditions, and there is no visible change. Therefore, any pH value within the range of about 5.0 to 8.0 can be selected to prepare anti-OX40 antibody preparations.

Example 3. Formulation of stable anti-OX40 antibody formulations

Four liquid preparations of anti-OX40 antibodies were formulated with a pH of about 7.0. The components of the liquid preparations are shown in Table 14. Replace the prepared anti-OX40 antibody sample solution by ultrafiltration with the prepared buffers of these 4 preparations, and then make the anti-OX40 antibody in the sample solution about 25mg/ml, add polysorbate 80, sterilize and filter, and sterile aliquot To a 15 R vial, 4ml/bottle, freeze-dry the sample, stopper and cap, perform a high temperature test, and check the stability.

The lyophilized preparation was placed under the conditions of 40°C±2°C and 25°C±2°C, and samples were taken at 0 days, 2 weeks, 4 weeks, and 3 months. The lyophilized preparation sample was reconstituted with sterile water to a volume close to the volume before lyophilization, and the protein content of the lyophilized preparation after reconstitution was determined to be about 26.0 mg/ml. Observed at the sampling time point, the appearance of the reconstituted samples, and whether there are visible foreign bodies; an ultraviolet visible spectrophotometer (produced by Shimadzu, Japan, model UV-1800) was used to determine the protein content in each sample; turbidity ; The purity (%) of each sample was determined by size exclusion high performance liquid chromatography (SEC-HPLC); the charge variant of anti-OX40 antibody in each sample was determined by cation exchange chromatography (CEX HPLC) ( %). Similar results were obtained for various antibody preparations. The results of the preparation of the antibody ADI-20112 (IgG1 type) are listed below.

(1) Appearance, whether there are visible foreign objects

Four formulations were observed for 3 months under the temperature conditions of 40℃±2℃ and 25℃±2℃.

The results showed that the appearance of the four preparations and the detection of visible foreign bodies were all qualified.

(2) Protein content

Under the temperature conditions of 40℃±2℃ and 25℃±2℃, the protein content of the four preparations did not change. See Table 15 for details.

(3) Turbidity

Under the temperature conditions of 40°C±2°C and 25°C±2°C, the turbidity results of the four preparations are shown in Table 16 and Figure 5. Compared with the turbidity on day 0, there was no significant change.

(4) Purity

Purity (SEC-HPLC method): The results are shown in Table 17 and Figure 6.

Purity (non-reduced CE-SDS method): Under the conditions of 40°C±2°C and 25°C±2°C, the purity of each preparation did not change significantly. See Table 18 for details.

Purity (reduced CE-SDS method): Under the conditions of 40 ℃ ± 2 ℃ and 25 ℃ ± 2 ℃, the purity of each preparation did not change significantly. See Table 19 for details.

(5) Charge variant

Under the condition of 40℃±2℃, the charge variants of the anti-OX40 antibody did not change significantly at the 3rd month of the sample of Formulation 1 compared to that of Day 0; the acidic components of the samples of Formulation 2 and Formulation 3 were relatively The changes on day 0 were 4.1% and 2.3%, respectively; the sample of Formulation 4 at 3 months had a change of 2.6% relative to the main components on day 0. The specific results are shown in Table 20 and Figures 7-9.

Under the condition of 25°C±2°C, the samples of each preparation did not change significantly in the charge variants of the anti-OX40 antibody relative to day 0 at 3 months.

It can be seen from the above experimental results that all formulations 1-4 have an acceptable degree of stability. Preparation 2 is slightly inferior to preparation 1 in terms of turbidity, purity (SEC-HPLC method) and charge variants (CEX-HPLC method); each charge variant of preparation 3 and preparation 4 (CEX-HPLC method) is stable The sex is slightly lower than that of Formulation 1. The stability indexes of Formulation 1 are better than other formulations.

The exemplary embodiments of the present invention are described above, and those skilled in the art should understand that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed in the text.

<110> INNOVENT BIOLOGICS (SUZHOU) CO., LTD.

<120> Preparations containing anti-OX40 antibodies, preparation methods and uses thereof

<160> 172

<170> PatentIn version 3.3

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<223> Antibody ADI-20112, ADI-20113, ADI-25650, ADI-25651, ADI-25652, ADI-25653 and ADI-25654

VH CDR3

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<223> Antibody ADI-20057, ADI-23504, ADI-23507, ADI-23509, ADI-20112, ADI-25650, ADI- 25651, ADI-25652, VL CDR1 of ADI-25653, ADI-25654 and ADI-20118

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<210> 97

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-23515

<400> 97

<210> 98

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-23518

<400> 98

<210> 99

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-23519

<400> 99

<210> 100

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-20048

<400> 100

<210> 101

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-20096

<400> 101

<210> 102

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-20051

<400> 102

<210> 103

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-20065, ADI-20066

<400> 103

<210> 104

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH of antibody ADI-20118

<400> 104

<210> 105

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20057, ADI-23504, ADI-23507, ADI-23509

<400> 105

<210> 106

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20112, ADI-25650, ADI-25651, ADI-25652, ADI-25653, ADI-25654

<400> 106

<210> 107

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20113

<400> 107

<210> 108

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20078, ADI-23515, ADI-23518, ADI-23519

<400> 108

<210> 109

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20048

<400> 109

<210> 110

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20096

<400> 110

<210> 111

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20051

<400> 111

<210> 112

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20065

<400> 112

<210> 113

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20066

<400> 113

<210> 114

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20118

<400> 114

<210> 115

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20057, ADI-23504, ADI-23507, ADI-23509

<400> 115

<210> 116

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20112, ADI-25650, ADI-25651, ADI-25652, ADI-25653, ADI-25654

<400> 116

<210> 117

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20113

<400> 117

<210> 118

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20078, ADI-23515, ADI-23518, ADI-23519

<400> 118

<210> 119

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20048

<400> 119

<210> 120

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20096

<400> 120

<210> 121

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20051

<400> 121

<210> 122

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20065

<400> 122

<210> 123

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20066

<400> 123

<210> 124

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of antibody ADI-20118

<400> 124

<210> 125

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20057 in the form of IgG1

<400> 125

<210> 126

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20057 in the form of IgG2

<400> 126

<210> 127

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence of antibody ADI-23504 in the form of IgG1

<400> 127

<210> 128

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence of antibody ADI-23504 in the form of IgG2

<400> 128

<210> 129

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23507 in the form of IgG1

<400> 129

<210> 130

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-23507 in the form of IgG2

<400> 130

<210> 131

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23509 in the form of IgG1

<400> 131

<210> 132

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23509 in the form of IgG2

<400> 132

<210> 133

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of antibodies ADI-20112 and ADI-20113 in the form of IgG1

<400> 133

<210> 134

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of antibodies ADI-20112 and ADI-20113 in the form of IgG2

<400> 134

<210> 135

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25650 in the form of IgG1

<400> 135

<210> 136

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25650 in the form of IgG2

<400> 136

<210> 137

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25651 in the form of IgG1

<400> 137

<210> 138

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25651 in the form of IgG2

<400> 138

<210> 139

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-25652 in the form of IgG1

<400> 139

<210> 140

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25652 in the form of IgG2

<400> 140

<210> 141

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25653 in the form of IgG1

<400>141

<210> 142

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25653 in the form of IgG2

<400> 142

<210> 143

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25654 in the form of IgG1

<400> 143

<210> 144

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-25654 in the form of IgG2

<400> 144

<210> 145

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-20078 in the form of IgG1

<400> 145

<210> 146

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20078 in the form of IgG2

<400> 146

<210> 147

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23515 in the form of IgG1

<400> 147

<210> 148

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23515 in the form of IgG2

<400> 148

<210> 149

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-23518 in the form of IgG1

<400> 149

<210> 150

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23518 in the form of IgG2

<400> 150

<210> 151

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23519 in the form of IgG1

<400> 151

<210> 152

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-23519 in the form of IgG2

<400> 152

<210> 153

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20048 in the form of IgG1

<400> 153

<210> 154

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-20048 in the form of IgG2

<400> 154

<210> 155

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-20096 in the form of IgG1

<400> 155

<210> 156

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of the antibody ADI-20096 in the form of IgG2

<400> 156

<210> 157

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20051 in the form of IgG1

<400> 157

<210> 158

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20051 in the form of IgG2

<400> 158

<210> 159

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20065 and ADI-20066 in the form of IgG1

<400> 159

<210> 160

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20065 and ADI-20066 in the form of IgG2

<400> 160

<210> 161

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20118 in the form of IgG1

<400> 161

<210> 162

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain amino acid sequence of the antibody ADI-20118 in the form of IgG2

<400> 162

<210> 163

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20057, ADI-23504, ADI-23507, ADI-23509

<400> 163

<210> 164

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20112, ADI-25650, ADI-25651, ADI-25652, ADI-25653, ADI-25654

<400> 164

<210> 165

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequences of antibodies ADI-20078, ADI-23515, ADI-23518, ADI-23519

<400> 165

<210> 166

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20048

<400> 166

<210> 167

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20096

<400> 167

<210> 168

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20051

<400> 168

<210> 169

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20065

<400> 169

<210> 170

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20066

<400> 170

<210> 171

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20118

<400> 171

<210> 172

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of antibody ADI-20113

<400> 172

Claims (17)

A liquid antibody preparation having a pH of 5.0-8.0, comprising (i) an anti-OX40 antibody or an antigen-binding fragment thereof, the anti-OX40 antibody or an antigen-binding fragment thereof comprising the HCDR1 of the amino acid sequence of SEQ ID NO: 4, SEQ ID The combination of HCDR2 of the amino acid sequence of NO: 22, and HCDR3 of the amino acid sequence of SEQ ID NO: 38, and LCDR1 of the amino acid sequence of SEQ ID NO: 45, the amino acid of SEQ ID NO: 51 The combination of LCDR2 of the sequence and LCDR3 of the amino acid sequence of SEQ ID NO: 58; (ii) a buffer selected from the group consisting of phosphate, citrate, citrate solvate, succinic acid, three Methylolaminomethane and their combinations; (iii) stabilizers selected from sucrose, trehalose, mannitol and combinations thereof; and (iv) surfactants selected from plonic , Polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate-20. The liquid antibody preparation as described in item 1 of the scope of patent application, wherein the concentration of the anti-OX40 antibody or antigen-binding fragment thereof in the liquid antibody preparation is 10-100 mg/mL. The liquid antibody preparation as described in item 1 of the scope of patent application, wherein the concentration of the buffer in the liquid antibody preparation is 1-20 mg/ml. The liquid antibody preparation according to the first item of the patent application, wherein the buffer is sodium citrate or sodium citrate dihydrate. The liquid antibody preparation as described in item 1 of the scope of patent application, wherein the concentration of the stabilizer in the liquid antibody preparation is 20-100 mg/ml. The liquid antibody preparation as described in item 1 of the scope of patent application, wherein the concentration of the surfactant in the liquid antibody preparation is 0.1-2 mg/ml. The liquid antibody preparation according to item 1 of the scope of patent application, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein (a) the heavy chain may The variable region VH includes the amino acid sequence of SEQ ID NO: 90; and/or (b) the light chain variable region VL includes the amino acid sequence of SEQ ID NO: 116. The liquid antibody preparation according to item 7 of the scope of patent application, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain and/or a light chain, wherein the heavy chain comprises SEQ ID NO: 133 or 134, The light chain comprises SEQ ID NO:164. The liquid antibody preparation as described in any one of items 1 to 8 of the scope of patent application, which contains 20-30 mg/ml anti-OX40 monoclonal antibody, 4-6 mg/ml sodium citrate or sodium citrate dihydrate, 40- 60mg/ml sucrose, 0.6-0.8mg/ml polysorbate 80, pH 6.0-7.0. The liquid antibody preparation described in item 9 of the scope of patent application, which contains 25mg/ml anti-OX40 monoclonal antibody, 5.88mg/ml sodium citrate dihydrate, 50.00mg/ml sucrose, 0.7mg/ml polysorbate 80 , The pH is 7.0. The liquid antibody preparation according to any one of items 1 to 8 of the scope of patent application, which comprises 25mg/ml anti-OX40 monoclonal antibody, 2.36mg/ml succinic acid, 50.00mg/ml mannitol, 0.7mg/ml Polysorbate 80 has a pH of 7.0. The liquid antibody preparation according to any one of items 1 to 8 of the scope of patent application, which comprises 25 mg/ml anti-OX40 monoclonal antibody, 20 mmol/L phosphate, 50.00 mg/ml sucrose, and 0.7 mg/ml polysorbate Ester 80, pH 7.0. The liquid antibody preparation according to any one of items 1 to 8 of the scope of patent application, which comprises 25mg/ml anti-OX40 monoclonal antibody, 2.42mg/mlTris, 50.00mg/ml trehalose, 0.7mg/ml polysorbate Ester 80, pH 7.0. A solid antibody preparation obtained by solidifying the liquid antibody preparation as described in any one of items 1 to 13 in the scope of the patent application. A liquid antibody preparation according to any one of the scope of patent application 1 to 13 or the use of the solid antibody preparation described in clause 14 of the scope of patent application for preparing activated T cells or inducing T cell-mediated in a subject The anti-tumor activity or the delivery device that enhances the body’s immune response or pre-filled syringes or drugs. The use of a liquid antibody preparation according to any one of items 1 to 13 of the scope of patent application or the use of a solid antibody preparation according to item 14 of the scope of patent application for the preparation of a delivery device or a pre-filled syringe or a pre-filled syringe for treating cancer of a subject drug. The use according to item 16 of the scope of patent application, wherein the cancer is lung cancer, liver cancer, gastric cancer, or colon cancer.

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