WO2020063668A1

WO2020063668A1 - Formulation containing anti-ox40 antibody, preparation method therefor, and use thereof

Info

Publication number: WO2020063668A1
Application number: PCT/CN2019/107829
Authority: WO
Inventors: 汪音爵; 曹魏; 周凯松
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2018-09-25
Filing date: 2019-09-25
Publication date: 2020-04-02
Also published as: TW202033179A; CN111683681B; TWI726426B; CN111683681A

Abstract

Disclosed is a formulation containing anti-OX40 antibody, and particularly a medical formulation of an antibody, antigen-binding fragment, buffer agent, stabilizer, and/or surfactant that contains molecules that specifically bind with OX40. Also disclosed are uses of the formulations in disease treatment and prevention.

Description

Preparation containing anti-OX40 antibody, preparation method and application thereof

Technical field

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

Background technique

Co-stimulatory molecules are a type of cell surface molecules other than antigen receptors or antigen ligands that are required for lymphocytes to respond effectively to antigens. By specifically binding to co-stimulatory ligands, lymphocyte-mediated co-stimulation reaction. Co-stimulatory molecules have been shown to enhance T cell expansion, effector function, and survival in vitro; and to enhance human T cell retention and antitumor activity in vivo.

OX40 (also known as CD134, TNFRSF4, and ACT35) is a costimulatory molecule and has been shown to promote costimulatory signals to T cells, leading to enhanced cell proliferation, survival, effector function, and migration (Gramaglia, et al. People, Ox-40: Ligand: a potent costimulatory molecule for the primary CD4T cell response. J Immunol. 1998; 161: 6510–6517; Gramaglia I et al. The OX40 Costimulatory Receptor Determines the the development of the CDC J Immunol. 2000; 165: 3043–3050).

Anti-OX40 antibodies that specifically bind OX40 as OX40 agonists 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.

Antibody formulations must be formulated not only in a manner that makes the antibody suitable for administration to a subject, but also in a manner that maintains its stability during storage and subsequent use. For example, if an antibody is not properly formulated in a liquid, the antibody in a liquid solution tends to decompose, aggregate, or undergo undesired chemical modification. The stability of the antibody in the antibody preparation depends on the buffer, stabilizer, surfactant, etc. used in the preparation.

Antibodies against OX40 are one example of a formulation that needs to be properly formulated to treat or prevent a disease. Although some anti-OX40 antibodies are known, there remains a need in the art for novel pharmaceutical formulations containing anti-OX40 antibodies that are sufficiently stable and suitable for administration to a subject.

Summary of invention

The present invention satisfies the aforementioned needs by providing a pharmaceutical formulation containing an antibody that specifically binds to OX40.

In one aspect, the invention provides a liquid antibody formulation 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 (eg, a human OX40 molecule), such as a polyclonal 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 an antigen-binding fragment thereof as defined herein.

In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the invention is about 0.1-50 mg / ml. In one embodiment, the concentration of the buffering agent 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, 8 mg / ml.

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

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

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

In one embodiment, the concentration of the surfactant in the liquid antibody formulation of the invention is about 0.01-5 mg / ml. In one embodiment, the concentration of the surfactant in the liquid antibody formulation of the present invention is 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.

In one embodiment, the surfactant is a non-ionic 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 of the liquid formulation is about 5.0, 6.0, 7.0, 8.0.

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

In a preferred embodiment, the liquid antibody formulation of the invention comprises

(i) about 1-150 mg / mL of anti-OX40 antibody or antigen-binding fragment thereof;

(ii) about 0.1-50 mg / ml of citrate, citrate hydrate, such as sodium citrate, sodium citrate dihydrate, as a buffer;

(iii) about 10-200 mg / ml of sucrose as a stabilizer, and

(iv) about 0.01-5 mg / ml of polysorbate-80 as a surfactant;

Wherein the pH of the liquid preparation is about 5.0-8.0.

(i) about 10-100 mg / mL of anti-OX40 antibody or antigen-binding fragment thereof;

(ii) about 1-20 mg / ml sodium citrate or sodium citrate dihydrate as a buffering agent;

(iii) about 20-100 mg / ml sucrose as a stabilizer, and

(iv) about 0.1-2 mg / ml of polysorbate-80 as a surfactant;

Wherein the pH of the liquid preparation is about 5.0-8.0.

(i) about 20-30 mg / mL of anti-OX40 antibody or antigen-binding fragment thereof;

(ii) about 4-6 mg / ml sodium citrate or sodium citrate dihydrate as a buffering agent;

(iii) about 40-60 mg / ml sucrose as a stabilizer, and

(iv) about 0.6-0.8 mg / ml of polysorbate-80 as a surfactant;

Wherein the pH of the liquid preparation is about 6.0-7.0.

In another aspect, the present invention provides a solid antibody preparation obtained by subjecting the liquid antibody preparation of the present invention to a curing treatment. 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 for injection.

The solid antibody preparation can be reconstituted in an appropriate 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 vehicle is selected from water for injection, organic solvents for injection, including but not limited to oil for injection, ethanol, propylene glycol, and the like, or a combination thereof.

In one embodiment, a liquid or solid formulation comprising an anti-OX40 antibody of the present invention is at about -80 ° C to about 45 ° C, such as -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃ for at least 10 days, at least 20 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, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 After at least 24 months, at least 36 months, or longer, the size of the anti-OX40 antibody or its antigen-binding fragment is reduced by no more than 10%, for example, not more than 5%, by size exclusion high performance liquid chromatography. , 4%, 3%, 2%, 1%, 0.5%, or 0.1%.

In one embodiment, a liquid or solid formulation comprising an anti-OX40 antibody of the present invention is at about -80 ° C to about 45 ° C, such as -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃ for at least 10 days, at least 20 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, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 At least 24 months, at least 36 months, or longer, detected by non-reduced sodium lauryl sulfate capillary electrophoresis (CE-SDS) and / or reduced CE-SDS, anti-OX40 antibody Or the purity of the antigen-binding fragment thereof is not more than 10%, for example, not more than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%.

In one embodiment, a liquid or solid formulation comprising an anti-OX40 antibody of the present invention is at about -80 ° C to about 45 ° C, such as -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃ for at least 10 days, at least 20 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, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 At least 24 months, at least 36 months, or longer, the cation exchange high-performance liquid chromatography (CEX-HPLC) test showed that the charge variants of the anti-OX40 antibody or its antigen-binding fragment did not change more than 10%, such as no more than 5%, 4%, 3%, 2%.

In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof in the liquid formulation of the present invention is capable of high affinity, for example, at 10 ^-7 M or less, preferably at 10 ^-8 M to 10 ^-12 M. K _D specifically binds OX40, and thus mediates a co-stimulatory response to an 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 contains

(i) in VH shown as SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 CDRs of the three complementary determining regions,

(ii) HCDR1 selected from the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, and 17 SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 of the amino acid sequence of HCDR2, and selected from SEQ ID NO: a combination of HCDR3 in the amino acid sequence of 37, 38, 39, 40, 41, 42, 43, and 44; 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 changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of;

and / or

(b) the VL contains

(i) three CDRs in VL as 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 selected from the amino acid sequences of SEQ ID NO: 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 changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of.

In still another preferred embodiment, the anti-OX40 antibody 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 heavy chain variable region VH comprises and is 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 have an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity Its composition

and / or

(b) the light chain variable region VL comprises at least 90%, 91%, 92%, and an amino acid sequence selected from the group consisting of SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123, and 124, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical amino acid sequences or consist of them.

In still 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, 154 , 155, 156, 157, 158, 159, 160, 161, and 162 have an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% Identity or consist thereof, said light chain comprising at least 90%, 91%, 92% of the amino acid sequence selected from the group consisting of SEQ ID NO: 163, 164, 165, 166, 167, 168, 169, 170, 171 and 172 Or consists of an amino acid sequence that is%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical.

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

In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe comprising a liquid antibody formulation or a solid antibody formulation of the present invention as described in various embodiments of this specification, such as for intravenous or intramuscular injection .

In one embodiment, the 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 subject of the invention as described in various embodiments of the present specification. A step of a liquid or solid antibody preparation, said delivery being performed, for example, by a delivery device using a pre-filled syringe.

The invention further provides the use of a liquid antibody preparation or a solid antibody preparation of the invention for the preparation of a delivery device that activates T cells in a subject or induces T cell-mediated antitumor activity or enhances the body's immune response (such as , Pre-filled syringes) or drugs, particularly for treating a disease in a subject, such as cancer, such as lung cancer (eg, non-small cell lung cancer), liver cancer, stomach cancer, or colon cancer.

The present invention further provides a subject under test by administering a liquid antibody preparation or solid antibody preparation of the invention or a delivery device (e.g., a pre-filled syringe) or a drug comprising the liquid antibody preparation or solid antibody preparation of the invention to a subject. A method for activating T cells or inducing T cell-mediated antitumor activity or enhancing an immune response in a subject.

The present invention further provides a subject to be treated by administering the liquid antibody preparation or solid antibody preparation of the invention or a delivery device (e.g., a pre-filled syringe) or a medicament comprising the liquid antibody preparation or solid antibody preparation to the subject. Methods of cancer, such as lung cancer (such as non-small cell lung cancer), liver cancer, stomach cancer, or colon cancer.

Other embodiments of the present invention will be apparent from the detailed description which follows.

Brief description of the drawings

The preferred embodiments of the invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, the drawings show a currently preferred embodiment. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

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

Fig. 2 shows a charge variant profile of an anti-OX40 antibody placed under heat stress for 10 days at pH 6.0.

Figure 3. shows a charge variant profile of an anti-OX40 antibody placed under heat stress for 10 days at pH 7.0.

Figure 4. shows a charge variant profile of an anti-OX40 antibody placed under heat stress for 10 days at pH 8.0.

Fig. 5 is a graph showing a change in turbidity of an anti-OX40 antibody preparation of the present invention after being left at a temperature of 40 ° C ± 2 ° C.

Fig. 6 is a graph showing the change in purity measured by the SEC-HPLC method after the anti-OX40 antibody preparation of the present invention was left to stand at a temperature of 40 ° C ± 2 ° C.

FIG. 7 is a graph showing the change in the acidic component 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 was left to stand at a temperature of 40 ° C. ± 2 ° C. FIG.

FIG. 8 is a graph showing the main component change of an anti-OX40 antibody charge variant measured by a CEX-HPLC method after the anti-OX40 antibody preparation of the present invention was left to stand at a temperature of 40 ° C. ± 2 ° C. FIG.

FIG. 9 is a graph showing changes 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 was left to stand at a temperature of 40 ° C. ± 2 ° C. FIG.

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 terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

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

I. Antibody preparations

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

As used herein, the term "anti-OX40 antibody formulation" 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 an active ingredient is suitable for therapeutic or prophylactic administration to human or non-human animals. According to the invention, the anti-OX40 antibody as an active ingredient in the preparation specifically binds to the OX40 molecule, and the resulting signalling promotes a co-stimulatory signal to T cells, leading to enhanced cell proliferation, survival, effector function, and migration.

The antibody preparation of the present invention may be sterile, homogeneous and / or isotonic, and the antibody preparation may be directly prepared as an aqueous liquid preparation, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized The formulation is reconstituted (ie, reconstituted) by dissolution and / or suspension 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.

The use of antibodies as active ingredients of drugs has been widely used, including products such as HERCEPTIN ^™ (trastuzumab), RITUXAN ^™ (rituximab), SYNAGIS ^™ (palizumab), and the like. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art.

A "sterile" formulation is a formulation that is sterile or free or essentially free of living microorganisms and their spores.

The term "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 capable of being obtained by a freeze-drying treatment of 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 (eg, a lyophilized preparation) in a physiologically acceptable solution.

In specific embodiments, the anti-OX40 antibody formulations of the present invention exhibit low to undetectable levels of antibody aggregation or degradation or chemical modification during manufacturing, preparation, transportation, and long-term storage, so that there is little or no antibody The biological activity of OX40 antibody is lost, showing high stability. As used herein, the term "stable" antibody formulation is an antibody in a formulation that 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 100% maintain its chemical structure after storage for a specific time, it is usually maintained at about 90%, 95%, 96%, 97%, 98% after storage for a specific time. Or about 99% of the antibody structure or function, the antibody formulation is considered "stable." In some embodiments, an anti-OX40 antibody formulation of the 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 subjecting the antibody formulation to various stress tests. These tests can indicate the extreme conditions that the formulated antibody preparations may encounter during manufacturing, storage, or transportation, or they can indicate that the instability of the antibodies in the antibody preparations is accelerated under extreme conditions not during manufacturing, storage, or transportation. condition. For example, fill the formulated anti-OX40 antibody preparation into a 5 mL glass bottle for shaking stress, freeze / thaw cycle (ie, freeze-thaw cycle) stress; fill the formulated anti-OX40 antibody preparation into a glass vial to verify that 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, and more preferably 25 ° C.

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

After a long storage time, if the formulation is checked for appearance, color and / or clarity, or for example by UV light scattering or size exclusion chromatography, the antibodies in the formulation do not show aggregation, precipitation and / or Cloudiness; or showing very little aggregation, precipitation, and / or cloudiness, then the antibody "maintains its physical stability" in the formulation. The accumulation of antibodies in the formulation can potentially lead to an increased immune response in patients, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of antibodies in a formulation.

In one embodiment, the stability of a 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, where the percentage of non-aggregated and non-decomposed antibodies in the formulation is greater The higher, the more stable the formulation. The percentage of non-aggregated and non-decomposed antibodies can be determined by size exclusion chromatography (eg, size exclusion high performance liquid chromatography).

When the phrase "acceptable level of stability" is used herein, it means that at least about 92% of non-aggregated and non-decomposed anti-OX40 antibodies are detected in the formulation after storage at a specific temperature for a specific time. In some embodiments, stored at a particular 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 The acceptable level of stability indicates that 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 About 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-decomposed anti-OX40 antibodies. When evaluating stability, the specific temperature at which the pharmaceutical formulation is stored can be any temperature from about -80 ° C to about 45 ° C, such as at about -80 ° C, about -30 ° 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-decomposed anti-OX40 antibodies are detected Form, the pharmaceutical formulation is considered stable. If at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-decomposed anti-OX40 antibody forms are detected after storage at about 25 ° C for 3 months, The pharmaceutical formulation is considered stable. If at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the non-aggregated and non-decomposed anti-OX40 antibody forms are detected after storage at about 5 ° C for 9 months, The pharmaceutical formulation is considered stable.

In addition, after a long storage time, if the chemical structure of the antibody in the formulation is complete, the antibody "maintains its chemical stability" in the formulation. Most chemical instability results from the formation of covalently modified forms of antibodies (eg, charge variants of antibodies). In some embodiments, after storage of the antibody formulation, changes in each charge variant component of the anti-OX40 antibody are detected.

In one embodiment, the charge variant of the anti-OX40 antibody in the antibody formulation is determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, peaks eluting from the CEX-HPLC column earlier than the retention time of the main peak are labeled as "acidic peaks", while those eluting from the CEX-HPLC column later than the retention time of the main peak. The peaks are marked as "basic peaks". In the CEX-HPLC method, the percentage of acidic components is determined by the ratio of the area of the acidic peak to the sum of the area of the main peak, the acidic peak and the basic peak; the percentage of the main component is the area of the main peak and the main peak, and the acidic peak and basicity. The ratio of the sum of peak areas is determined; the percentage of basic components is determined by the ratio of the area of the basic peak to the sum of the area of the main peak, the acid peak, and the area of the basic peak. Without being bound by theory, it is believed that deamidation of antibodies can make the antibodies more negatively charged and thus more acidic than non-deamidated antibodies (see, for example, Robinson, N., Protein, Deamidation, PNAS, 2002 (April 16, 1999, 99 (8): 5283-5288). When the term "acceptable degree of stability" is used herein, it means that up to about 25% of the antibody in the formulation is in a more acidic form after storage at a specific temperature for a specific time. In some embodiments, stored at a particular 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 Acceptable levels of stability 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 are expressed in Up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in an acidic form in the formulation after storage at a specific temperature for a specific time. When stability is evaluated, the temperature at which the pharmaceutical formulation is stored can be any temperature from about -80 ° C to about 45 ° C, such as at about -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 4 ° C. ° C-8 ° C, about 5 ° C, about 25 ° C, or about 45 ° C. For example, if stored at -80 ° C, -30 ° C, or -20 ° C 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% of the antibody in a more acidic form, the pharmaceutical preparation can be Considered stable. If after storage at 5 ° C 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% of the antibody is in a more acidic form, then Pharmaceutical formulations can also be considered stable. If after storage at 25 ° C 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% of the antibody in a more acidic form, the pharmaceutical preparation It can also be considered stable. If less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% are detected after storage at 37 ° C for 28 days , 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form, Pharmaceutical formulations can also be considered stable. If less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% are detected after storage at 40 ° C for 28 days , 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form, Pharmaceutical formulations can also be considered stable.

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

In some embodiments, a stable anti-OX40 antibody formulation of the 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 antibodies or antigen-binding fragments thereof that specifically bind to OX40 molecules

The antibody preparation of the present invention may comprise an anti-OX40 antibody or an antigen-binding fragment thereof that specifically binds to an OX40 molecule. As used herein, the term "OX40" is known as a costimulatory molecule whose activation can lead to enhanced cell proliferation, survival, effector function, and migration. Anti-OX40 antibodies are disclosed in the prior art as OX40 agonists. For example, WO 2012/027328 discloses the amino acid sequences of the heavy chain and light chain variable regions of anti-OX40 antibody mAb 106-222 and humanized 106-222 (Hu106); anti-OX40 antibody mAb 119-122 and Amino acid sequences of the humanized 119-122 (Hu119) heavy chain and light chain variable regions. In addition, U.S. Patent 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 the proliferation of effector T lymphocytes, and promoting the immune response against tumor cells expressing tumor-associated antigen (TAA). The documents are incorporated herein by reference in their entirety.

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

The term "anti-OX40 antibody", "anti-OX40", "OX40 antibody" or "OX40-binding antibody" refers to an antibody capable of binding the OX40 molecule with sufficient affinity so that the antibody can be used as a target A therapeutic and / or prophylactic agent for the 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 a radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (K _D ) of the anti-OX40 antibody is ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM, or ≦ 0.001 nM. Also in some embodiments, the anti-OX40 antibody is capable of specifically binding OX40 with a high affinity, for example with a K _{D of} 10 ^-7 M or less, preferably with a K _{D of} 10 ^-8 M to 10 ^-12 M, and This mediates a co-stimulatory response. In this context, when referring to "anti-OX40 antibodies", antigen-binding fragments of anti-OX40 antibodies are also included.

An "antigen-binding fragment" of an antibody refers to a molecule different from an intact antibody, which contains a portion of the intact antibody and binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab') 2; diabody; linear antibody; single chain antibody (e.g., scFv); single domain antibody; 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 a portion of an antigen (eg, OX40) that specifically interacts with an antibody molecule.

A "complementarity determining region" or "CDR region" or "CDR" is a sequence that is highly variable in an antibody variable domain and forms a structurally defined loop ("hypervariable loop") and / or contains antigen-contacting residues ( "Antigen contact point"). CDRs are primarily responsible for binding to epitopes. The CDRs of the heavy and light chains are commonly referred to as 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, while 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 including: For example: Chothia (Chothia et al. (1989) Nature 342: 877-883, based on the three-dimensional structure of antibodies and the topology of CDR loops, Al-Lazikani et al., "Standard conformations for the canonical structure of immunoglobulins", Journal of molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interface, 4th Edition, USDepartment of Health and Human Services, National Institute of Health (1987)), AbM (University of Bath), Contact (University College, London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr/ on the World Wide Web), and affinity propagation clustering based on the use of a large number of crystal structures North CDR definition.

It should be noted, however, that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may differ. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining an antibody with a specific CDR sequence defined in the present invention, the scope of the antibody also encompasses antibodies whose variable region sequences contain the specific CDR sequences, but due to the application of different protocols (e.g. Different assignment system rules or combinations) resulting in its claimed CDR boundary being different from the specific CDR boundary defined by the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the smallest overlapping regions can be determined, thereby providing a "minimal binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be clear to those skilled in the art, the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Accordingly, the invention also contemplates any variant of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.

The term "conservative amino acid residue substitutions" are those in which amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues with similar side chains has been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged polar side chains (e.g., , Glycine, asparagine, 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). Therefore, one or more amino acid residues of an anti-OX40 antibody can be replaced by other amino acid residues from the same side chain family, and the function of the altered antibody can be tested, especially the same binding characteristics as the OX40 molecule. The change in charge performance on the CDR surface is expected to affect the interface between the antibody and the solvent. Therefore, non-conservative amino acid residue substitutions have an unpredictable effect on maintaining or improving the stability of the antibody in solution.

"Antibodies or antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterozygous, and chimeric , Humanized (especially CDR-grafted), deimmunized, or human antibodies, Fab fragments, Fab 'fragments, F (ab') ₂ fragments, fragments generated from the Fab expression library, Fd, Fv, two Sulfide-linked Fv (dsFv), single chain antibody (e.g. scFv), diabody or tetrabody (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), Nanobody (nanobody) (also known as a single domain antibody), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against antibodies of the invention), and epitope-binding fragments of any of the foregoing.

A "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, the selection of human immunoglobulin VL or VH sequences is from a subtype of a variable domain sequence.

"Antibody in IgG form" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of different types of antibodies are different. For example, an antibody in the form of IgG1 means that the Ig domain of the 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 a human or human cell or derived from a non-human source that utilizes a human antibody library or other human Antibody coding sequence. This definition of a human antibody explicitly excludes humanized antibodies that include non-human antigen-binding residues.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, where all or substantially all CDRs correspond to those of a non-human antibody, and all or substantially all FR corresponds to those of human antibodies. A humanized antibody may optionally comprise at least a portion of a human antibody-derived antibody constant region. 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, eg, an isolated antibody that specifically binds OX40 is substantially free of antibodies that specifically bind to an antigen other than OX40 .

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

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the sequences are aligned (and gaps are introduced if necessary) to obtain the maximum percent sequence identity without regard to any conservative substitutions Following the portion of sequence identity, the percentage of amino acid residues in the candidate sequence to the same amino acid residues in the reference polypeptide sequence. Sequence alignments can be performed using various methods in the art 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 appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences 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 to, for example, 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 depending on the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation. In some embodiments, the antibody formulation is a liquid formulation, 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 invention relates to a formulation having a high concentration of an anti-OX40 antibody, for example, containing 40-150 mg / mL of an anti-OX40 antibody. It is known in the art that such high concentration antibody preparations can be diluted prior to injection, for example if a lower therapeutic antibody concentration is required for a particular therapeutic or prophylactic intervention or when treating smaller weight patients including children. A suitable concentration may 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 formulations of the invention described herein in the various embodiments are stable. In one embodiment, the anti-OX40 in the antibody formulation of the invention is stored at about -80 ° C, -30 ° C, -20 ° C, 5 ° C, 25 ° C, 37 ° C, 40 ° C, or 45 ° C for 6 months. The purity of the antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, as determined by size exclusion chromatography. In one embodiment, the anti-OX40 in the antibody formulation of the present invention is stored at about -80 ° C, -30 ° C, -20 ° C, 5 ° C, 25 ° C, 37 ° C, 40 ° C, or 45 ° C for 6 months. At least 50% of the antibody is in a non-basic and non-acidic form (ie, a main peak or a major charge form), as determined by cation exchange chromatography.

Exemplary anti-OX40 antibodies that can be included in the antibody formulations of the 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, each of which contains the sequences shown in the table below.

In a preferred embodiment, the anti-OX40 antibody in the antibody preparation of the invention comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein

(a) the VH contains

and / or

(b) the VL contains

In yet another 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,

and / or

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 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, 154 , 155, 156, 157, 158, 159, 160, 161, and 162 have an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% Identity or consist thereof, said light chain comprising at least 90%, 91%, 92% of the amino acid sequence selected from the group consisting of Or consists of an amino acid sequence that is%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical.

(ii). Buffering agent

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

In one embodiment, an antibody formulation of the invention comprises such a buffer or pH adjuster to provide improved pH control. The liquid formulation of the 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, an antibody of the invention has a pH of about 5.0, 6.0, 7.0, 8.0.

In one embodiment, the concentration of the buffering agent contained in the antibody formulation 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, 8 mg / ml.

(iii). Stabilizer

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

For the sugar as a stabilizer, it includes, but is not limited to, monosaccharides, such as sugar, 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 sorbitol (glucositol) ), 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, this includes salts, for example, NaCl.

(iv) .Surfactants

As used herein, the term "surfactant" refers to an organic substance having 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 non-ionic surfactant, such as an alkyl poly (ethylene oxide). Specific non-ionic surfactants that can be included in the formulations of the invention include, for example, polysorbates such as polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate -20; Plonick et al.

The amount of the non-ionic surfactant contained in the antibody preparation of the present invention may vary depending on the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation. In certain 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 ml of Polysorbate-80 or Plonic.

Other contemplated excipients 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 use in the formulations of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, edited by Rowe et al, American Pharmaceuticals (2003); and Remington: The Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005) ".

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

II. Target diseases and disorders

The antibody preparation of the present invention comprising an anti-OX40 antibody can be used to treat, ameliorate or prevent a variety of diseases or conditions. Anti-OX40 antibody-containing formulations are particularly useful for treating, ameliorating, or preventing cancer, immune-related diseases, and T-cell dysfunction diseases.

For example, an antibody formulation of the invention comprising an anti-OX40 antibody can be used to treat, ameliorate or prevent cancer. The cancers include, but are not limited to, B-cell lymphoma (including low / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, Advanced Immune Cellular NHL, Advanced Lymphoblastic NHL, Advanced Small Angioblastic NHL, Bulk 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 myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD) And abnormal vascular 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 lower NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and / or lymphoma, small lymph Cellular lymphoma, B-cell chronic lymphocytic leukemia and / or prelymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prelymphocytic lymphoma, immune cell tumor and / or lymphoplasmic ( lymphoplasmacytic lymphoma, lymphocytoplasmic lymphoma, marginal B-cell lymphoma, marginal spleen lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, Hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low / follicular lymphoma, intermediate / follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate diffuse NHL, Diffuse large B-cell lymphoma, aggressive NHL (including aggressive front-line NHL and aggressive recurrent NHL), recurrent or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma Primary exudative lymphoma, advanced immunoblast NHL, advanced lymphoblast NHL, advanced small non-nucleated fibroblast NHL, bulky disease NHL, Burkitt lymphoma, precursor (Peripheral) Large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

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

III. Administration of antibody formulations to a subject or patient

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

Therapeutic effects may also include reducing physical symptoms. The optimal effective amount and concentration of antibodies for any particular subject will depend on a number of factors, including the patient's age, weight, health and / or gender, the nature and extent of the disease, the activity of the specific antibody, Its clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation. For specific cases, the effective amount delivered can be determined within the judgment of the clinician. For the purposes of the present invention, an 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, such as HERCEPTIN ^TM 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 administered at 375 mg / m ² weekly; SYNAGIS ^TM is administered intramuscularly at 15 mg / kg body weight;

The invention also provides a formulation of the invention as described herein in various embodiments for use as a medicament, for example, for delivering an anti-OX40 antibody to a mammal, or for treating, preventing or ameliorating one of the diseases and conditions described above Or more.

The mammal is preferably a human, but may also be, for example, a horse or a cow or a dog or a cat. These antibodies are ideally those that match the target species, for example, human anti-OX40 antibodies for humans, horse anti-OX40 antibodies for horses, and dog anti-OX40 antibodies for dogs. If a native host antibody is not available, this can be accomplished by transferring CDR residues (usually, one or more framework residues) from the donor antibody to the recipient framework from the host species. An antibody specifically transfers, for example, to humanization. Equine, bovine, canine and feline antibodies are known in the art. The antibody will bind OX40 of the target species, but it can also cross-react with OX40 from other species.

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

The following examples are described to assist in understanding the invention. The embodiments are not intended and should not be interpreted in any way as limiting the scope of protection of the invention.

Examples

Materials and Method

1.1. Chemicals used in the study of lyophilized preparations

1.2. Equipment used

1.3. Test items and test methods for preparation stability

The following items were tested for the antibody preparation: (1) the appearance and presence of visible foreign bodies; (2) the protein content of the preparation by the ultraviolet method (UV method); (3) the pH value was measured using a pH meter; (4) the detection Turbidity of the preparation; (5) Determination of non-aggregated and non-decomposed anti-OX40 antibodies in antibody preparations by size exclusion chromatography, for example, size-exclusion chromatography-HPLC (SEC-HPLC) Percentage; (6) Determination of charge variants of anti-OX40 antibodies in antibody preparations by cation exchange chromatography, for example, cation-exchange chromatography-HPLC (CEX-HPLC); (7) by twelve The purity of the antibody preparation was determined by sodium alkyl 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) guard column

Mobile phase: 20mmol / L phosphate (PB) + 150mmol / L NaCl + 200mmol / L Arg, pH6.8

Flow rate: 0.5ml / min

Column temperature: 25 ° C

Detection wavelength: 280nm

Injection volume: 50 μl

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:

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: reduced from 100% A to 60% A in 25 min

Column temperature: 35 ° C

Injection volume: 50 μl

Detection wavelength: 280nm

Capillary electrophoresis with sodium lauryl sulfate (CE-SDS)

The antibody preparation sample was diluted with ultrapure water to 10.0 mg / ml, and 10 μl of the diluted sample was taken, and a pH 6.5 sample buffer solution was sequentially added thereto (the sample buffer solution was obtained by pipetting 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, US, article number 390953) 2μl, 250mmol / L N-ethylmaleimide (NEM) 5μl, After thorough mixing, heat at 70 ° C for 10 minutes, and transfer to the sample tube after cooling. CE-SDS analysis was performed using 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 criteria for formulation stability

Example 1. Preparation and purification of anti-OX40 antibodies

New anti-OX40 antibodies that specifically bind to OX40 were prepared and purified according to CN201710185399.9 and CN201710185400.8, with the antibody names shown in Table 6 below, and their sequence characteristics are summarized in Tables 1 to 5 above. .

Table 6 Anti-OX40 antibodies

抗OX40抗体名称Anti-OX40 antibody name
ADI-20057ADI-20057
ADI-23504(ADI-20057后代)ADI-23504 (offspring of ADI-20057)
ADI-23507(ADI-20057后代)ADI-23507 (ADI-20057 offspring)
ADI-23509(ADI-20057后代)ADI-23509 (offspring of ADI-20057)
ADI-20112ADI-20112
ADI-25650(ADI-20112后代)ADI-25650 (offspring of ADI-20112)
ADI-25651(ADI-20112后代)ADI-25651 (offspring of ADI-20112)
ADI-25652(ADI-20112后代)ADI-25652 (offspring of ADI-20112)
ADI-25653(ADI-20112后代)ADI-25653 (offspring of ADI-20112)
ADI-25654(ADI-20112后代)ADI-25654 (offspring of ADI-20112)

ADI-20078ADI-20078
ADI-23515(ADI-20078后代)ADI-23515 (offspring of ADI-20078)
ADI-23518(ADI-20078后代)ADI-23518 (offspring of ADI-20078)
ADI-23519(ADI-20078后代)ADI-23519 (offspring of ADI-20078)
ADI-20048ADI-20048
ADI-20096ADI-20096
ADI-20051ADI-20051
ADI-20065ADI-20065
ADI-20066ADI-20066
ADI-20118ADI-20118
ADI-20113ADI-20113

Example 2. Effect of the pH of the buffer on the stability of anti-OX40 antibodies

This example evaluates the effect of the pH of the buffer on the stability of the anti-OX40 antibody. Buffers were prepared at different pH values according to Table 7 below. The concentration of each of the above-mentioned anti-OX40 antibodies in the buffer solution of each pH value was 15 mg / ml. After filtration, aliquot and perform the following measurement.

Table 7. pH values of buffers

A. Heat stress (40 ℃ ± 2 ℃) experiment

Each of the above samples containing 15 mg / ml of anti-OX40 antibody was placed in a 40 ° C ± 2 ° C constant temperature and humidity box, and samples were taken on day 0, day 1, 5 and 10. At the sampling time, the appearance of each sample and the presence of visible foreign matter were observed; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800); and efficient through size exclusion The purity (%) of each sample was measured by liquid chromatography (SEC-HPLC) method; and the charge variant (%) of each sample was measured by cation exchange high performance liquid chromatography (CEX) HPLC. The results are shown below.

(1) Appearance and presence of visible foreign matter

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

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

After being left for 10 days at a temperature of 40 ° C ± 2 ° C and a pH of about 8.0, the samples of each group appeared slightly cloudy.

(2) Protein content

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

Table 8. Protein content results of anti-OX40 antibody samples (UV method, mg / ml)

N / A means this item is not set.

(3) Purity

The purity (SEC-HPLC method) of each sample only accelerated slightly at 40 ± 2 ° C for 10 days.

At pH 5.0, the change in sample purity is mainly due to the degradation of antibody proteins after high temperature acceleration; at pH 7.0, the change in sample purity is mainly due to the aggregation of antibody proteins after high temperature acceleration; at pH 8 Under .0 conditions, the initial purity of the antibody protein in the sample was slightly lower, mainly due to the aggregation of the protein.

Table 9 lists the results of measuring the protein content of a sample 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 stability of the anti-OX40 antibody in the pH 6.0 or pH 7.0 buffer was better.

Table 9. Purity results of anti-OX40 antibodies (SEC-HPLC method,%)

(4) Charge variant

The charge variants of each anti-OX40 antibody sample changed at high temperature (40 ± 2 ° C). Among acid variants of the antibody, at pH 8.0, the acid component of the anti-OX40 antibody ADI-20112 (IgG1 type) sample Significantly increased, samples with pH 7.0 followed, samples with pH 5.0 and pH 6.0 had the smallest change; for the main component of the antibody, the main component of the sample with pH 7.0 had the smallest change; for basic variants of the antibody, each The basic components of the sample under pH conditions have a consistent decrease compared to the basic components at day 0. The results are shown in Figures 1-4 of the specification.

B. Oscillation Stress Experiment

Each of the above samples containing 15 mg / ml of anti-OX40 antibody was shaken at 650 rpm in the dark at 25 ° C, and samples were taken on day 0,

day

1, 3, and 5 days. At the sampling time, the appearance of each sample and the presence of visible foreign matter were observed; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800); and by the SEC-HPLC method The purity (%) of each sample was measured.

The results are shown below.

(1) Appearance and presence of visible foreign matter

When the pH is about 5.0, 6.0, 7.0, 8.0 and the shaking is up to 5 days, the appearance and visible foreign matter of each group of samples are 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 measurement results of the antibody ADI-20112 (IgG1 type) sample.

Table 10. Protein content results of anti-OX40 antibody samples (UV method, mg / ml)

N / A means this item is not set.

(3) Purity

When shaking was performed for 5 days under conditions of pH of about 5.0, 6.0, 7.0, and 8.0, the purity of each group of samples was measured. The results showed that the purity of each group of samples was not significantly affected under shaking conditions.

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

Table 11. Purity results of anti-OX40 antibody samples under shaking (SEC-HPLC method,%)

N / A means this item is not set.

C. Freeze-thaw test

Each of the above samples containing 15 mg / ml of anti-OX40 antibody was frozen (-30 ° C or lower) and thawed (using a water bath at a temperature of 25 ° C), and samples were taken at the 0th, 3rd, and 6th cycles. At the sampling time, the appearance of each sample and the presence of visible foreign matter were observed; the protein content of each sample was measured using an ultraviolet visible spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800); The purity (%) of each sample was measured.

The results are shown below.

(1) Appearance and presence of visible foreign matter

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

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

(2) Protein content

Under freeze-thaw conditions, the protein content of each group of samples was not significantly affected. Table 12 lists the protein content measurement results of the antibody ADI-20112 (IgG1 type) sample.

Table 12. Protein content results of anti-OX40 antibody samples (UV method, mg / ml)

N / A means this item is not set.

(3) Purity

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

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

Table 13. Purity results of anti-OX40 antibody samples under freeze-thaw cycles (SEC-HPLC method,%)

N / A means this item is not set.

From the above experimental results, it can be seen that no obvious changes in appearance, protein content, and purity were observed in the anti-OX40 antibody samples in buffers with a pH of about 5.0, 6.0, 7.0, and 8.0 when subjected to various stress conditions. Foreign matter is visible, therefore, any pH value in the range of about 5.0 to 8.0 can be selected to formulate an anti-OX40 antibody preparation.

Example 3. Formulation of a Stable Anti-OX40 Antibody Formulation

Four liquid preparations with anti-OX40 antibodies were prepared, with a pH of about 7.0. The components of the liquid preparations are shown in Table 14. The previously prepared anti-OX40 antibody sample solution was replaced with the prepared buffer solution of the four preparations, and then the anti-OX40 antibody solution in the sample solution was about 25 mg / ml. After adding polysorbate 80, the bacteria were filtered and sterilized. Packed in a 15R vial, 4ml / bottle, freeze-dried the sample, tested by high temperature after stoppering and capping, and tested for stability.

Table 14. Components of the anti-OX40 antibody formulation

The lyophilized preparation was placed under 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 that before lyophilization, and the protein content of the lyophilized preparation after reconstitution was determined to be about 26.0 mg / ml. At the sampling time point, the appearance of each sample after reconstitution and the presence of visible foreign matter were observed; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800); Turbidity; the purity (%) of each sample was determined by size exclusion high performance liquid chromatography (SEC-HPLC) method; the charge variant of anti-OX40 antibody in each sample was determined by cation exchange chromatography (CEX) HPLC method ( %). Similar results were obtained for formulations prepared for different antibodies. The results of the antibody ADI-20112 (IgG1 type) preparation are listed below.

(1) Appearance and presence of visible foreign matter

The four formulations were observed for up to 3 months at a temperature of 40 ° C ± 2 ° C and 25 ° C ± 2 ° C.

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

(2) Protein content

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

Table 15. Protein content results of anti-OX40 antibody preparations (UV method, mg / ml)

(3) Turbidity

Under the 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, and no significant change occurred with respect to the turbidity at day 0.

Table 16. Turbidity results of anti-OX40 antibody preparations (OD350nm method)

(4) Purity

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

Table 17. Purity results of anti-OX40 antibody preparations (SEC-HPLC method,%)

Purity (non-reducing 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.

Table 18. Purity results of lyophilized preparation screening studies (non-reduced CE-SDS method,%)

Purity (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 19 for details.

Table 19. Purity results of lyophilized preparation screening studies (reduced CE-SDS method,%)

(5) Charge variant

At 40 ° C ± 2 ° C, the charge variants of anti-OX40 antibodies in the sample of Formulation 1 at the third month did not change significantly compared with that of Day 0; the acidic components of the samples of Formulation 2 and Formulation 3 were relatively The change at day 0 was 4.1% and 2.3%, respectively. At 3 months, the sample of Formulation 4 had a change of 2.6% of the main component with respect to day 0. The specific results are shown in Table 20 and Figures 7-9.

At 25 ° C ± 2 ° C, the samples of each preparation showed no significant change in the charge variants of anti-OX40 antibody at 3 months compared with day 0.

Table 20. Charge variant results of anti-OX40 antibodies in the study of lyophilized preparations (CEX-HPLC method,%)

It can be seen from the above experimental results that Formulations 1-4 have an acceptable degree of stability. Formulation 2 was slightly inferior to Formulation 1 in terms of turbidity, purity (SEC-HPLC method) and charge variant (CEX-HPLC method) indicators; Formulation 3 and Formulation 4 were stable for each charge variant (CEX-HPLC method). Slightly lower than Formulation 1. Each stability index of Formulation 1 was better than other formulations.

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

Claims

A liquid antibody preparation having a pH of about 5.0-8.0, for example, a liquid antibody preparation having a pH of about 5.0, 6.0, 7.0, 8.0, comprising

(i) an anti-OX40 antibody or an antigen-binding fragment thereof;

(ii) a buffer,

(iii) stabilizers, and

(iv) Surfactants.
The liquid antibody preparation according to claim 1, characterized in that the concentration of the anti-OX40 antibody or antigen-binding fragment thereof in the liquid antibody preparation is 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.
The liquid antibody preparation according to claim 1 or 2, characterized in that the concentration of the buffering agent in the liquid antibody preparation 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, 8 mg / ml.
The liquid antibody preparation according to claim 1, wherein the buffering agent is selected from the group consisting of phosphate, citrate, citrate solvate, succinic acid, trimethylolaminomethane, and Their combination is more preferably citrate, citrate hydrate, for example, sodium citrate, sodium citrate dihydrate.
The liquid antibody preparation according to any one of claims 1 to 4, characterized in that the concentration of the stabilizer in the liquid antibody preparation is about 10-200 mg / ml, preferably about 20-100 mg / ml, such as about 30, 40, 50, 60, 70, 80, 90 mg / ml.
The liquid antibody preparation according to any one of claims 1 to 5, characterized in that the stabilizer is selected from the group consisting of sucrose, trehalose, mannitol and combinations thereof, and more preferably sucrose.
The liquid antibody preparation according to any one of claims 1-6, characterized in that the concentration of the surfactant in the liquid antibody preparation is about 0.01-5 mg / ml, preferably 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.
The liquid antibody preparation according to any one of claims 1 to 7, characterized in that the surfactant is a non-ionic surfactant, such as Plonik, polysorbate-80, polysorbate-60 , Polysorbate-40, or polysorbate-20.
The liquid antibody preparation according to any one of claims 1 to 8, characterized in that the anti-OX40 antibody or antigen-binding fragment thereof is capable of high affinity, for example, at 10 -7 M or less, preferably at 10 An anti-OX40 antibody or an antigen-binding fragment thereof that specifically binds OX40 and thus mediates a co-stimulatory response, K D of -8 M to 10 -12 M.
The liquid antibody preparation according to claim 9, characterized in that 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 VH contains

(i) in VH shown as SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 CDRs of the three complementary determining regions,

(ii) HCDR1 selected from the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, and 17 SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 of the amino acid sequence of HCDR2, and selected from SEQ ID NO: a combination of HCDR3 in the amino acid sequence of 37, 38, 39, 40, 41, 42, 43, and 44; 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 changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of;

and / or

(b) the VL contains

(i) three CDRs in VL as 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 selected from the amino acid sequences of SEQ ID NO: 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 changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of.
The liquid antibody preparation according to claim 10, 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 VH contains

(i) three complementarity determining regions CDRs in VH as shown in SEQ ID NO: 90, 91, 92, 93, 94 and 95,

(ii) HCDR1 selected from the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, and 8, HCDR2 selected from the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 26, and 27, and selected from A combination of HCDR3 in the amino acid sequence of SEQ ID NO: 38; 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 changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of;

and / or

(b) the VL contains

(i) three CDRs in the VL shown in SEQ ID NO: 116,

(ii) a combination of LCDR1 having the amino acid sequence of SEQ ID NO: 45, LCDR2 having the amino acid sequence of SEQ ID NO: 51, and LCDR3 having the amino acid sequence of SEQ ID NO: 58; 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 changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of.
The liquid antibody preparation according to claim 10, 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 variable region VH comprises and is 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 have an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity Its composition

and / or

(b) the light chain variable region VL comprises at least 90%, 91%, 92%, and an amino acid sequence selected from the group consisting of SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123, and 124, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical amino acid sequences or consist of them.
The liquid antibody preparation according to claim 12, characterized in that 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 variable region VH comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence selected from SEQ ID NO: 90, 91, 92, 93, 94, and 95. , 96%, 97%, 98% or 99% identity or 100% identity amino acid sequence or consist of it;

and / or

(b) the light chain variable region VL comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or an amino acid sequence selected from SEQ ID NO: 116; An amino acid sequence that is 99% identical or 100% identical or consists of it.
The liquid antibody preparation according to claim 12, characterized in that the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises and is selected from the group consisting of SEQ ID NOs: 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, 154, 155, 156, 157, 158, 159, 160, 161 and 162 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % Or 99% identity or consisting of a light chain comprising at least 90% of an amino acid sequence selected from SEQ ID 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequences or consist of them.
The liquid antibody preparation according to claim 14, characterized in that the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises an amino acid selected from the group consisting of SEQ ID NOs: 133 and 134 A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or consisting of a light chain comprising SEQ ID NO: 164 Or has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity.
The liquid antibody preparation according to claim 1, comprising about 20-30 mg / ml anti-OX40 monoclonal antibody, about 4-6 mg / ml sodium citrate or sodium citrate dihydrate, about 40-60 mg / ml sucrose, about 0.6-0.8mg / ml polysorbate 80, pH is about 6.0-7.0, for example, it contains about 25-26mg / ml anti-OX40 monoclonal antibody ADI-20112, about 5.5-6.0mg / ml (for example about 5.88mg / ml) sodium citrate dihydrate, about 50.00 mg / ml sucrose, about 0.7 mg / ml polysorbate 80, and a pH of about 7.0.
The liquid antibody formulation according to any one of 1-16 claims, characterized in that said anti-OX40 antibody is a polyclonal antibody or a monoclonal antibody, or a combination of both, preferably, the anti-OX40 antibody is a monoclonal antibody .
A solid antibody preparation obtained by curing the liquid antibody preparation according to any one of claims 1 to 17, said curing being performed by, for example, a crystallization method, a spray drying method, or a freeze drying method, said solid antibody The formulation is, for example, in the form of a lyophilized powder for injection.
A delivery device comprising a liquid antibody preparation according to any one of claims 1-17 or a solid antibody preparation according to claim 18.
A pre-filled syringe comprising a liquid antibody preparation according to any one of claims 1-17 or a solid antibody preparation according to claim 18 for intravenous or intramuscular injection.
Use of a liquid antibody preparation according to any one of claims 1-17 or a solid antibody preparation according to claim 18 for the preparation of a delivery device or pre-filled syringe or medicament for delivering an anti-OX40 antibody to a mammal.
Use of a liquid antibody preparation according to any one of claims 1-17 or a solid antibody preparation according to claim 18 for the preparation of activated T cells or induced T cell-mediated anti-tumor activity or enhancement of the body in a subject Immune response delivery device or pre-filled syringe or drug.
Use of a liquid antibody preparation according to any one of claims 1-17 or a solid antibody preparation according to claim 18 for the preparation of a delivery device or pre-filled syringe or medicament for treating cancer in a subject, for example, the cancer is Lung cancer (such as non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer.