CN111683681B

CN111683681B - Formulations comprising anti-OX 40 antibodies, methods of making, and uses thereof

Info

Publication number: CN111683681B
Application number: CN201980006061.XA
Authority: CN
Inventors: 汪音爵; 曹魏; 周凯松
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Sherpa Biotechnology Suzhou Co ltd
Priority date: 2018-09-25
Filing date: 2019-09-25
Publication date: 2023-03-24
Anticipated expiration: 2039-09-25
Also published as: TW202033179A; CN111683681A; TWI726426B; WO2020063668A1

Abstract

Formulations comprising anti-OX 40 antibodies, particularly pharmaceutical formulations comprising antibodies and/or antigen-binding fragments that specifically bind OX40 molecules, buffers, stabilizers, and surfactants are disclosed. In addition, the invention also discloses the disease treatment or prevention application of the preparations.

Description

Formulations comprising anti-OX 40 antibodies, methods of making, and uses thereof

Technical Field

The present invention relates to the field of antibody formulations. More specifically, the invention relates to pharmaceutical formulations comprising antibodies that specifically bind to OX40 (hereinafter also referred to as "anti-OX 40 antibodies") and/or antigen-binding fragments thereof, methods for preparing such pharmaceutical formulations, and therapeutic and/or prophylactic uses of such pharmaceutical formulations.

Background

Costimulatory molecules are a class of cell surface molecules, other than antigen receptors or antigen ligands, required by lymphocytes to mount an effective immune response to an antigen, which mediates the costimulatory response by lymphocytes through specific binding to the costimulatory ligand. Costimulatory molecules have been shown to 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 ACT 35) is a costimulatory molecule and OX40 signalling has been shown to promote costimulatory signaling to T cells, leading to enhanced cell proliferation, survival, effector function and migration (Gramaglia I et al, OX-40ligand.

anti-OX 40 antibodies that specifically bind OX40 as OX40 agonists are described in, for example, WO 2012/027328, U.S. 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-OX 40 antibody formulations that can be used to treat, prevent or delay various cancers, immune related diseases and T cell dysfunctional diseases.

Antibody formulations must be formulated in a manner that not only renders the antibody suitable for administration to a subject, but also maintains its stability during storage and subsequent use. For example, if an antibody is not properly formulated in a liquid, the antibody in the liquid solution is prone to decomposition, aggregation, or undesirable chemical modification, etc. The stability of an antibody in an antibody formulation depends on the buffers, stabilizers, surfactants, and the like used in the formulation.

Antibodies to OX40 are one example of diseases that need to be appropriately formulated to treat or prevent the disease. Although some anti-OX 40 antibodies are known, there remains a need in the art for novel pharmaceutical formulations containing anti-OX 40 antibodies that are sufficiently stable and suitable for administration to a subject.

Disclosure of Invention

The present invention meets the above needs by providing pharmaceutical formulations containing antibodies that specifically bind to OX40.

In one aspect, the invention provides a liquid antibody preparation comprising (i) an anti-OX 40 antibody or antigen-binding fragment thereof; (ii) A buffering agent, (iii) a stabilizing agent, and (iv) a surfactant.

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

In one embodiment, the anti-OX 40 antibody is any antibody that binds an OX40 molecule (e.g., a human OX40 molecule), such as a polyclonal antibody, a monoclonal antibody, or a combination of the two. Preferably, in one embodiment, the anti-OX 40 antibody is a monoclonal antibody. In one embodiment, the anti-OX 40 antibody or antigen-binding fragment thereof is an anti-OX 40 antibody or antigen-binding fragment thereof defined herein.

In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 0.1-50mg/ml. In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 1-20mg/ml, e.g., 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 the group consisting of phosphate, citrate solvate, succinic acid, tris and combinations thereof, more preferably citrate, citrate hydrate, e.g., sodium citrate dihydrate.

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

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

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

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

In one embodiment, the liquid formulation has a pH of about 5.0 to 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, more preferably a subcutaneous injection.

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

(i) About 1-150mg/mL of an anti-OX 40 antibody or antigen-binding fragment thereof;

(ii) Citrate, citrate hydrate, e.g., sodium citrate dihydrate, as a buffer, in an amount of about 0.1-50 mg/ml;

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

(iv) Polysorbate-80 as a surfactant at about 0.01-5 mg/ml;

wherein the pH of the liquid formulation is about 5.0-8.0.

(i) About 10-100mg/mL of an anti-OX 40 antibody or antigen-binding fragment thereof;

(ii) About 1-20mg/ml sodium citrate or sodium citrate dihydrate as a buffer;

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

(iv) Polysorbate-80 as a surfactant at about 0.1-2 mg/ml;

wherein the pH of the liquid formulation is about 5.0-8.0.

(i) About 20-30mg/mL of an anti-OX 40 antibody or antigen-binding fragment thereof;

(ii) About 4-6mg/ml sodium citrate or sodium citrate dihydrate as a buffer;

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

(iv) Polysorbate-80 as a surfactant at about 0.6-0.8 mg/ml;

wherein the pH of the liquid formulation is about 6.0-7.0.

In another aspect, the present invention provides a solid antibody preparation obtained by subjecting a liquid antibody preparation of the present invention to a curing treatment. The solidification treatment is carried out by, for example, crystallization, spray drying or freeze drying. In a preferred embodiment, the solid antibody formulation is, for example, in the form of a lyophilized powder injection.

Solid antibody formulations can be reconstituted in a suitable vehicle to form reconstituted formulations of the invention prior to use. The reconstituted formulation is also a liquid antibody formulation of the invention. In one embodiment, the suitable 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 combinations thereof.

In one embodiment, a liquid or solid formulation of the invention comprising an anti-OX 40 antibody reduces purity of the anti-OX 40 antibody or antigen-binding fragment thereof by no more than 10%, such as no more than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% as measured by size exclusion high performance liquid chromatography after storage at about-80 ℃ to about 45 ℃, such as-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, 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 months, at least 24 months, at least 36 months, or longer.

In one embodiment, a liquid or solid formulation of the invention comprising an anti-OX 40 antibody reduces the purity of the anti-OX 40 antibody or antigen-binding fragment thereof by no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% as measured by non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method and/or reduced CE-SDS method after storage at about-80 ℃ to about 45 ℃, e.g., about-80 ℃, about-30 ℃, about-20 ℃, about-0 ℃, 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 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.

In one embodiment, a liquid or solid formulation of the invention comprising an anti-OX 40 antibody changes no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, or more, as measured by cation exchange high performance liquid chromatography (CEX-HPLC), after storage at about-80 ℃ to about 45 ℃, e.g., about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, 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 months, at least 24 months, at least 36 months, or more, as measured by cation exchange high performance liquid chromatography (CEX-HPLC).

In one embodiment, the anti-OX 40 antibody or antigen-binding fragment thereof in the liquid formulation of the invention can be with high affinity, e.g., at 10 ^-7 M or less, preferably at 10 ^-8 M to 10 ^-12 K of M _D An anti-OX 40 antibody or antigen-binding fragment thereof that specifically binds OX40 and thereby mediates a costimulatory response.

In a preferred embodiment, the anti-OX 40 antibody or antigen-binding fragment thereof in the liquid formulation of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein

(a) Said VH comprises

(i) 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 in VH,

(ii) A combination of an HCDR1 selected from the amino acid sequences of

SEQ ID NOs

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17, an HCDR2 selected from the amino acid sequences of

SEQ ID NOs

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36, and an HCDR3 selected from the amino acid sequences of

SEQ ID NOs

37, 38, 39, 40, 41, 42, 43 and 44; or

(iii) (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in each of the three CDRs relative to the sequence of (i) or (ii);

and/or

(b) Said VL comprising

(i) Three CDRs in VL as shown in SEQ ID NOs 115, 116, 117, 118, 119, 120, 121, 122, 123 and 124,

(ii) A combination of LCDR1 of an amino acid sequence selected from

SEQ ID NOS

45, 46, 47, 48, 49 and 50, LCDR2 of an amino acid sequence selected from

SEQ ID NOS

51, 52, 53, 54, 55 and 56, and LCDR3 of an amino acid sequence selected from

SEQ ID NOS

57, 58, 59, 60, 61, 62, 63, 64, 65 and 66; or

(iii) (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDRs, respectively, relative to the sequence of (i) or (ii).

In yet another preferred embodiment, the anti-OX 40 antibody in the liquid formulation of the 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 or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98, 99, 100, 101, 102, 103 and 104% identity or 100% identity to an amino acid sequence selected from the group consisting of

SEQ ID NOs

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%;

and/or

(b) The light chain variable region VL comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence selected from SEQ ID NOs 115, 116, 117, 118, 119, 120, 121, 122, 123 and 124.

In yet another preferred embodiment, the anti-OX 40 antibody in the liquid formulation of the invention comprises a heavy chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence 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, and/or a light chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 163, 164, 165, 166, 167, 168, 169, 170, 171 and 172.

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

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

In one embodiment, the invention relates to a method of delivering an anti-OX 40 antibody to a subject, e.g., a mammal, comprising the step of administering to the subject, e.g., a mammal, a liquid antibody formulation or a solid antibody formulation of the invention as described in various embodiments herein, e.g., by using a pre-filled syringe delivery device.

The invention further provides the use of a liquid antibody formulation or a solid antibody formulation of the invention for the preparation of a delivery device (e.g. a pre-filled syringe) or medicament for activating T cells or inducing a T cell-mediated anti-tumour activity or enhancing an immune response in a subject, in particular for the treatment of a disease, for example cancer, such as lung cancer (e.g. non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, in a subject.

The invention further provides a method of activating T cells or inducing T cell-mediated anti-tumor activity or enhancing an immune response in a subject by administering to the subject a liquid antibody formulation or a solid antibody formulation of the invention or a delivery device (e.g., a pre-filled syringe) or a medicament comprising the liquid antibody formulation or the solid antibody formulation.

The invention further provides a method of treating a disease, e.g., cancer, e.g., lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, in a subject by administering to the subject a liquid or solid antibody formulation of the invention or a delivery device (e.g., a pre-filled syringe) or a medicament comprising the liquid or solid antibody formulation.

Other embodiments of the invention will be apparent by reference to the detailed description that follows.

Drawings

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

FIG. 1 shows a charge variant profile of anti-OX 40 antibody placed under heat stress for 10 days at pH 5.0.

FIG. 2 shows a charge variant profile of anti-OX 40 antibody incubated for 10 days under heat stress at pH 6.0.

FIG. 3 shows a charge variant profile of anti-OX 40 antibody incubated for 10 days under heat stress at pH 7.0.

FIG. 4 shows a charge variant profile of anti-OX 40 antibody incubated for 10 days under heat stress at pH 8.0.

FIG. 5 is a graph showing the turbidity change after exposure of an anti-OX 40 antibody formulation of the invention to temperature conditions of 40 ℃. + -. 2 ℃.

FIG. 6 is a graph showing the change in purity of an anti-OX 40 antibody formulation of the invention as measured by SEC-HPLC after placement at a temperature of 40 ℃. + -. 2 ℃.

FIG. 7 is a graph showing the change in the acidic component of charge variants of anti-OX 40 antibodies as determined by CEX-HPLC after placement of anti-OX 40 antibody formulations of the invention at a temperature of 40 ℃. + -. 2 ℃.

FIG. 8 is a graph showing the change in major composition of charge variants of anti-OX 40 antibodies as determined by CEX-HPLC after exposure of an anti-OX 40 antibody formulation of the invention to a temperature of 40 ℃. + -. 2 ℃.

FIG. 9 is a graph showing the change in basic composition of charge variants of anti-OX 40 antibodies as determined by CEX-HPLC after placement of anti-OX 40 antibody formulations of the invention at a temperature of 40 ℃. + -. 2 ℃.

Detailed Description

Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. In addition, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless defined otherwise, 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 the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. The terms "comprises" or "comprising" are intended to be inclusive of the stated elements, integers, or steps, but not to exclude any other elements, integers or steps.

I. Antibody formulations

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 that does not contain additional components that are unacceptably toxic to a subject to whom the preparation will be administered. Such antibody preparations are generally sterile. "pharmaceutically acceptable" excipients are those excipients which can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.

The term "anti-OX 40 antibody preparation" as used herein means a combination of at least one anti-OX 40 antibody as an active ingredient and at least one inactive ingredient. After such combination, the anti-OX 40 antibody as active ingredient is suitable for therapeutic or prophylactic administration to a human or non-human animal. According to the invention, anti-OX 40 antibody as active ingredient in the preparation binds specifically to OX40 molecules, the resulting signaling promotes costimulatory signaling of T cells, resulting in enhanced cell proliferation, survival, effector function and migration.

The antibody formulations of the invention may be sterile, homogeneous and/or isotonic, and may be prepared directly as liquid formulations in aqueous form, e.g., ready-to-use prefilled syringes, or as lyophilized formulations for reconstitution (i.e., reconstitution) by dissolution and/or suspension in a physiologically acceptable solution immediately prior to use. In some embodiments, the anti-OX 40 antibody formulation is in the form of a liquid formulation, a lyophilized formulation, or a reconstituted formulation.

The use of antibodies as active ingredients in pharmaceuticals is now widespread, including the product HERCEPTIN ^TM (trastuzumab), RITUXAN ^TM (Rituximab), SYNAGIS ^TM (palivizumab) and the like. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art.

A "sterile" formulation is one that is sterile or free or substantially 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 formulation" refers to a composition obtained or obtainable by a freeze-drying process of a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

The term "reconstituted formulation" refers to a liquid formulation resulting from dissolving and/or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

In particular embodiments, the anti-OX 40 antibody formulations of the invention exhibit undetectable levels of antibody aggregation or degradation or chemical modification during manufacturing, manufacture, shipping, and long term storage, resulting in little to no loss of biological activity of the anti-OX 40 antibody, exhibiting high stability. As used herein, the term "stable" antibody formulation is one in which the antibody in the formulation retains an acceptable degree of physical and/or chemical stability after storage under specified conditions. Although an antibody contained in an antibody preparation does not 100% maintain its chemical structure after a particular time of storage, an antibody preparation is generally considered "stable" if about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function is maintained after a particular time of storage. In some embodiments, the anti-OX 40 antibody formulations of the invention substantially retain their physical and chemical stability after storage. The storage time is typically 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 formulation. These tests may indicate extreme conditions that a formulated antibody preparation may encounter during manufacturing, storage or transportation, and may also indicate conditions that accelerate the instability of the antibodies in the antibody preparation under extreme conditions not during manufacturing, storage or transportation. For example, formulated anti-OX 40 antibody formulations were filled into 5mL glass vials for shaking stress, freeze/thaw cycling (i.e., freeze-thaw cycling) stress; the formulated anti-OX 40 antibody formulation was filled into glass vials to examine antibody stability under high temperature stress.

The term "room temperature" as used herein means a temperature of from 15 ℃ to 30 ℃, preferably from 20 ℃ to 27 ℃, more preferably 25 ℃.

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 (e.g., 40 ℃).

After a long storage time, the antibodies in the preparation do not show aggregation, precipitation and/or turbidity if the preparation is checked for appearance, color and/or clarity, or determined, for example, by UV light scattering or by size exclusion chromatography; or exhibit very little aggregation, precipitation, and/or turbidity, the antibody "retains its physical stability" in the formulation. Safety issues arise due to aggregation of antibodies in the formulation which can potentially lead to increased immune responses in the patient. Therefore, there is a need to minimize or prevent aggregation of antibodies in a formulation.

In one embodiment, the stability of the formulation is measured by determining the percentage of non-aggregated and non-disintegrated antibodies in the formulation after storage at a specific temperature for a specific time, wherein the higher the percentage of non-aggregated and non-disintegrated antibodies in the formulation, the higher the stability of the formulation. The percentage of non-aggregated and non-disintegrated 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 is meant that at least about 92% of non-aggregated and non-resolved anti-OX 40 antibodies are detected in the formulation after storage at a particular temperature for a particular time. In some embodiments, an acceptable degree of stability after storage 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 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 represents at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% non-aggregated and non-degraded anti-OX 40 antibody. When evaluating stability, the particular temperature at which the pharmaceutical formulation is stored can be any temperature from about-80 ℃ to about 45 ℃, e.g., about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ℃ to 8 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 37 ℃, about 40 ℃, about 42 ℃, or about 45 ℃. For example, a pharmaceutical formulation is considered stable if, after 3 months of storage at about 40 ℃, at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-disintegrated forms of anti-OX 40 antibody are detected. A pharmaceutical formulation is considered stable if at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-resolved forms of anti-OX 40 antibody are detected after 3 months of storage at about 25 ℃. A pharmaceutical formulation is considered stable if at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of non-aggregated and non-disintegrated form of anti-OX 40 antibody is detected after 9 months of storage at about 5 ℃.

In addition, an antibody "retains its chemical stability" in a formulation if its chemical structure is intact in the formulation after a long storage time. Most of the chemical instability stems from the formation of covalently modified forms of antibodies (e.g., charge variants of antibodies). In some embodiments, changes in the charge variant components of the anti-OX 40 antibody are detected after storage of the antibody preparation.

In one embodiment, charge variants of the anti-OX 40 antibody in the antibody preparation are 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 are labeled as "basic peaks". In the CEX-HPLC method, the acidic component percentage is determined by the ratio of the acidic peak area to the sum of the main peak, acidic peak and basic peak area; the main component percentage is determined by the ratio of the main peak area to the sum of the main peak, the acidic peak and the basic peak area; the percentage of basic component is determined by the ratio of the area of the basic peak to the sum of the main peak, the acidic peak and the area of the basic peak. Without being bound by theory, it is believed that Deamidation of an antibody may cause the antibody to become more negatively charged and thus more acidic relative to a non-deamidated antibody (see, e.g., robinson, n., protein Deamidation, PNAS, 16.4.2002, 99 (8): 5283-5288). The term "acceptable degree of stability" as used herein means that at most about 25% of the antibody is in a more acidic form in the formulation after storage at a particular temperature for a particular time. In some embodiments, an acceptable degree of stability after storage 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 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, indicates that at most 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 after storage for a particular time at a particular temperature. When evaluating stability, the temperature at which the pharmaceutical formulation is stored can be any temperature from about-80 ℃ to about 45 ℃, e.g., at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ℃ to 8 ℃, about 5 ℃, about 25 ℃, or about 45 ℃. For example, a pharmaceutical formulation may be considered stable if 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 is in a more acidic form after 3 months of storage at-80 ℃, -30 ℃ or-20 ℃. A pharmaceutical formulation may also be considered stable if 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 after 9 months of storage at 5 ℃. A pharmaceutical formulation may also be considered stable if 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 after 28 days of storage at 25 ℃. A pharmaceutical formulation may 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%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is detected in a more acidic form. A pharmaceutical formulation may also be considered stable if after 28 days of storage at 40 ℃, 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 detected in a more acidic form.

In some embodiments, the stable anti-OX 40 antibody formulations of the invention are administered to a subject parenterally. As used herein, the term "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, sub-epidermal (subericular), intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In a specific embodiment, an anti-OX 40 antibody formulation of the invention is administered subcutaneously to a subject.

In some embodiments, a stable anti-OX 40 antibody formulation of the invention comprises: (i) An anti-OX 40 antibody or antigen-binding fragment thereof that specifically binds to an OX40 molecule; (ii) a buffering agent; (iii) (iii) a stabilizer, and (iv) a surfactant, the anti-OX 40 antibody formulation having a pH of about 5.0 to about 8.0.

(i) An anti-OX 40 antibody or antigen-binding fragment thereof that specifically binds to an OX40 molecule

An antibody preparation of the invention can comprise an anti-OX 40 antibody or antigen-binding fragment thereof that specifically binds to an OX40 molecule. As used herein, the term "OX 40" is known to be a costimulatory molecule, the activation of which can lead to enhanced cell proliferation, survival, effector function and migration. anti-OX 40 antibodies are disclosed in the prior art as OX40 agonists. For example, the amino acid sequences of the heavy and light chain variable regions of anti-OX 40 antibody mAb 106-222 and humanized 106-222 (Hu 106) are disclosed in WO 2012/027328; amino acid sequences of heavy and light chain variable regions of anti-OX 40 antibody mAb 119-122 and humanized 119-122 (Hu 119). In addition, anti-OX 40 antibodies that are OX40 agonists are also disclosed in U.S. Pat. No. 7,959,925, PCT publication No. WO 2006/121810, and Chinese patent application Nos. CN201710185399.9 and CN 201710185400.8. The anti-OX 40 antibody is capable of activating OX40, thereby inducing effector T lymphocyte proliferation, promoting an immune response against tumor cells expressing a Tumor Associated Antigen (TAA). The entire contents of which are incorporated herein by reference.

The term "co-stimulatory molecule" refers to a corresponding binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response (e.g., without limitation, proliferation) of the T cell. Costimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to the antigen receptor or its ligand. In an embodiment of the invention, the co-stimulatory molecule is an OX40 molecule.

The terms "anti-OX 40 antibody", "anti-OX 40", "OX 40 antibody", or "an antibody that binds OX40" refer to an antibody that is capable of binding OX40 molecule with sufficient affinity such that the antibody can be used as a therapeutic and/or prophylactic agent that targets OX40 molecules. In one embodiment, the anti-OX 40 antibody binds to an unrelated, non-OX 40 protein to less than about 10% of the binding of the antibody to OX40 as measured, for example, by Radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (K) of an anti-OX 40 antibody _D ) Less than or equal to 1 mu M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM. In still other embodiments, the anti-OX 40 antibody is capable of binding with high affinity, e.g., at 10 ^-7 M or less, preferably at 10 ^-8 M to 10 ^-12 K of M _D Specifically binds to OX40 and thereby mediates a costimulatory response. In this context, when referring to an "anti-OX 40 antibody," antigen-binding fragments of anti-OX 40 antibodies are also included.

An "antigen-binding fragment" of an antibody refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an 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; a diabody; a linear antibody; single chain antibodies (e.g., scFv); a single domain antibody; a bivalent or bispecific antibody or fragment thereof; camelid antibodies; and bispecific or multispecific antibodies formed from antigen-binding fragments.

As used herein, the term "epitope" refers to a portion of an antigen (e.g., OX 40) that specifically interacts with an antibody molecule.

"complementarity determining regions" or "CDR regions" or "CDRs" are regions of antibody variable domains that are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al (1989) Nature 342-883, al-Lazikani et al, "Standard constraints for the conditional structures of Immunological tissues", journal of Molecular Biology,273, 927-948 (1997)), kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. department of Health and Services, national Institutes of Health (1987)), abM (version of balance), unity (University Collection), international Munonglutics database (IMG) (in International science of biological databases), and Mass Spectrometry (CDR/CDR) using the same.

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 differ. I.e., the CDR sequences of the same antibody variable region defined under different assignment systems differ. Thus, where reference is made to an antibody defined with a particular CDR sequence as defined herein, the scope of the antibody also encompasses an antibody whose variable region sequences comprise the particular CDR sequence but whose claimed CDR boundaries differ from the particular CDR boundaries as defined herein due to the application of different protocols (e.g., different assignment system rules or combinations).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. The region of minimum overlap can be determined using at least two of the Kabat, chothia, abM, contact, and North methods, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be appreciated by those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure and protein folding of the antibody. Thus, the present invention also contemplates variants of any of the CDRs given herein. For example, in a variant of one CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues according to Kabat or Chothia definition may be replaced by conserved amino acid residues.

The term "conservative amino acid residue substitutions" are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues of an anti-OX 40 antibody can be replaced by other amino acid residues from the same side chain family, and the altered antibody can be tested for function, particularly the same binding properties as the OX40 molecule. Changes in the charge appearance of the CDR surfaces are expected to affect the interface between the antibody and the solvent, and therefore, non-conservative amino acid residue substitutions have unpredictable effects on maintaining or improving the stability of the antibody in solution.

Suitable "antibodies or antigen-binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (particularly grafted with CDRs), deimmunized, or human antibodies, fab fragments, fab 'fragments, F (ab') ₂ Fragments, fragments produced by Fab expression libraries, fd, fv, disulfide linked Fv (dsFv), single chain antibodies (e.g., scFv), diabodies or tetrabodies (Holliger p. Et al (1993) proc. Natl.acad.sci.u.s.a.90 (14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the 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, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences.

"IgG form of an antibody" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are the same for all antibodies of the same type, and differ between antibodies of different types. For example, an antibody in the form of an IgG1 refers to an antibody whose heavy chain constant region Ig domain is the Ig domain of IgG 1.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source, using a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"humanized" antibodies refer to chimeric antibodies 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, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

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

The term "specifically binds" or similar terms, 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 a dissociation constant of at least about 10 ^-7 M or less, preferably at 10 ^-8 M to 10 ^-12 K of M _D Specifically binds to OX40 and thereby mediates a costimulatory response.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are the same amino acid residues in the reference polypeptide sequence, after the sequences are aligned (and gaps introduced, if necessary) to achieve the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. 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. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may 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 antibody or antigen-binding fragment thereof included in an antibody formulation of the invention may vary with the particular desired characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is used. In some embodiments, the antibody formulation is a liquid formulation, which may contain about 1-150mg/mL, preferably about 10-100mg/mL, e.g., about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60mg/mL of the anti-OX 40 antibody or antigen-binding fragment thereof.

In one embodiment, the invention relates to formulations having high concentrations of anti-OX 40 antibody, e.g., containing 40-150 mg/mL of anti-OX 40 antibody. It is known in the art that such high concentration antibody formulations may be diluted prior to injection, for example if lower antibody concentrations are required for a particular therapeutic or prophylactic intervention or when treating smaller weight patients, including children. A suitable concentration may be 25mg/mL or 10mg/mL. Alternatively, the original formulation may be produced at such low concentrations.

In addition, the anti-OX 40 antibody formulations of the invention described herein in the various embodiments are stable. In one embodiment, the anti-OX 40 antibody in an antibody preparation of the invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, as determined by size exclusion chromatography, after storage at about-80 ℃, -30 ℃, -20 ℃, 5 ℃,25 ℃, 37 ℃, 40 ℃, or 45 ℃ for 6 months. In one embodiment, at least 50% of the anti-OX 40 antibody in an antibody preparation of the invention is in a non-basic and non-acidic form (i.e., the main peak or major charge form) after 6 months of storage at about-80 ℃, -30 ℃, -20 ℃, 5 ℃,25 ℃, 37 ℃, 40 ℃, or 45 ℃, as determined by cation exchange chromatography.

Exemplary anti-OX 40 antibodies that can be included in the antibody formulations of the invention are anti-OX 40 antibodies 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-8, or ADI-2353, each comprising the sequences shown in the table 201120112011 20113, disclosed in CN201710185399.9 and CN201710185400.8, for example.

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

In a preferred embodiment, the anti-OX 40 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) Said VH comprises

(ii) A combination of an HCDR1 selected from the amino acid sequences of

SEQ ID NOs

37, 38, 39, 40, 41, 42, 43 and 44; or

and/or

(b) Said VL comprising

(ii) A combination of LCDR1 of an amino acid sequence selected from

SEQ ID NOS

45, 46, 47, 48, 49 and 50, LCDR2 of an amino acid sequence selected from

SEQ ID NOS

51, 52, 53, 54, 55 and 56, and LCDR3 of an amino acid sequence selected from

SEQ ID NOS

57, 58, 59, 60, 61, 62, 63, 64, 65 and 66; or

(iii) (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in each of the three CDRs relative to the sequence of (i) or (ii).

In yet another preferred embodiment, the anti-OX 40 antibody in the antibody preparation of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,

SEQ ID NOs

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%;

and/or

In yet another preferred embodiment, the anti-OX 40 antibody in the antibody preparation of the invention comprises a heavy chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence 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, and/or a light chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 163, 164, 165, 166, 167, 168, 169, 170, 171 and 172.

(ii) Buffer agent

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

In one embodiment, the antibody formulation of the invention comprises such a buffer or pH adjuster 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 antibodies of the invention have a pH of about 5.0, 6.0, 7.0, 8.0.

In one embodiment, the buffer is included in an antibody formulation of the invention at a concentration of about 0.1-50mg/ml, preferably about 1-20mg/ml, e.g., 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 (b)

Suitable stabilizers for use in the present invention may function, for example, as 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 a sugar.

For sugars as stabilizers, they include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and the like, and combinations thereof. For example, the sugar may be sucrose, trehalose, raffinose, maltose, sorbitol, or mannitol. Preferably, the sugar is sucrose.

For ionic stabilizers, it includes salts, e.g., naCl.

(iv) A surfactant

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

In one embodiment, the surfactant in the liquid formulation of the present invention is a non-ionic surfactant, for example, an alkyl poly (ethylene oxide). Specific nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates, such as polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate-20; pluronic, and the like.

The amount of nonionic surfactant included in an antibody formulation of the invention can vary with the particular desired characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is used. In certain embodiments, the formulation may contain polysorbate-80 or pluronic at a concentration of about 0.01-5mg/ml, preferably about 0.1-2mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mg/ml.

Other contemplated excipients that may be utilized in the antibody liquid formulations of the present invention include, for example, flavoring agents, antimicrobial agents, sweetening agents, antioxidants, antistatic agents, gelatin, and the like. These and additional known Pharmaceutical Excipients and/or additives suitable for use in The formulations of The present invention are well known in The art, for example, as listed in "The Handbook of Pharmaceutical Excipients, 4 th edition, edited by Rowe et al, american Pharmaceuticals Association (2003); and Remington the Science and Practice of Pharmacy, 21 st edition, edited by Gennaro, lippincott Williams & Wilkins (2005) ".

Furthermore, an antibody formulation according to the invention as described herein in various embodiments is stable such that the anti-OX 40 antibody is greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, as measured by SEC-HPLC, even after 4 weeks, 1 month or 3 months of storage at 25 ℃ or 40 ℃.

Target diseases and disorders

The antibody formulations of the invention comprising anti-OX 40 antibodies of the invention can be used to treat, ameliorate or prevent a variety of diseases or disorders. Formulations comprising anti-OX 40 antibodies are particularly useful for treating, ameliorating, or preventing cancer, immune-related diseases, and T cell dysfunctional diseases.

For example, an antibody preparation of the invention comprising an anti-OX 40 antibody can be used to treat, ameliorate or prevent cancer. Such cancers include, but are not limited to, B-cell lymphomas (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulk disease) NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Waldenstrom's macroglobulinemia), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses (phakomatoses), edema (such as associated with brain tumors), B-cell proliferative disorders, and Meigs's syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, anterior (front line) low grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic (lymphoplasmacytic) lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone (extranodal marginal zone) -MALT lymphoma, nodal marginal zone (nodal marginal zone) lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive (aggressive) NHL (including aggressive frontier NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small anucleate NHL, storage disease (bulk disease) NHL, burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, granulomatosis like and/or Sezary (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 (e.g., non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer.

Administration of an antibody formulation to a subject or patient

The antibody formulations of the invention can be administered to a subject or patient. Administration is typically by infusion or by syringe. Accordingly, the invention provides a delivery device (e.g. a syringe) comprising an antibody formulation of the invention (e.g. a pre-filled syringe). The patient will receive an effective amount of an anti-OX 40 antibody as the primary active ingredient, i.e., an amount sufficient to treat, ameliorate, or prevent the disease or disorder of interest.

Therapeutic effects may also include reduction of physiological symptoms. The optimal effective amount and concentration of antibody for any particular subject will depend upon a variety of factors including the age, weight, health and/or sex of the patient, the nature and extent of the disease, the activity of the particular antibody, its clearance by the body, and also includes any possible other treatments administered in combination with the antibody preparation. For a particular situation, the effective amount delivered may be determined within the judgment of a clinician. For the purposes of the present invention, an effective dose may be from about 0.005mg/kg body weight to about 50mg/kg body weight, or from about 0.05mg/kg body weight to about 10mg/kg body weight. Known antibody-based drugs provide guidance in this regard, e.g., HERCEPTIN ^TM An initial loading dose of 4mg/kg body weight and a weekly maintenance dose of 2mg/kg body weight; RITUXAN ^TM At 375mg/m per week ² Administration; SYNAGIS ^TM Administered intramuscularly at 15mg/kg body weight; and so on.

The invention also provides formulations of the invention as described herein in various embodiments for use as a medicament, e.g., for delivering anti-OX 40 antibodies to a mammal, or for treating, preventing, or ameliorating one or more of the diseases and disorders described above.

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 desirably raised against the target species, e.g., human anti-OX 40 antibodies for humans, equine anti-OX 40 antibodies for horses, canine anti-OX 40 antibodies for dogs, etc. If a native host antibody is not available, antibody-specific transfer from one species to another, for example by humanization, can be achieved by transfer of CDR residues (typically also one or more framework residues) from a donor antibody to a recipient framework from the host species. Equine, bovine, canine, and feline antibodies are known in the art. The antibody will bind to OX40 of the target species, but it can also cross-react with OX40 from other species.

The dosage may be a single dose schedule or a multiple dose schedule.

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.

Examples

Materials and methods

1.1. Chemicals used in the study of lyophilized formulations

1.2. Instrumentation used

1.3. Detection item and detection method for stability of preparation

The following items were tested for antibody formulations: (1) detecting the appearance and the presence of visible foreign matter; (2) Determining the protein content of the formulation by ultraviolet method (UV method); (3) measuring the pH value by using a pH meter; (4) detecting the turbidity of the preparation; (5) Determining the percentage of non-aggregated and non-degraded anti-OX 40 antibody in the antibody preparation by size exclusion chromatography, e.g., size-exclusion high performance liquid chromatography (SEC-HPLC); (6) Determining charge variants of the anti-OX 40 antibody in the antibody preparation by cation exchange chromatography, e.g., cation exchange high performance liquid chromatography (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 analysis of antibody formulations, the following parameters were used:

a chromatographic column: TSK-gel SuperSW mAb HR (7.8X 300mm,4 μm) type analytical column, TSK-gel SuperSW (6.0X 40mm,4 μm) protective column

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

Flow rate: 0.5ml/min

Column temperature: 25 deg.C

Detection wavelength: 280nm

Sample introduction volume: 50 μ l

Temperature of a sample injection tray: about 10 deg.C

Operating time: 30 minutes

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

In the CEX-HPLC method for analysis of antibody preparations, the following parameters were used:

a chromatographic column: thermo Scientific ProPacTM WCX-10 weak cation analytical column

A mobile phase A:10mmol/L PB

Mobile phase B10 mmol/L PB,200mmol/L NaCl

Gradient: 25min from 100% to 60% A

Column temperature: 35 deg.C

Sample introduction volume: 50 μ l

Detection wavelength: 280nm

Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method

Diluting an antibody preparation sample to 10.0mg/ml with ultrapure water, taking 10 mu l of the diluted sample, sequentially adding 85 mu l of pH6.5 sample buffer solution (prepared by sucking 200 mu l of pH6.5 citric acid-phosphate buffer solution, adding 47 mu l of 10% SDS and adding ultrapure water to 1000 mu l) into the sample buffer solution, 2 mu l of 10kDa internal standard (Beckman, cat # 390953) and 5 mu l of 250mmol/L N-ethylmaleimide (NEM), fully mixing, heating at 70 ℃ for 10 minutes, cooling and transferring to a sample tube. CE-SDS analysis was performed using a Beckman PA800 Plus capillary electrophoresis apparatus at-5 kV for 20 seconds, at-15 kV and-16.5 kV for separation, and for 35 and 36 minutes for analysis, respectively.

Judgment standard of preparation stability

Example 1 preparation and purification of anti-OX 40 antibodies

Novel anti-OX 40 antibodies that specifically bind OX40 were prepared and purified as described in CN201710185399.9 and CN201710185400.8, having antibody names shown in table 6 below, respectively, the sequence characteristics of which are summarized in tables 1-5 above.

TABLE 6 anti-OX 40 antibodies

Example 2 Effect of pH of buffer on anti-OX 40 antibody stability

This example evaluates the effect of buffer pH on anti-OX 40 antibody stability. Buffers at different pH values were prepared according to table 7 below. The concentration of each of the above anti-OX 40 antibodies in each pH buffer was 15mg/ml. Filtered, packed and measured as follows.

TABLE 7 pH of the buffer

A. Heat stress (40 ℃ C. + -. 2 ℃ C.) test

Each of the above samples containing 15mg/ml of anti-OX 40 antibody was placed in a 40 ℃. + -. 2 ℃ incubator and sampled at 0 day, 1 day, 5 days, and 10 days. The appearance of each sample, the presence of visible foreign matter, was observed at the time of sampling; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (model UV-1800, manufactured by shimadzu, japan); the purity (%) of each sample was determined by size exclusion high performance liquid chromatography (SEC-HPLC); the charge variants (%) of each sample were determined by cation exchange high performance liquid chromatography (CEX HPLC). The results are shown below.

(1) Appearance, presence or absence of visible foreign matter

After the samples are placed for 1 day or 5 days at the temperature of 40 +/-2 ℃ and the pH value of about 5.0, 6.0, 7.0 and 8.0, the appearance and the detection of visible foreign matters of the samples of each group are qualified.

After the samples are placed for 10 days at the temperature of 40 +/-2 ℃ and the pH value of about 5.0, 6.0 and 7.0, the appearance and the detection of visible foreign matters of the samples of each group are qualified.

After 10 days at 40 ℃. + -. 2 ℃ pH of about 8.0, the samples of each group were slightly cloudy.

(2) Protein content

The protein content of each group of samples was unchanged at 40 ℃. + -. 2 ℃, and the results of the protein content determination in the samples of antibody ADI-20112 (IgG 1 type) are listed in Table 8.

TABLE 8 protein content results for anti-OX 40 antibody samples (UV method, mg/ml)

N/A indicates that the item is not set.

(3) Purity of

The purity of each sample (SEC-HPLC method) decreased only slightly at 40. + -. 2 ℃ accelerated for 10 days.

Under the condition of pH5.0, the reason for the change of the purity of the sample is mainly that the antibody protein is degraded after the high-temperature acceleration; under the condition of pH7.0, the reason for the change of the purity of the sample is mainly that the antibody protein aggregates after the high-temperature acceleration; at pH8.0, the initial purity of the antibody protein in the sample is slightly lower, mainly due to aggregation of the protein.

Table 9 lists the results of protein content determination for samples in which antibody ADI-20112 (IgG 1 type) was obtained. When the anti-OX 40 antibody ADI-20112 was formulated in buffer at pH6.0 or pH7.0, higher purity was observed, indicating better stability of the anti-OX 40 antibody in buffers at pH6.0 or pH 7.0.

TABLE 9 purity results for anti-OX 40 antibody (SEC-HPLC method,%)

(4) Charge variants

The charge variants of each anti-OX 40 antibody sample change under the high temperature (40 +/-2 ℃), wherein, for the acid variants of the antibodies, at pH8.0, the acid components of the anti-OX 40 antibody ADI-20112 (IgG 1 type) sample are obviously increased, and the samples at pH7.0 have the smallest changes, namely the samples at pH5.0 and pH 6.0; for the major component of the antibody, the change in the major component of the sample at ph7.0 was minimal; for the alkaline variants of the antibodies, the alkaline composition of the samples was consistently reduced at each pH relative to the alkaline composition at day 0, with the results shown in FIGS. 1-4 of the specification.

B. Experiment of oscillatory stress

Each of the above samples containing 15mg/ml of anti-OX 40 antibody was shaken at 650 rpm in a dark place at 25 ℃ and sampled at day 0, day 1, day 3, and day 5. The appearance of each sample, the presence of visible foreign matter, was observed at the time of sampling; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (model UV-1800, manufactured by shimadzu, japan); the purity (%) of each sample was determined by SEC-HPLC method.

The results are shown below.

(1) Appearance, presence or absence of visible foreign matter

The samples of each group were qualified for visual appearance and detection of visible foreign matter when shaken up to 5 days at pH of about 5.0, 6.0, 7.0, 8.0.

(2) Protein content

Under shaking conditions, the protein content of each group of samples was not significantly affected, and table 10 lists the results of protein content determination for samples in which antibody ADI-20112 (IgG 1 type) was present.

TABLE 10 protein content results for anti-OX 40 antibody samples (UV method, mg/ml)

N/A indicates that the item is not set.

(3) Purity of

The purity of each set of samples was determined by shaking at pH of about 5.0, 6.0, 7.0, 8.0 for 5 days. The results show that the purity of each set of samples was not significantly affected under shaking conditions.

Table 11 shows the results of purity measurement of the antibody ADI-20112 (IgG 1 type) sample.

TABLE 11 purity results of anti-OX 40 antibody samples under shaking (SEC-HPLC method,%)

N/A indicates that the entry is not set.

C. Freezing and thawing stress experiment

Each of the above samples containing 15mg/ml of anti-OX 40 antibody was subjected to cycles of freezing (-30 ℃ C. Or less) and thawing (using a water bath at a temperature of 25 ℃ C.) and sampled at 0, 3, and 6 cycles. The appearance of each sample, the presence of visible foreign matter, was observed at the time of sampling; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (model UV-1800, manufactured by shimadzu, japan); the purity (%) of each sample was determined by SEC-HPLC method.

The results are shown below.

(1) Appearance, presence or absence of visible foreign matter

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

After 3 cycles of freezing and thawing under the condition of pH of about 8.0, the appearance and visible foreign matter of each group of samples are qualified. After 6 cycles of freeze-thaw at ph8.0, the sample began to appear cloudy.

(2) Protein content

Protein content was not significantly affected in each set of samples under freeze-thaw conditions, and the results of the protein content determination of antibody ADI-20112 (IgG 1 type) samples are listed in table 12.

TABLE 12 protein content results (UV method, mg/ml) for anti-OX 40 antibody samples

N/A indicates that the item is not set.

(3) Purity of

The purity of each set of samples was determined by performing a freeze-thaw cycle at a pH of about 5.0, 6.0, 7.0, 8.0. The results show that the purity of each set of samples was not significantly affected under freeze-thaw conditions.

Table 13 lists the results of purity determination of the antibody ADI-20112 (IgG 1 type) samples therein.

TABLE 13 purity results for anti-OX 40 antibody samples under freeze-thaw cycles (SEC-HPLC method,%)

N/A indicates that the item is not set.

As can be seen from the above experimental results, the anti-OX 40 antibody samples were not observed to have significant changes in appearance, protein content and purity, nor visible foreign matter, after being subjected to various stress conditions in the buffers having pH values of about 5.0, 6.0, 7.0, 8.0, and therefore, any one of the pH values in the range of about 5.0 to 8.0 could be selected to formulate the anti-OX 40 antibody preparation.

Example 3 formulation of Stable anti-OX 40 antibody formulations

A 4 liquid formulation of anti-OX 40 antibody, pH about 7.0, was formulated, the components of which are shown in table 14. And (3) performing ultrafiltration replacement on the anti-OX 40 antibody sample solution prepared before by using the buffer solution of the 4 prepared preparations, then enabling the anti-OX 40 antibody in the sample solution to be about 25mg/ml, adding polysorbate 80, performing sterile filtration, performing sterile subpackaging into 15R penicillin bottles or 4 ml/bottles, performing freeze drying on the samples, performing high-temperature tests after capping, and detecting the stability.

TABLE 14 Components of anti-OX 40 antibody formulations

The lyophilized formulation was placed at 40 ℃. + -. 2 ℃ and 25 ℃. + -. 2 ℃ and sampled at 0 days, 2 weeks, 4 weeks and 3 months. A sample of the lyophilized formulation was reconstituted with sterile water to a volume near that before lyophilization and the protein content of the lyophilized formulation after reconstitution was determined to be about 26.0mg/ml. The appearance and the presence of visible foreign matter of each re-dissolved sample at the sampling time point were observed; the protein content in each sample was measured using an ultraviolet visible spectrophotometer (model UV-1800, manufactured by shimadzu, japan); turbidity; the purity (%) of each sample was determined by size exclusion high performance liquid chromatography (SEC-HPLC); the charge variants (%) of the anti-OX 40 antibody in each sample were determined by cation exchange chromatography (CEX HPLC). Similar results were obtained for each of the different antibody preparations. The results of the antibody ADI-20112 (IgG 1-type) preparations are listed below.

(1) Appearance, presence or absence of visible foreign matter

4 formulations were observed at 40 ℃. + -. 2 ℃ and 25 ℃. + -. 2 ℃ for up to 3 months.

The results show that the appearance and the detection of visible foreign matters of the 4 preparations are all qualified.

(2) Protein content

The protein content of the 4 formulations did not change at both 40 ℃. + -. 2 ℃ and 25 ℃. + -. 2 ℃ as detailed in Table 15.

TABLE 15 protein content results for anti-OX 40 antibody formulations (UV method, mg/ml)

(3) Turbidity of water

The turbidity results for the 4 formulations at 40 ℃. + -. 2 ℃ and 25 ℃. + -. 2 ℃ are shown in Table 16 and FIG. 5, and no significant change occurred with respect to the turbidity at day 0.

TABLE 16 turbidity results (OD 350 nm method) of anti-OX 40 antibody formulations

(4) Purity of

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

TABLE 17 purity results for anti-OX 40 antibody formulations (SEC-HPLC method,%)

Purity (non-reduced CE-SDS method): under the conditions of 40 +/-2 ℃ and 25 +/-2 ℃, the purity of each preparation is not obviously changed. See table 18 for details.

TABLE 18 Freeze-dried preparation screening study purity results (non-reduced CE-SDS method,%)

Purity (reduced CE-SDS method): under the conditions of 40 +/-2 ℃ and 25 +/-2 ℃, the purity of each preparation is not obviously changed. See table 19 for details.

TABLE 19 Freeze-dried preparation screening study purity results (reduced CE-SDS method,%)

(5) Charge variants

At 40 ℃. + -. 2 ℃ the samples of formulation 1 did not show a significant change at month 3 in each charge variant of the anti-OX 40 antibody relative to day 0; the change in the acidic component of the samples of formulation 2 and 3 with respect to day 0 was 4.1%, 2.3%, respectively; the change in the main component at 3 months for the sample of formulation 4 relative to day 0 was 2.6%. The results are shown in Table 20 and FIGS. 7-9.

At 25 ℃. + -. 2 ℃ samples of each formulation did not significantly change at 3 months for each charge variant of the anti-OX 40 antibody relative to day 0.

TABLE 20 Charge variant results for anti-OX 40 antibody in the study of lyophilized formulations (CEX-HPLC method,%)

As can be seen from the above experimental results, formulations 1-4 all had an acceptable degree of stability. Formulation 2 was slightly inferior to formulation 1 in turbidity, purity (SEC-HPLC method) and charge variant (CEX-HPLC method) detection indices; the charge variants (CEX-HPLC) of formulations 3 and 4 were slightly less stable than formulation 1. All stability indexes of the preparation 1 are superior to those of other preparations.

Having described exemplary embodiments of the invention, it will be understood by those skilled in the art that this disclosure is illustrative only, and that various other substitutions, adaptations and modifications may be made within the scope of the invention. Accordingly, the present invention is not limited to the specific embodiments recited herein.

Claims

1. A liquid antibody formulation having a pH of 6.0-7.0 comprising

(i) 20-30mg/ml of an anti-OX 40 antibody or antigen-binding fragment thereof comprising a combination of HCDR1 of the amino acid sequence of SEQ ID NO:4, HCDR2 of the amino acid sequence of SEQ ID 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, LCDR2 of the amino acid sequence of SEQ ID NO. 51, and LCDR3 of the amino acid sequence of SEQ ID NO. 58;

(ii) A buffer, said buffer being 4-6mg/ml sodium citrate or sodium citrate dihydrate;

(iii) A stabilizer, wherein the stabilizer is 40-60mg/ml sucrose; and

(iv) A surfactant which is polysorbate 80 at 0.6-0.8 mg/ml.

2. Liquid antibody formulation according to claim 1, characterized in that the anti-OX 40 antibody or antigen binding fragment thereof is an anti-OX 40 antibody or antigen binding fragment thereof capable of specifically binding OX40 with a high affinity and thereby mediating a co-stimulatory response, wherein the high affinity is at 10 ^-7 K of M or less _D Specifically binds to OX40.

3. The liquid antibody formulation of claim 2, wherein the anti-OX 40 antibody or antigen binding fragment thereof is at 10 ^- ⁸ M to 10 ^-12 K of M _D Specifically binds to OX40 and thereby mediates a costimulatory response.

4. Liquid antibody formulation according to claim 2, characterized in that the anti-OX 40 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 comprises the amino acid sequence of SEQ ID NO 90; and

(b) The VL comprises the amino acid sequence of SEQ ID NO: 116.

5. The liquid antibody formulation of claim 4, characterized in that the anti-OX 40 antibody or antigen-binding fragment thereof comprises a heavy chain and/or a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 133 or 134 and the light chain comprises the amino acid sequence of SEQ ID NO. 164.

6. A liquid antibody formulation according to any one of claims 1-5, which comprises 25mg/ml anti-OX 40 monoclonal antibody, 5.88mg/ml sodium citrate dihydrate, 50.00mg/ml sucrose, 0.7mg/ml polysorbate 80, at a pH of 7.0.

7. A solid antibody formulation obtained by solidifying the liquid antibody formulation of any one of claims 1 to 6.

8. The solid antibody formulation of claim 7, wherein the solidification is carried out by crystallization, spray drying or freeze drying.

9. The solid antibody formulation of claim 7, wherein said solid antibody formulation is in the form of a lyophilized powder injection.

10. A delivery device comprising a liquid antibody formulation according to any one of claims 1 to 6 or a solid antibody formulation according to any one of claims 7 to 9.

11. A pre-filled syringe comprising the liquid antibody formulation of any one of claims 1-6 or the solid antibody formulation of any one of claims 7-9 for intravenous or intramuscular injection.

12. Use of a liquid antibody formulation according to any one of claims 1-6 or a solid antibody formulation according to any one of claims 7-9 for the manufacture of a delivery device or a pre-filled syringe or a medicament for the treatment of cancer in a subject.

13. The use of claim 12, wherein the cancer is lung cancer, liver cancer, stomach cancer, or colon cancer.

14. The use of claim 13, wherein the lung cancer is non-small cell lung cancer.