CN114146174A

CN114146174A - anti-PD-L1/OX 40 bispecific antibody preparation and preparation method and application thereof

Info

Publication number: CN114146174A
Application number: CN202110841167.0A
Authority: CN
Inventors: 朱兴贵; 马一冬; 汪音爵
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2020-07-24
Filing date: 2021-07-23
Publication date: 2022-03-08
Anticipated expiration: 2041-07-23
Also published as: CN114146174B

Abstract

The present invention relates to formulations comprising an anti-PD-L1/OX 40 bispecific antibody, in particular to pharmaceutical formulations comprising an anti-PD-L1/OX 40 bispecific antibody, a buffer, a stabilizer and a surfactant. Furthermore, the invention relates to the therapeutic or prophylactic use of these formulations.

Description

anti-PD-L1/OX 40 bispecific antibody preparation and preparation method and application thereof

Technical Field

The present invention relates to the field of anti-PD-L1/OX 40 bispecific antibody formulations. More particularly, the present invention relates to pharmaceutical formulations, in particular stable liquid formulations, lyophilized formulations and reconstituted stable liquid formulations, comprising an anti-PD-L1/OX 40 bispecific antibody, as well as methods for the preparation of said pharmaceutical formulations, and therapeutic and/or prophylactic uses of said pharmaceutical formulations.

Background

The stability of the medicine is one of the important indexes for ensuring the effectiveness and the safety of the medicine. Obtaining a good formulation is a key condition to ensure that the drug maintains its effectiveness and safety over the shelf life. However, due to the complexity of the antibody itself and its degradation pathway, it is currently not possible to predict the formulation conditions required to optimize antibody stability. It is particularly contemplated that different antibodies will typically have very different CDR sequences, different antibody structures, and that these sequence and structure differences will result in different antibodies having different stability properties in solution. Therefore, based on the stringent requirements for safety and efficacy of human antibodies, it is necessary to optimize the optimal formulation for each antibody individually.

PD-L1 is over-expressed in most cancer tissues, and PD-L1 is over-expressed, so that the expression of cell cycle checkpoint proteins and cell proliferation related proteins can be regulated by inhibiting RAS and a PI3K/AKT signal channel, and finally the T cell proliferation is inhibited. OX40 (also known as CD134 or TNFRSF4) is a TNFR family member that is expressed predominantly on activated T cells, mainly CD4+ effector T cells and CD8+ effector and regulatory T cells (tregs). OX40 is overexpressed on CD4+ effector T cells as well as CD8+ effector T cells and regulatory T cells in many solid tumors (e.g., melanoma, lung, and renal cancers). In mouse models, anti-PD-L1/OX 40 bispecific antibodies have better tumor-inhibiting effects, see for example: PCT/CN 2020/073959.

At present, an anti-PD-L1/OX 40 bispecific antibody preparation is urgently needed, the quality of the traditional Chinese medicine in the long-term storage process of the medicine can be ensured, the preparation process is safe and reliable, the formula is simple, and the injection is convenient.

Disclosure of Invention

Summary of The Invention

The present invention meets the above-described needs by providing pharmaceutical formulations comprising anti-PD-L1/OX 40 bispecific antibodies. The antibody formulations of the invention exhibit excellent stability against a variety of stability-affecting factors, such as temperature, repeated freeze-thaw, shaking.

In one aspect, therefore, the present invention provides a liquid antibody formulation comprising (i) an anti-PD-L1/OX 40 bispecific antibody; (ii) a buffering agent, (iii) a stabilizing agent, and (iv) a surfactant. Preferably, the composition further comprises a chelating agent.

In one embodiment, an anti-PD-L1/OX 40 bispecific antibody consists of peptide chain #1 and peptide chain #2 as follows:

formula (I):

peptide chain # 1: VH-CH 1-Fc-X-VHH; and

formula (II):

peptide chain # 2: VL-CL;

wherein:

VH represents a heavy chain variable region;

CH1 represents heavy chain constant region domain 1;

fc comprises heavy chain constant region domains CH2, CH3, and optionally CH 4;

x may be absent or, when present, represents a linker;

VHH represents a single domain antigen binding site;

VL represents a light chain variable region;

CL represents a light chain constant region;

optionally, a hinge region is present between CH1 and the Fc.

In one embodiment, the PD-L1/OX40 bispecific antibody of the invention comprises the sequences shown in the following table:

anti-PD-L1/OX 40 bispecific antibody peptide chain #1 (exemplary polypeptide chain of formula (I))	SEQ ID NO:1
		anti-OX 40 antibody ADI-20057VH (exemplary VH of formula (I))	SEQ ID NO:2
CH1 (exemplary CH1 of formula (I))	SEQ ID NO:3
		Fc (exemplary Fc of formula (I))	SEQ ID NO:4
Joint (X of exemplary formula (I))	SEQ ID NO:5
		anti-PD-L1 Single Domain antibodies (exemplary VHH of formula (I))	SEQ ID NO:6
anti-PD-L1/OX 40 bispecific antibody peptide chain #2 (exemplary polypeptide chain of formula (II))	SEQ ID NO:7
		anti-OX 40 antibody ADI-20057VL (VL of exemplary formula (II))	SEQ ID NO:8
CL (CL of exemplary formula (II))	SEQ ID NO:9
		anti-PD-L1 Single Domain antibody CDR1 (exemplary VHH CDR1 of formula (I))	SEQ ID NO:10
anti-PD-L1 Single Domain antibody CDR2 (exemplary VHH CDR2 of formula (I))	SEQ ID NO:11
		anti-PD-L1 Single Domain antibody CDR3 (exemplary VHH CDR3 of formula (I))	SEQ ID NO:12
OX40 antibody part VHCDR1 (exemplary HCDR1 of VH of formula (I))	SEQ ID NO:13
		OX40 antibody part VHCDR2 (exemplary HCDR2 of VH of formula (I))	SEQ ID NO:14
OX40 antibody part VHCDR3 (exemplary HCDR3 of VH of formula (I))	SEQ ID NO:15
		OX40 antibody part VLCDR1 (LCDR 1 for VL of exemplary formula (II))	SEQ ID NO:16
OX40 antibody part VLCDR2 (LCDR 2 for VL of exemplary formula (II))	SEQ ID NO:17
		OX40 antibody part VLCDR3 (LCDR 3 for VL of exemplary formula (II))	SEQ ID NO:18
anti-OX 40 antibody heavy chain (exemplary VH-CH1-Fc of formula (I))	SEQ ID NO:19

Wherein:

in one embodiment, the anti-PD-L1/OX 40 bispecific antibody comprises at least one polypeptide chain #1 and one polypeptide chain # 2. Preferably, an antibody molecule of the invention comprises two (e.g. identical) polypeptide chains #1 and two (e.g. identical) polypeptide chains # 2.

In one embodiment, the VH-CH1-Fc-X-VHH of the anti-PD-L1/OX 40 bispecific antibody polypeptide chain #1 comprises SEQ ID NO:1 or a sequence having at least 90% identity thereto, and the VL-CL of polypeptide chain #2 comprises the amino acid sequence of SEQ ID NO:7 or a sequence having at least 90% identity thereto.

Preferably, the anti-PD-L1/OX 40 bispecific antibody is an anti-PD-L1/OX 40 bispecific antibody disclosed in PCT application No. PCT/CN2020/073959 (International application date: 1/23/2020).

In one embodiment, the anti-PD-L1/OX 40 bispecific antibody is an anti-PD-L1/OX 40 bispecific antibody recombinantly expressed in HEK293 cells or CHO cells.

In one embodiment, the concentration of anti-PD-L1/OX 40 bispecific antibody in a liquid antibody formulation of the invention is about 1-150 mg/ml. In another embodiment, the concentration of anti-PD-L1/OX 40 bispecific antibody in a liquid antibody formulation of the invention is about 10-100mg/ml, preferably 20-60mg/ml, especially about 40 mg/ml. In other embodiments, the concentration of anti-PD-L1/OX 40 bispecific antibody in a liquid antibody formulation of the invention is about 10,15,20,25,30, 35, 40,50, 60, 70,80,90 or 100 mg/ml.

In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 5-50 mM. In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 5-30mM, e.g., about 5,10,15,20,25,30 mM. In one embodiment, the buffer is a histidine buffer, a citric acid buffer, an acetic acid buffer, a phosphate buffer, preferably the buffer is a histidine buffer. In one embodiment, the buffer is a histidine buffer, preferably the buffer consists of histidine and histidine hydrochloride. In a preferred embodiment, the buffer is about 5-30mM histidine buffer, e.g., 10-20mM, such as about 10mM histidine.

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

In one embodiment, the stabilizing agent is selected from the group consisting of a polyol (e.g., sorbitol, mannitol, or a combination thereof), a saccharide (e.g., sucrose, trehalose, maltose, or a combination thereof), an amino acid (e.g., arginine hydrochloride, methionine, glycine, proline, and combinations or salts thereof), and any combination thereof. In one embodiment, the stabilizer comprises about 20-80mg/ml sorbitol, e.g., 20,25,30, 35, 40, 45, 50,55,60, 70,80 mg/ml sorbitol.

In one embodiment, the concentration of surfactant in the liquid antibody formulation of the invention is about 0.1-1 mg/ml. In one embodiment, the concentration of surfactant in a liquid antibody formulation of the invention is about 0.2-0.8mg/ml, e.g., about 0.2,0.3,0.4,0.5,0.6,0.7,0.8 mg/ml.

In one embodiment, the surfactant is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate-based surfactants, poloxamers, polyethylene glycols. In one embodiment, the surfactant is selected from polysorbate-based surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the invention is polysorbate 80.

In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the invention is about 0.005-0.05 mg/ml. In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the invention is about 0.008-0.018mg/ml, for example about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml.

In one embodiment, the chelating agent is a carboxylic acid type chelating agent. In one embodiment, the chelating agent is selected from disodium edetate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, dihydroxyethylglycine. In one embodiment, the chelating agent is selected from disodium edetate.

In one embodiment, the liquid formulation has a pH of about 5.0 to 6.0. In some embodiments, the pH of the liquid formulation is any of about 5.0 to 6.0, such as about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0. Preferably, the pH of the formulation is 5.5 ± 0.2.

In one embodiment, the liquid antibody formulation of the invention comprises:

(i) about 1-150mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, e.g. 10-100mg/ml, preferably 20-60mg/ml, e.g. 10,15,20,25,30, 35, 40,50, 60, 70,80,90 or 100mg/ml antibody protein;

(ii) about 5-50mM histidine buffer, citric acid buffer, preferably histidine buffer, e.g. 5-30mM, e.g. 5,10,15,20,25,30 mM;

(iii) about 20-80mg/ml sorbitol, sucrose, preferably sorbitol, e.g., 30,40,50, 60 mg/ml;

(iv) about 0.1-1mg/ml polysorbate 80, e.g., 0.1,0.2.0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0 mg/ml; and

(v) optionally, about 0.005-0.05mg/ml of edetate disodium, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml.

Wherein the pH of the liquid formulation is about 5.0-6.0, e.g., about 5.5.

For example, the liquid antibody formulation may comprise

(i) About 20-60mg/ml anti-PD-L1/OX 40 bispecific antibody protein, e.g., 20,30,40,50,55,60 mg/ml;

(ii) about 10-20mM histidine buffer, e.g., 20mM histidine buffer;

(iii) about 20-80mg/ml sorbitol, e.g., 20,25,30, 35, 40, 45, 50,55,60, 70,80 mg/ml sorbitol;

(iv) about 0.2-0.8mg/ml, such as 0.2,0.3,0.4,0.5,0.6,0.7,0.8mg/ml, such as 0.3-0.6mg/ml polysorbate 80; and

(v) optionally about 0.008-0.018mg/ml of edetate disodium, e.g. about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml.

Wherein the pH of the liquid formulation is about 5.0-6.0, e.g., about 5.5.

In a preferred embodiment, the liquid antibody formulation comprises:

(i) about 40mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, 10mM histidine, 50mg/ml sorbitol, 0.2mg/ml polysorbate 80, 0.01mg/ml disodium edetate pH 5.5; or

(ii) About 40mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, 10mM histidine, 50mg/ml sorbitol, 0.5mg/ml polysorbate 80, 0.01mg/ml disodium edetate pH 5.5; or

(iii) About 40mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, 10mM histidine, 50mg/ml sorbitol, 0.7mg/ml polysorbate 80, 0.01mg/ml disodium edetate pH 5.5;

the liquid formulations of the present invention can be stably stored for a long period of time, for example, at least 24 months or longer. In one embodiment, the liquid formulation of the present invention may be stored at about-80 ℃ to about 45 ℃, e.g., -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, and is stable.

In one embodiment, the liquid formulation of the present invention is stable for storage for at least 24 months. In one embodiment, the liquid formulation of the present invention is stable at least 40 ℃. In one embodiment, the liquid formulation of the present invention remains stable for at least 3 months, preferably at least 12 months, more preferably at least 24 months at about 2 ℃ to 8 ℃. In one embodiment, the liquid formulation of the invention remains stable for at least 2 months, preferably at least 3 months, more preferably at least 6 months at room temperature or, for example, about 25 ℃. In one embodiment, the liquid formulation of the present invention remains stable at about 40 ℃ for at least 2 weeks, preferably at least 1 month.

In one embodiment, stability of the formulation may be indicated by detecting changes in appearance, visible foreign matter, protein content, turbidity, purity, and/or charge variants of the formulation. In one embodiment, the stability of the liquid formulation of the present invention may be tested in a forced experiment of high temperature stress, e.g. after storage at 40 ℃ ± 2 ℃ for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, e.g. after storage at 25 ℃ ± 2 ℃ for at least 1 month or 2 months, or in a long term experiment, e.g. after storage at 5 ℃ ± 3 ℃ for at least 2 months or 3 months, or in a shaking experiment (e.g. shaking at room temperature, protected from light for 5 days at 650 r/min), and/or in a freeze-thaw experiment (e.g. repeated freeze-thaw 6 times at-20 ℃/2-8 ℃). In one embodiment, the stability of the liquid formulation of the invention is tested with respect to initial values, e.g. initial values on day 0 of storage, or initial values before shaking or freeze-thaw experiments.

In one embodiment, the stability of the liquid formulations of the present invention is visually inspected after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the liquid formulations of the present invention remain clear to slightly opalescent in appearance, as colorless to pale yellow liquids, and free of foreign objects. In one embodiment, no visible foreign matter is present in the formulation upon visual inspection under a clarity detector. In a fruitIn an embodiment, the stability of the liquid formulation of the invention is checked by measuring the change in protein content after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the rate of change of protein content is not more than 20%, preferably not more than 10%, such as 7-8%, more preferably not more than 5%, 2% or 1% relative to the initial value, for example by ultraviolet spectrophotometry (UV) method. In one embodiment, the stability of the liquid formulations of the invention is checked after storage, or after shaking experiments, or after freeze-thaw experiments by determining the change in turbidity of the liquid formulations of the invention, wherein e.g. by OD_350mmThe method detects that the variation value is not more than 0.06, preferably not more than 0.05, more preferably not more than 0.04, not more than 0.02 relative to the initial value. In one embodiment, the stability of the liquid formulations of the invention is checked by determining the change in purity of the liquid formulations of the invention after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the change in monomer purity (or main peak change) is no more than 10%, such as no more than 5%, 4%, 3%, such as no more than 2%, preferably no more than 1% relative to the initial value by the iCIEF method. In one embodiment, the stability of the liquid formulation of the invention is checked by determining the change in purity of the liquid formulation of the invention after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the change in monomer purity (or main peak change) is reduced by no more than 10%, such as no more than 5%, 4%, 3%, 2% or 1% relative to the initial value by non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. In one embodiment, the stability of the liquid formulations of the invention is tested by the iCIEF method after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the sum of the variation values of the charge variants (main component, acidic component and basic component) of the antibody with respect to the initial value is not more than 50%, such as not more than 40%, 30%, 20%, 10%, 5%, and/or the variation value of the main component is not more than 20%, 15%, 10%, 8%, 5%. In one embodiment, the stability of the liquid formulations of the invention is tested by direct ELISA after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the stability of the liquid formulations of the invention is determined relative to the initial value,the relative binding activity of the antibody is 70-130%, e.g., 70,80,90,93,95, 98,100,103,105,108,110,115,120,125, 130%, preferably 90-110%.

In one embodiment, the liquid formulation of the invention is stable after storage, e.g. after storage at 25 ℃ for at least 2 months, or after storage at 40 ℃ ± 2 ℃ for 1 month, preferably having one or more of the following characteristics: relative to the initial value on day 0 of storage,

(i) a major peak change value of less than 1% as measured by SEC-HPLC method, and/or a formulation having a purity of greater than 96%, preferably greater than 97%, 98%;

(ii) a major peak variation of less than 2% as measured by the non-reduced CE-SDS method, and/or a formulation having a purity of greater than 96%, preferably greater than 97%, 98%;

(iii) the sum of the variation values of the components (main component, acidic component and basic component) of the anti-PD-L1/OX 40 bispecific antibody protein in the preparation is not more than 40% and/or the variation value of the main component is not more than 20% as measured by the iCIEF method,

for example, no more than about 40% (e.g., no more than 35%, 30%, 25%, 20%, 15%, 10%) or no more than 20% (e.g., no more than 15%, 12%, 10%, 8%) of the sum of the change values after 1 month of storage at 40 ℃. + -. 2 ℃, or

For example, no more than about 20% (e.g., no more than 15%, 14%, 13%, 12%) or no more than about 15% (e.g., no more than 10%, 8%, 7%, 6%, 5%) of the sum of the change values after 2 months of storage at 25 ℃;

(iv) the relative binding activity of the anti-PD-L1/OX 40 bispecific antibody protein in the preparation, as measured by ELISA, is 70% to 130%, e.g., 90,93,95,98,100,103,105,108,110,115, 120%, e.g., 90% to 110%;

in a preferred embodiment, the liquid formulation of the invention is stable under shaking and/or repeated freezing and thawing.

Preferably, the formulation is stable under shaking or repeated freezing and thawing, e.g. for 5 days under conditions of shaking at room temperature in the dark at 650r/min or after 6 repeated freezing and thawing at-20 ℃/2-8 ℃, with one or more of the following characteristics:

(i) a major peak change value of less than 1% as measured by SEC-HPLC method, and/or a formulation having a purity of greater than 96%, preferably greater than 97%, 98%, 99%;

(ii) major peak variation, as measured by the non-reduced CE-SDS method, is less than 1%, and/or the formulation has a purity of greater than 96%, preferably greater than 97%, 98%;

(iii) the sum of the variation values of the components (main component, acidic component and basic component) of the anti-PD-L1/OX 40 bispecific antibody protein in the preparation as measured by iCIEF does not exceed 2%;

(iv) the relative binding activity of the anti-PD-L1/OX 40 bispecific antibody protein in the formulation, as measured by ELISA, is 70% -130%, e.g., 90% -110%;

in one aspect, the liquid formulation of the present invention is a pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.

In another aspect, the present invention provides ー solid antibody preparations obtained by subjecting the 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 aspect, the invention provides a delivery device comprising a liquid antibody formulation or a solid antibody formulation of the invention. 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, e.g. for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.

In yet another aspect, the invention provides a method of delivering an anti-PD-L1/OX 40 bispecific antibody protein to a subject, e.g., a mammal, comprising the step of administering to the subject a liquid antibody formulation or a solid antibody formulation of the invention, e.g., by a delivery device using a pre-filled syringe.

In a further aspect, the invention provides the use of a liquid antibody formulation or a solid antibody formulation of the invention for the manufacture of a delivery device (e.g. a pre-filled syringe) or a medicament for the treatment or prevention of a disease in a subject, including an autoimmune disease, an inflammatory disease, an infection, a tumour, a T cell dysfunctional disease, e.g. where the tumour is a cancer, e.g. PD-1, PD-L1 or PD-L2 with an elevated expression level and/or OX40 with a reduced expression level or activity, e.g. colon or rectal cancer or colorectal cancer or lung cancer.

The invention also provides a method of treating a disease, such as a tumor, 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.

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 set forth herein as such may vary. In addition, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Definition of

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. For the purposes of the present invention, the following terms are defined below.

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 that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

The term "and/or" when used to connect two or more selectable items should be understood to mean either one of the selectable items or any two or more of the selectable items.

As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

As used herein, the terms "anti-OX 40 antibody," "anti-OX 40," "OX 40 antibody," or "antibody that binds to OX 40" refer to an antibody that is capable of binding to a (human or monkey) OX40 protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting (human or monkey) OX 40.

The terms "programmed cell death 1 ligand 1", "PD-L1", "programmed death ligand 1", "cluster of differentiation 274", "CD 274", or "B7 homolog 1" as used herein refer to any native PD-L1 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats).

The term "antibody" is used herein in the broadest sense to refer to a protein that comprises an antigen binding site, encompassing natural and artificial antibodies of various structures, including, but not limited to, intact antibodies and antigen-binding fragments of antibodies.

A "bispecific" antibody refers to an antibody having two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. In one embodiment, a bispecific antibody has binding specificity for a first antigen and a second antigen.

The term "antibody preparation" refers to a preparation in a form that allows the biological activity of an antibody as an active ingredient to be exerted effectively, and that does not contain other components having unacceptable toxicity to the subject to which the preparation is to be administered. Such antibody preparations are generally sterile. Typically, pharmaceutically acceptable excipients are included in the antibody formulation. A "pharmaceutically acceptable" excipient is one that can be reasonably administered to a subject mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration and may, for example, be acceptable for injection.

The term "anti-PD-L1/OX 40 bispecific antibody preparation" is also referred to herein simply as "the antibody preparation of the present invention", which means a preparation comprising an anti-PD-L1/OX 40 bispecific antibody protein as an active ingredient and a pharmaceutically acceptable excipient. The anti-PD-L1/OX 40 bispecific antibody protein as an active ingredient is suitable for therapeutic or prophylactic administration to human or non-human animals after combining the anti-PD-L1/OX 40 bispecific antibody protein with a pharmaceutically acceptable excipient. The antibody formulations of the invention may be prepared, for example, 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-PD-L1/OX 40 bispecific antibody protein formulation is in the form of a liquid formulation.

By "stable" antibody formulation is meant that the antibodies in the formulation retain an acceptable degree of physical and/or chemical stability after storage under specified conditions, or after shaking, or after repeated freeze-thawing. Although an antibody contained in an antibody preparation may not maintain its chemical structure 100% after storage, shaking, or repeated freeze-thawing, 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. In some particular embodiments, the anti-PD-L1/OX 40 bispecific antibody protein formulations of the invention exhibit as little as undetectable antibody aggregation or degradation or chemical modification during manufacture, transport and long term storage, thereby exhibiting high stability with little or even no loss of biological activity of the anti-PD-L1/OX 40 bispecific antibody protein. In some embodiments, the anti-PD-L1/OX 40 bispecific antibody protein formulations of the present invention substantially retain their physical and chemical stability after storage, shaking, and/or repeated freeze-thawing. Preferably, the liquid formulation of the present invention may be stable at room temperature or at 40 ℃ for at least 2 weeks, and/or at 25 ℃ for at least 2 months, and/or at 2-8 ℃ for at least 24 months. Preferably, the liquid preparation of the invention can be stable after shaking for 5 days at room temperature and in the dark at 650r/min and/or after repeated freeze-thawing for 1-6 times at-20 ℃/2-8 ℃.

A variety of analytical techniques are known in the art for determining the stability of proteins, see, e.g., Peptide and Protein Drug Delivery,247- & 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs (1991) and Jones, A.Adv.drug Delivery Rev.10:29-90 (1993). Stability can be measured at selected temperatures and selected storage times. For example, the storage time may be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test may be used. In some embodiments, the stability test is performed by performing various stress tests on the antibody formulation. These tests may represent extreme conditions that a formulated antibody preparation may encounter during manufacturing, storage or transportation, and may also represent conditions that may accelerate the instability of the antibodies in the antibody preparation during non-manufacturing, storage or transportation. For example, a formulated anti-PD-L1/OX 40 bispecific antibody protein formulation can be filled into glass vials to test antibody stability under high temperature stress. For another example, the stability of the antibody can be checked after filling the formulated anti-PD-L1/OX 40 bispecific antibody protein preparation into glass vials and shaking for 5 days under ambient light conditions of 650 r/min. For another example, the stability of the antibodies was examined after filling the formulated anti-PD-L1/OX 40 bispecific antibody protein preparation into glass vials and repeated freeze-thawing at-20 ℃/2-8 ℃ for 1-6 times.

After a period of storage time, or after a period of shaking, or after repeated freeze-thawing for a plurality of times, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or exhibit very little aggregation, precipitation, turbidity, and/or denaturation, the antibody can be considered to "retain its physical stability" in the formulation. Safety issues can result 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. Light scattering methods can be used to detect visible aggregates in the agent. SEC-HPLC can be used to determine soluble aggregates in the formulation. In addition, the appearance, color and/or clarity of the formulation can be visually inspected, or by OD₃₅₀The stability of the formulation is indicated by turbidity of the formulation measured by nm method or purity of the formulation measured by non-reducing CE-SDS method. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomer in the formulation after storage at a particular temperature for a particular time or after shaking or after repeated freeze-thawing, wherein the higher the percentage of antibody monomer in the formulation, the higher the stability of the formulation.

An "acceptable degree" of physical stability may mean that at least about 90% of anti-PD-L1/OX 40 bispecific antibody protein monomers are detected in the formulation after storage at a particular temperature for a particular time, after shaking or after repeated freeze-thawing. In some embodiments, an acceptable degree of physical 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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of anti-PD-L1/OX 40 bispecific antibody protein monomers. When assessing physical stability, the particular temperature at which the pharmaceutical formulation is stored may 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 ℃, about 35 ℃, about 37 ℃, about 40 ℃, about 42 ℃, or about 45 ℃. For example, a pharmaceutical formulation is considered stable if at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-L1/OX 40 bispecific antibody protein monomer is detected after 1 month or 4 weeks of storage at about 40 ℃ ± 2 ℃. A pharmaceutical formulation is considered stable if after 2 months of storage at about 25 ℃, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-L1/OX 40 bispecific antibody protein monomer is detected. A pharmaceutical formulation is considered stable if after 9 months of storage at about 5 ℃, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of anti-PD-L1/OX 40 bispecific antibody protein monomers are detected.

An antibody may be considered to "maintain its chemical stability" in a formulation if the antibody in the formulation does not show significant chemical changes after a period of storage, or after a period of shaking, or after repeated freeze-thawing for multiple times. Most chemical instability results from the formation of covalently modified forms of antibodies (e.g., charge variants of antibodies). Basic variants can be formed, for example, by aspartic acid isomerization, N-and C-terminal modifications; acidic variants can be produced by deamidation, sialylation and saccharification. Chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody. For example, charge variants of the antibody in the formulation can be detected by electropolymerization electrophoresis (iCIEF) using an image of capillary tubes or the like. In one embodiment, the stability of the formulation is measured by determining the percent change in charge of the antibody in the formulation after storage at a particular temperature for a particular time or after shaking or repeated freezing and thawing for a plurality of times, wherein the smaller the change, the higher the stability of the formulation.

An "acceptable degree" of chemical stability can mean a percent change in the charge variant (e.g., major component or acidic component or basic component) in the formulation of no more than 40%, e.g., no more than 30%, no more than 20%, after storage at a particular temperature for a particular time, or after shaking for a period of time, or after repeated freeze-thawing for a number of times; or the sum of the percentage change values of the charge variants (main component, acidic component and basic component) does not exceed 60%, for example 50%, 30%. In some embodiments, an acceptable degree of chemical stability may be manifested as a percent change in the principal component charge variant of no more than about 50%, 40%, 30%, 20%, or 15% 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; or the sum of the percent change values of the charge variants does not exceed about 60%, 50%, or 30%. When assessing chemical stability, the temperature at which the pharmaceutical formulation is stored may 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 the percent change in principal component charge variants after 24 months of storage at 5 ℃ is 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%. A pharmaceutical formulation may also be considered stable if the percentage change in principal component charge variants after storage at 25 ℃ for 2 months has a value of 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%. A pharmaceutical formulation may also be considered stable if the percent change value of the major component charge variants is less than about 50%, 40%, 30%, 20%, 16%, 15%, 14%, 13%, 12%, 10%, 5% or 4% after 1 month of storage at 40 ℃.

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.

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

"stress conditions" refers to an environment that is chemically and/or physically hostile to an antibody protein, which may lead to unacceptable destabilization of the antibody protein, e.g., high temperature, shaking, freezing and thawing. By "high temperature stress" is meant that the antibody formulation is stored at room temperature or even higher (e.g., 40 ℃. + -. 2 ℃) for a period of time. The stability of the antibody formulations can be checked by high temperature stress accelerated tests.

As used herein, the term "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular (subepithelial), intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, the stable anti-PD-L1/OX 40 bispecific antibody protein formulations of the invention are administered parenterally to a subject. In one embodiment, the anti-PD-L1/OX 40 bispecific antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

I. Antibody formulations

The present invention provides a stable liquid antibody formulation comprising (i) an anti-PD-L1/OX 40 bispecific antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, the antibody formulation having a pH of about 5.0-6.0. In a preferred embodiment, the liquid antibody formulation of the invention is in the form of an injectable formulation.

(i) anti-PD-L1/OX 40 bispecific antibodies

An "anti-PD-L1/OX 40 bispecific antibody" refers to an antibody that specifically binds to PD-L1 and a molecule that specifically binds to OX 40.

peptide chain # 1:

VH-CH 1-Fc-X-VHH; and

peptide chain # 2:

VL-CL；

wherein:

VH represents a heavy chain variable region;

CH1 represents heavy chain constant region domain 1;

fc comprises heavy chain constant region domains CH2, CH3, and optionally CH 4;

x may be absent or, when present, represents a linker;

VHH represents a single domain antigen binding site;

VL represents a light chain variable region;

CL represents a light chain constant region;

optionally, a hinge region is present between CH1 and the Fc.

In one embodiment, the VH-CH1-Fc-X-VHH of the anti-PD-L1/OX 40 bispecific antibody polypeptide chain #1 comprises SEQ ID NO:1 or a sequence having at least 90% identity thereto, and the VL-CL of polypeptide chain #2 comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:7 or a sequence having at least 90% identity thereto.

In some embodiments, the anti-PD-L1/OX 40 bispecific antibody in the antibody formulations of the invention is an IgG format antibody. An "IgG-form 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 IgG format, and differ between antibodies of different IgG formats. For example, an antibody in the form of IgG2 refers to a heavy chain constant region, Ig, domain that is the Ig domain of IgG 2.

In a preferred embodiment, the anti-PD-L1/OX 40 bispecific antibody in the antibody preparation of the invention is as disclosed in PCT application No. PCT/CN2020/073959 (International application date: 1/23/2020).

In one embodiment, the anti-PD-L1/OX 40 bispecific antibody is an IgG2 type antibody produced by recombinant expression from CHO cells and purified. Preferably, the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity.

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, for example about 10,15,20,25,30, 35, 40,50, 60, 70,80,90 or 100mg/mL of anti-PD-L1/OX 40 bispecific antibody.

(ii) Buffering agent

Buffers are agents that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffering agent used in the formulations of the present invention may control the pH of the formulations of the present invention in the pH range of about 5.0 to 6.0, for example, a pH of about 5.0 to 5.5. In some specific embodiments, an antibody formulation of the invention has a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8. For example, the antibody formulation of the invention has a pH of 5.0-6.02, preferably a pH of 5.5.

In some embodiments, the formulations of the present invention comprise a buffer system selected from: a histidine-histidine hydrochloride buffer system, a citric acid-sodium citrate buffer system, an acetic acid-sodium acetate buffer system and a phosphate buffer system, preferably the histidine-histidine hydrochloride buffer system.

In some embodiments, the buffer used in the formulations of the present invention is a histidine buffer, in particular a buffer system consisting of histidine and histidine hydrochloride. In some embodiments, the concentration of histidine in the histidine buffer of the present invention is about 5-50mM, particularly about 5-30mM, e.g., about 5,10,15,20,25,30 mM. In one embodiment, the formulation of the invention comprises about 10mM histidine.

(iii) Stabilizer

Suitable stabilizers for use in the present invention may be selected from sugars, polyols and amino acids and combinations thereof. Examples of sugars that act as stabilizers include, but are not limited to, sucrose, trehalose, maltose, and combinations thereof. Examples of polyols as stabilizers include, but are not limited to, sorbitol, mannitol, or combinations thereof. Examples of amino acids that act as stabilizers include, but are not limited to, arginine hydrochloride, methionine, glycine, proline, and combinations thereof.

In one embodiment, the stabilizing agent is selected from the group consisting of a polyol (e.g., sorbitol, mannitol, or a combination thereof), a saccharide (e.g., sucrose, trehalose, maltose, or a combination thereof), an amino acid (e.g., arginine hydrochloride, methionine, glycine, proline, and combinations or salts thereof), and any combination thereof. In one embodiment, the stabilizer comprises about 20-80mg/ml sorbitol, e.g., 20,25,30, 35, 40, 45, 50,55,60, 70,80 mg/ml sorbitol. In one embodiment, the formulation of the present invention comprises about 50mg/ml sorbitol.

(iv) Surface active agent

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-20, polysorbate-80, polysorbate-60, or polysorbate-40; poloxamers, and the like. In a preferred embodiment, polysorbate-80 is included in the liquid formulation of the present invention as a surfactant.

In some embodiments, surfactants that may be used in the liquid formulations of the present invention include, but are not limited to, polysorbate-based surfactants (e.g., polysorbate 80, polysorbate 20), poloxamers, and polyethylene glycols.

The amount of surfactant contained in an antibody formulation of the invention may vary with the particular intended characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is used. In preferred embodiments, the formulation may contain about 0.01-5mg/ml, preferably about 0.1-1mg/ml, for example about 0.2,0.3,0.4,0.5,0.6,0.7,0.8, 0.9,1.0mg/ml of a surfactant, especially polysorbate-80, preferably about 0.5mg/ml of polysorbate-80.

(v) Chelating agents

As used herein, the term "chelating agent" refers to a compound that is capable of forming a chelate with a central atom, with the stability of the complex being greatly increased as a result of the formation of the chelate.

In one embodiment, the chelate compound in the liquid formulation of the present invention is a carboxylic acid type chelate agent. In one embodiment, the chelating agent is selected from disodium edetate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, dihydroxyethylglycine. In one embodiment, the chelating agent is selected from disodium edetate.

(vi) Other excipients

Other excipients are optionally included in the antibody liquid formulations of the invention. Such other excipients include, for example, anti-microbial agents, anti-static agents, antioxidants, 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 set forth 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, eds. Gennaro, Lippincott Williams & Wilkins (2005) ".

Preparation of the formulations

The present invention provides stable formulations comprising anti-PD-L1/OX 40 bispecific antibody proteins. The anti-PD-L1/OX 40 bispecific antibody proteins used in the formulations of the invention can be prepared using techniques known in the art for the production of antibodies. For example, the antibody can be recombinantly produced. In a preferred embodiment, the antibodies of the invention are recombinantly produced in 293 cells or CHO cells.

The use of antibodies as active ingredients of pharmaceuticals is now widespread. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al (knowledge purification of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99(2008) 599-613.) describe a monoclonal antibody three-column purification method using ion exchange chromatography (anionic IEX and/or cationic CEX chromatography) after a protein A capture step. Kelley et al (Weak chromatography for exchange purification of monomeric antibiotics, Biotechnology and Bioengineering 101(2008) 553-566) describe a two-column purification method in which a weakly-partitioning anion exchange resin is used after protein A affinity chromatography.

In general, recombinantly produced monoclonal antibodies can be purified using conventional purification methods to provide a drug substance with sufficient reproducibility and moderate purity for use in the formulation of antibody preparations. For example, after secretion of the antibody from the recombinant expression cells into the culture medium, the supernatant from the expression system can be concentrated using a commercially available protein concentration filter, such as the Amicon's ultrafiltration device. Thereafter, purification of the antibody can be performed using means such as chromatography, dialysis, and affinity purification. Protein a is adapted as an affinity ligand for the purification of antibodies of the IgG1, IgG2 and IgG4 types. Other antibody purification methods, such as ion exchange chromatography, may also be used. After obtaining an antibody of sufficient purity, a formulation comprising the antibody can be prepared according to methods known in the art.

For example, the preparation can be carried out by the following steps: (1) after fermentation is finished, centrifuging and clarifying the fermentation liquor to remove impurities such as cells and the like so as to obtain a supernatant; (2) capture of antibodies using affinity chromatography (e.g., protein a columns with specific affinity for IgG1, IgG2, and IgG 4-type antibodies); (3) performing virus inactivation; (4) refining and purifying (CEX cation exchange chromatography can be adopted generally) to remove impurities in the protein; (5) virus filtration (to reduce virus titer by, e.g., more than 4log 10); (6) ultrafiltration/diafiltration (which may be used to replace the protein in a formulation buffer that facilitates its stabilization and concentration to a suitable concentration for injection). See, e.g., B.Minow, P.Rogge, K.Thompson, BioProcess International, Vol.10, No.6,2012, pp.48-57.

Method for analyzing preparation

Biologicals stability studies typically include real-time stability studies under actual storage conditions (long-term stability studies), accelerated stability studies, and forced condition experimental studies. The stability research needs to search and optimize the research conditions according to the research purpose and the characteristics of the product; aiming at various influencing factors (such as temperature, repeated freezing and thawing, vibration and the like), a stability research scheme such as long-term, accelerated and/or forced condition tests and the like is established. Accelerated and forced condition testing facilitates understanding of product stability during short term deviations from storage conditions and extreme conditions, and provides supporting data for the determination of expiration date and storage conditions.

During storage, shaking or repeated freeze-thawing of the antibody preparation, aggregation, degradation or chemical modification of the antibodies may occur, leading to antibody heterogeneity (including size heterogeneity and charge heterogeneity) as well as aggregates and fragments, etc., thereby affecting the quality of the antibody preparation. Therefore, monitoring of the stability of the antibody formulation is necessary.

Various methods are known in the art for testing the stability of antibody formulations. For example, the purity of the antibody preparation can be analyzed and the aggregation level of the antibody can be evaluated by methods such as reduced CE-SDS, non-reduced CE-SDS and SEC-HPLC; charge variants in antibody preparations can be analyzed by capillary isoelectric focusing electrophoresis (cIEF), imaged capillary isoelectric focusing electrophoresis (iCIEF), ion exchange chromatography (IEX), and the like. In addition, the stability of the formulation can be rapidly judged by visually inspecting the appearance of the formulation. OD may also be used_350nmThe method detects turbidity change of the preparation, and the method can give related solubilityAnd information on the amount of insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) may be used to detect changes in protein content in the formulation.

The non-reduced CE-SDS method is a method for measuring the purity of an antibody using a capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge caused by SDS binding, which is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on the size or hydrodynamic radius of the molecules can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies. Generally, in the non-reduced CE-SDS method, a test sample is mixed with an SDS sample buffer and iodoacetamide. Thereafter, the mixture may be incubated at 68-72 ℃ for about 10-15 minutes, cooled to room temperature and the centrifuged supernatant used for analysis. And detecting the migration of the protein by adopting an ultraviolet detector to obtain an electrophoresis spectrogram. Antibody preparation purity can be calculated as the percentage of the peak area of the IgG main peak to the sum of all peak areas. For further description of the CE-SDS method, see, e.g., Richard R, et al, Application of CE SDS gel in reduction of biochemical anti-based products, electrophosphoresis, 2008,29, 3612-3620.

Size exclusion high performance liquid chromatography, SEC-HPLC, is another important method for antibody standardization and quality control. The method mainly separates molecules according to the size or the hydrodynamic radius difference of the molecules. By SEC-HPLC, antibodies can be isolated in three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram that is the sum of all peak areas. By SEC-HPLC, the percentage of antibody monomer in the formulated product can be measured, giving information on the content of soluble aggregates and shears. For further description of the SEC-HPLC method, see, e.g., J.Pharm.Scien.,83:1645-1650, (1994); pharm. Res.,11:485 (1994); J.pharm.Bio.anal.,15:1928 (1997); J.pharm.Bio.anal., 14:1133-1140 (1986). In addition, see also examplesFor example, R.Yang et al, High resolution section of recombinant monoclonal antibodies by size exclusion ultra-High performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and biological Analysis (2015),http://dx.doi.org/10.1016/ j.jpba.2015.02.032(ii) a And Alexandre Goyon et al, Protocols for the analytical characterization of thermal monoclonal antibodies.I-Non-characterizing chromatographic techniques, Journal of Chromatography,http://dx.doi.org/ 10.1016/j.jchromb.2017.05.010。

charge variants of antibodies in an antibody preparation can be determined by iCIEF. In this assay, peaks eluting from the iCIEF column earlier than the retention time of the main peak (or main component) are labeled as "acidic peaks" (or acidic components), while those eluting from the iCIEFC column later than the retention time of the main peak are labeled as "basic peaks" (or basic components).

Accelerated stability studies can be used to examine the stability properties of products, facilitating the screening of stable pharmaceutical formulation formats. For example, a sample of the formulation may be placed at an elevated temperature, e.g., about 40 ℃. + -. 2 ℃ and 25 ℃. + -. 2 ℃ for accelerated stability studies. In addition, shaking experiments or repeated freeze-thaw experiments can be performed to test the stability properties of the product. For example, shaking experiments are performed at room temperature in a dark place at 650r/min for 1-5 days. For example, repeated freeze-thaw experiments may be performed using a freeze-thaw cycle in which a product is frozen at-30 ℃ or lower for one day and then thawed at room temperature, wherein the repeated freeze-thaw cycles may be performed for 1 to 6 times. The detection indicators of product stability may include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method). In addition, the efficacy or biological activity of the antibody can be detected. For example, the ability of an antibody to bind to its antigenic molecule in a formulation can be tested. Various methods are known to those skilled in the art for quantifying the specific binding of an antibody to an antigen, such as immunoassay tests, ELISA, and the like.

Use of the formulation

The antibody preparations of the invention comprising an anti-PD-L1/OX 40 bispecific antibody protein of the invention have the effect of preventing or treating autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunctional diseases, e.g. tumors which are cancers, e.g. PD-1, PD-L1 or PD-L2 with elevated expression levels and/or cancers of OX40 with reduced expression levels or activity, e.g. colon or rectal cancer or colorectal cancer or lung cancer.

The invention also provides the use of a formulation of the invention in the manufacture of a medicament for delivering an anti-PD-L1/OX 40 bispecific antibody protein to a mammal. The invention also provides methods of using the formulations of the invention for treating or preventing one or more of the above-mentioned diseases and disorders. Preferably, the mammal is a human.

The antibody formulations of the invention can be administered to a subject or patient in a variety of ways. For example, administration may be by infusion or by syringe. Accordingly, in one aspect, 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-PD-L1/OX 40 bispecific antibody protein as the main active ingredient, i.e. an amount sufficient to treat, ameliorate or prevent the disease or disorder of interest.

The therapeutic effect may include a reduction in 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 the clinician.

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 in any way as, limiting the scope of the invention.

Acronym descriptions

Acronyms	Full scale
		ELISA	Enzyme linked immunosorbent assay
iCIEF	Imaging capillary isoelectric focusing electrophoresis
		nrCE-SDS	Non-reduced sodium dodecyl sulfate capillary gel electrophoresis
SEC-HPLC	Size exclusion high performance liquid chromatography
		HPLC-FLD	High performance liquid chromatography-fluorescence detection

Examples

An anti-PD-L1/OX 40 bispecific antibody was an antibody developed autonomously by Nedak biopharmaceutical (Suzhou) Inc., and disclosed in PCT application No. PCT/CN 2020/073959.

In order to develop a simple and easy-to-use injection preparation prescription suitable for the long-term stable storage of the fully human antibody, the influence of different pH values, different stabilizing agents and the content of a surfactant on the protein quality of the antibody is investigated through a 40 ℃ forced stability experiment, and finally, a preparation prescription favorable for the stability of the antibody is screened. The materials and methods used throughout the study were as follows: materials and methods

1.1. Materials used in the formulation study of the present invention

Note: N/A indicates not applicable.

1.2. The apparatus used in the formulation research of the present invention

1.3. Detection item and detection method for stability of preparation

The detection items in the whole research process mainly comprise: (1) detecting the appearance and the presence of visible foreign matter; (2) the protein content of the preparation was determined by the ultraviolet method (UV method); (3) by OD₃₅₀Detecting the turbidity of the preparation by a nm method; (4) the purity of the antibody preparation was determined by size exclusion high performance liquid chromatography (SEC-HPLC) and expressed as the area of the main peak as a percentage of the sum of all peak areas; (5) the purity of the antibody formulation was determined by non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS), expressed as the percentage of the area of the main peak to the sum of all peak areas; (6) determining the charge variants in the antibody preparation by the iCIEF method, expressed as a percentage of the main component, the acidic component and the basic component; (7) determining the relative binding activity of the anti-PD-L1/OX 40 bispecific antibody in the antibody preparation against the anti-PD-L1/OX 40 bispecific antibody antigen by a direct ELISA method; (8) polysorbate 80 content of the antibody formulation was determined by HPLC-FLD.

Detection of visible foreign matter

According to the method described in the pharmacopoeia of the people's republic of China (2015 edition, Sanyo: Chinese pharmaceutical science and technology publisher. 2015), the samples were inspected for visible impurities using a clarity detector (Tianjin Daitan production, model YB-2) and an insoluble particle detector (Tianjin Daitan production, model GWJ-8).

Protein content determination

The protein content in the sample was measured using an ultraviolet spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800) or a multichannel microspectrophotometer (manufactured by Thermo, USA, model Nanodrop 8000).

Turbidity measurement

The turbidity of the sample was determined by measuring the absorbance of the sample at 350nm using an ultraviolet spectrophotometer (model UV-1800, Shimadzu, Japan).

Purity (SEC-HPLC method)

Separating with size exclusion chromatography column, wherein the mobile phase is phosphate buffer solution (3.12 g sodium dihydrogen phosphate dihydrate, 8.77 g sodium chloride and 34.84g arginine are weighed, the pH is adjusted to 6.8 by hydrochloric acid after the ultrapure water is dissolved, and the volume is adjusted to 1000ml), and the column protective solution is 0.05% (w/v) NaN₃The sample amount is 50 mul, the flow rate is 0.5 ml/min, the collection time is 30min, the column temperature is 25 ℃, and the detection wavelength is 280 nm. A sample to be tested is diluted to 2mg/ml with ultrapure water to be used as a test solution. The preparation buffer was diluted in the same manner as described above to prepare a blank solution. 50 mul of each of the blank solution and the sample solution are injected into a liquid chromatograph, and detection is started.

Purity (non-reduced CE-SDS method)

And (3) detecting by adopting a capillary gel electrophoresis method. The capillary tube is an uncoated capillary tube, the inner diameter of the capillary tube is 50 mu m, the total length of the capillary tube is 30.2cm, and the effective length of the capillary tube is 20.2 cm. Before electrophoresis, the capillary column was washed with 0.1mol/L NaOH, 0.1mol/L HCl, ultrapure water, and electrophoresis gel at 70 psi. Diluting a sample to be detected to 2.0mg/mL with a proper amount of ultrapure water, taking 50 mu L of the diluted sample to be placed in a 1.5mL centrifuge tube, respectively adding 45 mu L of sample buffer solution with pH 6.5 (0.32 g of citric acid monohydrate and 2.45g of disodium hydrogen phosphate dodecahydrate are weighed and dissolved in 45mL of ultrapure water, fixing the volume to 50mL, preparing a citric acid-phosphate buffer solution, precisely taking 200 mu L of the buffer solution, adding 80 mu L of 10% (w/v) sodium dodecyl sulfate solution, adding water to 1mL, uniformly mixing, obtaining 1 mu L of internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, product number: 390953) and 5 mu L of 250mmol/L NEM solution (62 mg of N-ethylmaleimide is weighed and dissolved in 2mL of ultrapure water), fully mixing, heating at 70 +/-2 ℃ for 10 +/-2 minutes, after cooling to room temperature, the sample was transferred to a sample bottle as a test solution. The same volume of the buffer solution was used as the sample, and the same procedure was followed to obtain a blank solution. Sample introduction conditions of the sample are as follows: -5kV 20 seconds; separation voltage: 15kV 35 min. The capillary column temperature was controlled at 25 ℃ and the detection wavelength was 220 nm.

Charge variants (iCIEF method)

Isoelectric point and charge variants (iCIEF method) are adopted for detection. A premixed solution is prepared by using 770 mu l of 3M urea-0.5% MC solution, 44 mu l of ampholyte (pH 3-10), 55 mu l of 5M NDSB solution, 22 mu l of 0.5M Taurine solution, 2.2 mu l of pI Marker 6.14 and 2.2 mu l of pI Marker 9.46 under the condition ratio, and the detection is carried out at the UV detection wavelength of 280 nm. And calculating the contents of the main component, the acidic component and the alkaline component according to an area normalization method.

Polysorbate 80 content (HPLC-FLD method)

Detecting by fluorescence detection chromatography (HPLC-FLD method). Using 0.15mol/L sodium chloride, 0.05mol/L tris, pH 8.0, 5% acetonitrile, 5.0 μmol/L N-phenyl-1-naphthylamine, 15ppm 23 polyoxyethylene fatty alcohol ether, isocratic elution, flow rate: 1.5 ml/min; collecting time: 3 minutes; upper pressure limit: 100 bar; sample introduction amount: 10 mu l of the mixture; column temperature: 30 ℃; detection wavelength: 350nm for excitation light and 420nm for emission light. The polysorbate 80 content was calculated according to the standard curve method.

Relative binding Activity (direct ELISA method)

96-well plate wells were coated with human OX40 antigen (from Sino) and human PD-L1 antigen (from Promega) at 0.5. mu.g/ml, 100. mu.l/well, overnight at 4 ℃. After washing the plate, blocking solution (2% BSA-PBST, 300. mu.l/well) was added and blocked for 2h at 37 ℃. The test sample diluted in the gradient was added to the microplate from which the blocking solution was discarded at 100. mu.l/well, and only 100. mu.l of the diluent (2% BSA-PBST) was added to each well of the negative control, followed by incubation at 37 ℃ for 60min in a constant temperature incubator. After washing the plate, a HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, cat # A80-104P, USA) diluted with 2% BSA-PBST was added as a secondary antibody (100000-fold dilution, 100. mu.l/well) and reacted at 37 ℃ for 30 min. After washing the plate, 100. mu.l of TMB buffer was added to each wellColor solution, after developing for 10min, adding 100 μ l of 1mol/L H per well₂SO₄The reaction was terminated. The OD value at 450nm was measured using 620nm as a reference wavelength. The concentration value of each concentration gradient sample is taken as an abscissa, the OD450 nm-OD620 nm value of each gradient sample is taken as an ordinate, and the EC is calculated by applying Prism four-parameter fitting₅₀Reflecting the binding activity of the antibody to each antigen. Relative binding Activity (%) (EC of test article)₅₀EC of reference₅₀) 100%, wherein the reference is a stable anti-PD-L1/OX 40 bispecific antibody without any stress treatment.

Example 1 preparation and purification of anti-PD-L1/OX 40 bispecific antibody

An anti-PD-L1/OX 40 bispecific antibody was obtained as described in PCT application No. PCT/CN 2020/073959. The antibody has the amino acid sequence of SEQ ID NO:1 and the peptide chain #1 sequence of SEQ ID NO:7, peptide chain #2 sequence, bispecific antibody. PCT application number PCT/CN2020/073959 is incorporated herein by reference in its entirety.

Briefly, the antibody was recombinantly expressed in CHO cells and purified by filtration, chromatography, viral inactivation, filtration, and the like.

Example 2 pH screening assay

2.1 Experimental procedures

In this example, the influence of pH (5.0-7.0) on the stability of the purified anti-PD-L1/OX 40 bispecific antibody of example 1 was examined, and a total of 5 pH values of 5.0, 5.5, 6.0, 6.5 and 7.0 were designed.

Preparing 10mM histidine and 5% (w/v) sorbitol buffer, adjusting pH to 5.0, 5.5, 6.0, 6.5 and 7.0 with hydrochloric acid, ultrafiltering and replacing anti-PD-L1/OX 40 bispecific antibody into the above buffers with different pH values, and adjusting the protein content of the sample to about 30 mg/ml; and polysorbate 80 was added to a final concentration of about 0.2 mg/ml; filtering, subpackaging into 2R penicillin bottles, plugging and capping. The samples are placed at 40 +/-2 ℃ for stability investigation, and the specific scheme is shown in the table.

TABLE 1 Pre-prescription study protocol

Note: (1) the point is sampled as indicated by √. (2) And after sampling at the time points, putting the samples into an ultra-low temperature refrigerator for freezing and checking, and freezing and checking according to requirements.

2.2 criteria of judgment

According to the knowledge of the product and the precision of the instrument and the method, a judgment standard that the quality of the sample detection index value is unchanged from the initial value is set for judging whether the sample is changed or not, and the table 2 shows the specific.

TABLE 2 judgment criteria for unchanged mass

Detecting items	Unchanged judgment standard
		Appearance of the product	Clear to microemulsion, colorless to light yellow liquid, no foreign matter
Visible foreign body	Meets the general rule 0904 of pharmacopoeia of the people's republic of China (2015 edition, three parts)
		Protein content (UV method)	The change rate is less than or equal to 10 percent
Turbidity (OD350nm method)	Variation value is less than or equal to 0.02
		Purity (SEC-HPLC method)	The variation value of the main peak is less than or equal to 1 percent
Purity (nrCE-SDS method)	The variation value of the main peak is less than or equal to 2 percent
		Charge variants (iCIEF method)	The change value of the main component and the acid-base components is less than or equal to 2 percent
Relative binding Activity (direct ELISA method)	70～130％
		Polysorbate 80 content (HPLC-FLD method)	Should be 0.1-0.3 mg/ml

2.3 results of the experiment

The results of the pre-prescription study are detailed in the table. The result shows that the sample is placed for 1 month at the temperature of 40 +/-2 ℃, and the appearance and visible foreign matters of the sample under each pH condition are qualified; protein content, polysorbate 80, purity (SEC-HPLC method and nrCE-SDS method) and relative binding activity were not significantly changed. All samples were out of judgment for turbidity. The charge variant-acidic component of each sample is obviously increased, and the main component is obviously reduced; the alkaline components of the samples did not change significantly under the conditions except for the pH 5.0 and pH 5.5 conditions; the sample charge variation-the magnitude of the change in the major component is relatively small at pH 5.5. Finally, the pH 5.5 was determined for the prescription screening experiment.

TABLE 3 Pre-prescription study results

Example 3 prescription screening experiment

3.1 Experimental procedures

In the example, the influence of different buffer systems (histidine buffer system and citric acid buffer system) and auxiliary materials (sorbitol, sucrose, arginine hydrochloride and edetate disodium) on the stability of the anti-PD-L1/OX 40 bispecific antibody protein is mainly considered, 6 prescriptions are designed in total, and the detailed prescription information is shown in Table 4.

Each formulation buffer was prepared as per Table 4, and the anti-PD-L1/OX 40 bispecific antibody protein was ultrafiltered and replaced into the corresponding formulation buffer. After the replacement is finished, regulating the protein content of each prescription to be about 40mg/ml, and adding polysorbate 80 to ensure that the final concentration is about 0.2 mg/ml; filtering, subpackaging into penicillin bottles, plugging and capping. The stability of the above samples was investigated at 40 ℃. + -. 2 ℃ and the detailed experimental conditions and sampling schedule are shown in Table 5.

TABLE 4 prescription information Table

Note: the pH of the prescription 1-6 is adjusted by hydrochloric acid.

TABLE 5 stability test protocol

Note: (1) indicates that only 0 day and 1 month samples were tested. (2) The point is sampled as indicated by √. (3) And after sampling at the time points, putting the samples into an ultra-low temperature refrigerator for freezing to be detected, and thawing and inspecting according to requirements.

3.2 criteria of judgment

See table 2 for criteria for the determination.

3.3 results of the experiment

The experimental results for prescription determination are detailed in

Table (7). The result shows that the sample is placed for 1 month at 40 +/-2 ℃, and the appearance and visible foreign matters of the samples in other places are qualified except the prescription 6 which is unqualified in appearance and is not detected; protein content, polysorbate 80 and relative binding activity were not significantly changed. Except for the prescription 2, the turbidity of the other prescriptions exceeds the judgment standard. The purity of prescription 3, prescription 4 and prescription 5 (SEC-HPLC method) was reduced, mainly manifested by increased protein aggregates and fragments, and no significant change occurred in the remaining prescriptions. The purities (nrCE-SDS method) of the prescription 2, the prescription 3 and the prescription 4 are all reduced, mainly represented by the increase of protein polymers and fragments, and the other prescriptions are not obviously changed. The charge variant-acidic component of each prescription is obviously increased, the main component is obviously decreased, but the change amplitude of the charge variant-acidic component and the main component of the prescription 2 is relatively small.

Prescription determination experimental results show that prescription 2 is superior to other prescriptions in turbidity, purity (SEC-HPLC method) and charge variants (iCIEF method). Finally, prescription 2 was selected for subsequent experiments.

TABLE 5 prescription determination test results

Example 4 polysorbate 80 concentration study

4.1 Experimental procedure

In the present example, the influence of different polysorbate 80 concentrations on the stability of the anti-PD-L1/OX 40 bispecific antibody protein was mainly examined, and 3 prescriptions were designed, and the detailed prescription information is shown in the table.

The formulation buffer was prepared as shown and the anti-PD-L1/OX 40 bispecific antibody protein was ultrafiltered and replaced into the formulation buffer. After the replacement is finished, adjusting the protein content of each prescription to about 40mg/ml, and adding polysorbate 80 with different final concentrations; filtering, subpackaging into penicillin bottles, plugging and capping. The stability of the above samples was investigated at 40 ℃. + -. 2 ℃ and the detailed experimental conditions and sampling schedules are shown in the table.

TABLE 6 prescription information Table

Serial number	Prescription information
		Prescription 2	10mM histidine, 50mg/ml sorbitol, 0.2mg/ml polysorbate 80, 0.01mg/ml disodium edetate pH 5.5
Prescription 7	10mM histidine, 50mg/ml sorbitol, 0.5mg/ml polysorbate 80, 0.01mg/ml disodium edetate pH 5.5
		Prescription 8	10mM histidine, 50mg/ml sorbitol, 0.7mg/ml polysorbate 80, 0.01mg/ml disodium edetate pH 5.5

TABLE 7 stability study protocol

Note: only the last point is detected, and the sample at the middle point is frozen and preserved after being sampled and is not detected for the moment.

4.2 criteria of judgment

The judgment criteria are specifically shown in the table.

4.3 results of the experiment

The results of the polysorbate 80 concentration study are shown in the table. The results show that the appearances and visible foreign matters of all prescriptions under all conditions are qualified; protein content, purity (SEC-HPLC method and nrCE-SDS method) and charge variants were not significantly changed, and there was no significant difference between the formulas.

TABLE 8 polysorbate 80 concentration study results

And finally selecting the prescription 7 as an anti-PD-L1/OX 40 bispecific antibody preparation prescription by combining the experimental results and the experience of a preparation prescription development platform. The anti-PD-L1/OX 40 bispecific antibody formulation was determined to have a formula composition of: 40.0mg/ml recombinant anti-tumor necrosis factor receptor superfamily member 4(OX40) and anti-programmed death ligand 1(PD-L1) bispecific antibody, 10mM histidine, 50.00mg/ml sorbitol, 0.01mg/ml disodium edetate, 0.5mg/ml polysorbate 80, pH 5.5.

EXAMPLE 5 formulation stability Studies

5.1 stability Studies and protocols

Referring to NMPA (original CFDA) technical guidelines for bioproduct stability research, ICH Q5C stability tests of biotechnology/bioproduct and other related guidelines, accelerated stability research and forced condition test research on three batches of process and quality representative finished products of the bispecific antibody of the recombinant anti-tumor necrosis factor receptor superfamily member 4(OX40) and the anti-programmed death ligand 1(PD-L1) are respectively carried out.

The stability study sample information is shown in table 9. The actual storage temperature of the finished product of the anti-PD-L1/OX 40 bispecific antibody is 5 +/-3 ℃, the packaging container is a penicillin bottle, and the packaging specification is 200mg (5 ml)/bottle; the packaging container and the closed system for the stability study of the finished product of the anti-PD-L1/OX 40 bispecific antibody are completely consistent with the packaging material and the loading of the finished product which are actually stored. The stability inspection conditions, inspection time points, inspection indexes and quality standards are shown in tables 10 and 11.

TABLE 9 sample information

Finished product batch number	Production process	Scale of	Protein content
				HH20200101	Pilot plant test process	300L	41.1mg/ml
HH20200102	Pilot plant test process	300 L	41.5mg/ml
				HH20200103	Pilot plant test process	300 L	40.7mg/ml

TABLE 10 formulation stability test protocol

Research projects	Examination conditions	Survey time point
			Accelerated stability study	25℃±2℃/60％RH±5％RH	Month 0,1 and 2
Forced Condition test	40℃±2℃/75％RH±5％RH	Week 0,2, 4

TABLE 11 quality standards for finished anti-PD-L1/OX 40 bispecific antibodies

5.2 stability test results

The stability results under accelerated and forced conditions are shown in tables 12 and 13, respectively. The stability data and the prescription screening results show that the trends of the test results are consistent, and the established prescription can meet the storage stability of the anti-PD-L1/OX 40 bispecific antibody.

TABLE 12 accelerated stability study results (25 ℃. + -. 2 ℃)

Note: N/A indicates that the time point is free of the check item.

TABLE 13 results of the forced stability study (40 ℃. + -. 2 ℃)

Note: N/A indicates that the time point is not detected

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

Sequence listing

<110> Xinda biopharmaceuticals (Suzhou) Limited

<120> anti-PD-L1/OX 40 bispecific antibody preparation and preparation method and application thereof

<130> P32703CN009

<140> 2021108411670

<141> 2021-07-23

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<151> 2020-07-24

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

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Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

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Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

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Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

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Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

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Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

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Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

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Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

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Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

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Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

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Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

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Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

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Ala Ala Ser Ala Tyr Thr Ile Ser Arg Asn Ser Met Gly Trp Phe Arg

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Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Glu Ser Asp

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Gly Ser Thr Ser Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

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Leu Asp Asn Ser Lys Asn Thr Leu Tyr Leu Glu Met Asn Ser Leu Arg

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Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Pro Lys Val Gly Leu

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Gly Pro Arg Thr Ala Leu Gly His Leu Ala Phe Met Thr Leu Pro Ala

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Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

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Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

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Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

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Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

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Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

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Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

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Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

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Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

115 120 125

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

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Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

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Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

165 170 175

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

180 185 190

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Tyr Thr Ile Ser Arg Asn

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Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val

35 40 45

Ala Ala Ile Glu Ser Asp Gly Ser Thr Ser Tyr Ser Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Leu Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Ala Pro Lys Val Gly Leu Gly Pro Arg Thr Ala Leu Gly His Leu Ala

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Phe Met Thr Leu Pro Ala Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val

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Thr Val Ser Ser

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

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Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

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Phe Asn Arg Gly Glu Cys

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

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Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

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<220>

<223> LCDR2

<400> 17

Asp Ala Ser Asn Leu Glu Thr

1 5

<210> 18

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3

<400> 18

Gln Gln Ser Ala Asn Tyr Pro Tyr Thr

1 5

<210> 19

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH-CH1-Fc

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Claims

1. A liquid antibody formulation comprising

(i) anti-PD-L1/OX 40 bispecific antibody proteins;

(ii) a buffering agent, a water-soluble polymer,

(iii) a stabilizer, and

(iv) a surfactant, a water-soluble surfactant and a water-soluble surfactant,

wherein the anti-PD-L1/OX 40 bispecific antibody protein consists of the following polypeptide chain #1 and polypeptide chain # 2:

polypeptide chain # 1:

VH-CH 1-Fc-X-VHH; and

polypeptide chain # 2:

VL-CL；

wherein:

VH represents a heavy chain variable region;

CH1 represents heavy chain constant region domain 1;

fc comprises heavy chain constant region domains CH2, CH3, and optionally CH 4;

x may be absent or, when present, represents a linker;

VHH represents a single domain antigen binding site;

VL represents a light chain variable region;

CL represents a light chain constant region;

optionally, a hinge region is present between CH1 and Fc; or

The anti-PD-L1/OX 40 bispecific antibody comprises at least one polypeptide chain #1 and one polypeptide chain #2, preferably, two polypeptide chains #1 and two polypeptide chains # 2; or

The VHH comprises Complementarity Determining Regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR 3; wherein said VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID No. 10; the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID No. 11; the VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID No. 12;

and/or the VH comprises or consists of Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO. 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO. 15;

and/or the VL comprises or consists of Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO 17; LCDR3 comprising or consisting of the amino acid sequence of SEQ ID No. 18;

preferably, the liquid antibody formulation has a pH of about 5.0 to 6.0, e.g., a pH of 5.5. + -. 0.2, preferably a pH of 5.5.

2. The liquid antibody formulation according to claim 1, characterized in that the concentration of anti-PD-L1/OX 40 bispecific antibody protein in the liquid antibody formulation is about 1-100 mg/mL, preferably about 10-70 mg/mL, such as about 10,15,20,25,30, 35, 40,50,55,60, 70 mg/mL.

3. Liquid antibody formulation according to claim 1 or 2, characterized in that,

the liquid antibody preparation comprises a buffering agent selected from a histidine-histidine hydrochloride buffering system, a citric acid-sodium citrate buffering system, an acetic acid-sodium acetate buffering system and a phosphate buffering system,

preferably, the buffer in the liquid antibody formulation is selected from histidine, histidine hydrochloride and combinations thereof;

preferably, the buffer is at a concentration of about 5-50mM, preferably about 5-30mM, e.g., about 5,10,15,20,25,30 mM.

4. The liquid antibody formulation of any one of claims 1-3, characterized in that the stabilizing agent is selected from the group consisting of polyols (e.g., sorbitol, mannitol, and combinations thereof), saccharides (e.g., sucrose, trehalose, maltose, and combinations thereof), amino acids (e.g., arginine methionine hydrochloride glycine proline, and combinations thereof), and any combination thereof,

preferably, the stabilizer is sorbitol;

preferably, the stabilizer is about 20-80mg/ml, e.g., about 20,25,30, 35, 40, 45, 50,55,60, 70,80 mg/ml sorbitol.

5. The liquid antibody formulation according to any one of claims 1 to 4, characterized in that the surfactant is selected from polysorbate based surfactants, poloxamers, polyethylene glycols or combinations thereof, preferably polysorbate-80, preferably the concentration of the surfactant is about 0.1-1mg/ml, preferably about 0.2-0.8mg/ml, such as about 0.2,0.3,0.4,0.5,0.6,0.7,0.8 mg/ml.

6. The liquid antibody formulation according to any one of claims 1 to 4, characterized in that it further comprises a chelating agent selected from the group consisting of edetate disodium, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, dihydroxyethylglycine, preferably, the chelating agent is selected from edetate disodium;

preferably, the chelating agent is about 0.005-0.05mg/ml, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018mg/ml edetate disodium.

7. The liquid antibody formulation according to any one of claims 1-6, characterized in that the anti-PD-L1/OX 40 bispecific antibody comprises polypeptide chain #1 and polypeptide chain #2, wherein the VH-CH1-Fc-X-VHH of polypeptide chain #1 comprises SEQ ID No. 1 or a sequence having at least 90% identity thereto and the VL-CL of polypeptide chain #2 comprises an amino acid sequence having at least 90% identity to SEQ ID No. 7 or a sequence having at least 90% identity thereto.

8. The liquid antibody formulation of any one of claims 1-7, characterized in that the anti-PD-L1/OX 40 bispecific antibody is recombinantly expressed in 293 cells or CHO cells.

9. Liquid antibody formulation according to any one of claims 1 to 8, characterized in that the liquid formulation is an injection, preferably for subcutaneous or intravenous injection, or an infusion, for example for intravenous infusion.

10. The liquid antibody formulation of any one of claims 1-9, comprising:

(v) optionally, about 0.005-0.05mg/ml of edetate disodium, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml;

wherein the pH of the liquid formulation is about 5.0-6.0, e.g., about 5.5;

for example, the liquid antibody formulation may comprise

(ii) about 10mM histidine buffer;

(iv) about 0.2-0.8mg/ml, e.g., 0.2,0.3,0.4,0.5,0.6,0.7,0.8mg/ml, e.g., 0.3-0.6mg/ml polysorbate 80; and

(v) optionally, about 0.008-0.018mg/ml of disodium edetate, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml;

wherein the pH of the liquid formulation is about 5.0-6.0, e.g., about 5.5;

in a preferred embodiment, the liquid antibody formulation comprises:

(i) about 40mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, 10mM histidine, 50mg/ml sorbitol, 0.2mg/ml polysorbate 80, 0.01mg/ml disodium edetate, pH 5.5; or

(ii) About 40mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, 10mM histidine, 50mg/ml sorbitol, 0.5mg/ml polysorbate 80, 0.01mg/ml disodium edetate, pH 5.5; or

(iii) About 40mg/ml of anti-PD-L1/OX 40 bispecific antibody protein, 10mM histidine, 50mg/ml sorbitol, 0.7mg/ml polysorbate 80, 0.01mg/ml disodium edetate, pH 5.5.

11. Liquid antibody formulation according to any one of claims 1 to 10, characterized in that the formulation is stable after storage, e.g. after storage at 25 ℃ for at least 2 months, or after storage at 40 ℃ ± 2 ℃ for 1 month, preferably having one or more of the following characteristics:

(iii) as measured by the iCIEF method, the sum of the change values of the components (main component, acidic component and basic component) of the anti-PD-L1/OX 40 bispecific antibody protein in the formulation does not exceed 40% and/or the change value of the main component does not exceed 20% relative to the initial value on day 0 of storage, e.g., the sum of the change values does not exceed about 40% (e.g., does not exceed 30%) or the change value of the main component does not exceed about 20% (e.g., does not exceed 15%) after 1 month of storage at 40 ℃ ± 2 ℃, or the sum of the change values does not exceed about 20% (e.g., about 15%) or the change value of the main component does not exceed about 15% (e.g., does not exceed about 10%) after 2 months of storage at 25 ℃;

(iv) the relative binding activity of the anti-PD-L1/OX 40 bispecific antibody protein in the formulation, as measured by ELISA, relative to the initial value on day 0 of storage, is 70% -130%, e.g., 90% -110%;

more preferably, the formulation is stable under shaking and/or repeated freeze-thawing.

12. A solid antibody formulation obtained by solidifying a liquid antibody formulation according to any one of claims 1 to 11, for example in the form of a freeze-dried powder injection.

13. A delivery device comprising the liquid antibody formulation of any one of claims 1-12 or the solid antibody formulation of claim 13.

14. A pre-filled syringe comprising the liquid antibody formulation of any one of claims 1-12 or the solid antibody formulation of claim 13 for intravenous or intramuscular injection.

15. Use of a liquid antibody formulation according to any one of claims 1-12 or a solid antibody formulation of claim 13 for the preparation of a delivery device or a pre-filled syringe or a medicament for increasing the expression level of PD-1, PD-L1 or PD-L2 and/or having a reduced expression level of OX40 in a subject.

16. Use of a liquid antibody formulation according to any one of claims 1-12 or a solid antibody formulation according to claim 13 for the manufacture of a delivery device or a pre-filled syringe or a medicament for the treatment or prevention of an autoimmune disease, an inflammatory disease, an infection, a tumour, a T cell dysfunctional disease in a subject, e.g. the tumour is a cancer of the gastrointestinal tract, e.g. colon cancer.