CN114146174B

CN114146174B - Anti-PD-L1/OX 40 bispecific antibody preparation and preparation method and application thereof

Info

Publication number: CN114146174B
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: 2024-05-24
Anticipated expiration: 2041-07-23
Also published as: CN114146174A

Abstract

The present invention relates to formulations comprising anti-PD-L1/OX 40 bispecific antibodies, in particular to pharmaceutical formulations comprising anti-PD-L1/OX 40 bispecific antibodies, buffers, stabilizers and surfactants. Furthermore, the invention relates to the use of these formulations for the treatment or prophylaxis of diseases.

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

Background

Drug stability is one of the important indicators to ensure drug effectiveness and safety. Obtaining a good formulation prescription is a key condition to ensure that the drug remains effective and safe over the shelf life. However, due to the complexity of the antibody itself and its degradation pathways, it is currently not possible to make predictions about the formulation conditions required to optimize antibody stability. In particular, it is contemplated that different antibodies typically have very different CDR sequences, different antibody structures, and that these sequence and structural differences may result in different antibodies having different stability properties in solution. Thus, based on stringent requirements on safety and effectiveness 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 over-expression can regulate and control cell cycle checkpoint protein and cell proliferation related protein expression by inhibiting RAS and PI3K/AKT signal paths, and finally, the inhibition of T cell proliferation is caused. OX40 (also known as CD134 or TNFRSF 4) is a TNFR family member expressed mainly on activated T cells, mainly cd4+ effector T cells and cd8+ effector T cells 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 cancer, and kidney cancer). In a mouse model, anti-PD-L1/OX 40 bispecific antibodies have better tumor inhibition, see for example: PCT/CN2020/073959.

At present, an anti-PD-L1/OX 40 bispecific antibody preparation is urgently needed, which can ensure the quality of the medicine in the long-term storage process, 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 containing anti-PD-L1/OX 40 bispecific antibodies. The antibody formulations of the present invention exhibit excellent stability against a variety of stability-affecting factors (such as temperature, repeated freeze thawing, shaking).

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

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

Formula (I):

peptide chain #1: VH-CH1-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 CH4;

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 the light chain constant region;

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

In one embodiment, a PD-L1/OX40 bispecific antibody of the present 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 (VH of exemplary formula (I))	SEQ ID NO:2
CH1 (CH 1 of exemplary formula (I))	SEQ ID NO:3
		Fc (Fc of exemplary 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 (exemplified by VL of formula (II))	SEQ ID NO:8
CL (CL of exemplary formula (II))	SEQ ID NO:9
		Anti-PD-L1 single domain antibody CDR1 (VHH CDR1 of exemplary formula (I))	SEQ ID NO:10
Anti-PD-L1 single domain antibody CDR2 (VHH CDR2 of exemplary formula (I))	SEQ ID NO:11
		Anti-PD-L1 single domain antibody CDR3 (VHH CDR3 of exemplary formula (I))	SEQ ID NO:12
OX40 antibody portion VHCDR1 (HCDR 1 of an exemplary VH of formula (I))	SEQ ID NO:13
		OX40 antibody portion VHCDR2 (HCDR 2 of an exemplary VH of formula (I))	SEQ ID NO:14
OX40 antibody portion VHCDR3 (HCDR 3 of an exemplary VH of formula (I))	SEQ ID NO:15
		OX40 antibody portion VLCDR1 (LCDR 1 of an exemplary VL of formula (II))	SEQ ID NO:16
OX40 antibody portion VLCDR2 (LCDR 2 of an exemplary VL of formula (II))	SEQ ID NO:17
		OX40 antibody portion VLCDR3 (LCDR 3 of an exemplary VL of 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, an anti-PD-L1/OX 40 bispecific antibody comprises at least one polypeptide chain #1 and one polypeptide chain #2. Preferably, the 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 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 day: 23 of 1 month 2020).

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

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

In one embodiment, the concentration of buffer in the liquid antibody formulation of the present invention is about 5-50mM. In one embodiment, the concentration of buffer in the liquid antibody formulation of the invention is about 5-30mM, e.g., about 5, 10, 15, 20, 25, 30mM. In one embodiment, the buffer is a histidine buffer, a citric acid buffer, an acetic acid buffer, a phosphoric acid 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 present invention is about 20-80mg/ml. In one embodiment, the concentration of the stabilizing agent in the liquid antibody formulation of the present invention is about 30-60mg/ml, e.g., about 30, 40, 50, 60mg/ml.

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

In one embodiment, the concentration of surfactant in the liquid antibody formulation of the present invention is about 0.1-1mg/ml. In one embodiment, the concentration of surfactant in the 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, 0.8mg/ml.

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

In one embodiment, the concentration of chelating agent in the liquid antibody formulations of the invention is about 0.005-0.05mg/ml. In one embodiment, the concentration of chelating agent in the liquid antibody formulations of the invention is about 0.008-0.018mg/ml, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018mg/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, aminotriacetic acid, diethylenetriamine pentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethyl ethylenediamine triacetic acid, dihydroxyethyl glycine. In one embodiment, the chelating agent is selected from disodium edentate.

In one embodiment, the liquid formulation has a pH of about 5.0 to about 6.0. In some embodiments, the pH of the liquid formulation is any of about 5.0 to 6.0, for example 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 20-60 mg/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. 5,10,15,20,25,30mM;

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

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

(V) Optionally, about 0.005-0.05mg/ml of disodium edentate, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018mg/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 of anti-PD-L1/OX 40 bispecific antibody protein, e.g. 20,30,40,50,55,60mg/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, 80mg/ml sorbitol;

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

(V) Optionally, about 0.008-0.018mg/ml of disodium edentate, e.g., about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018mg/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 edentate pH 5.5; or (b)

(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 edentate pH 5.5; or (b)

(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 edentate pH 5.5;

The liquid formulations of the present invention may be stable for long periods of storage, 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 ℃, such as-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃, or about 45 ℃ for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, and is stable.

In one embodiment, the liquid formulation of the present invention may be stable 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 at about 2 ℃ to 8 ℃ for at least 3 months, preferably at least 12 months, more preferably at least 24 months. In one embodiment, the liquid formulation of the present invention remains stable for at least 2 months, preferably at least 3 months, more preferably at least 6 months at room temperature or e.g. 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, the stability of a formulation may be indicated by detecting changes in the appearance, visible foreign matter, protein content, turbidity, purity, and/or charge variants of the formulation. In one embodiment, the stability of the liquid formulations of the invention may be tested in a forced assay 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 assay, e.g. after storage at 25 ℃ ± 2 ℃ for at least 1 month or 2 months, or in a long-term assay, e.g. after storage at 5 ℃ ± 3 ℃ for at least 2 months or 3 months, or in a shaking assay (e.g. shaking for 5 days at room temperature, light-shielding 650 r/min), and/or in a freeze-thawing assay (e.g. repeated freeze-thawing 6 times at-20 ℃/2-8 ℃). In one embodiment, the stability of the liquid formulation of the invention is measured relative to an initial value, for example, an initial value stored on day 0, or an initial value prior to shaking or freeze-thawing experiments.

In one embodiment, the stability of the liquid formulation of the present invention is visually inspected after storage, or after shaking experiments, or after freeze-thawing experiments, wherein the liquid formulation of the present invention remains clear to micro-opalescent in appearance, colorless to pale yellow liquid, and free of foreign substances. In one embodiment, the formulation is visually inspected under a clarity detector for the presence of no visible foreign matter. In one embodiment, the stability of the liquid formulation according to the invention is checked after storage, or after shaking experiments, or after freeze-thawing experiments, by measuring the change in protein content, wherein the rate of change of the protein content is not more than 20%, preferably not more than 10%, e.g. 7-8%, more preferably not more than 5%,2% or 1% relative to the initial value, for example by ultraviolet spectrophotometry (UV). In one embodiment, the stability of the liquid formulation of the invention is checked by measuring the change in turbidity of the liquid formulation of the invention after storage, or after shaking experiments, or after freeze-thawing experiments, wherein the change value, relative to the initial 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, as measured for example by OD _350mm method. In one embodiment, the stability of the liquid formulation according to the invention is checked by measuring the change in purity of the liquid formulation according to the invention after storage, or after shaking experiments, or after freeze-thawing experiments, wherein the change in monomer purity (or the change in main peak) is not more than 10%, such as not more than 5%, 4%, 3%, such as not more than 2%, preferably not 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 measuring the change in purity of the liquid formulation of the invention after storage, or after shaking experiments, or after freeze thawing experiments, wherein the change in purity of the monomer (or the change in main peak) is reduced by no more than 10%, e.g. no more than 5%, 4%, 3%,2% or 1%, relative to the initial value by non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. In one embodiment, the stability of the liquid formulation of the invention is measured by the iCIEF method after storage, or after shaking experiments, or after freeze-thawing experiments, wherein the sum of the values of the changes in the charge variants (main, acidic and basic) of the antibody relative to the initial value is not more than 50%, e.g. not more than 40%, 30%, 20%, 10%, 5%, and/or the value of the change in the main component is not more than 20%,15%,10%,8%,5%. In one embodiment, the stability of the liquid formulation of the invention is tested by direct ELISA after storage, or after shaking experiments, or after freeze thawing experiments, wherein 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% relative to the initial value.

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: with respect to storing the initial value on day 0,

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

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

(iii) The sum of the values of the changes 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 value of the change of the main component is not more than 20% as measured by the iCIEF method,

For example, the sum of the values of the changes after 1 month of storage at 40 ℃ + -2 ℃ is not more than about 40% (e.g., not more than 35%,30%, 25%,20%,15%, 10%) or the value of the change in the principal component is not more than 20% (e.g., not more than 15%,12%,10%, 8%), or

For example, a total of no more than about 20% (e.g., no more than 15%,14%,13%, 12%) or a principal component change of no more than about 15% (e.g., no more than 10%,8%,7%,6%, 5%) 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 is 70% to 130%, e.g., 90,93,95,98,100,103,105,108,110,115,120%, e.g., 90% to 110%, as measured by ELISA;

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

Preferably, the formulation is stable under shaking or under repeated freeze thawing, e.g. shaking for 5 days at room temperature in the absence of light at 650r/min or after repeated freeze thawing 6 times at-20 ℃/2-8 ℃, having 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 the formulation has a purity of greater than 96%, preferably greater than 97%, 98%, 99%;

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

(iii) The sum of the values of the changes 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 2% as measured by iCIEF;

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

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 a solid antibody preparation obtained by subjecting the liquid antibody preparation of the present invention to a curing treatment. The solidification treatment is performed 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 for injection. The solid antibody formulations may be reconstituted in a suitable vehicle prior to use to form the reconstituted formulations of the invention. 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 or solid antibody formulation of the invention, e.g., by using a pre-filled syringe delivery device.

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

The invention also provides a method of treating a disease, such as a tumor as described above, 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 medicament comprising the liquid or solid antibody formulation.

Other embodiments of the present invention will become apparent by consideration of the detailed description which follows.

Detailed Description

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

Definition of the definition

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 numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and/or" when used in connection with two or more selectable items is understood to mean any one of the selectable items or any two or more of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited 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.

The term "anti-OX 40 antibody", "anti-OX 40", "OX40 antibody" or "OX40 binding antibody" as used herein refers to an antibody that is capable of binding (human or monkey) OX40 protein or fragment thereof with sufficient avidity that the antibody can be used as a diagnostic and/or therapeutic agent in targeted (human or monkey) OX 40.

The terms "programmed cell death 1 ligand 1", "PD-L1", "programmed death ligand 1", "cluster of differentiation 274", "CD274" or "B7 homolog 1" as used herein refer to any natural 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 its broadest sense to refer to a protein comprising an antigen binding site, and encompasses natural and artificial antibodies of various structures, including but not limited to whole antibodies and antigen binding fragments of antibodies.

"Bispecific" antibodies refer to antibodies 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, the 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 effectively exerted and that does not contain other components that have unacceptable toxicity to the subject to whom the preparation is to be administered. Such antibody formulations are typically sterile. Typically, the antibody formulation comprises a pharmaceutically acceptable excipient. A "pharmaceutically acceptable" excipient is a test agent 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 excipient is compatible with the mode of administration and may be, for example, injection acceptable.

The term "anti-PD-L1/OX 40 bispecific antibody preparation" also referred to herein simply as "antibody preparation of the invention" means a preparation comprising an anti-PD-L1/OX 40 bispecific antibody protein as an active ingredient and comprising 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 a human or non-human animal after combining the anti-PD-L1/OX 40 bispecific antibody protein with a pharmaceutically acceptable excipient. The antibody formulations of the invention may be, for example, prepared as liquid formulations in aqueous form, e.g., ready-to-use pre-filled syringes, or as lyophilized formulations, which are reconstituted (i.e., reconstituted) 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 antibody in the formulation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions, or after shaking, or after repeated freeze thawing. Although the antibodies contained in an antibody preparation may not maintain 100% of their chemical structure 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 specific embodiments, the anti-PD-L1/OX 40 bispecific antibody protein formulations of the invention exhibit little to no detectable aggregation or degradation or chemical modification of the antibodies during manufacture, preparation, transport, and long term storage, and thus little or even no loss of biological activity of the anti-PD-L1/OX 40 bispecific antibody protein, exhibiting high stability. In some embodiments, the anti-PD-L1/OX 40 bispecific antibody protein formulations of the 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 formulation of the present invention may be stable after shaking for 5 days at room temperature in the absence of light at 650r/min and/or after repeated freeze thawing 1-6 times at-20 ℃/2-8 ℃.

A variety of analytical techniques are known in the art for determining the stability of proteins, see for example 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). for measuring stability 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, stability testing is performed by performing various stress tests on antibody formulations. These tests may represent extreme conditions that formulated antibody formulations may encounter during manufacture, storage or transport, as well as conditions that may accelerate the instability of the antibodies in the antibody formulations during non-manufacture, storage or transport. 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 antibodies can be checked after filling the formulated anti-PD-L1/OX 40 bispecific antibody protein formulation into a glass vial and shaking at room temperature in the absence of light for 650r/min for 5 days. For another example, the formulated anti-PD-L1/OX 40 bispecific antibody protein formulation is filled into glass vials and after repeated freeze thawing 1-6 times at-20deg.C/2-8deg.C, the antibody is checked for stability.

After a period of storage, or after shaking for a period of time, or after repeated freeze thawing, the formulation 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 arise because aggregation of antibodies in the formulation can potentially lead to increased immune responses in the patient. Thus, there is a need to minimize or prevent aggregation of antibodies in a formulation. Light scattering methods can be used to measure visible aggregates in a customization agent. SEC-HPLC can be used to determine soluble aggregates in a formulation. In addition, the stability of the formulation may be indicated by visual inspection of the appearance, color and/or clarity of the formulation, or by detecting the turbidity of the formulation by the OD ₃₅₀ nm method, or by determining the purity of the formulation by the 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 after storage at a particular temperature for a particular time, at least about 90% of the anti-PD-L1/OX 40 bispecific antibody protein monomer is detected in the formulation after shaking or after repeated freeze thawing. In some embodiments, an acceptable level 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, indicates at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-L1/OX 40 bispecific antibody protein monomer. When assessing physical stability, the particular temperature at which the pharmaceutical formulation is stored may be any temperature from about-80 ℃ to about 45 ℃, for example, 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 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 2 months of storage at about 25 ℃. 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 9 months of storage at about 5 ℃.

An antibody can be considered to "retain its chemical stability" in a formulation if it does not show a significant chemical change after a period of storage, or after shaking for a period of time, or after repeated freeze-thawing for a number of times. Most chemical instabilities result from the formation of covalently modified forms of antibodies (e.g., charge variants of antibodies). For example, by aspartic acid isomerization, N-and C-terminal modifications, basic variants can be formed; acidic variants can be produced by deamidation, sialylation and saccharification. Chemical stability can be assessed by detecting and/or quantifying the chemically altered form of the antibody. For example, capillary isoelectric focusing Jiao Dianyong (iCIEF) can be imaged to detect charge variants of antibodies in the formulation. In one embodiment, the stability of a formulation is measured by determining the percent change in the charge variant of an antibody in the formulation after storage at a specified temperature for a specified time or after shaking or repeated freeze thawing, wherein the smaller the change the higher the stability of the formulation.

An "acceptable degree" of chemical stability may mean that the percentage change of the charge variant (e.g., the main component or the acidic or basic component) in the formulation after storage at a specific temperature for a specific time, or after shaking for a period of time, or after repeated freeze-thawing, is no more than 40%, e.g., no more than 30%, no more than 20%; or the sum of the percentage change values of the charge variants (main component, acidic component and basic component) is not more than 60%, for example not more than 50%, not more than 30%. In some embodiments, an acceptable degree of chemical stability may be exhibited by a percent change in the primary component charge variant of no more than about 50, 40, 30, 20, or 15% after storage at a specified 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 is no more than about 60%, 50%, or 30%. When evaluating chemical stability, the temperature at which the pharmaceutical formulation is stored may be any temperature from about-80 ℃ to about 45 ℃, for example, stored 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 value of the major component charge variant 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%. Pharmaceutical formulations may also be considered stable if the percent change value of the major component charge variant after storage at 25 ℃ for 2 months is 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%. Pharmaceutical formulations may also be considered stable if the percent change value of the major component charge variant after 1 month of storage at 40 ℃ is less than about 50%, 40%, 30%, 20%, 16%, 15%, 14%, 13%, 12%, 10%, 5% or 4%.

The term "lyophilized formulation" refers to a composition obtained or obtainable by a lyophilization 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 obtained by dissolving and/or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

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

"Stress conditions" refers to environments that are chemically and/or physically unfavorable for an antibody protein, which may lead to unacceptable destabilization of the antibody protein, e.g., high temperature, shaking, freeze thawing. By "high temperature stress" is meant that the antibody preparation is stored at room temperature or even higher temperatures (e.g., 40 ℃ + -2 ℃) for a period of time. The stability of the antibody preparation can be checked by a high temperature stress acceleration test.

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 (subcuticular), intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection, and infusion. In some embodiments, the stabilized anti-PD-L1/OX 40 bispecific antibody protein formulations of the invention are administered to a subject parenterally. In one embodiment, the anti-PD-L1/OX 40 bispecific antibody protein preparations of the invention are administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

I. Antibody formulations

The present invention provides stable liquid antibody formulations 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

By "anti-PD-L1/OX 40 bispecific antibody" is meant an antibody that is capable of specifically binding to PD-L1 and a molecule that specifically binds to OX 40.

peptide chain #1:

VH-CH1-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 CH4;

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 the light chain constant region;

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

In one embodiment, the VH-CH1-Fc-X-VHH of 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 an 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 antibodies in the antibody formulations of the invention are antibodies in IgG format. An "antibody of the IgG form" refers to an IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are identical 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 an Ig domain whose heavy chain constant region Ig domain is IgG 2.

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

In one embodiment, the anti-PD-L1/OX 40 bispecific antibody is an IgG 2-type antibody produced by recombinant expression in CHO cells and purified. Preferably, the antibodies in the liquid formulations of the invention exhibit 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 intended characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is to be used. In some embodiments, the antibody formulation is a liquid formulation, which may contain about 1-150mg/mL, preferably about 10-100mg/mL, e.g., about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100mg/mL of an anti-PD-L1/OX 40 bispecific antibody.

(Ii) Buffering agents

Buffers are agents that can maintain the pH of a solution within an acceptable range. In some embodiments, buffers used in the formulations of the present invention may control the pH of the formulations of the present invention to a pH in the range of about 5.0 to 6.0, for example, a pH of about 5.0 to 5.5. In some specific embodiments, the antibody formulations of the invention have 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 formulation of the present invention comprises a buffer system selected from the group consisting of: histidine-histidine hydrochloride buffer system, citric acid-sodium citrate buffer system, acetic acid-sodium acetate buffer system, phosphate buffer system, preferably histidine-histidine hydrochloride buffer system.

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

(Iii) Stabilizing agent

Suitable stabilizers for use in the present invention may be selected from the group consisting of sugars, polyols and amino acids and combinations thereof. Sugar as a stabilizer includes, but is not limited to, sucrose, trehalose, maltose, and combinations. The polyol used as the stabilizer includes, but is not limited to, sorbitol, mannitol, or a combination thereof. For amino acids as stabilizers, including but not limited to arginine, arginine hydrochloride, methionine, glycine, proline, and combinations thereof.

In one embodiment, the stabilizer is selected from the group consisting of polyols (e.g., sorbitol, mannitol, or combinations thereof), sugars (e.g., sucrose, trehalose, maltose, or combinations thereof), amino acids (e.g., arginine hydrochloride, methionine, glycine, proline, and combinations or salts thereof), and any combinations thereof. In one embodiment, the stabilizer comprises about 20-80mg/ml sorbitol, e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80mg/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 consist of groups of opposite solubility tendencies, typically oil-soluble hydrocarbon chains and water-soluble ionic groups.

In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic 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 as a surfactant in the liquid formulation of the present invention.

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

The amount of surfactant included in the antibody formulations of the present invention may vary with the particular intended characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is to be 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 surfactant, especially polysorbate-80, preferably about 0.5mg/ml polysorbate-80.

(V) Chelating agent

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

In one embodiment, the chelate in the liquid formulation of the present invention is a carboxylic acid type chelator. In one embodiment, the chelating agent is selected from disodium edetate, aminotriacetic acid, diethylenetriamine pentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethyl ethylenediamine triacetic acid, dihydroxyethyl glycine. In one embodiment, the chelating agent is selected from disodium edentate.

(Vi) Other excipients

Other excipients are optionally included in the liquid formulations of antibodies of the invention. Such other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, gelatin, and the like. These and other known pharmaceutical excipients and/or additives suitable for use in the formulations of the present invention are well known in the art, for example, as set forth in "The Handbook of Pharmaceutical Excipients, 4 th edition, rowe et al, american Pharmaceuticals Association (2003); and Remington THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, gennaro, eds., lippincott Williams & Wilkins (2005) ".

II preparation of the preparation

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 may be prepared using techniques known in the art for producing antibodies. For example, antibodies 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 (Maximizing productivity 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 the protein a capture step. Kelley et al (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies,Biotechnology and Bioengineering 101(2008)553–566) describe a two-column purification method in which a weakly partitioned 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 drug substances with sufficient reproducibility and moderate purity for formulation of antibody preparations. For example, after secretion of antibodies from recombinant expression cells into the culture medium, the supernatant from the expression system may be concentrated using a commercially available protein concentration filter, such as an Amicon ultrafiltration device. Thereafter, purification of the antibody can be performed using, for example, chromatography, dialysis, affinity purification, and the like. Protein a is suitable as an affinity ligand for purifying 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 preparation comprising the antibody may be prepared according to methods known in the art.

For example, the preparation can be carried out by the following steps: (1) Centrifuging and clarifying the fermentation liquor after fermentation is finished to remove impurities such as cells and the like so as to obtain a supernatant; (2) Capturing antibodies using affinity chromatography (e.g., protein a columns with specific affinity for IgG1, igG2, and IgG4 type antibodies); (3) performing viral inactivation; (4) Refining and purifying (CEX cation exchange chromatography can be generally adopted) to remove impurities in the protein; (5) Virus filtration (to reduce virus titer by, for example, 4log10 or more); (6) Ultrafiltration/diafiltration (which may be used to replace the protein in a formulation buffer that facilitates its stabilization and concentrate 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.

III analysis method of preparation

Biological stability studies generally include real-time stability studies under actual storage conditions (long-term stability studies), accelerated stability studies, and forced condition test studies. Stability research is to search and optimize research conditions according to research purposes and characteristics of products; stability research schemes such as long-term, acceleration and/or forced condition tests are formulated aiming at various influencing factors (such as temperature, repeated freezing and thawing, vibration and the like). Accelerated and forced condition testing is useful for understanding the stability of the product in short term deviations from storage conditions and in extreme cases, and for providing supportive data for the determination of expiration date and storage conditions.

During storage, shaking or repeated freeze-thawing of an antibody preparation, the antibodies may undergo aggregation, degradation or chemical modification, resulting in 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 antibody preparation stability is necessary.

Various methods are known in the art for detecting the stability of antibody preparations. 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; the charge variants in the antibody preparation may be analyzed by capillary isoelectric focusing electrophoresis (cIEF), imaging 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. The turbidity change of the formulation can also be detected using the OD _350nm method, which gives information about the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) can be used to detect changes in protein content in the formulation.

The non-reducing CE-SDS method is a method for measuring the purity of an antibody by using a capillary as a separation channel. In CE-SDS, protein migration is driven by 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 molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of intact antibodies for variability. In general, in the non-reducing CE-SDS method, a sample to be tested is mixed with SDS sample buffer and iodoacetamide. Thereafter, the mixture may be incubated at 68-72℃for about 10-15 minutes, and the supernatant centrifuged after cooling to room temperature for analysis. And detecting the migration of the protein by adopting an ultraviolet detector, and obtaining an electrophoresis spectrogram. Antibody preparation purity can be calculated as the percentage of peak area of the main IgG 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 development of biopharmaceutical antibody-based products, Electrophoresis,2008,29,3612-3620.

Size exclusion high performance liquid chromatography, SEC-HPLC, is another important method for antibody standards and quality control. The method is mainly used for separating molecules according to the size of the molecules or the difference of hydrodynamic radius. 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 over the sum of all peak areas. The percentage of antibody monomer in the formulation product can be measured by SEC-HPLC method, giving information on the content of soluble aggregates and shears. For further description of SEC-HPLC methods, see, for example, ,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). additionally, also see, for example, R.Yang et al ,High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC),Journal of Pharmaceutical and Biomedical Analysis(2015),http://dx.doi.org/10.1016/j.jpba.2015.02.032; and Alexandre Goyon et al ,Protocols for the analytical characterization of therapeutic monoclonal antibodies.I–Non-denaturing chromatographic techniques,Journal of Chromatography,http://dx.doi.org/10.1016/j.jchromb.2017.05.010.

The charge variant of an antibody 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 screening of stable pharmaceutical formulation forms. For example, the formulation samples may be subjected to accelerated stability studies at elevated temperatures, e.g., about 40 ℃ ± 2 ℃, 25 ℃ ± 2 ℃. In addition, shaking experiments or repeated freeze thawing experiments can be performed to test the stability properties of the product. For example, the shaking experiment was performed by shaking at 650r/min for 1-5 days at room temperature in the absence of light. For example, a freeze-stored product at a temperature below-30 ℃ for one day may be thawed at room temperature as a freeze-thawing cycle, and repeated freeze-thawing experiments may be performed, wherein 1 to 6 repeated freeze-thawing cycles may be performed. The detection indicators of the product stability can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reducing CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method). In addition, the efficacy or biological activity of the antibodies can be detected. For example, the ability of an antibody in a formulation to bind to its antigen molecule can be detected. Various methods are known to those skilled in the art for quantifying specific binding of antibodies to antigens, such as immunoassay tests, ELISA, and the like.

IV use of the formulation

The antibody formulations of the invention comprising an anti-PD-L1/OX 40 bispecific antibody protein of the invention have the ability to prevent or treat autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunctional diseases, e.g., where the tumor is a cancer, e.g., a cancer of 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 or colorectal 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 the treatment or prevention of one or more of the above diseases and disorders. Preferably, the mammal is a human.

The antibody formulations of the invention may 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 primary active ingredient, i.e., an amount sufficient to treat, ameliorate or prevent the disease or disorder of interest.

Therapeutic effects may include reducing physiological symptoms. The optimal effective amount and concentration of antibody for any particular subject will depend on 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, the rate of clearance by the body, and also any possible other treatment administered in combination with the antibody formulation. For a particular case, the effective amount delivered can be determined within the discretion 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.

Abbreviation description

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

Examples

Anti-PD-L1/OX 40 bispecific antibodies, an antibody developed independently of Xindabio-pharmaceuticals (Suzhou) Inc., are disclosed in PCT application No. PCT/CN 2020/073959.

In order to develop a simple and easy-to-use injection preparation prescription suitable for long-term stable storage of the fully human antibody, the influence of different pH values, different stabilizers and the content of a surfactant on the protein quality of the antibody is examined through a forced stability experiment at 40 ℃, and finally, the 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 that: N/A indicates inapplicability.

1.2. The instrument and equipment used in the research of the preparation of the invention

1.3. Detection item and detection method for preparation stability

The detection project in the whole research process mainly comprises the following steps: (1) detecting the appearance and the presence or absence of a visible foreign object; (2) Measuring the protein content in the preparation by Ultraviolet (UV) method; (3) Detecting turbidity of the preparation by an OD ₃₅₀ nm method; (4) The purity of the antibody preparation was determined by size exclusion high performance liquid chromatography (SEC-HPLC) and expressed as the percentage of the area of the main peak to the sum of all peak areas; (5) Purity of the antibody preparation was determined by non-reducing sodium dodecyl sulfate capillary electrophoresis (non-reducing CE-SDS) and expressed as the percentage of the area of the main peak to the sum of all peak areas; (6) Determining the charge variant in the antibody preparation by the iCIEF method, expressed as percentages 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 to the anti-PD-L1/OX 40 bispecific antibody antigen in the antibody preparation by a direct ELISA method; (8) The polysorbate 80 content of the antibody preparation was determined by HPLC-FLD.

Visible foreign matter detection

The samples were inspected for visible foreign matter by using a clarity detector (model YB-2, manufactured by Tianjin on the day) and an insoluble particle detector (model GWJ-8, manufactured by Tianjin on the day) according to the method described in the national pharmacopoeia Committee, the pharmacopoeia of the people's republic of China (2015 edition, three general rules 0904 "visible foreign matter inspection method", beijing: chinese medical science and technology Press.2015).

Protein content determination

The protein content in the sample was measured using an ultraviolet spectrophotometer (model UV-1800, manufactured by shimadzu) or a multichannel micro-spectrophotometer (model Nanodrop 8000, manufactured by Thermo U.S.A.).

Turbidity measurement

The absorbance of the sample at 350nm was measured using an ultraviolet spectrophotometer (model UV-1800, manufactured by shimadzu corporation) to determine the turbidity of the sample.

Purity (SEC-HPLC method)

The mixture was separated by using a size exclusion chromatography column, wherein the mobile phase was phosphate buffer (3.12 g of sodium dihydrogen phosphate dihydrate, 8.77 g g of sodium chloride and 34.84g of arginine were weighed, the pH was adjusted to 6.8 with hydrochloric acid after dissolution in ultrapure water and the volume was adjusted to 1000 ml), the column protection liquid was 0.05% (w/v) NaN ₃, the sample injection amount was 50. Mu.l, the flow rate was 0.5 ml/min, the acquisition time was 30 minutes, the column temperature was 25℃and the detection wavelength was 280nm. The sample to be measured was diluted to 2mg/ml with ultrapure water as a sample solution. The preparation buffer was diluted in the same manner as above to prepare a blank solution. 50 μl of each of the blank solution and the sample solution was injected into the liquid chromatograph, and detection was started.

Purity (non-reduction CE-SDS method)

And detecting by adopting a capillary gel electrophoresis method. The capillary is an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2cm and an effective length of 20.2cm. The capillary column was rinsed with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi of electrophoresis gel, respectively, prior to electrophoresis. Diluting a sample to be tested to 2.0mg/mL by using a proper amount of ultrapure water, taking 50 μl of the diluted sample into a 1.5 mL centrifuge tube, respectively adding 45 μl of a sample buffer solution with the pH of 6.5 (weighing 0.32g of citric acid monohydrate and 2.45g of disodium hydrogen phosphate dodecahydrate, dissolving in 45mL of ultrapure water, fixing the volume to 50mL, preparing a citric acid-phosphate buffer solution, precisely weighing 200 μl of the buffer solution, adding 80 μl of 10% (w/v) sodium dodecyl sulfate solution, adding water to 1mL, mixing uniformly, obtaining 1 μl of an internal standard (10 kDa protein, 5 mg/mL) (Beckman Coulter, product number: 390953) and 5 μl of a 250mmol/L NEM solution (weighing 62mg of N-ethylcis-butyl-maleimide, dissolving in 2mL of ultrapure water), heating for 10+ -2 minutes at 70+ -2 ℃ after fully mixing uniformly, cooling to room temperature, and transferring to a sample bottle to serve as a sample solution. The same volume of preparation buffer as the sample was used, and the same procedure was followed as described above to prepare an empty solution. Sample introduction conditions: -5kv for 20 seconds; separation voltage: 15kV for 35 minutes. The capillary column temperature is controlled at 25 ℃, and the detection wavelength is 220nm.

Charge variants (iCIEF method)

Isoelectric point and charge variant (iCIEF method) were used for detection. A premix was prepared with a ratio of 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 PI MARKER 6.14, 2.2. Mu. L PI MARKER 9.46, and detected at a 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)

Detection was performed by fluorescence detection chromatography (HPLC-FLD). Isocratic elution with 0.15mol/L sodium chloride, 0.05mol/L tris, pH 8.0,5% acetonitrile, 5.0. Mu. Mol/L N-phenyl-1-naphthylamine, 15ppm 23 polyoxyethylene fatty alcohol ether, flow rate: 1.5ml/min; acquisition time: 3 minutes; upper pressure limit: 100bar; sample injection amount: 10 μl; column temperature: 30 ℃; detection wavelength: excitation light is 350nm, and emission light is 420nm. The polysorbate 80 content was calculated according to the standard curve method.

Relative binding Activity (direct ELISA method)

Human OX40 antigen (from Sino) and human PD-L1 antigen (from Promega) were each coated with 96-well ELISA plates at 0.5. Mu.g/ml, 100. Mu.l/well, and 4℃overnight. After washing the plates, the plates were blocked with blocking solution (2% BSA-PBST, 300. Mu.l/well) at 37℃for 2h. The test sample was added to the blocked plate at 100. Mu.l/well, and a negative control was set by adding only 100. Mu.l of the dilution (2% BSA-PBST) per well, and incubated in a 37℃incubator for 60min. After washing the plate, an HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, cat. No. A80-104P) diluted with 2% BSA-PBST was added as a secondary antibody (100000-fold dilution, 100. Mu.l/well) and reacted at 37℃for 30min. After washing the plate, 100. Mu.l of TMB developing solution was added to each well, and after development for 10 minutes, 100. Mu.l of 1mol/L H ₂SO₄ was added to each well to terminate the reaction. OD at 450nm was measured with 620nm as reference wavelength. And (3) taking the concentration value of each concentration gradient sample as an abscissa, the OD450 nm-OD620 nm value of each gradient sample as an ordinate, and calculating the binding activity of the EC ₅₀ reflecting the antibody and each antigen by using Prism four-parameter fitting. Relative binding activity (%) = (EC ₅₀ of test sample/EC ₅₀ of reference sample) 100% wherein the reference sample 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 antibodies

Anti-PD-L1/OX 40 bispecific antibodies were obtained as described in PCT application No. PCT/CN 2020/073959. The antibody has the sequence shown in SEQ ID NO:1 and SEQ ID NO:7, a bispecific antibody. PCT application No. PCT/CN2020/073959 is incorporated herein by reference in its entirety.

Briefly, antibodies are expressed recombinantly in CHO cells and purified by filtration, chromatography, virus inactivation, filtration, and the like.

Example 2 pH screening assay

2.1 Experimental procedure

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

Preparing 10mM histidine, 5% (w/v) sorbitol buffer solution, respectively adjusting pH to 5.0, 5.5, 6.0, 6.5 and 7.0 by hydrochloric acid, ultrafiltering and substituting the anti-PD-L1/OX 40 bispecific antibody into the buffer solutions with different pH values, and adjusting the protein content of the sample to about 30mg/ml; and polysorbate 80 was added to a final concentration of about 0.2mg/ml; filtering, packaging into 2R penicillin bottles, adding plugs, and capping. The stability of the samples is examined under the condition of 40+/-2 ℃, and specific schemes are shown in the table.

TABLE 1 Pre-prescription study protocol

Note that: and (1) v represents this point sampling. (2) And after sampling at the time point, the samples are firstly placed into an ultralow temperature refrigerator for freezing and checking, and the frozen samples are subjected to frozen and checking as required.

2.2 Criterion for judging

Based on the knowledge of the product and the precision of the instrument and the method, a judging 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 specific is shown in Table 2.

TABLE 2 criterion for unchanged quality

Detecting items	Unchanged judgment standard
		Appearance of	Clear to micro-opalescent, colorless to pale yellow liquid, and no foreign matters
Visible foreign matter	Conforms to the general rule 0904 of the 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 (OD 350nm method)	The change value is less than or equal to 0.02
		Purity (SEC-HPLC method)	The change value of the main peak is less than or equal to 1 percent
Purity (nrCE-SDS method)	The change 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 each component of acid and alkali 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 Experimental results

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

TABLE 3 Pre-prescription study results

EXAMPLE 3 prescription screening experiments

3.1 Experimental procedure

The present example mainly examined the effect of different buffer systems (histidine buffer system and citric acid buffer system) and excipients (sorbitol, sucrose, arginine hydrochloride and disodium edentate) on the stability of the anti-PD-L1/OX 40 bispecific antibody protein, 6 prescriptions were designed altogether, and detailed prescription information is given in table 4.

Each prescription buffer was formulated as in Table 4, and the anti-PD-L1/OX 40 bispecific antibody protein was ultrafiltration replaced into the corresponding prescription buffer. After the replacement, the protein content of each prescription is regulated to about 40mg/ml, and polysorbate 80 is added to make the final concentration of the protein about 0.2mg/ml; filtering, packaging into penicillin bottles, adding plugs, and capping. The above samples were subjected to stability studies at 40 ℃ + -2℃and the detailed experimental conditions and sampling schedules are shown in Table 5.

TABLE 4 prescription information form

Note that: the pH of each of the formulations 1 to 6 was adjusted with hydrochloric acid.

TABLE 5 stability investigation protocol

Note that: (1) means that only 0 day and 1 month samples were tested. And (2) v represents the point sampling. (3) And after sampling at the time point, the samples are firstly placed into an ultralow temperature refrigerator for freezing and checking, and the samples are thawed and checked as required.

3.2 Criterion for judging

The criteria are specifically shown in table 2.

3.3 Experimental results

The prescription determination experiment results are detailed in

And (3) a table. The result shows that the prescription is placed for 1 month at 40+/-2 ℃ and the appearance of the sample at the other parts is qualified except that the prescription 6 is not detected due to unqualified appearance; the protein content, polysorbate 80 and relative binding activity were not significantly changed. Except prescription 2, the turbidity of the other prescriptions exceeds the judging standard. The purity (SEC-HPLC method) of each of formulas 3,4 and 5 was reduced, mainly as the protein polymers and fragments increased, with the remaining formulas unchanged significantly. The purity (nrCE-SDS method) of recipe 2, recipe 3 and recipe 4 all decrease, which is mainly represented by increased protein polymers and fragments, and the remaining recipes are not changed significantly. The charge variant-acid component of each prescription is obviously increased, and the main component is obviously reduced, but the change range of the charge variant-acid component and the main component of the prescription 2 is relatively smaller.

The results of the prescription determination experiments show that the prescription 2 is superior to other prescriptions in terms of turbidity, purity (SEC-HPLC method) and charge variant (iCIEF method). The final selected prescription 2 was developed for subsequent experiments.

TABLE 5 prescription determination test results

EXAMPLE 4 Polysorbate 80 concentration Studies

4.1 Experimental procedure

This example mainly examined the effect of different polysorbate 80 concentrations on the stability of anti-PD-L1/OX 40 bispecific antibody protein, and a total of 3 prescriptions were designed, with detailed prescription information as shown in the table.

Prescription buffers were prepared as per the table and anti-PD-L1/OX 40 bispecific antibody proteins were ultrafiltration replaced into the prescription buffers. After the replacement is completed, the protein content of each prescription is regulated to about 40mg/ml, and polysorbate 80 with different final concentrations is added; filtering, packaging into penicillin bottles, adding plugs, and capping. The stability of the samples is examined at 40+/-2 ℃, and detailed experimental conditions and sampling plans are shown in the table.

TABLE 6 prescription information form

Sequence number	Prescription information
		Prescription 2	10MM histidine, 50mg/ml sorbitol, 0.2mg/ml polysorbate 80,0.01mg/ml disodium edentate pH 5.5
Prescription 7	10MM histidine, 50mg/ml sorbitol, 0.5mg/ml polysorbate 80,0.01mg/ml disodium edentate pH 5.5
		Prescription 8	10MM histidine, 50mg/ml sorbitol, 0.7mg/ml polysorbate 80,0.01mg/ml disodium edentate pH 5.5

TABLE 7 stability investigation protocol

Note that: only the last spot is detected, the intermediate sample is frozen after sampling, and the detection is not carried out.

4.2 Criterion for judging

The judgment criteria are specifically shown in the table.

4.3 Experimental results

The results of polysorbate 80 concentration studies are shown in the table. The results show that the appearance and the visible foreign matters of all prescriptions under all conditions are qualified; the 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 prescriptions.

TABLE 8 Polysorbate 80 concentration study results

Combining the experimental results and the experience of the formulation prescription development platform, the final prescription 7 is selected as the anti-PD-L1/OX 40 bispecific antibody formulation prescription. The anti-PD-L1/OX 40 bispecific antibody preparation formulation was formulated as follows: 40.0mg/ml of recombinant anti-tumor necrosis factor receptor superfamily member 4 (OX 40) 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 investigation of stability of formulations

5.1 Stability investigation samples and protocols

Referring to the guidelines of NMPA (original CFDA), biological product stability study technical guidelines (trial), ICH Q5C, biological technology/biological product stability test, and the like, accelerated stability study and forced condition test study were respectively conducted on three batches of processes and quality representative finished products of the recombinant anti-tumor necrosis factor receptor superfamily member 4 (OX 40) and the anti-programmed death ligand 1 (PD-L1) bispecific antibody.

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

TABLE 9 sample information

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

TABLE 10 formulation stability investigation protocol

Study item	Investigation of conditions	Time point of investigation
			Accelerated stability study	25℃±2℃/60％RH±5％RH	Month 0, 1,2
Forced condition test	40℃±2℃/75％RH±5％RH	Week 0, 2, 4

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

5.2 Stability investigation 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, the trend of each test result is consistent, which indicates that the established prescription can meet the storage stability of the anti-PD-L1/OX 40 bispecific antibody.

TABLE 12 results of acceleration stability studies (25 ℃ C.+ -. 2 ℃ C.)

Note that: N/A indicates that this test item is absent at this time point.

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

Note that: N/A indicates that the time point has no such test item

While exemplary embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these disclosures are exemplary only, and that various other substitutions, adaptations, and modifications may be made within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments set forth herein.

Sequence listing

<110> Xinda biopharmaceutical (Suzhou) Co., ltd

<120> Anti-PD-L1/OX 40 bispecific antibody formulations, methods of making and uses 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

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 Gly

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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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

115 120

<210> 3

<211> 101

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<223> CH1

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys

100

<210> 4

<211> 223

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

130 135 140

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

145 150 155 160

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

195 200 205

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

210 215 220

<210> 5

<211> 10

<212> PRT

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<220>

<223> Joint

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 6

<211> 132

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 6

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

1 5 10 15

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

20 25 30

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Ser Ser

130

<210> 7

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide chain #2

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

100 105

<210> 9

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> CL

<400> 9

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

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 10

<211> 10

<212> PRT

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<220>

<223> VHH CDR1

<400> 10

Ala Tyr Thr Ile Ser Arg Asn Ser Met Gly

1 5 10

<210> 11

<211> 16

<212> PRT

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<220>

<223> VHH CDR2

<400> 11

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

1 5 10 15

<210> 12

<211> 24

<212> PRT

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<220>

<223> VHH CDR3

<400> 12

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

1 5 10 15

Met Thr Leu Pro Ala Leu Asn Tyr

20

<210> 13

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 13

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5 10

<210> 14

<211> 17

<212> PRT

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<220>

<223> HCDR2

<400> 14

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

1 5 10 15

Gly

<210> 15

<211> 14

<212> PRT

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<220>

<223> HCDR3

<400> 15

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

1 5 10

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 16

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 17

<211> 7

<212> PRT

<213> Artificial Sequence

<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 preparation comprising

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

(ii) A buffer selected from a histidine-hcl histidine buffer system;

(iii) A stabilizer selected from sorbitol, sucrose, arginine hydrochloride;

(iv) A surfactant selected from polysorbate-80; and

(V) A chelating agent selected from disodium edentate;

Wherein the anti-PD-L1/OX 40 bispecific antibody protein consists of two polypeptide chains #1 and two polypeptide chains # 2:

Polypeptide chain #1:

VH-CH1-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 CH4;

x represents a linker comprising or consisting of the amino acid sequence of SEQ ID NO. 5;

VHH represents a single domain antigen binding site;

VL represents a light chain variable region;

CL represents the light chain constant region;

a hinge region exists between CH1 and Fc;

VHH comprises Complementarity Determining Regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3; wherein the VHH CDR1 consists of the amino acid sequence of SEQ ID NO. 10; the VHH CDR2 consists of the amino acid sequence of SEQ ID NO. 11; the VHH CDR3 consists of the amino acid sequence of SEQ ID NO. 12;

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

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

The liquid antibody formulation has a pH of 5.0-6.0.

2. The liquid antibody formulation of claim 1, wherein the pH of the liquid antibody formulation is 5.5.

3. The liquid antibody formulation of claim 1, wherein the concentration of anti-PD-L1/OX 40 bispecific antibody protein in the liquid antibody formulation is 1-100 mg/ml.

4. The liquid antibody formulation of claim 3, wherein the concentration of anti-PD-L1/OX 40 bispecific antibody protein in the liquid antibody formulation is 10-70 mg/mL.

5. The liquid antibody formulation of claim 1, wherein the buffer in the liquid antibody formulation is selected from the group consisting of histidine, histidine hydrochloride, and combinations thereof.

6. The liquid antibody formulation of claim 5, wherein the buffer is present at a concentration of 5-50 mM.

7. The liquid antibody formulation of claim 6, wherein the buffer is present at a concentration of 5-30 mM.

8. The liquid antibody formulation of claim 1, wherein the stabilizer is sorbitol.

9. The liquid antibody formulation of claim 8, wherein the concentration of the stabilizing agent is 20-80 mg/ml.

10. The liquid antibody formulation of claim 1, wherein the concentration of the surfactant is 0.1-1 mg/ml.

11. The liquid antibody formulation of claim 10, wherein the concentration of the surfactant is 0.2-0.8 mg/ml.

12. The liquid antibody formulation of claim 1, wherein the concentration of the chelating agent is 0.005-0.05 mg/ml.

13. The liquid antibody formulation of claim 1, wherein the anti-PD-L1/OX 40 bispecific antibody protein comprises polypeptide chain #1 and polypeptide chain #2, wherein VH-CH1-Fc-X-VHH of polypeptide chain #1 comprises the sequence of SEQ ID No. 1, and VL-CL of polypeptide chain #2 comprises the sequence of SEQ ID No. 7.

14. The liquid antibody formulation of claim 1, wherein the anti-PD-L1/OX 40 bispecific antibody protein is recombinantly expressed in 293 cells or CHO cells.

15. The liquid antibody formulation of claim 1, wherein the liquid formulation is an injection.

16. The liquid antibody formulation of claim 15, wherein the injection is for subcutaneous or intravenous injection, or is an infusion.

17. The liquid antibody formulation of claim 1, comprising:

(i) 1-150 mg/ml of an anti-PD-L1/OX 40 bispecific antibody protein;

(ii) 5-50 mM histidine buffer;

(iii) Sorbitol and sucrose in 20-80 mg/ml;

(iv) 0.1-1 mg/ml polysorbate 80; and

(V) 0.005-0.05mg/ml edetate disodium;

wherein the pH of the liquid formulation is from 5.0 to 6.0.

18. The liquid antibody formulation of claim 17, comprising:

(i) 10-100 mg/ml of an anti-PD-L1/OX 40 bispecific antibody protein;

(ii) 5-50 mM histidine buffer;

(iii) Sorbitol and sucrose in 20-80 mg/ml;

(iv) 0.1-1 mg/ml polysorbate 80; and

(V) 0.005-0.05mg/ml edetate disodium;

wherein the pH of the liquid formulation is from 5.0 to 6.0.

19. The liquid antibody formulation of claim 18, comprising:

(i) 20-60 mg/ml of an anti-PD-L1/OX 40 bispecific antibody protein;

(ii) 5-50 mM histidine buffer;

(iii) Sorbitol 20-80 mg/ml;

(iv) 0.1-1 mg/ml polysorbate 80; and

(V) 0.005-0.05mg/ml edetate disodium;

wherein the pH of the liquid formulation is from 5.0 to 6.0.

20. The liquid antibody formulation of claim 19, comprising:

(i) 20-60 mg/ml of an anti-PD-L1/OX 40 bispecific antibody protein;

(ii) 10 mM histidine buffer;

(iii) 20-80 mg/ml sorbitol;

(iv) 0.2-0.8 mg/ml polysorbate 80; and

(V) 0.008-0. mg/ml disodium edetate;

wherein the pH of the liquid formulation is from 5.0 to 6.0.

21. The liquid antibody formulation of claim 20, comprising:

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

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

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

22. The liquid antibody formulation of any one of claims 1 to 21, which is stable after storage at 25 ℃ for at least 2 months or 40 ℃ ± 2 ℃ for 1 month.

23. The liquid antibody formulation of claim 22, wherein the liquid antibody formulation has 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 the formulation has a purity of greater than 96%;

(ii) A major peak change value of less than 2% as measured by non-reducing CE-SDS method, and/or the formulation has a purity of greater than 96%;

(iii) The sum of the values of the changes of the components of the anti-PD-L1/OX 40 bispecific antibody protein in the preparation is not more than 40% and/or the value of the changes of the main component is not more than 20%, the sum of the values of the changes of the components is not more than 40% or the value of the changes of the main component is not more than 20% after 1 month of storage at 40 ℃ + -2 ℃, or the sum of the values of the changes of the components is not more than 20% or the value of the changes of the main component is not more than 15% after 2 months of storage at 25 ℃ relative to the initial value of the day 0 of storage measured by the iCIEF method; the components comprise a main component, an acidic component and an alkaline component;

(iv) Relative binding activity of anti-PD-L1/OX 40 bispecific antibody protein in the formulation was 70% -130% relative to initial value on day 0 of storage as measured by ELISA;

The formulation is stable under shaking and/or repeated freeze thawing.

24. A solid antibody preparation obtained by solidifying the liquid antibody preparation according to any one of claims 1 to 21, which is in the form of a lyophilized powder for injection.

25. A delivery device comprising the liquid antibody formulation of any one of claims 1-21 or the solid antibody formulation of claim 24.

26. A pre-filled syringe comprising the liquid antibody formulation of any one of claims 1-21 or the solid antibody formulation of claim 24 for intravenous or intramuscular injection.

27. Use of the liquid antibody formulation according to any one of claims 1-21 or the solid antibody formulation according to claim 24 for the preparation of a delivery device or pre-filled syringe or medicament for the treatment or prophylaxis of a tumor in a subject, the tumor being colon cancer.