Anti-PD-L1 / anti-4-1BB bispecific antibody preparation

Abstract

The present invention provides an anti-PD-L1 / anti-4-1BB bispecific antibody formulation, as well as methods for its production and use. In one aspect, a stable aqueous pharmaceutical formulation comprising an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is provided.

Description

Related Applications

[0001] This application claims priority based on a PCT application filed with the China National Intellectual Property Administration on July 6, 2022, with application number PCT / CN2022 / 104038 and invention title "Anti-PD-L1 / Anti-4-1BB Bispecific Antibody Preparation", the entire content of which is incorporated herein by reference.

Technical Field

[0002] The present invention relates to an anti-PD-L1 / anti-4-1BB bispecific antibody preparation, as well as methods for its production and use.

Background Art

[0003] Antibodies are complex molecules like other protein therapeutics. Usually, large amounts of antibodies have to be used in pharmaceutical formulations to achieve a therapeutically effective amount in mammals. Antibodies are larger and more complex than conventional organic and inorganic therapeutics, which has posed special problems for antibody formulation. To maintain the biological activity of antibodies, they must be protected from degradation while preserving the integrity of the conformation of the amino acid sequence in the formulation. Also, monoclonal and polyclonal antibodies may be particularly relatively unstable. Although there are many available formulations, no method or system applicable to all antibodies has yet been found. Several factors to be considered during formulation development have been reported (Wang et al., "Antibody Structure, Instability, and Formulation", Journal of Pharmacy 96.1 (2007): p. 1-26).

[0004] There are many possible factors that can affect the stability of antibodies. In fact, when the antibody structure is heterogeneous, it becomes more complicated to prepare stable formulations containing these antibodies. Furthermore, excipients included in antibody formulations are preferably those that minimize any potential immune response. Such stable antibody formulations are still in demand.

Summary of the Invention

[0005] The present invention relates to anti-PD-L1 / anti-4-1BB bispecific antibody (especially BH3120) formulations, and methods for their production and use. BH3120 can bind to PD-L1 and 4-1BB simultaneously for cancer treatment. In this application, a BH3120 formulation with good thermal stability is provided. According to the formulations described in this application, by retaining the anti-PD-L1 / anti-4-BB bispecific antibody in a liquid solution, it is possible to make it less likely for antibody degradation, aggregation, or undesirable chemical modifications to occur.

[0006] In a uniform phase, the present invention relates to a stable aqueous pharmaceutical formulation comprising an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof and a buffer, and in some embodiments, the pH of the formulation is from about 5.0 to about 6.5. In some embodiments, the pH of the formulation is from about 5.5 to about 6.0. In some embodiments, the pH of the formulation is about 5.6 or about 5.8.

[0007] In some embodiments, the pH of the formulation is from about 5.6 to about 5.8.

[0008] In some embodiments, the pH of the formulation is from about 5.8 to about 6.0.

[0009] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is capable of binding to human PD-L1 and / or human 4-1BB. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is from about 10 mg / ml to about 100 mg / ml. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is from about 20 mg / ml to about 80 mg / ml. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is about 50 mg / ml. In some embodiments, the buffer solution contains a buffer selected from the group consisting of acetic acid, sodium acetate, tartrate, hydrogen chloride, sodium dihydrogen phosphate, and combinations thereof. In some embodiments, the buffer solution contains acetic acid and / or sodium acetate. In some embodiments, the formulation contains an acetic acid / sodium acetate buffer solution having a concentration of about 10 mM to about 100 mM. In some embodiments, the formulation contains an acetic acid / sodium acetate buffer solution having a concentration of about 10 mM or about 15 mM. In some embodiments, the formulation further contains an isotonic agent selected from the group consisting of trehalose, sucrose, proline, glycine, arginine, alanine, glutamic acid, methionine, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, and combinations thereof. In some embodiments, the isotonic agent is trehalose, arginine, or a combination thereof. In some embodiments, the formulation contains arginine at about 10 mM to about 200 mM. In some embodiments, the formulation contains arginine at about 100 mM or about 140 mM. In some embodiments, the arginine is in the hydrochloride form of arginine (arginine-HCl). In some embodiments, the formulation contains trehalose at about 10 mM to about 300 mM. In some embodiments, the formulation contains trehalose at about 130 mM.In some embodiments, the formulation further comprises a surfactant selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 (Pluronic (R) F68), and combinations thereof. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the formulation comprises from about 0.01 mg / ml to about 10 mg / ml of polysorbate 80. In some embodiments, the formulation comprises about 0.2 mg / ml of polysorbate 80.

[0010] In a uniform phase, the present invention relates to a stable aqueous pharmaceutical formulation comprising an anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, an isotonic agent at a concentration of about 100 mM to about 300 mM, a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml, and a buffer system. In some embodiments, the pH of the formulation is from about 5.0 to about 6.5. In some embodiments, the pH of the formulation is from about 5.5 to about 6.0. In some embodiments, the buffer system comprises one or more buffers selected from the group consisting of acetic acid, sodium acetate, tartrate, hydrogen chloride, and sodium dihydrogen phosphate. In some embodiments, the buffer system comprises an acetic acid / sodium acetate buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, the isotonic agent is trehalose, sucrose, proline, glycine, arginine, alanine, glutamic acid, methionine, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, or combinations thereof. In some embodiments, the isotonic agent is trehalose, arginine (e.g., arginine-HCl), or combinations thereof. In some embodiments, the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 (Pluronic (R)F68) and combinations thereof. In some embodiments, the surfactant is polysorbate 80.

[0011] In the homogeneous phase, the present invention relates to a stable aqueous pharmaceutical formulation comprising, or consisting of, an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 200 mM, optionally trehalose at a concentration of about 10 mM to about 300 mM, polysorbate 80 at a concentration of about 0.01 mg / ml to about 10 mg / ml, and an acetic acid / sodium acetate buffer at a concentration of about 10 mM to about 100 mM, wherein in some embodiments, the pH of the formulation is about 5.5 to about 6.0. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 20 mg / ml, about 50 mg / ml, or about 80 mg / ml. In some embodiments, the concentration of the alanine is about 100 mM or about 140 mM. In some embodiments, the concentration of the trehalose is about 130 mM. In some embodiments, the concentration of the polysorbate 80 is about 0.2 mg / ml. In some embodiments, the concentration of the acetic acid / sodium acetate buffer is about 10 mM or about 15 mM.

[0012] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1). Here, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDR) 1, 2, and 3. In some embodiments, the VH1 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 15, the VH1 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 16, and the VH1 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 17. Also here, the first light chain variable region (VL1) comprises CDR1, 2, and 3. In some embodiments, the VL1 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 18, the VL1 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 19, and the VL1 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 20. Furthermore, here, in some embodiments, VH1 and VL1 can interact to form an antigen-binding site that binds to PD-L1.

[0013] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2). Here, the second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2, and 3. In some embodiments, the VH2 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 21, the VH2 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 22, and the VH2 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 23. Also here, the second light chain variable region (VL2) comprises CDRs 1, 2, and 3. In some embodiments, the VL2 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 24, the VL2 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 25, and the VL2 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 26. Still further here, in some embodiments, the VH2 and VL2 can interact to form an antigen-binding site that binds to 4-1BB.

[0014] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and a second heavy chain variable region (VH2) and a second light chain variable region (VL2). Here, the first heavy chain variable region (VH1) comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 6, the first light chain variable region (VL1) comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 2, and VH1 and VL1 in some embodiments can interact to form an antigen-binding site that binds to PD-L1. Also here, the second heavy chain variable region (VH2) comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 12, the second light chain variable region (VL2) comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 10, and VH2 and VL2 in some embodiments can interact to form an antigen-binding site that binds to 4-1BB.

[0015] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof comprises a first heavy chain constant region (CH1) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 8, and a first light chain constant region (CL1) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4, and a second heavy chain constant region (CH2) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 14, and a second light chain constant region (CL2) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4. However, CH1 and CL1 in some embodiments can interact to form an antigen-binding site that binds to PD-L1, and CH2 and CL2 in some embodiments can interact to form an antigen-binding site that binds to 4-1BB.

[0016] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain and a first light chain, and a second heavy chain and a second light chain. Here, the first heavy chain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 27, the first light chain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 28, and the first heavy chain and the first light chain in some embodiments can interact to bind to PD-L1. Also here, the second heavy chain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 29, the second light chain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 30, and the second heavy chain and the second light chain in some embodiments can interact to bind to 4-1BB.

[0017] In some embodiments, the formulation has long-term stability.

[0018] In one aspect, the present invention relates to a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of the formulation described herein. In some embodiments, the subject has leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, or bone cancer. In some embodiments, the subject is human.

[0019] In one aspect, the present invention relates to an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, comprising: (a) an anti-PD-L1 arm comprising a first heavy-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 6 and a first light-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2; and an anti-4-1BB arm comprising a second heavy-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12 and a second light-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12; (b) 10 mM or 15 mM acetic acid / sodium acetate; (c) 100 mM or 140 mM arginine (e.g., arginine-HCl); (d) optionally 130 mM trehalose; and (e) 0.2 mg / ml polysorbate 80, or consisting of or substantially consisting of these components, and relates to a formulation of an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof that exhibits long-term stability, wherein the pH of the formulation in some embodiments is 5.5 to 6.0 (e.g., 5.6 or 5.8). In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 10 mg / ml to about 100 mg / ml. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 20 mg / ml to about 80 mg / ml. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 20 mg / ml, about 50 mg / ml, or about 80 mg / ml.

[0020] As used herein, the term "cancer" refers to cells having the ability of autonomous growth. Examples of such cells include cells in an abnormal state or situation characterized by rapid proliferative cell growth. This term includes cancerous growths such as tumors, carcinogenic processes, metastatic tissues, and malignant transformed cells, tissues or organs, regardless of the histopathological type or stage of invasion, and further includes malignant tumors of various organ systems such as the respiratory system, cardiovascular system, renal system, reproductive system, hematological system, nervous system, hepatic system, gastrointestinal system, endocrine system, etc., and is also intended to include adenocarcinomas including malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers and small intestine cancers. "Spontaneous" cancer includes any cancer that is not experimentally induced by transplanting cancer cells into the body of the subject, for example, a spontaneously occurring cancer, a cancer caused by the subject's exposure to a carcinogen, a cancer caused by the insertion of a recombinant cancer gene or the knockout of a tumor suppressor gene, and a cancer caused by infection (e.g., viral infection). The term "cancer" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. This term also includes carcinosarcomas, which include malignant tumors composed of cancerous and sarcomatous tissues. "Adenocarcinoma" refers to a cancer that is derived from glandular tissue or tumor cells therein and forms a distinguishable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from mesenchymal tissue. The term "hematopoietic neoplastic disease" includes diseases associated with proliferative / neoplastic cells derived from hematopoietic organs. Hematopoietic neoplastic diseases can arise from myeloid, lymphoid or erythroid or their precursor cells.

[0021] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, mouse antibodies, and the like. In some embodiments, the antibody can contain the Fc region of a mouse antibody or a human antibody. The term "antibody" also includes derivatives such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0022] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a heavy-chain variable domain or a light-chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab’, F(ab’)2, and Fv fragments.

[0023] Monoclonal antibodies as used herein specifically include "chimeric" antibodies. As used herein, the term "chimeric antibody" refers to an antibody (immunoglobulin) and fragments of such an antibody in which, as long as they exhibit the desired biological activity, a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species. Usually, a chimeric antibody refers to an antibody whose light-chain and heavy-chain genes are constructed from antibody variable-region and constant-region genes belonging to different species, for example, by genetic engineering. For example, the variable fragment of a mouse monoclonal antibody gene can be bound to a human constant fragment. In some embodiments, the antigen-binding site is derived from a mouse and the Fc portion is derived from a human.

[0024] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., the heavy or light chain variable domains of mammalian immunoglobulins) that can specifically bind to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0025] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human, or non-human that is treated according to the methods of the present invention. In the present invention, veterinary and non-veterinary applications are contemplated. In some embodiments, the subject is a mammal (such as a non-human mammal). The subject can include, but is not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, giant pandas, and primates. For example, non-human primates (such as monkeys, chimpanzees, gorillas, etc.), rodents (such as rats, mice, sand mice, hamsters, ferrets, rabbits, etc.), lagomorphs, pigs (such as pigs, mini-pigs, etc.), horses, dogs, cats, cows, other livestock, farm, and zoo animals. In some embodiments, the subject is a human.

[0026] As used herein, when referring to an antibody, the phrases "specifically binds" and "binds specifically to" mean that the antibody preferably interacts with its target molecule (such as PD-L1 or 4-1BB) compared to other molecules, as it depends on the presence of a specific structure (i.e., an antigenic determinant cluster or epitope) on the target molecule. In other words, this reagent does not bind to all molecules in the general sense, but rather discriminates and binds to molecules containing a specific structure. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to the PD-L1 molecule may be referred to as a PD-L1-specific antibody or an anti-PD-L1 antibody.

[0027] As used herein, the term "about" or "approximately" generally means within 20%, within 10%, 5%, 4%, 3%, 2%, or 1% of a given numerical value or range. That a given numerical value is approximate means that, in the absence of a clear statement to the contrary, it can be presumed to be a term such as "about" or "approximately".

[0028] As used herein, the term "sugar" refers to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, but are not limited to, sucrose, glucose, dextrose, trehalose, and the like.

[0029] As used herein, the terms "long-term storage" or "long-term stability" should be understood to mean that the pharmaceutical composition can be stored with a minimum quality retention period of 3 months or more, 6 months or more, preferably 1 year or more, and most preferably at least 2 years.

[0030] As used herein, the term "stable" should be understood to mean that the antibody contained in the pharmaceutical composition loses no more than 30%, preferably 20%, more preferably 15%, still more preferably 10%, and most preferably 5% of its activity relative to the activity of the composition at the start of storage.

[0031] As used herein, the term "substantially free of" means that a substance is either absent or present in only a small or trace amount that has no substantial effect on the properties of the composition. When no mention is made of the amount of a substance, it should be understood to mean "an undetectable amount".

[0032] As used herein, the term "pharmaceutically acceptable carrier" refers to any conventional non-toxic solid, semi-solid, liquid filler, diluent, encapsulating material, formulation aid, or excipient. A pharmaceutically acceptable carrier is non-toxic to the subject at the dosages and concentrations used and is compatible with the other components of the formulation.

[0033] As used herein, the terms "pharmaceutical composition" and "formulation" are used interchangeably.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. This specification describes methods and materials for the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0035] Other features and advantages of the present invention will become more apparent from the following detailed description, the accompanying drawings, and the claims.

Mode for Carrying Out the Invention

[0036] Antibodies, like other proteins, are prone to various physical and chemical degradations. Antibody instability can be observed in liquid, frozen, and lyophilized states. In many cases, multiple degradation pathways can occur simultaneously, and the degradation mechanism can change depending on stress conditions. These degradation pathways are classified into two types: physical instability and chemical instability.

[0037] Antibodies have similar structures, but their behaviors appear to be different. The present invention provides formulations of anti-PD-L1 / anti-4-1BB bispecific antibodies, as well as methods for their production and use. In one aspect, the present invention provides a stable aqueous formulation of an anti-PD-L1 / anti-4-1BB antibody that can be stored for a long time.

[0038] Antibody Formulation The present invention relates to a formulation of an anti-PD-L1 / anti-4-1BB bispecific antibody. The formulation of the present invention comprises an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, and may exist in the form of a liquid and a lyophilized powder. Further, such a formulation can include a buffer, an isotonic agent, a surfactant, a stabilizer, and other excipients.

[0039] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof can be any anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof described herein.

[0040] In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody described herein in the formulation is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg / ml. In some embodiments, the concentration does not exceed 50, 60, 70, 80, 90, 100 or 200 mg / ml. In some embodiments, the antibody or antigen-binding fragment thereof is from about 5 mg / ml to about 200 mg / ml, from about 5 mg / mL to about 180 mg / mL, from about 5 mg / mL to about 160 mg / mL, from about 5 mg / mL to about 140 mg / mL, from about 5 mg / mL to about 120 mg / mL, from about 5 mg / mL to about 100 mg / mL, from about 5 mg / mL to about 80 mg / mL, from about 5 mg / mL to about 60 mg / mL, from about 5 mg / mL to about 40 mg / mL, from about 5 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 200 mg / mL, from about 10 mg / mL to about 180 mg / mL, from about 10 mg / mL to about 160 mg / mL, from about 10 mg / mL to about 140 mg / mL, from about 10 mg / mL to about 120 mg / mL, from about 10 mg / mL to about 100 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 20 mg / mL, from about 20 mg / mL to about 200 mg / mL, from about 20 mg / mL to about 180 mg / mL, from about 20 mg / mL to about 160 mg / mL, from about 20 mg / mL to about 140 mg / mL, from about 20 mg / mL to about 120 mg / mL, from about 20 mg / mL to about 100 mg / mL, from about 20 mg / mL to about 80 mg / mL, from about 20 mg / mL to about 60 mg / mL, from about 20 mg / mL to about 40 mg / mL, from about 20 mg / mL to about 30 mg / mL, from about 30 mg / mL to about 200 mg / mL, from about 30 mg / mL to about 180 mg / mL, from about 30 mg / mL to about 160 mg / mL, from about 30 mg / mL to about 140 mg / mL, from about 30 mg / mL to about 120 mg / mL, from about 30 mg / mL to about 100 mg / mL, from about 30 mg / mL to about 80 mg / mL, from about 30 mg / mL to about 60 mg / mL, from about 30 mg / mL to about 40 mg / mL, from about 40 mg / mL to about 200 mg / mL, from about 40 mg / mL to about 180 mg / mL, from about 40 mg / mL to about 160 mg / mL, from about 40 mg / mL to about 140 mg / mL,About 40 mg / mL to about 120 mg / mL, about 40 mg / mL to about 100 mg / mL, about 40 mg / mL to about 80 mg / mL, about 40 mg / mL to about 60 mg / mL, about 40 mg / mL to about 50 mg / mL, about 50 mg / mL to about 200 mg / mL, about 50 mg / mL to about 180 mg / mL, about 50 mg / mL to about 160 mg / mL, about 50 mg / mL to about 140 mg / mL, about 50 mg / mL to about 120 mg / mL, about 50 mg / mL to about 100 mg / mL, about 50 mg / mL to about 80 mg / mL, about 50 mg / mL to about 60 mg / mL, about 60 mg / mL to about 200 mg / mL, about 60 mg / mL to about 180 mg / mL, about 60 mg / mL to about 160 mg / mL, about 60 mg / mL to about 140 mg / mL, about 60 mg / mL to about 120 mg / mL, about 60 mg / mL to about 100 mg / mL, about 60 mg / mL to about 80 mg / mL, about 60 mg / mL to about 70 mg / mL, about 70 mg / mL to about 200 mg / mL, about 70 mg / mL to about 180 mg / mL, about 70 mg / mL to about 160 mg / mL, about 70 mg / mL to about 140 mg / mL, about 70 mg / mL to about 120 mg / mL, about 70 mg / mL to about 100 mg / mL, about 70 mg / mL to about 80 mg / mL, about 80 mg / mL to about 200 mg / mL, about 80 mg / mL to about 180 mg / mL, about 80 mg / mL to about 160 mg / mL, about 80 mg / mL to about 140 mg / mL, about 80 mg / mL to about 120 mg / mL, about 80 mg / mL to about 100 mg / mL, about 80 mg / mL to about 90 mg / mL, about 90 mg / mL to about 200 mg / mL, about 90 mg / mL to about 180 mg / mL, about 90 mg / mL to about 160 mg / mL, about 90 mg / mL to about 140 mg / mL, about 90 mg / mL to about 120 mg / mL, about 90 mg / mL to about 100 mg / mL, about 100 mg / mL to about 200 mg / mL, about 100 mg / mL to about 180 mg / mL, about 100 mg / mL to about 160 mg / mL, about 100 mg / mL to about 140 mg / mL, about 100 mg / mL to about 120 mg / mL, about 120 mg / mL to about 200 mg / mL, about 120 mg / mL to about 180 mg / mL, about 120 mg / mL to about 160 mg / mL, about 120 mg / mL to about 140 mg / mL, about 140 mg / mL to about 200 mg / mL, about 140 mg / mL to about 180 mg / mL,It is present in the formulation in an amount of about 140 mg / mL to about 160 mg / mL, about 160 mg / mL to about 200 mg / mL, about 160 mg / mL to about 180 mg / mL, or about 180 mg / mL to about 200 mg / mL. In some embodiments, the concentration is about 50 mg / mL to about 500 mg / mL, about 25 mg / mL to about 400 mg / mL, about 50 mg / mL to about 250 mg / mL, about 5 mg / mL to about 280 mg / mL, about 5 mg / mL to about 200 mg / mL, about 5 mg / mL to about 125 mg / mL, about 5 mg / mL to about 75 mg / mL, about 5 mg / mL to about 50 mg / mL, or about 5 mg / mL to about 25 mg / mL. In some embodiments, the concentration is about 1 to about 100 mg / ml, about 5 to about 100 mg / ml, about 10 to about 100 mg / ml, about 20 to about 100 mg / ml, about 30 to about 100 mg / ml, about 40 to about 100 mg / ml, about 50 to about 100 mg / ml, or about 10 to about 50 mg / ml. For example, the antibody or antigen-binding fragment thereof is present in the formulation in an amount of about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, about 200 mg / mL, about 205 mg / mL, about 210 mg / mL, about 215 mg / mL, about 220 mg / mL, about 225 mg / mL, about 230 mg / mL, about 235 mg / mL, about 240 mg / mL, about 245 mg / mL, or about 250 mg / mL.

[0041] In alternative embodiments, the antibody or antigen-binding fragment thereof is present in the formulation in an amount of about 5 to about 10 mg / mL, about 11 to about 20 mg / mL, about 21 to about 30 mg / mL, about 31 to about 40 mg / mL, about 41 to about 50 mg / mL, about 51 to about 75 mg / mL, about 76 to about 100 mg / mL, about 101 to about 125 mg / mL, about 126 to about 150 mg / mL, about 151 to about 175 mg / mL, about 176 to about 200 mg / mL, about 201 mg / mL to about 225 mg / mL, about 226 mg / mL to about 250 mg / mL, about 5 to about 30 mg / mL, about 10 to about 50 mg / mL, about 10 to about 20 mg / mL, or about 10 to about 30 mg / mL. In some embodiments, the concentration is 20, 50 or 80 mg / ml.

[0042] Since the concentration of the antibody in the formulation is relatively high, an anti-PD-L1 / anti-4-1BB bispecific antibody formulation was developed to enhance the stability of these antibodies in the formulation.

[0043] Buffer The formulation can include various buffers such as acetic acid, sodium acetate, tartrate, sodium dihydrogen phosphate, and combinations thereof. The buffer can maintain a physiologically appropriate pH. In addition, the buffer can enhance the isotonicity and chemical stability of the formulation.

[0044] In some embodiments, the buffer is present in the formulation at a concentration of about 10 mM to 100 mM, such as about 10 mM to about 15 mM. For example, the buffer is present in the formulation at a concentration of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, or about 100 mM.

[0045] In certain embodiments, the pH of the formulation is 6.5 or less. In certain embodiments, the pH of the formulation is 6.0 or less. In certain embodiments, the pH of the formulation is 5.0 or greater. In certain embodiments, the pH of the formulation is 5.5 or greater.

[0046] In some embodiments, the pH of the formulation is from about 5.0 to about 6.5. In some embodiments, the pH of the formulation is from about 5.0 to about 6.4, from about 5.0 to about 6.3, from about 5.0 to about 6.2, from about 5.0 to about 6.1, from about 5.0 to about 6.0, from about 5.0 to about 5.9, from about 5.0 to about 5.8, from about 5.0 to about 5.7, from about 5.0 to about 5.6, from about 5.0 to about 5.5, from about 5.0 to about 5.4, from about 5.0 to about 5.3, from about 5.0 to about 5.2, from about 5.0 to about 5.1, from about 5.1 to about 6.5, from about 5.1 to about 6.4, from about 5.1 to about 6.3, from about 5.1 to about 6.2, from about 5.1 to about 6.1, from about 5.1 to about 6.0, from about 5.1 to about 5.9, from about 5.1 to about 5.8, from about 5.1 to about 5.7, from about 5.1 to about 5.6, from about 5.1 to about 5.5, from about 5.1 to about 5.4, from about 5.1 to about 5.3, from about 5.1 to about 5.2, from about 5.2 to about 6.5, from about 5.2 to about 6.4, from about 5.2 to about 6.3, from about 5.2 to about 6.2, from about 5.2 to about 6.1, from about 5.2 to about 6.0, from about 5.2 to about 5.9, from about 5.2 to about 5.8, from about 5.2 to about 5.7, from about 5.2 to about 5.6, from about 5.2 to about 5.5, from about 5.2 to about 5.4, from about 5.2 to about 5.3, from about 5.3 to about 6.5, from about 5.3 to about 6.4, from about 5.3 to about 6.3, from about 5.3 to about 6.2, from about 5.3 to about 6.1, from about 5.3 to about 6.0, from about 5.3 to about 5.9, from about 5.3 to about 5.8, from about 5.3 to about 5.7, from about 5.3 to about 5.6, from about 5.3 to about 5.5, from about 5.3 to about 5.4, from about 5.4 to about 6.5, from about 5.4 to about 6.4, from about 5.4 to about 6.3, from about 5.4 to about 6.2, from about 5.4 to about 6.1, from about 5.4 to about 6.0, from about 5.4 to about 5.9, from about 5.4 to about 5.8, from about 5.4 to about 5.7, from about 5.4 to about 5.6, from about 5.4 to about 5.5, from about 5.5 to about 6.5, from about 5.5 to about 6.4, from about 5.5 to about 6.3, from about 5.5 to about 6.2, from about 5.5 to about 6.1, from about 5.5 to about 6.0, from about 5.5 to about 5.9, from about 5.5 to about 5.8, from about 5.5 to about 5.7, from about 5.5 to about 5.6, from about 5.6 to about 6.5, from about 5.6 to about 6.4, from about 5.6 to about 6.3, from about 5.6 to about 6.2, from about 5.6 to about 6.1, from about 5.6 to about 6.0, from about 5.6 to about 5.9, from about 5.6 to about 5.8, from about 5.6 to about 5.7, from about 5.7 to about 6.5, from about 5.7 to about 6.4, from about 5.7 to about 6.3, from about 5.7 to about 6.2, from about 5.7 to about 6.1, from about 5.7 to about 6.0, from about 5.7 to about 5.9, from about 5.7 to about 5.8, from about 5.8 to about 6.5, from about 5.8 to about 6.4, from about 5.8 to about 6.3, from about 5.from about 8 to about 6.2, from about 5.8 to about 6.1, from about 5.8 to about 6.0, from about 5.8 to about 5.9, from about 5.9 to about 6.5, from about 5.9 to about 6.4, from about 5.9 to about 6.3, from about 5.9 to about 6.2, from about 5.9 to about 6.1, from about 5.9 to about 6.0, from about 6.0 to about 6.5, from about 6.0 to about 6.4, from about 6.0 to about 6.3, from about 6.0 to about 6.2, from about 6.0 to about 6.1, from about 6.1 to about 6.5, from about 6.1 to about 6.4, from about 6.1 to about 6.3, from about 6.1 to about 6.2, from about 6.2 to about 6.5, from about 6.2 to about 6.4, from about 6.2 to about 6.3, from about 6.3 to about 6.5, from about 6.3 to about 6.4, or from about 6.4 to about 6.5. In some embodiments, the pH is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0047] The pH of the formulation can be measured by any method known to those skilled in the art. One method for measuring pH is to use a pH meter with a microelectrode. The pH of the formulation can be adjusted using any method known in the art. Exemplary chemicals for changing the pH of the formulation are hydrochloric acid (HCl) and sodium hydroxide (NaOH).

[0048] In some embodiments, the acetate buffer described herein is an acetic acid / sodium acetate buffer. In some embodiments, the buffer is an acetic acid / sodium acetate buffer. In some embodiments, the buffer described herein contains acetic acid / sodium acetate at about 5 - 100 mM (e.g., about 5 - 90 mM, about 5 - 80 mM, about 5 - 70 mM, about 5 - 60 mM, about 5 - 50 mM, about 5 - 40 mM, about 5 - 30 mM, about 5 - 20 mM, about 5 - 10 mM, about 10 - 90 mM, about 10 - 80 mM, about 10 - 70 mM, about 10 - 60 mM, about 10 - 50 mM, about 10 - 40 mM, about 10 - 30 mM, about 10 - 20 mM, about 20 - 100 mM, about 20 - 90 mM, about 20 - 80 mM, about 20 - 70 mM, about 20 - 60 mM, about 20 - 50 mM, about 20 - 40 mM, about 20 - 30 mM, about 30 - 100 mM, about 30 - 90 mM, about 30 - 80 mM, about 30 - 70 mM, about 30 - 60 mM, about 30 - 50 mM, about 30 - 40 mM, about 40 - 100 mM, about 40 - 90 mM, about 40 - 80 mM, about 40 - 70 mM, about 40 - 60 mM, about 40 - 50 mM, about 50 - 100 mM, about 50 - 90 mM, about 50 - 80 mM, about 50 - 70 mM, about 50 - 60 mM, about 60 - 100 mM, about 60 - 90 mM, about 60 - 80 mM, about 60 - 70 mM, about 70 - 100 mM, about 70 - 90 mM, about 70 - 80 mM, about 80 - 100 mM, about 80 - 90 mM, or about 90 - 100 mM). When the concentration of the acetic acid / sodium acetate buffer is mentioned, the concentration is the number of moles of acetic acid and sodium acetate divided by the volume. In some embodiments, the buffer described herein contains acetic acid / sodium acetate at about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. In some embodiments, the formulation substantially does not contain other buffers (e.g., citrate buffer, succinate buffer, histidine buffer, or phosphate buffer).

[0049] As shown in the present invention, according to the formulations described herein, the stability of the anti-PD-L1 / anti-4-1BB bispecific antibody described herein can be significantly improved. For example, when the desired formulation contains an anti-PD-L1 / anti-4-1BB bispecific antibody and an acetic acid / sodium acetate buffer, and the pH of the formulation is 5.0 to 6.5 (e.g., 5.6 or 5.8), the stability of this formulation is significantly improved and no visible foreign matters are observed.

[0050] In some embodiments, the formulations described herein contain an anti-PD-L1 / anti-4-1BB bispecific antibody (10 to 100 mg / ml), 10 to 100 mM acetic acid / sodium acetate, 10 to 200 mM arginine-HCl, and 0.01 mg / ml to 10 mg / ml polysorbate 80, and its pH is 5.5 to 6.0. In some embodiments, the formulation further contains 100 to 300 mM trehalose.

[0051] In some embodiments, the formulations described herein contain an anti-PD-L1 / anti-4-1BB bispecific antibody, 10 to 20 mM acetic acid / sodium acetate, 100 to 200 mM arginine-HCl, 0.1 mg / ml to 0.5 mg / ml polysorbate 80, and its pH is 5.5 to 6.0. In some embodiments, the formulation further contains 100 to 150 mM trehalose.

[0052] Surfactant The formulation may optionally contain a surfactant. The surfactant may be a stabilizer. The surfactant / stabilizer is a compound that interacts with biomolecules and / or common pharmaceutical excipients in the formulation and has a stabilizing effect. Surfactants typically protect the active agent from stress at the air / solution interface and stress at the solution / surface, which may cause protein aggregation. Examples of surfactants include, but are not limited to, polysorbates, glycerin, dicarboxylic acids, oxalic acid, succinic acid, fumaric acid, phthalic acid, and combinations thereof. For example, surfactants containing non-ionic or ionic detergents can be used, provided they are pharmaceutically acceptable and suitable for administration to the subject. In some embodiments, the surfactant is a polysorbate. Examples of polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

[0053] In some embodiments, the surfactant is present in the formulation in an amount of about 0.01 mg / ml to about 1 mg / ml. For example, the surfactant is present in the formulation in amounts of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, and about 1 mg / ml. In some specific embodiments, the surfactant is present in the formulation in an amount of about 0.03 mg / ml to about 0.5 mg / ml, about 0.04 mg / ml to about 0.25 mg / ml, or about 0.1 mg / ml to about 0.2 mg / ml, for example, about 0.2 mg / ml.

[0054] In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. For example, polysorbate 80 can be present in an amount of about 0.01 mg / ml to about 1 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, about 0.1 mg / ml to about 0.3 mg / ml, for example, about 0.2 mg / ml. In alternative embodiments, polysorbate 80 is present in amounts of about 0.01 mg / ml to about 1 mg / ml, about 0.05 mg / ml to about 0.5 mg / ml, and about 0.1 mg / ml to about 0.2 mg / ml. In another alternative embodiment, polysorbate 80 is present in amounts of about 0.01 mg / ml to about 1 mg / ml, about 0.05 mg / ml to about 0.5 mg / ml, and about 0.1 mg / ml to about 0.3 mg / ml, for example, about 0.2 mg / ml. In some embodiments, the formulations described herein comprise polysorbate 80 in an amount of about 0.01 mg / ml to about 10 mg / ml (e.g., about 0.01 mg / ml to about 9 mg / ml, about 0.01 mg / ml to about 8 mg / ml, about 0.01 mg / ml to about 7 mg / ml, about 0.01 mg / ml to about 6 mg / ml, about 0.01 mg / ml to about 5 mg / ml, about 0.01 mg / ml to about 4 mg / ml, about 0.01 mg / ml to about 3 mg / ml, about 0.01 mg / ml to about 2 mg / ml, about 0.01 mg / ml to about 1 mg / ml, about 0.01 mg / ml to about 0.9 mg / ml, about 0.01 mg / ml to about 0.8 mg / ml, about 0.01 mg / ml to about 0.7 mg / ml, about 0.01 mg / ml to about 0.6 mg / ml, about 0.01 mg / ml to about 0.5 mg / ml, about 0.01 mg / ml to about 0.4 mg / ml, about 0.01 mg / ml to about 0.3 mg / ml, about 0.01 mg / ml to about 0.2 mg / ml).

[0055] Polysorbate 20 may be present in an amount of about 0.01 mg / ml to about 1 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, about 0.1 mg / ml to about 0.3 mg / ml, for example, about 0.2 mg / ml. In alternative embodiments, polysorbate 20 is present in an amount of about 0.01 mg / ml to about 1 mg / ml, about 0.05 mg / ml to about 0.5 mg / ml, or about 0.1 mg / ml to about 0.2 mg / ml. In another alternative embodiment, polysorbate 20 is present in an amount of about 0.01 mg / ml to about 1 mg / ml, about 0.05 mg / ml to about 0.5 mg / ml, and about 0.1 mg / ml to about 0.3 mg / ml, for example, about 0.2 mg / ml.

[0056] Isotonic agent The formulations of the present invention may further comprise an isotonic agent. Typically, an isotonic agent is used to adjust or maintain the osmotic pressure of a formulation to approximate that of a body fluid such as blood or plasma. Also, an isotonic agent can maintain the level of an antibody or antigen-binding fragment in a formulation. As used herein, the term "tonicity" refers to the behavior of biological components in a fluid environment or solution. An isotonic solution has the same osmotic pressure as plasma and can be intravenously injected into a subject without changing the osmotic pressure of the subject's plasma. In practice, in certain embodiments, the isotonic agent is present in an amount sufficient to render the formulation suitable for intravenous injection. Typically, an isotonic agent is also used as a filler or stabilizer. Thus, an isotonic agent enables an antibody or its antigen-binding fragment to overcome various stresses such as freezing and shear. Examples of isotonic agents include, but are not limited to, sugars, saccharides, glycerin, sorbitol, mannitol, sodium chloride, potassium chloride, magnesium chloride, and other inorganic salts. As will be well known to those of skill in the art, other isotonic agents can be used, and any pharmaceutically acceptable isotonic agent is suitable for administration to a subject.

[0057] In some embodiments, the isotonic agent is a sugar. Examples of sugars include glucose, sucrose, maltose, trehalose, dextrose, xylitol, fructose, and mannitol. In some embodiments, the formulations described herein contain an isotonic agent at about 10 - 500 mM (e.g., about 10 - 450 mM, about 10 - 400 mM, about 10 - 350 mM, about 10 - 300 mM, about 10 - 250 mM, about 10 - 200 mM, about 10 - 150 mM, about 10 - 100 mM, about 10 - 50 mM, about 50 - 500 mM, about 50 - 450 mM, about 50 - 400 mM, about 50 - 350 mM, about 50 - 300 mM, about 50 - 250 mM, about 50 - 200 mM, about 50 - 150 mM, about 50 - 100 mM, about 100 - 500 mM, about 100 - 450 mM, about 100 - 400 mM, about 100 - 350 mM, about 100 - 300 mM, about 100 - 250 mM, about 100 - 200 mM, about 100 - 150 mM, about 150 - 500 mM, about 150 - 450 mM, about 150 - 400 mM, about 150 - 350 mM, about 150 - 300 mM, about 150 - 250 mM, about 150 - 200 mM, about 200 - 500 mM, about 200 - 450 mM, about 200 - 400 mM, about 200 - 350 mM, about 200 - 300 mM, about 200 - 250 mM, about 250 - 500 mM, about 250 - 450 mM, about 250 - 400 mM, about 250 - 350 mM, about 250 - 300 mM, about 300 - 500 mM, about 300 - 450 mM, about 300 - 400 mM, about 300 - 350 mM, about 350 - 500 mM, about 350 - 450 mM, about 350 - 400 mM, about 400 - 500 mM, about 400 - 450 mM, or about 450 - 500 mM).

[0058] In some embodiments, the isotonic agent is sucrose or trehalose. In some embodiments, the isotonic agent is sucrose. In some embodiments, the concentration of trehalose is about 10 - 300 mM (for example, about 10 - 50 mM, about 50 - 100 mM, about 100 - 110 mM, about 110 - 120 mM, about 120 - 130 mM, about 130 - 140 mM, about 140 - 150 mM, about 150 - 160 mM, about 160 - 170 mM, about 170 - 180 mM, about 180 - 190 mM, about 190 - 200 mM, about 210 - 220 mM, about 220 - 230 mM, about 230 - 240 mM, about 240 - 250 mM, about 250 - 260 mM, about 260 - 270 mM, about 270 - 280 mM, about 280 - 290 mM, or about 290 - 300 mM).

[0059] In some embodiments, the formulations described herein contain about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about 300 mM of trehalose.

[0060] In some embodiments, the formulation can contain one or more isotonic agents. For example, the formulation may contain one or more of the isotonic agents at the above concentrations. In a specific embodiment, the formulation can contain trehalose and arginine (for example, arginine - HCl). The concentrations of trehalose and arginine in it are 50 - 300 mM respectively (for example, about 50 - 250 mM, about 50 - 200 mM, about 50 - 150 mM, about 100 - 150 mM, about 100 - 140 mM, or about 100 - 130 mM).

[0061] In some embodiments, the osmotic pressure of the formulation is about 200 mOsm / kg to about 500 mOsm / kg, about 250 mOsm / kg to about 450 mOsm / kg, about 200 mOsm / kg to about 350 mOsm / kg, about 230 mOsm / kg to about 330 mOsm / kg, about 250 mOsm / kg to about 300 mOsm / kg, about 260 mOsm / kg to about 280 mOsm / kg, about 350 mOsm / kg to about 450 mOsm / kg, about 380 mOsm / kg to about 420 mOsm / kg, for example, about 270 mOsm / kg or about 450 mOsm / kg. In some embodiments, the formulation is substantially isotonic, i.e., has substantially the same osmotic pressure as mammalian (e.g., human) blood. The osmotic pressure can be measured by any method known to those skilled in the art, for example, using a vapor pressure or a freezing-point osmometer. The osmotic pressure of the formulations described herein can be adjusted, for example, by one or more of the isotonic agents described herein.

[0062] Amino acid The formulation can optionally further contain an amino acid. In some embodiments, the amino acid is also an isotonic agent. In some embodiments, the amino acid is a stabilizer. Examples of amino acids include, but are not limited to, glycine, alanine, asparagine, lysine, serine, tyrosine, cysteine, glutamine, methionine, arginine, and proline. In some embodiments, the amino acid is in its hydrochloride form. In some embodiments, the amino acid is the L-enantiomer. In some embodiments, the amino acid is the D-enantiomer. In some embodiments, the amino acid is a mixture of the L- and D-enantiomers.

[0063] In some embodiments, the amino acid is present in the formulation in an amount of 10 - 500 mM (e.g., about 10 - 450 mM, about 10 - 400 mM, about 10 - 350 mM, about 10 - 300 mM, about 10 - 250 mM, about 10 - 200 mM, about 10 - 150 mM, about 10 - 100 mM, about 10 - 50 mM, about 50 - 500 mM, about 50 - 450 mM, about 50 - 400 mM, about 50 - 350 mM, about 50 - 300 mM, about 50 - 250 mM, about 50 - 200 mM, about 50 - 150 mM, about 50 - 100 mM, about 100 - 500 mM, about 100 - 450 mM, about 100 - 400 mM, about 100 - 350 mM, about 100 - 300 mM, about 100 - 250 mM, about 100 - 200 mM, about 100 - 150 mM, about 150 - 500 mM, about 150 - 450 mM, about 150 - 400 mM, about 150 - 350 mM, about 150 - 300 mM, about 150 - 250 mM, about 150 - 200 mM, about 200 - 500 mM, about 200 - 450 mM, about 200 - 400 mM, about 200 - 350 mM, about 200 - 300 mM, about 200 - 250 mM, about 250 - 500 mM, about 250 - 450 mM, about 250 - 400 mM, about 250 - 350 mM, about 250 - 300 mM, about 300 - 500 mM, about 300 - 450 mM, about 300 - 400 mM, about 300 - 350 mM, about 350 - 500 mM, about 350 - 450 mM, about 350 - 400 mM, about 400 - 500 mM, about 400 - 450 mM or about 450 - 500 mM).

[0064] Exemplary amino acids include arginine (e.g., arginine-HCl). In some embodiments, the concentration of arginine is about 10-200 mM (e.g., about 10-50 mM, about 50-100 mM, about 100-110 mM, about 110-120 mM, about 120-130 mM, about 130-140 mM, about 140-150 mM, about 150-160 mM, about 160-170 mM, about 170-180 mM, about 180-190 mM, or about 190-200 mM). In some embodiments, the formulations described herein contain about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM of arginine.

[0065] Other excipients Furthermore, the formulation can include other excipients including, but not limited to, water for injection, diluents, solubilizers, emollients, additional buffers, inorganic or organic salts, antioxidants, etc. However, in some embodiments, the formulation does not include other excipients except for the above excipients. Other pharmaceutically acceptable carriers, excipients or stabilizers as described in Remington’s Pharmaceutical Sciences 16th edition, Osol,A.Ed.(1980) may be included in the formulation as long as they do not adversely affect the desired properties of the formulation. In a specific embodiment, the formulation is substantially free of preservatives, but in an alternative embodiment, a preservative can be added as needed. For example, a cryoprotectant or cryoprotectants may be included in a lyophilized formulation.

[0066] In some embodiments, the formulation may also include a metal chelating agent and / or an antioxidant, as well as other pharmaceutically acceptable excipients. Suitable metal chelating agents include, for example, methylamine, ethylenediamine, deferoxamine, trientine, histidine, malate, phosphonate compounds such as etidronic acid, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and the like. Suitable antioxidants include, for example, citric acid, uric acid, ascorbic acid, lipoic acid, glutathione, tocopherol, carotene, lycopene, cysteine, and the like.

[0067] Exemplary formulations In one aspect, the present invention provides a stable aqueous pharmaceutical formulation having a pH of about 5.0 to about 6.5, comprising an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof and a buffer.

[0068] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of at least 10 mg / ml; b) a stabilizer at a concentration of about 10 mM to about 500 mM; c) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml; and d) a buffer system, or consists of or consists essentially of these components, wherein the pH of the formulation is about 5.0 to about 6.5.

[0069] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml; b) an isotonic agent at a concentration of about 10 mM to about 500 mM; c) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml; and d) comprising a buffer system, consisting of these components, or consisting essentially of these components, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0070] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: e) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and f) an amino acid at a concentration of about 10 mM to about 500 mM, and g) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml, and h) comprising a buffer system, consisting of these components, or consisting essentially of these components, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0071] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 500 mM, and c) comprising an acetic acid / sodium acetate buffer, consisting of these components, or consisting essentially of these components, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0072] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 500 mM, and c) trehalose at a concentration of about 10 mM to about 500 mM, and d) comprising an acetic acid / sodium acetate buffer, consisting of these components, or consisting essentially of these components, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0073] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml, and c) an acetic acid / sodium acetate buffer, or consists of or consists essentially of these components, wherein the pH of the formulation is about 5.0 to about 6.5.

[0074] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 500 mM, and c) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml (e.g., about 0.2 mg / ml), and d) an acetic acid / sodium acetate buffer, or consists of or consists essentially of these components, wherein the pH of the formulation is about 5.0 to about 6.5.

[0075] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 200 mM, and c) polysorbate 80 at a concentration of about 0.1 mg / ml to about 0.5 mg / ml (e.g., about 0.2 mg / ml), and d) an acetic acid / sodium acetate buffer at a concentration of about 10 to 100 mM, or consists of or consists essentially of these components, wherein the pH of the formulation is about 5.5 to about 6.0.

[0076] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) An anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) Arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 200 mM, and c) Trehalose at a concentration of about 10 mM to about 300 mM, and d) Polysorbate 80 at a concentration of about 0.1 mg / ml to about 0.3 mg / ml (e.g., about 0.2 mg / ml), and e) An acetic acid / sodium acetate buffer at a concentration of about 10 - 100 mM, or consists of or consists essentially of these components, wherein the pH of the formulation is about 5.5 to about 6.0.

[0077] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) An anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) Arginine (e.g., arginine-HCl) at a concentration of about 140 mM, and c) Polysorbate 80 at a concentration of about 0.2 mg / ml, and d) An acetic acid / sodium acetate buffer at a concentration of about 10 mM, or consists of or consists essentially of these components, wherein the pH of the formulation is about 5.5 to about 6.0 (e.g., 5.6).

[0078] In some embodiments, the stable aqueous pharmaceutical formulation comprises the following components: a) An anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, and b) Arginine (e.g., arginine-HCl) at a concentration of about 100 mM, and c) Trehalose at a concentration of about 130 mM, and d) Polysorbate 80 at a concentration of about 0.2 mg / ml, and e) containing, consisting of, or consisting essentially of an acetic acid / sodium acetate buffer having a concentration of about 15 mM, wherein the pH of the formulation is from about 5.5 to about 6.0 (e.g., 5.8).

[0079] In some embodiments, the formulation is substantially free of any sugar or any sugar other than trehalose. In some embodiments, the formulation is substantially free of other tonicity agents. In some embodiments, the formulation is substantially free of other buffering agents. In some embodiments, the formulation is substantially free of any amino acid or any amino acid other than arginine.

[0080] Stability The formulations described herein are stable upon storage. The formulations can be stored under refrigerated (e.g., 2 - 8°C) or frozen (e.g., -20°C, -70°C or -80°C) conditions. The desired formulation is stable at least about 1, 2, 3, or 4 weeks or more at about 4°C, about 25°C, about 40°C, or room temperature, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, usually at least about 12, 18, or 24 months or more. In some embodiments, they are stable at least about 6 months at about 25°C.

[0081] Stability can be measured by evaluating the physical stability, chemical stability, and / or biological activity of the antibody or its antigen-binding fragment in the formulation during compounding and after storage at a predetermined temperature. The physical and / or chemical stability of a liquid formulation can be evaluated qualitatively and / or quantitatively by a variety of different methods (see, for example, Analytical Techniques for Biopharmaceutical Development, Rodriguez-Diaz et al., eds., Informa Healthcare (2005)). For example, evaluation of aggregate formation (e.g., using size exclusion (or gel filtration) chromatography (SEC), size exclusion high performance liquid chromatography (SEC-HPLC), reverse phased chromatography-ultra performance liquid chromatography (RPC-UPLC), matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOFMS), ultracentrifugation analysis, light scattering (photon correlation spectroscopy, dynamic light scattering (DLS), multi-angle laser light scattering (MALLS)), flow-based microscopic imaging, electrical impedance (Coulter) counter, light obscuration or other liquid particle counting systems, measurement of turbidity or observation of the appearance of the sample or density gradient centrifugation, and / or visual inspection); evaluation of charge variants using cation exchange chromatography (Vlasak and Ionescu, Curr. Pharm. Biotechnol. 9:468-481 (2008) and Harris et al., J. Chromatogr. B Biomed. Sci. Appl.(see 752:233-245(2001)), ion exchange high performance liquid chromatography (IEX-HPLC), isoelectric focusing (IEF) such as capillary isoelectric focusing (cIEF) and capillary zone electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry; comparison of fragments, full-length and multimeric (i.e., dimeric, trimeric, etc.) antibodies by SDS-PAGE or SEC analysis; evaluation of peptide patterns (such as trypsin and LYS-), biological activity or antigen-binding function of antibodies, etc. Evaluation of biological activity or antigen-binding function, for example, binding of anti-PD-L1 / anti-4-1BB bispecific antibody to PD-1, PD-L1, or inhibition of binding between PD-1 and PD-L1, binding of anti-PD-L1 / anti-4-1BB bispecific antibody to 4-1BB, 4-1BBL, or inhibition of binding between 4-1BB and 4-1BBL can be evaluated using various techniques available to those skilled in the art.

[0082] Stability can be measured at a selected temperature for a selected period. In the uniform phase, the formulation is stable at about 40 °C for at least about 1, 2, 3, or 4 weeks, at least about 2 months, at least about 3 months, or at least about 6 months, at least about 9 months, at least 12 months, or at least 18 months. During this period, the formulation hardly precipitates and / or the decrease in protein concentration is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5%. In some embodiments, after 4 weeks at 40 °C, the decrease in protein concentration is less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In some embodiments, the decrease in protein concentration does not exceed 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 mg / ml. In some embodiments, the change in protein purity (e.g., measured by SEC-HPLC, RPC-UPLC, and / or CE-SDS methods) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5%. In some embodiments, the main peak ratio of charge isomers (e.g., measured by IEX-HPLC method) is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0083] In one aspect, the formulation is stable at about 25°C for at least 1 month, at least 2 months, at least about 3 months, at least 6 months, at least 9 months, at least 12 months, or at least 18 months. During this period, the formulation hardly precipitates and / or the decrease in protein concentration is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5%. In some embodiments, after 6 months at 25°C, the decrease in protein concentration is less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In some embodiments, the decrease in protein concentration does not exceed 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 or 5 mg / ml. In some embodiments, the change in protein purity (e.g., measured by SEC-HPLC, RPC-UPLC, and / or CE-SDS methods) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5%. In some embodiments, the main peak ratio of charge isomers (e.g., measured by IEX-HPLC method) is less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0084] Instability can involve any one or more of aggregation (e.g., non-covalent soluble aggregation (due to hydrophobic or charge interactions), covalent soluble aggregation (e.g., rearrangement / scrambling of disulfide bonds), insoluble aggregation (due to protein denaturation at liquid / air or liquid / solid interfaces), deamidation (e.g., deamidation of Asn), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), denaturation, shear / hydrolysis / breakage (e.g., breakage of hinge regions), formation of succinimide, N-terminal elongation, C-terminal processing, differences in glycosylation, etc.).

[0085] In some embodiments, the formulation after storage (e.g., about 4 weeks at about 40 ± 2 °C or about 6 months at about 25 ± 2 °C) is colorless and substantially free of visible particulates. In some embodiments, the formulation has substantially no precipitate after storage (e.g., about 4 weeks at about 40 ± 2 °C or about 6 months at about 25 ± 2 °C).

[0086] In some embodiments, the decrease in protein concentration after storage (e.g., about 4 weeks at about 40 ± 2 °C or about 6 months at about 25 ± 2 °C) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. In some embodiments, the change in protein purity (e.g., as measured by SEC-HPLC, RPC-UPLC, and / or CE-SDS methods) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

[0087] In some embodiments, the decrease in protein concentration after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 freeze-thaw cycles (e.g., after at least 1, 2, 3, 4, 5, or 6 hours at about -70 to about -80 °C followed by 1 cycle at 25 °C) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5%. In some embodiments, the change in protein purity (e.g., as measured by SEC-HPLC, RPC-UPLC, and / or CE-SDS methods) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

[0088] In some embodiments, after rocking for at least 1 day, 2 days, 3 days, 4 days, or 5 days (e.g., at about 100 rpm, about 150 rpm, about 200 rpm, about 210 rpm, about 220 rpm, about 230 rpm, about 240 rpm, about 250 rpm, about 260 rpm, about 270 rpm, about 280 rpm, about 290 rpm, about 300 rpm, about 350 rpm, about 400 rpm, about 450 rpm, or about 500 rpm), the decrease in protein concentration is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5%. In some embodiments, the change in protein purity (e.g., measured by SEC-HPLC, RPC-UPLC, and / or CE-SDS methods) is less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

[0089] Also, the formulation may be sterile and can be achieved according to procedures known to those skilled in the art before or after the manufacture of the formulation to produce a sterile pharmaceutical formulation suitable for the subject.

[0090] Anti-PD-L1 / anti-4-1BB bispecific antibody and antigen-binding fragment thereof One aspect of the present invention relates to a bispecific antibody comprising a first antigen-binding functional region that specifically binds to PD-L1 and a second antigen-binding functional region that specifically binds to 4-1BB. Here, the first antigen-binding functional region that specifically binds to the PD-L1 is (a) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, (b) an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, (c) an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and comprising a (A) heavy chain variable region, (a) an LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, (b) an LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, (c) an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 20, and comprising a (B) light chain variable region.

[0091] In some embodiments, the second antigen-binding functional region that specifically binds to 4-1BB of the bispecific antibody is (a) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 21, (b) an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, (c) an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, and an (A) heavy chain variable region, (a) an LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, (b) an LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, (c) an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26, and a (B) light chain variable region.

[0092] In some embodiments, the first antigen-binding functional region that specifically binds to PD-L1 of the bispecific antibody is (a) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, (b) an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, (c) an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and an (A) heavy chain variable region, (a) an LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, (b) an LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, (c) an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 20, and a (B) light chain variable region, and the second antigen-binding functional region that specifically binds to 4-1BB is (a) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 21, (b) an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, (c) an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, and an (A) heavy chain variable region, (a) an LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, (b) an LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, (c) an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region (B) containing the same.

[0093] Six CDRs of the first antigen-binding functional region that specifically binds to PD-L1 as shown in SEQ ID NOs: 15 to 20, and six CDRs of the second antigen-binding functional region that specifically binds to 4-1BB as shown in SEQ ID NOs: 21 to 26 are the CDRs of the heavy and light chain variable regions of a monoclonal antibody obtained by the inventor using hybridoma technology with human PD-L1 and 4-1BB as antigens. The monoclonal antibody has higher biological activities such as higher binding specificity and antitumor activity compared to the PD-L1 antibody and 4-1BB antibody known in the prior art.

[0094] The six CDR sequences of the first antigen-binding functional region that specifically binds to PD-L1 each have at least 70%, for example at least 75%, 80%, 85%, 90%, 95% or more identity with the sequences shown in SEQ ID NOs: 15 to 20, and may retain the biological activity of the corresponding parental sequence, or may contain one or more, for example 1, 2, 3 or more amino acid deletions, substitutions, and / or additions compared to the sequences shown in SEQ ID NOs: 15 to 20, and may retain the biological activity of the corresponding parental sequence.

[0095] The six CDR sequences of the second antigen-binding functional region that specifically binds to 4-1BB each have at least 70%, for example at least 75%, 80%, 85%, 90%, 95% or more identity with the sequences shown in SEQ ID NOs: 21 to 26, and may retain the biological activity of the corresponding parental sequence, or may contain one or more, for example 1, 2, 3 or more amino acid deletions, substitutions, and / or additions compared to the sequences shown in SEQ ID NOs: 21 to 26, and may retain the biological activity of the corresponding parental sequence.

[0096] In some embodiments, the antigen-binding functional region that specifically binds to the PD-L1 comprises a heavy-chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 that are the same as HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 6, and a light-chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 that are the same as LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2.

[0097] In some embodiments, the antigen-binding functional region that specifically binds to the 4-1BB comprises a heavy-chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 that are the same as HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 12, and a light-chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 that are the same as LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 10.

[0098] In some embodiments, the antibody or antigen-binding fragment described herein can comprise a light-chain variable region comprising one, two, or three CDRs having 0, 1, or 2 amino acid insertions, deletions, or substitutions as described herein. In some embodiments, the antibody or antigen-binding fragment thereof described herein can comprise a heavy-chain variable region comprising one, two, or three CDRs having 0, 1, or 2 amino acid insertions, deletions, or substitutions as described herein.

[0099] The insertion, deletion, or substitution may be within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on the Kabat numbering system. In some embodiments, the CDR is determined based on the Chothia numbering system.

[0100] In some embodiments, the first antigen-binding functional region that specifically binds to PD-L1 of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 6; or has at least 70%, such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence shown in SEQ ID NO: 6 and retains the biological activity of the corresponding parental sequence; or compared with the sequence shown in SEQ ID NO: 6, contains one or more, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more amino acid deletions, substitutions and / or additions and retains the biological activity of the corresponding parental sequence, and a heavy chain variable region, and comprises the amino acid sequence shown in SEQ ID NO: 2; or has at least 70%, such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence shown in SEQ ID NO: 2 and retains the biological activity of the corresponding parental sequence; or compared with the sequence shown in SEQ ID NO: 2, contains one or more, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more amino acid deletions, substitutions and / or additions and retains the biological activity of the corresponding parental sequence, and a light chain variable region.

[0101] In some embodiments, the second antigen-binding functional region that specifically binds to 4-1BB of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 12; or has at least 70%, such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence shown in SEQ ID NO: 12 and retains the biological activity of the corresponding parental sequence; or compared with the sequence shown in SEQ ID NO: 12, contains one or more, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more amino acid deletions, substitutions and / or additions and retains the biological activity of the corresponding parental sequence, and a heavy chain variable region, and comprises the amino acid sequence shown in SEQ ID NO: 10; or has at least 70%, such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence shown in SEQ ID NO: 10 and retains the biological activity of the corresponding parental sequence; or compared with the sequence shown in SEQ ID NO: 10, contains one or more, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more amino acid deletions, substitutions and / or additions and retains the biological activity of the corresponding parental sequence, and a light chain variable region.

[0102] In some embodiments, the first antigen-binding functional region that specifically binds to PD-L1 of the bispecific antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2, and the second antigen-binding functional region that specifically binds to 4-1BB comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10.

[0103] One skilled in the art would know that the combination of amino acid sequences should follow the biological law, that is, the light chain and the heavy chain or their variable regions, their antigen-binding fragments (such as SEQ ID NO: 6 and SEQ ID NO: 2, and SEQ ID NO: 12 and SEQ ID NO: 10) should be compatible with each other.

[0104] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region of the bispecific antibody are selected from Fab fragments, scFv fragments, and variable domain fragments Fv.

[0105] In some embodiments, both the first antigen-binding functional region and the second antigen-binding functional region of the bispecific antibody are Fab fragments.

[0106] In some embodiments, the Fab fragment of the bispecific antibody comprises a different first heavy chain variable region and a different second heavy chain variable region, and a different first light chain variable region and a different second light chain variable region.

[0107] In some embodiments, one of the first antigen-binding functional region and the second antigen-binding functional region of the bispecific antibody is a Fab fragment and the other is an scFv fragment.

[0108] In some embodiments, the bispecific antibody comprises a first Fc chain and a second Fc chain, and a first antigen-binding functional region that can specifically bind to PD-L1 and a second antigen-binding functional region that can specifically bind to 4-1BB, wherein both the first Fc chain and the second Fc chain are immunoglobulin G Fc fragments containing amino acid substitutions, and the first Fc chain and the second Fc chain together form a heterodimer capable of binding to an Fc receptor, and here again, the first Fc chain and the second Fc chain are each linked to the first antigen-binding functional region and the second antigen-binding functional region via a covalent bond or a linker, and either one of the first Fc chain and the second Fc chain contains amino acid substitutions at positions 366 and 399, and the other contains amino acid substitutions at positions 351, 407, and 409. Here, the positions of the amino acids are numbered according to the Kabat EU indexing system.

[0109] In some embodiments, the amino acid substitutions of the first Fc chain and the second Fc chain of the bispecific antibody are as follows. a) L351G, L351Y, L351V, L351P, L351D, L351E, L351K or L351W; b) T366L, T366P, T366W or T366V; c) D399C, D399N, D399I, D399G, D399R, D399T or D399A; d) Y407L, Y407A, Y407P, Y407F, Y407T or Y407H; and e) K409C, K409P, K409S, K409F, K409V, K409Q or K409R.

[0110] In some embodiments, the amino acid substitutions of the bispecific antibody are as follows. a) Substitution of T366L and D399R in either the first Fc chain or the second Fc chain, and substitution of L351E, Y407L and K409V in the other; b) Substitution of T366L and D399C in either the first Fc chain or the second Fc chain, and substitution of L351G, Y407L and K409C in the other; c) Substitution of T366L and D399C in either the first Fc chain or the second Fc chain, and substitution of L351Y, Y407A and K409P in the other; d) Substitution of T366P and D399N in either the first Fc chain or the second Fc chain, and substitution of L351V, Y407P and K409S in the other; e) Substitution of T366W and D399G in either the first Fc chain or the second Fc chain, and substitution of L351D, Y407P and K409S in the other; f) Substitution of T366P and D399I in either the first Fc chain or the second Fc chain, and substitution of L351P, Y407F and K409F in the other; g) Substitution of T366V and D399T in either the first Fc chain or the second Fc chain, and substitution of L351K, Y407T and K409Q in the other; h) Substitution of T366L and D399A in either the first Fc chain or the second Fc chain, and substitution of L351W, Y407H and K409R in the other.

[0111] In some embodiments, the amino acid substitutions of the bispecific antibody include the following. a) Substitution of T366L and K409V in either the first Fc chain or the second Fc chain, and substitution of L351E, Y407L and D399R in the other; b) Substitution of T366L and K409C in either the first Fc chain or the second Fc chain, and substitution of L351G, Y407L and D399C in the other; c) Substitution of T366L and K409P in either the first Fc chain or the second Fc chain, and substitution of L351Y, Y407A and D399C in the other; d) Substitution of T366P and K409S in either the first Fc chain or the second Fc chain, and substitution of L351V, Y407P and D399N in the other; e) Substitution of T366W and K409S in either the first Fc chain or the second Fc chain, and substitution of L351D, Y407P and D399G in the other; f) Substitution of T366P and K409F in either the first Fc chain or the second Fc chain, and substitution of L351P, Y407F and D399I in the other; g) Substitution of T366V and K409Q in either the first Fc chain or the second Fc chain, and substitution of L351K, Y407T and D399T in the other; h) Substitution of T366L and K409R in either the first Fc chain or the second Fc chain, and substitution of L351W, Y407H and D399A in the other.

[0112] In some embodiments, the substitution in either the first Fc chain or the second Fc chain of the bispecific antibody is T366L and D399R, and the substitution in the other is L351E, Y407L and K409V.

[0113] In some embodiments, in a solution in which a reducing agent is present (and does not contain a polypeptide other than the above-mentioned Fc chain and antigen-binding functional region), the weight ratio of the homodimer formed by the first Fc chain of the bispecific antibody and the first antigen-binding functional region linked thereto via a covalent bond, and the homodimer formed by the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond is less than 50%, for example less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20% or less, based on the total amount of all polypeptide chains.

[0114] In some embodiments, the bispecific antibody comprises a first heavy chain / first light chain pair that specifically binds to PD-L1, wherein the first heavy chain has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6 and a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 8, and the first light chain has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 4.

[0115] In some embodiments, the bispecific antibody comprises a second heavy chain / second light chain pair that specifically binds to 4-1BB, wherein the second heavy chain has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12 and a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 14, and the second light chain has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 and a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 4.

[0116] A second aspect of the present invention relates to an isolated polynucleotide encoding a bispecific antibody in the form of a heterodimer as described above.

[0117] In some embodiments, the nucleotide sequence encoding the amino acid sequence of the first antigen-binding functional region comprises the nucleotide sequences shown in SEQ ID NOs: 1 and 5.

[0118] In some embodiments, the nucleotide sequence encoding the amino acid sequence of the second antigen-binding functional region comprises the nucleotide sequences shown in SEQ ID NOs: 11 and 9.

[0119] In some embodiments, the nucleotide sequences encoding the amino acid sequences of the first light chain and the second light chain both comprise the nucleotide sequence shown in SEQ ID NO: 3.

[0120] In some embodiments, the nucleotide sequence encoding the amino acid sequence of either the first heavy chain or the second heavy chain comprises the nucleotide sequence shown in SEQ ID NO: 7, and the nucleotide sequence encoding the amino acid sequence of the other heavy chain comprises the nucleotide sequence shown in SEQ ID NO: 13.

[0121] The third aspect of the present invention relates to a recombinant expression vector containing the isolated polynucleotide as described above.

[0122] In some embodiments, the expression vector is the plasmid vector X0GC obtained by modifying from the pCDNA vector.

[0123] The fourth aspect of the present invention relates to a host cell containing the isolated polynucleotide as described above or the recombinant expression vector as described above.

[0124] In some embodiments, the host cell is human embryonic kidney cell HEK293 or HEK293T, HEK293E, HEK293F obtained by modifying from HEK293 cells; Chinese hamster ovary cell CHO or CHO-S, CHO-dhfr obtained by modifying from CHO cells -, CHO / DG44, ExpiCHO; Escherichia coli or Escherichia coli BL21, BL21(DE3), Rosetta, Origami obtained by modifying Escherichia coli; Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha obtained by modifying yeast; insect cells or cells High5, SF9 obtained by modifying insect cells; plant cells; mammalian mammary gland cells; somatic cells.

[0125] The fifth aspect of the present invention relates to a composition comprising a bispecific antibody as described above, or an isolated polynucleotide as described above, or a recombinant expression vector as described above, or a host cell as described above, and a pharmaceutically acceptable carrier.

[0126] The sixth aspect of the present invention relates to a method for producing a bispecific antibody as described above, comprising the following steps. 1) A step of expressing the isolated polynucleotide as described above or the recombinant expression vector as described above in a host cell, respectively; 2) A step of reducing the proteins expressed in the host cell, respectively; 3) A step of mixing the reduced proteins and then oxidizing the mixture.

[0127] In some embodiments, the host cell is HEK293T, HEK293E, HEK293F obtained by modifying human embryonic kidney cells HEK293 or HEK293 cells; CHO-S, CHO-dhfr obtained by modifying Chinese hamster ovary cells CHO or CHO cells -, selected from CHO / DG44, ExpiCHO; Escherichia coli or E. coli BL21, BL21(DE3), Rosetta, Origami obtained by modifying Escherichia coli; Pichia, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha obtained by modifying yeast; insect cells or cells High5, SF9 obtained by modifying insect cells; plant cells; mammalian mammary gland cells; somatic cells.

[0128] In some embodiments, the reduction step includes: 1) performing a reduction reaction in the presence of a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine, and other chemical derivatives; and 2) removing the reducing agent. In some embodiments, the reducing agent is dithiothreitol at a concentration of 0.1 mM or higher, such as 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM or higher, and the reaction is carried out at 4°C for at least 3 hours, such as 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours or longer.

[0129] In some embodiments, the oxidation step further includes performing an oxidation reaction in air or in the presence of an oxidizing agent selected from L-dehydroascorbic acid, cystamine, and their chemical derivatives. In some embodiments, the oxidizing agent is L-dehydroascorbic acid at a concentration of 0.5 mM or higher, such as 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.5 mM or higher, and the reaction is carried out at 4°C for at least 5 hours, such as 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or longer.

[0130] In some embodiments, the method further includes an isolation and purification step. In some embodiments, the isolation and purification include cation exchange resin, anion exchange resin, reverse phase chromatography, affinity chromatography, size exclusion chromatography, and combinations thereof.

[0131] The seventh aspect of the present invention relates to the use of the bispecific antibody as described above, and / or the isolated polynucleotide as described above, and / or the recombinant expression vector as described above, and / or the host cell as described above, and / or the composition as described above in the manufacture of a medicament for preventing and / or treating a target disease.

[0132] The eighth aspect of the present invention relates to the bispecific antibody as described above, and / or the isolated polynucleotide as described above, and / or the recombinant expression vector as described above, and / or the host cell as described above, and / or the composition as described above, which is used as a medicament for preventing and / or treating a target disease.

[0133] The ninth aspect of the present invention relates to a method for preventing and / or treating a disease, which includes the step of administering to a subject in need the bispecific antibody as described above, and / or the isolated polynucleotide as described above, and / or the recombinant expression vector as described above, and / or the host cell as described above, and / or the composition as described above.

[0134] In some embodiments, the subject is a mammal, preferably a human.

[0135] In some embodiments, the disease is selected from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, and bone cancer.

[0136] In other words, according to the present invention, a novel anti-PD-L1 / anti-4-1BB bispecific antibody is designed, which has the characteristics of natural IgG and shows a very stable natural antibody structure-like heterodimer form without heavy chain-light chain mismatches. The bispecific antibody produced by the present invention can bind to two target molecules, PD-L1 and 4-1BB, simultaneously, and when applied to the treatment of complex diseases, it can exert better effects than a single therapeutic agent and has fewer side effects. In addition, compared with the combination therapy of multiple drugs, since the bispecific antibody is a single therapeutic molecule, it not only facilitates use for patients and medical staff, but also simplifies the complex new drug development process.

[0137] In some embodiments, the details of the antibodies or antigen-binding fragments thereof described herein are described in PCT application number PCT / CN2022 / 070624, which is hereby incorporated by reference in its entirety.

[0138] Therapy The antibodies or antigen-binding fragments thereof described in the present invention, or formulations containing said antibodies or antigen-binding fragments thereof, can be used for various therapeutic purposes. In one aspect, according to the present invention, there is provided a method for treating cancer in a subject, a method for reducing the rate of increase in tumor volume over time in a subject, a method for reducing the risk of metastasis, or a method for reducing the risk of occurrence of additional metastases in a subject. In some embodiments, according to the treatment, the progression of cancer can be arrested, alleviated, delayed, or suppressed. In some embodiments, the treatment can result in a decrease in the number, severity, and / or duration of one or more symptoms of cancer in the subject.

[0139] In one aspect, the present invention features a method comprising administering, in a therapeutically effective amount, an antibody or antigen-binding fragment thereof disclosed herein, or a formulation comprising the antibody or antigen-binding fragment thereof disclosed herein, to a subject in need thereof (e.g., a subject having or identified or diagnosed as having cancer). In some embodiments, the cancer is selected from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, and bone cancer.

[0140] As used herein, "effective amount" means an amount or dosage sufficient to obtain a beneficial or desired result, including preventing, alleviating, delaying, or suppressing the progression of a disease (e.g., cancer). The effective amount can vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and thus can be determined on an individual basis.

[0141] The effective amount can be administered in one or more doses. For example, an effective amount of an antibody or antigen-binding fragment thereof is an amount sufficient to improve, halt, stabilize, reverse, suppress, alleviate, and / or delay the progression of cancer in a patient, or an amount sufficient to improve, halt, stabilize, reverse, and / or delay in vitro the growth of cells (e.g., biopsy cells, any cancer cells or cell lines described herein (e.g., cancer cell lines)). As understood in the art, the effective amount of an antibody or antigen-binding fragment thereof, or a formulation comprising the antibody or antigen-binding fragment thereof, can vary depending on other factors such as the patient's medical history and the type (and / or dosage) of antibody used.

[0142] Kit Kits containing the formulations described herein are also provided. The kit can also include one or more containers containing pharmaceutically acceptable excipients and can include other materials necessary from a business and user perspective, such as filters, needles, syringes, etc. Instructions related to the kit can typically be included in a commercially available package for the treatment, prevention, or diagnosis of the product, which includes, for example, information regarding indications, usage, dosage, manufacture, administration, contraindications, and / or warnings related to the use of such treatment, prevention, and diagnostic products. The kit may also be labeled with a label that is any type of data carrier containing information (e.g., leaflet, seal, chip, printed matter, or barcode). In some embodiments, the methods of use described herein can be included on the label or in the label. The kit can further include, but is not limited to, a device for administering the formulation, particularly a device containing the formulation, i.e., a prefilled device such as a prefilled syringe or a prefilled autoinjector. The kit can also include a container containing the formulation, i.e., a prefilled container such as a prefilled vial, cartridge, sachet, or ampule.

[0143] The formulation can be included in a container, package, or dispenser, together with the method of administration. The present invention further provides a method for manufacturing an antibody or an antigen-binding fragment thereof for various uses described herein.

[0144] Examples The present invention is further illustrated in the following examples. These examples do not limit the scope of the present invention described in the claims.

[0145] Methods and Materials Develop a long-term stable preservation formulation of a recombinant anti-PD-L1 / anti-4-1BB bispecific antibody injection as described in the PCT application number PCT / CN2022 / 070624. To ensure the quality within the shelf life (for example, at least 24 months), an experiment was designed to screen the formulation containing the anti-PD-L1 / anti-4-1BB bispecific antibody and evaluate the effects of different excipients on the stability of the antibody formulation. The materials and methods used in the experiment are as follows.

[0146]

Table 1

[0147]

Table 2

[0148]

Table 3

[0149] The stability of the test samples in different formulations was measured in the following ways.

[0150] Appearance According to the relevant methods described in the "Chinese Pharmacopoeia", the appearance of the test samples was observed. See 122-129 of the "Pharmacopoeia of the People's Republic of China" (2020 Edition). Specifically, as the examiner, the visual acuity in distance and near vision should reach 4.9 or above (corrected visual acuity is 5.0 or above), and there should be no color blindness. The examiner can adjust the position so that the test sample is at the clear vision distance (the clear vision distance from the test sample to the human eye, usually 25 cm). The test sample was placed under scattered sunlight and inspected and observed horizontally against a white background. Then, the examiner judged whether the color and transparency of the test sample were similar to that of water in the same container and whether there was any obvious turbidity or precipitation.

[0151] Protein concentration The ultraviolet-visible absorption spectrum of a protein is an absorption spectrum generated by the occurrence of electronic energy level transitions after some groups in the molecule absorb ultraviolet and / or visible light. Protein molecules contain aromatic amino acids with conjugated double bonds such as tyrosine and leucine. They have a maximum absorbance at a wavelength of approximately 280 nm, and within a certain range, their absorbance is proportional to the protein concentration. From Lambert-Beer's law (A = ε×b×c, where A is the absorbance, ε is the extinction coefficient, b is the detection optical path, and c represents the protein concentration of the sample), the protein concentration can be determined.

[0152] The protein concentration was measured using an ultraviolet-visible spectrophotometer (GE, Ultrospec® 8000). Before measurement, the ultraviolet-visible spectrophotometer was turned on and preheated for 30 minutes. The test sample was appropriately diluted with water or the corresponding buffer solution so that the sample signal was between 0.200 and 0.800. After adjusting to zero with the buffer solution or water, the appropriately diluted sample was placed in a test cell for measurement.

[0153] pH The pH was measured using a pH meter. Before measurement, the test sample and calibration buffer solution were taken out and preheated to room temperature. The pH meter was turned on and the appropriate calibration mode was selected. The approximate pH range of the test sample was estimated and the standard buffer solution to be used was selected. The electrode was washed with deionized water or ultrapure water, and the remaining water was wiped off and dried. After confirming that the calibration slope was between 95% and 105% and the deviation was approximately 0 ± 30 mV, the pH value of the sample was measured. The electrode was washed with deionized water or ultrapure water, and the remaining water was wiped off and dried. Next, the electrode was immersed in the sample solution and the "Read" button was pressed. After gently shaking the sample solution until the reading became stable, the pH value of the sample was recorded.

[0154] SEC-HPLC Size-exclusion high performance liquid chromatography (SEC-HPLC) is an important method for the quality control of antibodies. This method can separate molecules based on differences in molecular size and hydrodynamic radius. By SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (HMW), main peak (mainly antibody monomer), and low molecular weight form (LMW). SEC-HPLC can determine the percentage of antibody monomers in a sample and provide information on the content of soluble aggregates and fragments.

[0155] The SEC-HPLC method described in this specification uses an XBridge BEH SEC 200 column. Specifically, a 100 mM PB + 100 mM NaCl solution (pH approximately 6.7 ± 0.1) was prepared as the mobile phase, and isocratic elution was performed. The injection volume was 10 μl, the flow rate was 0.8 ml / min, the collection time was 25 minutes, the column temperature was 25 °C, and the detection wavelength was 280 nm. Detection was performed using a measurement solution prepared by diluting the test sample to 1 mg / ml with the mobile phase.

[0156] IEX-HPLC Ion exchange high performance liquid chromatography (IEX-HPLC), especially cation exchange high performance liquid chromatography (CEX-HPLC), is usually used to measure antibody charge variants. In this measurement method, peaks eluted from the column earlier than the retention time of the main peak were designated as "acidic peaks", and peaks eluted from the column later than the retention time of the main peak were designated as "alkaline peaks". The IEX-HPLC method was used for measurement with a MAbPac (registered trademark) SCX-10 column. Mobile phase A: 20 mM MES, 10 mM NaCl, pH 5.6 ± 0.1, and mobile phase B: 20 mM MES, 300 mM NaCl, pH 5.6 ± 0.1. The injection volume was 40 μl, the flow rate was 0.6 ml / min, the collection time was 62 minutes, the column temperature was 25 °C, and the detection wavelength was 280 nm. Detection was performed using a measurement solution prepared by diluting the test sample to 1 mg / ml with mobile phase A.

[0157] Capillary Electrophoresis - SDS (CE - SDS) (Reducing / Non - reducing) Capillary electrophoresis - SDS (CE - SDS) is a method for measuring antibody purity using a capillary as the separation channel. In CE - SDS, protein migration is driven by the surface charge due to SDS binding, which is proportional to the molecular weight of the protein. Since all SDS - protein complexes have a similar mass - to - charge ratio, electrophoretic separation can be achieved based on molecular size or hydrodynamic radius in the capillary's molecular sieve gel matrix. This method is widely used to detect the purity of antibodies. Usually, in the non - reducing CE - SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. Next, the mixture is incubated at 68 - 72 °C for about 10 - 15 minutes. After cooling to room temperature, the mixture is centrifuged and the supernatant is taken for analysis. Protein migration is detected by an ultraviolet detector to obtain an electrophoretic spectrum. The purity of the antibody can be determined as the percentage of the peak area of the IgG main peak in the sum of all peak areas. In the reducing CE - SDS method, the test sample is mixed with SDS sample buffer and 2 - mercaptoethanol. Next, the mixture is incubated at 68 - 72 °C for about 10 - 15 minutes. After cooling to room temperature, the mixture is centrifuged and the supernatant is taken for analysis. The purity of the antibody can be determined as the percentage of the sum of the peak areas of the light chain peak and the heavy chain peak in the total sum of all peak areas.

[0158] The capillary is a fused silica capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before performing electrophoresis, the capillary column was equilibrated using 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel. When the protein concentration is less than 10 mg / ml and the salt concentration is higher than 50 mM, the sample must be replaced with water or other low-salt buffer by ultrafiltration to avoid the adverse effects of high salt concentration. 100 μg (less than 45 μl) of the sample was transferred to a 0.5 ml microcentrifuge tube, and sample buffer was added until the total volume reached 95 μl. Next, 2 μl of internal standard substance (10 kDa), 5 μl of iodoacetamide solution (250 mM), or 5 μl of 2-mercaptoethanol was added. After thorough mixing, the centrifuge tube was centrifuged at 300 g for 1 minute. After centrifugation, the centrifuge tube was sealed with parafilm, then incubated in a 70 °C water bath for 10 minutes and cooled to room temperature for at least 3 minutes. Then, 100 μl of the sample was transferred to a 200 μl micro-sample tube, and all bubbles were removed. The sample was stored in a universal bottle and the lid was closed. As the detection conditions, sample injection: 5 kV, 40 seconds, isolation voltage: 15 kV, 40 minutes. The capillary column temperature was maintained at 25 °C, and the detection wavelength was 214 nm.

[0159] RPC-UPLC In reversed-phase ultra-high performance liquid chromatography (RPC-UPLC), the samples were detected using a MAbPac® RP column (analytical 4 μm, 2.1 × 100 mm, PN: 088647, Thermo) and a Waters® ultra-high pressure liquid system (grade H or above). Mobile phase A was 0.1% TFA / aqueous solution, and mobile phase B was 0.1% TFA / acetonitrile solution. The injection volume was 5 μL, the detection wavelength was 214 nm (ultraviolet), the column temperature was 70 °C, and the injector temperature was approximately 2 - 8 °C. Each test sample diluted to 1 mg / ml with water was used as the measurement solution for detection.

Example

[0160] Example 1. Preparation and purification of anti-PD-L1 / anti-4-1BB bispecific antibody The same experiment as described in the examples of PCT application number PCT / CN2022 / 070624 was conducted, and BH3120, an anti-PD-L1 / anti-4-1BB bispecific antibody, was prepared and purified. The anti-PD-L1 / anti-4-1BB bispecific antibody comprises an anti-PD-L1 arm and an anti-4-1BB arm. The anti-PD-L1 arm comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 6, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 8, a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 4. The anti-4-1BB arm comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 12, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 14, a light chain variable region having the amino acid sequence shown in SEQ ID NO: 10, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 4.

Example

[0161] Example 2. Preparation of Antibody Formulation Based on the experimental results described in the examples of PCT application number PCT / CN2022 / 070624, the possible components and concentrations in the BH3120 formulation were estimated. Elements including pH, acetate, arginine-HCL, and trehalose were considered. The elements considered in the experiment and their concentration ranges are shown in the following table.

[0162]

Table 4

[0163]

Table 5

Example

[0164] Example 3. Confirmation of Antibody Preparation BH3120 protein samples were prepared, measured under different experimental conditions, and the stability of the proteins in different formulations was compared. The experiments were conducted as shown in the following table.

[0165]

Table 6

[0166] Incubation Test at 40°C The protein samples were placed in a constant-temperature incubator at 40°C and left for 4 weeks in the dark. Sampling was carried out at 0 week, 1 week, 2 weeks, and 4 weeks respectively to detect the changes in each test item. As a result of leaving each formulation sample at 40°C for 0 week, 1 week, 2 weeks, and 4 weeks, all were colorless and transparent liquids, and there was no obvious turbidity or precipitation. The changes in protein concentration, pH, purity, and charge isomers are shown in the following table. As a result, it was found that the change in protein concentration within 4 weeks at 40°C was less than 10%, the decrease in SEC-HPLC purity within 2 weeks at 40°C was less than 3%, and the decrease in the main peak ratio of charge isomers within 4 weeks at 40°C was less than 10%. It was also found that the decrease in purity of non-reduced and reduced CE-SDS was less than 3% within 4 weeks at 40°C.

[0167]

Table 7

[0168] Freeze-Thaw Cycle Test at -70°C The sample dispensed into the vial was left at -70°C for at least 4 hours to be completely frozen. Next, the sample was completely thawed at room temperature or 25°C (one cycle). Sampling was performed after cycles 1, 3, and 5 to detect changes in each test item. As a result, after the freeze-thaw cycle, the sample was a colorless and transparent liquid, and there was no obvious turbidity or precipitation. There were also no obvious changes in other test items. The detailed results are shown in the table below.

[0169]

Table 8

[0170] Oscillation test at 250 rpm The dispensed sample was fixed to the shaker in the dark and shaken at a speed of 250 rpm. Sampling was performed after 1 day, 3 days, and 5 days to detect changes in each test item. As a result, under these stirring conditions, the sample was a colorless and transparent liquid, and there was no obvious turbidity or precipitation. There were also no significant changes in other test items. The detailed results are shown in the table below.

[0171]

Table 9

[0172] Incubation test at 25°C The protein sample was placed in an incubator at 25°C and left for 6 months in the dark. Sampling was performed at 0 month, 1 month, 2 months, 3 months, and 6 months, respectively, to detect changes in each test item (appearance, protein concentration, pH, purity (detected by SEC-HPLC, RPC-UPLC, and CE-SDS methods), and charge variants (detected by IEX-HPLC)). As a result, under these test conditions, the sample was a colorless and transparent liquid, and there was no obvious turbidity or precipitation. The detailed results are shown in the following table. As a result, it was found that within 6 months at 25°C, the change in protein concentration was less than 10%, the decrease in SEC-HPLC purity within 6 months at 25°C was less than 3.5%, and the decrease in the main peak ratio of charge variants within 6 months at 25°C was less than 15%. It was also found that within 6 months at 25°C, the purity decrease of non-reduced and reduced CE-SDS was less than 3%.

[0173]

Table 10-1

Table 10-2

[0174] In short, all test formulations BH3120 showed good thermal stability. As expected, the experimental results have shown that these formulations can more effectively suppress the formation of acidic peaks.

[0175] Other Embodiments Although the present invention has been described in detail by combining specific embodiments, the above description does not limit the scope of the present invention and is used as an exemplary description. The scope of the present invention is defined by the appended claims. Any other aspects, advantages, and improvements are all within the scope of the claims.

[0176] The sequences disclosed in the specification are shown in Table 11 below.

[0177]

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Claims

1. A stable aqueous pharmaceutical formulation comprising an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof and a buffer, having a pH of about 5.0 to about 6.

5.

2. The formulation according to claim 1, wherein the pH of the formulation is about 5.5 to about 6.

0.

3. The formulation according to claim 1, wherein the pH of the formulation is about 5.6 to about 5.8, or about 5.8 to about 6.

0.

4. The formulation according to any one of claims 1 to 3, wherein the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is capable of binding to human PD-L1 and / or human 4-1BB.

5. The formulation according to any one of claims 1 to 4, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 10 mg / ml to about 100 mg / ml.

6. The formulation according to any one of claims 1 to 5, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 20 mg / ml to about 80 mg / ml.

7. The formulation according to any one of claims 1 to 6, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 50 mg / ml.

8. The formulation according to any one of claims 1 to 7, wherein the buffer contains a buffering agent selected from the group consisting of acetic acid, sodium acetate, tartrate, hydrogen chloride, sodium dihydrogen phosphate, and combinations thereof.

9. The formulation according to claim 8, wherein the buffer contains acetic acid and / or sodium acetate.

10. The formulation according to any one of claims 1 to 9, wherein the formulation contains an acetic acid / sodium acetate buffer having a concentration of about 10 mM to about 100 mM.

11. The formulation according to any one of claims 1 to 10, wherein the formulation contains an acetic acid / sodium acetate buffer having a concentration of about 10 mM or about 15 mM.

12. The formulation according to any one of claims 1 to 11, further comprising an isotonic agent selected from the group consisting of trehalose, sucrose, proline, glycine, arginine, alanine, glutamic acid, methionine, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, and combinations thereof.

13. The formulation according to claim 12, wherein the isotonic agent is trehalose, arginine, or a combination thereof.

14. The formulation according to any one of claims 1 to 13, wherein the formulation contains from about 10 mM to about 200 mM of arginine.

15. The formulation according to any one of claims 1 to 14, wherein the formulation contains about 100 mM or about 140 mM of arginine.

16. The formulation according to any one of claims 12 to 15, wherein the arginine is in the form of arginine hydrochloride (arginine-HCl).

17. The formulation according to any one of claims 1 to 16, wherein the formulation contains from about 10 mM to about 300 mM of trehalose.

18. The formulation according to any one of claims 1 to 17, wherein the formulation contains about 130 mM of trehalose.

19. Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80, SDS, Poloxamer 188 (Pluronic (R) F68) and a surfactant selected from the group consisting of combinations thereof, the formulation according to any one of claims 1 to 18.

20. The formulation according to claim 19, wherein the surfactant is polysorbate 80.

21. The formulation according to any one of claims 1 to 20, wherein the formulation contains from about 0.01 mg / ml to about 10 mg / ml of polysorbate 80.

22. The formulation according to any one of claims 1 to 21, wherein the formulation contains about 0.2 mg / ml of polysorbate 80.

23. An anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof at a concentration of about 10 mg / ml to about 100 mg / ml, An isotonic agent at a concentration of about 100 mM to about 300 mM, A surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml, and A buffer system, and a stable aqueous pharmaceutical formulation having a pH of about 5.0 to about 6.

5.

24. The formulation according to claim 23, having a pH of about 5.5 to about 6.

0.

25. The formulation according to claim 23 or 24, wherein the buffer system contains one or more buffering agents selected from the group consisting of acetic acid, sodium acetate, tartrate, hydrogen chloride, and sodium dihydrogen phosphate.

26. The formulation according to any one of claims 23 to 25, wherein the buffer system contains an acetic acid / sodium acetate buffer at a concentration of about 10 mM to about 100 mM.

27. The formulation according to any one of claims 23 to 26, wherein the isotonic agent is trehalose, sucrose, proline, glycine, arginine, alanine, glutamic acid, methionine, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, or a combination thereof.

28. The formulation according to claim 27, wherein the isotonic agent is trehalose, arginine (e.g., arginine-HCl), or a combination thereof.

29. The surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 (Pluronic (R) F68), and combinations thereof, the formulation according to any one of claims 23 to 28.

30. The preparation according to claim 29, wherein the surfactant is polysorbate 80.

31. An anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof having a concentration of about 10 mg / ml to about 100 mg / ml, arginine (e.g., arginine-HCl) having a concentration of about 10 mM to about 200 mM, optionally trehalose having a concentration of about 10 mM to about 300 mM, polysorbate 80 having a concentration of about 0.01 mg / ml to about 10 mg / ml, and an acetic acid / sodium acetate buffer having a concentration of about 10 mM to about 100 mM, or consisting of these components, and having a pH of about 5.5 to about 6.0, a stable aqueous pharmaceutical preparation.

32. The preparation according to claim 31, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 20 mg / ml, about 50 mg / ml, or about 80 mg / ml.

33. The preparation according to claim 31 or 32, wherein the concentration of the alanine is about 100 mM or about 140 mM.

34. The preparation according to any one of claims 31 to 33, wherein the concentration of the trehalose is about 130 mM.

35. The preparation according to any one of claims 31 to 34, wherein the concentration of the polysorbate 80 is about 0.2 mg / ml.

36. The preparation according to any one of claims 31 to 35, wherein the concentration of the acetic acid / sodium acetate buffer is about 10 mM or about 15 mM.

37. The anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is a first heavy chain variable region (VH1) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 15, the VH1 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 16, and the VH1 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 17, a first heavy chain variable region (VH1), A first variable light chain region (VL1) comprising CDR1, 2, 3, wherein the VL1 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 18, the VL1 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 19, and the VL1 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

20. The formulation according to any one of claims 1 to 36, wherein the VH1 and VL1 can interact to form an antigen-binding site that binds to PD-L1.

38. The anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof A second variable heavy chain region (VH2) comprising complementarity-determining regions (CDR) 1, 2, 3, wherein the VH2 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 21, the VH2 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 22, and the VH2 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

23. A second variable light chain region (VL2) comprising CDR1, 2, 3, wherein the VL2 CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 24, the VL2 CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 25, and the VL2 CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

26. The formulation according to any one of claims 1 to 37, wherein the VH2 and VL2 can interact to form an antigen-binding site that binds to 4-1BB.

39. The anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof Comprises a first variable heavy chain region (VH1) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 6 and a first variable light chain region (VL1) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2, and Comprises a second variable heavy chain region (VH2) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12 and a second variable light chain region (VL2) comprising an amino acid sequence having at least 90% identity with SEQ ID NO:

10. The VH1 and VL1 can interact to form an antigen-binding site that binds to PD-L1. The formulation according to any one of claims 1 to 38, wherein the VH2 and VL2 can interact to form an antigen-binding site that binds to 4-1BB.

40. The anti-PD-L1 / anti-4-1BB bispecific antibody or its antigen-binding fragment is comprising a first heavy chain constant region (CH1) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 8, and a first light chain constant region (CL1) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4, and comprising a second heavy chain constant region (CH2) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 14, and a second light chain constant region (CL2) comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4, the CH1 and CL1 can interact to form an antigen-binding site that binds to PD-L1, The formulation according to any one of claims 1 to 39, wherein the CH2 and CL2 can interact to form an antigen-binding site that binds to 4-1BB.

41. The formulation according to any one of claims 1 to 40, wherein the formulation has long-term stability.

42. A method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of the formulation according to any one of claims 1 to 41.

43. The method according to claim 42, wherein the subject suffers from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, or bone cancer.

44. The method according to claim 42 or 43, wherein the subject is human.

45. An anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, comprising an anti-PD-L1 arm comprising a first heavy-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 6 and a first light-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2, and an anti-4-1BB arm comprising a second heavy-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12 and a second light-chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12, (b) acetic acid / sodium acetate at 10 mM or 15 mM, (c) arginine (e.g., arginine-HCl) at 100 mM or 140 mM, (d) optional trehalose at 130 mM, (e) polysorbate 80 at 0.2 mg / ml, or a formulation of an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof that contains, consists of, or consists essentially of these components and exhibits long-term stability, wherein the pH of the formulation is from 5.5 to 6.0 (e.g., 5.6 or 5.8). **Claim 46** The formulation according to claim 45, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is from about 10 mg / ml to about 100 mg / ml. **Claim 47** The formulation according to claim 45 or 46, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is from about 20 mg / ml to about 80 mg / ml. **Claim 48** The formulation according to any one of claims 45 to 47, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof is about 20 mg / ml, about 50 mg / ml, or about 80 mg / ml.

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