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

Abstract

Anti-PD-L1 / anti-4-1BB bispecific antibody formulations and methods of making and using the same are provided. In one aspect, 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.

Description

[0001] This application claims priority to the PCT application with application number PCT / CN2022 / 104038 filed with the State Intellectual Property Office of China on July 6, 2022, and invention name “Anti-PD-L1 / Anti-4-1BB Bispecific Antibody Preparation”, the entire contents of which are incorporated into this application by reference. Technical Field

[0002] The present invention relates to an anti-PD-L1 / anti-4-1BB bispecific antibody preparation and a preparation and use method thereof. Background Art

[0003] Like other protein therapeutic agents, antibodies are complex molecules. Usually, a large amount of antibodies have to be used in pharmaceutical preparations to reach a therapeutically effective dose in mammals. Since antibodies are larger and more complex than traditional organic and inorganic drugs, special problems are brought to the preparation of antibodies. In order for antibodies to maintain biological activity, preparations must maintain the conformational integrity of the amino acid sequence while protecting the antibodies from degradation. In addition, monoclonal and polyclonal antibodies are particularly likely to be relatively unstable. Although there are a large number of preparations to choose from, no method or system is suitable for all antibodies. Several factors that need to be considered in formulation development have been reported in the literature (Wang et al., "Antibody structure, instability, and formulation." Journal of pharmaceutical sciences 96.1 (2007): 1-26).

[0004] Many factors can affect the stability of antibodies. In fact, antibody structures can be heterogeneous, which makes the preparation of stable formulations of these antibodies more complicated. In addition, the excipients included in the antibody formulation should preferably minimize any potential immune response. There is still a need for stable formulations of such antibodies. Summary of the invention

[0005] The present invention relates to anti-PD-L1 / anti-4-1BB bispecific antibody (particularly BH3120) preparations and methods for preparing and using the same. BH3120 can be combined with PD-L1 and 4-1BB at the same time and used for cancer treatment. The present application provides a BH3120 preparation with good thermal stability. The preparation described in the present application can keep the anti-PD-L1 / anti-4-BB bispecific antibody in a liquid solution, making it difficult to decompose, aggregate or undergo undesirable chemical modification.

[0006] In one aspect, 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 about 5.0 to about 6.5. In some embodiments, the pH of the formulation is 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 its antigen-binding fragment can bind 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 its antigen-binding fragment 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 its antigen-binding fragment 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 its antigen-binding fragment is about 50 mg / ml. In some embodiments, the buffer comprises a buffer selected from acetic acid, sodium acetate, tartrate, hydrogen chloride, sodium dihydrogen phosphate, and a combination thereof. In some embodiments, the buffer comprises acetic acid and / or sodium acetate. In some embodiments, the formulation comprises an acetic acid / sodium acetate buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, the preparation comprises an acetic acid / sodium acetate buffer having a concentration of about 10 or about 15 mM. In some embodiments, the preparation further comprises a tension agent selected from trehalose, sucrose, proline, glycine, arginine, alanine, glutamic acid, methionine, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, and combinations thereof. In some embodiments, the tension agent is trehalose, arginine, or a combination thereof. In some embodiments, the preparation comprises about 10 mM to about 200 mM arginine. In some embodiments, the preparation comprises about 100 mM or about 140 mM arginine. In some embodiments, the arginine is a hydrochloride form of arginine (arginine-HCl). In some embodiments, the preparation comprises about 10 mM to about 300 mM trehalose. In some embodiments, the preparation comprises about 130 mM trehalose. In some embodiments, the formulation further comprises a surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 ( F68) and combinations thereof. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the formulation comprises 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 one aspect, 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 at a concentration of about 10 mg / ml to about 100 mg / ml; a tonicity 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, wherein in some embodiments, the pH of the formulation is about 5.0 to about 6.5. In some embodiments, the pH of the formulation is about 5.5 to about 6.0. In some embodiments, the buffer system comprises one or more buffers selected from 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 to about 100 mM. In some embodiments, the tonicity agent is trehalose, sucrose, proline, glycine, arginine, alanine, glutamic acid, methionine, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, or a combination thereof. In some embodiments, the tonicity agent is trehalose, arginine (e.g., arginine-HCl), or a combination thereof. In some embodiments, the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 ( F68) or a combination thereof. In some embodiments, the surfactant is polysorbate 80.

[0011] In one aspect, 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 acetic acid / sodium acetate buffer at a concentration of about 10 to about 100 mM, 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 its antigen-binding fragment is about 20 mg / ml, about 50 mg / ml, or about 80 mg / ml. In some embodiments, the concentration of the arginine is about 100 mM or about 140 mM. In some embodiments, the concentration of trehalose is about 130 mM. In some embodiments, the concentration of polysorbate 80 is about 0.2 mg / ml. In some embodiments, the concentration of 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), the first heavy chain variable region (VH1) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 15, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 16, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 17; the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, in some embodiments, the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 18, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 19, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 11. NO:20 has an amino acid sequence with at least 80% identity. In some embodiments, the VH1 and VL1 can interact with each other 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), the second heavy chain variable region (VH2) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 21, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 22, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 23; 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 at least 80% identical to SEQ ID NO: 24, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 25, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: NO:26 has an amino acid sequence with at least 80% identity. In some embodiments, the VH2 and VL2 can interact with each other 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 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), the first heavy chain variable region (VH1) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 6, the first light chain variable region (VL1) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, in some embodiments, VH1 and VL1 can interact with each other to form an antigen binding site that binds to PD-L1; the second heavy chain variable region (VH2) comprises an amino acid sequence at least 90% identical to SEQ ID NO: 12, the second light chain variable region (VL2) comprises an amino acid sequence at least 90% identical to SEQ ID NO: 10, in some embodiments, the VH2 and VL2 can interact with each other 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 antigen-binding fragment thereof comprises a first heavy chain constant region and a first light chain constant region and a second heavy chain constant region and a second light chain constant region, the first heavy chain constant region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, and the first light chain constant region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4; the second heavy chain constant region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, and the second light chain constant region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4.

[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, the first heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 27, the first light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 28, and in some embodiments, the first heavy chain and the first light chain can interact with each other and bind to PD-L1; the second heavy chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, and the second light chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 30, and in some embodiments, the second heavy chain and the second light chain can interact with each other and 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 a formulation described herein. In some embodiments, 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. In some embodiments, the subject is human.

[0019] In one aspect, the present invention relates to a formulation of an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof exhibiting long-term stability, comprising, consisting of, or consisting essentially of: (a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof comprising an anti-PD-L1 arm and an anti-4-1BB arm, wherein the anti-PD-L1 arm comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6, and the first light chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2; the anti-4-1BB arm comprises a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO: 12, and the second light chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13. NO: 10 amino acid sequences with at least 90% identity; (b) 10mM or 15mM acetic acid / sodium acetate; (c) 100mM or 140mM arginine (e.g., arginine-HCl); (d) optionally 130mM trehalose; and (e) 0.2mg / ml polysorbate 80, in some embodiments, the pH of the formulation 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 its antigen-binding fragment is about 10mg / ml to about 100mg / ml. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or its antigen-binding fragment is about 20mg / ml to about 80mg / ml. In some embodiments, the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or its antigen-binding fragment is about 20mg / ml, about 50mg / ml, or about 80mg / ml.

[0020] As used herein, the term "cancer" refers to a cell with autonomous growth ability. Examples of such cells include cells with abnormal states or conditions characterized by rapid proliferation of cell growth. The term is intended to include cancerous growths, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasion stage. Malignant tumors of various organ systems are also included, such as the respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, liver system, gastrointestinal system, and endocrine system; and adenocarcinomas, which include malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, and small intestinal cancers. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers caused by insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancers 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. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinoma tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor of a mesenchymal cell derivative. The term "hematopoietic neoplastic disease" includes diseases involving hyperplastic / / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic diseases can arise from the bone marrow, lymphoid or erythroid lineages or their precursor cells.

[0021] As used herein, the term "antibody" refers to any antigen binding molecule containing at least one (e.g., one, two, three, four, five or six) complementary 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 specific binding to an epitope. Non-limiting examples of antibodies include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, mouse antibodies, etc. In some embodiments, the antibody may 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, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0022] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody 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 described herein specifically include "chimeric" antibodies. As used herein, the term "chimeric antibody" refers to such antibodies (immunoglobulins) and fragments of such antibodies: a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in the antibody derived from a particular species, and the remainder of the chain is identical or homologous to the corresponding sequence in the antibody derived from another species, as long as it exhibits the desired biological activity. Typically, chimeric antibodies are antibodies whose light chain and heavy chain genes have been constructed from antibody variable and constant region genes belonging to different species by, for example, genetic engineering. For example, the variable fragments of the genes from mouse monoclonal antibodies can be connected to human constant fragments. In some embodiments, the antigen binding site is derived from mice, and the Fc portion is derived from humans.

[0024] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of mammalian immunoglobulin heavy or light chains) that is capable of specifically binding 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 to which the treatment according to the method described in the present invention is provided. The present invention contemplates veterinary and non-veterinary applications. In some embodiments, the object is a mammal (e.g., a non-human mammal). The object may include, but is not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, giant pandas and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other domestic, farm and zoo animals. In some embodiments, the object is a person.

[0026] As used herein, when referring to antibodies, the phrases "specifically bind" and "specifically bind to..." mean that the antibody preferably interacts with its target molecule (e.g., PD-L1 or 4-1BB) compared to other molecules because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the agent recognizes and binds to molecules including a specific structure, rather than all molecules in general. 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 a 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 "approximately" or "about" shall generally mean within 20%, within 10%, within 5%, 4%, 3%, 2% or within 1% of a given value or range. The numerical values ​​given are approximate, which means that if not explicitly stated, the term "approximately" or "about" can be inferred.

[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 term "long-term storage" or "long-term stability" should be understood to mean that the pharmaceutical composition can be stored for a minimum stable shelf life of three months or more, six months or more, and preferably one year or more, most preferably at least two years.

[0030] As used herein, the term "stable" for long-term storage is understood to mean that the antibody contained in the pharmaceutical composition does not lose more than 30%, preferably 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to the composition at the beginning of storage.

[0031] As used herein, the term "substantially free" means that no substance is present or only a very small trace amount of the substance is present, which has no substantial effect on the properties of the composition. If the amount of a substance is not mentioned, it should be understood as "undetectable amount".

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

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

[0034] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail.

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

[0036] Similar to other proteins, antibodies are susceptible 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 the stress conditions. These degradation pathways fall into two major categories - physical and chemical instability.

[0037] Although the antibodies have similar structures, the behaviors of the antibodies appear to be different. The present invention provides anti-PD-L1 / anti-4-1BB bispecific antibody formulations and methods for preparing and using the same. On the one hand, the present invention provides a stable aqueous formulation of anti-PD-L1 / anti-4-1BB antibodies that allows long-term storage.

[0038] Antibody preparations

[0039] The present invention relates to anti-PD-L1 / anti-4-1BB bispecific antibody preparations. The preparations of the present invention may be present in the form of liquid and lyophilized powder, comprising anti-PD-L1 / anti-4-1BB bispecific antibodies or antigen-binding fragments thereof. In addition, such preparations may include buffers, tension agents, surfactants, stabilizers and some other excipients.

[0040] In some embodiments, the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof may be any anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment as described herein.

[0041] 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 present in the formulation in an amount of about 5 mg / mL to about 200 mg / mL, about 5 mg / mL to about 180 mg / mL, about 5 mg / mL to about 160 mg / mL, about 5 mg / mL to about 140 mg / mL, about 5 mg / mL to about 120 mg / mL, about 5 mg / mL to about 100 mg / mL, about 5 mg / mL to about 80 mg / mL, about 5 mg / mL to about 60 mg / mL, about 5 mg / mL to about 40 mg / mL, about 5 mg / mL to about 20 mg / mL, about 1 0 mg / mL to about 200 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 100 mg / mL, about 10 mg / mL to about 80 mg / mL, about 10 mg / mL to about 60 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 20 mg / mL, about 20 mg / mL to about 200 mg / mL, about 20 mg / mL about 20 mg / mL to about 180 mg / mL, about 20 mg / mL to about 160 mg / mL, about 20 mg / mL to about 140 mg / mL, about 20 mg / mL to about 120 mg / mL, about 20 mg / mL to about 100 mg / mL, about 20 mg / mL to about 80 mg / mL, about 20 mg / mL to about 60 mg / mL, about 20 mg / mL to about 40 mg / mL, about 20 mg / mL to about 30 mg / mL, about 30 mg / mL to about 200 mg / mL, about 30 mg / mL to about 180 mg / mL, about 30 mg / mL to about 160 mg / mL g / mL, about 30 mg / mL to about 140 mg / mL, about 30 mg / mL to about 120 mg / mL, about 30 mg / mL to about 100 mg / mL, about 30 mg / mL to about 80 mg / mL, about 30 mg / mL to about 60 mg / mL, about 30 mg / mL to about 40 mg / mL, about 40 mg / mL to about 200 mg / mL, about 40 mg / mL to about 180 mg / mL, about 40 mg / mL to about 160 mg / mL, 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 / 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 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 1 180 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, about 140 mg / mL to about 160 mg / mL,In some embodiments, the concentration is 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 can be 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. / 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.

[0042] In alternative embodiments, the antibody or antigen-binding fragment may be 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.

[0043] Since antibodies have relatively high concentrations in formulations, anti-PD-L1 / anti-4-1BB bispecific antibody formulations were developed to improve the stability of these antibodies in formulations.

[0044] Buffer

[0045] The formulation may include a variety of buffers, such as acetic acid, sodium acetate, tartrate, sodium dihydrogen phosphate and combinations thereof. The buffer maintains a physiologically suitable pH. In addition, the buffer enhances the isotonicity and chemical stability of the formulation.

[0046] In some embodiments, the buffer is present in the formulation at a concentration of about 10 mM to about 100 mM, such as about 10 mM to about 15 mM. For example, the buffer can be 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, 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51mM , about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61mM, about 62mM, about 63mM, about 64mM, about 65mM, about 66mM, about 67mM, about 68mM, about 69mM, about 70mM, about 71mM, about 72mM, about 73mM, about 74mM, about 75mM, about 76mM mM, about 77mM, about 78mM, about 79mM, about 80mM, about 81mM, about 82mM, about 83mM, about 84mM, about 85mM, about 86mM, about 87mM, about 88mM, about 89mM, about 90mM, about 91mM, about 92mM, about 93mM, about 94mM, about 95mM, about 96mM, about 97mM, about 98mM, about 99mM or about 100mM.

[0047] In certain embodiments, the pH of the formulation is at or below pH 6.5. In certain embodiments, the pH of the formulation is at or below pH 6.0. In certain embodiments, the pH of the formulation is at or above pH 5.0. In certain embodiments, the pH of the formulation is at or above pH 5.5.

[0048] In some embodiments, the pH of the formulation is from about 5.0 to about 6.5. In some embodiments, the pH 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 , about 5.1 to about 5.6, about 5.1 to about 5.5, about 5.1 to about 5.4, about 5.1 to about 5.3, about 5.1 to about 5.2, about 5.2 to about 6.5, about 5.2 to about 6.4, about 5.2 to about 6.3, about 5.2 to about 6.2, about 5.2 to about 6.1, about 5.2 to about 6.0, about 5.2 to about 5.9, about 5.2 to about 5.8, about 5.2 to about 5.7, about 5.2 to about 5.6, about 5.2 to about 5.5, about 5.2 to about 5.4, about 5.2 to about 5.3, about 5.3 to about 6.5, about 5.3 to about 6.4, about 5.3 to about 6.3, about 5.3 to about 6.2, about 5.3 to about 6.1, about 5.3 to about 6.0, about 5.2 to about 5.9, about 5.2 to about 5.8, about 5.2 to about 5.7 .3 to about 5.9, about 5.3 to about 5.8, about 5.3 to about 5.7, about 5.3 to about 5.6, about 5.3 to about 5.5, about 5.3 to about 5.4, about 5.4 to about 6.5, about 5.4 to about 6.4, about 5.4 to about 6.3, about 5.4 to about 6.2, about 5.4 to about 6.1, about 5.4 to about 6.0, about 5.4 to about 5.9, about 5.4 to about 5.8, about 5.4 to about 5.7, about 5.4 to about 5.6, about 5.4 to about 5.5, about 5.5 to about 6.5, about 5.5 to about 6.4, about 5.5 to about 6.3, about 5.5 to about 6.2, about 5.5 to about 6.1, about 5.5 to about 6.0, about 5.5 to about 5.9, about 5.5 to about 5.8 about 5.8, about 5.5 to about 5.7, about 5.5 to about 5.6, about 5.6 to about 6.5, about 5.6 to about 6.4, about 5.6 to about 6.3, about 5.6 to about 6.2, about 5.6 to about 6.1, about 5.6 to about 6.0, about 5.6 to about 5.9, about 5.6 to about 5.8, about 5.6 to about 5.7, about 5.7 to about 6.5, about 5.7 to about 6.4, about 5.7 to about 6.3, about 5.7 to about 6.2, about 5.7 to about 6.1, about 5.7 to about 6.0, about 5.7 to about 5.9, about 5.7 to about 5.8, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1, about 5.8 to about 6.0, about 5.8 to about 5.9, about 5.9 to about 6.5, about 5.9 to about 6.4, about 5.9 to about 6.3, about 5.9 to about 6.2, about 5.9 to about 6.1, about 5.9 to about 6.0, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about 6.0 to about 6.1, about 6.1 to about 6.5, about 6.1 to about 6.4, about 6.1 to about 6.3, about 6.1 to about 6.2, about 6.2 to about 6.5, about 6.2 to about 6.4, about 6.2 to about 6.3, about 6.3 to about 6.5, about 6.3 to about 6.4, or 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.

[0049] The pH of the formulation can be measured by any means known to those skilled in the art. One way to measure the pH value is to use a pH meter with a microelectrode. The pH of the formulation can be adjusted using any means known in the art. Exemplary chemicals for changing the pH of the formulation are hydrochloric acid (HCl) and sodium hydroxide (NaOH).

[0050] 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 comprises 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 10-15 mM, about 10-20 mM, about 10-30 mM, about 10-40 mM, about 10-50 mM, about 10-60 mM, about 10-70 mM, about 10-15 mM, about 10-20 mM, about 10-20 mM, about 10-30 mM, about 10-40 mM, about 10-50 mM, about 10-20 mM, about 10-15 mM, about 10-20 mM, about 10-30 mM, about 10-40 mM, about 10-50 mM, about 10-60 mM, about 10-15 mM, about 10-20 mM, about 10-15 mM, about 10-20 mM, about 10-20 mM, about 10-30 mM, about 10-30 mM, about 10- M, about 10-60mM, about 10-50mM, about 10-40mM, about 10-30mM, about 10-20mM, about 20-100mM, about 20-90mM, about 20-80mM, about 20-70mM, about 20-60mM, about 20-50mM, about 20-40mM, about 20-30mM, about 30-100mM , about 30-90mM, about 30-80mM, about 30-70mM, about 30-60mM, about 30-50mM, about 30-40mM, about 40-100mM, about 40-90mM, about 40-80mM, about 40-70mM, about 40-60mM, about 40-50mM, about 50-100mM, about 50-90mM, In some embodiments, the buffer described herein comprises 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, about 100 mM, about 150 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, or about 100 mM of acetic acid / sodium acetate. When referring to the concentration of acetic acid / sodium acetate buffer, the concentration means the number of moles of acetic acid and sodium acetate together divided by the volume. In some embodiments, the buffer described herein comprises 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 of acetic acid / sodium acetate. In some embodiments, the formulation is substantially free of any other buffer (eg, citrate buffer, succinate buffer, histidine buffer, or phosphate buffer).

[0051] As shown in the present invention, the formulations described herein provide significant improvements in the stability of the anti-PD-L1 / anti-4-1BB bispecific antibodies described herein. For example, when the intended formulation includes anti-PD-L1 / anti-4-1BB bispecific antibodies and acetic acid / sodium acetate buffer, wherein the pH of the formulation is 5.0-6.5 (e.g., 5.6 or 5.8), the formulation provides significant improvements in stability, wherein no visible foreign matter is observed.

[0052] In some embodiments, the formulation described herein comprises anti-PD-L1 / anti-4-1BB bispecific antibody (10-100 mg / ml), 10-100 mM acetic acid / sodium acetate, 10-200 mM arginine-HCl and 0.01 mg / ml-10 mg / ml polysorbate 80, and its pH is 5.5-6.0. In some embodiments, the formulation further comprises 100-300 mM trehalose.

[0053] In some embodiments, the formulation described herein includes anti-PD-L1 / anti-4-1BB bispecific antibodies, 10-20 mM acetic acid / sodium acetate, 100-200 mM arginine-HCl, 0.1 mg / ml-0.5 mg / ml polysorbate 80, and its pH is 5.5-6.0. In some embodiments, the formulation further comprises 100-150 mM trehalose.

[0054] Surfactants

[0055] The preparation may optionally include a surfactant. The surfactant may be a stabilizer. The surfactant / stabilizer is a chemical compound that interacts with the biomolecules and / or general pharmaceutical excipients in the preparation and acts as a stabilizer. Surfactants generally protect active ingredients from stress caused by the air / solution interface and stress caused by the solution / surface, which can cause protein aggregation. Surfactants may include, but are not limited to, polysorbates, glycerol, dicarboxylic acids, oxalic acid, succinic acid, fumaric acid, phthalic acid and combinations thereof. Surfactants may be used, including, for example, nonionic or ionic detergents, as long as they are pharmaceutically acceptable, i.e., suitable for application to an object. In some embodiments, the surfactant is a polysorbate. Examples of polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0056] In some embodiments, surfactant is present in the preparation in an amount of about 0.01 mg / ml to about 1 mg / ml. For example, surfactant can be present in the preparation in the following amount: 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, such as about 0.2 mg / ml.

[0057] In some embodiments, surfactant is polysorbate 20 or polysorbate 80.For example, polysorbate 80 can exist with the amount of about 0.01mg / ml to about 1mg / ml, about 0.1mg / ml to about 0.5mg / ml, about 0.1mg / ml to about 0.3mg / ml, for example, about 0.2mg / ml.In alternative embodiments, polysorbate 80 exists with the amount of about 0.01mg / ml to about 1mg / ml, about 0.05mg / ml to about 0.5mg / ml and about 0.1mg / ml to about 0.2mg / ml.In other alternative embodiments, polysorbate 80 exists with the amount of about 0.01mg / ml to about 1mg / ml, about 0.05mg / ml to about 0.5mg / ml and about 0.1mg / ml to about 0.3mg / ml, for example, about 0.2mg / ml. In some embodiments, the formulations described herein comprise from about 0.01 mg / ml to about 10 mg / ml (e.g., from about 0.01 mg / ml to about 9 mg / ml, from about 0.01 mg / ml to about 8 mg / ml, from about 0.01 mg / ml to about 7 mg / ml, from about 0.01 mg / ml to about 6 mg / ml, from about 0.01 mg / ml to about 5 mg / ml, from about 0.01 mg / ml to about 4 mg / ml, from about 0.01 mg / ml to about 3 mg / ml, from 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) of polysorbate 80.

[0058] Polysorbate 20 can be with about 0.01mg / ml to about 1mg / ml, about 0.1mg / ml to about 0.5mg / ml, about 0.1mg / ml to about 0.3mg / ml, for example, the amount of about 0.2mg / ml exists.In alternative embodiments, polysorbate 20 is with about 0.01mg / ml to about 1mg / ml, about 0.05mg / ml to about 0.5mg / ml or about 0.1mg / ml to about 0.2mg / ml amount exists.In other alternative embodiments, polysorbate 20 is with about 0.01mg / ml to about 1mg / ml, about 0.05mg / ml to about 0.5mg / ml and about 0.1mg / ml to about 0.3mg / ml, for example, the amount of about 0.2mg / ml exists.

[0059] Tension Agents

[0060] The preparation of the present invention may also include a tonicity agent. Typically, a tonicity agent is used to adjust or maintain the osmotic pressure of a preparation so that it is closer to the osmotic pressure of a body fluid (e.g., blood or plasma). A tonicity agent may also maintain the level of an antibody or antigen binding fragment in a preparation. As used herein, the term "tonicity" refers to the behavior of a biological component in a fluid environment or solution. An isotonic solution has the same osmotic pressure as plasma and can be intravenously infused into a subject without changing the osmotic pressure of the subject's plasma. In fact, in certain embodiments, a tonicity agent is present in an amount sufficient to make the preparation suitable for intravenous infusion. Typically, a tonicity agent also acts as a filler or stabilizer. Therefore, a tonicity agent may enable an antibody or its antigen binding fragment to overcome a variety of stresses, such as freezing and shearing. A tonicity agent may include, but is not limited to, sugars, carbohydrates, glycerol, sorbitol, mannitol, sodium chloride, potassium chloride, magnesium chloride, and other inorganic salts. Those skilled in the art will appreciate that other tonicity agents may be used, as long as they are pharmaceutically acceptable, i.e., suitable for application to a subject.

[0061] In certain embodiments, the tonicity agent is a sugar. Examples of sugars include glucose, sucrose, maltose, trehalose, dextrose, xylitol, fructose, and mannitol. In some embodiments, the formulations described herein comprise 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 50-40 ... 0-350mM, about 50-300mM, about 50-250mM, about 50-200mM, about 50-150mM, about 50-100mM, about 100-500mM, about 100-450mM, about 100-400mM, about 100-350mM, about 100-300mM, about 100-250mM, about 100-200mM, about 100-150mM, about 150-50 0mM, about 150-450mM, about 150-400mM, about 150-350mM, about 150-300mM, about 150-250mM, about 150-200mM, about 200-500mM, about 200-450mM, about 200-400mM, about 200-350mM, about 200-300mM, about 200-250mM, about 250-500mM, about 250-4 or about 450-500 mM).

[0062] In some embodiments, the tonicity agent is sucrose or trehalose. In some embodiments, the tonicity agent is sucrose. In some embodiments, the concentration of trehalose is about 10-300 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, 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).

[0063] In some embodiments, the formulations described herein comprise 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 trehalose.

[0064] In some embodiments, the formulation may include one or more tonicity agents. For example, the formulation may include one or more of the above tonicity agents at the above concentrations. In certain specific embodiments, the formulation may include trehalose and arginine (e.g., arginine-HCl), wherein the concentrations of trehalose and arginine are each 50-300 mM (e.g., 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).

[0065] In some embodiments, the osmotic pressure of the formulation is about 200mOsm / kg to about 500mOsm / kg, about 250mOsm / kg to about 450mOsm / kg, about 200mOsm / kg to about 350mOsm / kg, about 230mOsm / kg to about 330mOsm / kg, about 250mOsm / kg to about 300mOsm / kg, about 260mOsm / kg to about 280mOsm / kg, about 350mOsm / kg to about 450mOsm / kg, about 380mOsm / kg to about 420mOsm / kg, for example, about 270mOsm / kg or about 450mOsm / kg. In some embodiments, the formulation is substantially isotonic, i.e., has substantially the same osmotic pressure as the blood of a mammal (e.g., a human). Osmolality can be measured by any means known to those skilled in the art, such as using a vapor pressure or freezing-type osmometer.The osmolality of the formulations described herein can be adjusted, for example, by one or more tonicity agents described herein.

[0066] Amino Acids

[0067] The formulation may also optionally include an amino acid. In some embodiments, the amino acid is also a tension agent. In some embodiments, the amino acid is a stabilizer. Examples of amino acids include, but are not limited to, glycine, alanine, aspartic acid, lysine, serine, tyrosine, cysteine, glutamine, methionine, arginine, and proline. In some embodiments, the amino acid is in the form of its hydrochloride salt. In some embodiments, the amino acid is an L-enantiomer. In some embodiments, the amino acid is a D-enantiomer. In some embodiments, the amino acid is a mixture of L- and D-enantiomers.

[0068] In some embodiments, the amino acid is present at 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 mM, about 50-300mM, about 50-250mM, about 50-200mM, about 50-150mM, about 50-100mM, about 100-500mM, about 100-450mM, about 100-400mM, about 100-350mM, about 100-300mM, about 100-250mM, about 100-200mM, about 100-150mM, about 150-500mM, about 150-450mM, about 150-400mM, about 150-350mM, about 150-300mM, about 150-250mM, about 150-200mM, about 200-500mM, about 200-450mM, about 200-400mM, about 200-350mM, about 200-300mM, about 200-250mM, about 250-500mM, about 250-450mM, About 250-400mM, about 250-350mM, about 250-300mM, about 300-500mM, about 300-450mM, about 300-400mM, about 300-350mM, about 350-500mM, about 350-450mM, about 350-400mM, about 400-500mM, about 400-450mM or about 450-500mM) is present in the formulation.

[0069] An exemplary amino acid is 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 comprise 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 arginine.

[0070] Other excipients

[0071] In addition, the preparation may include other excipients, including but not limited to water for injection, diluents, solubilizers, soothing agents, additional buffers, inorganic or organic salts, antioxidants, etc. However, in some embodiments, in addition to those mentioned above, the preparation does not contain other excipients. The preparation may include other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980), provided that it does not adversely affect the desired properties of the preparation. In a specific embodiment, the preparation is substantially free of preservatives, but in alternative embodiments, preservatives may be added as needed. For example, a cryoprotectant or a lyoprotectant may be included in a lyophilized preparation.

[0072] In some embodiments, the formulation may also include metal chelators and / or antioxidants, as well as other pharmaceutically acceptable excipients. Suitable metal chelators include, for example, methylamine, ethylenediamine, deferoxamine, trientine, histidine, malate, phosphonate compounds, such as etidronic acid, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), etc. Suitable antioxidants include, for example, citric acid, uric acid, ascorbic acid, lipoic acid, glutathione, tocopherol, carotene, lycopene, cysteine, etc.

[0073] Exemplary Formulations

[0074] In one aspect, the present invention provides a stable aqueous pharmaceutical formulation comprising an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof and a buffer, wherein the pH of the formulation is about 5.0 to about 6.5.

[0075] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0076] 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;

[0077] b) a stabilizer at a concentration of about 10 mM to about 500 mM;

[0078] c) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml; and

[0079] d) a buffer system, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0080] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0081] 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;

[0082] b) a tonicity agent at a concentration of about 10 mM to about 500 mM;

[0083] c) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml; and

[0084] d) a buffer system, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0085] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0086] e) 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;

[0087] f) an amino acid at a concentration of about 10 mM to about 500 mM;

[0088] g) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml; and

[0089] h) a buffer system, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0090] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0091] 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;

[0092] b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 500 mM;

[0093] c) acetic acid / sodium acetate buffer, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0094] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0095] 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;

[0096] b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 500 mM;

[0097] c) trehalose at a concentration of about 10 mM to about 500 mM;

[0098] d) acetic acid / sodium acetate buffer, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0099] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0100] 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;

[0101] b) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml;

[0102] c) acetic acid / sodium acetate buffer, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0103] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0104] 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;

[0105] b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 500 mM;

[0106] c) a surfactant at a concentration of about 0.01 mg / ml to about 10 mg / ml (e.g., about 0.2 mg / ml);

[0107] d) acetic acid / sodium acetate buffer, wherein the pH of the formulation is from about 5.0 to about 6.5.

[0108] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0109] 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;

[0110] b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 200 mM;

[0111] 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);

[0112] d) an acetic acid / sodium acetate buffer having a concentration of about 10-100 mM, wherein the pH of the formulation is about 5.5 to about 6.0.

[0113] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0114] 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;

[0115] b) arginine (e.g., arginine-HCl) at a concentration of about 10 mM to about 200 mM;

[0116] c) trehalose at a concentration of about 10 mM to about 300 mM;

[0117] 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);

[0118] e) an acetic acid / sodium acetate buffer having a concentration of about 10-100 mM, wherein the pH of the formulation is about 5.5 to about 6.0.

[0119] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0120] 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;

[0121] b) arginine (e.g., arginine-HCl) at a concentration of about 140 mM;

[0122] c) polysorbate 80 at a concentration of about 0.2 mg / ml;

[0123] d) an acetic acid / sodium acetate buffer at a concentration of about 10 mM, wherein the pH of the formulation is about 5.5 to about 6.0 (e.g., 5.6).

[0124] In some embodiments, the stable aqueous pharmaceutical formulation comprises, consists of, or consists essentially of:

[0125] 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;

[0126] b) arginine (e.g., arginine-HCl) at a concentration of about 100 mM;

[0127] c) trehalose at a concentration of about 130 mM;

[0128] d) polysorbate 80 at a concentration of about 0.2 mg / ml;

[0129] e) acetic acid / sodium acetate buffer at a concentration of about 15 mM, wherein the pH of the formulation is about 5.5 to about 6.0 (e.g., 5.8).

[0130] 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 any other tonicity agent. In some embodiments, the formulation is substantially free of any other buffer. In some embodiments, the formulation is substantially free of any amino acid or any amino acid other than arginine.

[0131] stability

[0132] The preparations described herein are stable when stored. The preparations can be refrigerated (e.g., 2-8°C) or frozen (e.g., at -20°C, -70°C, or -80°C) for storage. It is expected that the preparations are stable at about 4°C, about 25°C, about 40°C, or room temperature for at least about 1, 2, 3, or 4 weeks or longer; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, and generally at least about 12, 18, or 24 months or longer. In some embodiments, it is stable at about 25°C for at least about 6 months or longer.

[0133] Stability can be tested by evaluating the physical stability, chemical stability, and / or biological activity of the antibody or antigen-binding fragment thereof in the formulation upon formulation and after storage at a specified temperature. The physical and / or chemical stability of a liquid formulation can be qualitatively and / or quantitatively evaluated in a variety of different ways (see, e.g., Analytical Techniques for Biopharmaceutical Development, Rodriguez-Diaz et al. eds. Informa Healthcare (2005)), including evaluation of aggregate formation (e.g., using size exclusion chromatography (SEC), size exclusion high performance liquid chromatography (SEC-HPLC), reverse phase chromatography-ultra performance liquid chromatography (RPC-UPLC), matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), analytical ultracentrifugation, light scattering (photon correlation spectroscopy, dynamic light scattering (DLS), multi-angle laser light scattering (MLS), and the like). lightscattering, MALLS)), flow-based microscopy imaging, electronic impedance (Coulter) counting, light obscuration or other liquid particle counting systems, by measuring turbidity, by observing the appearance of the test sample, density gradient centrifugation and / or by visual inspection); by using cation exchange chromatography to assess charge heterogeneity (see Vlasak and Ionescu, Curr. Pharm. Biotechnol. 9:468-481 (2008) and Harris et al. J. Chromatogr. B Biomed. Sci. Appl.752:233-245 (2001)), ion exchange high performance liquid chromatography (IEX-HPLC), isoelectric focusing (IEF) such as capillary technique (cIEF) or capillary zone electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE or SEC analysis to compare fragmented, intact and multimeric (i.e., dimer, trimer, etc.) antibodies; peptide mapping (e.g., tryptic or LYS-, etc.); evaluation of the biological activity or antigen binding function of the antibody; etc. The biological activity or antigen binding function (e.g., the binding of the anti-PD-L1 / anti-4-1BB bispecific antibody to PD-1, PD-L1, or the inhibition of the binding between PD-1 and PD-L1; the binding of the anti-PD-L1 / anti-4-1BB bispecific antibody to 4-1BB, 4-1BBL, or the inhibition of the binding between 4-1BB and 4-1BBL) can be evaluated using a variety of techniques available to the skilled person. .

[0134] Stability can be measured at a selected temperature and within a selected time period. On the one hand, 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, at least about 6 months, at least about 9 months, at least about 12 months, or at least about 18 months. During this period, the formulation is substantially free of any precipitation, and / or the reduction 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 reduction 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 protein concentration is reduced by no more 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 mg / ml. 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, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5%. In some embodiments, the main peak ratio of the charge isomer (e.g., as measured by IEX-HPLC) is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0135] 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 about 6 months, at least about 9 months, at least about 12 months, or at least about 18 months. During this period, the formulation is substantially free of any precipitation, 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 protein concentration is reduced by no more 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 mg / ml. 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, 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., as measured by IEX-HPLC) is less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0136] Instability may involve any one or more of the following: aggregation (e.g., non-covalent soluble aggregation (caused by hydrophobic or charge interactions), covalent soluble aggregation (e.g., disulfide bond rearrangement / mispairing), insoluble aggregation (caused by protein denaturation at liquid / air and liquid / solid interfaces), deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), denaturation, shearing / hydrolysis / cleavage (e.g., hinge region cleavage), succinimide formation, N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0137] 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 any visible foreign matter. In some embodiments, the formulation is substantially free of any precipitation after storage (e.g., about 4 weeks at about 40±2°C or about 6 months at about 25±2°C).

[0138] In some embodiments, the decrease in protein concentration after storage (e.g., about 4 weeks at about 40±2°C or 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%.

[0139] 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., at about -70 to about -80°C for at least 1, 2, 3, 4, 5, or 6 hours at 25°C as 1 cycle) 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%.

[0140] In some embodiments, 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% after at least 1, 2, 3, 4, or 5 days of shaking (e.g., 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). 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%.

[0141] Additionally, the formulation may be sterile, and this can be achieved either before or after preparation of the formulation according to procedures known to those skilled in the art to produce a sterile pharmaceutical formulation suitable for administration to a subject.

[0142] Anti-PD-L1 / anti-4-1BB bispecific antibodies and antigen-binding fragments

[0143] 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, wherein the first antigen-binding functional region that specifically binds to PD-L1 comprises:

[0144] (A) a heavy chain variable region comprising

[0145] (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 15,

[0146] (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and

[0147] (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17; and

[0148] (B) a light chain variable region comprising

[0149] (a) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18,

[0150] (b) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and

[0151] (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO:20.

[0152] In some embodiments, the second antigen-binding functional region of the bispecific antibody that specifically binds to 4-1BB comprises:

[0153] (A) a heavy chain variable region comprising

[0154] (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 21,

[0155] (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and

[0156] (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 23; and

[0157] (B) a light chain variable region comprising

[0158] (a) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24,

[0159] (b) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and

[0160] (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0161] In some embodiments, the first antigen-binding functional region of the bispecific antibody that specifically binds to PD-L1 comprises

[0162] (A) a heavy chain variable region comprising

[0163] (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 15,

[0164] (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and

[0165] (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17; and

[0166] (B) a light chain variable region comprising

[0167] (a) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18,

[0168] (b) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and

[0169] (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 20; and

[0170] The second antigen binding functional region that specifically binds to 4-1BB comprises:

[0171] (A) a heavy chain variable region comprising

[0172] (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 21,

[0173] (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and

[0174] (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 23; and

[0175] (B) a light chain variable region comprising

[0176] (a) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24,

[0177] (b) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and

[0178] (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0179] The 6 CDRs in the first antigen binding functional region specifically binding to PD-L1 as shown in SEQ ID NO: 15-20 and the 6 CDRs in the second antigen binding functional region specifically binding to 4-1BB as shown in SEQ ID NO: 21-26 are the CDRs of the heavy chain and light chain variable regions of the monoclonal antibodies obtained by the inventors using hybridoma technology with human PD-L1 and 4-1BB as antigens. The monoclonal antibodies are different from the PD-L1 antibodies and 4-1BB antibodies known in the prior art and have higher biological activity, such as higher binding specificity, anti-tumor activity, etc.

[0180] The six CDR sequences of the first antigen-binding functional region that specifically binds to PD-L1 may have at least 70%, for example, at least 75%, 80%, 85%, 90%, 95% or higher identity with the sequences shown in SEQ ID NOs: 15-20, and retain the biological activity of the corresponding parent sequences; 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: 15-20, and retain the biological activity of the corresponding parent sequences.

[0181] The six CDR sequences of the second antigen-binding functional region that specifically binds to 4-1BB may have at least 70%, for example, at least 75%, 80%, 85%, 90%, 95% or higher identity with the sequences shown in SEQ ID NOs: 21-26, and retain the biological activity of the corresponding parent sequences; 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: 21-26, and retain the biological activity of the corresponding parent sequences.

[0182] In some embodiments, the antigen-binding functional region that specifically binds to PD-L1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 identical to HCDR1, HCDR2 and HCDR3 of SEQ ID NO:6; and the light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO:2.

[0183] In some embodiments, the antigen binding functional region that specifically binds to 4-1BB comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 identical to HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 12; and the light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10.

[0184] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable region containing one, two or three CDRs as described herein with zero, one or two amino acid insertions, deletions or substitutions. In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable region containing one, two or three CDRs as described herein with zero, one or two amino acid insertions, deletions or substitutions.

[0185] The insertions, deletions and substitutions 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 scheme. In some embodiments, the CDR is determined based on the Chothia numbering scheme.

[0186] In some embodiments, the first antigen-binding functional region of the bispecific antibody that specifically binds to PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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 parent sequence; or contains one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acid deletions, substitutions and / or additions compared to the sequence shown in SEQ ID NO:6, and retains the biological activity of the corresponding parent sequence; the light chain variable region 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:6, and retains the biological activity of the corresponding parent sequence. The sequence shown in NO:2 has at least 70%, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity, and retains the biological activity of the corresponding parent sequence; or contains one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acid deletions, substitutions and / or additions compared to the sequence shown in SEQ ID NO:2, and retains the biological activity of the corresponding parent sequence.

[0187] In some embodiments, the second antigen-binding functional region of the bispecific antibody that specifically binds to 4-1BB comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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 parent sequence; or contains one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acid deletions, substitutions and / or additions compared to the sequence shown in SEQ ID NO: 12, and retains the biological activity of the corresponding parent sequence; the light chain variable region 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: 12, and retains the biological activity of the corresponding parent sequence. The sequence shown in NO:10 has at least 70%, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity, and retains the biological activity of the corresponding parent sequence; or contains one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acid deletions, substitutions and / or additions compared to the sequence shown in SEQ ID NO:10, and retains the biological activity of the corresponding parent sequence.

[0188] In some embodiments, the first antigen-binding functional region of the bispecific antibody that specifically binds to PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region comprises 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 and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:12, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10.

[0189] Those skilled in the art will know that the combination of amino acid sequences should follow biological laws, that is, the light chain and heavy chain or their variable regions, and their antigen-binding fragments (e.g., SEQ ID NO: 6 and SEQ ID NO: 2, and SEQ ID NO: 12 and SEQ ID NO: 10) should match each other.

[0190] In some embodiments, the first antigen-binding region and the second antigen-binding region of the bispecific antibody are selected from a Fab fragment, a scFv fragment, and a variable domain fragment Fv.

[0191] In some embodiments, the first antigen-binding region and the second antigen-binding region of the bispecific antibody are both Fab fragments.

[0192] 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.

[0193] In some embodiments, one of the first antigen-binding region and the second antigen-binding region of the bispecific antibody is a Fab fragment, and the other is a scFv.

[0194] 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.

[0195] Wherein, the first Fc chain and the second Fc chain are both immunoglobulin G Fc fragments containing amino acid substitutions, and the first Fc chain and the second Fc chain together constitute a heterodimer that can bind to an Fc receptor;

[0196] wherein the first Fc chain and the second Fc chain are connected to the first antigen-binding functional region and the second antigen-binding functional region respectively through a covalent bond or a linker; and

[0197] wherein one of the first Fc chain or the second Fc chain comprises amino acid substitutions at positions 366 and 399, and the other comprises amino acid substitutions at positions 351, 407 and 409, wherein amino acid positions are numbered according to the Kabat EU index numbering system.

[0198] In some embodiments, the amino acid substitutions of the first Fc chain and the second Fc chain of the bispecific antibody are as follows,

[0199] a) L351G, L351Y, L351V, L351P, L351D, L351E, L351K or L351W;

[0200] b) T366L, T366P, T366W or T366V;

[0201] c) D399C, D399N, D399I, D399G, D399R, D399T or D399A;

[0202] d) Y407L, Y407A, Y407P, Y407F, Y407T or Y407H; and

[0203] e) K409C, K409P, K409S, K409F, K409V, K409Q or K409R.

[0204] In some embodiments, the amino acid substitutions of the bispecific antibody include:

[0205] a) substitutions T366L and D399R on the first or second Fc chain and substitutions L351E, Y407L and K409V on the other;

[0206] b) substitutions T366L and D399C on the first or second Fc chain and substitutions L351G, Y407L and K409C on the other;

[0207] c) substitutions T366L and D399C on the first or second Fc chain and substitutions L351Y, Y407A and K409P on the other;

[0208] d) substitutions T366P and D399N on the first or second Fc chain and substitutions L351V, Y407P and K409S on the other;

[0209] e) substitutions T366W and D399G on the first or second Fc chain and L351D, Y407P and K409S on the other;

[0210] f) substitutions T366P and D399I on the first or second Fc chain and substitutions L351P, Y407F and K409F on the other;

[0211] g) substitutions T366V and D399T on the first or the second Fc chain and substitutions L351K, Y407T and K409Q on the other;

[0212] h) Substitutions T366L and D399A on the first or the second Fc chain and substitutions L351W, Y407H and K409R on the other.

[0213] In some embodiments, the amino acid substitutions of the bispecific antibody include:

[0214] a) substitutions T366L and K409V on the first or second Fc chain and substitutions L351E, Y407L and D399R on the other;

[0215] b) substitutions T366L and K409C on the first or second Fc chain and substitutions L351G, Y407L and D399C on the other;

[0216] c) substitutions T366L and K409P on the first or second Fc chain and substitutions L351Y, Y407A and D399C on the other;

[0217] d) substitutions T366P and K409S on the first or second Fc chain and substitutions L351V, Y407P and D399N on the other;

[0218] e) substitutions T366W and K409S on the first or second Fc chain and L351D, Y407P and D399G on the other;

[0219] f) substitutions T366P and K409F on the first or second Fc chain and substitutions L351P, Y407F and D399I on the other;

[0220] g) substitutions T366V and K409Q on the first or second Fc chain and substitutions L351K, Y407T and D399T on the other;

[0221] h) Substitutions T366L and K409R on the first or the second Fc chain and substitutions L351W, Y407H and D399A on the other.

[0222] In some embodiments, the substitutions on the first or second Fc chain of the bispecific antibody are T366L and D399R, and the substitutions on the other are L351E, Y407L, and K409V.

[0223] In some embodiments, in a solution containing a reducing agent (in which no other polypeptides are present except for the Fc chain and the antigen-binding functional region described below), the weight ratio of the homodimer formed by the first Fc chain of the bispecific antibody and the first antigen-binding functional region covalently linked thereto to the homodimer formed by the second Fc chain of the bispecific antibody and the second antigen-binding functional region covalently linked thereto is less than 50% of the total amount of all polypeptide chains, for example, less than 45%, 40%, 35%, 30%, 25%, 20% or less.

[0224] 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 of SEQ ID NO:6 and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:8; and the first light chain has a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:4.

[0225] 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; 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.

[0226] A second aspect of the present invention relates to an isolated polynucleotide encoding a bispecific antibody in heterodimeric form as described above.

[0227] In some embodiments, the nucleotide sequence encoding the amino acid sequence of the first antigen-binding domain comprises the nucleotide sequences shown in SEQ ID NOs: 1 and 5.

[0228] In some embodiments, the nucleotide sequence encoding the amino acid sequence of the second antigen-binding domain comprises the nucleotide sequences shown in SEQ ID NOs: 11 and 9.

[0229] 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.

[0230] In some embodiments, the nucleotide sequence encoding the amino acid sequence of 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 comprises the nucleotide sequence shown in SEQ ID NO:13.

[0231] The third aspect of the present invention relates to a recombinant expression vector comprising the isolated polynucleotide as described above.

[0232] In some embodiments, the expression vector is a plasmid vector XOGC modified from a pCDNA vector.

[0233] A fourth aspect of the present invention relates to a host cell comprising the isolated polynucleotide as described above or the recombinant expression vector as described above.

[0234] In some embodiments, the host cell is selected from human embryonic kidney cells HEK293 or HEK293T, HEK293E, HEK293F modified from HEK293 cells; hamster ovary cells CHO, or CHO-S, CHO-dhfr modified from CHO cells; -, CHO / DG44, ExpiCHO; Escherichia coli, or E. coli BL21, BL21(DE3), Rosetta, Origami modified from E. coli; yeast, or Pichia, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha modified from yeast; insect cells, or High5, SF9 cells modified from insect cells; plant cells; mammalian mammary cells; somatic cells.

[0235] The fifth aspect of the present invention relates to a composition comprising the bispecific antibody as described above, or the isolated polynucleotide as described above, or the recombinant expression vector as described above, or the host cell as described above, and a pharmaceutically acceptable carrier.

[0236] A sixth aspect of the present invention relates to a method for preparing the bispecific antibody as described above, comprising the following steps:

[0237] 1) expressing the isolated polynucleotide as described above or the recombinant expression vector as described above in a host cell, respectively;

[0238] 2) respectively reducing the proteins expressed in the host cells; and

[0239] 3) The reduced proteins are mixed and the mixture is then oxidized.

[0240] In some embodiments, the host cell is selected from human embryonic kidney cells HEK293 or HEK293T, HEK293E and HEK293F modified from HEK293 cells; hamster ovary cells CHO, or CHO-S, CHO-dhfr modified from CHO cells; - , CHO / DG44, ExpiCHO; Escherichia coli, or E. coli BL21, BL21(DE3), Rosetta, Origami modified from E. coli; yeast, or Pichia, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha modified from yeast; insect cells, or High5, SF9 cells modified from insect cells; plant cells; mammalian mammary cells; somatic cells.

[0241] In some embodiments, the reduction step comprises 1) performing a reduction reaction in the presence of a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine, cysteine ​​or other chemical derivatives thereof; 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.

[0242] In some embodiments, the oxidation step is carried out in air or in the presence of an oxidizing agent selected from L-dehydroascorbic acid, cystamine or a chemical derivative thereof. In some embodiments, the oxidizing agent is L-dehydroascorbic acid at a concentration of 0.5 mM or more, such as 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.5 mM or more, 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 more.

[0243] In some embodiments, the method further comprises the steps of separation and purification. In some embodiments, the separation and purification comprises cation resin exchange, anion resin exchange, reverse phase chromatography, affinity chromatography, size exclusion chromatography and combinations thereof.

[0244] 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 preparation of a medicament for preventing and / or treating a disease in a subject.

[0245] 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 for use as a drug for preventing and / or treating a disease of a subject.

[0246] The ninth aspect of the present invention relates to a method for preventing and / or treating a disease, which comprises administering the bispecific antibody as described above and / or the separated bispecific antibody 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 to a subject in need thereof.

[0247] In some embodiments, the subject is a mammal, preferably a human subject.

[0248] In some embodiments, the disease is selected from the following tumors: 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 and bone cancer.

[0249] In other words, the present invention designs a novel anti-PD-L1 / anti-4-1BB bispecific antibody, which is in the form of a natural antibody structure-like heterodimer, has the characteristics of natural IgG and has no heavy chain and light chain mismatch, and is therefore a highly stable heterodimer form of anti-PD-L1 / anti-4-1BB bispecific antibody. The bispecific antibody prepared by the present invention can bind to two target molecules PD-L1 and 4-1BB at the same time, and when applied to the treatment of complex diseases, it has better efficacy and fewer side effects than a single therapeutic agent. In addition, compared with the combined treatment of multiple drugs, the bispecific antibody, as a single therapeutic molecule, not only facilitates the use of patients and medical staff, but also simplifies the complex development process of new drugs.

[0250] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are described in detail in PCT Application No. PCT / CN2022 / 070624, which is incorporated herein by reference in its entirety.

[0251] Treatment

[0252] The antibodies or antigen binding fragments thereof of the present invention or preparations comprising the antibodies or antigen binding fragments thereof can be used for a variety of therapeutic purposes. On the one hand, the invention provides methods for treating cancer in a subject, methods for reducing the rate at which tumor volume increases over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis occurring in a subject. In some embodiments, the treatment may stop, slow down, delay or inhibit cancer progression. In some embodiments, the treatment may result in a reduction in the quantity, severity and / or duration of one or more symptoms of cancer in a subject.

[0253] In one aspect, the invention features a method comprising administering to a subject in need thereof (e.g., a subject suffering from, or identified or diagnosed with, cancer) a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein or a formulation comprising an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the cancer is selected from the following tumors: 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, and bone cancer.

[0254] As used herein, "effective amount" means an amount or dosage sufficient to achieve a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease (e.g., cancer). The effective amount will vary according to the age and weight of the subject to which the antibody, antigen binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or its composition will be administered, the severity of the symptoms, and the route of administration, and therefore administration can be determined on an individual basis.

[0255] The effective amount can be administered once or more. For example, the effective amount of an antibody or antigen binding fragment is enough to improve, terminate, stabilize, reverse, inhibit, slow down and / or delay the amount of the patient's cancer progression, or is enough to improve, terminate, stabilize, reverse, slow down and / or delay the amount of cells (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro proliferation. As understood in the art, the effective amount of an antibody or antigen binding fragment or a preparation comprising an antibody or its antigen binding fragment can be especially according to the patient's medical history and other factors, such as the antibody type (and / or dosage) used and change.

[0256] Reagent test kit

[0257] Also provided is a kit comprising the preparation described herein. The kit may also include one or more containers comprising pharmaceutically acceptable excipients, and include other materials desired from a commercial and user perspective, including filters, needles and syringes. Related to the kit may be instructions generally included in the commercial packaging of the treatment, prevention or diagnosis product, which contain such information: for example, about the indications, uses, dosages, manufacture, administration, contraindications and / or warnings for the use of such treatment, prevention or diagnosis products. The kit may also be labeled, and the label may be any type of data carrier (e.g., leaflets, stickers, chips, printed matter or barcodes) containing information. In certain embodiments, the method of use discussed herein may be included in or on the label. The kit may also include a device for applying the preparation, and in particular a device containing the preparation, i.e. a pre-filled device, such as, but not limited to, a pre-filled syringe or a pre-filled automatic syringe. The kit may also include a container containing the preparation, i.e., a pre-filled container, such as a pre-filled vial (vial), a cylindrical vessel, a pouch or an ampoule.

[0258] The formulation may be included in a container, pack or dispenser together with instructions for administration. The present invention also provides methods for preparing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0259] Example

[0260] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0261] Methods and Materials

[0262] In order to develop a formulation for long-term stable storage of a recombinant anti-PD-L1 / anti-4-1BB bispecific antibody injection as described in PCT application No. PCT / CN2022 / 070624 and to ensure product quality during its shelf life (e.g., at least 24 months), an experiment was designed to screen formulations including anti-PD-L1 / anti-4-1BB bispecific antibodies. The effects of different excipients on the stability of antibody formulations were evaluated. The materials and methods used in the experiment are as follows:

[0263] Table 1. Materials used in formulation studies

[0264] name factory Part Number Sodium acetate Sigma-Aldrich 71183-250G Acetic acid Sigma-Aldrich 33209-1L Sodium hydroxide Sigma-Aldrich 6203 Trehalose Sigma-Aldrich T9531 Trehalose Sinopharm Group 63012666 L-Arginine monohydrochloride Merck 1.01544.1000 Polysorbate 80 Sigma-Aldrich P1754-25ML

[0265] Table 2. Instruments used in formulation studies

[0266] name factory model Centrifuge Eppendorf 5424R / 5430R / ST-16R Balance METTLER TOLEDO XSE204 Ultrapure water system Millipore Milli-Q pH Meter METTLER TOLEDO S470 Constant temperature incubator BOXUN BSP-400 Tangential flow ultrafiltration concentration system (TFF system) Millipore <![CDATA[Labscale TM TFF]]> UV spectrophotometer GE Ultrospec 8000 HPLC Agilent 1200,1260 series HPLC / UPLC Waters Acquity H-class Capillary electrophoresis system (CE system) Beckman Coulter CESI 8000Plus Automatic osmometer Gonotec Osmomat auto Vortex Scientific Industries VORTEX-GENIE2

[0267] Table 3. Test samples

[0268]

[0269]

[0270] The stability of the test samples in different formulations was tested by the method described below.

[0271] Appearance

[0272] Observe the appearance of the test sample according to the relevant methods described in the Chinese Pharmacopoeia. "Pharmacopoeia of the People's Republic of China". Pharmacopoeia of the People's Republic of China (2020): 122-129. Specifically, the inspector should have 4.9 or above in both long-distance and near-distance vision tests (corrected visual acuity 5.0 or above), and should not be color blind. The inspector can adjust the position so that the test sample is at the clear visual distance of the eye (referring to the clear observation distance from the test sample to the human eye, usually 25cm). Take the test sample and place it in diffuse sunlight and examine it horizontally against a white background. The inspector then judges whether the color and clarity of the test sample are similar to those of water in the same container, and whether there is obvious turbidity or precipitation.

[0273] Protein concentration

[0274] The UV-Vis absorption spectrum of protein is an absorption spectrum caused by the transition of electronic energy levels after certain groups in the molecule absorb ultraviolet and / or visible light. Protein molecules contain aromatic amino acids such as tyrosine and tryptophan with conjugated double bonds. It has a maximum absorbance at a wavelength of about 280nm, and its absorbance is proportional to the protein concentration within a certain range. Based on the Beer-Lambert law (A=a×b×c, where A represents absorbance, a represents extinction coefficient, b represents detection pathlength, and c represents the protein concentration of the sample), the protein concentration can be calculated.

[0275] UV-visible spectrophotometer (GE, Ultrospec TM 8000) to determine the protein concentration. Before testing, turn on the UV-visible spectrophotometer power and preheat for 30 minutes. Dilute the sample to be tested with water or corresponding buffer solution so that the sample signal value is between 0.200-0.800. After zeroing with water or buffer, add the appropriately diluted sample to the detection cell for testing.

[0276] pH

[0277] Use a pH meter to test the pH value. Before testing, take out the sample to be tested and the calibration buffer and equilibrate them to room temperature. Turn on the power of the pH meter and select the correct calibration mode. Confirm the approximate pH range of the sample to be measured, and then select the standard buffer solution to be used. Rinse the electrode with deionized or ultrapure water and absorb the moisture. After confirming that the calibration slope is between 95-105% and the deviation is about 0±30mV, measure the pH value of the sample. Rinse the electrode with deionized or ultrapure water and gently absorb the remaining moisture. After that, insert the electrode into the sample solution and press the "Read" button. Gently shake the sample solution until it is balanced and stable, and then read the sample pH value.

[0278] SEC-HPLC

[0279] Size exclusion high performance liquid chromatography, i.e. SEC-HPLC, is an important method for antibody quality control. The method is based on the size or hydrodynamic radius difference of the molecule to separate the molecule. Through SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (high molecular weight form, HMW), main peak (mainly antibody monomer) and low molecular weight form (low molecular weight form, LMW). Through SEC-HPLC, the percentage of antibody monomers in the sample can be measured, providing the content information of soluble aggregates and fragments.

[0280] The SEC-HPLC method described in this article uses an XBridge BEH SEC 200 column. Specifically, a 100mM PB+100mM NaCl solution (pH should be 6.7±0.1) was prepared as the mobile phase for isocratic elution. The injection volume was 10μl, the flow rate was 0.8ml / min, the acquisition time was 25 minutes, the column temperature was 25°C, and the detection wavelength was 280nm. The sample to be tested was diluted to 1mg / ml with the mobile phase and used as the test solution for detection.

[0281] IEX-HPLC

[0282] Ion exchange high performance liquid chromatography (IEX-HPLC), especially cation exchange high performance liquid chromatography (CEX-HPLC), is often used to determine the charge variants of antibodies. In this assay, peaks eluting from the column earlier than the retention time of the main peak are labeled as "acidic peaks", while those eluting from the column later than the retention time of the main peak are labeled as "basic peaks". The IEX-HPLC method uses MAbPac TM SCX-10 column was used for detection, mobile phase A: 20mM MES, 10mM NaCl, pH5.6±0.1; mobile phase B: 20mM MES, 300mM NaCl, pH 5.6±0.1. The injection volume was 40μl, the flow rate was 0.6ml / min, the acquisition time was 62 minutes, the column temperature was 25℃, and the detection wavelength was 280nm. The sample to be tested was diluted to 1mg / ml with mobile phase A buffer as the test solution for detection.

[0283] CE-SDS(R / NR)

[0284] CE-SDS is a method for measuring the purity of antibodies using a capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge caused by SDS binding, which is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on the size or hydrodynamic radius of the molecule can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to detect the purity of antibodies. Generally, in the non-reduced CE-SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. Thereafter, 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 migration of the protein is detected using a UV detector to obtain an electrophoretic spectrum. The purity of the antibody can be calculated as the percentage of the peak area of ​​the main IgG peak to the sum of all peak areas. In the reduced CE-SDS method, the test sample is mixed with SDS sample buffer and 2-mercaptoethanol. The mixture is then 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. Antibody purity can be calculated as the percentage of the sum of the light chain peak area and the heavy chain peak area to the sum of all peak areas.

[0285] The capillary is a fused silica capillary with an inner diameter of 50 μm. The total length is 30.2 cm and the effective length is 20.2 cm. Before electrophoresis, the capillary column is equilibrated with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel in sequence. If the protein concentration is less than 10 mg / mL and the salt concentration is greater than 50 mM, the sample must be replaced by ultrafiltration into water or other low-salt buffer to avoid adverse effects caused by excessive salt concentration. Pipette 100 μg of sample (less than 45 μL) into a 0.5 mL microcentrifuge tube, add sample buffer to a total volume of 95 μL, and then add 2 μL of internal standard (10 kDa), 5 μL of iodoacetamide solution (250 mM) or 5 μl of 2-mercaptoethanol. After thorough mixing, the centrifuge tube is centrifuged at 300 × g for 1 min. After centrifugation, the centrifuge tube is sealed with a sealing film, then placed in a 70°C water bath for 10 min and cooled at room temperature for at least 3 min. After that, transfer 100 μL of sample to a 200 μL micro-sample tube and remove any bubbles present. Place the sample in a universal bottle and cover it with a cap. The following conditions are used for detection: sample injection: 5 kV, 40 seconds; separation voltage: 15 kV, 40 minutes. The capillary column temperature is controlled at 25 °C and the detection wavelength is 214 nm.

[0286] RPC-UPLC

[0287] Reversed-phase ultra-performance liquid chromatography (RPC-UPLC) using MAbPac TMRP column (analytical 4 μm, 2.1 × 100 mm, PN: 088647, Thermo) and Waters TM The samples were tested by ultra-high pressure liquid phase system (H class plus). Mobile phase A was 0.1% TFA / water solution; mobile phase B was 0.1% TFA / acetonitrile solution. The injection volume was 5 μL; the detection wavelength was 214 nm (UV); the column oven temperature was 70°C; the injector temperature was about 2-8°C. Each sample to be tested was diluted with water to 1 mg / ml as the test solution for testing.

[0288] Example 1. Preparation and purification of anti-PD-L1 / anti-4-1BB bispecific antibodies

[0289] The same experiment was performed as described in the example of PCT application No. PCT / CN2022 / 070624 to prepare and purify BH3120, which is an anti-PD-L1 / anti-4-1BB bispecific antibody, comprising an anti-PD-L1 arm and an anti-4-1BB arm, wherein the anti-PD-L1 arm comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:6, a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:8, a light chain variable region with an amino acid sequence as shown in SEQ ID NO:2, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:4; the anti-4-1BB arm comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:12, a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:14, a light chain variable region with an amino acid sequence as shown in SEQ ID NO:10, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:4.

[0290] Example 2: Preparation of antibody preparation

[0291] Based on the experimental results described in the examples of PCT application No. PCT / CN2022 / 070624, the possible components and concentrations in the BH3120 formulation were estimated. The factors to be investigated include: pH, acetate, arginine-HCl and trehalose. The factors investigated in the experiment and their concentration ranges are shown in the following table.

[0292] Table 4 Main factors and scope of investigation

[0293] factor scope pH 5.0～6.5 Acetate 10~100mM Trehalose 0~275mM Arginine-HCl 0~140mM

[0294] Prepare the corresponding blank formulation buffer (without antibody protein and polysorbate 80). BH3120 protein is centrifuged by ultrafiltration to replace the buffer, and the protein concentration is adjusted to 20mg / mL, 50mg / mL or 80mg / mL. Add polysorbate 80 to a final concentration of 0.2mg / mL. After further filtration and sterilization, the sample is dispensed in a cillin bottle (about 0.2mL / bottle). The stability of the sample is investigated under the conditions of 40°C and 25°C, freeze-thaw cycles (-70°C to room temperature) and shaking (250rpm). The exemplary compositions of the two preparations are shown in the table below.

[0295] Table 5. Composition of the formulations

[0296]

[0297] Example 3: Confirmation of antibody preparation

[0298] BH3120 protein samples were prepared and tested under different experimental conditions. Protein stability in different formulations was compared. The experiments were performed as shown in the table below.

[0299] Table 6. Experiments to test the stability of BH3120 protein in different formulations

[0300]

[0301] 40℃ incubation test

[0302] The protein samples were placed in a constant temperature incubator at 40°C in the dark for 4 weeks. Samples were collected at 0 weeks, 1 week, 2 weeks, and 4 weeks to detect changes in each test item. After being placed at 40°C for 0 weeks, 1 week, 2 weeks, and 4 weeks, each preparation sample was a colorless transparent liquid with no obvious turbidity or precipitation. The changes in protein concentration, pH, purity, and charge isomers are shown in the following table. The results showed that the protein concentration changed by less than 10% within 4 weeks at 40°C, the SEC-HPLC purity decreased by less than 3% within 2 weeks at 40°C, and the main peak ratio of charge isomers decreased by less than 10% within 4 weeks at 40°C. Within 4 weeks at 40°C, the purity of non-reduced and reduced CE-SDS decreased by less than 3%.

[0303] Table 7. Results of preparation confirmation at 40°C

[0304]

[0305]

[0306] -70℃ freeze-thaw cycle test

[0307] The samples in vials were placed at -70°C for at least 4 hours to completely freeze them. After that, the samples were completely thawed at room temperature or 25°C (as a cycle). After 1, 3, and 5 cycles, samples were taken to detect changes in each test item. The results showed that after the freeze-thaw cycle, the samples were all colorless and transparent liquids, without obvious turbidity or precipitation, and there were no obvious changes in other test items. The specific results are shown in the table below.

[0308] Table 8. Formulation validation results after freeze-thaw cycles

[0309]

[0310]

[0311] 250rpm shaking test

[0312] The subpackaged samples were fixed on a shaker in the dark and shaken at 250rpm. Samples were taken after 1 day, 3 days, and 5 days to detect changes in each test item. The results showed that under these shaking conditions, the samples were all colorless and transparent liquids without obvious turbidity or precipitation. There were no significant changes in other test items. The specific results are shown in the following table.

[0313] Table 9. Formulation validation results under shaking conditions

[0314]

[0315]

[0316] 25℃ incubation test

[0317] The protein samples were placed in a constant temperature incubator at 25°C and protected from light for 6 months. Samples were taken at 0 month, 1 month, 2 months, 3 months, and 6 months to detect changes in various test items (e.g., appearance, protein concentration, pH, purity (detected by SEC-HPLC, RPC-HPLC and CE-SDS), and charge isomers (detected by IEX-HPLC)). The results showed that under these test conditions, the samples were colorless and transparent liquids without obvious turbidity or precipitation. The specific results are shown in the table below. The results showed that the protein concentration changed by less than 10% within 6 months at 25°C, the SEC-HPLC purity decreased by less than 3.5% within 6 months at 25°C, and the main peak ratio of charge isomers decreased by less than 15% within 6 months at 25°C. Within 6 months at 25°C, the non-reduced and reduced CE-SDS purities decreased by less than 3%.

[0318] Table 10. Results of formulation validation at 25°C

[0319]

[0320]

[0321] In summary, all tested BH3120 formulations exhibited good thermal stability. As expected, the experimental results showed that the formulations could better suppress the formation of acidic peaks.

[0322] Other Implementations

[0323] It should be understood that although the invention has been described in conjunction with its specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and improvements are within the scope of the claims.

[0324] The sequences disclosed in the specification are shown in Table 11.

[0325] Table 11. Sequences disclosed in the specification

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

Claims

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

5.

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

0.

3. The formulation of claim 1 or 2, wherein the pH of the formulation is from about 5.6 to about 5.8 or from about 5.8 to about 6.

0.

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

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

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

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

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

9. The formulation of claim 8, wherein the buffer comprises acetic acid and / or sodium acetate.

10. The formulation of any one of claims 1-9, wherein the formulation comprises acetic acid / sodium acetate buffer at a concentration of about 10 mM to about 100 mM.

11. The formulation of any one of claims 1-10, wherein the formulation comprises acetic acid / sodium acetate buffer at a concentration of about 10 or about 15 mM.

12. The formulation of any one of claims 1-11, further comprising a tonicity 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 of claim 12, wherein the tonicity agent is trehalose, arginine, or a combination thereof.

14. The formulation of any one of claims 1-13, wherein the formulation comprises about 10 mM to about 200 mM arginine.

15. The formulation of any one of claims 1-14, wherein the formulation comprises about 100 mM or about 140 mM arginine.

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

17. The formulation of any one of claims 1-16, wherein the formulation comprises about 10 mM to about 300 mM trehalose.

18. The formulation of any one of claims 1-17, wherein the formulation comprises about 130 mM trehalose.

19. The formulation of any one of claims 1 to 18, further comprising a surfactant selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 ( F68) and combinations thereof.

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

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

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

23. 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; a tonicity 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 wherein the pH of the formulation is from about 5.0 to about 6.

5.

24. The formulation of claim 23, wherein the pH of the formulation is from about 5.5 to about 6.

0.

25. The formulation of claim 23 or 24, wherein the buffer system comprises 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 of any one of claims 23-25, wherein the buffer system comprises an acetic acid / sodium acetate buffer at a concentration of about 10 to about 100 mM.

27. The formulation of any one of claims 23-26, wherein the tonicity 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 of claim 27, wherein the tonicity agent is trehalose, arginine (eg, arginine-HCl), or a combination thereof.

29. The formulation of any one of claims 23-28, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, SDS, poloxamer 188 ( F68) or a combination thereof.

30. The formulation of claim 29, wherein the surfactant is polysorbate 80.

31. A stable aqueous pharmaceutical formulation comprising or consisting of: 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; 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 having a concentration of about 10 to about 100 mM, wherein the pH of the formulation is from about 5.5 to about 6.

0.

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

33. The formulation of claim 31 or 32, wherein the concentration of arginine is about 100 mM or about 140 mM.

34. The formulation of any one of claims 31-33, wherein the concentration of trehalose is about 130 mM.

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

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

37. The formulation of any one of claims 1-36, wherein the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises: a first heavy chain variable region (VH1) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 15, the VH1 CDR2 comprises an amino acid sequence at least 80% identical to SEQ ID NO: 16, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 17; a first light chain variable region (VL1) comprising CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 18, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 19, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 20; The VH1 and VL1 can interact with each other to form an antigen binding site that binds to PD-L1.

38. The formulation of any one of claims 1-37, wherein the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises: a second heavy chain variable region (VH2) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 21, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 22, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 23; and a second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:24, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:25, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:26; The VH2 and VL2 can interact with each other to form an antigen binding site that binds to 4-1BB.

39. The formulation of any one of claims 1-38, wherein the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises: a first heavy chain variable region (VH1) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 6, and a first light chain variable region (VL1) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the VH1 and VL1 can interact with each other to form an antigen binding site that binds to PD-L1; and A second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein the second heavy chain variable region (VH2) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12, and the second light chain variable region (VL2) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, wherein the VH2 and VL2 can interact with each other to form an antigen binding site that binds to 4-1BB.

40. The formulation of any one of claims 1-39, wherein the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof comprises: a first heavy chain constant region comprising an amino acid sequence at least 90% identical to SEQ ID NO:8 and a first light chain constant region comprising an amino acid sequence at least 90% identical to SEQ ID NO:4; and A second heavy chain constant region and a second light chain constant region, wherein the second heavy chain constant region comprises an amino acid sequence at least 90% identical to SEQ ID NO:14, and the second light chain constant region comprises an amino acid sequence at least 90% identical to SEQ ID NO:

4.

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

42. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of the formulation of any one of claims 1-41.

43. The method of claim 42, wherein the subject has 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 of claim 42 or 43, wherein the subject is a human.

45. A formulation of an anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof exhibiting long-term stability, comprising, consisting of, or consisting essentially of: (a) an anti-PD-L1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, comprising an anti-PD-L1 arm and an anti-4-1BB arm; the anti-PD-L1 arm comprises a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:6, and the first light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2; the anti-4-1BB arm comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:12, and the second light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10; (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, The pH of the formulation is 5.5-6.0 (eg, 5.6 or 5.8).

46. ​​The formulation of claim 45, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is about 10 mg / ml to about 100 mg / ml.

47. The formulation of claim 45 or 46, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is about 20 mg / ml to about 80 mg / ml.

48. The formulation of any one of claims 45-47, wherein the concentration of the anti-PD-L1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is about 20 mg / ml, about 50 mg / ml, or about 80 mg / ml.

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