Formulations containing anti-PD-1 / HER2 bispecific antibodies, and methods for preparing and using the same

Abstract

The present invention relates to formulations comprising an anti-PD-1 / HER2 bispecific antibody, particularly pharmaceutical formulations comprising an anti-PD-1 / HER2 bispecific antibody, a buffer, a stabilizer, and a surfactant, and further relates to the use of these formulations to treat or prevent diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody formulations. More specifically, the present invention relates to pharmaceutical formulations, particularly stable liquid formulations, comprising recombinant anti-programmed death receptor 1 (PD-1) and anti-human epidermal growth factor receptor 2 (HER2) bispecific antibodies (also called anti-PD-1 / HER2 bispecific antibodies), as well as methods for preparing said pharmaceutical formulations and therapeutic and / or prophylactic uses of said pharmaceutical formulations. [Background technology]

[0002] Overexpression of human epidermal growth factor receptor 2 (HER2) (also known as NEU, ERBB-2, CD340, or p185) has been linked to various cancers, including breast cancer, ovarian cancer, gastric cancer, uterine cancer, melanoma, and cholangiocarcinoma. For example, overexpression of HER2 has been observed in invasive and metastatic breast cancers, as well as breast cancers with high recurrence rates and / or poor patient outcomes.

[0003] One approach to treating HER2-overexpressing cancers is to use anti-HER2 antibodies that inhibit HER2 signaling. For example, trastuzumab is a therapeutic anti-HER2 antibody that blocks HER2-mediated intracellular signaling and is widely used to treat HER2-overexpressing tumors. Unfortunately, its antitumor effects in clinical applications have generally been less favorable than preclinical studies. In conventional techniques, anti-HER2 antibodies are usually administered in combination with chemotherapy drugs (Slamon DJ et al., N Engl J Med, 344:783-792, 2001).

[0004] In recent years, research into immune checkpoint molecules has revealed that the activation of the inhibitory signal pathway of immune checkpoints prevents T lymphocytes from effectively killing tumors (Yao S, Zhu Y and Chen L. Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2):130-146), which is one of the reasons why drugs that only target tumor cells (such as trastuzumab) have poor anti-tumor effects.

[0005] Programmed cell death protein-1 (PD-1) is a key immune checkpoint protein. It is a 55-kDa type I transmembrane protein inducibly expressed primarily on activated T cells, but is also expressed on B cells, NK cells, monocytes, and DCs. Two cell surface glycoprotein ligands for PD-1 have been identified: programmed cell death protein ligand 1 (PD-L1) and programmed cell death protein ligand 2 (PD-L2). PD-1 ligands are highly expressed on many cancer cells. Engagement of PD-1 with its ligand can lead to T cell apoptosis, immune unresponsiveness, T cell "exhaustion," and IL-10 secretion. Blocking the PD1 pathway can therefore restore T cell function in cancer patients (Sheridan, Nature Biotechnology 30 (2012) 729-730). Monoclonal antibodies against PD-1 have been described, such as nivolumab from Bristol-Myers Squibb (BMS) and pembrolizumab from Merck. (登録商標) ) is a fully humanized IgG4 antibody molecule, and is the first (登録商標)) is a humanized IgG4 antibody molecule. When this anti-PD-1 monoclonal antibody binds to PD-1 on T lymphocytes, it can block the binding of PD-1 to its ligands PD-L1 and PD-L2, thereby promoting T lymphocyte activation, proliferation, and production of immune-stimulating cytokines such as IL-2, and relieving the inhibition of PD-1-mediated immunosurveillance of antitumor T lymphocytes.

[0006] In light of the importance of the immune checkpoint molecule PD-1 in regulating immune responses, the present inventors have conducted extensive research and have developed an anti-PD-1 / HER2 bispecific antibody that targets both PD-1 and HER2, which can target HER2 in tumor cells while simultaneously activating T lymphocytes, thereby improving anti-tumor activity and reducing side effects. The patent application number for the anti-PD-1 / HER2 bispecific antibody is PCT / CN2018 / 075851 (filing date: February 8, 2018), in which an anti-PD-1 / HER2 bispecific antibody consisting of an anti-PD-1 half antibody and an anti-HER2 half antibody is constructed and expressed. When tumor-bearing mice were inoculated with HCC1954 human breast cancer cells into immunodeficient NCG mice, the anti-PD-1 / HER2 bispecific antibody was administered. Compared with the administration of anti-HER2 monoclonal antibody or anti-PD-1 monoclonal antibody, the anti-PD-1 / HER2 bispecific antibody exhibited significantly improved antitumor activity and significantly reduced tumor volume.

[0007] There is a need in the art for anti-PD-1 / HER2 bispecific antibody formulations that can treat, prevent, or delay various diseases associated with the HER2 signaling pathway and the PD-1 signaling pathway, and which have good stability such that when formulated into a liquid, the anti-PD-1 / HER2 bispecific antibody in the liquid solution does not easily decompose, aggregate, or undergo undesired chemical modifications. Summary of the Invention

[0008] To address the above needs, the present invention provides a pharmaceutical formulation comprising an anti-PD-1 / HER2 bispecific antibody protein that specifically binds to PD-1 and HER2. The antibody formulation of the present invention can be formulated to suitably administer the antibody to a subject and can maintain its stability during storage and subsequent use.

[0009] In one aspect, the present invention provides a liquid antibody formulation comprising: (i) an anti-PD-1 / HER2 bispecific antibody protein; (ii) a buffering agent; (iii) a stabilizer; and (iv) a surfactant.

[0010] The anti-PD-1 / HER2 bispecific antibody protein in the antibody formulations of the invention comprises a first half antibody comprising a first VH / VL unit that specifically binds PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds HER2. In some embodiments, the anti-PD-1 / HER2 bispecific antibody protein comprises at least about 10 7 M -1 , preferably about 10 8 M -1 , and more preferably about 10 9 M -1 and inhibiting the binding of PD-1 to its ligand by binding to PD-1 on the surface of T lymphocytes with an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 , and more preferably about 10 9 M -1 By binding to HER2 on the surface of tumor cells with an affinity constant of 100 or higher, they can block HER2-mediated intracellular signal transduction and exert anti-tumor effects.

[0011] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein is the recombinant anti-PD-1 / HER2 bispecific antibody protein disclosed in PCT Application No. PCT / CN2018 / 075851 (filing date: February 8, 2018), the entire contents of which are hereby incorporated by reference for purposes of this application. In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising a first VH / VL unit that specifically binds to PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds to HER2, wherein the first VH / VL unit comprises all of the heavy chain and light chain CDRs contained in the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:12 / SEQ ID NO:10, and the second VH / VL unit comprises all of the heavy chain and light chain CDRs contained in the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2.

[0012] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising a first VH / VL unit that specifically binds PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds HER2, wherein the first VH / VL unit is at least 90%, 91%, 92%, or 93% identical to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 12 / SEQ ID NO: 10, or the paired heavy chain variable region sequence / light chain variable region sequence. , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2, and the second VH / VL unit comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2.

[0013] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising, in N to C orientation, the heavy chain sequences of SEQ ID NO: 12 and SEQ ID NO: 14, or heavy chain sequences at least 90%, 95%, 98% or 99% identical thereto, and the light chain sequences of SEQ ID NO: 10 and SEQ ID NO: 4, or light chain sequences with at least 90%, 95%, 98% or 99% identity thereto; and a second half antibody comprising, in N to C orientation, the heavy chain sequences of SEQ ID NO: 6 and SEQ ID NO: 8, or heavy chain sequences with at least 90%, 95%, 98% or 99% identity thereto, and the light chain sequences of SEQ ID NO: 2 and SEQ ID NO: 4, or light chain sequences with at least 90%, 95%, 98% or 99% identity thereto.

[0014] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein is produced by HEK293 cells, or HEK293T, HEK293F, or HEK293E cells obtained by modifying HEK293 cells, or CHO cells, or CHO-S, CHO-dhfr, or CHO-dhfr cells obtained by modifying CHO cells. - , CHO / DG44, or ExpiCHO cells.

[0015] In one embodiment, the concentration of the anti-PD-1 / HER2 bispecific antibody protein in the liquid antibody formulation of the present invention is about 1 to 150 mg / mL. In another embodiment, the concentration of the anti-PD-1 / HER2 bispecific antibody protein in the liquid antibody formulation of the present invention is about 10 to 100 mg / mL. In other embodiments, the concentration of the anti-PD-1 / HER2 bispecific antibody protein in the liquid antibody formulation of the present invention is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL.

[0016] In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 5 to 50 mM, or about 10 to 30 mM, for example, about 10, 15, 20, 25, or 30 mM.

[0017] In one embodiment, the buffering agent is selected from histidine, histidine hydrochloride, and combinations thereof.

[0018] In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 50 to 500 mM, or about 100 to 400 mM, for example, about 100, 150, 200, 250, 300, 350, or 400 mM.

[0019] In one embodiment, the stabilizer is selected from polyols (eg, sorbitol), sugars (eg, sucrose, trehalose), and any combination thereof.

[0020] In another embodiment, the stabilizer is selected from a combination of a polyol (e.g., sorbitol), a sugar (e.g., sucrose, trehalose), or any combination thereof with an antioxidant. In one embodiment, the total concentration of stabilizers in the liquid antibody formulation is about 50 to 500 mM, preferably about 100 to 400 mM, for example, about 100, 150, 200, 250, 300, 350, or 400 mM, of which the concentration of the antioxidant is about 1 to 50 mM, preferably about 5 to 40 mM, for example, about 5, 10, 20, 30, or 40 mM. In one embodiment, the antioxidant is methionine.

[0021] In one embodiment, the concentration of the surfactant in the liquid antibody formulation of the present invention is about 0.1 to 1 mg / mL, or about 0.2 to 0.8 mg / mL, for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg / mL.

[0022] In one embodiment, the surfactant is a non-ionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants. In one specific embodiment, the surfactant in the liquid antibody formulation of the invention is polysorbate 80.

[0023] In one embodiment, the pH value of the liquid formulation is about 5.0 to 6.5. In some embodiments, the pH value of the liquid formulation is any value between about 5.0 and 6.5, such as about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, or 6.4.

[0024] In one embodiment, the liquid formulation is a pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection, hi one embodiment, the liquid formulation is an intravenous infusion.

[0025] In one embodiment, the liquid antibody formulation of the invention comprises: (i) about 1 to 150 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 5 to 50 mM of histidine and / or histidine hydrochloride; (iii) about 50 to 500 mM of sorbitol, sucrose, trehalose, and any combination thereof; or a combination of sorbitol, sucrose, trehalose, or any combination thereof with methionine at a concentration of about 1 to 50 mM, for a total concentration of about 50 to 500 mM; (iv) about 0.1 to 1 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0026] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) about 10 to 100 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 10 to 30 mM of histidine and / or histidine hydrochloride; (iii) about 100 to 400 mM sorbitol, sucrose, and / or trehalose, or a combination of sorbitol, sucrose and / or trehalose and methionine at a concentration of about 5 to 40 mM, with a total concentration of about 100 to 400 mM; (iv) about 0.2 to 0.8 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0027] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) about 20 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 10 mM histidine; (iii) about 50 mg / mL of sorbitol; (iv) about 0.3 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0028] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 50 mg / mL of sorbitol; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0029] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 80 mg / mL of sucrose; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0030] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 80 mg / mL of trehalose; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0031] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 80 mg / mL sucrose and about 1.49 mg / mL methionine; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0032] In one preferred embodiment, the liquid antibody formulation of the invention comprises: (i) approximately 42 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; and (ii) about 0.85 mg / mL of histidine and about 3.17 mg / mL of histidine hydrochloride; (iii) about 80 mg / mL of sucrose; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid preparation is about 5.0 to 6.5, preferably about 5.5.

[0033] In another aspect, the present invention provides a solid antibody formulation obtained by subjecting the liquid antibody formulation of the present invention to a solidification treatment. The solidification treatment is carried out by, for example, a crystallization method, a spray-drying method, or a freeze-drying method. In a preferred embodiment, the solid antibody formulation is in the form of, for example, a freeze-dried powder for injection. The solid antibody formulation can be reconstituted in a suitable solvent before use to form a reconstituted formulation of the present invention. The reconstituted formulation is also a liquid antibody formulation of the present invention. In one embodiment, the suitable solvent is selected from water for injection, an organic solvent for injection, including, but not limited to, oil for injection, ethanol, propylene glycol, etc., or a combination thereof.

[0034] The liquid formulations of the present invention can be stably stored for an extended period of time, for example, at least 24 months or more. In one embodiment, the liquid formulations of the present invention can be stably stored at temperatures from about −80° C. to about 45° C., for example, −80° C., about −30° C., about −20° C., about 0° C., about 5° C., about 25° C., about 35° C., about 38° C., about 40° C., about 42° C., or about 45° C., for a period of at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.

[0035] In one embodiment, the liquid formulation of the present invention can be stably stored for at least 24 months. In yet another embodiment, the liquid formulation of the present invention is stable at at least 40°C. In yet another embodiment, the liquid formulation of the present invention is stably stored at about 2°C to 8°C for at least 3 months, preferably at least 12 months, and more preferably 24 months. In one embodiment, the liquid formulation of the present invention is stably stored at room temperature or, for example, about 25°C for at least 2 months, preferably at least 3 months, and more preferably 6 months. In yet another embodiment, the liquid formulation of the present invention is stably stored at about 40°C for at least 2 weeks, and preferably at least 1 month.

[0036] In one embodiment, the stability of a formulation after storage may be indicated by detecting changes in the appearance, visible extraneous matter, protein content, turbidity, purity, and / or charge variants of the formulation. In one embodiment, the stability of a liquid formulation of the invention may be detected in a high temperature stress experiment, e.g., after storage at 40°C ± 2°C for at least 1 week, 2 weeks, or preferably 1 month, or in an accelerated experiment, e.g., after storage at 25°C ± 2°C for at least 1 month or 2 months, or in a long-term experiment, e.g., after storage at 5°C ± 3°C for at least 2 months or 3 months.

[0037] In one embodiment, the stability of the liquid formulation of the present invention after storage is examined visually, where the liquid formulation of the present invention remains clear to slightly opalescent in appearance, is a colorless to pale yellow liquid, and is free of foreign matter. In one embodiment, the formulation is free of visible foreign matter when examined visually with a transparency detector. In one embodiment, the stability of the liquid formulation of the present invention after storage is examined by measuring the change in protein content, where, for example, by ultraviolet spectrophotometry (UV), the rate of change in protein content is 20% or less, preferably 10% or less, e.g., 7-8%, and more preferably 5% or less, relative to the initial value on day 0 of storage. In one embodiment, the stability of the liquid formulation of the present invention is examined by measuring the change in turbidity of the liquid formulation of the present invention after storage, where, for example, OD 350nmWhen detected by a method, the change from the initial value on day 0 of storage is 0.06 or less, preferably 0.06 or less, and more preferably 0.04 or less. In one embodiment, the stability of the liquid formulation of the present invention is examined by measuring the change in purity of the liquid formulation of the present invention after storage, whereby, when measured by size exclusion high performance liquid chromatography (SEC-HPLC), the change in purity of the monomer is 10% or less, e.g., 5%, 4%, or 3% or less, e.g., 1-2% or less, preferably 1% or less, relative to the initial value on day 0 of storage. In one embodiment, the stability of the liquid formulation of the present invention is examined by measuring the change in purity of the liquid formulation of the present invention after storage, whereby, when measured by non-reduced and / or reduced sodium lauryl sulfate capillary electrophoresis (CE-SDS), the change in purity of the monomer is reduced by 10% or less, e.g., 5%, 4%, or 3% or less. In one embodiment, the stability of the liquid formulation of the present invention is measured by imaging capillary isoelectric focusing (iCIEF) after storage, whereby the combined change in the antibody charge variants (major component, acidic component, and basic component) relative to the initial value on day 0 of storage is 50% or less, for example, 40%, 30%, 20%, 10%, or 5% or less. In one embodiment, the stability of the liquid formulation of the present invention is detected by cation exchange high performance liquid chromatography (CEX-HPLC) after storage, whereby the combined change in the antibody charge variants (major component, acidic component, and basic component) relative to the initial value on day 0 of storage is 40% or less, for example, 38%, 36%, 34%, 32%, or 30% or less.

[0038] In one embodiment, the formulation is stable after storage, for example after at least 24 months at 2-8°C, or after at least 3 months at room temperature, or after 1 month at 40°C ± 2°C, and preferably has the following properties: (i) the formulation has a purity of greater than 90%, preferably greater than 95%, 96%, 97%, 98%, or 99%, as measured by SEC-HPLC; (ii) the formulation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, or 98%, as measured by reduced or non-reduced CE-SDS methods; (iii) when measured by the iCIEF method, the total change in the components (main component, acidic component, and basic component) of the anti-PD-1 / HER2 bispecific antibody protein in the formulation is 50% or less, for example, 40%, 30%, 20%, 10%, or 5% or less, relative to the initial value on day 0 of storage; (iv) the relative binding activity of the anti-PD-1 / HER2 bispecific antibody protein in the formulation is 70% to 130%, e.g., 70%, 80%, 90%, 100%, 110%, 120%, or 130%, relative to the initial value on day 0 of storage, as measured by ELISA; The present invention has one or more of the following characteristics.

[0039] In one aspect, the present invention provides a delivery device comprising a liquid or solid antibody formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe containing the liquid or solid antibody formulation of the present invention, and is used, for example, for intravenous, subcutaneous, intradermal, or intramuscular injection, or intravenous infusion.

[0040] In yet another aspect, the invention provides a method for delivering an anti-PD-1 / HER2 bispecific antibody protein to a subject, e.g., a mammal, comprising administering to the subject a liquid or solid antibody formulation of the invention, wherein the delivery is accomplished by a delivery device, e.g., utilizing a pre-filled syringe.

[0041] In yet another aspect, the invention provides the use of a liquid or solid antibody formulation of the invention for the manufacture of a delivery device (e.g., a pre-filled syringe) or medicament for treating, preventing, or delaying a disorder associated with the HER2 signaling pathway and the PD-1 signaling pathway in a subject, such as various hematological diseases and solid tumors, including, but not limited to, 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, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, and melanoma.

[0042] Other embodiments of the present invention will become apparent upon reference to the following detailed description. Preferred embodiments of the invention, which will now be described in detail, will be better understood when read in conjunction with the following drawings: For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, but it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0043] [Figure 1] 1 illustrates the structure of an anti-PD-1 / HER2 bispecific antibody comprising an anti-PD-1 half antibody molecule and an anti-HER2 half antibody molecule. [Figure 2] Figure 1 shows a trend graph of turbidity change in anti-PD-1 / HER2 bispecific antibody formulations measured by OD350nm method after incubation at 40°C ± 2°C for various periods at pH 5.0, 5.5, 6.0, and 6.5. On the horizontal axis, TO represents 0 days, 1W represents 1 week, 2W represents 2 weeks, and 1M represents 1 month. [Figure 3] Figure 1 shows a trend graph of the change in protein purity of anti-PD-1 / HER2 bispecific antibody formulations measured by SEC-HPLC after incubation at pH 5.0, 5.5, 6.0, and 6.5 at 40°C ± 2°C for different periods of time. On the horizontal axis, TO represents 0 days, 1W represents 1 week, 2W represents 2 weeks, and 1M represents 1 month. [Figure 4] Figure 1 shows a trend graph of the change in protein purity for anti-PD-1 / HER2 bispecific antibody formulations measured by non-reducing CE-SDS assay after incubation at 40°C ± 2°C at pH 5.0, 5.5, 6.0, and 6.5 for different periods of time. On the horizontal axis, TO represents 0 days, 1W represents 1 week, 2W represents 2 weeks, and 1M represents 1 month. [Figure 5] Figure 1 shows a trend graph of the change in protein purity for anti-PD-1 / HER2 bispecific antibody formulations measured by reduced CE-SDS assay after incubation at 40°C ± 2°C at pH 5.0, 5.5, 6.0, and 6.5 for different periods of time. On the horizontal axis, TO represents 0 days, 1W represents 1 week, 2W represents 2 weeks, and 1M represents 1 month. [Figure 6]This figure shows the trend graph of changes in the major components of charge variants in anti-PD-1 / HER2 bispecific antibody formulations measured by iCIEF after incubation at 40°C ± 2°C for different periods of time at pH 5.0, 5.5, 6.0, and 6.5. On the horizontal axis, TO represents 0 days, 1W represents 1 week, 2W represents 2 weeks, and 1M represents 1 month. [Figure 7] Figure 1 shows the time course of the major components of charge variants measured by the iCIEF method after storage of anti-PD-1 / HER2 bispecific antibody formulations with different stabilizers (Formulations 1-4) at approximately 40°C for 0 days, 1 week, 2 weeks, and 4 weeks. On the horizontal axis, TO represents 0 days, 1W represents 1 week, 2W represents 2 weeks, 4W represents 4 weeks, F1 represents Formulation 1, F2 represents Formulation 2, F3 represents Formulation 3, and F4 represents Formulation 4. [Figure 8] This graph shows the time course of the major components of charge variants measured by the iCIEF method after storage of anti-PD-1 / HER2 bispecific antibody formulations with different stabilizers (Formulations 1-4) at 25°C ± 2°C for 0 days, 1 week, 2 weeks, and 4 weeks. On the horizontal axis, TO represents 0 days, 1M represents 1 month, 2M represents 2 months, F1 represents Formulation 1, F2 represents Formulation 2, F3 represents Formulation 3, and F4 represents Formulation 4. DETAILED DESCRIPTION OF THE INVENTION

[0044] Before describing the present invention in detail, it is to be understood that this invention is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary, and the terminology used herein is not limiting, but rather for the purpose of describing particular embodiments.

[0045] definition Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined below.

[0046] The term "about," when used in conjunction with a numerical value, is meant to include numbers in a range from 5% less than the stated numerical value as a lower limit, to 5% more than the stated numerical value as an upper limit.

[0047] The term "and / or", when used in conjunction with two or more options, should be understood to refer to any one of the options or to any two or more of the options.

[0048] As used herein, the term "comprise" or "comprises" means including the stated element, integer, or step, but not excluding any other element, integer, or step. As used herein, the term "comprise" or "comprises" also includes cases where the stated element, integer, or step is the only element, integer, or step, unless otherwise specified. For example, when referring to "comprising" an antibody variable region of a specific sequence, it is intended to include an antibody variable region consisting of that sequence.

[0049] As used herein, the term "antibody" is used in the broadest sense to mean a protein that contains an antigen-binding site and covers natural and artificial antibodies of various structures, including, but not limited to, intact antibodies and antigen-binding fragments of antibodies.

[0050] The terms "whole antibody," "full-length antibody," "complete antibody," and "complete antibody" are used herein to refer interchangeably to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected to each other via disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity-determining regions (CDRs)) interposed by conserved regions (framework regions (FRs))). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various effector functions.

[0051] The term "humanized" antibody is a chimeric antibody that contains amino acid residues from non-human HVRs and human FRs. In some embodiments, all or nearly all of the HVRs (e.g., CDRs) contained in a humanized antibody correspond to those HVRs of a non-human antibody, and all or nearly all of the FR regions correspond to those FRs of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0052] The term "half antibody" or "half polymer" refers to a monovalent antigen-binding polypeptide. In some embodiments, a half antibody or half polymer comprises a VH / VL unit and optionally at least a portion of an immunoglobulin constant domain. In some embodiments, a half antibody or half polymer comprises one immunoglobulin heavy chain, or an antigen-binding fragment thereof, linked to one immunoglobulin light chain. In some embodiments, a half antibody or half polymer is monospecific, i.e., binds to a single antigen or epitope. In some specific embodiments, a half antibody binds to HER2 and does not bind to PD-1. In some specific embodiments, a half antibody binds to PD-1 and does not bind to HER2. Those skilled in the art will readily appreciate that a half antibody may have an antigen-binding domain consisting of a single variable domain (e.g., from Camelidae).

[0053] The term "VH / VL unit" refers to the antigen-binding region of an antibody that includes at least one VH CDR and at least one VL CDR. In some embodiments, a VH / VL unit includes at least one, at least two, or all three VH CDRs and at least one, at least two, or all three VL CDRs. In certain embodiments, a VH / VL unit further includes at least a portion of a framework region (FR). In some embodiments, a VH / VL unit includes three VH CDRs and three VL CDRs. In some embodiments, a VH / VL unit includes at least one, at least two, at least three, or all four VH FRs and at least one, at least two, at least three, or all four VL FRs.

[0054] As used herein, the term "bispecific antibody" refers to an antibody that contains antigen-binding domains that specifically bind to epitopes on two different biological molecules. Unless otherwise specified, the names of the bispecific antibodies listed refer to the order of the antigens bound by the bispecific antibody in random order. That is, in some embodiments, the terms "anti-PD-1 / HER2 bispecific antibody" and "anti-HER2 / PD-1 bispecific antibody" may be used interchangeably. In some embodiments, a bispecific antibody comprises two half antibodies, each comprising a single heavy chain variable region and, optionally, at least a portion of a heavy chain constant region, and a single light chain variable region and, optionally, at least a portion of a light chain constant region. In some embodiments, a bispecific antibody comprises two half antibodies, each comprising a single heavy chain variable region and a single light chain variable region, but not more than one single heavy chain variable region or more than one single light chain variable region. In some embodiments, a bispecific antibody comprises two half antibodies, each comprising a single heavy chain variable region and a single light chain variable region, wherein the first half antibody binds to a first antigen but not to a second antigen, and the second half antibody binds to the second antigen but not to the first antigen.

[0055] The term "antibody formulation" refers to a preparation in which the biological activity of the active ingredient, an antibody, can be effectively exerted, and which does not contain other ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. These antibody formulations are generally sterile. Antibody formulations usually contain a pharmaceutically acceptable excipient. A "pharmaceutically acceptable" excipient is a reagent that can be appropriately administered to a mammalian subject so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient depends on the mode of administration, and may be, for example, a concentration acceptable for injection.

[0056] The term "anti-PD-1 / HER2 bispecific antibody formulation," also abbreviated herein as "antibody formulation of the present invention," refers to a preparation containing an anti-PD-1 / HER2 bispecific antibody protein as an active ingredient and a pharmaceutically acceptable excipient. The combination of the anti-PD-1 / HER2 bispecific antibody protein with a pharmaceutically acceptable excipient renders the active ingredient, the anti-PD-1 / HER2 bispecific antibody protein, suitable for therapeutic or prophylactic administration to humans or non-human animals. The antibody formulations of the present invention may be prepared as aqueous liquid formulations, such as ready-to-use pre-filled syringes, or as lyophilized formulations that are reconstituted (i.e., redissolved) by dissolving and / or suspending in a physiologically acceptable solution immediately before use. In some embodiments, the anti-PD-1 / HER2 bispecific antibody protein formulation is in a liquid formulation form.

[0057] A "stable" antibody formulation is one in which the antibody in the formulation maintains an acceptable degree of physical and / or chemical stability after storage under specified conditions. While the antibody contained in the antibody formulation may not maintain 100% of its chemical structure after storage for a specified period, an antibody formulation is generally considered "stable" if it maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function after storage for a specified period. In some specific embodiments, the anti-PD-1 / HER2 bispecific antibody protein formulations of the present invention exhibit undetectable levels of antibody aggregation, degradation, or chemical modification during production, preparation, transportation, and long-term storage, thereby exhibiting high stability with minimal or no loss of biological activity of the anti-PD-1 / HER2 bispecific antibody protein. In some embodiments, the anti-PD-1 / HER2 bispecific antibody protein formulations of the present invention substantially maintain their physical and chemical stability after storage. Preferably, the liquid formulations of the present invention are stable at room temperature or at 40°C for at least 2 weeks, and / or stable at 25°C for at least 2 months, and / or stable at 2-8°C for at least 24 months.

[0058] Several analytical techniques are known in the art for measuring protein stability; see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A., Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature and a selected storage time. For example, the storage time may be selected based on the expected shelf life of the formulation. In some cases, accelerated stability testing may be employed. In some embodiments, stability testing is performed by subjecting the antibody formulation to various stress tests. These tests may represent the stress conditions that a prepared antibody formulation may encounter during manufacturing, storage, or transportation, or may represent conditions that may accelerate antibody instability in the antibody formulation during non-manufacturing, storage, or transportation. For example, to verify antibody stability under high-temperature stress, a formulated anti-PD-1 / HER2 bispecific antibody protein formulation may be filled into a glass vial.

[0059] An antibody is considered to "maintain its physical stability" in a formulation if the formulation exhibits no or very little aggregation, precipitation, turbidity, and / or denaturation after storage for a certain period of time. Aggregation of antibodies in a formulation poses a safety issue because it can potentially increase the immune response of patients. Therefore, it is necessary to minimize or prevent aggregation of antibodies in formulations. Light scattering methods can be used to measure visible aggregates in formulations. SEC can be used to measure soluble aggregates in formulations. Additionally, formulations can be visually inspected for appearance, color, and / or clarity, or by OD spectroscopy. 350nm The stability of a formulation can be demonstrated by measuring the turbidity of the formulation using a CE-SDS method or by measuring the purity of the formulation using a non-reducing CE-SDS method. In one embodiment, the stability of a formulation is measured by measuring the percentage of antibody monomer in the formulation after storage at a predetermined temperature for a predetermined period of time, where the greater the percentage of antibody monomer in the formulation, the more stable the formulation.

[0060] "Acceptable" physical stability can refer to at least about 92% of the anti-PD-1 / HER2 bispecific antibody protein monomer measured in the formulation after storage at a given temperature for a given period of time. In some embodiments, acceptable physical stability refers to at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-PD-1 / HER2 bispecific antibody protein monomer after storage at a given temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When assessing physical stability, the predetermined temperature at which the pharmaceutical formulation is stored may be any temperature between about −80° C. and about 45° C., for example, about −80° C., about −30° C., about −20° C., about 0° C., about 4° C. to 8° C., about 5° C., about 25° C., about 35° C., about 37° C., about 40° C., about 42° C., or about 45° C. For example, a pharmaceutical formulation is considered stable if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-PD-1 / HER2 bispecific antibody protein monomer is detected after storage at about 40° C.±2° C. for one month or four weeks. A pharmaceutical formulation is considered stable if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-PD-1 / HER2 bispecific antibody protein monomer is detectable after two months of storage at about 25° C. A pharmaceutical formulation is considered stable if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-PD-1 / HER2 bispecific antibody protein monomer is detectable after nine months of storage at about 5° C.

[0061] An antibody is considered to "maintain its chemical stability" in a formulation if it does not exhibit significant chemical changes after storage for a certain period of time. Most chemical instability results from the formation of covalently modified forms of the antibody (e.g., antibody charge variants). For example, basic variants can be formed by aspartic acid isomerization, N- and C-terminal modifications, and acidic variants can be formed by deamidation, sialylation, and glycation. Chemical stability can be assessed by measuring and / or quantifying the chemical modification forms of the antibody. For example, antibody charge variants in a formulation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing (iCIEF). In one embodiment, the stability of a formulation is measured by measuring the percentage change in antibody charge variants in the formulation after storage for a certain period of time at a certain temperature, where the smaller the change, the more stable the formulation.

[0062] An "acceptable" degree of chemical stability can indicate a change in the percentage of charge variants (e.g., major component, acidic component, or basic component) in the formulation after storage at a given temperature for a given period of time of 50% or less, e.g., 30% or less, 20% or less. In some embodiments, an acceptable degree of chemical stability can be indicated by a change in the percentage of major component charge variants of about 50%, 40%, 30%, 20%, 15% or less after storage at a given temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When assessing chemical stability, the pharmaceutical formulation may be stored at any temperature between about −80° C. and about 45° C., for example, at about −80° C., about −30° C., about −20° C., about 0° C., about 4° C. to 8° C., about 5° C., about 25° C., or about 45° C. For example, after 24 months of storage at 5° C., a pharmaceutical formulation is considered stable if the change in the percentage of major component charge variants is less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. A pharmaceutical formulation is also considered stable if the percentage change in the major component charge variant is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% after two months of storage at 25° C. A pharmaceutical formulation is also considered stable if the percentage change in the major component charge variant is less than about 50%, 40%, 30%, 20%, 10%, 5%, or 4% after one month of storage at 40° C.

[0063] The term "lyophilized formulation" refers to a composition obtained or obtainable by the process of freeze-drying a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0064] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

[0065] As used herein, the term "room temperature" refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, and more preferably 25°C.

[0066] "Harsh conditions" refer to an environment that is chemically and / or physically unfavorable to an antibody protein, which can cause the antibody protein to become unstable. "High temperature harsh" refers to storing an antibody formulation at room temperature or higher (e.g., 40°C ± 2°C) for a certain period of time. The stability of an antibody formulation can be examined by a high temperature harsh accelerated test.

[0067] As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, a stable anti-PD-1 / HER2 bispecific antibody protein formulation of the invention is administered parenterally to a subject. In one embodiment, an anti-PD-1 / HER2 bispecific antibody protein formulation of the invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

[0068] I. Antibody Preparations The present invention provides a stable liquid antibody formulation having a pH of about 5.0 to 6.5, comprising (i) an anti-PD-1 / HER2 bispecific antibody protein, (ii) a buffering agent, (iii) a stabilizer, and (iv) a surfactant. In a preferred embodiment, the liquid antibody formulation of the present invention is in the form of an injectable formulation.

[0069] (i) anti-PD-1 / HER2 bispecific antibody protein The "anti-PD-1 / HER2 bispecific antibody protein" in the antibody formulations of the present invention comprises a first half antibody comprising a first VH / VL unit that specifically binds to PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds to HER2. In some embodiments, the anti-PD-1 / HER2 bispecific antibody protein comprises at least about 10 7 M -1 , preferably about 10 8 M -1 , and more preferably about 10 9 M -1 or higher and can bind to PD-1 on the surface of T lymphocytes with an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 , and more preferably about 10 9 M -1 By being able to bind to HER2 on the surface of tumor cells with an affinity constant of 100 or higher, the antibody can be used as a therapeutic and / or prophylactic agent that bispecifically targets PD-1 and HER2 molecules.

[0070] The VH / VL units that specifically bind to PD-1 or HER2 comprise six CDRs derived from any of the anti-PD-1 antibodies reported in the prior art and future anti-PD-1 antibody VH / VL units, or sequences with one, two, three, four, five, six, or more amino acid mutations (e.g., amino acid substitutions or deletions) in one or more of the six CDRs; or six CDRs derived from any of the anti-HER2 antibodies reported in the prior art and future anti-HER2 antibody VH / VL units, or sequences with one, two, three, four, five, six, or more amino acid mutations (e.g., amino acid substitutions or deletions) in one or more of the six CDRs.

[0071] In one embodiment, the first VH / VL unit that specifically binds PD-1 of the anti-PD-1 / HER2 bispecific antibody protein comprises all six heavy and light chain CDRs contained in the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 12 / SEQ ID NO: 10 derived from an anti-PD-1 half antibody, or sequences with one, two, three, four, five, six, or more amino acid mutations (e.g., amino acid substitutions or deletions) in one or more of the six CDRs.

[0072] In one embodiment, the second VH / VL unit that specifically binds HER2 of the anti-PD-1 / HER2 bispecific antibody protein comprises all six heavy and light chain CDRs contained in the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2 derived from an anti-HER2 half antibody, or sequences with one, two, three, four, five, six, or more amino acid mutations (e.g., amino acid substitutions or deletions) in one or more of the six CDRs.

[0073] The terms "CDR" or "complementarity-determining region" or "CDR region" (which may be used interchangeably herein with hypervariable region "HVR") refer to amino acid regions in an antibody variable region that are primarily responsible for binding to an antigen epitope. Heavy and light chain CDRs are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. Many methods for determining the CDR sequences of a given VH, VL, or VHH amino acid sequence are well known in the art. For example, the Kabat complementarity-determining region (CDR), determined by sequence variability, is the most commonly used (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the location of the structural rings (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs are a trade-off between the Kabat HVRs and the Chothia structural rings and are used by Oxford Molecular's AbM antibody modeling software. The "Contact" HVRs are based on analysis of available complex crystal structures.

[0074] The amino acid mutation, e.g., amino acid substitution, is preferably a conservative amino acid substitution. "Conservative amino acid substitution" refers to an amino acid change in which a specific amino acid is replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. In one embodiment of the present invention, in one preferred aspect, the conservative substitution residue is taken from Conservative Substitution Table A below, and preferably is a preferred substitution residue shown in Table A.

[0075] [Table 1]

[0076] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising a first VH / VL unit that specifically binds PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds HER2, wherein the first VH / VL unit is at least 90%, 91%, 92%, or 93% identical to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 12 / SEQ ID NO: 10, or the paired heavy chain variable region sequence / light chain variable region sequence. , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2, and the second VH / VL unit comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2.

[0077] The type of heavy chain constant region of the first half antibody and the second half antibody in the anti-PD-1 / HER2 bispecific antibody protein is not particularly limited, but is preferably the heavy chain constant region of an IgG1, IgG2, or IgG4 immunoglobulin, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical). More preferably, the heavy chain constant region is the heavy chain constant region of a human IgG1 immunoglobulin, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0078] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a heavy chain constant region used in IgG1 (e.g., human IgG1). In another embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a heavy chain constant region used in IgG4 (e.g., human IgG4). For example, the Fc domains of the two heavy chains of the anti-PD-1 / HER2 bispecific antibody each contain hinge regions with "CPPC" amino acid residues and / or Y349C and S354C (according to the "EU numbering system" of Kabat), respectively, allowing the anti-PD-1 half antibody and the anti-HER2 half antibody to form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the anti-PD-1 half antibody and the anti-HER2 half antibody.

[0079] In one embodiment, the anti-PD-1 half antibody and / or the anti-HER2 half antibody of the anti-PD-1 / HER2 bispecific antibody protein comprise amino acid mutations in the Fc domain that affect antibody effector function. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the amino acid mutations are in the CH2 domain of the Fc region; for example, the anti-PD-1 / HER2 bispecific antibody protein comprises amino acid substitutions at positions 234 and 235 (EU numbering system) in the anti-PD-1 half antibody and / or anti-HER2 half antibody Fc region. In a specific embodiment, the amino acid substitutions are L234A and L235A (also referred to as "LALA mutations").

[0080] In another embodiment, the light chain of the anti-PD-1 / HER2 bispecific antibody protein comprises a kappa light chain constant region or a lambda light chain constant region, e.g., a human kappa light chain constant region or a human lambda light chain constant region.

[0081] In one embodiment, the Fc domains of the two heavy chains of the anti-PD-1 / HER2 bispecific antibody protein each contain a protrusion ("knob") or a cavity ("hole"), and the protrusion or cavity in one heavy chain Fc domain can fit into the cavity or protrusion in the other heavy chain Fc domain, thereby forming a stable "knob-in-hole" bond between the two heavy chains. In one embodiment, one of the two heavy chains contains the amino acid substitution T366W, and the other heavy chain contains the amino acid substitutions T366S, L368A, and Y407V (EU numbering system). This allows the protrusion in one chain to fit into the cavity in the other chain, contributing to the correct pairing of the two heavy chains of the anti-PD-1 / HER2 bispecific antibody protein.

[0082] In one embodiment, the immunoglobulin CH1 domain and CL domain of the heavy and light chains of each half of the anti-PD-1 / HER2 bispecific antibody protein comprise a protrusion or cavity, respectively, and the protrusion or cavity in the CH1 domain can be placed in the cavity or protrusion in the CL domain, respectively, so that the heavy and light chains of each half also form a stable "knobs-in-hole" bond.

[0083] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising, from N to C orientation, the heavy chain sequences of SEQ ID NO: 12 and SEQ ID NO: 14, or heavy chain sequences at least 90%, 95%, 98% or 99% identical thereto, and the light chain sequences of SEQ ID NO: 10 and SEQ ID NO: 4, or light chain sequences with at least 90%, 95%, 98% or 99% identity thereto; and a second half antibody comprising, from N to C orientation, the heavy chain sequences of SEQ ID NO: 6 and SEQ ID NO: 8, or heavy chain sequences with at least 90%, 95%, 98% or 99% identity thereto, and the light chain sequences of SEQ ID NO: 2 and SEQ ID NO: 4, or light chain sequences with at least 90%, 95%, 98% or 99% identity thereto.

[0084] As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. The "percentage of sequence identity" can be calculated by the following method: Two best-matched sequences are compared within a comparison window, and the number of positions in the two sequences containing the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is determined to obtain the number of matching positions. The number of matching positions is then divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to generate a percentage of sequence identity. The best-matching performed to determine the percent sequence identity can be achieved by various methods known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for matching sequences, including any algorithms needed to achieve maximum matching within the full length sequences being compared or within a target sequence region.

[0085] The anti-PD-1 / HER2 bispecific antibody protein in the antibody formulation of the present invention can simultaneously bind to PD-1 and HER2 proteins while maintaining the affinity constants of the parent antibodies, thereby enabling it to block the HER2 signaling pathway and the PD-1 signaling pathway. Therefore, it can be used to treat, prevent, or delay various diseases or disorders associated with the HER2 signaling pathway and / or the PD-1 signaling pathway.

[0086] In a preferred embodiment, the anti-PD-1 / HER2 bispecific antibody protein of the present invention is the recombinant anti-PD-1 / HER2 bispecific antibody protein disclosed in PCT Application No. PCT / CN2018 / 075851 (filing date: February 8, 2018), which includes a fully human anti-PD-1 half antibody and a humanized anti-HER2 half antibody, of which the fully human anti-PD-1 half antibody has heavy chain sequences, from N to C, of ​​SEQ ID NOs: 12 and 14, and light chain sequences, from N to C, of ​​SEQ ID NOs: 10 and 4; and the humanized anti-HER2 half antibody has heavy chain sequences, from N to C, of ​​SEQ ID NOs: 6 and 8, and light chain sequences, from N to C, of ​​SEQ ID NOs: 2 and 4.

[0087] In one embodiment, the anti-PD-1 / HER2 bispecific antibody protein is produced by HEK293 cells, or HEK293T, HEK293F, or HEK293E cells obtained by modifying HEK293 cells, or CHO cells, or CHO-S, CHO-dhfr, or CHO-dhfr cells obtained by modifying CHO cells. - , CHO / DG44, or ExpiCHO cells and purified. Preferably, the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity. When the anti-PD-1 / HER2 bispecific antibody was administered to tumor-bearing mice, which were obtained by inoculating immunodeficient NCG mice with HCC1954 human breast cancer cells, the anti-PD-1 / HER2 bispecific antibody showed significantly improved anti-tumor activity and significantly reduced tumor volume compared to the anti-PD-1 monoclonal antibody or anti-HER2 monoclonal antibody.

[0088] The amount of anti-PD-1 / HER2 bispecific antibody protein contained in the antibody formulations of the present invention can vary depending on the particular desired characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is being used. In some embodiments, the antibody formulations are liquid formulations and may contain about 1 to 150 mg / mL, preferably about 10 to 100 mg / mL, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL of anti-PD-1 / HER2 bispecific antibody protein.

[0089] (ii) Buffer A buffer is a reagent capable of maintaining the pH value of a solution within an acceptable range. In some embodiments, the buffer used in the formulations of the present invention is capable of controlling the pH of the formulations of the present invention within a pH range of about 5.0 to 6.5, for example, at about pH 5.5. In some specific embodiments, the antibody formulations of the present invention have a pH of about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, or 6.4.

[0090] In some embodiments, the buffer used in the formulation of the present invention is selected from histidine, histidine hydrochloride, and combinations thereof. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 5 to 50 mM. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 10 to 30 mM, e.g., about 10, 15, 20, 25, or 30 mM.

[0091] In one embodiment, the buffer used in the formulations of the present invention is about 10 mM histidine, hi another embodiment, the buffer used in the formulations of the present invention is about 20 mM histidine.

[0092] In yet another embodiment, the buffer used in the formulations of the present invention is a combination of about 5.5 mM histidine and about 15 mM histidine hydrochloride.

[0093] (iii) stabilizers Suitable stabilizers for use in the present invention may be selected from sugars, polyols, and combinations thereof. Additionally, the stabilizer of the present invention may include an antioxidant.

[0094] The saccharide stabilizer may be a disaccharide, trisaccharide, or polysaccharide, and the saccharide may be selected from, but is not limited to, sucrose, dextroglucose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, and dextran. In one embodiment, the saccharide stabilizer is sucrose and / or trehalose.

[0095] The stabilizing polyol may be selected from, but is not limited to, mannitol, sorbitol, and xylitol, hi one embodiment, the stabilizing polyol is sorbitol.

[0096] In some embodiments, the stabilizer sugar and / or polyol is present in the liquid formulation of the present invention at a concentration of about 50-500 mM, preferably about 100-400 mM, for example, about 100, 150, 200, 250, 300, 350, 400 mM.

[0097] Antioxidants that can be further included in the stabilizer of the present invention include, but are not limited to, homocysteine, cysteine, cystathionine, methionine, glutathione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine, and glutathione. When an antioxidant is included, the total concentration of the stabilizer is about 50 to 500 mM, preferably about 100 to 400 mM, for example, about 100, 150, 200, 250, 300, 350, or 400 mM, and the concentration of the antioxidant is about 1 to 50 mM, preferably about 5 to 40 mM, for example, about 5, 10, 20, 30, or 40 mM.

[0098] In one embodiment, the liquid formulation of the present invention contains sorbitol as a stabilizer. The amount of sorbitol in the liquid formulation of the present invention may be about 50 to 400 mM, for example, about 50, 100, 150, 200, 250, 300, 350, or 400 mM.

[0099] In one embodiment, the liquid formulation of the present invention contains sucrose as a stabilizer. The amount of sucrose in the liquid formulation of the present invention may be about 50 to 300 mM, for example, about 50, 100, 150, 200, 250, or 300 mM.

[0100] In one embodiment, the liquid formulation of the present invention contains trehalose as a stabilizer. The amount of trehalose in the liquid formulation of the present invention may be about 50 to 300 mM, for example, about 50, 100, 150, 200, 250, or 300 mM.

[0101] In one embodiment, the liquid formulation of the present invention contains a combination of sucrose and methionine as a stabilizer, in which the total concentration of the stabilizers is about 50 to 500 mM, preferably about 100 to 400 mM, for example, about 100, 150, 200, 250, 300, 350, or 400 mM, and the concentration of methionine is about 1 to 50 mM, preferably about 5 to 40 mM, for example, about 5, 10, 20, 30, or 40 mM.

[0102] (iv) surfactants As used herein, the term "surfactant" refers to organic substances with amphiphilic structures, i.e., they consist of groups with opposite solubility tendencies, generally consisting of an oil-soluble hydrocarbon chain and a water-soluble ionic group.

[0103] In one embodiment, the surfactant in the liquid formulation of the present invention is a non-ionic surfactant, such as an alkylpoly(oxylene). Specific non-ionic surfactants that can be included in the formulation of the present invention include, for example, polysorbates such as polysorbate 20, polysorbate 80, polysorbate 60, polysorbate 40, and pluronics. In one preferred embodiment, the liquid formulation of the present invention includes polysorbate 80 as the surfactant.

[0104] The amount of surfactant included in the antibody formulations of the present invention can vary depending on the particular desired characteristics of the formulation, the particular environment, and the particular purpose for which the formulation will be used. In some preferred embodiments, the formulation may contain about 0.1 to 1 mg / mL, preferably about 0.2 to 0.8 mg / mL, e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg / mL, of polysorbate surfactant (e.g., polysorbate 80).

[0105] (v) Other excipients The antibody liquid formulation of the present invention may or may not contain other excipients. For example, the antibody liquid formulation of the present invention may contain a tonicity modifier. The tonicity modifier is selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.

[0106] These excipients, other known pharmaceutical excipients, and / or additives used in the formulations of the present invention are known in the art, and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, edited by Rowe et al., American Pharmaceuticals Association (2003)" and "Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)."

[0107] II. Preparation of the Formulation The present invention provides stable formulations comprising anti-PD-1 / HER2 bispecific antibody proteins. The anti-PD-1 / HER2 bispecific antibody proteins used in the formulations of the present invention can be prepared by techniques known in the art for producing antibodies. For example, the anti-PD-1 / HER2 bispecific antibody proteins can be recombinantly prepared. In a preferred embodiment, the anti-PD-1 / HER2 bispecific antibody proteins of the present invention are produced using recombinant human PD-1 / HER2 antibodies (HEK293 cells, HEK293T, HEK293F, or HEK293E cells derived from HEK293 cells, or CHO cells, CHO-S, CHO-dhfr, or CHO-dhfr cells derived from CHO cells). - , CHO / DG44, or ExpiCHO cells, and recombinantly prepared anti-PD-1 / HER2 bispecific antibody proteins, for example, as described in PCT Application No. PCT / CN2018 / 075851.

[0108] Currently, antibodies are widely used as active ingredients in medicines. Techniques for purifying therapeutic antibodies to pharmaceutical levels are known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613) describe a three-column antibody purification method that utilizes ion exchange chromatography (anion IEX and / or cation CEX chromatography) after a protein A capture step. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553-566) describe a two-column purification method in which the resin is exchanged with a weakly partitioning anion after protein A affinity chromatography.

[0109] In general, recombinantly produced antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient repeatability and appropriate purity for preparing antibody formulations. For example, after the antibody is secreted into the culture medium from recombinant expression cells, the supernatant from the expression system can be concentrated using a commercially available protein concentration filter, such as an Amicon ultrafiltration device. The antibody can then be purified by, for example, chromatography, dialysis, affinity purification, etc. Protein A is used as an affinity ligand to purify IgG1, IgG2, and IgG4 antibodies. Other antibody purification methods, such as ion exchange chromatography, may also be used. After obtaining an antibody of sufficient purity, a formulation containing the antibody can be prepared using methods known in the art.

[0110] For example, the preparation may include the following steps: (1) centrifuging the fermentation broth after fermentation is complete to remove impurities such as cells and obtain a supernatant; (2) capturing antibodies by affinity chromatography (e.g., a protein A column with specific affinity for IgG1, IgG2, and IgG4 antibodies); (3) inactivating the virus; (4) purifying the protein (typically using cation exchange chromatography); (5) filtering the virus (to reduce the viral titer by, for example, 4 log or more); and (6) ultrafiltration / osmotic filtration (which can be used to replace the protein with a formulation buffer that contributes to its stability and concentrate it to a concentration appropriate for injection). See, for example, B. Minow, P. Rogge, and K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.

[0111] III. Methods for Analyzing Formulations During storage of antibody formulations, antibody aggregation, degradation, or chemical modification may occur, leading to antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates, fragments, etc., which may affect the quality of the antibody formulation. Therefore, it is necessary to monitor the stability of antibody formulations.

[0112] Several methods are known in the art for measuring the stability of antibody formulations. For example, methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody formulations and evaluate the level of antibody aggregation. Capillary isoelectric focusing (CIEF), imaging capillary isoelectric focusing (iCIEF), and ion exchange chromatography (IEX) can be used to analyze charge variants in antibody formulations. Furthermore, the stability of a formulation can be quickly determined by visually detecting the appearance of the formulation. OD, which can provide information on the amount of soluble and insoluble aggregates, can also be used. 350nmThe method can also detect changes in the turbidity of the formulation. Also, ultraviolet spectrophotometry (UV method) can detect changes in the protein content of the formulation.

[0113] The non-reducing CE-SDS method is a method for measuring antibody purity using a capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge due to SDS binding, which is directly proportional to the protein's molecular weight. Because all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method is widely applied to monitor the purity of denatured intact antibodies. In general, in the non-reducing CE-SDS method, the sample is mixed with SDS sample buffer and iodoacetamide. The mixture is then incubated at 68-72°C for approximately 10-15 minutes, cooled to room temperature, centrifuged, and the supernatant is used for analysis. Protein migration is detected using a UV detector, and an electropherogram is obtained. The purity of an antibody preparation can be calculated as the percentage of the IgG main peak area relative to the sum of all peak areas. For further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

[0114] Size-exclusion high-performance liquid chromatography (SEC-HPLC) is another important method used for antibody standardization and quality control. This method primarily separates molecules based on differences in molecular size or hydrodynamic radius. SEC-HPLC can separate antibodies into three major forms: a high molecular weight form (HMMS), a main peak (mainly antibody monomer), and a low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area relative to the sum of all peak areas in the chromatography. SEC-HPLC can measure the percentage of antibody monomer in the formulation product and provide information on the content of soluble aggregates and shear products. For further descriptions of SEC-HPLC methods, see, for example, J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986). Further, reference may be made to, for example, R. Yang et al., "High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC)," Journal of Pharmaceutical and Biomedical Analysis (2015), http: / / dx.doi.org / 10.1016 / j.jpba.2015.02.032, and Alexandre Goyon et al., "Protocols for the analytical characterization of therapeutic monoclonal antibodies. I-Non-denaturing chromatographic techniques," Journal of Chromatography, http: / / dx.doi.org / 10.1016 / j.jchromb.2017.05.010.

[0115] Imaging capillary isoelectric focusing (iCIEF) can be used to analyze antibody charge heterogeneity. This method can provide a quantitative distribution profile of charge variants. iCIEF achieves the purpose of molecular separation based on the charge difference (apparent pI value) of molecules in a pH gradient. In iCIEF, the separation column is typically a short capillary (e.g., a silica capillary with a length of 5 cm and an inner diameter of 100 μm). Proteins are focused in the capillary column at high voltage, and the focusing is monitored online in real time by a whole-column imaging detection system operated at 280 nM. One advantage of this technique is that the whole-column imaging detection system can simultaneously record various charge variants of an antibody sample. In iCIEF, a sample is typically mixed with urea and an iCIEF buffer, which contains methylcellulose, pI molecular weight standards, and ampholytes. Then, after the sample is focused for a certain period of time on an iCIEF column, such as an iCE280 analyzer (Protein Simple, Santa Clara, CA), the absorbance at 280 nm is measured to obtain chromatography focused on the antibody charge variants. In iCIEF chromatography, protein-related peaks eluted before the main peak (i.e., the major component) are classified as acidic components, while protein-related peaks eluted after the main peak are classified as basic components. The relative amounts of the major component, acidic component, and basic component can be expressed as a percentage of the total peak area.For further description of iCIEF, see, for example, Salas-Solano O et al., Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study, J Sep Sci. 2012 Nov; 35(22): 3124-9. doi: 10.1002 / jssc.201200633. Epub 2012 Oct 15, and Dada OO et al., Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation, Electrophoresis. 2015 Nov; 36(21-22): 2695-2702. doi: 10.1002 / elps.201500219. Epub 2015 Sep 18.

[0116] Charge variants of antibodies in antibody formulations can also be measured by cation exchange high-performance liquid chromatography (CEX-HPLC). In this measurement method, a peak eluted from a CEX-HPLC column earlier than the retention time of the main peak is labeled as an "acidic peak," and a peak eluted from a CEX-HPLC column later than the retention time of the main peak is labeled as a "basic peak."

[0117] Accelerated stability studies can be used to examine the stability properties of a product and contribute to screening for stable pharmaceutical formulations. For example, accelerated stability studies can be performed by subjecting formulation samples to elevated temperatures, e.g., about 40°C ± 2°C or 25°C ± 2°C. Detection parameters can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method), and charge variants (iCIEF method, CEX-HPLC method).

[0118] Alternatively, the efficacy or biological activity of the antibody can be measured. For example, the binding ability of the antibody to its antigen molecules (HER2 molecule and PD-1 molecule) in the formulation can be measured. Those skilled in the art are familiar with several methods available for quantifying specific binding between an antibody and an antigen, such as immunoassay tests and ELISA.

[0119] The anti-PD-1 / HER2 bispecific antibody protein formulations of the invention are stable. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, for example, after storage at 5°C ± 3°C for 3 months, the purity of the anti-PD-1 / HER2 bispecific antibody protein in the antibody formulations of the invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater, as measured by size exclusion chromatography or non-reducing CS-SDS. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, for example, after storage at 5°C ± 3°C for 3 months, at least 60%, preferably at least 65%, of the anti-PD-1 / HER2 bispecific antibody protein in the antibody formulation of the invention is in a non-basic and non-acidic form (i.e., the main peak or predominantly charged form) as measured by iCIEF methods.

[0120] IV. Use of the Preparation The antibody formulations of the invention, comprising the anti-PD-1 / HER2 bispecific antibody proteins of the invention, can be used to treat, prevent, or delay various diseases or disorders associated with the HER2 signaling pathway and / or the PD-1 signaling pathway. As used herein, "diseases or disorders associated with the HER2 signaling pathway" and / or "diseases or disorders associated with the PD-1 signaling pathway" refer to diseases or disorders that can be treated (e.g., ameliorated) or prevented by the anti-PD-1 / HER2 bispecific antibody protein formulations of the invention. Any disease or disorder that could benefit from treatment with the antibody formulations of the invention is applicable to the present invention.

[0121] Formulations of the invention comprising the anti-PD-1 / HER2 bispecific antibody protein can be used to prevent or treat a variety of hematological diseases and solid tumors in a subject, including, but not limited to, leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, and melanoma.

[0122] The invention further provides the use of a formulation of the invention in the preparation of a medicament for delivering an anti-PD-1 / HER2 bispecific antibody protein to a mammal, preferably a human, or for treating, preventing, or ameliorating one or more of the above diseases and disorders.

[0123] The antibody formulations of the invention can be administered to a subject or patient in a variety of ways. For example, administration may be by infusion or by syringe. Accordingly, in one aspect, the invention provides a delivery device (e.g., a syringe) containing an antibody formulation of the invention (e.g., a pre-filled syringe). The patient receives an effective amount of the anti-PD-1 / HER2 bispecific antibody protein as the primary active ingredient, which is an amount sufficient to treat, ameliorate, or prevent a disease or disorder.

[0124] Therapeutic effects can include the alleviation of physiological symptoms. The optimal effective dose and concentration of antibody used for any particular subject will depend on various factors, including the patient's age, weight, physical condition, and / or sex, the nature and extent of the disease, the activity of the specific antibody, the body's clearance of the antibody, and any other possible treatments administered in combination with the antibody formulation. The effective amount to be delivered in a specific case is within the discretion of the clinician. Depending on the indication being treated, an effective dosage may be from about 0.005 mg / kg body weight to about 50 mg / kg body weight, or from about 0.1 mg / kg body weight to about 20 mg / kg body weight. The use of known antibody-based pharmaceuticals can provide some guidance in this regard. The dosage may be a single dose or a multiple dose regimen.

[0125] The following examples are set forth to aid in the understanding of the present invention, and are not intended to, and should not be construed as, limiting the scope of the invention as claimed in any manner.

[0126] Explanation of Abbreviations CE-SDS: Sodium lauryl sulfate capillary gel electrophoresis ELISA: Enzyme-linked immunosorbent assay iCIEF: Imaging capillary isoelectric focusing SEC-HPLC: Size-exclusion high-performance liquid chromatography [Example]

[0127] To develop a formulation that would ensure long-term storage stability for a recombinant anti-programmed death receptor 1 (PD-1) and anti-human epidermal growth factor receptor 2 (HER2) bispecific antibody injection so that quality could be controlled over the product's shelf life (at least 24 months), a formulation screening study was designed to investigate the effects of different auxiliary materials on the stability of the anti-PD-1 / HER2 bispecific antibody formulation. The materials and methods used in the study are as follows:

[0128] Materials and Methods [Table 2]

[0129] [Table 3]

[0130] 1.3. Test items and methods for stability of formulations The following items were detected for the antibody formulation: (1) the appearance and presence or absence of visible foreign matter were detected; (2) the protein content of the formulation was measured by ultraviolet (UV) light; (3) the turbidity was measured by detecting absorbance at 350 nm; and (4) size-exclusion chromatography, e.g., size-exclusion high-performance liquid chromatography (SLC). The purity of the antibody formulation (expressed as the percentage of the monomer area relative to the sum of all peak areas) was measured by chromatography-HPLC (SEC-HPLC). (5) The purity of the antibody formulation (expressed as the percentage of the monomer area relative to the sum of all peak areas) was measured by reduced sodium lauryl sulfate capillary gel electrophoresis (reduced CE-SDS) and / or non-reduced sodium lauryl sulfate capillary gel electrophoresis (non-reduced CE-SDS). (6) The charge variants (expressed as the percentage of the main component, acidic component, and basic component) in the antibody formulation were measured by imaging capillary isoelectric focusing (iCIEF). (7) The relative binding activity of the anti-PD-1 / HER2 bispecific antibody in the antibody formulation to the PD-1 antigen and the HER2 antigen was measured by immunoassay, such as direct ELISA.

[0131] Measurement of visible foreign matter Visible foreign matter in the samples was inspected using a transparency detector (manufactured by Tianjin Tiandatanfa, model number YB-2) according to the method described in the State Pharmacopoeia Commission, Pharmacopoeia of the People's Republic of China (2015 edition, Four Parts General Rule 0904 "Testing Method for Visible Foreign Matter", Beijing: China Pharmaceutical Science and Technology Press, 2015).

[0132] Protein content measurement The protein content in the samples was measured using an ultraviolet spectrophotometer (Shimadzu, Japan, model UV-1800).

[0133] Turbidity measurement The absorbance of the sample was measured at 350 nm using an ultraviolet spectrophotometer (Shimadzu, Japan, model number UV-1800) to confirm the turbidity of the sample.

[0134] Purity (SEC-HPLC method) Separation was performed using a volume exclusion chromatography column. The mobile phase was phosphate buffer (3.12 g of sodium dihydrogen phosphate dihydrate, 8.77 g of sodium chloride, and 34.84 g of arginine were weighed, dissolved in ultrapure water, adjusted to pH 6.8 with hydrochloric acid, and the volume was adjusted to 1000 mL). The chromatography column protection solution was 0.05% (w / v) NaN3. The injection volume was 50 μL, the flow rate was 0.5 mL / min, the sampling time was 30 minutes, the column temperature was 25°C, and the detection wavelength was 280 nm. The sample to be measured was diluted to 2 mg / mL with ultrapure water to prepare the test solution. The formulation buffer was diluted in the same manner as above and used as the blank solution. 50 μL of each of the blank solution and the test solution was poured into the liquid chromatograph, and detection began.

[0135] Purity (reduced CE-SDS method) Detection was performed by capillary gel electrophoresis. The capillary was uncoated, with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Prior to electrophoresis, the capillary column was washed at 70 psi with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel. The sample to be measured was diluted to 2.0 mg / mL with an appropriate amount of ultrapure water. 50 μL of the diluted sample was transferred to a 1.5 mL centrifuge tube, and 45 μL of pH 6.5 sample buffer solution (0.32 g of citric acid monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate were weighed and dissolved in 45 mL of ultrapure water, and the volume was adjusted to 50 mL to prepare a citrate-phosphate buffer solution. 200 μL of the buffer solution was precisely measured, and 80 μL of 10% (w / v) sodium lauryl sulfate solution was added. Water was added to make the total volume 1 mL, and the mixture was mixed uniformly). 1 μL of internal standard solution (10 kDa protein, 5 mg / mL) (Beckman Coulter, product number: 390953) and 5 μL of β-mercaptoethanol were added, and the mixture was thoroughly mixed uniformly. After thorough mixing, the mixture was heated at 70±2°C for 10±2 minutes, cooled to room temperature, and transferred to a sample bottle to prepare the test sample solution. A blank solution was prepared by taking the same volume of formulation buffer as the test sample and operating in the same manner as above. Sample injection conditions: -5 kV for 20 seconds, separation voltage: -15 kV for 35 minutes. The temperature of the capillary column was controlled at 25°C, and the detection wavelength was 220 nm.

[0136] Purity (non-reduced CE-SDS method) Detection was performed by capillary gel electrophoresis. The capillary was uncoated, with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Prior to electrophoresis, the capillary column was washed at 70 psi with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel. The sample to be measured was diluted to 2.0 mg / mL with an appropriate amount of ultrapure water, and 50 μL of the diluted sample was placed in a 1.5 mL centrifuge tube. 45 μL of pH 6.5 sample buffer solution (0.32 g of citric acid monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate were weighed and dissolved in 45 mL of ultrapure water, and the volume was adjusted to 50 mL to obtain a citrate-phosphate buffer solution. 200 μL of the buffer solution was precisely measured, and 80 μL of 10% (w / v) sodium lauryl sulfate solution was added. Water was added to make the total volume 1 mL, and the mixture was mixed uniformly), and 1 μL of internal standard solution (10 kDa protein, 5 mg / mL) (Beckman 5 μl of 250 mmol / L NEM solution (62 mg of N-ethylmaleimide was weighed and dissolved in 2 mL of ultrapure water) was added to each well and mixed thoroughly. After heating at 70 ± 2°C for 10 ± 2 minutes, the mixture was cooled to room temperature and transferred to a sample bottle to prepare the test solution. A blank solution was obtained by taking the same volume of formulation buffer as the test sample and operating in the same manner as above. Sample injection conditions: -5 kV for 20 seconds, separation voltage: -15 kV for 35 minutes. The capillary column temperature was controlled at 25°C, and the detection wavelength was 220 nm.

[0137] Charge variants (iCIEF method) Detection was performed using imaging capillary isoelectric focusing (iCIEF). The capillary had an inner diameter of 100 μm and a total length of 5 cm. Prior to sample electrophoresis, the capillary column was rinsed with 0.5% methylcellulose solution (MC solution) and ultrapure water. A vacuum injection method was used, with a prefocusing voltage and time of 1.5 kV for 1 minute, a focusing voltage and time of 3 kV for 8 minutes, an injection time of 55 seconds, a sample plate temperature of 10°C, and a detection wavelength of 280 nm. The cathodic stabilizer was a 500 mmol / L arginine solution, and the 0.5% MC solution reduced adhesion between the protein and the capillary. The test sample was diluted with water to 1.0 mg / mL, and 20 μl of the diluted test sample solution was taken. 78 μl of premixed solution (70 μl of MC solution with a pI of 0.5%, 4 μl of ampholytes (pH 3-10), 2 μl of cathodic stabilizer, 1 μl of pI 5.85 marker, and 1 μl of pI 9.99 marker) was added to the diluted solution and mixed thoroughly to obtain the sample solution to be measured. Injection analysis was performed, and the contents of the main component, acidic component, and basic component were calculated using the area normalization method.

[0138] Relative binding activity (direct ELISA method) Antigens (recombinant human PD-1 purchased from Sinobiological, product number 10377-H08H) were diluted to 0.5 μg / mL in CBS to detect the relative binding activity of the anti-PD-1 end of the anti-PD-1 / HER2 bispecific antibody to PD-1, and human HER2 / ErbB2 protein (His Tag) purchased from Sinobiological, product number 10004-H08H-100 was used to detect the relative binding activity of the anti-HER2 end of the anti-PD-1 / HER2 bispecific antibody to HER2) and coated overnight at 4°C in 96-well ELISA plates at 100 μl / well. After washing, the plates were sealed with sealing solution (2% BSA-PBST, 300 μl / well) at 37°C for 2 hours. Anti-PD-1 / HER2 bispecific antibody was diluted to 3 μg / mL in 2% BSA-PBST and then diluted in a 3-fold gradient to 11 concentrations (0.05 to 3000 ng / mL). 100 μl of the diluted sample was added to an ELISA plate, from which the sealing solution had been removed. A negative control was set up by adding only 100 μl of the dilution solution (2% BSA-PBST) per well. The plate was then incubated at 37°C for 60 min in an incubator. After washing, HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, USA, product number A80-104P) diluted in 2% BSA-PBST was added as the secondary antibody (1:100,000, 100 μl / well) and incubated at 37°C for 30 min. After washing the plate, 100 μl of TMB coloring solution was added per well. After 10 minutes of color development, 100 μl of 1 mol / L H2SO4 was added per well to stop the reaction. OD values ​​were measured at 450 nm with 620 nm as the reference wavelength. EC values ​​were calculated using Prism 4 parameter fitting, with the concentration values ​​of each concentration gradient sample on the horizontal axis and the OD values ​​from 450 nm to 620 nm of each gradient sample on the vertical axis. 50 was calculated to reflect the binding activity of the antibody to each antigen.

[0139] Example 1. Preparation and purification of anti-PD-1 / HER2 bispecific antibodies Anti-PD-1 / HER2 bispecific antibodies were prepared and purified as described in PCT Application No. PCT / CN2018 / 075851.

[0140] Specifically, an X0GC expression vector (see Chinese Patent Application No. 200780038403.3 for the construction of the X0GC expression vector) containing the heavy and light chains of an anti-human PD-1 antibody was constructed, in which the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:9, the amino acid sequence is set forth in SEQ ID NO:10, the nucleotide sequence of the light chain constant region is set forth in SEQ ID NO:3, the amino acid sequence is set forth in SEQ ID NO:4, the nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:11, the amino acid sequence is set forth in SEQ ID NO:12, and the nucleotide sequence of the heavy chain constant region is set forth in SEQ ID NO:13, the amino acid sequence is set forth in SEQ ID NO:14.

[0141] X0GC expression vectors containing anti-human HER2 antibody heavy and light chains were constructed, respectively, wherein the light chain variable region nucleotide sequence is shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2, the light chain constant region nucleotide sequence is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, the heavy chain variable region nucleotide sequence is shown in SEQ ID NO: 5, the amino acid sequence is shown in SEQ ID NO: 6, the heavy chain constant region nucleotide sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.

[0142] The expression vector containing the heavy and light chains of the anti-human PD-1 antibody was cultured in 293F cells (FreeStyle TM The antibodies were transfected into 293-F Cells (product number R79007, Invitrogen), expressed, purified, and then subjected to a reduction process to obtain anti-human PD-1 half antibody molecules containing one heavy chain and one light chain.

[0143] Similarly, an expression vector containing the heavy and light chains of the anti-human HER2 antibody was transformed into 293F cells (FreeStyle TM The antibodies were transfected into 293-F Cells (product number R79007, Invitrogen), expressed, purified, and then subjected to a reduction process to obtain an anti-human HER2 half antibody molecule containing one heavy chain and one light chain.

[0144] Reduced anti-PD-1 half antibody molecules and reduced anti-HER2 half antibody molecules were mixed in an equimolar ratio and allowed to recombine for 24 hours at 4°C to yield a solution of heterodimeric bispecific antibodies containing anti-PD-1 half antibody molecules and anti-HER2 half antibody molecules. The solution was concentrated by ultrafiltration using an ultrafiltration concentrator tube and then purified at 4°C using an AKTA explorer 100 protein purification system (GE Healthcare) and a Source 15S ion chromatography column (16 mm ID, 17 mL, GE Healthcare) to yield an anti-PD-1 / HER2 bispecific antibody with a purity of 99.96%.

[0145] Example 2. One test to examine the effect of pH on formulation stability This example investigated the stability of formulations containing anti-PD-1 / HER2 bispecific antibodies at pH 5.0 to 6.5. A total of four pH values ​​were designed: 5.0, 5.5, 6.0, and 6.5.

[0146] 2.1 Experimental procedure A 10 mM histidine, 5% (w / v) sorbitol buffer solution was prepared and adjusted to pH 5.0, 5.5, 6.0, and 6.5 with dilute hydrochloric acid. The purified anti-PD-1 / HER2 bispecific antibody from Example 1 was then ultrafiltered and substituted into these solutions with different pH values. After the substitution was complete, the bispecific antibody protein content in the sample was adjusted to approximately 20 mg / mL, and polysorbate 80 was added to a final concentration of 0.30 mg / mL. The sample was then filtered and dispensed into vials, which were then plugged and capped. The stability of each sample was tested at 40°C ± 2°C; the specific experimental method is shown in Table 1.

[0147] [Table 4]

[0148] 2.2 Judgment criteria To determine whether the sample had changed, criteria were established for determining whether there was any change in quality by comparing the sample detection index value with the initial value based on product recognition and the precision of the equipment and method. Details are shown in Table 2.

[0149] [Table 5]

[0150] 2.3 Experimental results of the formulation screening test (1) Appearance and visible foreign matter After being stored at 40°C ± 2°C for one month, the samples at pH 5.0, pH 5.5, pH 6.0 and pH 6.5 passed the test for both appearance and visible foreign matter.

[0151] (2) Protein content The protein content of each sample after being stored at pH 5.0, 5.5, 6.0 and 6.5 at 40°C ± 2°C for different times is shown in Table 3. As can be seen from the results, there was no significant change in any of the samples at each pH value after being stored at 40°C ± 2°C for one month.

[0152] [Table 6]

[0153] (3) Turbidity The turbidity detection results for each sample after being stored at 40°C ± 2°C for different periods of time at pH 5.0, 5.5, 6.0, and 6.5 are shown in Table 4, and the change trends are shown in Figure 2. As can be seen from the results, after being stored at 40°C ± 2°C for one month, the turbidity of each pH sample increased, and the higher the pH, the faster the turbidity change rate.

[0154] [Table 7]

[0155] (4) Purity After being stored at pH 5.0, 5.5, 6.0, and 6.5 at 40°C ± 2°C for different periods of time, the protein purity of each sample was measured by SEC-HPLC. The results are shown in Table 5, and the change trends are shown in Figure 3. As can be seen from the results, there was no obvious change in the purity of any of the samples at different pH values ​​after one month at 40°C ± 2°C.

[0156] [Table 8]

[0157] After storing samples at pH 5.0, 5.5, 6.0, and 6.5 at 40°C ± 2°C for different periods of time, the protein purity of each sample was measured using the non-reducing CE-SDS method. The results are shown in Table 6, and the change trends are shown in Figure 4. As can be seen from the results, after one month of storage at 40°C ± 2°C, the purity of the samples at each pH value decreased by 2.6%, 2.5%, 2.5%, and 3.2%, respectively, compared to the purity of the sample stored at day 0.

[0158] [Table 9]

[0159] After storing samples at pH 5.0, 5.5, 6.0, and 6.5 at 40°C ± 2°C for different periods of time, the protein purity of each sample was measured using the reduced CE-SDS method. The results are shown in Table 7, and the change trends are shown in Figure 5. As can be seen from the results, after one month at 40°C ± 2°C, the purity of the samples at each pH value decreased by 0.4%, 0.7%, 0.6%, and 1.4%, respectively, compared to the purity of the sample stored at day 0.

[0160] [Table 10]

[0161] (5) Charge variants After storing samples at pH 5.0, 5.5, 6.0, and 6.5 at 40°C ± 2°C for different periods of time, the charge variants of each sample were measured using the iCIEF method. The results are shown in Table 8, and the change trends are shown in Figure 6. As can be seen from the results, after one month at 40°C ± 2°C, the samples at each pH value showed significant changes in the main components, acidic components, and basic components. The higher the pH value, the faster the main components of the sample decreased and the faster the acidic components increased.

[0162] [Table 11]

[0163] (6) Relative binding activity After incubation at 40°C ± 2°C for different periods of time at pH 5.0, 5.5, 6.0, and 6.5, the relative binding activity of each sample was measured by direct ELISA. The results are shown in Table 9. As can be seen, after two weeks at 40°C ± 2°C, the relative binding activity of each sample to PD-1 antigen and HER2 antigen was higher than 70%, while the relative binding activity of the anti-HER2 end of the pH 6.0 and pH 6.5 samples significantly decreased, falling below 70%. After one month at 40°C ± 2°C, the relative binding activity of each sample to PD-1 antigen remained higher than 70%, and only the relative binding activity of the pH 6.0 and pH 6.5 samples to HER2 antigen was lower than 70%, but still higher than 50%.

[0164] [Table 12]

[0165] The results of the above-described tests on the effect of pH on formulation stability showed that after two weeks of storage at pH 5.0-6.5 and 40°C ± 2°C, the sample appearance and visible foreign matter were acceptable, there was no significant change in protein content, and there was no obvious change in the relative binding activity to HER2 antigen and PD-1 antigen. After one month of storage at pH 5.0-6.5 and 40°C ± 2°C, the sample appearance and visible foreign matter were acceptable, there was no significant change in protein content, and there was no obvious change in the relative binding activity to PD-1 antigen. It was clear that the relative binding activity of the anti-PD-1 / HER2 bispecific antibody to the HER2 antigen decreased only at pH 6.0 and pH 6.5, but was still greater than 50%. In subsequent examples, pH 5.5 was selected from the pH range of 5.0-6.5 for experiments.

[0166] Example 3. Formulation screening test 3.1 Stabilizer screening test The effect of different stabilizers, including sorbitol as a polyol, sucrose and trehalose as sugars, and methionine as an antioxidant, on the stability of formulations containing anti-PD-1 / HER2 bispecific antibodies was investigated.

[0167] 3.1.1 Stabilizer screening test process A total of four formulations were designed, and detailed formulation information is shown in Table 10. Buffer solutions for each formulation were prepared according to Table 10, and the anti-PD-1 / HER2 bispecific antibody was ultrafiltered and substituted with the respective formulation solutions. After filtration, the protein content of each formulation was adjusted to approximately 50.0 mg / mL, and polysorbate 80 was added to a final polysorbate 80 concentration of 0.20 mg / mL. The solution was filtered and dispensed into vials, which were then capped with plugs. Stability was tested for each sample at 40°C, 25°C, and 5°C; the specific method is shown in Table 11. Detection parameters included appearance, visible foreign matter, protein content, purity (SEC-HPLC, non-reducing CE-SDS), and charge variants (iCIEF).

[0168] [Table 13]

[0169] [Table 14]

[0170] 3.1.2 Judgment criteria Details of the criteria are shown in Table 2 in Example 2. 3.1.3 Stabilizer screening test

[0171] (1) Appearance and visible foreign matter As is clear from the results of observations at 40°C for one month, at 25°C ± 2°C for two months, and at 5°C ± 3°C for three months, the samples prepared using each formulation passed the test in terms of both appearance and visible foreign matter.

[0172] (2) Protein content The protein content of the samples from each formulation was measured after one month at 40°C, two months at 25°C ± 2°C, and three months at 5°C ± 3°C, and the results are shown in Table 12. As can be seen from the results, there was no change in the protein content of any of the four formulations under the three different temperature conditions of 40°C, 25°C ± 2°C, and 5°C ± 3°C.

[0173] [Table 15]

[0174] (3) Purity Purity (SEC-HPLC method): The results are shown in Table 13. As can be seen from the results, after 4 weeks at 40°C, there was no significant change in the purity of any of the samples from each formulation; after 2 months at 25°C ± 2°C, there was no obvious change in the purity of any of the samples from each formulation; and after 3 months at 5°C ± 3°C, there was no obvious change in the purity of any of the samples from each formulation.

[0175] [Table 16]

[0176] Purity (non-reduced CE-SDS method, %): The results are shown in Table 14. As can be seen from the results, after 4 weeks at 40°C, there was no significant change in the purity of any of the samples from each formulation; after 2 months at 25°C ± 2°C, there was no significant change in the purity of any of the samples from each formulation; and after 3 months at 5°C ± 3°C, there was no significant change in the purity of any of the samples from each formulation.

[0177] [Table 17]

[0178] Purity (reduced CE-SDS method): The results are shown in Table 15. As can be seen from the results, after 4 weeks at 40°C, there was no significant change in the purity of any of the samples from each formulation, after 2 months at 25°C ± 2°C, there was no significant change in the purity of any of the samples from each formulation, and after 3 months at 5°C ± 3°C, there was no significant change in the purity of any of the samples from each formulation.

[0179] [Table 18]

[0180] (4) Charge mutants (iCIEF method) Charge variants (iCIEF method): The results are shown in Table 16. The change trends of the main components of charge variants for each formulation at 40°C and 25°C ± 2°C are shown in Figures 7 and 8, respectively.

[0181] As is clear from the results, after four weeks of testing at 40°C, the main components, acidic components, and basic components of the charge mutants of each formulation underwent significant changes, with the main components decreasing and the acidic components increasing, and the change trends were nearly consistent, with no significant differences between formulations 1 to 4. After two months of accelerated testing at 25°C ± 2°C, the main components, acidic components, and basic components of the charge mutants of each formulation underwent significant changes, with the main components decreasing and the acidic components increasing, and the change trends were nearly consistent, with no significant differences between formulations 1 to 4. After three months of testing at 5°C ± 3°C, no significant changes were observed in the main components, acidic components, and basic components of the charge mutants of each formulation.

[0182] [Table 19]

[0183] As is clear from the results of the formulation determination experiment, the change trends were relatively consistent among formulations 1 to 4, with no obvious differences between them. Considering the simplicity of the formulation, there was no obvious difference in protein protection between formulations 1, 2, and 3, which all use a single stabilizer. However, in consideration of the subsequent development of the lyophilized formulation, formulation 2 was selected. Considering the subsequent production safety, the buffer system in formulation 2 was adjusted from concentrated hydrochloric acid to histidine and histidine hydrochloride to adjust the pH. The following experiments were further conducted to ensure the stability of the formulation after preparation.

[0184] Example 4. Experiment to confirm the formulation 4.1 Formula design and experimental method Formulation 5 was designed, and the details of formulation 5 are shown in Table 17.

[0185] [Table 20]

[0186] The formulation validation experiment method is shown in Table 18. [Table 21]

[0187] 4.2 Experimental results The results of the 40°C forced incubation experiment are shown in Table 19. After 4 weeks at 40°C, the appearance, visible foreign matter, and biological activity were all acceptable, and there were no significant changes in either the protein content or purity (SEC-HPLC method and CE-SDS method). Only the main component of the charge variant (iCIEF method) decreased by 16.0%, while the acidic component increased by 14.0% and the basic component increased by 2.0%.

[0188] [Table 22]

[0189] As is clear from the results, the recombinant fully human anti-programmed death receptor 1 (PD-1) and humanized anti-human epidermal growth factor receptor 2 (HER2) bispecific antibody of the present invention at 42.0 mg / mL in formulation 5 (histidine 0.85 mg / mL, histidine hydrochloride 3.17 mg / mL, sucrose 80.00 mg / mL, polysorbate 80 0.2 mg / mL, pH 5.5) showed a change trend that was relatively consistent with that of formulation 2 in Example 3.

[0190] The optimal formulation was determined to be approximately 42.0 mg / mL of recombinant fully human anti-programmed cell death receptor 1 (PD-1) and humanized anti-human epidermal growth factor receptor 2 (HER2) bispecific antibody, 0.85 mg / mL of histidine, 3.17 mg / mL of histidine hydrochloride, 80.00 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, and pH 5.5.

[0191] While exemplary embodiments of the present invention have been described above, those skilled in the art should understand that these disclosures are exemplary only and that various other substitutions, adaptations and modifications can be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments enumerated herein. Some aspects of the invention are described below. 1. (i) an anti-PD-1 / HER2 bispecific antibody protein; (ii) a buffer; and (iii) a stabilizer; and (iv) a surfactant, The anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising a first VH / VL unit that specifically binds to PD-1, and a second half antibody comprising a second VH / VL unit that specifically binds to HER2, wherein the first VH / VL unit comprises all of the heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 12 / SEQ ID NO: 10, and the second VH / VL unit comprises all of the heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 6 / SEQ ID NO: 2, Preferably, the liquid antibody formulation has a pH value of about 5.0 to 6.5, for example, about 5.0, 5.5, 6.0, or 6.5. 2. The liquid antibody formulation according to Item 1, wherein the concentration of the anti-PD-1 / HER2 bispecific antibody protein in the liquid antibody formulation is about 1 to 150 mg / mL, preferably about 10 to 100 mg / mL, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL. 3. The liquid antibody formulation according to item 1 or 2, wherein the buffer in the liquid antibody formulation is selected from histidine, histidine hydrochloride, and a combination thereof, and the concentration of the buffer is preferably about 5 to 50 mM, and more preferably about 10 to 30 mM, for example, about 10, 15, 20, 25, or 30 mM. 4. The liquid antibody formulation according to any one of items 1 to 3, wherein the stabilizer is selected from a polyol (e.g., sorbitol), a sugar (e.g., sucrose, trehalose), and any combination thereof, and the concentration of the stabilizer is preferably about 50 to 500 mM, and more preferably about 100 to 400 mM, for example, about 100, 150, 200, 250, 300, 350, or 400 mM. 5. The liquid antibody formulation according to any one of items 1 to 3, wherein the stabilizer is selected from a combination of a polyol (e.g., sorbitol), a sugar (e.g., sucrose, trehalose), or any combination thereof with an antioxidant; the total concentration of the stabilizers is preferably about 50 to 500 mM, and more preferably about 100 to 400 mM, for example, about 100, 150, 200, 250, 300, 350, or 400 mM; the concentration of the antioxidant is about 1 to 50 mM, preferably about 5 to 40 mM, for example, about 5, 10, 20, 30, or 40 mM; and the antioxidant is, for example, methionine. 6. The liquid antibody formulation according to any one of items 1 to 5, wherein the surfactant in the liquid antibody formulation is selected from polysorbate surfactants, and is preferably polysorbate 80. 7. The liquid antibody formulation according to any of items 1 to 6, wherein the concentration of the surfactant is about 0.1 to 1 mg / mL, preferably about 0.2 to 0.8 mg / mL, for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg / mL. 8. The anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising a first VH / VL unit that specifically binds to PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds to HER2, wherein the first VH / VL unit is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11111223 ... 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO:6 / SEQ ID NO:2, 2. The liquid antibody formulation of item 1, wherein the first half antibody comprises, in N to C orientation, the heavy chain sequences of SEQ ID NO: 12 and SEQ ID NO: 14, or heavy chain sequences having at least 90%, 95%, 98% or 99% identity thereto, and the light chain sequences of SEQ ID NO: 10 and SEQ ID NO: 4, or light chain sequences having at least 90%, 95%, 98% or 99% identity thereto, and the second half antibody comprises, in N to C orientation, the heavy chain sequences of SEQ ID NO: 6 and SEQ ID NO: 8, or heavy chain sequences having at least 90%, 95%, 98% or 99% identity thereto, and the light chain sequences of SEQ ID NO: 2 and SEQ ID NO: 4, or light chain sequences having at least 90%, 95%, 98% or 99% identity thereto. 9. The anti-PD-1 / HER2 bispecific antibody protein is produced by culturing HEK293 cells, or HEK293T, HEK293F, or HEK293E cells obtained by modifying HEK293 cells, or CHO cells, or CHO-S or CHO-dhfr cells obtained by modifying CHO cells. - 9. The liquid antibody formulation of any of items 1 to 8, wherein the liquid antibody formulation is recombinantly expressed in CHO / DG44 or ExpiCHO cells. 10. The liquid antibody formulation according to any one of items 1 to 9, wherein the liquid formulation is an injection, preferably used for subcutaneous injection or intravenous injection, or an infusion, for example used for intravenous infusion. 11. (i) about 1 to 150 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 5 to 50 mM of histidine and / or histidine hydrochloride; (iii) about 50 to 500 mM of sorbitol, sucrose, trehalose, and any combination thereof; or a combination of sorbitol, sucrose, trehalose, or any combination thereof with methionine at a concentration of about 1 to 50 mM, for a total concentration of about 50 to 500 mM; (iv) about 0.1 to 1 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; For example, the liquid antibody formulation may comprise: (i) about 10 to 100 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 10 to 30 mM of histidine and / or histidine hydrochloride; (iii) about 100 to 400 mM sorbitol, sucrose, and / or trehalose, or a combination of sorbitol, sucrose and / or trehalose and methionine at a concentration of about 5 to 40 mM, for a total concentration of about 100 to 400 mM; (iv) about 0.2 to 0.8 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; Alternatively, the liquid antibody formulation may comprise: (i) about 20 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 10 mM histidine; (iii) about 50 mg / mL of sorbitol; (iv) about 0.3 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 50 mg / mL of sorbitol; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 80 mg / mL of sucrose; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 80 mg / mL of trehalose; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 20 mM histidine; (iii) about 80 mg / mL sucrose and about 1.49 mg / mL methionine; (iv) about 0.2 mg / mL of polysorbate 80; The pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5; Alternatively, the liquid antibody formulation may comprise: (i) approximately 42 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; and (ii) about 0.85 mg / mL of histidine and about 3.17 mg / mL of histidine hydrochloride; (iii) about 80 mg / mL of sucrose; (iv) about 0.2 mg / mL of polysorbate 80; 11. The liquid antibody formulation according to any one of items 1 to 10, wherein the pH value of the liquid formulation is about 5.0 to 6.5, preferably about 5.5. 12. The formulation is stable after storage, for example, after at least 24 months at 2-8°C, or after at least 3 months at room temperature, or after 1 month at 40°C ± 2°C, and preferably has one of the following properties: (i) the formulation has a purity of greater than 90%, preferably greater than 95%, 96%, 97%, 98%, or 99%, as measured by SEC-HPLC; (ii) the formulation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, or 98%, as measured by reduced or non-reduced CE-SDS methods; (iii) when measured by the iCIEF method, the total change in the components (main component, acidic component, and basic component) of the anti-PD-1 / HER2 bispecific antibody protein in the formulation is 50% or less, for example, 40%, 30%, 20%, 10%, or 5% or less, relative to the initial value on day 0 of storage; (iv) The liquid antibody formulation of any of Items 1 to 11, having one or more of the following characteristics: the relative binding activity of the anti-PD-1 / HER2 bispecific antibody protein in the formulation is 70% to 130%, e.g., 70%, 80%, 90%, 100%, 110%, 120%, or 130%, relative to the initial value on day 0 of storage, as measured by ELISA. 13. A solid antibody formulation obtained by solidifying the liquid antibody formulation according to any one of items 1 to 12, for example, in the form of a lyophilized powder for injection. 14. A delivery device comprising the liquid antibody formulation according to any one of items 1 to 12 or the solid antibody formulation according to item 13. 15. A pre-filled syringe for intravenous or intramuscular injection, comprising the liquid antibody formulation according to any one of items 1 to 12 or the solid antibody formulation according to item 13. 16. Use of the liquid antibody formulation according to any one of items 1 to 12 or the solid antibody formulation according to item 13 in the preparation of a medicament for treating, preventing, or delaying a disorder associated with the HER2 signaling pathway and the PD-1 signaling pathway, wherein the disorder is, for example, various hematological diseases and solid tumors, including, but not limited to, 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, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, and melanoma.

Claims

1. (i) an anti-PD-1 / HER2 bispecific antibody protein; and (ii) a buffering agent; and (iii) a stabilizer; and (iv) a surfactant, The anti-PD-1 / HER2 bispecific antibody protein comprises a first half antibody comprising a first VH / VL unit that specifically binds to PD-1 and a second half antibody comprising a second VH / VL unit that specifically binds to HER2, as shown in FIG. 1 , wherein the first VH / VL unit is the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 12 / SEQ ID NO: 10, and the second VH / VL unit is the paired heavy chain variable region sequence / light chain variable region sequence of SEQ ID NO: 6 / SEQ ID NO: 2, the first half antibody comprising, in N to C orientation, the heavy chain sequences of SEQ ID NO: 12 and SEQ ID NO: 14, and, in N to C orientation, the light chain sequences of SEQ ID NO: 10 and SEQ ID NO: 4, and the second half antibody comprising, in N to C orientation, the heavy chain sequences of SEQ ID NO: 6 and SEQ ID NO: 8, and, in N to C orientation, the light chain sequences of SEQ ID NO: 2 and SEQ ID NO: 4, The liquid antibody formulation comprises: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 3.1 mg / mL of histidine; and (iii) about 50 mg / mL of sorbitol; (iv) about 0.2 mg / mL of polysorbate 80; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 3.1 mg / mL of histidine; and (iii) about 80 mg / mL sucrose; and (iv) about 0.2 mg / mL of polysorbate 80; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 3.1 mg / mL of histidine; and (iii) about 80 mg / mL trehalose; (iv) about 0.2 mg / mL of polysorbate 80; Alternatively, the liquid antibody formulation may comprise: (i) about 50 mg / mL of an anti-PD-1 / HER2 bispecific antibody protein; (ii) about 3.1 mg / mL of histidine; and (iii) about 80 mg / mL sucrose and about 1.49 mg / mL methionine; (iv) about 0.2 mg / mL of polysorbate 80; Alternatively, the liquid antibody formulation may comprise: (i) about 42 mg / mL of anti-PD-1 / HER2 bispecific antibody protein; and (ii) about 0.85 mg / mL histidine and about 3.17 mg / mL histidine hydrochloride; (iii) about 80 mg / mL sucrose; and (iv) about 0.2 mg / mL of polysorbate 80, wherein the liquid antibody formulation has a pH value of about 5.0 to 6.

0.

2. The anti-PD-1 / HER2 bispecific antibody protein is produced by HEK293 cells or HEK293T, HEK293F, or HEK293E cells obtained by modifying HEK293 cells, and CHO cells or CHO-S, CHO-dhfr, or CHO-dhfr cells obtained by modifying CHO cells. － 2. The liquid antibody formulation of claim 1, wherein the antibody is recombinantly expressed in CHO / DG44, CHO / DG44, or ExpiCHO cells.

3. The liquid antibody formulation of claim 1 , wherein the liquid formulation is an injection and is used for subcutaneous or intravenous injection, or an infusion and is used for intravenous infusion.

4. 10. The liquid antibody formulation of claim 1, wherein the pH value of the liquid formulation is about 5.

5.

5. The formulations are stable after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ± 2°C for 1 month, and have the following properties: (i) the formulation has a purity of greater than 90% as measured by SEC-HPLC; (ii) the preparation has a purity of greater than 90% as measured by reduced or non-reduced CE-SDS methods; (iii) when measured by the iCIEF method, the total change in the major component, acidic component, and basic component of the anti-PD-1 / HER2 bispecific antibody protein in the formulation is 50% or less compared to the initial value on day 0 of storage; (iv) the relative binding activity of the anti-PD-1 / HER2 bispecific antibody protein in the formulation is 70% to 130% of the initial value on day 0 of storage, as measured by ELISA.

6. A solid antibody formulation in the form of a lyophilized powder for injection, obtained by solidifying the liquid antibody formulation according to any one of claims 1 to 5.

7. A delivery device comprising the liquid antibody formulation of any one of claims 1 to 5 or the solid antibody formulation of claim 6.

8. A pre-filled syringe for intravenous or intramuscular injection, comprising the liquid antibody formulation according to any one of claims 1 to 5 or the solid antibody formulation according to claim 6.

9. Use of the liquid antibody formulation of any one of claims 1 to 5 or the solid antibody formulation of claim 6 in the preparation of a medicament for treating, preventing or delaying a disorder associated with the HER2 signaling pathway and the PD-1 signaling pathway.

10. The use according to claim 9, wherein the disorder is selected from various hematological diseases and solid tumors.

11. The use of claim 10, wherein the various hematological diseases and solid tumors are selected from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, and melanoma.

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