RU2829156C2 - Composition containing anti-pd-1/her2 bispecific antibody, method for preparation and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a liquid pharmaceutical composition containing an anti-PD-1/HER2 bispecific antibody, a buffer, a stabilizer and a surfactant. Also disclosed is a solid composition of the antibody in the form of a lyophilised powder, obtained by solidifying said liquid composition.

EFFECT: invention provides anti-PD-1/HER2 bispecific antibody protein stabilization during storage.

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Description

AREA OF TECHNOLOGY

The present invention relates to the field of antibody-based compositions. More particularly, the present invention relates to a pharmaceutical composition, in particular to a stable liquid composition, comprising a recombinant bispecific antibody against programmed death protein 1 (PD-1) receptor and human epidermal growth factor receptor 2 (HER2) (also known as an anti-PD-1/HER2 bispecific antibody), a method for producing this pharmaceutical composition, as well as therapeutic and/or prophylactic use of this pharmaceutical composition.

LEVEL OF TECHNOLOGY

Overexpression of human epidermal growth factor receptor 2 (HER2) (also known as NEU, ERBB-2, CD340, or p185) is associated with a variety of malignancies, including breast cancer, ovarian cancer, gastric cancer, uterine cancer, melanoma, and cholangiocarcinoma. For example, HER2 is overexpressed in invasive and metastatic breast cancer and in breast cancers with a high recurrence rate and/or poor patient prognosis.

One approach to the treatment of HER2-overexpressing cancers is to use anti-HER2 antibodies that inhibit HER2-mediated signaling. An example of a therapeutic anti-HER2 antibody that blocks HER2-mediated intracellular signaling is trastuzumab, which is widely used to treat HER2-overexpressing tumors. Unfortunately, when such antibodies are used clinically, their antitumor effect is often weaker than in preclinical studies. In the prior art, anti-HER2 antibodies are usually used in combination with chemotherapeutic drugs and the like (Slamon DJ et al., N Engl J Med, 344:783-792, 2001).

In recent years, studies of immune checkpoint molecules have shown that activation of inhibitory immune checkpoint signaling pathways causes the inability of T cells to effectively kill tumor cells (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 leads, in one aspect, to the weak antitumor effect of drugs that act exclusively on target molecules on tumor cells (e.g., trastuzumab).

Programmed cell death protein 1 (PD-1) is an important immune checkpoint protein and a type I transmembrane protein with a molecular weight of 55 kDa. It is expressed primarily upon induction on the surface of activated T lymphocytes; it is also expressed on cells such as B lymphocytes, natural killer (NK) cells, monocytes, and dendritic cells (DCs). PD-1 has been shown to have two cell surface glycoprotein ligands, programmed death protein ligand 1 (PD-L1) and programmed death protein ligand 2 (PD-L2). PD-1 ligands are highly expressed on many tumor cells. Binding of PD-1 to PD-1 ligand results in T-lymphocyte apoptosis, lack of immune response, T-lymphocyte depletion and IL-10 secretion, etc.; thus, blocking PD1-dependent pathway can restore T-lymphocyte function in cancer patients (Sheridan, Nature Biotechnology, 30(2012), 729-730). Monoclonal antibodies against PD-1 have been developed, such as nivolumab by Bristol-Myers Squibb (BMS) and pembrolizumab by Merck. Nivolumab (trade name OPDIVO ^® ) is a fully humanized IgG4 antibody molecule, and pembrolizumab (trade name KEYTRUDA ^® ) is a humanized IgG4 antibody molecule. Anti-PD-1 monoclonal antibodies, by binding to PD-1 on T lymphocytes, can inhibit the binding of PD-1 to its ligands PD-L1 and PD-L2, thereby inducing the activation and proliferation of T lymphocytes and the production of cytokines involved in immune activation, such as IL-2, and attenuating the suppression of immune monitoring of T lymphocytes with antitumor activity through PD-1.

In view of the importance of the immune checkpoint molecule PD-1 in modulating immune responses, the inventors conducted a thorough study and developed an anti-PD-1/HER2 bispecific antibody targeting PD-1 and HER2, which is capable of simultaneously targeting HER2 on tumor cells and activating T lymphocytes, thus suggesting the advantage of reducing side effects and enhancing antitumor activity. A patent application for this anti-PD-1/HER2 bispecific antibody has been filed under patent number PCT/CN2018/075851 (filed on February 8, 2018), which describes the construction and expression of an anti-PD-1/HER2 bispecific antibody consisting of an anti-PD-1 half antibody and an anti-HER2 half antibody. The anti-PD-1/HER2 bispecific antibody was administered to tumor-bearing mice obtained by inoculating HCC1954 human breast cancer cells into NCG immunodeficient mice, and the results of the experiments showed that, compared with the anti-HER2 monoclonal antibody or the anti-PD-1 monoclonal antibody, the anti-PD-1/HER2 bispecific antibody has significantly increased antitumor activity and is able to markedly reduce tumor volume.

There is a need in the art for creating anti-PD-1/HER2 bispecific antibody compositions that can be used to treat, prevent or slow down the development of various diseases associated with the HER2-dependent signaling pathway or the PD-1-dependent signaling pathway, wherein the compositions should have good stability in the case where the anti-PD-1/HER2 bispecific antibody is manufactured as a liquid preparation, and the anti-PD-1/HER2 bispecific antibody in a liquid solution does not have a tendency to decompose, aggregate or undergo unwanted chemical modification.

BRIEF SUMMARY OF THE INVENTION

The present invention satisfies the above-mentioned need for pharmaceutical compositions comprising an anti-PD-1/HER2 bispecific antibody protein that specifically binds to PD-1 and HER2. The antibody composition disclosed in this document allows for the preparation of an antibody preparation in such a way that it is suitable for administration to a subject, and the antibody remains stable during storage as well as during subsequent use.

In one aspect, the present invention provides a liquid antibody composition comprising (i) an anti-PD-1/HER2 bispecific antibody protein; (ii) a buffer; (iii) a stabilizer; and (iv) a surfactant.

The anti-PD-1/HER2 bispecific antibody protein in the antibody composition disclosed herein consists of a first half-antibody and a second half-antibody, wherein the first half-antibody comprises a first VH/VL unit specifically binding to PD-1, and the second half-antibody comprises a second VH/VL unit specifically binding to HER2. In some embodiments, the anti-PD-1/HER2 bispecific antibody protein is capable of 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 or higher, thereby blocking the binding of PD-1 to its ligands and causing activation or proliferation of T lymphocytes and the production of cytokines involved in immune activation such as IL-2; and is capable of binding to HER2 on the surface of tumor cells 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 or higher, thereby blocking intracellular signaling mediated by HER2 and exerting an antitumor effect.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein is a recombinant anti-PD-1/HER2 bispecific antibody protein as disclosed in PCT Application No. PCT/CN2018/075851 (filed February 8, 2018), the contents of which are incorporated herein by reference in their entirety for the purposes of this application. In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein consists of a first half-antibody and a second half-antibody, wherein the first half-antibody comprises a first VH/VL unit that specifically binds to PD-1, and the second half-antibody comprises 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/light chain variable region sequences set forth in 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/light chain variable region sequences set forth in SEQ ID NO: 6/SEQ ID NO: 2.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein comprises a first half-antibody and a second half-antibody, wherein the first half-antibody comprises a first VH/VL unit specifically binding to PD-1 and the second half-antibody comprises a second VH/VL unit specifically binding to HER2, wherein the first VH/VL unit comprises the variable heavy chain/variable light chain paired sequences set forth in SEQ ID NO: 12/SEQ ID NO: 10 or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said variable heavy chain/variable light chain paired sequences, and the second VH/VL unit comprises the paired sequences variable heavy chain/variable light chain sequences as shown in SEQ ID NO: 6/SEQ ID NO: 2 or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said variable heavy chain/variable light chain paired sequences.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein consists of a first half-antibody and a second half-antibody, wherein the first half-antibody comprises the heavy chain sequences of SEQ ID NO: 12 and SEQ ID NO: 14 or heavy chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction and comprises the light chain sequences of SEQ ID NO: 10 and SEQ ID NO: 4 or light chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction, and the second half-antibody comprises the heavy chain sequences of SEQ ID NO: 6 and SEQ ID NO: 8 or heavy chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction and comprises 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 in the N to C direction.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein is an anti-PD-1/HER2 bispecific antibody protein that is recombinantly expressed in HEK293, HEK293T, HEK293F, or HEK293E cells modified from HEK293 cells, or in CHO, CHO-S, CHO- ^dhfr- , CHO/DG44, or ExpiCHO cells modified from CHO cells.

In one embodiment, the concentration of the anti-PD-1/HER2 bispecific antibody protein in the liquid antibody composition disclosed herein is about 1-150 mg/mL. In another embodiment, the concentration of the anti-PD-1/HER2 bispecific antibody protein in the liquid antibody composition disclosed herein is about 10-100 mg/mL. In other embodiments, the concentration of the anti-PD-1/HER2 bispecific antibody protein in the liquid antibody composition disclosed herein is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/mL.

In one embodiment of the invention, the concentration of the buffer in the liquid antibody composition disclosed herein is about 5-50 mM. In one embodiment of the invention, the concentration of the buffer in the liquid antibody composition disclosed herein is about 10-30 mM, such as about 10, 15, 20, 25, or 30 mM.

In one embodiment of the invention, the buffer is selected from histidine, histidine hydrochloride, or a combination thereof.

In one embodiment, the concentration of the stabilizer in the liquid antibody composition disclosed herein is about 50-500 mM. In one embodiment, the concentration of the stabilizer in the liquid antibody composition disclosed herein is about 100-400 mM, such as about 100, 150, 200, 250, 300, 350, or 400 mM.

In one embodiment of the invention, the stabilizer is selected from a polyol (e.g., sorbitol), a saccharide (e.g., sucrose or trehalose), and a combination thereof.

In another embodiment, the stabilizer is selected from combinations of a polyol (e.g., sorbitol), a saccharide (e.g., sucrose or trehalose), and any combination thereof with an antioxidant. In one embodiment, the total concentration of the stabilizer in the liquid antibody composition is about 50-500 nM, preferably about 100-400 mM, such as about 100, 150, 200, 250, 300, 350, or 400 mM, and the concentration of the antioxidant is about 1-50 mM, preferably about 5-40 mM, such as about 5, 10, 20, 30, or 40 mM. In one embodiment, the antioxidant is methionine.

In one embodiment, the concentration of surfactant in the liquid antibody composition disclosed herein is about 0.1-1 mg/mL. In one embodiment, the concentration of surfactant in the liquid antibody composition disclosed herein is about 0.2-0.8 mg/mL, such as about 0.2; 0.3; 0.4; 0.5; 0.6; 0.7, or 0.8 mg/mL.

In one embodiment of the invention, the surfactant is a nonionic surfactant. In one embodiment of the invention, the surfactant is selected from polysorbate surfactants. In one particular embodiment of the invention, the surfactant in the liquid antibody composition disclosed herein is polysorbate-80.

In one embodiment, the pH of the liquid composition is about 5.0-6.5. In some embodiments, the pH of the liquid composition may be any value in the range of 5.0-6.5; for example, about 5.0; 5.2; 5.4; 5.6; 5.8; 6.0; 6.2, or 6.4.

In one embodiment of the invention, the liquid composition is a pharmaceutical composition, preferably an injectable dosage form, and more preferably a composition for subcutaneous injection or intravenous injection. In one embodiment of the invention, the liquid composition is a dosage form for intravenous infusion.

In one embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 1-150 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) about 5-50 mM histidine and/or histidine hydrochloride;

(iii) about 50-500 mM sorbitol, sucrose, trehalose, or any combination thereof, or

a combination of sorbitol, sucrose, trehalose, and any combination thereof with methionine at a total concentration of about 50-500 mM, wherein the methionine concentration is about 1-50 mM; and

(iv) approximately 0.1-1 mg/ml polysorbate-80, while

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 10-100 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) about 10-30 mM histidine and/or histidine hydrochloride;

(iii) about 100-400 mM sorbitol, sucrose and/or trehalose, or

a combination of sorbitol, sucrose and/or trehalose with methionine at a total concentration of about 100-400 mM, wherein the methionine concentration is about 5-40 mM; and

(iv) approximately 0.2-0.8 mg/ml polysorbate-80, while

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 20 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) approximately 10 mM histidine;

(iii) about 50 mg/ml sorbitol; And

(iv) approximately 0.3 mg/ml polysorbate 80, with

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 50 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) approximately 20 mM histidine;

(iii) about 50 mg/ml sorbitol; And

(iv) approximately 0.2 mg/ml polysorbate 80, with

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 50 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) approximately 20 mM histidine;

(iii) approximately 80 mg/ml sucrose; And

(iv) approximately 0.2 mg/ml polysorbate 80, with

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 50 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) approximately 20 mM histidine;

(iii) approximately 80 mg/ml trehalose; And

(iv) approximately 0.2 mg/ml polysorbate 80, with

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 50 mg/mL of the anti-PD-1/HER2 bispecific antibody protein;

(ii) approximately 20 mM histidine;

(iii) approximately 80 mg/mL sucrose and approximately 1.49 mg/mL methionine; and

(iv) approximately 0.2 mg/ml polysorbate 80, with

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In one preferred embodiment of the invention, the liquid antibody composition disclosed herein comprises:

(i) approximately 42 mg/mL of anti-PD-1/HER2 bispecific antibody protein;

(ii) about 0.85 mg/ml histidine and about 3.17 mg/ml histidine hydrochloride;

(iii) approximately 80 mg/ml sucrose; And

(iv) approximately 0.2 mg/ml polysorbate 80, with

The pH of the liquid composition is about 5.0-6.5; preferably about 5.5.

In another aspect, the present invention provides a solid antibody composition obtained by converting the liquid antibody composition disclosed herein into a solid form. The procedure for converting into a solid form is carried out, for example, by crystallization, spray drying or freeze drying. In one preferred embodiment, the solid antibody composition is, for example, a lyophilized powder for injection. The solid antibody composition can be converted into a suitable solvent before use to obtain a reconstituted composition of the present invention. The reconstituted composition is also a liquid antibody composition disclosed herein. In one embodiment, the suitable solvent is selected from water for injection, organic solvents for injection (including, but not limited to, oil for injection, ethanol, propylene glycol, etc.), and a combination thereof.

The liquid composition disclosed in this document can be stored without loss of stability for a long period of time, such as for at least 24 months or longer. In one embodiment of the invention, the liquid composition disclosed herein may remain stable after storage at a temperature of from about -80 °C to about 45 °C, such as at -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 at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months or longer.

In one embodiment of the invention, the liquid composition disclosed herein can be stored without loss of stability for at least 24 months.

In another embodiment of the invention, the liquid composition disclosed herein remains stable at a temperature of at least 40°C. In yet another embodiment of the invention, the liquid composition disclosed herein remains stable at a temperature of about 2-8°C for at least 3 months, preferably at least 12 months and more preferably at least 24 months. In one embodiment of the invention, the liquid composition disclosed herein remains stable at room temperature or, for example, at a temperature of about 25°C for at least 2 months, preferably at least 3 months and more preferably at least 6 months. In yet another embodiment of the invention, the liquid composition disclosed herein remains stable at a temperature of about 40°C for at least 2 weeks, preferably at least 1 month.

In one embodiment of the invention, the stability of the composition after storage can be judged by the change in its appearance, the presence of visible particles, the protein content, the turbidity, the purity of the composition and/or the content of charged isoforms. In one embodiment of the invention, an accelerated high temperature stress test can be used to determine the stability of the liquid composition disclosed herein, such as after storage at 40±2°C for at least 1 week, 2 weeks or, preferably, 1 month, or an accelerated test, such as after storage at 25±2°C for at least 1 month or 2 months, or a long-term test, such as after storage at 5±3°C for at least 2 months or 3 months.

In one embodiment of the invention, to determine the stability after storage of the liquid composition disclosed in this document, a visual inspection thereof is carried out, wherein the liquid composition disclosed in this document remains a transparent or slightly opalescent liquid without color or slightly yellow color, containing no visible particles. In one embodiment of the invention, upon visual inspection of the composition on a transparency detector, no visible particles are detected in it. In one embodiment of the invention, the stability of the liquid composition disclosed in this document is studied after storage by determining the changes in the protein content, wherein the rate of change in the protein content is no more than 20%, preferably no more than 10%, such as 7-8%, and more preferably no more than 5%, relative to the initial value on day 0 of the storage period, for example, when measured by UV spectrophotometry. In one embodiment of the invention, the stability of the liquid composition disclosed herein is studied after storage by determining the change in turbidity of the liquid composition disclosed herein, wherein the change is no more than 0.06, preferably no more than 0.05 and more preferably no more than 0.04 relative to the initial value on day 0 of the storage period, for example by measuring the optical density OD ₃₅₀ nm. In one embodiment of the invention, the stability of the liquid composition disclosed herein is studied after storage by determining the change in purity of the liquid composition disclosed herein, wherein the change in monomer purity is no more than 10%, such as no more than 5%, 4% or 3%, such as 12%, preferably no more than 1% relative to the initial value on day 0 of the storage period, when measured, for example, by size exclusion high performance liquid chromatography (size exclusion HPLC). In one embodiment of the invention, the stability of the liquid composition disclosed herein is studied after storage by determining the change in purity of the liquid composition disclosed herein, wherein the monomer purity decreases by no more than 10%, such as no more than 5%, 4%, or 3%, when measured by sodium dodecyl sulfate capillary electrophoresis (SDS-CE) under reducing and/or non-reducing conditions. In one embodiment of the invention, the stability of the liquid composition disclosed herein is studied after storage by imaging-guided capillary isoelectric focusing (iCIEF), wherein the total change in the content of charged isoforms (major component, acidic component, and basic component) of the antibody is no more than 50%, such as no more than 40%, 30%, 20%, 10%, or 5%, relative to the initial value at day 0 of the storage period. In one embodiment of the invention, the stability of the liquid composition disclosed herein is studied after storage by high performance cation exchange liquid chromatography (COX-HPLC), wherein the overall change in the content of charged isoforms (major component, acidic component or basic component) of the antibody is no more than 40%, such as no more than 38%, 36%, 34%, 32% or 30%, relative to the initial value on day 0 of the storage period.

In one embodiment of the invention, the composition remains stable after storage, for example at a temperature of 2-8°C for at least 24 months, at room temperature for at least 3 months, or at a temperature of 40±2°C for 1 month, and preferably has one or more of the following characteristics:

(i) the purity is greater than 90%, preferably greater than 95%, 96%, 97%, 98% or 99%, as measured by size exclusion HPLC;

(ii) the purity is greater than 90%, preferably greater than 92%, 94%, 96% or 98% when measured by the CE-SDS method under reducing or non-reducing conditions;

(iii) the overall change in the content of the components (major component, acidic component and basic component) of the anti-PD-1/HER2 bispecific antibody protein in the composition is ≤50%, such as ≤40%, 30%, 20%, 10% or 5%, relative to the initial value at day 0 of the storage period, as measured by the iCIEF method; and

(iv) the relative binding activity of the anti-PD-1/HER2 bispecific antibody protein in the composition is 70-130%, such as 70%, 80%, 90%, 100%, 110%, 120% or 130%, relative to the initial value at day 0 of the storage period when measured by ELISA.

In one aspect, the subject of the present invention is a delivery device comprising a liquid antibody composition or a solid antibody composition as disclosed herein. In one embodiment, the delivery device as disclosed herein is in the form of a pre-filled syringe comprising a liquid antibody composition or a solid antibody composition as disclosed herein, for example for intravenous, subcutaneous, intradermal or intramuscular injection or intravenous infusion.

In another aspect, the present invention provides a method of delivering an anti-PD-1/HER2 bispecific antibody protein to a subject, such as a mammal, which comprises administering a liquid antibody composition or a solid antibody composition disclosed herein to the subject, wherein the delivery is carried out, for example, using a delivery device in the form of a pre-filled syringe.

In another aspect, the present invention provides the use of a liquid antibody composition or a solid antibody composition disclosed herein for the preparation of a delivery device (e.g., a pre-filled syringe) or a medicament for treating, preventing, or slowing the progression of a disease associated with HER2 and PD-1 mediated signaling pathways in a subject, wherein the disease includes, for example, various blood diseases and solid tumors, including, but not limited to, leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, and melanoma.

Other embodiments of the present invention will be apparent from the detailed description provided herein.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The preferred embodiments of the present invention, described in detail below, will be more easily understood when read in conjunction with the figures below. For purposes of illustrating the present invention, embodiments of the present invention that are currently preferred are shown in the figures. However, it should be understood that the present invention is not limited to the precise arrangements and means of carrying out the invention shown in the figures.

FIG. 1 illustrates the structure of an anti-PD-1/HER2 bispecific antibody consisting of an anti-PD-1 half-antibody molecule and an anti-HER2 half-antibody molecule.

FIG. 2 illustrates the dynamics of turbidity change in samples of the anti-PD-1/HER2 bispecific antibody composition after storage at pH 5.0; 5.5; 6.0 and 6.5 and a temperature of 40±2°C for different periods of time, as determined by OD _{350 nm} . On the abscissa, T0 corresponds to day 0, 1W corresponds to 1 week, 2W corresponds to 2 weeks, and 1M corresponds to 1 month.

FIG. 3 illustrates the dynamics of protein purity changes in samples of the anti-PD-1/HER2 bispecific antibody composition after storage at pH 5.0; 5.5; 6.0 and 6.5 and a temperature of 40 ± 2 °C for different periods of time as determined by the size-exclusion HPLC method. On the abscissa axis, T0 corresponds to day 0, 1W corresponds to 1 week, 2W corresponds to 2 weeks, and 1M corresponds to 1 month.

FIG. 4 illustrates the dynamics of protein purity changes in samples of the bispecific antibody composition against PD-1/HER2 after storage at pH 5.0; 5.5; 6.0 and 6.5 and a temperature of 40 ± 2 °C for different periods of time when determined by the CE-SDS method under non-reducing conditions. On the abscissa, T0 corresponds to day 0, 1W corresponds to 1 week, 2W corresponds to 2 weeks, and 1M corresponds to 1 month.

FIG. 5 illustrates the dynamics of protein purity changes in samples of the anti-PD-1/HER2 bispecific antibody composition after storage at pH 5.0; 5.5; 6.0 and 6.5 and a temperature of 40 ± 2 °C for different periods of time when determined by the CE-SDS method under reducing conditions. On the abscissa, T0 corresponds to day 0, 1W corresponds to 1 week, 2W corresponds to 2 weeks, and 1M corresponds to 1 month.

FIG. 6 illustrates the dynamics of the change in the content of the charged isoform (the main component) in samples of the anti-PD-1/HER2 bispecific antibody composition after storage at pH 5.0; 5.5; 6.0 and 6.5 and a temperature of 40 ± 2 ° C for different periods of time as determined by the iCIEF method. On the abscissa axis, T0 corresponds to day 0, 1W corresponds to 1 week, 2W corresponds to 2 weeks, and 1M corresponds to 1 month.

FIG. 7 illustrates the change in the content of the charged isoform (major component) over time in anti-PD-1/HER2 bispecific antibody formulations containing different stabilizers (formulations 14) after storage at 40°C for 0 days, 1 week, 2 weeks, and 4 weeks, as determined by the iCIEF method. On the abscissa, T0 corresponds to day 0, 1W corresponds to 1 week, 2W corresponds to 2 weeks, 4W corresponds to 4 weeks; F1 corresponds to formulation 1, F2 corresponds to formulation 2, F3 corresponds to formulation 3, and F4 corresponds to formulation 4.

FIG. 8 illustrates the change in the content of the charged isoform (major component) over time in anti-PD-1/HER2 bispecific antibody formulations containing different stabilizers (formulations 14) after storage at 25±2°C for 0 days, 1 week, 2 weeks, and 4 weeks, as determined by the iCIEF method. On the abscissa, T0 corresponds to day 0, 1M corresponds to 1 month, and 2M corresponds to 2 months; F1 corresponds to formulation 1, F2 corresponds to formulation 2, F3 corresponds to formulation 3, and F4 corresponds to formulation 4.

DETAILED DESCRIPTION

Before describing the present invention in detail, it should be emphasized that the present invention is not limited to any particular methods or experimental conditions given in this document, as the methods and conditions may vary. It should further be emphasized that the terms used in this document are intended only to describe particular embodiments of the invention and are not intended to limit its scope.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purposes of this disclosure, the following terms are defined below.

The term "about" when used in combination with a numerical value is intended to cover numerical values ranging from a lower limit of 5% less than the stated numerical value to an upper limit of 5% more than the stated numerical value.

The term "and/or" when used to connect two or more options shall be understood to refer to any one of the options or to any two or more options.

As used in this document, the terms "comprise" or "comprising" mean the inclusion of the described elements, integers, or steps, but not the exclusion of any other elements, integers, or steps. As used in this document, the terms "comprise" or "comprising," unless otherwise specified, also refer to the situation where the whole consists of the described elements, integers, or steps. For example, if a variable region of an antibody is said to "comprise" a certain sequence, the variable region of the antibody is also meant to consist of the certain sequence.

The term "antibody" as used herein in its broadest sense refers to a protein containing an antigen-binding site and encompasses both natural and artificial antibodies of various structures, including, but not limited to, intact antibodies and antigen-binding fragments of antibodies.

The terms "whole antibody", "full-length antibody", "complete antibody" and "intact antibody" are used interchangeably herein and refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The constant region of each heavy chain is comprised of 3 domains: CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The constant region of each light chain is comprised of a single CL domain. The VH region and VL region can be further divided into hypervariable regions (complementarity determining regions or CDRs) and relatively conserved regions (framework regions or FRs) located between them. Each VH or VL consists of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit a number of effector functions.

The term "humanized" refers to a chimeric antibody comprising amino acid residues from non-human HVR regions and amino acid residues from non-human FR regions. In some embodiments, all or substantially all of the HVRs (e.g., CDRs) in a humanized antibody correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. "Humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

The term "half-antibody" or "hemimer" refers to a monovalent antigen-binding polypeptide. In some embodiments, a half-antibody or hemimer comprises a VH/VL unit and, optionally, at least a portion of an immunoglobulin constant domain. In some embodiments, a half-antibody or hemimer comprises a single immunoglobulin heavy chain linked to an immunoglobulin light chain, or an antigen-binding fragment thereof. In some embodiments, a half-antibody or hemimer 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 appreciate that a half-antibody can comprise an antigen-binding domain consisting of a single variable domain, such as that derived from animals of the Camelidae family.

The term "VH/VL unit" refers to an antigen-binding portion 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 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.

As used herein, the term "bispecific antibody" includes antigen-binding domains that specifically bind to epitopes on two different biological molecules. Unless otherwise specified, the order of the antigens bound by the bispecific antibody in the named bispecific antibody is arbitrary. 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, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises one heavy chain variable region and, optionally, at least a portion of a heavy chain constant region, and comprises one light chain variable region and, optionally, at least a portion of a light chain constant region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises one heavy chain variable region and one light chain variable region, but does not comprise more than one heavy chain variable region and does not comprise more than one light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises one heavy chain variable region and one light chain variable region, and wherein the first half-antibody binds to a first antigen but does not bind to a second antigen, and the second half-antibody binds to a second antigen but does not bind to the first antigen.

The term "antibody composition" refers to a preparation in a form in which the antibody exhibits its biological activity as an active ingredient intended to exert a beneficial effect, wherein the preparation does not contain other components that exert an unacceptable toxic effect in the subject to which the composition is to be administered. Such antibody compositions are typically sterile. Typically, the antibody composition includes a pharmaceutically acceptable excipient. A "pharmaceutically acceptable" excipient is one that can reasonably be administered to a mammalian subject such that an effective dose of the active ingredient used in the composition can be delivered to the subject. The concentration of the excipient is selected taking into account the route of administration and, for example, may be suitable for injection.

The term "anti-PD-1/HER2 bispecific antibody composition" as used herein as "the antibody composition disclosed herein" also refers to a preparation comprising an anti-PD-1/HER2 bispecific antibody protein as an active ingredient and a pharmaceutically acceptable excipient. The anti-PD-1/HER2 bispecific antibody protein as an active ingredient is suitable for therapeutic or prophylactic use in a human or non-human animal after combining the anti-PD-1/HER2 bispecific antibody protein with a pharmaceutically acceptable excipient. The antibody composition disclosed herein can be formulated, for example, as an aqueous liquid composition, for example in a ready-to-use pre-filled syringe, or as a lyophilized composition to be reconstituted (i.e., re-dissolved) by dissolving and/or suspending in a physiologically acceptable solution immediately prior to use. In some embodiments of the invention, the anti-PD-1/HER2 bispecific antibody protein composition is prepared in the form of a liquid composition.

A "stable" antibody composition is one in which the antibody retains an acceptable degree of physical and/or chemical stability after storage under specified conditions. Although the antibody in an antibody composition may not retain 100% of its chemical structure after storage for a specified period of time, the antibody composition is considered "stable" if the antibody, after storage for a specified period of time, typically retains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function. In some specific embodiments, the anti-PD-1/HER2 bispecific antibody protein composition disclosed herein exhibits evidence of aggregation, degradation, or chemical modification of the antibody during manufacturing, formulation, transportation, or long-term storage, resulting in little or no loss of biological activity of the anti-PD-1/HER2 bispecific antibody protein and indicating its high stability. In some embodiments, the anti-PD-1/HER2 bispecific antibody protein composition disclosed herein substantially retains physical and chemical stability after storage. Preferably, the liquid composition disclosed herein may remain stable at room temperature or at 40°C for at least 2 weeks and/or at 25°C for at least 2 months and/or at 28°C for at least 24 months.

Various assays for determining protein stability are known in the art, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be determined for a particular temperature and for a particular storage period. For example, the storage period can be selected based on the expected shelf life of the composition. Alternatively, an accelerated stability study can be conducted. In some embodiments, the stability study is conducted using various stress tests to which the antibody composition is subjected. These tests can be conducted under extreme conditions to which the prepared antibody composition may be subjected during manufacturing, storage, or transportation, and they can also represent conditions that may contribute to accelerated loss of antibody stability in the antibody composition during non-manufacturing operations, storage, or transportation. For example, a finished anti-PD-1/HER2 bispecific antibody protein composition can be placed in a glass vial to study the stability of the antibody under high temperature stress conditions.

An antibody may be considered "physically stable" in a composition if, after storage for a period of time, the composition shows no detectable aggregation, precipitation, turbidity and/or denaturation, or very little detectable aggregation, precipitation, turbidity and/or denaturation. Aggregation of antibodies in a composition poses safety concerns because it may potentially result in a more pronounced immune response in a patient. Accordingly, aggregation of antibodies in a composition should be minimized or prevented. Light scattering-based methods may be used to detect visible aggregates in a composition. Size exclusion chromatography (SEC) may be used to detect soluble aggregates in a composition. In addition, the stability of a composition may be assessed by visual inspection of the appearance, color and/or clarity of the composition, or by determining the turbidity of the composition by optical density (OD _{350 nm} ), or by determining the purity of the composition by CE-SDS under non-reducing conditions. In one embodiment of the invention, the stability of the composition is determined by measuring the percentage of monomeric antibody in the composition after storage at a certain temperature for a certain period of time, wherein the higher the percentage of monomeric antibody in the composition, the higher the stability of the composition.

An “acceptable degree” of physical stability is defined as at least approximately 92% of the monomeric protein of the anti-PD-1/HER2 bispecific antibody being recovered after storage at a specified temperature for a specified period of time. In some embodiments, an acceptable degree of physical stability corresponds to a monomeric protein content of the anti-PD-1/HER2 bispecific antibody of at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after storage at a certain 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 specific temperature at which the pharmaceutical composition is stored can be any value in the range from about -80 °C to about 45 °C, such as about -80 °C, about -30 °C, about -20 °C, about 0 °C, about 4-8 °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, the pharmaceutical composition is considered stable if, after storage at a temperature of about 40 ± 2 °C for 1 month or 4 weeks, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the monomeric protein of the anti-PD-1/HER2 bispecific antibody is detected. The pharmaceutical composition is considered stable if, after storage at about 25°C for 2 months, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the monomeric protein of the anti-PD-1/HER2 bispecific antibody is detected. The pharmaceutical composition is considered stable if, after storage at about 5°C for 9 months, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the monomeric protein of the anti-PD-1/HER2 bispecific antibody is detected.

An antibody is considered to be "chemically stable" in a composition if the antibody in the composition does not undergo significant chemical changes after storage for a specified period of time. In most cases, chemical instability arises due to the formation of covalently modified forms of the antibody (e.g., charged isoforms of the antibody). Basic isoforms can be formed, for example, by aspartic acid isomerization, as well as N- and C-terminal modifications; acidic isoforms can be formed by deamination, sialylation, and glycosylation. Chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody. For example, charged isoforms of the antibody in a composition can be detected by cation exchange chromatography (CEC) or image-guided capillary isoelectric focusing (iCIEF). In one embodiment of the invention, the stability of a composition is determined by measuring the percentage change in the content of charged isoforms of the antibody in the composition after storage at a certain temperature for a certain period of time, wherein the smaller the degree of change, the higher the stability of the composition.

An "acceptable degree" of chemical stability can be said to exist if the percentage change in the content of charged isoforms (e.g., a major component, an acid component, or a basic component) in the composition after storage at a given temperature for a given period of time does not exceed 50%, for example, does not exceed 30% or 20%. In some embodiments, an acceptable degree of chemical stability may correspond to a percentage change in the content of the charged isoform (major component) of no more than about 50%, 40%, 30%, 20%, or 15% after storage at a certain 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 temperature at which the pharmaceutical composition is stored may be any temperature from about -80°C to about 45°C, such as about -80°C, about -30°C, about -20°C, about 0°C, about 4-8°C, about 5°C, about 25°C, or about 45°C. For example, a pharmaceutical composition may be considered stable if the percentage change in the content of the charged isoform (major component) after storage at 5°C for 24 months 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 composition may be considered stable if the percentage change in the content of the charged isoform (major component) after storage at 25°C for 2 months is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. A pharmaceutical composition may be considered stable if the percentage change in the content of the charged isoform (major component) after storage at 40°C for 1 month is less than about 50%, 40%, 30%, 20%, 10%, 5%, or 4%.

The term "lyophilized composition" refers to a composition that is obtained or obtainable by freeze-drying a liquid composition. Preferably, it is a solid composition whose water content is less than 5%, preferably less than 3%.

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

The term “room temperature” as used in this document refers to a temperature of 15-30°C, preferably 20-27°C, and more preferably 25°C.

The term "stress conditions" refers to environmental conditions that are unfavorable for the chemical and/or physical stability of antibody proteins and may cause unacceptable destabilization of antibody proteins. The term "high temperature stress" refers to storage of an antibody composition at room temperature or at a higher temperature (e.g., 40 ± 2 °C) for a certain period of time. The stability of an antibody composition can be studied in an accelerated high temperature stress test.

The term "parenteral administration" as used herein refers to any administration other than enteral and local administration, typically administration by injection or infusion, including but 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, the stable anti-PD-1/HER2 bispecific antibody protein composition disclosed herein is administered to a subject parenterally. In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein composition disclosed herein is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

I. Antibody Composition

The present invention provides a stable liquid antibody composition comprising (i) an anti-PD-1/HER2 bispecific antibody protein, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, wherein the pH of the antibody composition is about 5.0-6.5. In one preferred embodiment, the liquid antibody composition disclosed herein is in an injectable form.

(i) Anti-PD-1/1IER2 bispecific antibody protein

The "anti-PD-1/HER2 bispecific antibody protein" in the antibody composition disclosed herein consists of a first half-antibody and a second half-antibody, wherein the first half-antibody comprises a first VH/VL unit that specifically binds to PD-1, and the second half-antibody comprises a second VH/VL unit that specifically binds to HER2. In some embodiments, the anti-PD-1/HER2 bispecific antibody protein is capable of 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 or higher, and is capable of binding to HER2 on the surface of tumor cells 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 or higher, such that the antibody can be used as a therapeutic agent and/or prophylactic agent having the ability to bispecifically target PD-1 and HER2 molecules.

A VH/VL unit that specifically binds to PD-1 or HER2 comprises 6 CDRs from a VH/VL unit derived from an anti-PD-1 antibody described in the prior art and an anti-PD-1 antibody to be developed in the future, or sequences having one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) compared to one or more CDRs of the 6 CDRs; or comprises 6 CDRs from a VH/VL unit derived from an anti-HER2 antibody described in the prior art and an anti-HER2 antibody to be developed in the future, or sequences having one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) compared to one or more CDRs of the 6 CDRs.

In one embodiment of the invention, the first VH/VL unit of the anti-PD-1/HER2 bispecific antibody protein that specifically binds to PD-1 comprises all 6 CDRs of the heavy and light chains contained in the paired heavy chain variable region/light chain variable region sequences set forth in SEQ ID NO: 12/SEQ ID NO: 10 derived from an anti-PD-1 half-antibody, or in sequences having one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) compared to one or more CDRs of the 6 CDRs.

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

The terms "CDR," "complementarity determining region," or "CDR region" (used interchangeably with the term "hypervariable region" (HVR) herein) refer to the amino acid region within the variable region of an antibody that is primarily responsible for binding to an antigen epitope. The CDRs of the heavy and light chains are typically designated CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. Various schemes for determining the sequence of CDRs for a given VH, VL, or VHH amino acid sequence are known in the art. For example, the Kabat method, based on sequence variability, is most commonly used to determine complementarity determining regions (CDRs) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Chothia method is based on the locations of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The HVRs defined by the AbM method represent a compromise between the Kabat-defined HVRs and the Chothia structural loops and are used in the Oxford Molecular AbM antibody modeling software. The definition of the "contact" HVRs is based on the analysis of available complex crystal structures.

Amino acid changes, such as amino acid substitutions, are preferably conservative amino acid substitutions. The term "conservative amino acid substitution" refers to an amino acid change in which the amino acid is replaced by a chemically similar amino acid. Conservative substitution tables showing functionally similar amino acids are well known to those skilled in the art. In any of the embodiments disclosed herein, in one preferred aspect, the conservatively substituted residue is taken from conservative substitution table A below, preferably preferred substituted residues are shown in table A.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein comprises a first half-antibody and a second half-antibody, wherein the first half-antibody comprises a first VH/VL unit specifically binding to PD-1 and the second half-antibody comprises a second VH/VL unit specifically binding to HER2, wherein the first VH/VL unit comprises the variable heavy chain/variable light chain paired sequences set forth in SEQ ID NO: 12/SEQ ID NO: 10 or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said variable heavy chain/variable light chain paired sequences, and the second VH/VL unit comprises the paired sequences variable heavy chain/variable light chain sequences as shown in SEQ ID NO: 6/SEQ ID NO: 2 or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said variable heavy chain/variable light chain paired sequences.

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

In one embodiment, the anti-PD-1/HER2 bispecific antibody protein comprises an IgG1 heavy chain constant region (e.g., human IgG1). In another embodiment, the anti-PD-1/HER2 bispecific antibody protein comprises an IgG4 heavy chain constant region (e.g., human IgG4). For example, each of the Fc domains of the two heavy chains of the anti-PD-1/HER2 bispecific antibody comprises a hinge region comprising the amino acid residues "CPPC" and/or the Fc domains comprise the Y349C and S354C substitutions (according to "EU numbering" according to Kabat), respectively, and thus the anti-PD-1 half-antibody and the anti-HER2 half-antibody form interchain disulfide bonds in the Fc regions, which ensures the stabilization of the correct pairing of the anti-PD-1 half-antibody and the anti-HER2 half-antibody.

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 comprises amino acid mutations in the Fc domain that affect the effector function of the antibody. In one specific embodiment, the effector function is antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the amino acid mutations are present 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) of the Fc region of the anti-PD-1 half-antibody and/or the anti-HER2 half-antibody. In one specific embodiment, the amino acid substitutions are L234A and L235A (also referred to as "LALA mutations").

In another embodiment of the invention, 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, such as a human kappa light chain constant region or a human lambda light chain constant region.

In one embodiment of the invention, the two heavy chains of the anti-PD-1/HER2 bispecific antibody protein comprise a knob and a cavity in their Fc domains, respectively, or, conversely, a knob (or cavity) in the Fc domain of one heavy chain can be placed into a cavity (or knob) in the Fc domain of another heavy chain such that the two heavy chains form a stable knob-in-hole association with each other. In one embodiment of the invention, one of the two heavy chains comprises the amino acid substitution T366W and the other heavy chain comprises the amino acid substitutions T366S, L368A and Y407V (EU numbering). In this way, the knob in one chain can be placed into a cavity in the other chain, which facilitates proper pairing of the two heavy chains of the anti-PD-1/HER2 bispecific antibody protein.

In one embodiment of the invention, the CH1 domain of the immunoglobulin and the CL domain of the heavy chain and light chain in each half-antibody of the anti-PD-1/HER2 bispecific antibody protein comprise a bulge and a cavity, respectively, or, conversely, a bulge (or cavity) in the CH1 domain may be placed within a cavity (or bulge) in the CL domain such that the heavy chain and light chain in each half-antibody also form a stable knob-into-hole association with each other.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein consists of a first half-antibody and a second half-antibody, wherein the first half-antibody comprises the heavy chain sequences of SEQ ID NO: 12 and SEQ ID NO: 14 or heavy chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction and comprises the light chain sequences of SEQ ID NO: 10 and SEQ ID NO: 4 or light chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction, and the second half-antibody comprises the heavy chain sequences of SEQ ID NO: 6 and SEQ ID NO: 8 or heavy chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction and comprises the light chain sequences of SEQ ID NO: 2 and SEQ ID NO: 4 or a light chain sequence having at least 90%, 95%, 98% or 99% identity thereto in the N to C direction.

As used herein, the term "sequence identity" refers to the extent to which sequences are identical at nucleotide or amino acid levels within a comparison window. "Percentage sequence identity" may be calculated using the following steps: comparing two optimally aligned sequences within the comparison window; determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, and I) or amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to yield the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size); and multiplying the result by 100 to yield the percent sequence identity. Optimal alignment for determining percent sequence identity can be achieved by various methods known in the art, for example using publicly available computer programs such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in the art can determine appropriate sequence alignment parameters, including algorithms needed to optimally align compared sequences over a range of full-length sequences or target regions of sequences.

The anti-PD-1/HER2 bispecific antibody protein in the antibody composition disclosed in this document is capable of simultaneously binding to PD-1 and HER2 proteins and maintaining the affinity constant of each parent antibody, thereby blocking the HER2-dependent signaling pathway and the PD-1-dependent signaling pathway, and therefore this antibody composition can be used to treat, prevent or slow down the development of various diseases or disorders associated with the HER2-dependent signaling pathway and/or the PD-1-dependent signaling pathway.

In one preferred embodiment, the anti-PD-1/HER2 bispecific antibody protein disclosed herein is a recombinant anti-PD-1/HER2 bispecific antibody protein disclosed in PCT Application No. PCT/CN2018/075851 (filed February 8, 2018), which comprises a fully human anti-PD-1 half-antibody and a humanized anti-HER2 half-antibody, wherein the heavy chain of the fully human anti-PD-1 half-antibody has the sequence of SEQ ID NO: 12 and SEQ ID NO: 14 in the N to C direction, and the light chain has the sequence of SEQ ID NO: 10 and SEQ ID NO: 4 in the N to C direction; The heavy chain of the humanized anti-HER2 half antibody has the sequence of SEQ ID NO: 6 and SEQ ID NO: 8 in the N to C direction, and the light chain has the sequence of SEQ ID NO: 2 and SEQ ID NO: 4 in the N to C direction.

In one embodiment of the invention, the anti-PD-1/HER2 bispecific antibody protein is produced with recombinant expression in HEK293, HEK293T, HEK293F or HEK293E cells obtained by modification from HEK293 cells and in CHO, CHO-S, CHO-dhfr ^- , CHO/DG44 or ExpiCHO cells obtained by modification from CHO cells, after which the bispecific antibody protein is purified. Preferably, the antibody in the liquid composition disclosed herein exhibits significant antitumor activity. The anti-PD-1/HER2 bispecific antibody was administered to tumor-bearing mice obtained by inoculating HCC1954 human breast cancer cells into NCG immunodeficient mice, and the results of the experiments showed that, compared with the anti-PD-1 monoclonal antibody or the anti-HER2 monoclonal antibody, the anti-PD-1/HER2 bispecific antibody has significantly increased antitumor activity and is able to markedly reduce tumor volume.

The amount of anti-PD-1/HER2 bispecific antibody protein in the antibody composition disclosed herein may vary depending on the particular characteristics of the composition desired, the particular environmental conditions, and the particular purpose for which the composition is used. In some embodiments, the antibody composition is a liquid composition that may contain about 1-150 mg/mL, preferably about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/mL, of anti-PD-1/HER2 bispecific antibody protein.

(ii) Buffer

Buffers are reagents that are capable of maintaining the pH of a solution within an acceptable range. In some embodiments, a buffer in a composition disclosed herein is capable of maintaining the pH of the composition disclosed herein within the range of about 5.0-6.5; for example, about 5.5. In some embodiments, the pH of an antibody composition disclosed herein is about 5.0; 5.2; 5.4; 5.6; 5.8; 6.0; 6.2, or 6.4.

In some embodiments, the buffer of the composition disclosed herein is selected from histidine, histidine hydrochloride, and a combination thereof. In one embodiment, the concentration of the buffer in the liquid antibody composition disclosed herein is about 5-50 mM. In one embodiment, the concentration of the buffer in the liquid antibody composition disclosed herein is about 10-30 mM, such as about 10, 15, 20, 25, or 30 mM.

In one embodiment of the invention, the buffer in the composition disclosed herein is about 10 mM histidine. In another embodiment of the invention, the buffer in the composition disclosed herein is about 20 mM histidine.

In another embodiment of the invention, the buffer in the composition disclosed herein is a combination of about 5.5 mM histidine and about 15 mM histidine hydrochloride.

(iii) Stabilizer

Stabilizers suitable for use in the present invention may be selected from a saccharide, a polyol, and a combination thereof. In addition, the stabilizer of the present invention may further comprise an antioxidant.

The saccharide used as a stabilizer may be a disaccharide, a trisaccharide, and a polysaccharide, and the saccharide may be selected from, but not limited to: sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, and glucan. In one embodiment of the invention, the saccharide used as a stabilizer is sucrose and/or trehalose.

The polyol used as a stabilizer may be selected from, but not limited to: mannitol, sorbitol, and xylitol. In one embodiment of the invention, the polyol used as a stabilizer is sorbitol.

In some embodiments of the invention, the saccharide and/or polyol used as a stabilizer is present in the liquid composition disclosed herein at a concentration of about 50-500 mM, preferably about 100-400 mM, such as about 100, 150, 200, 250, 300, 350 or 400 mM.

Antioxidants that may also be included in the stabilizers of the present invention are selected, inter alia, from homocysteine, cysteine, cystathionine, methionine, glutathione and peptides comprising any of the amino acids homocysteine, cysteine, cystathionine, methionine and glutathione. In case an antioxidant is included in the composition, the total concentration of the stabilizer is about 50-500 nM, preferably about 100-400 mM, such as about 100, 150, 200, 250, 300, 350 or 400 mM, wherein the concentration of the antioxidant is about 1-50 mM, preferably about 5-40 mM, such as about 5, 10, 20, 30 or 40 mM.

In one embodiment of the invention, the liquid composition disclosed herein comprises sorbitol as a stabilizer. Sorbitol may be present in the liquid composition disclosed herein in an amount of about 50-400 mM, such as about 50, 100, 150, 200, 250, 300, 350 or 400 mM.

In one embodiment of the invention, the liquid composition disclosed herein comprises sucrose as a stabilizer. Sucrose may be present in the liquid composition disclosed herein in an amount of 50-300 mM, such as about 50, 100, 150, 200, 250 or 300 mM.

In one embodiment of the invention, the liquid composition disclosed herein comprises trehalose as a stabilizer. Trehalose may be present in the liquid composition disclosed herein in an amount of about 50-300 mM, such as about 50, 100, 150, 200, 250 or 300 mM.

In one embodiment of the invention, the liquid composition disclosed herein comprises a combination of sucrose and methionine as a stabilizer. In this combination, the total concentration of the stabilizer is about 50-500 nM, preferably about 100-400 mM, such as about 100, 150, 200, 250, 300, 350 or 400 mM, wherein the concentration of methionine is about 1-50 mM, preferably about 5-40 mM, such as about 5, 10, 20, 30 or 40 mM.

(iv) Surfactant

The term "surfactant" as used in this document refers to an organic substance with an amphiphilic structure, that is, its structure contains groups that are opposite in their preferred dissolution environment, typically a fat-soluble hydrocarbon chain and a water-soluble ionic group.

In one embodiment of the invention, the surfactant in the liquid composition disclosed herein is a non-ionic surfactant, such as an alkyl poly(ethylene oxide). Particular non-ionic surfactants that can be included in the composition include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60 or polysorbate-40, Pluronic, and the like. In one preferred embodiment, the liquid composition disclosed herein comprises polysorbate-80 as a surfactant.

The amount of surfactant in the antibody composition disclosed herein may vary depending on the particular characteristics desired of the composition, the particular environmental conditions, and the particular purpose for which the composition is used. In some preferred embodiments, the composition may comprise a polysorbate surfactant (e.g., polysorbate 80) at a concentration of about 0.1-1 mg/mL, preferably about 0.2-0.8 mg/mL, such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg/mL.

(v) Other excipients

The liquid antibody composition disclosed herein may or may not contain other excipients. For example, the liquid antibody composition disclosed herein further comprises a tonicity regulator. The tonicity regulator may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.

These and other known pharmaceutical excipients and/or additives suitable for use in the composition disclosed herein are well 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)".

II. Preparation of the composition

The present invention provides a stable composition comprising an anti-PD-1/HER2 bispecific antibody protein. The anti-PD-1/HER2 bispecific antibody protein used in the composition disclosed herein can be produced using antibody production techniques known in the art. For example, the anti-PD-1/HER2 bispecific antibody protein can be produced by recombinant methods. In one preferred embodiment, the anti-PD-1/HER2 bispecific antibody protein disclosed herein is produced by recombinant expression in HEK293, HEK293T, HEK293F or HEK293E cells modified from HEK293 cells and in CHO, CHO-S, CHO- ^dhfr- , CHO/DG44 or ExpiCHO cells modified from CHO cells. For example, the anti-PD-1/HER2 bispecific antibody protein is produced by a recombinant method as described in PCT Application No. PCT/CN2018/075851.

Nowadays, antibodies are very often used as active ingredients in pharmaceuticals. Methods for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599–613) describe a three-column purification method for antibodies that uses ion exchange chromatography (anionic IEC and/or cationic IEC) after capture on a protein A column. 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 that uses chromatography on a weakly partitioning anion exchange resin after protein A affinity chromatography.

In general, antibodies produced by recombinant methods can be purified using conventional purification methods to obtain a drug substance with sufficient reproducibility and acceptable purity suitable for the preparation of antibody compositions. For example, after secretion of the antibody from recombinant expression cells into a culture medium, 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 methods such as chromatography, dialysis, and affinity purification. Protein A can be used as an affinity ligand for the purification of IgG1, IgG2, and IgG4 antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. Once an antibody of sufficient purity is obtained, a composition containing the antibody can be prepared according to methods known in the art.

For example, the preparation may be accomplished using the following steps: (1) post-fermentation removal of impurities, particularly cells, from the fermentation broth by centrifugation and clarification to obtain a supernatant; (2) antibody capture using affinity chromatography (e.g., a protein A column having specific affinity for IgG1, IgG2, and IgG4 antibodies); (3) virus inactivation; (4) purification (which may typically include cation exchange chromatography, CEC) to remove impurities in the protein; (5) virus filtration (to reduce the viral titer, e.g., by more than 4 log10); and (6) ultrafiltration/diafiltration (which may be used to ensure that the protein is transferred to a formulation buffer that maintains its stability and to concentrate the solution to a concentration suitable for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol.10, No. 6, 2012, pp.48-57.

III. Method of composition analysis

During storage of antibody compositions, antibodies may undergo aggregation, degradation or chemical modification, which leads to antibody heterogeneity (including size heterogeneity and charge heterogeneity), formation of aggregates or fragments, etc., which may affect the quality of antibody compositions. Therefore, it is necessary to monitor the stability of antibody compositions.

For studying the stability of antibody compositions, there are various methods known in the art. For example, the purity of the antibody composition can be analyzed and the content of antibody aggregates can be estimated by methods such as CE-SDS under reducing conditions, CE-SDS under non-reducing conditions and size-exclusion HPLC; the content of charged isoforms in the antibody composition can be analyzed by capillary electrophoresis with isoelectric focusing (cIEF), capillary isoelectric focusing under imaging (iCIEF), ion exchange chromatography (IEC) and the like. In addition, the stability of the composition can be quickly estimated by visually inspecting the appearance of the composition. The change in turbidity of the composition can also be determined by measuring OD _{350 nm} , which provides information on the amount of soluble and insoluble aggregates. The change in protein content in the composition can also be determined using the ultraviolet spectrophotometry method (UV spectrophotometry method).

Non-reducing SDS-CE is a method for determining the purity of antibodies using a capillary as a separation channel. In non-reducing SDS-CE, protein migration is determined by the surface charge, which depends on the amount of bound SDS and is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass/charge ratios, the molecular sieve matrix gel within the capillary allows electrophoretic separation based on the size or hydrodynamic radius of the molecules. This method is widely used to monitor the purity of denatured intact antibodies. Typically, in non-reducing SDS-CE, the sample to be tested is mixed with sample buffer containing SDS and iodoacetamide. The mixture may then be incubated at 68-72°C for approximately 10-15 min and then cooled to room temperature, followed by centrifugation of the supernatant for analysis. Migration of proteins is detected using an ultraviolet detector to obtain an electrophoresis. The purity of the antibody composition can be quantitatively expressed as a percentage of the area of the main IgG peak out of the total area of all peaks. A more detailed description of the CE-SDS method is given, for example, in the article by Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

Size-exclusion high-performance liquid chromatography (SEC-HPLC) is another important method for standardization and quality control of antibodies. In this method, molecules are separated primarily on the basis of differences in their sizes or hydrodynamic radii. With SEC-HPLC, antibodies can be separated into three main fractions: the high-molecular-weight fraction (HMWF), the main peak (mainly monomeric antibody), and the low-molecular-weight fraction (LMWF). The purity of the antibody can be quantitatively expressed as the percentage of the area of the main peak out of the total area of all peaks in the chromatogram. SEC-HPLC measures the percentage of monomeric antibody in a composition, from which the content of soluble aggregates and degradation products can be inferred. A more detailed description of SEC-HPLC is given, for example, in the articles J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal, 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986). In addition to these articles, also see, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.02.032 and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies I, -Non-denaturing chromatographic techniques, Journal of Chromatography, http://dx.doi.org/10.1016/j.jchromb.2017.05.010.

The imaging-guided capillary isoelectric focusing (iCIEF) method can be used to analyze the charge heterogeneity of antibodies. This method can quantify the distribution of charged isoforms. In iCIEF, molecules are separated based on their charge differences in a pH gradient (apparent pI). In iCIEF, the separation column is typically a short capillary (e.g., a 5 cm long, 100 μm ID quartz capillary), the proteins are focused in the capillary column under high voltage, and their focusing is monitored online in real time using an imaging system with whole-column detection at 280 nm. The advantage of this method is that the whole-column detection system allows for the simultaneous detection of the different charged isoforms present in the antibody sample. Typically, in iCIEF analysis, the sample is mixed with urea and iCIEF buffer containing methylcellulose, pI and molecular weight standards, and ampholytes. Then, after focusing the sample for a specified period of time in an iCIEF analyzer, such as the iCE280 analyzer (Protein Simple, Santa Clara, CA, USA), the absorbance at 280 nm is measured using an iCIEF column, such as the ProtionSimple iCIEF preassembled column, to obtain a spectrum of the focused charged antibody isoforms. In the iCEIF spectrum, protein-related peaks that elute before the major peak (i.e., the major component) are defined as acidic components, and protein-related peaks that elute after the major peak are defined as basic components. The relative amounts of the major component, the acidic component, and the basic component can be expressed as a percentage of the total peak area. A more detailed description of the iCIEF method is given, for example, in the articles by 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.

Charged antibody isoforms in an antibody composition can also be determined by cation-exchange high-performance liquid chromatography (CEX-HPLC). In this method, peaks that elute from the CEX-HPLC column before the retention time of the main peak are referred to as "acid peaks" and peaks that elute from the CEX-HPLC column after the retention time of the main peak are referred to as "main peaks".

Accelerated stability studies can be used to test the stability of drugs and facilitate the screening of stable pharmaceutical compositions. For example, accelerated stability studies can be performed by storing samples of the composition at elevated temperatures, such as approximately 40 ± 2°C or 25 ± 2°C. Parameters tested can include appearance, visible particulate matter, protein content, turbidity, purity (size exclusion HPLC and CE-SDS under non-reducing conditions), and charged isoform content (iCIEF and KOC-HPLC).

In addition, the effectiveness or biological activity of the antibody can be analyzed. For example, the ability of the antibody in the composition to bind to its antigen molecules (the HER2 molecule and the PD-1 molecule) can be tested. Those skilled in the art are familiar with various methods for quantifying the specific binding of an antibody to an antigen, such as enzyme immunoassays, such as ELIS A.

The anti-PD-1/HER2 bispecific antibody protein composition disclosed herein is stable. In one embodiment, the purity of the anti-PD-1/HER2 bispecific antibody protein in the antibody composition disclosed herein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or higher 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, such as after storage at 5±3°C for 3 months, as measured by size exclusion chromatography or CE-SDS under non-reducing conditions. In one embodiment of the invention, at least 60%, preferably at least 65%, of the anti-PD-1/HER2 bispecific antibody protein in the antibody composition disclosed herein is in the non-basic and non-acidic isoforms (i.e., the major peak or major charged isoform) 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, such as after storage at 5±3°C for 3 months, as measured by the iCIEF method.

IV. Use of composition

An antibody composition comprising the anti-PD-1/HER2 bispecific antibody protein disclosed herein can be used to treat, prevent or slow the progression of various diseases or disorders associated with the HER2-dependent signaling pathway and/or the PD-1-dependent signaling pathway. "Diseases or disorders associated with the HER2-dependent signaling pathway" and/or "diseases or disorders associated with the PD-1-dependent signaling pathway" as used herein refer to diseases or disorders that can be treated (e.g., ameliorated) or prevented with the anti-PD-1/HER2 bispecific antibody protein composition disclosed herein. Any disease or disorder that can benefit from treatment with the antibody composition disclosed herein is suitable for the present invention.

The composition comprising the anti-PD-1/HER2 bispecific antibody protein disclosed herein can be used to prevent or treat various blood diseases or solid tumors, including, but not limited to, leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma and melanoma.

The present invention also provides the use of the composition disclosed herein for the preparation of a medicament for delivering the anti-PD-1/HER2 bispecific antibody protein to a mammal or for treating, preventing or ameliorating one or more of the diseases and disorders described above. Preferably, the mammal is a human.

The antibody composition disclosed herein can be administered to a subject or patient in a variety of ways. For example, administration can be by infusion or by syringe. Accordingly, in one aspect, the present invention provides a delivery device (e.g., a syringe) comprising the antibody composition disclosed herein (e.g., a pre-filled syringe). The patient will receive an effective amount of the anti-PD-1/HER2 bispecific antibody protein as the primary active ingredient, i.e., an amount sufficient to treat, prevent, or improve the disease or disorder of interest.

The therapeutic effect may include relief of physiological symptoms. The optimal effective amount and concentration of the antibody for any particular subject will depend on various factors, including the age, weight, health and/or sex of the patient, the nature and extent of the disease, the activity of the particular antibody, its clearance from the body, as well as any other possible drugs used in combination with the antibody composition. In a particular case, the effective amount to be delivered can be determined by the physician based on his/her judgment. Depending on the disease to be treated, the effective dose can vary from about 0.005 mg/kg body weight to about 50 mg/kg body weight, or from 0.1 mg/kg body weight to about 20 mg/kg body weight. In this regard, experience with known antibody drugs can provide some guidance. The dosage can be administered single or multiple times.

In order to facilitate the understanding of the present invention, the following examples are provided below. These examples are not intended to limit the scope of legal protection of the present invention and should not be interpreted as such in any way.

Abbreviations

CE-SDS: capillary electrophoresis with sodium dodecyl sulfate ELIS A: enzyme-linked immunosorbent assay

iCIEF: image-guided capillary isoelectric focusing Size-exclusion HPLC: size-exclusion high-performance liquid chromatography Examples

In order to develop a formulation suitable for long-term stable storage of the injectable solution of the recombinant bispecific antibody against programmed death protein 1 (PD-1) receptor and human epidermal factor receptor 2 (HER2), and to ensure controlled quality of the drug throughout its shelf life (at least 24 months), a screening study of formulations was designed to study the effect of various excipients on the stability of the anti-PD-1/HER2 bispecific antibody formulation. The materials and methods used in these studies are listed below.

Materials and methods

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

Note: NP means "not applicable".

1.2. Instruments and equipment

1.3. Parameters under study and methods for studying the stability of the composition

The following parameters of the antibody formulation were determined: (1) appearance and presence of visible particles; (2) protein content of the formulation, which was determined by ultraviolet spectrophotometry (UV spectrophotometry); (3) turbidity, which was determined by measuring absorbance at 350 nm; (4) purity of the antibody formulation, which was determined by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography (SEC-HPLC)) and expressed as a percentage of the monomer peak area relative to the sum of all peak areas; (5) the purity of the antibody composition, which was determined by reducing sodium dodecyl sulfate capillary electrophoresis (SDS-CE) and/or non-reducing sodium dodecyl sulfate capillary electrophoresis (NRE-SDS) and was expressed as a percentage of the monomer peak area relative to the sum of all peak areas; (6) the content of charged isoforms in the antibody composition, which was determined by imaging-guided capillary isoelectric focusing (iCIEF) and was expressed as a percentage of the major component, the acidic component, and the basic component; (7) the relative binding activity of the anti-PD-1/HER2 bispecific antibody in the antibody composition to PD-1 and HER2 antigens, which was determined by an enzyme-linked immunosorbent assay, such as a direct ELISA.

Detection of visible particles

Visible particles in the sample were detected with a transparency detector (model YB-2, Tianda Tianfa, Tianjin) using the method described in The Pharmacopoeia of the People's Republic of China National Pharmacopoeia Committee (2015 edition, Volume IV

General Rules 0904 "Visible Particle Test"), Beijing, China Medical Science Press, 2015.

Determination of protein content

The protein content of the sample was determined using a spectrophotometer in the ultraviolet region of the spectrum (model UV-1800, Shimadzu, Japan).

Determination of turbidity

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

Purity (size exclusion HPLC)

The separation was performed using a phosphate buffered SEC column (3.12 g sodium dihydrogen phosphate dihydrate, 8.77 g sodium chloride, and 34.84 g arginine dissolved in ultrapure water, adjusting the pH to 6.8 by adding hydrochloric acid, and making the volume 1000 mL) as the mobile phase. A 0.05% (w/v) NaN ₃ solution was used as a guard solution for the chromatographic column, the injected solution volume was 50 μL, the flow rate was 0.5 mL/min, the collection time was 30 min, the column temperature was 25°C, and the detection wavelength was 280 nm. For use as a test solution, the sample was diluted to a concentration of 2 mg/mL with ultrapure water. To prepare a blank solution, the composition buffer was diluted as described above. The blank solution and the test solution were separately injected into the liquid chromatograph in an amount of 50 µl per determination.

Purity (KE-DSN under reducing conditions)

The study was carried out by capillary gel electrophoresis. An uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm and an effective length of 20.2 cm was used. Before electrophoresis, the capillary column was washed with 0.1 mol/L sodium hydroxide solution, 0.1 mol/L hydrochloric acid and ultrapure water, and the electrophoresis gel was washed at a pressure of 70 psi. The sample was diluted to a concentration of 2.0 mg/mL with an appropriate amount of ultrapure water. 50 μl of the diluted sample were transferred to a 1.5 ml centrifuge tube and 45 μl of sample buffer pH 6.5, 1 μl of internal standard (10 kDa protein, 5 mg/ml; Beckman Coulter, cat. no. 390953) and 5 μl of β-mercaptoethanol were added. The sample buffer was prepared as follows: 0.32 g of citric acid monohydrate and 2.45 g of disodium phosphate dodecahydrate were dissolved in 45 ml of ultrapure water and the volume was adjusted to 50 ml to obtain citrate-phosphate buffer; 80 μl of 10% (w/v) sodium dodecyl sulfate solution were added to 200 μl of the resulting buffer, the volume was adjusted to 1 ml with water and the mixture was mixed well. The mixture was mixed well, heated to 70±2°C for 10±2 min, cooled to room temperature and transferred to a sample vial for further use as a test solution. The same volume of formulation buffer as the sample was treated in the same manner as described above to obtain a blank solution. Sample injection conditions were -5 kV for 20 s; separation voltage was -15 kV for 35 min. The capillary column temperature was maintained at 25°C and the detection wavelength was 220 nm.

Purity (KE-DSN under non-reducing conditions)

The study was carried out by capillary gel electrophoresis. An uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm and an effective length of 20.2 cm was used. Before electrophoresis, the capillary column was washed with 0.1 mol/L sodium hydroxide solution, 0.1 mol/L hydrochloric acid and ultrapure water, and the electrophoresis gel was washed at a pressure of 70 psi. The sample was diluted to a concentration of 2.0 mg/mL with an appropriate amount of ultrapure water. Fifty microliters of the diluted sample were transferred to a 1.5-mL centrifuge tube and 45 microliters of sample buffer pH 6.5, 1 microliter of internal standard (10 kDa protein, 5 mg/mL; Beckman Coulter, Cat. No. 390953), and 5 microliters of 250 mmol/L NEM solution (62 mg N-ethylmaleimide dissolved in 2 mL ultrapure water) were added. The sample buffer was prepared as follows: 0.32 g citric acid monohydrate and 2.45 g disodium phosphate dodecahydrate were dissolved in 45 mL ultrapure water and the volume was diluted to 50 mL to obtain a citrate phosphate buffer; To 200 μl of the resulting buffer, 80 μl of 10% (w/v) sodium dodecyl sulfate solution were added, the volume was brought to 1 ml with water and the mixture was mixed well. The mixture was mixed well, heated to a temperature of 70 ± 2 °C for 10 ± 2 min, cooled to room temperature and transferred to a sample vial for further use as a test solution. The composition buffer of the same volume as the sample was treated in the same manner as described above to obtain a blank solution. Sample injection conditions: -5 kV for 20 s; separation voltage: -15 kV for 35 min. The capillary column temperature was maintained at 25 °C and the detection wavelength was 220 nm.

Charged isoforms (iCIEF)

The determination was performed by the imaging-guided capillary isoelectric focusing (iCIEF) method. A capillary with an internal diameter of 100 μm and a total length of 5 cm was used. Before electrophoresis, the capillary column was washed with 0.5% methylcellulose solution (hereinafter abbreviated as "MC solution") and ultrapure water. The sample was injected under vacuum, pre-focusing was performed at 1.5 kV for 1 min, focusing was performed at 3 kV for 8 min, the sample injection time was 55 s, the sample tray temperature was 10 °C, and the detection wavelength was 280 nm. A 500 mmol/L arginine solution was used as a cathode stabilizer, and a 0.5% MC solution was added to reduce the adhesion between the protein and the capillary. The sample was diluted to a concentration of 1.0 mg/mL with water, 20 μL of the diluted sample was added to 78 μL of the premixed solution, and the mixture was mixed well to obtain a test solution. The premixed solution contained the following components: 70 μL of 0.5% MC solution (pI), 4 μL of ampholyte (pH 3-10), 2 μL of cathode stabilizer, 1 μL of pI 5.85 marker, and 1 μL of pI 9.99 marker. The test solution was injected into the chromatograph for analysis, and the contents of the major component, acidic component, and basic component were calculated using the area normalization method.

Relative binding activity (direct ELISA)

The antigen was diluted to a concentration of 0.5 mg/mL with CBS, and the wells of a 96-well microplate were coated with the antigen by adding the resulting solution to them in an amount of 100 μL/well and incubated at 4°C overnight (to determine the relative binding activity of the anti-PD-1 component of the anti-PD-1/HER2 bispecific antibody to PD-1, recombinant human PD-1 from Sinobiological (catalog No.: 10377-H08H) was used; to determine the relative binding activity of the anti-HER2 component of the anti-PD-1/HER2 bispecific antibody to HER2, human HER2/ErbB2 protein (with His-tag) from Sinobiological (catalog No.: 10004-H08H-100) was used). After washing, the wells of the microplate were blocked with blocking solution (2% BSA-PBST, 300 μg/well) at 37°C for 2 h. Anti-PD-1/HER2 bispecific antibody was diluted to a concentration of 3 μg/ml with 2% BSA-PBST and serial 3-fold dilutions were made to obtain 11 concentrations (0.05-3000 ng/ml). After removing the blocking solution, the solutions obtained from the sample dilution series were added to the wells of the microplate at a volume of 100 μl/well. Only 100 μl of the dilution solution (2% BSA-PBST) were added to the wells with the negative control. The plate was incubated at 37°C for 60 min in a thermostatically controlled incubator. After washing the plate, goat anti-human IgG Fc-horseradish peroxidase (HRP)-conjugated antibody (BETHYL, USA, catalog No.: A80-104P) diluted in 2% BSA-PBST was added to the wells as a secondary antibody (100,000-fold dilution, 100 μl/well), and the reaction was carried out at 37°C for 30 min. After washing the plate, 100 μl of TMB chromogenic solution were added to the wells, and after 10 min, the chromogenic reaction was stopped by adding 100 μl of 1 mol/ _{l H2SO4} solution to each well. The optical density (OD) value was measured at 450 nm using 620 nm as a reference wavelength. Taking the sample concentration values at all concentration gradients as the values on the abscissa axis, and the OD _{450 nm} - OD _{620 nm} values of the sample at all concentration gradients as the values on the ordinate axis, the EC ₅₀ values, reflecting the binding activity of the antibody to each antigen, were calculated using a four-parameter approximation using the Prism program.

Example 1. Production and purification of anti-PD-1/HER2 bispecific antibody

The anti-PD-1/HER2 bispecific antibody was prepared and purified as described in PCT Application No. PCT/CN2018/075851.

Specifically, X0GC expression vectors containing a heavy chain and a light chain of an anti-human PD-1 antibody, respectively, were constructed (the construction of X0GC expression vectors is described in Chinese Patent Application No. 200780038403.3), wherein the nucleotide sequence of the light chain variable region has SEQ ID NO: 9, and the amino acid sequence thereof has SEQ ID NO: 10; the nucleotide sequence of the light chain constant region has SEQ ID NO: 3, and the amino acid sequence thereof has SEQ ID NO: 4; the nucleotide sequence of the heavy chain variable region has SEQ ID NO: 11, and the amino acid sequence thereof has SEQ ID NO: 12; the nucleotide sequence of the heavy chain constant region has SEQ ID NO: 13, and the amino acid sequence thereof has SEQ ID NO: 14.

Expression vectors X0GC were constructed containing a heavy chain and a light chain of an anti-human HER2 antibody, respectively, wherein the nucleotide sequence of the light chain variable region has SEQ ID NO: 1, and the amino acid sequence thereof has SEQ ID NO: 2; the nucleotide sequence of the light chain constant region has SEQ ID NO: 3, and the amino acid sequence thereof has SEQ ID NO: 4; the nucleotide sequence of the heavy chain variable region has SEQ ID NO: 5, and the amino acid sequence thereof has SEQ ID NO: 6; the nucleotide sequence of the heavy chain constant region has SEQ ID NO: 7, and the amino acid sequence thereof has SEQ ID NO: 8.

Expression vectors containing the heavy chain and light chain of human anti-PD-1 antibody, respectively, were transfected into 293F cells (FreeStyle™ 293-F cells, Cat. No. R79007, Invitrogen) and then expressed, purified, and recovered to yield an anti-PD-1 half antibody molecule containing one heavy chain and one light chain.

Similarly, expression vectors containing the heavy chain and light chain of the human anti-HER2 antibody, respectively, were transfected into 293F cells (FreeStyle™ 293-F cells, Cat. No. R79007, Invitrogen) and then expressed, purified, and recovered to produce an anti-HER2 half antibody molecule containing one heavy chain and one light chain.

The reconstituted anti-PD-1 half-antibody molecules and the reconstituted anti-HER2 half-antibody molecules were mixed at an equimolar ratio and subjected to recombination reaction at 4°C for 24 h to obtain a bispecific antibody solution containing heterodimers of the anti-PD-1 half-antibody molecule and the anti-HER2 half-antibody molecule. The solution was concentrated by ultrafiltration using an ultrafiltration concentration tube and then purified at 4°C using an AKTA explorer 100 type protein purification system (GE Healthcare) and a Source 15S ion chromatography column (16 mm i.d., 17 ml, GE Healthcare) to obtain the anti-PD-1/HER2 bispecific antibody with a purity of 99.96%.

Example 2. Study of the effect of pH on the stability of the composition (I)

In this example, the stability of anti-PD-1/HER2 bispecific antibody formulations was studied, the pH of which varied from 5.0 to 6.5. A total of 4 different pH values were used, namely: 5.0; 5.5; 6.0 and 6.5.

2.1. Experimental methods

A 10 mM histidine-5% (w/v) sorbitol buffer was prepared and its pH was adjusted to 5.0; 5.5; 6.0 and 6.5 with diluted hydrochloric acid. The purified anti-PD-1/HER2 bispecific antibody obtained in Example 1 was transferred to solutions with different pH values by buffer exchange using ultrafiltration. The bispecific antibody protein content in the samples after buffer exchange was adjusted to approximately 20 mg/mL, and then polysorbate 80 was added to a final polysorbate 80 concentration of 0.30 mg/mL. The solutions were filtered and distributed into vials, which were then stoppered and capped. The stability of the samples was studied at 40±2°C; the specific experimental scheme is given in Table 1.

Note: (1) x indicates the sampling performed at that time point. (2) After sampling at the indicated time points, the samples were first placed in an ultra-low temperature freezer for storage and frozen at -80°C until subsequent testing, and then thawed as needed for testing.

2.2. Criteria

Based on the known characteristics of the preparation, as well as the accuracy of the device and method, criteria were established for the absence of changes in the parameters of the test sample compared to the initial values, which made it possible to determine whether the sample had changed (see Table 2 for details).

2.3. Experimental results of the screening study (I) of the compositions

(1) Appearance and visible particles

After storage at 40±2°C for one month, samples with pH 5.0; 5.5; 6.0 and 6.5 still met the standard in terms of appearance and visible particles.

(2) Protein content

The results of protein content determination in samples with pH 5.0; 5.5; 6.0 and 6.5 after storage at 40±2°C for different periods of time are shown in Table 3. These results show that samples with different pH did not change significantly after storage at 40±2°C for 1 month.

(3) Turbidity

The results of turbidity determination in samples with pH 5.0; 5.5; 6.0 and 6.5 after storage at 40±2°C for different periods of time are shown in Table 4, and the dynamics of turbidity change is shown in FIG. 2. The results show that the turbidity of samples with different pH after storage at 40±2°C for 1 month increased, and the higher the pH value, the higher the degree of turbidity change.

(4) Cleanliness

After storage at 40±2°C for different periods of time, the protein purity in samples with pH 5.0; 5.5; 6.0 and 6.5 was determined by size-exclusion HPLC. The results are shown in Table 5, and the dynamics of the change are shown in FIG. 3. These results show that the purity of samples with different pH after testing at 40±2°C for 1 month did not change significantly.

After storage at 40±2°C for different periods of time, the protein purity of samples with pH 5.0; 5.5; 6.0 and 6.5 was determined by CE-SDS under non-reducing conditions. The results are shown in Table 6 and the dynamics of the change are shown in FIG. 4. From these results, it is evident that, compared with the purity of the sample on day 0, the purity values of samples with different pH after testing at 40±2°C for 1 month decreased by 2.6%; 2.5%; 2.5% and 3.2%, respectively.

After storage at 40±2°C for different periods of time, the protein purity of samples with pH 5.0; 5.5; 6.0 and 6.5 was determined by CE-SDS under reducing conditions. The results are shown in Table 7, and the dynamics of the change is shown in FIG. 5. From these results it is evident that, compared with the purity of the sample on day 0, the purity values of samples with different pH after testing at 40±2°C for 1 month decreased by 0.4%; 0.7%; 0.6% and 1.4%, respectively.

(5) Charged isoforms

After storage at 40±2°C for different periods of time, the content of charged isoforms in samples with pH 5.0; 5.5; 6.0 and 6.5 was determined by iCIEF. The results are shown in Table 8, and the dynamics of the change is shown in FIG. 6. These results show that after the study at 40±2°C for 1 month, the content of the main component, as well as the acidic and basic components in the samples with different pH did not change significantly. The higher the pH value, the faster the content of the main component in the sample decreased and the faster the content of the acidic component increased.

(6) Relative binding activity

After storage at 40±2°C for different periods of time, the relative binding activity in the samples with pH 5.0; 5.5; 6.0 and 6.5 was determined by direct ELISA. The results are shown in Table 9. The results show that: after the study at 40±2°C for 2 weeks, the relative binding activity to PD-1 antigen and to HER2 antigen for each sample was more than 70%; the relative binding activity of the anti-HER2 component in the samples with pH 6.0 and 6.5 decreased markedly and in both cases was less than 70%; after the study at 40±2°C for 1 month, the relative binding activity to PD-1 antigen for each sample was still more than 70% and only in the case of the samples with pH 6.0 and 6.5 the relative binding activity to HER2 antigen was below 70% but still above 50%.

The results obtained in the study of the effect of pH on the stability of the formulations show that: after storing anti-PD-1/HER2 bispecific antibodies at pH 5.0-6.5 at 40±2°C for 2 weeks, the samples continued to meet the standard in terms of appearance and visible particles, their protein content did not change significantly, and the relative binding activity to the HER2 antigen and PD-1 antigen also did not change significantly; in addition, after storing anti-PD-1/HER2 bispecific antibodies at pH 5.0-6.5 at 40±2°C for one month, the samples continued to meet the standard in terms of appearance and visible particles, their protein content did not change significantly, and the relative binding activity to the PD-1 antigen also did not change significantly, and the relative binding activity of the anti-PD-1/HER2 bispecific antibody to the HER2 antigen under storage conditions at pH 6.0 and 6.5 decreased, but was still above 50%. Thus, for the experiments described in the examples below, pH 5.5 was chosen from pH 5.0-6.5.

Example 3. Screening study of compositions

3.1 Screening study of stabilizers

The ability of various stabilizers (sorbitol as a polyol; sucrose and trehalose as saccharides; methionine as an antioxidant, etc.) to influence the stability of a composition containing an anti-PD-1/HER2 bispecific antibody was studied.

3.1.1. Stabilizer Screening Methods

Four formulations were prepared as listed in Table 10. The buffers included in the formulations were prepared according to Table 10, and the anti-PD-1/HER2 bispecific antibody was transferred into the solutions of the corresponding formulations by buffer exchange using ultrafiltration. The protein content of each formulation solution after buffer exchange was adjusted to approximately 50.0 mg/mL, and then polysorbate 80 was added to a final polysorbate 80 concentration of 0.20 mg/mL. The solutions were filtered and distributed into vials, which were then stoppered and capped. The stability of the samples was studied at temperatures of 40°C, 25°C, and 5°C; The specific study design is shown in Table 11. The parameters studied included appearance, visible particle content, protein content, purity (SEC-HPLC and CE-SDS), and content of charged isoforms (iCIEF).

3.1.2. Evaluation criteria

The specific evaluation criteria are given in Table 2 in Example 2.

3.1.3. Screening study of stabilizers (1) Appearance and visible particles

The stability parameters were determined for 1 month when stored at 40°C, 2 months when stored at 25±2°C and 3 months when stored at 5±3°C; the results showed that all formulation samples met the standard in terms of appearance and visible particles.

(2) Protein content

The stability parameters were determined for 1 month in case of storage at 40°C, for 2 months in case of storage at 25±2°C and for 3 months in case of storage at 5±3°C; the results of protein content determination in samples of all formulations are shown in Table 12. The results show that the protein content of the four formulations did not change under storage conditions at different temperatures of 40°C, 25±2°C and 5±3°C.

(3) Cleanliness

Purity (SEC-HPLC): the results are shown in Table 13. The results show that: in samples of all compositions after testing at a temperature of 40°C for 4 weeks, the purity did not change significantly; in samples of all compositions after testing at a temperature of 25±2°C for 2 months, the purity did not change significantly; in samples of all compositions after testing at a temperature of 5±3°C for 3 months, the purity did not change significantly.

Purity (CE-SDS under non-reducing conditions): the results are presented in Table 14. The results show that: in samples of all compositions after testing at a temperature of 40°C for 4 weeks, the purity did not change significantly; in samples of all compositions after testing at a temperature of 25±2°C for 2 months, the purity did not change significantly; in samples of all compositions after testing at a temperature of 5±3°C for 3 months, the purity did not change significantly.

Purity (CE-SDS under reducing conditions): the results are presented in Table 15. The results show that: in samples of all compositions after testing at a temperature of 40°C for 4 weeks, the purity did not change significantly; in samples of all compositions after testing at a temperature of 25±2°C for 2 months, the purity did not change significantly; in samples of all compositions after testing at a temperature of 5±3°C for 3 months, the purity did not change significantly.

(4) Charged isoforms (iCIEF)

Charged isoforms (iCIEF): the results are shown in Table 16. The dynamics of changes in the content of the charged isoform (the main component) in samples of different compositions stored at a temperature of 40°C and 25±2°C are shown in FIG. 7 and FIG. 8, respectively.

The results show that: after the study at a temperature of 40°C for 4 weeks, the content of the main component, as well as the content of the acidic and basic components of the charged isoforms in the samples of all compositions changed significantly: the content of the main component decreased, and the content of the acidic component increased; however, the dynamics of changes in the samples were almost the same, and no significant differences were observed between compositions 1-4. After the accelerated test with storage at a temperature of 25±2°C for 2 months, the content of the main component, as well as the content of the acidic and basic components of the charged isoforms in the samples of all compositions changed significantly: the content of the main component decreased, and the content of the acidic component increased; however, the dynamics of changes in the compositions were almost the same, and no significant differences were observed between compositions 1-4. After the study at a temperature of 5±3°C for 3 months, the content of the main component, as well as the content of the acidic and basic components of the charged isoforms in the samples of all compositions did not change significantly.

The results of the formulation evaluation experiment show that formulations 14 are relatively reproducible in terms of the dynamics of changes in stability parameters, and no significant differences are observed between them. In terms of formulation simplicity, formulations 1, 2, and 3, which contain only one stabilizer, did not differ significantly in their ability to protect the protein, and formulation 2 was selected, taking into account the subsequent development of a lyophilized dosage form. To ensure the safety of further production, concentrated hydrochloric acid, which is part of formulation 2 as a component of the buffer system and used to adjust the pH, was replaced by histidine and histidine hydrochloride. To ensure the stability of the composition of the updated formulation, the following experiment was conducted.

Example 4: Experiment to confirm the stability of the composition

4.1. Development of composition and experimental design

Composition 5 was developed and the detailed preparation of composition 5 is shown in Table 17.

The experimental design for confirming the composition is shown in Table 18.

4.2. Experimental results

The results of the forced test (storage at 40°C) are shown in Table 19. After storage at 40°C for 4 weeks, the sample met the standard for appearance, visible particles and biological activity, the protein content and purity (SEC-HPLC and CE-SDS) did not change significantly; and only the content of the major component (charged isoform) (iCIEF) changed (decreased by 16.0%), the content of the acidic component (increased by 14.0%) and the content of the basic component (increased by 2.0%).

The results show that in terms of the dynamics of change in stability parameters, the composition disclosed in this document, containing 42.0 mg/ml of a bispecific antibody comprising a recombinant fully human half-antibody against the programmed death protein 1 (PD-1) receptor and a humanized half-antibody against the human epidermal growth factor receptor 2 (HER2) of composition 5 (0.85 mg/ml histidine, 3.17 mg/ml histidine hydrochloride, 80.00 mg/ml sucrose, 0.2 mg/ml polysorbate-80, pH 5.5), generally coincides with the composition of composition 2 from example 3.

Thus, it was determined that the most preferred composition of the composition is the following composition: approximately 42.0 mg/ml of a bispecific antibody comprising a recombinant fully human half-antibody against the receptor of programmed death protein 1 (PD-1) and a humanized half-antibody against the receptor of human epidermal growth factor 2 (HER2), 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, pH 5.5.

Illustrative embodiments of the present invention have been described above. Those skilled in the art will understand that these embodiments are provided for illustrative purposes only and that various other substitutions, adaptations and modifications thereof may be made without departing from the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments of the invention listed herein.

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SEQUENCE LIST

<110> Innovent Biologics (Suzhou) Co., Ltd.; Beijing Hanmi Pharm. Co.,Ltd.

<120> COMPOSITION CONTAINING ANTI-PD-1/HER2 BISPECIFIC ANTIBODY,

METHOD OF OBTAINING IT AND ITS USE

<130>

<160> 14

<170> PatentIn, version 3.3

<210> 1

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain variable region of human anti-HER2 antibody

<400> 1

gacattcaga tgactcagag cccttcttca ctgtcagctt ccgtgggcga cagagtcact 60

atcacctgcc gcgcaagtca ggatgtgaac accgcagtcg cctggtacca gcagaagcct 120

ggcaaagctc caaagctgct gatctacagc gcatctttcc tgtattctgg agtgcccagt 180

aggtttagtg ggtcacggtc cggtaccgac ttcacactga ctatctccag cctgcagcct 240

gaggattttg ccacatacta ttgccagcag cactatacca caccccctac tttcggccag 300

ggaaccaaag tggagatcaa g 321

<210> 2

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain variable region of human anti-HER2 antibody

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 3

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain constant region of human anti-HER2 antibody

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cgaactgtgg ccgctccaag cgtcttcatt tttccaccct ctgacgaaca gctgaagtca 60

gggacagctt ccgtggtctg tctgctgaac aatttttacc ccagggaggc caaagtgcag 120

tggaaggtcg ataacgctct gcagagcgga aattctcagg agagtgtgac agaacaggac 180

tcaaaagatt ccacttatag cctgtctagt accctgacac tgtccaaggc agactacgaa 240

aagcataaag tgtatgcctg tgaggtcaca catcagggtc tgtcaagccc cgtcactaag 300

tccttcaatc gtggcgaatg c 321

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<223> Light chain constant region of human anti-HER2 antibody

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

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

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

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

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

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

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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<212> DNA

<213> Artificial sequence

<220>

<223> Human anti-HER2 antibody heavy chain variable region

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gaggtgcagc tggtcgaaag tggggggtggg ctggtgcagc caggcggatc actgaggctg 60

tcctgcgccg ctagcggctt caacatcaaa gacacctata ttcactgggt ccgacaggca 120

ccagggaagg gtctggaatg ggtggctcgt atctacccta caaatggtta cactagatat 180

gccgactccg tgaaaggccg gtttactatt tctgctgata ccagtaagaa cacagcatac 240

ctgcagatga atagcctgag ggctgaggat accgcagtgt actattgctc tcggtggggg 300

ggtgacggct tctacgctat ggattattgg ggccagggaa ctctggtcac cgtgtccagc 360

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<212> PRT

<213> Artificial sequence

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<223> Human anti-HER2 antibody heavy chain variable region

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

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<223> Heavy chain constant region of human anti-HER2 antibody

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gcttcaacaa aaggaccttc cgtgtttcca ctggcaccct ctagtaagag tacttcagga 60

ggaaccgcag cactgggatg tctggtgaag gactacttcc cagagcccgt caccgtgtct 120

tggaacagtg gagcactgac ctccggggtc catacatttc ctgccgtgct gcagtcatcc 180

ggtctgtata gcctgagctc tgtggtcaca gtcccaagtt catccctggg cacccagaca 240

tacatctgca acgtgaatca caaaccttcc aatactaagg tcgacaagaa agtggaacca 300

aaaagctgtg ataagactca tacctgccca ccttgtcctg caccagagct gctgggaggt 360

ccaagcgtgt tcctgtttcc acccaagccc aaagacacac tgatgatttc tcgcacaccc 420

gaagtcactt gtgtggtcgt ggacgtgtcc cacgaggatc ctgaagtcaa gttcaactgg 480

tacgtggatg gcgtcgaggt gcataatgct aagaccaaac ccagagagga acagtacaac 540

agcacctatc gcgtcgtgtc tgtcctgaca gtgctgcacc aggattggct gaacggaaag 600

gagtacaagt gcaaagtgag caacaaggcc ctgcccgctc ctatcgagaa gaccatttct 660

aaggctaaag gccagcctag agaaccacag gtgtatacag agcctccaag tcgcgacgag 720

ctgacaaaaa accaggtctc cctgacttgt ctggtgaagg gattctaccc tagcgatatc 780

gcagtggagt gggaatctaa tgggcagcca gaaaacaatt ataagaccac accccctgtg 840

ctggactcag atggttcctt ctttctgctg agtgtgctga ccgtggacaa gtccaggtgg 900

cagcagggga acgtcttttc ctgcagcgtg atgcatgagg ccctgcacaa tcattacaca 960

cagaaatctc tgagtctgtc accaggaaag 990

<210> 8

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain constant region of human anti-HER2 antibody

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

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

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Glu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Leu Ser Val Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 9

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain variable region of anti-human PD-1 antibody

<400> 9

gacatccaga tgacccagtc ccctagcagc gtgagcgctt ccgtgggcga cagggtgacc 60

atcacctgca gggcctccca gggcatctcc tcctggctgg cctggtatca acagaagccc 120

ggcaaggccc ccaagctgct gatctccgct gcctcctccc tgcagtccgg agtgccttcc 180

aggttcagcg gttctggctc cggcaccgac ttcaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag gccaaccacc tgcctttcac cttcggcggc 300

ggcaccaagg tggagatcaa g 321

<210> 10

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain variable region of anti-human PD-1 antibody

<400> 10

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

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

35 40 45

Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn His Leu Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain variable region of anti-human PD-1 antibody

<400> 11

caggtgcagc tggtgcagtc cggagccgag gtgaagaagc ccggctcctc cgtgaaggtg 60

tcctgcaagg cctccggcgg caccttctcc tccaccgcca tctcctgggt gaggcaggct 120

cctggccagg gactggagtg gatgggaggc atctggccct ccttcggcac agcctcctac 180

gcccagaagt tccagggcag ggtgaccatc accgccgacg agagcacctc caccgcctac 240

atggagctga gctccctgag gtccgaggac accgccgtgt actactgtgc cagggccgag 300

tactcctcca ccggcatctt cgactactgg ggccagggca ccctggtgac agtgtcctcc 360

<210> 12

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain variable region of anti-human PD-1 antibody

<400> 12

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ala Glu Tyr Ser Ser Thr Gly Ile Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain constant region of anti-human PD-1 antibody

<400> 13

gcttcaacaa aaggaccttc cgtgtttcca ctggcaccct ctagtaagag tacttcagga 60

ggaaccgcag cactgggatg tctggtgaag gactacttcc cagagcccgt caccgtgtct 120

tggaacagtg gagcactgac ctccggggtc catacatttc ctgccgtgct gcagtcatcc 180

ggtctgtata gcctgagctc tgtggtcaca gtcccaagtt catccctggg cacccagaca 240

tacatctgca acgtgaatca caaaccttcc aatactaagg tcgacaagaa agtggaacca 300

aaaagctgtg ataagactca tacctgccca ccttgtcctg caccagagct gctgggaggt 360

ccaagcgtgt tcctgtttcc acccaagccc aaagacacac tgatgatttc tcgcacaccc 420

gaagtcactt gtgtggtcgt ggacgtgtcc cacgaggatc ctgaagtcaa gttcaactgg 480

tacgtggatg gcgtcgaggt gcataatgct aagaccaaac ccagagagga acagtacaac 540

agcacctatc gcgtcgtgtc tgtcctgaca gtgctgcacc aggattggct gaacggaaag 600

gagtacaagt gcaaagtgag caacaaggcc ctgcccgctc ctatcgagaa gaccatttct 660

aaggctaaag gccagcctag agaaccacag gtgtatacac tgcctccaag tcgcgacgag 720

ctgacaaaaa accaggtctc cctgctgtgt ctggtgaagg gattctaccc tagcgatatc 780

gcagtggagt gggaatctaa tgggcagcca gaaaacaatt ataagaccac accccctgtg 840

ctgcggtcag atggttcctt ctttctgtac agtaaactga ccgtggacaa gtccaggtgg 900

cagcagggga acgtcttttc ctgcagcgtg atgcatgagg ccctgcacaa tcattacaca 960

cagaaatctc tgagtctgtc accaggaaag 990

<210> 14

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain constant region of anti-human PD-1 antibody

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

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

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Arg Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

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Claims (38)

1. A liquid antibody composition for stabilizing the anti-PD-1/HER2 bispecific antibody protein during storage, comprising: (i) anti-PD-1/HER2 bispecific antibody protein; (ii) buffer; (iii) stabilizer and (iv) a surfactant; wherein the anti-PD-1/HER2 bispecific antibody protein comprises a first half-antibody and a second half-antibody, wherein the first half-antibody comprises a first VH/VL unit that specifically binds to PD-1 and the second half-antibody comprises a second VH/VL unit that specifically binds to HER2, wherein the first VH/VL unit comprises the variable heavy chain/variable light chain paired sequences set forth in SEQ ID NO: 12/SEQ ID NO: 10 and the second VH/VL unit comprises the variable heavy chain/variable light chain paired sequences set forth in SEQ ID NO: 6/SEQ ID NO: 2; and wherein the liquid antibody composition contains: (i) 42 mg/ml anti-PD-1/HER2 bispecific antibody protein; (ii) 0.85 mg/ml histidine and 3.17 mg/ml histidine hydrochloride; (iii) 80 mg/ml sucrose; And (iv) 0.2 mg/ml polysorbate 80; or The liquid antibody composition contains: (i) 50 mg/ml anti-PD-1/HER2 bispecific antibody protein; (ii) 20 mM histidine; (iii) 50 mg/ml sorbitol; And (iv) 0.2 mg/ml polysorbate 80; or The liquid antibody composition contains: (i) 50 mg/ml anti-PD-1/HER2 bispecific antibody protein; (ii) 20 mM histidine; (iii) 80 mg/ml sucrose; And (iv) 0.2 mg/ml polysorbate 80; or The liquid antibody composition contains: (i) 50 mg/ml anti-PD-1/HER2 bispecific antibody protein; (ii) 20 mM histidine; (iii) 80 mg/ml trehalose; And (iv) 0.2 mg/ml polysorbate 80; or The liquid antibody composition contains: (i) 50 mg/ml anti-PD-1/HER2 bispecific antibody protein; (ii) 20 mM histidine; (iii) 80 mg/ml sucrose and 1.49 mg/ml methionine; and (iv) 0.2 mg/ml polysorbate 80; the pH of the liquid composition is 5.0-6.0. 2. The liquid antibody composition of claim 1, wherein the anti-PD-1/HER2 bispecific antibody protein comprises a first half-antibody and a second half-antibody, wherein the first half-antibody comprises the heavy chain sequences shown in SEQ ID NO: 12 and SEQ ID NO: 14, or heavy chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction, and comprises the light chain sequences shown in SEQ ID NO: 10 and SEQ ID NO: 4, or light chain sequences sharing at least 90%, 95%, 98%, or 99% identity thereto in the N to C direction, and the second half-antibody comprises the heavy chain sequences shown in SEQ ID NO: 6 and SEQ ID NO: 8, or heavy chain sequences sharing at least 90%, 95%, 98%, or 99% identity in the N to C direction and includes the light chain sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, or light chain sequences having at least 90%, 95%, 98% or 99% identity thereto in the N to C direction. 3. A liquid antibody composition according to any one of claims 1, 2, wherein the anti-PD-1/HER2 bispecific antibody protein is recombinantly expressed in HEK293, HEK293T, HEK293F or HEK293E cells obtained by modification from HEK293 cells, and in CHO, CHO-S, CHO-dhfr ^- , CHO/DG44 or ExpiCHO cells obtained by modification from CHO cells. 4. A liquid antibody composition according to any one of claims 1 to 3, wherein the liquid composition is intended for injection, preferably for subcutaneous or intravenous injection, or for infusion, for example for intravenous infusion. 5. A liquid antibody composition according to any one of claims 1 to 4, wherein the pH of the liquid antibody composition is 5.5. 6. A solid antibody composition obtained by converting a liquid antibody composition according to any one of claims 1-5 into a solid form, wherein the solid composition is in the form of a lyophilized powder, and is intended to stabilize the anti-PD-1/HER2 bispecific antibody protein during storage. 7. A pre-filled syringe containing a liquid antibody composition according to any one of claims 1 to 5 or a solid antibody composition according to claim 6, wherein the solid antibody composition is reconstituted by dissolution in a physiologically acceptable solution immediately prior to use as an intravenous or intramuscular injection.