Anti-pd-1 antibody pharmaceutical compositions and uses thereof

Abstract

The present application provides a stable anti-PD-1 antibody pharmaceutical composition and uses thereof. The pharmaceutical composition contains a buffer and an anti-PD-1 antibody or an antigen-binding fragment thereof; wherein the concentration of the anti-PD-1 antibody or the antigen-binding fragment thereof is about 100-250 mg / mL, and comprises LCDR1, LCDR2 and LCDR3 with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; wherein the pH of the pharmaceutical composition is about 5.0-6.5. The present application also provides an injection containing the pharmaceutical composition and uses of the pharmaceutical composition and the injection in the preparation of a medicament for treating a disease or a disorder by eliminating, inhibiting or reducing the activity of PD-1.

Description

Technical Field

[0001] This invention relates to the field of therapeutic pharmaceutical compositions, and more particularly to anti-PD-1 antibody pharmaceutical compositions and their uses. Background Technology

[0002] Immune escape is a characteristic feature of cancer. Ahmadzadeh, M. et al., Blood, 114:1537-44, disclosed that tumor-specific T lymphocytes are commonly found in the tumor microenvironment, draining lymph nodes, and peripheral blood; however, due to the immunosuppressive network present in the tumor microenvironment, they are usually unable to control tumor progression. CD8+ tumor-infiltrating T lymphocytes (TILs) typically express activation-induced inhibitory receptors, including CTLA-4 and PD-1, while tumor cells frequently express immunosuppressive ligands, including PD-1 ligand 1 (PD-L1, also called B7-H1 or CD274), which inhibits T cell activation and effector function. In the inhibitory mechanism, PD-1 and its ligands have become an important pathway for tumor cells to suppress activated T cells in the tumor microenvironment.

[0003] Programmed death receptor 1 (PD-1) plays a crucial role in immune regulation and the maintenance of peripheral tolerance. PD-1 is primarily expressed in activated T cells and B cells, where its function is to inhibit lymphocyte activation—a normal peripheral tissue tolerance mechanism of the immune system to prevent immune overreaction. However, activated T cells infiltrating the tumor microenvironment highly express PD-1, and inflammatory factors secreted by activated leukocytes induce tumor cells to highly express PD-1 ligands PD-L1 and PD-L2. This leads to persistent activation of the PD-1 pathway in activated T cells within the tumor microenvironment, suppressing T cell function and preventing them from killing tumor cells. Therapeutic PD-1 antibodies can block this pathway, partially restoring T cell function and enabling activated T cells to continue killing tumor cells.

[0004] Over the past decade, PD-1 / PD-L1 pathway blockade has been proven to be an effective way to induce durable antitumor responses in various cancer indications. Monoclonal antibodies (mAbs) that block the PD / PD-L1 pathway can enhance the activation and effector function of tumor-specific T cells, reduce tumor burden, and improve survival rates.

[0005] Antibody drug formulations should be long-term stable, containing a safe and effective amount of the drug. Due to the special structure and properties of antibodies, antibody drugs require a stable environment during preparation, storage, and transportation. Different types of proteins and antibodies have different physicochemical properties and degradation reactions; therefore, the formulations of buffer solutions, excipients, etc., for antibody drug formulations are also different.

[0006] To improve patient compliance and ease of administration in cancer patients, subcutaneous (SC) injection is a preferred method. However, the high doses required to achieve the desired effect necessitate the preparation of high-concentration formulations. However, high-concentration antibody formulations often present numerous challenges. For example, their high viscosity makes aspiration and injection difficult, resulting in significant drug residues in containers and syringes, leading to large dosage deviations and injection site pain. Furthermore, the high viscosity can cause serious process problems during production, such as requiring extremely high pressures for concentration and filtration, or even preventing the drug from passing through filter membranes altogether. Additionally, the high concentration of antibody proteins in the formulation is prone to aggregation, causing instability, the formation of insoluble microparticles, increased immunogenicity, and increased side effects.

[0007] Therefore, there is still a need in this field to develop a high-concentration antibody formulation targeting human programmed death receptor 1 to meet the manufacturing and clinical application requirements for high antibody concentration, long-term stability, non-aggregation, and low viscosity. Summary of the Invention

[0008] The pharmaceutical composition described in this invention is a highly stable pharmaceutical composition containing an antibody that specifically binds to PD-1. In particular, by selecting appropriate buffer systems and pH, optimizing stabilizers and surfactants, and conducting pharmacokinetic and pharmacodynamic studies, this invention has developed a high-concentration antibody formulation that can be administered subcutaneously and exhibits long-term stability, no aggregation, and ultra-low viscosity.

[0009] The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; and (2) an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the aforementioned anti-PD-1 antibody or its antigen-binding fragment comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0011] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is selected from murine antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, humanized antibodies or their antigen-binding fragments, preferably humanized antibodies or their antigen-binding fragments.

[0012] In some embodiments, the aforementioned anti-PD-1 antibody or its antigen-binding fragment includes a light chain variable region as shown in SEQ ID NO:7 and a heavy chain variable region as shown in SEQ ID NO:8.

[0013] In some embodiments, the anti-PD-1 antibody described above comprises a light chain amino acid sequence as shown in SEQ ID NO:9 and a heavy chain amino acid sequence as shown in SEQ ID NO:10.

[0014] In some embodiments, the concentration of the anti-PD-1 antibody or its antigen-binding fragment in the above-mentioned pharmaceutical composition is about 100 to 250 mg / mL, preferably about 150 to 250 mg / mL, more preferably about 150 to 200 mg / mL; more preferably, the concentration of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment is about 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 175 mg / mL, 180 mg / mL, 185 mg / mL, 190 mg / mL, 195 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, preferably about 180 mg / mL, 185 mg / mL, 190 mg / mL or 195 mg / mL.

[0015] In some embodiments, the pH of the above-mentioned pharmaceutical composition is about 5.0 to 6.5, preferably about 5.5 to 6.2, more preferably 5.9 to 6.1, and even more preferably about 6.0.

[0016] In some formulations, the osmotic pressure of the above-mentioned pharmaceutical composition is in the range of 260 to 320 mOsm / kg, preferably in the range of 290 to 310 mOsm / kg.

[0017] In some formulations, the viscosity of the above-mentioned pharmaceutical composition is ≤8.0 cP when measured at about 25°C.

[0018] In some embodiments, the buffer solution is selected from one or more of acetate buffer, citrate buffer, and histidine buffer; preferably, the buffer solution is histidine buffer.

[0019] In some formulations, the histidine buffer is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, preferably histidine-histidine hydrochloride buffer.

[0020] In some formulations, the histidine buffer is a histidine-histidine hydrochloride buffer. In some formulations, the histidine-histidine hydrochloride buffer is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some formulations, the histidine buffer is prepared from 1–30 mM L-histidine and 1–30 mM L-histidine monohydrochloride. In some formulations, the histidine buffer is prepared from histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some formulations, the histidine buffer is prepared from histidine and histidine hydrochloride in a molar ratio of approximately 1:1. In some formulations, the histidine buffer is prepared from histidine and histidine hydrochloride in a molar ratio of approximately 1:3. In some formulations, the histidine preparation is a histidine buffer with a pH of approximately 5.5, prepared from approximately 4.5 mM L-histidine and approximately 15.5 mM L-histidine monohydrochloride. In some formulations, the histidine preparation is a histidine buffer with a pH of approximately 5.5, consisting of approximately 7.5 mM L-histidine and approximately 22.5 mM L-histidine monohydrochloride. In other formulations, the histidine preparation is a histidine buffer with a pH of approximately 6.0, consisting of approximately 10 mM histidine and approximately 10 mM histidine hydrochloride.

[0021] In some embodiments, the histidine buffer is a histidine-histidine acetate buffer, preferably with a molar ratio of 1:1 to 1.5:1, preferably with a pH of 6.0 ± 0.3, preferably about 6.0, and preferably containing 10–15 mM histidine and 10–15 mM histidine acetate.

[0022] In some embodiments, the above-mentioned buffer solution is an acetate buffer, preferably an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, more preferably an acetate-sodium acetate buffer. In some embodiments, the acetate buffer is prepared from 1-30 mM acetic acid and 1-30 mM sodium acetate. In some embodiments, the acetate buffer is prepared from acetic acid and sodium acetate in a molar ratio of about 1:2.1. In some embodiments, the acetate buffer is prepared from acetic acid and sodium acetate in a molar ratio of about 1:5.7. In some embodiments, the acetate buffer is an acetate buffer with a pH of about 5.0 prepared from about 6.5 mM acetic acid and about 13.5 mM sodium acetate. In some embodiments, the acetate buffer is an acetate buffer with a pH of about 5.5 prepared from about 3 mM acetic acid and about 17 mM sodium acetate.

[0023] In some embodiments, the above-mentioned buffer solution is a citrate buffer, preferably a citrate-sodium citrate buffer. In some embodiments, the citrate buffer is prepared from 1–30 mM citric acid and 1–30 mM sodium citrate. In some embodiments, the citrate buffer is prepared from citric acid and sodium citrate in a molar ratio of about 1:1 to 1:4. In some embodiments, the citrate buffer is a citrate buffer with a pH of about 6.0 prepared from about 5.0 mM citric acid and about 15.0 mM sodium citrate. In some embodiments, the citrate buffer is a citrate buffer with a pH of about 6.0 prepared from about 10 mM citric acid and about 10 mM sodium citrate.

[0024] In some embodiments, the concentration of the buffer solution is about 5 to 100 mM, preferably about 10 to 50 mM, more preferably about 10 to 30 mM; more preferably about 15 to 25 mM. Non-limiting examples of the buffer solution concentration are about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 45 mM, 50 mM, or any two values ​​within these ranges as endpoints, preferably about 15 mM, 20 mM, or 25 mM.

[0025] In some embodiments, the pH of the buffer solution is about 5.0 to 6.5, preferably about 5.5 to 6.5, more preferably about 5.5 to 6.2, and even more preferably about 5.9 to 6.1. Non-limiting examples of the pH of the buffer solution are about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.9, 6.0, or 6.1.

[0026] In some embodiments, the above-mentioned pharmaceutical composition further includes a stabilizer selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine, and trehalose; preferably, the arginine salt is arginine hydrochloride.

[0027] In some embodiments, the concentration of the stabilizer is about 10 to 400 mM, preferably about 100 to 250 mM, more preferably about 120 to 220 mM, and more preferably about 130 to 180 mM. Non-limiting examples of the stabilizer concentration are about 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 145 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, or any two values ​​within these ranges as endpoints, preferably about 140 mM, 150 mM, or 160 mM.

[0028] In some embodiments, the stabilizer is arginine or arginine salt at a concentration of about 120–220 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30–100 mM and sucrose at a concentration of about 100–180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30–100 mM and glycine at a concentration of about 50–150 mM; preferably, the stabilizer is arginine or arginine salt at a concentration of about 130–180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30–70 mM and sucrose at a concentration of about 110–170 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30–70 mM and glycine at a concentration of about 80–120 mM; preferably, the arginine salt is arginine hydrochloride.

[0029] In some formulations, the stabilizer mentioned above is arginine or arginine salt. In some embodiments, the stabilizer is arginine or arginine salt at a concentration of about 30-250 mM. The concentration of the arginine or arginine salt is preferably about 100-250 mM, more preferably about 120-220 mM, more preferably about 130-180 mM, and more preferably about 140-160 mM. Non-limiting examples of the concentration of the arginine or arginine salt are about 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, and preferably about 135 mM, 140 mM, 145 mM, 150 mM, or 155 mM. Preferably, the arginine salt is arginine hydrochloride.

[0030] In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100 to 300 mM, preferably about 150 to 300 mM, more preferably about 200 to 280 mM, and non-limiting examples of the sucrose concentration are about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably about 220 mM.

[0031] In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100-300 mM, preferably about 150-300 mM, more preferably about 200-280 mM, and non-limiting examples of trehalose concentrations are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably about 220 mM.

[0032] In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is sodium chloride with a concentration of about 30 to 200 mM, preferably about 50 to 190 mM, more preferably about 100 to 180 mM, more preferably about 120 to 170 mM, more preferably about 130 to 150 mM, and non-limiting examples of the sodium chloride concentration are about 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, preferably about 135 mM or 140 mM.

[0033] In some embodiments, the stabilizer is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100 to 300 mM, preferably about 150 to 300 mM, more preferably about 200 to 280 mM, and non-limiting examples of mannitol concentrations are about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably about 240 mM.

[0034] In some embodiments, the stabilizer is sorbitol. In some embodiments, the stabilizer is sorbitol at a concentration of about 100 to 300 mM, preferably about 150 to 300 mM, more preferably about 200 to 280 mM, and non-limiting examples of sorbitol concentrations are about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably about 240 mM.

[0035] In some embodiments, the stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the stabilizer is a combination of about 30–200 mM sodium chloride and about 30–200 mM mannitol, preferably a combination of about 30–100 mM sodium chloride and about 100–180 mM mannitol, more preferably a combination of about 30–70 mM sodium chloride and about 120–180 mM mannitol. Non-limiting examples of the stabilizer include a combination of about 50 mM sodium chloride and about 140 mM mannitol, or a combination of about 50 mM sodium chloride and about 150 mM mannitol.

[0036] In some embodiments, the stabilizer is a combination of arginine hydrochloride and sucrose. In some embodiments, the stabilizer is a combination of about 30–200 mM arginine hydrochloride and about 30–200 mM sucrose, preferably a combination of about 30–100 mM arginine hydrochloride and about 100–180 mM sucrose, more preferably a combination of about 30–70 mM arginine hydrochloride and about 110–170 mM sucrose. Non-limiting examples of the stabilizer include a combination of about 50 mM arginine hydrochloride and about 130 mM sucrose, a combination of about 50 mM arginine hydrochloride and about 140 mM sucrose, or a combination of about 50 mM arginine hydrochloride and about 150 mM sucrose.

[0037] In some embodiments, the stabilizer is a combination of arginine hydrochloride and glycine. In some embodiments, the stabilizer is a combination of about 30–200 mM arginine hydrochloride and about 30–200 mM glycine, preferably a combination of about 30–100 mM arginine hydrochloride and about 50–150 mM glycine, more preferably a combination of about 30–70 mM arginine hydrochloride and about 80–120 mM glycine. Non-limiting examples of the stabilizer include a combination of about 50 mM arginine hydrochloride and about 100 mM glycine, or a combination of about 50 mM arginine hydrochloride and about 110 mM glycine.

[0038] In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizer is a combination of about 30–200 mM sodium chloride and about 30–200 mM sucrose, preferably a combination of about 30–100 mM sodium chloride and about 100–180 mM sucrose, more preferably a combination of about 30–70 mM sodium chloride and about 100–150 mM sucrose. Non-limiting examples of the stabilizer include a combination of about 50 mM sodium chloride and about 120 mM sucrose, or a combination of about 50 mM sodium chloride and about 130 mM sucrose.

[0039] In some embodiments, the stabilizer is a combination of sodium chloride and trehalose. In some embodiments, the stabilizer is a combination of about 30–200 mM sodium chloride and about 30–200 mM trehalose, preferably a combination of about 40–150 mM sodium chloride and about 40–180 mM trehalose, more preferably a combination of about 40–100 mM sodium chloride and about 80–160 mM trehalose. Non-limiting examples of the stabilizer include a combination of about 50 mM sodium chloride and about 120 mM trehalose, or a combination of about 50 mM sodium chloride and about 140 mM trehalose.

[0040] In some embodiments, the pharmaceutical composition further includes a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188.

[0041] In some formulations, the surfactant is selected from polysorbate 80.

[0042] In some formulations, the surfactant mentioned above is selected from polysorbate 20.

[0043] In some embodiments, the concentration of the surfactant, calculated by w / v, is about 0.001% to 0.1%, preferably about 0.01% to 0.1%, more preferably about 0.02% to 0.08%, and more preferably about 0.02% to 0.06%; as a non-limiting example, the concentration of the surfactant is about 0.02%, 0.04%, or 0.08%, preferably about 0.04%.

[0044] In some formulations, the pharmaceutical composition comprises, or is composed of, any one of the components shown in (1) to (8) below:

[0045] (1)(a) about 150–250 mg / mL of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment; (b) about 10–30 mM histidine buffer, pH about 5.5–6.5; (c) about 120–220 mM arginine or arginine salt; and (d) about 0.01%–0.1% (w / v) of polysorbate 80; or

[0046] (2)(a) about 150–250 mg / mL of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment; (b) about 10–30 mM histidine buffer at a pH of about 5.5–6.5; (c) a stabilizer, said stabilizer being a combination of about 30–100 mM arginine hydrochloride and about 100–180 mM sucrose; and (d) about 0.01%–0.1% (w / v) of polysorbate 80; or

[0047] (3)(a) about 150–250 mg / mL of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment; (b) about 10–30 mM histidine buffer at a pH of about 5.5–6.5; (c) a stabilizer, said stabilizer being a combination of about 30–100 mM arginine hydrochloride and about 50–150 mM glycine; and (d) about 0.01%–0.1% (w / v) of polysorbate 80; or

[0048] (4)(a) approximately 150–200 mg / mL of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment; (b) approximately 10–30 mM acetate buffer, pH approximately 5.5–6.0; (c) approximately 130–180 mM of arginine or arginine hydrochloride; and (d) approximately 0.02%–0.08% (w / v) of polysorbate 80; or

[0049] (5)(a) about 150–200 mg / mL of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment; (b) about 10–30 mM acetate buffer, pH about 5.5–6.0; (c) a stabilizer, said stabilizer being a combination of about 30–70 mM arginine hydrochloride and about 110–170 mM sucrose; and (d) about 0.02%–0.08% (w / v) of polysorbate 80; or

[0050] (6)(a) about 150 to 200 mg / mL of the above-mentioned anti-PD-1 antibody or its antigen-binding fragment; (b) about 10 to 30 mM acetate buffer, pH about 5.5 to 6.0; (c) a stabilizer, said stabilizer being a combination of about 30 to 70 mM arginine hydrochloride and about 80 to 120 mM glycine; and (d) about 0.02% to 0.08% (w / v) of polysorbate 80;

[0051] (7)(a) approximately 180 mg / mL of anti-PD-1 antibody, said anti-PD-1 antibody comprising the light chain amino acid sequence as shown in SEQ ID NO:9 and the heavy chain amino acid sequence as shown in SEQ ID NO:10; (b) approximately 20 mM histidine buffer at a pH of approximately 6.0; (c) approximately 140 mM arginine hydrochloride; and (d) approximately 0.02% (w / v) of polysorbate 80; or

[0052] (8)(a) about 180 mg / mL of anti-PD-1 antibody, said anti-PD-1 antibody comprising the light chain amino acid sequence as shown in SEQ ID NO:9 and the heavy chain amino acid sequence as shown in SEQ ID NO:10; (b) about 20 mM histidine buffer at a pH of about 6.0; (c) about 150 mM of arginine hydrochloride; and (d) about 0.04% (w / v) of polysorbate 80.

[0053] In some embodiments, the present invention provides a pharmaceutical composition comprising a buffer solution, an anti-PD-1 antibody or an antigen-binding fragment thereof, a stabilizer, and a surfactant; wherein the anti-PD-1 antibody comprises a light chain amino acid sequence as shown in SEQ ID NO:9 and a heavy chain amino acid sequence as shown in SEQ ID NO:10; the concentration of the anti-PD-1 antibody or the antigen-binding fragment thereof is 150–200 mg / mL; the pH of the pharmaceutical composition is 5.9–6.1, and the osmotic pressure is in the range of 260–320 mOsm / kg, preferably 290–310 mOsm / kg. Preferably, in the pharmaceutical composition, the buffer solution is a histidine buffer with a concentration of 15–25 mM and a pH of approximately 5.9–6.1, preferably approximately 6.0. Preferably, in the pharmaceutical composition, the stabilizer is approximately 140–160 mM, preferably approximately 150 mM, of arginine hydrochloride. Preferably, the surfactant in the pharmaceutical composition is polysorbate 80, and its concentration is preferably 0.02-0.06% (w / v), more preferably about 0.04% (w / v). Preferably, the viscosity of the above pharmaceutical composition measured at about 25°C is ≤8.0 cP, more preferably ≤7.0 cP.

[0054] In some embodiments, the pharmaceutical composition described in any of these embodiments is a liquid formulation or a lyophilized formulation.

[0055] In some formulations, the pharmaceutical composition is a liquid preparation.

[0056] In some formulations, the liquid or lyophilized formulations described above are stable at 2–8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

[0057] In some formulations, the aforementioned liquid or lyophilized formulations are stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

[0058] The present invention also provides an injection comprising the pharmaceutical composition described in any of the embodiments herein and a sodium chloride solution or a glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85 to 0.9% (w / v); preferably, the concentration of the glucose solution is about 5 to 25% (w / v), more preferably about 5 to 10% (w / v); preferably, the concentration of the anti-PD-1 antibody in the injection is about 0.5 to 50 mg / mL, more preferably about 0.5 to 20 mg / mL; the pH of the injection is about 5.0 to 6.5, preferably about 5.5 to 6.2.

[0059] In some formulations, the aforementioned pharmaceutical composition or injection is administered via subcutaneous injection.

[0060] The present invention also provides the use of the pharmaceutical composition or injection described in any of the embodiments herein in the preparation of a medicament for treating a disease or condition by eliminating, inhibiting or reducing PD-1 activity.

[0061] The present invention also provides pharmaceutical compositions or injections as described in any of the embodiments herein, which treat diseases or conditions by eliminating, inhibiting or reducing PD-1 activity.

[0062] The present invention also provides a method for treating a disease or condition by eliminating, inhibiting or reducing PD-1 activity, comprising administering to a subject in need a pharmaceutical composition or injection as described in any of the embodiments herein.

[0063] In some protocols, the aforementioned diseases or conditions are selected from cancer, infectious diseases, or inflammatory diseases.

[0064] This invention also provides a method for reducing the viscosity of a high-concentration antibody drug formulation, wherein the antibody concentration in the antibody drug formulation is ≥150 mg / mL, such as in the range of 150–250 mg / mL. The method includes using arginine hydrochloride, sodium chloride, or sucrose and arginine hydrochloride as stabilizers and using histidine buffer as a buffer to prepare the high-concentration antibody drug formulation. Preferably, the amount of arginine hydrochloride used is such that its concentration in the prepared antibody drug formulation is approximately 100–200 mM, more preferably approximately 140–160 mM. Preferably, the amount of sodium chloride used is such that its concentration in the prepared antibody drug formulation is approximately 100–200 mM, more preferably approximately 140–160 mM. Preferably, when using a mixture of sucrose and arginine hydrochloride as a stabilizer, the amount of sucrose used is such that its concentration in the prepared antibody drug formulation is approximately 100–180 mM, preferably approximately 110–150 mM; and the amount of arginine hydrochloride used is such that its concentration in the prepared antibody drug formulation is approximately 30–80 mM, preferably approximately 30–60 mM. Preferably, the pH value of the histidine buffer used is 5.0–6.5, preferably 5.5–6.2, more preferably 5.9–6.1. Preferably, the amount of histidine buffer used is such that its concentration in the prepared antibody drug formulation is 15–25 mM, preferably approximately 20 mM. Preferably, the antibody is the anti-PD-1 antibody described in any embodiment herein. Preferably, the method for reducing the viscosity of the high-concentration antibody drug formulation can reduce the viscosity of the prepared antibody drug formulation to below approximately 8.0 cP (measured at approximately 25°C). In some embodiments, the method further includes adding the surfactant described in any of the embodiments herein, preferably 0.02 to 0.06% (w / v) of polysorbate 80, to the antibody drug formulation.

[0065] In some embodiments, the present invention also provides the use of arginine hydrochloride, sodium chloride, or a sucrose-arginine-hydrochloride-histidine buffer as a stabilizer in reducing the viscosity of high-concentration antibody drug formulations, or in the preparation of high-concentration antibody drug formulations with reduced viscosity. Preferably, the stabilizer, histidine buffer, antibody, and antibody concentration are as described in any embodiment herein. Preferably, the application can reduce the viscosity of the high-concentration antibody drug formulation to below about 8.0 cP (measured at about 25°C). Attached Figure Description

[0066] Figure 1 First round of formulation screening—SEC-HPLC purity trend chart under high temperature test.

[0067] Figure 2 First round of formulation screening—CEX-HPLC purity trend chart under high temperature test.

[0068] Figure 3 Second round of formulation screening—SEC-HPLC purity trend chart under high temperature test.

[0069] Figure 4 Second round of formulation screening—CEX-HPLC purity trend chart under high temperature test.

[0070] Figure 5 Third round of formulation screening—SEC-HPLC purity trend chart under high temperature test.

[0071] Figure 6 Third round of formulation screening—CEX-HPLC purity trend chart under high temperature test.

[0072] Figure 7 The mean blood drug concentration-time change curves for groups A and B.

[0073] Figure 8 The curve showing the inhibitory effect of the subcutaneous injection formulation on the growth of MC38 tumors transplanted from hPD-1 humanized mice. Detailed Implementation

[0074] Definitions and Explanations

[0075] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that this invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting. All references cited herein, including patents, patent applications, papers, textbooks, and the like, and references cited therein, are incorporated herein by reference in their entirety to the extent that they have not already been cited. If one or more of the incorporated references and similar materials differ from or contradict this application, including but not limited to defined terminology, usage of terminology, described techniques, and the like, this application shall prevail.

[0076] Unless otherwise expressly stated, the singular forms “a,” “an,” and “the” used in this specification and the appended claims include plural references. Thus, for example, reference to “a polypeptide” includes a combination of two or more polypeptides, etc.

[0077] The terms "pharmaceutical composition" or "formulation" refer to a mixture containing one or more antibodies described herein, along with other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0078] The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a lyophilized formulation in a weighted suspension. The liquid formulation of the present invention is stable during storage, and its stability is independent of lyophilization (or other state-changing methods, such as spray drying).

[0079] The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In some formulations, aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and / or freezing to maintain stability (e.g., chemical and / or physical stability and / or biological activity).

[0080] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (such as consistency, improved stability) and / or adjust osmotic pressure. Common examples of excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

[0081] The term “about” as used in this application, when referring to measurable values ​​(such as quantity, duration, etc.), is intended to cover variations of ±20% or ±10% relative to a specific value, including ±5%, ±1%, and ±0.1%, because such variations are suitable for carrying out the disclosed methods.

[0082] The term "buffer solution with a pH of about 5.0 to 6.5" refers to a reagent that, through the action of its acid / base conjugate components, enables a solution containing the reagent to resist pH changes. The buffer solution used in the formulations of this invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

[0083] In this document, examples of “buffer solutions” that control the pH within this range include acetic acid, acetate (e.g., sodium acetate), succinic acid, succinate (e.g., sodium succinate), gluconic acid, histidine, histidine salts (e.g., histidine hydrochloride), methionine, citric acid, citrate, phosphate, citrate / phosphate, imidazole, combinations thereof, and other organic acid buffers.

[0084] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, such as histidine buffers containing histidine and histidine hydrochloride; the histidine buffers of the present invention also include histidine buffers containing histidine and acetate (such as sodium or potassium salts).

[0085] "Citrate buffer," also known as "citric acid buffer," is a buffer solution containing citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, and magnesium citrate. The preferred citrate buffer is sodium citrate buffer.

[0086] "Acetate buffer" is a buffer solution containing acetate ions. Examples of acetate buffer solutions include acetate-sodium acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc. The preferred acetate buffer solution is acetate-sodium acetate buffer.

[0087] "Succinate buffer" is a buffer solution containing succinate ions. Examples of succinate buffer solutions include sodium succinate-sodium succinate, potassium succinate-potassium succinate, calcium succinate-calcium succinate, magnesium succinate-magnesium succinate, etc. A preferred succinate buffer solution is sodium succinate-sodium succinate buffer.

[0088] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (e.g., arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opins, alanine, strombine, and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (BSA), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAase A. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, and sucrose, can also play a role in controlling osmotic pressure. The stabilizers specifically used in this invention are selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine, glycine, and proline. Amino acids can exist in their D- and / or L- forms, but are typically L-forms. Amino acids can exist in any suitable salt, such as hydrochloride, like arginine hydrochloride. Preferred stabilizers include sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, and arginine hydrochloride-sucrose.

[0089] The term "surfactant" generally refers to agents that protect proteins, such as antibodies, from air / solution interface-induced stress and solution / surface-induced stress to reduce antibody aggregation or minimize particulate formation in formulations. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (pluronic), and sodium dodecyl sulfate (SDS). Unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" refer to mass-volume concentration (w / v), such as "about 0.04% polysorbate 80," where "0.04%" means "0.04 g of polysorbate 80 in 100 mL of liquid."

[0090] The term "viscosity" as used in this article can refer to either "kinematic viscosity" or "absolute viscosity." Kinematic viscosity is a measure of the resistance to flow produced by a fluid under the influence of gravity. Absolute viscosity, sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is L. 2 / T, where L is length and T is time. Kinematic viscosity is typically expressed in centistokees (cSt). The SI unit for kinematic viscosity is mm. 2 / s, or lcSt. Absolute viscosity is expressed in centipoise (cP). The SI unit for absolute viscosity is millipascal-second (mPa·s), where 1 cP = 1 mPa·s.

[0091] For the liquid formulations of the present invention, the term "low viscosity level" as used herein will refer to an absolute viscosity below about 15 centipoise (cP). For example, if the formulation exhibits an absolute viscosity of about 15 cP, about 14 cP, about 13 cP, about 12 cP, about 11 cP, about 10 cP, about 9 cP, about 8 cP, or lower when measured using standard viscosity measurement techniques, then the liquid formulation of the present invention will be considered to have "low viscosity". For the liquid formulations of the present invention, the term "medium viscosity level" as used herein will refer to an absolute viscosity between about 35 cP and about 15 cP. For example, if the formulation exhibits an absolute viscosity of about 34 cP, about 33 cP, about 32 cP, about 31 cP, about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP, about 21 cP, about 20 cP, about 19 cP, 18 cP, about 17 cP, about 16 cP, or about 15 cP when measured using standard viscosity measurement techniques, then the liquid formulation of the present invention will be considered to have "medium viscosity". In some embodiments, the pharmaceutical compositions of the present invention may exhibit ultra-low levels of viscosity of about 7 cP or less. In some embodiments, a comparison of the viscosities of different excipients has revealed that arginine or its salts can achieve significantly better viscosity, stability, and efficacy than other excipients. In some embodiments, a comparison of buffer systems has revealed that the histidine buffer system has significantly better viscosity, stability, and efficacy than other buffer systems.

[0092] The term "isotonic" means that the formulation has an osmotic pressure that is essentially the same as that of human blood. Isotonic formulations generally have an osmotic pressure of approximately 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmoremeter.

[0093] The term "stable" formulation refers to a formulation in which the antibody substantially retains its physical and / or chemical stability and / or biological activity during the manufacturing process and / or storage. A pharmaceutical formulation may be considered stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after a certain period of storage. In some cases, maintaining approximately 90%, 95%, 96%, 97%, 98%, or 99% of the antibody structure or function after a certain period of storage can also be considered "stable." Various analytical techniques for measuring protein stability are available in this field and are reviewed in *Peptide and Protein Drug Delivery*, 247–301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29–90 (both incorporated herein by reference).

[0094] After a formulation has been stored at a certain temperature for a certain period of time, its stability can be measured by determining the percentage of remaining natural antibodies (and other methods). Among other methods, the percentage of natural antibodies can be measured by size exclusion chromatography (e.g., size exclusion high-performance liquid chromatography [SEC-HPLC]), where "natural" refers to non-aggregated and non-degraded antibodies. In some formulations, protein stability is determined by the percentage of monomeric proteins in a solution with a low percentage of degradation (e.g., fragmentation) and / or aggregated proteins. In some formulations, the formulation can be stored stably at room temperature, about 25–30°C, or 40°C 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, with a maximum of about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies.

[0095] Stability can be measured (and by other methods) by determining the percentage of antibody (“acidic form”) that migrates in the more acidic fraction of the main antibody fraction (“major charged form”) during ion exchange, wherein stability is inversely proportional to the percentage of acidic antibody. Among other methods, the percentage of “acidified” antibody can be measured by ion exchange chromatography (e.g., cation exchange high-performance liquid chromatography [CEX-HPLC]). In some embodiments, acceptable stability means that after storage at a certain temperature for a certain period of time, the detectable acidic antibody form does not exceed approximately 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. The storage period prior to stability measurement may be 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 stability, the permissible storage temperature for the pharmaceutical preparation may be any temperature within the range of about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 2–8°C, about 5°C, about 25°C, or about 40°C.

[0096] If an antibody does not show substantially no signs of aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or upon measurement by UV light scattering or size exclusion chromatography, then the antibody “maintains its physical stability” in the pharmaceutical composition. Aggregation is the process by which individual molecules or complexes covalently or nonvalently associate to form aggregates. Aggregation can proceed to the extent that visible precipitates are formed.

[0097] The stability of a formulation, such as its physical stability, can be assessed using methods known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is partly an inherent property of proteins dissolved in solution and is typically measured by turbidimetry and quantified in turbidimetric units (NTU).

[0098] The turbidity level, which varies with the concentration of one or more components in the solution (e.g., protein and / or salt concentration), is also referred to as the “emulsification” or “emulsive appearance” of the formulation. The turbidity level can be calculated by referring to a standard curve generated using a suspension of known turbidity. Reference standards used to determine the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia (European Pharmacopoeia, 4th edition, “Directorate for the Quality of Medicine of the Council of Europe” (EDQM), Strasbourg, France). According to the European Pharmacopoeia, a clear solution is defined as a solution with a turbidity less than or equal to that of a reference suspension having a turbidity of approximately 3 according to the European Pharmacopoeia. Turbidimetric turbidity measurements can detect Rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

[0099] An antibody is considered to retain its biological activity as defined below if its chemical stability at a given time point is sufficient to ensure that the antibody retains its chemical stability in the pharmaceutical composition. Chemical stability can be assessed, for example, by detecting or quantifying chemical changes in the antibody. Chemical changes can include size changes (e.g., shortening), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.

[0100] If the antibody in the pharmaceutical composition is biologically active for its intended purpose, then the antibody "retains its biological activity" in the pharmaceutical composition. For example, if, after storage at temperatures such as 5°C, 25°C, and 45°C for a certain period of time (e.g., 1 to 12 months), the anti-PD-1 antibody contained in the formulation has an affinity for binding to PD-1 that is at least 90%, 95%, or greater than the antibody binding affinity before the storage, then the formulation of the present invention can be considered stable. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.

[0101] In the context of this invention, in a pharmacological sense, a "therapeutic effective amount" or "effective amount" of an antibody refers to an amount effective in preventing, treating, or alleviating symptoms of an impairment for which the antibody can be effectively treated. In this invention, a "therapeutic effective amount" or "therapeutic effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease regression, said disease regression being demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability caused by disease suffering. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting human efficacy, or by determining the activity of the drug in an in vitro assay. A therapeutically effective amount of a drug includes a "preventatively effective amount," which is any amount of the drug that, when given alone or in combination with other therapeutic agents to a subject at risk of disease or a subject experiencing disease relapse, inhibits the development or recurrence of the disease.

[0102] The terms "subject" or "patient" are intended to include mammalian organisms. Examples of subjects / patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.

[0103] The terms “administration,” “giving,” and “treatment” refer to the introduction of a composition containing a therapeutic agent into a subject using any of the various methods or delivery systems known to those skilled in the art. Routes of administration for anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes, such as injection or infusion. “Parenteral administration” refers to administration by injection other than enteral or local administration, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intra-tracheal, intracapsular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, and intrasternal injections and infusions, as well as intracorporeal electroporation.

[0104] Anti-PD-1 antibody

[0105] The term "antibody" as used herein should be understood to include both the complete antibody molecule and its antigen-binding fragment. The term "antigen-binding portion" or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment") of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to human PD-1 or its epitopes. Therefore, it is used in the broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0106] The term "isolated antibody" refers to the purified state of a binding compound, and in this context, it means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the binding compound described herein.

[0107] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0108] The terms "mouse antibody" or "hybridoma antibody" in this disclosure refer to monoclonal antibodies against human PD-1 prepared in accordance with the knowledge and skills in the art. Preparation involves injecting the test subject with PD-1 antigen, followed by isolation of a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0109] The term "chimeric antibody" refers to an antibody possessing a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies originate from different species. Typically, the variable domain is derived from antibodies from rodents, etc. ("parental antibodies"), while the constant domain sequence is derived from human antibodies, making the resulting chimeric antibody less likely to induce an adverse immune response in human subjects compared to parental rodent antibodies.

[0110] The term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, humanized antibodies contain at least one, and usually two, variable domains, where all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or almost all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant region (Fc) of human immunoglobulins.

[0111] The term "full-length antibody" or "complete antibody molecule" refers to an immunoglobulin molecule containing four peptide chains: two heavy (H) chains (approximately 50–70 kDa in full length) and two light (L) chains (approximately 25 kDa in full length) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0112] The term "CDR" refers to the complementarity-determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are named HCDR1, HCDR2, and HCDR3, or LCDR1, LCDR2, and LCDR3, for each heavy and light chain variable region. The precise boundaries of these CDRs are defined differently depending on the system.

[0113] The precise amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined using any of many known schemes, including those based on the three-dimensional structure of the antibody and the topology of the CDR loop: Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997); and those based on antibody sequence variability: Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, Department of Health and Human Services, National Institutes of Health (1987), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids). Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering of a large number of crystal structures. The CDR of the antibodies of the present invention can be determined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0114] As used herein, “antigen-binding fragment” includes fragments of antibodies or derivatives thereof, typically comprising at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof typically retains at least 10% of the antigen-binding activity of the parent antibody. Preferably, the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of an antibody may include conserved or non-conserved amino acid substitutions (referred to as “conserved variants” or “functionally conserved variants” of the antibody) that do not significantly alter its biological activity.

[0115] The anti-PD-1 antibody or its antigen-binding fragment described in this invention includes any one of the anti-PD-1 antibodies or its antigen-binding fragments described in application number CN201310258289.2, the entire contents of which are incorporated herein by reference. In some embodiments, the CDR sequence of the antibody used in the methods and compositions of this invention includes humanized antibody clone 38 described in CN201310258289.2.

[0116] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment used in the methods and compositions of the present invention comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0117] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment used in the methods and compositions of the present invention is selected from murine antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, humanized antibodies or their antigen-binding fragments, preferably humanized antibodies or their antigen-binding fragments.

[0118] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment used in the methods and compositions of the present invention comprises a light chain variable region as shown in SEQ ID NO:7 and a heavy chain variable region as shown in SEQ ID NO:8.

[0119] In some embodiments, the anti-PD-1 antibody used in the methods and compositions of the present invention comprises a light chain amino acid sequence as shown in SEQ ID NO:9 and a heavy chain amino acid sequence as shown in SEQ ID NO:10.

[0120] In some embodiments, the non-limiting, exemplary antibody used in the examples herein is Toripalimab (a humanized IgG4 mAb having the structure described in WHO Drug Information (Vol. 32, No. 2, pp. 372-373 (2018)) and containing the heavy and light chain amino acid sequences shown in SEQ ID NO: 9 and 10).

[0121] The amino acid sequences of SEQ ID NO: 1-10 mentioned in this article are shown in the table below:

[0122]

[0123]

[0124] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment used in the methods and compositions of the present invention is a humanized antibody or chimeric antibody, and may include a human constant region. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions; preferably, the anti-PD-1 antibody or its antigen-binding fragment suitable for the methods and compositions of the present invention comprises a heavy chain constant region of human IgG1 or IgG4 isotypes, more preferably a human IgG4 constant region. In some embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or its antigen-binding fragment contains an S228P mutation, which replaces a serine residue in the hinge region with a proline residue normally present at the corresponding position in the IgG1 isotype antibody.

[0125] In some embodiments, the present invention provides a method for preparing an anti-PD-1 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0126] This invention provides mammalian host cells for expressing the recombinant antibodies of this invention, including a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammal host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines exhibit high expression levels.

[0127] In one embodiment, the present invention provides a method for preparing an anti-PD-1 antibody, wherein the method includes, when an expression vector is introduced into a mammalian host cell, producing the antibody by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably by secreting the antibody into the culture medium in which the host cell grows. The antibody can be recovered from the culture medium using standard protein purification methods.

[0128] Antibodies expressed in different cell lines or in transgenic animals may have different glycosylations. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are part of the invention, regardless of their glycosylation. Similarly, in some embodiments, non-fucosylated antibodies are advantageous because they generally have stronger efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structure is a normal component of native human serum IgG.

[0129] Pharmaceutical preparations

[0130] The pharmaceutical composition described in this invention is a high-concentration, highly stable, and ultra-low viscosity pharmaceutical composition containing an antibody that specifically binds to PD-1. The clinical need for subcutaneous (SC) administration of high-dose protein drugs (>100 mg / mL) typically introduces additional technical development challenges regarding manufacturing, analytical testing, stability, and delivery. A common property of high-concentration protein formulations is high viscosity, which is directly due to the reversible self-association of the protein. High viscosity can also pose additional clinical development challenges due to high injection force (increased pain at the injection site) and can also alter the pharmacokinetic properties of the drug. Therefore, an important aspect of product development is the search for formulations with low viscosity. This invention, through the selection of appropriate buffer systems and pH, optimization of stabilizers and surfactants, and pharmacokinetic and pharmacodynamic studies, develops a high-concentration antibody formulation suitable for subcutaneous administration, exhibiting long-term stability, no aggregation, and ultra-low viscosity.

[0131] The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; and (2) an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0132] The anti-PD-1 antibody or its antigen-binding fragment in the pharmaceutical composition of the present invention is as described in any embodiment of the "Anti-PD-1 Antibody" section of this application.

[0133] For example, the anti-PD-1 antibody or its antigen-binding fragment in the pharmaceutical composition of the present invention comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Preferably, the above-mentioned anti-PD-1 antibody or its antigen-binding fragment is selected from murine antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, humanized antibodies or their antigen-binding fragments, and preferably humanized antibodies or their antigen-binding fragments. Preferably, the above-mentioned anti-PD-1 antibody or its antigen-binding fragment comprises a light chain variable region as shown in SEQ ID NO:7, and a heavy chain variable region as shown in SEQ ID NO:8. More preferably, the above-mentioned anti-PD-1 antibody comprises a light chain amino acid sequence as shown in SEQ ID NO:9, and a heavy chain amino acid sequence as shown in SEQ ID NO:10.

[0134] The concentration of the anti-PD-1 antibody or its antigen-binding fragment in the pharmaceutical composition of the present invention is about 100 to 250 mg / mL, preferably about 150 to 250 mg / mL, and more preferably about 150 to 200 mg / mL.

[0135] The pH of the pharmaceutical composition of the present invention is about 5.0 to 6.5, preferably about 5.5 to 6.2, and more preferably about 6.0.

[0136] The buffer solution in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer, and histidine buffer; preferably, the buffer solution is histidine buffer. Preferably, the histidine buffer solution is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer. Preferably, the concentration of the buffer solution is about 5–100 mM, more preferably about 10–50 mM, more preferably about 10–30 mM; more preferably about 15–25 mM. Preferably, the pH of the buffer solution is about 5.0–6.5, more preferably about 5.5–6.5, more preferably about 5.5–6.2.

[0137] Therefore, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer with a pH of about 5.5 to 6.5, at a concentration of about 10 to 30 mM in the pharmaceutical composition; and an anti-PD-1 antibody or antigen-binding fragment thereof as described in any of the preceding embodiments at a concentration of about 150 to 250 mg / mL, especially the humanized antibody clone 38 or antigen-binding fragment thereof described herein.

[0138] In some embodiments, the pharmaceutical composition of the present invention further includes a stabilizer selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine, and trehalose; preferably, the arginine salt is arginine hydrochloride. Preferably, the concentration of the stabilizer is about 10–400 mM, more preferably about 100–250 mM, more preferably about 120–220 mM, and more preferably about 130–180 mM. Preferably, the stabilizer is arginine or arginine salt at a concentration of about 120-220 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30-100 mM and sucrose at a concentration of about 100-180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30-100 mM and glycine at a concentration of about 50-150 mM; preferably, the stabilizer is arginine or arginine salt at a concentration of about 130-180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30-70 mM and sucrose at a concentration of about 110-170 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30-70 mM and glycine at a concentration of about 80-120 mM; preferably, the arginine salt is arginine hydrochloride.

[0139] Therefore, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer solution with a pH of about 5.5 to 6.5, at a concentration of about 10 to 30 mM in the pharmaceutical composition; an anti-PD-1 antibody or its antigen-binding fragment thereof described in any of the preceding embodiments at a concentration of about 150 to 250 mg / mL, particularly the humanized antibody clone 38 or its antigen-binding fragment described herein; and a stabilizer of about 100 to 250 mM, preferably selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine, and trehalose, preferably arginine salt being arginine hydrochloride. Preferably, the stabilizer is arginine or arginine salt at a concentration of about 120 to 220 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30 to 100 mM and sucrose at a concentration of about 100 to 180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 30 to 100 mM and glycine at a concentration of about 50 to 150 mM.

[0140] In some embodiments, the pharmaceutical composition further includes a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188. Preferably, the concentration of the surfactant, calculated as w / v, is about 0.001% to 0.1%, more preferably about 0.01% to 0.1%, and even more preferably about 0.02% to 0.08%.

[0141] Therefore, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer solution with a pH of about 5.5 to 6.5, at a concentration of about 10 to 30 mM in the pharmaceutical composition; an anti-PD-1 antibody or its antigen-binding fragment thereof described in any of the preceding embodiments at a concentration of about 150 to 250 mg / mL, especially the humanized antibody clone 38 or its antigen-binding fragment described herein; a stabilizer of about 100 to 250 mM, preferably, the stabilizer being arginine or arginine salt at a concentration of about 120 to 220 mM; or the stabilizer being a combination of arginine hydrochloride at a concentration of about 30 to 100 mM and sucrose at a concentration of about 100 to 180 mM; or the stabilizer being a combination of arginine hydrochloride at a concentration of about 30 to 100 mM and glycine at a concentration of about 50 to 150 mM; and about 0.01% to 0.1% polysorbate 80, w / v.

[0142] The pharmaceutical compositions of the present invention are liquid formulations or lyophilized formulations.

[0143] The osmotic pressure of the pharmaceutical composition of the present invention is in the range of 260 to 320 mOsm / kg, preferably in the range of 290 to 310 mOsm / kg.

[0144] The viscosity of the pharmaceutical composition of the present invention is ≤8.0 cP when measured at about 25°C.

[0145] Medical uses and methods

[0146] The present invention also provides the use of the pharmaceutical composition or injection described in any of the embodiments herein in the preparation of a medicament for treating a disease or condition by eliminating, inhibiting or reducing PD-1 activity.

[0147] The present invention also provides pharmaceutical compositions or injections as described in any of the embodiments herein, which treat diseases or conditions by eliminating, inhibiting or reducing PD-1 activity.

[0148] The present invention also provides a method for treating a disease or condition by eliminating, inhibiting or reducing PD-1 activity, comprising administering to a subject in need a pharmaceutical composition or injection as described in any of the embodiments herein.

[0149] In some schemes, the aforementioned disease or condition is selected from cancer, infectious disease, or inflammatory disease: the cancer is preferably selected from colon cancer, neuroendocrine tumor, esophageal cancer, nasopharyngeal carcinoma, sarcoma, melanoma, urothelial carcinoma, and non-small cell lung cancer.

[0150] Example

[0151] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. The present invention has been described in detail herein, and its specific implementation has also been disclosed. For those skilled in the art, various changes and modifications to the specific embodiments of the present invention will be obvious without departing from the spirit and scope of the invention. Any modifications, equivalent substitutions, improvements, etc., should be included within the protection scope of the present invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional methods and materials in the art.

[0152] The detection methods used in the embodiments include:

[0153] (1) Appearance

[0154] Visual inspection was used to check the appearance. The light intensity of the clarity meter was maintained between 1000 lx and 1500 lx. The sample was held at eye level, and gently shaken or inverted to avoid air bubbles. Visual inspection was performed against both black and white backgrounds. Results were recorded for color, opalescence, and visible foreign matter.

[0155] (2) Protein content

[0156] Protein concentrations were detected using Nanodrop and SoloVPE. When using Nanodrop, the percentage extinction factor (E1%) was set at 1.416 (mg / mL). -1 cm -1 Rinse the detector three times with ultrapure water, then add 3 μL of ultrapure water to the detection well and click "calibrate," using the ultrapure water as a blank for calibration. After blank calibration, measure the sample by adding 3 μL of sample to the detection well, clicking the "detect" button, and recording the instrument's detection data. Measure each sample in triplicate, with each solution measured once.

[0157] When using SoloVPE assay, the percentage extinction factor (E1%) was set at 1.416 (mg / mL). -1 cm -1 After calibrating with ultrapure water as a blank, measure the sample by adding 120 μL of sample to the cuvette, clicking the "Detect" button, and recording the instrument's detection data.

[0158] (3) SEC-HPLC purity

[0159] SEC-HPLC purity was determined using an HPLC system (Waters e2695 instrument) equipped with a SEC column (TSK gel G3000SWXL, 7.8*300mm, 5μm). The mobile phase consisted of 50mM phosphate, 300mM Na2SO4, and pH 7.0±0.2. Peak area normalization was used for quantitative analysis. The peak area percentages of monomers, polymers, and fragments were calculated separately. The peak area percentage of monomers was used as the sample purity, and the peak area percentages of polymers and fragments were used as the contents of polymers and fragments, respectively. Chromatographic parameters are shown in Table 1 below.

[0160] Table 1: SEC-HPLC Chromatographic Parameters

[0161] Chromatographic conditions Chromatographic parameters chromatographic column TSK gel G3000SWXL,7.8*300mm,5μm detector wavelength 280nm Autosampler temperature 5±3℃ Column temperature 25±2℃ Flow rate 0.5 mL / min Injection volume 25μL Operating mode Isocratic elution Washout time 30min

[0162] (4) Purity of R-CE-SDS

[0163] The purity determination of antibody preparations using the reduction CE-SDS electrophoresis method utilizes a high-voltage direct current electric field as the driving force and a capillary as the separation channel. The pre-filled gel forms a molecular sieve within the capillary. Sodium dodecyl sulfate eliminates the charge effect between different protein molecules, and the reducing agent β-mercaptoethanol cleaves disulfide bonds in the sample. Samples of different molecular sizes move at different speeds within the capillary, thus enabling separation. The sample is diluted to 1 mg / mL with loading buffer (SDS-MW Sample Buffer). 95 μL of the loading buffer (SDS-MW Sample Buffer) is mixed with 5 μL of β-mercaptoethanol and vortexed as a blank control. 95 μL of the test sample (1 mg / mL) is mixed with 5 μL of β-mercaptoethanol, centrifuged at 3000 rpm at room temperature for 30 seconds, incubated at 70±2℃ for 15±2 min, cooled to room temperature, and centrifuged at 6000 rpm at room temperature for 1 min. The separation time is 40 min, and the result is detected using a capillary electrophoresis system (Beckman). The purity of the sample is calculated by summing the purity of the heavy chain (HC), non-glycosylated heavy chain (NGHC), and light chain (LC).

[0164] (5) Purity of NR-CE-SDS

[0165] Antibody preparation purity detection using non-reducing CE-SDS electrophoresis utilizes a high-voltage direct current electric field as the driving force and a capillary as the separation channel. The pre-filled gel forms a molecular sieve within the capillary. Sodium dodecyl sulfate is used to treat the sample to eliminate the charge effect between different protein molecules, allowing for the separation of samples with different molecular sizes and migration speeds within the capillary. The addition of an alkylating agent to the test solution effectively reduces component diffusion, resulting in sharp peaks, high separation efficiency, and ensuring the sample remains in a non-reduced state. Dilute the sample to 1 mg / mL with SDS-MW Sample Buffer; take 95 μL of SDS-MW Sample Buffer and add 5 μL of 0.8 M iodoacetamide solution, vortex to mix, and use as a blank control; take 95 μL of test sample (1 mg / mL), add 5 μL of 0.8 M iodoacetamide solution, centrifuge at 3000 rpm at room temperature for 30 sec, incubate at 70±2℃ for 5±1 min, cool to room temperature, centrifuge at 6000 rpm at room temperature for 1 min, and detect using a capillary electrophoresis apparatus (Beckman).

[0166] (6) CEX-HPLC purity

[0167] CEX-HPLC purity was determined using an HPLC system (Waterse2695 instrument) equipped with a ProPac WCX-10 column (4*250mm). The mobile phase composition was: Phase A: 10mM sodium dihydrogen phosphate dihydrate solution (pH 4.7±0.2); Phase B: 10mM disodium hydrogen phosphate dodecahydrate solution (pH 9.2±0.2). The percentages of the main peak, acidic peak, and basic peak were calculated using the peak area normalization method. If automatic integration could not obtain reasonable integration results, manual integration was used. Detailed chromatographic parameters are shown in Table 2 below.

[0168] Table 2: CEX-HPLC chromatographic parameters

[0169]

[0170]

[0171] (7) Cell activity

[0172] In this method, PD-1 Jurkat T cells were used as effector cells, and CHO engineered cells overexpressing PD-L1 were used as target cells. T cell antigen receptors on Jurkat effector cells can bind to antigens on CHO target cells, inhibiting NFAT luciferase reporter gene expression. PD-1 monoclonal antibodies can bind to PD-1 on Jurkat T cells, thereby blocking the interaction between PD-1 on Jurkat T cells and PD-L1 on CHO target cells and promoting T cell activation, leading to NFAT luciferase reporter gene activation. The ability of PD-1 monoclonal antibodies to bind to PD-1 molecules was assessed by adding luciferase detection reagent and using chemiluminescence immunoassay to detect the signal strength of NFAT-Luciferase expression in T cells-Jurkat cells.

[0173] (8) Binding activity

[0174] This method employs an indirect approach, using human PD-1 as the antigen coated onto a 96-well plate. The PD-1 monoclonal antibody binds to human PD-1. Biotinylated mouse anti-human IgG4 specifically binds to the PD-1 monoclonal antibody bound to the solid-phase antigen (human PD-1). Horseradish peroxidase-conjugated streptavidin binds to the biotinylated mouse anti-human IgG4. The horseradish peroxidase-conjugated streptavidin catalyzes the TMB to turn blue under hydrogen peroxide, with the blue intensity positively correlated with the amount of horseradish peroxidase-conjugated streptavidin bound. The blue color turns yellow after termination with 2M hydrochloric acid. The absorbance (OD value) is measured at 450nm / 620nm using a microplate reader. The effective binding activity (EC50) of the PD-1 monoclonal antibody is evaluated using a standard curve.

[0175] (9) MFI Subvisible Particle Detection

[0176] MFI sub-visible particle detection was performed using a particle detector (MFI5100). Since the sample was a high concentration, it needed to be diluted before loading. After dilution, the sample was gently and thoroughly mixed to avoid air bubbles. Then, 1.3 mL of sample was taken from the sample plate using a pyrogen-free pipette tip in a clean bench and placed onto the sample plate. The sample plate was then tightly covered with clean aluminum foil. After moving the sample from the clean bench to the corresponding working plate of the instrument, the sample loading position was entered into the instrument, and the detection sequence was run. As the sample flowed through the flow cell, sub-visible particles were photographed and counted by a miniature camera.

[0177] (10) HIAC sub-visible particle detection

[0178] Sub-visible particle detection is performed using HIAC. After gently and thoroughly mixing the sample to avoid air bubbles, it is placed in the sample tank. The instrument's robotic arm automatically collects the sample, and as the sample flows through the sensor, the sub-visible particles are counted using the photoresist method.

[0179] (11) Viscosity

[0180] Viscosity was measured using a viscometer (manufacturer: RheoSense, model: MicroVisc) at approximately 25°C and a shear rate of approximately 1000-2000 s. -1 .

[0181] The abbreviations used in the following examples are explained as follows: "hr" represents hour, "W" represents week, "M" represents month, "C" represents number of freeze-thaw cycles, "FT" represents freeze-thaw cycle, "RT" represents room temperature, and "T0" represents the initial test of the prescription sample before it is laid out.

[0182] Example 1: Preliminary screening experiment of buffer system and stabilizer

[0183] In liquid pharmaceutical compositions, the buffer system and pH closely affect antibody stability. Each antibody with unique physicochemical properties has an optimal type of buffer and pH. This embodiment aims to preliminarily screen for the best buffer system and stabilizer, so that the anti-PD-1 antibody disclosed in this invention has optimal stability for clinical application.

[0184] 1.1 Experimental Procedure

[0185] This example uses the antibody toripalimab. The sample was prepared using Millipore Pellicon3 0.11m... 2 The membrane was concentrated to approximately 180 mg / mL using UF / DF ultrafiltration. The sample was then dialyzed into the corresponding formulation shown in Table 3, and the final concentration was adjusted to approximately 180 mg / mL. The corresponding concentration of polysorbate 80(II) was then added. The solution was aseptically filled into 2R vials (2.0 mL / vial) in a laminar flow hood for stability sampling and testing.

[0186] Table 3: Formulation information from the first round of formulation screening – preliminary screening experiments of buffer system and stabilizer

[0187]

[0188] Note: " / " indicates none.

[0189] 1.2 Experimental Results

[0190] 1.2.1 Appearance and Viscosity Results

[0191] According to the results in Table 4, no obvious foreign matter or obvious opalescence was found in any of the formulations at T0. After three and five freeze-thaw cycles, the appearance of the samples did not change significantly; after being placed under high temperature and long-term conditions for one month, the appearance did not change significantly; the FS1-1, FS1-3 and FS1-6 formulations had higher viscosity.

[0192] Table 4: First Round of Prescription Screening—Sample Appearance Data

[0193]

[0194] 1.2.2 SEC Purity Results

[0195] according to Figure 1 The SEC-HPLC purity trend chart and the SEC-HPLC purity results in Table 5 show that, after being stored at high temperature for 1 month (1M), the purity of formulations FS1-4 decreased significantly, while the polymer content increased significantly. The purity of the remaining formulations did not show significant changes. After being stored under long-term conditions for 1M and subjected to five freeze-thaw cycles, the SEC purity of all formulations did not show significant changes.

[0196] Table 5: First Round of Formulation Screening—SEC-HPLC Data

[0197]

[0198] 1.2.3 R-CE-SDS Purity Results

[0199] According to the R-CE-SDS purity results in Table 6, the purity of R-CE-SDS in all samples decreased after being placed at high temperature for 1 minute. However, the purity of R-CE-SDS in all samples did not show significant changes after long-term storage for 1 minute and five freeze-thaw cycles.

[0200] Table 6: First Round Prescription Screening—R-CE-SDS Data

[0201]

[0202]

[0203] 1.2.4 NR-CE-SDS Purity Results

[0204] According to the NR-CE-SDS purity results in Table 7, after being placed under high temperature for 1M, the NR-CE-SDS purity of all samples decreased significantly, with the purity of FS1-4 and FS1-5 formulas decreasing relatively faster; after being placed for a long period of 1M and subjected to five freeze-thaw cycles, the NR-CE-SDS purity of all samples did not show significant changes.

[0205] Table 7: First Round Prescription Screening—NR-CE-SDS Data

[0206]

[0207] 1.2.5 CEX-HPLC purity results

[0208] according to Figure 2 The CEX-HPLC purity trend chart and the CEX-HPLC purity results in Table 8 show that after being stored at high temperature for 1 M, the purity of all formulations decreased significantly, and a significant increase in acid peaks was observed. The purity of formulations FS1-3 decreased the fastest. After long-term storage for 1 M and five freeze-thaw cycles, no significant decrease in CEX purity was observed in any of the formulations.

[0209] Table 8: First Round of Formulation Screening—CEX-HPLC Data

[0210]

[0211]

[0212] 1.2.6 Combining activity results

[0213] According to the binding activity results in Table 9, the binding activity did not change significantly after being placed under high temperature or long-term conditions for 1 M and after five freeze-thaw cycles.

[0214] Table 9: First Round of Formulation Screening—Combined with Activity Data

[0215]

[0216] 1.2.7 Cell viability results

[0217] According to the cell viability results in Table 10, the cell viability of all samples did not change significantly after being placed under high temperature or long-term conditions for 1M and after five freeze-thaw cycles.

[0218] Table 10: First Round of Prescription Screening—Cell Viability Data

[0219]

[0220] 1.2.8 Results of Subvisible Particles

[0221] According to the subvisible particle detection results in Table 11, no significant increase in subvisible particles was observed in any prescription after being placed at high temperature for 1 minute; after being placed at long-term conditions for 1 minute and repeated freeze-thaw cycles 5 times, the number of subvisible particles in all prescriptions did not change significantly.

[0222] Table 11: First Round of Prescription Screening—Subvisible Particle Data

[0223]

[0224] 1.3 Conclusions of the First Round of Prescription Screening

[0225] From the appearance results, no significant differences were found among the formulations. Regarding viscosity, formulations FS1-1, FS1-3, and FS1-6 had higher viscosities. From the SEC-HPLC results, formulation FS1-4 produced more aggregates. From the NR-CE-SDS purity results, formulations FS1-4 and FS1-5 had lower purity. From the CEX-HPLC purity results, the CEX purity of formulation FS1-3 decreased more rapidly. No differences were observed among the formulations in the NR-CE-SDS purity, binding activity, and cell activity results. Regarding sub-visible particles, formulation FS1-4 produced more sub-visible particles, while other formulations showed no significant differences.

[0226] In summary, formulations FS1-2 and FS1-5 showed better overall performance, therefore sucrose and arginine hydrochloride were selected as stabilizers for the next round of screening experiments.

[0227] Example 2: Comparative Study of pH, Excipient Screening, and Low-Concentration Formulation

[0228] To further investigate the effects of different excipients on antibody stability, we selected one of the following excipients—sodium chloride, sucrose, arginine hydrochloride, glycine, or mannitol—for comparative testing. We examined the effects of these different excipients on stability in a pH 6.0, 20 mM histidine buffer system, a pH 5.5, 20 mM histidine buffer system, and a pH 6.0, 20 mM citrate buffer system, under an antibody toripalimab concentration of 180 mg / mL.

[0229] 2.1 Experimental Procedure

[0230] This example uses the antibody toripalimab. The sample was prepared using Millipore Pellicon3 0.11m... 2 The membrane was concentrated to approximately 180 mg / mL using UF / DF ultrafiltration. The sample was then dialyzed into the corresponding formulation shown in Table 12, and the final concentration was adjusted to approximately 180 mg / mL. The corresponding concentration of polysorbate 80(II) was then added. The solution was aseptically filled into 2R vials (2.0 mL / vial) in a laminar flow hood for stability sampling and testing.

[0231] Table 12: Prescription information from the second round of prescription screening – pH, excipient screening, and low-concentration prescription comparison study.

[0232]

[0233] Note: " / " indicates none.

[0234] 2.2 Experimental Results

[0235] 2.2.1 Appearance and Concentration Results

[0236] According to the results in Table 13, after being placed under high temperature and long-term conditions for 1 M, the protein content of all samples did not change significantly; after being placed under high temperature, accelerated or long-term conditions for 1 M, no obvious foreign matter was found in the samples, and no obvious opalescence was found; the viscosity results of FS2-2 and FS2-6 formulations were better.

[0237] Table 13: Second Round of Prescription Screening—Protein Content Data

[0238]

[0239]

[0240] 2.2.2 SEC-HPLC purity results

[0241] according to Figure 3 The SEC-HPLC purity trend chart and the SEC-HPLC purity results in Table 14 show that the SEC purity of all samples decreased after being placed under high temperature conditions for 1 M, with the SEC purity of formulations FS2-4, FS2-5, and FS2-7 decreasing more rapidly; no significant change in SEC purity was observed in any of the samples after being placed under accelerated or long-term conditions for 1 M.

[0242] Table 14: Second Round Formulation Screening—SEC-HPLC Purity Data

[0243]

[0244]

[0245] 2.2.3 R-CE-SDS Purity Results

[0246] According to the R-CE-SDS purity results in Table 15, the purity of all samples decreased after being placed at a high temperature of 40℃ for 1 M; however, no significant changes in purity were observed in any sample after being placed at an accelerated temperature of 25℃ or under long-term conditions for 1 M. There were no differences between the groups of samples.

[0247] Table 15: Second Round of Formulation Screening—R-CE-SDS Purity Data

[0248]

[0249] 2.2.4 NR-CE-SDS Purity Results

[0250] According to the NR-CE-SDS purity results in Table 16, after being placed under high temperature (40℃), accelerated temperature (25℃), or long-term conditions for 1 M, the purity of all samples did not show significant changes.

[0251] Table 16: Second Round of Formulation Screening—NR-CE-SDS Purity Data

[0252]

[0253]

[0254] 2.2.5 CEX-HPLC purity results

[0255] according to Figure 4 The CEX-HPLC purity trend chart and the CEX-HPLC results in Table 17 show that after being placed under high temperature conditions for 1 M, the CEX-HPLC purity of all samples decreased, with the purity of the FS2-4 formulation decreasing more rapidly; after being placed under accelerated and long-term conditions for 1 M, the CEX-HPLC purity of all samples did not change significantly.

[0256] Table 17: Second Round of Formulation Screening—CEX-HPLC Purity Data

[0257]

[0258]

[0259] 2.2.6 Cell viability results

[0260] According to the cell viability results in Table 18, after being placed at high temperature, accelerated conditions and long-term conditions for 1M and subjected to three and five freeze-thaw cycles, no significant changes in cell viability were observed in any of the samples.

[0261] Table 18: Second Round of Prescription Screening—Cell Viability Data

[0262]

[0263] 2.2.7 Combining activity results

[0264] According to the binding activity results in Table 19, after being placed under high temperature, accelerated conditions and long-term conditions for 1M and subjected to three and five freeze-thaw cycles, the binding activity of all samples did not show significant changes.

[0265] Table 19: Second Round of Formulation Screening—Combined with Activity Data

[0266]

[0267] 2.2.8 Results of Subvisible Particles

[0268] According to the subvisible particle results in Table 20, after being placed under accelerated or long-term conditions for 1 M, no significant changes were observed in the subvisible particles of all samples.

[0269] Table 20: Second Round of Prescription Screening—Subvisible Particle Data

[0270]

[0271]

[0272] 2.3 Conclusions of the Second Round of Prescription Screening

[0273] Based on protein content, appearance, R-CE-SDS purity results, NR-CE-SDS purity results, CEX-HPLC purity results, cell viability, binding activity results, and sub-visible particle results, no significant differences were found among the formulations. Regarding viscosity, formulations FS2-2 and FS2-6 were superior. However, based on SEC-HPLC purity results, formulations FS2-4, FS2-5, and FS2-7 showed a rapid decrease in purity. FS2-4, a low-concentration formulation originally determined for intravenous injection (CN application number 201610628048.6), was found to be unsuitable for high-concentration antibody formulations.

[0274] In summary, the final formulation selected was FS2-2 (20mM histidine buffer, pH 6.0, containing 140mM arginine hydrochloride). To maintain an osmotic pressure of around 300mOsm / kg for better use in subcutaneous injection, the formulation was further adjusted by increasing the arginine hydrochloride content to 150mM, and the Tween concentration of this formulation was further investigated.

[0275] Example 3: Surfactant Screening Experiment

[0276] Surfactants are commonly added to liquid formulations to protect proteins, such as antibodies, from air / solution interface-induced stress and solution / surface-induced stress during storage, thereby reducing antibody aggregation or minimizing particulate formation in the formulation and contributing to the stability of the antibody's physicochemical properties. The effects of different concentrations of polysorbate 80 on the stability of a formulation containing 20 mM histidine buffer and 180 mg / mL of the antibody toripalimab were investigated.

[0277] 3.1 Experimental Procedure

[0278] This example uses the antibody toripalimab. The sample was prepared using Millipore Pellicon3 0.11m... 2 The membrane was concentrated to approximately 180 mg / mL using UF / DF ultrafiltration. The sample was then dialyzed into the corresponding formulation shown in Table 21, and the final concentration was adjusted to approximately 180 mg / mL. The corresponding concentration of polysorbate 80(II) was then added. The solution was aseptically filled into 1 mL pre-filled syringes and 1.1 mL vials in a laminar flow hood for stability sampling and testing.

[0279] Table 21: Formulation Information in the Third Round Screening - Surfactant Screening Experiment

[0280]

[0281] 3.2 Experimental Results

[0282] 3.2.1 Appearance and Concentration Results

[0283] According to the results in Table 22, the protein content of all samples did not change significantly; no obvious foreign matter or obvious opalescence was found in the appearance of any of the samples.

[0284] Table 22: Third Round of Formulation Screening—Protein Content and Appearance Data

[0285]

[0286] 3.2.2 SEC-HPLC purity results

[0287] according to Figure 5 The SEC-HPLC purity trend chart and the SEC-HPLC purity results in Table 23 show that no significant changes in purity were observed in any of the samples.

[0288] Table 23: Third Round of Formulation Screening—SEC-HPLC Purity Data

[0289]

[0290]

[0291] 3.2.3 R-CE-SDS Results

[0292] According to the R-CE-SDS purity results in Table 24, no significant changes were observed in any of the samples.

[0293] Table 24: Third Round of Formulation Screening—R-CE-SDS Purity Data

[0294]

[0295] 3.2.4 NR-CE-SDS Results

[0296] According to the NR-CE-SDS purity results in Table 25, no significant changes were observed in any of the samples.

[0297] Table 25: Second Round of Formulation Screening—NR-CE-SDS Purity Data

[0298]

[0299] 3.2.5 CEX-HPLC purity results

[0300] according to Figure 6 The CEX-HPLC purity trend chart and the CEX-HPLC purity results in Table 26 show that no significant changes in purity were observed in any of the samples.

[0301] Table 26: Third Round of Formulation Screening—CEX-HPLC Purity Data

[0302]

[0303] 3.2.6 Combining activity results

[0304] According to the binding activity results in Table 27, no significant changes in activity were observed in any of the samples.

[0305] Table 27: Third Round of Formulation Screening—Combined with Activity Data

[0306]

[0307] 3.2.7 Cell viability results

[0308] According to the cell viability results in Table 28, no significant changes in viability were observed in any of the samples.

[0309] Table 28: Third Round of Prescription Screening—Cell Viability Data

[0310]

[0311] 3.2.8 Results of Subvisible Particles

[0312] According to the subvisible particle results in Table 29, no significant changes were observed in any of the samples.

[0313] Table 29: Third Round of Prescription Screening—Subvisible Particle Data

[0314]

[0315] 3.3 Conclusions of the Third Round of Prescription Screening

[0316] No significant differences were observed in the appearance, concentration, purity, activity, and subvisible particles of the samples, indicating good stability of the formulation. Tween can promote solubility, and for high-concentration formulations, increasing the Tween concentration can effectively mitigate the increase in the number of subvisible particles; therefore, a 0.04% formulation was ultimately selected.

[0317] Example 4: Experiment on Influencing Factors

[0318] 4.1 Experimental Procedure

[0319] This example uses the antibody toripalimab. The sample was prepared using Millipore Pellicon3 0.11m... 2 The membrane was ultrafiltered and concentrated to a concentration of approximately 180 mg / mL. The sample was then dialyzed into the corresponding formulation 2 shown in Table 21, and the final concentration was adjusted to approximately 180 mg / mL. Then, the corresponding concentration of polysorbate 80(II) was added. The solution was aseptically filled into 1 mL pre-filled syringes in a laminar flow hood, 1.1 mL / vial. An influencing factor experiment was conducted, and the specific procedure is shown in Table 30.

[0320] Table 30: Experimental Design of Influencing Factors

[0321]

[0322] 4.2 Experimental Results

[0323] 4.2.1 Summary of Horizontal Shaking Test Results

[0324] According to the results in Table 31, no significant changes were found in protein concentration, appearance, SEC-HPLC purity, R-CE-SDS purity, NR-CE-SDS purity, CEX-HPLC purity, binding activity, and cell viability for any of the samples.

[0325] Table 31: Summary of shaking test results

[0326]

[0327]

[0328] 4.2.2 Freeze-thaw test results

[0329] According to the results in Table 32, no significant changes were found in protein concentration, appearance, SEC-HPLC purity, R-CE-SDS purity, NR-CE-SDS purity, CEX-HPLC purity, binding activity, and cell viability for any of the samples.

[0330] Table 32: Summary of Freeze-Thaw Experiment Results

[0331]

[0332] 4.2.3 Results of the light irradiation experiment

[0333] According to the results in Table 33, no significant changes were found in protein concentration, appearance, SEC-HPLC purity, R-CE-SDS purity, NR-CE-SDS purity, CEX-HPLC purity, binding activity, and cell viability for any of the samples.

[0334] Table 33: Summary of Light Irradiation Experiment Results

[0335]

[0336]

[0337] In summary, through investigation of different buffer systems, different pH conditions, different antibody concentrations, and different excipient compositions, we determined the optimal formulation to be: 20 mM histidine buffer (pH 6.0), 150 mM arginine hydrochloride, and 0.04% polysorbate 80 (II) by controlling the target pH range of 5.9 to 6.1 and the osmotic pressure range of 260 to 320 mOsm / kg.

[0338] Example 5: Long-term stability study of the formulation

[0339] Liquid pharmaceutical products containing therapeutic antibodies typically require storage at 2-8°C, making high stability during long-term storage crucial. Based on the screening results above, the formulation used was approximately 180 mg / mL of toripalimab antibody, 20 mM histidine buffer (pH 6.0), 150 mM arginine hydrochloride, and 0.04% polysorbate 80 (II). We used this formulation for subsequent production and long-term stability studies.

[0340] Two batches of finished products were selected and stored at 2–8°C for 6 months before analysis and testing. Stability was assessed using the following parameters: (a) visual appearance; (b) pH; (c) CE-SDS (sodium dodecyl sulfate capillary electrophoresis) to determine antibody molecular weight; (d) SEC-HPLC to measure antibody monomers and aggregates; (e) CEX-HPLC to measure antibody major charge, acidic charge, or basic charge content; (f) ELISA to determine antibody binding activity; and (g) protein content.

[0341] The results showed that the two batches of finished products exhibited no significant changes in appearance, pH value, protein content, purity (SEC-HPLC, CEX-HPLC, R-CE-SDS, NR-CE-SDS), and biological activity, as detailed in Table 34. These results indicate that the two batches of finished products demonstrated excellent stability during storage at 2-8℃ for 0-6 months.

[0342] Table 34: Long-term stability data of formulations

[0343]

[0344] Example 6: Accelerated stability study of the formulation

[0345] Two batches of the finished product were selected and stored at 25±2℃ and 60±5% relative humidity (RH) for 0-6 months before analysis and testing of each sample. The formulation consisted of approximately 180 mg / mL of toripalimab antibody, 20 mM histidine buffer (pH 6.0), 150 mM arginine hydrochloride, and 0.04% polysorbate 80 (II).

[0346] As shown in Table 35, the finished product formulation has high stability against protein degradation, and the degradation kinetic parameters measured at 25±2℃ meet the requirements for storage at room temperature for up to 6 months.

[0347] Table 35: Accelerated stability data of formulations

[0348]

[0349] Example 7: Comparative pharmacokinetic study of subcutaneous and intravenous formulations in cynomolgus monkeys

[0350] 7.1 Experimental Objective

[0351] Different doses of the subcutaneous formulation of JS001 (toripalimab) were injected subcutaneously into cynomolgus monkeys. The preliminary pharmacokinetic characteristics of the subcutaneous formulation of JS001 (formulation FS2-2 in Example 2) were evaluated by detecting the drug concentration in serum. At the same time, an intravenous administration group was set up. The intravenous formulation of JS001 was formulated as follows: approximately 40 mg / mL JS001, approximately 20 mM citrate buffer (pH approximately 6.0), approximately 150 mM mannitol, approximately 50 mM sodium chloride, and approximately 0.02% polysorbate 80. The pharmacokinetic characteristics of JS001 under different routes of administration were evaluated, and the bioavailability was preliminarily investigated.

[0352] 7.2 Preparation method of the test sample

[0353] The required amount of test sample should be calculated based on the recent weight of the cynomolgus monkeys, the dosage, and the drug content. For the intravenous formulation: dilute to 0.8 mg / mL under sterile conditions using 0.9% sodium chloride injection. This experiment requires immediate intravenous infusion into the test animals over 30 minutes; a constant-rate infusion pump is recommended. For the subcutaneous formulation: dilute to the required concentration using a placebo (prepared by Junmeng), administer at a volume of 0.5 mL / kg via injection into the lateral thigh of the hind limb.

[0354] 7.3 Animal selection, dosing design, and grouping

[0355] This experiment used cynomolgus monkeys, randomly divided into groups of 3 animals each, with animal weights ranging from 2.5 to 5 kg. The grouping and drug administration details for the cynomolgus monkeys are shown in Table 36. Group B received a single dose of 4 mg / kg, administered subcutaneously as JS001; while Group A received an intravenous dose of JS001 at 4 mg / kg.

[0356] Table 36: Experimental grouping and drug administration in cynomolgus monkeys

[0357]

[0358] Note: 1. sc: subcutaneous injection; 2. iv: intravenous injection; 3. NA: none.

[0359] 7.4 Blood Sample Collection and Results

[0360] Whole blood samples (approximately 1 mL of PK blood) were collected from the vein of the non-drug-treated limb of cynomolgus monkeys. After collection, the blood samples were placed in labeled sample tubes and placed in an ice box to allow the blood to coagulate naturally. Then, the samples were centrifuged at 1500×g for 10 minutes at 2-8℃. Serum was separated and placed into EP tubes labeled with sample numbers. PK samples were aliquoted into two tubes: one for testing and one for backup. After processing, samples were stored at -60 to -90℃. Samples were transported to the bioanalytical department via cold chain logistics after collection, with relevant transportation and temperature control records maintained; samples were kept frozen during transport. Pharmacokinetic parameters were calculated using a non-compartmental model in WinNonlin v 6.4 (Pharsight Inc.) software. AUC (0-t) The calculation method is Linear Trapezoidal Linear Interpolation. Report the elimination rate constant K. el Half-life t 1 / 2 Peak time T max Peak concentration C max Drug exposure AUC (0-t) Apparent volume of distribution V d System clearance rate (CLs), mean residence time (MRT), and other parameters were recorded. The mean serum drug concentrations of the two groups are shown in Table 37, and the mean serum drug concentration-time curves for both groups are shown in [Table 37]. Figure 7 The mean serum pharmacokinetic results for the two groups are shown in Table 38.

[0361] Table 37: Mean serum drug concentrations in groups A and B

[0362]

[0363] Note: NA indicates not detected, hr indicates hours.

[0364] Table 38: Pharmacokinetic parameters of cynomolgus monkeys (mean ± standard deviation)

[0365]

[0366]

[0367] Note: NA means none, hr means hour; aa means P<0.01, aaa means P<0.001, group A vs B.

[0368] The results showed that in cynomolgus monkeys, a single subcutaneous injection of the test drug JS001 at a dose of 4 mg / kg resulted in an AUC(0-t) of 11800±700 hr*μg / mL; a single intravenous injection of the test drug JS001 at a dose of 4 mg / kg resulted in an AUC(0-t) of 11800±700 hr*μg / mL. (0-t) The concentration was 12300±1910hr*μg / mL; the bioavailability of JS001 after subcutaneous injection was 95.9%; the average blood concentration-time change curves of the test drug JS001 after subcutaneous and intravenous injection were basically consistent; therefore, the pharmacokinetic effects of developing the intravenous formulation of JS001 into a subcutaneous formulation are comparable, while the subcutaneous formulation can improve the compliance and convenience of administration for cancer patients.

[0369] Example 8: Inhibitory effect of subcutaneous injection formulation on the growth of MC38 tumors transplanted from hPD-1 humanized mice.

[0370] 8.1 Test Objective

[0371] The antitumor effect of the subcutaneous injection formulation JS001 (toripalimab) of the present invention (formulation FS2-2 in Example 2) in a mouse subcutaneous transplantation model of colon cancer MC38 was evaluated.

[0372] 8.2 Testing Process

[0373] Six- to seven-week-old female hPD-1 humanized mice (Biocytok Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with 1 x 10⁻⁶ oz. 6 MC38 cells (0.1 ml / cell). The average tumor volume was approximately 134 mm. 3 Twenty-four animals were selected and randomly divided into four groups of six animals each, based on tumor volume. The groups were a negative control group receiving normal saline and a JS001-FS2-2 treatment group receiving FS2-2 preparation at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively.

[0374] Mice were administered the drug on the same day of administration, via subcutaneous injection in the neck for all groups. The drugs were administered twice weekly for six consecutive weeks, ending the experiment three days after the last administration. Tumor volume and body weight were measured twice weekly, and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated (TGI% = [1 - (Ti - T0) / (Vi - V0)] × 100%). (Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the negative control group on day i of administration; V0: mean tumor volume in the negative control group on day 0 of administration).

[0375] The results are as follows Figure 8 As shown in the figure. Results showed that on day 21 after the start of drug administration, the mean tumor volume in the negative control group was 1501 ± 122 mm. 3 At doses of 1, 3, and 10 mg / kg, the mean tumor volume of JS001-FS2-2 was 661 ± 108 mm. 3 578±75mm 3 531±184mm 3 The TGI% were 61.5%, 67.5%, and 71.0%, respectively. This indicates that JS001-FS2-2 significantly inhibited the growth of MC38 tumor volume in hPD-1 humanized mice at doses of 1, 3, and 10 mg / kg, exhibiting a good dose-response effect.

Claims

1. A pharmaceutical composition comprising: (1) 10-30 mM of a histidine buffer; (2) an anti-PD-1 antibody or an antigen-binding fragment thereof; wherein the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a light chain variable region as set forth in SEQ ID NO: 7, and a heavy chain variable region as set forth in SEQ ID NO: 8, and the concentration of the anti-PD-1 antibody or the antigen-binding fragment thereof is 150-200 mg / mL; (3) 130-180 mM of arginine hydrochloride; and (4) 0.02-0.08% of polysorbate 80 calculated by w / v; wherein the pH of the pharmaceutical composition is 5.5-6.5, and the osmotic pressure of the pharmaceutical composition is in the range of 260-320 mOsm / kg.

2. The pharmaceutical composition of claim 1, wherein, The pH of the pharmaceutical composition is 5.5-6.

2.

3. The pharmaceutical composition of claim 2, wherein The pH of the pharmaceutical composition is 5.9-6.

1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The histidine buffer is selected from a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein The anti-PD-1 antibody comprises a light chain amino acid sequence as set forth in SEQ ID NO: 9, and a heavy chain amino acid sequence as set forth in SEQ ID NO:

10. 6.The pharmaceutical composition of any one of claims 1-3, comprising the components as set forth in (1) or (2) below, respectively: (1) (a) 180 mg / mL of an anti-PD-1 antibody comprising a light chain amino acid sequence as set forth in SEQ ID NO: 9, and a heavy chain amino acid sequence as set forth in SEQ ID NO: 10; (b) 20 mM histidine buffer, pH 6.0; (c) 140 mM arginine hydrochloride; and (d) 0.02% of polysorbate 80; or (2) (a) 180 mg / mL of an anti-PD-1 antibody comprising a light chain amino acid sequence as set forth in SEQ ID NO: 9, and a heavy chain amino acid sequence as set forth in SEQ ID NO: 10; (b) 20 mM of a histidine buffer, pH 6.0; (c) 150 mM of arginine hydrochloride; and (d) 0.04% of polysorbate 80. 7.An injection comprising the pharmaceutical composition of any one of claims 1-6 and a 0.9% w / v sodium chloride solution.

8. The injectable composition of claim 7, wherein the injectable composition is a solution. The pH of the injection is 5.5-6.

5.

9. The injectable composition of claim 8, wherein the injectable composition is a solution. The pH of the injection is 5.5-6.

2. 10.Use of the pharmaceutical composition of any one of claims 1-6 or the injection of any one of claims 7-9 in the manufacture of a medicament for treating a disease or a disorder by eliminating, inhibiting or reducing the activity of PD-1, wherein the disease or disorder is a cancer, wherein the cancer is selected from colon cancer, neuroendocrine tumor, esophageal cancer, nasopharyngeal carcinoma, sarcoma, melanoma, uroepithelial carcinoma or non-small cell lung cancer.

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