Combined pharmaceutical composition for treating colorectal cancer

Abstract

The invention belongs to the field of biological medicines, and relates to a combined pharmaceutical composition for treating colorectal cancer. The invention provides a combined medicine composition which comprises an anti-PD-L1 antibody and anlotinib, and the medicine composition has good anti-colorectal cancer activity.

Description

[0001] This application is a divisional application of the invention patent application with the invention name “Combination drug composition for treating colorectal cancer”, the application date of July 3, 2020, and the application number of 202010636341.3. Technical Field

[0002] The present application belongs to the field of biomedicine and relates to a combination drug composition for treating colorectal cancer. Background Art

[0003] Tyrosine kinases are a group of enzymes that catalyze the phosphorylation of protein tyrosine residues. They play an important role in intracellular signal transduction. They are involved in the regulation, signal transmission and development of normal cells, and are also closely related to the proliferation, differentiation, migration and apoptosis of tumor cells. Many receptor tyrosine kinases are related to the formation of tumors. According to the difference in their extracellular region structure, they can be divided into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), etc.

[0004] PD-L1 (Programmed death-ligand 1), also known as CD247 and B7-H1, is a ligand of programmed death molecule 1 (PD-1). PD-L1 is highly expressed on the surface of many tumor cells, and the malignancy of tumors and poor prognosis are closely related to the expression level of PD-L1. In the tumor microenvironment, PD-L1 on the surface of cancer cells inhibits the activation and proliferation of T cells by binding to PD-1 or CD80 on the surface of T cells, promotes the exhaustion or anergy of effector T cells, induces T cell apoptosis, and stimulates helper T cells to differentiate into regulatory T cells, thereby preventing T cells from killing tumor cells. Anti-PD-L1 antibodies can block the interaction between PD-L1, PD-1 and CD80, so that the related negative regulatory signals cannot be activated and transmitted, thereby avoiding the inhibition of the activity of effector T cells in the tumor microenvironment, so that T cells can play the role of killing and inhibiting tumor cells. Because anti-PD-L1 antibodies can act directly on tumor tissues, they have high specificity and safety.

[0005] Colorectal cancer (CRC) is one of the common malignant tumors of the digestive tract with high incidence and mortality.

[0006] WO2016022630 discloses a class of PD-L1 antibodies that have a high affinity for PD-L1, can significantly inhibit the interaction between PD-L1 and PD-1 on the cell surface, and significantly promote T cells to secrete IL-2 and INF-γ.

[0007] Although there are many treatment options for patients with proliferative diseases (cancer), there remains a need for more effective therapeutic agents for clinical use, particularly the use of more than one drug in combination. SUMMARY OF THE INVENTION

[0009] In one aspect, the present application provides a combination drug combination comprising an anti-PD-L1 antibody and anlotinib.

[0010] On the other hand, the present application provides a combination pharmaceutical composition for treating colorectal cancer, which includes an anti-PD-L1 antibody and anlotinib.

[0011] Further, anlotinib is in the form of a free base or in the form of a pharmaceutically acceptable salt thereof. For example, the pharmaceutically acceptable salt of anlotinib may be a hydrochloride or a dihydrochloride.

[0012] Further, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain CDR1 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; a heavy chain CDR2 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5; a heavy chain CDR3 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6; a light chain CDR1 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 10; a light chain CDR2 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 11; and a light chain CDR3 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 12. Furthermore, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain CDR1 region selected from SEQ ID NO: 1 or SEQ ID NO: 4; a heavy chain CDR2 region selected from SEQ ID NO: 2 or SEQ ID NO: 5; a heavy chain CDR3 region selected from SEQ ID NO: 3 or SEQ ID NO: 6; a light chain CDR1 region selected from SEQ ID NO: 7 or SEQ ID NO: 10; a light chain CDR2 region selected from SEQ ID NO: 8 or SEQ ID NO: 11; and a light chain CDR3 region selected from SEQ ID NO: 9 or SEQ ID NO: 12. Further, the anti-PD-L1 antibody comprises: a heavy chain CDR1 region having an amino acid sequence as shown in SEQ ID NO: 1, a heavy chain CDR2 region having an amino acid sequence as shown in SEQ ID NO: 2, and a heavy chain CDR3 region having an amino acid sequence as shown in SEQ ID NO: 3; and a light chain CDR1 region having an amino acid sequence as shown in SEQ ID NO: 7, a light chain CDR2 region having an amino acid sequence as shown in SEQ ID NO: 8, and a light chain CDR3 region having an amino acid sequence as shown in SEQ ID NO: 9. Further, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain variable region having at least 80% homology to the amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14; and a light chain variable region having at least 80% homology to the amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 16.Furthermore, the anti-PD-L1 antibody comprises: a variable heavy chain selected from hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1 or hu5G11-hIgG4 humanized antibody, and a variable light chain selected from hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1 or hu5G11-hIgG4 humanized antibody.

[0013] Furthermore, the above-mentioned combination pharmaceutical composition of the present application is packaged in the same kit, and the kit also includes instructions for the combined use of PD-L1 antibody and anlotinib to treat colorectal cancer.

[0014] Furthermore, the present application provides a combination pharmaceutical composition, which includes a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody and a pharmaceutical composition containing 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib in a single dose, wherein the pharmaceutical composition containing the anti-PD-L1 antibody is a single dose or multiple doses.

[0015] Furthermore, the present application provides a combination pharmaceutical composition, which includes a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody provided in a multiple-dose form and a pharmaceutical composition containing a single dose of 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib.

[0016] Furthermore, the present application provides a combination pharmaceutical composition, which is a preparation suitable for administration within a single treatment cycle (e.g., a treatment cycle of 21 days), including a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody and a pharmaceutical composition containing 84 to 168 mg of anlotinib.

[0017] Further, the present application provides a combination pharmaceutical composition, comprising an anti-PD-L1 antibody and anlotinib in a weight ratio of (0.35-29):1, preferably (3.5-29):1, more preferably (3.5-14.5):1, and most preferably (7-14.5):1. Wherein, the anti-PD-L1 antibody and anlotinib can be packaged separately or together. And wherein, anlotinib can be packaged in multiple equal parts (e.g., 2 equal parts, 7 equal parts, 14 equal parts, 28 equal parts or more equal parts); the anti-PD-L1 antibody can be packaged in a single equal part or multiple equal parts (e.g., 2 equal parts, 4 equal parts or more equal parts).

[0018] On the other hand, the present application also provides the use of the combined pharmaceutical composition in the preparation of a drug for treating colorectal cancer. The present application also provides a method for treating colorectal cancer, which comprises administering an effective amount of the combined pharmaceutical composition of the present application to an individual in need. The present application also provides the use of the combined pharmaceutical composition for treating colorectal cancer. The combined pharmaceutical composition comprises an anti-PD-L1 antibody and anlotinib.

[0019] On the other hand, the present application also provides the use of an anti-PD-L1 antibody and anlotinib in combination for the preparation of a drug for treating colorectal cancer. The present application also provides a method for treating colorectal cancer, comprising administering an effective amount of an anti-PD-L1 antibody and anlotinib to an individual in need. The present application also provides the use of an anti-PD-L1 antibody and anlotinib in combination for the treatment of colorectal cancer. Alternatively, the present application also provides a combination of an anti-PD-L1 antibody and anlotinib for the treatment of colorectal cancer.

[0020] Further, the anti-PD-L1 antibody and anlotinib are each in the form of a pharmaceutical composition, which can be administered simultaneously, sequentially or intermittently. Further, the anti-PD-L1 antibody is administered once a week, every 2 weeks, every 3 weeks, or every 4 weeks; preferably, the anti-PD-L1 antibody is administered at a dose of 600 to 2400 mg each time. Further, anlotinib is administered at a dose of 6 mg, 8 mg, 10 mg or 12 mg once a day, for 2 consecutive weeks and 1 week of rest.

[0021] In addition, the present application provides a kit for treating colorectal cancer, the kit comprising a pharmaceutical composition of an anti-PD-L1 antibody and a pharmaceutical composition of anlotinib, and instructions for using the anti-PD-L1 antibody and anlotinib in combination to treat colorectal cancer.

[0022] Furthermore, the above-mentioned kit is a kit suitable for administration within a single treatment cycle (e.g., a treatment cycle of 21 days), comprising a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody and a pharmaceutical composition containing 84 to 168 mg of anlotinib. Summary of the invention

[0023] In one aspect, the present application provides a combination drug combination comprising an anti-PD-L1 antibody and anlotinib.

[0024] On the other hand, the present application provides a combination pharmaceutical composition for treating colorectal cancer, which includes an anti-PD-L1 antibody and anlotinib.

[0025] In some embodiments of the present application, the combination pharmaceutical composition includes a pharmaceutical composition of an anti-PD-L1 antibody and a pharmaceutical composition of anlotinib.

[0026] In some embodiments of the present application, the combination pharmaceutical composition is packaged in the same kit, which also includes instructions for the combined use of PD-L1 antibody and anlotinib to treat colorectal cancer.

[0027] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, comprising a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody and 6 to 12 mg of anlotinib, wherein the pharmaceutical composition is a single dose or multiple doses.

[0028] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, comprising a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody and a pharmaceutical composition in a single dose form containing 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib, wherein the pharmaceutical composition of the anti-PD-L1 antibody is a single dose or multiple doses.

[0029] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, comprising a pharmaceutical composition in the form of multiple doses containing 600 to 2400 mg of an anti-PD-L1 antibody and a pharmaceutical composition in the form of a single dose containing 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib.

[0030] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, comprising an anti-PD-L1 antibody and anlotinib in a weight ratio of (0.35-29):1, preferably (3.5-29):1, more preferably (3.5-14.5):1, and most preferably (7-14.5):1. Wherein, the anti-PD-L1 antibody and anlotinib can be packaged separately or together. And wherein, anlotinib can be packaged in multiple equal parts (e.g., 2 equal parts, 7 equal parts, 14 equal parts, 28 equal parts or more equal parts).

[0031] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, comprising a pharmaceutical composition of an anti-PD-L1 antibody and a pharmaceutical composition of anlotinib, wherein the anti-PD-L1 antibody is prepared to be suitable for administering 600 to 2400 mg of the anti-PD-L1 antibody to an individual in need in the form of a single dose or multiple doses each time, and the pharmaceutical composition of anlotinib is prepared to be suitable for administering 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib to an individual in need in the form of a single dose every day for 14 consecutive days.

[0032] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, which includes an anti-PD-L1 antibody pharmaceutical composition having an anti-PD-L1 antibody concentration of 10-60 mg / mL and a pharmaceutical composition in single-dose form containing 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib.

[0033] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, which includes an anti-PD-L1 antibody pharmaceutical composition having an anti-PD-L1 antibody concentration of 10 mg / mL and a pharmaceutical composition in single-dose form containing 8 mg and / or 10 mg and / or 12 mg of anlotinib.

[0034] In some embodiments, a combination pharmaceutical composition for treating colorectal cancer is provided, comprising a pharmaceutical composition in the form of multiple doses containing 1200 mg of an anti-PD-L1 antibody and a pharmaceutical composition in the form of a single dose containing 8 mg and / or 10 mg and / or 12 mg of anlotinib.

[0035] On the other hand, the present application also provides the use of the combined pharmaceutical composition in the preparation of a drug for treating colorectal cancer. The present application also provides a method for treating colorectal cancer, which comprises administering an effective amount of the combined pharmaceutical composition of the present application to an individual in need. The present application also provides the use of the combined pharmaceutical composition for treating colorectal cancer. The combined pharmaceutical composition comprises an anti-PD-L1 humanized monoclonal antibody and anlotinib.

[0036] On the other hand, the present application also provides the use of a combination of an anti-PD-L1 antibody and anlotinib in the preparation of a drug for treating colorectal cancer. The present application also provides a method for treating colorectal cancer, comprising administering an effective amount of a combination of an anti-PD-L1 antibody and anlotinib to an individual in need. The present application also provides the use of a combination of an anti-PD-L1 antibody and anlotinib in the treatment of colorectal cancer.

[0037] On the other hand, the present application also provides the use of anti-PD-L1 antibodies and anlotinib in the preparation of a drug for treating colorectal cancer. The present application also provides a method for treating colorectal cancer, comprising administering an effective amount of anti-PD-L1 antibodies and anlotinib to an individual in need. The present application also provides the use of anti-PD-L1 antibodies and anlotinib in the treatment of colorectal cancer.

[0038] On the other hand, the present application provides a kit for treating colorectal cancer, comprising a pharmaceutical composition of an anti-PD-L1 antibody and a pharmaceutical composition of anlotinib, and instructions for using the anti-PD-L1 antibody and anlotinib in combination to treat colorectal cancer.

[0039] In another aspect, the present application also provides an anti-PD-L1 antibody for treating colorectal cancer. The present application also provides a method for treating colorectal cancer, comprising administering an effective amount of the anti-PD-L1 antibody of the present application to an individual in need. The present application also provides the use of the anti-PD-L1 antibody for treating colorectal cancer. The present application also provides the use of the anti-PD-L1 antibody in the preparation of a medicament for treating colorectal cancer.

[0040] Dosage / treatment regimen of combined pharmaceutical compositions

[0041] In one aspect, the present invention provides a combination pharmaceutical composition comprising an anti-PD-L1 antibody and anlotinib.

[0042] In some embodiments of the present application, in the above-mentioned uses or treatment methods, the anti-PD-L1 antibody and anlotinib are each in the form of a pharmaceutical composition, which can be administered simultaneously, sequentially or intermittently.

[0043] In some embodiments of the present application, in the above-mentioned uses or treatment methods, the anti-PD-L1 antibody and anlotinib are each administered in an intermittent manner. In some embodiments, the antibody and anlotinib are administered in the same or different dosing regimens. In some embodiments, they are administered in different dosing regimens.

[0044] In some embodiments of the present application, the use or treatment method, the anti-PD-L1 antibody can be administered once a week (q1w), every 2 weeks (q2w), every 3 weeks (q3w), or every 4 weeks (q4w). In a specific embodiment, the anti-PD-L1 antibody is administered once every 3 weeks. In some embodiments, the anti-PD-L1 antibody is administered at a dose of 600 to 2400 mg each time.

[0045] The anlotinib can be administered at a dosage of 6 mg, 8 mg, 10 mg or 12 mg once daily, with a continuous medication schedule of 2 weeks and a 1-week break.

[0046] In some embodiments, the anti-PD-L1 antibody and anlotinib have the same or different treatment cycles, respectively. In some specific embodiments, the anti-PD-L1 antibody and anlotinib have the same treatment cycle, for example, one treatment cycle is every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

[0047] In some embodiments of the present application, in the use or treatment method, 21 days is a treatment cycle, PD-L1 antibody is administered on the first day of each cycle, and anlotinib is administered every day on days 1-14 of each cycle. In a specific embodiment, PD-L1 antibody is administered once on the first day of each cycle, and anlotinib is administered once a day on days 1-14 of each cycle.

[0048] In some embodiments of the present application, in the use or treatment method, the anti-PD-L1 antibody may include 0.01 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 15 mg / kg, 0.1 to 10 mg / kg, 1 to 15 mg / kg, 1 to 20 mg / kg, 1 to 3 mg / kg, 3 to 10 mg / kg, 3 to 15 mg / kg, 3 to 20 mg / kg , 3 to 30 mg / kg, 10 to 20 mg / kg, or 15 to 20 mg / kg; or 60 mg to 2400 mg, 90 mg to about 1800 mg, 120 mg to 1500 mg, 300 mg to 900 mg, 600 mg to 900 mg, 300 mg to 1200 mg, 600 mg to 1200 mg, or 900 mg to 1200 mg.

[0049] In some embodiments of the use or treatment method, 21 days is a treatment cycle, 1200 mg of PD-L1 antibody is administered on the first day of each cycle, and 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib is administered daily on days 1 to 14 of each cycle.

[0050] In some embodiments of the present application, in one treatment cycle every three weeks, an anti-PD-L1 antibody and anlotinib are administered to an individual in need at a weight ratio of (0.35-29):1, preferably (3.5-29):1, more preferably (3.5-14.5):1, and most preferably (7-14.5):1, wherein the anti-PD-L1 antibody and anlotinib are administered in a single dose and multiple doses, respectively.

[0051] Anti-PD-L1 antibody pharmaceutical composition

[0052] In some embodiments of the present application, the single dose or single dose form of the anti-PD-L1 antibody pharmaceutical composition comprises 300 mg or 600 mg of the anti-PD-L1 antibody.

[0053] In some embodiments of the present application, the total dose of the anti-PD-L1 antibody pharmaceutical composition is 600 to 2400 mg. In some embodiments, the total dose of the anti-PD-L1 antibody pharmaceutical composition includes an anti-PD-L1 antibody selected from 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, or a range formed by any of the above values. In some embodiments, the total dose of the anti-PD-L1 antibody pharmaceutical composition is preferably 600 to 2100 mg, or 900 mg to 1500 mg of anti-PD-L1 antibody.

[0054] In some embodiments of the present application, the anti-PD-L1 antibody pharmaceutical composition comprises one or more of a buffer, an isotonicity regulator, a stabilizer and / or a surfactant. In particular, the anti-PD-L1 antibody pharmaceutical composition comprises 1-150 mg / mL anti-PD-L1 antibody (monoclonal antibody), 3-50 mM buffer, 2-150 mg / mL isotonicity regulator / stabilizer and 0.01-0.8 mg / mL surfactant, and the pH is about 4.0-6.8.

[0055] In some embodiments of the present application, the anti-PD-L1 antibody pharmaceutical composition is calculated by w / v, and the anti-PD-L1 monoclonal antibody concentration is about 5-150 mg / mL; preferably about 10-60 mg / mL; more preferably about 10-30 mg / mL. In some specific embodiments, the mass volume concentration of the anti-PD-L1 monoclonal antibody is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL or about 120 mg / mL, preferably about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL or about 60 mg / mL, more preferably about 10 mg / mL, about 20 mg / mL or about 30 mg / mL. In some embodiments, the anti-PD-L1 monoclonal antibody has a mass volume concentration of about 10 mg / mL. In other embodiments, the anti-PD-L1 monoclonal antibody has a mass volume concentration of about 30 mg / mL. In other embodiments, the anti-PD-L1 monoclonal antibody has a mass volume concentration of about 60 mg / mL.

[0056] In some embodiments of the present application, the buffer is a histidine salt buffer. The histidine salt buffer concentration is about 5-30mM, preferably about 10-25mM, more preferably about 10-20mM, and most preferably about 10-15mM. In some specific embodiments, the histidine salt buffer is about 5mM, about 10mM, about 15mM, about 20mM, about 25mM or about 30mM. In some embodiments, the histidine salt buffer is about 10mM. In other embodiments, the histidine salt buffer is about 15mM. In other embodiments, the histidine salt buffer is about 20mM. Wherein, the histidine salt buffer comprises histidine and hydrochloric acid.

[0057] In some embodiments of the present application, calculated by w / v, the isotonic regulator / stabilizer is about 20-150mg / mL of sucrose, preferably about 40-100mg / mL of sucrose, more preferably about 60-80mg / mL of sucrose. In some specific schemes, the concentration of the sucrose is about 40mg / mL, 50mg / mL, 60mg / mL, 70mg / mL, 80mg / mL, 90mg / mL or 100mg / mL. In some specific embodiments, the concentration of the sucrose is about 60mg / mL. In some specific embodiments, the concentration of the sucrose is about 70mg / mL. In some specific embodiments, the concentration of the sucrose is about 80mg / mL. In some specific embodiments, the concentration of the sucrose is about 90mg / mL.

[0058] In some embodiments of the present application, the surfactant is selected from polysorbate 80, polysorbate 20, and poloxamer 188; preferably polysorbate 80 or polysorbate 20; more preferably polysorbate 80. In some embodiments, the concentration of the surfactant is about 0.05-0.6 mg / mL, preferably about 0.1-0.4 mg / mL, and more preferably about 0.2-0.3 mg / mL, calculated by w / v.

[0059] In some embodiments of the present application, the surfactant is about 0.01-0.8 mg / mL of polysorbate 80 or polysorbate 20, calculated on a w / v basis. In some specific embodiments, the surfactant is about 0.05-0.6 mg / mL of polysorbate 80, preferably about 0.1-0.4 mg / mL of polysorbate 80, more preferably about 0.2-0.3 mg / mL of polysorbate 80, and most preferably about 0.2 mg / mL of polysorbate 80. In some embodiments, the content of polysorbate 80 in the pharmaceutical composition is about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL or 0.6 mg / mL; preferably, the content of polysorbate 80 in the pharmaceutical composition is about 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL or 0.5 mg / mL; more preferably, the content of polysorbate 80 in the pharmaceutical composition is about 0.2 mg / mL, 0.3 mg / mL or 0.4 mg / mL; optimally, the content of polysorbate 80 in the pharmaceutical composition is about 0.2 mg / mL. In some embodiments, the content of polysorbate 80 in the pharmaceutical composition is about 0.1 mg / mL. In other embodiments, the content of polysorbate 80 in the pharmaceutical composition is about 0.2 mg / mL. In some embodiments, the content of polysorbate 80 in the pharmaceutical composition is about 0.3 mg / mL. In other embodiments, the pharmaceutical composition contains about 0.4 mg / mL of polysorbate 80. In some embodiments, the pharmaceutical composition contains about 0.5 mg / mL of polysorbate 80.

[0060] In some embodiments of the present application, the pharmaceutical composition is an aqueous solution. In some embodiments, the pH value of the aqueous solution is selected from 4.0-6.8; preferably 4.5-6.5; more preferably 5.5-6.0; most preferably 5.5. In some embodiments, the pH value of the aqueous solution is about 4.5, about 4.8, about 5.0, about 5.2, about 5.4, about 5.5, about 5.6, about 5.8 or about 6.0, preferably about 5.0, about 5.2, about 5.4, about 5.5 or about 5.6, more preferably about 5.5. In some embodiments, the pH value of the aqueous solution is about 5.0. In some embodiments, the pH value of the aqueous solution is about 5.2. In some embodiments, the pH value of the aqueous solution is about 5.4. In some embodiments, the pH value of the aqueous solution is about 5.5. In some embodiments, the pH value of the aqueous solution is about 5.6. In some embodiments, the pH value of the aqueous solution is about 5.8. In some embodiments, the pH value of the aqueous solution is about 6.0.

[0061] In some specific embodiments of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody at a mass volume concentration of about 20 mg / mL, (b) sucrose at a mass volume concentration of about 70 mg / mL, (c) polysorbate 80 at a mass volume concentration of about 0.1 mg / mL, (d) histidine at a molar concentration of about 20 mM, (e) an appropriate amount of optional hydrochloric acid, and the pH value of the composition is adjusted to about 5.0. In a specific embodiment of the present invention, the pharmaceutical composition comprises: (a) an anti-PD-L1 monoclonal antibody at a mass volume concentration of about 20 mg / mL, (b) sucrose at a mass volume concentration of about 70 mg / mL, (c) polysorbate 80 at a mass volume concentration of about 0.1 mg / mL, (d) histidine at a molar concentration of about 20 mM, (e) an appropriate amount of optional hydrochloric acid, and the pH value of the composition is adjusted to about 5.0.

[0062] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody at a mass volume concentration of approximately 10 mg / mL, (b) sucrose at a mass volume concentration of approximately 80 mg / mL, (c) polysorbate 80 at a mass volume concentration of approximately 0.2 mg / mL, (d) histidine at a molar concentration of approximately 10 mM, and (e) optionally an appropriate amount of hydrochloric acid to adjust the pH of the composition to approximately 5.5.

[0063] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody having a mass volume concentration of approximately 50 mg / mL, (b) sucrose having a mass volume concentration of approximately 80 mg / mL, (c) polysorbate 80 having a mass volume concentration of approximately 0.3 mg / ml, (d) histidine having a molar concentration of approximately 10 mM, and (e) optionally an appropriate amount of hydrochloric acid to adjust the pH value of the composition to approximately 5.5.

[0064] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody having a mass volume concentration of approximately 100 mg / mL, (b) sucrose having a mass volume concentration of approximately 80 mg / mL, (c) polysorbate 80 having a mass volume concentration of approximately 0.5 mg / mL, (d) histidine having a molar concentration of approximately 10 mM, and (e) optionally an appropriate amount of hydrochloric acid to adjust the pH value of the composition to approximately 5.5.

[0065] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody at a mass volume concentration of approximately 30 mg / mL, (b) sucrose at a mass volume concentration of approximately 80 mg / mL, (c) polysorbate 80 at a mass volume concentration of approximately 0.2 mg / mL, (d) histidine at a molar concentration of approximately 10 mM, and (e) optionally an appropriate amount of hydrochloric acid to adjust the pH of the composition to approximately 5.5.

[0066] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody having a mass volume concentration of approximately 60 mg / mL, (b) sucrose having a mass volume concentration of approximately 80 mg / mL, (c) polysorbate 80 having a mass volume concentration of approximately 0.2 mg / mL, (d) histidine having a molar concentration of approximately 10 mM, and (e) optionally an appropriate amount of hydrochloric acid to adjust the pH value of the composition to approximately 5.5.

[0067] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 antibody having a mass volume concentration of approximately 10 mg / mL, (b) sucrose having a mass volume concentration of approximately 70 mg / mL, (c) polysorbate 80 having a mass volume concentration of approximately 0.4 mg / mL, (d) histidine having a molar concentration of approximately 20 mM, and (e) an appropriate amount of acetic acid, and the pH value of the composition is adjusted to approximately 6.5.

[0068] In another specific embodiment of the present application, the pharmaceutical composition comprises: (a) an anti-PD-L1 monoclonal antibody having a mass volume concentration of approximately 10 mg / mL, (b) sucrose having a mass volume concentration of approximately 80 mg / mL, (c) polysorbate 80 having a mass volume concentration of approximately 0.2 mg / mL, (d) histidine having a molar concentration of approximately 20 mM, and (e) optionally an appropriate amount of hydrochloric acid to adjust the pH value of the composition to approximately 5.5.

[0069] In another specific embodiment of the present application, the pharmaceutical composition is a water-soluble injection, and the water-soluble injection includes but is not limited to a water-soluble preparation that has not been lyophilized or a water-soluble preparation reconstituted by a lyophilized powder. In some embodiments, the pharmaceutical composition is an aqueous solution. In other schemes, the pharmaceutical composition is a lyophilized preparation. The lyophilized preparation refers to an aqueous solution undergoing a lyophilization process to prepare a preparation, and lyophilization is a stabilization process in which the substance is first frozen, and then the amount of solvent is first reduced by sublimation (primary drying process), and then the amount of solvent is reduced by desorption (secondary drying process) until the amount of solvent is a value that no longer supports biological activity or chemical reaction. The lyophilized preparation of the present invention can also be dried by other methods known in the art, such as spray drying and bubbling drying (bubbledrying).

[0070] Anlotinib pharmaceutical composition

[0071] In some embodiments of the present application, the single dose or single dose form of anlotinib pharmaceutical composition comprises 6 mg, 8 mg, 10 mg, or 12 mg of anlotinib.

[0072] In some embodiments of the present application, according to the treatment cycle of 2 weeks of administration and 1 week of rest, the total dose of the anlotinib pharmaceutical composition administered in each cycle includes 84 to 168 mg. In some schemes, the total dose of the anlotinib pharmaceutical composition includes a range selected from 84 mg, 112 mg, 140 mg, 168 mg or any of the above values. In some schemes, the total dose of the anlotinib pharmaceutical composition preferably includes 112 mg to 168 mg.

[0073] Anti-PD-L1 antibodies

[0074] In some embodiments of the present application, the anti-PD-L1 antibody is the antibody in WO2016022630 or CN107001463A.

[0075] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain CDR1 region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; a heavy chain CDR2 region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5; : a heavy chain CDR3 region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous to the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:6; a light chain CDR1 region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:10; NO:11 has a light chain CDR2 region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous to the amino acid sequence shown in SEQ ID NO:11; and a light chain CDR3 region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous to the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:12.

[0076] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain CDR1 region selected from SEQ ID NO: 1 or SEQ ID NO: 4; a heavy chain CDR2 region selected from SEQ ID NO: 2 or SEQ ID NO: 5; a heavy chain CDR3 region selected from SEQ ID NO: 3 or SEQ ID NO: 6; a light chain CDR1 region selected from SEQ ID NO: 7 or SEQ ID NO: 10; a light chain CDR2 region selected from SEQ ID NO: 8 or SEQ ID NO: 11; a light chain CDR3 region selected from SEQ ID NO: 9 or SEQ ID NO: 12.

[0077] In some embodiments of the present application, the isolated anti-PD-L1 antibody described herein comprises: a heavy chain CDR1 region having an amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 region having an amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 region having an amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 region having an amino acid sequence shown in SEQ ID NO: 7, a light chain CDR2 region having an amino acid sequence shown in SEQ ID NO: 8, and a light chain CDR3 region having an amino acid sequence shown in SEQ ID NO: 9.

[0078] Each CDR region described herein and its various variants described above can specifically recognize and bind to PD-L1, thereby effectively blocking the signal transduction between PD-L1 and PD-1.

[0079] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain variable region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous to the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14; and a light chain variable region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous to the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0080] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain variable region as shown in SEQ ID NO: 13; and a light chain variable region as shown in SEQ ID NO: 15.

[0081] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain variable region as shown in SEQ ID NO: 14; and a light chain variable region as shown in SEQ ID NO: 16.

[0082] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain amino acid sequence as shown in SEQ ID NO: 17; and a light chain amino acid sequence as shown in SEQ ID NO: 18.

[0083] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain amino acid sequence as shown in SEQ ID NO: 19; and a light chain amino acid sequence as shown in SEQ ID NO: 20.

[0084] In some embodiments of the present application, the anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain amino acid sequence as shown in SEQ ID NO:21; and a light chain amino acid sequence as shown in SEQ ID NO:18.

[0085] In a specific embodiment, the anti-PD-L1 humanized monoclonal antibody provided herein comprises a conservative substitution variant selected from one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21. The anti-PD-L1 humanized monoclonal antibody comprising the conservative substitution variant retains the ability to specifically recognize and bind to PD-L1.

[0086] In some embodiments of the present application, the anti-PD-L1 antibody may be an IgG1 or IgG4 antibody.

[0087] In some embodiments of the present application, the anti-PD-L1 antibody is an IgG1 antibody. In some embodiments, the anti-PD-L1 antibody is a glycosylated IgG1 antibody.

[0088] In some embodiments of the present application, the anti-PD-L1 antibody comprises a heavy chain complementary determining region (CDR) selected from 13C5 or 5G11 antibodies, and a light chain complementary determining region selected from 13C5 or 5G11 antibodies. In one embodiment, the anti-PD-L1 antibody of the present invention comprises a variable heavy chain selected from ch5G11-hIgG1, ch5G11-hIgG4, ch13C5-hIgG1, ch13C5-hIgG4 chimeric antibodies, and a variable light chain selected from ch5G11-hIgG1, ch5G11-hIgG4, ch13C5-hIgG1, ch13C5-hIgG4 chimeric antibodies. In one embodiment, the anti-PD-L1 antibody of the present invention comprises a variable heavy chain selected from a humanized antibody of hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1 or hu5G11-hIgG4, and a variable light chain selected from a humanized antibody of hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1 or hu5G11-hIgG4. Reference may be made to the records in patent documents WO2016022630 or CN107001463A: the HCDR1 sequence of 13C5, ch13C5-hIgG1, ch13C5-hIgG4, hu13C5-hIgG1 or hu13C5-hIgG4 is SYGMS (SEQ ID NO: 4), the HCDR2 sequence is SISSGGSTYYPDSVKG (SEQ ID NO: 5), the HCDR3 sequence is GYDSGFAY (SEQ ID NO: 6), the LCDR1 sequence is ASQSVSTSSSSFMH (SEQ ID NO: 10), the LCDR2 sequence is YASNLES (SEQ ID NO: 11), and the LCDR3 sequence is QHSWEIPYT (SEQ ID NO: 12). NO:12); the HCDR1 sequence of 5G11, ch5G11-hIgG1, ch5G11-hIgG4, hu5G11-hIgG1 or hu5G11-hIgG4 is TYGVH (SEQ ID NO:1), the HCDR2 sequence is VIWRGVTTDYNAAFMS (SEQ ID NO:2), the HCDR3 sequence is LGFYAMDY (SEQ ID NO:3), the LCDR1 sequence is KASQSVSNDVA (SEQ ID NO:7), the LCDR2 sequence is YAANRYT (SEQ ID NO:8), and the LCDR3 sequence is QQDYTSPYT (SEQ ID NO:9).

[0089] In some embodiments of the present application, the anti-PD-L1 antibody in the drug combination may be selected from one or more. As used in the present application, the term "multiple" may be more than one, for example, two, three, four, five or more. For example, in some embodiments of the present application, the anti-PD-L1 antibody is selected from a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 15, or selected from a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 16, or selected from a combination of the above. For another example, the anti-PD-L1 antibody is selected from a heavy chain amino acid sequence as shown in SEQ ID NO: 17 and a light chain amino acid sequence as shown in SEQ ID NO: 18, or selected from a heavy chain amino acid sequence as shown in SEQ ID NO: 19 and a light chain amino acid sequence as shown in SEQ ID NO: 20, or selected from a heavy chain amino acid sequence as shown in SEQ ID NO: 21 and a light chain amino acid sequence as shown in SEQ ID NO: 18, or selected from a combination of the above optional multiple.

[0090] Anlotinib

[0091] As used in this application, the chemical name of Anlotinib is 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropylamine, which has the following structural formula:

[0092]

[0093] As used in this application, the anlotinib includes its non-salt form (e.g., free base) and its pharmaceutically acceptable salt, and the non-salt form or salt is included in the protection scope of this application. For example, the pharmaceutically acceptable salt of anlotinib can be a hydrochloride or a dihydrochloride. The dosage of anlotinib or its salt involved in this application, unless otherwise specified, is based on the molecular weight of anlotinib free base.

[0094] Colorectal cancer

[0095] In some embodiments of the present application, the colorectal cancer is selected from colon cancer or rectal cancer.

[0096] Mode of administration

[0097] The following contents do not limit the administration method of the drug combination of the present application.

[0098] The components of the pharmaceutical composition of the present application can be administered independently, or part or all of them can be administered together in various suitable ways, including, but not limited to, oral or parenteral (by intravenous, intramuscular, topical or subcutaneous route). In some embodiments, the components of the pharmaceutical combination of the present application can be administered independently, or part or all of them can be administered together orally or by injection, such as intravenous injection or intraperitoneal injection.

[0099] The components in the pharmaceutical composition of the present application can be each independently, or some or all of them together can be in suitable dosage forms, including, but not limited to, tablets, lozenges, pills, capsules (such as hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions and sustained-release preparations for oral or parenteral administration.

[0100] The components in the pharmaceutical combination of the present application may contain pharmaceutically acceptable carriers and / or excipients independently of each other, or part or all of them may contain pharmaceutically acceptable carriers and / or excipients together.

[0101] The pharmaceutical combination of the present application may further comprise an additional therapeutic agent. In one embodiment, the additional therapeutic agent may be a small cell cancer therapeutic agent known in the art.

[0102] Technical Effects

[0103] The use of the above-mentioned combined pharmaceutical composition of the present application is helpful for:

[0104] (1) Producing a better therapeutic effect in reducing tumor growth or even eliminating tumors compared to administering any of the drugs in the combination alone;

[0105] (2) provide for administration of a smaller amount of the drug in the combination than when either drug is administered alone;

[0106] (3) provide a treatment that is well tolerated in patients and has fewer adverse effects and / or complications than either drug given alone;

[0107] (4) provide better disease control rates among treated patients;

[0108] (5) provide longer survival (e.g., median survival, progression-free survival, or overall survival) among treated patients;

[0109] (6) Providing a longer survival (e.g., median survival, progression-free survival, or overall survival) for the treated patients compared to standard chemotherapy;

[0110] (7) provide a longer duration of disease remission (DOR); and / or

[0111] (8) Compared with the single administration of any drug in the combination, the combination has good inhibitory activity against colorectal cancer and exhibits a more excellent anti-tumor synergistic effect.

[0112] Definition and Description

[0113] Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this application, it is intended to refer to the corresponding commodity or its active ingredient.

[0114] As used herein, the term "combination pharmaceutical composition" refers to a combination of two or more active ingredients (administered in the form of each active ingredient itself, or in the form of its own pharmaceutically acceptable salt or ester derivatives, prodrugs or compositions) administered simultaneously or sequentially. In this article, the terms "combination pharmaceutical composition" and "drug combination" are used interchangeably.

[0115] As used herein, the term "antibody" refers to a binding protein having at least one antigen binding domain. The antibodies and fragments thereof of the present invention may be whole antibodies or any fragments thereof. Therefore, the antibodies and fragments of the present invention include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, as well as immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab)' fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv), and other antibody fragments known in the art. Antibodies and fragments thereof may also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The anti-PD-L1 antibodies and fragments thereof disclosed herein may be of IgG1, IgG2, IgG3, or IgG4 isotypes. The term "isotype" refers to the antibody species encoded by the heavy chain constant region gene. In one embodiment, the anti-PD-L1 antibodies and fragments thereof disclosed herein are of IgG1 or IgG4 isotypes. The PD-L1 antibodies and fragments thereof of the present invention may be derived from any species, including but not limited to mice, rats, rabbits, primates, llamas, and humans. The PD-L1 antibody and its fragments can be chimeric antibodies, humanized antibodies or complete human antibodies. In one embodiment, the anti-PD-L1 antibody is an antibody produced by a hybridoma cell line derived from a mouse. Therefore, in one embodiment, the anti-PD-L1 antibody is a murine antibody. In another embodiment, the anti-PD-L1 antibody is a chimeric antibody. In another embodiment, the chimeric antibody is a mouse-human chimeric antibody. In another embodiment, the antibody is a humanized antibody. In another embodiment, the antibody is derived from a murine antibody and is humanized.

[0116] "Humanized antibodies" are antibodies that contain complementary determining regions (CDRs) derived from non-human antibodies; and framework regions and constant regions derived from human antibodies. For example, the anti-PD-L1 antibodies provided herein may include CDRs derived from one or more murine antibodies and human framework regions and constant regions. Therefore, in one embodiment, the humanized antibodies provided herein bind to the same epitope on PD-L1 as the murine antibodies from which the CDRs of the antibodies are derived. Exemplary humanized antibodies are provided herein. Additional anti-PD-L1 antibodies or variants thereof comprising heavy chain CDRs and light chain CDRs provided herein can be produced using any human framework sequence and are also included in the present invention. In one embodiment, framework sequences suitable for use in the present invention include those framework sequences that are structurally similar to the framework sequences provided herein. Additional modifications may be made in the framework region to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications; point mutations to reduce immunogenicity or remove T cell epitopes; or mutations returned to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including back mutations to germline sequences. For example, in one embodiment, one or more amino acids in the human framework region of the VH and / or VL of the humanized antibodies provided herein are back-mutated to the corresponding amino acids in the parent murine antibody. For example, for the VH and VL of humanized 5G11 and humanized 13C5, several sites of the framework amino acids of the above-mentioned template human antibodies are back-mutated to the corresponding amino acid sequences in mouse 5G11 and 13C5 antibodies. In one embodiment, the amino acids at positions 53 and / or 60 and / or 67 of the light chain variable region are back-mutated to the corresponding amino acids found at the positions in the light chain variable regions of mouse 5G11 or 13C5. In another embodiment, the amino acids at positions 24 and / or 28 and / or 30 and / or 49 and / or 73 and / or 83 and / or 94 of the heavy chain variable region are back-mutated to the corresponding amino acids found at the positions in the heavy chain variable regions of mouse 5G11 or 13C5. In one embodiment, the humanized 5G11 antibody comprises a light chain variable region in which the amino acid at position 60 is mutated from Ser (S) to Asp (D), and the amino acid at position 67 is mutated from Ser (S) to Tyr (Y); and a heavy chain variable region in which the amino acid at position 24 is mutated from Phe (F) to Val (V), the amino acid at position 49 is mutated from Ala (A) to Gly (G), the amino acid at position 73 is mutated from Thr (T) to Asn (N), and the amino acid at position 83 is mutated from Thr (T) to Asn (N).In one embodiment, the humanized 13C5 antibody comprises a light chain variable region in which the amino acid at position 53 is mutated from Tyr (Y) to Lys (K); and a heavy chain variable region in which the amino acid at position 28 is mutated from Thr (T) to Ile (I), the amino acid at position 30 is mutated from Ser (S) to Arg (R), the amino acid at position 49 is mutated from Ser (S) to Ala (A), and the amino acid at position 94 is mutated from Tyr (Y) to Asp (D). Additional or alternative back mutations can be made in the framework region of the humanized antibodies provided herein to improve the properties of the antibodies. The present invention also includes humanized antibodies that bind to PD-L1 and include framework modifications corresponding to the exemplary modifications described herein relative to any suitable framework sequence, as well as other framework modifications that otherwise improve antibody properties.

[0117] The present invention provides an isolated antibody or fragment thereof that binds to PD-L1, wherein the antibody is produced by a hybridoma selected from the group consisting of hybridomas referred to herein as 13C5 and 5G11. Therefore, the present invention also includes hybridomas 13C5, 5G11, and any hybridoma that produces the antibodies disclosed herein. The present invention also provides isolated polynucleotides encoding antibodies and fragments thereof provided herein. The present invention also includes expression vectors comprising isolated polynucleotides, and host cells comprising the expression vectors.

[0118] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity with other antigens (such as PD-1 molecules from different species). In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0119] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition (i.e., antibody molecules whose primary sequences are substantially identical and which exhibit a single binding specificity and affinity for a particular epitope). mAb is an example of an isolated antibody. mAbs can be produced by hybridoma technology, recombinant technology, transgenic technology or other techniques known to those skilled in the art.

[0120] The antibodies and antigen-binding fragments thereof disclosed herein are specific for PD-L1. In one embodiment, the antibody or and its fragment is specific for PD-L1. In one embodiment, the antibodies and fragments provided herein bind to human or primate PD-L1, but do not bind to PD-L1 from any other mammal. In another embodiment, the antibody or and its fragment does not bind to mouse PD-L1. The terms "human PD-L1", "hPD-L1" and "huPD-L1" and the like are used interchangeably herein and refer to variants or isoforms of human PD-L1 and human PD-L1. "Specific to" means that the antibody and its fragment bind to PD-L1 with greater affinity than any other target.

[0121] The term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic, partially or completely stabilizing or curing a disease and / or side effects resulting from a disease. As used herein, "treat" encompasses any treatment of a patient's disease that: (a) inhibits the symptoms of a disease, i.e., prevents its development; or (b) alleviates the symptoms of a disease, i.e., causes regression of the disease or symptoms.

[0122] The term "effective amount" means (i) treating or preventing a specific disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a specific disease, condition or disorder, or (iii) preventing or delaying the onset of one or more symptoms of a specific disease, condition or disorder described herein. The amount of active substance (e.g., antibody or compound of the present application) constituting a "therapeutically effective amount" may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent or therapeutic agent combination to induce a desired response in the individual. The effective amount may also be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0123] The term "administer" means that a composition comprising a therapeutic agent is physically introduced into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of immune checkpoint inhibitors (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies) include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In certain embodiments, the immune checkpoint inhibitor (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) is administered by a non-parenteral route, and in certain embodiments, is administered orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, e.g., intranasally, vaginally, rectally, sublingually or topically. Administration can also be performed, e.g., once, multiple times, and / or over one or more extended periods of time.

[0124] Unless otherwise indicated, the term "dose" is used to refer to the dose administered to a patient regardless of the patient's weight or body surface area (BSA). For example, a 60 kg person and a 100 kg person would receive the same dose of antibody (e.g., 240 mg anti-PD-1 antibody).

[0125] The term "weight-based dose" mentioned herein refers to a dose administered to a patient calculated based on the patient's weight. For example, when a patient with a body weight of 60 kg requires 3 mg / kg of anti-PD-1 antibody, one can draw an appropriate amount of anti-PD-1 antibody (i.e., 180 mg) from a fixed-dose formulation of the anti-PD-1 antibody at one time.

[0126] Anlotinib can be administered by a variety of routes, including but not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, by inhalation, vaginal, intraocular, by topical administration, subcutaneous, intrafat, intraarticular, intraperitoneal and intrathecal. In some specific embodiments, it is administered orally. The amount of anlotinib administered can be determined according to the severity of the disease, the response of the disease, any treatment-related toxicity, the age and health status of the patient. For example, the daily dose of anlotinib administered can be 2 mg to 20 mg, and in some embodiments, the daily dose of a compound of Formula I or a pharmaceutically acceptable salt thereof can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 mg. Anlotinib can be administered once or more daily. In some embodiments, anlotinib is administered once a day in an oral solid formulation.

[0127] The dosing regimen of anlotinib can be comprehensively determined based on the activity, toxicity and patient tolerance of the drug. Preferably, anlotinib is administered in an interval administration manner. The interval administration includes an administration period and a withdrawal period, and anlotinib can be administered once or more per day during the administration period. For example, the ratio of the administration period to the withdrawal period in days is 2:0.5-5, preferably 2:0.5-3, more preferably 2:0.5-2, and more preferably 2:0.5-1. In some embodiments, the drug is administered for 2 consecutive weeks and stopped for 2 weeks. In some embodiments, the drug is administered for 2 consecutive weeks and stopped for 1 week. In some embodiments, the drug is administered for 5 consecutive days and stopped for 2 days. For example, anlotinib can be administered orally at a dose of 6 mg, 8 mg, 10 mg or 12 mg once a day, and the drug is administered for 2 consecutive weeks and stopped for 1 week.

[0128] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0129] The term "pharmaceutically acceptable salt" includes salts formed by alkali ions and free acids or salts formed by acid ions and free bases, for example, hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formates, acetates, trifluoroacetates, fumarates, oxalates, maleates, citrates, succinates, methanesulfonates, benzenesulfonates or p-toluenesulfonates, preferably hydrochloride, hydrobromide, sulfate, formates, acetates, trifluoroacetates, fumarates, maleates, methanesulfonates, p-toluenesulfonates, sodium salts, potassium salts, ammonium salts, amino acid salts, etc. In the present application, when forming a pharmaceutically acceptable salt, the molar ratio of the free acid to the alkali ion is about 1:0.5 to 1:5, preferably 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8. In the present application, when a pharmaceutically acceptable salt is formed, the molar ratio of the free base to the acid ion is about 1:0.5 to 1:5, preferably 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8.

[0130] The term "subject" refers to a mammal. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

[0131] The term "about" should be understood to include within three standard deviations of the mean or within the standard tolerance range in a particular field. In certain embodiments, about should be understood to mean a variation of no more than 0.5. "About" modifies all values ​​listed thereafter. For example, "about 1, 2, 3" means "about 1", "about 2", "about 3".

[0132] As used herein, "combination" or "combined use" means that two or more active substances can be administered to an individual in need thereof simultaneously, each as a single preparation, or sequentially in any order, each as a single preparation.

[0133] The term "single dose" refers to the smallest packaging unit containing a certain amount of medicine. For example, if a box of medicine contains seven capsules, each capsule is a single dose; or each bottle of injection is a single dose.

[0134] The term "multiple doses" consists of a plurality of single doses.

[0135] The term "pharmaceutical composition" refers to a mixture of one or more active ingredients or their pharmaceutical combination of the present application and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound or its pharmaceutical combination of the present application to an individual in need.

[0136] The term "mM" means millimole / liter.

[0137] Herein, unless otherwise specified, the terms "comprise, comprises and comprising" or equivalents are open-ended expressions, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0138] For the purpose of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. These publications are provided only because they are disclosed prior to the filing date of the present application. All statements regarding the dates of these documents or the representations of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that the publications are part of the common general knowledge in the art in any country. DETAILED DESCRIPTION

[0139] For the sake of clarity, the present invention is further described with examples, but the examples do not limit the scope of the present application. All reagents used in the present application are commercially available and can be used without further purification. The anti-PD-L1 antibody in the embodiment is prepared according to the method described in Example 2 of WO2016022630, and after affinity chromatography, the eluate containing the antibody is obtained according to a conventional antibody purification method.

[0140] Example 1 Anti-tumor experiment on MC-38 / PD-L1 mouse model

[0141] Anti-PD-L1 humanized monoclonal antibody injection hu5G11-hIgG1: The anti-PD-L1 humanized monoclonal antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 13 and a light chain variable region sequence as shown in SEQ ID NO: 15. The heavy chain amino acid sequence of the anti-PD-L1 humanized monoclonal antibody in Example 1 is shown in SEQ ID NO: 17, and the light chain amino acid sequence is shown in SEQ ID NO: 18. The anlotinib is selected from anlotinib dihydrochloride.

[0142] MC-38 / PD-L1 mouse colon cancer subcutaneous transplant tumor, concentration 2×10 6 / mL*0.2mL / mouse, inoculated under sterile conditions under the right armpit of C57BL / 6 mice (shaved at the inoculation site), and passaged by plug method. The C57BL / 6 mice weighed 16-18g, were female, and were raised in an SPF environment. The animals were randomly divided into 4 groups the day after subcutaneous tumor plug inoculation:

[0143] i. Control group;

[0144] ii. Single-drug group: anlotinib;

[0145] iii. Single-drug group: anti-PD-L1 humanized monoclonal antibody;

[0146] iv. Combination group: anti-PD-L1 humanized monoclonal antibody + anlotinib;

[0147] There were 12 rats in the control group and 6 rats in each of the other groups.

[0148] The dosing regimen is shown in Table 1:

[0149] Table 1 Dosage regimen

[0150]

[0151]

[0152] ip intraperitoneal injection;

[0153] ig gavage;

[0154] qd: once daily administration;

[0155] qod: Administer once every other day.

[0156] The control group and the single-drug group were given a solvent (normal saline) or drug (dissolved in normal saline) at a volume of 10 mL / kg. When the combination group was given two drugs at the same time, the volume of each drug was 10 mL / kg. Starting from the sixth day of administration, the body weight and tumor diameter were measured every three days, and the performance of the animals was observed every day.

[0157] On day 0, mice were divided into groups, including 12 mice in the control group and 6 mice in each of the other groups. On day 1, drugs were prepared and administered by gavage (ig) and intraperitoneal (ip). Administration was stopped on day 22. During the experiment, tumor volumes were measured on days 6, 9, 12, 15, 18, 21, 24, and 27, and the experiment was terminated on day 28.

[0158] The tumor volume and tumor inhibition rate were calculated using the following formula:

[0159] Tumor volume (TV) = (length × width 2 ) / 2.

[0160] Tumor inhibition rate (TGI) = (1-tumor weight of treatment group / tumor weight of control group) × 100%.

[0161] The results are shown in Table 2 , which show that the combination of anti-PD-L1 humanized monoclonal antibody and anlotinib can inhibit the growth of colorectal cancer.

[0162] Table 2 Experimental results of MC-38 / PD-L1 subcutaneous transplanted tumors in mice

[0163]

[0164] *P<0.05vs. control group, **P<0.01vs. control group.

[0165] The above results showed that the tumor inhibition rate of the combination group reached 78.2%, which was significantly higher than that of the anlotinib monotherapy group and the anti-PD-L1 derived monoclonal antibody monotherapy group, indicating that the combination of anlotinib and anti-PD-L1 derived monoclonal antibody has a good inhibitory effect on colorectal cancer.

Claims

1. A combination pharmaceutical composition for treating colorectal cancer, comprising an anti-PD-L1 antibody and anlotinib.

2. The combined pharmaceutical composition according to claim 1 or 2, characterized in that: The combined pharmaceutical composition includes an anti-PD-L1 antibody pharmaceutical composition and anlotinib pharmaceutical composition.

3. The combined pharmaceutical composition according to claim 1 or 2, characterized in that: The combination drug composition is packaged in the same kit, which also includes instructions for the combined use of PD-L1 antibody and anlotinib to treat colorectal cancer.

4. The combined pharmaceutical composition according to claim 1 or 2, characterized in that: The combination pharmaceutical composition includes a pharmaceutical composition containing 600 to 2400 mg of an anti-PD-L1 antibody and a pharmaceutical composition with a single dose of 6 mg, 8 mg, 10 mg and / or 12 mg of anlotinib.

5. Use of the combination pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for treating colorectal cancer.

6. Use of anti-PD-L1 antibodies and anlotinib in the preparation of drugs for treating colorectal cancer.

7. The use according to claim 5 or 6, characterized in that The anti-PD-L1 antibody and anlotinib are each in the form of a pharmaceutical composition, which can be administered simultaneously, sequentially or intermittently; or the anti-PD-L1 antibody can be administered once a week, every 2 weeks, every 3 weeks, or every 4 weeks; or anlotinib can be administered once a day at a dose of 6 mg, 8 mg, 10 mg or 12 mg, for 2 consecutive weeks and 1 week of rest.

8. The combination pharmaceutical composition according to any one of claims 1 to 4, or the use according to any one of claims 5 to 7, characterized in that: The anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain CDR1 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; a heavy chain CDR2 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5; a heavy chain CDR3 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6; a light chain CDR1 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 10; a light chain CDR2 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 11; and a light chain CDR3 region that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO:

12.

9. The combination pharmaceutical composition according to any one of claims 1 to 4, or the use according to any one of claims 5 to 7, characterized in that: The anti-PD-L1 antibody comprises the following amino acid sequence: a heavy chain CDR1 region selected from SEQ ID NO: 1 or SEQ ID NO: 4; a heavy chain CDR2 region selected from SEQ ID NO: 2 or SEQ ID NO: 5; a heavy chain CDR3 region selected from SEQ ID NO: 3 or SEQ ID NO: 6; a light chain CDR1 region selected from SEQ ID NO: 7 or SEQ ID NO: 10; a light chain CDR2 region selected from SEQ ID NO: 8 or SEQ ID NO: 11; and a light chain CDR3 region selected from SEQ ID NO: 9 or SEQ ID NO:

12.

10. The combination pharmaceutical composition according to any one of claims 1 to 4, or the use according to any one of claims 5 to 7, wherein the colorectal cancer is selected from colon cancer or rectal cancer.

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