IL-21-Anti-albumin Single Domain Antibody Fusion Protein Pharmaceutical Composition and Its Use

Abstract

The present invention relates to an IL-21-anti-albumin single domain antibody fusion protein pharmaceutical composition and its use. The pharmaceutical composition contains an IL-21-anti-albumin single domain antibody fusion protein, a buffer, and may further contain at least one stabilizer, and optionally may also contain a surfactant. The IL-21-anti-albumin single domain antibody fusion protein has high stability, and both the single agent and its combination with a PD-1 monoclonal antibody exhibit excellent anti-tumor activity.

Description

Technical Field

[0001] The present invention relates to the field of therapeutic pharmaceutical compositions. In particular, the present invention relates to the field of pharmaceutical preparations, the pharmaceutical composition containing an IL-21 - anti - albumin single - domain antibody fusion protein, and the use of the IL-21 fusion protein in the treatment of tumors and enhancing the anti - tumor activity of immune checkpoint inhibitors. Background Art

[0002] Cytokine therapy is an effective strategy for stimulating the immune system to induce an immune response against diseases such as cancer or infection. However, cytokines administered to patients usually have a short half - life. For example, interleukin - 21 can stimulate various immune cells such as T cells, B cells, and NK cells and enhance their anti - tumor activity. It has been reported that the half - life of recombinant IL - 21 after intravenous administration is only about one to three hours (see Schmidt H, Clin. Cancer Res. November 1, 2010; Vol. 16, No. 21: pp. 5312 - 5319).

[0003] Immune escape is one of the characteristics of cancer. Ahmadzadeh, M. et al., Blood, 114:1537 - 44 discloses that tumor - specific T lymphocytes are often present in the tumor microenvironment, draining lymph nodes, and peripheral blood, but due to the network of immunosuppressive mechanisms present in the tumor microenvironment, they usually cannot control tumor progression. CD8 + Tumor - infiltrating T lymphocytes (TIL) usually express activation - induced inhibitory receptors, including CTLA - 4 and PD - 1, while tumor cells often express immunosuppressive ligands, including programmed death - ligand 1 (PD - L1, also called B7 - H1 or CD274), which inhibits T - cell activation and effector functions. Among the inhibitory mechanisms, PD - 1 and its ligand have become an important way for tumor cells to utilize to inhibit activated T cells in the tumor microenvironment.

[0004] In the past decade, PD - 1 / PD - L1 pathway blockade has been proven to be an effective way to induce durable anti - tumor responses in various cancer indications. Monoclonal antibodies (mAbs) that block the PD / PD - L1 pathway can enhance the activation and effector functions of tumor - specific T cells, reduce tumor burden, and improve survival rates. Between 2014 and 2017, the FDA has approved 2 anti - PD1 monoclonal antibodies (nivolumab) and 3 anti - PD - L1 monoclonal antibodies (atezolizumab, avelumab, and durvalumab) for the treatment of human tumors.

[0005] Although remarkable effects have been observed in 10 - 30% of cancer patients receiving immune checkpoint inhibitor therapy, a large proportion of patients are initially resistant to anti-PD-1 monoclonal antibody therapy, and more than half of the patients who initially respond will eventually relapse with tumors. Therefore, expanding the beneficiary population and improving the efficacy through synergistic combinations of immunotherapies have received extensive attention in the industry. IL-21 is a member of the γ-chain cytokine family, which can stimulate CD8 + T cell expansion and increase cytotoxicity, enhance T cell-dependent B cell proliferation and antibody production, promote NK cell differentiation and activation, and reduce Treg cells. Recombinant human IL-21 has been shown to have antitumor activity as a single therapy or in combination with targeted therapies or monoclonal antibodies in preclinical and clinical studies. For example, phase I and II clinical trials have been conducted using recombinant IL-21 (rIL-21) for metastatic melanoma, renal cell carcinoma (RCC), colorectal cancer, and non-Hodgkin lymphoma. However, recent studies have shown that, like other cytokines, the antitumor effect of IL-21 is limited by its short half-life in clinical trials.

[0006] Therefore, there is a need to develop new cytokine therapeutics that can effectively treat diseases and explore their effects when combined with immune checkpoint inhibitors. Summary of the Invention

[0007] The pharmaceutical composition disclosed in the present invention is a highly stable pharmaceutical composition containing an IL-21 - anti-albumin single-domain antibody fusion protein (IL-21 fusion protein). In particular, the present invention discovers that the combination containing trehalose has unexpected characteristics, namely high stability.

[0008] The half-life and exposure level in the blood of the IL-21 - anti-albumin single-domain antibody fusion protein pharmaceutical composition of the present invention are significantly superior to those of recombinant interleukin 21, and it shows superior antitumor activity both as a single agent and in combination with anti-PD-1 monoclonal antibody in a mouse subcutaneous xenograft tumor model.

[0009] The present invention discovers that, compared with rhIL-21 (recombinant human IL-21), a nanobody targeting human serum albumin (HSA) fused to the C-terminus of human IL-21, a fusion protein herein referred to as JS-EC21, has a significantly prolonged half-life and increased exposure in cynomolgus monkeys (t 1 / 2 and AUC are nearly 10 times and 50 times higher than those of rhIL-21, respectively). In addition, JS-EC21 significantly enhances the antitumor effects of anti-PD-1 monoclonal antibody or TIGIT monoclonal antibody and provides long-term protective antitumor immunity. The combination of JS-EC21 and TIGIT monoclonal antibody can induce significant enrichment of the KEGG signaling pathway related to tumor immune response and increase the expression levels of genes related to CD8 + T cell and NK cell cytotoxicity.

[0010] On the one hand, the present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an IL-21 - anti - albumin single - domain antibody fusion protein.

[0011] In some embodiments, the above - mentioned IL-21 - anti - albumin single - domain antibody fusion protein comprises: (a) the cytokine IL-21, and (b) a single - domain antibody (sdAb) that specifically binds albumin. The above - mentioned IL-21 comprises the amino acid sequence shown in SEQ ID NO:1, and the above - mentioned single - domain antibody comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0012] In some embodiments, the above - mentioned single - domain antibody (sdAb) comprises the amino acid sequence shown in SEQ ID NO:5.

[0013] In some embodiments, the above - mentioned single - domain antibody is fused to the C - terminus of the cytokine, and the above - mentioned cytokine and single - domain antibody are directly linked.

[0014] In some embodiments, the concentration of the IL-21 - anti - albumin single - domain antibody fusion protein in the above - mentioned pharmaceutical composition is about 0.1 - 100 mg / mL, preferably about 0.2 - 20 mg / mL, preferably about 0.2 - 10 mg / mL, preferably about 0.5 - 5 mg / mL, preferably about 1 - 5 mg / mL, more preferably about 0.5 - 1 mg / mL; more preferably, the concentration of the above - mentioned IL-21 - anti - albumin single - domain antibody fusion protein is about 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL or 10 mg / mL, preferably about 1 mg / mL.

[0015] In some embodiments, the above - mentioned buffer is selected from one or more of acetate buffer, citrate buffer, and histidine buffer; preferably, the above - mentioned buffer is selected from one or more of acetate - sodium acetate buffer, citrate - sodium citrate buffer, histidine - acetate buffer, and histidine - hydrochloride buffer; preferably, the above - mentioned buffer is acetate - sodium acetate buffer and histidine - acetate buffer; more preferably, the above - mentioned buffer is acetate - sodium acetate buffer.

[0016] In some embodiments, the above buffer is a histidine buffer. Preferably, the above histidine buffer is selected from histidine - hydrochloride buffer or histidine - acetate buffer, and histidine - hydrochloride buffer is preferred. In some embodiments, the above histidine - hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L - histidine and L - histidine monohydrochloride. In some embodiments, the histidine buffer is made of 1 - 30 mM of L - histidine and 1 - 30 mM of L - histidine monohydrochloride. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride with a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride with a molar ratio of 1:1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride with a molar ratio of 1:3. In some embodiments, the histidine preparation is a histidine buffer with a pH of about 5.0, made of about 1.7 mM of L - histidine and about 18.3 mM of L - histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of about 5.5, made of about 4.5 mM of L - histidine and about 15.5 mM of L - histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of about 5.5, made of about 7.5 mM of L - histidine and about 22.5 mM of L - histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of about 6.0, made of about 15 mM of histidine and about 15 mM of histidine hydrochloride.

[0017] In some embodiments, the above histidine buffer is a histidine - acetate buffer. Preferably, the molar ratio of the two is about 1:1 to 1.5:1. Preferably, the pH of such a buffer is 5.5 ± 0.3, preferably about 5.5. Preferably, such a buffer contains 15 - 20 mM of histidine and 12 - 15 mM of acetic acid.

[0018] In some embodiments, the above buffer is an acetate buffer. Preferably, the acetate buffer is an acetic acid-sodium acetate buffer or an acetic acid-potassium acetate buffer, and an acetic acid-sodium acetate buffer is preferred. In some embodiments, the acetate buffer is made of 1 - 30 mM acetic acid and 1 - 30 mM sodium acetate. In some embodiments, the acetate buffer is made of acetic acid and sodium acetate with a molar ratio of about 2:3. In some embodiments, the acetate buffer is made of acetic acid and sodium acetate with a molar ratio of about 1:2.1. In some embodiments, the acetate buffer is made of acetic acid and sodium acetate with a molar ratio of about 1:5.7. In some embodiments, the acetate buffer is an acetate buffer with a pH of about 4.8 made of about 8 mM acetic acid and about 12 mM sodium acetate. In some embodiments, the acetate buffer is an acetate buffer with a pH of about 5.0 made of 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 made of about 3 mM acetic acid and about 17 mM sodium acetate.

[0019] In some embodiments, the above buffer is a citrate buffer. Preferably, the citrate buffer is a citric acid-sodium citrate buffer.

[0020] In some embodiments, the concentration of the above buffer is about 1 - 200 mM, preferably about 5 - 200 mM, preferably about 10 - 50 mM, preferably about 10 - 30 mM; preferably about 10 - 20 mM. Non-limiting examples of the buffer concentration are about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM or 180 mM, or a range formed by any two values within these ranges as endpoints. Preferably, it is 10 mM, 15 mM, 20 mM or 30 mM.

[0021] In some embodiments, the pH of the above buffer is about 4.0 - 6.5, preferably about 4.0 - 6.0, preferably about 4.5 - 6.0, preferably about 4.5 - 5.5, preferably about 4.5 - 5.1, preferably about 4.5 - 5.0, preferably about 4.7 - 5.0. Non-limiting examples of the pH of the buffer are about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 6.0. Preferably, it is about 4.7 or 4.8.

[0022] In some embodiments, the above-mentioned pharmaceutical composition further comprises a stabilizer, and the stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, and trehalose. Preferably, the stabilizer is trehalose.

[0023] In some embodiments, the concentration of the stabilizer is about 10 mM - 400 mM, preferably 20 mM - 300 mM, more preferably 30 mM - 200 mM.

[0024] In some embodiments, the stabilizer is mannitol at a concentration of about 100 - 300 mM; or the stabilizer is sucrose at a concentration of about 100 - 300 mM; or the stabilizer is trehalose at a concentration of about 100 - 300 mM; or the stabilizer is a combination of about 30 - 200 mM of sodium chloride and about 30 - 200 mM of mannitol; or the stabilizer is a combination of about 30 - 200 mM of sodium chloride and about 30 - 200 mM of sucrose; preferably, the stabilizer is trehalose at about 100 - 300 mM; more preferably, the stabilizer is trehalose at about 200 - 280 mM.

[0025] In some embodiments, the stabilizer is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100 - 300 mM, and the concentration of the mannitol is preferably about 150 - 300 mM, preferably about 200 - 280 mM. Non-limiting examples of the concentration of the mannitol are about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 240 mM.

[0026] In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100 - 300 mM, and the concentration of the sucrose is preferably about 150 - 300 mM, preferably about 200 - 280 mM. Non-limiting examples of the concentration of the sucrose are about 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 235 mM.

[0027] In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100 - 300 mM, and the concentration of the trehalose is preferably about 150 - 300 mM, preferably about 200 - 280 mM. Non-limiting examples of the concentration of the trehalose are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 235 mM.

[0028] In some embodiments, the above stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the above stabilizer is a combination of about 30 - 200 mM of sodium chloride and about 30 - 200 mM of mannitol, preferably a combination of about 40 - 150 mM of sodium chloride and about 40 - 180 mM of mannitol, more preferably a combination of about 40 - 100 mM of sodium chloride and about 40 - 150 mM of mannitol. Non - limiting examples of the above stabilizer are a combination of about 50 mM of sodium chloride and about 120 mM of mannitol, or a combination of about 50 mM of sodium chloride and about 140 mM of mannitol.

[0029] In some embodiments, the above stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the above stabilizer is a combination of about 30 - 200 mM of sodium chloride and about 30 - 200 mM of sucrose, preferably a combination of about 40 - 150 mM of sodium chloride and about 40 - 180 mM of sucrose, more preferably a combination of about 40 - 100 mM of sodium chloride and about 40 - 150 mM of sucrose. A non - limiting example of the above stabilizer is a combination of about 50 mM of sodium chloride and about 140 mM of sucrose.

[0030] In some embodiments, the above pharmaceutical composition further comprises a surfactant, and the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer 188.

[0031] In some embodiments, the above surfactant is selected from polysorbate 80.

[0032] In some embodiments, the above surfactant is selected from polysorbate 20.

[0033] In some embodiments, calculated by w / v, the concentration of the above surfactant is about 0.001% - 0.1%, preferably about 0.01% - 0.1%, more preferably about 0.02% - 0.08%, most preferably about 0.01% - 0.05%; as non - limiting examples, the concentration of the above surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.

[0034] In some embodiments, the above IL - 21 fusion protein comprises: (a) cytokine IL - 21, and (b) a single - domain antibody (sdAb) that specifically binds albumin, wherein the above IL - 21 comprises the amino acid sequence shown in SEQ ID NO:1, and the sdAb comprises the amino acid sequence shown in SEQ ID NO:5.

[0035] In some embodiments, the above IL - 21 - anti - albumin single - domain antibody fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0036] In some embodiments, the above-mentioned pharmaceutical composition comprises the components shown in any one of the following (1)-(6), wherein the IL-21-anti-albumin single-domain antibody fusion protein is as described in any embodiment of the present invention:

[0037] (1) (a) An IL-21-anti-albumin single-domain antibody fusion protein at about 0.5 mg / mL - 5 mg / mL; (b) about 10 - 30 mM acetate buffer with a pH of about 4.5 - 5.5 (preferably 4.5 - 5.0); (c) about 100 - 300 mM trehalose; (d) and about 0.01% - 0.1% polysorbate 80; or

[0038] (2) (a) An IL-21-anti-albumin single-domain antibody fusion protein at about 0.5 mg / mL - 5 mg / mL; (b) about 10 - 30 mM acetate buffer with a pH of about 4.5 - 5.5 (preferably 4.5 - 5.0); (c) about 100 - 300 mM sucrose; (d) and about 0.01% - 0.1% polysorbate 80; or

[0039] (3) (a) An IL-21-anti-albumin single-domain antibody fusion protein at about 0.5 mg / mL - 5 mg / mL; (b) about 10 - 30 mM acetate buffer with a pH of about 4.5 - 5.5 (preferably 4.5 - 5.0); (c) about 100 - 300 mM mannitol; (d) and about 0.01% - 0.1% polysorbate 80; or

[0040] (4) (a) An IL-21-anti-albumin single-domain antibody fusion protein at about 0.5 mg / mL - 5 mg / mL; (b) about 10 - 30 mM histidine buffer with a pH of about 4.5 - 5.5 (preferably 4.5 - 5.0); (c) about 30 - 200 mM sodium chloride and about 30 - 200 mM mannitol; (d) and about 0.01% - 0.1% polysorbate 80; or

[0041] (5) (a) An IL-21-anti-albumin single-domain antibody fusion protein at about 1 mg / mL; (b) about 20 mM acetate buffer with a pH of about 4.8; (c) about 235 mM trehalose; (d) and about 0.2% polysorbate 80; or

[0042] (6) (a) An IL-21-anti-albumin single-domain antibody fusion protein at about 1 mg / mL; (b) about 20 mM acetate buffer with a pH of about 4.8; (c) about 235 mM sucrose; (d) and about 0.2% polysorbate 80.

[0043] In some embodiments, the above-mentioned pharmaceutical composition is a liquid preparation or a freeze-dried preparation.

[0044] In some embodiments, the above-mentioned pharmaceutical composition is a liquid preparation.

[0045] In some embodiments, the above-mentioned liquid preparation or freeze-dried preparation is 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.

[0046] In some embodiments, the above-mentioned aqueous solution or freeze-dried preparation is stable at 40 °C for at least 7 days, at least 14 days or at least 28 days.

[0047] In yet another aspect, the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating or preventing cancer; preferably, the above-mentioned cancer includes mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer and colorectal cancer.

[0048] In yet another aspect, the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating or preventing inflammatory diseases.

[0049] In yet another aspect, the present invention provides the use of an IL-21 fusion protein (also known as an IL-21 - anti-albumin single-domain antibody fusion protein), or the above-mentioned pharmaceutical composition, or a combination of an IL-21 fusion protein and an immune checkpoint modulator or a combination of the above-mentioned pharmaceutical composition and an immune checkpoint modulator in the preparation of a medicament for preventing or treating tumors, or the use of an IL-21 - anti-albumin single-domain antibody fusion protein or the above-mentioned pharmaceutical composition in the preparation of a medicament for enhancing the anti-tumor activity of an immune checkpoint modulator.

[0050] In yet another aspect, the present invention provides a method for preventing or treating tumors, which comprises administering an effective amount of an IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or a combination of an IL-21 fusion protein and an immune checkpoint modulator or a combination of the above-mentioned pharmaceutical composition and an immune checkpoint modulator to an individual in need.

[0051] In yet another aspect, the present invention provides an IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or a combination of an IL-21 fusion protein and an immune checkpoint modulator or a combination of the above-mentioned pharmaceutical composition and an immune checkpoint modulator, which is used for treating or preventing tumors.

[0052] In some embodiments, the tumor is an IL-21-related tumor, or an immune checkpoint-related tumor.

[0053] In some embodiments, the tumor is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, kidney cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-Hodgkin lymphoma, esophageal cancer, gastric cancer and colorectal cancer.

[0054] In some embodiments, the tumor is colorectal cancer.

[0055] In one or more embodiments, the IL-21-single domain antibody against albumin fusion protein disclosed by the present invention comprises: (a) cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, wherein IL-21 comprises the amino acid sequence as shown in SEQ ID NO:1, and the single domain antibody (sdAb) comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0056] In one or more embodiments, the single domain antibody (sdAb) disclosed by the present invention comprises the amino acid sequence as shown in SEQ ID NO:5.

[0057] In one or more embodiments, the single domain antibody disclosed by the present invention is fused to the C-terminus of the cytokine, and the cytokine and the single domain antibody are directly linked.

[0058] In one or more embodiments, the IL-21 fusion protein disclosed by the present invention comprises the amino acid sequence as shown in SEQ ID NO:6.

[0059] In one or more embodiments, the IL-21 fusion protein disclosed by the present invention is used in combination with an immune checkpoint modulator

[0060] In one or more embodiments, the immune checkpoint modulators disclosed by the present invention are selected from PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27, and B7H4 immune checkpoint inhibitors.

[0061] In one or more embodiments, the immune checkpoint modulator disclosed by the present invention is an anti-PD-1 antibody.

[0062] In one or more embodiments, the anti-PD-1 antibody disclosed by the present invention comprises:

[0063] (1) light chain complementarity determining regions with amino acid sequences as shown in SEQ ID NO:7, 8, and 9, and heavy chain complementarity determining regions with amino acid sequences as shown in SEQ ID NO:10, 11, and 12; or

[0064] (2) a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:14; or

[0065] (3) A light chain with an amino acid sequence as shown in SEQ ID NO: 15, and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 16.

[0066] In one or more embodiments, the anti-PD-1 antibodies disclosed in the present invention are selected from one or more of nivolumab, pembrolizumab, toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Cemiplimab; preferably toripalimab.

[0067] In some embodiments, compared with the use of the IL-21 fusion protein or the anti-PD-1 antibody alone, the IL-21 fusion protein disclosed in the present invention in combination with the anti-PD-1 antibody has one or more of the following properties:

[0068] (1) The proportion of CD3 + T cells in immune cells is significantly increased;

[0069] (2) The proportion of CD8 + T cells in T cells is significantly increased;

[0070] (3) The proportion of CD8 + T cells in the proliferative state (Ki 67 + ) is significantly increased; and

[0071] (4) The proportion of NK cells in immune cells is significantly increased.

[0072] In one or more embodiments, the immune checkpoint modulator disclosed in the present invention is an anti-TIGIT antibody.

[0073] In one or more embodiments, the anti-TIGIT antibodies disclosed in the present invention are selected from: Tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, and BMS-986207.

[0074] In some embodiments, compared with the use of the IL-21 fusion protein or the anti-TIGIT antibody alone, the IL-21 fusion protein disclosed in the present invention in combination with the anti-TIGIT antibody has one or more of the following properties:

[0075] (1) Significantly increase the expression level of interferon γ;

[0076] (2) Significantly increase the expression level of granzyme A;

[0077] (3) Significantly increase the expression level of granzyme B;

[0078] (4) Significantly increase the expression level of perforin-1;

[0079] (5) Enrich more KEGG signaling pathways with differential gene expression; and

[0080] (6) Most of the significantly enriched KEGG signaling pathways induced are mediated by the IL-21 fusion protein.

[0081] In yet another aspect, the present invention provides a pharmaceutical combination comprising:

[0082] (1) An immune checkpoint modulator; and

[0083] (2) An IL-21 - anti-albumin single domain antibody fusion protein or the pharmaceutical composition as described above;

[0084] In yet another aspect, the present invention provides a kit, which comprises:

[0085] (1) One or more single-dose units of the IL-21 fusion protein and one or more single-dose units of the immune checkpoint modulator, wherein the IL-21 fusion protein is as disclosed herein; preferably, wherein the immune checkpoint modulator is as disclosed herein; or

[0086] (2) One or more single-dose units of the pharmaceutical combination as disclosed herein.

[0087] In one or more embodiments, the kit disclosed by the present invention further comprises instructions for indicating the usage method of the pharmaceutical composition therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 : Inhibitory effect of the fusion protein JS-EC21 of the present invention on MC38 tumors.

[0089] Figure 2 : Pharmacokinetic curves of low-dose, medium-dose, and high-dose JS-EC21 in cynomolgus monkeys.

[0090] Figure 3 : Inhibitory effect of the fusion protein JS-EC21 of the present invention combined with anti-PD-1 monoclonal antibody on MC38 tumors.

[0091] Figure 4 : Changes in tumor volume of mice after administration of the fusion protein JS-EC21 of the present invention alone or in combination with toripalimab.

[0092] Figure 5 : Changes in tumor weight of mice after administration of the fusion protein JS-EC21 of the present invention alone or in combination with toripalimab.

[0093] Figure 6 : Changes in immune cells in tumors of mice after the fusion protein JS-EC21 and toripalimab were administered alone or in combination in the present invention.

[0094] Figures 4 - 6 Among them, the difference analysis between the two groups was performed by two-tailed unpaired Student’s t test. * indicates P < 0.05; ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference. Detailed implementation manners

[0095] Definitions and explanations

[0096] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course, changes can be made to the above. It should also be understood that the terms used in the present application are only for describing specific implementation manners and are not intended to be limiting.

[0097] Unless the content is otherwise clearly stated, the singular forms "a", "an" and "the" used in this specification and the appended claims include plural referents. Thus, for example, reference to "a polypeptide" includes combinations of two or more polypeptides and the like.

[0098] The term "pharmaceutical composition" or "formulation" refers to a mixture containing one or more antibodies described herein and other components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and thus exert biological activity.

[0099] The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a lyophilized formulation with heavy suspension. The liquid formulation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state change methods, such as spray drying).

[0100] The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and / or freezing to maintain stability (such as chemical and / or physical stability and / or biological activity).

[0101] The term "excipient" refers to a reagent that can be added to a formulation to provide desired properties (such as consistency, improved stability) and / or adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants and polymers.

[0102] As used herein, "about" in reference to a measurable value such as an amount, a duration, and the like is intended to cover variations of ±20% or ±10% relative to the specific value, including ±5%, ±1%, and ±0.1% because such variations are appropriate for carrying out the disclosed methods.

[0103] The term "buffer solution with a pH of about 4.0 - 6.5" refers to a reagent that, through the action of its acid / base conjugate components, enables the solution containing the reagent to resist pH changes. The buffer solution used in the preparation of the present invention may have a pH within the range of about 4.0 to about 6.5, or within the range of about 4.5 to about 6.5, or within the range of about 4.5 to about 5.5.

[0104] In this context, examples of "buffer solutions" that control the pH within this range include acetates (such as sodium acetate), succinates (such as sodium succinate), gluconic acid, histidine, histidine hydrochloride, methionine, citrates, phosphates, citrate / phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.

[0105] "Histidine buffer solution" is a buffer solution containing histidine ions. Examples of histidine buffer solutions include histidine and salts of histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, such as a histidine buffer solution containing histidine and histidine hydrochloride; the histidine buffer solution of the present invention also includes a histidine buffer solution containing histidine and acetate (such as sodium salt or potassium salt) or glacial acetic acid.

[0106] "Citrate buffer solution" is a buffer solution including citrate ions. Examples of citrate buffer solutions include citric acid - sodium citrate, citric acid - potassium citrate, citric acid - calcium citrate, citric acid - magnesium citrate, etc. The preferred citrate buffer solution is citric acid - sodium citrate buffer solution.

[0107] "Acetate buffer solution" is a buffer solution including acetate ions. Examples of acetate buffer solutions include acetic acid - sodium acetate, acetic acid - potassium acetate, acetic acid - calcium acetate, acetic acid - magnesium acetate, etc. The preferred acetate buffer solution is acetic acid - sodium acetate buffer solution.

[0108] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or the 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 (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines, alanopine, octopine, strombine, and N-oxide of trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizers specifically used in the present invention are selected from one or more of polyols, amino acids, salts, and sugars. The preferred salt is sodium chloride, the preferred sugars are sucrose and trehalose, the preferred polyols are sorbitol and mannitol. The preferred amino acids are arginine or its salts (such as arginine hydrochloride), glycine, and proline. The preferred stabilizers are 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, arginine hydrochloride-sucrose, more preferably arginine hydrochloride, sodium chloride-sucrose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, and even more preferably sucrose or trehalose.

[0109] The term "surfactant" generally includes reagents that protect proteins such as antibodies from stress induced by the air / solution interface and the solution / surface interface to reduce antibody aggregation or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkyl phenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). In this article, unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" both refer to the mass / volume concentration (w / v), such as "about 0.02% polysorbate 80" where "0.02%" means "0.02 g of polysorbate 80 is contained in 100 mL of liquid".

[0110] The term "isotonic" means that the preparation has an osmotic pressure substantially the same as human blood. Isotonic preparations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0111] A "stable" preparation is one in which the antibody therein substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or upon storage. A pharmaceutical preparation can be stable even if the contained antibody does not maintain its 100% chemical structure or biological function after storage for a certain period of time. In some cases, after storage for a certain period of time, maintaining about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the antibody structure or function can also be considered "stable". Various analytical techniques for measuring protein stability are available in the art and are reviewed in "Peptide and Protein Drug Delivery" 247 - 301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29 - 90 (both incorporated by reference).

[0112] After storage of the preparation at a certain temperature for a certain period of time, its stability can be measured by determining the percentage of the remaining native antibody therein (among other methods). Among other methods, the percentage of the native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC - HPLC]), where "native" means non - aggregated and non - degraded. In some protocols, the stability of the protein is determined by the percentage of monomeric protein in a solution with a low percentage of degraded (e.g., fragmented) and / or aggregated protein. In some protocols, the preparation can be stably stored 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 no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

[0113] The stability can be measured by determining the percentage of the antibody (“acidic form”) that migrates in a fraction that is more acidic in this antibody main fraction (“major charged form”) during ion exchange (among other methods), where the stability is inversely proportional to the percentage of the acidic form antibody. Among other methods, the percentage of “acidified” antibody can be measured by ion exchange chromatography (such as cation exchange high performance liquid chromatography [CEX-HPLC]). In some embodiments, an acceptable level of stability means that when the formulation is stored at a certain temperature for a certain period of time, the detectable acidic form of the antibody is at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%. The certain period of time stored before measuring stability can 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 evaluating stability, the certain temperature at which the pharmaceutical formulation is allowed to be stored can be any temperature within the range of about -80°C to about 45°C, such as stored 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.

[0114] The above-mentioned antibody “maintains its physical stability” in the pharmaceutical composition if the antibody does not substantially show signs such as aggregation, precipitation, and / or denaturation when visually inspected for color and / or clarity or measured by UV light scattering or by size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the extent of forming visible precipitates.

[0115] The stability of the formulation, such as physical stability, can be evaluated by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of the formulation is partly an inherent property of the protein dissolved in the solution and is usually measured by nephelometry and quantified in nephelometric turbidity units (NTU).

[0116] The turbidity level that varies with, for example, the concentration of one or more components in a solution (such as protein and / or salt concentration) is also referred to as the "opalescence" or "opalescent appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia standards (European Pharmacopoeia, Fourth Edition, "Directorate for the Quality of Medicine of the Council of Europe" (EDQM), Strasbourg, France). According to the European Pharmacopoeia standards, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension having a turbidity of approximately 3 according to the European Pharmacopoeia standards. Turbidity measurements by nephelometry 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.

[0117] An antibody "maintains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time point is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be evaluated, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical alterations can include size alterations (such as truncation), which can be evaluated 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 alterations include charge alterations (such as those occurring as a result of deamidation or oxidation), which can be evaluated by, for example, ion exchange chromatography.

[0118] An antibody "maintains its biological activity" in a pharmaceutical composition if the antibody is biologically active for its intended purpose. For example, if after storage of the formulation at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (such as 1 to 12 months), the affinity of the IL-21 fusion protein contained in the formulation for binding to IL-17A is at least 90%, 95% or more of the antibody binding affinity before such storage, then the formulation of the present invention can be considered stable. The binding affinity can also be determined using, for example, ELISA or surface plasmon resonance techniques.

[0119] In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount that is effective in the prevention or treatment or alleviation of the symptoms of a disorder that the antibody can effectively treat.

[0120] In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" or "effective amount" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease, where the regression of the disease is demonstrated by a decrease in the severity of the disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of injury or disability caused by the disease affliction. The ability of a drug or therapeutic agent to promote the regression of a disease 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 predictive of human efficacy, or by measuring the activity of the agent in in vitro assays.

[0121] A therapeutically effective amount of a drug includes a "preventively effective amount", i.e., any amount of the drug that, when administered alone or in combination with other therapeutic drugs (such as anti-tumor agents) to a subject at risk of developing a disease (such as at risk of developing cancer) or a subject with a recurrence of a disease (such as having a recurrence of cancer), inhibits the development or recurrence of the disease (such as cancer).

[0122] The terms "subject", "patient", or "individual" 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 present invention, the subject is a human.

[0123] The terms "administer", "give", and "treat" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of an anti-PD-1 antibody include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to a mode of administration that is generally by injection, other than enteral or topical administration, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intramedullary, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion, as well as in vivo electroporation.

[0124] The term "antibody" is used in its broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, provided that they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0125] As used herein, the term "full-length antibody" or "intact antibody molecule" refers to an immunoglobulin molecule containing four peptide chains, two heavy (H) chains (about 50-70 kDa when full-length) and two light (L) chains (about 25 kDa when full-length), which are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into complementarity determining regions (CDRs) with high variability and intervening regions that are more conserved and are called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Amino acids are typically assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991): Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 341:878-883.

[0126] The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0127] As used herein, the term "CDR" refers to the complementarity determining regions within the variable sequences of an antibody. There are three CDRs present in each of the variable regions of the heavy and light chains, which are named HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each of the heavy and light chain variable regions. The exact boundaries of these CDRs are defined differently according to different systems. The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or delimited according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are inserted between flanking extensions (termed framework regions (FRs)), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant region. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0128] As used herein, "antigen-binding fragment" includes fragments of an antibody or derivatives thereof, typically including 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; diabodies; 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 its derivative typically retains at least 10% of the antigen-binding activity of the parent antibody. Preferably, the binding fragment or its derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

[0129] Unless otherwise specified, the CDRs of the antibodies of the present invention can have their boundaries determined by those skilled in the art according to any protocol in the art (e.g., different assignment systems or combinations).

[0130] As used herein, the "framework region" or "FR" refers to the immunoglobulin variable region excluding the CDR regions.

[0131] A "chimeric antibody" refers to an antibody and fragments thereof, wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species (such as human) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (such as mouse) or belonging to another antibody class or subclass, provided that it exhibits the desired biological activity.

[0132] A "human antibody" refers to an antibody that contains only human immunoglobulin sequences. If a human antibody is produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell, it may contain murine carbohydrate chains. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively.

[0133] A "humanized antibody" refers to an antibody form that contains sequences from non-human (such as murine) antibodies and human antibodies. Such antibodies contain the minimal sequence derived from the non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FR regions are the FR regions of human immunoglobulin. A humanized antibody optionally further includes at least a portion of the immunoglobulin constant region (Fc) (usually the human immunoglobulin constant region).

[0134] "Fv" is the smallest antibody fragment that contains a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly and non-covalently associated. When these two domains fold, they generate six hypervariable loops (3 loops each from the heavy and light chains), which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or a half-Fv that includes only the three CDRs specific for a particular antigen) has the ability to recognize and bind an antigen, but with a lower affinity than the entire binding site.

[0135] The term "heavy chain antibody" or "HCAb" refers to a functional antibody that includes a heavy chain but lacks the light chain that is commonly present in a 4-chain antibody. It is known that camelids (such as camels, llamas, or alpacas) produce HCAb.

[0136] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). The sdAb is capable of binding to an antigen alone without pairing with a corresponding CDR-containing polypeptide. In some cases, the single domain antibody is engineered from camelid HCAb, and its heavy chain variable domain is referred to herein as "VHH" (variable domain of the heavy chain of heavy chain antibody). Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hammers-Casterman et al., Nature, Vol. 363: pp. 446-448, 1993; Greenberg et al., Nature, Vol. 374: pp. 168-173, 1995; Hassanzadeh-Ghassaeh et al., Nanomedicine (Lond), Vol. 8: pp. 1013-1026, 2013). The basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0137] Unless otherwise specified, the numbering of residues in immunoglobulin heavy chains herein refers to the EU index numbering of Kabat et al. "Kabat's EU index" refers to the residue numbering of human IgG1 EU antibody.

[0138] "Therapeutic anti-PD-1 monoclonal antibody" refers to an antibody that specifically binds to the mature form of a specific PD-1 expressed on the surface of certain mammalian cells. Mature PD-1 has no pre-secretory leader sequence, or leader peptide. The terms "PD-1" and "mature PD-1" are used interchangeably herein and, unless otherwise clearly defined or clearly apparent from the context, should be understood to refer to the same molecule.

[0139] As described herein, a therapeutic anti-human PD-1 antibody or anti-hPD-1 antibody refers to a monoclonal antibody that specifically binds to mature human PD-1.

[0140] "Isolated antibody or its antigen-binding fragment" refers to a purified state and in that case the specified molecule substantially does not contain other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials (such as cell debris or growth medium).

[0141] As used herein, the term "cytokine" shall be understood to mean any protein or peptide, its analogs or functional fragments, which are capable of stimulating or inducing a cytolytic immune response against a preselected cell type (e.g., cancer cells or virus-infected cells) in mammals. Accordingly, a variety of cytokines are contemplated for inclusion in the present application. Useful cytokines include, for example, tumor necrosis factor (TNF), interleukin (IL), lymphokine (L), colony stimulating factor (CSF), interferon (IFN), including species variants, truncated analogs thereof that are capable of stimulating or inducing such cytolytic immune response. Useful tumor necrosis factors include, for example, TNFα. Useful lymphokines include, for example, LT. Useful colony stimulating factors include, for example, GM-CSF and M-CSF. Useful interleukins include, for example, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21, IL22 and IL-33. Useful interferons include, for example, IFN-α, IFN-β and IFN-γ. The term "cytokine" shall also be understood to include any variants of wild-type cytokines (such as IL-21, IL-7, IL-15, etc.), which include modifications that retain at least an important portion (such as at least about 50%) of any of their desired functions.

[0142] "Immune checkpoint" refers to a series of molecules expressed on immune cells that can regulate the degree of immune activation and play an important role in preventing the occurrence of autoimmune diseases (abnormal immune function that attacks normal cells). Common immune checkpoints include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27 and B7H4. The immune checkpoint modulators described herein refer to reagents that can specifically bind to these immune checkpoints and enhance, attenuate or block their biological activities, such as inhibitors. Such reagents particularly include antibodies or antigen-binding fragments thereof that specifically bind to these immune checkpoints. The immune checkpoint-related tumors described herein refer to tumors that can be prevented and treated by binding to immune checkpoints and weakening or blocking their biological activities. In a particularly preferred embodiment, the immune checkpoint-related tumors described herein are PD-1 or PD-L1 or TIGIT-related tumors.

[0143] The "adverse event" (AE) described herein is any adverse and generally unintended or undesired sign, symptom or disease associated with the use of a medical treatment. For example, an adverse event may be associated with the activation of the immune system or the expansion of immune system cells in response to treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different severity levels.

[0144] "Tumor burden" refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells in the whole body or the total size of the tumor. Tumor burden can be measured by a variety of methods known in the art, such as using calipers after the tumor is removed from the subject, or measuring its size using imaging techniques (such as ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scan) when the tumor is in the body.

[0145] The term "tumor size" refers to the total size of the tumor, which can be measured as the length and width of the tumor. Tumor size can be measured by a variety of methods known in the art, such as using calipers after the tumor is removed from the subject, or measuring its size using imaging techniques (such as bone scan, ultrasound, CT or MRI scan) when the tumor is in the body.

[0146] The terms "subject", "individual", "patient" include any living organism, preferably an animal, more preferably a mammal (such as a rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. The terms "subject" and "patient" can be used interchangeably herein.

[0147] "Treating" cancer as described herein refers to administering to a subject having or diagnosed with cancer a treatment regimen (such as administering an anti-PD-1 antibody) as described herein to achieve at least one positive treatment effect (such as, a decrease in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell infiltration into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). Positive treatment effects in cancer can be measured in a variety of ways (see W.A. Weber, J. Nucl. Med., 50:1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI criteria, T / C ≤ 42% is the minimum level of anti-tumor activity. T / C (%) = median treated tumor volume / median control tumor volume × 100. PFS (also called "time to tumor progression") refers to the length of time during and after treatment that the cancer does not grow, and includes the amount of time the patient experiences CR or PR and the amount of time the patient experiences SD. DFS refers to the length of time during and after treatment that the patient remains disease-free. OS refers to an increase in life expectancy compared to an untreated individual or patient. The treatment regimen of the inventive combination for effectively treating cancer patients can vary according to a variety of factors (such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject). Although the embodiments of the present invention may not achieve an effective positive treatment effect in every subject, they should be effective and achieve a positive treatment effect in a statistically significant number of subjects.

[0148] The terms "mode of administration", "administration regimen" are used interchangeably and refer to the dosage and timing of each therapeutic agent in the inventive combination.

[0149] As used herein, the terms "cancer" or "malignant tumor" refer to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth, and proliferation lead to the formation of malignant tumors, which invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0150] After IL-21 binds to its receptor, it can regulate the proliferation of B cells, differentiate into plasma cells for immune response, and promote the proliferation and differentiation of CD8 + T cells and NK cells, and enhance the killing activity of NK cells, thereby achieving tumor treatment or prevention. Therefore, the "IL-21-related tumors" described herein refer to tumors that benefit from the administered IL-21 or IL-21 fusion protein in the treatment or prevention of tumors.

[0151] Examples of tumors or cancers suitable for treatment or prevention using the methods, drugs, and kits of the present invention particularly refer to tumors or cancers that benefit from IL-21, or immune checkpoint-related tumors, especially PD-1 or PD-L1 or TIGIT-related tumors, including but not limited to mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, kidney cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-Hodgkin lymphoma, esophageal cancer, gastric cancer, and colorectal cancer. In a particularly preferred embodiment, the tumors / cancers disclosed in the present invention refer to colorectal cancer.

[0152] In the following paragraphs, various aspects of the present invention are described in further detail.

[0153] IL-21 fusion protein

[0154] The IL-21 - anti-albumin single-domain antibody fusion protein (IL-21 fusion protein) disclosed in the present invention includes any of the IL-21 fusion proteins described in WO2019246004, the entire content of which is incorporated herein by reference. In some embodiments, the amino acid sequence of the antibody used in the methods and compositions of the present invention includes the amino acid sequence of the IL-21 fusion protein P798 described in WO2019246004.

[0155] The non-limiting, exemplary antibody used in the examples herein is selected from the IL-21 - anti-albumin single-domain antibody fusion protein P798 (JS-EC21) described in WO2019246004, and its amino acid sequence is shown as follows:

[0156] Amino acid sequence of IL-21:

[0157] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:1)

[0158] Amino acid sequence of single-domain antibody:

[0159] EVQLVESGGGLVQPGGSLRLSCAAS GSTWSINT LAWYRQAPGKQRDLVAR ISSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC YAQSTWYPPS WGQGTLVTVSS(SEQ ID NO:5)

[0160] HCDR1: GSTWSINT(SEQ ID NO:2)

[0161] HCDR2: ISSGGST(SEQ ID NO:3)

[0162] HCDR3: YAQSTWYPPS(SEQ ID NO:4)

[0163] Full-length amino acid sequence of IL-21-single-domain anti-albumin antibody fusion protein JS-EC21:

[0164] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSEVQLVESGGGLVQPGGSLRLSCAASGSTWSINTLAWYRQAPGKQRDLVARISSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYAQSTWYPPSWGQGTLVTVSS(SEQ ID NO:6)

[0165] Anti-PD-1 antibody

[0166] As used herein, the term "PD-1 antibody" refers to any chemical compound or biological molecule that binds to the PD-1 receptor and blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. In any method of treatment, medicament, and use of the present invention for treating a human individual, the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD-1. The amino acid sequence of human PD-1 can be found in NCBI locus number: NP_005009. The amino acid sequences of human PD-L1 and PD-L2 can be found in NCBI locus numbers: NP_054862 and NP_079515, respectively.

[0167] As used herein, when referring to an "anti-PD-1 antibody", unless otherwise specified or described, the term includes its antigen-binding fragment.

[0168] The anti-PD-1 antibody or its antigen-binding fragment suitable for any use, therapy, medicament, and kit described in the present invention binds to PD-1 with high specificity and high affinity, blocks the binding of PD-L1 / 2 to PD-1, and inhibits PD-1 signal transduction, thereby achieving an immunosuppressive effect. In any use, therapy, medicament, and kit disclosed herein, the anti-PD-1 antibody includes the full-length antibody itself, as well as antigen-binding portions or fragments that bind to the PD-1 receptor and exhibit functional characteristics similar to those of the intact Ab in inhibiting ligand binding and upregulating the immune system. In some embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody that cross-competes with toripalimab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody is murine, chimeric, humanized, or human Ab or its antigen-binding fragment. In certain embodiments for treating a human individual, the Ab is a humanized Ab.

[0169] 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 suitable for any use, therapy, medicament, and kit described in the present invention comprises a heavy chain constant region of the human IgG1 or IgG4 isotype, more preferably the human IgG4 constant region. In some embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody contains the S228P mutation, which replaces the serine residue in the hinge region with a proline residue that is normally present at the corresponding position in an IgG1 isotype antibody.

[0170] In some embodiments, the anti-PD-1 antibody disclosed in the present invention includes any anti-PD-1 antibody or its antigen-binding fragment described in WO2014206107, and all the content disclosed therein is incorporated herein by reference. In some embodiments, the anti-PD-1 antibodies that can be used for any use, therapy, drug, and kit disclosed in the present invention are selected from the humanized antibodies 38, 39, 41, and 48 described in WO2014206107. In a preferred embodiment, the anti-PD-1 antibody that can be used for any use, therapy, drug, and kit disclosed in the present invention is toripalimab (which is described in WHO Drug Information (Vol. 32, No. 2, pp. 372-373 (2018))).

[0171] The non-limiting, exemplary antibodies used in the examples herein are selected from the humanized antibody toripalimab described in WO2014206107, and its amino acid CDR sequences, variable region sequences, and full-length amino acid sequences are as follows (KABAT):

[0172] Toripalimab light chain variable region (SEQ ID NO:13)

[0173] DVVMTQSPLSLPVTLGQPASISC RSSQSIVHSNGNTYLE WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLT FGQGTKLEIK

[0174] LCDR1: RSSQSIVHSNGNTYLE (SEQ ID NO:7)

[0175] LCDR2: KVSNRFS (SEQ ID NO:8)

[0176] LCDR3: FQGSHVPLT (SEQ ID NO:9)

[0177] Toripalimab heavy chain variable region (SEQ ID NO:14)

[0178] QGQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPIHGLEWIG VIESETGGTAYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR EGITTVATTYYWYFDV WGQGTTVTVSS

[0179] HCDR1: DYEMH (SEQ ID NO:10)

[0180] HCDR2: VIESETGGTAYNQKFKG (SEQ ID NO:11)

[0181] HCDR3: EGITTVATTYYWYFDV (SEQ ID NO:12)

[0182] Toripalimab light chain (SEQ ID NO:15)

[0183] DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0184] Toripalimab heavy chain (SEQ ID NO:16)

[0185] QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0186] Preferably, in any embodiment of the uses, therapies, drugs, and kits disclosed in the present invention, the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, and its light-chain CDRs are the amino acids shown in SEQ ID NO: 7, 8, and 9, and its heavy-chain CDRs are the amino acids shown in SEQ ID NO: 10, 11, and 12.

[0187] More preferably, in any embodiment of the uses, therapies, drugs, and kits disclosed in the present invention, the anti-PD-1 antibody comprises: (a) a light-chain variable region comprising SEQ ID NO: 13, and (b) a monoclonal antibody comprising a heavy-chain variable region of SEQ ID NO: 14.

[0188] Even more preferably, in any embodiment of the uses, therapies, drugs, and kits disclosed in the present invention, the anti-PD-1 antibody is a monoclonal antibody comprising: (a) a light chain comprising SEQ ID NO: 15, and (b) a heavy chain comprising SEQ ID NO: 16.

[0189] The anti-PD-1 antibodies that can be used in any of the uses, therapies, drugs, and kits disclosed in the present invention also include Nivolumab and Pembrolizumab that have been approved by the FDA.

[0190] In certain embodiments, the anti-PD-1 antibodies that can be used in any of the uses, therapies, drugs, and kits disclosed in the present invention also include anti-PD-L1 monoclonal antibodies that specifically bind to PD-L1 to block the binding of PD-L1 to PD-1, such as nivolumab, pembrolizumab, toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Cemiplimab.

[0191] Anti-TIGIT antibody

[0192] The term "TIGIT", which stands for T cell Ig and ITIM domain protein, refers to any native TIGIT from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed TIGIT as well as any form of TIGIT or any fragment thereof produced by intracellular processing. The term also includes variants of native TIGIT, such as splice variants or allelic variants. In one embodiment, TIGIT refers to the full-length or a fragment thereof (such as a mature fragment lacking the signal peptide) of human and cynomolgus monkey TIGIT. In one embodiment, human TIGIT refers to the mature TIGIT (amino acid residues 1-21 being the leader peptide) that is identical to the sequence of amino acid residues 22-244 of Genbank accession number NP_776160.2. In one embodiment, human TIGIT refers to the extracellular domain of TIGIT that is identical to the sequence of amino acid residues 22-141 of Genbank accession number NP_776160.2. In one embodiment, cynomolgus monkey (Macaca fascicularis) TIGIT refers to the mature TIGIT that is identical to the sequence of amino acid residues 22-245 of Genbank accession number XP_005548158.1.

[0193] The terms "anti-TIGIT antibody", "anti-TIGIT", "TIGIT antibody", or "antibody that binds TIGIT" refer to an antibody that is capable of binding to the TIGIT protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting TIGIT.

[0194] As used herein, when referring to an "anti-TIGIT antibody", the term includes its antigen-binding fragments, unless otherwise specified or described.

[0195] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof disclosed in the present invention includes any anti-TIGIT antibody or antigen-binding fragment thereof described in Application No. PCT / CN2020 / 101883 (WO2021 / 008523), and all the content disclosed therein is incorporated herein by reference. In some embodiments, the CDR sequences of the anti-TIGIT antibody used in the methods, uses, and compositions of the present invention include the CDR sequences from the antibodies hu20 or hu3 described in PCT / CN2020 / 101883. In some embodiments, the light chain variable region and heavy chain variable region of the anti-TIGIT antibody used in the methods, uses, and compositions of the present invention include the light chain variable region and heavy chain variable region from the antibodies hu20 or hu3 described in PCT / CN2020 / 101883. In some embodiments, the anti-TIGIT antibody used in the methods, uses, and compositions of the present invention is the antibody hu20 or hu3 described in PCT / CN2020 / 101883.

[0196] In some embodiments, the anti-TIGIT antibodies disclosed in the present invention are selected from: Tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, or BMS-986207.

[0197] Pharmaceutical preparation

[0198] The pharmaceutical composition disclosed in the present invention is a highly stable pharmaceutical composition containing an IL-21-anti-albumin single-domain antibody fusion protein. The present invention discovers that the combination of trehalose can significantly improve the stability of the pharmaceutical composition.

[0199] The pharmaceutical composition disclosed in the present invention is sensitive to pH. When the pH range is controlled at 4.5 - 5.1, the pharmaceutical composition disclosed in the present invention has high stability.

[0200] The present invention provides a pharmaceutical composition comprising: (1) a buffer; (2) an IL-21-anti-albumin single-domain antibody fusion protein.

[0201] The IL-21 fusion protein in the pharmaceutical composition of the present invention comprises: (a) cytokine IL-21, and (b) a single-domain antibody (sdAb) that specifically binds to albumin. The above IL-21 includes the amino acid sequence shown in SEQ ID NO:1, and the above single-domain antibody comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 respectively. Preferably, the fusion protein in the pharmaceutical composition of the present invention comprises cytokine IL-21 with the amino acid sequence shown in SEQ ID NO:1 and sdAb with the amino acid sequence shown in SEQ ID NO:5; more preferably, the fusion protein in the pharmaceutical composition of the present invention comprises the amino acid sequence shown in SEQ ID NO:6 or consists of the same.

[0202] In the pharmaceutical composition of the present invention, the concentration of the IL-21 - anti-albumin single-domain antibody fusion protein is about 0.1 - 100 mg / mL, preferably about 0.2 - 20 mg / mL, preferably about 0.2 - 10 mg / mL, preferably about 0.5 - 5 mg / mL, more preferably 1 - 5 mg / mL; more preferably, the concentration of the above IL-21 - anti-albumin single-domain antibody fusion protein is about 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL or 10 mg / mL, preferably about 1 mg / mL.

[0203] The buffer in the pharmaceutical composition of the present invention can be selected from acetate buffer, citrate buffer, and histidine buffer, which provides a pH of 4.0 to 6.5, preferably 4.5 to 5.5, more preferably 4.8 ± 0.3, and more preferably about 4.8 for the pharmaceutical composition of the present invention. On the other hand, the pH of the buffer used in the pharmaceutical composition of the present invention can be 4.0 - 6.5, preferably 4.5 - 5.5, more preferably 4.8 ± 0.3, and more preferably about 4.8.

[0204] The particularly preferred buffer solution in the pharmaceutical composition of the present invention is an acetate buffer solution. Preferably, the above-mentioned acetate buffer solution is an acetic acid-sodium acetate buffer solution or an acetic acid-potassium acetate buffer solution, and an acetic acid-sodium acetate buffer solution is preferred. In some embodiments, the acetate buffer solution is made of 1-30 mM acetic acid and 1-30 mM sodium acetate. In some embodiments, the acetate buffer solution is made of acetic acid and sodium acetate with a molar ratio of about 2:3. In some embodiments, the acetate buffer solution is made of acetic acid and sodium acetate with a molar ratio of about 1:2.1. In some embodiments, the acetate buffer solution is made of acetic acid and sodium acetate with a molar ratio of about 1:5.7. In some embodiments, the acetate buffer solution is an acetate buffer solution with a pH of about 4.8 made of about 8 mM acetic acid and about 12 mM sodium acetate. In some embodiments, the acetate buffer solution is an acetate buffer solution with a pH of about 5.0 made of about 6.5 mM acetic acid and about 13.5 mM sodium acetate. In some embodiments, the acetate buffer solution is an acetate buffer solution with a pH of about 5.5 made of about 3 mM acetic acid and about 17 mM sodium acetate.

[0205] Therefore, the pharmaceutical composition of the present invention may contain: an acetic acid-sodium acetate buffer solution with a pH of 4.5-5.5 (preferably 4.5-5.0), and its concentration in the pharmaceutical composition is 10-30 mM; and 0.5-5 mg / mL of the IL-21-anti-albumin single-domain antibody fusion protein in any of the previous embodiments, preferably JS-EC21 disclosed herein.

[0206] In some embodiments, the pharmaceutical composition of the present invention further contains a stabilizer. Preferably, the above-mentioned stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, and trehalose. Preferably, the above-mentioned stabilizer is trehalose. The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10 mM-400 mM, preferably 20 mM-300 mM, and more preferably 30 mM-200 mM. In some embodiments, the above-mentioned stabilizer is mannitol with a concentration of about 100-300 mM, preferably 200-300 mM; or the above-mentioned stabilizer is sucrose with a concentration of about 100-300 mM, preferably 200-300 mM; or the above-mentioned stabilizer is trehalose with a concentration of about 100-300 mM, preferably 200-300 mM; or the above-mentioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol; or the above-mentioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose. Preferably, the above-mentioned stabilizer is about 100-300 mM trehalose or 100-300 mM sucrose; more preferably, the above-mentioned stabilizer is about 200-280 mM trehalose.

[0207] Thus, in some embodiments, the pharmaceutical composition of the present invention comprises: an acetic acid-sodium acetate buffer solution with a pH of 4.5 - 5.5 (preferably 4.5 - 5.0), and its concentration in the pharmaceutical composition is 10 - 30 mM; 0.5 - 5 mg / mL of the IL-21-anti-albumin single-domain antibody fusion protein of any of the previous embodiments, preferably JS-EC21 disclosed herein; and 20 mM - 300 mM of a stabilizer. Preferably, the above stabilizer includes one or more of sodium chloride, mannitol, sucrose, and trehalose, preferably 100 - 300 mM of trehalose or 100 - 300 mM of sucrose, and more preferably 200 - 280 mM of trehalose.

[0208] In some embodiments, the pharmaceutical composition of the present invention further comprises a surfactant. The preferred surfactants are selected from polysorbate 80, polysorbate 20, and poloxamer 188. The most preferred surfactant is polysorbate 80. Calculated as w / v, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% - 0.1%, preferably about 0.02% - 0.08%, and preferably about 0.02% - 0.04%. As a non-limiting example, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.04%, or 0.08%, preferably 0.02%.

[0209] Thus, in some embodiments, the pharmaceutical composition of the present invention comprises: an acetic acid-sodium acetate buffer solution with a pH of 4.5 - 5.5 (preferably 4.5 - 5.0), and its concentration in the pharmaceutical composition is 10 - 30 mM; 0.5 - 5 mg / mL of the IL-21-anti-albumin single-domain antibody fusion protein of any of the previous embodiments, preferably JS-EC21 disclosed herein; and 20 mM - 300 mM of a stabilizer. Preferably, the above stabilizer includes one or more of sodium chloride, mannitol, sucrose, and trehalose, preferably 100 - 300 mM of trehalose or 100 - 300 mM of sucrose, and more preferably 200 - 280 mM of trehalose; and polysorbate 80 at a concentration of 0.02% - 0.04% calculated as w / v.

[0210] The pharmaceutical composition of the present invention can be a liquid preparation or a freeze-dried preparation.

[0211] Drug combination

[0212] The present invention also provides a drug combination, which contains the IL-21 fusion protein described herein and an immune checkpoint modulator. The immune checkpoint modulator disclosed in the present invention can be as described in any of the foregoing embodiments; preferably, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-TIGIT antibody. In this drug combination, the above-mentioned anti-IL-21 fusion protein and immune checkpoint modulator can be provided in the form of a mixture (i.e., in the form of a pharmaceutical composition), or each can be provided in the form of an independent preparation. When in the form of an independent preparation, each preparation contains a pharmaceutically acceptable carrier in addition to the active ingredient. When provided in the form of a pharmaceutical composition, the pharmaceutical composition usually contains a pharmaceutically acceptable carrier in addition to the active ingredient.

[0213] The drug combination of the present invention may also contain one or more additional therapeutic agents. The additional therapeutic agent can be (for example) a chemotherapeutic agent, a biotherapeutic agent, an immunogenic agent (such as an attenuated cancer cell, a tumor antigen, an antigen-presenting cell (such as a dendritic cell pulsed with a tumor-derived antigen or nucleic acid), an immunostimulatory cytokine (such as IL-2, IFN-γ, GM-CSF), and a cell transfected with a gene encoding an immunostimulatory cytokine (such as but not limited to GM-CSF)).

[0214] Therapeutic methods and uses

[0215] The present invention provides the use of the IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or the combination of the IL-21 fusion protein and the immune checkpoint modulator, or the combination of the above-mentioned pharmaceutical composition and the immune checkpoint modulator in the preparation of a drug for preventing or treating tumors. The present invention also provides the use of the above-mentioned pharmaceutical composition or IL-21 fusion protein in a drug for enhancing or improving the anti-tumor activity of the immune checkpoint modulator.

[0216] The present invention provides a method for preventing or treating tumors, which comprises administering an effective amount of the IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or the combination of the IL-21 fusion protein and the immune checkpoint modulator, or the combination of the above-mentioned pharmaceutical composition and the immune checkpoint modulator to an individual in need. The effective amount includes a prophylactically effective amount and a therapeutically effective amount.

[0217] The present invention provides the IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or the combination of the IL-21 fusion protein and the immune checkpoint modulator, or the combination of the above-mentioned pharmaceutical composition and the immune checkpoint modulator, which is used for treating or preventing tumors.

[0218] The tumors disclosed by the present invention may be as described in any of the foregoing embodiments. Preferably, the tumors described herein are tumors that benefit from IL-21 treatment, or are PD-1 or TIGIT-related tumors, preferably selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, renal cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-Hodgkin lymphoma, esophageal cancer, gastric cancer, and colorectal cancer; preferably, the tumors are metastatic melanoma, renal cell carcinoma, colorectal cancer, and non-Hodgkin lymphoma, more preferably colorectal cancer.

[0219] The immune checkpoint modulators disclosed by the present invention may be as described in any of the foregoing embodiments; preferably, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-TIGIT antibody.

[0220] In one or more embodiments, the difference analysis between the two groups of the present invention is performed by two-tailed unpaired Student's t test, * indicates P < 0.05; ** indicates P < 0.01, *** indicates P < 0.001, ns indicates no significant difference; P < 0.05 indicates a significant increase or significant elevation.

[0221] In one or more embodiments, compared with the use of IL-21 fusion protein or anti-PD-1 antibody alone, the combination of the IL-21 fusion protein and anti-PD-1 antibody disclosed by the present invention has one or more properties selected from the following:

[0222] (1) The proportion of CD3 + T cells in immune cells is significantly increased;

[0223] (2) The proportion of CD8 + T cells in T cells is significantly increased;

[0224] (3) The proportion of CD8 + T cells in the proliferative state (Ki 67 + ) is significantly increased; and

[0225] (4) The proportion of NK cells in immune cells is significantly increased.

[0226] Therefore, in some embodiments, the present application provides the use of the combination of the IL-21 fusion protein and anti-PD-1 antibody disclosed herein in the preparation of a reagent for one or more of the following uses:

[0227] (1) Increasing the proportion of CD3 + T cells in immune cells;

[0228] (2) Increasing the proportion of CD8 + T cells in T cells;

[0229] (3) Increase the proportion of CD8 + T cells in the proliferative state (Ki 67 + ); and

[0230] (4) Increase the proportion of NK cells among immune cells.

[0231] In a preferred embodiment, the reagent is used for the treatment or prevention of the tumors disclosed herein.

[0232] In one or more embodiments, compared with the use of the IL-21 fusion protein or the anti-TIGIT antibody alone, the combination use of the IL-21 fusion protein and the anti-TIGIT antibody of the present invention has one or more properties selected from the following:

[0233] (1) Significantly increase the expression level of interferon γ;

[0234] (2) Significantly increase the expression level of granzyme A;

[0235] (3) Significantly increase the expression level of granzyme B;

[0236] (4) Significantly increase the expression level of perforin 1;

[0237] (5) Enrich more KEGG signaling pathways with differential gene expression; and

[0238] (6) Most of the significantly enriched KEGG signaling pathways induced are mediated by the IL-21 fusion protein.

[0239] Therefore, in some embodiments, the present application provides the use of the combination of the IL-21 fusion protein disclosed herein and the above anti-TIGIT antibody in the preparation of a reagent for one or more of the following uses:

[0240] (1) Increase the expression level of interferon γ in a subject;

[0241] (2) Increase the expression level of granzyme A in a subject;

[0242] (3) Increase the expression level of granzyme B in a subject; and

[0243] (4) Increase the expression level of perforin 1 in a subject.

[0244] In a preferred embodiment, the reagent is used for the treatment or prevention of the tumors disclosed herein.

[0245] The preferred IL-21 fusion protein for the tumors disclosed in the present invention may be as described in any of the embodiments herein, and is preferably composed of: (a) cytokine IL-21, and (b) a single-domain antibody (sdAb) that specifically binds to albumin. The cytokine IL-21 includes the amino acid sequence shown in SEQ ID NO:1, and the single-domain antibody (sdAb) contains HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 respectively; more preferably, it is composed of the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO:5; more preferably, it is composed of the amino acid sequence shown in SEQ ID NO:6.

[0246] The preferred anti-PD-1 antibody for the tumors disclosed in the present invention may be as described in any of the embodiments herein, and is more preferably an antibody with light chain CDRs having the amino acids shown in SEQ ID NOs: 7, 8, and 9, and heavy chain CDRs having the amino acids shown in SEQ ID NOs: 10, 11, and 12, more preferably a monoclonal antibody containing the light chain variable region shown in SEQ ID NO:13 and the heavy chain variable region shown in SEQ ID NO:14, more preferably a monoclonal antibody containing the light chain shown in SEQ ID NO:15 and the heavy chain shown in SEQ ID NO:16, and most preferably the humanized antibodies 38, 39, 41, and 48 described in WO2014206107, and most preferably toripalimab.

[0247] In a particularly preferred embodiment, the present invention provides a method for preventing or treating tumors, which method comprises administering to a tumor patient a therapeutically effective amount of an IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or a combination of an IL-21 fusion protein and an anti-PD-1 antibody or a combination of the above-mentioned pharmaceutical composition and an anti-PD-1 antibody; preferably, the tumor patient is a colorectal cancer patient; preferably, the IL-21 fusion protein contains the amino acid sequence shown in SEQ ID NO:6; preferably, the anti-PD-1 antibody is toripalimab.

[0248] In a particularly preferred embodiment, the present invention provides the use of an IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or a combination of an IL-21 fusion protein and an anti-PD-1 antibody or a combination of the above-mentioned pharmaceutical composition and an anti-PD-1 antibody in the preparation of a medicament for preventing or treating tumors; preferably, the tumor patient is a colorectal cancer patient; preferably, the IL-21 fusion protein contains the amino acid sequence shown in SEQ ID NO:6; preferably, the anti-PD-1 antibody is toripalimab.

[0249] Kit

[0250] The present invention provides a kit, which comprises:

[0251] (1) One or more single-dose units of an IL-21 fusion protein and one or more single-dose units of an immune checkpoint modulator, wherein the IL-21 fusion protein is as described herein; preferably, the immune checkpoint modulator is as described herein; or

[0252] (2) One or more single-dose units of a pharmaceutical composition as described herein.

[0253] In one or more embodiments, the kit disclosed by the present invention further comprises instructions for indicating the usage method of the pharmaceutical composition.

[0254] Medical uses and methods

[0255] The present invention further provides a pharmaceutical composition disclosed in any embodiment of the present invention for treating or preventing cancer, the use of the pharmaceutical composition disclosed in any embodiment of the present invention in the preparation of a medicament for treating or preventing cancer, and a method of treating or preventing cancer by administering a therapeutically effective amount of the pharmaceutical composition disclosed in any embodiment of the present invention to an individual or patient in need thereof.

[0256] In the present invention, the diseases suitable for treatment and prevention by the pharmaceutical composition of the present invention include but are not limited to mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer, and colorectal cancer.

[0257] The present invention will be specifically described below by way of examples, which do not mean any limitation to the present invention. The methods and materials used in the examples are conventional methods and materials in the art unless otherwise specified.

[0258] The present invention uses the following abbreviations:

[0259] His-tag represents a histidine tag;

[0260] HRP represents horseradish peroxidase;

[0261] TMB represents 3,3',5,5'-tetramethylbenzidine;

[0262] HBSS represents Hank's Balanced Salt Solution, that is, Hank's balanced salt solution;

[0263] HEPES represents N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, a Good's buffer reagent.

[0264] BID represents one dose, twice a day

[0265] CDR stands for Complementary Determining Region

[0266] DFS stands for Disease Free Survival

[0267] FR stands for Framework Region

[0268] IgG stands for Immunoglobulin G

[0269] Q2W stands for one dose every two weeks

[0270] QD stands for one dose every day

[0271] The capillary micro differential scanning fluorimetry (DSF) of the present invention

[0272] Use a capillary to automatically fill with the sample and place it in the corresponding numbered position. Set the scanning temperature at 20°C - 95°C and the heating rate at 1°C / min. After the instrument operation is completed, process and analyze the data.

[0273] The appearance detection method of the present invention

[0274] The appearance is detected by visual inspection. Ensure that the light intensity of the clarity detector is maintained between 1000 lx - 1500 lx. Keep the sample at the same horizontal level as the eyes, and gently shake or invert it to avoid generating air bubbles. Conduct visual inspection in front of a black background and a white background respectively. Observe the color, opalescence and visible foreign matters.

[0275] The protein content detection method of the present invention

[0276] The protein concentration is detected using an ultraviolet spectrophotometer. Set the percentage extinction coefficient (E1%) at 1.527 (g / ml) -1 cm -1 . Use a BIO MATE 3S instrument. After cleaning the cuvette with ultrapure water, add 150 μL of ultrapure water, click to measure, and use ultrapure water for blank correction. Each sample is measured in duplicate solutions in parallel and each solution is measured 3 times repeatedly, and the average value is taken. Calculate the protein concentration of the corresponding sample through the extinction coefficient and OD value.

[0277] The size exclusion chromatography (SEC - HPLC) detection method of the present invention

[0278] The purity of SEC - HPLC is detected using an HPLC (Waters e2695 instrument) equipped with a SEC column (Waters Xbrige BEH 125A 7.8 * 300 mm, 3.5 μm). The mobile phase composition is 50 mM phosphate buffer, 300 mM sodium chloride, 300 mM arginine, pH 6.8 ± 0.2. The relative percentages of the main peak (monomer), polymer and fragment are calculated by the area normalization method. See the following table for details:

[0279] Chromatographic conditions Chromatographic parameters Detector wavelength 280 nm Autosampler temperature 25±3℃ Column temperature 5±3℃ Flow rate 0.50 ml / min Injection volume 25 μl Run mode Isocratic mode Run time 30.00 min

[0280] The non-reducing capillary electrophoresis (NR-CE-SDS) detection method of the present invention

[0281] The purity of NR-CE-SDS was detected by a capillary electrophoresis instrument (Maurice instrument) equipped with a CE-SDS cartridge. Take 50 μl of the dilution solution (1× sample buffer Maurice CE-SDS), add 5.0 μl of 0.8 M iodoacetamide and mix well, then add 50 μl of the sample (1 mg / ml), and vortex to mix evenly; at the same time, take 50 μl of the dilution solution, add 5.0 μl of 0.8 M iodoacetamide and mix well, incubate at 70 °C for 5 min, and calculate by the area normalization method. The percentage of the main peak in the sum of the areas of all corrected peaks is the main peak purity.

[0282] The reducing capillary electrophoresis (R-CE-SDS) detection method of the present invention

[0283] The purity of R-CE-SDS was detected by a capillary electrophoresis instrument (Maurice instrument) equipped with a CE-SDS cartridge. Take 50 μl of the dilution solution (1× sample buffer Maurice CE-SDS), add 5.0 μl of 2-mercaptoethanol and mix well, then add 50 μl of the sample (1 mg / ml), and vortex to mix evenly; at the same time, take 50 μl of the dilution solution, add 5.0 μl of 2-mercaptoethanol and mix well, incubate at 70 °C for 15 min, and calculate by the area normalization method. The percentage of the main peak in the sum of the areas of all corrected peaks is the main peak purity.

[0284] The imaging capillary isoelectric focusing (iCIEF) detection method of the present invention

[0285] iCIEF was detected by a capillary electrophoresis instrument (Maurice instrument) equipped with an iCIEF cartridge. After the sample was diluted and treated with the iCIEF mixture, it was detected using a capillary electrophoresis instrument (Maurice). Components with different isoelectric points were focused at different positions to achieve the effects of focusing and separation.

[0286] The iCIEF mixture was prepared as follows:

[0287] Reagent name Volume required per sample (μL) 1% MC (methylcellulose) 35 Pharmalyte Mix (ampholyte) 4 pI Marker Mix (isoelectric point marker) 1 500 mM Arg (arginine) 2 8 M urea 50

[0288] Mix 8 μl of the sample solution (1 mg / mL) with 92 μL of the iCIEF mixture, centrifuge to remove air bubbles, and perform instrumental analysis and detection.

[0289] The binding activity detection method of the present invention

[0290] Dilute the recombinant human IL-21R protein His tag (Acro, catalog number ILR-H5226) to 4.0 μg / ml for coating, and incubate it statically in a constant temperature incubator at 37 ± 2 °C for 90 min. Wash the plate and block it with 2% skim milk. Add test samples at different concentrations (starting from 4 μg / mL, diluted in 3-fold serial dilutions), incubate for 1 hour and wash the plate. Then incubate with HRP-conjugated rabbit anti-camel VHH antibody (GenScript, catalog number A01861-200) diluted 1:5000 for 1 hour, and then incubate with HRP substrate TMB (Sigma, catalog number T2885) for 30 minutes for color development to detect the binding signal of the test samples. Use the slope curve fitting of the logarithm (agonist) to the response variable (GraphPad Prism) to determine EC 50 。

[0291] The method for detecting cell activity of the present invention

[0292] On the first day, dilute the test sample to 30 μg / ml (6× analysis concentration) with the test sample analysis buffer (HBSS + 10 nM HEPES), and perform 3-fold serial dilutions at 12 concentrations. Adjust the Pfeiffer cell density to 4.0×10 6 cells / ml, add 25 μl / well to a 96-well round bottom plate, and then add 5 μl / well of the pre-diluted test sample to the 96-well round bottom plate. Incubate in an incubator at 37.0 °C and 5.0% carbon dioxide for 30 min. Add 10 μl of lysis buffer (STAT3 phospho-Y705 kit) to each well and lyse at room temperature for 30 min. Then aspirate 16 μl of the cell lysate mixture from each well into a HTRF 96-well small volume plate, and add 4 μl of the mixed antibody (STAT3 phospho-Y705 kit). Incubate in the dark at room temperature for 18 ± 2 h. On the second day, read the plate with a multimode microplate reader and perform data analysis and processing with GraphPad Prism software. The calculation formula is: Ratio 665 / 620 = OD 665nm / OD620nm * 10000. Specific embodiments

[0294] To better understand the content of the present invention, the following specific examples are provided for further illustration, but the specific embodiments are not intended to limit the content of the present invention. The content of all references cited throughout this application is hereby incorporated by reference in its entirety.

[0295] Example 1: Protein concentration screening experiment

[0296] In liquid pharmaceutical compositions, the protein concentration closely affects the stability of antibodies, and each fusion protein with unique physicochemical properties has an optimal concentration. This example aims to screen an optimal protein concentration to enable the IL-21 fusion protein disclosed in the present invention to have optimal stability for clinical applications.

[0297] In this example, histidine buffers (histidine - acetic acid) with pH 4.5 and pH 5.0 were used to investigate the stability of IL-21 - fusion proteins at different concentrations. The IL-21 fusion protein with a concentration of 4.0 mg / ml (number JS-EC21) was dialyzed and exchanged into the corresponding buffer three times, with each exchange time being greater than 4 hours. After adjusting to the target concentration, Tween 80 was added, and the mixture was filtered and filled into 2R vials with a specification of 0.5 ml / vial for stability sampling and detection. IL-21 - fusion proteins with concentrations of 1 mg / ml, 3 mg / ml, and 5 mg / ml were screened respectively, as shown in Table 1 specifically.

[0298] The samples were placed under accelerated conditions (25 ± 2°C) and long-term conditions (5 ± 3°C), and were taken out for analysis and detection at week 0, week 2, and week 4. The main pathways of protein degradation are the formation of aggregates, cleavage products, and charged variants. The evaluation indicators include: 1. The percentage of protein monomers and aggregated forms determined by size exclusion chromatography (SEC-HPLC); 2. CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method to detect the purity of the protein under reducing and non-reducing conditions; 3. ELISA method to detect antibody binding activity and cell activity. The effects of different concentrations on the stability of IL-21 - fusion proteins were investigated, and the results are shown in Tables 2 - 6.

[0299] Table 1 Prescription information in the protein concentration and pH screening experiment

[0300]

[0301] 1.1 SEC-HPLC screening results

[0302] According to the SEC-HPLC results in Table 2, after being placed under accelerated conditions for 4 weeks, the protein aggregate content increased in Formulations FS1-3 and FS1-4, while there were no obvious changes in Formulations FS1-1 and FS1-2; after being placed under long-term conditions for 4 weeks, there were no obvious changes in the purity of SEC-HPLC protein monomers.

[0303] Table 2 SEC-HPLC data in the protein concentration and pH screening experiment

[0304]

[0305]

[0306] 1.2 NR-CE-SDS Screening Results

[0307] According to the NR-CE-SDS results in Table 3, after being placed under accelerated conditions for 4 weeks, the protein purity in all formulations decreased slightly; after being placed under long-term conditions for 4 weeks, no obvious purity changes were observed in all samples.

[0308] Table 3 CE-SDS Data in Protein Concentration and pH Screening Experiments

[0309]

[0310] 1.3 R-CE-SDS Screening Results

[0311] According to the R-CE-SDS results in Table 4, after being placed under accelerated conditions for 4 weeks, the R-CE-SDS purity of the protein in Formulations FS1-3 and FS1-4 decreased; after being placed under long-term conditions for 4 weeks, no obvious purity changes were observed in all samples.

[0312] Table 4 CE-SDS Data in Protein Concentration and pH Screening Experiments

[0313]

[0314] 1.4 Biological Activity Screening Results

[0315] According to the activity screening results in Table 5, after being placed under accelerated and long-term conditions for 4 weeks, no obvious changes were observed in the binding activity and cell activity of all samples.

[0316] Table 5 Activity Data in Protein Concentration and pH Screening Experiments

[0317]

[0318]

[0319] Judging from the SEC-HPLC results, in the histidine buffer solution at pH 5.0, for the samples with high protein concentrations of 3 mg / ml and 5 mg / ml (Formulations FS1-3 and FS1-4), after being placed under accelerated conditions for 4 weeks, the SEC-HPLC monomer purity decreased significantly, while there was no significant change at the low protein concentration of 1 mg / ml. There were no obvious differences in the performance of R-CE-SDS purity and NR-CE-SDS purity among different formulations. Judging from the activity results, there were no obvious changes in the binding activity and cell activity of the samples in each formulation. The above results show that the product is sensitive to protein concentration. Therefore, a protein concentration of 1 mg / ml was selected for the next round of formulation screening.

[0320] Example 2: Buffer System and pH Screening

[0321] To further explore the effects of different buffer systems and pH on the stability of the IL-21-fusion protein JS-EC21 of the present invention, acetate buffer, histidine buffer, and citrate buffer were screened, with the pH range from 4.7 to 6.5. The IL-21-fusion protein (designated JS-EC21) was dialyzed against the corresponding buffer, and the sample was in the corresponding formulation after buffer exchange. The final protein concentration was approximately 1 mg / ml, and it was aseptically filled into 2R vials with a specification of 2.5 ml / vial for stability testing (as shown in Table 6).

[0322] The samples were placed in accelerated conditions (25 ± 2°C) and long-term conditions (5 ± 3°C) respectively, and were taken out for analysis and testing at week 0, week 1, week 2, week 4, and week 8. The effects of different buffers and pH on the stability of the IL-21-fusion protein were investigated, and the results are shown in Tables 7 - 13.

[0323] The evaluation indicators include: 1. Determination of the Tm value (melting temperature) of the protein by DSC (capillary micro differential scanning fluorimetry); 2. Visual inspection of the appearance; 3. Determination of the protein content by ultraviolet spectrophotometry; 4. Determination of the percentages of protein monomers, aggregates, and fragments by size exclusion chromatography (SEC-HPLC); 5. Detection of the percentages of the main charge, acidic charge, or basic charge of the protein by iCIEF (imaging capillary isoelectric focusing); 6. Detection of the purity of the protein by CE-SDS (sodium dodecyl sulfate capillary electrophoresis) under reducing and non-reducing conditions; 7. Detection of antibody binding activity and cell activity by ELISA.

[0324] Table 6 Experimental protocol for buffer system and pH screening

[0325]

[0326]

[0327] 2.1 DSF screening results

[0328] According to the DSF results in Table 7, as the pH increased in the 8 formulations, the Tm value showed a downward trend, and FS2-1, FS2-2, FS2-3, and FS2-4 were slightly higher than the other 4 groups.

[0329] Table 7 DSF data in buffer system and pH screening

[0330]

[0331] 2.2 Appearance and protein concentration screening results

[0332] According to the results in Table 8, white dots were found to precipitate in the two groups of FS2-7 and FS2-8 at T0; after being placed for 8 weeks under accelerated conditions or long-term conditions, only the two groups of FS2-1 and FS2-3 showed no abnormal appearance.

[0333] According to the results in Table 9, the five groups of FS2-4, FS2-5, FS2-6, FS2-7 and FS2-8 terminated the detection during the stability sample preparation process due to abnormal appearance; for the remaining three groups, after being placed for 8 weeks under accelerated conditions or long-term conditions, no change in protein content was observed.

[0334] Appearance results in buffer system and pH screening in Table 8

[0335]

[0336] Note: " / " indicates not determined.

[0337] Protein content results in buffer system and pH screening in Table 9

[0338]

[0339] Note: " / " indicates not determined.

[0340] 2.3 SEC-HPLC purity screening results

[0341] According to the SEC-HPLC results in Table 10, the four groups of FS2-4, FS2-6, FS2-7 and FS2-8 terminated the detection due to abnormal appearance; for the remaining four groups, after being placed for 8 weeks under accelerated conditions or long-term conditions, no significant change was observed in the SEC-HPLC protein monomer purity.

[0342] SEC-HPLC data in buffer system and pH screening in Table 10

[0343]

[0344]

[0345] Note: " / " indicates not determined.

[0346] 2.4 NR / R-CE-SDS purity screening results

[0347] According to the NR / R-CE-SDS purity results in Table 11, among them, the four groups of samples FS2-4, FS2-6, FS2-7 and FS2-8 terminated the detection due to abnormal appearance; for the remaining four groups, after being placed for 8 weeks under accelerated conditions and long-term conditions, no significant change in protein purity was observed in all samples.

[0348] CE-SDS data in buffer system and pH screening in Table 11

[0349]

[0350] Note: " / " indicates not determined.

[0351] 2.5iCIEF screening results

[0352] According to the iCIEF results in Table 12, among which the detection of four groups of samples FS2-4, FS2-6, FS2-7 and FS2-8 was terminated due to abnormal appearance; after being placed for 8 weeks under accelerated conditions, an increase in acidic peaks occurred in the remaining four groups, and no differences were observed among groups; after being placed for 8 weeks under long-term conditions, no significant changes occurred in all samples.

[0353] iCIEF results in buffer system and pH screening in Table 12

[0354]

[0355] Note: " / " indicates not determined.

[0356] 2.6 Biological activity screening results

[0357] According to the activity results in Table 13, after being placed for 8 weeks under accelerated and long-term conditions, among which the detection of three groups of samples FS2-6, FS2-7 and FS2-8 was terminated due to abnormal appearance; no obvious changes occurred in the binding activity and cell activity of the remaining 5 groups.

[0358] Activity results in buffer system and pH screening in Table 13

[0359]

[0360]

[0361] Note: " / " indicates not determined.

[0362] Based on the comprehensive analysis of various data, from the DSF results, FS2-1, FS2-2, FS2-3, and FS2-4 are slightly higher than the other 4 groups; from the appearance results, after being placed for 8 weeks under accelerated conditions, only the formulations of FS2-1 and FS2-3 did not show appearance changes; from the SEC-HPLC purity results, NR / R-CE-SDS purity results, iCIEF results and activity results, no significant differences were observed between the formulations of FS2-1 and FS2-3. Based on the above results, this product is sensitive to pH. Generally speaking, the buffer pH shows the best performance in the range of 4.5 - 5.0. Therefore, 20 mM acetic acid - sodium acetate buffer, pH 4.5 - 5.0 is selected to carry out the next round of formulation screening.

[0363] Example 3: Screening experiment of stabilizer and surfactant

[0364] To further explore the effects of different stabilizers (excipients) and surfactants on the stability of the IL-21 fusion protein JS-EC21 of the present invention, sucrose, trehalose or mannitol was selected as the stabilizer for comparative testing.

[0365] The IL-21 fusion protein was subjected to UF / DF buffer exchange using a Millipore Pellicon3 0.11m 2 membrane, diluted to place the sample in the corresponding formulation, with a protein concentration of approximately 1 mg / ml. The specific formulation information is shown in Table 14. The samples were filled into 2R vials and 6R vials with specifications of 2.0 ml / vial and 3.0 ml / vial respectively, and subjected to stability sampling and testing.

[0366] Each formulated preparation was placed under accelerated (25 ± 2°C) and long-term (5 ± 3°C) conditions, and analyzed and tested at week 0, week 2, and week 4 respectively; under freeze-thaw (-40°C / RT) conditions, the samples were tested for 3 and 5 cycles of repeated freeze-thaw; under shaking (250 rpm, RT) conditions, analyzed and tested on the 1st day and the 3rd day respectively; under light (5000 lux, RT) conditions, analyzed and tested on the 5th day and the 10th day respectively. The effects of different stabilizers and surfactants on the stability of the IL-21 fusion protein were investigated, and the results are shown in Tables 15 - 19.

[0367] The evaluation indicators include: 1. Visual inspection of appearance; 2. Detection of protein content by ultraviolet spectrophotometry; 3. Determination of the percentages of protein monomers, aggregates, and fragments by size exclusion chromatography (SEC-HPLC); 4. Measurement of the main charge, acidic charge, or basic charge content of the antibody by cation exchange chromatography (CEX-HPLC); 5. Detection of protein purity by CE-SDS (sodium dodecyl sulfate capillary electrophoresis) under reducing and non-reducing conditions; 6. Detection of antibody binding activity by ELISA.

[0368] Table 14 Screening experiment plan for stabilizers and surfactants

[0369]

[0370] 3.1 Screening results of appearance and protein content

[0371] According to the results in Table 15, after being placed under accelerated conditions and long-term conditions for 4 weeks, the protein content of all samples did not show significant changes; no abnormal appearance was observed; after 3 days under shaking conditions, no abnormal appearance was observed; after 10 days under white light irradiation, no abnormal appearance was observed; after 3 cycles of freeze-thaw, a few white dots appeared in the FS3-1 formulation.

[0372] Table 15 Results of protein content and appearance in the screening of stabilizers and surfactants

[0373]

[0374] Note: " / " indicates not determined.

[0375] 3.2 SEC-HPLC Screening Results

[0376] According to the SEC purity results in Table 16, no obvious change in monomer content occurred under accelerated, long-term, freeze-thaw, shaking, and light conditions.

[0377] SEC-HPLC Purity in the Screening of Stabilizers and Surfactants - Table 16

[0378]

[0379] 3.3 NR / R-CE-SDS Screening Results

[0380] According to the NR-CE-SDS purity results in Table 17, after the 3 groups of samples were placed for 4 weeks under accelerated conditions, the protein purities of NR-CE-SDS and R-CE-SDS both decreased, but no inter-group differences were shown; after being placed for 4 weeks under long-term conditions, 5 freeze-thaw cycles, 3 days of shaking, or 10 days of light exposure, no obvious changes in the protein purities of NR-CE-SDS or R-CE-SDS were observed.

[0381] Screening of Stabilizers and Surfactants - NR / R-CE-SDS Purity Results - Table 17

[0382]

[0383] 3.4 CEX-HPLC Screening Results

[0384] According to the CEX-HPLC results in Table 18, after the 3 groups of samples were placed for 4 weeks under accelerated conditions, an increase in acidic peaks occurred in all of them, but no inter-group differences were shown. Under light conditions, the increase in acidic peaks of the FS3-2 formulation was relatively obvious. After being placed for 4 weeks under long-term conditions, 5 freeze-thaw cycles, 3 days of shaking, or 10 days of light exposure, no significant changes were observed in the CEX-HPLC results of all samples.

[0385] Screening of Stabilizers and Surfactants - CEX-HPLC Data - Table 18

[0386]

[0387] 3.5 Biological Activity Screening Results

[0388] According to the biological activity measurement results in Table 19, after being placed for 4 weeks under high temperature, accelerated, or long-term conditions, as well as 5 freeze-thaw cycles, 3 days of shaking, or 10 days of light exposure, no significant changes occurred in the binding activities of all samples.

[0389] Table 19 Screening of Stabilizers and Surfactants - Binding Activity Results

[0390]

[0391] From the appearance results, a little white precipitate appeared in FS3-1 under freeze-thaw conditions; from the results of protein content, SEC-HPLC purity, NR / R-CE purity, CEX, and activity, there were no significant differences among the three groups of formulations. Based on the above results, the IL-21 fusion protein of the present invention showed good stability under the conditions of acetate-sodium acetate buffer (pH 4.5 - 5.0), trehalose or sucrose, and polysorbate 80 as stabilizers.

[0392] Example 4: Pharmacokinetics of IL-21 Fusion Protein

[0393] 1. Test Purpose

[0394] To evaluate the differences in the pharmacokinetic properties of the IL-21 - anti-albumin single-domain antibody fusion protein JS-EC21 of the present invention and recombinant human IL-21 by intraperitoneal administration to mice. The IL-21 - anti-albumin single-domain antibody fusion protein JS-EC21 in this example was formulated according to Prescription FS3-3.

[0395] 2. Administration and Blood Sample Preparation

[0396] Twelve female Balb / c mice were selected and randomly divided into 2 groups, with 6 animals in each group. They were intraperitoneally injected with JS-EC21 at 0.15 mg / kg and recombinant human IL-21 at 0.15 mg / kg, respectively. Before administration, and at 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h after administration, mouse blood was collected into EDTA-K2 anticoagulant tubes, centrifuged at 3500 rpm for 10 minutes, and the plasma was collected and stored in a -80°C refrigerator for future testing.

[0397] 3. PK Detection Process

[0398] a. Dilute anti-human IL-21, 250×Cap.Ab (invitrogen, 88 - 8218-88) as the antigen to 1×, coat a 96-well plate at 100 μl / well, and incubate overnight at 2 - 8°C.

[0399] b. Add 1×ELISA dilution at 200 μL / well and block at room temperature for 1 hour. Wash the plate 4 times with 1×PBST at 300 μl / well.

[0400] c. Prepare the standard curve samples of JS-EC21 with blank mouse plasma in the range of 312.5 ng / mL - 20 μg / mL; prepare the standard curve samples of recombinant human IL-21 with blank mouse plasma in the range of 156.3 ng / mL - 20 μg / mL; dilute the sample to be tested with blank mouse plasma within the standard curve range. Finally, dilute the above standard curve samples and the sample to be tested uniformly 5-fold with 1×ELISA diluent and add 100 μL per well. Incubate at room temperature for 2 hours and then wash the plate.

[0401] d. Add the anti-human IL-21 250x Det.Ab (invitrogen, 88 - 8218 - 88) diluted to 1× at 100 μL per well, incubate at room temperature for 1 hour and then wash the plate.

[0402] e. Add the avidin-HRP (invitrogen, 88 - 8218 - 88) diluted to 1× at 100 μL per well, incubate at room temperature for 0.5 hour and then wash the plate.

[0403] f. Add the 1×TMB chromogenic substrate (invitrogen, 88 - 8218 - 88) at 100 μL per well, incubate in the dark at room temperature for 15 minutes, and then add 2M hydrochloric acid solution at 100 μL per well to terminate the reaction.

[0404] g. Detect the absorbance at 450 nm / 570 nm on an enzyme-linked immunosorbent assay reader, and analyze the blood drug concentration of the test sample using the 4-parameter model of SoftMax Pro 5.4.1.

[0405] h. Use DAS 3.0 software, select the non-compartmental model statistical moment, and perform PK parameter fitting on the data obtained in step g.

[0406] 4. Test Results

[0407] As shown in Table 20, when the in-vivo administration dose of the present invention in mice is 0.15 mg / kg, the half-life of IL-21-fusion protein JS-EC21 is 15.5 h, Cmax is 185.2 μg / L, and AUC (0-t) is 3368.1 μg / L*h; the half-life of recombinant human IL-21 is 2.7 h, Cmax is 3.1 μg / L, and AUC (0-t) is 11.5 μg / L*h. The drug exposure of the IL-21-fusion protein JS-EC21 of the present invention is greater and the half-life is longer, which is significantly superior to recombinant human IL-21.

[0408] Table 20 PK analysis table of single intraperitoneal administration of the fusion protein JS-EC21 of the present invention and recombinant human IL-21 in mice

[0409]

[0410] Note: AUC (0-t) : Area under the curve of the drug concentration-time curve; t 1 / 2z : Terminal elimination half-life; C max : Peak drug concentration.

[0411] Example 5: Inhibitory effect of IL-21 fusion protein alone on the growth of murine MC38 subcutaneous transplanted tumors

[0412] 1. Test purpose

[0413] To evaluate the antitumor effect of JS-EC21 of the present invention in a murine MC38 subcutaneous transplantation model and compare it with recombinant human IL-21 (rhIL-21). In this example, the IL-21-single domain antibody against albumin fusion protein JS-EC21 was formulated according to prescription FS3-3.

[0414] 2. Test procedure

[0415] Female C57BL / 6WT mice at 6-8 weeks of age (ZhaoYan (Suzhou) New Drug Research Center Co., Ltd.) were subcutaneously inoculated with 1 x 10 6 (cells / 0.1 mL) of MC38 cells on the right dorsal side. When the tumor size was approximately 91 mm 3 , the mice were randomly divided into 5 groups (n = 8 / group), namely:

[0416] Solvent control group: Normal saline;

[0417] Treatment groups:

[0418] JS-EC21, 0.45 mg / kg;

[0419] JS-EC21, 1.25 mg / kg;

[0420] JS-EC21, 3.75 mg / kg;

[0421] Recombinant human IL-21, 0.625 mg / kg.

[0422] The administration route for all groups was intraperitoneal injection, administered 2 times a week for 6 consecutive times, and the experiment ended 3 days after the last administration. The tumor volume and body weight were measured 2 times a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor growth inhibition rate TGI was calculated. TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100%, where Ti: the average tumor volume of the treatment group on the i-th day of administration, T0: the average tumor volume of the treatment group on the 0-th day of administration; Vi: the average tumor volume of the solvent control group on the i-th day of administration, V0: the average tumor volume of the solvent control group on the 0-th day of administration.

[0423] 3. Test Results

[0424] As Figure 1 shown, on the 21st day after drug administration, the average tumor volume in the normal saline group was 2446 mm 3 . In the treatment groups, the average tumor volume in the JS-EC21 (0.42 mg / kg) group was 1482 mm 3 , and the TGI was 41.0%; the average tumor volume in the JS-EC21 (1.25 mg / kg) group was 599 mm 3 , and the TGI was 78.4%; the average tumor volume in the JS-EC21 (3.75 mg / kg) group was 390 mm 3 , and the TGI was 87.3%. The average tumor volume in the recombinant human IL-21 (0.625 mg / kg) group was 1698 mm 3 , and the TGI was 31.8%. Compared with the normal saline group, administration of 1.25 mg / kg and 3.75 mg / kg JS-EC21 significantly inhibited tumor growth in mice, and showed a good dose-effect. The tumor inhibitory effect of 1.25 mg / kg JS-EC21 was significantly better than that of equimolar recombinant human IL-21 (0.625 mg / kg).

[0425] Example 6: Pharmacokinetics of IL-21 Fusion Protein JS-EC21 in Cynomolgus Monkeys

[0426] 1. Test Objectives

[0427] To compare the pharmacokinetics of the JS-EC21 fusion protein of the present invention and rhIL-21 in cynomolgus monkeys, including half-life (t 1 / 2 ) and AUC, etc.

[0428] 2. Test Procedures

[0429] A total of 18 cynomolgus monkeys (6 in each group, half male and half female, monkey age 3.8 - 4.3 years old) were randomly assigned to the low-dose, medium-dose, and high-dose groups of the fusion protein JS-EC21. Through intravenous infusion for about 10 minutes, the animals were intravenously injected with 0.05 mg / kg in the low-dose group, 0.15 mg / kg in the medium-dose group, and 0.5 mg / kg in the high-dose group. Before dosing, at 5 minutes after dosing, after the infusion was completed, and at 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 120 hours (D6), and 168 hours (D8) after the start of dosing, blood samples were collected from the three dose groups. All blood samples were separated into serum. The concentration of JS-EC21 in the serum was determined using a validated ELISA method, with a lower limit of quantification of 2 ng / mL. Non-compartmental model analysis (NCA) in WinNonlin 8.0 was used to determine the pharmacokinetic parameters.

[0430] The cynomolgus monkeys were provided by Huazhen Laboratory Animal Breeding Center (Guangzhou, China). The Institutional Animal Care and Use Committee (IACUC) of Zhaoyan Laboratory (Suzhou) Company, IACUC serial number: ACU20-2031.

[0431] 3. Test Results

[0432] It has been reported in the literature that within the evaluated dose range of 0.1 - 0.5 mg / kg, the pharmacokinetics of rhIL-21 in cynomolgus monkeys seems to be dose-dependent and shows a short elimination half-life (0.4 - 0.8 h). To compare with rhIL-21, the pharmacokinetic characteristics of cynomolgus monkeys after a single intravenous infusion of JS-EC21 at low dose (0.05 mg / kg), medium dose (0.15 mg / kg), and high dose (0.5 mg / kg) were then determined. As Figure 2 shown, the serum concentrations of JS-EC21 in the low-dose, medium-dose, and high-dose groups were in a dose-proportional relationship. As shown in Table 21, when the dose was 0.5 mg / kg, the half-life of JS-EC21 was approximately 7.08 hours, much longer than the half-life of rIL-21 (0.81 hours). In addition, the exposure of JS-EC21 in cynomolgus monkeys was significantly improved, and Cmax and AUC were nearly 6 times and 50 times higher than those of rhIL-21, respectively. These data indicate that compared with rhIL-21, the half-life of the engineered fusion protein JS-EC21 was significantly prolonged and the exposure was significantly increased.

[0433] Table 21 Pharmacokinetic parameters of cynomolgus monkeys after intravenous administration of JS-EC21

[0434]

[0435] Note: t 1 / 2, terminal elimination half-life; T max , time to maximum concentration; C max , peak drug concentration; AUC last , area under the drug concentration-time curve from the dosing time to the last sample collection; V d , apparent volume of distribution; CL, clearance; MRT, mean residence time.

[0436] a Data were from the published paper Waggie KS, Holdren MS, Byrnes-Blake K, et al., Preclinical safety, pharmacokinetics, and pharmacodynamics of recombinant human interleukin-21 in cynomolgus macaques (Macaca fascicularis), International journal of toxicology, 2012; 31(4): 303-316.

[0437] b No data.

[0438] Example 7: Inhibitory effect of the combination of IL-21 fusion protein and anti-PD-1 monoclonal antibody on the growth of subcutaneous transplanted MC38 tumors in mice

[0439] 1. Test purpose

[0440] To evaluate the anti-tumor effect of the combination of JS-EC21 of the present invention and anti-PD-1 monoclonal antibody in a subcutaneous transplantation model of MC38 tumors in mice.

[0441] 2. Test procedure

[0442] Female hPD-1 humanized mice (6-8 weeks old, Beijing Biocytogen Co., Ltd.) were subcutaneously inoculated with 1×10 6 (cells / 0.1 mL) of MC38 cells on the right dorsal side. When the tumor size was approximately 91 mm 3 , the mice were randomly divided into 5 groups (n = 7 / group), namely:

[0443] Solvent control group: anti-KLH hIgG4, 0.3 mg / kg;

[0444] Treatment groups:

[0445] Anti-PD-1 monoclonal antibody, 0.3 mg / kg;

[0446] JS-EC21, 1 mg / kg;

[0447] PD-1 monoclonal antibody + JS-EC21, 0.3 mg / kg + 1 mg / kg;

[0448] Anti-KLH hIgG4 + JS-EC21, 0.3 mg / kg + 1 mg / kg.

[0449] In this example, the IL-21-anti-albumin single domain antibody fusion protein JS-EC21 was formulated according to prescription FS3-3; the PD-1 monoclonal antibody was Toripalimab, which is a humanized IgG4 mAb with the structure disclosed in WHO Drug Information (Vol. 32, No. 2, pp. 372-373 (2018)) and contains the light and heavy chain amino acid sequences shown in SEQ ID NO: 15 and 16.

[0450] All groups were administered by intraperitoneal injection, twice a week for 6 consecutive times, and the experiment ended 3 days after the last administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor growth inhibition rate TGI was calculated. TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100%, where Ti: the average tumor volume of the treatment group on the i-th day of administration, T0: the average tumor volume of the treatment group on the 0-th day of administration; Vi: the average tumor volume of the solvent control group on the i-th day of administration, V0: the average tumor volume of the solvent control group on the 0-th day of administration.

[0451] 3. Test results

[0452] As Figure 3 shown, on the 21st day after administration, the average tumor volume of the anti-KLH hIgG4 group was 2008 mm 3 . The average tumor volume of the PD-1 monoclonal antibody group was 1229 mm 3 , and the TGI was 41.3%. The average tumor volume of the JS-EC21 group was 1216 mm 3 , and the TGI was 42.0%. The average tumor volume of the PD-1 monoclonal antibody combined with JS-EC21 group was 673 mm 3 , and the TGI was 70.8%. The experimental results showed that the IL-21-fusion protein JS-EC21 combined with PD-1 in the present invention had a significant tumor inhibitory effect, and the drug effect was superior to that of the PD-1 antibody (p < 0.001) or single drug treatment of JS-EC21 (p < 0.05), showing a certain synergistic effect.

[0453] Example 8: Inhibitory effect of the combination of IL-21 fusion protein and Toripalimab on the growth of hPD-1 humanized mouse MC38 subcutaneous transplanted tumors and its mechanism of action

[0454] 1. Test Purpose

[0455] To evaluate the anti-tumor effect of the combination of JS-EC21 and toripalimab in a mouse MC38 subcutaneous xenograft model and the pharmacodynamic mechanism of their combination.

[0456] 2. Test Procedure

[0457] Female hPD-1 humanized mice (6 - 8 weeks old, Beijing Biocytogen Co., Ltd.) were subcutaneously inoculated with 1 x 10 6 (cells / 0.1 mL) of MC38 cells on the right dorsal side. When the tumor size was approximately 91 mm 3 , the mice were randomly divided into 5 groups, with 7 mice in each group (see Table 22), and drug administration was started (see Table 23). Toripalimab was self-made by Shanghai Junshi Biosciences Co., Ltd. The administration route for all groups was intraperitoneal injection, twice a week for 6 consecutive times, and the experiment ended 3 days after the last administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor growth inhibition rate (TGI) was calculated. TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100%, where Ti is the average tumor volume of the treatment group on the i-th day of drug administration, T0 is the average tumor volume of the treatment group on the 0-th day of drug administration; Vi is the average tumor volume of the vehicle control group on the i-th day of drug administration, and V0 is the average tumor volume of the vehicle control group on the 0-th day of drug administration. Among them, Group 1 in Table 22 was the vehicle control group, and Groups 2, 3, and 4 were the treatment groups.

[0458] Table 22 Mouse Grouping and Drug Administration Parameters

[0459] Group Drug Dosing dose Number of animals Dosing method 1 hIgG4 isotype control 0.3 mg / kg 7 ip 2 JS-EC21 1 mg / kg 7 ip 3 Toripalimab 0.3 mg / kg 7 ip 4 JS-EC21 + Toripalimab 1 + 0.3 mg / kg 7 ip

[0460] Table 23 Drug Administration Schedule

[0461]

[0462]

[0463] On the 21st day after administration, the mouse tumors were immediately removed and transferred into a 50 mL centrifuge tube placed on ice, and pre-cooled PBS solution was added to cover the tumors. Preparation of single cells: Take an equal amount (about 0.5 g) of tumor tissue, cut the tumor into small pieces of 2-4 mm, and prepare a single cell suspension from the tumor tissue using Tumor Dissociation Kit (mouse: 130-096-730); then enrich TIL cells using CD45 (TIL) MicroBeads (mouse: 130-110-618). Take an appropriate amount of the above cells, add 100 μL of detection antibody application solution to each tube (dilute the PE anti-mouse CD8a / FITC anti-mouse CD3e / Pacific Blue anti-mouse NK1.1 mother liquor at a ratio of 1:200 with PBS to obtain the detection antibody application solution, freshly prepared). Incubate at 4°C for 30 min; after the reaction time, centrifuge and wash once with 1 mL of PBS solution at 500 g for 5 min; add 400 μL of 1×FOXP3 Fixtion / Perm Buffer to each tube for fixation and membrane permeabilization, protect from light at room temperature for 20-60 min, centrifuge and wash once at 600 g for 5 min; add 400 μL of 1×FOXP3 Perm Buffer, centrifuge and wash once at 600 g for 5 min; add 100 μL of detection antibody application solution to each tube (dilute the APC anti-mouse Ki-67 mother liquor at a ratio of 1:200 with 1×FOXP3 Perm Buffer to obtain the detection antibody application solution, freshly prepared), incubate at 4°C for 1-2 h; after the reaction time, centrifuge and wash twice with 1 mL of PBS solution at 600 g for 5 min; detect CD8 + / Ki-67 + T cell ratio.

[0464] 3. Test results

[0465] As Figure 4 shown, on the 21st day after administration, the average tumor volume of the hIgG4 isotype control group was 2008 mm 3 . The average tumor volume of the toripalimab group was 1229 mm 3 , and the TGI was 41.3%. The average tumor volume of the JS-EC21 group was 1216 mm 3 , and the TGI was 42.0%. The average tumor volume of the toripalimab combined with JS-EC21 group was 673 mm 3 , significantly lower than that of the toripalimab group (p < 0.001) and the JS-EC21 group (p < 0.05), and the TGI was 70.8%. As Figure 5As shown, the tumor weight on the 21st day after weighing and dosing was significantly lower in the toripalimab combined with JS-EC21 group than in the toripalimab group (p < 0.001) and the JS-EC21 group (p < 0.05). Therefore, these experimental results indicate that the combination of the IL-21-fusion protein JS-EC21 and toripalimab has a significant tumor inhibitory effect, and the drug efficacy is superior to that of toripalimab or JS-EC21 monotherapy, with a certain synergistic effect.

[0466] Flow cytometry was used to study the immunocyte typing of tumor tissues. For the proportion of CD3 + T cells in immune cells (CD45 + ), as shown in Figure 6 (A), the combined dosing group was significantly higher than the toripalimab dosing group (p < 0.01) or the JS-EC21 dosing group (p < 0.01); for the proportion of CD8 + T cells in CD3 + T cells, as shown in Figure 6 (B), the combined dosing group was significantly higher than the toripalimab dosing group (p < 0.05) or the JS-EC21 dosing group (p < 0.01); for the proportion of CD8 + T cells in the proliferative state (Ki67 + ), as shown in Figure 6 (C), the combined dosing group was significantly higher than the toripalimab dosing group (p < 0.05) or the JS-EC21 dosing group (p < 0.05); for the proportion of NK cells in immune cells (CD45 + ), as shown in Figure 6 (D), the combined dosing group was significantly higher than the toripalimab dosing group (p < 0.05) or the JS-EC21 dosing group (p < 0.05). These research results indicate that the combination of toripalimab and JS-EC21 can significantly increase the proportion of CD8 + T cells and NK cells infiltrating tumors, thereby enhancing the anti-tumor immune response. Sequence Listing <110> Shanghai Junshi Biosciences Co., Ltd. Suzhou Junmeng Biosciences Co., Ltd. <120> IL-21-Anti-albumin Single Domain Antibody Fusion Protein Pharmaceutical Composition and Its Use <130> MTI220059 <150> CN2021102350207 <151> 2021-03-03 <150> CN2021110383043 <151> 2021-09-06 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of IL-21 <400> 1 Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile 1 5 10 15 Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30 Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45 Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55 60 Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser 65 70 75 80 Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys 85 90 95 Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys 100 105 110 Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His 115 120 125 Gly Ser Glu Asp Ser 130 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 2 Gly Ser Thr Trp Ser Ile Asn Thr 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 3 Ile Ser Ser Gly Gly Ser Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 4 Tyr Ala Gln Ser Thr Trp Tyr Pro Pro Ser 1 5 10 <210> 5 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of single domain antibody <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Trp Ser Ile Asn 20 25 30 Thr Leu Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Arg Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Ala Gln Ser Thr Trp Tyr Pro Pro Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 6 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Full - length amino acid sequence of IL - 21 - anti - albumin single - domain antibody fusion protein JS - EC21 <400> 6 Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile 1 5 10 15 Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30 Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45 Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55 60 Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser 65 70 75 80 Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys 85 90 95 Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys 100 105 110 Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His 115 120 125 Gly Ser Glu Asp Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 145 150 155 160 Thr Trp Ser Ile Asn Thr Leu Ala Trp Tyr Arg Gln Ala Pro Gly Lys 165 170 175 Gln Arg Asp Leu Val Ala Arg Ile Ser Ser Gly Gly Ser Thr Tyr Tyr 180 185 190 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 195 200 205 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Tyr Ala Gln Ser Thr Trp Tyr Pro Pro Ser Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 7 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 8 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 9 Phe Gln Gly Ser His Val Pro Leu Thr 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 10 Asp Tyr Glu Met His 1 5 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 11 Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 12 Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe Asp Val 1 5 10 15 <210> 13 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Toripalimab light chain variable region <400> 13 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 14 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Variable region of the heavy chain of toripalimab <400> 14 Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Ile His Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 15 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Toripalimab light chain <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 16 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Toripalimab heavy chain <400> 16 Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Ile His Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450

Claims

1. A pharmaceutical composition, comprising: (1) A buffer solution, which is a 10 - 30 mM sodium acetate buffer solution or a 10 - 30 mM histidine buffer solution, with a pH of 4.5 - 5.0; (2) An IL-21 - anti - albumin single - domain antibody fusion protein; Wherein the IL-21 - anti - albumin single - domain antibody fusion protein comprises: (a) cytokine IL-21, and (b) a single - domain antibody (sdAb) that specifically binds to albumin. The amino acid sequence of the IL-21 is the amino acid sequence shown in SEQ ID NO: 1, and the single - domain antibody (sdAb) comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 respectively; (3) A stabilizer, which is 200 - 280 mM trehalose or 200 - 280 mM sucrose; (4) A surfactant, which is 0.01 - 0.05% polysorbate 80.

2. The pharmaceutical composition according to claim 1, wherein the single - domain antibody is fused to the C - terminus of the cytokine, and the cytokine and the single - domain antibody are directly linked. The amino acid sequence of the single - domain antibody (sdAb) is the amino acid sequence shown in SEQ ID NO:

5.

3. The pharmaceutical composition according to claim 1 or 2, wherein the buffer solution is a 10 - 30 mM acetic acid - sodium acetate buffer solution.

4. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the buffer solution is 4.7 or 4.

8.

5. The pharmaceutical composition according to claim 1 or 2, wherein the stabilizer is 200 - 280 mM trehalose.

6. The pharmaceutical composition according to claim 1 or 2, wherein, the IL-21 - anti - albumin single - domain antibody fusion protein comprises: (a) cytokine IL-21, and (b) a single - domain antibody (sdAb) that specifically binds to albumin. The amino acid sequence of the IL-21 is the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the single - domain antibody (sdAb) is the amino acid sequence shown in SEQ ID NO:

5.

7. The pharmaceutical composition according to claim 1 or 2, wherein the amino acid sequence of the IL-21 - anti - albumin single - domain antibody fusion protein is the amino acid sequence shown in SEQ ID NO:

6.

8. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the IL-21 - anti - albumin single - domain antibody fusion protein is 0.1 - 2 mg / mL.

9. The pharmaceutical composition according to claim 1 or 2, which comprises the components shown in the following (1) or (2): (1) (a) 1 mg / mL of IL-21 - anti - albumin single - domain antibody fusion protein; (b) 20 mM acetic acid - sodium acetate buffer solution, with a pH of 4.8; (c) 235 mM of trehalose; (d) and 0.02% of polysorbate 80; or (2) (a) IL-21-anti-albumin single domain antibody fusion protein at 1 mg / mL; (b) 20 mM acetic acid-sodium acetate buffer, pH 4.8; (c) 235 mM sucrose; (d) and 0.02% polysorbate 80.

10. Use of the pharmaceutical composition according to any one of claims 1-9, or a combination of the pharmaceutical composition according to any one of claims 1-9 and an immune checkpoint modulator, in the preparation of a drug for preventing or treating colorectal cancer, or use of the pharmaceutical composition according to any one of claims 1-9 in the preparation of a drug for enhancing the anti-colorectal cancer activity of an immune checkpoint modulator; the immune checkpoint modulator is an anti-PD-1 antibody, and the anti-PD-1 antibody is toripalimab.

11. Pharmaceutical combination, comprising: (1) an immune checkpoint modulator; and (2) the pharmaceutical composition according to any one of claims 1-9; wherein, the immune checkpoint modulator is an anti-PD-1 antibody, and the anti-PD-1 antibody is toripalimab.

12. A drug for preventing or treating a tumor, the drug comprising the pharmaceutical composition according to any one of claims 1-9 and an immune checkpoint modulator, or the pharmaceutical combination according to claim 11, the immune checkpoint modulator is an anti-PD-1 antibody, and the anti-PD-1 antibody is toripalimab, and the drug is formulated for administration to an individual in need.

13. A kit, comprising: one or more single drug dose units of the pharmaceutical combination according to claim 11.

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