CN115006517A

CN115006517A - IL-21-anti-albumin single domain antibody fusion protein pharmaceutical composition and application thereof

Info

Publication number: CN115006517A
Application number: CN202210203928.4A
Authority: CN
Inventors: 刘洪川; 刘辉; 张静; 孟琴; 刘沛想; 李秀鹏; 赵文亭; 冯辉; 姚盛
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2021-03-03
Filing date: 2022-03-03
Publication date: 2022-09-06
Also published as: WO2022184148A1

Abstract

The invention relates to an IL-21-anti-albumin single domain antibody fusion protein pharmaceutical composition and application thereof. The pharmaceutical composition comprises IL-21-anti-albumin single domain antibody fusion protein and buffer solution, and also can comprise at least one stabilizer, and optionally can also comprise a surfactant. The IL-21-anti-albumin single-domain antibody fusion protein has high stability, and the single drug and the combined use of the single drug and the PD-1 monoclonal antibody show excellent anti-tumor activity.

Description

IL-21-anti-albumin single domain antibody fusion protein pharmaceutical composition and application thereof

Technical Field

The present invention relates to the field of therapeutic pharmaceutical compositions. In particular, the invention relates to the field of pharmaceutical preparations, the pharmaceutical composition contains IL-21-anti-albumin single domain antibody fusion protein, and the use of the IL-21 fusion protein in treating tumors and enhancing the anti-tumor activity of immune checkpoint inhibitors.

Background

Cytokine therapy is an effective strategy to stimulate the immune system to induce an immune response against a disease (e.g., cancer or infection). However, cytokines administered to patients typically 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. Recombinant IL-21 has been reported to have a half-life of only about one to three hours following intravenous administration (see Schmidt H, Clin. cancer Res.2010, 11/1/16; Vol. 21: 5312-5319).

Immune escape is one of the characteristics of cancer. Ahmadzadeh, m. et al, Blood,114:1537-44, disclose that tumor-specific T lymphocytes are frequently present in the tumor microenvironment, draining lymph nodes and peripheral Blood, but are often unable to control tumor progression due to the network of immunosuppressive mechanisms present in the tumor microenvironment. CD8 ⁺ Tumor infiltrating T lymphocytes (TILs) typically express activation-induced inhibitory receptors, including CTLA-4 and PD-1, while tumor cells often express immunosuppressive ligands, including PD-1 ligand 1(PD-L1, also called B7-H1 or CD274), which inhibit T cell activation and effector functions. Among the inhibitory mechanisms, PD-1 and its ligands have become an important pathway for tumor cells to utilize it to inhibit activated T cells in the tumor microenvironment.

In the last decade, blocking of the PD-1/PD-L1 pathway has proven to be an effective way to induce a durable anti-tumor response in various cancer indications. Monoclonal antibodies (mAbs) blocking 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-PD 1 monoclonal antibody (nivolumab) and 3 anti-PD-L1 monoclonal antibody (atezolizumab, avelumab, and durvalumab) for the treatment of human tumors.

Although observed in 10-30% of cancer patients receiving immune checkpoint inhibitor treatmentTo a surprising effect, a significant proportion of patients were initially resistant to anti-PD-1 mab treatment, and more than half of the patients who initially responded eventually relapse into tumors. Therefore, there is a great deal of interest in the industry to expand the beneficiary population and improve efficacy through the synergistic combination of immunotherapy. IL-21 is a member of the gamma chain cytokine family and stimulates CD8 ⁺ T cell expansion and increased cytotoxicity, enhanced T cell-dependent B cell proliferation and antibody production, promoted NK cell differentiation and activation, and reduced Treg cells. Recombinant human IL-21 has been demonstrated in non-clinical and clinical studies to produce anti-tumor activity as a monotherapy or in combination with targeted therapies or monoclonal antibodies. For example, phase I and II clinical trials have been conducted on metastatic melanoma, Renal Cell Carcinoma (RCC), colorectal cancer, and non-Hodgkin's lymphoma using recombinant IL-21 (rIL-21). 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.

Therefore, there is a need to develop new cytokine therapeutics that can effectively treat diseases, and to investigate the effects of their combination with immune checkpoint inhibitors.

Disclosure of Invention

The invention discloses a pharmaceutical composition which is a high-stability pharmaceutical composition containing IL-21-anti-albumin single-domain antibody fusion protein (IL-21 fusion protein). In particular, the present inventors have found that combinations comprising trehalose have the surprising feature of having high stability.

The half-life and the exposure level in blood of the IL-21-anti-albumin single-domain antibody fusion protein pharmaceutical composition are obviously superior to those of recombinant interleukin 21, and the single drugs and the combination of the single drugs and PD-1 monoclonal antibody show excellent anti-tumor activity in a mouse subcutaneous transplantation tumor model.

The invention discovers that compared with rhIL-21 (recombinant human IL-21), the half life of the fusion protein called JS-EC21 formed by fusing the nano antibody targeting Human Serum Albumin (HSA) to the C terminal of the human IL-21 is remarkably prolonged and the exposure is remarkably increased in cynomolgus monkeys (t _1/2 And AUC approximately 10-fold and 50-fold higher than rhIL-21, respectively). Furthermore, JS-EC21 significantly enhanced PD-1The monoclonal antibody or TIGIT monoclonal antibody has antitumor effect and long-term protective antitumor immunity. The combination of JS-EC21 with TIGIT mab induced significant enrichment of the KEGG signaling pathway associated with tumor immune response and increased CD8 ⁺ Expression levels of T cell and NK cytotoxicity related genes.

In one aspect, the present invention provides a pharmaceutical composition comprising: (1) a buffer solution; and (2) IL-21-anti-albumin single domain antibody fusion proteins.

In some embodiments, the IL-21-anti-albumin single domain antibody fusion protein comprises: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, said IL-21 comprising an amino acid sequence as set forth in SEQ ID NO:1, said single domain antibody comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

In some embodiments, the single domain antibody (sdAb) comprises the amino acid sequence set forth as SEQ ID No. 5.

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

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

In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, and a histidine buffer; preferably, the buffer is selected from one or more of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, histidine-acetic acid buffer and histidine-hydrochloride buffer; preferably, the buffer solution is acetic acid-sodium acetate buffer solution and histidine-acetic acid buffer solution; more preferably, the buffer is an acetic acid-sodium acetate buffer.

In some embodiments, the buffer is a histidine buffer, preferably the histidine buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer. In some embodiments, the histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of 1-30mM L-histidine and 1-30mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1: 3. In some embodiments, the histidine formulation is: histidine buffer with a pH of about 5.0 was prepared from about 1.7mM L-histidine and about 18.3mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer with a pH of about 5.5 was prepared from about 4.5mM L-histidine and about 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine formulation is: histidine buffer with a pH of about 5.5 made from about 7.5mM L-histidine and about 22.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer at about pH 6.0 was prepared with about 15mM histidine and about 15mM histidine hydrochloride.

In some embodiments, the histidine buffer is a histidine-acetic acid buffer, preferably in a molar ratio of about 1:1 to about 1.5:1, preferably such a buffer has a pH of 5.5 ± 0.3, preferably about 5.5, and preferably such a buffer contains 15-20mM histidine and 12-15mM acetic acid.

In some embodiments, the buffer is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer. In some embodiments, the acetate buffer is made up of 1-30mM acetic acid and 1-30mM sodium acetate. In some embodiments, the acetic acid buffer is made of acetic acid and sodium acetate in a molar ratio of about 2: 3. In some embodiments, the acetic acid buffer is made of acetic acid and sodium acetate in a molar ratio of about 1: 2.1. In some embodiments, the acetic acid buffer is made of acetic acid and sodium acetate in a molar ratio of about 1: 5.7. In some embodiments, the acetate buffer is: an acetate buffer of about pH 4.8 made from about 8mM acetic acid and about 12mM sodium acetate. In some embodiments, the acetate buffer is: an acetate buffer at about pH5.0 made from about 6.5mM acetic acid and about 13.5mM sodium acetate. In some embodiments, the acetate buffer is: an acetate buffer of about pH 5.5 made with about 3mM acetic acid and about 17mM sodium acetate.

In some embodiments, the buffer is a citric acid buffer, and preferably, the citric acid buffer is a citric acid-sodium citrate buffer.

In some embodiments, the buffer is at a concentration of about 1mM to about 200mM, preferably about 5mM to about 200mM, preferably about 10mM to about 50mM, preferably about 10mM to about 30 mM; preferably about 10-20mM, and the above buffer concentration is, by way of non-limiting example, about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, or 180mM, or any two of these ranges, preferably 10mM, 15mM, 20mM, or 30 mM.

In some embodiments, the pH of the buffer is about 4.0 to about 6.5, preferably about 4.0 to about 6.0, preferably about 4.5 to about 5.5, preferably about 4.5 to about 5.1, preferably about 4.5 to about 5.0, preferably about 4.7 to about 5.0, and 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 about 4.7 or 4.8.

In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, and trehalose, preferably, the stabilizer is trehalose.

In some embodiments, the concentration of the above stabilizing agent is about 10mM to 400mM, preferably 20mM to 300mM, more preferably 30mM to 200 mM.

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

In some embodiments, the stabilizing agent is mannitol. In some embodiments, the stabilizing agent is mannitol at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, non-limiting examples of the concentration of mannitol are about 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240 mM.

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

In some embodiments, the stabilizing agent is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100mM, preferably about 150mM, 300mM, preferably about 200mM, 280mM, non-limiting examples of trehalose concentrations are about 180mM, 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 235 mM.

In some embodiments, the stabilizing agent is a combination of sodium chloride and mannitol. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol, preferably a combination of about 40-150mM sodium chloride and about 40-180mM mannitol, preferably a combination of about 40-100mM sodium chloride and about 40-150mM mannitol, non-limiting examples of the stabilizing agent are a combination of about 50mM sodium chloride and about 120mM mannitol, or a combination of about 50mM sodium chloride and about 140mM mannitol.

In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose, preferably a combination of about 40-150mM sodium chloride and about 40-180mM sucrose, preferably a combination of about 40-100mM sodium chloride and about 40-150mM sucrose, a non-limiting example of the stabilizing agent is a combination of about 50mM sodium chloride and about 140mM sucrose.

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

In some embodiments, the surfactant is selected from polysorbate 80.

In some embodiments, the surfactant is selected from polysorbate 20.

In some embodiments, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.1%, preferably from about 0.02% to about 0.08%, preferably from about 0.01% to about 0.05%, calculated as w/v; by way of non-limiting example, the concentration of the above surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.

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

In some embodiments, the IL-21-anti-albumin single domain antibody fusion protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO 6.

In some embodiments, the above pharmaceutical composition comprises a component according to any one of the following (1) to (6), wherein the IL-21-anti-albumin single domain antibody fusion protein is according to any one of the embodiments of the present invention:

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

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

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

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

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

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

In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

In some embodiments, the pharmaceutical composition is a liquid formulation.

In some embodiments, the liquid formulation or lyophilized formulation described above is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the aqueous solution or lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

In yet another aspect, the present invention also provides the use of the above pharmaceutical composition for the preparation of a medicament for the treatment or prevention of cancer; preferably, the above cancers include mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer and colorectal cancer.

In yet another aspect, the present invention also provides the use of the above pharmaceutical composition for the preparation of a medicament for the treatment or prevention of inflammatory diseases.

In yet another aspect, the invention provides the use of an IL-21 fusion protein (also known as IL-21-anti-albumin single domain antibody fusion protein), or the pharmaceutical composition described above, or a combination of an IL-21 fusion protein and an immune checkpoint modulator, or the pharmaceutical composition described above and an immune checkpoint modulator, in the manufacture of a medicament for the prevention or treatment of a tumor, or the use of an IL-21-anti-albumin single domain antibody fusion protein, or the pharmaceutical composition described above, in the manufacture of a medicament for enhancing the anti-tumor activity of an immune checkpoint modulator.

In yet another aspect, the invention provides a method for preventing or treating a tumor comprising administering to an individual in need thereof an effective amount of an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an immune checkpoint modulator, or a combination of a pharmaceutical composition as described above and an immune checkpoint modulator.

In a further aspect, the invention provides an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an immune checkpoint modulator, or a combination of a pharmaceutical composition as described above and an immune checkpoint modulator, for use in the treatment or prevention of a tumor.

In some embodiments, the tumor is a tumor associated with IL-21, or is a tumor associated with an immune checkpoint.

In some embodiments, the tumor is selected from the group consisting of mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, renal cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-hodgkin's lymphoma, esophageal cancer, gastric cancer, and colorectal cancer.

In some embodiments, the tumor is colorectal cancer.

In one or more embodiments, the IL-21-anti-albumin single domain antibody fusion proteins disclosed herein comprise: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, IL-21 comprising the amino acid sequence shown as SEQ ID NO:1, the single domain antibody (sdAb) comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown as SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

In one or more embodiments, the single domain antibodies (sdabs) disclosed herein comprise the amino acid sequence set forth as SEQ ID NO: 5.

In one or more embodiments, the single domain antibodies disclosed herein are fused to the C-terminus of the cytokine, and the cytokine and single domain antibody are directly linked.

In one or more embodiments, the IL-21 fusion proteins disclosed herein comprise an amino acid sequence as set forth in SEQ ID NO 6.

In one or more embodiments, the IL-21 fusion proteins disclosed herein are used in combination with immune checkpoint modulators

In one or more embodiments, the immune checkpoint modulator disclosed herein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27, and B7H4 immune checkpoint inhibitors.

In one or more embodiments, the immune checkpoint modulator disclosed herein is an anti-PD-1 antibody.

In one or more embodiments, the anti-PD-1 antibodies disclosed herein comprise:

(1) the amino acid sequence is shown as SEQ ID NO: 7.8 and 9, and the amino acid sequence is as shown in SEQ ID NO: 10. 11 and 12; or

(2) A light chain variable region having an amino acid sequence shown as SEQ ID NO. 13 and a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 14; or

(3) A light chain with an amino acid sequence shown as SEQ ID NO. 15 and a heavy chain with an amino acid sequence shown as SEQ ID NO. 16.

In one or more embodiments, the anti-PD-1 antibodies disclosed herein are selected from one or more of nivolumab, pembrolizumab, toreplilimuab, sintilizumab, Camrelizumab, tiselizumab, semiplimab; preferably a toriplalimab.

In some embodiments, the IL-21 fusion proteins disclosed herein have one or more properties selected from the group consisting of:

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

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

(3) proliferation status (Ki 67) ⁺ ) CD8 (1) ⁺ The proportion of T cells is significantly increased; and

(4) the proportion of NK cells in immune cells is obviously improved.

In one or more embodiments, the immune checkpoint modulator disclosed herein is an anti-TIGIT antibody.

In one or more embodiments, the anti-TIGIT antibodies disclosed herein are selected from the group consisting of: tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, and BMS-986207.

(1) significantly increases the expression level of interferon gamma;

(2) significantly increasing the expression level of granzyme a;

(3) significantly increasing the expression level of granzyme B;

(4) significantly increases the expression level of perforin 1;

(5) enriching for more KEGG signaling pathways with differential gene expression; and

(6) the majority of the significantly enriched KEGG signaling pathway induced was mediated by the IL-21 fusion protein.

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

(1) an immune checkpoint modulator; and

(2) an IL-21-anti-albumin single domain antibody fusion protein or a pharmaceutical composition as described above;

in yet another aspect, the present invention provides a kit comprising:

(1) one or more single drug dosage units of an IL-21 fusion protein and one or more single drug dosage units of an immune checkpoint modulator, wherein the IL-21 fusion protein is as disclosed herein; preferably wherein the immune checkpoint modulator is as disclosed herein; or

(2) A pharmaceutical combination as disclosed herein of one or more single-dose pharmaceutical dosage units.

In one or more embodiments, the disclosed kits further comprise instructions for indicating a method of use of the pharmaceutical compositions therein.

Drawings

FIG. 1: the fusion protein JS-EC21 has the effect of inhibiting MC38 tumor.

FIG. 2: pharmacokinetic profiles of JS-EC21 at low, medium and high doses in cynomolgus monkeys.

FIG. 3: the fusion protein JS-EC21 combines with the PD-1 monoclonal antibody to inhibit the MC38 tumor.

FIG. 4 is a schematic view of: the fusion protein JS-EC21 and Terepril monoclonal antibody of the invention can change the tumor volume of mice after being singly administrated or combined.

FIG. 5: the weight of the mouse tumor changes after the fusion protein JS-EC21 and the Terepril monoclonal antibody are singly administrated or combined.

FIG. 6: the fusion protein JS-EC21 and Terepril monoclonal antibody of the invention can be used for changing immune cells in the tumor of mice after being singly administered or combined.

In FIGS. 4-6, the difference analysis between the two groups was performed by two-tailed unpaired students't test, which indicates P < 0.05; denotes P <0.01, denotes P <0.001, ns denotes no significant difference.

Detailed Description

Definitions and explanations

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

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

The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable upon storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).

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

The term "excipient" refers to an agent that can be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of ± 20% or ± 10% from the particular value, including ± 5%, ± 1% and ± 0.1%, as such variations are suitable for performing the disclosed methods.

The term "buffer pH of about 4.0-6.5" refers to an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in the range of about 4.0 to about 6.5, or a pH in the range of about 4.5 to about 5.5.

Examples of "buffers" that control the pH within this range herein include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconic acid, histidine hydrochloride, methionine, citrates, phosphates, citrate/phosphates, imidazole, acetic acid, acetates, citrates, combinations thereof, and other organic acid buffers.

"histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include salts of histidine and histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like, such as histidine buffer containing histidine and histidine hydrochloride; histidine buffers of the present invention also include histidine buffers comprising histidine and an acetate salt (e.g., sodium or potassium salt) or glacial acetic acid.

A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. The preferred citrate buffer is a citric acid-sodium citrate buffer.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is an acetic acid-sodium acetate buffer.

The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), octopine (opines), alanine, octopine (strombine), and the N-oxide of Trimethylamine (TMAO), human serum albumin (hsa), albumin (bsa), α -bovine serum albumin, globulin, α -lactalbumin, GABA, lysozyme, myoglobin, ovalbumin, and rnasea. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., may also act to control osmotic pressure. The stabilizer used in the present invention is one or more selected from the group consisting of a polyol, an amino acid, a salt and a sugar. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or its salts (e.g. arginine hydrochloride), glycine, proline. 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, more preferably sucrose or trehalose.

The term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of the antibodies 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 (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), Sodium Dodecyl Sulfate (SDS). Herein, unless otherwise specified, the terms "polysorbate 20 concentration" and "polysorbate 80 concentration" both refer to mass volume concentrations (w/v), such as "0.02%" in "about 0.02% polysorbate 80" which means "0.02 g polysorbate 80 in 100mL of liquid".

The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using an osmometer of the vapor pressure or subfreezing type.

The term "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. A pharmaceutical preparation may be stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a period of time may 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-: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ 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, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

Stability can be measured by determining (among other methods) the percentage of antibody that migrates ("acidic form") in the fraction where this main fraction of antibody ("predominantly charged form") is acidic during ion exchange, where stability is inversely proportional to the percentage of acidic form antibody. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in acidic form can be detected in the formulation after storage of the formulation at a temperature and for a time. The certain time period of storage prior to measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in the pharmaceutical composition if it shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such a matte measurement correlates with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by nephelometry and is measured in Nephelometric Turbidity Units (NTU).

The level of turbidity which varies with, for example, the concentration of one or more components in the solution (e.g., protein and/or salt concentration) is also referred to as the "opacification" or "opacified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of a pharmaceutical composition may be based on the "European Pharmacopoeia" standards (European Pharmacopoeia), fourth edition, "European commission for Quality of Medicine instructions" (EDQM), Strasbourg, France). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

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

An antibody in a pharmaceutical composition "retains its biological activity" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation at a temperature, e.g., 5 ℃,25 ℃, 45 ℃ or the like, for a period of time (e.g., 1 to 12 months), the formulation contains an IL-21 fusion protein that binds IL-17A with at least 90%, 95% or more of the binding affinity of the antibody prior to such storage. Binding affinity can also be measured using, for example, ELISA or plasmon resonance techniques.

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 effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody is effective to treat.

In the present invention, a "therapeutically effective amount" or a "therapeutically effective dose" or an "effective amount" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or the prevention of injury or disability resulting from the affliction of the disease. The ability of a drug or therapeutic to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by assaying the activity of the agent in an in vitro assay.

A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of a drug that inhibits the development or recurrence of a disease (e.g., cancer) when administered to a subject at risk of developing a disease (e.g., at risk of developing cancer) or a subject with a disease recurrence (e.g., having a cancer recurrence), either alone or as combined with other therapeutic drugs (e.g., antineoplastic agents).

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

The terms "administering," "administering," and "treating" 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 of skill in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "parenteral administration" refers to modes of administration other than enteral or topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and via in vivo electroporation.

The term "antibody" is used in its broadest sense and encompasses a variety of 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, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or antigen-binding fragment thereof.

The term "full length antibody" or "whole antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in length) and two light (L) chains (about 25kDa in length) 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 3 domains, CH1, CH2, and CH 3. 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 regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region consists of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. 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.; version 5; 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: 901-883 or Chothia et al, (1989) Nature 341: 878-883.

The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may 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).

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently for different systems. CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or demarcated according to the convention 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, called 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 are not involved 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 major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain).

As used herein, an "antigen-binding fragment" includes a fragment of an antibody or derivative thereof, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof typically retains at least 10% of the antigen binding activity of the parent antibody. Preferably, the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or 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 (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity.

Unless otherwise indicated, the CDRs of the antibodies of the invention can be bounded by one of skill in the art according to any protocol in the art (e.g., different assignment systems or combinations).

As used herein, "framework region" or "FR" refers to immunoglobulin variable regions that do not include CDR regions.

"chimeric antibody" refers to antibodies and fragments thereof as follows: wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity.

"human antibody" refers to an antibody comprising only human immunoglobulin sequences. A human antibody may contain murine carbohydrate chains if it is produced in a mouse, mouse cells, or a hybridoma derived from a mouse cell. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

"humanized antibody" refers to antibody forms containing sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain a minimal sequence derived from a single side of a non-human immunoglobulin. Typically, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition site and the antigen binding site. The fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. These two domains fold to create six hypervariable loops (3 loops each for 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 half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity than the entire binding site.

The term "heavy chain antibody" or "HCAb" refers to a functional antibody that includes a heavy chain but lacks the light chain typically found in 4 chain antibodies. Camelids (such as camels, llamas or alpacas) are known to produce hcabs.

The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three Complementarity Determining Regions (CDRs). sdabs are capable of binding to an antigen alone without pairing with the corresponding CDR-containing polypeptide. In some cases, the single domain antibody is engineered from a camelidae HCAb, and its heavy chain variable domain is referred to herein as a "VHH" (variable domain of the heavy chain of a heavy chain antibody). Camelidae sdabs are one of the smallest antigen-binding antibody fragments known (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-Ghassah et al, Nanomedicine (Lond), Vol.8: pp.1013-1026, 2013). The basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

The numbering of residues in immunoglobulin heavy chains herein is by reference to the EU index numbering of Kabat et al, unless otherwise indicated. The "EU index of Kabat" refers to the residue numbering of the human IgG1 EU antibody.

"therapeutic anti-PD-1 monoclonal antibody" refers to an antibody that specifically binds to the mature form of a particular PD-1 expressed on the surface of certain mammalian cells. Mature PD-1 lacks a pre-secretory leader sequence, or leader peptide. The terms "PD-1" and "mature PD-1" are used interchangeably herein and, unless otherwise specifically defined or clear from context, should be understood to be the same molecule.

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.

An "isolated antibody or antigen-binding fragment thereof" refers to a molecule that is in a purified state and in this case is designated as being substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials (such as cell debris or growth media).

As used herein, the term "cytokine" is understood to refer to any protein or peptide, analog or functional fragment thereof, that is capable of stimulating or inducing a cytocidal immune response in a mammal against a preselected cell type (e.g., a cancer cell or a virally infected cell). Thus, it is contemplated that a variety of cytokines may be incorporated into the present application. Useful cytokines include, for example, Tumor Necrosis Factor (TNF), Interleukins (IL), lymphokines (L), Colony Stimulating Factor (CSF), Interferons (IFN), including species variants, truncated analogs thereof capable of stimulating or inducing such a cytocidal immune response. Useful tumor necrosis factors include, for example, TNF α. 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" should also be understood to include any variant of a wild-type cytokine (such as IL-21, IL-7, IL-15, etc.) which includes at least a significant portion (such as at least about 50%) of its modification and maintenance of any desired function.

"immune checkpoint" refers to a series of molecules expressed on immune cells that modulate the degree of immune activation, which play an important role in preventing the development of an autoimmune (aberrant immune function, attack on normal cells) effect. Common immune checkpoints include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27, and B7H 4. As used herein, immune checkpoint modulators refer to agents, such as inhibitors, that specifically bind to these immune checkpoints and enhance, attenuate or block their biological activity. Such agents include, inter alia, antibodies or antigen-binding fragments thereof that specifically bind to these immune checkpoints. An immune checkpoint-associated tumor as described herein refers to a tumor whose biological activity can be attenuated or blocked by binding to an immune checkpoint, thereby effecting the prevention and treatment of the tumor. In particularly preferred embodiments, the immune checkpoint-related tumor described herein is a PD-1 or PD-L1 or TIGIT-related tumor.

An "adverse effect" (AE) as referred to herein is any adverse and often unintentional or undesirable sign, symptom or disease associated with the use of medical treatment. For example, adverse reactions may be associated with activation of the immune system or expansion of immune system cells in response to therapy. The medical treatment may have one or more related AEs, and each AE may have the same or different severity level.

"tumor burden" refers to the total amount of tumor mass distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor throughout the body. Tumor burden can be determined by a variety of methods known in the art, such as measuring its size using calipers after the tumor is removed from the subject, or while in vivo using imaging techniques such as ultrasound, bone scans, Computed Tomography (CT), or Magnetic Resonance Imaging (MRI) scans.

The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, for example, measuring its dimensions using calipers after the tumor is removed from the subject, or while in vivo using imaging techniques such as bone scans, ultrasound, CT, or MRI scans.

The terms "subject", "individual", "object" include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. The terms "subject" and "patient" are used interchangeably herein.

As used herein, "treating" cancer refers to employing a treatment regimen described herein (e.g., administration of an anti-PD-1 antibody) to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject having or diagnosed with cancer. 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, T/C.ltoreq.42% is the minimum level of anti-tumor activity according to the NCI standard. 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 during which cancer does not grow, and includes the amount of time a patient experiences CR or PR and the amount of time a patient experiences SD. DFS refers to the length of time during and after treatment that a patient is still disease free. OS refers to an extension of life expectancy compared to an initial or untreated individual or patient. The treatment regimen for a combination of the invention effective in treating a cancer patient can vary depending on a variety of factors such as the disease state, age, weight of the patient and the ability of the therapy to elicit an anti-cancer response in the subject. Although embodiments of the invention may not achieve an effective positive therapeutic effect in each subject, a positive therapeutic effect should be effective and achieved in a statistically significant number of subjects.

The terms "mode of administration", "dosing regimen", which are used interchangeably, refer to the dosage and time of use of each therapeutic agent in the combination of the invention.

Herein, the term "cancer" or "malignancy" refers to a wide variety of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distal parts of the body through the lymphatic system or blood stream.

IL-21 binds to its receptor and regulates B cell proliferation, differentiates into plasma cells to perform immune response, and promotes CD8 ⁺ Proliferation and differentiation of T cells and NK cells, and increase killing activity of NK cells, thereby realizing tumor treatment or prevention. Thus, as used herein, a "tumor associated with IL-21" refers to a tumor that would benefit from administration of IL-21 or an IL-21 fusion protein in the treatment or prevention of the tumor.

Examples of tumors or cancers suitable for treatment or prevention with the methods, medicaments and kits of the present invention refer especially to tumors or cancers benefiting 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, breast, ovarian, melanoma, kidney, pancreatic, lymphoma, leukemia, head and neck, liver, non-hodgkin's lymphoma, esophageal, gastric and colorectal cancers. In a particularly preferred embodiment, the tumor/cancer disclosed herein is colorectal cancer.

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

IL-21 fusion proteins

The IL-21-anti-albumin single domain antibody fusion proteins (IL-21 fusion proteins) disclosed herein include any of the IL-21 fusion proteins described in publication number WO2019246004, the entire disclosure 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 invention comprises the amino acid sequence from IL-21 fusion protein P798 described in WO 2019246004.

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, the amino acid sequence of which is shown below:

amino acid sequence of IL-21:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:1)

amino acid sequence of single domain antibody:

EVQLVESGGGLVQPGGSLRLSCAASGSTWSINTLAWYRQAPGKQRDLVARISSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYAQSTWYPPSWGQGTLVTVSS(SEQ ID NO:5)

HCDR1：GSTWSINT(SEQ ID NO:2)

HCDR2：ISSGGST(SEQ ID NO:3)

HCDR3：YAQSTWYPPS(SEQ ID NO:4)

full-length amino acid sequence of IL-21-anti-albumin single domain antibody fusion protein JS-EC 21:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSEVQLVESGGGLVQPGGSLRLSCAASGSTWSINTLAWYRQAPGKQRDLVARISSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYAQSTWYPPSWGQGTLVTVSS(SEQ ID NO:6)

anti-PD-1 antibodies

Herein, a "PD-1 antibody" refers to any chemical compound or biomolecule that binds to the PD-1 receptor, 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 of the inventive methods of treatment, medicaments and uses for treating a human subject, 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 human PD-1 amino acid sequence can be found at NCBI locus number: NP _ 005009. Human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbers: NP-054862 and NP-079515.

Herein, when referring to an "anti-PD-1 antibody," unless otherwise indicated or described, the term includes antigen-binding fragments thereof.

The anti-PD-1 antibody or antigen binding fragment thereof suitable for any use, therapy, medicament and kit described in the invention binds PD-1 with high specificity and high affinity, blocks the binding of PD-L1/2 and PD-1, and inhibits PD-1 signal transduction, thereby achieving immunosuppressive effects. In any of the uses, therapies, medicaments and kits 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 properties similar to those of an 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 for binding to human PD-1 with tereprinimab. In other embodiments, the anti-PD-1 antibody is a murine, chimeric, humanized, or human Ab or an antigen-binding fragment thereof. In certain embodiments for treating a human subject, the Ab is a humanized Ab.

In some embodiments, the constant region is selected from the group consisting of a human IgG1, IgG2, IgG3, and IgG4 constant region; preferably, the anti-PD-1 antibodies suitable for use in any of the uses, therapies, medicaments and kits described herein comprise a heavy chain constant region of human IgG1 or IgG4 isotype, more preferably a human IgG4 constant region. In some embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody comprises the S228P mutation that replaces a serine residue in the hinge region with a proline residue that is typically present at the corresponding position of an IgG1 isotype antibody.

In some embodiments, the anti-PD-1 antibodies disclosed herein include any one of the anti-PD-1 antibodies described in publication No. WO2014206107 or an antigen-binding fragment thereof, the entire disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits disclosed herein are selected from the humanized antibodies 38, 39, 41 and 48 described in WO 2014206107. In a preferred embodiment, the anti-PD-1 antibody useful in any of the uses, therapies, medicaments and kits disclosed herein is tereprinimab (toriplalimab), a polypeptide having WHO Drug Information (vol 32, phase 2, page 372-373 (2018)).

The non-limiting, exemplary antibody used in the examples herein is selected from the humanized antibody tereprinimab described in WO2014206107, whose amino acid CDR sequences, variable region sequences and full-length amino acid sequences are as follows (KABAT):

terepril monoclonal antibody light chain variable region (SEQ ID NO:13)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK

LCDR1：RSSQSIVHSNGNTYLE(SEQ ID NO:7)

LCDR2：KVSNRFS(SEQ ID NO:8)

LCDR3：FQGSHVPLT(SEQ ID NO:9)

Terapril Single heavy chain variable region (SEQ ID NO:14)

QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSS

HCDR1：DYEMH(SEQ ID NO:10)

HCDR2：VIESETGGTAYNQKFKG(SEQ ID NO:11)

HCDR3：EGITTVATTYYWYFDV(SEQ ID NO:12)

Terepril monoclonal antibody light chain (SEQ ID NO:15)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Terepril monoclonal antibody heavy chain (SEQ ID NO:16)

QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Preferably, in any one of the embodiments of the uses, therapies, medicaments and kits disclosed herein, the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, with the light chain CDRs of SEQ ID NO: 7.8 and 9, and the heavy chain CDR is SEQ ID NO: 10. 11 and 12.

More preferably, in any one of the embodiments of the uses, therapies, medicaments and kits disclosed herein, the anti-PD-1 antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO:13, and (b) a light chain variable region comprising SEQ ID NO:14 in the heavy chain variable region of seq id no.

Further preferably, in any one of the uses, therapies, medicaments and kits disclosed herein, the anti-PD-1 antibody is a polypeptide comprising: (a) comprises the amino acid sequence of SEQ ID NO:15, and (b) a light chain comprising SEQ ID NO:16, or a monoclonal antibody to the heavy chain of seq id no.

anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits disclosed herein also include Nivolumab and Pembrolizumab that have been approved by the FDA.

In certain embodiments, anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits disclosed herein 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, toriplalimab, sinilimab, Camrelizumab, tislellizumab, cemipimab.

anti-TIGIT antibodies

The term "TIGIT", collectively referred to as T cell immunoglobulin and ITIM domain proteins (T cell Ig and ITIM domain), 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 produced by intracellular processing or any fragment thereof. Human TIGIT refers to a TIGIT extracellular domain identical to the Genbank accession NP _776160.2 amino acid residues 22-141 sequence. In one embodiment, cynomolgus monkey (Wacaca fascicularis) TIGIT refers to mature TIGIT that is identical to the Genbank accession number XP — 005548158.1 amino acid residues 22-245 sequence.

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

Herein, when referring to "anti-TIGIT antibody," the term includes antigen binding fragments thereof unless otherwise indicated or described.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof disclosed herein include any of the anti-TIGIT antibodies or antigen-binding fragments thereof described in application No. PCT/CN2020/101883(WO2021/008523), the entire disclosure of which is incorporated herein by reference. In some embodiments, the CDR sequences of the anti-TIGIT antibodies used in the methods, uses and compositions of the invention comprise CDR sequences from antibody hu20 or hu3 described in PCT/CN 2020/101883. In some embodiments, the light chain variable region and the heavy chain variable region of an anti-TIGIT antibody for use in the methods, uses and compositions of the present invention comprise the light chain variable region and the heavy chain variable region from antibody hu20 or hu3 described in PCT/CN 2020/101883. In some embodiments, the anti-TIGIT antibody used in the methods, uses and compositions of the invention is antibody hu20 or hu3 described in PCT/CN 2020/101883.

In some embodiments, the anti-TIGIT antibodies disclosed herein are selected from the group consisting of: tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, or BMS-986207.

Pharmaceutical preparation

The invention discloses a pharmaceutical composition which is a high-stability pharmaceutical composition containing IL-21-anti-albumin single-domain antibody fusion protein. The invention discovers that the stability of the pharmaceutical composition can be obviously improved by the combination of trehalose.

The pharmaceutical composition disclosed by the invention is sensitive to pH, and has higher stability when the pH range is controlled to be 4.5-5.1.

The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) IL-21-anti-albumin single domain antibody fusion proteins.

The IL-21 fusion protein in the pharmaceutical composition of the invention comprises: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, said IL-21 comprising the amino acid sequence set forth in SEQ ID NO:1, said single domain antibody comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth 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 invention comprises cytokine IL-21 with an amino acid sequence as shown in SEQ ID NO. 1, and sdAb with an amino acid sequence as shown in SEQ ID NO. 5; more preferably, the fusion protein in the pharmaceutical composition of the invention comprises or consists of the amino acid sequence shown as SEQ ID NO 6.

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

The buffer in the pharmaceutical composition of the present invention may be selected from the group consisting of an acetate buffer, a citrate buffer and a histidine buffer to provide a pH of 4.0 to 6.5, preferably 4.5 to 5.5, more preferably 4.8 ± 0.3, more preferably about 4.8 to the pharmaceutical composition of the present invention. In another aspect, the buffer used in the pharmaceutical composition of the present invention may have a pH of 4.0 to 6.5, preferably 4.5 to 5.5, more preferably 4.8 ± 0.3, more preferably about 4.8.

The buffer solution particularly preferred in the pharmaceutical composition of the present invention is an acetic acid buffer solution, and preferably, the acetic acid buffer solution is an acetic acid-sodium acetate buffer solution or an acetic acid-potassium acetate buffer solution, and preferably, an acetic acid-sodium acetate buffer solution. In some embodiments, the acetate buffer is made of 1-30mM acetic acid and 1-30mM sodium acetate. In some embodiments, the acetic acid buffer is made of acetic acid and sodium acetate in a molar ratio of about 2: 3. In some embodiments, the acetic acid buffer is made of acetic acid and sodium acetate in a molar ratio of about 1: 2.1. In some embodiments, the acetic acid buffer is made of acetic acid and sodium acetate in a molar ratio of about 1: 5.7. In some embodiments, the acetate buffer is: an acetate buffer at about pH 4.8 made from about 8mM acetic acid and about 12mM sodium acetate. In some embodiments, the acetate buffer is: an acetate buffer at about pH5.0 made from about 6.5mM acetic acid and about 13.5mM sodium acetate. In some embodiments, the acetate buffer is: an acetate buffer of about pH 5.5 made with about 3mM acetic acid and about 17mM sodium acetate.

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

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

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

In some embodiments, the pharmaceutical compositions of the present invention further comprise a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. The concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% to about 0.1%, preferably about 0.02% to about 0.08%, preferably about 0.02% to about 0.04%, in w/v. By way of non-limiting example, the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.04% or 0.08%, preferably 0.02%.

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

The pharmaceutical composition of the present invention may be a liquid formulation, or a lyophilized formulation.

Pharmaceutical combination

The invention also provides a pharmaceutical combination comprising an IL-21 fusion protein as described herein and an immune checkpoint modulator. The immune checkpoint modulator of the present disclosure may be as described in any of the preceding embodiments; preferably, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-TIGIT antibody. In such a pharmaceutical combination, the anti-IL-21 fusion protein and the immune checkpoint modulator may be provided as a mixture (i.e., as a pharmaceutical composition) or each may be provided as separate formulations. When present as separate formulations, each formulation may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. When provided in the form of a pharmaceutical composition, the pharmaceutical composition will generally contain, in addition to the active ingredient, a pharmaceutically acceptable carrier.

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

Methods of treatment and uses

The invention provides an IL-21 fusion protein, or the pharmaceutical composition, or the combination of the IL-21 fusion protein and an immune checkpoint modulator, or the combination of the pharmaceutical composition and the immune checkpoint modulator, for use in the preparation of a medicament for the prevention or treatment of a tumor. The invention also provides the use of the pharmaceutical composition or the IL-21 fusion protein in a medicament for enhancing or improving the anti-tumor activity of an immune checkpoint modulator.

The present invention provides a method for preventing or treating a tumor comprising administering to an individual in need thereof an effective amount of an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an immune checkpoint modulator, or a combination of a pharmaceutical composition as described above and an immune checkpoint modulator. The effective amount includes a prophylactically effective amount and a therapeutically effective amount.

The present invention provides an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an immune checkpoint modulator, or a combination of a pharmaceutical composition as described above and an immune checkpoint modulator, for use in the treatment or prevention of a tumor.

The tumors disclosed herein can be as described in any of the embodiments above. Preferably, the tumor described herein is a tumor benefiting from IL-21 treatment, or is a PD-1 or TIGIT-associated tumor, preferably selected from mesothelioma, lung, breast, ovarian, melanoma, renal, pancreatic, lymphoma, leukemia, head and neck, liver, non-hodgkin lymphoma, esophageal, gastric and colorectal; preferably, the tumor is metastatic melanoma, renal cell carcinoma, colorectal cancer and non-hodgkin's lymphoma, more preferably colorectal cancer.

The immune checkpoint modulator of the present disclosure may be as described in any of the preceding embodiments; preferably, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-TIGIT antibody.

In one or more embodiments, the differential analysis between the two groups of the invention is performed by two-tailed unpaired students't test, denoted P < 0.05; denotes P <0.01, denotes P <0.001, ns denotes no significant difference; p <0.05 indicates a significant increase or a significant increase.

In one or more embodiments, the IL-21 fusion proteins disclosed herein have one or more properties selected from the group consisting of:

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

(4) the proportion of NK cells in the immune cells is obviously improved.

Thus, in some embodiments, the present application provides the use of an IL-21 fusion protein disclosed herein in combination with an anti-PD-1 antibody in the preparation of an agent for one or more of the following uses:

(1) increasing CD3 in immune cells ⁺ The proportion of T cells;

(2) increasing CD8 in T cells ⁺ The proportion of T cells;

(3) increase proliferation status (Ki 67) ⁺ ) CD8 (1) ⁺ The proportion of T cells; and

(4) increasing the proportion of NK cells in the immune cells.

In preferred embodiments, the agents are used in the treatment or prevention of tumors as disclosed herein.

In one or more embodiments, the IL-21 fusion protein of the invention has one or more properties selected from the group consisting of:

(1) significantly increases the expression level of interferon gamma;

(2) the expression level of granzyme A is obviously increased;

(3) the expression level of granzyme B is obviously increased;

(4) significantly increased expression levels of perforin 1;

Thus, in some embodiments, the present application provides for the use of an IL-21 fusion protein disclosed herein in combination with an anti-TIGIT antibody described above in the preparation of an agent for one or more of the following uses:

(1) increasing the expression level of interferon gamma in a subject;

(2) increasing the expression level of granzyme a in the subject;

(3) increasing the expression level of granzyme B in the subject; and

(4) increasing the expression level of perforin 1in the subject.

Preferred IL-21 fusion proteins for use in the disclosed tumors may be as described in any embodiment herein, preferably comprising: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, the cytokine IL-21 comprising an amino acid sequence as set forth in SEQ ID NO:1, the single domain antibody (sdAb) comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; more preferably an amino acid sequence comprising the amino acid sequence shown as SEQ ID NO. 1 and the amino acid sequence shown as SEQ ID NO. 5; more preferably, the polypeptide comprises an amino acid sequence shown as SEQ ID NO. 6.

Preferred anti-PD-1 antibodies for use in the disclosed tumors can be as described in any embodiment herein, more preferably comprise light chain CDRs of SEQ ID NO: 7.8 and 9, and the heavy chain CDRs are SEQ ID NOs: 10. 11 and 12, more preferably an antibody comprising the amino acids set forth in SEQ ID NOs: 13 and the variable region of the light chain shown in SEQ ID NO:14, more preferably a monoclonal antibody comprising the heavy chain variable region of SEQ ID NO:15 and SEQ ID NO:16, more preferably the humanized antibodies 38, 39, 41 and 48 described in WO2014206107, most preferably terlipril mab.

In a particularly preferred embodiment, the present invention provides a method for the prophylaxis or treatment of a tumor, which method comprises administering to a patient having a tumor a therapeutically effective amount of an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an anti-PD-1 antibody, or a combination of a pharmaceutical composition as described above and an anti-PD-1 antibody; preferably, the tumor patient is a colorectal cancer patient; preferably, the IL-21 fusion protein comprises an amino acid sequence as set forth in SEQ ID NO 6; preferably, the anti-PD-1 antibody is tereprinimab.

In a particularly preferred embodiment, the present invention provides the use of an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an anti-PD-1 antibody, or a combination of a pharmaceutical composition as described above and an anti-PD-1 antibody, for the manufacture of a medicament for the prevention or treatment of a tumor; preferably, the tumor patient is a colorectal cancer patient; preferably, the IL-21 fusion protein comprises an amino acid sequence as set forth in SEQ ID NO 6; preferably, the anti-PD-1 antibody is tereprinimab.

Reagent kit

The present invention provides a kit comprising:

(1) one or more single drug dosage units of an IL-21 fusion protein and one or more single drug dosage 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

(2) One or more single unit pharmaceutical dosage units of a pharmaceutical composition as described herein.

In one or more embodiments, the kits disclosed herein further comprise instructions for indicating a method of use of the pharmaceutical compositions.

Medical uses and methods

The invention also provides a pharmaceutical composition disclosed in any embodiment of the invention for use in the treatment or prevention of cancer, the use of a pharmaceutical composition disclosed in any embodiment of the invention in the manufacture of a medicament for the treatment or prevention of cancer, and a method of administering a therapeutically effective amount of a pharmaceutical composition disclosed in any embodiment of the invention to a subject or patient in need thereof to treat or prevent cancer.

In the present invention, 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.

The present invention will be specifically described below by way of examples, which are not intended to limit the present invention in any way. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

The invention employs the following abbreviations:

his-tag represents a histidine tag;

HRP stands for horse radish peroxidase;

TMB represents 3,3',5,5' -tetramethylbenzidine;

HBSS stands for Hank's Balanced Salt Solution, i.e., Hank's Balanced Salt Solution;

HEPES stands for N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid, Good's buffer reagent.

BID represents one dose, 2 times daily

CDR stands for complementarity determining region

DFS stands for disease-free survival

FR represents a framework region

IgG stands for immunoglobulin G

Q2W represents one dose every two weeks

QD represents one dose per day

The invention discloses a capillary micro differential scanning fluorescence method (DSF)

Automatically absorbing the sample fully by using a capillary tube, putting the sample into a corresponding numbering position, scanning the set temperature of 20-95 ℃, and increasing the temperature at the rate of 1 ℃/min. The instrument runs to end processing and analyzing data.

The invention relates to an appearance detection method

The appearance was checked by visual inspection. Ensuring that the illumination intensity of the clarity detector is kept between 1000lx and 1500 lx. The sample is kept at the same level of the eye, gently shaken or inverted to avoid air bubbles. Visual inspection was performed before black and white background, respectively. Color, opalescence and visible foreign matter were observed.

The protein content detection method of the invention

Protein concentration was measured using an ultraviolet spectrophotometer. The percent extinction coefficient (E1%) was set at 1.527(g/ml) ^-1 cm ^-1 . A BIO MATE 3S instrument was used, the cuvette was washed with ultrapure water, 150. mu.L of ultrapure water was added, and click measurement was performed with blank correction using ultrapure water. 2 solutions were assayed in parallel for each sample and 3 replicates per solution were taken and averaged. And calculating the protein concentration of the corresponding detection product according to the extinction coefficient and the OD value.

The invention relates to a size exclusion chromatography (SEC-HPLC) detection method

SEC-HPLC purity was determined by HPLC (Waters e2695 instrument) fitted with a SEC column (Waters Xbridge BEH 125A 7.8 x 300mm,3.5 μm). The mobile phase composition was 50mM phosphate buffer, 300mM sodium chloride, 300mM arginine, pH 6.8. + -. 0.2. The relative percentages of the main peak (monomer), mer and fragment were calculated using area normalization. Details are given in the following table:

chromatographic conditions	Chromatographic parameters
		Wavelength of detector	280nm
Auto sampler temperature	25±3℃
		Column temperature
	5±3℃
		Flow rate of flow	0.50ml/min
Sample introduction volume	25μl
		Mode of operation	Isocratic mode
Run time	30.00min

The invention relates to a non-reduction capillary electrophoresis (NR-CE-SDS) detection method

The purity of NR-CE-SDS was measured by a capillary electrophoresis apparatus (Maurice apparatus) equipped with a CE-SDS cartridge. Taking 50 ul of diluent (1 Xsample buffer solution Maurice CE-SDS), adding 5.0 ul of 0.8M iodoacetamide, mixing evenly, then adding 50 ul of sample (1mg/ml), mixing evenly by vortex; and simultaneously taking 50 mu l of the diluent, adding 5.0 mu l of 0.8M iodoacetamide, uniformly mixing, incubating for 5min at 70 ℃, and calculating according to an area normalization method, wherein the percentage of the main peak to the sum of all corrected peak areas is the purity of the main peak.

The invention relates to a detection method of reduced capillary electrophoresis (R-CE-SDS)

The purity of R-CE-SDS was measured by a capillary electrophoresis apparatus (Maurice apparatus) equipped with a CE-SDS cartridge. Taking 50 ul of diluent (1 Xsample buffer solution Maurice CE-SDS), adding 5.0 ul of 2-mercaptoethanol, mixing evenly, adding 50 ul of sample (1mg/ml), and mixing evenly by vortex; and simultaneously taking 50 mu l of diluent, adding 5.0 mu l of 2-mercaptoethanol, uniformly mixing, incubating for 15min at 70 ℃, and calculating according to an area normalization method, wherein the percentage of the main peak to the sum of all corrected peak areas is the purity of the main peak.

The invention provides an imaging capillary isoelectric focusing (iCIEF) detection method

The iCIEF was detected using a capillary electrophoresis apparatus (Maurice apparatus) equipped with an iCIEF cartridge. The sample was diluted with the iCIEF mixture and detected using a capillary electrophoresis apparatus (Maurice). The different isoelectric point components are focused at different locations to achieve focusing and separation.

The following is formulated for iCIEF mixture:

name of reagent	Volume required for each sample (. mu.L)
		1% MC (methyl cellulose)	35
Pharmalyte Mix (ampholyte)	4
		pI Marker Mix (isoelectric Marker)	1
500mM Arg (arginine)	2
		8M Urea	50

And mixing 8 mu L of sample solution (1mg/mL) with 92 mu L of iCIEF mixed solution, centrifuging to remove bubbles, and performing instrumental analysis and detection.

The method for detecting binding Activity of the present invention

The recombinant human IL-21R protein His tag (Acro Corp., product number ILR-H5226) was diluted to 4.0. mu.g/ml for coating, and incubated at 37. + -. 2 ℃ for 90min in an incubator. Plates were washed and blocked with 2% skim milk. Different concentrations of the test sample (starting from 4. mu.g/mL, 3-fold gradient dilution) were added, incubated for 1 hour and the plates washed. Then incubated with HRP-conjugated rabbit anti-camel VHH antibody (GenScript, Inc., cat # A01861-200) diluted at 1:5000 for 1 hour, and then incubated with HRP substrate TMB (Sigma, cat # T2885) for 30 minutes to develop color, and the binding signal of the sample to be detected was detected. Slope curve fitting (GraphPad Prism) EC using log (agonist) versus response variable ₅₀ 。

The method for detecting cell activity of the present invention

The first day the test samples were diluted to 30. mu.g/ml (6 × assay concentration) with test sample assay buffer (HBSS +10nM HEPES), 3-fold gradient dilution, 12 concentrations. Pfeiffer cell density was adjusted to 4.0X 10 ⁶ Adding 25 mul/well of each cell/ml into a 96-well round bottom plate, adding 5 mul/well of a pre-diluted sample to be detected into the 96-well round bottom plate, and incubating for 30min in a 5.0% carbon dioxide incubator at 37.0 ℃. After 10. mu.l of lysis buffer (STAT3 Pho-Y705 kit) was added to each well and lysed at room temperature for 30min, 16. mu.l of cell lysis mixture was pipetted into HTRF 96 well plates of small volume, 4. mu.l of pooled antibody (STAT3 Pho-Y705 kit) was added and incubated at room temperature for 18. + -.2 h in the absence of light. The next day, data analysis was performed using multifunctional microplate reader read plates and GraphPad Prism software. The calculation formula is as follows: ratio 665/620-OD 665nm/OD 620nm 10000.

Detailed Description

For better understanding of the present invention, the following description is given with reference to specific examples, but the present invention is not limited to the specific embodiments. The contents of all references cited throughout this application are expressly incorporated herein by reference.

Example 1: protein concentration screening experiment

In the liquid pharmaceutical composition, the protein concentration closely affects the stability of the antibody, and each fusion protein having unique physicochemical properties has an optimum concentration. This example is directed to screening for an optimal protein concentration that provides the disclosed IL-21 fusion proteins with optimal stability for clinical use.

This example uses histidine buffers (histidine-acetic acid) at pH4.5 and pH5.0 to examine the stability of IL-21-fusion proteins at different concentrations. And dialyzing the IL-21 fusion protein (with the number of JS-EC21) with the concentration of 4.0mg/ml to change the solution to a corresponding buffer solution for three times, wherein the solution changing time is more than 4 hours each time, adjusting to the target concentration, adding Tween 80, filtering and filling into a 2R penicillin bottle with the specification of 0.5 ml/bottle, and performing stability lofting and detection. 1mg/ml, 3mg/ml and 5mg/ml of IL-21-fusion protein were selected, respectively, as shown in Table 1.

The samples were placed under accelerated conditions (25. + -. 2 ℃ C.) and prolonged conditions (5. + -. 3 ℃ C.), and taken out at

weeks

0, 2 and 4 for analytical examination. The major pathways for protein degradation are the formation of aggregates, cleavage products and charged variants. The evaluation indexes include: 1. size exclusion chromatography (SEC-HPLC) determines the percentage of protein monomer to aggregated form; CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method, under reducing and non-reducing conditions, detecting the purity of the protein; and 3, detecting the antibody binding activity and the cell activity by an ELISA method. The effect of different concentrations on the stability of IL-21-fusion proteins was examined and the results are shown in tables 2-6.

Table 1 protein concentration and prescription information in pH screening experiments

1.1SEC-HPLC screening results

According to SEC-HPLC results in Table 2, after 4 weeks of accelerated conditions, the contents of protein aggregates in prescription FS1-3 and prescription FS1-4 are increased, and the contents of prescription FS1-1 and prescription FS1-2 are not obviously changed; after being placed for 4 weeks under long-term conditions, the purity of the SEC-HPLC protein monomer is not obviously changed.

TABLE 2SEC-HPLC DATA IN PROTEIN CONCENTRATION AND PH SCREENING EXPERIMENTS

1.2NR-CE-SDS screening results

According to the results of NR-CE-SDS in Table 3, the protein purity in all the prescriptions was slightly decreased by standing for 4 weeks under accelerated conditions; after standing for 4 weeks under long-term conditions, no significant purity changes occurred in all samples.

TABLE 3 CE-SDS data in protein concentration and pH screening experiments

1.3 results of R-CE-SDS screening

According to the results of R-CE-SDS in Table 4, the purity of protein R-CE-SDS in prescription FS1-3 and prescription FS1-4 was decreased after 4 weeks of accelerated condition storage; after standing for 4 weeks under long-term conditions, no significant purity changes occurred in all samples.

TABLE 4 CE-SDS data in protein concentration and pH screening experiments

1.4 results of biological Activity screening

According to the results of the activity screening in Table 5, all samples showed no significant change in binding activity and cell activity after 4 weeks of accelerated and prolonged storage.

TABLE 5 protein concentration and Activity data in pH screening experiments

From the SEC-HPLC results, in histidine buffer pH5.0, samples with high protein concentrations of 3mg/ml and 5mg/ml (formulas FS1-3 and FS1-4) were left under accelerated conditions for 4 weeks, with a significant decrease in SEC-HPLC monomer purity, while the low protein concentration of 1mg/ml did not change significantly. There was no significant difference in the R-CE-SDS purity and NR-CE-SDS purity for the different formulations. From the activity results, the binding activity and cell activity of each formula was not significantly changed. The above results show that the product is sensitive to protein concentration, so a protein concentration of 1mg/ml was selected for the next round of prescription screening.

Example 2: buffer system and pH screening

To further explore the effect of different buffer systems and pH on the stability of the IL-21-fusion protein JS-EC21 of the present invention, acetate, histidine and citrate buffers were screened, with pH ranging from 4.7 to 6.5. And (3) carrying out corresponding buffer solution replacement on the IL-21-fusion protein (No. JS-EC21) dialysis bag, enabling the sample to be in a corresponding prescription after the solution replacement, enabling the final protein concentration to be about 1mg/ml, carrying out sterile filling on 2R penicillin bottles with the specification of 2.5 ml/bottle, and carrying out stability detection (shown in table 6).

The samples were placed in accelerated conditions (25. + -. 2 ℃) and in long-term conditions (5. + -. 3 ℃) and were taken out at week 0, week 1, week 2, week 4 and week 8, respectively, for analysis and examination. The effect of different buffers and pH on the stability of the IL-21-fusion protein was examined and the results are shown in tables 7-13.

The evaluation indexes include: measuring the Tm value (melting temperature) of the protein by DSC (capillary micro differential scanning fluorescence); 2. visual appearance was observed; 3. measuring the protein content by an ultraviolet spectrophotometry; 4. size exclusion chromatography (SEC-HPLC) to determine the percentage of protein monomers, aggregates and debris; detecting the percentage of the main charge, acidic charge or basic charge of the protein by an iCIEF (imaging capillary isoelectric focusing) method; CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method, under reducing and non-reducing conditions, detecting the purity of the protein; and 7, detecting the binding activity of the antibody and the activity of the cells by an ELISA method.

TABLE 6 buffer System and pH screening protocol

2.1DSF screening results

According to the DSF results in Table 7, the Tm values of the formulations of group 8 tended to decrease as the pH increased, with FS2-1, FS2-2, FS2-3 and FS2-4 being slightly higher than those of the remaining group 4.

TABLE 7 buffer system and DSF data in pH screening

2.2 appearance and protein concentration screening results

According to the results in the table 8, white spots are found to be separated out when the two groups of FS2-7 and FS2-8 are in T0; after being placed for 8 weeks under accelerated conditions or long-term conditions, only two groups of FS2-1 and FS2-3 have no abnormal appearance.

According to the results in Table 9, the detection was terminated during the stability lofting process by five groups of FS2-4, FS2-5, FS2-6, FS2-7 and FS2-8 due to appearance abnormality; the rest three groups are placed for 8 weeks under accelerated condition or long-term condition, and no change of protein content is seen.

TABLE 8 external appearance results in buffer systems and pH screening

Note: "/" indicates not measured.

TABLE 9 results of protein content in buffer systems and pH screens

Note: "/" indicates not measured.

2.3SEC-HPLC purity screening results

According to the SEC-HPLC results in Table 10, four groups of FS2-4, FS2-6, FS2-7 and FS2-8 were terminated due to appearance abnormality; the other four groups are placed for 8 weeks under accelerated conditions or long-term conditions, and the SEC-HPLC protein monomer purity has no obvious change.

TABLE 10 SEC-HPLC DATA IN BUFFER SYSTEMS AND PH SCREENING

Note: "/" indicates not measured.

2.4NR/R-CE-SDS purity screening results

According to the NR/R-CE-SDS purity results in Table 11, four groups among samples FS2-4, FS2-6, FS2-7 and FS2-8 terminated the detection due to appearance abnormality; the other four groups are placed under accelerated conditions and long-term conditions for 8 weeks, and no obvious change of protein purity occurs in all samples.

TABLE 11 CE-SDS data in buffer systems and pH screening

Note: "/" indicates not measured.

2.5iCIEF screening results

According to the iCIEF results in Table 12, four groups of samples FS2-4, FS2-6, FS2-7 and FS2-8 terminate the test due to appearance abnormality; the other four groups are placed for 8 weeks under the accelerated condition, the acid peak is increased, and no difference is seen among the groups; all samples did not change significantly after 8 weeks of long term storage.

TABLE 12 iCIEF results in buffer systems and pH screens

Note: "/" indicates not measured.

2.6 results of biological Activity screening

According to the activity results in table 13, the test was terminated by three groups of samples FS2-6, FS2-7 and FS2-8 due to appearance abnormality, placed under accelerated and long-term conditions for 8 weeks; no significant changes in binding activity and cellular activity occurred in the remaining 5 groups.

TABLE 13 buffer systems and Activity results in pH Screen

Note: "/" indicates not measured.

By combining various data analysis, from the DSF result, FS2-1, FS2-2, FS2-3 and FS2-4 are slightly higher than the rest 4 groups; from the appearance results, only the two prescriptions FS2-1 and FS2-3 showed no appearance change after being placed under the accelerated condition for 8 weeks; no significant differences were seen between the two formulations FS2-1 and FS2-3 as seen from the SEC-HPLC purity results, NR/R-CE-SDS purity results, iCIEF results and activity results. Based on the above results, the product was sensitive to pH, and overall buffer pH was optimized in the range of 4.5-5.0. Therefore, 20mM acetic acid-sodium acetate buffer was selected, pH 4.5-5.0 for the next round of prescription screening.

Example 3: stabilizer and surfactant screening experiments

In order to further explore the influence of different stabilizers (auxiliary materials) and surfactants on the stability of the IL-21-fusion protein JS-EC21, sucrose, trehalose or mannitol are selected as the stabilizers to be compared and tested.

IL-21-fusion proteins Using Millipore Pellicon 30.11m ² The membrane was subjected to UF/DF exchange and diluted to bring the sample to the corresponding recipe with a protein concentration of about 1mg/ml, the specific recipe information is shown in Table 14. And filling the sample into a 2R penicillin bottle and a 6R penicillin bottle, wherein the specifications are respectively 2.0 ml/bottle and 3.0 ml/bottle, and performing stability lofting and detection.

The preparation of each prescription is respectively placed at the accelerating (25 +/-2 ℃) condition and the long-term (5 +/-3 ℃) condition, and is respectively taken out at the 0 th week, the 2 nd week and the 4 th week for analysis and detection; freezing and thawing (-40 ℃/RT) condition, detecting repeated freezing and thawing for 3 times and 5 times; performing analysis detection on the 1 st day and the 3 rd day respectively under shaking (250rpm, RT) conditions; the assay was performed on day 5 and day 10 under light (5000lux, RT) conditions. The effect of different stabilizers and surfactants on the stability of the IL-21-fusion protein was examined and the results are shown in tables 15-19.

The evaluation indexes include: 1. visual inspection of the appearance; 2. detecting the protein content by an ultraviolet spectrophotometry; 3. size exclusion chromatography (SEC-HPLC) to determine the percentage of protein monomers, aggregates and debris; 4. cation exchange chromatography (CEX-HPLC) measures antibody major charge, acidic charge, or basic charge content; CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method, under reducing and non-reducing conditions, detecting the purity of the protein; and 6, detecting the antibody binding activity by an ELISA method.

TABLE 14 stabilizer and surfactant screening protocol

3.1 appearance and protein content screening results

According to the results in table 15, no significant change in protein content occurred in all samples after 4 weeks of storage under accelerated and prolonged conditions; no appearance abnormality is found; no abnormal appearance is found after shaking for 3 days; no abnormal appearance is seen after the white light irradiation for 10 days; after 3 freeze-thaw cycles, FS3-1 prescription developed a few white spots.

TABLE 15 protein content and appearance results in stabilizer and surfactant screening

Note: "/" indicates not measured.

3.2SEC-HPLC screening results

According to the SEC purity results in Table 16, no significant change in monomer content occurred over accelerated, long-term, freeze-thaw, shaking, and light conditions.

TABLE 16 SEC-HPLC purities in stabilizer and surfactant screening

3.3NR/R-CE-SDS screening results

According to the purity results of NR-CE-SDS in Table 17, when 3 groups of samples were left under accelerated conditions for 4 weeks, the protein purity of both NR-CE-SDS and R-CE-SDS was decreased, but no difference between groups was observed; after being placed for 4 weeks under long-term conditions, 5 times of freeze-thaw cycles, 3 days of shaking or 10 days of illumination, no obvious change of the purity of the NR-CE-SDS or R-CE-SDS protein is seen.

TABLE 17 stabilizer and surfactant screening-NR/R-CE-SDS purity results

3.4CEX-HPLC screening results

According to the CEX-HPLC results in Table 18, 3 groups of samples all showed an increase in the acidic peak after 4 weeks of storage under accelerated conditions, but showed no difference between the groups. Under the illumination condition, the increase of the prescription acidity peak of FS3-2 is relatively obvious. After the sample is placed for 4 weeks under long-term conditions, 5 times of freeze-thaw cycles, 3 days of shaking or 10 days of illumination, the CEX-HPLC results of all samples have no obvious change.

TABLE 18 stabilizer and surfactant screening-CEX-HPLC data

3.5 biological Activity screening results

According to the biological activity assay results in table 19, no significant change in binding activity occurred in all samples after 4 weeks of high temperature, accelerated or prolonged storage, and 5 freeze-thaw cycles, 3 days of shaking or 10 days of light exposure.

TABLE 19 stabilizer and surfactant screening-binding Activity results

From the appearance result, FS3-1 has a few white spots separated out under the condition of freeze thawing; the three formulas showed no significant differences in terms of protein content, SEC-HPLC purity, NR/R-CE purity and CEX and activity results. 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.

Example 4: pharmacokinetics of IL-21 fusion proteins

1. Purpose of testing

Intraperitoneal administration of mice was performed to evaluate the difference between pharmacokinetic properties of the IL-21-anti-albumin single domain antibody fusion protein JS-EC21 and recombinant human IL-21. This example IL-21-anti-albumin single domain antibody fusion protein JS-EC21 was formulated as prescribed FS 3-3.

2. Administration and blood sample preparation

12 Balb/c female mice were selected, randomly divided into 2 groups of 6 animals each, and were intraperitoneally injected with 0.15mg/kg JS-EC21 group and 0.15mg/kg recombinant human IL-21 group, respectively. Before administration, 0.5h, 2h, 6h, 24h, 48h, 72h and 96h after administration, collecting mouse blood in an EDTA-K2 anticoagulation tube, centrifuging at 3500rpm for 10 minutes, collecting plasma, and storing in a refrigerator at-80 ℃ for testing.

3. PK detection process

a. Anti-human IL-21,250 × Cap. Ab (invitrogen, 88-8218-88) was diluted 1X with antigen, coated in 96-well plates at 100. mu.l/well, and incubated overnight at 2-8 ℃.

b. Add 1 XELISA diluent at 200. mu.L/well and block for 1 hour at room temperature. Plates were washed 4 times with 1 XPBST at 300. mu.l/well.

c. Preparing JS-EC21 standard curve samples by using blank mouse plasma, wherein the range is 312.5ng/mL-20 mu g/mL; preparing a recombinant human IL-21 standard curve sample by using blank mouse plasma, wherein the range is 156.3ng/mL-20 mu g/mL; the sample to be tested is diluted with blank mouse plasma to within the standard curve range. Finally, the standard curve sample and the sample to be tested were diluted 5-fold in a single way using 1 × ELISA diluent, and loaded at 100 μ L/well. After 2 hours incubation at room temperature the plates were washed.

d. Add anti-human IL-21250. times. Det. Ab (invitrogen, 88-8218-88) diluted to 1 Xwell at 100. mu.L/well, incubate for 1 hour at room temperature, and wash the plate.

e. avidin-HRP (invitrogen, 88-8218-88) diluted to 1 Xwas added at 100. mu.L/well, and the plate was washed after incubation at room temperature for 0.5 hour.

f. 1 XTMB chromogenic substrate (invitrogen, 88-8218-88) was added at 100. mu.L/well, and after incubation for 15 minutes at room temperature in the dark, 2M hydrochloric acid solution was added at 100. mu.L/well to terminate the reaction.

g. The absorbance at 450nm/570nm is measured on a microplate reader, and the blood concentration of the detected product is analyzed by a 4-parameter model of SoftMax Pro5.4.1.

h. And (3) selecting a non-compartmental model statistical moment by using DAS 3.0 software, and performing PK parameter fitting on the data obtained in the step g.

4. Test results

As shown in Table 20, the half-life of the IL-21-fusion protein JS-EC21 was 15.5h, Cmax was 185.2. mu.g/L, and AUC was obtained when the mice of the present invention were administered with a dose of 0.15mg/kg in vivo _(0-t) 3368.1 μ g/L × h; method for producing recombinant human IL-21The half life is 2.7h, the Cmax is 3.1 mu g/L, and the AUC _(0-t) 11.5 μ g/L × h. The IL-21-fusion protein JS-EC21 has larger drug exposure and longer half-life, and is obviously superior to recombinant human IL-21.

TABLE 20 analysis table of PK analysis of mice with single-dose intraperitoneal administration of the fusion protein JS-EC21 of the invention and recombinant human IL-21

Note: AUC _(0-t) : area under the drug concentration-time curve; t is t _1/2z : a terminal elimination half-life; c _max : the peak concentration of the drug.

Example 5: inhibition of growth of mouse MC38 subcutaneous graft tumor by IL-21 fusion protein alone

1. Purpose of testing

The anti-tumor effect of the JS-EC21 of the invention in the mouse MC38 subcutaneous transplantation model was evaluated and compared with recombinant human IL-21 (rhIL-21). This example IL-21-anti-albumin single domain antibody fusion protein JS-EC21 was formulated as prescribed FS 3-3.

2. Test procedure

6-8 week old female C57BL/6WT mice (SHOWA DERIVED NEW MEDICINE SCHOOL CO., LTD.) were inoculated subcutaneously on the right back with 1X 10 cells ⁶ (one/0.1 mL) MC38 cells. When the tumor size is about 91mm ³ When mice were randomly divided into 5 groups (n-8/group), each of which was:

solvent control group: physiological saline;

treatment groups:

JS-EC21，0.45mg/kg；

JS-EC21，1.25mg/kg；

JS-EC21，3.75mg/kg；

recombinant human IL-21, 0.625 mg/kg.

All groups were administered by intraperitoneal injection 2 times a week, 6 times a week, and 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition rate TGI was calculated. TGI (%) - (1- (Ti-T0)/(Vi-V0)) x 100%, wherein Ti: mean tumor volume at day i of dosing for treatment group, T0: mean tumor volume at day 0 of dosing for treatment groups; and Vi: mean tumor volume at day i of dosing for the solvent control group, V0: mean tumor volume at day 0 of dosing for the solvent control group.

3. Test results

As shown in FIG. 1, on day 21 after the administration, the mean tumor volume in the saline group was 2446mm ³ . In the treatment group, the JS-EC21(0.42mg/kg) group had an average tumor volume of 1482mm ³ TGI 41.0%; the average tumor volume in the JS-EC21(1.25mg/kg) group was 599mm ³ TGI is 78.4%; the average tumor volume in the JS-EC21(3.75mg/kg) group was 390mm ³ The TGI was 87.3%. The mean tumor volume of the recombinant human IL-21(0.625mg/kg) group was 1698mm ³ The TGI was 31.8%. Compared with the normal saline group, JS-EC21 of 1.25mg/kg and JS-EC 3.75mg/kg significantly inhibited tumor growth of mice and showed good dose effect. JS-EC21 with the concentration of 1.25mg/kg has obviously better tumor inhibition effect than recombinant human IL-21 with the same molar mass (0.625 mg/kg).

Example 6: pharmacokinetics of IL-21 fusion protein JS-EC21 in cynomolgus monkeys

1. Purpose of testing

Comparison of the pharmacokinetics, including half-lives (t) of the JS-EC21 fusion protein of the invention and rhIL-21 in cynomolgus monkeys _1/2 ) And AUC, etc.

2. Test procedure

A total of 18 cynomolgus monkeys (6 per group, half female and half, monkey age 3.8-4.3 years) were randomly assigned to the fusion protein JS-EC21 low, medium and high dose groups. Animals were injected intravenously at 0.05mg/kg in the low dose group, 0.15mg/kg in the medium dose group, and 0.5mg/kg in the high dose group by intravenous infusion for about 10 minutes. Blood samples were collected from 3 dose groups before dosing, 5 minutes after dosing, after infusion was completed, and 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 dosing began. All blood samples were separated as serum. The JS-EC21 concentration in serum was determined using a validated ELISA method with a lower limit of quantitation of 2 ng/mL. Pharmacokinetic parameters were determined using non-compartmental analysis (NCA) in WinNonlin 8.0.

The cynomolgus monkey is provided by the Huazhen laboratory animal breeding center (Guangzhou, China). Institutional Animal Care and Use Committee (IACUC) IACUC, Inc. of Showa laboratory (Suzhou) Serial No: ACU 20-2031.

3. Test results

The pharmacokinetics of rhIL-21 in cynomolgus monkeys have been reported in the literature to appear dose-dependent within the estimated dose range of 0.1-0.5mg/kg and to exhibit a short elimination half-life (0.4-0.8 h). For comparison with rhIL-21, pharmacokinetic profiles of cynomolgus monkeys were next determined following a single intravenous infusion of JS-EC21 at low (0.05mg/kg), medium (0.15mg/kg) and high (0.5mg/kg) doses. As shown in fig. 2, the serum concentrations of JS-EC21 were in dose proportion for the low, medium and high dose groups. As shown in Table 21, the half-life of JS-EC21 was approximately 7.08 hours, much longer than the half-life of rIL-21 (0.81 hours) at a dose of 0.5 mg/kg. In addition, the exposure of JS-EC21 in cynomolgus monkeys was significantly improved, and Cmax and AUC were approximately 6-fold and 50-fold higher than rhIL-21, respectively. These data indicate that the half-life of the engineered fusion protein JS-EC21 is significantly extended and exposure is significantly increased compared to rhIL-21.

TABLE 21 pharmacokinetic parameters for cynomolgus intravenous administration with JS-EC21

Remarks t _1/2 Terminal elimination half-life; t is _max Time of maximum concentration; c _max The drug peak concentration; AUC _last Area under the drug concentration-time curve from the time of administration to the last sample acquisition; v _d Apparent volume of distribution; CL, clearance; MRT, mean residence time.

Data from published articles Waggie KS, Holdren MS, Byrnes-Blake K, et al, preliminary safety, pharmacokinetics, and pharmacodynamics of recombinant human interleukin-21in cyclomolgus macaques (Macaca fascicularis), International journal of society, 2012; 31(4):303-316.

b no data.

Example 7: combined application of IL-21 fusion protein and PD-1 monoclonal antibody on inhibiting growth of mouse MC38 subcutaneous transplantation tumor

1. Purpose of testing

The anti-tumor effect of JS-EC21 and PD-1 monoclonal antibody in a mouse MC38 subcutaneous transplantation model is evaluated.

2. Test procedure

6-8 weeks old female hPD-1 humanized mice (Jiangsu Gene biotechnology, Periosis) were inoculated subcutaneously on the right dorsal side with 1X 10 ⁶ (one/0.1 mL) MC38 cells. When the tumor size is about 91mm ³ When mice were randomly divided into 5 groups (n-7/group), each of which was:

solvent control group: anti-KLH hIgG4, 0.3 mg/kg;

treatment groups:

PD-1 monoclonal antibody, 0.3 mg/kg;

JS-EC21，1mg/kg；

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

anti-KLH hIgG4+ JS-EC21, 0.3mg/kg +1 mg/kg.

The IL-21-anti-albumin single-domain antibody fusion protein JS-EC21 is prepared according to the prescription FS 3-3; the PD-1 mAb is Teraprimab (Torpialimab), which is a polypeptide having the structure disclosed in WHO Drug Information (Vol.32, No. 2, pp.372-373 (2018)) and comprising the sequence SEQ ID NO:15 and 16, and a humanized IgG4 mAb of the light and heavy chain amino acid sequences set forth.

All groups were administered by intraperitoneal injection 2 times a week, 6 times a week, and 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition TGI was calculated. TGI (%) - (1- (Ti-T0)/(Vi-V0)) x 100%, wherein Ti: mean tumor volume at day i of dosing for treatment group, T0: mean tumor volume at day 0 of dosing for treatment groups; and Vi: mean tumor volume at day i of dosing for the solvent control group, V0: mean tumor volume at day 0 of dosing for the solvent control group.

3. Test results

As shown in FIG. 3, on day 21 after administration, the mean tumor volume of the anti-KLH hIgG4 group was 2008mm ³ . The mean tumor volume of the PD-1 monoclonal antibody group was 1229mm ³ The TGI was 41.3%. The mean tumor volume of the JS-EC21 group was 1216mm ³ And TGI of 42.0%. The mean tumor volume of the group of PD-1 monoclonal antibody combined with JS-EC21 was 673mm ³ And TGI of 70.8%. Experimental results show that the IL-21-fusion protein JS-EC21 has a remarkable tumor inhibition effect when combined with PD-1, and the drug effect is superior to that of PD-1 antibody (p is less than 0.001) or JS-EC21 single-drug therapy (p is less than 0.05), and the IL-21-fusion protein JS-EC21 has a certain synergistic effect.

Example 8: inhibition effect of combined use of IL-21 fusion protein and teripril monoclonal antibody (Torpialimab) on growth of hPD-1 humanized mouse MC38 subcutaneous transplantation tumor and action mechanism thereof

1. Purpose of testing

The anti-tumor effect of JS-EC21 and Terepril monoclonal antibody in a mouse MC38 subcutaneous transplantation tumor model and the combined drug effect mechanism thereof are evaluated.

2. Test procedure

6-8 weeks old female hPD-1 humanized mice (Jiangsu Gene biotechnology, Inc., Boiosaeus picture) were inoculated subcutaneously on the right back at 1X 10 ⁶ (one/0.1 mL) MC38 cells. When the tumor size is about 91mm ³ At this time, the mice were randomly divided into 5 groups of 7 mice each (see table 22), and administration was started (see table 23). The Terepril monoclonal antibody is self-made by Shanghai Junshi organisms. All groups were administered by intraperitoneal injection 2 times a week, 6 times a week, and 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition rate TGI was calculated. TGI (%) ([ 1- (Ti-T0)/(Vi-V0)]X 100%, wherein Ti: mean tumor volume at day i of dosing for treatment group, T0: mean tumor volume at day 0 of dosing for treatment groups; and Vi: mean tumor volume at day i of dosing for the solvent control group, V0: tumor volumes of solvent control groups on day 0 of administrationThe value is obtained. Wherein group 1in table 22 is the solvent control group and

groups

2, 3 and 4 are the treatment groups.

TABLE 22 mouse grouping and dosing parameters

Group of	Medicine	Administration dosage	Number of animals	Method of administration
					1	hIgG4 isotype control	0.3mg/kg	7	ip
2	JS-EC21	1mg/kg	7	ip
					3	Terepril monoclonal antibody	0.3mg/kg	7	ip
4	JS-EC21+ Terepril monoclonal antibody	1+0.3mg/kg	7	ip

TABLE 23 dosing schedules

On day 21 post-dose, mice were tumor-removed and immediately transferred to a 50mL centrifuge tube placed on ice, and pre-cooled PBS solution was added to the tumor-free. Preparation of single cells: an equal amount (about 0.5g) of Tumor tissue is taken, the Tumor is cut into 2-4mm small pieces, and the Tumor tissue is prepared into single cell suspension by the Tumor Disocation Kit (mouse: 130-096-730); then TIL cells were enriched using CD45(TIL) MicroBeads (mouse: 130-110-618). Taking a proper amount of the cells, adding 100 microliters of detection antibody application solution (diluting PE anti-mouse CD8a/FITC anti-mouse CD3e/Pacific Blue anti-mouse NK1.1 mother solution with PBS according to the proportion of 1:200 to obtain detection antibody application solution, and incubating for 30min at 4 ℃; after the action time is reached, centrifuging and washing 1 time by 1mL of PBS solution and 500g for 5 min; adding 400 microliters of 1 XFOXP 3 Fixtion/Perm Buffer into each tube, fixing and breaking the membrane, and centrifugally washing for 1 time at room temperature for 20-60min in a dark place at 600g for 5 min; adding 400 microliter 1 XFOXP 3 Perm Buffer, 600g, centrifuging for 5min and washing for 1 time; adding 100 microliters of detection antibody application solution into each tube (diluting the APC anti-mouse Ki-67 mother solution by using 1 XFOXP 3 Perm buffer according to the proportion of 1:200 to obtain detection antibody application solution, and incubating for 1-2h at 4 ℃; after the action time is reached, centrifuging and washing for 2 times by 1mL of PBS solution and 600g for 5 min; machine detection CD8 of BD flow cytometer ⁺ /Ki-67 ⁺ T cell ratio.

3. Test results

As shown in FIG. 4, the mean tumor volume of the hIgG4 isotype control group was 2008mm on day 21 post-dose ³ . The mean tumor volume of the Terepril mab group was 1229mm ³ TGI was 41.3%. The mean tumor volume of the JS-EC21 group was 1216mm ³ And TGI of 42.0%. The mean tumor volume of the Tereprinimab combined JS-EC21 group is 673mm ³ Significantly lower than that of Terepril monoclonal group (p < 0.001) and JS-EC21 group (p < 0.05), and 70.8% of TGI. As shown in FIG. 5, tumor weights were weighed at day 21 after dosing, and the Tereprinimab combined JS-EC21 group was significantly lower than the Tereprinimab group (p < 0.001) and the JS-EC21 group (p < 0.05). Therefore, the experimental results show that the IL-21-fusion protein JS-EC21 has a remarkable tumor inhibition effect when being used together with the Terepril monoclonal antibody, and the drug effect is superior to that of the Terepril monoclonal antibody or JS-EC21 single drug treatment, and has a certain synergistic effect.

Immunocytotyping studies of tumor tissue using flow cytometry for immune cells (CD 45) ⁺ ) Medium CD3 ⁺ The ratio of T cells, as shown in FIG. 6(A), was significantly higher in the combination group than in the Tereprinimab group (p < 0.01) or in the JS-EC21 group (p < 0.01); for CD3 ⁺ CD8 in T cells ⁺ The ratio of T cells, as shown in FIG. 6(B), was significantly higher in the combination group than in the case of Tereprinimab (p < 0.05) or JS-EC21 (p < 0.01); for the cell in a proliferative state (Ki 67) ⁺ ) CD8 (1) ⁺ The ratio of T cells, as shown in FIG. 6(C), was significantly higher in the combination group than in the case of Tereprinimab (p < 0.05) or JS-EC21 (p < 0.05); for immune cells (CD 45) ⁺ ) The proportion of NK cells in the group administered with combination was significantly higher than that in the group administered with Tereprinimab (p < 0.05) or JS-EC21 (p < 0.05), as shown in FIG. 6 (D). The research results show that the combination of Teraprimab and JS-EC21 can significantly increase the CD8 of tumor infiltration ⁺ The ratio of T cells to NK cells, thereby enhancing the anti-tumor immune response.

Sequence listing

<110> Shanghai Junshi bio-medical science and technology GmbH

SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

<120> IL-21-anti-albumin single domain antibody fusion protein pharmaceutical composition and use thereof

<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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> HCDR1

<400> 10

Asp Tyr Glu Met His

1 5

<210> 11

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<220>

<223> Terepril monoclonal antibody 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 (Artificial Sequence)

<220>

<223> Terapril Single anti heavy chain variable region

<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 (Artificial Sequence)

<220>

<223> Teraprimab 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 (Artificial Sequence)

<220>

<223> Terapril mab 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; and

(2) an IL-21-anti-albumin single domain antibody fusion protein;

wherein the IL-21-anti-albumin single domain antibody fusion protein comprises: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, the IL-21 comprising an amino acid sequence set forth in SEQ ID NO:1, the single domain antibody (sdAb) comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

2. The pharmaceutical composition of claim 1, wherein the single domain antibody is fused to the C-terminus of the cytokine and the single domain antibody are directly linked;

preferably, the single domain antibody (sdAb) comprises the amino acid sequence shown as SEQ ID No. 5.

3. The pharmaceutical composition of claim 1 or 2, wherein the buffer is selected from one or more of an acetate buffer, a citrate buffer, and a histidine buffer; preferably, the buffer is selected from one or more of acetic acid-sodium acetate buffer, acetic acid-potassium acetate buffer, citric acid-sodium citrate buffer, histidine-acetic acid buffer and histidine-hydrochloride buffer; more preferably, the buffer is histidine-acetic acid buffer and acetic acid-sodium acetate buffer, preferably acetic acid-sodium acetate buffer.

4. The pharmaceutical composition according to any of claims 1 to 3, wherein the buffer has a concentration of about 1 to about 200mM, preferably about 5 to about 200mM, preferably about 10 to about 50mM, preferably about 10 to about 30mM, preferably about 10 to about 20 mM; preferably, the pH of the buffer is about 4.0 to about 6.5, preferably about 4.5 to about 5.5, preferably 4.5 to about 5.1, preferably about 4.5 to about 5.0, preferably about 4.7 to about 5.0, more preferably about 4.7 or 4.8.

5. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, and trehalose; preferably, the stabilizer is trehalose.

6. The pharmaceutical composition of claim 5, wherein the stabilizer is at a concentration of about 10mM-400mM, preferably 20mM-300mM, more preferably 30mM-200 mM; preferably, the stabilizing agent is mannitol at a concentration of about 100-300mM, preferably at a concentration of about 150-300mM, preferably at a concentration of about 200-280mM, preferably at a concentration of about 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM or 280mM, more preferably at a concentration of 240 mM; or the stabilizer is sucrose at a concentration of about 100-300mM, preferably the concentration of sucrose is about 150-300mM, preferably about 200-280mM, preferably about 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, more preferably 235 mM; or the stabilizer is trehalose at a concentration of about 100-300mM, preferably at a concentration of about 150-300mM, preferably at a concentration of about 200-280mM, preferably at a concentration of about 180mM, 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, more preferably 235 mM; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose; preferably, the stabilizer is trehalose at about 100 and 300 mM; more preferably, the stabilizer is trehalose at about 200 mM.

7. The pharmaceutical composition of any one of claims 1-6, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20, or poloxamer 188, preferably the surfactant is polysorbate 80.

8. The pharmaceutical composition of claim 7, wherein the surfactant concentration is about 0.001% to about 0.1%, preferably about 0.01% to about 0.1%, preferably about 0.02% to about 0.08%, preferably about 0.01% to about 0.05%.

9. The pharmaceutical composition of any one of claims 1-8, wherein the IL-21-anti-albumin single domain antibody fusion protein comprises: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, wherein the IL-21 comprises an amino acid sequence set forth as SEQ ID NO:1, and the single domain antibody (sdAb) comprises an amino acid sequence set forth as SEQ ID NO: 5.

10. The pharmaceutical composition of any one of claims 1-9, wherein the IL-21-anti-albumin single domain antibody fusion protein comprises an amino acid sequence set forth in SEQ ID No. 6.

11. The pharmaceutical composition of any one of claims 1-10, wherein the IL-21-anti-albumin single domain antibody fusion protein concentration is about 0.1-100mg/mL, preferably about 0.2-20mg/mL, preferably about 0.2-10mg/mL, preferably about 0.5-5mg/mL, preferably about 1-5mg/mL, more preferably about 0.5-1 mg/mL; more preferably, the IL-21-anti-albumin single domain antibody fusion protein has a concentration of about 0.5mg/mL, 0.8mg/mL, 1mg/mL, 1.2mg/mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, or 10mg/mL, preferably about 1 mg/mL.

12. The pharmaceutical composition according to any one of claims 1 to 11, which comprises a component represented by any one of the following (1) to (6):

(1) (a) about 0.5mg/mL to 5mg/mL of an IL-21-anti-albumin single domain antibody fusion protein; (b) about 10-30mM acetic acid-sodium acetate buffer, pH about 4.5-5.5; (c) about 100mM trehalose; (d) and about 0.01% -0.1% polysorbate 80; or

(2) (a) about 0.5mg/mL to 5mg/mL of an IL-21-anti-albumin single domain antibody fusion protein; (b) about 10-30mM acetic acid-sodium acetate buffer, pH about 4.5-5.5; (c) about 100mM sucrose; (d) and about 0.01% -0.1% polysorbate 80; or

(3) (a) about 0.5mg/mL to 5mg/mL of an IL-21-anti-albumin single domain antibody fusion protein; (b) about 10-30mM acetic acid-sodium acetate buffer, pH about 4.5-5.5; (c) about 100mM mannitol; (d) and about 0.01% -0.1% polysorbate 80; or

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

(5) (a) about 1mg/mL of an IL-21-anti-albumin single domain antibody fusion protein; (b) about 20mM acetic acid-sodium acetate buffer, pH about 4.8; (c) about 235mM trehalose; (d) and about 0.02% polysorbate 80; or

(6) (a) about 1mg/mL of an IL-21-anti-albumin single domain antibody fusion protein; (b) about 20mM acetic acid-sodium acetate buffer, pH about 4.8; (c) about 235mM sucrose; (d) and about 0.02% polysorbate 80.

Use of an IL-21-anti-albumin single domain antibody fusion protein, or a pharmaceutical composition according to any one of claims 1-12, or a combination of an IL-21-anti-albumin single domain antibody fusion protein and an immune checkpoint modulator, or a combination of a pharmaceutical composition according to any one of claims 1-12 and an immune checkpoint modulator, in the manufacture of a medicament for the prevention or treatment of a tumor, or use of an IL-21-anti-albumin single domain antibody fusion protein, or a pharmaceutical composition according to any one of claims 1-12, in the manufacture of a medicament for enhancing the anti-tumor activity of an immune checkpoint modulator;

preferably, the IL-21-anti-albumin single domain antibody fusion protein comprises: (a) a cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds albumin, the IL-21 comprising an amino acid sequence as set forth in SEQ ID NO:1, the single domain antibody (sdAb) comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively;

preferably, the single domain antibody (sdAb) comprises the amino acid sequence set forth as SEQ ID No. 5;

preferably, the single domain antibody is fused to the C-terminus of the cytokine and the single domain antibody are directly linked;

preferably, the IL-21-anti-albumin single domain antibody fusion protein comprises an amino acid sequence shown as SEQ ID NO. 6;

preferably, the IL-21-anti-albumin single domain antibody fusion protein is used in combination with an immune checkpoint modulator;

preferably, the immune checkpoint modulator is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27, and B7H4 immune checkpoint inhibitors;

preferably, the immune checkpoint modulator is selected from an anti-PD-1 antibody or an anti-TIGIT antibody;

preferably, the anti-PD-1 antibody is selected from one or more of nivolumab, pembrolizumab, toriplalimab, Sintillimuab, Camrelizumab, Tislelizumab and Cemiplimelimab; preferably a toriplalimab;

preferably, the anti-PD-1 antibody comprises:

(3) A light chain with an amino acid sequence shown as SEQ ID NO. 15 and a heavy chain with an amino acid sequence shown as SEQ ID NO. 16;

preferably, the anti-TIGIT antibody is selected from: tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, and BMS-986207;

preferably, the IL-21-anti-albumin single domain antibody fusion protein used in combination with an anti-PD-1 antibody has one or more properties selected from the group consisting of:

(1) CD3 in immune cells ⁺ The proportion of T cells is obviously improved;

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

(3) proliferation status (Ki 67) ⁺ ) CD8 (1) ⁺ The proportion of T cells is obviously improved; and

(4) the proportion of NK cells in immune cells is obviously improved;

preferably, the IL-21-anti-albumin single domain antibody fusion protein used in combination with an anti-TIGIT antibody has one or more properties selected from the group consisting of:

(1) significantly increases the expression level of interferon gamma;

(2) significantly increasing the expression level of granzyme a;

(3) significantly increasing the expression level of granzyme B;

(4) significantly increases the expression level of perforin 1;

(5) enriching more KEGG signal paths with differential gene expression; and

(6) the induced most significantly enriched KEGG signaling pathway is mediated by IL-21-anti-albumin single domain antibody fusion protein;

preferably, the tumor is a tumor associated with IL-21, or is an immune checkpoint-related tumor;

preferably, the tumor is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, renal cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-hodgkin's lymphoma, esophageal cancer, gastric cancer, and colorectal cancer;

preferably, the tumor is colorectal cancer.

14. A pharmaceutical combination comprising:

(1) an immune checkpoint modulator; and

(2) an IL-21-anti-albumin single domain antibody fusion protein or a pharmaceutical composition according to any one of claims 1-12;

wherein the IL-21-anti-albumin single domain antibody fusion protein is according to claim 13;

preferably, the immune checkpoint modulator is as claimed in claim 13.

15. A method of preventing or treating a tumor, the method comprising administering to an individual in need thereof an effective amount of the IL-21-anti-albumin single domain antibody fusion protein of claim 13, or the IL-21-anti-albumin single domain antibody fusion protein of claim 13 and the immune checkpoint modulator of claim 13, or the pharmaceutical composition of claims 1-12 and the immune checkpoint modulator of claim 13, or the pharmaceutical combination of claim 14.

16. A kit, comprising:

(1) one or more single drug dosage units of an IL-21-anti-albumin single domain antibody fusion protein and one or more single drug dosage units of an immune checkpoint modulator; wherein the IL-21-anti-albumin single domain antibody fusion protein is according to claim 13; preferably, the immune checkpoint modulator is as claimed in claim 13; or

(2) The pharmaceutical combination of claim 14 in one or more single unit pharmaceutical dosage units.