Combined composition of modified cytokine and CD40 agonist and anti-tumor application of combined composition

Abstract

The invention provides a combined composition containing cell factors with immunomodulatory effects, such as modified IL-2 molecules or IL-7, IL-12, IL-15, IL-18, IL-21 and the like, and a CD40 agonistic antibody. The tumor growth inhibition rate of the combined composition after combined use is obviously superior to that of any single drug. The combined composition provided by the invention has an obvious anti-tumor synergistic effect.

Description

Combination composition of modified cytokine and CD40 agonist and its anti-tumor use

[0001] This application claims priority to the prior application with patent application number 202310992589.7 filed with the State Intellectual Property Office of China on August 8, 2023, entitled “Combined composition of modified cytokines and CD40 agonists and their anti-tumor use”, the full text of which is incorporated into this application by reference. Technical Field

[0002] The present invention relates to the field of medical technology, and in particular to the combination of two immunotherapy drugs, in particular a combination composition of a modified cytokine and a CD40 agonist and its anti-tumor use. Background Art

[0003] Cancer is a major public health problem worldwide, posing a serious threat to human health, with increasing morbidity and mortality rates.

[0004] With the advancement of surgery, radiotherapy, chemotherapy, targeted drugs, and immunotherapy, cancer treatment outcomes have significantly improved. However, it is undeniable that even with early diagnosis and timely treatment, the efficacy of malignant tumors remains far from satisfactory. For example, even after radical surgery for colon cancer, a considerable number of cases still develop liver metastasis several years, or even 10 years later. For some tumors, overall treatment efficacy has not improved significantly. For example, despite extensive efforts over the past 20 years, there has been no significant breakthrough in improving long-term survival rates for small cell lung cancer. Pancreatic cancer is one of the most malignant solid tumors, with an overall 5-year survival rate of only 7% to 9%, primarily due to the lack of effective early diagnosis methods. Effective treatment options are lacking for locally advanced and metastatic pancreatic cancer, which accounts for 70% to 80% of pancreatic cancer cases. Even for locally resectable pancreatic cancer, some patients cannot benefit from direct surgery due to their extremely adverse biological behavior. Traditional treatments, primarily surgery, radiotherapy, and chemotherapy, have reached a bottleneck in improving efficacy. The emergence of targeted drugs has provided new options for cancer treatment, but this has also led to the emergence of clinical drug resistance. Immunotherapy, such as immune checkpoint inhibitors, has been a research hotspot in recent years, achieving significant breakthroughs in melanoma, kidney cancer, lung cancer, and liver cancer. However, the low proportion of patients who benefit from these drugs remains a challenge that needs to be addressed in the future. Therefore, clinical cancer treatment still requires new, safe, and effective therapeutic agents to benefit more patients.

[0005] IL-2 has a molecular weight of approximately 15.5 kDa and is mainly produced by activated CD4 + T cells, CD8 +Secreted by T cells, NK cells and activated dendritic cells (DC). IL-2 receptors are mainly composed of IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-2 enhances CD8 + T cell and NK cell cytotoxicity, CD4 + IL-2 plays a crucial role in T cell differentiation and maintaining regulatory T cell function. IL-2 was approved by the FDA in 1992 and 1998 for the treatment of metastatic renal cell carcinoma and metastatic melanoma, respectively. However, IL-2 is a bidirectional regulatory cytokine, capable of activating both regulatory T cells (Tregs) and effector T cells. Therefore, IL-2's clinical applications encompass both tumorigenesis and autoimmunity. Because Tregs highly express CD25, IL-2 preferentially activates them. Increased Treg levels are associated with suppressed lymphocyte activation and poor clinical anti-tumor efficacy. Furthermore, because IL-2 is rapidly metabolized and cleared by the kidneys, its half-life in vivo is only tens of minutes. To maintain effective drug concentrations, high doses of IL-2 are required, which can cause severe toxicities, including vascular leak syndrome, pulmonary edema, hypotension, and cardiotoxicity. The applicant modified IL-2 and obtained a series of modified IL-2, which extended the half-life of IL-2 and solved the defect of IL-2 in activating Treg. For details, see patent CN114380919A. This molecule is currently undergoing Phase II clinical research in China.

[0006] CD40, a member of the tumor necrosis factor receptor superfamily, is primarily expressed on the surface of antigen-presenting cells such as dendritic cells, B cells, and macrophages. Expression has also been reported on endothelial cells, fibroblasts, smooth muscle cells, and certain tumor cells. By binding to its ligand, CD40L, CD40 can upregulate the expression of the histocompatibility complex on the surface of dendritic cells and other cells, increase the expression of the costimulatory molecules CD80 / CD86, enhance IL-12 secretion, and promote immune system activation. CD40 agonists have demonstrated significant anti-tumor effects in preclinical oncology efficacy trials, and several molecules have advanced to clinical development, including Seagen's SEA-CD40, Apexigen's sotigalimab, Celldex Therapeutics' CDX-1140, Roche's selicrelumab, and Alligator Bioscience's mitazalimab. However, the clinical efficacy of CD40 agonists has been disappointing, with significant toxicities such as hepatotoxicity, thrombocytopenia, and cytokine release syndrome. Despite years of development, no product has reached market.

[0007] Summary of the Invention

[0008] In order to improve the above technical problems, the present invention combines a CD40 agonist with a modified cytokine, which can significantly enhance the efficacy of the CD40 agonist. The combination of the two drugs has a significant synergistic anti-tumor effect.

[0009] The present invention provides a combined composition, comprising:

[0010] (i) a modified cytokine or a protein heterodimer comprising a modified cytokine;

[0011] (ii) Anti-CD40 agonistic antibodies.

[0012] According to an embodiment of the present invention, (i) the modified cytokine is selected from cytokines having immunomodulatory effects.

[0013] According to an embodiment of the present invention, (i) the modified cytokine is selected from modified cytokines such as IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0014] According to an embodiment of the present invention, the mass ratio of components (i) to (ii) is 100:1-1:100, preferably 50:1-1:50, and more preferably 10:1-1:10, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0015] According to an embodiment of the present invention, the dosage of component (i) is 0.1 mg / kg-20 mg / kg, preferably 0.5 mg / kg-10 mg / kg, for example 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg.

[0016] According to an embodiment of the present invention, the dosage of component (ii) is 0.01 mg / kg-20 mg / kg, preferably 0.05 mg / kg-10 mg / kg, for example 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg.

[0017] According to an embodiment of the present invention, in the combination composition, components (i) and (ii) are each administered in the form of a pharmaceutical composition.

[0018] According to an embodiment of the present invention, in the combination composition, components (i) and (ii) can be administered simultaneously, sequentially or intermittently.

[0019] According to an embodiment of the present invention, component (ii) includes but is not limited to Selicrelumab, APX005M, ChiLob7 / 4, ADC-1013, SEA-CD40, CDX-1140.

[0020] According to an embodiment of the present invention, component (ii) optionally further comprises its ligand CD40L or an engineered CD40L molecule.

[0021] According to an embodiment of the present invention, the administration route of the combination composition includes but is not limited to intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, etc.

[0022] According to an embodiment of the present invention, component (i) is selected from modified wild-type or biased cytokines such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, etc.

[0023] According to an embodiment of the present invention, component (i) is selected from:

[0024] 1) Aldesleukin, NKTR-214, THOR-707, ALKS4230, ANV419;

[0025] 2) a modified IL-2 molecule, wherein the modification comprises replacing the region of the IL-2 molecule that mediates the binding of the IL-2 molecule to the IL2Rα with the region of the IL-15 molecule that mediates the binding of the IL-15 molecule to the IL15Rα, wherein the region of the IL-15 molecule that mediates the binding of the IL-15 molecule to the IL15Rα comprises 9 or more amino acid residues;

[0026] 3) A protein heterodimer comprising a modified IL-2 molecule.

[0027] In certain embodiments, the region of the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα is a continuous amino acid sequence.

[0028] In certain embodiments, the region of the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα is a discontinuous amino acid sequence.

[0029] In certain embodiments, the region of the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα comprises one or more discontinuous amino acid sites.

[0030] In certain embodiments, the region of the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα is a continuous amino acid sequence.

[0031] In certain embodiments, the region of the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα is a discontinuous amino acid sequence.

[0032] In certain embodiments, the region of the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα comprises one or more discontinuous amino acid sites.

[0033] In certain embodiments, the modification is capable of reducing the affinity of the modified IL-2 molecule for IL2Rα.

[0034] In certain embodiments, the modification is capable of abolishing the affinity of the modified IL-2 molecule for IL2Rα.

[0035] In certain embodiments, the affinity is measured by biolayer interferometry (BLI).

[0036] In certain embodiments, the region mediating the binding of IL-2 molecules to IL2Rα includes loop M or a variant thereof, and the loop M comprises the sequence shown in SEQ ID NO.1.

[0037] In certain embodiments, the region that mediates the binding of IL-2 molecules to IL2Rα includes loop N or a variant thereof, and the loop N comprises the sequence shown in SEQ ID NO.5.

[0038] In certain embodiments, the region that mediates the binding of IL-2 molecules to IL2Rα includes helix B or a variant thereof, and the helix B comprises the sequence shown in SEQ ID NO.2.

[0039] In certain embodiments, the region mediating the binding of IL-2 molecules to IL2Rα includes loop M or its variant, helix B or its variant, wherein the loop M comprises the sequence shown in SEQ ID NO.1, and the helix B comprises the sequence shown in SEQ ID NO.2.

[0040] In certain embodiments, the region mediating the binding of IL-2 molecules to IL2Rα includes helix B or a variant thereof, loop N or a variant thereof, wherein the helix B comprises the sequence shown in SEQ ID NO.2, and the loop N comprises the sequence shown in SEQ ID NO.5.

[0041] In certain embodiments, the region mediating the binding of IL-2 molecules to IL2Rα includes loop M or its variant, loop N or its variant, wherein loop M comprises the sequence shown in SEQ ID NO.1, and loop N comprises the sequence shown in SEQ ID NO.5.

[0042] In certain embodiments, the region mediating the binding of IL-2 molecules to IL2Rα comprises, from N-terminus to C-terminus, loop M or its variant, helix B or its variant, loop N or its variant, wherein loop M comprises the sequence shown in SEQ ID NO.1, helix B comprises the sequence shown in SEQ ID NO.2, and loop N comprises the sequence shown in SEQ ID NO.5.

[0043] In certain embodiments, the region that mediates the binding of IL-15 molecule to IL15Rα includes loop m or a variant thereof, and the loop m comprises the sequence shown in SEQ ID NO.8.

[0044] In certain embodiments, the region that mediates the binding of IL-15 molecules to IL15Rα includes loop n or a variant thereof, and the loop n comprises the sequence shown in SEQ ID NO.12.

[0045] In certain embodiments, the region that mediates the binding of IL-15 molecules to IL15Rα comprises helix b or a variant thereof, and the helix b comprises the sequence shown in SEQ ID NO.9.

[0046] In certain embodiments, the region mediating the binding of IL-15 molecule to IL15Rα includes loop m or its variant, helix b or its variant, wherein loop m comprises the sequence shown in SEQ ID NO.8, and helix b comprises the sequence shown in SEQ ID NO.9.

[0047] In certain embodiments, the region mediating the binding of IL-15 molecules to IL15Rα includes helix b or a variant thereof, loop n or a variant thereof, wherein the helix b comprises the sequence shown in SEQ ID NO.9, and the loop n comprises the sequence shown in SEQ ID NO.12.

[0048] In certain embodiments, the region mediating the binding of IL-15 molecules to IL15Rα includes loop m or its variant, loop n or its variant, wherein loop m comprises the sequence shown in SEQ ID NO.8, and loop n comprises the sequence shown in SEQ ID NO.12.

[0049] In certain embodiments, the region mediating the binding of IL-15 molecules to IL15Rα comprises, from N-terminus to C-terminus, loop m or its variant, helix b or its variant, loop n or its variant, wherein loop m comprises the sequence shown in SEQ ID NO.8, helix b comprises the sequence shown in SEQ ID NO.9, and loop n comprises the sequence shown in SEQ ID NO.12.

[0050] In certain embodiments, the modification comprises replacing loop M or its variant within the region mediating IL-2 molecule binding to IL2Rα with loop m or its variant within the region mediating IL-15 molecule binding to IL15Rα.

[0051] In certain embodiments, the modification comprises replacing helix B or a variant thereof in the region mediating IL-2 molecule binding to IL2Rα with helix b or a variant thereof in the region mediating IL-15 molecule binding to IL15Rα.

[0052] In certain embodiments, the modification comprises replacing loop N or a variant thereof in the region mediating IL-2 molecule binding to IL2Rα with loop n or a variant thereof in the region mediating IL-15 molecule binding to IL15Rα.

[0053] In certain embodiments, the modification comprises replacing loop M or its variant within the region that mediates binding of IL-2 molecules to IL2Rα with loop m or its variant within the region that mediates binding of IL-15 molecules to IL15Rα, and replacing helix B or its variant within the region that mediates binding of IL-2 molecules to IL2Rα with helix b or its variant within the region that mediates binding of IL-15 molecules to IL15Rα.

[0054] In certain embodiments, the modification comprises replacing helix B or its variant in the region that mediates binding of IL-2 molecules to IL2Rα with helix b or its variant in the region that mediates binding of IL-15 molecules to IL15Rα, and replacing loop N or its variant in the region that mediates binding of IL-2 molecules to IL2Rα with loop n or its variant in the region that mediates binding of IL-15 molecules to IL15Rα.

[0055] In certain embodiments, the modification comprises replacing loop M or its variant within the region that mediates binding of IL-2 molecules to IL2Rα with loop m or its variant within the region that mediates binding of IL-15 molecules to IL15Rα, and replacing loop N or its variant within the region that mediates binding of IL-2 molecules to IL2Rα with loop n or its variant within the region that mediates binding of IL-15 molecules to IL15Rα.

[0056] In certain embodiments, the modification comprises replacing loop M or its variant in the region that mediates binding of IL-2 molecules to IL2Rα with loop m or its variant in the region that mediates binding of IL-15 molecules to IL15Rα, replacing helix B or its variant with helix b or its variant in the region that mediates binding of IL-15 molecules to IL15Rα, and replacing loop N or its variant in the region that mediates binding of IL-2 molecules to IL2Rα with loop n or its variant in the region that mediates binding of IL-15 molecules to IL15Rα.

[0057] In certain embodiments, the modification enables the modified IL-2 molecule to bind to IL15Rα or the sushi domain of IL15Rα.

[0058] In certain embodiments, the modification further comprises replacing loop X (loop X) or a variant thereof in the IL-2 molecule with loop X (loop X) or a variant thereof in the IL-15 molecule, wherein loop X comprises the sequence shown in SEQ ID NO.10, and loop X comprises the sequence shown in SEQ ID NO.3.

[0059] In certain embodiments, the modification further comprises replacing helix C or its variant in the IL-2 molecule with helix C or its variant in the IL-15 molecule, wherein the helix C comprises the sequence shown in SEQ ID NO.11, and the helix C comprises the sequence shown in SEQ ID NO.4.

[0060] In certain embodiments, the substitutions increase the stability of the modified IL-2 molecule compared to a modified IL-2 molecule not comprising the substitutions.

[0061] In certain embodiments, the modification further comprises amino acid substitution in the helix D region of the IL-2 molecule, wherein the substituted amino acid is capable of forming an aromatic ring interaction with the 43rd phenylalanine (F) in the helix b to stabilize the hydrophobic core, wherein the position of the amino acid is based on the amino acid sequence of the natural IL-15 molecule and is determined according to the EU index of KABAT numbering; the helix D comprises the sequence shown in SEQ ID NO.6.

[0062] In certain embodiments, the amino acid sequence of the natural IL-15 molecule is shown as SEQ ID NO.13.

[0063] In certain embodiments, the amino acid substitution comprises W121F, wherein the amino acid position is determined based on the amino acid sequence of the native IL-2 molecule according to the EU index using KABAT numbering.

[0064] In certain embodiments, the amino acid sequence of the natural IL-2 molecule is shown as SEQ ID NO.7.

[0065] In certain embodiments, the modified IL-2 molecule comprises a sequence that is 75% or greater identical to the sequence of SEQ ID NO: 14. The 75% or greater identity can be 80%, 85%, 90% or greater, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0066] In certain embodiments, the modified IL-2 molecule comprises the sequence shown in SEQ ID NO.14.

[0067] In certain embodiments, the modified IL-2 molecule comprises a sequence that is 75% or greater identical to the sequence of SEQ ID NO: 27. The 75% or greater identity can be 80%, 85%, 90% or greater, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0068] In certain embodiments, the modified IL-2 molecule comprises the sequence shown in SEQ ID NO. 27.

[0069] In certain embodiments, 3) the protein heterodimer is selected from a protein heterodimer comprising a modified IL-2 molecule and IL15R α or a variant thereof, and a protein heterodimer comprising a modified IL-2 molecule and the sushi domain of the IL15R α receptor or a variant thereof.

[0070] In certain embodiments, 3) the protein heterodimer is selected from a protein heterodimer comprising the above-mentioned modified IL-2 molecule and IL15R α or a variant thereof, and a protein heterodimer comprising the above-mentioned modified IL-2 molecule and the sushi domain of the IL15R α receptor or a variant thereof.

[0071] In certain embodiments, the IL15R α comprises the sequence shown in SEQ ID NO.16.

[0072] In certain embodiments, the sushi domain comprises the sequence shown in SEQ ID NO. 15 or SEQ ID NO. 20.

[0073] In certain embodiments, the protein heterodimer does not bind to the IL2R α receptor and has a higher affinity for IL2R β than for IL-2 molecules, including native IL-2 molecules and functional variants thereof.

[0074] In certain embodiments, the functional variant of the native IL-2 molecule comprises the modified IL-2 molecule.

[0075] In certain embodiments, the IL15Rα variant or the sushi domain variant comprises an Fc fragment.

[0076] In certain embodiments, the Fc fragment is located at the C-terminus of the IL15Rα or the sushi domain.

[0077] In certain embodiments, the Fc comprises any one of human IgG1Fc, human IgG4Fc and mouse IgG2aa.1Fc.

[0078] In certain embodiments, the Fc comprises the sequence shown in SEQ ID NO.18, SEQ ID NO.24 or SEQ ID NO.25.

[0079] In certain embodiments, the protein heterodimer comprises the sequence shown in SEQ ID NO. 14 or SEQ ID NO. 27 and the sequence shown in SEQ ID NO. 19, SEQ ID NO. 21, or SEQ ID NO. 22. In certain embodiments, the protein heterodimer is a complex of IL-2 / 15 chimera 1 (SEQ ID NO. 14 or SEQ ID NO. 27) and sushi-hIgG4Fc (SEQ ID NO. 21).

[0080] The present invention also provides use of the above combination composition in preparing drugs for treating and / or preventing tumors / cancers.

[0081] According to an embodiment of the present invention, the tumor / cancer includes but is not limited to the following types: pancreatic cancer, prostate cancer, colon cancer, melanoma, and lung adenocarcinoma.

[0082] The present invention also provides a method for treating and / or preventing tumors / cancers, comprising administering a therapeutically effective amount of the above-mentioned combination composition to a patient.

[0083] According to an embodiment of the present invention, tumors / cancers include but are not limited to the following types: pancreatic cancer, prostate cancer, colon cancer, melanoma, lung adenocarcinoma.

[0084] The present invention also provides a medicine kit for treating tumors / cancers, comprising the above-mentioned combination composition.

[0085] According to an embodiment of the present invention, tumors / cancers include but are not limited to the following types: pancreatic cancer, prostate cancer, colon cancer, melanoma, lung adenocarcinoma. Beneficial effects

[0086] The present invention provides a combination composition comprising a modified IL-2 molecule and a CD40 agonist antibody. The combination composition significantly inhibits tumor growth compared to either drug alone. The combination composition provided by the present invention exhibits significant synergistic anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 shows the tumor inhibitory effect of CY002-060 combined with CD40 antibody in the mouse pancreatic cancer Pan02 model.

[0088] Figure 2 shows the tumor inhibitory effect of CY002-060 combined with CD40 antibody in the mouse prostate cancer RM-1 model.

[0089] FIG3 shows the tumor inhibition effect of CY002-060 combined with CD40 antibody in the mouse colon cancer CT26 model.

[0090] FIG4 shows the tumor inhibitory effect of CY002-060 combined with CD40 antibody in the mouse melanoma B16F10 model.

[0091] FIG5 shows the tumor inhibition effect of CY002-060 combined with CD40 antibody in the mouse colon cancer MC38 model. DETAILED DESCRIPTION

[0092] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0093] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0094] CY002-060 was prepared according to Example 1 of patent CN114380919A, specifically a complex of IL-2 / 15 chimera 1 (SEQ ID NO. 14) and sushi-hIgG4Fc (SEQ ID NO. 21).

[0095] The preparation of CY002-062 refers to Example 1 of patent CN114380919A, specifically a complex of IL-2 / 15 chimera 1 (SEQ ID NO. 27) and sushi-hIgG4Fc (SEQ ID NO. 21).

[0096] The anti-CD40 antibody was anti-mouse CD40 antibody, clone FGK4.5, purchased from Bioxcell.

[0097] Example 1 Tumor Inhibitory Effect of CY002-060 Combined with CD40 Antibody in the Mouse Pancreatic Cancer Pan02 Model

[0098] Pan02 cells were cultured in DMEM medium containing 10% fetal bovine serum. Cells were collected during the exponential growth phase and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation. 7-week-old female C57BL / 6J mice were selected and 3×10 cells were subcutaneously inoculated per mouse. 6 On day 10 after inoculation (D10), the tumor volume reached approximately 100 mm 3At the time of the study, mice were randomly divided into 4 groups according to the size of the tumor, with 6 mice in each group, and were intraperitoneally injected with: (1) CY002-060 3 mg / kg, (2) anti-CD40 antibody 2 mg / kg, (3) CY002-060 3 mg / kg, and anti-CD40 antibody 2 mg / kg 48 hours later, and (4) negative control saline. All groups were administered once a week for 3 times according to the above regimen. During the administration period, the size of the tumor was measured regularly with a vernier caliper, and its long diameter and short diameter were measured. The volume was calculated as follows: tumor volume = 0.5 × long diameter × short diameter 2 All experimental results are expressed as mean tumor volume ± SEM (standard error of the mean).

[0099] Tumor growth curves are shown in Figure 1. At the end of the experiment (D35), tumor growth inhibition (TGI) was calculated as follows: TGI = (1 - tumor volume of the treatment group / tumor volume of the negative control group) × 100%. The TGI for 3 mg / kg CY002-060 alone was 20.7%, the TGI for 2 mg / kg anti-CD40 antibody was 68.5%, and the TGI for the combination of CY002-060 and anti-CD40 antibody was 100%.

[0100] According to Kim Jong-gyun's method, Q = E a+b / (Ea+Eb-Ea*Eb), where E a+b represents the effect of the two drugs when administered together, and Ea and Eb represent the effects of the drugs when administered alone. When Q = 1, it is additive, Q > 1, it is synergistic, and Q < 1 is antagonistic. The calculated value is Q > 1.

[0101] Example 2 Tumor Inhibitory Effect of CY002-060 Combined with CD40 Antibody in the Mouse Prostate Cancer RM-1 Model

[0102] RM-1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. Cells were collected during the exponential growth phase and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation. 7-week-old female C57BL / 6J mice were selected and subcutaneously inoculated with 1×10 6 On day 8 after inoculation (D8), the tumor volume grew to approximately 75 mm 3The mice were randomly divided into 4 groups according to the size of the tumor, with 6 mice in each group. On the day of grouping, they were intraperitoneally injected with: (1) CY002-060 3 mg / kg, (2) anti-CD40 antibody 2 mg / kg, (3) CY002-060 3 mg / kg and anti-CD40 antibody 2 mg / kg, and (4) negative control saline. All groups were given the drug once a week for 3 times. During the drug administration period, the size of the tumor was measured regularly with a vernier caliper to measure its long diameter and short diameter. The volume was calculated as follows: tumor volume = 0.5 × long diameter × short diameter 2 .

[0103] All experimental results are expressed as mean tumor volume ± SEM (standard error of the mean). Tumor growth curves are shown in Figure 2. At the end of the experiment (D22), tumor growth inhibition (TGI) was calculated as follows: TGI = (1 - tumor volume of the treatment group / tumor volume of the negative control group) × 100%. The TGI for 3 mg / kg CY002-060 alone was 7.1%, the TGI for 2 mg / kg anti-CD40 antibody was 48.9%, and the TGI for the combination of CY002-060 and anti-CD40 antibody was 85.7%.

[0104] According to the method described in Example 1, Q>1 was calculated.

[0105] Example 3 Tumor Inhibitory Effect of CY002-060 Combined with CD40 Antibody in Mouse Colon Cancer CT26 Model

[0106] CT26 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. Cells were collected during the exponential growth phase and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation. Seven-week-old female BALB / c mice were selected and subcutaneously inoculated with 5×10 6 On day 7 after inoculation (D7), the tumor volume reached approximately 85 mm 3 At the time of the study, mice were randomly divided into 4 groups according to the tumor volume, with 6 mice in each group. On D8, they were intraperitoneally injected with: (1) CY002-060 3 mg / kg, (2) anti-CD40 antibody 2 mg / kg, (3) CY002-060 3 mg / kg and anti-CD40 antibody 2 mg / kg, and (4) negative control saline. All groups were given the drug once a week for 2 times. During the drug administration period, the tumor size was measured regularly with a vernier caliper to measure its long diameter and short diameter. The volume was calculated as follows: tumor volume = 0.5 × long diameter × short diameter 2 .

[0107] All experimental results are expressed as mean tumor volume ± SEM (standard error of the mean). Tumor growth curves are shown in Figure 3. At the end of the experiment (D21), tumor growth inhibition (TGI) was calculated as follows: TGI = (1 - tumor volume of the treatment group / tumor volume of the negative control group) × 100%. The TGI for 3 mg / kg CY002-060 alone was 36.9%, for 2 mg / kg anti-CD40 antibody was 34.1%, and for the combination of CY002-060 and anti-CD40 antibody was 64.5%.

[0108] According to the method described in Example 1, Q>1 was calculated.

[0109] Example 4 Tumor Inhibitory Effect of CY002-060 Combined with CD40 Antibody in Mouse Melanoma B16F10 Model

[0110] B16F10 cells were cultured in DMEM medium containing 10% fetal bovine serum. Cells were collected during the exponential growth phase and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation. 7-week-old female C57BL / 6J mice were selected and subcutaneously inoculated with 2×10 5 On day 8 after inoculation (D8), the tumor volume reached approximately 85 mm 3 Mice were randomly divided into 4 groups according to tumor volume, with 6 mice in each group. On the day of grouping, they were intraperitoneally injected with: (1) CY002-060 3 mg / kg, (2) anti-CD40 antibody 2 mg / kg, (3) CY002-060 3 mg / kg and anti-CD40 antibody 2 mg / kg, and (4) negative control saline. All groups were given the drug once a week for 2 times. During the drug administration period, the size of the tumor was measured regularly with a vernier caliper to measure its long diameter and short diameter. The volume was calculated as follows: tumor volume = 0.5 × long diameter × short diameter 2 .

[0111] All experimental results are expressed as mean tumor volume ± SEM (standard error of the mean). Tumor growth curves are shown in Figure 4. At the end of the experiment (D18), tumor growth inhibition (TGI) was calculated as follows: TGI = (1 - tumor volume of the treatment group / tumor volume of the negative control group) × 100%. The TGI for 3 mg / kg CY002-060 alone was 17.3%, the TGI for 2 mg / kg anti-CD40 antibody was 44.0%, and the TGI for the combination of CY002-060 and anti-CD40 antibody was 65.6%.

[0112] According to the method described in Example 1, Q>1 was calculated.

[0113] Example 5 Tumor Inhibitory Effect of CY002-060 Combined with CD40 Antibody in Mouse Colon Cancer MC38 Model

[0114] MC38 cells were cultured in DMEM medium containing 10% fetal bovine serum. Cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation. 7-week-old female C57BL / 6J mice were selected and subcutaneously inoculated with 1×10 6 On day 7 after inoculation (D7), the tumor volume reached approximately 85 mm 3 The mice were randomly divided into 4 groups according to the size of the tumor, with 8 mice in each group. On the day of grouping, they were intraperitoneally injected with: (1) CY002-060 1.5 mg / kg, (2) anti-CD40 antibody 2 mg / kg, (3) CY002-060 1.5 mg / kg and anti-CD40 antibody 2 mg / kg, and (4) negative control saline. All groups were given the drug twice a week for a total of 5 times. During the drug administration period, the tumor size was measured regularly with a vernier caliper to measure its long diameter and short diameter. The volume was calculated as follows: tumor volume = 0.5 × long diameter × short diameter 2 .

[0115] All experimental results are expressed as mean tumor volume ± SEM (standard error of the mean). Tumor growth curves are shown in Figure 5. At the end of the experiment (D21), tumor growth inhibition (TGI) was calculated as follows: TGI = (1 - tumor volume of the treatment group / tumor volume of the negative control group) × 100%. The TGI for 1.5 mg / kg CY002-060 alone was 65.5%, the TGI for 2 mg / kg anti-CD40 antibody was 85%, and the TGI for the combination of CY002-060 and anti-CD40 antibody was 100%.

[0116] According to the method described in Example 1, Q>1 was calculated.

[0117] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A combined composition, comprising: (i) a modified cytokine or a protein heterodimer comprising a modified cytokine; (ii) anti-CD40 agonistic antibodies; Preferably, (i) the modified cytokine is selected from cytokines having immunomodulatory effects; Preferably, (i) the modified cytokine is selected from modified cytokines such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, etc.

2. The combined composition according to claim 1, characterized in that The mass ratio of component (i) to component (ii) is 100:1-1:100, preferably 50:1-1:50, more preferably 10:1-1:10, such as 3:0.5, 3:1, etc.; and / or, component (i) is administered at a dosage of 0.1 mg / kg-20 mg / kg, preferably 0.5 mg / kg-10 mg / kg, for example 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg; and / or, component (ii) is administered at a dosage of 0.01 mg / kg-20 mg / kg, preferably 0.05 mg / kg-10 mg / kg, for example 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg; and / or, in the combination composition, components (i) and (ii) are each administered in the form of a pharmaceutical composition; And / or, in the combination composition, components (i) and (ii) may be administered simultaneously, sequentially or at intervals; And / or, the administration route of the combination composition includes but is not limited to intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, etc.

3. The combined composition according to claim 1 or 2, characterized in that: Component (ii) includes but is not limited to Selicrelumab, APX005M, ChiLob7 / 4, ADC-1013, SEA-CD40, CDX-1140; Preferably, component (ii) optionally further comprises its ligand CD40L or modified CD40L molecule.

4. The combined composition according to any one of claims 1 to 3, characterized in that: Component (i) is selected from modified wild-type or biased cytokines such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, etc.; Preferably, component (i) is selected from: 1) Aldesleukin, NKTR-214, THOR-707, ALKS4230, ANV419; 2) a modified IL-2 molecule, wherein the modification comprises replacing the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to the IL2Rα with the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to the IL15Rα, wherein the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to the IL15Rα comprises more than 9 amino acid residues; 3) A protein heterodimer, wherein the protein heterodimer comprises a modified IL-2 molecule.

5. The combined composition according to claim 4, characterized in that: 2) In the modified IL-2 molecule, The region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα is a continuous amino acid sequence; Preferably, the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα is a discontinuous amino acid sequence; Preferably, the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα comprises one or more discontinuous amino acid sites; Preferably, the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα is a continuous amino acid sequence; Preferably, the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα is a discontinuous amino acid sequence; Preferably, the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα comprises one or more discontinuous amino acid sites; Preferably, the modification is capable of reducing the affinity of the modified IL-2 molecule to IL2Rα; Preferably, the modification is capable of eliminating the affinity of the modified IL-2 molecule to IL2Rα; Preferably, the affinity is measured by biolayer interferometry (BLI); Preferably, the region mediating the binding of IL-2 molecule to IL2Rα includes loop M or a variant thereof, and the loop M comprises the sequence shown in SEQ ID NO.1; Preferably, the region mediating the binding of IL-2 molecules to IL2Rα includes loop N or a variant thereof, and the loop N comprises a sequence as shown in SEQ ID NO.5; Preferably, the region mediating the binding of IL-2 molecules to IL2Rα comprises helix B or a variant thereof, and the helix B comprises the sequence shown in SEQ ID NO.2; Preferably, the region mediating the binding of IL-2 molecules to IL2Rα includes loop M or its variant, helix B or its variant, the loop M comprises the sequence shown in SEQ ID NO.1, and the helix B comprises the sequence shown in SEQ ID NO.2; Preferably, the region mediating the binding of IL-2 molecules to IL2Rα comprises helix B or a variant thereof, loop N or a variant thereof, wherein the helix B comprises the sequence shown in SEQ ID NO.2, and the loop N comprises the sequence shown in SEQ ID NO.5; Preferably, the region mediating the binding of IL-2 molecules to IL2Rα includes loop M or its variant, loop N or its variant, the loop M comprises the sequence shown in SEQ ID NO.1, and the loop N comprises the sequence shown in SEQ ID NO.5; Preferably, the region mediating the binding of IL-2 molecules to IL2Rα comprises, from N-terminus to C-terminus, loop M or its variant, helix B or its variant, loop N or its variant, wherein loop M comprises the sequence shown in SEQ ID NO.1, helix B comprises the sequence shown in SEQ ID NO.2, and loop N comprises the sequence shown in SEQ ID NO.5; Preferably, the region mediating the binding of IL-15 molecule to IL15Rα includes loop m or a variant thereof, and the loop m comprises the sequence shown in SEQ ID NO.8; Preferably, the region mediating the binding of IL-15 molecule to IL15Rα includes loop n or a variant thereof, and the loop n comprises the sequence shown in SEQ ID NO.12; Preferably, the region mediating the binding of IL-15 molecule to IL15Rα comprises helix b or a variant thereof, and the helix b comprises the sequence shown in SEQ ID NO.9; Preferably, the region mediating the binding of IL-15 molecule to IL15Rα comprises loop m or its variant, helix b or its variant, the loop m comprises the sequence shown in SEQ ID NO.8, and the helix b comprises the sequence shown in SEQ ID NO.9; Preferably, the region mediating the binding of IL-15 molecules to IL15Rα comprises helix b or a variant thereof, loop n or a variant thereof, wherein the helix b comprises the sequence shown in SEQ ID NO.9, and the loop n comprises the sequence shown in SEQ ID NO.12; Preferably, the region mediating the binding of IL-15 molecule to IL15Rα comprises loop m or its variant, loop n or its variant, wherein loop m comprises the sequence shown in SEQ ID NO.8, and loop n comprises the sequence shown in SEQ ID NO.12; Preferably, the region mediating the binding of IL-15 molecules to IL15Rα comprises, from N-terminus to C-terminus, loop m or its variant, helix b or its variant, loop n or its variant, wherein loop m comprises the sequence shown in SEQ ID NO.8, helix b comprises the sequence shown in SEQ ID NO.9, and loop n comprises the sequence shown in SEQ ID NO.12; Preferably, the modification comprises replacing loop M or its variant in the region mediating the binding of IL-2 molecule to IL2Rα with loop m or its variant in the region mediating the binding of IL-15 molecule to IL15Rα; Preferably, the modification comprises replacing helix B or its variant in the region mediating the binding of IL-2 molecules to IL2Rα with helix b or its variant in the region mediating the binding of IL-15 molecules to IL15Rα; Preferably, the modification comprises replacing loop N or its variant in the region mediating the binding of IL-2 molecule to IL2Rα with loop n or its variant in the region mediating the binding of IL-15 molecule to IL15Rα; Preferably, the modification comprises replacing loop M or its variant in the region that mediates binding of IL-2 molecule to IL2Rα with loop m or its variant in the region that mediates binding of IL-15 molecule to IL15Rα, and replacing helix B or its variant in the region that mediates binding of IL-2 molecule to IL2Rα with helix b or its variant in the region that mediates binding of IL-15 molecule to IL15Rα; Preferably, the modification comprises replacing helix B or its variant in the region mediating binding of IL-2 molecules to IL2Rα with helix b or its variant in the region mediating binding of IL-15 molecules to IL15Rα, and replacing loop N or its variant in the region mediating binding of IL-2 molecules to IL2Rα with loop n or its variant in the region mediating binding of IL-15 molecules to IL15Rα; Preferably, the modification comprises replacing loop M or its variant in the region that mediates binding of IL-2 molecule to IL2Rα with loop m or its variant in the region that mediates binding of IL-15 molecule to IL15Rα, and replacing loop N or its variant in the region that mediates binding of IL-2 molecule to IL2Rα with loop n or its variant in the region that mediates binding of IL-15 molecule to IL15Rα; Preferably, the modification comprises replacing loop M or its variant in the region that mediates binding of IL-2 molecule to IL2Rα with loop m or its variant in the region that mediates binding of IL-15 molecule to IL15Rα, replacing helix B or its variant with helix b or its variant in the region that mediates binding of IL-15 molecule to IL15Rα, and replacing loop N or its variant in the region that mediates binding of IL-2 molecule to IL2Rα with loop n or its variant in the region that mediates binding of IL-15 molecule to IL15Rα; Preferably, the modification enables the modified IL-2 molecule to bind to IL15Rα or the sushi domain of IL15Rα; Preferably, the modification further comprises replacing loop X or its variant in the IL-2 molecule with loop X or its variant in the IL-15 molecule, wherein loop X comprises the sequence shown in SEQ ID NO.10, and loop X comprises the sequence shown in SEQ ID NO.3; Preferably, the modification further comprises replacing helix C or its variant in the IL-2 molecule with helix C or its variant in the IL-15 molecule, wherein the helix C comprises the sequence shown in SEQ ID NO.11, and the helix C comprises the sequence shown in SEQ ID NO.4; Preferably, the substitution is capable of increasing the stability of the modified IL-2 molecule compared to a modified IL-2 molecule not comprising the substitution; Preferably, the modification further comprises replacing an amino acid in the helix D region of the IL-2 molecule, wherein the replaced amino acid can form an aromatic ring interaction with the 43rd phenylalanine (F) in the helix b to stabilize the hydrophobic core, wherein the position of the amino acid is based on the amino acid sequence of the natural IL-15 molecule and is determined according to the EU index of KABAT numbering; the helix D comprises the sequence shown in SEQ ID NO.6; Preferably, the amino acid sequence of the natural IL-15 molecule is as shown in SEQ ID NO.13; Preferably, the amino acid substitution comprises W121F, wherein the position of the amino acid is determined based on the amino acid sequence of the natural IL-2 molecule according to the EU index with KABAT numbering; Preferably, the amino acid sequence of the natural IL-2 molecule is as shown in SEQ ID NO.

7.

6. The combined composition according to claim 4 or 5, characterized in that: 2) In the modified IL-2 molecule, The modified IL-2 molecule comprises a sequence having 75% or more identity to the sequence shown in SEQ ID NO. 14; the 75% or more identity may be 80%, 85%, 90% or more identity, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; Preferably, the modified IL-2 molecule comprises the sequence shown in SEQ ID NO.14; Preferably, the modified IL-2 molecule comprises a sequence having 75% or more identity with the sequence shown in SEQ ID NO.

27. The above 75% or more identity may be 80%, 85%, 90% or 95% or more identity. Uniqueness, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; Preferably, the modified IL-2 molecule comprises the sequence shown in SEQ ID NO.

27.

7. The combined composition according to claim 4, characterized in that: 3) the protein heterodimer is selected from a protein heterodimer comprising a modified IL-2 molecule and IL15Rα or a variant thereof, and a protein heterodimer comprising a modified IL-2 molecule and a sushi domain of an IL15Rα receptor or a variant thereof; Preferably, 3) the protein heterodimer is selected from a protein heterodimer comprising the above-mentioned modified IL-2 molecule and IL15Rα or a variant thereof, and a protein heterodimer comprising the above-mentioned modified IL-2 molecule and the sushi domain of the IL15Rα receptor or a variant thereof; Preferably, the IL15Rα comprises the sequence shown in SEQ ID NO.16; Preferably, the sushi domain comprises the sequence shown in SEQ ID NO.15 or SEQ ID NO.20; Preferably, the protein heterodimer does not bind to the IL2Rα receptor and has a higher affinity to IL2Rβ than to the IL-2 molecule, wherein the IL-2 molecule includes a natural IL-2 molecule and a functional variant thereof; Preferably, the functional variant of the native IL-2 molecule comprises the modified IL-2 molecule; Preferably, the IL15Rα variant or the sushi domain variant comprises an Fc fragment; Preferably, the Fc fragment is located at the C-terminus of the IL15Rα or the sushi domain; Preferably, the Fc comprises any one of human IgG1Fc, human IgG4Fc and mouse IgG2aa.1Fc; Preferably, the Fc comprises a sequence as shown in SEQ ID NO.18, SEQ ID NO.24 or SEQ ID NO.25; Preferably, the protein heterodimer comprises a sequence as shown in SEQ ID NO.14 or SEQ ID NO.27, and a sequence as shown in SEQ ID NO.19, SEQ ID NO.21 or SEQ ID NO.22; Preferably, the protein heterodimer is a complex of IL-2 / 15 chimera 1 (SEQ ID NO.14 or SEQ ID NO.27) and sushi-hIgG4Fc (SEQ ID NO.21).

8. Use of the combination composition according to any one of claims 1 to 7 in the preparation of drugs for treating and / or preventing tumors / cancers; Preferably, the tumor / cancer includes but is not limited to the following types: pancreatic cancer, prostate cancer, colon cancer, melanoma, lung adenocarcinoma.

9. A method for treating and / or preventing tumors / cancers, the method comprising administering to a patient a therapeutically effective amount of the combination composition according to any one of claims 1 to 7; Preferably, the tumor / cancer includes, but is not limited to, the following types: pancreatic cancer, prostate cancer, colon cancer, melanoma, lung adenocarcinoma.

10. A drug kit for treating tumors / cancers, comprising the combination composition according to any one of claims 1 to 7; Preferably, the tumor / cancer includes, but is not limited to, the following types: pancreatic cancer, prostate cancer, colon cancer, melanoma, lung adenocarcinoma.

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