Bifunctional protein targeting PD-1 and TGF [beta] and application of bifunctional protein in treatment of cancer in combination with radiotherapy

Abstract

The invention relates to a bifunctional protein targeting PD-1 and TGF [beta] and application of the bifunctional protein in cancer treatment by combining radiotherapy. Specifically, the present invention relates to the use of a bifunctional protein targeting PD-1 and TGF [beta] in the preparation of a medicament for treating an individual suffering from cancer, the bifunctional protein targeting PD-1 and TGF [beta] comprising: (1) an anti-PD-1 antibody or an antigen binding fragment thereof; (2) an immunoglobulin Fc structural domain; and (3) a TGF-beta R II extracellular domain. The invention further relates to application of the PD-1 and TGF beta targeting bifunctional protein in preparation of drugs for treating or preventing lung diseases or symptoms, especially radiation-induced lung injury, caused by radiotherapy of individuals.

Description

Technical Field

[0001] The present invention relates to a bifunctional protein targeting PD-1 and TGFβ and use thereof in treating cancer in combination with radiotherapy. Background Art

[0002] The incidence and mortality of malignant tumors, especially thoracic tumors, are increasing every year. Radiotherapy (RT) is an important treatment for thoracic tumors, but it has a bidirectional effect on the tumor immune microenvironment (TME). Radiation-induced DNA damage in tumor cells induces type I or type II IFN responses, which can enhance innate immunity. At the same time, the death of immunogenic cells leads to the release of damage-associated molecular patterns (DAMPs), inducing MHC I / II upregulation and promoting antigen presentation, thereby enhancing adaptive immunity. However, in addition to the immunostimulatory effect, the expression of PD-L1 on the surface of tumor cells increases after radiotherapy, activating the PD-1 / PD-L1 pathway, leading to the exhaustion of cytotoxic T cells and causing immunosuppression.

[0003] Immune checkpoints are a type of inhibitory signaling molecules in the immune system that prevent tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens. Studies have found that one of the reasons why tumor cells can evade the body's immune system and proliferate uncontrollably is that they utilize the inhibitory signaling pathway of immune checkpoints, thereby inhibiting the activity of T lymphocytes, making it impossible for T lymphocytes to effectively kill tumors.

[0004] Programmed death 1 (PD-1) is a member of the CD28 superfamily. PD-1 is an important immune checkpoint protein and is currently an important target for tumor immunotherapy. PD-1 is expressed on activated T cells, B cells, and myeloid cells. It has two ligands, namely programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). PD-L1 interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses. The expression of PD-L1 protein can be detected in many human tumor tissues. The microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells. The expressed PD-L1 is conducive to the occurrence and growth of tumors and induces the apoptosis of anti-tumor T cells. PD-1 / PD-L1 pathway inhibitors can block the binding of PD-1 and PD-L1, block negative regulatory signals, restore T cell activity, and thus enhance the immune response. Therefore, immune regulation targeting PD-1 / PD-L1 is of great significance for tumor suppression.

[0005] The PD-1 / PD-L1 pathway is one of the immune checkpoints. In the immune microenvironment, blocking the PD-1 / PD-L1 pathway with antibodies targeting PD-1 or PD-L1 is used to block negative regulatory signals, restore T cell activity, thereby enhancing the immune response and more effectively improving the effect of inhibiting tumor occurrence and development.

[0006] Given that RT has immunomodulatory effects, and immune checkpoint inhibitors (ICIs) can improve tumor cell-induced T cell exhaustion after radiotherapy, and the combination of RT and ICI can enhance antigen presentation, thereby exerting synergistic anti-tumor effects, such as abscopal effects, the combination therapy of RT and ICI has attracted attention. However, normal tissue toxicity caused by combined therapy has become one of the main limitations of clinical application, especially when thoracic radiotherapy is combined with ICI, the risk of acute / chronic radiation lung injury (RILI) is increased. Therefore, an important part of advancing the development of immunotherapy is to overcome the toxicity of combined therapy.

[0007] Transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-β signals through a heterotetrameric receptor complex composed of two type I and two type II transmembrane serine / threonine kinase receptors. Transforming growth factor-β (TGFβ) is a pleiotropic cytokine with immunomodulatory properties, such as limitation and termination of inflammatory and allergic immune responses. TGFβ has been implicated in inflammatory, malignant, infectious, and autoimmune diseases, as well as osteoporosis and fibrosis, including cirrhosis and systemic sclerosis. Specifically, persistently high levels of TGFβ in tumors are associated with immune tolerance, angiogenesis, metastasis, and increased tumor extracellular matrix deposition, all of which may drive cancer progression and resistance to therapy.

[0008] As one of the most important cytokine categories, the TGF-β family plays an important role in a variety of pathophysiological processes including embryonic development, cell proliferation, and maintenance of immune homeostasis through the combination of Smads activated by TGF-β receptors and various lineage-determining transcription factors. During tumor progression, the functions of the TGF-β signaling pathway to inhibit immune responses and promote epithelial-mesenchymal transition (EMT) can be controlled by tumor cells, thereby promoting tumor proliferation, migration, and immune evasion. In addition, the TGF-β classical pathway plays a central role in the occurrence and development of RILI, manifested as pneumonia in the acute / subacute phase and fibrosis in the chronic phase, with a clinical incidence of 10-30%, affecting treatment progress and patient prognosis. After radiotherapy, the latent associated peptide-TGF-β complex deposited in the extracellular matrix (ECM) undergoes reactive oxygen species (ROS)-induced conformational changes, releasing activated TGF-β signaling, promoting CAF heterogeneity and radiation resistance and participating in the development of RILI.

[0009] In view of the synergistic effect of radiotherapy and ICI and the key role of TGF-β signaling in tumor immune escape and RILI, and the current lack of research on bifunctional antibodies targeting PD-1 and TGF-β, there is still a need to explore new strategies for the combination of ICI, TGF-β signaling and radiotherapy in systemic and local treatment. Summary of the invention

[0010] The present invention provides a bifunctional protein targeting PD-1 and TGFβ for use in treating, preventing and / or diagnosing diseases associated with PD-1 activity or TGFβ activity. The bifunctional protein provided by the present invention can be used as an independent therapy or in combination with other therapies (such as radiotherapy) for the treatment of cancer.

[0011] Specifically, the present invention provides a use of a bifunctional protein targeting PD-1 and TGFβ in the preparation of a medicament for treating an individual suffering from cancer, wherein the bifunctional protein targeting PD-1 and TGFβ comprises: (1) an anti-PD-1 antibody or an antigen-binding fragment thereof; (2) an immunoglobulin Fc domain; and (3) a TGF-βRⅡ extracellular domain.

[0012] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ is administered alone or in combination with radiotherapy.

[0013] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ is administered alone; preferably, the individual to whom it is administered alone has previously received radiotherapy.

[0014] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ is administered in combination with radiation therapy.

[0015] In some embodiments, the radiation therapy comprises stereotactic body radiation therapy (SBRT) or whole chest irradiation (WTI).

[0016] In some embodiments, the cancer is a breast tumor.

[0017] In some embodiments, the cancer is selected from lung cancer, breast cancer, or colon cancer.

[0018] In some embodiments, the individual has a disease or condition induced by radiation therapy.

[0019] In some embodiments, the disease or condition caused by radiation therapy is a disease or condition of the lung. In some embodiments, the disease or condition of the lung is radiation lung injury. In some embodiments, the radiation lung injury includes radiation pneumonitis and radiation pulmonary fibrosis.

[0020] In some embodiments, the individual has only a primary tumor, or has both a primary tumor and a secondary tumor; preferably, the individual has both a primary tumor and a secondary tumor.

[0021] The present invention also provides a use of a bifunctional protein targeting PD-1 and TGFβ in the preparation of a drug for treating or preventing a lung disease or condition caused by radiotherapy in an individual, wherein the bifunctional protein targeting PD-1 and TGFβ comprises: (1) an anti-PD-1 antibody or an antigen-binding fragment thereof; (2) an immunoglobulin Fc domain; and (3) a TGF-βRⅡ extracellular domain (ECD).

[0022] In some embodiments, the disease or disorder is radiation-induced lung injury, preferably, the radiation-induced lung injury is selected from radiation pneumonitis (RP) or radiation-induced pulmonary fibrosis (RIPF).

[0023] In some embodiments, the individual has only a primary tumor, or has both a primary tumor and a secondary tumor; preferably, the individual has both a primary tumor and a secondary tumor.

[0024] The present invention also provides the use of a bifunctional protein targeting PD-1 and TGFβ in the preparation of a drug for achieving one or more of the following purposes in an individual: inhibiting tumor growth and prolonging patient survival; increasing the number of tumor-infiltrating cytotoxic lymphocytes in the tumor; reducing myeloid-derived suppressor cells; increasing the CD8+T / CD4+T cell ratio; inhibiting tumor angiogenesis; downregulating the expression of molecules related to ECM, cell adhesion and cell migration; reducing EGFR signaling in tumor tissue; weakening immunosuppressive features such as ECM formation, tumor migration and angiogenesis, and compensating for the negative impact of RT-induced increased ECM expression on TME; enhancing TNF signals and enhancing inflammatory responses in tumors; alleviating fibrosis in TME, especially It is fibrosis caused by radiotherapy; inhibits the regulation of inflammatory mediators of calcium signaling pathway and TRP channel; reduces calcium ion binding and calmodulin binding; reduces CAMK2A expression; inhibits TGF-β1 / Smad2 pathway, reduces the proportion of fibroblasts and thus reduces pulmonary fibrosis; inhibits inflammasome expression in neutrophils; reduces neutrophil-mediated chronic inflammatory lung injury; reduces the proportion of neutrophils and the formation of ROS-induced neutrophil extracellular traps to reduce the pro-fibrotic effect; wherein the bifunctional protein targeting PD-1 and TGFβ comprises: (1) anti-PD-1 antibody or antigen-binding fragment thereof; (2) immunoglobulin Fc domain; and (3) TGF-βRⅡ extracellular domain. In some embodiments, the individual is an individual who has not received radiotherapy before, or an individual who has received radiotherapy before, or an individual who is about to receive radiotherapy.

[0025] In any of the aforementioned embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-PD-1 antibody are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; preferably, the bifunctional protein targeting PD-1 and TGFβ comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; more preferably, the bifunctional protein targeting PD-1 and TGFβ comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10.

[0026] In any of the aforementioned embodiments, the amino acid sequence of the TGFβRⅡ extracellular domain is shown in SEQ ID NO:11.

[0027] In any of the foregoing embodiments, the bifunctional protein targeting PD-1 and TGFβ comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, a light chain with an amino acid sequence as shown in SEQ ID NO: 10, and a TGFβRⅡ extracellular domain with an amino acid sequence as shown in SEQ ID NO: 11; wherein the heavy chain amino acids and the TGFβRⅡ extracellular domain amino acids are in the form of (GGGGS) 4 The two peptide segments are connected to form the first subunit of the bifunctional protein; preferably, the amino acid sequence of the first subunit is as shown in SEQ ID NO:12. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : JS201 combined with radiotherapy inhibits tumor growth in mice and prolongs their survival. A: Structural diagram of JS201. B: Flow chart of treatment of mice. C, D: Anti-tumor growth effect diagram of each group in LLC model. E, F: Anti-tumor growth effect diagram of each group in CT26.WT model. G, H: Anti-tumor growth effect diagram of each group in 4T1 model.

[0029] Figure 2 : JS201 combined with radiotherapy reverses the immunosuppressive microenvironment. A-D: IHC results of each group. E: Gene heat map of JS201 group and anti-PD-1 group. F, G: GO database enrichment analysis of JS201 group and anti-PD-1 group. H: DisGeNET analysis of JS201 group and anti-PD-1 group. I: Analysis result of Reactome pathway. J: KEGG enrichment analysis result. K: Gene heat map of JS201 group and JS201+RT group. L, M: KEGG enrichment analysis result. N: DisGeNET analysis of JS201 group and JS201+RT group. O: Analysis result of Reactome pathway. P: GO analysis result.

[0030] Figure 3 :The remote effect of JS201 combined with radiotherapy. A: Schematic diagram of mouse model construction. B: Graph of the inhibitory effect of each group in the primary tumor model. C: Graph of the inhibitory effect of each group in the secondary tumor model. D: Graph of in vivo imaging results of each group of mice. E: Tumor size of each group after treatment in the primary tumor model. F: Tumor size of each group after treatment in the secondary tumor model. G~J: Flow cytometry results of myeloid-derived suppressor cells in primary and secondary tumors and spleen of LLC-luc tumor-bearing mice. K~N: Flow cytometry results of CD4+ and CD8+ T cells in primary and secondary tumors and spleen of LLC-luc tumor-bearing mice.

[0031] Figure 4:JS201 alleviates acute / subacute radiation pneumonitis and inhibits calcium signaling-related pathways in RP development. A: Schematic diagram of the construction of the mouse acute radiation lung injury model. B: Chest CT results of mice. C: HE staining results of the left lung tissue of mice. D: Myeloperoxidase (MPO) staining results of mice. E~G: ImageJ quantitative analysis results. H: RNA sequencing results of the transcriptome of lung tissue of mice with acute RILI model. I: KEGG enrichment results. J: GO enrichment results. K: Venn diagram of pathway genes related to calcium signaling and inflammation enriched in GO, KEGG and Reactome databases. L: Heat map of 5 genes. M: Expression results of CAMK2A in the acute RP stage. N: IHC results of CAMK2A protein expression in different groups. O: Western blot results of CAMK2A protein expression in different groups.

[0032] Figure 5 : JS201 alleviates radiation-induced pulmonary fibrosis (RIPF) and prolongs survival. A: Schematic diagram of the construction of the chronic RIPF model in mice. B: Weekly weight change curve of each group of mice after radiotherapy. C: Percent survival curve of each group of mice after radiotherapy. D: Chest CT of mice 6 months after RT. E: HYP content in lung tissue of each group of mice. F: HE staining analysis of structural changes in lung tissue of each group of mice, Masson and Sirius red staining analysis of fibrosis. G: Ashcroft score of each group of mice. H: Masson staining result of each group of mice. I: Sirius red staining result of each mouse.

[0033] Figure 6 : JS201 reduces the proportion of fibroblasts and inhibits the TGF-β1 / Smad2 pathway. A: TSNE results of lung tissue. B: Cell annotation and analysis. C: Result of the proportion of similar cells between groups. D: DEG analysis results of fibroblasts between the two groups. E: Heat map of significant DEGs of each cell type in the anti-PD-1 group. F: Heat map of significant DEGs in the JS201 group. G: KEGG enrichment results. H: GO enrichment results. I: KEGG enrichment results. J: GO enrichment results. K, L: TSNE analysis results. M: Expression of 9 genes in 7 clusters. N, O: Western blot of lung tissue proteins.

[0034] Figure 7: JS201 reduced the proportion of neutrophils and the formation of ROS-induced neutrophil extracellular traps (NETs). A: Heat map of DEGs between neutrophils. B: Reactome enrichment result map. C: GO enrichment result map of DEGs in neutrophils. D: TSNE descending clustering result map of neutrophils in the two groups. E: Neutrophils were divided into four subgroups based on gene expression profiles. F, G: Comparison and analysis results between clusters (F) and groups (G) for 7 genes. H: Heat map of total DEGs between the two groups. I~K: DHE staining results of frozen bronchoalveolar lavage fluid (BALF) and lung tissue samples of RIPF mice. L~O: Cytokine quantification results of mouse serum. P: Western blot of related proteins. Q: CITH3 (green) and MPO (red) homologous double-labeling immunofluorescence assay results.

[0035] Figure 8 : In vivo biosafety evaluation of JS201. A: JS201 administered in vivo did not cause significant damage to major organs. B-D: There were no significant differences in serum ALT, AST and Cr among the three groups of patients. E: Schematic diagram of the action of JS201.

[0036] Fig. 9 : RNA-seq results of LLC tumor tissues. Result plots of the percentage of genes enriched in GO (A), DisGeNET (B), Reactome (C), and KEGG (D) for downregulated DEGs in LLC tumors treated with JS-201 compared with anti-PD1 antibody treatment. Result plots of the percentage of genes enriched in Reactome (E), KEGG (F), GO (G), and DisGeNET (H) for upregulated DEGs in LLC tumors in the combination treatment group compared with JS-201.

[0037] Fig.10 : RNA-Seq results of lung tissues 1 month after radiotherapy. A: Total DEGs of mRNA between JS-201 and anti-PD-1 antibody groups. B: Total DEGs of lncRNA between JS-201 and anti-PD-1 antibody groups. C: Heat map of significant DEGs between JS-201 and anti-PD-1 antibody groups, n=3 for each group. D, E: GO (D) and KEGG (E) enrichment of significant DEGs of lncRNA. F: Reactome enrichment results of DEGs of mRNA. G, H: ATP2A1 expression (G) and CASQ1 expression (H) in GSE174196 database. Student's t-test was used to calculate p-values. lncRNA refers to long noncoding RNA; mRNA, transcribed RNA.

[0038] Fig.11: Single-cell RNA-Seq results of lung tissues 6 months after radiotherapy. A, B: UMAP results (A) and cell annotation (B) of all clusters in the JS-201 and anti-PD-1 groups, n=1 for each group. C: Cell markers used for annotation. D, E: UMAP results of the original cluster of fibroblasts (D) and clusters based on characteristic gene expression (E). F: KEGG enrichment results of downregulated DEGs in neutrophils between the JS-201 group and the anti-PD-1 group. G, H: UMAP results of the original cluster of neutrophils (G) and clusters based on characteristic gene expression (H).

[0039] Fig.12 : Construction, quantification and validation results of JS-201. A: Structure of JS-201. B: SDS-PAGE results of intact JS-201 and reduced JS-201. C: The ability of JS-201 to bind to PD-1. D: The ability of JS-201 to bind to TGF-β1. E: The ability of JS-201 to bind to TGF-β2. F: The ability of JS-201 to bind to TGF-β3. G: The ability of JS-201 to bind to TGF-β1 and PD-1 simultaneously. H: The ability of JS-201 to bind to TGF-β2 and PD-1 simultaneously. I: The ability of JS-201 to bind to TGF-β3 and PD-1 simultaneously.

[0040] The illustrative but non-limiting explanations of some of the terms used in the above drawings are as follows:

[0041] “Day” or “D” stands for “day”; “month” or “m” stands for “month”; “ns” stands for “no significant difference”; “Anti-PD-1” stands for anti-PD-1 antibody; “Anti-mPD-1” stands for anti-mouse PD-1 antibody; “NS (different from lowercase “ns”)” stands for “normal saline” or “normal saline group”; “control” stands for “control” or “control group”; “Group” stands for “group”; “value” stands for “value”; “P value” stands for “P value”; “corrected P value” stands for “corrected P value”; “size” stands for “size”; “Rich Factor” stands for “enrichment factor”; “Term” stands for “term”; “description” stands for “description”; “count” stands for “count”; “gene count” stands for “gene count”; “ratio” stands for “ratio”; “cluster” stands for “cluster”; “extracellular “matrix” stands for “extracellular matrix”; “MDSC” stands for “myeloid-derived suppressor cells”; “external encapsulating structure” stands for “external encapsulating structure”; “response to wounding” stands for “response to wounding”; “cell adhesion” stands for “cell adhesion”; “biological adhesion” stands for “biological adhesion”; “cell migration” stands for “cell migration”; “tissue development” stands for “tissue development”; “locomotion” stands for “movement”; “anatomical structure formation involved in morphogenesis” stands for “anatomical structure morphogenesis” stands for “anatomical structure morphogenesis”; “regulation of multicellular organismal process” stands for “regulation of multicellular organismal process”; “cellular response to chemical stimulus” stands for “cellular response to chemical stimulus”; “animal organ development” stands for “animal organ development”; “system development” stands for “system development”; and “extracellular region” stands for “extracellular region”.“multicellular organism development” stands for “multicellular organism development”; “cell differentiation” stands for “cell differentiation”; “anatomical structure development” stands for “anatomical structure development”; “developmental process” stands for “developmental process”; “cell periphery” stands for “cell periphery”; “myocardial infarction” stands for “myocardial infarction”; “coronary heart disease” stands for “coronary heart disease”; “neoplasm metastasis” stands for “neoplasm metastasis”; “breast carcinoma” stands for “breast cancer”; “hypertensive disease” stands for “hypertensive disease”; “heart failure” stands for “heart failure”; “asthma” stands for “asthma”; “adenocarcinoma” stands for “adenocarcinoma”; “cardiovascular diseases” stands for “cardiovascular diseases”; “degenerative polyarthritis” stands for “degenerative polyarthritis”; “atherosclerosis” stands for “atherosclerosis”; “coronary artery disease” stands for “coronary artery disease”; “endometriosis” stands for “endometriosis”; “secondary malignant neoplasm of lymph node” stands for “secondary malignant neoplasm of lymph node”; “mammary neoplasm” stands for “breast neoplasm”; “inflammation” stands for “inflammation”; “squamous cell carcinoma” stands for “squamous cell carcinoma”; “adenoma” stands for “adenoma”; “psoriasis” stands for “psoriasis”; “dermatological disorders” stands for “dermatological disorders”; “type Ⅰ hemidesmosome assembly” stands for “type Ⅰ hemidesmosome assembly”; “histidine catabolism” stands for “histidine catabolism”; “erythrocytes take up oxygen and release carbon dioxide” stands for “erythrocytes take up oxygen and release carbon dioxide”; “EGFR interacts with phospholipase C-gamma” stands for “EGFR interacts with phospholipase C-gamma”;“signaling by EGFR” stands for “EGFR signaling”; “GRB2 events in EGFR signaling” stands for “GRB2 events in EGFR signaling”; “elastic fibre formation” stands for “elastic fibre formation”; “MET activates PTK2 signaling” stands for “MET activates PTK2 signaling”; “regulation of TLR by endogenous ligand” stands for “regulation of TLR by endogenous ligand”; “a tetrasaccharide linker sequence is required for GAG synthesis” stands for “a tetrasaccharide linker sequence is required for GAG synthesis”; “formation of the confirmed envelope” stands for “formation of the confirmed envelope”; “straited muscle contraction” stands for “straited muscle contraction”; “HS-GAG biosynthesis” stands for “HS-GAG biosynthesis”; “chemokine receptor bind chemokines” stands for “chemokine receptor binds chemokines”; “assembly of collagen fibris and other multimeric structures” stands for “assembly of collagen fibris and other multimeric structures”;

[0042] “degradation of the extracellular matrix” stands for “degradation of the extracellular matrix”; “RAF / MAP kinase cascade” stands for “RAF / MAP kinase cascade”; “PIP3 activates AKT signaling” stands for “PIP3 activates AKT signaling”; “platelet degranulation” stands for “platelet degranulation”; “PI5P, PP2Aand IER3 regulate PI3K / AKT signaling” stands for “PI5P, PP2A and IER3 regulate PI3K / AKT signaling”; “tumor free” stands for “tumor free”; “1m after RT” / “1month after RT”, “2m after RT” / “2month after RT”, “3m after RT” / “3month after RT” stand for “1 month after radiotherapy”, “2 months after radiotherapy” and “3 months after radiotherapy” respectively; “calcium signaling pathway” stands for “calcium signaling pathway”; “cardiac muscle contraction” stands for “myocardial contraction”; “arrhythmogenic right “ventricularcardiomyopathy(ARVC)” stands for “arrhythmogenic right ventricular cardiomyopathy”; “oxytocin signalingpathway” stands for “oxytocin signaling pathway”; “apelin signaling pathway” stands for “apelin signaling pathway”; “linoleic acid metabolism” stands for “linoleic acid metabolism”; “alpha-linoleic acidmetabolism” stands for “alpha-linoleic acid metabolism”; “protein digestion and absorption” stands for “protein digestion and absorption”; “hypertrophic cardiomyopathy(HCM)” stands for “hypertrophic cardiomyopathy”; “tyrosinemetabolism” stands for “tyrosine metabolism”; “calcium ion binding” stands for “calcium ion binding”; “actinbinding” stands for “actin binding”; “structural constituent of muscle” stands for “structural constituent of muscle”; “tropomyocin binding” stands for “tropomyocin”; and “ion channel activity” stands for “ion channel activity”.“phospholipase A2 activity” stands for “phospholipase A2 activity”; “titin binding” stands for “titin binding”; “calcium-dependent phospholipase A2 activity” stands for “calcium-dependent phospholipase A2 activity”; “actin filament binding” stands for “actin filament binding”; “sequence-specific double-stranded DNA binding” stands for “sequence-specific double-stranded DNA binding”; “T cell” stands for “T cell”; “Th2 cell” stands for “Th2 cell”; “unknown” stands for “unknown”; “lipofibroblast” stands for “lipofibroblast”; “progenitor cell” stands for “progenitor cell”; “macrophage 1” stands for “type 1 macrophage”; “endothelial cell” stands for “endothelial cell”; “monocyte” stands for “monocyte”; “neutrophil” stands for “neutrophil”; “fibroblast” stands for “fibroblast”; “alveolar epithelial cell 1” stands for “type 1 alveolar epithelial cell”; “pericyte” stands for “pericyte”; “metabolism of xenobiotics by cytochrome P450” stands for “metabolism of xenobiotics by cytochrome P450”; “TGF-beta signaling pathway” stands for “TGF-β signaling pathway”; “glutathione metabolism” stands for “glutathione metabolism”; “fluid shearstress and atherosclerosis” stands for “fluid shear stress and atherosclerosis”; “protein processing in endoplasmic reticulum” stands for “protein processing in endoplasmic reticulum”; “relaxin signaling pathway” stands for “relaxin signaling pathway”; “axon guidance” stands for “axon guidance”; “pathways in cancer” stands for “signaling pathways in cancer”; “regulation of actin cytoskeleton” stands for “actin cytoskeleton regulation”; “dilated cardiomyopathy” stands for “dilated cardiomyopathy”; “complement and coagulation cascades” stands for “complement and coagulation cascades”;“amoebiasis” stands for “amoebiasis”; “proteoglycans in cancer” stands for “proteoglycans in cancer”; “PI3K-Akt signaling pathway” stands for “PI3K-Akt signaling pathway”; “focal adhesion” stands for “focal adhesion”; “ECM-receptor interaction” stands for “ECM-receptor interaction”; “collagen binding” stands for “collagen binding”; “integrin binding” stands for “integrin binding”; “extracellular matrix structural constituent” stands for “extracellular matrix structural constituent”; “sulfur compound binding” stands for “sulfur compound binding”; “heparin” stands for “heparin binding”; “glycosaminoglycan binding” stands for “glycosaminoglycan binding”; “growth factor binding” stands for “growth factor binding”; “contractile fiber part” stands for “contractile fiber part”; “actincytoskeleton” stands for “actin cytoskeleton”; “extracellular matrix component” stands for “extracellular matrix component”; “cell-substrate “junction” stands for “cell-substrate junction”; “anchoring junction” stands for “anchoring junction”; “adherens junction” stands for “adherence junction”; “proteinaceous extracellular matrix” stands for “proteinaceous extracellular matrix”; “negative regulation of locomotion” stands for “negative regulation of locomotion”; “tissue morphogenesis” stands for “tissue morphogenesis”; “regulation of cellular component” stands for “regulation of cellular component”; “wound healing” stands for “wound healing”; “blood vessel morphogenesis” stands for “blood vessel morphogenesis”; “cellular response to growth factor stimulation” stands for “cellular response to growth factor stimulation”; “extracellular structure of organization” stands for “extracellular structure of organization”;“extracellular matrix organization” stands for “extracellular matrix organization”; “small cell lung cancer” stands for “small cell lung cancer”; “malaria” stands for “malaria”; “relaxin signaling pathway” stands for “relaxin signaling pathway”; “pertussis” stands for “pertussis”; “glutathione metabolism” stands for “glutathione metabolism”; “staphylococcus aureus infection” stands for “Staphylococcus aureus infection”; “human papillomavirus infection” stands for “human papillomavirus infection”; “drug metabolism-cytochrome P450” stands for “drug metabolism-cytochrome P450”; “proteoglycans in cancer” stands for “proteoglycans in cancer”; “pathways in cancer” stands for “pathways in cancer”; “platelet-derived growth factor binding” stands for “platelet-derived growth factor”; “insulin-like growth factor binding” stands for “insulin-like growth factor”; “banded collagen fibril” stands for “banded collagen fibril”; “fibrillar collagen “Trimer” stands for “fibrillar collagen trimer”; “complex of collagen trimers” stands for “complex of collagen trimers”; “basement membrane” stands for “basement membrane”; “cell-substrate adhesion” stands for “cell-substrate adhesion”; “response to elevated platelet cytosolic Ca; 2+ " stands for "in response to elevated platelet cytoplasmic Ca 2+"Response of RHO GTPases to WASPs and WAVEs" stands for "RHO GTPases activate WASPs and WAVEs"; "EPHB-mediated forward signaling" stands for "EPHB-mediated forward signaling"; "inflammasomes" stands for "inflammasomes"; "cell surface interactions at the vascular wall" stands for "cell surface interactions at the vascular wall"; "signaling by VEGF" stands for "VEGF signaling"; "VEGFA-VEGFR2 pathway" stands for "VEGFA-VEGFR2 pathway"; "recycling pathway of L1" stands for "recycling pathway of L1"; "gap junction trafficking and regulation" stands for "gap junction trafficking"; "Fc gamma receptor (FCGR) dependent phagocytosis" stands for "Fc-γ receptor-dependent phagocytosis"; "developmental biology" stands for "developmental biology"; "signaling by Rho “GTPases” stands for “Rho GTPase signaling pathway”; “platelet activation, signaling and aggregation” stands for “platelet activation, signaling and aggregation”; “RHOGTPases activate IQGAPs” stands for “RHO GTPase activates IQGAPs”; “RHO GTPase effectors” stands for “RHO GTPase effectors”; “hemostasis” stands for “heme-copper terminal oxidase activity”; “cytochrome-c oxidase activity” stands for “cytochrome-c oxidase activity”; “electron carrier activity” stands for “electron carrier activity”; “hydrogenion transmembrane transporter activity” stands for “hydrogen ion transmembrane transporter activity”; “carbohydrate binding” stands for “carbohydrate binding”; and “cadherin binding” stands for “cadherin binding”.“Protease binding” stands for “protease binding”; “cell adhesion molecule binding” stands for “cell adhesion molecule binding”; “structural molecule activity” stands for “structural molecule activity”; “structural constituent of cytoskeleton” stands for “structural component of cytoskeleton”; “serine / threonineprotein kinase complex” stands for “serine / threonineprotein kinase complex”; “cyclin-dependentprotein kinase holoenzyme complex” stands for “cyclin-dependent protein kinase holoenzyme complex”; “cytosolic part” stands for “cytoplasmic part”; “anchoring junction” stands for “anchoring junction”; “myelinsheath” stands for “myelin sheath”;

[0043] “purine ribonucleoside triphosphate metabolic process” stands for “purine ribonucleoside triphosphate metabolic process”; “supramolecular fiber organization” stands for “supramolecular fiber organization”; “homotypic cell-cell adhesion” stands for “homotypic cell-cell adhesion”; “regulation of multi-organism process” stands for “regulation of multi-organism process”; “response to tumor cell” stands for “response to drug” stands for “response to drug”; “cytolysis” stands for “cytolysis”; “platelet activation” stands for “platelet activation”; “BALF” stands for “bronchoalveolar lavage fluid”; “reninsecretion” stands for “renin secretion”; “melanogenesis” stands for “melanogenesis”; “aldosteronesynthesis and secretion” stands for “aldosterone synthesis and secretion”; “estrogen signaling pathway” stands for “estrogen signaling pathway”;

[0044] “formation of the cornified envelope” stands for “formation of the cornified envelope”; “chagas disease (American trypanosomiasis)” stands for “Chagas disease (American trypanosomiasis)”; “toll-like receptor signaling pathway” stands for “toll-like receptor signaling pathway”; “legionellosis” stands for “legionellosis”; “rheumatoid arthritis” stands for “rheumatoid arthritis”; “taxis” stands for “tropism”; “chemotaxis” stands for “chemotaxis”; “arteriosclerosis” stands for “atherosclerosis”; “pre-eclampsia” stands for “preeclampsia”;

[0045] “diabetic nephropathy” stands for “diabetic nephropathy”; “sepsis” stands for “sepsis”; “idiopathic pulmonary fibrosis” stands for “idiopathic pulmonary fibrosis”; “serine-type endopeptidase inhibitor activity” stands for “serine-type endopeptidase inhibitor activity”; “sphingolipid de novo biosyhthesis” stands for “sphingolipid synthesis from scratch”; “graft-versus-host disease” stands for “graft-versus-host disease”; “tuberculosis” stands for “tuberculosis”; “phagosome” stands for “phagosome”; and “apoptosis” stands for “cell apoptosis”.

[0046] “Leukocyte transendothelial migration” stands for “leukocyte transendothelial migration”; “bacterial invasion of epithelial cells” stands for “bacterial invasion of epithelial cells”; and “salmonella infection” stands for “Salmonella infection”. DETAILED DESCRIPTION

[0047] The present invention relates to a method for treating cancer, which comprises administering a bifunctional protein targeting PD-1 and TGFβ and radiotherapy to an individual.

[0048] the term

[0049] In order to make the present invention easier to understand, some technical terms are specifically defined below. Unless otherwise explicitly stated elsewhere in this article, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0050] "Antibody" as described herein refers to any form of antibody that can achieve the desired biological activity or binding activity. Therefore, it is used in the broadest sense, but is not limited to monoclonal antibodies, polyclonal antibodies, bi(multi)specific antibodies, humanized full-length human antibodies, chimeric antibodies, and single-domain antibodies from camels. "Antibody" specifically binds to antigens and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, and the light chain constant region comprises a constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conservative regions called framework regions (FRs). Generally speaking, from the N-terminus to the C-terminus, both the light chain and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids are usually assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991): Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia et al., (1987) J Mol. Biol. 196: 901-917 or Chothia et al., (1989) Nature 341: 878-883. Unless otherwise expressly indicated, "CDR" as used herein refers to the complementarity determining region of an immunoglobulin variable region defined using the Kabat numbering system.

[0051] The carboxyl terminal portion of the heavy chain can define the constant region primarily responsible for effector function. Typically, human light chains are divided into kappa chains and lambda chains. Human heavy chains are generally divided into μ, δ, γ, α, or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG subclasses are well known to those skilled in the art and include, but are not limited to, IgG1, IgG2, IgG, and IgG4.

[0052] Herein, antibodies include: naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0053] Herein, "antibody fragment" or "antigen binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability of a full-length antibody to specifically bind to an antigen, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; nanobodies, and bispecific (multi-) antibodies formed by antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the antigen-binding fragment typically retains at least 10% of the antigen-binding activity of the parent antibody. Preferably, the antigen-binding fragment 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 an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies).

[0054] As used herein, "chimeric antibodies" refer to antibodies and fragments thereof in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a specific species (such as humans) or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species (such as mice) or belonging to another antibody class or subclass, as long as it exhibits the desired biological activity.

[0055] As used herein, "human antibodies" refer to antibodies that contain only human immunoglobulin sequences. If the human antibody is produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, it may contain murine carbohydrate chains. Similarly, "mouse antibodies" or "rat antibodies" refer to antibodies that contain only mouse or rat immunoglobulin sequences, respectively.

[0056] Herein, "humanized antibody" refers to an antibody form containing sequences from non-human (such as murine) antibodies and human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Typically, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulins. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc) (typically a human immunoglobulin constant region).

[0057] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring the antibody to be produced by any particular method.

[0058] Herein, "programmed death receptor-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" used herein includes human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1.

[0059] Herein, "therapeutic anti-PD-1 monoclonal antibody" refers to an antibody that specifically binds to a mature form of a specific PD-1 expressed on the cell surface of certain mammals (especially humans). Mature PD-1 has no pre-secretory leader sequence, or leader peptide. The terms "PD-1" and "mature PD-1" are used interchangeably herein and should be understood to refer to the same molecule unless otherwise explicitly defined or clearly apparent from the context.

[0060] Herein, "transforming growth factor-β (TGF-β)" belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-β transmits signals through a heterotetrameric receptor complex, which is composed of two type I and two type II transmembrane serine / threonine kinase receptors. These type I and type II receptors are transmembrane proteins that consist of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signal transduction, and type II receptors are essential for binding ligands and expression of type I receptors. Type I and type II receptors form a stable complex after ligand binding, causing type II receptors to phosphorylate type I receptors. TGFβ has three mammalian isoforms, TGFβ1, TGFβ2, and TGFβ3, each of which has different functions in vivo. The binding of TGFβ to TGFβRII is a key step in initiating the activation of the TGFβ signaling pathway, causing SMAD2 phosphorylation and the translocation of the activated SMAD2 / SMAD4 complex to the nucleus to regulate gene expression.

[0061] As used herein, "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which results in the selective damage, destruction, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0062] As used herein, the term "cancer" or "malignancy" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth division, and growth lead to the formation of malignant tumors, which invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. Examples of cancers suitable for treatment or prevention using the methods, medicaments, and kits of the present invention include, but are not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas. More specific examples of cancer include squamous cell carcinoma, myeloma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, nasopharyngeal cancer, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0063] As used herein, the term "tumor mutation burden (TMB)" refers to the total number of somatic gene coding errors, base substitutions, gene insertions or deletions detected per million bases. In some embodiments of the present invention, the tumor mutation burden (TMB) is estimated by analyzing somatic mutations (including coding base substitutions and megabase insertions of the panel sequences studied).

[0064] In this article, the term "thoracic tumor" refers to the classification of thoracic tumors according to the classification standards published by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO Classification of Tumours, 5th Edition, Volume 5), including lung tumors, pleural and pericardial tumors, cardiac tumors, thoracic mesenchymal tumors, thymic tumors, germ cell tumors of the mediastinum, haematolymphoid tumors of the mediastinum, ectopic tumors of thyroid and parathyroid origin, and metastasis of tumors to the above sites.

[0065] Specifically, herein, lung tumors include, but are not limited to, bronchial papilloma, sclerosing pneumocytoma, alveolar adenoma, papillary adenoma, bronchiolar adenoma / ciliary mucosal tumor, mucinous cystadenoma, mucinous gland adenoma, precursor gland lesion, atypical adenomatous hyperplasia, adenocarcinoma in situ, microinvasive adenocarcinoma, invasive non-mucinous adenocarcinoma, invasive mucinous adenocarcinoma, colloid adenocarcinoma, undifferentiated adenocarcinoma, intestinal adenocarcinoma, squamous dysplasia and carcinoma in situ, squamous cell carcinoma, lymphoepithelial carcinoma, large cell carcinoma, adenosquamous carcinoma, pleomorphic carcinoma, pulmonary blastoma, sarcoma, NUT lung cancer, SMARCA-deficient undifferentiated tumor of the chest, pleomorphic adenoma, adenoid cystic carcinoma, epithelial myoepithelial carcinoma, mucoepidermoid carcinoma, salivary gland hyalinization Clear cell carcinoma, myoepithelioma and myoepithelioma, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia, carcinoid / neuroendocrine tumor, small cell lung cancer, large cell neuroendocrine carcinoma, melanoma, meningioma, pulmonary hamartoma, pulmonary chondroma, diffuse pulmonary lymphangiomatosis, pleural pulmonary blastoma, pulmonary artery intimal sarcoma, congenital peribronchial myofibroblastic tumor, primary pulmonary myxoid sarcoma with EWSR-CREB fusion, lymphangioleiomyomatosis, PEComa, mucosa-associated lymphoid tissue lymphoma, diffuse large B-cell lymphoma, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, pulmonary Langerhans cell histiocytosis, Erdheim–Chester disease.

[0066] Herein, pleural and pericardial tumors include, but are not limited to, adenomatous tumors located in the pleura and pericardium, well-differentiated papillary mesothelioma, mesothelioma in situ, localized mesothelioma, diffuse mesothelioma, primary effusion lymphoma, and chronic inflammation-associated diffuse large B-cell lymphoma.

[0067] As used herein, cardiac tumors include, but are not limited to, papillary fibroelastoma located in the heart, cardiac myxoma, cardiac fibroma, cardiac rhabdomyoma, adult cell rhabdomyoma, atrial septal cardiac lipoma and lipomatous hypertrophy, atrioventricular valve fatty hamartoma, mature cardiomyocyte hamartoma, mesenchymal cardiac hamartoma, cardiac angioma, conduction system hamartoma, atrioventricular node cystic tumor, cardiac angiosarcoma, cardiac leiomyosarcoma, cardiac undifferentiated pleomorphic sarcoma, other sarcomas that may involve the heart, cardiac diffuse large B-cell lymphoma, and cardiac fibrin-associated diffuse large B-cell lymphoma.

[0068] As used herein, the term "thoracic mesenchymal tumor" includes, but is not limited to, lipoma, thymolipoma, liposarcoma, ligamentous tumor, solitary fibrous tumor, calcifying fibroma, inflammatory myofibroblastic tumor, myxofibrosarcoma, hemangioma, lymphangioma, epithelioid hemangioendothelioma, angiosarcoma, rhabdomyosarcoma, paraganglioma, granular cell tumor, neurilemoma, malignant peripheral nerve sheath tumor, peripheral neuroblastic tumor, synovial sarcoma, and undifferentiated small round cell sarcoma located in the thoracic mesenchyme.

[0069] As used herein, thymic tumors include, but are not limited to, type A thymoma (including atypical subtypes), type AB thymoma, type B1 thymoma, type B2 thymoma, type B3 thymoma, micronodular thymoma with lymphoid stroma, metaplastic thymoma, lipofibroadenoma, squamous cell carcinoma, basaloid carcinoma, lymphoepithelial carcinoma, thoracic nut carcinoma, clear cell carcinoma, low-grade adenocarcinoma, mucoepidermoid carcinoma, thymic carcinoma with adenoid cystic carcinoma-like features, intestinal adenocarcinoma, adenocarcinoma NOS, adenosquamous carcinoma, sarcomatoid carcinoma, undifferentiated carcinoma, thymic carcinoma NOS, neuroendocrine tumors, carcinoid / neuroendocrine tumors, small cell carcinoma, and large cell neuroendocrine carcinoma.

[0070] Herein, mediastinal germ cell tumors include but are not limited to seminoma located in the mediastinum, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, mixed germ cell tumor, germ cell tumor with somatic solid malignancies, and germ cell tumor with related hematological malignancies.

[0071] Herein, mediastinal hematolymphoid tumors include, but are not limited to, primary mediastinal large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, T-lymphocytic leukemia / lymphoma, classical Hodgkin's lymphoma, mediastinal gray zone lymphoma, and follicular dendritic cell sarcoma located in the mediastinum.

[0072] Herein, ectopic tumors derived from the thyroid gland and parathyroid gland include but are not limited to ectopic tumors derived from the thyroid gland and parathyroid gland.

[0073] As used herein, the term "immunotherapy" refers to treating a subject who has a disease or is at risk of infection or recurrence of a disease by methods that include inducing, enhancing, suppressing or otherwise modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, slowing down or preventing the onset, progression, severity or recurrence of symptoms, complications or conditions, or biochemical indicators associated with a disease.

[0074] As used herein, "administering," "giving," 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 skilled in the art. Routes of administration of the antibody may include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to administration other than enteral or topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion, as well as electroporation in vivo.

[0075] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability caused by disease affliction. The ability of a therapeutic agent to promote regression of disease can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays.

[0076] Herein, a therapeutically effective amount of a drug includes a "prophylactically effective amount," ie, any amount of a drug that inhibits the development or recurrence of cancer when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer or a subject having cancer recurrence.

[0077] As used herein, "patient," "patient," or "subject" refers to any single subject, typically a mammal, including humans and other mammals such as horses, cows, dogs, or cats, who is in need of medical treatment or is participating in a clinical trial, epidemiological study, or used as a control.

[0078] The "RECIST 1.1 efficacy criteria" described herein refer to the definitions described in Eisenhauver et al., EA et al., Eur. J Cancer 45:228-247 (2009) for target damage or non-target damage based on the background of the measured response. Before immunotherapy, it was the most commonly used standard for evaluating the efficacy of solid tumors. However, with the advent of the immune era, many problems that have never appeared in tumor evaluation have emerged. Therefore, based on the emerging phenomenon caused by immunotherapy itself, in 2016, the RECIST working group revised the existing "RECIST v.1.1" and proposed a new judgment standard, namely the "irRECIST standard" described herein, which aims to better evaluate the efficacy of immunotherapy drugs.

[0079] The term "ECOG" scoring standard is an indicator of the patient's general health status and tolerance to treatment based on his or her physical strength. The ECOG physical status scoring standard is scored as follows: 0, 1, 2, 3, 4, and 5. A score of 0 means that the ability to move is completely normal, with no difference from the ability to move before the onset of the disease. A score of 1 means that the patient can move freely and engage in light physical activities, including general housework or office work, but cannot engage in heavier physical activities.

[0080] "Sustained response" refers to a sustained therapeutic effect after cessation of treatment with a therapeutic agent or combination therapy described herein. In some embodiments, the sustained response has a duration at least the same as the duration of treatment or at least 1.5, 2.0, 2.5, or 3 times the duration of treatment.

[0081] "Tissue section" refers to a single portion or piece of a tissue sample, such as a thin slice of tissue cut from a sample of normal tissue or a tumor.

[0082] "Treatment" of cancer as described herein refers to the use of the treatment regimen described herein (such as the administration of anti-PD-1 antibodies) to subjects suffering from cancer or diagnosed with cancer 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 to peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). Positive therapeutic effects in cancer can be measured in a variety of ways (see WA Weber, J. Nucl. Med., 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI standard, T / C≦42% is the minimum level of anti-tumor activity. It is considered that T / C (%) = median treated tumor volume / median control tumor volume × 100. In some embodiments, the therapeutic effect achieved by the combination of the present invention is any one of PR, CR, OR, PFS, DFS and OS. PFS (also called "time to tumor progression") refers to the length of time that cancer does not grow during and after treatment, and includes the amount of time the patient experiences CR or PR and the amount of time the patient experiences SD. DFS refers to the length of time during and after treatment that the patient is still disease-free. OS refers to the extension of life expectancy compared with the initial or untreated individual or patient. In some embodiments, the response to the combination of the present invention is any one of PR, CR, PFS, DFS, OR or OS, which is assessed using RECIST 1.1 efficacy criteria. The treatment regimen of the combination of the present invention for effectively treating cancer patients can be changed according to multiple factors (such as the ability of the patient's disease state, age, body weight and therapy to stimulate the anti-cancer response of the subject). Although embodiments of the present invention may not achieve effective positive therapeutic effects in each subject, it should be effective and achieve positive therapeutic effects in statistically significant numbers of subjects.

[0083] The terms "administration method" and "administration regimen" are used interchangeably to refer to the dosage and timing of each therapeutic agent in the combination of the present invention.

[0084] The term "immunohistochemistry (IHC)" refers to a method of determining antigens (peptides and proteins) in tissue cells by using the principle of specific binding of antigens to antibodies, and conducting localization, qualitative and relative quantitative research on the colorants (fluorescein, enzymes, metal ions, isotopes) labeled with antibodies through chemical reactions. In some embodiments of the present invention, before treatment with anti-PD-1 antibodies, PD-L1 is detected in tumor tissue samples of the subject, and the staining experiment is performed using Roche's anti-human PD-L1 antibody SP142 (Cat No: M4422). In some embodiments, a membrane staining intensity of tumor cells ≥ 1% is defined as PD-L1 positive.

[0085] An "adverse reaction" (AE) as described herein is any unfavorable and generally unintended or undesirable sign, symptom or disease associated with the use of a medical treatment. For example, an adverse reaction may be associated with activation of the immune system or expansion of immune system cells in response to a treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity.

[0086] As used herein, "tumor burden" refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of a tumor throughout the body. Tumor burden can be determined by a variety of methods known in the art, such as measuring the size of a tumor using calipers after it has been removed from a subject, or using imaging techniques such as ultrasound, bone scans, computed tomography (CT), or magnetic resonance imaging (MRI) scans while in vivo.

[0087] As used herein, the term "tumor size" refers to the overall 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, such as measuring the size of a tumor using calipers after it has been removed from a subject, or using imaging techniques such as bone scans, ultrasound, CT or MRI scans while in vivo.

[0088] Bifunctional protein targeting PD-1 and TGFβ

[0089] In this article, the bifunctional proteins targeting PD-1 and TGFβ include:

[0090] (i) antibodies or antigen-binding fragments thereof that block the PD-1 / PD-L1 pathway;

[0091] (ii) an immunoglobulin Fc domain; and

[0092] (iii) TGFβRⅡ extracellular domain (ECD).

[0093] In some embodiments, the antibody that blocks the PD-1 / PD-L1 pathway is an anti-PD-1 antibody.

[0094] In some embodiments, the bifunctional protein of the present invention is effectively connected from N-terminus to C-terminus in the order of (i), (ii) and (iii); (iii), (i) and (ii); or (iii), (ii) and (i); preferably, (i) and (ii) are connected by a hinge region, (i) and (iii) as well as (ii) and (iii) are connected by a connecting peptide, the amino acid sequence of the connecting peptide contains glycine residues and serine residues or consists of glycine residues and serine residues, and the length is 2-31 amino acid residues; preferably, each is independently (GGGGS)nG, wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.

[0095] In some embodiments, the bifunctional protein of the present invention is effectively connected from the N-terminus to the C-terminus in the order of (i), (ii) and (iii). Preferably, (i) and (ii) are connected by a hinge region, and (ii) and (iii) are connected by a connecting peptide. The amino acid sequence of the connecting peptide contains or consists of glycine residues and serine residues, and the length is 2-31 amino acid residues; preferably, each is independently (GGGGS)nG, wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.

[0096] In some embodiments, the bifunctional protein of the present invention comprises an anti-PD-1 antibody and a TGFβRⅡ extracellular domain (ECD) operably linked to the C-terminus of each of the two heavy chains of the antibody.

[0097] In some embodiments, the bifunctional protein of the present invention is a heterotetrameric glycoprotein composed of two bifunctional protein first subunits and two bifunctional protein second subunits bonded by disulfide bonds.

[0098] The present invention uses a monoclonal antibody targeting PD-1 as the molecular part of the bifunctional protein that blocks the PD-1 / PD-L1 pathway, which is used to block negative regulatory signals, restore T cell activity, and thus enhance immune response; and uses the TGFβRⅡ extracellular domain as the immunomodulatory molecular part of the bifunctional protein to weaken the immune tolerance of cancer cells. Inhibiting the PD-1 / PD-L1 pathway on the basis of targeting and neutralizing TGFβ in the tumor microenvironment can restore T cell activity, enhance immune response, and more effectively improve the inhibition of tumor occurrence and development.

[0099] In some embodiments, the bifunctional protein of the invention is 10 -8 M or less, for example, 10 -9 M to 10 -12 The dissociation constant (KD) of 10 -8 M or less, for example, 10 -9 M to 10 -12 The dissociation constant (KD) of M specifically binds to TGFβ1 / TGFβ2 / TGFβ3 molecules respectively.

[0100] In some embodiments, the amino acid sequence of the TGFβRⅡ extracellular domain is shown in SEQ ID NO:11.

[0101] In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-PD-1 antibody are shown in SEQ ID NOs: 1 to 6, respectively. In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-PD-1 antibody are shown in SEQ ID NOs: 7 and 8, respectively. In some embodiments, the amino acid sequences of the heavy chain and light chain of the anti-PD-1 antibody are shown in 9 and 10, respectively. In some embodiments, the TGFβRⅡ extracellular domain is connected by a peptide (GGGGS) n G is connected to the C-terminus of the anti-PD-1 antibody heavy chain; wherein n is independently selected from 3, 4, 5 or 6, preferably 4 or 5.

[0102] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ described herein includes any one of the bifunctional proteins targeting PD-1 and TGFβ described in application number CN202011038252.5, and all the contents disclosed therein are incorporated herein by introduction.

[0103] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ used in the methods and compositions of the present invention is the bifunctional protein targeting PD-1 and TGFβ described in CN 202011038252.5, whose heavy chain amino acid sequence is as shown in SEQ ID NO:9, and the TGFβRⅡ extracellular domain shown in SEQ ID NO:11 is connected to the C-terminus of SEQ ID NO:9 through the GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO:13) peptide segment to form the first subunit of the bifunctional protein (whose amino acid sequence is shown in SEQ ID NO:12), and the light chain amino acid sequence is as shown in SEQ ID NO:10, forming the second subunit of the bifunctional protein.

[0104] The specific sequences of SEQ ID NO:1 to SEQ ID NO:12 are as follows:

[0105] SEQ ID NO: 1 (HCDR1): DYEMH.

[0106] SEQ ID NO: 2 (HCDR2): VIESETGGTAYNQKFKG.

[0107] SEQ ID NO: 3 (HCDR3): EGITTVATTYYWYFDV.

[0108] SEQ ID NO: 4 (LCDR1): RSSQSIVHSNGNTYLE.

[0109] SEQ ID NO: 5 (LCDR2): KVSNRFS.

[0110] SEQ ID NO: 6 (LCDR3): FQGSHVPLT.

[0111] SEQ ID NO:7 (PD-1 antibody heavy chain variable region):

[0112] QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVI ESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATT YYWYFDVWGQGTTVTVSS.

[0113] SEQ ID NO: 8 (PD-1 antibody light chain variable region):

[0114] DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLE IK.

[0115] SEQ ID NO:9 (heavy chain of PD-1 antibody):

[0116] QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL。

[0117] SEQ ID NO:10 (Light chain of PD-1 antibody or the second subunit of bifunctional protein JS-TZO3):

[0118] DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0119] SEQ ID NO:11 (Extracellular domain (ECD) of TGF-βRⅡ, TGFRECD):

[0120] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE TFFMCSCSSDECNDNIIFSEEYNTSNPD。

[0121] SEQ ID NO: 12 (the first subunit of the bifunctional protein JS-TZO3: heavy chain of PD-1 antibody + linker + TGFRECD):

[0122] .

[0123] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ used in the methods and compositions of the present invention is selected from a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a humanized antibody or an antigen-binding fragment thereof, preferably a humanized antibody or an antigen-binding fragment thereof.

[0124] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ used in the methods and compositions of the present invention is a humanized antibody or a chimeric antibody and may include a human constant region. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions; preferably, the bifunctional protein targeting PD-1 and TGFβ suitable for the methods and compositions described in the present invention comprises a heavy chain constant region of human IgG1 or IgG4 isotype, more preferably a human IgG4 constant region. In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the bifunctional protein targeting PD-1 and TGFβ provided herein to produce an Fc region variant, such as introducing an S228P mutation into the sequence of the IgG4 heavy chain constant region of the bifunctional protein targeting PD-1 and TGFβ, which replaces the serine residue in the hinge region with a proline residue that is usually present at the corresponding position of an IgG1 isotype antibody.

[0125] Medical uses and methods

[0126] Provided herein are bifunctional proteins targeting PD-1 and TGFβ for use in treating, preventing and / or diagnosing diseases such as cancer associated with PD-1 activity and / or TGFβ activity in a subject, and methods for treating, preventing and / or diagnosing diseases such as cancer associated with PD-1 activity and / or TGFβ activity in a subject. The bifunctional protein can be used as an independent therapy or in combination with other therapies (e.g., radiotherapy) for the treatment of cancer.

[0127] Preferably, the cancer is as described above. Preferably, the cancer is a breast tumor. In some embodiments, the cancer is lung cancer, breast cancer or colon cancer.

[0128] Preferably, the subject described herein has previously received radiotherapy. Preferably, the subject has other diseases or conditions caused by radiotherapy, such as diseases or conditions of the lung, more specifically radiation lung injury, including radiation pneumonitis (RP) or radiation pulmonary fibrosis (RIPF).

[0129] In some embodiments, the subject has only a primary tumor, or both a primary tumor and a secondary tumor.

[0130] The treatment and prevention described in this article can achieve one or more of the following effects: inhibit tumor growth and prolong patient survival; increase the number of tumor-infiltrating cytotoxic lymphocytes in the tumor; inhibit tumor angiogenesis; reduce fibrosis in the TME, especially fibrosis caused by radiotherapy; reduce inflammatory infiltration and tissue damage.

[0131] In some embodiments, the treatment and prevention described herein can achieve one or more of the following effects: downregulating the expression of molecules related to ECM, cell adhesion and cell migration; reducing EGFR signaling in tumor tissues; weakening immunosuppressive features such as ECM formation, tumor migration and angiogenesis, compensating for the negative impact of RT-induced increased ECM expression on TME; enhancing TNF signaling and enhancing inflammatory responses within tumors; inhibiting inflammatory mediator regulation of calcium signaling pathways and TRP channels; reducing calcium ion binding and calmodulin binding; reducing CAMK2A expression; inhibiting the TGF-β1 / Smad2 pathway, reducing the proportion of fibroblasts and thus alleviating pulmonary fibrosis; inhibiting the expression of inflammasomes in neutrophils.

[0132] In some embodiments, the treatment and prevention described herein can achieve one or more of the following effects: reducing myeloid-derived suppressor cells (MDSC); increasing the CD8+T / CD4+T cell ratio; reducing natural killer cell-mediated cytotoxicity; alleviating neutrophil-mediated chronic inflammatory lung injury; reducing the proportion of neutrophils and the formation of ROS-induced neutrophil extracellular traps, thereby reducing profibrotic effects.

[0133] In some embodiments, the treatment and prevention described herein can achieve one or more of the following effects: no significant damage to the heart, liver, and kidneys; no effect on liver and kidney function; and reduced acute and chronic pulmonary toxicity induced by RT. Therefore, in some embodiments, the present invention provides the use of the bifunctional protein targeting PD-1 and TGFβ described herein in the preparation of a drug for achieving any one or more of the above effects in an individual. Preferably, the application is combined with radiotherapy; in some embodiments, the individual has previously received radiotherapy and / or is about to receive radiotherapy. In some embodiments, the present invention provides a method for achieving any one or more of the above effects, the method comprising administering to a subject in need, such as a cancer patient described herein, a therapeutically or preventively effective amount of a bifunctional protein targeting PD-1 and TGFβ as described in any embodiment of the present invention; preferably, the method also includes administering radiotherapy to the subject; in some embodiments, the individual has previously received radiotherapy and / or is about to receive radiotherapy. In some embodiments, the individual is an individual as described in any embodiment herein, such as an individual suffering from only a primary tumor or a primary tumor and a secondary tumor at the same time, and preferably, the tumor is associated with PD-1 activity and / or TGFβ activity, more preferably a chest tumor.

[0134] In this article, radiotherapy has the well-known meaning of this article, which refers to a local treatment method for treating tumors using radiation. Radiation includes α, β, and γ rays produced by radioisotopes and x-rays, electron beams, proton beams, and other particle beams produced by various x-ray therapy machines or accelerators. Radiotherapy can be three-dimensional radiotherapy or four-dimensional radiotherapy. Radiotherapy dose distribution can be point dose, volume dose distribution, and dose intensity modulation in volume dose distribution. Radiotherapy techniques can be stereotactic radiotherapy (SRT), whole chest irradiation (WTI), and stereotactic radiosurgery (SRS). Stereotactic radiotherapy (SRT) includes three-dimensional conformal radiotherapy (3DCRT) and three-dimensional conformal intensity modulated radiotherapy (IMRT); stereotactic radiosurgery (SRS) includes X-knife, gamma knife (Y knife), and Cyber ​​Knife. The dose and regimen of radiotherapy can be determined by skilled technicians according to different patients, diseases, etc.

[0135] In some embodiments, the bifunctional protein targeting PD-1 and TGFβ described herein is used in combination with radiotherapy to treat or prevent diseases associated with PD-1 activity and / or TGFβ activity, such as cancer, especially lung cancer, breast cancer or colon cancer.

[0136] In some embodiments, the present invention provides the use of the bifunctional protein targeting PD-1 and TGFβ described herein in the preparation of a drug for treating or preventing radiation lung injury in cancer patients receiving radiotherapy. In some embodiments, the radiation lung injury includes radiation pneumonitis (RP) and radiation pulmonary fibrosis (RIPF). In some embodiments, the subject suffers only from a primary tumor, or suffers from a primary tumor and a secondary tumor at the same time. The patient may be a patient who has previously received radiotherapy and has the radiation lung injury, or may be a patient who is about to receive radiotherapy to prevent radiation lung injury.

[0137] In some embodiments, provided herein is a method for treating or preventing radiation lung injury in patients receiving radiotherapy, the method comprising administering to the patient a step of treating or preventing an effective amount of a bifunctional protein targeting PD-1 and TGFβ or a pharmaceutical composition thereof as described in any embodiment herein. In some embodiments, the radiation lung injury includes radiation pneumonitis (RP) and radiation pulmonary fibrosis (RIPF). In some embodiments, the subject suffers only from a primary tumor, or suffers from a primary tumor and a secondary tumor at the same time. The patient may be a patient who has previously received radiotherapy and has the radiation lung injury, or may be a patient who is about to receive radiotherapy to prevent radiation lung injury.

[0138] Pharmaceutical composition

[0139] In the methods and uses described herein, the bifunctional protein of PD-1 and TGFβ can be used in the form of a pharmaceutical composition. The pharmaceutical composition may contain a bifunctional protein as described herein and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition described herein is particularly suitable for use in combination with radiotherapy for the treatment or prevention of diseases associated with PD-1 activity and / or TGFβ activity as described herein, such as cancer.

[0140] Pharmaceutical compositions comprising the bifunctional proteins described herein can be prepared by mixing the bifunctional protein having the desired degree of purity with one or more pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)).

[0141] The pharmaceutical compositions described herein may also include one or more other active ingredients, which are required for the specific indications being treated, preferably those active ingredients with complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, and the active ingredients are appropriately combined in an amount effective for the intended use.

[0142] Example

[0143] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. It will be obvious to those skilled in the art that various changes and improvements will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. should be included in the protection scope of the present invention. The methods and materials used in the examples are conventional methods and materials in the art unless otherwise stated.

[0144] Methods and Materials

[0145] Experimental animals

[0146] Adult female C57BL / 6 mice and BALB / c mice were obtained from Hubei Provincial Experimental Animal Center and Hubei Beient Biotechnology Co., Ltd. (Hubei, China). All mice were 7–8 weeks old and weighed 18–20 g. Five mice were housed per cage under standard laboratory conditions, with an unlimited supply of sterile food and water. All animal experiments were performed under specific pathogen-free grade conditions in accordance with the guidelines of the International Guiding Principles for Animal Research and approved by the Ethics Committee of Huazhong University of Science and Technology (Hubei, China). The IACUC number is 3224.

[0147] Material

[0148] Reagents

[0149] The murine bispecific antibody JS-201 and the murine anti-PD-1 antibody JS-001 were provided by Shanghai Junshi Biopharmaceuticals Co., Ltd. (Shanghai, China). Both antibodies were diluted to 1 mg / ml with 0.9% sterile saline and used in in vivo experiments.

[0150] Cell lines and culture media

[0151] The cell lines used in this study, including LLC cells (lung cancer cells), 4T1 cells (breast cancer cells), and CT26.WT cells (colon cancer cells), were obtained from the Laboratory of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. LLC luciferase-positive (LLC-luc) cell lines were established by stably transducing lentivirus containing luciferase GV260 into the parent LLC cell line and were obtained from Shanghai Gene Gene Co., Ltd. (Shanghai, China). LLC and LLC-Luc cells were cultured in DMEM medium (HyClone, Utah, USA), and CT26.WT and 4T1 cells were cultured in RPMI-1640 medium (HyClone). When culturing cells, 10% (w / v) fetal bovine serum (Gibco, New York, USA) and 1% (w / v) penicillin-streptomycin (Gibco) were added to the culture medium, and the cells were incubated in a clean environment at 37°C and 5% carbon dioxide.

[0152] Establishment of animal model and treatment of animals

[0153] Tumor-bearing animal models

[0154] For the establishment of primary single-focal tumor models, 50 μl LLC cells were plated at 1×10 6 The cells were inoculated subcutaneously in the right hind flank of C57BL / 6 mice at the same dose, location and method as CT26 and 4T1 cells inoculated into BALB / c mice. Nine mice were set up in each group for LLC and CT26 tumor treatment experiments, and seven mice were set up in each group for 4T1 tumor treatment experiments.

[0155] In the double tumor lesion experiment to explore the abscopal effect, 1×10 6 LLC cells were inoculated subcutaneously on the right anterior flank of C57BL / 6 mice as primary lesions. 5 LLC-luc cells were inoculated subcutaneously in the right posterior flank as secondary lesions (n=10).

[0156] The tumor-bearing mice were randomly divided into the following five groups, with an equal number of mice in each group: 0.9% saline (NS) group, anti-PD-1 group, radiotherapy (RT) group, JS201 group, and JS201 and radiotherapy combined group (JS201+RT). 7-10 days after modeling, when the primary tumor volume reached 100mm 3 Treatment was started at 1:00 pm. 10 mg / kg of the drug was injected intraperitoneally at D0, D3, D6 and D9 of the treatment. For the RT and JS201+RT groups, 8 Gy of radiotherapy was given only to the primary site at D4, and a 0.5 cm compensation membrane was given. Radiotherapy was performed using an RS2000 X-ray Biological Research irradiator (160 kV, 25 mA; Rad Source Technologies, Inc., Suwanee, GA, USA). Tumor volume was measured every three days. When the tumor volume of the mouse exceeded about 2000 mm 3 When the tumor volume reached 1.37 × 1.34, the mice were euthanized. The formula for calculating tumor volume is volume = length × width 2 / 2.

[0157] RILF Model

[0158] Adult female C57BL / 6 mice were anesthetized, fixed, and covered with a 1 cm thick compensation film. After each group received a whole chest irradiation of 16 Gy / 1F, different treatments were given regularly according to the group. The mice were randomly divided into 4 groups: blank control group, 0.9% saline group, anti-PD-1 group, and JS201 group. The mice in the blank control group were not irradiated or treated.

[0159] In the acute / subacute RILF test, 10 mg / kg of drug was administered by intraperitoneal injection at D0, D3, D6, and D9 after radiotherapy. Another group of acute RILI test mice (n=6) for RNA sequencing were euthanized 1 month after radiotherapy. The modeling and treatment procedures of each group were consistent.

[0160] In the chronic RILI trial, after the acute / subacute phase of administration, treatment was performed every two weeks from week 13 to week 26. The body weight and survival rate of mice (n=15) were recorded weekly, and the observation endpoint was set at week 26, 6 months after radiotherapy. In view of the long observation period and high mortality rate, a batch of RIPF model mice (n=9) was subsequently added for tissue collection. The modeling and treatment procedures of each group were consistent.

[0161] In vivo imaging

[0162] Tumor-bearing mice were intraperitoneally injected with 10 μl / g body weight of D-luciferin potassium (MB1834, Meilun Biotechnology, Shanghai, China) at a concentration of 15 mg / ml, and then anesthetized with 1.5% pentobarbital. The fluorescent real-time imaging of mice was detected and analyzed by IVIS Spectrum Imaging System (Caliper Life Sciences, Massachusetts, USA).

[0163] Micro-computed tomography

[0164] In RILF and IPF experiments, mice were anesthetized with 1.5% pentobarbital and fixed in an appropriate posture. Chest computed tomography images were observed and reconstructed by a micro-CT scanner (SkyScan, 1176, Brooke, MA, USA) to evaluate lung inflammation and fibrosis.

[0165] Cytokine assay

[0166] The expression of TGF-β1 and MPO-DNA complex in mouse serum and bronchoalveolar lavage fluid was quantified by ELISA to reflect the content of NETs. MPO-DNA complex (MM-1177M2, ELISA, Jiangsu, China) and TGF-β1 (EK981, Lianke Biotechnology, Hangzhou, China) ELISA kits were used for experimental detection according to the manufacturer's instructions.

[0167] Flow cytometry

[0168] Primary tumors, secondary tumors, and spleens were removed en bloc from euthanized mice, and the distribution of immune cells in the tumor microenvironment was evaluated by flow cytometry (n=3). The obtained tissues were minced and digested with type IV collagenase, hyaluronidase, and DNase at 37°C for 1 hour. After rinsing and filtering, the tissues were added to the red blood cell lysate, centrifuged (1200 rpm, 5 minutes), and resuspended in PBS to obtain a cell suspension. Afterwards, the BV510 Zombie Aqua TMThe cell suspension was stained using the Fixable Viability Kit (423101, BioLegend, CA, USA), blocked with anti-mouse CD16 / 32 (101320, BioLegend), and light-stained with the following fluorescent antibodies: APC / CY7 anti-mouse CD45 (103115, BioLegend), PE anti-mouse CD3 (100205, BioLegend), BV421 anti-mouse CD4 (116023, BioLegend), PerCP / CY5.5 anti-mouse CD8a (100733, BioLegend), FITC anti-mouse CD11b (101205, BioLegend), and PE / CY7 anti-mouse Ly-6G / Ly-6C (108416, BioLegend) antibodies. Flow cytometry was performed using a CytoFLEX LX flow cytometer (Beckman, CA, USA) and analyzed using FlowJo v10.8.1 software.

[0169] Determination of Hydroxyproline (HYP) Content

[0170] The HYP content in lung tissue was detected to reflect the metabolism of collagen in fibrotic lesions. The detection process was strictly carried out according to the instructions of the kit purchased from Nanjing Jiancheng Bioengineering Institute (A030-2-1, Nanjing, China). Three mice were randomly selected from each group, and a part of lung tissue was cut out for detection, and two secondary wells were made for the cut lung tissue.

[0171] Histopathology and Immunohistochemistry (IHC)

[0172] Completely resected LLC tumors from tumor-bearing mouse models and lung tissues from RILF models were fixed in 4% paraformaldehyde for 24-48 hours. The tissues were then dehydrated in ethanol, embedded in paraffin, and cut into 4 μm thick sections. Before staining, paraffin sections were dewaxed in xylene, absolute ethanol, and 75% ethanol, followed by rinsing in distilled water.

[0173] HE (BH0001), Masson (BH0002) and Sirius red (B00039) staining kits were purchased from Boerfu Biotechnology (Hubei, China). Pathological staining was performed using the corresponding kits according to the manufacturer's instructions. Sirius red-stained sections were observed under a polarized light microscope. The Ashcroft scoring criteria were used to evaluate the HE staining of lung tissue in the RILF model to assess the severity of fibrotic lesions in different treatment groups. ImageJ software was used for visual quantitative analysis.

[0174] In IHC experiments, after antigen retrieval, 3% hydrogen peroxide blocking and 3% BSA sealing, the sections were incubated with the following primary antibodies: anti-CAMK2A (1:800, A22611, Ibotek, Hubei, China), anti-CD8 (1:1000, ab217344, Abcam, Cambridge, UK), anti-CD31 (1:2000, ab182981, Abcam) and anti-MPO (1:1000, GB11224, Sevier Bio, Hubei, China). After secondary antibody incubation, the sections were developed with DAB and the nuclei were stained before dehydration and sealing. Images were taken by general microscope.

[0175] Immunofluorescence

[0176] The frozen lung tissues and bronchial perfusate of RILF model mice were sliced ​​at 4 μm thickness. DHE staining was performed using a reactive oxygen species detection kit (S0033S, Bio-Time, Shanghai, China) and DAPI staining solution (B0025, Boerfu Biotech) according to the manufacturer's instructions.

[0177] Lung tissues of the RILF model fixed with 4% paraformaldehyde were embedded in paraffin, sliced ​​at 4 μm, dewaxed and rinsed. Homologous double-labeled IF was performed using anti-histone H3 (citrulline R2+R8+R17, CITH3) antibody (1:2000, ab281584, Abcam) and anti-MPO antibody (1:1000, GB11224, Sewell Bio) as primary antibodies. At the end of the secondary antibody incubation, cell nuclei were stained with DAPI and the sections were sealed after removing tissue autofluorescence. Images were taken with a fluorescence microscope.

[0178] Western blot

[0179] Protein extraction was performed on unfixed lung tissue from RILF model mice. Lung tissue was added to RIPA (G2002, Sevier Bio) and PMSF (G2007, Sevier Bio), crushed, ultrasonicated and centrifuged, and boiled at 100°C with 5× loading buffer (G2013, Sevier Bio). Electrophoresis was performed using a one-step PAGE gel rapid preparation kit (12%) (E304-01, Novozyme, Nanjing, China). Protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA) and blocked with 5% BSA or fat-free milk for 1 hour. The PVDF membrane was incubated with the following primary antibodies at 4 °C overnight: anti-COL3A1 (1:1000, 66887S, CST, MA, USA), anti-COL1A2 (1:1000, A5786, Ibotek), anti-Smad2 (1:1000, ab40855, Abcam), anti-phospho-Smad2 (Ser467) (1:1000, ab280888, Abcam) m), anti-GAPDH (1:10000, A00227-1, Boster, Hubei, China), anti-CAMK2A (1:1000, A22611, Abcam), anti-α-SMA (1:1000, A17910, Abcam), anti-TGF-β1 (1:1000, A2124, Abcam) and anti-CITH3 (1:1000, ab281584, Abcam). After incubation with the appropriate secondary antibodies for 1 hour at room temperature, the blots were developed using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA) and the protein bands were observed. The blot signals were detected using the G:BOX Chemi X system (Syngene, Cambridge, UK).

[0180] RNA-seq assay

[0181] Total RNA was treated with RQ1 DNase (M6101, Promega, Beijing, China) to remove DNA. The absorbance at 260nm / 280nm was measured using SmartSpecPlus to determine the purity and amount of purified RNA. The integrity of the RNA was further verified by 1.5% agarose gel electrophoresis. 1 μg of total RNA was taken from each sample for RNA-seq library preparation. mRNA was captured with VAHTS mRNA capture beads (N401, Novozyme). Treated with RQ1 DNase to remove DNA, the purified RNA was then prepared by directional RNA-seq library preparation of VAHTSUniversal V8 RNA-seq Library Prep Kit for Illumina (NR605, Novozyme). The polyadenylated mRNA was purified and fragmented, and the fragmented mRNA was converted into double-stranded cDNA. After completing end repair and tailing with A, the DNA was connected to the adapter (N323, Novozyme). After the ligation products were purified and 300-500 bp fragments were isolated, the ligation products were amplified and purified, and after quantitative determination, they were stored at -80° for subsequent sequencing. The dUTP-labeled strands were not amplified to facilitate strand-specific sequencing. Libraries for high-throughput sequencing were prepared according to the manufacturer's instructions and applied to the Illumina NovaSeq 6000 system (Illumina, USA) for 150 nt paired-end sequencing.

[0182] Single-cell RNA-seq assay

[0183] The 10x Genomics Cell Preparation Guide describes best practices and general protocols for washing, counting, and concentrating cells from cell suspensions (>100,000 total cells) for preparation of 10x Genomics single cell protocols. The cell suspension was loaded onto a Chromium microfluidic chip with 3' chemistry and encoded using the 10x Chromium Controller (10xGenomics, DE, USA). RNA encoding the cells was then reverse transcribed and sequencing libraries were constructed using the Chromium Single Cell 3' Kit (10x Genomics, DE, USA) according to the manufacturer's instructions. Illumina sequencing was performed according to the manufacturer's instructions (Novaseq 6000, Illumina, USA).

[0184] In vivo biosafety evaluation

[0185] ALT, AST and Cr in the serum of mice in all treatment groups were detected using Chemray 240 automatic biochemical analyzer (Rayto Science, Shenzhen, China) to evaluate the effects of JS201 on liver and kidney function. The hearts, livers and kidneys of mice treated with JS201 were fixed with 4% paraformaldehyde, sliced ​​and stained with HE to observe the effects of JS201 on important organs.

[0186] JS-201 affinity detection at the PD-1 end

[0187] Mouse PD-1 protein was diluted with PBS and coated on an ELISA plate (200 ng / well). After incubation and blocking, serially diluted bispecific antibody JS-201 or control mouse IgG protein / anti-PD-1 antibody and TGF-βtrap were added and incubated at 37°C for 1 hour. After incubation with HRP-labeled mouse IgG antibody, the color development substrate was added and the absorbance was detected at a wavelength of 450 nm after stopping the color development.

[0188] JS-201 affinity test on TGF-β

[0189] Mouse TGF-β1, TGF-β2, and TGF-β3 proteins were diluted with PBS and coated on ELISA plates (200 ng / well). After incubation and blocking, serially diluted bispecific antibody JS-201 or control mouse IgG protein / anti-PD-1 antibody and TGF-βtrap were added and incubated at 37°C for 1 hour. After incubation with HRP-labeled mouse IgG antibody, color reaction substrate was added and the absorbance was detected at a wavelength of 450 nm after stopping the color development.

[0190] Double antigen sandwich ELISA

[0191] TGF-β1, TGF-β2 and TGF-β3 were used to coat 96-well plates overnight. After washing and blocking, serially diluted JS-201 or control mouse IgG, anti-PD-1 antibody and TGF-βtrap were added to the culture plate and incubated at 37°C for 1 hour. After washing, PD-1-Biotin (200 ng / ml, 100 μl per well) was added and incubated for 1 hour. The test plate was washed and incubated with peroxidase-labeled streptavidin (1:1000). According to the standard ELISA protocol, the simultaneous binding of JS-201 with TGF-β and PD-1 was detected by absorbance value.

[0192] Statistical analysis

[0193] Data were analyzed using GraphPad Prism version 8.0. Results are expressed as mean ± standard deviation (SD) or mean ± standard error (SEM). Statistical significance was defined as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, respectively.

[0194] Example 1: JS201 combined with radiotherapy inhibits tumor growth and improves survival in mice

[0195] The structure of JS201 is as follows Figure 1 (A) As shown. In the single lesion tumor burden study, three tumor-bearing models that are poorly responsive to ICB (immune checkpoint inhibitor blockade therapy) were used to evaluate the efficacy of the combination therapy: the Lewis lung carcinoma (LLC) model of lung cancer, the CT26 model of colon cancer, and the 4T1 model of breast cancer. Mice were treated as Figure 1 (B) The observation endpoint of the survival curve was set at 60 days after the start of treatment or when the tumor volume reached 2000mm 3 hour.

[0196] In the LLC model, on day 15 after treatment, the tumor burden of mice in the JS201 group was significantly reduced compared with the anti-PD-1 group (p<0.0001) and the NS group (p<0.0001). The JS201 and RT combination group had a stronger anti-tumor growth effect than the JS201 group (p<0.0001) or the local RT group (p<0.0001). Figure 1 , C). A similar trend of anti-monofocal tumor effect can also be seen in CT26 (JS201 group vs anti-PD-1 group, p < 0.0001; JS201 group vs NS group, p < 0.0001; JS201 + RT combination group vs RT group, p < 0.0001; JS201 + RT combination group vs JS201 group, p = 0.0039; Figure 1 , E) Neutralization of 4T1 tumor model (JS201 group vs anti-PD-1 group, p = 0.0376; JS201 group vs NS group, p = 0.0004; JS201 + RT combination group vs RT group, p < 0.0001; JS201 + RT combination group vs JS201 group, p = 0.0245; Figure 1 , observed in G).

[0197] In the LLC model, the overall survival of the JS201 group was significantly prolonged compared with the anti-PD-1 group (p=0.0012) and the NS group (p<0.0001), and the survival benefit of the combination therapy group was more obvious (JS201+RT group vs RT group, p=0.0380; JS201+RT group vs JS201 group, p=0.0335; Figure 1, D). For the WT26 model, the JS201 group had a significant survival benefit compared with the NS group (p = 0.0019), but did not show a significant survival advantage compared with the anti-PD-1 group. Figure 1 , F), the survival of the radiotherapy and JS201 combination group was significantly prolonged. The survival curve of the 4T1 model was similar to that of the CT26 model (JS201 group vs NS group, p = 0.0284; JS201 + RT combination group vs anti-PD-1 group, p = 0.0420; JS201 + RT combination group vs NS group, p = 0.0085; Figure 1 , H).

[0198] Example 2: JS201 combined with radiotherapy reverses the immunosuppressive microenvironment

[0199] After treatment, mice bearing LLC tumors were killed, and the tumors were removed as a whole for pathological staining and immunohistochemistry (IHC). The results of CD8 IHC showed that the number of tumor-infiltrating cytotoxic lymphocytes in the JS201 group was significantly increased compared with the NS group (p<0.0001) and the anti-PD-1 group (p=0.0023), and the combination of JS201 and RT further enhanced this effect (JS201+RT group vs RT group, p=0.0020; JS201+RT group vs JS201 group, p=0.0205; Figure 2 , B). CD31 is a biomarker of vascular endothelial cells and is used to reflect tumor angiogenesis. The results showed that JS201 and combined treatment had significant anti-tumor angiogenesis activity compared with the anti-PD-1 group (p = 0.0184, Figure 2 , C). Sirius red staining showed that JS201 significantly reduced fibrosis in the TME compared with the NS group (p<0.0001) and the anti-PD-1 group (p<0.0001). The increased fibrosis in the TME caused by radiotherapy (JS201+RT group vs JS201, p=0.0199) could be alleviated by the combination of JS201 (JS201+RT group vs radiotherapy group, p=0.0001; Figure 2 , D).

[0200] LLC tumor tissues (3 in each group) were harvested in full from the anti-PD-1 group, JS201 group, and JS201+RT combination group and analyzed by transcriptome RNA sequencing to monitor the effects of various therapies on TME. Gene heatmaps comparing the JS201 group with the anti-PD-1 group are shown in Figure 2. Figure 2 As shown in (E), there are 1920 significantly differentially expressed genes (DEGs). GO database enrichment analysis showed that compared with the anti-PD-1 group ( Figure 2, FG), the expression of molecules related to ECM (FDR=0), cell adhesion (FDR=9.26E-25), and cell migration (FDR=4.70E-22) were significantly downregulated in the JS201 group compared with the control group, which was consistent with the reduced ECM receptor interaction in the KEGG enrichment analysis (FDR=7.17E-05, Figure 2 , J). The enrichment results in DisGeNET showed that compared with the anti-PD-1 group, the expression of genes related to disease development was significantly downregulated in the JS201 group, including tumor metastasis (FDR = 9.22E-17), adenocarcinoma (FDR = 9.22E-17), secondary lymph node malignancy (FDR = 1.92E-09), and tumor angiogenesis (FDR = 1.46E-08) ( Figure 2 In addition, analysis of the Reactome pathway revealed that EGFR signaling was significantly reduced in tumor tissues treated with JS201 compared with tissues treated with anti-PD-1 (FDR = 0.026, Figure 2 , I), which is a signaling pathway that has been shown to be involved in tumor migration and proliferation after TGF-β activation. Fig. 9 (AD) shown.

[0201] Figure 2 The gene heat map of (K) shows the comparison of 1612 significantly expressed DEGs in the JS201+RT group and the JS201 group, showing the biphasic effect of RT on TME. The results of KEGG enrichment analysis showed that compared with the use of JS201 alone ( Figure 2 Compared with LM, combined radiotherapy enhanced the expression of genes in the TNF signaling pathway (FDR=0.0004) and ECM receptor interaction (FDR=0.0006) pathways, which is consistent with the Reactome enrichment results ( Figure 2 , O). In addition, DisGeNET enrichment results showed that the combination of JS201 and RT would aggravate the inflammatory response (FDR = 1.26E-18) and promote fibrosis in tumor tissue (FDR = 1.24E-14) ( Figure 2 , N), which is consistent with the GO term analysis ( Figure 2 , P) in ECM (FDR = 0) and inflammatory response (FDR = 5.63E-25) up-regulated results, as well as IHC results. The enriched gene percentage results are shown in Fig. 9 (EH). The above results indicate that compared with anti-PD-1, JS201 attenuated immunosuppressive features such as ECM formation, tumor migration and angiogenesis, and compensated for the negative effects of RT-induced ECM on TME. The beneficial effect of combined radiotherapy for single lesions is mainly reflected in the enhancement of inflammatory response within the tumor by enhancing TNF signaling.

[0202] Example 3: Abscopal effect of JS201 combined with radiotherapy

[0203] Refer to previous studies to establish Figure 3 (A) The dual-lesion LLC-luc tumor burden model was used to evaluate the abscopal effect of the combined therapy. The growth curve of the primary tumor inoculated in the anterior flank of mice showed that the anti-tumor effect of the combined treatment group was significantly increased compared with the JS201 group (p = 0.0346) and the RT group (p < 0.0001). Figure 3 , B). The secondary tumor growth curve showed that the JS201 group showed a significant anti-tumor effect on distal lesions compared with the NS group (p = 0.0025), but there was no significant difference compared with the anti-PD-1 group. Compared with the anti-PD-1 group (p = 0.0011), the JS201 + RT combination group showed a significant advantage in distal lesions, and was also superior to the JS201 group (p = 0.0172) and the RT group (p = 0.0015) ( Figure 3 , C). Five mice were randomly selected from each group for in vivo imaging to evaluate tumor growth and migration. The results showed that JS201 reduced the density and fluorescence intensity of primary tumors relative to anti-PD-1 and NS, but the combination treatment group showed better anti-tumor effects on both primary and secondary tumors, indicating that the combination treatment significantly enhanced the abscopal effect ( Figure 3 , D). Figure 3 (E and F) To avoid the possible influence of luciferin injection on flow cytometry (FCM) during live imaging, other mice that did not undergo live imaging were selected for FCM detection.

[0204] Example 4: JS201 combined with radiotherapy optimizes immune cell infiltration in primary and secondary tumors

[0205] Flow cytometry was used to detect immune cell infiltration in primary and secondary tumors and spleen of LLC-luc tumor-bearing mice (n=3), including myeloid-derived suppressor cells (MDSC) ( Figure 3 , G) and CD4+ and CD8+ T cells ( Figure 3 , K). MDSC is an important immunosuppressive cell in tumor immune escape. Compared with the NS group (p = 0.0460) and the anti-PD-1 group (p = 0.0492), the MDSC of the primary tumor in the JS201 group was significantly reduced, while in the combined treatment group, the promoting effect of RT on MDSC was slowed down (p = 0.0281) ( Figure 3 JS201 similarly compensated for RT-induced immunosuppression in secondary tumors (p = 0.0029) and spleen (p = 0.0079, Figure 3Although the reduction of MDSC by JS201 was not significant compared with the anti-PD-1 group in secondary tumors, there was a significant difference between the JS201 and radiotherapy group and the anti-PD-1 group (p = 0.0035, Figure 3 , J).

[0206] The ratio of CD8+T to CD4+T cells was used to evaluate the activity of cytotoxic reactions in the TME. The results showed that there was no significant difference between the JS201 group and the anti-PD-1 group. Compared with the anti-PD-1 group, the CD8+T / CD4+T level in the combination group was significantly increased (p=0.0014), and was also significantly higher than that in the RT group (p=0.0276, Figure 3 , L). Similar trends were observed in secondary tumors (JS201+RT group vs anti-PD-1 group, p=0.0164; JS201+RT group vs RT group, p=0.0276; Figure 3 , M), but no significant trend was observed in spleen ( Figure 3 , N). The above results indicate that the combination of JS201 and RT enhances the abscopal effect by increasing the CD8+T / CD4+T cell ratio in primary and secondary tumors and reducing MDSCs in primary and secondary tumors.

[0207] Example 5: JS201 alleviates acute / subacute radiation pneumonitis (RP)

[0208] Acute / subacute RP mouse models were established using a single dose of 16 Gy / 1F whole thoracic irradiation (15 mice per group), and CT scanning and tissue staining were performed at observation time points, set at 1, 2, and 3 months after RT (3 mice per observation point), as Figure 4 (A) Radiological results showed that both the NS group and the PD-1 group had obvious fused patchy shadows, solid lesions, and a large amount of exudates, while only a slight increase in bronchovascular shadows, a small amount of exudates, and scattered streaky lesions were observed in the JS201 group ( Figure 4 , B).

[0209] Left lung tissues of mice (n=3) were isolated for HE and myeloperoxidase (MPO) staining. HE results showed that lung tissues in the NS and PD-1 groups showed obvious inflammatory infiltration 1 month after radiotherapy, which was further aggravated 2 months after radiotherapy, showing overall abnormal lung tissue structure, accompanied by a wide area of ​​solid tissue, alveolar collapse and atrophy and poor structure; these effects were slightly alleviated 3 months after radiotherapy. The JS201 group showed less inflammatory infiltration and tissue damage at the observation site than the NS and anti-PD-1 groups ( Figure 4 , C). MPO staining can reflect the changes and severity of neutrophil infiltration at different times and in different groups, and its trend is consistent with the HE results ( Figure 4 , D). ImageJ quantitative analysis showed that the MPO positive area rate in the JS201 group was significantly higher than that in the blank control group (1 month, p = 0.0106; 2 months, p = 0.0003; 3 months, p = 0.0064), but significantly lower than that in the NS group (1 month, p < 0.0001; 2 months, p = 0.00101; 3 months, p = 0.0312) and the anti-PD-1 group (1 month, p < 0.0001; 2 months, p = 0.0466; 3 months, p = 0.0069), and the same trend was maintained at the three time points ( Figure 4 , EG).

[0210] Example 6: JS201 inhibits calcium ion signaling-related pathways in the development of RP

[0211] Lung tissues were collected separately from acute RILI model mice (n=3) for transcriptome RNA sequencing to investigate the mechanism by which the JS201 group alleviated radiation pneumonitis compared with anti-PD-1. Figure 4 (H) A total of 920 significantly expressed DEGs were shown, of which 749 genes were significantly downregulated in the JS201 group ( Fig.10 , A). KEGG enrichment results showed that calcium signaling pathway (padj = 0.0002) and inflammatory mediator regulation of TRP channels (padj = 0.0189) were significantly inhibited after JS201 treatment compared with anti-PD-1 treatment ( Figure 4 , I). GO enrichment results also showed that calcium ion binding (padj = 0) and calmodulin binding (padj = 0.0025) were significantly reduced in the JS201 group ( Figure 4 , J), indicating that calcium signaling-related pathways may play an important role in the development of RP. Enrichment analysis of lncRNA showed similar results ( Fig.10 , BE).

[0212] To further detect the key genes in the progression of RP, a Venn diagram of pathway genes related to calcium signaling and inflammation enriched in the GO, KEGG, and Reactome databases was drawn ( Fig.10 , F), such as Figure 4 (K) A total of 5 genes were confirmed, and their heat maps are shown in Figure 4 (L) Subsequent verification of the differential expression of 5 genes in the GSE174196 public database showed that CAMK2A expression (p = 0.024) was significantly upregulated in the acute RP stage ( Figure 4 , M), while other genes were not expressed or had no significant differences in expression ( Fig.10, G, H). Lung tissues were isolated from acute RP mouse models (n = 3) for IHC and western blot assays to verify the expression of CAMK2A protein in different groups. IHC results showed that 1 month after radiotherapy, CAMK2A expression in lung tissues of the NS group and anti-PD-1 group was significantly increased compared with the control group, while this phenomenon was significantly attenuated in the JS201 group ( Figure 4 , N). Western blot results showed a similar trend and were also consistent with the trend of TGF-β1 expression ( Figure 4 , O), indicating that CAMK2A plays a proinflammatory role in RP and may be associated with TGF-β signaling.

[0213] Example 7: JS201 alleviates radiation-induced pulmonary fibrosis (RIPF) and prolongs survival

[0214] A single dose of 16 Gy whole-thoracic irradiation was used to establish a chronic RIPF mouse model (15 mice in each group). The observation time point was set at 6 months after RT. JS201 was administered according to the instructions provided by the merchant. The specific process is as follows Figure 5 (A) The effect of RIPF on mice can be reflected by the weight change curve and survival percentage curve recorded weekly. Statistical analysis showed that the weight loss of mice in the JS201 group was significantly lower than that in the anti-PD-1 group (p = 0.0002). Compared with the NS group, although there was no significant difference, the weight curve showed a rebound trend within 6 weeks before the observation endpoint, which was opposite to the continuous downward trend of the NS group ( Figure 5 , B). In addition, JS201 significantly prolonged the survival of RIPF mice compared with NS mice (p=0.0402, Figure 5 , C).

[0215] Figure 5 (D) shows the chest CT of mice 6 months after RT. The left lung was collected for pathological staining, and the right lung was used for hydroxyproline (HYP) determination to evaluate the collagen metabolism in the lung tissue. The statistical results showed that the HYP content in the lung tissue of mice in the JS201 group was not significantly different from that in the blank control group, but was significantly lower than that in the NS (p = 0.0078) and anti-PD-1 groups (p = 0.0023) groups ( Figure 5 , E). HE staining analysis to evaluate structural changes in lung tissue and Masson and Sirius red staining analysis to evaluate fibrosis. Figure 5 (F) Ashcroft score can be used to evaluate HE outcomes, and the score showed that RIPF in the JS201 group was significantly more severe than that in the control group (p = 0.0001), but significantly milder than that in the NS group (p = 0.0002) and the anti-PD-1 group (p = 0.0010). Figure 5, G), and this conclusion was consistent with Masson staining (JS201 group vs control group, p < 0.0001; JS201 group vs NS group, p = 0.0005; JS201 group vs anti-PD-1 group, p < 0.0001; Figure 5 , H) and picrosirius red staining were consistent (JS201 group vs control group, p < 0.0001; JS201 group vs NS group, p < 0.0001; JS201 group vs PD-1 group, p < 0.0001; Figure 5 , I).

[0216] Example 8: JS201 reduces the proportion of fibroblasts and inhibits the TGF-β1 / Smad2 pathway

[0217] One mouse was randomly selected from each of the anti-PD-1 group and the JS201 group, and the lung tissue was completely removed for single-cell RNA sequencing to elucidate the possible mechanism of JS201 in alleviating RIPF compared with anti-PD-1. The results of 23 clusters of TSNE of lung tissues collected from the two groups are shown in Figure 2. Figure 6 (A) As shown. Cell markers were used to annotate and analyze single-cell RNA-seq results ( Fig.11 , C). Marker annotation and corresponding UMAP results are shown in Fig.11 (A, B). After cell annotation, a total of 12 types of cells and their functions were analyzed. The results of the proportion of similar cells in each group are shown in Figure 6 (C) shows that the cell ratio in each group is Fig.11 (D) as shown.

[0218] The DEGs of fibroblasts between the two groups were analyzed to detect functional differences, and the heat map is shown in Figure 6 As shown in (D). Since there were few DEGs in fibroblasts between groups, no meaningful enrichment results were obtained, so intra-group DEG enrichment analysis was subsequently performed to explore cell functions. The heat map of significant DEGs for each cell type in the anti-PD-1 group is shown in Figure 6 (E) Heat map of significant DEGs in the JS201 group Figure 6 (F) KEGG enrichment results of 708 significant DEGs showed that in the anti-PD-1 group, the TGF-β signaling pathway (padj = 0.0231) and ECM receptor interaction (padj = 7.53E-11) in fibroblasts were significantly enhanced compared with other cells ( Figure 6 , G). Correspondingly, GO enrichment results showed that in the anti-PD-1 group, fibroblasts had enhanced collagen binding (padj=5.52E-09) and ECM (padj=2.17E-38) ( Figure 6 , H). For the JS201 group, KEGG enrichment results ( Figure 6 , I) showed that ECM-receptor interaction increased significantly (padj=6.56E-10), and GO enrichment results ( Figure 6 , J) also showed a significant increase in collagen binding (padj = 7.44E-10) and ECM (padj = 2.50E-51), but no significant difference was found in the TGF-β signaling pathway (padj = 0.0912). Further TSNE analysis of fibroblasts between the two groups was performed ( Figure 6 , K), the expression of nine genes in these seven clusters, Col3a1, Col1a2, Col1a1, Tgfb1i1, Tgfbr2, Smad2, Smad4, FGF1, and Fgfr1, were analyzed. Figure 6 (M). According to the above gene expression, collagen production, TGF-β signaling and proliferation-related fibroblasts ( Figure 6 , L), divided fibroblasts into three subgroups. The results showed that compared with anti-PD-1 treatment, JS201 inhibited the TGF-β signaling pathway and alleviated lung fibrosis by reducing the proliferation and proportion of fibroblasts rather than changing their function. UMAP clustering results are shown in Fig.11 As shown in (D, E).

[0219] Lung tissues of individual RIPF model mice were isolated to extract proteins for western blot to verify the expression of TGF-β / Smad pathway in the four groups. The results showed that after radiotherapy, TGF-β1 was significantly increased in the NS group and anti-PD-1 group compared with the control group, thereby activating downstream molecules, increasing phosphorylated Smad2, and increasing collagen I / III expression, while the fibrosis process and the expression of corresponding key molecules in the JS201 group were significantly weakened ( Figure 6 The above results indicate that JS201 slows down RIPF by limiting the size and collagen production of fibroblasts through inhibiting the TGF-β / Smad pathway.

[0220] Example 9: JS201 reduces the proportion of neutrophils, ROS induction and the formation of neutrophil extracellular traps (NETs)

[0221] Single-cell sequencing showed that the proportion of neutrophils in the JS201 group was significantly lower than that in the anti-PD-1 group ( Figure 6 , C). In addition to the cell ratio, the functional differences of the neutrophil populations between the two groups were also determined. The heat map of DEGs between neutrophils is shown in Figure 7 (A) Reactome enrichment results showed that JS201 significantly inhibited the expression of inflammasome (padj = 0.0047) and NLRP3 inflammasome (padj = 0.0384) in neutrophils ( Figure 7, B). In addition, the KEGG enrichment results showed that the expression of natural killer cell-mediated cytotoxicity-related genes in the JS201 group was significantly reduced compared with the anti-PD-1 group (padj=0.0005, Fig.11 , F), indicating that JS201 alleviated neutrophil-mediated chronic inflammatory lung injury. The GO enrichment results of DEGs in neutrophils showed that compared with the anti-PD-1 group, the supramolecular fibrous organization (padj = 0.0027), ECM (padj = 5.73E-05), and inflammatory response (padj = 0.0290) in the JS201 group were significantly reduced ( Figure 7 , C), indicating that JS201 weakened the profibrotic effect of neutrophils. The TSNE descending clustering results of the two groups of neutrophils are shown in Figure 7 (D) and the UMAP results are shown in Fig.11 , G. A total of 7 genes were analyzed, and inter-cluster ( Figure 7 , F) and between groups ( Figure 7 , G) were compared and analyzed: Cox6c and Cox7c related to oxidative stress, Lgals1 related to fibrosis, Gzma and Gzmb related to cytotoxicity, and Cybb and Cyba related to NADPH oxidase. Based on the above gene expression profiles, neutrophils were divided into four subgroups ( Figure 7 , E; Fig.11 , H), the results showed that the JS201 group significantly reduced neutrophil subsets associated with cytotoxicity, NOX2, and fibrosis compared with the anti-PD-1 group.

[0222] To investigate the mechanism by which neutrophils promote RIPF, the total DEGs between the two groups were analyzed. As shown in the heat map ( Figure 7 , H), the expression of genes related to ROS-induced NETs was significantly increased in the anti-PD-1 group. Given that in the Western blot validation results, the expression of Cybb / NOX2 protein, which can induce ROS, was higher in the anti-PD-1 group compared with the JS201 group (padj = 3.28E-28) Figure 7 , P), so frozen bronchoalveolar lavage fluid (BALF) and lung tissue (n = 3) samples of RIPF mice were used for DHE staining to verify the infiltration of ROS. The results showed that the ROS infiltration in BALF and lung of the JS201 group was significantly lower than that of the NS group (lung, p = 0.0002; BALF, p = 0.0022) and the anti-PD-1 group (lung, p = 0.0004; BALF, p = 0.0036) ( Figure 7, IK). The expression of NET-specific marker CITH3 was determined by Western blotting, while the expression of MPO-DNA complex and TGF-β1 in serum and BALF was determined by ELISA (n=3). The results showed that the expression of TGF-β1 and CITH3 in lung tissues of NS group and anti-PD-1 group after radiotherapy was significantly higher than that of control group, while this effect was alleviated in JS201 group ( Figure 7 , NO). In addition, the results of serum cytokine quantification showed that after RT treatment, the levels of TGF-β1 and MPO-DNA complex in the JS201 group were significantly lower than those in the NS group (TGF-β1, p<0.0001; MPO-DNA complex, p=0.0015) and the anti-PD-1 group (TGF-β1, p=0.0286; MPO-DNA complex, p=0.0012), while in BALF, TGF-β1 (JS201 group vs NS group <0.0001; JS201 group vs anti-PD-1 group, p=0.0097) and MPO-DNA complex (JS201 group vs. NS group, p<0.0001; JS201 group vs anti-PD-1 group, p<0.0001) showed similar trends ( Figure 7 , LO). In addition, homologous double-labeling immunofluorescence assays were performed for CITH3 (green) and MPO (red) to track NETs in lung tissue. The results showed that NETs with reticular structures were visible in the thickened alveolar septa, marked by arrows, which simultaneously expressed MPO and CITH3. Compared with the control group, severe neutrophil infiltration could be found in the lung interstitium in the NS group and the anti-PD-1 group. In contrast, there was a certain degree of interstitial neutrophil infiltration in the JS201 group, but there was no significant release of NETs ( Figure 7 , Q). The above results indicate that JS201 alters the size and function of neutrophils to attenuate RIPF.

[0223] Example 10: In vivo biosafety evaluation of JS201

[0224] Considering the potential adverse effects of whole chest irradiation on the heart, tumor-bearing mice that did not undergo thoracic radiotherapy were selected for in vivo biosafety assessment of JS201 (n=4-6). The hearts, livers, and kidneys of the mice were completely separated and HE stained. The myocardial fibers of the mice were neatly arranged, the structures of the liver lobules and kidney capsules were clear, and there was no acute damage such as exudation, congestion, and inflammatory cell infiltration in the tissues, indicating that JS201 administered in vivo did not cause significant damage to the major organs ( Figure 8 , A).

[0225] Serum glutathione aminotransferase (ALT), glutamate oxaloacetate transaminase (AST), and creatinine (Cr) levels were measured in mice in the NS group, anti-PD-1 group, and JS201 group 15 days after the start of treatment. The results showed that there were no significant differences in serum ALT, AST, and Cr among the three groups, indicating that JS201 administered in vivo did not affect liver and kidney function ( Figure 8 , BD).

[0226] In summary, if Figure 8 As shown in (E), the above results indicate that JS201 targets PD-1 and neutralizes TGF-β1 signaling in a competitive binding manner, and the combination therapy with a single high-dose RT improves the anti-tumor efficacy by optimizing the negative features of the TME while alleviating RT-induced acute and chronic lung toxicity.

[0227] Example 11: Functional evaluation of JS201 binding to PD-1 and TGF-β in vitro

[0228] We used non-reducing and reducing SDS-PAGE to verify the structure of JS-201: in the non-reducing state, JS-201 appeared as a long chain with a molecular weight of about 180KD; after DTT opened the disulfide bond, JS-201 appeared as a long chain with a molecular weight of 70-100KD and a short chain with a molecular weight of 25-35KD. Fig.12 (B) As shown. We used enzyme-linked immunosorbent assay and double sandwich antigen detection to verify the ability of JS-201 to bind to the targets PD-1 and TGF-β, and compared it with anti-PD-1 monoclonal antibody, TGF-βtrap and blank mouse IgG antibody, where TGF-βtrap was constructed by fusion of blank mouse IgG protein with TGF-βRII extracellular domain. The results showed that the ability of JS-201 to bind to PD-1 alone was similar to that of anti-PD-1 monoclonal antibody, and the binding ability reached a peak at a concentration of 104ng / ml ( Fig.12 , C); JS-201 alone has a stronger ability to bind to TGF-β1 than TGF-βtrap, and the binding capacity is close to the peak when the concentration reaches 104ng / ml ( Fig.12 , D); The ability of JS-201 to bind TGF-β2 alone is similar to that of TGF-βtrap, and the binding capacity reaches a peak when the concentration reaches 105-106ng / ml ( Fig.12 , E); The ability of JS-201 to bind TGF-β3 alone is slightly weaker than that of TGF-βtrap, and the binding capacity reaches a peak when the concentration reaches 106ng / ml ( Fig.12 , F). In the double sandwich ELISA experiment, JS-201 was confirmed to bind to PD-1 and TGF-β at the same time, and the binding ability was significantly enhanced when the concentration exceeded 103ng / ml ( Fig.12, GI), while PD-1 monoclonal antibody and TGF-βtrap cannot bind to the above targets at the same time, proving that JS-201 has excellent dual-target binding ability.

Claims

1. Use of a bifunctional protein targeting PD-1 and TGFβ in the preparation of a medicament for treating an individual suffering from cancer, wherein the bifunctional protein targeting PD-1 and TGFβ comprises: (1) anti-PD-1 antibodies or antigen-binding fragments thereof; (2) an immunoglobulin Fc domain; and (3) TGF-βRⅡ extracellular domain.

2. The use according to claim 1, wherein the bifunctional protein targeting PD-1 and TGFβ is administered alone or in combination with radiotherapy; Preferably, the bifunctional protein targeting PD-1 and TGFβ is administered alone; Preferably, the individual has previously received radiotherapy; Preferably, the radiation therapy comprises stereotactic body radiation therapy (SBRT) or whole chest irradiation (WTI).

3. The use according to any one of claims 1 to 2, wherein the cancer is a breast tumor; preferably, the cancer is selected from lung cancer, breast cancer or colon cancer.

4. The use according to any one of claims 1 to 3, wherein the individual has a disease or condition caused by radiotherapy, or the individual suffers from a primary tumor only, or the individual suffers from both a primary tumor and a secondary tumor; preferably, the individual suffers from both a primary tumor and a secondary tumor.

5. The use according to claim 4, wherein the disease or condition caused by radiotherapy is a disease or condition of the lung; preferably, the disease or condition of the lung is radiation lung injury; preferably, the radiation lung injury is radiation pneumonitis or radiation pulmonary fibrosis.

6. Use of a bifunctional protein targeting PD-1 and TGFβ in the preparation of a drug for treating or preventing a lung disease or condition caused by radiotherapy in an individual, wherein the bifunctional protein targeting PD-1 and TGFβ comprises: (1) an anti-PD-1 antibody or an antigen-binding fragment thereof; (2) an immunoglobulin Fc domain; and (3) a TGF-βRⅡ extracellular domain; preferably, the disease or condition is radiation lung injury; preferably, the radiation lung injury is selected from radiation pneumonitis or radiation pulmonary fibrosis.

7. The use according to claim 6, wherein the individual suffers from a primary tumor only, or suffers from a primary tumor and a secondary tumor at the same time; preferably, the individual suffers from a primary tumor and a secondary tumor at the same time.

8. Use of a bifunctional protein targeting PD-1 and TGFβ in the preparation of a drug for achieving one or more of the following purposes in an individual: Inhibit tumor growth and prolong patient survival; Increases the number of tumor-infiltrating cytotoxic lymphocytes in tumors; Reduce myeloid-derived suppressor cells; Increase the CD8+T / CD4+T cell ratio; Anti-tumor angiogenesis; downregulates the expression of molecules related to ECM, cell adhesion, and cell migration; Reduce EGFR signaling in tumor tissues; Attenuate immunosuppressive features such as ECM formation, tumor migration, and angiogenesis, compensating for the negative effects of RT-induced increased ECM expression on TME; Enhances TNF signaling and increases inflammatory responses within tumors; Reduce fibrosis in the TME, especially fibrosis caused by radiation therapy; Inhibits the regulation of inflammatory mediators of calcium signaling pathways and TRP channels; Reduces calcium ion binding and calmodulin binding; Reduce CAMK2A expression; Inhibit TGF-β1 / Smad2 pathway, reduce the proportion of fibroblasts and thus alleviate lung fibrosis; Inhibits inflammasome expression in neutrophils; Alleviate neutrophil-mediated chronic inflammatory lung injury; and Reduce the proportion of neutrophils and the formation of ROS-induced neutrophil extracellular traps, thereby alleviating the profibrotic effect; The bifunctional protein targeting PD-1 and TGFβ comprises: (1) an anti-PD-1 antibody or an antigen-binding fragment thereof; (2) an immunoglobulin Fc domain; and (3) a TGF-βRⅡ extracellular domain.

9. The use according to any one of claims 1 to 8, wherein: The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-PD-1 antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; preferably, the bifunctional protein targeting PD-1 and TGFβ comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; more preferably, the bifunctional protein targeting PD-1 and TGFβ comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10; and / or The amino acid sequence of the TGFβRⅡ extracellular domain is shown in SEQ ID NO:

11.

10. The use according to any one of claims 1 to 8, wherein the bifunctional protein targeting PD-1 and TGFβ comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, a light chain with an amino acid sequence as shown in SEQ ID NO: 10, and a TGFβRⅡ extracellular domain with an amino acid sequence as shown in SEQ ID NO: 11; wherein, The Fc domain in the heavy chain amino acids and the TGFβRⅡ extracellular domain amino acids are connected by a (GGGGS)4G peptide segment.

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