Protein molecule and its uses

Abstract

The present application relates to a protein molecule comprising IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor and a second factor, and uses thereof, wherein said first factor and said second factor are each independently selected from the following group: IL2, GMCSF, IL7, IL15, IL21, and FLT3L, and all of said IL12a or a functional fragment thereof, said IL12b or a functional fragment thereof, wherein said first factor and said second factor are located on the same polypeptide chain. The protein molecule can be used in the treatment of tumors.

Description

Technical Field

[0001] This application relates to the field of biopharmaceuticals, and more specifically, to protein molecules and their uses.

Background Art

[0002] Tumors pose a serious threat to human health. In recent years, immunotherapy has shown great potential as a new treatment method in tumor treatment. Cytokine is a very important immune signal in the body, and cytokine fusion protein technology has become another focus of current tumor immunotherapy. This is achieved by fusing two or more cytokines using genetic engineering techniques based on the fact that they are functionally the same or related but each has a different target. However, the current results of using cytokine fusion protein technology in tumor treatment are still not satisfactory, and there is still much room for improvement.

Summary of the Invention

[0003] This application provides a protein molecule comprising IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor and a second factor, wherein the first factor and the second factor are independently selected from the group consisting of IL2, GMCSF, IL7, IL15, IL21 and FLT3L, and IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, the first factor and the second factor are located on the same polypeptide chain. In a specific embodiment, the first factor is different from the second factor.

[0004] In certain embodiments, the first factor and the second factor are selected from the following groups: for the first factor being IL2 and the second factor being GMCSF; for the first factor being IL7 and the second factor being CMCSF; for the first factor being IL15 and the second factor being GMCSF; for the first factor being IL21 and the second factor being GMCSF; for the first factor being IL2 and the second factor being FLT3L; for the first factor being IL7 and the second factor being FLT3L; for the first factor being IL15 and the second factor being FLT3L; for the first factor being IL21 and the second factor being FLT3L; for the first factor being GMCSF and the second factor being IL2; for the first factor being GMCSF and the second factor being IL7; for the first factor being GMCSF and the second factor being IL15; for the first factor being GMCSF and the second factor being IL21; for the first factor being FLT3L and the second factor being IL2; for the first factor being FLT3L and the second factor being IL7; for the first factor being FLT3L and the second factor being IL15; and for the first factor being FLT3L and the second factor being IL21.

[0005] In certain embodiments, the IL12a or a functional fragment thereof, the IL12b or a functional fragment thereof, the first factor and / or the second factor are of mammalian origin.

[0006] In certain embodiments, the IL12a or a functional fragment thereof, the IL12b or a functional fragment thereof, the first factor and the second factor are derived from the same species. In certain embodiments, the same species is a human. In certain embodiments, the IL12b or a functional fragment thereof is located at the N-terminus of the polypeptide chain. In certain embodiments, the IL12a or a functional fragment thereof is located at the C-terminus of the IL12b or a functional fragment thereof.

[0007] In certain embodiments, the C-terminus of the IL12b or a functional fragment thereof and the N-terminus of the IL12a or a functional fragment thereof are directly or indirectly linked. In certain embodiments, the first factor is located at the C-terminus of the IL12a or a functional fragment thereof. In certain embodiments, the N-terminus of the first factor is directly or indirectly connected to the C-terminus of the IL12a or a functional fragment thereof.

[0008] In certain embodiments, the protein molecule consists of, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the first factor, and the second factor. In certain embodiments, the second factor is located at the C-terminus of the IL12a or a functional fragment thereof. In certain embodiments, the N-terminus of the second factor is directly or indirectly connected to the C-terminus of the IL12a or a functional fragment thereof.

[0009] In certain embodiments, the protein molecule consists of, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the second factor, and the first factor.

[0010] In certain embodiments, the protein molecule includes any one of the amino acid sequences selected from the following group: SEQ ID NO: 32-48.

[0011] In certain embodiments, the protein molecule further includes a targeting moiety, and the targeting moiety, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the first factor, and the second factor are located on the same polypeptide chain. In certain embodiments, the targeting moiety is located at the C-terminus of the IL12a or a functional fragment thereof. In certain embodiments, the N-terminus of the targeting moiety is directly or indirectly linked to the C-terminus of the IL12a or a functional fragment thereof.

[0012] In certain embodiments, the protein molecule comprises, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the targeting moiety, the first factor, and the second factor.

[0013] In certain embodiments, the protein molecule comprises, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the targeting moiety, the second factor, and the first factor. In certain embodiments, the targeting moiety is located at the C-terminus of the first factor. In certain embodiments, the N-terminal side of the targeting moiety is directly or indirectly connected to the C-terminal side of the first factor.

[0014] In certain embodiments, the protein molecule comprises, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the second factor, the first factor, and the targeting moiety.

[0015] In certain embodiments, the protein molecule comprises, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the first factor, the targeting moiety, and the second factor. In certain embodiments, the targeting moiety is located at the C-terminus of the second factor. In certain embodiments, the N-terminal side of the targeting moiety is directly or indirectly connected to the C-terminal side of the second factor.

[0016] In certain embodiments, the protein molecule comprises, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the first factor, the second factor, and the targeting moiety.

[0017] In certain embodiments, the protein molecule comprises, in order from the N-terminus to the C-terminus, the IL12b or a functional fragment thereof, the IL12a or a functional fragment thereof, the second factor, the targeting moiety, and the first factor. In certain embodiments, the number of the target moieties is one or more. In certain embodiments, the target moieties are the same. In certain embodiments, the target moieties are different. In certain embodiments, the targeting moiety can specifically target a tumor-associated antigen.

[0018] In certain embodiments, the tumor-associated antigen is selected from the group consisting of: the EDB structural domain of fibronectin, the EDA structural domain of fibronectin, and / or necrotic regions. In certain embodiments, the targeting moiety consists of an antibody or an antigen-binding fragment thereof.

[0019] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, F(ab)2, dAb, isolated complementarity-determining region CDR, Fv, and scFv. In certain embodiments, the antigen-binding fragment is scFv. In certain embodiments, the targeting moiety consists of any one of the amino acid sequences selected from the group shown below: SEQ ID NO: 1-9. In certain embodiments, the indirect linkage is linked by a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the linker links the target moiety and the linker contains a thrombin cleavage site. In certain embodiments, the linker consists of any one of the amino acid sequences selected from the group shown below: SEQ ID NO: 114-117.

[0020] In certain embodiments, the protein molecule includes any one of the amino acid sequences selected from the following group: SEQ ID NO: 49 - 71. This application provides a nucleotide molecule encoding the protein molecule.

[0021] In certain embodiments, the nucleotide molecule includes any one of the nucleotide sequences selected from the following group: SEQ ID NO: 73 - 112. This application provides a vector containing the described nucleotide molecule. This application provides a cell that expresses the protein molecule, or contains the nucleotide molecule, or contains the vector. This application provides a method for preparing the protein molecule, which includes the step of culturing the cell. This application provides a pharmaceutical composition containing the protein molecule. This application provides the use of the protein molecule and the pharmaceutical composition in the preparation of an anti - tumor agent. In certain embodiments, the tumor consists of solid tumors. In certain embodiments, the tumor consists of melanoma.

[0022] This application provides a method for preventing, alleviating or treating a tumor, which includes administering the heterodimer and / or the pharmaceutical composition to a subject in need thereof. In certain embodiments, the tumor consists of solid tumors. In certain embodiments, the tumor consists of melanoma.

[0023] Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of the present application allows for modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention related to the present application. Therefore, the descriptions in the drawings and specifications of the present application are merely exemplary and not limiting.

Brief Description of the Drawings

[0024] The specific features related to the present application are set forth in the appended claims. By referring to the following embodiments and drawings, the features and advantages of the invention targeted by the present application can be better understood. The brief description of the drawings is as follows.

Figure 1

Figure 2

Figure 3-20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

[0025] The following specific embodiments show the modes in which this application is implemented, and other advantages and effects in this application can be easily understood by those skilled in the art as disclosed in this specification.

[0026] For the purposes of the present application, the term "protein molecule" is considered to belong to the term "cytokine fusion protein", but this term generally means a fusion protein obtained by fusing two or more cytokines by genetic recombination technology. They have the inherent biological activity of the constituent factors, or significantly enhance a part of that activity, and furthermore, due to the complementary and synergistic effects of their biological activities, they exhibit complex biological functions that are impossible with a simple pairing of single cytokines, and may even produce some new structural and biological functions.

[0027] In the present application, the term "polypeptide chain" generally means a chain structure containing a plurality of peptide bonds formed by the mutual binding of a plurality of amino acids. In the present application, the polypeptide chain may include IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor and a second factor, and the first factor and the second factor may each be independently selected from the group consisting of IL2, GMCSF, IL7, IL15, IL21 and FLT3L. In certain embodiments, the polypeptide chain may further include one or more targeting moieties.

[0028] In the context of the present application, the term "targeting moiety" means a class of moieties that act on specific tissues or cells. For example, the targeting moiety can specifically target tumor-associated antigens. In the context of the present application, the targeting moiety consists of an antibody or an antigen-binding fragment thereof.

[0029] In the present application, the term "tumor-associated antigen" (TAA) means an antigen molecule present on tumor cells or normal cells. The tumor-associated antigen can include embryonic proteins, glycoprotein antigens and squamous epithelial antigens. The tumor-associated antigen is selected from the group consisting of the EDB structural domain of fibronectin, the EDA structural domain of fibronectin, and necrotic regions of cells.

[0030] In the present application, the term "antigen-binding fragment" generally means a fragment having antigen-binding activity. In the present application, the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')2, F(ab)2, dAb, isolated complementarity-determining region CDR, Fv, and scFv.

[0031] In the present application, the term "antibody" means a polypeptide molecule capable of specifically recognizing and / or neutralizing a specific antigen. The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light chains and two identical heavy chains. In the case of IgG, each light chain can be linked to each heavy chain by a covalent disulfide bond, while the two heavy chains can be linked to each other by a disulfide bond. Each heavy chain has a variable structural domain (VH) at the N-terminus, followed by three (in the case of α and γ types) or four (in the case of μ and ε types) constant structural domains (CH). In the present application, the terms "IL12a", "IL12b", "IL2", "GMCSF", "IL7", "IL15", "IL21", "FLT3L" can be regarded as "cytokines". "Cytokine" generally means a small protein with a wide range of biological activities, which is synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) or specific non-immune cells (such as endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokine plays an important role in controlling cell-cell interactions, cell growth and differentiation. In the present application, the cytokine may be selected from one or more groups of interleukin ((Interleukin, IL), FMS-related tyrosine kinase 3 ligand (FLT3L), and colony stimulating factor (Colony Stimulating Factor, CSF)). The interleukin is usually a cytokine produced by lymphocytes, monocytes, or other non-monocytes. In the present application, the interleukin may be selected from one or more groups of IL12, IL2, IL7, IL15, IL21. In the present application, the colony stimulating factor generally refers to a cytokine that stimulates the formation of cell colonies of different hematopoietic stem cells in a semi-solid culture medium. In the context of the present application, the colony stimulating factor is considered to be granulocyte macrophage colony stimulating factor (GMCSF).

[0032] In the present application, the term "IL12" generally refers to interleukin-12, which can play an important regulatory role in cell-cell interactions, immune regulation, hematopoiesis, and inflammatory processes. The molecule of IL12 usually consists of two subunits, a p40 subunit (40 kd) and a p35 subunit (35 kd), which are bound by disulfide bonds. In the present application, IL12 containing the p35 subunit (35 kd) may be denoted as IL12a, and IL12 containing the p40 subunit (40 kd) may be denoted as IL12b. For example, the p35 subunit of mouse-derived IL12 (mIL12) may contain an amino acid sequence as shown in SEQ ID NO:16, and the p40 subunit may contain an amino acid sequence as shown in SEQ ID NO:17. Also, the p35 subunit of human-derived IL12 (hIL12) may consist of the amino acid sequence shown in SEQ ID NO:18, and the p40 subunit may consist of the amino acid sequence shown in SEQ ID NO:19.

[0033] In the present application, the term "IL2" generally refers to interleukin-2, which plays an important regulatory role in cell-cell interactions, immune regulation, hematopoiesis, and inflammatory processes. For example, mouse-derived IL2 (mIL2) may contain the amino acid sequence shown in SEQ ID NO:20. For example, human-derived IL2 (hIL2) may contain an amino acid sequence as shown in SEQ ID NO:21.

[0034] In the present application, the term "IL15" generally refers to interleukin-15, which plays an important regulatory role in cell-cell interactions, immune regulation, hematopoiesis, and inflammatory processes. For example, mouse-derived IL15 (mIL15) may contain the amino acid sequence shown in SEQ ID NO:22. Also, human-derived IL15 (hIL15) may contain the amino acid sequence shown in SEQ ID NO:23.

[0035] In this application, the term "IL7" generally refers to interleukin-7, which plays an important regulatory role in cell-cell interactions, immune regulation, hematopoiesis, and inflammatory processes. For example, mouse-derived IL7 (mIL7) may contain the amino acid sequence shown in SEQ ID NO:24. Also, human-derived IL7 (hIL7) may contain an amino acid sequence as shown in SEQ ID NO:25.

[0036] In this application, the term "IL21" generally refers to interleukin-21, which plays an important regulatory role in cell-cell interactions, immune regulation, hematopoiesis, and inflammatory processes. For example, mouse-derived IL21 (mIL21) may contain the amino acid sequence shown in SEQ ID NO:26. Also, human-derived IL21 (hIL21) may contain an amino acid sequence as shown in SEQ ID NO:27.

[0037] In this application, the term "FLT3L" generally refers to FMS-related tyrosine kinase 3 ligand, which controls the proliferation and differentiation of non-erythroid hematopoietic stem cells, promotes the proliferation, differentiation, and maturation of pre-B lymphocytes, dendritic cells, NK cells, and cytotoxic T lymphocytes, and has important anti-tumor effects. For example, mouse-derived FLT3L (mFLT3L) may contain the amino acid sequence shown in SEQ ID NO:28. Also, human-derived FLT3L (hFLT3L) may contain an amino acid sequence as shown in SEQ ID NO:29.

[0038] In this application, the term "GMCSF" generally refers to granulocyte macrophage colony-stimulating factor. The GMCSF may have a 4α helix bundle structure. For example, mouse-derived GMCSF (mGMCSF) may contain an amino acid sequence as shown in SEQ ID NO:30. Also, human-derived GMCSF (hGMCSF) may contain an amino acid sequence as shown in SEQ ID NO:31.

[0039] In the present application, the term "functional fragment" generally refers to a fragment that retains a specific function. For example, an IL12a functional fragment refers to a fragment that retains the function of IL12a. For example, for the IL12a functional fragment, IL12a, fragment (GenBank: AIC49052.1) is used.) As another example, for the IL12b functional fragment, IL12b, fragment (GenBank: AIC54621.1) is used.)

[0040] In the present application, the term "direct linkage" is generally used in contrast to the term "indirect linkage" which means direct linkage. For example, the direct linkage may be a direct connection without a spacer between substances (such as the same or different cytokines and / or targeting moieties). The spacer may be a linker. For example, the linker may be a peptide linker. "Indirect linkage" generally refers to a situation where substances are not directly linked (such as the same or different cytokines and / or targeting fractions). For example, the indirect linkage may be a connection via a spacer. For example, the N-terminus of IL2 is directly or indirectly linked to the C-terminus of IL12b or its functional fragment via a linker. For example, the N-terminus of GMCSF is directly linked to the C-terminus of IL12b or its functional fragment or indirectly linked via a linker.

[0041] In the present application, the term "nucleotide molecule" generally refers to a biological macromolecular compound formed by polymerization of a large number of nucleotides, which is divided into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) according to its chemical composition. In the present application, the nucleotide molecule encodes the protein molecule, and it may contain a nucleotide sequence shown in any of SEQ ID NO: 73-112.

[0042] In the context of the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate within a suitable host cell and transfers the inserted nucleic acid molecule within the host cell and / or between host cells. The vector may mainly include vectors used for inserting DNA or RNA into cells, vectors used mainly for replicating DNA or RNA, and vectors used mainly for the expression of transcription and / or translation of DNA or RNA. The vector may further include vectors having the various functions described above. The vector may also be a polynucleotide that is transcribed or translated into a polypeptide when introduced into a suitable host cell. Usually, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product.

[0043] In the present application, the term "cell" generally refers to an individual cell, cell line, or cell culture that can contain, or has contained, the nucleotide molecules described herein or the vectors described herein, or can express the protein molecules described herein. The cell may be composed of the progeny of a single host cell. Due to natural mutations, accidental mutations, or intentional mutations, the daughter cells may not necessarily be identical morphologically or genetically to the original parent cell, but it is considered sufficient if they can express the protein molecules described in this application or can contain the nucleotide molecules and vectors described in this application. In the present application, by using a virus that expresses a vector to transfect B16(rtTA) tumor cells or 293A cells, cells that can express the protein molecules described in this application can be obtained.

[0044] In the present application, the term "tumor" generally refers to a mammalian physiological state characterized by unregulated cell growth and survival. For example, the tumor can constitute a solid tumor. Also, the tumor may be a melanoma.

[0045] In this application, the "solid tumor" generally refers to a tangible mass that can be diagnosed by clinical examinations (such as X-ray, CT scan, ultrasound, palpation, etc.). In this application, the solid tumor may be melanoma.

[0046] In this application, generally, the term "melanoma" is used to refer to a pigmented nevus with malignant changes. The melanoma may arise from nevi with the properties of border nevi or compound nevi, and exhibits symptoms such as sudden appearance, rapid growth, and increased color of the nevus. Protein molecule

[0047] In one aspect, the present application provides a protein molecule comprising IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor and a second factor, wherein the first factor and the second factor are each independently selected from the group consisting of IL2, GMCSF, IL7, IL15, IL21 and FLT3L, and the IL12a or a functional fragment thereof, the IL12b or a functional fragment thereof, the first factor and the second factor are located on the same polypeptide chain.

[0048] In this application, the protein molecule may be a cytokine fusion protein obtained by fusing two or more of the above cytokines, namely IL12, IL2, IL7, IL15, IL21, FLT3L and GMCSF, by genetic recombination technology. The protein molecule has both the unique biological activities of the factors constituting it, and can exert biological functions that are not present in a single cytokine due to the complementary and synergistic effects of biological activities, and can further generate some new structures and biological functions. In this application, the cytokine can be selected from IL12, IL2, IL7, IL15, IL21, FLT3L and GMCSF. In this application, the first factor and the second factor may be different.

[0049] In the present application, the first factor and the second factor may be selected from the following group: for the first factor being IL2 and the second factor being GMCSF; for the first factor being IL7 and the second factor being GMCSF; for the first factor being IL15 and the second factor being GMCSF; for the first factor being IL21 and the second factor being GMCSF; for the first factor being IL2 and the second factor being FLT3L; for the first factor being IL7 and the second factor being FLT3L; for the first factor being IL15 and the second factor being FLT3L; for the first factor being IL21 and the second factor being FLT3L; for the first factor being GMCSF and the second factor being IL2; for the first factor being GMCSF and the second factor being IL7; for the first factor being GMCSF and the second factor being IL15; for the first factor being GMCSF and the second factor being IL21; for the first factor being FLT3L and the second factor being IL2; for the first factor being FLT3L and the second factor being IL7; for the first factor being FLT3L and the second factor being IL15; and for the first factor being FLT3L and the second factor being IL21.

[0050] In the present application, the IL12a or its functional fragment, the IL12b or its functional fragment, the first factor and / or the second factor may be of mammalian origin.

[0051] In the present application, the IL12a or its functional fragment, the IL12b or its functional fragment, the first factor and the second factor may be derived from the same species, and the same species may be human.

[0052] In the present application, the IL12a or its functional fragment, the IL12b or its functional fragment, the first factor and the second factor may be of human origin, and the human-derived IL12a, IL12b, IL2, IL7, IL15, IL21, FLT3L and GMSCF may be denoted as hIL12a, hIL12b, hIL2, hIL7, hIL15, hIL21, hFLT3L and hGMSCF.

[0053] In the present application, the IL12a or its functional fragment, the IL12b or its functional fragment, the first factor and the second factor may be derived from a mouse, and the mouse-derived IL12a, IL12b, IL2, IL7, IL15, IL21, FLT3L and GMSCF may be denoted as mIL12a, mIL12b, mIL2, mIL7, mIL15, mIL21, mFLT3L and mGMSCF. In the present application, the IL12b or its functional fragment may be located at the N-terminus of the polypeptide chain. In the present application, the IL12a or its functional fragment may be located at the C-terminus of the IL12b or its functional fragment.

[0054] In the present application, the C-terminus of the IL12b or its functional fragment may be directly or indirectly linked to the N-terminus of the IL12a or its functional fragment. In the present application, the first factor may be located at the C-terminus of the IL12a or its functional fragment. In the present application, the N-terminal side of the first factor may be directly or indirectly linked to the C-terminal side of the IL12a or its functional fragment.

[0055] In the present application, the protein molecule may sequentially contain the IL12b or its functional fragment, the IL12a or its functional fragment, the first factor and the second factor from the N-terminus to the C-terminus. In the present application, the second factor may be located at the C-terminus of the IL12a or its functional fragment. In the present application, the N-terminus of the second factor may be directly or indirectly linked to the C-terminus of the IL12a or its functional fragment.

[0056] In the present application, the protein molecule may sequentially contain the IL12b or its functional fragment, the IL12a or its functional fragment, the second factor, and the first factor from the N-terminus to the C-terminus.

[0057] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF to form an IL12b-IL12a-IL2-IL2-GMCSF protein molecule.

[0058] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL7, and the C-terminus of IL7 is fused to the N-terminus of GMCSF to form an IL12b-IL12a-IL7-GMCSF protein molecule.

[0059] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL15, and the C-terminus of IL15 is fused to the N-terminus of GMCSF to form an IL12b-IL12a-IL15-GMCSF protein molecule.

[0060] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL21, and the C-terminus of IL21 is fused to the N-terminus of GMCSF to form an IL12b-IL12a-IL21-GMCSF protein molecule.

[0061] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of FLT3L to form an IL12b-IL12a-IL2-IL2-FLT3L protein molecule.

[0062] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL7, and the C-terminus of IL7 is fused to the N-terminus of FLT3L to form an IL12b-IL12a-IL7-FLT3L protein molecule.

[0063] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL15, and the C-terminus of IL15 is fused to the N-terminus of FLT3L to form an IL12b-IL12a-IL15-FLT3L protein molecule.

[0064] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL21, and the C-terminus of IL21 is fused to the N-terminus of FLT3L to form an IL12b-IL12a-IL21-FLT3L protein molecule.

[0065] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL2 to form an IL12b-IL12a-GMCSF-IL2 protein molecule.

[0066] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL7 to form an IL12b-IL12a-GMCSF-IL7 protein molecule.

[0067] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL15 to form an IL12b-IL12a-GMCSF-IL15 protein molecule.

[0068] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL21 to form an IL12b-IL12a-GMCSF-IL21 protein molecule.

[0069] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of FLT3L, and the C-terminus of FLT3L is fused to the N-terminus of IL2 to form an IL12b-IL12a-FLT3L-IL2 protein molecule.

[0070] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of FLT3L, and the C-terminus of FLT3L is fused to the N-terminus of IL7 to form an IL12b-IL12a-FLT3L-IL7 protein molecule.

[0071] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of FLT3L, and the C-terminus of FLT3L is fused to the N-terminus of IL15 to form an IL12b-IL12a-FLT3L-IL15 protein molecule.

[0072] For example, the protein molecule may have a structure in which the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of FLT3L, and the C-terminus of FLT3L is fused to the N-terminus of IL21 to form an IL12b-IL12a-FLT3L-IL21 protein molecule. In the present application, the protein molecule may contain any one of the amino acid sequences selected from the following group: SEQ ID NO: 32-48.

[0073] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF to form an mIL12b-mIL12a-mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.32).

[0074] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL7, and the C-terminus of mIL7 is fused to the N-terminus of mGMCSF to form an mIL12b-mIL12a-mIL7-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.33).

[0075] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL15, and the C-terminus of mIL15 is fused to the N-terminus of mGMCSF to form an mIL12b-mIL12a-mIL15-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.34).

[0076] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL21, and the C-terminus of mIL21 is fused to the N-terminus of mGMCSF to form an mIL12b-mIL12a-mIL21-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.35).

[0077] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mFLT3L to form an mIL12b-mIL12a-mIL2-mFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.36).

[0078] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL7, and the C-terminus of mIL7 is fused to the N-terminus of mFLT3L to form an mIL12b-mIL12a-mIL7-mFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.37).

[0079] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL15, and the C-terminus of mIL15 is fused to the N-terminus of mFLT3L to form an mIL12b-mIL12a-mIL15-mFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.38).

[0080] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL21, and the C-terminus of mIL21 is fused to the N-terminus of mFLT3L to form an mIL12b-mIL12a-mIL21-mFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.39).

[0081] For example, the protein molecule may have a structure in which the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mGMCSF, and the C-terminus of mGMCSF is fused to the N-terminus of mIL2 to form an mIL12b-mIL12a-mGMCSF-mIL2 protein molecule (its amino acid sequence may be shown in SEQ ID NO.40).

[0082] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL2, and the C-terminus of hIL2 is fused to the N-terminus of hGMCSF to form an hIL12b-hIL12a-hIL2-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.41).

[0083] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL7, and the C-terminus of hIL7 is fused to the N-terminus of hGMCSF to form an hIL12b-hIL12a-hIL7-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.42).

[0084] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL15, and the C-terminus of hIL15 is fused to the N-terminus of hGMCSF to form an hIL12b-hIL12a-hIL15-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.43).

[0085] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL21, and the C-terminus of hIL21 is fused to the N-terminus of hGMCSF to form an hIL12b-hIL12a-hIL21-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.44).

[0086] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL2, and the C-terminus of hIL2 is fused to the N-terminus of hFLT3L to form an hIL12b-hIL12a-hIL2-hFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.45).

[0087] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL7, and the C-terminus of hIL7 is fused to the N-terminus of hFLT3L to form an hIL12b-hIL12a-hIL7-hFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.46).

[0088] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL15, and the C-terminus of hIL15 is fused to the N-terminus of hFLT3L to form an hIL12b-hIL12a-hIL15-hFLT3L protein molecule (its amino acid sequence may be shown in SEQ ID NO.47).

[0089] For example, the protein molecule may have a structure in which the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL21, and the C-terminus of hIL21 is fused to the N-terminus of hFLT3L to form an hIL12b-hIL12a-hIL21-hFLT3L protein molecule (the amino acid sequence thereof may be shown in SEQ ID NO. 48).

[0090] In this application, mIL12b-mIL12a-mIL2-mGMCSF, mIL12b-mIL12a-mIL7-mGMCSF, mIL12b-mIL12a-mIL15-mGMCSF, mIL12b-mIL12a-mIL21-mGMCSF, mIL12b-mIL12a-mIL2-mFLT3L, mIL12b-mIL12a-mIL7-mFLT3L, mIL12b-mIL12a-mIL15-mFLT3L, mIL12b-mIL12a-mIL21-mFLT3L, mIL12b-mIL12a-mGMCSF-mIL2, hIL12b-hIL12a-hIL2-hGMCSF, hIL12b-hIL12a-hIL7-hGMCSF, hIL12b-hIL12a-hIL15-hGMCSF, hIL12b-hIL12a-hIL21-hGMCSF, hIL12b-hIL12a-hIL2-hFLT3L, hIL12b-hIL12a-hIL7-hFLT3L, hIL12b-hIL12a-hIL15-hFLT3L, and hIL12b-hIL12a-hIL21-hFLT3 can be abbreviated in order as follows: mIL12bIL12aIL2GMCSF, mIL12bIL12aIL7GMCSF, mIL12bIL12aIL15GMCSF, mIL12bIL12aIL21GMCSF, mIL12bIL12aIL2FLT3L, mIL12bIL12aIL7FLT3L, mIL12bIL12aIL15FLT3L, mIL12bIL12aIL21FLT3L, mIL12bIL12aGMCSFIL2, hIL12bIL12aIL2GMCSF, hIL12bIL12aIL7GMCSF, hIL12bIL12aIL15GMCSF, hIL12bIL12aIL21GMCSF, hIL12bIL12aIL2FLT3L, hIL12bIL12aIL7FLT3L, hIL12bIL12aIL15FLT3L, and hIL12bIL12aIL21FLT3L

[0091] In this application, the protein molecule may further include a targeting portion, and the targeting portion, the IL12b or its functional fragment, the IL12a or its functional fragment, the first factor, and the second factor are located on the same polypeptide chain.

[0092] In the present application, the number of the target portions may be 1 or more. The targeting portions may be the same or different, and the targeting portions can specifically target tumor-associated antigens.

[0093] In the present application, the tumor-associated antigen may be selected from the group consisting of the EDB structural domain of fibronectin, the EDA structural domain of fibronectin, and / or necrotic regions. In the present application, the targeting portion may be composed of an antibody or an antigen-binding fragment thereof.

[0094] In the present application, the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')2, F(ab)2, dAb, an isolated complementarity-determining region CDR, Fv, and scFv. In the present application, the antigen-binding fragment may be scFv.

[0095] In the present application, the targeting portion of the protein molecule is L19V L (the amino acid sequence thereof may be shown in SEQ ID NO.10), L19V H (the amino acid sequence thereof may be shown in SEQ ID NO.11), F8V L (the amino acid sequence thereof may be shown in SEQ ID NO.12), F8V H (the amino acid sequence thereof may be shown in SEQ ID NO.13), NHS76V L (the amino acid sequence thereof may be shown in SEQ ID NO.14), and NHS76V H (the amino acid sequence thereof may be shown in SEQ ID NO.15). In the present application, the targeting portion may be located at the C-terminus of the IL12a or a functional fragment thereof. In the present application, the N-terminus of the targeting moiety may be directly or indirectly linked to the C-terminus side of the IL12a or a functional fragment thereof.

[0096] In the present application, the protein molecule from the N-terminus to the C-terminus may sequentially include an IL12b or a functional fragment thereof, an IL12a or a functional fragment thereof, a targeting moiety, a first factor, and a second factor.

[0097] In the present application, the protein molecule from the N-terminus to the C-terminus may sequentially include an IL12b or a functional fragment thereof, an IL12a or a functional fragment thereof, a targeting moiety, a second factor, and a first factor. In the present application, the targeting moiety may be located at the C-terminus of the first factor. In the present application, the N-terminus side of the targeting moiety may be directly or indirectly connected to the C-terminus side of the first factor.

[0098] In the present application, the protein molecule from the N-terminus to the C-terminus may sequentially include an IL12b or a functional fragment thereof, an IL12a or a functional fragment thereof, a second factor, a first factor, and a targeting moiety.

[0099] In the present application, the protein molecule from the N-terminus to the C-terminus may sequentially include an IL12b or a functional fragment thereof, an IL12a or a functional fragment thereof, a first factor, a targeting moiety, and a second factor. In the present application, the targeting moiety may be located at the C-terminus of the second factor. In the present application, the N-terminus side of the targeting moiety may be directly or indirectly connected to the C-terminus side of the second factor.

[0100] In the present application, the protein molecule from the N-terminus to the C-terminus may sequentially include IL12b or a functional fragment thereof, IL12a or a functional fragment thereof, a first factor, a second factor, and a targeting portion.

[0101] In the present application, the protein molecule from the N-terminus to the C-terminus may sequentially include IL12b or a functional fragment thereof, IL12a or a functional fragment thereof, a second factor, a targeting portion, and a first factor. In the present application, the targeting portion may include any one of the amino acid sequences shown in the following group: SEQ ID NO: 1-9. In the present application, the indirect connection may be connected by a linker. In the present application, the linker may be a peptide linker. In the present application, the linker may be connected to the targeting portion, and the linker may include a thrombin cleavage site. In the present application, the linker may include any one of the amino acid sequences shown in the following group: SEQ ID NO: 114-117.

[0102] For example, the cytokines may be connected to each other by the linker. In the present application, IL12a, IL12b, IL2, IL7, IL15, IL21, FLT3L, and GMSCF may be connected to each other by the linker peptide. For example, the linker may include the amino acid sequence shown in any one of SEQ ID NO. 114 and SEQ ID NO. 116.

[0103] For example, the cytokine and the targeting moiety may be connected to each other by the linker. In the present invention, a linkage by the linker may be made between the targeting portion of the protein molecule and IL12a, IL12b, IL2, IL7, IL15, IL21, FLT3L, and GMSCF. For example, the linker may consist of an amino acid sequence shown in any one of SEQ ID NOs: 114-117.

[0104] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is fused to the N-terminus of L19V H and the C-terminus of L19V H is fused to the N-terminus of L19V L and the C-terminus of L19V L is fused to the N-terminus of L19V H and the C-terminus of L19V H is fused to the N-terminus of L19V L and the C-terminus of L19V L is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, whereby an IL12b-IL12a-L19V H -L19V L -L19V H -L19V L -IL2-GMCSF protein molecule may be formed.

[0105] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is fused to the N-terminus of F8V H and the C-terminus of F8V H is fused to the N-terminus of F8V L and the C-terminus of F8V L is fused to the N-terminus of F8V H and the C-terminus of F8V H is fused to the N-terminus of F8V L and the C-terminus of F8V L is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, whereby an IL12b-IL12a-F8V H -F8V L-An IL2-GMCSF protein molecule may be formed.

[0106] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of NHS76V H of H the C-terminus of which is fused to the N-terminus of NHS76V L of L the C-terminus of which is fused to the N-terminus of NHS76V H of H the C-terminus of which is fused to the N-terminus of NHS76V L of L the C-terminus of which is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-NHS76V H -NHS76V L -NHS76V H -NHS76V L -IL2-GMCSF protein molecule may be formed.

[0107] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of NHS76V H of H the C-terminus of which is fused to the N-terminus of F8V L of L the C-terminus of which is fused to the N-terminus of F8V H of H the C-terminus of which is fused to the N-terminus of F8V L of L the C-terminus of which is fused to the N-terminus of NHS76V, and the C-terminus of NHS76V is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-NHS76V H -F8V L -F8V H -NHS76V L -IL2-GMCSF protein molecule may be formed.

[0108] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of NHS76V H and the C-terminus of NHS76V H is fused to the N-terminus of L19V L and the C-terminus of L19V L is fused to the N-terminus of L19V H and the C-terminus of L19V H is fused to the N-terminus of NHS76V L and the C-terminus of NHS76V L is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-NHS76V H -L19V L -L19V H -NHS76V L -IL2-GMCSF protein molecule may be formed.

[0109] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of F8V H and the C-terminus of F8V H is fused to the N-terminus of NHS76V L and the C-terminus of NHS76V L is fused to the N-terminus of NHS76V H and the C-terminus of NHS76V H is fused to the N-terminus of F8V L and the C-terminus of F8V L is fused to the N-terminus of NHS76V H and the C-terminus of F8V L is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-F8V H -NHS76V L -NHS76V H -F8V L -IL2-IL2-GMCSF protein molecule may be formed.

[0110] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of F8V H and the C-terminus of F8VH The C-terminus of L is fused to the N-terminus of L The C-terminus of H is fused to the N-terminus of H The C-terminus of L is fused to the N-terminus of L The C-terminus of H -L19V L -L19V H -F8V L -IL2-GMCSF protein molecules may be formed.

[0111] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is H fused to the N-terminus of H The C-terminus of L is fused to the N-terminus of L The C-terminus of H is fused to the N-terminus of H The C-terminus of L is fused to the N-terminus of L The C-terminus of H -NHS76V L -NHS76V H -L19V L -IL2-GMCSF protein molecules may be formed.

[0112] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is H fused to the N-terminus of H The C-terminus of L is fused to the N-terminus of L The C-terminus of H is fused to the N-terminus of H The C-terminus of L is fused to the N-terminus ofL The C-terminus of [sequence name] is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-L19V H -F8V L -F8V H -L19V L -IL2-GMCSF protein molecule may be formed.

[0113] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of NHS76V H of H The C-terminus of L is fused to the N-terminus of F8V L The C-terminus of F8V H is fused to the N-terminus of F8V H The C-terminus of F8V L is fused to the N-terminus of NHS76V L The C-terminus of NHS76V is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-IL2-NHS76V H -F8V L -F8V H -NHS76V L -GMCSF protein molecule may be formed.

[0114] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL2, and the C-terminus of IL2 is fused to the N-terminus of F8V H of H The C-terminus of F8V L is fused to the N-terminus of F8V L The C-terminus of F8V H is fused to the N-terminus of F8V H The C-terminus of F8V L is fused to the N-terminus of F8V L The C-terminus of F8V is fused to the N-terminus of GMCSF, so that an IL12b-IL12a-IL2-F8V H -F8V L -F8V H -F8V L-A GMCSF protein molecule may be formed.

[0115] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL2, the C-terminus of IL2 is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of NHS76V H of, H the C-terminus of NHS76V L is fused to the N-terminus of F8V L the C-terminus of F8V H is fused to the N-terminus of F8V H the C-terminus of F8V L is fused to the N-terminus of NHS76V, resulting in the formation of an IL12b - IL12a - IL2 - GMCSF - NHS76V H -F8V L -F8V H -NHS76V L protein molecule.

[0116] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, the C-terminus of IL12a is fused to the N-terminus of IL2, the C-terminus of IL2 is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of F8V H of, H the C-terminus of F8V L is fused to the N-terminus of F8V L the C-terminus of F8V H is fused to the N-terminus of F8V H the C-terminus of F8V L is fused to the N-terminus of F8V, resulting in the formation of an IL12b - IL12a - IL2 - GMCSF - F8V H -F8V L -F8V H -F8V L protein molecule.

[0117] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is fused to the N-terminus of L19V H of, H the C-terminus of L19V LFused to the N-terminus of, L19V L The C-terminus of is L19V H Fused to the N-terminus of, L19V H The C-terminus of is L19V L Fused to the N-terminus of, L19V L The C-terminus of is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL2, resulting in IL12b-IL12a-L19V H- L19V L -L19V H -L19V L -GMCSF-IL2 protein molecules may be formed.

[0118] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is NHS76V H Fused to the N-terminus of, NHS76V H The C-terminus of is NHS76V L Fused to the N-terminus of, NHS76V L The C-terminus of is NHS76V H Fused to the N-terminus of, NHS76V H The C-terminus of is NHS76V L Fused to the N-terminus of, NHS76V L The C-terminus of is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL2, resulting in IL12b-IL12a-NHS76V H -NHS76V L -NHS76V H -NHS76V L -GMCSF-IL2 protein molecules may be formed.

[0119] For example, the structure of the protein molecule is such that the C-terminus of IL12b is fused to the N-terminus of IL12a, and the C-terminus of IL12a is NHS76V H Fused to the N-terminus of, NHS76V H The C-terminus of is F8V L Fused to the N-terminus of, F8V L The C-terminus of is F8V H Fused to the N-terminus of, F8V H The C-terminus of is NHS76V L Fused to the N-terminus of, NHS76V LThe C-terminus of [protein name] is fused to the N-terminus of GMCSF, and the C-terminus of GMCSF is fused to the N-terminus of IL2, so that an IL12b-IL12a-NHS76V H -F8V L -F8V H -NHS76V L -GMCSF-IL2 protein molecule may be formed. In the present application, the protein molecule may include any one of the amino acid sequences shown in the following group: SEQ ID NO: 49-71.

[0120] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mL19VHm, and the C-terminus of mL19V H is fused to the N-terminus of mL19V L and the C-terminus of mL19V L is fused to the N-terminus of mL19V H and the C-terminus of mL19V H is fused to the N-terminus of mL19V L and the C-terminus of mL19V L is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, so that an mIL12b-mIL12a-mL19V H -mL19V L -mL19V H -mL19V L -mIL2-mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 49) may be formed.

[0121] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mF8V H and the C-terminus of mF8V H is fused to the N-terminus of mF8V L and the C-terminus of mF8V L is fused to the N-terminus of mF8V H and the C-terminus of mF8V H is fused to the N-terminus of mF8V L and the C-terminus of mF8V LThe C-terminus of [protein name] is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mF8V H -mF8V L -mF8V H -mF8V L -mIL2-mGMCSF protein molecules (the amino acid sequence of which may be shown in SEQ ID NO.50) may be formed.

[0122] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, and the C-terminus of mIL12a is fused to the N-terminus of mNHS76V H of the N-terminus, and the C-terminus of mNHS76V H is fused to the N-terminus of mNHS76V L of the N-terminus, and the C-terminus of mNHS76V L is fused to the N-terminus of mNHS76V H of the N-terminus, and the C-terminus of mNHS76V H is fused to the N-terminus of mNHS76V L of the N-terminus, and the C-terminus of mNHS76V L is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mNHS76V H -mNHS76V L -mNHS76V H -mNHS76V L -mIL2-mGMCSF protein molecules (the amino acid sequence of which may be shown in SEQ ID NO.51) may be formed.

[0123] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, and the C-terminus of mIL12a is fused to the N-terminus of mNHS76V H of the N-terminus, and the C-terminus of mNHS76V H is fused to the N-terminus of mF8V L of the N-terminus, and the C-terminus of mF8V L is fused to the N-terminus of mF8V H of the N-terminus, and the C-terminus of mF8V H is fused to the N-terminus of mNHS76V L of the N-terminus, and the C-terminus of mNHS76V LThe C-terminus of [molecule name] is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mNHS76V H -mF8V L -mF8V H -mNHS76V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID No. 52) may be formed.

[0124] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mNHS76V H the N-terminus of, mNHS76V H the C-terminus of is fused to the N-terminus of mL19V L the N-terminus of, mL19V L the C-terminus of is fused to the N-terminus of mL19V H the N-terminus of, mL19V H the C-terminus of is fused to the N-terminus of mNHS76V L the N-terminus of, mNHS76V L the C-terminus of is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mNHS76V H -mL19V L -mL19V H -mNHS76V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 53) may be formed.

[0125] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mF8V H the N-terminus of, mF8V H the C-terminus of is fused to the N-terminus of mNHS76V L the N-terminus of, mNHS76V L the C-terminus of is fused to the N-terminus of mNHS76V H the N-terminus of, mNHS76V H the C-terminus of is fused to the N-terminus of mF8V L the N-terminus of, mF8V LThe C-terminus of [protein name] is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mF8V H -mNHS76V L -mNHS76V H -mF8V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.54) may be formed.

[0126] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, and the C-terminus of mIL12a is fused to the N-terminus of mF8V H and the C-terminus of mF8V H is fused to the N-terminus of mL19V L and the C-terminus of mL19V L is fused to the N-terminus of mL19V H and the C-terminus of mL19V H is fused to the N-terminus of mF8V L and the C-terminus of mF8V L is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mF8V H -mL19V L -mL19V H -mF8V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.55) may be formed.

[0127] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, and the C-terminus of mIL12a is fused to the N-terminus of mL19V H and the C-terminus of mL19V H is fused to the N-terminus of mNHS76V L and the C-terminus of mNHS76V L is fused to the N-terminus of mNHS76V H and the C-terminus of mNHS76V H is fused to the N-terminus of mL19V L and the C-terminus of mL19V LThe C-terminus of [protein name] is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of GMCSF, resulting in mIL12b-mIL12a-mL19V H -mNHS76V L -mNHS76V H -mL19V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.56) may be formed.

[0128] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mL19V H of, the N-terminus of mL19V H C-terminus is fused to the N-terminus of mF8V L of, the N-terminus of mF8V L C-terminus is fused to the N-terminus of mF8V H of, the N-terminus of mF8V H C-terminus is fused to the N-terminus of mL19V L of, the N-terminus of mL19V L The C-terminus of [protein name] is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mL19V H -mF8V L -mF8V H -mL19V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.57) may be formed.

[0129] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mNHS76V H of, the N-terminus of mNHS76V H C-terminus is fused to the N-terminus of mF8V L of, the N-terminus of mF8V L C-terminus is fused to the N-terminus of mF8V H of, the N-terminus of mF8V H C-terminus is fused to the N-terminus of mNHS76V L of, the N-terminus of mNHS76V LBy fusing the C-terminus of [protein name] with the N-terminus of mGMCSF, mIL12b-mIL12a-mNHS76V H -mF8V L -mF8V H -mNHS76V L -mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO.58) may be formed.

[0130] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, the C-terminus of mIL2 is fused to the N-terminus of mF8V H of, the C-terminus of mF8V H is fused to the N-terminus of mF8V L of, the C-terminus of mF8V L is fused to the N-terminus of mF8V H of, the C-terminus of mF8V H is fused to the N-terminus of mF8V L of, the C-terminus of mF8V L is fused to the N-terminus of mGMCSF, resulting in mIL12b-mIL12a-mIL2-mF8V H -mF8V L -mF8V H -mF8V L -mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO.59) may be formed.

[0131] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, the C-terminus of mGMCSF is fused to the N-terminus of mNHS76V H of, the C-terminus of mNHS76V H is fused to the N-terminus of mF8V L of, the C-terminus of mF8V L is fused to the N-terminus of mF8V H of, the C-terminus of mF8V H is fused to the N-terminus of mNHS76V L resulting in mIL12b-mIL12a-mIL2-mGMCSF-mNHS76VH -mF8V L -mF8V H -mNHS76V L A protein molecule (its amino acid sequence may be shown in SEQ ID NO. 60) is formed.

[0132] For example, the structure of the protein molecule is such that the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, and the C-terminus of mGMCSF is fused to the N-terminus of mF8V H of, the C-terminus of mF8V H is fused to the N-terminus of mF8V L of, the C-terminus of mF8V L is fused to the N-terminus of mF8V H of, the C-terminus of mF8V H is fused to the N-terminus of mF8V L By fusion of the C-terminus to the N-terminus, mIL12b - mIL12a - mIL2 - mGMCSF - mF8V H -mF8V L -mF8V H -mF8V L A protein molecule (its amino acid sequence may be shown in SEQ ID NO. 61) may be formed.

[0133] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hL19V H of, the C-terminus of hL19V H is fused to the N-terminus of hL19V L of, the C-terminus of hL19V L is fused to the N-terminus of hL19V H of, the C-terminus of hL19V H is fused to the N-terminus of hL19V L of, the C-terminus of hL19V L is fused to the N-terminus of hIL2, and by fusion of the C-terminus of hIL2 to the N-terminus of hGMCSF, hIL12b - hIL12a - hL19V H -hL19V L -hL19V H -hL19V L-hIL2-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.62) may be formed.

[0134] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hNHS76V H the C-terminus of hNHS76V H is fused to the N-terminus of hNHS76V L the C-terminus of hNHS76V L is fused to the N-terminus of hNHS76V H the C-terminus of hNHS76V H is fused to the N-terminus of hNHS76V L the C-terminus of hNHS76V L is fused to the N-terminus of hIL2, and the C-terminus of hIL2 is fused to the N-terminus of hGMCSF, resulting in the formation of an hIL12b-hIL12a-hNHS76V H -hNHS76V L -hNHS76V H -hNHS76V L -hIL2-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.63) may be formed.

[0135] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hNHS76V H the C-terminus of hNHS76V H is fused to the N-terminus of hF8V L the C-terminus of hF8V L is fused to the N-terminus of hF8V H the C-terminus of hF8V H is fused to the N-terminus of hNHS76V L the C-terminus of hNHS76V L is fused to the N-terminus of hIL2, and the C-terminus of hIL2 is fused to the N-terminus of hGMCSF, resulting in the formation of an hIL12b-hIL12a-hNHS76V H -hF8V L -hF8V H -hNHS76V L-hIL2-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 64) may be formed.

[0136] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL2, the C-terminus of hIL2 is fused to the N-terminus of hNHS76V H , the C-terminus of hNHS76V H is fused to the N-terminus of hF8V L , the C-terminus of hF8V L is fused to the N-terminus of hF8V H , the C-terminus of hF8V H is fused to the N-terminus of hNHS76V L , the C-terminus of hNHS76V L is fused to the N-terminus of hGMCSF, thereby forming hIL12b-hIL12a-hIL2-hNHS76V H -hF8V L -hF8V H -hNHS76V L -hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 65) is formed.

[0137] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL2, the C-terminus of hIL2 is fused to the N-terminus of hF8V H , the C-terminus of hF8V H is fused to the N-terminus of hF8V L , the C-terminus of hF8V L is fused to the N-terminus of hF8V H , the C-terminus of hF8V H is fused to the N-terminus of hF8V L , the C-terminus of hF8V L is fused to the N-terminus of hF8V H -hF8V L -hF8V H -hF8V L-hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 66) may be formed.

[0138] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hL19V H , the C-terminus of hL19V H is fused to the N-terminus of hL19V L , the C-terminus of hL19V L is fused to the N-terminus of hL19V H , the C-terminus of hL19V H is fused to the N-terminus of hL19V L , the C-terminus of hL19V L is fused to the N-terminus of hGMCSF, and the C-terminus of hGMCSF is fused to the N-terminus of hIL2, resulting in an hIL12b-hIL12a-hL19V H -hL19V L -hL19V H -hL19V L -hGMCSF-hIL2 protein molecule (its amino acid sequence may be shown in SEQ ID NO. 67) may be formed.

[0139] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hNHS76V H , the C-terminus of hNHS76V H is fused to the N-terminus of hNHS76V L , the C-terminus of hNHS76V L is fused to the N-terminus of hNHS76V H , the C-terminus of hNHS76V H is fused to the N-terminus of hNHS76V L , the C-terminus of hNHS76V L is fused to the N-terminus of hGMCSF, and the C-terminus of hGMCSF is fused to the N-terminus of hIL2, resulting in an hIL12b-hIL12a-hNHS76V H -hNHS76V L -hNHS76V H -hNHS76V LA protein molecule of -hGMCSF-hIL2 (the amino acid sequence thereof may be shown in SEQ ID NO.68) may be formed.

[0140] For example, the structure of the protein molecule is such that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hNHS76V H the C-terminus of hNHS76V H is fused to the N-terminus of hF8V L the C-terminus of hF8V L is fused to the N-terminus of hF8V H the C-terminus of hF8V H is fused to the N-terminus of hNHS76V L the C-terminus of hNHS76V L is fused to the N-terminus of hGMCSF, and the C-terminus of hGMCSF is fused to the N-terminus of hIL2, so that hIL12b-hIL12a-hNHS76V H -hF8V L -hF8V H -hNHS76V L -hGMCSF-hIL2 protein molecule (the amino acid sequence thereof may be shown in SEQ ID NO.69) may be formed.

[0141] In the present application, nnmIL12b-mIL12a-mL19V H -mL19V L -mL19V H -mL19V L -mIL2-mGMCSF, mIL12b-mIL12a-mF8V H -mF8V L -mF8V H -mF8V L -mIL2-mGMCSF, mIL12b-mIL12a-mNHS76V H -mNHS76V L -mNHS76V H -mNHS76V L -mIL2-mGMCSF, mIL12b-mIL12a-mNHS76V H -mF8V L -mF8V H -mNHS76V L-mIL2-mGMCSF, mIL12b-mIL12a-mNHS76V H -mL19V L -mL19V H -mNHS76V L -mIL2-mGMCSF, mIL12b-mIL12a-mF8V H -mNHS76V L -mNHS76V H -mF8V L -mIL2-mGMCSF, mIL12b-mIL12a-mF8V H -mL19V L -mL19V H -mF8V L -mIL2-mGMCSF, mIL12b-mIL12a-mL19V H -mNHS76V L -mNHS76V H -mL19V L -mIL2-mGMCSF, mIL12b-mIL12a-mL19V H -mF8V L -mF8V H -mL19V L -mIL2-mGMCSF, mIL12b-mIL12a-mIL2-mNHS76V H -mF8V L -mF8V H -mNHS76V L -mGMCSF, mIL12b-mIL12a-mIL2-mF8V H -mF8V L -mF8V H -mF8V L -mGMCSF, mIL12b-mIL12a-mIL2-mGMCSF-mNHS76V H -mF8V L -mF8V H -mNHS76V L 、mIL12b-mIL12a-mIL2-mGMCSF-mF8V H -mF8V L -mF8V H -mF8V L 、hIL12b-hIL12a-hL19V H -hL19V L -hL19V H-hL19V L -hIL2 - hGMCSF, hIL12b - hIL12a - hNHS76V H -hNHS76V L -hNHS76V H -hNHS76V L -hIL2 - hGMCSF, hIL12b - hIL12a - hNHS76V H -hF8V L -hF8V H -hNHS76V L -hIL2 - hGMCSF, hIL12b - hIL12a - hNHS76V H -hF8V L -hF8V H -hNHS76V L -hIL2 - hGMCSF, hIL12b - hIL12a - hIL2 - hF8V H -hF8V L -hF8V H -hF8V L -hGMCSF, hIL12b - hIL12a - hL19V H -hL19V L -hL19V H -hL19V L -hGMCSF - hIL2, hIL12b - hIL12a - hNHS76V H -hNHS76V L -hNHS76V H -hNHS76V L -hGMCSF - hIL2, hIL12b - hIL12a - hNHS76V H -hF8V L -hF8V H -hNHS76V L-hGMCSF-hIL2 can be abbreviated in order as follows: mIL12bIL12aDiaL19IL2GMCSF, mIL12bIL12aDiaF8IL2GMCSF, mIL12bIL12aDiaNHS76IL2GMCSF, mIL12bIL12aDiaNHS76F8IL2GMCSF, mIL12bIL12aDiaNHS76L19IL2GMCSF, mIL12bIL12aDiaF8NHS76IL2GMCSF, mIL12bIL12aDiaF8L19IL2GMCSF, mIL12bIL12aDiaL19NHS76IL2GMCSF, mIL12bIL12aDiaL19F8IL2GMCSF, mIL12bIL12aIL2DiaNHS76F8GMCSF, mIL12bIL12aIL2DiaF8GMCSF, mIL12bIL12aIL2GMCSFDiaNHS76F8, mIL12bIL12aIL2GMCSFDiaF8, hIL12bIL12aDiaL19IL2GMCSF, hIL12bIL12aDiaNHS76IL2GMCSF, hIL12bIL12aDiaNHS76F8IL2GMCSF, hIL12bIL12aIL2DiaNHS76F8GMCSF, hIL12bIL12aIL2DiaF8GMCSF, hIL12bIL12aDiaL19GMCSFIL2, hIL12bIL12aDiaNHS76GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2.

[0142] In addition, the proteins, polypeptides and / or amino acid sequences related to this application are understood to include at least the following ranges: variants or homologs having the same or similar functions as the said proteins or polypeptides.

[0143] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added to the amino acid sequence of the protein and / or the polypeptide (e.g., the protein molecule). For example, the functional variant may consist of a protein or polypeptide that already has amino acid changes due to at least 1, for example, 1 to 30, 1 to 20 or 1 to 10, for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially maintain the biological properties of the protein or polypeptide before the modification (e.g., substitution, deletion or addition). For example, the functional variant may maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the protein or polypeptide before the modification.

[0144] In the present application, the homolog may be, for example, a protein having at least about 80% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., the protein molecule). Or a polypeptide.

[0145] In the present application, homology is generally defined as the similarity, resemblance or relatedness between two or more sequences. The "percentage of sequence homology" can be calculated by comparing two sequences to be compared with a comparison window and determining the presence or absence of the same nucleobases (such as A, T, C, G, I, etc.) or the same amino acid residues (such as Ala, Pro, Ser, Thr, Gly, Val, etc.). The number of matching positions is determined from (Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, Met), and the number of matching positions is divided by the total number of positions (window size) within the comparison window, and the result is multiplied by 100 to calculate the percentage of sequence homology. For example, comparisons can be made using commonly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), etc. to determine the percentage of sequence homology. A person skilled in the art can determine appropriate parameters for comparing sequences, including the algorithms necessary to perform the maximum comparison within the range of the full-length sequence to be compared or within the target sequence region. The homology can also be determined by methods such as FASTA and BLAST. The description of the FASTA algorithm is in W.R. Pearson and D.J. Lipman, "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Person, "Rapid and sensitive protein similarity searches. Science", 227: 1435-1441, 1989. The description of the FASTA algorithm is in S. Altschul, W. Gish, W. Miller, E.W. Myers and D Lipman, "Basic local alignment search tool", Journal of Molecular Biology, 215: 403-410, 1990. On the other hand, the present application provides a nucleotide molecule encoding a protein molecule as described. In the present application, the nucleotide molecule consisting of any one of the nucleotide sequences selected from the following groups: SEQ ID NO: 73 - 112 is used. On the other hand, the present application provides a vector containing the nucleotide molecule.

[0146] For the purposes of the present application, methods for constructing vectors and plasmids, for example, methods for inserting a gene encoding a protein into a vector or plasmid, and methods for introducing a plasmid into a host cell, are well known to those skilled in the art and are described in many publications, such as Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. On the other hand, the present invention provides a cell that expresses the protein molecule, contains the nucleotide molecule, or contains the vector.

[0147] In the present application, the cell can be used for the treatment of tumors. In the present application, the tumor may consist of solid tumors. For example, the tumor may consist of melanoma. On the other hand, the present application provides a method for preparing the protein molecule, the method including the following step: culturing the cell. In the present application, the method can be carried out in the context that the protein molecule can be expressed. On the other hand, the present application provides a pharmaceutical composition containing the protein molecule.

[0148] In the present application, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier may contain a buffering agent, an antioxidant, a preservative, a low molecular weight peptide, a protein, a hydrophilic polymer, an amino acid, a saccharide, a chelating agent, a counter ion, a metal complex and / or a nonionic surfactant, etc. For example, the pharmaceutically acceptable carrier may contain an excipient. For example, the excipient may be selected from the group consisting of starch, dextrin, sucrose, lactose, magnesium stearate, calcium sulfate, carboxymethyl cellulose, talc, calcium alginate gel, chitosan and nanomicropheres, etc. For example, the pharmaceutically acceptable carrier may also be selected from the group consisting of a pH adjuster, an osmotic pressure adjuster, a solubilizing agent and a bacteriostatic agent.

[0149] In the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir.

[0150] In the present application, the combination of the drugs can be used to inhibit tumor growth. For example, the combined drug therapy of the present invention can suppress or delay the onset or progression of a disease, can reduce (or substantially eliminate) the tumor size by promoting the expression of cytokines, and / or can alleviate and / or stabilize the disease state.

[0151] In the present application, the combination of the drugs may contain a therapeutically effective amount of the protein molecule, the nucleic acid molecule, the vector and / or the host cell. The therapeutically effective amount means the amount necessary to (at least partially) prevent and / or treat a disease, a symptom, and its complications in a subject suffering from or at risk of developing a disease or a symptom (such as cancer). On the other hand, the present application provides the application of the protein molecule and the pharmaceutical composition in the preparation of an anti-malignant tumor agent. This application provides the protein molecule, the pharmaceutical composition, the nucleic acid molecule, the vector and / or the cell for treating tumors.

[0152] This application provides a method for alleviating or treating tumors, which may include administering the protein molecule, the drug combination, the nucleic acid molecule, the vector and / or the cell to a subject in need thereof.

[0153] In this application, the administration method may include oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir. In this application, the tumor may constitute a solid tumor. For example, the tumor may consist of melanoma.

[0154] Without being limited by any theory, the following embodiments are only intended to illustrate the protein molecule, preparation method, uses, etc. of the invention of this application, and are not intended to limit the scope of the invention of this application.

Examples

[0155] Reagents: DMEM medium, 1640 medium, fetal bovine serum (manufactured by Lifetechnologies), cell culture flasks and plates (manufactured by Corning), doxycycline (DOX) (manufactured by Shanghai Bioengineering Co. Restriction enzymes were purchased from Takara and NEB, ligase was purchased from NEB, DNA polymerase was purchased from Takara, plasmid extraction kits and gel recovery kits were purchased from OmegaBiotech, and primer synthesis was purchased from Shanghai Bioengineering Co., Ltd. respectively.

[0156] Example 1 Preparation of tumor cells expressing a modifiable protein 1.1 Construction of the initial expression vector pLentis-CMV-rtTA-IRES-Bsd The DNA sequence of the synthesis of rtTA (GenBank: ALK44378.1) with BamHI and EcoRI sites at both ends was synthesized, and the synthesized product was ligated into the vector pUC57. The pUC57 vector ligated with rtTA was digested as described. The digestion system was as follows: 6 μg of the pUC57 vector plasmid ligated with rtTA, 4 μl of digestion buffer, 1 μl of BamHI, 1 μl of EcoRI, and water was added to make a total volume of 40 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the rtTA fragment was recovered and used.

[0157] The vector pLentis-CMV-IRES-Bsd was placed in an EP tube and digested with the following digestion system: 2 μg of the pLentis-CMV-IRES-Bsd vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of EcoRI, and water was added to make a total volume of 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-CMV-IRES-Bsd vector fragment was recovered and used.

[0158] pLentis-CMV-IRES-Bsd and rtTA were ligated as follows: 2 μl of pLentis-CMV-IRES-Bsd, 2 μl of rtTA, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, 4.5 μl of water, and left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate, placed in LB medium, and cultured overnight at 37 °C with shaking. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was confirmed by enzymatic digestion whether the rtTA fragment was successfully ligated to the pLentis-CMV-IRES-Bsd vector. Then, the correct vector was sent for sequencing, and it was confirmed that the construction of the first expression vector pLentis-CMV-rtTA-IRES-Bsd was successful.

[0159] 1.2 Preparation of virus for the first expression vector pLentis-CMV-rtTA-IRES-Bsd 1) Count the digested and cultured 293FT cells, spread 3×106 cells / well in a 10-cm culture dish, and add 10 ml of culture medium. 2) Observe the cell state the next night. If the state is appropriately good, perform transfection. Add chloroquine to the culture plate to a final concentration of 25 μM. Take one tube, sterilize the water, add the following plasmids (5 μg of pMD2.G + 15 μg of pSPAX2 + 20 μg of pLentis-CMV-rtTA-IRES-Bsd) to make a total volume of 1045 μl, then add 155 μl of 2 M CaCl2 and mix well. Finally, shake while dropping 1200 μl of 2×HBS. After dropping, quickly add the mixed solution to the cell culture well and shake gently. 3) Observe the cells on the third morning and change the medium to 10 ml of fresh DMEM medium. 4) On the morning of the 5th day, observe the cell state, collect the supernatant of the culture dish, filter it through a 0.45 μm filter, then put it into a high-speed centrifuge tube and centrifuge at 50,000 g for 2 hours. Carefully discard the supernatant, wipe the liquid with blotting paper as much as possible, resuspend the precipitate in 500 μl of HBSS, dissolve it for 2 hours, then divide it into small tubes and store at -70 °C to obtain the first expression vector pLentis-CMV-rtTA-IRES-Bsd virus. 1.3 Infect the B16 tumor cells with the virus of the first expression vector pLentis-CMV-rtTA-IRES-Bsd.

[0160] Inoculate the digested and cultured mouse melanoma cells B16 into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, add 10 μl of the virus of the first expression vector pLentis-CMV-rtTA-IRES-Bsd and continue culturing in an incubator for 24 hours. Discard the supernatant and replace it with fresh medium to continue culturing. When the cells have completely grown, transfer this cell to a culture flask, add blasticidin at a concentration suitable for this cell and continue culturing. Replace the medium every 2 days and maintain the blasticidin concentration at 8 μg / ml. As a result of one week of screening, the surviving cells are cells that stably express the control protein, and this cell was named B16(rtTA).

[0161] Example 2 Influence of induction of green fluorescent protein (GFP) expression on tumor growth 2.1 Construction of an inducible expression vector encoding green fluorescent protein (GFP) The GFP gene was amplified by PCR reaction using primers with the GFP gene as a template, and the PCR conditions were carried out according to the instructions of PrimeStar HS DNA polymerase. The PCR product was subjected to agarose gel electrophoresis, recovered using a gel recovery kit, and then digested with BamHI and EcoRI. The digestion system was prepared by adding water to 30 μg of the PCR recovery product, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of EcoRI to a total volume of 40 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. The GFP gene fragment was recovered and stored for use after electrophoresis.

[0162] The controlled expression vector was enzymatically cleaved with the following system: 2 μg of pLentis-PTRE-MCS-PGK-PURO plasmid, 3 μl of enzyme cleavage buffer, 1 μl of BamHI and 1 μl of EcoRI, water was added to make the total volume 30 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the vector fragment was recovered and used. pLentis-PTRE-MCS-PGK-PURO and GFP were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of GFP, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of Escherichia coli receptor state. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. The plasmid was extracted from the cultured cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-GFP-PGK-PURO was successful.

[0163] 2.2 Preparation of cells for controlled expression of GFP The virus of the GFP expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-GFP-PGK-PURO was obtained.

[0164] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-GFP-PGK-PURO was added, and the culture was continued in an incubator for 24 hours. The supernatant was discarded and replaced with fresh medium for continuous culture. When the cells were fully grown, they were transferred to a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After culturing for 3 days, the surviving cells were those that could regulate the expression of GFP, and these were named B16(rtTA)-GFP.

[0165] 2.3 Effect of GFP expression control on tumor growth The logarithmically growing B16(rtTA)-GFP cells were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl of each was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the tumor growth was recorded. As shown in Figure 1, it was found that the induced expression of GFP had no effect on suppressing tumor growth (Figure 1, each line represents the tumor area situation in one mouse), and the survival curves of each group of mice are shown in Figure 2.

[0166] Example 3 Design of protein molecules The design includes the following protein molecules: pmIL12bIL12aDiaL19IL2GMCSF (the structure of which is shown in Figure 3), mIL12bIL12aDiaF8IL2GMCSF (the structure of which is shown in Figure 4), mIL12bIL12aDiaNHS76IL2GMCSF (the structure of which is shown in Figure 5). The protein molecules also include mIL12bIL12aDiaNHS76F8IL2GMCSF (the structure of which is shown in Figure 6), mIL12bIL12aDiaNHS76L19IL2GMCSF (the structure of which is shown in Figure 7), mIL12bIL12aDiaF8NHS76IL2GMCSF (the structure of which is shown in Figure 8), and mIL12bIL12aDiaF8L19IL2GMCSF (the structure of which is shown in Figure 9), mIL12bIL12aDiaL19NHS76IL2GMCSF (the structure of which is shown in Figure 10), mIL12bIL12aDiaL19F8IL2GMCSF (the structure of which is shown in Figure 11), and mIL12bIL12aIL2DiaNHS76F8GMCSF (the structure of which is shown in Figure 12), mIL12bIL12aIL2DiaF8GMCSF (the structure of which is shown in Figure 13), mIL12bIL12aIL2GMCSFDiaNHS76F8 (the structure of which is shown in Figure 14), and mIL12bIL12aIL2GMCSFDiaF8 (the structure of which is shown in Figure 15), hIL12bIL12aDiaL19IL2GMCSF (the structure of which is shown in Figure 16), hIL12bIL12aDiaNHS76IL2GMCSF (the structure of which is shown in Figure 17), and hIL12bIL12aDiaNHS76F8IL2GMCSF (the structure of which is shown in Figure 18), hIL12bIL12aIL2DiaF8GMCSF (the structure of which is shown in Figure 20), mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr, and hIL12bIL12aDiaL19GMCSFIL2, hIL12bIL12aDiaNHS76GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2, hIL12bIL12aIL2DiaNHS76F8GMCSF (the structure of which is shown in Figure 19), hIL12bIL12aIL2DiaNHS76F8GMCSF, hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr, mIL12bIL12aIL2GMCSF,mIL12bIL12aGMCSFIL2, mIL12bIL12aIL7GMCSF, mIL12bIL12aIL15GMCSF, mIL12bIL12aIL21GMCSF, mIL12bIL12aIL2FLT3L, mIL12bIL12aIL7FLT3L, mIL12bIL12aIL15FLT3L, mIL12bIL12aIL21FLT3L, hIL12bIL12aIL2GMCSF, hIL12bIL12aIL7GMCSF, hIL12bIL12aIL15GMCSF, hIL12bIL12aIL21GMCSF, hIL12bIL12aIL2FLT3L, hIL12bIL12aIL7FLT3L, hIL12bIL12aIL15FLT3L, hIL12bILhIL12bIL12aIL21FLT3L.

[0167] For example, Figure 3 shows the structure of the protein molecule mIL12bIL12aDiaL19IL2GMCSF, where the "m" in the formula indicates that it is a cytokine derived from a mouse. In the polypeptide chain, the protein molecule has mIL12bIL12aDiaL19IL2GMCSF, which is interleukin IL12b, interleukin IL12a, targeting moiety L19V H , L19V L , L19V H , L19V L , interleukin IL2, and granulocyte macrophage colony-stimulating factor GMCSF in this order, and the targeting moiety in the order of L19V H , L19V L , L19V H , L19V L may be represented by DiaL19.

[0168] For example, Figure 4 shows the structure of the protein molecule mIL12bIL12aDiaF8IL2GMCSF, where the "m" in the formula indicates that the cytokine is derived from a mouse. In this protein molecule, mIL12bIL12aDiaF8IL2GMCSF, in order from the N-terminus to the C-terminus, is interleukin IL12b, interleukin IL12a, targeting moiety F8V H , F8V L , F8VH 、F8V L 、 interleukin IL2, granulocyte macrophage colony-stimulating factor GMCSF, and are represented by a polypeptide chain, F8V H 、F8V L 、F8V H 、F8V L The targeting portion in the order of may be represented by DiaF8.

[0169] For example, Figure 5 shows the structure of the protein molecule mIL12bIL12aDiaNHS76IL2GMCSF, where the "m" in the formula indicates that the cytokine is of mouse origin. The protein molecule contains a polypeptide chain, and in the polypeptide chain, in order from the N-terminus to the C-terminus, interleukin IL12b, interleukin IL12a, the targeting portion NHS76V H 、NHS76V L 、NHS76V H 、NHS76V L 、 interleukin IL2, granulocyte IL2, and mIL12bIL12aDiaNHS76IL2GMCSF is represented. Granulocyte macrophage colony-stimulating factor GMCSF is NHS76V H 、NHS76V L 、NHS76V H 、NHS76V L The targeting fraction can be denoted by DiaNHS76 in the order of.

[0170] For example, Figure 6 shows the structure of the protein molecule mIL12bIL12aDiaNHS76F8IL2GMCSF, where the "m" in the formula indicates that the cytokine is of mouse origin. The protein molecule consists of a polypeptide chain, and mIL12bIL12aDiaNHS76F8IL2GMCSF, in the polypeptide chain, interleukin IL12b, interleukin IL12a, the targeting portion NHS76V H 、F8V L 、F8V H 、NHS76V L, interleukin IL2, granulocyte macrophage colony-stimulating factor GMCSF, is NHS76V H , F8V L , F8V H , NHS76V L are represented in the order of, and NHS76V H , F8V L , F8V H , NHS76V L The targeting parts of can be denoted as DiaNHS76F8.

[0171] Note that the protein molecules mIL12bIL12aDiaNHS76L19IL2GMCSF (whose structure is shown in Figure 7), mIL12bIL12aDiaF8NHS76IL2GMCSF (whose structure is shown in Figure 8), mIL12bIL12aDiaF8L19IL2GMCSF (whose structure is shown in Figure 9), mIL12bIL12aDiaL19NHS76IL2GMCSF (whose structure is shown in Figure 10), and mIL12bIL12aDiaL19F8IL2GMCSF (whose structure is shown in Figure 11) have structures similar to the above protein molecules (for example, mIL12bIL12aDiaNHS76F8IL2GMCSF of the protein molecule). Since only the targeting parts are different, the specific structures of these protein molecules will not be repeated here.

[0172] For example, Figure 12 shows the structure of the protein molecule mIL12bIL12aIL2DiaNHS76F8GMCSF. In the formula, "m" indicates that the cytokine is derived from a mouse. The protein molecule consists of a polypeptide chain. In the polypeptide chain of mIL12bIL12aIL2DiaNHS76F8GMCSF, there are interleukin IL12b, interleukin IL12a, interleukin IL2, the NHS76V of the targeting part H , F8V L , F8V H , NHS76V L , granulocyte macrophage colony-stimulating factor GMCSF in this order, and NHS76V H , F8VL , F8V H , NHS76V L The targeting part of L can be denoted as DiaNHS76F8.

[0173] Note that the structure of the protein molecule mIL12bIL12aIL2DiaF8GMCSF (the structure is shown in Figure 13) is similar to the structure of the protein molecule mIL12bIL12aIL2DiaNHS76F8GMCSF which only differs in the targeting part. Therefore, the specific structure of the protein molecule mIL12bIL12aIL2DiaF8GMCSF will not be repeated here.

[0174] For example, Figure 14 shows the structure of the protein molecule mIL12bIL12aIL2GMCSFDiaNHS76F8. The "m" in the formula indicates that the cytokine is of mouse origin. The protein molecule consists of polypeptide chains. mIL12bIL12aIL2GMCSFDiaNHS76F8 contains interleukin IL12b, interleukin IL12a, interleukin IL2, granulocyte macrophage colony-stimulating factor GMCSF, and the targeting part NHS76V H , F8V L , F8V H , NHS76V L The targeting parts can be denoted as DiaNHS76F8 in the order of H , F8V, L , F8V, H , NHS76V.

[0175] Note that the structure of the protein molecule mIL12bIL12aIL2GMCSFDiaF8 (the structure is shown in Figure 15) is similar to the structure of the protein molecule mIL12bIL12aIL2GMCSFDiaNHS76F8 which only differs in the targeting part. Therefore, the specific structure of the protein molecule mIL12bIL12aIL2GMCSFDiaF8 will not be repeated here.

[0176] For example, Figure 16 shows the structure of the protein molecule hIL12bIL12aDiaL19IL2GMCSF, where the "h" in the formula indicates that the cytokine is of human origin. The protein molecule consists of polypeptide chains, and hIL12bIL12aDiaL19IL2GMCSF, in the polypeptide chain, in order from the N-terminus to the C-terminus, is interleukin IL12b, interleukin IL12a, targeting moiety L19V H , L19V L , L19V H , L19V L , interleukin IL2, granulocyte macrophage colony-stimulating factor GMCSF, and the targeting moieties of L19V H , L19V L , L19V H , L19V L are represented by DiaL19 in order.

[0177] Note that the structure of the protein molecule hIL12bIL12aDiaNHS76IL2GMCSF (the structure is shown in Figure 17) is similar to the structure of the protein molecule hIL12bIL12aDiaL19IL2GMCSF, and only the targeting moiety is different. Therefore, the specific structure of the protein molecule hIL12bIL12aDiaNHS76IL2GMCSF will not be repeated here.

[0178] For example, Figure 18 shows the structure of the protein molecule hIL12bIL12aDiaNHS76F8IL2GMCSF, where the "h" in the formula indicates that the cytokine is of human origin. The protein molecule is such that hIL12bIL12aDiaNHS76F8IL2GMCSF is interleukin IL12b, interleukin IL12a, targeting moiety NHS76V H , F8V L , F8V H , NHS76V L , interleukin IL2, granulocyte macrophage colony-stimulating factor GMCSF, and NHS76V H , F8V L , F8V H , NHS76VL are represented in the polypeptide chain in the order of, NHS76V H , F8V L , F8V H , NHS76V L The targeting moiety can be represented by DiaNHS76F8 in the order of.

[0179] For example, FIG. 19 shows the structure of the protein molecule hIL12bIL12aIL2DiaNHS76F8GMCSF, where the "h" in the formula indicates that the cytokine is of human origin. The protein molecule is such that hIL12bIL12aIL2DiaNHS76F8GMCSF is interleukin IL12b, interleukin IL12a, interleukin IL2, targeting moiety NHS76V H , F8V L , F8V H , NHS76V L and granulocyte macrophage colony-stimulating factor GMCSF in the order of polypeptide chains, and the targeting moiety can be represented by DiaNHS76F8.

[0180] Note that the structure of the protein molecule hIL12bIL12aIL2DiaF8GMCSF (the structure is shown in FIG. 20) is similar to the structure of the protein molecule hIL12bIL12aIL2DiaNHS76F8GMCSF, and only the targeting moiety is different. Therefore, the specific structure of the protein molecule hIL12bIL12aIL2DiaF8GMCSF will not be repeated here.

[0181] Example 4 Effect of Inducible Expression of mIL12bIL12aDiaL19IL2GMCSF on Tumor Growth 4.1 Construction of mIL12bIL12aDiaL19IL2GMCSF Regulated Expression Vector The coding sequence of the mIL12bIL12aDiaL19IL2GMCSF gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was added with 5 μg of the mIL12bIL12aDiaL19IL2GMCSF plasmid, 4 μl of the digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 40 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaL19IL2GMCSF gene fragment was recovered and used.

[0182] The amino acid sequence of the mIL12bIL12aDiaL19IL2GMCSF protein molecule is shown in SEQ ID NO.49, and the nucleotide sequence encoding the mIL12bIL12aDiaL19IL2GMCSF is shown in SEQ ID NO.90.

[0183] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of the digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to make the total volume 30 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0184] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaL19IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaL19IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaL19IL2GMCSF-PGK-PURO was successful.

[0185] 4.2 Preparation of cells that regulatively express mIL12bIL12aDiaL19IL2GMCSF The virus of the mIL12bIL12aDiaL19IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaL19IL2GMCSF-PGK-PURO was obtained.

[0186] The digested and cultured B16(rtTA) tumor cells were inoculated into 6-well plates at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second control expression vector pLentis-PTRE-mIL12bIL12aDiaL19IL2GMCSF-PGK-PURO was added, and the cells were further cultured in an incubator for 24 hours. After 24 hours of culture, the supernatant was discarded and replaced with fresh medium. When the cells had completely grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After 3 days of culture, the surviving cells were those capable of controlling the expression of mIL12bIL12aDiaL19IL2GMCSF, and were named B16(rtTA)-mIL12bIL12aDiaL19IL2GMCSF.

[0187] 4.3 Effect of the induced expression of mIL12bIL12aDiaL19IL2GMCSF on tumor growth B16(rtTA)-mIL12bIL12aDiaL19IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl of the cell suspension was injected into the right dorsal side of 8- to 10-week-old female C57BL / 6 mice using a 1-ml syringe, one mouse per 50 μl. A total of 10 mice were used to grow tumors using water containing 2 g / L doxycycline. mIL12bscF8-IL2IL12aGMCSF was able to induce tumor regression in some mice, as shown in Figure 21.

[0188] Example 5 Effect of the induced expression of mIL12bIL12aDiaF8IL2GMCSF on tumor growth 5.1 Construction of the mIL12bIL12aDiaF8IL2GMCSF-adjusted expression vector The coding sequence of the mIL12bIL12aDiaF8IL2GMCSF gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was prepared by adding 5 μg of the mIL12bIL12aDiaF8IL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and adding water to make the total volume 40 μl, then leaving it at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaF8IL2GMCSF gene fragment was recovered and used.

[0189] The amino acid sequence of the mIL12bIL12aDiaF8IL2GMCSF protein molecule is shown in SEQ ID NO.50, and the nucleotide sequence encoding the mIL12bIL12aDiaF8IL2GMCSF is shown in SEQ ID NO.91.

[0190] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, adding water to make the total volume 30 μl, and leaving it at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0191] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaF8IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaF8IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaF8IL2GMCSF-PGK-PURO was successful.

[0192] 5.2 Preparation of cells that regulatively express mIL12bIL12aDiaF8IL2GMCSF The virus of the mIL12bIL12aDiaF8IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaF8IL2GMCSF-PGK-PURO was obtained.

[0193] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second control expression vector pLentis-PTRE-mIL12bIL12aDiaF8IL2GMCSF-PGK-PURO was added, and the cells were further cultured in an incubator for 24 hours. After 24 hours of culture, the supernatant was discarded and replaced with fresh medium. When the cells had completely grown, they were passaged into a culture flask, and puromycin at a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the surviving cells were those capable of controlling the expression of mIL12bIL12aDiaF8IL2GMCSF, and were named B16(rtTA)-mIL12bIL12aDiaF8IL2GMCSF.

[0194] 5.3 Effect of Inducible Expression of mIL12bIL12aDiaF8IL2GMCSF on Tumor Growth The B16(rtTA)-mIL12bIL12aDiaF8IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. A total of 10 mice were used to grow tumors with water containing 2 g / L doxycycline. As shown in Figure 22, mIL12bIL12aDiaF8IL2GMCSF was able to induce tumor regression in all mice.

[0195] Example 6 Effect of Inducible Expression of mIL12bIL12aDiaNHS76IL2GMCSF on Tumor Growth 6.1 Construction of mIL12bIL12aDiaNHS76IL2GMCSF Control Expression Vector The coding sequence of the mIL12bIL12aDiaNHS76IL2GMCSF gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of the mIL12bIL12aDiaNHS76IL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 40 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaNHS76IL2GMCSF gene fragment was recovered and used.

[0196] The amino acid sequence of the mIL12bIL12aDiaNHS76IL2GMCSF protein molecule is shown in SEQ ID NO.51, and the nucleotide sequence encoding the mIL12bIL12aDiaNHS76IL2GMCSF is shown in SEQ ID NO.92.

[0197] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0198] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaNHS76IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaNHS76IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was confirmed by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76IL2GMCSF-PGK-PURO was successful.

[0199] 6.2 Preparation of cells that regulate the expression of mIL12bIL12aDiaNHS76IL2GMCSF The virus of the mIL12bIL12aDiaNHS76IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76IL2GMCSF-PGK-PURO.

[0200] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above-described second control expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76IL2GMCSF-PGK-PURO virus was added, and the culture was continued in an incubator. After 24 hours, the supernatant was discarded, replaced with fresh medium, and the culture was continued. When the cells had completely grown, they were passaged into a culture flask, and puromycin at a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the surviving cells were those capable of controlling the expression of mIL12bIL12aDiaNHS76IL2GMCSF, and these cells were named B16(rtTA)--mIL12bIL12aDiaNHS76IL2GM.

[0201] 6.3 Effect of Induced Expression of mIL12bIL12aDiaNHS76IL2GMCSF on Tumor Growth Effect of Induced Expression of mIL12bIL12aDiaNHS76IL2GMCSF on Tumor Growth The B16(rtTA)-mIL12bIL12aDiaNHS76IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and tumors were grown using water containing 2 g / L doxycycline. Then, 50 μl / each, for a total of 10 injections, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 23, mIL12bIL12aDiaNHS76IL2GMCSF was able to induce tumor regression in all mice.

[0202] Example 7 Effect of Induced Expression of mIL12bIL12aDiaNHS76F8IL2GMCSF on Tumor Growth 7.1 Construction of mIL12bIL12aDiaNHS76F8IL2GMCSF-Regulated Expression Vector The coding sequence of the mIL12bIL12aDiaNHS76F8IL2GMCSF gene was synthesized to have digestion sites for BamHI, BglII, XhoI, and EcoRI at both ends. After digestion with BamHI or BglII and XhoI or EcoRI, 5 μg of the mIL12bIL12aDiaNHS76F8IL2GMCSF plasmid, 4 μl of enzyme digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water were added to make a total volume of 40 μl, and the mixture was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaNHS76F8IL2GMCSF gene fragment was recovered and used.

[0203] The amino acid sequence of the mIL12bIL12aDiaNHS76F8IL2GMCSF protein molecule is shown in SEQ ID NO.52, and the nucleotide sequence encoding the mIL12bIL12aDiaNHS76F8IL2GMCSF is shown in SEQ ID NO.93.

[0204] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI. Water was added to make a total volume of 30 μl, and the mixture was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0205] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaNHS76F8IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaNHS76F8IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and the second expression vector pLentis-PTRE- was determined. It was confirmed that the construction of mIL12bIL12aDiaNHS76F8IL2GMCSF-PGK-PURO was successful.

[0206] 7.2 Preparation of cells that regulatively express mIL12bIL12aDiaNHS76F8IL2GMCSF The virus of the mIL12bIL12aDiaNHS76F8IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76F8IL2GMCSF-PGK-PURO was obtained.

[0207] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second modified expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76F8IL2GMCSF-PGK-PURO was added, and the incubation was continued. After culturing for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. When the cells had completely grown, they were subcultured into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After continuing the culture for 3 days, the surviving cells were those capable of regulating the expression of mIL12bIL12aDiaNHS76F8IL2GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aDiaNHS76F8IL2GMCSF.

[0208] 7.3 Effect of Induced Expression of mIL12bIL12aDiaNHS76F8IL2GMCSF on Tumor Growth The B16(rtTA)-mIL12bIL12aDiaNHS76F8IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and after tumors had grown using water containing 2 g / L of a potent component, 50 μl per mouse, for a total of 10 mice, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 24, mIL12bIL12aDiaNHS76F8IL2GMCSF was able to induce tumor regression in all mice.

[0209] Example 8 Effect of Induced Expression of mIL12bIL12aDiaNHS76L19IL2GMCSF on Tumor Growth 8.1 Construction of the mIL12bIL12aDiaNHS76L19IL2GMCSF-Regulated Expression Vector Enzyme cleavage sites of BamHI, BglII, XhoI, and EcoRI were synthesized at both ends of the coding sequence of the mIL12bIL12aDiaNHS76L19IL2GMCSF gene and digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of the mIL12bIL12aDiaNHS76L19IL2GMCSF plasmid, 4 μl of the enzyme cleavage buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 40 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaNHS76L19IL2GMCSF gene fragment was recovered and used.

[0210] The amino acid sequence of the mIL12bIL12aDiaNHS76L19IL2GMCSF protein molecule is shown in SEQ ID NO.53, and the nucleotide sequence encoding the mIL12bIL12aDiaNHS76L19IL2GMCSF is shown in SEQ ID NO.94.

[0211] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of the digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to make the total volume 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0212] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaNHS76L19IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaNHS76L19IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and after confirming by enzymatic digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76L19IL2GMCSF-PGK-PURO was successful.

[0213] 8.2 Preparation of cells that regulatively express mIL12bIL12aDiaNHS76L19IL2GMCSF The virus of the aforementioned mIL12bIL12aDiaNHS76L19IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76L19IL2GMCSF-PGK-PURO. Digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second modified expression vector pLentis-PTRE-mIL12bIL12aDiaNHS76L19IL2GMCSF-PGK-PURO was added. After culturing in an incubator for 24 hours, the supernatant was discarded and replaced with fresh medium. When the cells had completely grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After culturing for 3 days, the surviving cells were cells capable of controlling the expression of mIL12bIL12aDiaNHS76L19IL2GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aDiaNHS76L19IL2GMCSF.

[0214] 8.3 Effect of mIL12bIL12aDiaNHS76L19IL2GMCSF expression induction on tumor growth B16(rtTA)-mIL12bIL12aDiaNHS76L19IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and tumors were grown using water containing 2 g / L. Then, 50 μl each, a total of 10, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 25, mIL12bIL12aDiaNHS76L19IL2GMCSF was able to induce tumor regression in all mice.

[0215] Example 9 Effect of inducible expression of mIL12bIL12aDiaF8NHS76IL2GMCSF on tumor growth 9.1 Construction of mIL12bIL12aDiaF8NHS76IL2GMCSF-adjusted expression vector The coding sequence of the mIL12bIL12aDiaF8NHS76IL2GMCSF gene was synthesized, and digestion sites for BamHI, BglII, XhoI, and EcoRI were provided at both ends. It was digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of the mIL12bIL12aDiaF8NHS76IL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 40 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaF8NHS76IL2GMCSF gene fragment was recovered and used.

[0216] The amino acid sequence of the mIL12bIL12aDiaF8NHS76IL2GMCSF protein molecule is shown in SEQ ID NO.54, and the nucleotide sequence encoding the mIL12bIL12aDiaF8NHS76IL2GMCSF is shown in SEQ ID NO.95.

[0217] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0218] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaF8NHS76IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaF8NHS76IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and after confirming by enzymatic digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaF8NHS76IL2GMCSF-PGK-PURO was successful.

[0219] 9.2 Preparation of cells that regulatively express mIL12bIL12aDiaF8NHS76IL2GMCSF The virus of the mIL12bIL12aDiaF8NHS76IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaF8NHS76IL2GMCSF-PGK-PURO was obtained.

[0220] 10 μl of the above virus of mIL12bIL12aDiaF8NHS76IL2GMCSF-PGK-PURO was added, and the incubator was continued. After culturing for 24 hours, the supernatant was discarded and replaced with fresh medium to continue culturing. When the cells were fully grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added and cultured for 3 days. As a result, the surviving cells were cells that could control the expression of mIL12bIL12aDiaF8NHS76IL2GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aDiaF8NHS76IL2GMCSF.

[0221] 9.3 Influence of the Induced Expression of mIL12bIL12aDiaF8NHS76IL2GMCSF on Tumor Growth B16(rtTA)-mIL12bIL12aDiaF8NHS76IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and after tumors grew using water containing 2 g / L of a potent component, 50 μl per piece, for a total of 10 pieces, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 26, mIL12bIL12aDiaF8NHS76IL2GMCSF was able to induce tumor regression in some mice.

[0222] Example 10 Effect of the Induced Expression of mIL12bIL12aDiaF8L19IL2GMCSF on Tumor Growth 10.1 Construction of the mIL12bIL12aDiaF8L19IL2GMCSF-Regulated Expression Vector Digestion sites of BamHI, BglII, XhoI, and EcoRI were synthesized at both ends of the mIL12bIL12aDiaF8L19IL2GMCSF gene coding sequence and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was composed of 5 μg of mIL12bIL12aDiaF8L19IL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water added to a total volume of 40 μl, and left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaF8L19IL2GMCSF gene fragment was recovered and used.

[0223] The amino acid sequence of the mIL12bIL12aDiaF8L19IL2GMCSF protein molecule is shown in SEQ ID NO.55, and the nucleotide sequence encoding the mIL12bIL12aDiaF8L19IL2GMCSF is shown in SEQ ID NO.96.

[0224] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to a total volume of 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0225] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaF8L19IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaF8L19IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaF8L19IL2GMCSF-PGK-PURO was successful.

[0226] 10.2 Preparation of cells that regulatively express mIL12bIL12aDiaF8L19IL2GMCSF The virus of the mIL12bIL12aDiaF8L19IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaF8L19IL2GMCSF-PGK-PURO was obtained.

[0227] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second control expression vector pLentis-PTRE-mIL12bIL12aDiaF8L19IL2GMCSF-PGK-PURO was added, and after continuing the culture in an incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. When the cells were fully grown, they were subcultured into a culture flask, and puromycin at a final concentration of 3 μg / ml was added to continue the culture for 3 days. The surviving cells were those capable of controlling the expression of mIL12bIL12aDiaF8L19IL2GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aDiaF8L19IL2GMCSF.

[0228] 10.3 Influence of mIL12bIL12aDiaF8L19IL2GMCSF expression induction on tumor growth The B16(rtTA)-mIL12bIL12aDiaF8L19IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and after growing tumors using water containing 2 g / L doxycycline, 50 μl / each, a total of 10 were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 27, mIL12bIL12aDiaF8L19IL2GMCSF was able to induce tumor regression in some mice.

[0229] Example 11 Effect of inducible expression of mIL12bIL12aDiaL19NHS76IL2GMCSF on tumor growth 11.1 Construction of the mIL12bIL12aDiaL19NHS76IL2GMCSF-adjusted expression vector The gene coding sequences of mIL12bIL12aDiaL19NHS76IL2GMCSF were synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII, and XhoI or EcoRI. The digestion system consisted of adding 5 μg of mIL12bIL12aDiaL19NHS76IL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water to make a total volume of 40 μl, and leaving it at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aDiaL19NHS76IL2GMCSF gene fragment was recovered and used.

[0230] The amino acid sequence of the mIL12bIL12aDiaL19NHS76IL2GMCSF protein molecule is shown in SEQ ID NO.56, and the nucleotide sequence encoding the said mIL12bIL12aDiaL19NHS76IL2GMCSF is shown in SEQ ID NO.97.

[0231] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make a total volume of 30 μl, and left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0232] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaL19NHS76IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaL19NHS76IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaL19NHS76IL2GMCSF-PGK-PURO was successful.

[0233] 11.2 Preparation of cells that regulatively express mIL12bIL12aDiaL19NHS76IL2GMCSF The virus of the mIL12bIL12aDiaL19NHS76IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaL19NHS76IL2GMCSF-PGK-PURO.

[0234] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second modified expression vector pLentis-PTRE-mIL12bIL12aDiaL19NHS76IL2GMCSF-PGK-PURO was added. After culturing in an incubator for 24 hours, the supernatant was discarded and replaced with fresh medium. When the cells had completely grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After culturing for 3 days, the surviving cells were those capable of controlling the expression of mIL12bIL12aDiaL19NHS76IL2GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aDiaL19NHS76IL2GMCSF.

[0235] 11.3 Effect of induced expression of mIL12bIL12aDiaL19NHS76IL2GMCSF on tumor growth The B16(rtTA)-mIL12bIL12aDiaL19NHS76IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and tumors were grown using water containing 2 g / L. Then, 50 μl each, for a total of 10 injections, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 28, mIL12bIL12aDiaL19NHS76IL2GMCSF was able to induce tumor regression in some mice.

[0236] Example 12 Effect of induced expression of mIL12bIL12aDiaL19F8IL2GMCSF on tumor growth 12.1 Construction of the mIL12bIL12aDiaL19F8IL2GMCSF-adjusted expression vector Digestion sites for BamHI, BglII, XhoI, and EcoRI were synthesized at both ends of the coding sequence of the mIL12bIL12aDiaL19F8IL2GMCSF gene, and digestion was performed with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of the mIL12bIL12aDiaL19F8IL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water added to a total volume of 40 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the mIL12bIL12aDiaL19F8IL2GMCSF gene fragment was recovered and used.

[0237] The amino acid sequence of the mIL12bIL12aDiaL19F8IL2GMCSF protein molecule is shown in SEQ ID NO.57, and the nucleotide sequence encoding the mIL12bIL12aDiaL19F8IL2GMCSF is shown in SEQ ID NO.98.

[0238] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, with water added to a total volume of 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0239] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aDiaL19F8IL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aDiaL19F8IL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aDiaL19F8IL2GMCSF-PGK-PURO was successful.

[0240] 12.2 Preparation of cells that regulatively express mIL12bIL12aDiaL19F8IL2GMCSF The virus of the mIL12bIL12aDiaL19F8IL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aDiaL19F8IL2GMCSF-PGK-PURO was obtained.

[0241] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second control expression vector pLentis-PTRE-mIL12bIL12aDiaL19F8IL2GMCSF-PGK-PURO was added, and the culture was continued in an incubator. After 24 hours, the supernatant was discarded, replaced with fresh medium, and the culture was continued. When the cells had completely grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added, and the culture was continued for 3 days. The surviving cells were those capable of controlling the expression of mIL12bIL12aDiaL19F8IL2GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aDiaL19F8IL2GMCSF.

[0242] 12.3 Effect of Inducible Expression of mIL12bIL12aDiaL19F8IL2GMCSF on Tumor Growth The B16(rtTA)-mIL12bIL12aDiaL19F8IL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and tumors were grown using water containing 2 g / L doxycycline. Then, 50 μl each, a total of 10, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. As shown in Figure 29, mIL12bIL12aDiaL19F8IL2GMCSF was able to induce tumor regression in some mice.

[0243] Example 13 Effect of Inducible Expression of mIL12bIL12aIL2DiaNHS76F8GMCSF on Tumor Growth 13.1 Construction of the mIL12bIL12aIL2D2DiaNHS76F8GMCSF Control Expression Vector The coding sequences of mIL12bIL12aDiaL19F8IL2GMCSF genes were synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of mIL12bIL12aIL2DiaNHS76F8GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 40 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. The mIL12bIL12aIL2DiaNHS76F8GMCSF gene fragment was recovered after electrophoresis and used.

[0244] The amino acid sequence of the mIL12bIL12aIL2DiaNHS76F8GMCSF protein molecule is shown in SEQ ID NO.58, and the nucleotide sequence encoding the mIL12bIL12aIL2DiaNHS76F8GMCSF is shown in SEQ ID NO.99.

[0245] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. The pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0246] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL2DiaNHS76F8GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL2DiaNHS76F8GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-PTRE-mIL12bIL12aIL2DiaNHS76F8GMCSF-PGK-PURO was successful.

[0247] 13.2 Preparation of cells that regulatively express mIL12bIL12aIL2DiaNHS76F8GMCSF The virus of the mIL12bIL12aIL2D2DiaNHS76F8GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL2DiaNHS76F8GMCSF-PGK-PURO was obtained.

[0248] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second modified expression vector pLentis-PTRE-mIL12bIL12aIL2DiaNHS76F8GMCSF-PGK-PURO was added, and the culture was continued in an incubator as follows. After 24 hours of culture, the supernatant was discarded and replaced with fresh medium to continue the culture. When the cells were fully grown, they were passaged into a culture flask, and puromycin at a final concentration of 3 μg / ml was added. After 3 days of continuous culture, the surviving cells were those capable of controlling the expression of mIL12bIL12aIL2DiaNHS76F8GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aIL2DiaNHS76F8GMCSF.

[0249] 13.3 Effect of induced expression of mIL12bIL12aIL2DiaNHS76F8GMCSF on tumor growth The B16(rtTA)-mIL12bIL12aIL2DiaNHS76F8GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8-10-week-old C57BL / 6 female mice using a 1 ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 30. The induction of mIL12bIL12aIL2DiaNHS76F8GMCSF expression significantly increased the survival rate of the mice.

[0250] Example 14 Effect of induced expression of mIL12bIL12aIL2DiaF8GMCSF on tumor growth 14.1 Construction of the mIL12bIL12aIL2DiaF8GMCSF-adjusted expression vector The coding sequences of the mIL12bIL12aIL2DiaF8GMCSF genes were synthesized with the enzyme cleavage sites of BamHI, BglII, XhoI, and EcoRI at both ends, and then digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of the mIL12bIL12aIL2DiaNHS76F8GMCSF plasmid, 4 μl of the enzyme cleavage buffer, 1 μl of BamHI, 1 μl of XhoI, and water added to make a total volume of 40 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the mIL12bIL12aIL2DiaF8GMCSF gene fragment was recovered and used.

[0251] The amino acid sequence of the mIL12bIL12aIL2DiaF8GMCSF protein molecule is shown in SEQ ID NO.59, and the nucleotide sequence encoding the mIL12bIL12aIL2DiaF8GMCSF is shown in SEQ ID NO.100.

[0252] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of the digestion buffer, 1 μl of BamHI, 1 μl of XhoI, water was added to make a total volume of 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0253] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL2DiaF8GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL2DiaF8GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the E. coli receptor state. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-mIL12bIL12aIL2DiaF8GMCSF-PGK-PURO was successful.

[0254] 14.2 Preparation of cells that regulatively express mIL12bIL12aIL2DiaF8GMCSF The virus of the mIL12bIL12aIL2DiaF8GMCSF expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL2DiaF8GMCSF-PGK-PURO.

[0255] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above-described second control expression vector pLentis-PTRE-mIL12bIL12aIL2DiaF8GMCSF-PGK-PURO virus was added, and the cells were further cultured in an incubator for 24 hours. After 24 hours of culture, the supernatant was discarded and replaced with fresh medium. When the cells had completely grown, they were passaged into a culture flask, and puromycin at a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the surviving cells were those capable of controlling the expression of mIL12bIL12aIL2DiaF8GMCSF, and they were named B16(rtTA)-mIL12bIL12aIL2DiaF8GMCSF.

[0256] 14.3 Effect of Induced Expression of mIL12bIL12aIL2DiaF8GMCSF on Tumor Growth The B16(rtTA)-mIL12bIL12aIL2DiaF8GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 31. The induced expression of mIL12bIL12aIL2DiaF8GMCSF significantly increased the survival rate of the mice.

[0257] Example 15 Effect of Induced Expression of mIL12bIL12aIL2GMCSFDiaNHS76F8 on Tumor Growth 15.1 Construction of mIL12bIL12aIL2GMCSFDiaNHS76F8 Control Expression Vector The coding sequence of the mIL12bIL12aIL2GMCSFDiaNHS76F8 gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of 5 μg of the mIL12bIL12aIL2GMCSFDiaNHS76F8 plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water added to make a total volume of 40 μl, which was left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL2GMCSFDiaNHS76F8 gene fragment was recovered and used.

[0258] The amino acid sequence of the mIL12bIL12aIL2GMCSFDiaNHS76F8 protein molecule is shown in SEQ ID NO.60, and the nucleotide sequence encoding the mIL12bIL12aIL2GMCSFDiaNHS76F8 is shown in SEQ ID NO.101.

[0259] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water added to make a total volume of 30 μl, which was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0260] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL2GMCSFDiaNHS76F8 were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL2GMCSFDiaNHS76F8, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-mIL12bIL12aIL2GMCSFDiaNHS76F8-PGK-PURO was successful.

[0261] 15.2 Preparation of cells that regulatively express mIL12bIL12aIL2GMCSFDiaNHS76F8 The virus of the mIL12bIL12aIL2GMCSFDiaNHS76F8 expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL2GMCSFDiaNHS76F8-PGK-PURO.

[0262] 10 μl of the above virus of mIL12bIL12aIL2GMCSFDiaNHS76F8-PGK-PURO was added, and the culture was continued in the incubator as follows. After culturing for 24 hours, the supernatant was discarded and replaced with fresh medium, and the culture was continued. After the cells had completely grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After continuing the culture for 3 days, the surviving cells were cells that could control the expression of mIL12bIL12aIL2GMCSFDiaNHS76F8, and these cells were named B16(rtTA)-mIL12bIL12aIL2GMCSFDiaNHS76F8.

[0263] 15.3 Effect of Induced Expression of mIL12bIL12aIL2GMCSFDiaNHS76F8 on Tumor Growth B16(rtTA)-mIL12bIL12aIL2GMCSFDiaNHS76F8 cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and injected into the right dorsal side of 8-10-week-old C57BL / 6 female mice at 50 μl per mouse using a 1 ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 32. The induced expression of mIL12bIL12aIL2GMCSFDiaNHS76F8 significantly increased the survival rate of the mice.

[0264] Example 16 Effect of Induced Expression of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr on Tumor Growth 16.1 Construction of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr Regulatory Expression Vector Linker 1 between IL2 and DiaNHS76F8 (the amino acid sequence is shown in SEQ ID NO.114) was replaced with linker 2 (the amino acid sequence is shown in SEQ ID NO.115), and linker 3 between DiaNHS76F8 and GMCSF (the amino acid sequence is shown in SEQ ID NO.116) was replaced with linker 4 (the amino acid sequence is shown in SEQ ID NO.116). Using the amino acid sequence shown in SEQ ID NO.117, the amino acid sequence of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr with thrombin cleavage sites in linker 2 and linker 4 was obtained.

[0265] The sequence encoding the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr gene was synthesized to have BamHI, BglII, XhoI, and EcoRI digestion sites at each end. After digestion with BamHI or BglII and XhoI or EcoRI, 5 μg of the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr plasmid, 4 μl of enzyme digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water were added to make a total volume of 40 μl, and the mixture was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr gene fragment was recovered and used.

[0266] The amino acid sequence of the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr protein molecule is shown in SEQ ID NO.70, and the nucleotide sequence encoding the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr is shown in SEQ ID NO.111.

[0267] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water was added to make a total volume of 30 μl, and the mixture was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0268] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and after confirming by enzymatic cleavage whether the fragment was ligated to the vector correctly, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector, pLentis-mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr-PGK-PURO, was successful.

[0269] 16.2 Preparation of cells that regulatively express mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr The virus of the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr-PGK-PURO.

[0270] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the above virus of the second modified expression vector pLentis-PTRE-mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr-PGK-PURO was added during the culture. After continuous culture in an incubator for 24 hours, the supernatant was discarded and replaced with fresh medium, and the culture was continued. When the cells had completely grown, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. After continuous culture for 3 days, the surviving cells were those capable of controlling the expression of IL12bIL12aIL2DiaNHS76F8GMCSF-Thr. These cells were named B16(rtTA)-mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr.

[0271] 16.3 Effect of induced expression of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr on tumor growth B16(rtTA)-mIL12bIL12aIL2GMCSFDiaNHS76F8 cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and after tumors grew using water containing 2 g / L of a potent component, 50 μl per mouse, for a total of 10 mice, were injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. Induced expression of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr significantly improved the survival rate of the mice, as shown in Figure 33.

[0272] Example 17 Construction of cells expressing hIL12bIL12aDiaL19GMCSFIL2, hIL12bIL12aDiaNHS76GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2, hIL12bIL12aIL2DiaNHS76F8GMCSF, hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr 17.1 Construction of a vector for controlling the expression of a target gene The vector pLentis-CMV-MCS-IRES-PURO was digested in an EP tube with the following system: 2 μg of pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to a total volume of 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector fragment was recovered and used.

[0273] Linker 1 between IL2 and DiaNHS76F8 (the amino acid sequence is shown in SEQ ID NO.114) was replaced with linker 2 (the amino acid sequence is shown in SEQ ID NO.115), and linker 3 between DiaNHS76F8 and GMCSF (the amino acid sequence is shown in SEQ ID NO.116) was replaced with linker 4 (the amino acid sequence is shown in SEQ ID NO.117) to obtain the amino acid sequence of hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr having thrombin cleavage sites in linker 2 and linker 4.

[0274] Synthesis of hIL12bIL12aDiaL19GMCSFIL2, hIL12bIL12aDiaNHS76GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2, hIL12bIL12aIL2DiaNHS76F8GMCSF, hIL12bIL12aIL2DiaNHS76F8GMCSF, and hIL12bIL12aILDiaNHS76F8GMCSF, respectively. The DNA sequence of hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr is synthesized by adding an enzyme cleavage site of BamHI or BglII to its 5'-end and an enzyme cleavage site of XhoI or EcoRI to its 3'-end. A plasmid having the target gene was synthesized in a system where 5 μg of plasmid, 4 μl of enzyme cleavage buffer, 1 μl of BamHI, and 1 μl of XhoI were added with water to make a total volume of 40 μl and left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the fragment was recovered and used.

[0275] The amino acid sequence of the hIL12bIL12aDiaL19GMCSFIL2 protein molecule is shown in SEQ ID NO.67, and the nucleotide sequence encoding the hIL12bIL12aDiaL19GMCSFIL2 is shown in SEQ ID NO.108.

[0276] The amino acid sequence of the hIL12bIL12aDiaNHS76GMCSFIL2 protein molecule is shown in SEQ ID NO.68, and the nucleotide sequence encoding the hIL12bIL12aDiaNHS76GMCSFIL2 is shown in SEQ ID NO.109.

[0277] The amino acid sequence of the hIL12bIL12aDiaNHS76F8GMCSFIL2 protein molecule is shown in SEQ ID NO.69, and the nucleotide sequence encoding the hIL12bIL12aDiaNHS76F8GMCSFIL2 is shown in SEQ ID NO.110.

[0278] The amino acid sequence of the hIL12bIL12aIL2DiaNHS76F8GMCSF protein molecule is shown in SEQ ID NO.65, and the nucleotide sequence encoding the hIL12bIL12aIL2DiaNHS76F8GMCSF is shown in SEQ ID NO.106.

[0279] The amino acid sequence of the hIL12bIL12aIL2D2DiaNHS76F8GMCSF-Thr protein molecule is shown in SEQ ID NO.71, and the nucleotide sequence encoding the hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr is shown in SEQ ID NO.112.

[0280] pLentis-CMV-MCS-IRES-PURO, hIL12bIL12aDiaL19GMCSFIL2, hIL12bIL12aDiaNHS76GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2, hIL12bIL12aDiaNHS76F8GMCSFIL2, hIL12bIL12aIL2DiaNHS76F8GMCSF, and hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr were each ligated as follows: 2 μl of pLentis-CMV-MCS-IRES-PURO, 2 μl of the gene fragment, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of E. coli. The next day, colonies were picked from the transformed plates and cultured overnight at 37°C with a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing to determine the success of the construct, and the vector pLentis-CMV expressing the target gene was obtained. hIL12bIL12aDiaL19GMCSFIL2-IRES-PURO, vector pLentis-CMV-hIL12bIL12aDiaNHS76GMCSFIL2-IRES-PURO, vector pLentis-CMV-. hIL12bIL12aDiaNHS76F8GMCSFIL2-IRES-PURO, vector pLentis-CMV-hIL12bIL12aIL2DiaNHS76F8GMCSF-IRES-PURO, vector pLentis-CMV-. hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr-IRES-PURO.

[0281] 17.2 Preparation of Virus for Expression Vector 1) The digested and cultured 293FT cells were counted, and 3×106 cells / well were spread on 10-cm culture dishes and divided into 5 plates with a culture volume of 10 ml. 2) On the night of the next day, observe the cell state. If the state is good, perform transfection. After making up G 6 μg + pSPAX2 15 μg + expression vector 20 μg to a total of 1045 μl, add 155 μl of 2 M CaCl2, mix well, and finally add 1200 μl of 2×HBS and add dropwise while shaking. After adding the drops, quickly add the mixed solution to the cell culture well and gently shake to mix. 3) On the morning of the third day, observe the cells and replace the medium with 10 ml of fresh DMEM medium. 4) On the morning of the fifth day, observe the cell state, collect the supernatant from the culture dish, filter it through a 0.45 μm filter, then put it into a high-speed centrifuge tube and centrifuge at 50,000 g for 2 hours. Carefully discard the supernatant, aspirate as much liquid as possible with blotting paper, then resuspend the precipitate in 200 μl of HBSS, dissolve it over 2 hours, divide it into small tubes and store at -70 °C.

[0282] 17.3 Transfection of 293A cells using the expression virus The digested cultured 293A cells were inoculated into a 6-well plate at a culture volume of 1 ml to a density of 105 cells / well. After 24 hours, add 10 μl of the virus expressing the above target gene, continue culturing in the incubator for 24 hours, then discard the supernatant, replace it with fresh medium and continue culturing. After the cells grew completely, passage them into a culture flask, add puromycin at a final concentration of 3 μg / ml and continue culturing. Replace the medium every 2 days to maintain the puromycin concentration. After one week of screening, the surviving cells are cells that stably express the above cytokines, and were named 293A(hIL12bIL12aDiaL19GMCSFIL2), 293A(hIL12bIL12aDiaNHS76GMCSFIL2), 293A(hIL12bIL12aDiaNHS76F8GMCSFIL2), 293A(hIL12bIL12aIL2DiaNHS76F8GMCSF), 293A(hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr) respectively.

[0283] The constructed expression cells were seeded at 5×104 cells per well in a 24-well plate and cultured for 96 hours. The supernatant was collected and the expression of protein molecules in the supernatant was detected using a Human IL12p70 ELISA kit operated according to the instructions. As shown in Figure 34, all of these cells successfully constructed cells expressing protein molecules and were able to produce a large amount of IL12p70.

[0284] Example 18 Expression of Double-stranded Fusion Protein mIL12aIL2-IL12bGMCSF 18.1 Construction of Expression Vector pLentis-CMV-mIL12aIL2-IL12bGMCSF-IRES-PURO The structure of the double-stranded fusion protein is shown in Figure 35. The coding sequence of the mIL12aIL2-IL12bGMCSF gene was synthesized, and the coding sequences of mIL12aIL2 and mIL12bGMCSF were ligated with the self-cleaving peptide T2A that would be cleaved into two peptide chains after expression. The synthesized IL12bGMCSF with BamHI and XhoI enzyme sites at the front and rear ends of the double-stranded fusion protein mIL12aIL2-IL12bGMCSF sequence was incorporated into the plasmid with the target gene in the following system: 5 μg mIL12aIL2-IL12bGMCSF plasmid, 4 μl of enzyme cleavage buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make a total volume of 40 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the fragment was recovered and used.

[0285] The amino acid sequence of the double-stranded fusion protein mIL12aIL2-IL12bGMCSF is shown in SEQ ID NO.72 and SEQ ID NO.118, and the nucleotide sequence encoding the mIL12aIL2-IL12bGMCSF is shown in SEQ ID NO.113.

[0286] The vector pLentis-CMV-MCS-IRES-PURO was placed in an EP tube and digested with the following system: 2 μg of pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI were added to water to a total volume of 30 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector fragment was recovered and used.

[0287] mIL12aIL2-IL12bGMCSF and pLentis-CMV-MCS-IRES-PURO were ligated as follows: 2 μl of pLentis-CMV-MCS-IRES-PURO vector fragment, 2 μl of gene fragment, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. The plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and it was confirmed by enzyme digestion whether the fragment was successfully ligated to the vector, and the correct vector nucleotide sequence was determined to confirm the success of the construction. The expression vector pLentis-CMV-mIL12aIL2-IL12bGMCSF-IRES-PURO was obtained.

[0288] 18.2 Preparation of the expression virus 1) The digested and cultured 293FT cells were counted, 3×106 cells / well were spread on a 10 cm culture dish, and 10 ml of culture solution was added. 2) On the night of the next day, observe the cell state. If the state is good, perform transfection. After making up G 6 μg + pSPAX2 15 μg + expression vector 20 μg to a total of 1045 μl, add 155 μl of 2 M CaCl2, mix well, and finally add 1200 μl of 2×HBS and add dropwise while shaking. After adding the drops, quickly add the mixture to the cell culture well and gently shake to mix. 3) On the morning of the third day, observe the cells and replace the medium with 10 ml of fresh DMEM medium.

[0289] 4) On the morning of the fifth day, observe the cell state, collect the supernatant from the culture dish, filter it through a 0.45 μm filter, then put it into a high-speed centrifuge tube and centrifuge at 50,000 g for 2 hours. Carefully discard the supernatant, aspirate as much liquid as possible with blotting paper, then resuspend the precipitate in 200 μl of HBSS, dissolve it over 2 hours, divide it into small tubes and store at -70 °C.

[0290] 18.3 Preparation of expressing cells The digested cultured 293A cells were inoculated into a 6-well plate at a cell density of 105 cells / well with a culture volume of 1 ml. After 24 hours, add 10 μl of the virus expressing the above target gene, continue culturing in the incubator for 24 hours, then discard the supernatant, replace it with fresh medium and continue culturing. When the cells are fully grown, transfer them to a culture flask, add puromycin at a final concentration of 3 μg / ml and continue culturing, and change the medium every 2 days to maintain the puromycin concentration. After one week of screening, the surviving cells are cells that stably express the described protein, and were named 293A-mIL12aIL2-IL12bGMCSF.

[0291] Spread the constructed expressing cells at 5×104 cells per well in a 24-well plate, culture for 96 hours, then collect the supernatant, and detect the expression level of the fusion protein in the supernatant using a Human IL12p70 ELISA kit operated according to the instructions. The results of the expression assay are shown in Figure 36.

[0292] Example 19 Expression of Protein Molecule mIL12bIL12aIL2GMCSF 19.1 Construction of Expression Vector The protein molecule mIL12bIL12aIL2GMCSF is schematically shown in Fig. 35. The DNA sequences corresponding to the synthetic genes are each linked to the cytokine by a linker 1 (the amino acid sequence of which is shown in SEQ ID NO. 114), forming a long fusion peptide chain with an IgG signal peptide at the front end of the fusion gene and enzyme cleavage sites of BamHI and XhoI at the front and rear ends of the synthetic sequence, respectively. The enzymatic assembly of the plasmid with the target gene was carried out by adding 5 μg of the plasmid, 4 μl of the enzyme digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water to make a total volume of 40 μl, and leaving it at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was carried out on a 1% agarose gel. After electrophoresis, the fragment was recovered and used.

[0293] The vector pLentis-CMV-MCS-IRES-PURO was placed in an EP tube and digested with the following system: 2 μg of the pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 μl of the digestion buffer, 1 μl of BamHI and 1 μl of XhoI were added to water to make a total volume of 30 μl, and left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was carried out on a 1% agarose gel. After electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector fragment was recovered and used.

[0294] The amino acid sequence of the mIL12bIL12aIL2GMCSF protein molecule is shown in SEQ ID NO. 32, and the nucleotide sequence encoding the mIL12bIL12aIL2GMCSF is shown in SEQ ID NO. 73.

[0295] mIL12bIL12aIL2GMCSF and pLentis-CMV-MCS-IRES-PURO were ligated as follows: 2 μl of pLentis-CMV-MCS-IRES-PURO vector fragment, 2 μl of gene fragment, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacteria using a plasmid extraction kit, and it was confirmed by enzymatic digestion whether the fragment was successfully ligated to the vector, and the nucleotide sequence of the correct vector was determined to confirm the success of the construction. The expression vector pLentis-CMV-mIL12bIL12aIL2GMCSF-IRES-PURO was obtained.

[0296] 19.2 Preparation of the expression virus 1) The digested and cultured 293FT cells were counted, and 3×106 cells / well were spread on a 10 cm culture dish and 10 ml of culture medium was added. 2) On the night of the next day, the cell state was observed, and if it was in good condition, transfection was performed. After making a total of 1045 μl of G 6 μg + pSPAX2 15 μg + expression vector 20 μg), 155 μl of 2 M CaCl2 was added and mixed well. Finally, 1200 μl of 2×HBS was added and dropped while shaking. After adding the drops, the mixed solution was quickly added to the cell culture well and gently shaken to mix. 3) On the morning of the third day, the cells were observed and the medium was replaced with 10 ml of fresh DMEM medium. 4) On the morning of the fifth day, the cell state was observed, the supernatant was collected from the culture dish, filtered through a 0.45 μm filter, then placed in a high-speed centrifuge tube and centrifuged at 50,000 g for 2 hours. The supernatant was carefully discarded, and as much liquid as possible was aspirated with blotting paper. Then, the precipitate was resuspended in 200 μl of HBSS, dissolved over 2 hours, aliquoted into small tubes, and stored at -70°C.

[0297] 19.3 Preparation of Expressed Cells The digested cultured 293A cells were inoculated into a 6-well plate at a culture volume of 1 ml to a density of 105 cells / well. After 24 hours, 10 μl of the virus expressing the target gene was added, and the culture was continued in an incubator for 24 hours. Then, the supernatant was discarded, replaced with fresh medium, and the culture was continued. When the cells had completely grown, they were transferred to a culture flask, puromycin was added at a final concentration of 3 μg / ml, and the culture was continued, with the medium being changed every two days to maintain the puromycin concentration. After one week of screening, the surviving cells were cells that stably expressed the described protein, and were named 293A-mIL12bIL12aIL2GMCSF.

[0298] The constructed expressed cells were spread at 5×104 cells per well in a 24-well plate and cultured for 96 hours. The supernatant was collected, and the expression of protein molecules in the supernatant was detected using a Human IL12p70 ELISA kit operated according to the instructions. The results of the expression test are shown in Figure 36. The yield of the protein molecule of the present invention was significantly higher than that of the double-stranded fusion protein.

[0299] Example 20 Effect of Induced Expression of mIL12bIL12aIL2GMCSF on Tumor Growth 20.1 Construction of mIL12bIL12aIL2GMCSF Regulated Expression Vector The coding sequence of the mIL12bIL12aIL2GMCSF gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was prepared by adding 5 μg of mIL12bIL12aIL2GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, adding water to make the total volume 40 μl, and leaving it at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL2GMCSF gene fragment was recovered and used.

[0300] The amino acid sequence of the mIL12bIL12aIL2GMCSF protein molecule is shown in SEQ ID NO.32, and the nucleotide sequence encoding the mIL12bIL12aIL2GMCSF is shown in SEQ ID NO.73.

[0301] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to make the total volume 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0302] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL2GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL2GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. The plasmid was extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzyme digestion whether the fragment was ligated to the vector normally, and the nucleotide sequence of the correct vector was determined to confirm the successful construction of the second expression vector pLentis-mIL12bIL12aIL2GMCSF-PGK-PURO.

[0303] 20.2 Preparation of cells that regulatively express mIL12bIL12aIL2GMCSF The virus of the mIL12bIL12aIL2GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis - PTRE - mIL12bIL12aIL2GMCSF - PGK - PURO was obtained.

[0304] Digested and cultured B16(rtTA) tumor cells were inoculated into a 6 - well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above - mentioned second - modified expression vector pLentis - PTRE - mIL12bIL12aIL2GMCSF - PGK - PURO was added, and the culture was continued in an incubator for 24 hours and then discarded. The supernatant was replaced with fresh medium and the culture was continued. When the cells grew larger, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the cells that could be controlled to express mIL12bIL12aIL2GMCSF survived, and these cells were named B16(rtTA) - mIL12bIL12aIL2GMCSF.

[0305] 20.3 Influence of the induced expression of mIL12bIL12aIL2GMCSF on tumor growth B16(rtTA) - mIL12bIL12aIL2GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8 - 10 - week - old C57BL / 6 female mice using a 1 - ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 37. The induced expression of mIL12bIL12aIL2GMCSF significantly increased the survival rate of the mice.

[0306] Example 21 Effect of the induced expression of mIL12bIL12aGMCSFIL2 on tumor growth 21.1 Construction of the mIL12bIL12aGMCSFIL2 regulated - expression vector The coding sequence of the mIL12bIL12aGMCSFIL2 gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was added with 5 μg of the mIL12bIL12aGMCSFIL2 plasmid, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and water was added to make the total volume 40 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aGMCSFIL2 gene fragment was recovered and used.

[0307] The amino acid sequence of the mIL12bIL12aGMCSFIL2 protein molecule is shown in SEQ ID NO.40, and the nucleotide sequence encoding the mIL12bIL12aGMCSFIL2 is shown in SEQ ID NO.81.

[0308] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to make the total volume 30 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0309] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aGMCSFIL2 were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aGMCSFIL2, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-mIL12bIL12aGMCSFIL2-PGK-PURO was successful.

[0310] 21.2 Preparation of cells that regulatively express mIL12bIL12aGMCSFIL2 The virus of the mIL12bIL12aGMCSFIL2 expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aGMCSFIL2-PGK-PURO was obtained.

[0311] Digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-mIL12bIL12aGMCSFIL2-PGK-PURO was added, and the culture was continued in an incubator for 24 hours and then discarded. The supernatant was replaced with fresh medium and the culture was continued. When the cells grew larger, they were subcultured into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the cells that could be controlled to express mIL12bIL12aGMCSFIL2 survived, and these cells were named B16(rtTA)-mIL12bIL12aGMCSFIL2.

[0312] 21.3 Effect of Induced Expression of mIL12bIL12aGMCSFIL2 on Tumor Growth B16(rtTA)-mIL12bIL12aGMCSFIL2 cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1-ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 38. The induced expression of mIL12bIL12aGMCSFIL2 significantly increased the survival rate of mice.

[0313] Example 22 Effect of Induced Expression of mIL12bIL12aIL7GMCSF on Tumor Growth 22.1 Construction of mIL12bIL12aIL7GMCSF Regulated Expression Vector The coding sequence of the mIL12bIL12aIL7GMCSF gene was synthesized, and digestion sites for BamHI, BglII, XhoI, and EcoRI were provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was added with 5 μg of mIL12bIL12aIL7GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and water was added to make the total volume 40 μl, and left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL7GMCSF gene fragment was recovered and used.

[0314] The amino acid sequence of the mIL12bIL12aIL7GMCSF protein molecule is shown in SEQ ID NO.33, and the nucleotide sequence encoding the mIL12bIL12aIL7GMCSF is shown in SEQ ID NO.74.

[0315] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make a total volume of 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0316] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL7GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL7GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the E. coli receptor state. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacteria using a plasmid extraction kit, and it was identified by an enzyme digestion method whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and the construction of the second expression vector pLentis-mIL12bIL12aIL7GMCSF-PGK-PURO was successful.

[0317] 22.2 Preparation of cells that regulate the expression of mIL12bIL12aIL7GMCSF The virus of the mIL12bIL12aIL7GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL7GMCSF-PGK-PURO was obtained.

[0318] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-mIL12bIL12aIL7GMCSF-PGK-PURO was added, and the culture was continued in an incubator for 24 hours and then discarded. The supernatant was replaced with fresh medium and the culture was continued. When the cells grew larger, they were passaged into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the cells that could be controlled to express mIL12bIL12aIL7GMCSF survived, and these cells were named B16(rtTA)-mIL12bIL12aIL7GMCSF.

[0319] 22.3 Effect of induced expression of mIL12bIL12aIL7GMCSF on tumor growth The B16(rtTA)-mIL12bIL12aIL7GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice at 50 μl / mouse using a 1 ml syringe. After tumor growth, 10 mice were fed with water containing 2 g / L doxycycline, and the survival rate was recorded as shown in Figure 39.

[0320] Example 23 Effect of induced expression of mIL12bIL12aIL15GMCSF on tumor growth 23.1 Construction of mIL12bIL12aIL15GMCSF regulated expression vector The coding sequences of mIL12bIL12aIL15GMCSF genes were synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was added with 5 μg of mIL12bIL12aIL15GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and water was added to make the total volume 40 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the mIL12bIL12aIL15GMCSF gene fragment was recovered after electrophoresis and used.

[0321] The amino acid sequence of the mIL12bIL12aIL15GMCSF protein molecule is shown in SEQ ID NO.34, and the nucleotide sequence encoding the mIL12bIL12aIL15GMCSF is shown in SEQ ID NO.75.

[0322] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to make the total volume 30 μl, and left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0323] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL15GMCSF were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL15GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-mIL12bIL12aIL15GMCSF-PGK-PURO was successful.

[0324] 23.2 Preparation of cells that regulatively express mIL12bIL12aIL15GMCSF The virus of the mIL12bIL12aIL15GMCSF expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL15GMCSF-PGK-PURO was obtained.

[0325] Digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-mIL12bIL12aIL15GMCSF-PGK-PURO was added, and the culture was continued in an incubator for 24 hours. The supernatant was discarded and replaced with fresh medium to continue the culture. After the cells had completely grown, they were subcultured into a culture flask, and puromycin with a final concentration of 3 μg / ml was added and the culture was continued for 3 days. The surviving cells were cells that could control the expression of mIL12bIL12aIL15GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aIL15GMCS.

[0326] 23.3 Influence of Inducible Expression of mIL12bIL12aIL15GMCSF on Tumor Growth B16(rtTA)-mIL12bIL12aIL15GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1-ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 40. The inducible expression of mIL12bIL12aIL15GMCSF significantly increased the survival rate of the mice.

[0327] Example 24 Effect of Inducible Expression of mIL12bIL12aIL21GMCSF on Tumor Growth 24.1 Construction of mIL12bIL12aIL21GMCSF Regulated Expression Vector The coding sequence of the mIL12bIL12aIL21GMCSF gene was synthesized, and digestion sites for BamHI, BglII, XhoI, and EcoRI were provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was added with 5 μg of mIL12bIL12aIL21GMCSF plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 40 μl, and left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL21GMCSF gene fragment was recovered and used.

[0328] The amino acid sequence of the mIL12bIL12aIL21GMCSF protein molecule is shown in SEQ ID NO.35, and the nucleotide sequence encoding the mIL12bIL12aIL21GMCSF is shown in SEQ ID NO.76.

[0329] The regulated expression vector pLentis - PTRE - MCS - PGK - PURO was digested with the following digestion system: 2 μg of the pLentis - PTRE - MCS - PGK - PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to make the total volume 30 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis - PTRE - MCS - PGK - PURO vector after electrophoresis was recovered and used.

[0330] pLentis - PTRE - MCS - PGK - PURO and mIL12bIL12aIL21GMCSF were ligated as follows: 2 μl of pLentis - PTRE - MCS - PGK - PURO, 2 μl of mIL12bIL12aIL21GMCSF, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzyme digestion whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis - mIL12bIL12aIL21GMCSF - PGK - PURO was successful.

[0331] 24.2 Preparation of cells that regulatively express mIL12bIL12aIL21GMCSF The virus of the mIL12bIL12aIL21GMCSF expression vector was prepared in the same way as the virus of the first expression vector, and the virus of the second expression vector pLentis - PTRE - mIL12bIL12aIL21GMCSF - PGK - PURO was obtained.

[0332] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-described second modified expression vector pLentis-PTRE-mIL12bIL12aIL21GMCSF-PGK-PURO was added, and the culture was continued in an incubator for 24 hours. The supernatant was discarded, replaced with fresh medium, and the culture was continued. When the cells had completely grown, they were subcultured into a culture flask, puromycin was added at a final concentration of 3 μg / ml, and the culture was continued for 3 days. As a result, the surviving cells were those capable of controlling the expression of mIL12bIL12aIL21GMCSF, and these cells were named B16(rtTA)-mIL12bIL12aIL21GMCSF. Effect of Induced Expression of mIL12bIL12aIL21GMCSF on Tumor Growth

[0333] B16(rtTA)-mIL12bIL12aIL21GMCSF cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 41. The induced expression of mIL12bIL12aIL21GMCSF significantly increased the survival rate of the mice.

[0334] Example 25 Effect of Induced Expression of mIL12bIL12aIL2FLT3L on Tumor Growth 25.1 Construction of mIL12bIL12aIL2FLT3L Regulatory Expression Vector The coding sequence of the mIL12bIL12aIL2FLT3L gene was synthesized, with digestion sites for BamHI, BglII, XhoI, and EcoRI provided at both ends, and digested with BamHI or BglII and XhoI or EcoRI. The digestion system was prepared by adding 5 μg of the mIL12bIL12aIL2FLT3L plasmid, 4 μl of the digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and adding water to make the total volume 40 μl, then leaving it at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL2FLT3L gene fragment was recovered and used.

[0335] The amino acid sequence of the mIL12bIL12aIL2FLT3L protein molecule is shown in SEQ ID NO.36, and the nucleotide sequence encoding the mIL12bIL12aIL2FLT3L is shown in SEQ ID NO.77.

[0336] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of the digestion buffer, 1 μl of BamHI and 1 μl of XhoI, adding water to make the total volume 30 μl, and leaving it at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0337] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL2FLT3L were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL2FLT3L, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by enzymatic digestion whether the fragment was ligated to the vector normally, and the nucleotide sequence of the correct vector was determined to confirm the successful construction of the second expression vector pLentis-mIL12bIL12aIL2FLT3L-PGK-PURO.

[0338] 25.2 Preparation of cells that regulatively express mIL12bIL12aIL2FLT3L The virus of the mIL12bIL12aIL2FLT3L expression vector was prepared in the same manner as the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL2FLT3L-PGK-PURO.

[0339] Digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-mIL12bIL12aIL2FLT3L-PGK-PURO was added, and the culture was continued in an incubator for 24 hours and then discarded. The supernatant was replaced with fresh medium and the culture was continued. When the cells grew larger, they were subcultured into a culture flask, and puromycin with a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the cells that could be controlled to express mIL12bIL12aIL2FLT3L survived, and these cells were named B16(rtTA)-mIL12bIL12aIL2FLT3L.

[0340] 25.3 Effect of Induced Expression of mIL12bIL12aIL2FLT3L on Tumor Growth B16(rtTA)-mIL12bIL12aIL2FLT3L cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1-ml syringe. After tumor growth, 10 mice were fed with water containing 2 g / L doxycycline, and the survival rate was recorded as shown in Figure 42. It was found that the induced expression of mIL12bIL12aIL2FLT3L significantly increased the survival rate of mice.

[0341] Example 26 Effect of Induced Expression of mIL12bIL12aIL7FLT3L on Tumor Growth 26.1 Construction of mIL12bIL12aIL7FLT3L Regulated Expression Vector The coding sequence of the mIL12bIL12aIL7FLT3L gene was synthesized to have BamHI, BglII and XhoI, EcoRI digestion sites at both ends, and the following digestion system for digestion with BamHI or BglII and XhoI or EcoRI: 5 μg of mIL12bIL12aIL7FLT3L plasmid, 4 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI were added, water was added to make the total volume 40 μl, and it was left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL7FLT3L gene fragment was recovered and used.

[0342] The amino acid sequence of the mIL12bIL12aIL7FLT3L protein molecule is shown in SEQ ID NO.37, and the nucleotide sequence encoding the mIL12bIL12aIL7FLT3L is shown in SEQ ID NO.78.

[0343] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, water was added to a total volume of 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and the pLentis-PTRE-MCS-PGK-PURO vector after electrophoresis was recovered and used.

[0344] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL7FLT3L were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL7FLT3L, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. The plasmid was extracted from the cultured bacterial cells using a plasmid extraction kit, and it was identified by the enzyme digestion method whether the fragment was successfully ligated to the vector. The correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-mIL12bIL12aIL7FLT3L-PGK-PURO was successful.

[0345] 26.2 Preparation of cells that regulatively express mIL12bIL12aIL7FLT3L The virus of the mIL12bIL12aIL7FLT3L expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL7FLT3L-PGK-PURO was obtained.

[0346] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-described second modified expression vector pLentis-PTRE-mIL12bIL12aIL7FLT3L-PGK-PURO was added, and the culture was continued in an incubator for 24 hours and then discarded. The supernatant was replaced with fresh medium and the culture was continued. When the cells grew larger, they were subcultured into a culture flask, and puromycin at a final concentration of 3 μg / ml was added. As a result of continuous culture for 3 days, the cells that could be controlled to express mIL12bIL12aIL7FLT3L survived, and these cells were named B16(rtTA)-mIL12bIL12aIL7FLT3L.

[0347] 26.3 Effect of induced expression of mIL12bIL12aIL7FLT3L on tumor growth The B16(rtTA)-mIL12bIL12aIL7FLT3L cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right back of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. After the tumors grew, 10 mice were fed with water containing 2 g / L doxycycline and the data was recorded. As shown in Figure 43, the survival rate of the mice was significantly improved by inducing the expression of mIL12bIL12aIL7FLT3L.

[0348] Example 27 Effect of induced expression of mIL12bIL12aIL15FLT3L on tumor growth 27.1 Construction of the mIL12bIL12aIL15FLT3L controlled expression vector The coding sequence of the mIL12bIL12aIL15FLT3L gene was synthesized, and digestion sites for BamHI, BglII, XhoI, and EcoRI were provided at both ends. It was digested with BamHI or BglII and XhoI or EcoRI. The digestion system was added with 5 μg of the mIL12bIL12aIL15FLT3L plasmid, 4 μl of the digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and water was added to make the total volume 40 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL15FLT3L gene fragment was recovered and used.

[0349] The amino acid sequence of the mIL12bIL12aIL15FLT3L protein molecule is shown in SEQ ID NO.38, and the nucleotide sequence encoding the mIL12bIL12aIL15FLT3L is shown in SEQ ID NO.79.

[0350] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of the digestion buffer, 1 μl of BamHI and 1 μl of XhoI, and water was added to make the total volume 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0351] pLentis - PTRE - MCS - PGK - PURO and mIL12bIL12aIL15FLT3L were ligated as follows: 2 μl of pLentis - PTRE - MCS - PGK - PURO, 2 μl of mIL12bIL12aIL15FLT3L, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase, and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the E. coli receptor state. Then, the ligated system was transformed into the E. coli receptor state. The next day, colonies were picked from the transformed plate and cultured overnight at 37°C on a shaker using LB medium. Plasmids were extracted from the cultured bacterial cells using a plasmid extraction kit. After confirming whether the fragment was successfully ligated to the vector by enzymatic digestion, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis - mIL12bIL12aIL15FLT3L - PGK - PURO was successful.

[0352] 27.2 Preparation of cells that express mIL12bIL12aIL15FLT3L in a regulated manner The virus of the mIL12bIL12aIL15FLT3L expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis - PTRE - mIL12bIL12aIL15FLT3L - PGK - PURO was obtained.

[0353] The digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-mIL12bIL12aIL15FLT3L-PGK-PURO was added, and the culture was continued in an incubator for 24 hours. The supernatant was discarded, replaced with fresh medium, and the culture was continued. When the cells had completely grown, they were subcultured into a culture flask, puromycin was added at a final concentration of 3 μg / ml, and the culture was continued for 3 days. The surviving cells were those that could control the expression of mIL12bIL12aIL15FLT3L, and these cells were named B16(rtTA)- and mIL12bIL12aIL15FLT3L.

[0354] 27.3 Effect of Induced Expression of mIL12bIL12aIL15FLT3L on Tumor Growth The B16(rtTA)-mIL12bIL12aIL15FLT3L cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and 50 μl per mouse was injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice using a 1 ml syringe. After tumor growth, water containing 2 g / L doxycycline was given to 10 mice, and the survival rate was recorded as shown in Figure 44. Induction of the expression of mIL12bIL12aIL15FLT3L significantly increased the survival rate of the mice.

[0355] Example 28 Effect of Induced Expression of mIL12bIL12aIL21FLT3L on Tumor Growth 28.1 Construction of mIL12bIL12aIL21FLT3L Regulated Expression Vector The coding sequence of the mIL12bIL12aIL21FLT3L gene was synthesized, and digestion sites for BamHI, BglII, XhoI, and EcoRI were provided at both ends. It was digested with BamHI or BglII and XhoI or EcoRI. The digestion system consisted of adding 5 μg of the mIL12bIL12aIL21FLT3L plasmid, 4 μl of digestion buffer, 1 μl of BamHI, and 1 μl of XhoI, and adding water to make the total volume 40 μl, and leaving it at 37 °C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the mIL12bIL12aIL21FLT3L gene fragment was recovered and used.

[0356] The amino acid sequence of the mIL12bIL12aIL21FLT3L protein molecule is shown in SEQ ID NO.39, and the nucleotide sequence encoding the mIL12bIL12aIL21FLT3L is shown in SEQ ID NO.80.

[0357] The regulated expression vector pLentis-PTRE-MCS-PGK-PURO was digested with the following digestion system: 2 μg of the pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 μl of digestion buffer, 1 μl of BamHI and 1 μl of XhoI, adding water to make the total volume 30 μl, and leaving it at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector was recovered and used.

[0358] pLentis-PTRE-MCS-PGK-PURO and mIL12bIL12aIL21FLT3L were ligated as follows: 2 μl of pLentis-PTRE-MCS-PGK-PURO, 2 μl of mIL12bIL12aIL21FLT3L, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the receptor state of Escherichia coli. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured cells using a plasmid extraction kit, and after confirming by enzyme digestion whether the fragment was successfully ligated to the vector, the correct vector was sent for sequencing, and it was confirmed that the construction of the second expression vector pLentis-mIL12bIL12aIL21FLT3L-PGK-PURO was successful.

[0359] 28.2 Preparation of cells that regulatively express mIL12bIL12aIL21FLT3L The virus of the mIL12bIL12aIL21FLT3L expression vector was prepared in the same manner as the virus of the first expression vector, and the virus of the second expression vector pLentis-PTRE-mIL12bIL12aIL21FLT3L-PGK-PURO was obtained.

[0360] Digested and cultured B16(rtTA) tumor cells were inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml. After 24 hours, 10 μl of the virus of the above-mentioned second modified expression vector pLentis-PTRE-mIL12bIL12aIL21FLT3L-PGK-PURO was added, and the culture was continued in an incubator for 24 hours. The supernatant was discarded, replaced with fresh medium, and the culture was continued. When the cells were fully grown, they were passaged into a culture flask, puromycin was added at a final concentration of 3 μg / ml, and the culture was continued for 3 days. The surviving cells were cells that could control the expression of mIL12bIL12aIL21FLT3L, and these cells were named B16(rtTA)-mIL12bIL12aIL21FLT3L.

[0361] 28.3 Effect of Induced Expression of mIL12bIL12aIL21FLT3L on Tumor Growth B16(rtTA)-mIL12bIL12aIL21FLT3L cells in the logarithmic growth phase were digested, diluted to 2×106 cells / ml with HBSS, and injected into the right dorsal side of 8- to 10-week-old C57BL / 6 female mice at 50 μl per mouse using a 1 ml syringe. After tumor growth, 10 mice were raised using water containing 2 g / L doxycycline, and the survival rate was recorded as shown in Figure 45. Induction of the expression of mIL12bIL12aIL21FLT3L significantly increased the survival rate of the mice.

[0362] Example 29 Intratumoral Injection of mIL12bIL12aIL2GMCSF in Mice with Lung Cancer 29.1 Preparation of mIL12bIL12aIL2GMCSF The constructed mIL12bIL12aIL2GMCSF-expressing cells 293A-mIL12bIL12aIL2GMCSF were subcultured in a 15 cm culture dish. When the cells were completely grown, the culture medium was changed to 30 ml of CDM4HEK293, and after continuous culture for 5 days, the supernatant was collected, filtered through a 0.45 μm filter, and reconstituted with 50 kd AMICON. Ultrafiltration of ULTRA-15 was concentrated 30 times to obtain 1 ml of protein solution, and the protein concentration was measured using an IL12p70 ELISA kit. The fractionated protein was stored at -20°C.

[0363] 29.2 Tumor Treatment Digested and cultured mouse lung cancer cells (LLC), 5×105 cells, were subcutaneously injected into the right half of C57BL / 6 mice, and treatment was started when the tumor length reached approximately 5 mm. 3 μg was taken according to the protein concentration, diluted to 35 μl with sterile water, then 65 μl of glycerol was added, and the mixture was gently blown with a pipette tip while mixing. The prepared protein solution was aspirated with a 29G insulin syringe and slowly injected into the tumor. To prevent the outflow of the solution, the needle was left in place for a while after injection. The injected mice were returned to the cage and the survival rate was recorded. Mice injected with 65% glycerol alone were used as controls. The experimental results are shown in Figure 46. The administration of mIL12bIL12aIL2GMCSF significantly improved the survival rate of mice.

[0364] Construction of cells expressing hIL12bIL12aIL2GMCSF, hIL12bIL12aIL7GMCSF, hIL12bIL12aIL15GMCSF, hIL12bIL12aIL21GMCSF, hIL12bIL12aIL2FLT3L, hIL12bIL12aIL7FLT3L, hIL12bIL12aIL15FLT3L, hIL12bIL12aIL21FLT3L in Example 30

[0365] The vector pLentis-CMV-MCS-IRES-PURO was placed in an EP tube and digested with the following system: 2 μg of plasmid, 3 μl of digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water was added to make a total volume of 30 μl, and it was left at 37 °C for 12 hours. The EP tube was taken out, 3.3 μl of 10× loading buffer was added, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the vector fragment was recovered and used.

[0366] The DNA sequences of hIL12bIL12aIL2GMCSF, hIL12bIL12aIL7GMCSF, hIL12bIL12aIL15GMCSF, hIL12bIL12aIL21GMCSF, hIL12bIL12aIL2FLT3L, hIL12bIL12aIL7FLT3L, hIL12bIL12aIL15FLT3L, and hIL12bIL12aIL21FLT3L were each synthesized, and when synthesizing, a BamHI or BglII enzyme cleavage site was added to the 5'-end and an XhoI or EcoRI enzyme cleavage site was added to the 3'-end. A plasmid having the target gene was formed in a system where 5 μg of plasmid, 4 μl of digestion buffer, 1 μl of BamHI, 1 μl of XhoI, and water were added to make a total volume of 40 μl and left at 37°C for 12 hours. The EP tube was taken out, 4.4 μl of 10× loading buffer was added, electrophoresis was performed on a 1% agarose gel, and after electrophoresis, the fragment was recovered and used.

[0367] The amino acid sequence of the hIL12bIL12aIL2GMCSF protein molecule is shown in SEQ ID NO.41, and the nucleotide sequence encoding the hIL12bIL12aIL2GMCSF is shown in SEQ ID NO.82.

[0368] The amino acid sequence of the hIL12bIL12aIL7GMCSF protein molecule is shown in SEQ ID NO.42, and the nucleotide sequence encoding the hIL12bIL12aIL7GMCSF is shown in SEQ ID NO.83.

[0369] The amino acid sequence of the hIL12bIL12aIL15GMCSF protein molecule is shown in SEQ ID NO.43, and the nucleotide sequence encoding the hIL12bIL12aIL15GMCSF is shown in SEQ ID NO.84.

[0370] The amino acid sequence of the hIL12bIL12aIL21GMCSF protein molecule is shown in SEQ ID NO.44, and the nucleotide sequence encoding the hIL12bIL12aIL21GMCSF is shown in SEQ ID NO.85.

[0371] The amino acid sequence of the hIL12bIL12aIL2FLT3L protein molecule is shown in SEQ ID NO.45, and the nucleotide sequence encoding the hIL12bIL12aIL2FLT3L is shown in SEQ ID NO.86.

[0372] The amino acid sequence of the hIL12bIL12aIL7FLT3L protein molecule is shown in SEQ ID NO.46, and the nucleotide sequence encoding the hIL12bIL12aIL7FLT3L is shown in SEQ ID NO.87.

[0373] The amino acid sequence of the hIL12bIL12aIL15FLT3L protein molecule is shown in SEQ ID NO.47, and the nucleotide sequence encoding the hIL12bIL12aIL15FLT3L is shown in SEQ ID NO.88.

[0374] The amino acid sequence of the hIL12bIL12aIL21FLT3L protein molecule is shown in SEQ ID NO.48, and the nucleotide sequence encoding the hIL12bIL12aIL21FLT3L is shown in SEQ ID NO.89. pLentis-CMV-MCS-IRES-PURO and hIL12bIL12aIL2GMCSF, hIL12bIL12aIL15GMCSF, hIL12bIL12aIL15GMCSF, hIL12bIL12aIL21GMCSF, hIL12bIL12aIL2FLT3L, hIL12bIL12aIL7FLT3L, hIL12bIL12aIL15FLT3L, hIL12bIL12aIL21FLT3L were ligated as follows: 2 μl of pLentis-CMV-MCS-IRES-PURO, 2 μl of gene fragment, 1 μl of ligase buffer, 0.5 μl of T4 DNA ligase and 4.5 μl of water. For ligation, it was left at room temperature for 4 hours. Then, the ligated system was used for the transformation of the E. coli receptor state. The next day, colonies were picked from the transformed plate and cultured overnight at 37 °C on a shaker using LB medium. Plasmids were extracted from the cultured bacteria using a plasmid extraction kit, and it was confirmed by enzymatic digestion whether the fragment was successfully ligated to the vector, and the correct base sequence of the vector was determined to confirm the success of the construction. The viral vectors expressing the target genes are pLentis-CMV-hIL12bIL12aIL2GMCSF-IRES-PURO, pLentis-CMV-hIL12bIL12aIL7GMCSF-IRES-PURO, pLentis-CMV-hIL12bIL12aIL15GMCSF-IRES-PURO, pLentis-CMV-hIL12bIL12aIL15GMCSF-IRES-PURO. IRES-PURO, pLentis-CMV-hIL12bIL12aIL21GMCSF-IRES-PURO, pLentis-CMV-hIL12bIL12aIL2FLT3L-IRES-PURO, pLentis-CMV-hIL12bIL12aIL7FLT3L-.IRES-PURO, pLentis-CMV-hIL12bIL12aIL12aIL15FLT3L-IRES-PURO, and pLentis-CMV-hIL12bIL12aIL21FLT3L-IRES-PURO were obtained.

[0375] The virus used for preparing the expression vector was prepared as follows. 1) The digested and cultured 293FT cells were counted, spread at 3×106 cells / well in a 10-cm culture dish, and divided into five plates with a culture volume of 10 ml. 2) On the next night, the cell state was observed, and if in good condition, transfection was performed. After making a total of 1045 μl of G 6 μg + pSPAX2 15 μg + expression vector 20 μg), 155 μl of 2 M CaCl2 was added and mixed well. Finally, 1200 μl of 2×HBS was added and dropped while shaking. After adding the drops, the mixed solution was quickly added to the cell culture well and gently shaken to mix. 3) On the morning of the third day, the cells were observed and the medium was replaced with 10 ml of fresh DMEM medium. 4) On the morning of the fifth day, the cell state was observed, the supernatant was collected from the culture dish, filtered through a 0.45-μm filter, placed in a high-speed centrifuge tube, centrifuged at 50,000 g for 2 hours, the supernatant was carefully discarded, and as much liquid as possible was aspirated with blotting paper. Then, the precipitate was resuspended in 200 μl of HBSS, dissolved over 2 hours, divided into small tubes, and stored at -70°C.

[0376] The cultured 293A cells were digested and inoculated into a 6-well plate at a density of 105 cells / well with a culture volume of 1 ml, and the 293A cells were transfected with the expression virus as follows. After 24 hours, 10 μl of the expression virus was added, and after continuous culture in an incubator for 24 hours, the supernatant was discarded and replaced with fresh medium for continued culture. After the cells had grown completely, they were passaged into a culture flask, and puromycin at a final concentration of 3 μg / ml was added for continued culture. The medium was changed every two days to maintain the puromycin concentration. After one week of screening, the surviving cells were 293A(hIL12bIL12aIL2GMCSF), 293A(hIL12bIL12aIL7GMCSF), 293A(hIL12bIL12aIL15GMCSF), 293A(hIL12bIL12aIL21GMCSF), 293A(hIL12bIL12aIL2FLT3L), 293A(hIL12bIL12aIL7FLT3L), 293A(hIL12bIL12aIL7FLT3L), 293A(hIL12bIL12aIL15FLT3L), 293A(hIL12bIL12aIL21FLT3L) cells that stably expressed cytokines.

[0377] The constructed expression cells were spread at 5×104 cells per well in a 24-well plate and cultured for 96 hours. The supernatant was collected, and the expression of protein molecules in the supernatant was detected using a Human IL12p70 ELISA kit operated according to the instructions. As a result, the construction of cells expressing the protein molecule of the present invention was successful (shown in Figure 47).

[0378] The foregoing detailed description is for purposes of illustration and example and is not intended to limit the scope of the appended claims. Currently, multiple variations of the embodiments recited in the present application are apparent to those skilled in the art and remain within the scope of the appended patent claims and their equivalent embodiments.

Claims

1. A protein molecule comprising IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor and a second factor, wherein the first factor and the second factor are each independently selected from the group consisting of IL2, GMCSF, IL7, IL15, IL21 and FLT3L, and the IL12a or a functional fragment thereof, the IL12b or a functional fragment thereof, the first factor and the second factor are located on the same polypeptide chain and comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NO:32 to SEQ ID NO:

48.

2. A protein molecule comprising IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor, a second factor, and a targeting moiety, wherein the first factor and the second factor are each independently selected from the group consisting of IL2, GMCSF, IL7, IL15, IL21 and FLT3L, and the IL12a or a functional fragment thereof, the IL12b or a functional fragment thereof, the first factor and the second factor are located on the same polypeptide chain and comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NO:49 to SEQ ID NO:

71.

3. A nucleotide molecule encoding the protein molecule according to claim 1 or 2.

4. A vector comprising the nucleotide molecule according to claim 3.

5. A cell expressing the protein molecule according to claim 1 or 2, or comprising the nucleotide molecule according to claim 3 or the vector according to claim 4.

6. A pharmaceutical composition comprising the protein molecule according to claim 1 or 2, or the nucleotide molecule according to claim 3.

7. The pharmaceutical composition according to claim 6 for the prevention, alleviation, or treatment of tumors.

Images